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Investigating the potential roles of three mammalian traits in female reproductive investment

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Investigating the potential roles of three mammalian traits in female reproductive investment

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Content Investigating the potential roles of three mammalian traits in female reproductive
investment
by
Michael James Lough-Stevens
A Dissertation presented to the
FACULTY OF USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR BIOLOGY)
MAY 2022
Copyright 2022 Michael Lough-Stevens
ii

Acknowledgements

I want to thank all members of the Matthew D. Dean lab past and present for their
advice and support during this seven-year PhD, especially Caleb Ghione, Dr. Sara
Keeble, Dr. Nicholas G. Schultz and first and foremost my advisor, thesis chair and
mentor Dr. Matthew D. Dean. My family has been infinitely supportive and encouraging,
thank you to my Mom Mary E. Lough, my Dad James E. Stevens, my sister Madeleine
C. Kestler and my brother-in-law Jordan Kestler, especially as my PhD has come to a
close at a stressful time for all. The rest of my committee has been stalwart and very
considerate, and I want to thank each of them: Dr. Ian M. Ehrenreich, Dr. Scott Kanoski,
and Dr. Sergey Nuzhdin, as well as separately Dr. Steven Finkle who sat on my
qualifying exam at the last minute.
Last and most importantly I dedicate this dissertation to my wife, Ida Ayu Sabrina Putri,
my light and my life through these past six years, after we met dancing as two grad
students, and hopefully dancing forever onwards into the future.

iii

Table of Contents
Acknowledgements .......................................................................................................................ii
List of Tables ................................................................................................................................. vi
List of Figures .............................................................................................................................. vii
List of Supplemental Figures ..................................................................................................... vii
Abstract ........................................................................................................................................ viii
Chapter 1: Introduction ................................................................................................................ 1
The Baubellum .......................................................................................................................... 1
1.1 Definition of the baubellum .......................................................................................... 1
1.2 Evolutionary hypotheses of the baubellum ................................................................ 2
1.3 Goals of Chapter 2 ........................................................................................................ 5
The Copulatory Plug ................................................................................................................ 6
1.4 Definition of the copulatory plug .................................................................................. 6
1.5 Evolutionary hypotheses of the copulatory plug ....................................................... 7
1.6 Goals of Chapter 3 ...................................................................................................... 12
Pseudopregnancy .................................................................................................................. 13
1.7 Definition of Pseudopregnancy ................................................................................. 13
1.8 Evolutionary Hypotheses of Pseudopregnancy ...................................................... 14
1.9 Goals of Chapter 4 ...................................................................................................... 15
Chapter 2 The baubellum is more developmentally and evolutionarily labile than the
baculum ....................................................................................................................................... 17
Abstract .................................................................................................................................... 17
Introduction .............................................................................................................................. 18
Materials and Methods .......................................................................................................... 20
2.1 Evolutionary patterns .................................................................................................. 20
2.2 Developmental patterns .............................................................................................. 23
Results ..................................................................................................................................... 24
2.3 Evolutionary patterns .................................................................................................. 24
2.4 Developmental patterns .............................................................................................. 31
Discussion ............................................................................................................................... 36
Acknowledgements ................................................................................................................ 41
Conflict of Interest .................................................................................................................. 41
Author Contributions .............................................................................................................. 41
Chapter 3 Male-derived copulatory plugs enhance implantation success in female Mus
musculus ...................................................................................................................................... 43
Abstract .................................................................................................................................... 43
iv

Introduction .............................................................................................................................. 44
Materials and Methods .......................................................................................................... 46
3.1 Ethics statement .......................................................................................................... 46
3.2 Animals .......................................................................................................................... 46
3.3 Vasectomies ................................................................................................................. 48
3.4 Inducing ovulation ........................................................................................................ 49
3.5 Identifying ejaculation based only on behavior ....................................................... 49
3.6 Experiment 1: 24-hour fertility (non-vasectomized males) .................................... 50
3.7 Experiment 2: implantation rates (vasectomized males) ...................................... 51
3.8 Experiment 3: progesterone assays (vasectomized and non-vasectomized
males)................................................................................................................................... 52
3.9 Experiment 4: differential abortion (non-vasectomized males) ............................ 54
Results ..................................................................................................................................... 54
weaning age ........................................................................................................................ 54
3.11 Behavioral scoring of ejaculations is reliable ........................................................ 56
ejaculation ........................................................................................................................... 56
implantation ......................................................................................................................... 59
3.14 Experiment 3: progesterone is equally elevated among females who receive
an ejaculate ......................................................................................................................... 61
3.15 of abortion ............................................................................................................................ 64
Discussion ............................................................................................................................... 64
3.16 Model 1: differences in ejaculate composition or time in situ may influence
implantation success ......................................................................................................... 65
... 66
3.18 Model 3: copulatory plugs may contribute to the threshold level of stimulation
required to facilitate implantation ..................................................................................... 66
3.19 Progesterone levels are only slightly correlated with defects in implantation .. 68
Conclusions ............................................................................................................................. 69
Supplementary Materials ...................................................................................................... 69
Acknowledgements ................................................................................................................ 70
Conflict of Interest .................................................................................................................. 70
Author Contributions .............................................................................................................. 70
Chapter 4 Pseudopregnancies are more severe in Metatheria than Eutheria ................. 71
Abstract .................................................................................................................................... 71
Introduction .............................................................................................................................. 71
v

Materials and Methods .......................................................................................................... 73
4.1 Using corpus luteum lifespan to quantify pseudopregnancy severity. ................ 73
4.2 Phenotype 1: the lifespan of CL in pseudopregnant females, normalized by
Gestation length in true pregnancies (CLG). ................................................................. 74
4.3 Phenotype 2: the lifespan of CL in pseudopregnant females, normalized by the
lifespan of CL in truly pregnant females (CLCL). .......................................................... 75
4.4 Phenotype 3: Peak Progesterone in pseudopregnant females, normalized by
peak progesterone in truly pregnant females (PP). ...................................................... 75
4.5 Ancestral state estimation; testing the “physiological cost” hypothesis .............. 76
4.6 Testing the “lost opportunity” hypothesis; seasonality, delayed
im ............................................................................... 76
4.7 Additional covariates: ovulation type and placental invasiveness ....................... 77
Results ..................................................................................................................................... 78
4.8 Phenotype 1: the lifespan of CL in pseudopregnant females, normalized by
Gestation length in true pregnancies (CLG). ................................................................. 78
Phenotype 2: the lifespan of CL in pseudopregnant females, normalized by the
lifespan of CL in truly pregnant females (CLCL). .......................................................... 81
4.10 Phenotype 3: Peak Progesterone in pseudopregnant females, normalized by
peak progesterone in truly pregnant females (PP). ...................................................... 82
4.11 Testing the “lost opportunity” hypothesis; seasonality, delayed
............................................................................... 83
Discussion ............................................................................................................................... 83
4.12 Why does pseudopregnancy occur? ...................................................................... 84
Chapter 5 Future Directions ..................................................................................................... 88
5.1 Research Impacts of this Dissertation .......................................................................... 88
5.2 Future Experiments ......................................................................................................... 91
5.2.1 The baubellum .......................................................................................................... 91
5.2.2 The copulatory plug ................................................................................................. 92
5.2.3 Pseudopregnancy .................................................................................................... 94
List of References .................................................................................................................... 100

List of Tables
TABLE 1 COMPARISON OF VARIATION IN BACULA VS. BAUBELLA LENGTHS.......................................................... 34
TABLE 2 DISTRIBUTION OF DEVELOPMENTAL STAGES 24 HOURS AFTER COPULATION ........................................ 58
TABLE 3 THE NUMBER OF FEMALES IMPLANTING CON-A BEADS ACCORDING TO CATEGORY. THE TOTAL NUMBER OF
UNIQUE MALES WAS 31 AND 31 TGM4- -INDIVIDUALS. ............................................................ 58

List of Figures
FIGURE 1 ...................................................................................................................................................... 20
FIGURE 2 ...................................................................................................................................................... 27
FIGURE 3 ...................................................................................................................................................... 28
FIGURE 4 ...................................................................................................................................................... 29
FIGURE 5 ...................................................................................................................................................... 32
FIGURE 6 ...................................................................................................................................................... 61
FIGURE 7 ...................................................................................................................................................... 63
FIGURE 8 ...................................................................................................................................................... 81

List of Supplemental Figures
SUPPLEMENTAL FIGURE 1 .............................................................................................................................. 96
SUPPLEMENTAL FIGURE 2 .............................................................................................................................. 97
SUPPLEMENTAL FIGURE 3 .............................................................................................................................. 98
SUPPLEMENTAL FIGURE 4 .............................................................................................................................. 99

Abstract

The evolution of female choice and its impact on sexually dimorphic structures,
behaviors, and physiology, have long fascinated biologists [1]. Female choice is defined
as the conscious and unconscious actions of females that influence their reproductive
decision-making and in turn male traits, thereby causing sexual selection. Striking
examples of sexual selection by female choice, first proposed by Darwin, may be flashy,
such as the male peacock’s long and colorful tail feathers, whose morphology and
visual displays are driven by the pre-copulatory mate choice of females. Since Darwin
there has been an explosion in theories and data to support the fact that pre-copulatory
sexual selection via female choice is widespread in animals. Yet this behavioral and
morphological sexual selection is likely dwarfed by female choice that occurs during
(pericopulatory) and after (post-copulatory) mating [2]. The goal of my PhD was to
analyze whether previously poorly understood and understudied sexually dimorphic
traits are the results of post-copulatory sexual selection in mammals. The baubellum is
a poorly understood bone in the clitoris of mammals. The copulatory plug is a male
seminal solid product that has previously been shown to counter a female’s access to
other males and for sperm retention, but its other possible functions have been left
relatively unexplored. Pseudopregnancy or false pregnancy is a poorly defined term for
pregnant but do not have any fertilized young.

ix

What are the connections between these three topics and why are they the
scope of my dissertation? All three of these concepts are potentially driven by female
choice, yet none of them have been analyzed under that hypothesis, nor explored in
enough depth to previously be tested under that hypothesis. My dissertation evaluated
three concepts in mammals (baubella, copulatory plugs, and pseudopregnancy) that on
the outset are all well known in the literature for over a century, but each are in fact still
not well-understood and require more vigorous evaluation and revision. In particular, the
central theme of my research has been investigating how these mammalian traits have
evolved potentially under post-copulatory sexual selection or female choice, especially
the copulatory plug and pseudopregnancy.
My introduction (Chapter 1) will put into context each of these three topics,
setting the stage first for baubella (a meta-analysis of its evolutionary and
developmental plasticity, the focus of Chapter 2), then the previously known functions
of, and our conceptual framework for, our investigation for studying copulatory plugs
(my experimental discovery of its novel function on implantation is the focus of Chapter
3), and then a meta-analysis of the ‘severity’ of pseudopregnancy, Chapter 4. Finally, I
show the potential impact of these publications and relevant future directions for
research in these fields (Chapter 5)
1

Chapter 1: Introduction

The Baubellum

1.1 Definition of the baubellum

The baubellum (also known as the os clitoris, see 2.1.1 Scoring baculum and
baubellum , bony structure projecting from the tip of the corpus cavernosum of the clitoris in
mammals (Figure 1). Before the publication of our manuscript its phylogenetic
distribution and developmental patterns had never been explored within a comparative
framework, although previous, more limited studies had attempted to make more
general conclusions (see 1.2 Evolutionary hypotheses of the baubellum). Although the
baubellum had been described sporadically in many single studies for several hundred
years, there had been no systematic tabulation of its presence and absence across
mammals. Moreover, while it has had widespread use as a morphological character for
species and population identification in clades, such as the family Sciuridae (Squirrels,
chipmunks, and their allies), the baubellum has never achieved the same level of
investigation as its developmentally homologous counterpart the baculum (os penis), a
[3-5]. The baculum has
received considerable attention to its morphology [6-43], development [44-56], function
[28, 57-66], and distribution and evolution [9, 58, 64, 66-68], yet similar studies and
explanations for the baubellum are lacking. This lack of studies on the baubellum may
2

have many reasons, likely tied to the dearth of information available regarding internal
anatomy of the mammalian clitoris [69-73], a topic of which is beyond the scope of this
study. Below I describe the main known hypotheses of the baubellum’s function prior to
the publication of our manuscript in Ecology and Evolution.

1.2 Evolutionary hypotheses of the baubellum

1.2.1 The baubellum is functional

The baubellum may exist because it has some previously unknown function.
Sexually dimorphic traits may evolve different functions if the ecological reproductive pressures differ for males and females. Although there is no global
hypothesis for baubellum function, two intriguing examples exist of possible functions
for baubella. The ‘Transient Masculization’ Hypothesis argues that the baubellum is part
of a suite of male-like features that immature females adopt to deter conspecific
aggression. Multiple species of mammals have previously been called ‘masculinized’
but what the function of this masculinization is, if any, often remains unknown [74-76]
(although Hyenas, who have extreme masculinization and do not have baubella, have
been extensively investigated [77-82])Hawkins et al. [83] discovered that immature
female fossa, cat-like predators from Madagascar, developed multiple male traits, such
as large genital bones (blocking the entrance of the vagina) [84], genital spines, and
genital secretions. These traits allow them to avoid adult female aggression and male
courtship until they establish their own territory, after which they lose all male traits,
3

including the partial or complete loss of the baubellum by adulthood. The Transient
Masculinization phenomenon has not been observed in other species, although the
baubellum in domestic dogs can enlarge the clitoris and block the entrance of the
vagina, due often to environmental exposure to hormones in gestation, but this is rare
and treated as pathological [85-88].
A second example is the ‘Stimulation’ hypothesis, which argues that the
baubellum comes into direct contact with the baculum during copulation and induce
[89, 90] studied male and female
bacula and baubella and noted that the penis would likely contact the ventral side of the
clitoris during copulation, and the presence of a baubellum would extend the clitoris and
the likelihood of contact and stimulation. In species with clitorises located within the
vaginal tract, such as tenrecs and dogs, the penis and clitoris can come into direct
contact during copulation, however tenrecs and dogs rarely, if ever, have baubella [85,
91].

1.2.2 The baubellum is non-functional and neutrally evolving

Males and females can have traits that diverge between males and females but
are only functional in one sex, and non-functional and non-harmful in the other. In
humans, both males and females possess nipples, but only the female nipple has the
mammary tissue required to make milk; male nipples are non-functional yet non-
harmful. The baubellum could function similarly, imparting no function to female
mammals but no significant cost. As an example, the ‘Non-Canalized Hypothesis
4

argues that the baubellum in the clitoris exists because of the evolution of the baculum
in the penis, therefore the baubellum is non-functional while the baculum is functional.
Patterson and Thaeler, Jr. [57] used this argument when deciding whether baculum
shapes and sizes were driven primarily by phylogeny or by their function, arguing that,
in a comparison of male bacula to female baubella, baubella should have equal
variance as bacula if the bones were phyletic, otherwise if bacula were functional then
bacula would have less variation than baubella because they were ‘canalized,’ i.e. the
target of selection. As a test of this hypothesis, they showed that in the family Sciuridae
bacula varied less than baubella within species, sometimes by an order of magnitude,
suggesting that the baculum was functional and the baubellum was merely the product
of ‘phyletic divergence.’ This is a problematic argument because the authors assume
that higher variance in baubella automatically means non-functionality, a dubious
assertion. Additionally, it was not possible to evaluate whether baubellum variance was
congruent with the definition of a neutrally evolving trait because the authors did not
build a phylogenetic distribution of baubella nor evaluated other types of data needed to
declare the baubellum non-functional. Lastly, there has been mixed evidence of
baubella being more variable than bacula for the family Sciuridae [92-101], because
even within sciurid species bacula and baubella can be remarkably similar in size and
shape [94]. Given that their data were limited to the family Sciuridae, this argument
cannot be extended to other mammalian groups and additionally this hypothesis.

5

1.2.3 The baubellum is deleterious

Lastly, it is possible that the baubellum is non-functional and also deleterious to
female reproduction and survival. This hypothesis would predict that females should
baubellum and have a lower chance of survival and ability to produce offspring than
females who do not have baubella.
It has been sporadically reported in the literature that in some species, such as
river otters and voles, not all females have a baubellum [45, 102], and in other species,
such as the walrus, the baubellum decreases in weight and length with age [103, 104].
It has never been tested whether possessing a baubellum changes a female’s lifetime
reproductive success or rate of survival in a species.

1.3 Goals of Chapter 2

The overall objective for our study is to test whether the baubellum is functional,
with an emphasis on comparing evolutionary and developmental trends to the male
baculum. We extended methods we developed for Schultz et al. [67] to estimate the
relative number of evolutionary transitions in baubellum character states. Our approach
additionally compiles baubellum developmental trajectories morphological data from
multiple unrelated species, a comparative approach which has never before been
attempted. As the title of our published manuscript indicates, we found that the
baubellum has evolved far more times than the baculum but also appears to be
6

phylogenetically and developmentally constrained by the presence or absence of the
baculum, as well as far more variable in shape and size, suggesting it is ultimately non-
functional.

The Copulatory Plug

1.4 Definition of the copulatory plug

In many mammals, a portion of the male ejaculate coagulates to form a solid
mass in vaginal tracts of females [105-107]. Widespread phylogenetically but not
systematically studied, the form and function of the copulatory plug remains unknown
for most mammalian species [108, 109]. Formed from the action of Transglutaminase 4
(TGM4) protein crosslinking Seminal Vesicle Secretion 2 (SVS2) protein, the mouse
knockouts of TGM4, SVS2, or PATE4 will result in the total loss of the copulatory plug
[105, 110-114], making mice (Mus musculus) an ideal model for dissecting the function
of copulatory plugs. In mice, this solid structure composed of secretions from the
seminal vesicles and prostate gland occupies the entire vagina, from the vaginal
opening until the cervix (the opening into paired uterine tracts) [115, 116], thus the
copulatory plug reliably blocks sperm from rival males and helping retain their own
sperm in the female’s uterus [106, 116, 117].
Before the publication of our paper there were fewer studies evaluating the
benefits of copulatory plugs for females (but see [109, 110, 118, 119]); the majority of
studies over the last 100 years have focused mainly on the benefits of copulatory plugs
to males. Its visibility and ability to remain up to 48 hours makes it ideal in the laboratory
7

setting for determining whether males and females have recently mated for a wide
variety of mammals [116, 120, 121]. A previous paper from our lab determined that
males who could not form plugs through genetic disruption are unable to effectively
impregnate females on a regular basis and raise the possibility that the absence of a
copulatory plug disrupted multiple stages of early pregnancy [110]. Below I describe the
main known hypotheses of the copulatory plug’s function prior to the publication of our
manuscript in Biology of Reproduction.

1.5 Evolutionary hypotheses of the copulatory plug

1.5.1 The ‘Chastity-Enforcement’ Hypothesis

The Chastity Enforcement hypothesis is that the copulatory plug from the first
male to mate with a female functions to delay or stop subsequent males from
successfully copulating with the same female, which improves first male reproductive
success by delaying the onset of sperm competition [106, 109, 117, 118, 120, 122-129].
Male copulatory plugs inhibit male rival reproductive success and acts effectively as a
form of passive mate guarding as rival males must first wedge aside or dislodge the first
copulatory plug with their teeth or their penis, giving more time for first male sperm
(which persists in the female vaginal tract for only several hours) to inseminate the
female’s eggs [106, 117, 120, 124, 125, 128].
This hypothesis predicts that larger rodent plugs are more effective at blocking
rival males [120], although deeper plugs last longer [116, 120]. In particular the anterior
8

tip of the copulatory plug positioned at the mouth of the cervix is the least likely to be
dislodged by rival males [120]. Plug size and shape varies with male seminal vesicle
size and seminal vesicle size with the degree of perceived sperm competition; second
males however do not adjust plug size in response to an already plugged female but will
ejaculate more sperm than first males in rodents [120, 130-132]. Said another way,
rodent male copulatory size is the product of genetics and the cues of sperm
competition encountered during seminal vesicle development, but not from individual
mating bouts against rival males.
While female mice mate multiply in the laboratory and in the wild, there is a
limited window of time (approximately 12-15 hours) when females are receptive to
mating, corresponding approximately to the time of estrous and the viability of her eggs
[109, 121, 133-139]. Importantly, female mice observe a 4 day estrous cycle, but if her
mating bout with one or more males does not lead to fertilization, she does not re-enter
estrous for 10-14 days (see 1.5.3 The ‘Implantation Induction’ Hypothesis for more
details) and if she is pregnant, she is unavailable for the duration of her pregnancy of
approximately 21 days [121], meaning there is a reproductive cost to males who do not
attempt to mate immediately with receptive females (females not in heat, whether
pregnant or not, rarely mate with males [137]). Note that female mice enter a post-
partum estrous, leading to pregnancy while nursing [121]. Ultimately this means that
males who encounter a female in heat will likely be under intense sperm competition
against rival males also seeking to mate with her.
Copulatory plugs in this context also function as a form of sexual conflict, as
while males may prefer to fertilize all of a female’s eggs, females potentially benefit from
9

multiple matings due to increased genetic diversity or ‘good genes’ of offspring as well
as potentially increased fertilization success from the physical stimulation of intercourse
and increased pup weaning success [118, 119, 140-145] (Chapter 3).
Additional conflict may come in the form of physical harm, as researchers
attempting to remove plugs have accidentally also removed the outer vaginal mucosal
lining, suggesting that second male mating attempts may leave similar damage [109,
146] (but see [145, 147]). Another indicator of conflict is that females naturally produced
counteracting proteases to break down the outer cornified layer of their vaginal tracts to
slough off copulatory plugs over time [105, 116] or even physically remove plugs
themselves using their mouths [106, 148]. Second male behavior also shifts to a more
aggressive state to counteract the plug, with increased number of thrusts and increased
time to ejaculation (although as a caveat this is due to the presence of a plug, second
male mating behavior does not alter if the female’s plug has been artificially removed,
see [145]), overall suggesting that these multiple plugs lead to a more stressful and
painful experience for females as well [120].

1.5.2 The ‘Sperm Motility’ and ‘Sperm Leakage’ Hypotheses

The Sperm Motility hypothesis and the Sperm Leakage hypothesis are that male
copulatory plugs enhance fertilization success in the absence of sperm competition
[106, 109, 110, 114, 149]. The Sperm Motility hypothesis specifically argues that
copulatory plugs push sperm and associated liquid seminal products past the vaginal
tract and deeper into the uterus than in its absence, while the Sperm Leakage
10

hypothesis argues that not all species show a post-coital contraction of the cervix so
copulatory plugs must act as a barrier to sperm leakage [106, 109, 110, 150, 151].
Sperm motility in rodents is due to multiple inputs, such as sperm capacitation, i.e. the
biochemical activation by enzymatic reactions of sperm flagellum undulation [111, 112,
149, 152, 153], the act of ejaculation itself, and the rapid expansion of the solidifying
copulatory plug by enzymatic reaction of Transglutaminase 4 (TGM4) [106].
However the sperm motility hypothesis has had mixed support, as dyes
embedded in gels administered to rat and mouse vaginal tracts immediately before
mating are not pushed transcervically by the presence of a plug [154], and artificial
insemination without the addition of a copulatory plug in some rodent species, such as
Guinea Pigs, are more than 50% likely to give birth [129]. Support for the sperm motility
hypothesis comes from data showing artificial removal of plugs and remating to a
second male does not negate first male reproductive success [145], immediate removal
of copulatory plugs in mice and guinea pigs does not impact single male mating
reproductive success [129, 155], and females mated to males who cannot form
copulatory plugs have less seminal products in their bilateral uteri and lower fertilization
success than females mated to males who form plugs, even in situations when first
males do not form plugs, and still receive little if any fertilization success when second
males who are vasectomized deposit copulatory plugs [110, 114, 117] (Chapter 3).
Support for sperm motility also is support against sperm leakage as the
immediate removal of plugs does not impact some rodent species’ reproductive
success, including mice [129, 145, 155], yet if males ejaculate without leaving a plug
there does appear to be significant leakage of semen out of the uteri into the vagina in
11

mice and rats and lower reproductive success [110, 114, 117, 156]. Similarly, in rats
uterine semen leaks into the vagina if the plug is lost naturally soon after deposition
[156, 157]. Overall, these results show that this mixed support for either hypothesis
could be the result of species-specific mechanisms for sperm delivery and retention and
may quickly evolve even within rodents.

1.5.3 The ‘Implantation Induction’ Hypothesis

The Implantation Induction hypothesis is that female mammals require a certain
level of stimulation by males to be able to implant fertilized eggs and the male
copulatory plug is potentially an important form of stimulation required for implantation
[109, 110].
Some species of rodents such as Guinea Pigs do not require male stimulation for
implantation [129], however most other rodents such as mice, rats, and hamsters need
varying amounts of male copulatory stimulation to achieve implantation [141, 158-163].
For example, sexually naïve female rats can become receptive to fertilized egg
implantation by cervical stimulation through insertion of a glass rod or vibrator, while
sexually experienced female rats engage in ‘paced mating’ where females will
repeatedly mate with the same male with breaks inbetween, and often required a
certain number of thrusts, but not male ejaculation, before their bodies can implant
embryos several days later [159-163]. Hamsters require either cervical stimulation by
vibrator or the stimulatory action of male thrusts to implant fertilized eggs, and like rats
do not require male ejaculation [158].
12

Domestic mice, like rats and hamsters, are primed for implantation by vibrators
and glass rods under certain numbers of insertions and for different lengths of time
[158], and when female mice engage in paced mating [142], demonstrating that female
mice likely respond to a certain combination of male stimulatory signals in order to
achieve implantation. Notably, female mice require ejaculation to prime her body for
implantation, a result not observed so far in other rodents. The copulatory plug is likely
an important part of the ‘code’ females receive from males to enter a physiologically
state of implantation.

1.6 Goals of Chapter 3

The objective of our study is to expand upon the previous publication from our
lab that showed the absence of copulatory plugs by a male genetic knockout mouse
strain negatively impacted female reproductive success in the absence of sperm
competition [110]. The pattern seen was fertile males who were unable to form
copulatory plugs through genetic manipulation were far less likely to sire offspring than
males with intact copulatory plugs. Females mated to males without plugs had far less
sperm counts in their uteri and oviducts, yet a normal number of oocytes were fertilized
when compared to males which make plugs, thus validating the previous sperm motility
and sperm leakage hypotheses. Given that fertilization does not seem impaired by the
lack of a plug, we have specifically sought to understand whether reproduction in the
absence of the copulatory plug fails because of an implantation or gestation defect. Our
approach therefore examines rates of implantation via our novel method of inserting
beads which mimic embryos into the uterine lining to evaluate whether the copulatory
13

plug does in fact impact positively implantation rates in addition to previously
documented functions of female chastity, sperm motility, and sperm leakage. As was
published in the journal Biology of Reproduction, we found females who mated with
males without copulatory plugs were significantly less likely to implant beads than
females who received copulatory plugs, showing that males stimulated females’
reproductive decision-making towards implantation, a potentially remarkable case of
female choice in mammals.

Pseudopregnancy

1.7 Definition of Pseudopregnancy

Pseudopregnancy (also known as non-pregnancy, false pregnancy or
pseudocyesis) are anatomical, physiological, and behavioral phenotypes that indicate a
female mammal is pregnant, but does not have embryos or fetuses [134, 164-169]. This
term is used currently in the literature to help determine whether females that appear
pregnant phenotypically are in fact pseudopregnant [134, 169]. The agricultural, pet,
and breeding communities are invested in preventing pseudopregnancy for
domesticated animals, such as domestic dogs, cats, ruminants and horses, especially in
species that are monoestrous (breed once per year) like dogs, at times even defining
the condition as clinical or pathological [170-174].
Yet lab mammals from multiple species of wild-derived rodents, carnivorans, and
marsupials have also been shown to experience pseudopregnancy [136, 158, 165, 175-
181], as have many wild animals [169, 182-187], suggesting that this condition is
widespread throughout the class Mammalia and not a disease or the result of selective
14

breeding. Over the last 100 years since the term pseudopregnancy was coined and
defined [134, 169], there has been considerable discrepancy in techniques and
diagnoses for defining pseudopregnancy; there is no review addressing the prevalence
of, or the physiological mechanisms that lead to, pseudopregnancy.
Additionally, there is considerable disagreement of what stimuli, male-involved or
not, that are required for inducing pseudopregnancy, which may be itself a reflection of
the dizzying array of mating systems and necessary physiological steps for pregnancy
that differ between clades of mammals [188-191]. Another confusion is the paucity of
explanations for the function, if any, of pseudopregnancy, with multiple authors
assuming it is a failure of one or more steps of pregnancy (but see 1.8.1 The ‘Shared
Cooperative Care’ Hypothesis).
As we had just finished our own analysis of the rate of pseudopregnancy due to
the presence or absence of copulatory plugs in mice in we were interested in answering
two main questions in Chapter 4: what is the lifespan of pseudopregnancy relative to
pregnancy, which we term ‘severity’ and describe in detail in , and what are the drivers
of pseudopregnancy severity? Below I discuss the known evolutionary hypotheses to
explain pseudopregnancy prior to the writing of Chapter 4.

1.8 Evolutionary Hypotheses of Pseudopregnancy

1.8.1 The ‘Shared Cooperative Care’ Hypothesis

The Shared Cooperative Care hypothesis is that pseudopregnancy evolved as a
mechanism for non-breeding females to nurse related females’ pups and for social
15

cohesion needed for survival. Most social canids (dogs; family Canidae) that have been
studied so far have a number of shared mechanisms of investment in juveniles, such as
defense, food provisioning, and playing [189]. Social hierarchies are likewise a better
guarantee for survival than attempting to hunt alone in these species. Most wild and
domestic canid species that have been studied are seasonally estrous (monestrous)
and typically if they do not breed within a certain time frame will automatically enter
pseudopregnancy without copulation with a male [170, 173, 174, 186].
What is remarkable is that non-breeding females are behaviorally discouraged
from breeding yet still undergo an obligatory pseudopregnancy simultaneous to the
breeding female’s pregnancy. This is costly because their pseudopregnancy lasts as
long as a regular pregnancy, they lactate and provide nursing for the breeding female’s
pups, and they do not re-enter estrous until the following year, losing any opportunities
for direct reproductive fitness. Under an inclusive fitness model, which posits that any
altruism is due to the close genetic relationships between themselves and breeder’s
pups, if the non-breeding females are related to either the dominant female or male,
they could gain indirect reproductive fitness. Additionally, each female raising their own
pups could decrease social cohesion and cause more frequent fracturing of the group.
Some authors argue this seemingly-tight relationship between pseudopregnancy and
indirect reproductive fitness means that the ancestor of all canids was social, as
pseudopregnancy would be selectively disadvantageous in solitary canids.

1.9 Goals of Chapter 4

16

The objective for our study was to follow up our publication of , which found that
male mice ejaculations and copulatory plugs were inducing pseudopregnancy, which we
assessed as a spike in blood serum progesterone, in females even in the absence of
viable fertilized eggs. We constructed a mammalian phylogeny of pseudopregnancy
‘severity’ (pseudopregnancy length divided by the length of pregnancy) and rigorously
assessed what ecological or male-induced best explain the lifespan of
pseudopregnancy through a meta-analysis of the scientific literature in Chapter 4.Our
results, which are in-prep for submission to publication, show that pseudopregnancy
severity is significantly greater in the subclass Metatheria than in the subclass Eutheria.
The differences between these clades are stark, as metatherians have short
pregnancies and even shorter lifetime placentas, while eutherians have lengthy
pregnancies and placentas, suggesting that the cost of pregnancy is so high in
eutherians that they have evolved far more mechanisms to detect and arrest the length
of pseudopregnancy than in metatherians. Indeed, we find through a literature review a
wide variety of independently-evolved physiological cues for sustaining pregnancy and
detecting the presence or absence of offspring that are lacking in metatherians.
17

Chapter 2 The baubellum is more developmentally and evolutionarily labile than the
baculum
Lough-Stevens, M., et al. (2018). "The baubellum is more developmentally and
evolutionarily labile than the baculum." Ecology and Evolution 8(2): 1073-1083.

Abstract

Understanding the evolutionary forces that influence sexual dimorphism is a
fundamental goal in biology. Here, we focus on one particularly extreme example of
sexual dimorphism. Many mammal species possess a bone in their penis called a
baculum. The female equivalent of this bone is called the baubellum and occurs in the
clitoris, which is developmentally homologous to the male penis. To understand the
ecies and analyzed their distribution in a phylogenetic
framework. The majority of species (N = 134) shared the same state in males and
females (both baculum and baubellum present or absent). However, the baubellum has
experienced significantly more transitions, and more recent transitions, so that the
remaining 29 species have a baculum but not a well-developed baubellum. Even in
species where both bones are present, the baubellum shows more ontogenetic
variability and harbors more morphological variation than the baculum. Our study
demonstrates that the baculum and baubellum are generally correlated across
mammals, but that the baubellum is more evolutionarily and developmentally labile than
the baculum. The accumulation of more evolutionary transitions, especially losses in the
baubellum, as well as noisier developmental patterns, suggests that the baubellum may
be nonfunctional, and lost over time.
18

Introduction
Sexual dimorphism, where the same trait takes on different states in the two sexes, is a
nearly ubiquitous phenomenon in nature, and understanding the evolutionary forces that
lead to sexual dimorphism is an important goal for evolutionary biology [70, 192].
The baculum is a highly unusual bone found in the penis—and the baubellum is a bone
found in the clitoris—of many mammalian species [21, 94]. As with many studies of
primary sexual traits, the baculum seems to accumulate morphological divergence more
rapidly than nonsexual morphologies [57, 66] consistent with a model of adaptive
evolution continuously driving morphological change. The baculum is presumed to be
adaptive because of its species-specific shape [9, 21, 30, 57], rapid evolution under
experimental evolution [62], and the influence of its shape on male reproductive
success [61, 62].
The evolutionary and developmental forces affecting the female baubellum, and how
they correlate with the baculum, remain poorly understood. In general, the baubellum is
much smaller and less morphologically defined than the baculum [63]. For example,
adult male walruses have the largest known baculum, while adult female walruses have
a much smaller and differently shaped baubellum (Figure 1). Other species like Eastern
gray squirrels have a baubellum that is similar in both size and shape to the male
baculum (Figure 1).
A recent study of approximately 1,000 mammalian species revealed that the male
baculum has been gained nine independent times and has been lost 10 independent
19

times [67]. These multiple independent transitions provide a unique opportunity to ask if
and how evolution and development of the baubellum correlate with the baculum. Here,
we analyze the presence/absence of the baubellum across 163 species, and present
five main findings. First, the presence/absence of the baculum/baubellum is identical in
134 of the 163 species. Second, in spite of this general correlation, the baubellum
showed significantly more evolutionary gains and losses than the baculum, such that
states did not match in 29 species. Third, these 29 species are always with a baculum
but without a baubellum—we observed no species that lack a baculum but possess a
baubellum. Fourth, the baubellum displayed much more variation in development than
the baculum, even disappearing with female age in some species. Fifth, the baubellum
showed significantly more morphological variation than the baculum. Overall, the
baubellum shows more evolutionary and developmental variation than the baculum,
indirectly arguing that the baubellum may be relatively nonfunctional.

20

Figure 1
Comparison of walrus/squirrel baculum/baubellum. Note the walrus baculum and baubellum are very different in both size and
shape, while the two bones are very similar in the Eastern gray squirrel. *Adapted from Fay, 1982; Burt, 1960; Layne, 1954

Materials and Methods

2.1 Evolutionary patterns
2.1.1 Scoring baculum and baubellum presence/absence
Presence/absence of the baculum of 1,143 species was taken from table S2 of Schultz
et al [67]. Presence/absence of the baubellum was scored through literature searching
and online museum records from August 2015 to January 2017. We were able to find
records for 185 species (Table S1). Our primary data came from searches in Google
Scholar (https://www.scholar.google.com) and Web of Science
(https://webofscience.com/), with the phrases baubellum, baubella, os clitoris, os
clitoridis, os glandis, ossicle, os genital/s, os genitale, clitoral bone, clitoris
bone, clitorisknochen, klitorisknochen, and cartilage clitoris.
We only scored baubella as present if it was (1) shown in photograph or illustration, (2)
summarized with measurements, or (3) described in qualitative terms. We scored
baubella as absent if it was (1) absent from photographed or illustrated genital
dissections, or (2) stated by authors that they were unable to find cartilage or bone upon
dissection. Interestingly, many species appear to be polymorphic, in which some but not
all females within a species have a baubellum, an issue we specifically address below.
21

Scoring a baubellum as absent is challenging. The baubellum is generally smaller than
the baculum, it is not present in every age class, or remains cartilaginous and difficult to
observe in some species [101, 104]. Nevertheless, we note its absence in one
extremely well-studied model system, the rat. Multiple detailed histological studies have
demonstrated that the rat lacks a baubellum [4, 53, 193-195], even though male rats
possess a prominent baculum.
2.1.2 Phylogenetic inference

A large molecular phylogeny of 3,707 mammalian species was taken from
supplementary file #1 of Schultz et al. [67] and was trimmed down to include only
species where both the baculum and the baubellum were scored, resulting in 163
species. We then applied stochastic mapping as implemented in the
function MAKE.SIMMAP of the R package PHYTOOLS [196, 197]. This is a powerful
approach to simulate trait evolution across a phylogenetic tree, while avoiding some of
the overly stringent assumptions of a strict parsimony framework. Essentially, character
state transitions are distributed across a tree according to an estimated transition rate
matrix, with the caveat that each iteration must be consistent with the observed trait
states [198, 199]. This same approach was employed by Schultz et al. [67] to model
baculum evolution. We summarized baculum and baubellum gains and losses from
1,000 iterations of stochastic mapping across each of the four strategies described
above, using only the 163 species for which both baculum and baubellum were scored.
Visual representations were made using the DENSITYMAP function of PHYTOOLS [197], as
well as customized scripts written in R (https://www.r-project.org), available upon
22

request. Branches where a transition occurred in at least 50% of the stochastic mapping
iterations were considered “high confidence” transitions.
From the stochastic mapping iterations, we also tested whether transition times differed
between baculum and baubellum, using a mixed effects model implemented in
the LMER function in the R package LME4 [200]. Using a likelihood ratio test and a chi-
square distribution with one degree of freedom, we tested whether a model that
included bone (baubellum vs. baculum) as a fixed effect explained differences in
transition times significantly better than a model that did not. For both models, iteration
number was included as a random effect because transition times within an iteration will
not be independent from each other.
2.1.3 “Polymorphic” species

Seventeen species were best classified as “polymorphic,” where some females had a
baubellum while others of the same age class did not, for example in domestic dogs
[201]. We implemented four different strategies of stochastic mapping to account for
alternative views of the polymorphic state. First, “polymorphic” was considered a third
state in addition to “present” or “absent.” Second, all polymorphic species were
assigned the state of “present,” which could be interpreted as a trait state that normally
develops but is incompletely penetrant or difficult to observe and occasionally
overlooked in the literature. Third, all polymorphic species were assigned the state of
“absent,” which could be interpreted as a trait state that normally does not develop. For
these second and third models, it is interesting to note that female rats, ferrets, and
dogs all develop baubella with additional administration of testosterone [4, 87, 88, 194,
23

195, 202], and it is possible that variation in hormonal profile explains polymorphism.
Lastly, “polymorphic” species were randomly assigned “present” or “absent,” which is
some combination of the second and third strategies. For the remainder of this
manuscript, these four strategies are referred to as “polymorphic,” “present,” “absent,”
and “random,” respectively. All four strategies give qualitatively the same answers (see
below).
2.2 Developmental patterns

2.2.1 Comparing the development of baubella with bacula

During our literature search, we uncovered five species where multiple males and
females from multiple age classes were assessed for the presence of both a baculum
and baubellum [45, 83, 84, 103, 104, 203-208]. Because the original data were not
available for most of these studies, we qualitatively compared them as growth curves.

2.2.2 Comparing within-species variability of baubella with bacula

Our literature search also uncovered 13 species with quantitative measurements of
bacula and baubella length from multiple males and females, all adults. We could
therefore compare the coefficients of variation (CV = standard deviation/mean) for
bacula and baubella. We tested whether the baubellum CV's differed significantly from
baculum CV's using a phylogenetically controlled paired t test, as implemented in
the PHYL.PAIREDTTEST function in the R package PHYTOOLS [197]. One of the 13
24

species, Parascalops breweri was represented by Talpa europaea on the phylogeny for
this test only. The other 12 were already represented in the phylogeny.
In addition to this global approach, we tested whether CV differed between the baubella
and the bacula within each species separately, using Feltz and Miller's [209] asymptotic
test for the equality of coefficients of variation, as well as Krishnamoorthy and Lee's
[210] modified signed-likelihood ratio test. These two approaches were implemented
with the functions ASYMPTOTIC_TEST2 and MSLR_TEST2, respectively, in the R
package CVEQUALITY [211]. We noticed several species where large differences in
baubellum CV versus baculum CV failed to produce statistical significance at p = .05,
and suspected this might be due to small sample sizes available from the literature. To
understand the sample size required for statistical significance, we computationally
increased sample size until statistical significance was observed.
Results

2.3 Evolutionary patterns

2.3.1 The baubellum shows more evolutionary transitions than the baculum

A total of 163 species had reliable data for both baculum and baubellum presence and
were also represented in a large mammalian phylogeny [67]. Of these, 117 had a
baubellum, 29 lacked one, and 17 were polymorphic (Figure 2, Table S1). In 134
species, the state of the baubellum matched the state of the baculum (Figure 2,
25

Table S1). However, it should be noted that a large proportion of these (51 of the 134
species) are derived from a single family, Sciuridae (squirrels and chipmunks), so the
generality of this pattern should be treated with caution. Sciurid bacula and baubella are
regularly used in taxonomy, and so these bones may have been investigated more than
in other families [98, 99]. All 29 species for which states did not match had a baculum
but lacked a well-developed baubellum (either baubellum absent or polymorphic).
26

27

Figure 2
Summary of 1,000 iterations of stochastic mapping for baubellum (left) and baculum (right). Colored circles at terminal nodes
indicate character state of each bone: present (red), absent (blue), or polymorphic (purple). Branches are colored according to the
average time spent in each state across the 1,000 iterations, on a scale ranging from present (red) through polymorphic (purple) to
absent (blue). Boxes on branches indicate “high confidence” character transitions, indicating the percentage of stochastic mapping
iterations where transitions occurred on those branches. Boxes on branches are colored according to the state to which the
character transitioned (red = present, blue = absent, purple = polymorphic). Note there are more transitions that tend to occur more
recently in the baubellum compared to the baculum. A “zoomable” version of this figure is provided in Fig. S1)

Under the “polymorphic” model, the baubellum showed significantly more evolutionary
transitions compared to the baculum (an average of 92.9 vs. 21.0 transitions,
respectively; Wilcoxon Rank Sum Test [WRST] p < 10
15
) (Figure 3 and Figure 4). The
other three models also showed significantly more transitions in the baubellum versus
the baculum (an average of 102.3 vs. 14.3, 55.1 vs. 14.4, and 28.0 vs. 13.5 baubellum
vs. baculum transitions for the “absent,” “random,” and “present” models, respectively,
WRST p < 10
15
in all three cases) (Figure 4). In sum, the baubellum has experienced
more evolutionary transitions than the baculum, regardless of how we scored
polymorphic species.
28

Figure 3
Summary of the average ± standard deviation number of baubellum (number above line) and baculum (number below line)
transitions between three states among 1,000 iterations of stochastic mapping. The baculum and baubellum are modeled as three
distinct morphological states: present, polymorphic, and absent. Note the baubellum experiences significantly more evolutionary
transitions than the baubellum across all transition types (see text)

29

Figure 4
Summary of the number of transitions experienced by the baubellum versus baculum between two different states. Each model
recodes polymorphic as present or absent. In all cases, the baubellum experienced significantly more transitions than the baculum
(see text)

In addition, baubellum transitions tended to occur more recently than baculum
transitions. For the “polymorphic” model, baubellum transitions occurred an average
27.9 million years ago versus 43.7 million years ago for the baculum. These results held
under the other three models (29.3 vs. 39.1, 27.9 vs. 39.2, and 28.2 vs. 41.4 million
years ago baubellum vs. baculum transitions for the “absent,” “random,” and “present”
2
> 997, df = 1, p < 10
in all four cases). Therefore, not
only has the baubellum experienced more transitions, but those transitions tended to
occur more recently than baculum transitions.
30

2.2.2 Many species with a well-developed baculum lacked a well-developed baubellum

Of 145 species with a well-developed baculum, 12 lacked a well-developed baubellum
(Figure 2, Table S1). These species were widely distributed across the phylogeny and
included two primates (Formosan rock macaque, Macaca cyclopis; Rhesus
macaque, Macaca mulatta), six rodents (Maya mouse, Peromyscus mayensis; Norway
rat, Rattus norvegicus; Brandt's vole, Lasiopodomys brandtii; Common vole, Microtus
arvalis; Spix's yellow-toothed cavy, Galea spixii; Eurasian beaver, Castor fiber), two
bats (Southern yellow bat, Lasiurus ega; Underwood's bonneted bat, Eumops
underwoodi), one carnivore (Wolverine, Gulo gulo), and one afrosoricid (Lesser
hedgehog tenrec, Echinops telfairi). By contrast, there were no species that had a
baubellum and lacked a baculum. This could be partially due to study bias, whereby
investigators are less likely to look for a baubellum in a species that has no record of a
baculum. In addition, eight species were scored as baculum present and baubellum
present, but their baubellum remained cartilaginous, unlike the baculum (Table S1).
An additional 17 species had a well-developed baculum but were polymorphic for the
baubellum. These species were also widely distributed, and included one primate
(Senegal galago, Galago senegalensis) four rodents (Guinea pig, Cavia porcellus;
California vole, Microtus californicus; Long-tailed vole, Microtus longicaudus; American
red squirrel, Tamiasciurus hudsonicus), ten carnivores (Eurasian otter, Lutra lutra;
Australian sea lion, Neophoca cinerea; North American raccoon, Procyon lotor;
Northern fur seal, Callorhinus ursinus; Domestic dog, Canis domesticus [C. lupus in
31

phylogeny]; Southern elephant seal, Mirounga leonina; European polecat, Mustela
putorius; Polar bear, Ursus maritimus; American mink, Neovison vison; Domestic
cat, Felis silvestris), and two bats (Greater horseshoe bat, Rhinolophus ferrumequinum;
Black mastiff bat, Molossus ater). The percentage of individuals with a baubellum in
polymorphic species varied, from one in 100 (1%) of adult female raccoons [55], to one
in two (50%) in black mastiff bats [23] (Table S1). In sum, many species with a well-
developed baculum lack a well-developed baubellum, but no species with a baubellum
lacked a baculum.
2.4 Developmental patterns

2.4.1 The baubellum showed more ontogenetic variation than the baculum

When present, the baculum generally grows steadily from birth to reproductive maturity
(Figure 5). The baubellum of two species (Weddell seal, Leptonychotes weddellii;
Golden hamster, Mesocritus auratus) showed similar developmental trajectories [204,
206, 208] (Figure 5). However, three additional species showed striking divergence in
developmental patterns (Figure 5). In one species (Northern river otter, Lontra
canadensis), the baubellum did not begin development until 2 years after birth [205], in
contrast to the male baculum which was present at birth and continued to grow
throughout the animal's life [45, 212]. In two species (Walrus, Odobenus rosmarus;
Fossa, Cryptoprocta fossa), baubellum size decreased with age, opposite the
developmental patterns of the baculum [83, 103, 104, 213].

32

Figure 5
Developmental trajectories of the baculum are consistent across species (top panel), compared to the baubellum in which multiple
different paths are observed (bottom panel)

2.4.2 Within species, the baubellum of adult females is more variable than the baculum
of adult males

The baubellum CV's were significantly larger than the baculum CV's across 13 species,
as judged by a phylogenetically controlled paired t test (t = 3.6, p = .005).
Across 13 species, 12 had a higher baubellum CV versus baculum CV, seven of which
were significantly higher by the asymptotic test and six of which were significantly higher
by the modified signed-likelihood ratio test (Table 1). Some of the nonsignificant results
seemed to arise because of small sample size. For example, even though the
33

baubellum of Spermophilus mexicanus had a CV more than three times that of the
baculum, the difference was not statistically significant, probably because only two
females and two males were sampled (Table 1). If we assume existing estimates of CV
were reasonably accurate for this species, we would have had to sample at least five
males and five females before detecting a significant difference under the asymptotic
test (Table 1). The higher baubellum CV was phylogenetically widespread, observed in
carnivores, primates, bats, moles, and rodents.
34

Sciurus niger 4 3.38 0.55 0.163+/-0.058 11 12.36 0.01 0.001+/-0 83.66 0.000‡ 68.8 0.000‡ - (Long and Frank, 1968)
Spermophilus mexicanus 2 2.15 0.78 0.362+/-0.181 2 4.45 0.49 0.111+/-0.056 1.01 0.315 0.76 0.385 5 Male: (Burt, 1960);
Female: (Layne, 1954)
Taxidea taxus 4 10.71 1.97 0.184+/-0.065 2 98.7† 9.87† 0.1+/-0.05† 0.38 0.538 0.52 0.471 13 Male: (Burt, 1960);
Female: (Long and Frank, 1968)
Urocyon cinereoargenteus 3 6.33 1.44 0.228+/-0.093 11 50.94 4.5 0.088+/-0.019 5.09 0.024‡ 2.05 0.152 - Male: (Long and Frank,
1968); Female: (Hildebrand, 1954)

Table 4 Distribution of developmental stages 24 hours after copulation
Table 5 Comparison of variation in bacula vs. baubella lengths
species N females mean females SD females CV females N males mean males SD males CV males asymptotic statistic asymptotic p value mslr statistic
mslr p value min sample size References§
Cryptoprocta ferox 4 5.5 3.3 0.6+/-0.212 17 72.2 9.6 0.133+/-0.023 23.76 0.000‡ 10.12 0.001‡ - (Hawkins et al. 2002)
Lemur catta 6 0.38 0.06 0.158+/-0.046 4 1.4 0.14 0.1+/-0.035 0.65 0.419 0.73 0.393 21 (Drea and Weil 2008)
Leptonychotes weddellii 5 35 6.67 0.191+/-0.06 58 186.3 38.9 0.209+/-0.019 0.05 0.820 0.18 0.668 512 (Smith 1966)
Lontra canadensis 2 10.35 10.11 0.977+/-0.488 55 94.92 4.46 0.047+/-0.004 412.61 0.000‡ 36.17 0.000‡ - Male: (Friley, 1949);
Female: (Scheffer, 1939)
Mormopterus planiceps 6 1.62 0.05 0.031+/-0.009 9 7.9 0.18 0.023+/-0.005 0.6 0.439 0.42 0.517 44 Male: (Krutzsch and
Crichton 1987); Female: (Crichton and Krutzsch 1987)
Mustela vison 6* 1.02 0.45 0.439+/-0.127 99 44.6 2.18 0.049+/-0.003 312.33 0.000‡ 98.42 0.000‡ - (Long and Shirek, 1970)
Parascalops breweri 2 0.68 0.11 0.157+/-0.079 2 0.68 0.05 0.072+/-0.036 0.53 0.465 0.36 0.549 9 (Sinclair, 2014)
Phoca vitulina 2 7.5 2.12 0.283+/-0.141 3 127.3 2.52 0.02+/-0.008 7.81 0.005‡ 4.4 0.036‡ - Male: (Mohr, 1962);
Female:(Scheffer, 1949)
Procyon lotor 3 13.84 6.58 0.476+/-0.194 36 102.9 0.06 0.001+/-0 1237.05 0.000‡ 204.6 0.000‡ - (Long and Frank, 1968)
Sciurus niger 4 3.38 0.55 0.163+/-0.058 11 12.36 0.01 0.001+/-0 83.66 0.000‡ 68.8 0.000‡ - (Long and Frank, 1968)
Spermophilus mexicanus 2 2.15 0.78 0.362+/-0.181 2 4.45 0.49 0.111+/-0.056 1.01 0.315 0.76 0.385 5 Male: (Burt, 1960);
Female: (Layne, 1954)
Taxidea taxus 4 10.71 1.97 0.184+/-0.065 2 98.7† 9.87† 0.1+/-0.05† 0.38 0.538 0.52 0.471 13 Male: (Burt, 1960);
Female: (Long and Frank, 1968)
Urocyon cinereoargenteus 3 6.33 1.44 0.228+/-0.093 11 50.94 4.5 0.088+/-0.019 5.09 0.024‡ 2.05 0.152 - Male: (Long and Frank,
1968); Female: (Hildebrand, 1954)

Table 6 Distribution of developmental stages 24 hours after copulation
Table 7 Comparison of variation in bacula vs. baubella lengths
species N females mean females SD females CV females N males mean males SD males CV males asymptotic statistic asymptotic p value mslr statistic
mslr p value min sample size References§
Cryptoprocta ferox 4 5.5 3.3 0.6+/-0.212 17 72.2 9.6 0.133+/-0.023 23.76 0.000‡ 10.12 0.001‡ - (Hawkins et al. 2002)
Lemur catta 6 0.38 0.06 0.158+/-0.046 4 1.4 0.14 0.1+/-0.035 0.65 0.419 0.73 0.393 21 (Drea and Weil 2008)
Leptonychotes weddellii 5 35 6.67 0.191+/-0.06 58 186.3 38.9 0.209+/-0.019 0.05 0.820 0.18 0.668 512 (Smith 1966)
Lontra canadensis 2 10.35 10.11 0.977+/-0.488 55 94.92 4.46 0.047+/-0.004 412.61 0.000‡ 36.17 0.000‡ - Male: (Friley, 1949);
Female: (Scheffer, 1939)
Mormopterus planiceps 6 1.62 0.05 0.031+/-0.009 9 7.9 0.18 0.023+/-0.005 0.6 0.439 0.42 0.517 44 Male: (Krutzsch and
Crichton 1987); Female: (Crichton and Krutzsch 1987)
Mustela vison 6* 1.02 0.45 0.439+/-0.127 99 44.6 2.18 0.049+/-0.003 312.33 0.000‡ 98.42 0.000‡ - (Long and Shirek, 1970)
Parascalops breweri 2 0.68 0.11 0.157+/-0.079 2 0.68 0.05 0.072+/-0.036 0.53 0.465 0.36 0.549 9 (Sinclair, 2014)
Phoca vitulina 2 7.5 2.12 0.283+/-0.141 3 127.3 2.52 0.02+/-0.008 7.81 0.005‡ 4.4 0.036‡ - Male: (Mohr, 1962);
Female:(Scheffer, 1949)
Procyon lotor 3 13.84 6.58 0.476+/-0.194 36 102.9 0.06 0.001+/-0 1237.05 0.000‡ 204.6 0.000‡ - (Long and Frank, 1968)
Sciurus niger 4 3.38 0.55 0.163+/-0.058 11 12.36 0.01 0.001+/-0 83.66 0.000‡ 68.8 0.000‡ - (Long and Frank, 1968)
Spermophilus mexicanus 2 2.15 0.78 0.362+/-0.181 2 4.45 0.49 0.111+/-0.056 1.01 0.315 0.76 0.385 5 Male: (Burt, 1960);
Female: (Layne, 1954)
Taxidea taxus 4 10.71 1.97 0.184+/-0.065 2 98.7† 9.87† 0.1+/-0.05† 0.38 0.538 0.52 0.471 13 Male: (Burt, 1960);
Female: (Long and Frank, 1968)
Urocyon cinereoargenteus 3 6.33 1.44 0.228+/-0.093 11 50.94 4.5 0.088+/-0.019 5.09 0.024‡ 2.05 0.152 - Male: (Long and Frank,
1968); Female: (Hildebrand, 1954)

35

N=number of specimens
SD=standard deviation

CV=coefficient of variation, with standard errors calculated as CV/sqrt(2N)

*50 females were dissected, baubellum was found in 6

†Mean taken from Burt 1960, standard deviation estimated at 10% of mean based on Long and Frank's (1968) statement that two male bacula were "nearly the same in length"
‡Bold indicates statistical significance at p<=0.05

§References: unless otherwise indicated, male and female data taken from same study. Full citations can be found in Supplementary Table 1.

36

Our finding that the baubellum showed more within-species variation than the baculum
is probably conservative because we based that inference on length measurements that
likely underestimated the amount of morphological variation in the baubellum. For
example, figure 1 of Long and Shirek [214] showed a collection of mink baubella that
vary dramatically not only in terms of length but also in overall shape, which the present
analyses do not capture. In fact, Long and Shirek [214] remarked of the baubellum that
“no other morphological structure known to us has such [high] variation.” In addition,
multiple studies have demonstrated the importance of baubellum shape in
distinguishing closely related species or subspecies that are otherwise morphologically
identical [97-99]. Unfortunately, the existing literature was not detailed enough for us to
quantify baubellum variation beyond length measurements.

Discussion

Sexual dimorphism is common in nature, and the evolutionary and developmental
contexts of sexual dimorphism have long-fascinated biologists [1, 215, 216]. A major
unsolved question is to what extent sexually dimorphic characters are constrained by
the shared genome of males and females [192]. Sexual dimorphism is expected to be
greatest in species where different optima can be reached via sex-specific expression of
the genome and response to selection. However, most traits are likely to be correlated
between sexes, placing significant constraint on the degree to which dimorphism can
evolve. At one extreme, a particular state may be beneficial in one sex, but harmful in
the other. In the absence of sex-specific modification of expression, the species will
37

evolve to a phenotypic compromise, where neither sex can reach its optima because of
counterselection in the other sex [192]. One evolutionary solution to such sexual conflict
is sex-specific expression of the genome, freeing each sex to evolve its own trait value,
or even for one sex to lose the trait if it is nonfunctional or deleterious.
Here, we investigate these issues using the baculum and baubellum as a model
system, with a focus on testing how strictly the two are correlated. Of 163 species, 134
(83.2%) shared states (both bones present, absent, or polymorphic), which may
demonstrate a strong evolutionary correlation (Figure 3). However, investigators may be
more likely to look for a baubellum if it is already known that a baculum exists in a
species, leading to potential study bias that inflates the correlation of the two states.
Nevertheless, the baubellum accumulated more evolutionary transitions than the
baculum, and these transitions occurred more recently, demonstrating the two are not
strictly correlated. Furthermore, the developmental and morphological variation of the
baubellum exceeds that of the baculum. Taken together, our study suggests that
baubellum is relatively free to accumulate variation and may not be functional in many
lineages.

Other bones, especially “free-floating” bones like the baculum and baubellum have been
gained and lost repeatedly in terrestrial vertebrates, but in almost all cases their
presence/absence is perfectly correlated between males and females. For example,
mammals have independently lost their clavicles a minimum of four times, and digits in
mammals have been independently lost dozens of times [217]. The patella has been
independently gained 4–6 times and lost twice in mammals [218], gained multiple times
38

in reptiles [219], and has variable presence in amphibians [220]. Even in the face of
these multiple independent transitions, clavicles, digits, and patella display the same
trait in males and females across species. So far, the intersection of sexual dimorphism
and bone losses and gains has only been observed in the mammalian bovids (Family
Bovidae) [221-223]. Female and male expression of horns are not perfectly correlated in
bovid evolution. Interestingly, similar to our study, there are no known species where
females have horns, but males do not. Bovid horns are sexually dimorphic in shape,
and the most comprehensive analyses conclude they function primarily in males for
intrasexual competition and for defense in females [221, 222]. The baculum and
baubellum thus represent a highly unusual case of widespread independently evolving
sexually dimorphic bones, with the baubellum demonstrating more evolutionary and
developmental lability compared to the baculum.

The proximate causes of sexual dimorphism in bacula and baubella appear to be linked
to hormonal profiles, or the sensitivity of individuals to various hormones. For example,
artificial administration of testosterone in dogs, ferrets, and rats leads to robust
development of the baubellum, even though very few female dogs and ferrets, and no
female rats, naturally develop one [4, 86, 88, 194, 201, 202, 224]. Interestingly,
castrating males prevented the baculum from reaching an adult stage in multiple
species [48, 52, 54, 56, 225], suggesting androgens are an important mechanistic link
between the development of both the baculum and the baubellum. A study that
compared skeletal growth in the forepaw and penis in castrated and noncastrated rats
concluded that growth factor Somatotropin positively affects bone development in the
39

forepaw but not the penis, and testosterone propionate by contrast affected bone
growth in the penis but not the forepaw [54]. In the most well-studied case, inducement
of the rat baubellum is time-dependent and dosage-dependent and is most effective
when administered before 10 days after birth. In laboratory-raised voles, Ziegler [102]
found that a baubellum-absent mother had some but not all offspring with baubella; it is
possible that natural variation in endogenous androgens or the maternal environment
explains such within-litter variance. The baculum and the baubellum appear to be more
androgen-sensitive than other bones in the skeletal system. If they are more sensitive at
the cellular level, these bones would serve as a model for understanding how
androgens affect early cell fate decisions in bone development.

Is sexual dimorphism of the baculum and baubellum influenced by the morphology of
the penis and clitoris, respectively? The development and evolution of the baubellum
cannot be understood without characterization of the soft tissue anatomy in which it
resides, namely the clitoris. However, few studies exist on the comparative anatomy of
the clitoris. In one study of 10 species (including primates, moles, and hedgehogs), the
internal structure of the clitoris and the baubellum differed greatly not only in size and
shape, but also whether they were distal or proximal to the urethra and vaginal opening
[226]. The position of the clitoris varied across 41 eutherian and marsupial species, from
deep within the vaginal tract to just inside the vaginal opening or cranial to the vaginal
opening [227]. Too few species overlap with our study, therefore it remains unknown
how baubellum and clitoral anatomy covary.
40

In addition to the anatomy of the surrounding soft tissue, behavioral data are required to
evaluate whether the baubellum is in fact functional. In many species, the clitoris
contains erectile bodies that engorge during copulation [73, 74, 228]. There is even
some speculation that engorgement of the clitoris alters access to the female's
reproductive tract, and without it copulation cannot occur [84, 229]. In males,
engorgement of the penis can lead to changes in the orientation of the baculum,
probably the result of hydrostatic pressure in the corpora cavernosa pressing against
the baculum [3, 28]. The stiffening of the corpus cavernosa in the clitoris might have
similar effects on the baubellum, which again might shed light on its potential role during
copulation. In some primates and pinnipeds, the clitoris and surrounding tissue can
undergo changes in color, shape, and/or size during seasonal estrous [230-233]. The
link to seasonal estrus suggests that the clitoris, and thus the baubellum, may play a
role in reproduction in these species. Juvenile female fossa has a very large baubellum
and clitoris that gives juvenile females a “masculinized” appearance [84], and it has
been speculated that this masculinized phenotype reduces male sexual harassment
and female territoriality [83]. Interestingly, female fossa lose their baubellum as they
age.

In conclusion, our study demonstrates that the baubellum is relatively free to
accumulate evolutionary transitions and developmental variation compared to the
baculum. At least in some species, these patterns suggest that the baubellum does not
play an important functional role and has become relatively unlinked to the character in
41

males, the baculum. In the future, additional anatomical, behavioral, and developmental
data may modify these conclusions in specific cases, but the overall trend appears to be
multiple cases of relaxed selection against a general background of developmental and
evolutionary correlation. These unusual bones provide a unique model system to
understand the evolutionary and developmental mechanisms that give rise to
morphological novelty and sexual dimorphism.
Acknowledgements

For helpful discussion, we thank Bruce Patterson (Field Museum) and Sara Keeble (U
Southern California). We thank Peter Ralph for statistical advice. This work was funded
by The Frederick and Dorothy Quimby Memorial Scholarship (MLS), NSF CAREER
award 1150259 (MDD) and NIH Grant R01GM098536 (MDD). Some of this work was
presented at “The Morphological Diversity of the Intromittent Organ” session at the 2016
Society of Integrative and Comparative Biology, funded by NSF grant #1545777 to
Brandon Moore and Dianne Kelly.

Conflict of Interest

None declared.

Author Contributions

42

MLS, NS, and MD were responsible for the conception of the project, literature review,
analysis, and writing of the manuscript. All authors have provided critical review and
approved submission of this manuscript.
43

Chapter 3 Male-derived copulatory plugs enhance implantation success in female Mus
musculus
Lough-Stevens, M., et al. (2021). "Male-derived copulatory plugs enhance implantation
success in female Mus musculus." Biology of Reproduction 104(3): 684-694.

Abstract
Among a wide diversity of sexually reproducing species, male ejaculates coagulate to
form what has been termed a copulatory plug. A number of functions have been
attributed to copulatory plugs, including the inhibition of female remating and the
promotion of ejaculate movement. Here we demonstrate that copulatory plugs also
influence the likelihood of implantation, which occurs roughly 4 days after copulation in
mice. Using a bead transfer method to control for differences in ejaculate retention and
fertilization rates, we show that implantation rates significantly drop among females
mated to genetically engineered males incapable of forming plugs (because they lack
functional transglutaminase 4, the main enzyme responsible for its formation).
Surprisingly, this result does not correlate with differences in circulating progesterone
levels among females, an important hormone involved in implantation. We discuss three
models that connect male-derived copulatory plugs to implantation success, including
the hypothesis that plugs contribute to a threshold amount of stimulation required for
females to become receptive to implantation.

44

Introduction

Across a huge diversity of sexually reproducing species, male ejaculates coagulate to
various degrees. In some cases, ejaculates form a solid structure referred to as a
“copulatory plug.” Biologists have long sought to understand the molecular basis,
functional role, and evolutionary significance of copulatory plugs (reviewed in [106]).
Multiple lines of evidence suggest copulatory plugs evolved as a means for one male to
inhibit remating by females [109, 117, 118, 125, 129-131]. However, copulatory plugs
also play important roles in non-competitive mating, including retention of the ejaculate
and sperm survival [110, 112, 114, 115, 149, 150, 156, 234-240]. Genetic engineering
experiments have enabled powerful experimental approaches aimed at understanding
the function of the copulatory plug. Such experiments have demonstrated that
transglutaminase 4 (TGM4) and seminal vesicle secretory protein 2 (SVS2) are required
for copulatory plug formation, with contributions from the protein prostate and testis
expressed 4 (PATE4) [110, 114, 149]. TGM4 is a catalytic enzyme that crosslinks SVS2
and is required to form the copulatory plug in rodents [241, 242]. In a previous study, we
copulatory plug and were significantly less likely to sire a litter after 2 weeks of being
paired with a female [110]. In the absence of a plug, their ejaculates were quickly lost
seemed to be a normal number of embryos each mating [110], leaving the reduction in
litter success unexplained. The overall goal of the present study was to understand
45

which post- on whether copulatory plugs influence the probability that females enter a physiological
state receptive for implantation, which in mice occurs approximately four and a half days
after copulation. In many mammalian species (including mice), copulatory stimulation is
required for implantation to proceed normally [134, 158, 165, 167, 238, 243-249]. This
stimulation can even be applied artificially; Diamond [141] showed that the number,
duration, and intervals between vaginal insertions of an artificial stimulator differentially
affected the probability of “behavioral pseudopregnancy,” an indication that the female
is primed for implantation. Interestingly, the parameters that most closely mimicked
natural mating behaviors of male mice were also the most successful at artificially
inducing pseudopregnancy, suggesting that females require a species-specific set of
copulatory cues to allow implantation. Consistent with the idea that some threshold level
of copulatory stimulation must be achieved prior to future reproductive effort, female
rodents demonstrate a behavior referred to as “paced mating,” where females are more
likely to return for more copulation bouts with a male if copulation is interrupted [142,
159-163]. Lastly, it is well known that artificial insemination in female mice almost
always has to be accompanied by mating with vasectomized males if implantation is to
succeed [121]. The molecular mechanisms that link copulatory stimulation with
downstream reproductive events are not well understood. However, it is known that in
mice, copulation induces differential gene expression and neurogenesis in female
brains [178, 250]. Given the relationship between copulatory stimulation and
subsequent pregnancy, we hypothesized that the lack of a copulatory plug leads to
reduced implantation success. Using a bead transfer approach, we demonstrate that
46

males. Surprisingly, this implantation defect occurred even though females mated to
les showed the characteristic surge in progesterone, the main hormone
required to initiate implantation and sustain pregnancy. Our study expands the
functional roles of copulatory plugs, suggesting they influence implantation several days
after copulation, but leave the molecular mechanism of this effect unknown. We discuss
several models that could link copulatory plugs with implantation success, including that
they contribute to copulatory stimulation required to shift female physiology toward a
state receptive for implantation.

Materials and Methods

3.1 Ethics statement

All experiments, animal husbandry, and personnel were approved by the University of
Southern California’s Institute of Animal Care and Use Committee under the protocols
#11777 and #11394. The goal of the four main experiments below was to evaluate

3.2 Animals

Mice were housed on a 14:10 h light:dark cycle with ad libidum access to water and
food. Three different strains of mice were used throughout. All female mice were
47

the
prostate- -
[251] and consists of a 7 kb “knockout first”
cassette inserted into the C knockout allele but was unnecessary in the present study. All male mice were
essentially genetically identical except for this 7 kb “knockout first” cassette that
spans exons 2–3 of TGM4. Seminal vesicle secretory protein 2 (SVS2) and PATE4 are
also involved in making functional copulatory plugs [112, 114], and there are available
knockout models for both. However, SVS2 has additional off-target effects, including the
influence of sperm capacitation [111, 112, 152] and protection of sperm from the uterine
environment [113, 153], which could confound our studies of plug function. PATE4 does
not completely eliminate the copulatory plug [114], making it a less ideal model to study
behavior [110]. Several additional features suggest that knocking TGM4 has minimal
off-target effects: TGM4 is only expressed in the male prostate [107, 252, 253], and the
only annotated domains are related to the cross-linking reaction necessary for plug
formation. Importantly, TGM4 has accumulated multiple loss-of-function mutations in
some species that do not form a plug [254], which is not predicted if this gene has
important functions outside the context of plug formation. To produce experimental
mice, we paired a single breeder male and single female together for 2 weeks, and then
48

separated them so that the dam could give birth in isolation. Pups were weaned at 21–
28 days of age and placed in cages of up to three siblings segregated by sex. All
experimental females were between 8–9 weeks old at time of experiment. Experimental
males were caged individually to avoid dominance interactions at the time of the
experiment [255, 256].

3.3 Vasectomies

Most of our experiments detailed below required males that were incapable of
fertilization. We vasectomized males at approximately 10 weeks of age using the scrotal
entry method [121]. Males were anesthetized by inhalation of 1.5–2% isoflurane
an analgesic. We applied eye lubricant, and then shaved the scrotum followed by three
alternative washes of betadine and 20% isopropyl alcohol. A small incision was made in
the scrotal region, and we carefully lifted each vas deferens out to cut and remove a
Vetbond glue. A vasectomy usually took 30 min, and animals generally woke and
became active 5–10 min after cessation of isoflurane. Animals recovered for at least 2
weeks prior to experiments.

49

3.4 Inducing ovulation

In most of our experiments detailed below, we artificially induced estrus in females
between 8–9 weeks of age. We intraperitoneally injected females with 5 IU Pregnant
Mare Serum Gonadotropin (BioVendor #RP1782725000) approximately 60 h before
copulation and then injected with 5 IU Human Chorionic Gonadotropin (hCG; Millipore
Sigma #230734) approximately 8–9 h before copulation and 7–8 h before the start of
their dark cycle. As much as possible, we controlled batch effects by assigning sibling
females to different conditions. For example, with three full sisters in a litter, one would
be used as a control. Of course, this was not always possible and depended on the
number of females weaned per litter.

3.5 Identifying ejaculation based only on behavior

Successful ejaculation is often identified by observing copulatory plugs [121], but
ot produce a copulatory plug. Therefore, we scored ejaculation
success only through behavioral observations and not by the presence of copulatory
method. We continuously observed males and females for a maximum of 4 h after
pairing. Pre-copulatory behaviors include chasing, mounting, intromissions, and bouts of
thrusting without ejaculation [257, 258]. Ejaculation was identified as sustained thrusting
50

that culminated in male and female freezing in position, often followed by the pair
collapsing to their side [259]. After ejaculation, males and females often appear
relatively uninterested in each other, occupying separate areas of the cage while
grooming themselves. All behavioral observations were done blind to genotype and all
females were checked for copulatory plugs for later quantification of our accuracy in
behavioral scoring of ejaculation.

3.6 Experiment 1: 24-hour fertility (non-vasectomized males)

We induced ovulation in FVB females, then individually paired them with either a
We dissected the oviducts, removed eggs from the ampullae, treated them with
hyaluronidase (Sigma #H4272) to remove the cumulus cells, and then transferred eggs
to a customized depression slide. Using a compound microscope, all eggs were scored
as having 2 pronuclei or in some stage of later cell division. Eggs that were undergoing
apoptosis or lacked a zona pellucida were considered damaged and excluded from
analysis. The remaining eggs were considered unfertilized. All scoring was done blind to
treatment. We used a generalized linear model with binomial variance (logit link) to test
whether the number of fertilized (two pronuclei stage or beyond) vs. unfertilized eggs
varied according to male genotype, using the glm function in R [260]. In an additional
analysis of only fertilized eggs, we used the same approach to test whether the number
of fertilized eggs reaching the two-cell stage varied according to male genotype.
Significance
51

3.7 Experiment 2: implantation rates (vasectomized males)

We induced ovulation in females then split them into three groups: mated to
but otherwise treated identically. We observed pairs until ejaculation, and then removed
females within 20 min, or in the case of unpaired controls moved them to a new cage.
success, we required a method that was independent of fertilization success. We
#60010) [261-263], whereby embryos are trans-cervically transferred into the uterus
using a specialized pipette tip. Instead of embryos, we transferred Concanavalin A
Sepharose (Con-A) beads (Sigma #C9017), a well-established method for assessing
the decidual response in the mouse uterus [164, 264-267]. Compared to natural
implantation, mothers who implant Con-A beads show no detectable difference in
uterine gene expression [268], suggesting they are a good proxy for testing whether the
uterus is receptive to implantation. Females were trans-cervically injected with 12–18
Con- -buffered solution 2.5 days
-flexible pipette tip fitted to a P-
[261-263, 269]. Implantation occurs approximately 4.5 days after ejaculation in female
mice [121, 133]. Four days after injecting beads (6.5 days after ejaculation), we injected
100 mL of 1% Chicago Blue Dye into one of the female’s two lateral tail veins, via a 27-
gauge needle [121, 270]. Circulating dye collects at implantation sites, enhancing
52

detectability. Approximately 3 min later, females were euthanized, and their uteri
dissected for scoring under a dissection microscope. To be scored as an implantation
site, the observer had to see the localization of dye as well as general decidualization of
the uterus [266]. Scoring was done blind to treatment. When implantation occurred, it
was normally a small number of beads (see Results); therefore, we scored
implantations as successful or not rather than by the number of beads implanted.
Among females receiving an ejaculate, we tested whether implantation success varied
account for the non-independence that arises because most males were used in
multiple crosses, we permuted genotype among males 10,000 times to build an
empirical null distribution of chi-squared values. To make the test one-tailed (because
success), we multiplied permuted chi- our empirical null distribution was centered on zero.

3.8 Experiment 3: progesterone assays (vasectomized and non-vasectomized males)

Progesterone is an essential reproductive hormone that regulates the initiation and
timing of implantation success in mammals, spiking in female mice at roughly 4.5 day
post-ejaculation and remaining high for at least 5 days afterward in female mice [139,
271]. For a subset of females from Experiment 2, we measured serum progesterone
through enzyme-linked immunosorbent assays (ELISA). At approximately 6.5 days after
53

ejaculation, a subset of females was injected with Chicago Blue dye, anesthetized with
1.5–2% isoflurane (FLURISO™) in pure oxygen and exsanguinated via cardiac
puncture with a 22 G needle [121]. This procedure typically yielded 600– blood. Blood was allowed to settle for a minimum of 20 min before being spun down for
12–15 min at 4 degrees Celsius at 2000 x g. Spun-down blood separated into two
distinct layers; the top serum layer was collected via pipet. Serum was aliquoted and
Samples were run on 96-well plates, with at least 8 wells of each plate consisting solely
of positive and negative controls as well as 16 wells to draw a standard curve following
the manufacturer’s recommendations (two replicates each of 1000, 500, 250, 125, 62.5,
ach sample was diluted to three different
concentrations. During the course of our experiment, we discovered that variability in
absorbance readings was related to both the progesterone level of the sample and the
dilutions applied during the ELISA assays. For example, replicates with low
progesterone and high dilutions—or high progesterone with low dilution—tended to yield
unstable estimates of progesterone. Therefore, we diluted serum of mated females
200X, 400X, and 500X, and unmated females 25X, 50X, and 100X. Each set of dilutions
was run in triplicate, for a total of nine absorbance reads per female. Raw absorbance
customized R scripts. Within individual blood samples, some dilution series were more
variable than others. We therefore calculated a weighted mean progesterone level per
individual female, with the contribution of each dilution factor weighted by the reciprocal
54

of its coefficient of variation (standard deviation divided by mean). All statistics are
based on this weighted mean.

3.9 Experiment 4: differential abortion (non-vasectomized males)

abortion among their mates rather than reduced implantation, we compared placental
scars to the number of pups born across a number of crosses. Placental scars are
melanized tissue left at implantation sites for several months after birth [272]. If females
abort their litters after implantation but before birth, we would observe more placental
scars than pups born. In this experiment, females were not induced to ovulate, but
rather paired with a non- separated. Females were allowed to give birth, then euthanized a week later. We
dissected out the uterus, pinned it onto to a petri dish and submerged it in hydrogen
peroxide for 2 h to bleach the tissue and highlight placental scars.

Results

3.10 age

55

did they differ in the likelihood that litters reached weaning age. This previous study was
based on almost 200 crosses (Table 2 of reference [[110]]). Here we included a greater
une 2018, under the
assumption that larger sample sizes would provide additional power. After being paired
= 296.7, df =
We previously reported that if a litter was born, there was no difference in
the likelihood that a litter reached weaning age or litter size [10]. With the larger dataset
ificantly less likely to reach
0.0005). The low success of crosses producing weaned litters is
backgrounds, where less than 50% of all crosses produce pups that survive to weaning
[273, 274] males sired a median 6 offspring, slightly but significantly fewer than the median 7
offspring sired by males sired fewer, smaller litters, that were less likely to survive to weaning. The
reduced probability more likely to neglect their litters.

56

3.11 Behavioral scoring of ejaculations is reliable

Ejaculation success was scored only via behavioral observations so that all crosses
could be we judged did not end in successful ejaculation, we observed a plug in 13 of them.
Figure 6)
are based on crosses that ended in successful ejaculation, where our accuracy was
high.

3.12

5.4, range = 0– – Table 2, Supplementary Data S1). There was no difference in the total number of eggs
–28;
–35) (two-tailed t-test = 0.93, df = 45.39, P = 0.36).
These results were consistent with our previous study, but here we carefully controlled
57

time since ejaculation [10]. We scored 307 fertilized eggs from 44 females (21 to
reached the two- Table 2, Supplementary Data S1). There were fewer total females in this second
analysis because eight females had zero fertilized eggs, of whom seven were mated to
could have resulted in their fertilized eggs not reaching as advanced of a developmental
ion and
developmental rate would confound any test of implantation success, further justifying
our use of the bead transfer experiments of Experiment 2.
58

Table 8 Distribution of developmental stages 24 hours after copulation
Male Total eggs Unfertilized (%) 2 pronuclei (%) >=2 Cells (%)
22 317 111 (35.0) 36 (11.4) 170 (53.6)
TGM4- - 30 325 224 (68.9) 43 (13.2) 58 (17.8)

Table 9 The number of females implanting Con-A beads according to category. The total number of unique males
was 31 TGM4+/+ and 31 TGM4-/-individuals.
Implanted

Genotype Ejaculation Plug Unique males Plug Unique males %
implanted

Alone 0 0 0 0 58 0 0

Yes 42 0 21 29 1 15 58.33

TGM4- - Yes - 25 18 - 39 15 39.06

8 0 8 5 35 20 16.67

TGM4- - - 3 3 - 28 18 9.67

59

3.13 Experiment 2: implantation

Of the 50 control females never exposed to males, none showed implanted Con-A
beads, as expected because female mice are not receptive to implantation without
copulation [121] (). Of 72 females who received an ejaculate from vasectomized
Table 3). Of 64 females
beads (Table 2 -value = 0.014,
Figure 6). A total of 50 unique males were used in these 135 crosses, each male
copulated with a mean of 2.72 (standard deviation [SD] = 2.58, range = 1–11) females.
Because all males for this experiment were vasectomized, differences in implantation
could not be due to differences in fertilization rate. In addition, all females received a
1.7, range = 13– – 11–18) (one- differences could not be due to variation in the number of beads transferred (number of
– –8;
Welch two-sample t-test =1.53, df = 62.3, P = 0.13). Females who were paired with
males but did not receive an ejaculation (scored behaviorally) had low implantation
Table 3 0.3, df = 1, P = 0.59). For females whom we scored as having not received an
60

implantations were incorrectly scored based on the presence of a copulatory plug.
Furthermore, among all 13 females who had a plug even though they were mis-scored
to the 58.33% implantation rate identified from females receiving an ejaculate from
accompanied by copulatory attempts, is not sufficient to induce implantation. Rather,
females receiving an ejaculate are much more likely to be receptive for implantation,
especially in cases where the ejaculate forms a copulatory plug (Table 3,
Supplementary Data S2).

61

Figure 6
likely

3.14 Experiment 3: progesterone is equally elevated among females who receive an
ejaculate

Across 42 plates’ worth of ELISA assays (Supplementary Data S3), the r2 of the
standard curve averaged 0.918 (SD = 0.085) (Supplementary Figure S1), indicating we
could reliably construct standard curves across plates. From serum samples taken from
98 females, we made a total of 333 dilutions in triplicate. Across 333 dilution sets, the
median coefficient of variation was 0.11, with 147 of these less than 0.1. These
62

coefficients of variation are reasonably low, indicating good repeatability in spite of the
known noisiness associated with ELISA assays. A total of 49 females tested received
an ejaculate from = 29) males, of whom 11 or 12 females, respectively, implanted beads successfully.
Among these 49 females, there was no significant difference in serum progesterone
level based on mate genotype or implantation success (two - genotype: F1,46 = 0.043, P = 0.84; implantation success: F1,46 = 0.96, P = 0.33).
However, across all five possible mating outcomes (females paired and mated to either
Figure 7). Tukey honest significant differences revealed that females receiving an
ejaculate had higher progesterone expression when compared to the other mating
outcomes (Figure 7), consistent with previous studies showing that mated females have
elevated progesterone [275, 276]. Control females showed uniformly low levels of
progesterone expression (Figure 7). In sum, females receiving an ejaculate showed an
upwards shift in circulating progesterone approximately 6.5 days after ejaculation,
regardless of their implantation success or the genotype of their mates. Although
tangential to our main line of investigation, we were interested in how these results
would change if we used nonvasectomized (intact) males. We repeated the experiment
with 42 females who received an ejaculate from a non- embryos. Among these 42 females, there was no significant difference in progesterone
level based on male genotype or implantation success (two -
63

genotype: F1,39 = 2.8, P = 0.1; implantation success: F1,39 = 0.46, P = 0.5). However,
progesterone levels varied across all five possible mating treatments (one- receiving an ejaculate had high progesterone compared to the other three groups
(Supplementary Figure S2). Interestingly, however, the progesterone levels were higher
among females mated to intact males vs. vasectomized males (compare
Supplementary Figure S2 vs. Figure 7, Supplementary Data S3).

Figure 7
Boxplot of progesterone levels estimated across 98 females from five different groups; Mated vs. Unmated indicates whether
ejaculation was scored within 4 h of pairing. All male mates were vasectomized. WT = = = of females per group. Gray hash marks indicate individual observations. Vertical line separates the two different groups
(homogeneous within each group) identified with Tukey HSD tests.

64

3.15 f
abortion

with an average size of 8.1 pups and an average of 9.0 scars. Thus, there were
generally more placental scars than pups born, indicating at least some offspring died
litter, and she had a single pup with a single placental scar; none of the other females
had placental scars, suggesting that their litters failed prior to implantation.

Discussion

Male-derived copulatory plugs promote implantation 4 days postcopulation. Females
suffered
reduced implantation success cannot be solely attributed to reduced volume of
ejaculate in the female reproductive tract or reduced fertilization (Table 2), because our
bead transfer experiments (Table 3, Figure 6) used vasectomized males (so all males
were sterile) and all females received the same amount of Con-A beads (which mimic
embryos). We now discuss three different models that could link male-derived
copulatory plugs to downstream implantation events.

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3.16 Model 1: differences in ejaculate composition or time in situ may influence
implantation success

differ from its main target protein, SVS2, causing it to precipitate into the copulatory plug [253,
277] an unusually high amount of soluble SVS2. SVS2 is required for copulatory plug
formation [113], but it has additional functions including regulation of sperm capacitation
[112, 234, 235], and protection from cytotoxic challenges [149, 236]. Seminal fluid
proteins have been shown to impact female reproductive biology. Some seminal fluid
proteins induce inflammatory responses and cytokine expression in the uterus [278-
280]. Cytokines and prostaglandins from male seminal fluid bind to receptors in the
female reproductive tract, and induce shifts in gene expression that, among other
functions, promote implantation [281, 282]. Seminal fluid also impacts reproductive
events in other species. Ovulation-
[283-286] is a neuropeptide in seminal fluid that triggers ovulation and upregulation of
reproductive hormones such as prolactin in induced ovulators (e.g., camelids, rabbits)
[286-292] and is present in the semen of some (e.g., bovids and cervids) but not all
(horses, pigs) spontaneous ovulators [285, 290, 293, 294]. Prepubertal female mice can

[295].
66

plasma progesterone and rates of pregnancy [294]. Although not all molecules
discussed here are part of the copulatory plug itself, the stoichiometry of ejaculated
protein molecules will likely differ when males cannot form a copulatory plug, which
could in turn differentially influence implantation success. In addition to different
female reproductive tract (uterus and oviducts), as ejaculates essentially “leak out” in
the absence of plugs [110]. It is therefore possible that molecular interactions simply
have less time to manifest themselves.

3.17

It is possible that copulatory plugs themselves store and deliver chemical or hormonal
signals to females via diffusion into the surrounding epithelial tissue. In mice, some
male-derived hormones enter the female’s bloodstream after copulation [296-298].
Under a model where copulatory plugs represent delivery mechanisms for such
hormones, females mated dosage of chemical signals to continue with implantation.

3.18 Model 3: copulatory plugs may contribute to the threshold level of stimulation
required to facilitate implantation

Many studies have linked copulatory stimulation to reproductive success [64, 299]. In
mice, the copulatory plug is very large and prominent, and glues into the cervix and
67

vaginal canal for 24–48 h after ejaculation[116]. Since it is often visible externally, the
copulatory plug in mice is likely to induce considerable mechanical stretch reception.
The copulatory plug may be an important component of the stimulation required to shifts
females toward implantation receptivity. Several follow-up experiments could more
precisely test this physical stimulation model. One could insert artificial plugs and
determine whether implantation occurs with stretch reception itself. Yang et al. [178]
demonstrated that gene expression in female brain regions shifts in response to
copulation. Our study predicts that such changes will be muted when females mate to
nerally, to males that provide inadequate copulatory
stimulation. Another prediction is that experimentally preventing copulatory stimulation,
through either application of anesthetics or ablation of critical nerves [300, 301], could
mimic the reduced implantation we observe here. Recently, it has been shown that
female mice with a history of reproduction show relatively early aging and decrease in
late-life reproductive potential compared to females housed with sterile males [302]. It
would be interesting to test whether these shifts in life history occur when males cannot
form a copulatory plug. If this third model is correct, then the copulatory plug may be
one of many traits assessed during female choice, which is simply the phenomenon by
which females nonrandomly invest in reproduction with particular males [2, 303-305].
Both copulatory plug size and resident time vary among mouse strains, and copulatory
plugs derived from males who have recently mated are smaller [116, 123], indicating
genetic and environmental variance that could potentially covary with implantation
success.

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3.19 Progesterone levels are only slightly correlated with defects in implantation

Under normal reproduction, progesterone shows a predictable cycle in the events
leading up to implantation and pregnancy in mice [275]. It begins at low physiological
levels during early pregnancy, peaking between 5 and 9 days after copulation to
promote decidualization and early gestation [306]. Low levels of progesterone are
associated with an inability to sustain pregnancies past 11 days [306]. Contrary to
expectation, females receiving an ejaculate showed this characteristic post-copulatory
increase in progesterone, regardless of the male’s genotype, vasectomy status, or
implantation success. Perhaps other hormones not measured here, like prolactin, better
explain for the differences in implantation. Females experience twice daily surges in
prolactin that maintain corpora lutea via increased expression of estradiol and
luteinizing hormone receptors, thus controlling female progesterone levels through
pregnancy [166, 302, 307, 308]. One possibility is that prolactin surges are winding
pregnancy are too low, postpartum maternal investment decreases because prolactin
normally drives maternal neurogenesis [250, 309, 310]. If copulatory plugs are affecting
prolactin levels, then it could explain both the reduced implantation and increased litter
shown to increase the accumulation of uterine fluid [311, 312], and it is possible that
increased uterine or oviductal fluid dynamics contributes to signaling related to
implantation. Female mating history also influences levels of prolactin [302]. Our results

69

considered normal levels of progesterone, their uteri were not always receptive to
implantation. It is also possible that progesterone defects occur at stages other than
those observed here.

Conclusions

Our study expands upon a growing list of functional roles of copulatory plugs. By using
a bead transfer approach that bypasses differences in ejaculate retention and
embryonic development, we demonstrate that females are less receptive to
implantation, and less likely to wean litters that are born, if their mates cannot form a
copulatory plug. We present three models to explain this phenomenon, including that
copulatory plugs contribute to a threshold level of stimulation required to trigger female
physiology toward a state of implantation. Perhaps most surprisingly, plug formation and
implantation success is separated by about 4 days in mice, implying long-term effects of
copulatory plugs.

Supplementary Materials

Supplementary material is available at BIOLRE online.

70

Acknowledgements

We thank Shelby Chickman and Juanna Xie for their assistance with data collection and
colony maintenance and the members of the Dean laboratory for their helpful feedback.
Ian Ehrenreich and Rachel Schell provided access to a plate reader. Three anonymous
reviewers and an associate editor greatly improved the manuscript.

Conflict of Interest

The authors have declared that no conflict of interest exists.

Author Contributions

M.L.S. and M.D. conceived and designed the research; M.L.S., C.G., M.U., A.H., and
C.S. performed the research; M.L.S. and M.D. conducted the analysis; and M.L.S. and
M.D. wrote the paper.

71

Chapter 4 Pseudopregnancies are more severe in Metatheria than Eutheria

Abstract

Female mammals cycle through multiple checkpoints during reproduction. All
else equal, females should avoid investment in the next stage if the current stage has
failed. Nevertheless, non-pregnant females from a diversity of mammalian species often
enter a physiological state identical to pregnancy, a phenomenon termed
“pseudopregnancy”. Here we quantify the severity of pseudopregnancy across
mammals by focusing on the lifetime of the corpus luteum, an endocrine gland that is
essential for implantation and pregnancy. We find that pseudopregnancies are more
“severe” (i.e., tend to last longer) among metatherians versus eutherians. We
hypothesize that the extended pregnancies of Eutherian species are energetically
costlier, and selection has favored the evolution of mechanisms to reduce the length of
pseudopregnancy.

Introduction

Female mammals exhibit extensive maternal investment through gestation and
nursing of their offspring [174, 313-319]. Females can discontinue their investment at
multiple stages, by blocking implantation, aborting offspring, or abandoning their litters
[308, 320-332], which can be important checkpoints if reproduction has failed. Yet
farmers, veterinarians, breeders, and evolutionary biologists alike have all described
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cases of “pseudopregnancy”, wher identical to truly pregnant females even though fertilization and implantation has not
occurred [166, 170-172, 174, 176, 333-354].
Because pseudopregnancy involves some level of maternal investment without
offspring, it likely represents a fitness cost for females, who would benefit from exiting
pseudopregnancy and returning to a reproductive state. However, some species (for
example, dogs), exhibit pseudopregnancies that last longer than a normal pregnancy,
while others (for example, humans) almost never appear pseudopregnant.
Pseudopregnancy has been described in many mammal species regardless of mating
system, seasonality, and number of estrous cycles per year [189, 320, 355-358].
We pose and assess two non-mutually exclusive hypotheses to explain the
variation in pseudopregnancy length across mammals, both of which rely on the
assumption that pseudopregnancy is costly. Under the “physiological cost” hypothesis,
pseudopregnancy represents a physiological cost that should be avoided, especially in
species where gestation is normally long. This hypothesis predicts that Eutherians (true
placental mammals with long gestation times) should have short pseudopregnancies
compared to Metatherians (marsupial mammals with short gestation times). Under the
“lost opportunity” hypothesis, pseudopregnancy represents lost opportunity, which
should be more costly in species where reproductive opportunities are relatively
confined in time or implantation is separated in time from fertilization. This hypothesis
predicts that pseudopregnancy should be short in species that breed seasonally,
implantation.
73

To assess these hypotheses, we searched the literature for data on the lifetime
of the corpus luteum (the temporary endocrine organ that sustains pregnancy) relative
to gestation length, and progesterone levels (a major hormone involved in pregnancy).
We show that Eutherians have shorter pseudopregnancies compared to Metatherians,
providing support for the “physiological cost” hypothesis, but no support for the “lost
opportunity” hypothesis. Interestingly, Eutherians have also evolved relatively elaborate
hormonal regulation of pregnancy, offering potential molecular mechanisms by which
Eutherians exit pseudopregnancy more quickly than Metatherians. Our study suggests
that the evolutionary shift towards increased investment in Eutherian gestation was
accompanied by selection that favored females that avoid pseudopregnancy, although
much of the variation remains unexplained.

Materials and Methods

4.1 Using corpus luteum lifespan to quantify pseudopregnancy severity.

We sought a biologically objective indication that a female has entered a state of
pregnancy or pseudopregnancy. We focused on the functional lifespan of corpora lutea
(CL), the temporary endocrine glands that form from a ruptured ovarian follicle. Corpora
lutea are sustained by angiogenesis and produce progesterone, which is correlated with
implantation success and gestation. Although there have been some attempts to find
alternative hormones that distinguish between pregnancy and pseudopregnancy (e.g.
relaxin), no other hormone has so far been better reported. More importantly, circulating
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hormones such as progesterone from the corpus luteum have been shown to affect the
mother’s brain, hormonal cycling, mammary development, uterine development, and
immune function. The presence of an active corpus luteum thus serves as a proxy for
an altered physiology for female mammals that is likely costly to produce and maintain.
Given that an active corpus luteum allows females to produce and alter
concentrations of hormones necessary for early pregnancy, we consider females who
maintain their corpora lutea but are not pregnant to be pseudopregnant in the current
study. We searched the literature for reports of functional corpora lutea in non-pregnant
females. We defined functional lifespan from histological data showing robust,
vascularized corpora lutea, or indirectly from the characteristic spike in progesterone
that they produce. We took care to ensure that females could not have been pregnant –
that is, the authors had to indicate that females were housed in isolation of males or
were housed with vasectomized males. In other words, the functional corpora lutea
observed could not have been due to a true pregnancy that was aborted and therefore
undetected by the researcher. We then quantified pseudopregnancy severity in three
main ways:

4.2 Phenotype 1: the lifespan of CL in pseudopregnant females, normalized by
Gestation length in true pregnancies (CLG).

First, we estimated the severity of pseudopregnancy as the functional lifespan of
CL from non-pregnant females, divided by the natural gestation length of that species,
also taken from the literature. We refer to this phenotype as CLG. In most cases, CLG
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ranged from 0 (pseudopregnancy does not occur) to 1 (pseudopregnancy persists for as
long as a normal gestation). In some cases, this metric exceeded 1, indicating
pseudopregnancies that persisted for longer than a normal pregnancy. We were able to
calculate CLG for 67 species.

4.3 Phenotype 2: the lifespan of CL in pseudopregnant females, normalized by the
lifespan of CL in truly pregnant females (CLCL).

In a complementary approach, we normalized CL lifespan in pseudopregnant
females by CL lifespan in truly pregnant females. We were able to calculate CLCL for
19 species, as it was comparatively rare to find studies of CL studies in pregnant
females, especially in non-domesticated, non-human species.

4.4 Phenotype 3: Peak Progesterone in pseudopregnant females, normalized by peak
progesterone in truly pregnant females (PP).

We compared the progesterone levels in pseudopregnant vs. pregnant females
that were directly compared within a paper. In early pregnancy, progesterone is
produced by CL, and later by the placenta. Therefore, phenotype #3 is a less direct
measurement of the CL. In addition, comparing progesterone levels is a more
qualitative assessment because the data we collected were oftentimes in the form of a
curve on a figure rather than raw numbers as the original data for all papers was not
available.
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4.5 Ancestral state estimation; testing the “physiological cost” hypothesis

To test whether pseudopregnancies are more severe in Methatherians compared
to Eutherians, we separately mapped pseudopregnancy severity onto the phylogeny
produced by Upham et al. [359], trimmed to match the species for which we were able
to gather each phenotype. We then estimated the ancestral states and their 95%
confidence intervals at the base of Methatherians vs. Eutherians, using the ACE function
in the R package APE.
A common approach to test for differences in phenotypic optima among groups is
to employ Ornstein-Uhlenbeck (OU) models. However, the two states we are analyzing
(Metatherian vs. Eutherian) only evolved once each, rendering these models
inapplicable because they require multiple derivations of a trait.

4.6 Testing the “lost opportunity” hypothesis; seasonality, delayed

All else equal, we expect pseudopregnancy to be more costly in a species that
nths exist
between copulation and implantation, or ovulates a single time per year. The reason
pseudopregnancy would be more costly in such species is that they must wait longer for
the next reproductive opportunity. We compared the degree of seasonality (one defined
period for breeding versus year-round breeding),
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development (one or more stages where the stages of pregnancy were paused by the
mother), and cyclicity (one versus multiple rounds of ovulation yearly).

4.7 Additional covariates: ovulation type and placental invasiveness

In addition to testing our two main hypotheses, we also tested for a relationship
between pseudopregnancy severity and ovulation type and placental invasiveness.
Mammalian species have two main pathways to ovulation which must be considered in
the current study. Spontaneous ovulators (e.g., humans, mice, raccoons, kangaroos) do
not require any male cues and will ovulate even if isolated from males.
Pseudopregnancy in spontaneous ovulators is probably easiest to study and report in
the literature, because functional corpora lutea in the absence of a male is an indication
of pseudopregnancy. It should be noted that even in spontaneous ovulators, male cues
can influence the timing and frequency of ovulation [322, 323, 327, 360].
In contrast, induced ovulators (e.g., cats, rabbits, alpacas, koalas) require male
cues to induce ovulation. In some cases, copulation itself is required to induce
ovulation. Pseudopregnancy will be more difficult to study in these species because the
investigator must expose the female to a male that induces ovulation but does not
fertilize her ova – for example by exposing females to vasectomized males. In some
species (belugas, tiger quolls, american and asian black bears), females undergo both
spontaneous and induced ovulation; in our study we call these species “facultative”
ovulators. One species (dwarf hamsters) requires male cues at two different stages: one
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to ovulate, and a second exposure to induce the activation of copora lutea. We
classified dwarf hamsters as induced ovulators.
Across species, placental morphology ranges from shallowly invasive
(epitheliochorial), moderately invasive (endotheliochorial), or strongly invasive
(hemochorial). We might predict that pregnancy would be costlier in species with
relatively invasive placenta. Similarly, we might predict that females from these species
have evolved more elaborate control over sustaining pregnancy, and are therefore able
to exit pseudopregnancy more quickly.

Results

All three phenotypes we gathered indicated that Metatherians experience more
severe pseudopregnancies than Eutherians. However, only phenotype 1 (CLG) yielded
enough data to make robust inferences.

4.8 Phenotype 1: the lifespan of CL in pseudopregnant females, normalized by
Gestation length in true pregnancies (CLG).

We quantified CLG from 67 species, including 22 Metatherian species and 45
Eutherian species (Figure 8). Due to the complexity of ovulation types across mammal
species, we parsed the data in multiple ways. Regardless of how we parsed the data,
Metatherians showed larger CLG than Eutherians (Figure 8).
79

The 67 species include 14 induced ovulators (3 Metatherian, 11 Eutherian), 47
spontaneous ovulators (17 Metatherian, 30 Eutherian), and 6 facultative ovulators (2
Metatherian, 4 Eutherian). For spontaneous and facultative ovulators, corpora lutea
lifespan was taken from experiments where females were separated from males. For
induced ovulators, data were taken from experiments where females were exposed to
sterile males that could induce ovulation but not fertilize embryos. Using ancestral state
reconstruction, the most recent common ancestor of Metatherians had higher CLG than
the most recent common ancestor of eutherians (metatherian CLG: 0.991, 95%
confidence interval [CI] 0.587-1.394; eutherian CLG: 0.267, 95% CI -0.027-0.561).
This pattern remained after analyzing spontaneous ovulators separately from
induced ovulators, regardless of whether facultative ovulators were included in either
group. For spontaneous ovulators, the Metatherian ancestor again showed higher CLG
compared to eutherians (metatherian CLG: 1.011, 95% CI 0.587-1.434; eutherian CLG:
0.255, 95% CI -0.051-0.560), even if we included facultative ovulators as spontaneous
ovulators (metatherian CLG: 1.017, 95% CI 0.588-1.446; eutherian CLG: 0.248, 95% CI
-0.059-0.554). For induced ovulators, the pattern remained qualitatively similar
(metatherian CLG: 0.880, 95% CI 0.298-1.461; eutherian CLG: 0.513, 95% CI 0.044-
0.982), even if we included facultative ovulators as induced ovulators (metatherian CLG:
0.886, 95% CI 0.346-1.427; eutherian CLG: 0.557, 95% CI -0.130-0.985). Unlike all
previous results, the results from induced ovulators have overlapping confidence
intervals between Metatherians and Eutherians, which could be related to many fewer
species with this form of ovulation (N=14 or 17 species in the two analyses of induced
ovulators).
80

It is interesting to note that in several species, CLG is greater than 1, meaning the
female remains in a pseudopregnant state for longer than a normal gestation. These
include both Metatherians (e.g., Northern Brown Bandicoots, Common Wombat, etc.)
and eutherians (multiple members of the dog family Canidae). In contrast, species in the
family Felidae showed pseudopregnancies roughly half the length of pregnancy, and the
orders Primates, Artiodactyla, and Perissodactyla all showed uniformly low
pseudopregnancy severity.

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Figure 8
Phylogeny of 67 Species of mammals color-coded by their ovulation type.

Phenotype 2: the lifespan of CL in pseudopregnant females, normalized by the lifespan
of CL in truly pregnant females (CLCL).

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Corpora lutea lifespan in truly pregnant females was much rarer to find in the
literature, and we were only able to collect data from 19 species (12 Eutherian and 7
Metatherian). Most of these were spontaneous ovulators (7 Eutherian, 7 Metatherian),
and we focus on these 14 species here. Consistent with the CLG phenotype described
above, the Metatherian ancestor had an estimated CLCL more than twice as high as the
Eutherian ancestor (Methatherian CLCL=0.923, 95% CI 0.429-1.418; Eutherian
CLCL=0.409, 95% CI -0.063-0.880), although the confidence intervals overlap greatly.

4.10 Phenotype 3: Peak Progesterone in pseudopregnant females, normalized by peak
progesterone in truly pregnant females (PP).

Data on progesterone were even rarer to find in the literature, and we were only
able to collect data from 12 species (7 Eutherian and 5 Metatherian). Eutherians and
metatherian progesterone levels at the beginning of either pseudopregnancy or
pregnancy increased at the same rate within a species , however, as progesterone
levels in pregnant levels hit some height characteristic to each species, pseudopregnant
eutherians and metatherians often had either lower or even a higher peak height of
progesterone: progesterone was lower eutherians and 2 , and
recorded; the remainder of the species had equal heights of
peak progesterone for both pregnancy and pseudopregnancy. As progesterone fell after
faster drops in their
hormonal levels unlike their pregnant counterparts, likely due to the shorter
pseudopregnancy length, while no such drop was observed for metatherians. In
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general, progesterone levels in pseudopregnancy in half of eutherians can be
categorized as identical to pregnant animals, and for the other half overall lower levels
of progesterone that has more rapid drop-off. Conversely, metatherian
pseudopregnancy may lead to either lower or higher progesterone peaks and no more
rapid fall-off to baseline than pregnant animals. Since these numbers are low, we do not
analyze them statistically. Nevertheless, it is interesting to note that Metatherian
pseudopregnancy again appears more “severe” in the sense that they always resemble
truly pregnant progesterone cycles.

4.11 Testing the “lost opportunity” hypothesis; seasonality, delayed

There was no evidence that pseudopregancy was less severe in i) species where
reproduction is concentrated to one time period (seasonal breeders), ii) species that
iii) species that undergo one estrus
cycle per year (monoestrus) (Supplementary Figures 1-4).
Lastely, we found a non-significant trend that more invasive placenta were
associated with decreased pseudopregnancy severity. It is possible that this pattern
would become more obvious after the addition of more species, and may depend on the
classification of placental invasiveness [361].
Discussion

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4.12 Why does pseudopregnancy occur?

Here we show that all mammals species studied show some level of
pseudopregnancy, which occurs when females behaviorally or physiologically resemble
pregnant females even though they are not pregnant. During pregnancy, females
experience changes to their anatomy, physiology, neurobiology, hormonal levels,
lifespan, immune system, vulnerability to predation [362-375]. Thus, pseudopregnancy
should be deleterious because these changes occur without the fitness payoff of
offspring. To be sure, it has been argued that in some species pseudopregnancy is
beneficial; for example, mongoose females regularly enter severe pseudopregnancies
that are correlated with foster care under a specific social organization [316]. However
beneficial pseudopregnancy appears to be rare. Our data provide support for the
“physiological cost” hypothesis, by which Eutherian species have evolved reduced
pseudopregnancy severity. This hypothesis posits that Eutherians have evolved
mechanisms to reduce the severity of pseudopregnancy given their relatively high
investment in pregnancy. In contrast, we find no support for the “lost opportunity”
hypothesis, suggesting that pseudopregnancy length does not predict the frequency of
missed reproductive opportunities in most species.
To fully understand the strength of selection that presumably acts against
pseudopregnancy, we must appreciate the frequency with which pseudopregnancy
occurs in nature. In an extreme case that it never occurs, then the severity measured
here is irrelevant because it is never exposed to selection. Very few studies have
attempted to study wild populations to see the rates of pseudopregnancy, however, a
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study of street cats that were to be hysterectomized found that at the time of surgery
197 females were pseudopregnant, 22 pregnant, and 26 had corpora albanica (post
pregnancy or pseudopregnancy), i.e., 80% were pseudopregnant, likely due to
previously vasectomized males in the wild population [354]. A study of coyotes shot with
their litters showed out of 51 animals with corpora lutea, only 1 was pseudopregnant,
the remainder had litters [376].
Why does pseudopregnancy occur at all? Because we find evidence of
pseudopregnancy in every mammal species studied, it must have existed in the most
recent common ancestor of placental mammals. Pseudopregnancy may represent an
unavoidable risk associated with pregnancy. The hormonal signaling associated with
maintaining a pregnancy begins prior to actual conception of offspring [165, 275, 300,
377] and may be impossible to turn off without risking reproductive failure when
fertilization has occurred. Said another way, females that completely avoid
pseudopregnancy might inadvertently experience elevated failure in the case of real
pregnancies.
The molecular mechanisms that govern pregnancy are complicated and often
show species-specific turnover [138, 271, 378-380]. Nevertheless, one general
observation is that Eutherian species tend to have more elaborate mechanisms of
hormonal control over pregnancy, offering a potential molecular mechanism for their
reduced pseudopregnancy severity. As argued above, the corpus luteum is a critical
endocrine gland required or at least correlated with a real pregnancy. Hormones related
to corpora lutea are generally classified into three categories: i) luteotrophic hormones
sustain corpus luteum development or maintenance, ii) luteolytic hormones lead to the
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degradation of the corpus luteum, and iii) anti-luteolytic hormones prevent the
degradation of the corpus luteum. All three of these hormonal classes have been found
in Eutherian species studied to date, while very few have been found in Metatherian
species – a difference that cannot simply be explained by bias in scientific effort [166,
191, 307, 334, 337, 381-403].
For example, Inbaraj et al. summarized 9 species’ distinct luteolytic pathways,
and from horses to cows to humans there are multiple non-overlapping hormones
involved in luteal regression (Table 1 of [403]), and hundreds of different genes
involved, with the domestic cows having for example 105 identified genes but humans
only 34 (Fig. 4 of [403]). By contrast, only one luteolytic hormone, Prostaglandin F4
(PGF4), has been definitively identified in Methatherians, which is structurally similar to
the eutherian analogue PGF2. As a second example, in Primates, while chorionic
gonadotropin is the key antiluteolytic hormone that inhibits the regression of the corpus
luteum [361, 404], two antiluteolytic hormones are known in the Eutherian order
Artiodactyla (cows, whales, etc.): interferon tau in most species studied to date [405],
and estrogen in pigs [406] and lastly in Perissodactyla horses have an additional,
unidentified, antiluteolytic hormone [407].
In conclusion, we demonstrate that Eutherian species have evolved
pseudopregnancies with reduced severities compared to Metatherian species, as
predicted by the “physiological cost” hypothesis. Given the relatively high investment of
pregnancy in Eutherians, we hypothesize that selection has favored the evolution of
relatively elaborate hormonal mechanisms that enable females to exit pseudopregnancy
earlier than Metatherian females and return to a reproductive state. Explaining the finer
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scale variation in Eutherian pseudopregnancy – ranging from almost nothing (for
example, primates) to almost as long as a normal gestation (for example, bears) to
some that last for longer than a normal pregnancy (for example, bandicoots) – remains
a goal for future studies of mechanisms governing maternal investment in placental
mammals.

Acknowledgements

We would like to thank Caleb Ghione for his helpful suggestions and

88

Chapter 5 Future Directions

5.1 Research Impacts of this Dissertation

Chapter 2 of my dissertation represented the first review of the baubellum in
female mammals from a historical, phylogenetic, developmental, and comparative
morphological approach, and additionally attempted to compare these various facets of
its biology to the biology of the far better-studied male mammalian genital bone, the
baculum. Although we demonstrated in Chapter 2 that the baubellum is not likely an
example of female choice, we still demonstrated that its linked development and
evolutionary history is likely an example of developmental constraint.
Developmental constraints can be anatomical, physiological, and behavioral
phenotypes that are maintained in a species despite potentially being maladaptive
because there is a constraint on the system that prevents selection [192, 408]. For
example, phenotypic traits such as nipples in male mammals, who do not make
mammary tissue, are maintained because they are essential for offspring survival [409].
What drives this developmental constraint and which sex “requires” the trait has
become a much larger question of interest to biologists in the last half century [192].
What are the drivers of evolutionary novelty and innovation has traditionally been given
greater consideration than the developmental constraints, despite both playing
important roles in understanding sexually dimorphic traits driven by selection, such as
female choice [303, 304, 308, 410, 411]. The conflicting interests of males and females
within their environment is largely understudied and has large implications for the
evolutionary trajectory of a species [412-414].
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We lastly found for genital bones that baubellum trait varied far more within a
species for females than males, likely driven by how ‘canalized’ the baculum is and the
baculum’s functionality in reproduction. Our research highlights how the investigation of
female comparative biology, especially morphological, can not only validate previous
research that has been done on their male counterparts but also produce new
information on potential mechanisms for the maintenance and evolution of novel
structures [71].
For Chapter 3 we found for the first time that male copulatory plugs influence the
rate of implantation in female mammals. This is a possible example of post-copulatory
female choice, as male stimulation of females during copulation in rodents is already
generally required for ovulation or for the corpus luteum to produce progesterone, a vital
hormone during pregnancy (see section 1.5.3 and Chapter 4 for further discussion). In
our case we asked whether the copulatory plug promoted either implantation or
gestation success by mating females with males genetically unable to form plugs and
judging implantation success using a novel NSET bead approach (see [110] and
sections 1.5.1 and 1.5.2 for the rationale of why we did not focus on the copulatory
plug’s effects on fertilization success). Our results showed that the absence of the plug
impairs the ability of females to implant embryos, even though they have physiologically
relevant levels of progesterone after mating, and that the absence of the copulatory plug
additionally delays fertilization and leads to slightly smaller litter sizes. This research
joins other recent studies showing that female choice does not stop after copulation,
and instead is an ongoing process during implantation and gestation [281, 282, 308,
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415]. We therefore isolated a novel yet unknown mechanism of either neuronal or
hormonal control of implantation in female mice.
Lastly in Chapter 4 we discovered that pseudopregnancy severity is higher in the
subclass Metatheria, who have short-term placental pregnancy with extended lactation,
than Eutheria, who have long-term placental pregnancy with relatively short lactation.
We additionally found that metatherians are generally uniform in their pseudopregnancy
severity, but eutherians are not only greater in their variability of pseudopregnancy
severity, but also appear to have far more mechanisms available for females to extend
and arrest pseudopregnancy. Intriguingly, but non-significantly, pseudopregnancy
severity appears to trend lower in species with more invasive placental types (see
supplemental figures 1-4).
Our results add to an already large literature of the hormonal and neuronal
networks controlling the steps of pregnancy in mammals (see [379] for a review of
delayed implantation and development for example). Unlike [379] we were unable to
uncover any sign of female choice, for example there was no pattern of increased
pseudopregnancy severity in species that required male-induced ovulation (see a recent
review of the evolutionary history of mammalian induced and spontaneous ovulation for
further information [416]). Our meta-analysis suggests that pseudopregnancy is a by-
product of the phenomenon of internal fertilization and pregnancy, and therefore cannot
be selected out without damaging a female’s ability to reproduce. Our literature review
shows that the benefits of pseudopregnancy appear limited to certain eutherian families
that operate under strict family-based social structures, with most females gaining only
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indirect reproductive success, meaning that pseudopregnancy again did not evolve to
benefit females across Mammalia.

5.2 Future Experiments

Understanding the evolutionary forces that drive female choice is a fundamental
goal in biology, and despite it being first postulated by Charles Darwin, post-copulatory
sexual selection has remained difficult to identify and study [417-420]. The fact that
females and males do not share common reproductive interests has driven sexually
dimorphic morphology, transcription, sex chromosomes, development, physiology, and
behavior through evolutionary time. These future possible experiments would help
further elucidate better the role of post-copulatory sexual selection or “cryptic” female
choice [304, 410, 421].

5.2.1 The baubellum
In Chapter 1 and Chapter 2 we demonstrated through our meta-analysis that the
baubellum is likely non-function from morphological, developmental, and phylogenetic
information. Yet as we argued in Chapter 2 (see DISCUSSION), one of the limitations of
investigating for potentially unknown functions of the baubellum is the poor
understanding of the clitoris’s function itself outside of humans [69, 72, 73, 422]. The
most straightforward way to investigate the baubellum’s functionality would be to study it
in a species where researchers could experimentally control its presence or absence.
Although there is no knockout model available for mice for either the baculum or
baubellum, the Norway Rat (Rattus norvegicus) presents a potential opportunity as
92

females do not naturally form baubella unless there is administration of testosterone
after birth [4]. Past studies in rats have shown that the baubellum can only be formed if
testosterone is administered before 10 days after birth, and as testosterone would have
significant off-target effects on the female’s genitalia and reproductive systems, I would
argue that microdoses of testosterone to the clitoris on one or more days after birth
might be sufficient for establishing a baubellum without impacting her reproduction. As
sexually naïve female rats can be induced into pseudopregnancy through stimulation
via a glass rod, we could first test whether the baubellum decreases or increases the
sensitivity of the glans clitoris for entering pseudopregnancy [159, 160, 423]. Another
avenue would be to compare the paced mating behavior of rats with or without baubella
and the subsequent rate of pseudopregnancy with vasectomized males, or the rate of
pregnancy and litter size with intact males [159-163, 423]. A final avenue for females
with and without baubella would be to investigate the activation of the early intermediate
transcription factor c-fos, which is associated with elevated brain activity, in the
ventromedial hypothalamus of rat brain, which has been previously shown to be
associated with the activation of hormonal pathways for pseudopregnancy and
pregnancy [159, 424-426]. The female rat could therefore represent a dynamic model
for studying the function of the baubellum, if any, as well as increasing our overall
knowledge of the function of the clitoris outside of humans.

5.2.2 The copulatory plug
In Chapter 1 and Chapter 2 we demonstrated experimentally for the first time that
the male copulatory plug increases the implantation rate of the female mouse, a
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previous unknown function that highlights the possibility that the copulatory plug
overcomes a stimulatory threshold that influences female choice (see DISCUSSION for
why we did not see support for our alternative hypotheses). Yet we were unable to
demonstrate what was the mechanism by which the copulatory plug stimulates the
female towards a physiological state required for implantation. By placing an artificial
plug made out of a polyurethane foam into a female vagina that would swell in size into
three mice, our unpublished preliminary data showed that one out of the three female
mice had a demonstrable increase in progesterone but did not implant artificial beads,
suggesting indeed that the mechanical stretching of the highly innervated vagina is
important, but perhaps not sufficient for implantation. One option would be to revisit
these artificial plug experiments with a greater number of trials, and with a slight twist:
chemically blocking the sensitivity of the female’s vagina. Previously lidocaine, a local
anesthesia that acts on nerve endings to block signaling, has been administered to the
medial amygdala of the brain and prevented pseudopregnancy in 50% of female rats. If
in fact the copulatory plug acts in a stimulatory fashion we should see no progesterone
rise or implantation take place if lidocaine is administered either topically swabbed onto
or injected into the vagina, and by using artificial plugs we test whether any chemical
aspect of the copulatory plug is necessary for implantation, as current literature is
conflicting [269, 296]. Finally, to address our hypothesis that copulatory plugs act as
suppositories that slowly release hormones over time, we could quantify the levels of
hormones present in a male copulatory plug after ejaculation using an ELISA kit.
Estrogen and Progesterone have already been shown to be transferrable in the semen
of male mice and bats to females using radioactive isotopes, but no study has ever
94

attempted to detect endogenous male hormone transfer to females in any mammal
[296, 427]. To quantify bias in our results, given that after ejaculation into a female’s
reproductive tract the copulatory plug would be coated with both female and male-
produced hormones, we would feed prior to the experiment stable isotope Nitrogen-15
to male mice, causing male hormones to be heavier and differentiable using gas
chromatography-mass spectroscopy (GCMS) [105, 428]. These studies would help to
better elucidate the mechanism by which females make reproductive decisions on the
basis of the male copulatory plug, and in turn improve our understanding of the types of
female stimulation required reproductive success and the ‘black box’ of female choice.

5.2.3 Pseudopregnancy
Finally in Chapter 1: Introduction and Chapter 4 Pseudopregnancies are more
severe in Metatheria than Eutheria I demonstrated that pseudopregnancy, which is the
physiological and anatomical steps of pregnancy without offspring, is significantly more
severe in metatherians than eutherians. This observation is noteworthy because
metatherians have both far shorter pregnancies than most eutherians but also far fewer
mechanisms for controlling pregnancy duration in general with respect to the corpus
luteum. Our goals for this chapter are to continue to add new species for which
information is available in the literature for their pseudopregnancy duration, corpus
luteum duration, pseudopregnancy progesterone profiles, and the associated
reproductive factors such as seasonality, diapause, placental invasiveness, and number
of estrous cycles. In particular we hope to use a more recently published metric of
placental invasiveness (see [361]), which would allow us to apply a more fine-scale
95

approach, as there are potential trends as seen in Supplemental Figure 3, but will
require additional species to test whether those trends are significant. Overall, we do
not expect in general our significant findings to change with the addition of more
species, but we do expect to undercover additional variation between families and
orders of mammals, hopefully creating a more general review of pseudopregnancy that
will be useful for researchers to better understand how mechanisms of
pseudopregnancy are natural, widespread, and worthy of equal standing as other topics
of a species’ reproduction.

96

Supplemental Figure 1
Phylogeny of 44 Species of mammals color-coded by whether they breed year-round
(aseasonal) or only at a certain period of year (seasonal).
97

Supplemental Figure 2
Phylogeny of 35 Species of mammals color-coded by whether they have reproductive
diapause type.

98

Supplemental Figure 3
Phylogeny of 46 Species of mammals color-coded by their placental type.
99

Supplemental Figure 4
Phylogeny of 48 Species of mammals color-coded by whether they have more than 1
(polyestrous) or a single estrous cycle (monestrous) per year.

100

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Asset Metadata

Creator Lough-Stevens, Michael James (author)

Core Title Investigating the potential roles of three mammalian traits in female reproductive investment

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Molecular Biology

Degree Conferral Date 2022-08

Publication Date 07/20/2022

Defense Date 03/09/2022

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag baubellum,copulatory plug,female choice,implantation,OAI-PMH Harvest,os clitoris,pseudopregnancy,relaxed selection

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Dean, Matthew D. (committee chair), Ehrenreich, Ian M. (committee member), Kanoski, Scott (committee member), Nuzhdin, Sergey (committee member)

Creator Email loughste@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC111373449

Unique identifier UC111373449

Legacy Identifier etd-LoughSteve-10870

Document Type Dissertation

Format application/pdf (imt)

Rights Lough-Stevens, Michael James

Type texts

Source 20220720-usctheses-batch-957 (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

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Repository Name University of Southern California Digital Library

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