University of Southern California Dissertations and Theses

The effect of vitamin E on the damage in the germinal epithelium in the testis of the rat caused by irradiation

(USC Thesis Other)

The effect of vitamin E on the damage in the germinal epithelium in the testis of the rat caused by irradiation

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content THE EFFECT OF VITMIN E ON THE DAMAGE IN THE GERMINAL EPITHELIUM IN THE TESTIS OF THE EAT CAUSED BY IRRADIATION A Thesis Presented to the Faculty of the Department of Zoology University of Southern California In Partial Fulfillment of the Requirements for the Degree Master of Arts by Sherwood C* Schwartz May 1939 UMI Number: EP67131 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will Indicate the deletion. DIWBmdlbn P^bi sbrtg UMI EP67131 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 This thesisj written by .......S . H E R W Q Q D . . . . C . . . . . . S C H W J m . T Z . . . ...... under the direction of hX^. Faculty Committee, and approved by a ll its members, has been presented to and accepted by the Council on Graduate Study and Research in partial fu lfill ment of the requirem ents fo r the degree of MASTER DE__ART8 D ean / Secretary D a te... Faculty Committee Chairman ACKNÛMiEDGEMENTS The author wishes to express his appreciation to Doctor Cornelius 0» Bailey for his assistance in irradiation and the use of his X-ray equipment; to Archer-Daniels-Midland Company for the supply of wheat germ oil concentrate and for helpful suggestions concerning the experimentation; and to Doctor Robert Rutherford of the College of Dentistry for the photomicrographs* TABLE OF CONTENTS CHAPTER PAGE I. INTRODUCTION........ 1 II. REVIEW OF LITERATURE............... 3 III. EXPLANATION . . . . . . . 20 IV. MATERIALS AND METHODS.......... 24 V. OBSERVATIONS .............................. 29 The Normal Structure of the Rat Testis ......... 29 The Preventive Effect of Vitamin E ........... 32 The Effect of 1/4 H.E.D. on the Rat Testis ...... 32 The Effect of 1/2 H.E.D. on the Rat Testis ...... 35 The Effect of 3/4 H.E.D. on the Rat Testis ...... 36 The Effect of 1 H.E.D. on the Rat Testis ....... 38 The Effect of Vitamin E on the Rat Testis ....... 40 The Regenerative Effect of Vitamin E . . . ....... 40 The Effect of 1/4 H.E.D. on the Rat Testis ....... 44 The Effect of 1/2 H.E.D. on the Rat Testis ...... 46 The Effect of 3/4 H.E.D. on the Rat Testis ...... 49 The Effect of 1 H.E.D. on the Rat Testis ...... 51 VI. DISCUSSION AND CONCLUSIONS ......... . 55 The Preventive Effect of Vitamin E . . ......... 55 The Regenerative Effect of Vitamin E . . ........ 58 VII. SUMMARY ........................ 62 LITERATURE CITED.......................................... 64 LIST OF FIGURES FIGURE PAGE 1. Photomicrograph of a cross section of a normal rat testis to show the normal structure of the seminiferous tubules . . 31 2. Photomicrograph of a cross section of a testis from a rat which had been fed 13 ce. of wheat germ oil concentrate. . . 31 3. Photomicrograph of a cross section of rat testis 3 days after it had received 1/4 H.E.D. X-ray. (Animal B-1) .... 34 4. Photomicrograph of a cross section of rat testis 3 days after it had received 1/4 H.E.D. X-ray, from the animal fed 13 cc. wheat germ oil before the irradiation (Animal A-l) . ... . . . , . . . . . . . . . . . . . . . . . . 34 5. , Photomicrograph of a cross section of rat testis 3 days after it had received l/2 H.E.D. X-ray. (Animal B-2) .... 37 6. Photomicrograph of a cross section of rat testis 3 days after it had received 1/2 H.E.D. X-ray, from the animal fed 13 cc. of wheat germ oil before the irradiation (Animal A-2) ................ 37 7. Pliotoiaicrograph of a cross section of rat testis 3 days after it had received 3/4 H.E.D. X-ray. (Animal B-3) . ♦ . 39 8. Photomicrograph of a cross section of rat testis 3 days after it had received 3/4 H.E.D. X-^ray, from the animal fed 13 cc. of wheat germ oil before the irradiation (Animal A-3)......................... 39 FIGURE PAGE 9* Photoraierograph of a cross section of rat testis 3 days after it had received 1 H.E.D. X-ray (Animal (B-4) .... 41 10. Photomicrograph of a cross section of rat testis 3 days after it had received 1 H.E.D. X-ray, from the animal fed 13 cc. of wheat germ oil before the irradiation (Animal A—4) . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 11. Photomicrograph of a cross section of a normal rat testis to show the structure of the seminiferous tubules .... 43 12. Photomicrograph of a cross section of a testis from a rat which had been fed 33 cc. of wheat germ oil concentrate . 43 13. Photomicrograph of a cross section of rat testis 39 days after it had received I/4 H.E.D. X-ray (Animal D-l) ...... 47 14. Photomicrograph of a cross section of rat testis 39 days after it had received I/4 H.E.D. X-ray, from the animal fed 33 cc. of wheat germ oil after the irradiation (Animal c-1) ................. ............... 47 15. Photomicrograph of a cross section of rat testis 39 days after it had received 1/2 H.E.D. X-ray (Animal D-2) ... 50 16. Photomicrograph of a cross section of rat testis 39 days after it had received I/2 H.E.D. X-ray, from the animal fed 33 cc. of wheat germ oil after the irradiation (Animal C-2) ................................. 50 FIGURES PAGE 17. Photomicrograph of a cross section of rat testis 39 days after it had received 3/4 H.E.D. X-ray (Animal D-3) .... 52 18. Photomicrograph of a cross section of rat testis 39 days after it had received 3/4 H.E.D. X-ray, from the animal fed 33 cc. of wheat germ oil after the irradiation. (Animal C-3)............ 52 19. Photomicrograph of a cross section of rat testis 39 days after it had received 1 H.E.D. X-ray (Animal D-4). .... 54 20. Photomicrograph of a cross section of rat testis 39 days , after it had received 1 H.E.D. X-ray from the animal fed 33 cc. of wheat germ oil after the irradiation (Animal C-i).......................................... 54 CHAPTER I INTRODUCTION Each year various rays, the most common of which are X-rays and electric welding rays, cause accidental sterilization in a number of individuals. In the case of X-rays, this sterilization has claimed both operators and patients. Although the total number of such instances is not large in absolute figures, the function lost is of such importance that it warrants scientific investigation, to attempt a determination of corrective measures. The specificity of vitamin E for the germinal epithelium has been known for many years. Until a few years ago, the problems investi gated in connection with vitamin E had been almost wholly of a deficiency nature. The effects of inadequate vitamin E in both sexes were carefully studied and described. However, of late, the direction of investigation has been turned toward the therapeutic value of vitamin E. At first this concerned itself with animals, especially those of great economic value such as the cow. In the past months, vitamin E has been employed as a curative means in some types of non-fertility in human beings, both female and male. Some measure of success has been attained in these cases of natural non-fertility by the use of wheat germ oil concentrate, which is the best known source of vitamin E. In this investigation, animals ex perimentally sterilized by X-ray, were employed to determine to what ex tent, if any, vitamin E could aid in conditions of ’ray sterility*. In addition to this major objective of a therapeutic nature. 2 there were two other important considerations. The first of these was to determine the specificity of vitamin E from a new approach. Instead of the usual deficiency identification, this would provide a possible means for testing positively the effect of vitamin E on the germinal epithelium. The second consideration involved a purely theoretical problem concerning avitaminosis and irradiation. Previous work had established a connection between irradiation and three other vitamins, B, C, and D. The question had been raised as to the possibility of irradiation being essentially a condition of avitaminosis. The procedure in this investigation lent it self to the application of these experiments to the evaluation of such a possibility. CHAPTER II REVIEW OF LITERATURE In 1922 Evans and Bishop published an account of a substance X, contained in natural foodstuffs, needed for the prevention or cure of sterility caused ty a purified diet. Barnett Sure (1923), who had been working on this same problem for several years, published his results shortly afterward, and suggested the term vitamin E for the substance which they had called X. This change was soon adopted. The investigations of the early workers in this field, includ ing Mattill (1923), had concerned the female rat exclusively. However, the effect of vitamin E deficiency on the male rat soon followed in a care ful study by Mason (1926). Beard (1926) verified the results on the male and female mouse. During the following decade, various other forms, in cluding man, were shown to be equally dependent upon the presence of vita min E in the diet for normal reproduction. In spite of its physiological importance, vitamin E has but few satisfactorily proven sources. Evans and Bishop (1922), Daniels (1925), Beard (1926), Sure (1927), Mason (1929), Smith and Nelson (1931), Vogt M/ller (1931), Leitch (1932), Hill and Burdett (1932), and Evans (1934) have suggested various substances as definite sources of vitamin E. How ever, in the latest comprehensive discussion of vitamin E, Bacharach (1938) stated that wheat germ oil, rice oil, lettuce leaves, cottonseed oil, and palm oil are the only certain sources of vitamin E. For the past few years attention has been directed to concen- 4 trates from these sources. The most valuable of these has been wheat germ oil concentrate. Evans (1932) has used this oil almost exclusive ly and most of the other workers have followed his example. Olcott (1931), Olcott and Mattill (1931), Emerson (1937), and Todd (1938), have also employed other concentrates satisfactorily. At present, synthetic chemical preparations of vitamin E (tocopherol) on a commercial scale are in progress and in a short time will probably replace the concentrates now used. There has been considerable difficulty comparing even approxi mately the few known vitamin E sources. The reason for this has been that tests for vitamin E are little more than qualitative at the present time. Sure (1927) , Verzar (1929), Blumberg (1935) > and Bacharach (1937) have suggested methods of estimation. Pacini and Linn (1936) have out lined a numerical evaluation plan. Yet none of these has been generally accepted. Since the discovery of vitamin E, many histological studies of the testes in E deficient animals have been made. A thorough investi gation of this condition in the rat was prepared by Mason (1933). He described the effect of vitamin E deficiency as a specific and character istic degeneration of the germinal epithelium of the testis. He found that vitamin E therapy did not help the tubules, except those slightly affected. It was this irreversible type of injury which appeared to be so characteristic for vitamin E deficiency. This offered additional proof that the absence of this factor affects the germinal cells of the testis in a manner entirely different from that produced by other vitamin 5 deficiencies. In an earlier work. Mason (1929) had described an initial period of fertility in males placed upon a diet deficient in Vitmain 1. The exact length of this period was evidently dependent upon the amount of vitamin E stored in the tissues of the animal. This period of initial fertility was followed by a definite and progressive degeneration of the germinal epithelium of the testis. The extent of this degeneration varied within rather wide limits in different tubules, or parts of tubules, in the same testis. Once this process had its start, the germinal epi thelium continued to degenerate even though the animal was supplied with great amounts of vitamin 1. Mattill, (1927), also referred to the ir reversibility of this condition produced by E deficiency. Simmonds (1928) found that there was a considerable variation in the degree to which individual male rats withstood the effects of diets low in vitamin E. Some remained potent much longer than others. Waddell (1931) described a sterility characterized by the complete dis appearance of the germinal epithelium, great loss in amount of testicular tissue, and pronounced edema. He attributed this condition to ferric chloride which he had added to the diet. Later in 1931, Waddell found that the action of ferric chloride is destructive to the vitamin E acti vity of the diet. Thus he was dealing essentially with a vitamin E de ficiency. Evans (1932), also called attention to the complete degener ation of the seminiferous tubules in the rat that accompanied loss of vitamin E. Only diminutive tubules devoid of all save Sertoli cells were left. Cure, he observed, by large doses of vitamin E, was rarely successful, and it seemed that the damage to the epithelium was irreparable. 6 Perhaps Korenchevsky (1933) has best summarized the histo logical effects of vitamin E deficiency. He divided the changes into several stages. In the first there is, essentially, a degeneration of the spermatozoa. These tend to occlude the lumen of the tubule. In the second stage, the spermatozoa are scarce or absent. The lumen of the tubule is reduced or entirely absent because of the swollen condition of the seminiferous cells. At this time vacuolated spaces appear be tween cells. In the third stage, giant cells appear, forming round or ovoid masses of fused cytoplasm of the spermatids. These contain from 2 to 40, or more, spermatid nuclei. The nuclei are degenerated and liquified and the giant ceUs soom assume a similar disintegrated appear ance. The spermatocytes and spermatogonia exhibit severe degeneration at about this time. Vacuolation, which appeared in the previous stage, becomes much more common. In the fourth stage, the tubules are greatly reduced and consist only of a syncytium of Sertoli cells having wrinkled nuclei outlines. The protoplasm has a fibrous structure. The inter stitial cells of the testis, in this stage as in the preceding stages, remain totally unchanged. Thus the first years of experimentation had resulted in the examination of results and the presentation of factual matter. Soon, however, questions as to the nature, causes, and reasons for these results were raised. Mattill (1938) stated that, according to Adamstone (1934), vitamin E is intimately connected with the nucleus during cell division 7 and probably exerts an indirect, controlling influence. However, Mason (1933), gave credit to Judasz-Schaffer for a fundamentally similar theory. The latter had suggested that vitamin E is essential for growth and pro liferation of cells. The only reason that the testis and the developing embryo are affected so severely is that they have such a high metabolic rate and rapid cell division. Lewis (1935) reported that it was Mason who suggested the cellular proliferation theory in relation to vitamin E. Mason, however, merely enlarged, clarified, and established this view, adapting it to his experimental evidence. It was he who also connected it more exactly with the nucleus. He postulated that it was necessary either for the maintenance of the normal plysico-chemical structure of the nuclear chromatin, or for some process involving the synthesis of the complex molecules of chromatin, or for some basic process of cell metabol ism. Mason (1933) also stated that the lack of vitamin E produces in areas of rapid cell proliferation, a more profound injury than is produced by the lack of any other known vitamin factor. Vitamin E cannot be re lated to apy specific tissue or even type of tissue. It is the rate of metabolic activity and the rate of cell proliferation of the tissue that is the determiner. In the following year, 1934, Adamstone subjected this parti cular problem to further intensive investigation. He found that chicks fed a prolonged vitamin E deficient diet developed characteristic patho logical lesions of the visceral organs. These lesions histologically represented foci of destruction and degeneration of normal tissues ac companied by replacement and invasion by new cell growths. The whole 8 series of effects was evidently due to a phenomenon of uncontrolled and unrestricted cell growth which simulated malignancy. Vitamin E is so essential to the maintenance of the normal nuclear condition in the cell that it acts Indirectly as a control over cell division. The whole series of effects appeared to he due to uncontrolled cell proliferation accompanying injury to the nucleus of the cell. Mason (1936), after collecting the various evidences in this concern, reported that the majority of experimental data available indi cates that vitamin E is specifically needed for nuclear activity and function of the cells in general. The lack of vitainin is first mani fested in those tissues such as the germinal epithelium of the testis and the developing fetus where cellular proliferation or division are es pecially rapid. This, of course, is merely a recapitulation of his, Judasz-Schaffer, and Adamstone*s work. In 1937, Haddow and Russell, working on experimental cancer development in mice induced by vitamin E feeding, made use of this cellular proliferation theory in the explanation of their results. The effect of a vitamin E deficiency in the female is quite different from the degeneration observed in the male. Beard (1926),Sure, Kik, and Walker (1929), Urner (1931), Vogt M/ller (1931), Evans (1932), and Mason (1933) have described vitamin E deficient females, in various animals. No degeneration of any sort is observable. There is no inter ference with the reproductive process until a few hours before the fetus should come to term. The resorption of the fetus at this time is the manifestation of the vitamin E deficiency. This has been proven by the 9 effectiveness of curative doses of vitamin E administered at any time up to a few hours before implantation, according to Evans (1932). Perhaps Vogt-M/Uer (1931) has best stated the case. In the female rat ovulation and implantation take place normally, while sterility is due to resorption of the implanted ovum at an early stage of pregnancy. Mattill (1938) stated that the difference in the effect on male and female may be due to the fact that the influence in the male is upon its own tissue, while in the female it is upon the fetus. This suggestion, of course, is based upon the cellular proliferation theory previously discussed. Bacharach, Allchome, and Glynn (1938) seem to feel that the period of vitamin E deficiency has an effect on the maternal organism much more deep seated than has been hitherto believed. Fertility is also influenced by vitamins other than vitamin E. Mattill (1927) described the effects of a vitamin B deficient diet in this relation. Here, however, the effects of a vitamin B deficiency upon fertility was probably due to a lowering of the general metabolism of the body. The absence of vitamin B to the extent of inducing complete paralysis, did not result in degenerated gonads. Motile sperm were found in the epididymis quite regularly, in one case several hours after death. These experiments of course, were carried out in the presence of adequate vitamin E. Moore and Samuels (1931) verified these observ ations, and found that the effect of the vitamin B deficiency upon fer tility was apparently on the endocrine secretion of the testis since the secondary sex characteristics were greatly degenerated. They also 10 proved that the effects of vitamin B deficiency on fertility were not direct but were effected through the hypophysis. Inadequate vitamin A is also responsible for a condition of sterility. The effect in this case, like that of vitamin 1, is upon the germinal epithelium of testis. However, when vitamin A deficiency is the cause, the damage is readily repairable (Evans 1932). Repro ductive ability is lost, but the germ tissue is not so severely degener ated. The deficiency seems to affect the entire body and therefore the reproductive processes, Wiile vitamin E is specific for the reproductive function itself. Pfeiffer (1937) found that toxemia, produced by injection of subiethal doses of diphtheria toxin, brings about degenerative changes that resemble the condition of the testis in vitamin E deficiency. The germinal epithelium is affected in much the same way except that com|>lete removal of the germinal epithelium may occur within 10 days from the time of injection. A functional relationship between vitamin E and the sex hormones has been studied, and heis been the point of contradiction in many investigations. Verzar (1929) found that an abundance of vitamin E had a stimulating effect on the sexual cycle in the female rat. He postulated tbat vitamin E may be a stimulating substance for the ovary, and suggested that the effect may be produced through the hypophysis. Diakov and Krizenecly (1933) attempted to reproduce Verzar* s results of premature sex development but were unable to verify his find ings. Instead they found that the intraperitoneal injections proved to 11 be toxic. The abdomen was distended, the skin of the abdomen was wet, and the animals lost a great amount of weight. The sexual organs not only showed no signs of maturity, but there was a tendency for the uterus and fallopiswi tubes to atrophy. Olcott and Mattill (1934), investi gating this same problem, discovered that vitamin E, when injected sub- eutaneously, had no effect on the ovaries, uterus, opening of the vagina, or Gornification in the immature rat. In 1936, Mason and Melamjy stated that claims relating to oestrogenic and gonadotropic actions of vitamin E in the endocrine activity of the sex glands had not been sub stantiated. Evans (1932) established the fact that excessive amounts of vitamin E had no effect in increasing the size of the litters in rats. Ifi/hen the rats were fed from 2 to 20 times the normal amount of vitamin E, the norm of the numbers in the litters was in no way disturbed. Weisner and Bacharach (1937) found that a vitamin E deficiency resulted in dif ferent sex behavior in different rats. They attributed this to a change in the erogenous function of the anterior pituitary. However, since it was discovered that the addition of wheat germ oil did not ilicit normal sex behavior, the effect, very possibly, may not have been wholly due to the absence of the vitamin E. X-ray, it was found, could also destroy germinal tissue in a manner similar to a vitamin E deficiency. As early as 1926 Oslund and Bachem had made a rather careful study of the degenerative effect of X-ray upon the tubules of the testis in the rat. They found that the extent of degeneration produced in the seminiferous epithelium depended 12 roughly on the amount of irradiation. However, the damage for a given dose after a given number of days was not constant. The most actively dividing cells were found to be most easily injured. The spermatocytes were the first to degenerate, and the spermatogonia were the next to be affected. Doses exceeding 3 H.E.D* (Human Erythemal Dose) degenerated all seminiferous epithelium except the Sertoli syncytium. This per sisted up to 7 1/2 H.E.D. At 8 H.E.D. death resulted. Whether the animals died immediately or after a certain interval was not stated. Warren (1928) agreed as to the relative insensitivity of the mature sperm cells, but according to his observations the spermatogonia were the cells most sensitive to X-ray. Mirskaia and Crew (1931) also described the destruction of the germinal tissue. In addition to the Sertoli or lining syncytium which had been found to be insensitive, they also noted the lack of X-ray effect on the interstitial cells. Asdell and Warren (1931) further verified the work of the preceding investigators. The greatest effect of the X-ray was upon the rapidly dividing cells, the spermatocytes and spermatogonia. The insensitivity of the mature sperma tozoa was once again observed. Witschi, Levine, and Hill (1932) reported the abnormal abundance of interstitial cells in the X-rayed testis, in addition to noting the absence of germinal tissue. Wintz (1932) divided the effects of the X-rays into three groups; the first consisted of the degeneration of the spermatogonia, the second, of depopulation of the seminal canal, and the third, of destruction of the interstitial cells. The first two, he found, were capable of repair, while the third effect was the same as 13 castration* The exact sensitivity of the interstitial cells, he added, was not safely known* Wintz also suggested that the accumulation of small doses played a very important role in irradiation. Schchegolov (1936) X-rayed the posterior third of one day old rats. The rays evidently halted the growth of the testis tubules. After the first week, the movement of the germinal cells was arrested and they became vacuolated. Their boundaries disappeared and their nuclei disintegrated and were mixed with the cytoplasm. Essenberg (1937) studied the effects of the rays on embryos. He irradiated the germinal crescent in embryo chicks. Although malformations were occasionally present, germinal tissue with non-lethal doses usually resembled the norm al after 6 days* All the germ and intermediate sex cells were present although the tissue as a whole was smaller than the non-irradiated. One 6 day old embryo with a lethal dose remained alive, but it looked more like a lump of rarified tissue than a chick embryo. Gatenby and Mirkerji (1929) made an intensive cytological study of the germinal cells after X-ray treatment. Each of the cells of the tubule epithelium, including the entire germ cycle, was carefully studied as to changes in vacuoles, Golgi apparatus, mitochondria, granules, and nuclear material. The explanation for the effect of X-ray, they suggest ed, was that the changes are due to an action on the lipoid granules in the Golgi apparatus. Bagg and Little (1924) attempted to bring about abnormalities in the offspring by the use of X-ray on the parents. In some cases ab normal young were obtained, but the process could not be controlled to any u great degree. Hooker's efforts (1925) to change the motility of the sperm by means of irradiation met with no success in doses less than 2 H.S.D. Beyond this miount no uniform results could be obtained. Asdell and Warren (1931) irradiated the sperm with very high doses of X-ray. No change could be recorded in the motility of the sperm or in the young which were produced by artificial ensemenation of these highly irradiated sperm. Barth (1929) produced a difference in the sex ratio of Drosophila by irradiation of the adult male parent. His experiments indicated that X-ray produced an excess of normal males. A possible explanation of this is that there might be a differential susceptibility of the X sperm and the Y sperm to X-ray. The X sperm evidently suffers a loss of functional activity. According to Barth (1929), Parkes found that after a certain period had elapsed from the time of X-ray, females were produced in a greater excess. The question of regeneration of epithelium after destruction by irradiation has received little attention. Oslund and Bachem (1926) found that regeneration would occur after approximately one month from "Indifferent" cells which they claimed were produced by the effect of the X-rays. The time required to regenerate the tissue in cases of extensive injury was about three months in the rat. Mirskaia and Crew (1931) studied regeneration in birds. They investigated the amount of recovery from a dose of about 5 1/2 H.E.D. by removing portions of the testis after 14, 24, 34, and 48 days. No regeneration could be observed at any time. The interstitial cells were greatly l:Qrpertrophied, but the 15 tubules were completely devoid of the germ cells. Hooker (1925) attempted to find a minimum effective dose necessary to establish permanent sterility. He also tried to discover fractional doses which might inhibit reproduction for arbitrarily chosen lengths of time. However, he found that different animals show differ ent resistance to irradiation of the testes. There was no consistency at all in the results. Hooker also showed that irradiating the entire body with the testes protected had no effect on the fertility of the animal. Snyder (Hooker, 1925) reported that a thick aluminum filter prevented X-ray sterilization no matter how long the exposure was. A thin filter, on the other hand, gave exactly the same results as no fil ter. The exact thicknesses were not given. Snyder found, however, that there was always an initial fertile period in the rat before the animal became sterile due to the irradiation. He explained this by assuming that mature sperm are not affected by roentgen ray exposure. Bagg and Little reported no such period of fertility in their animals after irradiation. Hooker's results could not be used in support of either Bagg and Little, or Snyder. Warren (1928) also described a period of initial fertility. He agreed with the explanation furnished by Snyder. Until time has elapsed for the maturation of the spermatogonia, the volume of the epididymis, the sperm resevoir, will continue fertility. The absence of that generation of sperm which should have developed from the sensitive germ cells that absorbed the effect of the X-rays is the cause of the infertility. Asdell and Warren (1931) reiterate and emphasize this explanation for the period 16 of initial fertility. Huhner (1935) reported several instances in which, in human beings, the X-rays had interfered with the spermatogenic function of the testicle, or destroyed it entirely. The irradiation had been administer ed for dermatological conditions about the thigh and perineum. Although 'adequate' protection had been employed by expert dermatologists, the patients, who had been previously fertile, became sterile. Some testi cles, it was found, were more sensitive to X-ray than were others. The exact dosages which brought about the sterility were not known. The gonads are more sensitive to X-ray than are other tissues of the body# As Hooker (1925) suggested, the probable reason for this is that young, rapidly dividing cells are more susceptible than are cells that have already reached maturity. The entire technique of X-ray treatments depends upon this principle. Growths, such as cancer, that are proliferating at abnormal rates are thus severely affected by rays while normal tissue remains unharmed. Marshak and Bollman (1936) expressed essentially this same view, in stating that the frequency of mitoses in a tissue is a factor in determining the susceptibility of that tissue to X-rays. The sensitivity of a tissue may be in part dependent upon the number of nuclei per unit volume. The effect of irradiation upon germinal tissue, which is so strikingly different than that upon other body tissue, could be fairly well explained by these considerations. In 1937 Martin and Moursund studied the effects of X-ray 17 upon the small intestine* They noted that the general appearance of the tissue and the type of degeneration of the villi resembled a vitamin B deficiency to a remarkable degree* Since X-ray and B deficiency produced such a similar result, it was thought that an over supply of vitamin B might counteract the effects of irradiation* Large quanti ties of vitamin B were administered and then the animals were irradiated. Up to 1 1/2 H.E.D. the vitamin aided considerably in preventing the effects of the X-rays. Beyond this amount of irradiation, little pro tection was afforded. In general, however, the results proved of such benefit that this procedure is now being applied clinically to human beings. In 1938, Carrie performed a similar experiment to that of Martin and Moursund using vitamin 0. He found that < a condition of leukopenia resulted in irradiated animals. By administering large oversupplies of vitamin C before the X-ray treatment, he was able to pre vent this condition. In addition, he was able to use vitamin C thera peutically after the irradiation had produced the leukopenia. Although vitamin D and X-ray have not been connected by any similar experiment, the close relationship between vitamin D and ultra violet rays has long been known. Ergosterol, after ultra-violet rays treatment, becomes a most potent source of vitamin D. The effect of the rays of the sun are also well known in connection with vitamin D (Clouse, 1932). In the field of vitamin E therapy, the name of Vogt-^Uer probably stands foremost. As early as 1931 he had suggested that 18 vitamin E preparations may be of value in the treatment of habitual abortion in women* Previous to this he had used wheat gei*m oil concen trates on farm animals in cases of habitual abortion* Mattill (1938) described one of these investigations in which 33 of 50 otherwise normal cows which had repeatedly failed to become pregnant were carried to full term by the injection of sterilised wheat germ oil. Dryerre (1933) studied abortion in sheep and prevented all but one of his cases of habitual abortion by the use of concentrates of vitamins A, D, and E. Results similar to these were also obtained Tutt (Mattill, 1938). Almost as soon as some success was achieved in the treatment of animals, attention was directed to possible therapeutic application to human beings. Vogt-M^ller (1931) described several instances of habitual abortion in human females which were cured by wheat germ oil administration* MaQueen-Williams (1934) stated that vitamin E appeared to be a means of treating a very distressing condition with some hope of success. Its use proved to be successful in 14 of 15 threatened abortions, and it also was found to be valuable if used throughout pregnancy in cases of habitual abortion. Watson and Tew (1936), in observations on 80 women, and Young (1937), also reported excellent results by the use of wheat germ oil for both of these conditions. Shute (1936) again confirmed the same general results in the human female. Moreover, he reported a seasonal rhythm in pregnancy difficulties which seemed to correlate with the green diets, rich in vitamin E, of the summer and autumn. This seasonal variation was equally true for the male, he discovered, and might have some bearing on male sterility. 19 Saphir (1936) could not show any experimental basis for the efficacy of vitamin E in the treatment of habitual abortion. The con centrates failed to produce any estrogenic, gonadotropic, or lutenizing effects in laboratory tests. Nevertheless, practical evidence as to its actual value in these cases has continued to mount. Moench (1936), who made an extensive study of male sterility of various kinds in human beings, found that corrective results could be obtained by wheat germ oil treatment. Most of the investigations, how ever, both before and after this, have been almost exclusively on the female. In BacharachVs review of vitamin E therapy (1938), he found that about 75 per cent of the cases of women with two or more previous miscarriages have been terminated successfully by increasing their vitamin E supply. This was far above any statistically expected percent age. Mattill (1938) warns that sterility is so various in form that no specific statements could carry much weight. However, many physicians have been quite successful, he adds, in the use of vitamin E concentrates. CHAPTER III EXPLANATION Practically all of the investigation concerning vitamin E has been of a more or less negative nature. %e effect of the lack of the vitamin has been studied rather than the effect of its presence. Vitamin E therapy has been employed in the case of habitual abortion, it is true, but these are naturally occurring phenomena and not laboratory controlled experiments. Furthermore, histological studies have not been made to determine the actual effect of this vitamin on the E deficient tissues. In the first part of these experiments, the positive specifi city of vitamin E for the germinal epithelium in the male was investigated, The epithelium of the testis tubules was first destroyed by X-ray, and then wheat germ concentrate was administered in large quantities to re store the degenerated epithelium. The X-rayed testis, it is known, i^ll regenerate of its own accord over a period of time. This, however, was controlled by the use of a second animal, also X-rayed, which did not re ceive vitamin E in more than usual dietary quantities. The difference in regeneration, since age and genetic differences were eliminated, could logically be attributed to the excess vitamin E that the first animal had received. Various degrees of irradiation were employed so that regener ation in respect to the amount of degeneration could be determined. Hhile the doses of X-ray varied, the time element and the wheat germ oil 21 treatment remained constant. Carrie (1938) showed that vitamin C, used in large quantities, could prevent the appearance of leukopenia following irradiation, and could also remedy the condition if it had already occurred. Martin and Moursund (1937, 1938) performed a very similar experiment, injecting large oversupplies of vitamin B in preventing disturbances of the small intestine. To explain their results, Martin and Moursund suggested the possibility that X-ray may be an avitaminosis. They noted that pellagra is due to an avitaminosis aggravated by sunlight, and so it might not be unreasonable to assume that the shorter wave lengths might havé a similar effect. The exact physiological action of vitamins in the cells of the body is at present unknown. Carrie has connected vitamin C and X- rays, Martin and Moursund have connected vitamin B and X-rays, and the connection of vitamin D with the sun’s rays and with ultra-violet rays has been known for some time. Might it not be possible that there is some fundamental tie between vitamins and rays? At present this must remain pure conjecture. The second half of this investigation, however, was concerned, in part, with a possible substantiation of such a view. The effects of vitamin E deficiency and X-ray of the testis are remarkably similar. The histological degenera.tion presents much the same picture in both cases. The most sensitive cells, according to most workers, are the spermatocytes and the spermatogonia in both X-ray and E deficiency. Both severe irradiation and extreme E deficiency shrink the tubules and gradually occlude the lumen with the degenerated 22 germ cells. Soon the epithelium separates entirely from the Sertoli syncytium which remains unaffected. Giant cells, large ovoid multi nucleated masses, appear in both X-rayed and E deficient testes* Neither irradiation nor lack of vitamin E has any apparent effect upon the inter stitial cells. Both conditions, on the contrary, tend to hyptrophy the interstitial elements. Thus, the histological similarity between irradi ation and vitamin E deficiency is evidenced in many ways. The theoretical nature of both of these processes furnishes a further link between them. Vitamin E, according to the generally accept ed theory, is essentially concerned with cellular proliferation and exerts some fundamental force upon the nuclear activities in tiie division of the cell. Vitamin E deficiency is thus first evidenced in those tissues un dergoing a rapid process of division. Therefore, it acts primarily and particularly upon the germinal cells, since these are ordinarily the most rapidly dividing cells in the body. X-ray depends likewise upon cellular proliferation for its action. It attacks those tissues most rapidly proliferating, and is therefore employed for cellular growths where division is occurring at an abnormal rate. As a matter of fact, this is the basis for its therapeutic use in tMs connection. Thus it is too that the gonads are the most sensitive cells of the body to irradiation. With these many evidences of similarity, it was thought that an experiment such as that with vitamin B and X-ray and C and X- ray was warranted with vitamin E and X-ray. The second part of the in vestigation was concerned with the value of vitamin E in regenerating 23 the tissue of the germinal epithelium which had been destroyed by irradiation. The first part was devoted to the effect of vitamin E in preventing the effects of irradiation upon the epithelium. The results, in both cases, might bear some significance on the possible theoretical connection of rays and vitamins. Of greater practical value, however, was the possible use of the results in a therapeutic manner. If vitamin E proved to be capable of preventing X-ray from affecting germinal tissue to any great extent, it could be administered to subjects and operators before the use of the rays and thus eliminate accidental sterilization. If vitamin E proved to be capable of regenerating germinal tissue at a much faster than natural rate, it could be used therapeutically after the damage had been done. CHAPTER IV MATERIALS AND METHODS In this investigation adult male rats were used. Since the experiment consisted of two more or less separate parts, the animals were divided into two sections. Each section consisted of eight rats, four experimental and four control. In order to eliminate ary genetic differences, each experimental animal had a littermate as a control. To further standardize results, there was a variation of no more than eight days in age between any of the animals. In the first section, each of the animals in the experimental unit. Group A, received a total of 13 cc. of wheat germ oil concentrate. This was administered 1/2 cc. daily in two I/4 cc. doses. The oil con tained a minimum of 40 Pacini-Linn units of vitamin E per gram. Intra muscular injection of the oil was first attempted, but because of the slow absorption of the oil and the irritation produced by frequent in jections, this technique was abandoned. The most satisfactory method was found to be feeding by means of a calibrated dropper. This proved to be quite accurate since the rats developed a desire for the oil and licked every bit of it from the dropper. The control unit of the first section. Group B, received no special attention, being fed the normal rat diet. Both groups of the first section were then X-rayed. The experimental and control littermate s were paired off and were X-rayed on the same board at the same time. Lead-rubber sheets covered the entire 25 rat except for the testes which were exposed to the entire effects of the rays. The first pair received I/4 H.E.D. (Human Erythemal Dose, or Skin Unit), the second, 1/2 H.E.D., the third, 3/4 H.E.D., and the fourth pair, 1 full H.E.D* The details of the X-ray treatment are as follows: voltage 121,000 distance 25 cm filter 2 mm aluminum filter In order for the effects of the X-ray to be clearly evidenced, three days were allowed to elapse after the irradiation. A shorter period would not show enough effects and a longer period might involve regeneration. The animals were killed by a blow on the head, since a slower death, by chloroform for example, would possibly change the deli cate germinal tissue. The testes were removed immediately and preserved in Bouin’s solution. In addition to the eight animals included in this section, another adult male rat of the same age, which had been fed 13 cc. of wheat germ oil but had not been irradiated, was used as a control. This was to determine any possible effect of the oil itself on the testes. In the second section, both the experimental group and the control group were first irradiated. Again, the littermates were paired off and X-rayed on the same board at the same time. The amounts of irradiation, the procedure, and the apparatus for the second section were identical with that of the first. 26 The experimental rats. Group C, then received 1 cc. of wheat germ oil daily in two l/2 cc. doses. The method of administration was the same as that used for the first group* The initial dose was given as soon as the animals were taken from the X-ray board. Each rat received a total of 33 cc. of the oil concentrate. After the irradiation, the control unit of the second section. Group D, received no oil nor any other special attention. Both the experimental and control groups were killed at the same time, the method being the same as for the first group. The testes were removed immediately and preserved in Bouin’s solution. One adult male rat of the same age, which had been fed 33 cc. of wheat germ oil but had not been irradiated, was used as a control. This was to determine any possible effects of this amount of the oil it self on the testes. In addition, several normal adult male rats of the same age were also employed as controls. This, basically, formed the standard for comparison of the rats in the investigation itself. The procedures for all of the controls did not vary from those described for the rats used in the actual investigation. A complete tabulation of the numbers, litters, birth dates, wheat germ oil treatment, irradiation treatment, death dates, control animal, and groupings, can be found in Chart 1. In the chart, the experimental unit of the first group is group A, the control unit is Group B, while in the second group the experimental unit is Group C, and the control unit is Group D. 27 In order to standardize the histological preparations and make the results comparable, all of the sections were made from the middle third of the left testis in each of the animals. All of the tissues were fixed in Bouin’s solution, dehydrated and cleared in the usual manner, and imbedded in paraffin. They were cut in cross section at 8, 6, and 4 micra. In order to observe as much differentiation as possible, several stains were employed. These included: Delafield’s Hematoxylin and Eosin Differential, Mallory’s Tripple Stain, Pichro- fuchsin, and a combination of Delafield’s Hematoxylin and Cyanin. Except for slight changes in the xylenes, the duration of the treatment in the various reagents was as usual: Bouin’s solution 48 hours 50^ Alcohol 1 hour 70^ Alcohol 24 hours B0% Alcohol 1 hour 90^ Alcohol 1 hour 95^ Alcohol 1 hour Absolute Alcohol 1 hour Absolute and Xylene 45 minutes Xylene 40 minutes Paraffin and Bayberry Wax (10-1 proportion) 18 hours (three changes) Imbed in Paraffin and Bayberry Wax Canada Balsam was used in mounting. Table showing the exact treatments received by the various groups in the investigation. - — CV ro fV rO 4 t oGc: j CQ cD m CÛ I a a a C 3 - Î3- o o o - CT- c r O - Cr- C r- o z - o- m n T C T l m co *T* ro m (O m C O T C O cn cn c r cn rl/ r O C M m (V cn n c % c r T rO N r i fX ry (V (V N CV CV CV C V (V oi c V m r Oi 'Y - 1 I V r cn cn cn cn cn <n cn ?n c^ a Q C k Q k a q ; c C k c i c i C\ Q k a <Z '? '-jj L U U j L U Uj m Ui U j L U Uj h i Uj U J Uj Uj & % % Z Z Z z Z Z z Z :Z Z z z Z ij Ç V x y c;? — c :^ - Z c :t Z - r 0 - c~ 0 - c r c r o cr o - o r o - Q - n - n - c y ~ c r m (t Ü r O rO c o C n C T ) CO m rO cn cn fO m 1 O:' o Q O o o O o 0) Q I o ( o 1 Q o o o X A ( \ 1 <\ 1 rx (X \ C V fX z C V cy 1 CV J , 1 cv CV I C V 1 CV 1 CV I cV fj o V o o o u o o V o m cn C O cn cn cn c^ cn O J u f J o V r > o u V o u u V i < J u lo C J V cy o cl o o o 1 ^ o - rr~' o - < 3 ~ Cr- % cr- ro o~ -d m m fO ro M > CV Ô f c u 3 o M 1 Y o cy O fV c\i 7 cn (ÿ 7 cn C V rx cn CV t " n c3 --------- O r . m O o ro u > m O o cn c r m cr m o - T < ll c Ü C M r \J ( \ r w fX < y c V r w o o o 1 0 1 3 cn < M rx r< C V CV V (X { ' i O o Do c^ O o O o O o O o O o O o Oo Oo O o O o ro rO m m m rO m ro T m c O m % 4 . , fO Z z . Oi m 4 Oo V "4- kn Oo ;û ^ - V 4 V ' t i C io o - C\J Oo cz- CV 'T) CO CVJ \ h CV r - CM kn cn C\f V - czi C-- C\J Tt- Tj- ’V ' N c - n — f\i m V - — f\J m V - ~r (M rp v~ ; d Œ a c r cO à ÛÛ CÛ k j O u o a CJ Ô à L f + OJ o - c\j evj o Tf- OJ LO C \J C5- C\i (3- OJ Tt- CU 4 - OJ c— k n Tt- OJ r - o "4- C vj o vO rvj cr* JD V - C\J t ^ Oo O J O o O V - nvi vj> vO v ~ C V J O r - Th CV Oo Oo V - c v Cxj L n \ 0 N~. oo 0 - o CM cn s CHAPTER V OBSERVATIONS As a basis for comparison of the various states of degener ation caused by irradiation, the normal histological structure of the seminiferous tubules must first be considered. The tubules of the testis in the rat, contrary to the con dition in the higher forms, are not divided into lobules by connective tissue sheaths. They are distributed throughout the testis in a regular fashion, and are neither concentrated at nor relegated to any particular portions. The interstitial elements, which make up the remainder of the essential structure of the testis, are likewise scattered homogeneous ly and appear as small groups of cells separating the seminiferous tubules, Normally, in histological cross section, the interstitial cells occur in groups of about 5 or 6, although fewer and greater numbers are occasion ally seen. Blood vessels may be frequently observed passing in these interstitial elements which represent the means by which the body fluids pass to the tubules. The outermost layer of the seminiferous tubule consists of a very thin connective tissue sheath called the basement membrane. The remainder of the tubule is referred to as the germinal epithelium. This epithelium represents the various stages of spermatogenesis and consists of consecutive and fairly concentric rings of germinal cells. These appear in the order of the developmental phases of the male sex elements. 30 The first layer of the epithelium is composed of Sertoli cells, sperma togonia, and occasionally, undifferentiated primary germ cells. The Sertoli cells, sometimes called the Sertoli syncytium, are not a part of the sex differentiating cells, but have a supporting and nutritional function in the epithelium. The layers which follow consist, respective ly, of spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, and spermatozoa. There is a great deal of variation in these stages of spermatogenesis in reference to any particular tubule, however. Usually, not all forms of germinal cells are present in a single cross section. In addition, a layer of one form of germ cell is rarely present as a single ring of cells. The term ’layer’ actually refers to a general placement or localization in the epithelium rather than a specific cellular row or line. The central portion of the tubule is a lumen which varies in size in accordance with the number of stages of spermatogenesis present in the tubule at that particular time. The tails of the spermatozoa can usually be seen as fine lines projecting into the lumen. The lumen is utilized in the removal of the mature sperm from the seminiferous tubules. In the testis from the control animal which had been fed 13 cc. of wheat germ oil over a period of 29 days, there appeared no deviations from the normal testis described above. Histologically, the interstitial elements and the tubules, with their variously differentiat ed germ cells, in no way differed from the normal appearance of these structures. Figures 1 and 2 represent typical cross sections of a normal testis and the 13 cc. control testis respectively. FIGURE 1 Photomicrograph of a cross section of a normal rat testis to show the normal structure of the seminiferous tubules. (x 200) b..••..basement membrane 1......1nterstitial cells l.«••..lumen p......primary germ layer psc.•••primary spermatocytes 5......5ertoli cell sg. ..*.spermatogonia ssc.... secondary spermatocytes St.....spermatid sz.•.••spermatozoa FIGURE 2 Photomicrograph of a cross section of a testis from a rat which had been fed 13 cc. of wheat germ oil concentrate. (z 200) v;'*. ipac. S- ssc- » " C i - A # # ;I: ‘ v-.“ , l * , S f c ' % ' : 3 # * » « ■ S :b' ■ ,vZ% 4*: Æ FIGURE 1 ■ b l • s - 7 7 ^ . K Z i m FIGURE 2 32 In order to avoid confusion in the designation of the testes in the investigation, the following rule will be adopted: the X-rayed testes from the animals treated with vitamin E will be called the treated testes, while those X-rayed testes which received no vitamin E will be called untreated. Although it is admitted that both groups of testes were actually treated, that is, with X-ray, only the testes treated with both X-ray and vitamin E will be referred to as treated testes. The normal control testis is from a perfectly normal rat which had received no special treatment of any kind. The 13 cc. control testis is from the rat which had been fed 13 cc. of wheat germ oil con centrate and had received no irradiation. The 33 cc. control testis is from the rat which had been fed 33 cc. of wheat germ oil concentrate and had received no irradiation. The Preventive Effect of Vitamin E: - It might be mentioned first that there was no observable gross difference between the irradi ated testes and normal testes. Immediately before they were treated for histological study, which was three days after irradiation, there was no external evidence of degeneration. Histologically, however, the effects of the X-ray had been well established at this time. The damage was correlated, in general, with the amount of irradiation. Tliis will be seen in the following descriptions of the effects of the various doses of X-ray and the results of the use of wheat germ oil for the prevention of these effects. Since l/4 H.E.D. is a very mild degree of irradiation, there 33 was only a sli^t amount of degeneration observable in the first set to be investigated. The spermatocytes seemed to be the cells which sus tained the greatest injury. The X-ray degenerated the nuclei to such an extent that they appeared homogeneous with the cytoplasm. The cells themselves had a tendency to assume a beaded appearance. The sperma togonia and Sertoli cells, on the other hand, were uninjured by the irradi ation. The spermatids and spermatozoa seemed likewise unaffected by the X-ray. The mature spermatozoa, however, were pulled away from the epithelium and began to occlude the lumen of the tubule. The inter stitial cells were unharmed, but the basement membranes were, in some in stances, ruffled away from the tubules. In general, it could be said that this amount of irradiation had only a slight effect upon the epithelium. Many of the tubules ex hibited none of the effects noted above, and were quite normal in appear ance. Although there seemed to be an indication that the centrally lo cated tubules were affected more severely than were the more superficially located tubules, nothing really definite could be said in this respect. Mitotic figures were noticeably less in evidence than in normal testes. The testis of the animal treated with wheat germ oil present ed the same picture of degeneration. Although occasional sections seemed to show that the vitamin E was quite beneficial, a study of a great many sections indicated that no generalities in this direction could be made. No reliable difference in either the number of tubules affected or the average degeneration in the tubules could be established. Figures 3 and 4 represent typical cross sections of the untreated and FIGURE 3 Photomicrograph of a cross section of rat testis 3 days after it had received 1/4 H.E.D. X-ray* (Animal B-l) (x 200) b.•••••basement membrane be.....beaded spermatocytes i. ....interstitial cells FIGURE 4 Photomicrograph of a cross section of rat testis 3 days after it had received I/4 H.E.D, X-ray, from the rat fed 13 cc. of wheat germ oil before the irradiation. (Animal A-l) (x 200) * A • F\GV)Pk.E. 3 • - y . 3 %%• Y? A # , f . . • < ■ • - ♦ , * V . j.,'• - * ' ' ' * > . . - <n. A * ' ' « ■ H . ' ^ r > - R ' K , ' 1. >7-/ a ' ^ ' j sy < i * jT/ r I * - . ' . » f w ' • . . » < £ > ' (#. CV' F16VJRF 4- 35 treated testes at 1/4 H.E.D. irradiation. The appearance of the tubules in the 1/2 H.E.D. irradiation testes differed but slightly from those doses with the l/4 H.E.D. There were really no definite characteristics by which the two could be un failingly identified. The degeneration had affected more tubules, but the amount of destruction in the tubules was in no great way different from the previous degree of irradiation. The difference in damage was quantitative rather than qualitative. In addition to the effects noted at l/4 H.E.D., which were also present in this case, the spermatogonia were also occasionally affected. The lumina of many of the tubules were almost entirely occlud ed. This occlusion consisted of spermatozoa separated from the Sertoli cells, spermatids, and the degenerate, beaded sperme-tocytes. The Sertoli cells were completely normal, and the interstitial cells were like wise unaffected by the X-rays. Basement membranes were quite frequently separated from the tubules. A few of the tubules appeared normal, but these were rather scarce as compared with the 1/4 H.E.D. testis. Mitotic figures were very rare, and active division of the germinal cells had apparently been halted. Again there was an indication that the most central tubules re ceived the greatest injury from the X-rays. At 1/2 H.E.D., the treated testis, in many sections, seemed much less affected than the testis from the animal which had not received the excess vitamin E. However, this difference was again not reliable, since in a number of sections there appeared to be no distinction between 36 them. It may be said, nevertheless, that although the treated tubules were not consistently less affected than the non-treated, it was rare that the non-treated testis showed less degeneration than the treated testis. Figures 5 and 6 represent typical cross sections of the untreat ed and treated testes at l/2 H.E.D. irradiation. At 3/4 H.E.D. there was a great increase in the amount of degeneration in the epithelium. For the first time the process of separation was in definite evidence. Separations appeared between the germ cells, disrupting the epithelium at many points. In some instances the second layer was entirely separated from the Sertoli syncytium. Many tubules were completely occluded by the degenerated germ cells which totally obliterated the lumen. The basement membranes, in most instances were entirely ruffled away from the tubules. Here there was evidence which showed quite clearly that the interior tubules, or those deeper from the surface, suffered more severe ly from the effects of the irradiation than did the peripheral tubules. In many cases, but for a few exceptions, all of the tubules that were most severely damaged were located near the central portion of the testis. The indication of beneficial results from the wheat germ oil treatment was again rather slight* However, in the treated testis some practically normal tubules could be observed. Occasionally, basement membranes could be seen which had not lifted away from the tubules at any point. Separations among germ cells were more rare than in the tubules of the testis which had received treatment. This improvement which FIGURE 5 Photomicrograph of a cross section of rat testis 3 days after it had received 1/2 H.E.D. X-ray. (Animal B-2) (x 200) b......basement membrane be.....beaded spermatocytes i .interstitial cells o....».occlusion FIGURE 6 Photomicrograph of a cross section of rat testis 3 days after it had received l/2 H.E.D. X-ray, from the rat fed 13 cc. of wheat germ oil before the irradiation. (Animal A-2) (x 200) C L %?e- • FIGURE 5 f ' \ i / j 4 » • ^ * ♦ • % *T*%* • . “ ♦•’ i I b e .Ay.% «r <: % < & ! FIGURE 6 3S might be attributed to vitamin E, however, had by no means prevented the effects of the X-ray* The little difference that possibly existed could not even be called a marked improvement* Figures 7 and 8 represent typical cross sections of the untreated and treated testes at 3/4- H*E*D* irradiation. At one full H*E*D* there was one very significant identifying feature* This was the appearance of giant cells* These were large ovoid masses made up of degenerated spermatids, and in some cases, it seemed, of spermatocytes. One of these multinucleated giant cells was measured and was found to be 5*24 miera long and 3*#5 micra wide* Most of the giant cells contained about 20 nuclei* The nuclei were atypical and seemed to be lacking in chromatin. There appeared to be a *cell* membrane around the entire fused mass. Large separations were found in the epithelium of a great many tubules* In a few cases the germinal cells were removed from the Sertoli syncytium* Basement membranes were in almost all instances separated from the tubules* The interstitial cells, at some few points, seemed to be a little more numerous than usual* No general hypertrophy of these interstitial elements could be observed, however* The beaded spermatocytes, degenerating spermatogonia, and occluded lumina were here again observed* The spermatids were quite severely affected, making up the mass of the giant cells for the most part. It was rather difficult to determine the exact effect of the X-rays upon the mature spermatozoa since they were entangled with the germinal cell debris in the lumen of the tubule* While they had been loosened from the epithelium by even FIGURE 7 Photomicrograph of a cross section of rat testis 3 days after it had received 3/4 H.E&D. X-ray, (Animal B-3) (x 200) b.,,..basement membrane d..degenerated germ cells 1....*interstitial cells 8.....separation FIGURE 8 Photomicrograph of a cross section of rat testis 3 days after it had received 3/4 H.E.D, X-ray, from the rat fed 13 cc. of wheat germ oil before the irradiation. (Animal A-3) (x 200) I s *> • \* V V 'mi üi^zt^yÿs # v r $ ' ^ s FIGURE 7 p ^ # ' ■ - . *t ^ * ■ ] < > - m . bv'*V>îiî**’ FIGURE 8 _ 1 40 the 1/4 H.E.D., their cellular structure seemed little affected at any of the doses of the irradiation. The Sertoli cells were definitely un harmed through all of the doses of irradiation, including the 1 H.E.D. The excess vitamin E appeared to have very slight definite value in this case. In no instance, in those sections examined, was a giant cell found in the testis of the animal which had received the wheat germ oil. Separations were a little less in evidence, and more nearly normal tubules could be observed. The epithelium presented approximately the same picture of degeneration, however, so that any effect attributed to the vitamin E could at best be called slight. Fig ures 9 and 10 show the typical structures in the untreated and treated testes at 1 full H.E.D. irradiation. The Regenerative Effect of Vitamin E: - Since the normal testis was described in connection with the preventive effect of vitamin E, such a description at this point would be merely unnecessary repitition and has therefore been omitted. The experimental animals in this part of the investigation received 33 cc. of wheat germ oil over a period of 39 days. There was a possibility that tlie wheat germ oil itself might have some effect upon the testis, so a control rat was also fed this amount of excess vita min E and its testes were compared with the normal. In the case of the 13 cc. control, it was noted previously, the wheat germ oil had produced no noticeable effect upon the testes. However, the 33 cc. did seem to alter the histological appearance of the seminiferous tubules. This FIGURE 9 Photomicrograph of a cross section of rat testis 3 days after it had received 1 H.E.D. X-ray. (Animal B-4) (x 200) b.... .basement membrane d.....degenerated germ cells g.....giant cell 1.....1nterstitial 8.....separation FIGURE 10 Photomicrograph of a cross section of rat testis 3 days after it had received 1 H.E.D. X-ray, from the rat fed 13 cc. of #ieat germ oil before the irradiation. (Animal A-4) (x 200) ^ •• '. < • - , i %r* T ' ( F , FIG U R E 9 d. , e 0-? f r: " ffT 15 ■ ' T i i A » rV^' FIGURE 10 42 alteration appeared in the spermatocytes of the germinal epithelium. These cells, which usually form the greatest portion of the epithelium, seemed to be far fewer than in the normal testis. There was no evidence of degeneration in this connection. The spermatocytes merely appeared quantitatively reduced. Figures 11 and 12 present typical cross sections of the normal and 33 cc. control testes. The explanation for this condition could be that the vitamin E had tended to hasten the process of spermatogenesis. The spermatocytes might have matured more rapidly into spermatids and thence to spermatozoa. Since the vitamin E could have no particular effect in removing the mature cells from the tubules, the result would be a greater number of both the least and most fully differentiated sex cells and a comparative scarcity of the transitional states. This explanation would fit the conditions as presented by the histological appearance of the control rat which had been fed the 33 cc. of wheat germ oil. The irradiated testes, which were removed from the rats 39 days after the X-ray treatment in this case, showed a marked gross degen eration in every instance. They appeared to be mere miniatures of the large normal rat testes. The average length of the irradiated testes was approximately 15*0 mm., and the average diameter at largest cross section was about 7.0 mm. In the normal rat testis these measurements are approximately 19.5 mm. and 9.0 mm. respectively. Generally, the irradiated testes averaged about half the normal testis size. There was very little difference in the measurements of the FIGURE 11 Photomicrograph of a cross section of a normal rat testis to show the normal structure of the seminiferous tubules. (x 200) b.»••.basement membrane 1..*..interstitial cells 1*....lumen p.....primary germ layer psc...primary spermatocytes 5.....5.rtoli cell sg....spermatogonia ssc...secondary spermatocytes St....spermatid 82....spermatozoa FIGURE 12 Photomicrograph of a cross section of a testis from a rat which had been fed 33 cc. of wheat germ oil concentrate. (x 200) - ' * • %* * . # # & . .*4 ♦ ■'•«.' FIGURE 11 î-m ")T? r -* -» M S f_ .- >■ FtGURE iZ 44 testes which had received the effect of the excess vitamin E after the irradiation and those from the rats which had not been fed the wheat germ oil. The average length and largest cross sectional diameter were 14*6 mm. and 6*6 mm. respectively for the untreated, while for the treated these were found to be 15*3 mm* and 7*4 mm* Although this difference cannot be entirely disregarded, it seems to be too small to be used as an indication or criterion of the effectiveness of the vita min E concentrate. Histologically, the degeneration caused by the irradiation of the testes was further evidenced. A description of the conditions caused by the X-ray treatment will now be presented. This will follow the progressive course of the degeneration from the 1/4 H.E.D. to the 1 H.E.D. The effects upon the untreated and the vitamin E treated testes, as pairs, will be considered consecutively for the various amounts of irradiation. Although 1/4 H.E.D. is not a very great amount of irradiation, the histological picture of the X-rayed testis, in spite of the fact that there had been almost a month and a half allowed for regeneration, was far from normal. The most noticeable abnormality was the presence of large vacuoles in the seminiferous epithelium. This vacuolation was very common, being present in about three quarters of the tubules. The vacuoles appeared mainly in the spermatocyte layers, although many were present in the spermatid and spermatogonia regions. Very infrequently a vacuole could be found which interrupted the Sertoli syncytium, but such instances were quite rare. 45 The spermatocytes were the most severely injured of the germinal cells. Sometimes cellular structure seemed to disappear and leave undifferentiable cytoplasm in its place. Spermatogonia occasion ally underwent degeneration, but never as badly as the spermatocytes. The Sertoli cells remained uninjured by the X-rays. The basement mem branes surrounding the tubules remained intact around the entire tubule in all but a few cases. The interstitial cells showed no evidence of having been affected by the irradiation. The lumina of some of the tubules were noticeably larger than normal, while the spermatids and spermatozoa were found in the epithelia less frequently than in the normal testis. This indicated, perhaps, that the degenerate spermatids and the spermatozoa, pulled loose from the Sertoli cells but not necessarily injured, had left the tubules. This would explain both the lack of the more mature sex cells and the abnormal size of the lumina observed in the irradiated testis. In some cases it appeared that degenerate spermatocytes had also passed from the tubules. Many of the tubules, however, still contained degenerated germ cell debris which tended to occlude the lumina. In the testis which had received 1/4 H.E.D. irradiation and had then been treated with wheat germ oil, there was a definite improvement over these effects. Only occasionally was there large vacuolation apparent. In addition, many tubules could be observed which were quite normal in appearance. Nevertheless, the effects of the irradiation had by no means been entirely irradicated. In some instances very degener ate tubules could be found. In most cases, hov/ever, the damage in the 46 treated testis was relegated exclusively to the spermatocytes. As in the untreated testis, basement membranes in all but a few tubules were completely intact. Spermatogonia, Sertoli cells, and the interstitial elements were also quite normal in appearance. The conditions of enlargement and occulsion of the lumina were here present as in the untreated testis. However, the frequency of occurrence was far less. While it should be emphasized that the treated testis was not perfectly normal, it was definitely more nearly normal than the untreated testis. Individual tubules in the specimen which had received the bene fits of excess vitamin E might be selected which could be demonstrated to be far more degenerate tlian arbitrarily selected tubules of the untreated testis. However, upon examination of many sections and a great number of tubules, on a general average, the treated specimen appeared more nearly normal, histologically. Figures 13 and 14 show the state of the untreated testis and the treated testis after regeneration from the 1/4 H.E.D. X-ray. The untreated testis which had received l/2 H.E.D. irradiation presented a very degenerate histological appearance. In many cases, vacuolations turned the seminiferous tubules into little more than a cellular network. The vacuoles were present in every type of epithelial cell region. Sertoli cells, spermatogonia, spermatids, spermatocytes, and spermatozoa, were all involved in the degeneration. The sperma togonia were frequently damaged and the Sertoli cells only occasionally affected by the X-rays. In most of the tubules, the spermatids and FIGURE 13 Photomicrograph of a cross section of rat testis 39 days after it had received 1/4 H.E.D. X-ray. (Animal D-1) (x 200) d.....degenerated germ cells 1.....1nterstitial cells r.....regenerated germ cells 5.....5ertoli syncytium V . . . . .vacuole FIGURE 14 Photomicrograph of a cross section of rat testis 39 days after it had received I/4 H.E.D. X-ray, from the animal fed 33 cc. of wheat germ oil after the irradiation. (Animal C-l) (x 200) 2a FIGURE. 13 m * F I G U R E 1 4 - 48 spermatozoa were noticeably absent and the lumina were abnormally large. The explanation for this is probably the same as was advanced for the same condition in the 1/4 H.E.D. specimens. In other tubules, degener ated germinal cells tended to occlude the lumen. Ruffling of the basement membranes away from the tubules was more common than in the previous amount of irradiation, although it still was not very frequent. The interstitial cells, instead of becoming hypertrophied as has been described by several authors, were very scarce in this irradiated testis. The number of interstitial elements was far reduced from the normal testis or that seen in the 1/4 H.E.D. irradiation. The general picture of degeneration at this l/2 H.E.D. X-ray treatment was very severe. Instead of cells, especially in the case of the spermatocytes, homogeneous cytoplasmic streaks were present in the epithelium in the most degenerated tubules. All semblance of cellular structure had disappeared. It should be noted, however, that this particular animal died several days before the rest of the animals under investigation were killed. Thus, regeneration could not have continued for as long a period of time as in the remainder of the animals. Since the cause of death ?;as unknown, it is possible that there was some foreign effect upon the testis tissue of the rat. Such a possibility is rather doubtful, however. In the treated testis at l/2 H.E.D. a much more normal con dition was evident. The process of vacuolation was no more advanced 49 than in the 1/4 H.E.D. untreated testis. The vacuoles were almost en tirely confined to the spermatocyte region of the epithelium. The spermatocytes occasionally assumed the beaded appearance usually result ing from irradiation of the testis. Thorough cytoplasmic degeneration was not present in the tubules of the treated testis, and cell structures were maintained practically throughout. Ruffling of the basement mem branes was approximately as frequent as in the untreated testis. The Sertoli and spermatogonia cells were only occasionally damaged severely. The spermatids were frequently injured. The interstitial cells appeared quite normal in structure and distribution, as compared with their almost total absence in the untreated testis described previously. The difference in appearance between treated and untreated testes was even more marked at l/2 H.E.D. than it had been at 1/4 H.E.D. The unfortunate early death of the untreated animal, however, casts a slight doubt upon these results. A typical cross section of these un treated and treated testes may be seen in figures 15 and 16. At 3/4 H.E.D. the picture of the degeneration of l/2 H.E.D. was repeated. The various criteria of irradiation damage remained qualitatively similar, although quantitatively the damage increased. Large sections of Sertoli syncytium were interrupted by the large vacuoles or spaces which pervaded the epithelial layers. A noticeable increase of ruffled basement membranes was observed. The interstitial cells appeared quite normal, being neither hypertrophied nor particularly scarce. The spermatocytes, spermatogonia, and Sertoli cells were again FIGURE 15 Photomicrograph of a cross section of rat testis 39 days after it had received 1/2 H.E.D* X-ray. (Animal D-2) (x 200) d.....dagenerated germ cells 1.....1nterstitial cells ffl.....mature sperm cells r.....regenerated germ cells 5.....5ertoli syncytium V . . . . .vacuole FIGURE 16 Photomicrograph of a cross section of rat testis 39 days after it had received I/2 H.E.D. X-ray, from the animal fed 33 cc. of wheat germ oil after the irradiation. (Animal C-2) (x 200) a F IG U R E IS 7 Y * *L' . , , f # #* L . "m FIGURE 16 51 affected in that particular order in respect to damage. Destruction of cell formation, as in the l/2 H.E.D. untreated testis, was once more in evidence. The shape of the tubules gave further indication of the increase in irradiation. As the X-ray treatment was made more severe, it could be readily observed that the tubules were progressively distorted from their characteristic round shape in cross section* While in some cases this might be due to mechanical defects in the preparation of the sections, most of these instances could be directly attributed to the effects of the X-rays. Indentations and grooves in the surface of the tubules appeared with progressive severity as the amounts of irradiation were increased. In the treated testis at 3/4 H.E.D., the histological appear ance was considerably better. In general, it appeared but little worse than the untreated testis at 1/4 H.E.D. There was a marked im provement over both the l/2 and 3/4 H.E.D. untreated testes. This does not imply, however, that this 3/4 H.E.D. treated testis was normal. Some very degenerate tubules could be found which exhibited practically all of the most severe effects of irradiation found in the untreated 1/2 and 3/4 H.E.D. It was only upon examination of a great number of tubules that this average improvement definitely exhibited itself. Figures 17 and 18 illustrate the differences as found in the untreated and treated testes regenerated from 3/4 H.E.D. X-ray. The 1 H.E.D. untreated testis differed but slightly from the 3/4 H.E.D. The degeneration, in both a qualitative and quantitative sense, appeared very similar in both instances. The vacuolation, the figure 17 Photomicrograph of a cross section of rat testis 39 days after it had received 3/4 H.E.D. X-ray. (Animal D-3) (x 200) d.....degenerated germ cells 1 . ...1nterstitial cells m.....mature sperm cells r.....regenerated germ cells 5.....5ertoli syncytium V . . . . .vacuole FIGURE 18 Photomicrograph of a cross section of rat testis 39 days after it had received 3/4 H.E.D. X-ray, from the animal fed 33 cc. of wheat germ oil after the irradiation. (Animal C-3) (x 200) : s 17 F I G U R E 18 53 separation of basement membranes from tubules, the condition of the various germ cells, Sertoli syncytium, and interstitial cells, all differed in no perceptible way from these same conditions in the 3/4 H.E.D* irradiated untreated testis. The appearance of the vitamin E treated testis at 1 H.E.D. presented the same histological picture as the untreated testis at this same amount of irradiation. Although, occasionally, tubules could be found which seemed to be far less injured than some of the untreated tubules, exactly the reverse condition could also be observed in other cases. In general, there was no reliable difference which might be attributed to the effect of the wheat germ oil at 1 H.E.D. X-ray treat ment. These points are brought out by Figures 19 and 20 which show the regeneration of the untreated and treated testes from 1 H.E.D. irradiation. To all practical purposes, there is no difference. FIGURE 19 Photomicrograph of a cross section of rat testis 39 days after it had received 1 H.E.D. X-ray. (Animal D-4) (x 200) d.....degenerated germ cells 1.....1nterstitial cells r.....regenerated germ cells 5.....5ertoli syncytium V . . . . .vacuole FIGURE 20 Photomicrograph of a cross section of rat testis 39 days after it had received 1 H.E.D, X-ray, from the animal fed 33 cc. of wheat germ oil after the irradiation. Animal C-4) (x 200) / • * FIGURE 19 ■ - "^.v - : , * . r . \ » l % » ' - F I G U R E S O CHAPTER VI DISCUSSION AND CONCLUSIONS In the following discussion there can be little reference to the findings of other workers. The reason for this is that practically no investigations bearing directly upon this problem have thus far been attempted. The more pertinent relative literature has been reviewed in a previous chapter. In this discussion, therefore, in the main, the findings of this investigation alone will receive major consideration. Preventive Effect of.Vitamin E: - Since the greatest value of the prevention of irradiation effects upon the testis lies in its possi ble therapeutic application, the first question to be answered is: what practical or actual prevention was accomplished in the experimental ani mals. An examination of the observations recorded at the I/4, I/2, 3/4 and 1 H.E.D. show that at no dosage was thera a completely definite improvement exhibited by the treated testes. There were indications of beneficial results produced by the wheat germ oil, but at no stage could absolute criteria be established. In very general terms it might be stated that average histological appearance showed little difference be tween treated and untreated testes. The degeneration exhibited through out was almost equivalent in all cases. The vitamin E appeared to have no greater or less preventive effect upon one layer of germinal cells than upon the others. The sperma 5 . 6 tocytes, which are the first to be affected by irradiation, were also the first to degenerate in the wheat germ oil treated testes- The sperma togonia, spermatids, and spermatozoa, in that respective order, were the next to be most seriously injured, although in some cases the spermatids seemed to be affected before the spermatogonia- The Sertoli syncytium, as a general rule, remained intact- Very rarely was there even a slight break in the syncytium- No particular change was evidenced by the inter stitial cells at any of the amounts of irradiation* Their number, posi tion, and appearance, as far as could be determined, remained quite normal throughout- Only the separations among the germinal cells and the formation of giant cells gave any really definite indication of the value of vitamin E- Sepai’ ations which appeared in the 3/% H-E-D- and 1 H-E-D- untreated testes, were almost totally missing from the 3/4 H-E-D- end were only occasionally present in the 1 H-E-D- of the treated series- The giant cells made their only appearance in the untreated 1 H-E-D- In none of the sections of the treated testis at 1 H-E-D. that were studied was a single giant cell found. Separation and giant cell formation, however, cannot form the basis for a general, statement concerning the efficacy of vitamin E treatment, since the remainder of the observations differed so slightly from the normal effects of irradiation- There is good reason to believe that the effects of irradiation as described above are not the most severe produced by those particular amounts of X-rays- In all probability, the effects of the X-ray treatment 57 are not complete by 3 or 4 - days after the irradiation* Degeneration continues for some time afterward* The exact time at which degeneration is at its worst and regeneration of the epithelial tissue is about to begin is not precisely knovm* These conditions, however, were thought to have little effect upon this part of the experimentation which was devoted to the prevention of the irradiation effects* Any difference would be exhibited as soon as the X-ray had taken noticeable effects upon the sem iniferous tubules. Immediately after irradiation the testes Show little histo logical sign of degeneration. However, three or four days after X-ray treatment the epithelium exhibits the definite injurious effects describ ed above* Therefore, this period of time was utilized in the experi mentation. It is very possible that this is not the optimal time at which to study the most sharply contrasting effects of the irradiation. Since this time is unknown, the arbitrary three days was used. Emphasis must be placed upon the number of sections and tubules examined throughout the consecutive amounts of irradiation. Particular tubules could very easily be chosen to show that vitamin E had definitely prevented the effects of the X-ray at each of the doses of irradiation. On the other hand, by arbitrary selection of tubules one could show that the feeding of wheat germ oil previous to irradiation had brought about a more severe degeneration than would normally occur. Interpretation of such results as were found, can only be made in t erms of generalities which must cover the average conditions as found in the various specimens. Strict adherence to specific sections could result in the utilization of 58 the results toward any end* For this reason, only typical conditions existing in large numbers of tubules have been stressed and form the basis for the comparisons and observations. The conclusion to be drawn from these attempts to prevent the effects of irradiation is that an over supply of wheat germ oil (vitamin E), as determined by histological study, has no particular practical value in the prevention of sterility caused by X-rays. The prevention of the appearance of giant cells, and the alleviation of the condition of separation tend to indicate that possibly some aid is given by the wheat germ oil. This help is of such a negligible nature that it could not indicate any advocation of the use of wheat germ oil (vitamin E) therapeutically to prevent the damage caused by irradiation of the testes. Further, these results do not tend to strengthen any concept of a fundamental connection between avitaminosis and irradiation. A simpler interpretation could easily explain any possible beneficial re sults obtained in this investigation. For example, the mere fact that there is occasional degeneration in a normal testis could explain the re sults. Vitamin E in excess might merely build up normal tubules and eliminate this slight degeneration. Thus, upon irradiation there will be some slight beneficial results due to the use of vitamin E. Not because vitamin E has in any way prevented the irradiation — but merely because the tubules were in better condition before the irradiation be cause of the excess wheat germ oil. 59 The Regenerative Effect of Vitamin E: - In the investigation of the regenerative effect of vitamin E after irradiation, a period of about six weeks was allowed to elapse after the X-ray treatment- The reason for this particular amount of time was that Oslund (1926) re ported that regeneration in the rat testis began after about a month, following mild irradiation. Since the X-ray treatments used in these experiments were rather mild, it was thought that after about six weeks regeneration should be fairly well advanced, at least in the I/4 H-E-D* and the 1/2 H-E-D. Therefore, if excess vitamin E had definite regen erating value, the treated testes should be practically normal after this period of time. It was found, however, that none of the untreated testes were regenerated to any marked degree. Only that testis which had received 1/4 H-E.D- appeared to be somewhat normal. The testes which had received 1/2, 3/4 and 1 H.E-D- were all degenerate. Vacuolation ran a progressive course tlirough the series. It affected the spermatocytes primarily but in the l/2 H.E.D. the spermatogonia were involved, and in the 3/4 and 1 H.E.D. the Sertoli syncytium was occasionally ruptured. Throughout the various amounts of irradiation, the spermatocytes, spermatogonia, spermatids, and spermatozoa, in that respective order, were most severely injured- Sertoli cells were sometimes affected in the 3/4 and 1 H.E.D. Basement membranes were practically intact at the lowest amount of irradi ation but became progressively ruffled away from the tubules as the irradiation increased. The interstitial cells remained normal in all instances except the 1/2 H-E-D- In this ease, the interstitial cells 60 remained normal in all instances except the 1/2 H.E.D- In this case, the interstitial cells were very scarce. Since this particular animal died several days before it was to be killed, this might have been due to a secondary cause. The results of the wheat germ oil treatment, as determined histologically, were more promising than in the case of the preventive effect- The treated 1-4 H-E-D- approximated normal to a much greater extent than did its untreated control- Vacuolation was rather infrequent and many seemingly normal tubules could be found- At l/2 H.E-D. there was a very great difference between the treated and the untreated testes, as has been noted in the observation. At 3/4 H.E.D. there was a fairly definite but not very marked difference caused by the wheat germ oil. At 1 H.E-D., however, there was no observable distinction between the treated and untreated testes. If the vitamin E were expected to have a regenerative effect, the results would probably be expected to fall somewhat as they actually have. In the lower amounts of irradiation there existed the greatest difference between the treated and untreated testes. In the 3/4 H.E.D. this improvement grew smaller, while at 1 H.E.D. the value of the wheat germ oil totally disappeared, apparently. Perhaps another two or three weeks of vitamin E treatment would have made the results sharper and more easily observable. The particular time used was decided upon after a consideration of the work previously done in this connection. This investigation was sufficient to give some indication. Further and more extensive work in this field. 61 both in regard to amounts of wheat germ oil and time allowed for regen eration, might very well lead to more clearly defined and standardized results* From these experiments on regeneration after irradiation, it would seem that wheat germ oil (vitamin E) has some definite specific action in hastening the repair of the seminiferous epithelium* This action is greater in the milder irradiation than in the higher amounts of X-ray* An experiment involving the breeding of wheat germ oil treat ed and untreated male rats after sterilization by irradiation would be advisable as a further step in determining the exact positive value of vitamin E. Since there is no present standard vitamin E unit, this might suggest a new method of standardization. A unit might be fixed as the amount of a certain concentrate of wheat germ oil necessary to make fertile a male rat sterilized by a certain amount of X-ray treat ment. The major objection to such a scheme would be the fact that various adult male rats might react differently to the same amounts of irradiation. Therapeutic application of wheat germ oil (vitamin E) in cases of sterilization due to X-ray, probably would result in a hasten ing effect upon the normal process of testicular regeneration* .Its use in this connection should be further investigated before attempting application to human beings who have been afflicted by accidental steril ization. CHAPTER VII SUMMARY The Preventive Effects of Vitamin E 1. Excess wheat germ oil (vitamin E) at most, has a very slight and un reliable effect in preventing damage to the germinal epithelium of normal rat testis caused by irradiation. 2. There was no reliable evidence to show that such an effect was more marked at any particular amount of irradiation, although the higher amounts seemed to indicate more characteristic differences. 3. This effect of excess vitamin E does not warrant the use of wheat germ oil therapeutically in attempting to prevent accidental "ray sterilization* in human beings. 4# The results obtained in this part of the investigation do not tend to substantiate any view that avitaminosis and irradiation are fundament ally connected* The Regenerative Effects of Vitamin E 1* Excess wheat germ oil (vitamin E) seems to have a definite effect in hastening regeneration of the germinal epithelium of the rat testis after damage by irradiation. 2. This effect cannot be extremely rapid since the wheat germ oil did not, in the time allowed in the experimentation, result in thorough cure or absolute normality of the germinal epithelium, as histologically determined* ( 6 3 3. The regenerative effect seemed to vary quiet regularly with the progressive amounts of irradiation. The testes which received the mildest irradiation showed the greatest improvement while those receiving the most severe irradiation showed the least. 4. This effect of excess vitamin E does warrant consideration for the therapeutic use of wheat germ oil in attempting to restore normality to the germinal epithelium more quickly after *ray sterilization* has occurred. $• The regenerative value of vitamin E in cases of damaged epithelium presents a new possibility for the standardization of a vitamin E unit# LITERATURE CITED Adaastone, F.B#, ”A Possible Relation of Vitamin E to Unrestricted Cell Division,** Science 80:450, 1934» . , **The Effects of Severe and Prolonged Vitamin E Deficiency in the Chick," Anat. JSgg., 60:36-37, 1934* (Abstract 24) Asdell, S.A., and S*L. Warren, "The Effect of High Voltage Roentgen Radiation (200 kv) upon the Fertility and Motility of the Sperm of the Rabbit," M# Roentgenol* and Rad, Ther.. 25:81-84, 1931* Bacharach, A*L*, "A Recent Research on Vitamin E," Nutr* Abstr* and Rev»* 7:811-822, 1938» ______, E» Allchorne, and H,E, Glynn, "The Influence of Vitamin E Deficiency on Implantation" under "Investigations into the Method of Estimating Vitamin E," Biochem. J*, 31:2287-2292, 1937* Bagg, H.J» and C.C» Little, "Hereditary Structural Defects in the Descend ants of Mice Exposed to Roentgen Ray Irradiation," ^» J* Anat». 33:119-145, 1924* Barth, L»G., "The Effect of X-rays on the Spermatozoa of Drosophila," Phvsiol. Zool.* 2:172-180, 1929* Beard, H.H,, "The Relation between Diet and Reproduction," under "Studies in the Nutrition of the White Mouse," Am* J* Phvsiol*. 75:682-695, 1926* Blumberg, H», "A Growth Deficiency Disease Curable by Wheat Germ Oil," J. Biol* Chem.. 108:227-238, 1936* Carried, C*, "Zur Behebung der Rontgenstrahlenleukopenie," Klin* Wchnschr** 17:163, 1938* Clouse, R.C., "Vitamin D," 1»A»M.A., 99:215-222, 1932* Daniels, A*L* and M*K* Hutton, "Fertility of the White Rat on Purified Rations," Proc* Soc* Exper* Biol* and Med*. 23:225t227, 1925* Diakov, F*A* and J* Krizenecty, "Vitamin E and Pituitary Hormone, I* Fail ure of Vitamin E Preparation to Induce Precodious Sexual Development," Proc* Soc. Exper. Biol» and Med*. 31:58, 1933* ______, "Vitamin E and Pituitary Hormone, II. Failure of Anterior Pituitary Hormone and Prolan A to Substitute Completely Vitamin E*," Proc* Soc. Exper. Biol, and Med.* 31:59, 1933* 65 Diyerre, H., "Vitamins and the Prevention of Abortion in Sheep," Nature. 132:752, 1933* Emerson, O.H., G.A. Emerson, A. Mohammad and H.M* Evans, "The Chemistry of Vitamin É," "Tocopherols from Various Sources," J* Biol. Chem.. 122:99-107, 1937. Essenberg, J.M., "An Attempt to Castrate the Chick Embryo with X-rays," Radiology. 28:352-356, 1937. Evans, H.M. and K.S. Bishop, "On the Existence of a Hitherto Unrecognized Dietary Factor Essential for Reproduction," Science 56:650-651, 1922. _________ , "Vitamin E," J.A.M.A., 99:469-475, 1932. _________ , "Vital Need of the Body for Certain Unsaturated Fatty Acids," Z* Biol. Chem.. 106:445-450, 1934* Gatenby, J.B. and S. Wigoder, "The Effect of X-Radiation on the Sperma togenesis of the Guinea Pig," Proc. Rov. Soc.. London. _s.B., 104:351-370, 1928. , R.N. Mirkerji and S.B. Wigoder, "The Effect of X-Radiation on the Spermatogenesis of Abraxas Grossulariata," Proc. Rov. Soc*. London. ^*B*, 105:446-468, 1929* Haddow, A* and H.M* Russell, "The Influence of Wheat Germ Oil in the Diet on the Induction of Tumours in Mice," J. Cancer. 29:363- 366, 1937. Hill, L. and E.F. Burdett, "Fertility of Bees and Vitamin E," Nature. 130:540, 1932. Hooker, D.R., "The Effect of Exposure to Roentgen Rays on Reproduction in Male Rats," J. Roentgenol, and Rad. Ther.. 14:327-336, 1925. Huhner, M., "Sterility and the X-rays," J.A.g.A., 104:1808-1809, 1935* Korenschevsky, V., "Sterility in Males on Diets Deficient in Vitamin A or Vitamin E," Proc. Rov. Soc. Med.. 26:1187-1192, 1933* Leitch, I., "Nutrition in Relation to Reproduction with Special Reference to Sterility," Nutr. Abstr. and Rev*. 2:451-458, 1932. Lewis, L.R., "The Relation of the Vitamins to Obstetrics," Am. J* Qbst. and Gynec.. 29:759-765, 1935* Marshak, A., and V.L. Bollman^ "The Absorption of X-rays by Sperm and Erythrocytes and Its Relation to the Susceptibility of Tissues to X-rays," Am. J. Cancer. 26;581-585, 1936. 66 Martin, C.L. and W.H. Monrsund, "Irradiation Sickness," Radiology. 30:227-284, 1933. , "The Treatment of Roentgen Sickness with Synthetic Vitamin Hydrocholoride," Am. J. Roentgenol, and Rad. Ther.. 33:620-624, 1937. Mason, K.E., "The Effect of Rinified Diets, and their Modifications on Growth and Testiculai* Degeneration in Male Rats," J. Nutrition. 1:311-334, 1928. _________ , "Differences in Testis Injury and Repair after Vitamin A Deficiency, Vitamin E Deficiency and Inanition," Am. J. Anat.. 52:153- 239, 1933* , , and R.M. Melampy, "Absence of Vitamin E in the Royal Jelly of Bees," Proc. Soc. Exper. Biol, and Med.. 35:459-463, 1936. Mattill, H.A., "The Relation of Vitamins B and E to Fertility in the Male Rat," Ain. J. Phvsiol.. 79:305-315, 1926. _________ , "Vitamin E," J.A.M.A., 110:1831-1337, 1933. McQueen-Williams, "Increased Gonadotropic. Content of Anterior Hypo thy ses for Vitamin E Deficient Ffâiale Rat." Anat»; , Red, 53:77, 1934 (Abstr. Sect) Mirskaia, L. and F.A.E. Crew, "The Effect of Destruction of the Sperma- togenic Tissue by X-rays upon Certain Secondary Gonadic Characters of the Cock," Quart. J. Exper. Phvsiol.. 21:135-133. Moench, G.L., "A Consideration of Some of the Aspects of Sterility," Am. J. Qbst. and Gvnec.. 32:4-6-415, 1936» Moore, Ç.R. and L.T. Samuels, "The Action of Testis Hormone in Correct ing Changes Induced in the Rat Prostate and Seminal Vesicles by Vitamin B Deficiency or Partial Inanition,*’ Am. J. Phvsiol.. 96:278- 287, 1931. Olcott, H.S. and H.A. Mattill, "The Unsaponifiable Lipids of Lettuce, II. Fractionation," J. Biol. Chem.. 93:59-64, 1931* "Vitamin E, I. Some Chemical and Physiological Properties," J. Biol. Chem.. 104:423-435, Oslund, R.M. and A. Bachem, "Germinal Epithelium in X-rayed Testes of Rats," Proc. Soc. Exper. Biol, and Med.. 23:761, 1925* Pacini, A.J. and D.R. Linn, "A Vitamin E Unit," J. Am. Pharm. A., 25: 206-207, 1936. 67 Pfeiffer, C.A., "Some Factors Influencing the Vitalization of the Ovarian Graft and the Production of Sex Hormones in the Male Rat," Endocrinology. 21:260-267, 1937. Saphir, W., "Vitamin E and the Gonads," Endocrinology. 20:107-108, 1937. Shchegolev, G.G., "Influence of X-ray Treatment on Some Phases of Post- Embryonic Development of Rat Testis," Biol. Abstr.. 10: #8015, 1936. Shute, E., "Early Diagnosis of Abruptio Placentae and Its Treatment with Vitamin E," Am. J. Qbst. and Gvnec.. 33:429-436, 1937. Simmonds, N. and J.E. Becker, and E.V. McCollum, "The Distribution of Vitamin E," J. Nutrition. 1:39-47, 1928. Smith, H.O. and V.E. Nelson, "Cod Liver Oil for Reproduction and Lactation." Proc. Soc. Exper. - Biol, and Med.. 28:393, 1930. Sure, B., "Dietary Requirements for Reproduction, II. The Existence of a Specific Vitamin for Reproduction," J. Biol. Chem.. 58:693-709, 1^23* . "Dietary Requirements for Reproduction, IX. Cod Liver Oil versus Wheat Germ Oil as Sources of Vitamin E," J. Biol. Chem. ^ 74:45-53, 1927. also Ixxx; "XI. Vitamin E Potency of Butter Fat," "VIII. Vitamin E Potency of Unsaponifiable Matter from Crude Cotton seed and Crude Corn Oils," Ibid. p. Ixxx. * M.C. Kik and D.J. Walker, "The Effects of Avitaminosis on Hematopoietic Function, III. Vitamin E Deficiency," J. Biol* Chem.. 83:401-408, 1929. Todd, A.R., F. Bergel, W. Waldmann, and T.S. Work, "Constituents of Vitamin E Concentrates from Rice and Wheat Germ Oils," Nature. 140: 361-362, 1937. . "Studies on Vitamin E, I. The Isolation of Some Crystalline Alcohols from the Unsaponifiable Matter of Rice and Wheat Germ Oils," Biochem* J., 31:2247-2256, 1937; II. "The Isolation of B Tocopherol from Wheat Germ Oil," Ibid. pp. 2257-2263. Urner, J.A., "The Intra-Uterine Changes in the Pregnant Albino Rat (Mus Norvegicus) Deprived of Vitamin E," Anat. Rec.. 50:175-187, 1931. Verzar, F., "The Influence of Diet on Internal Secretion," Proc. Staff. Meet. Mavo Clin.. 4:351-352, 1929. Vogt-M/ller, P., "Treatment of Habitual Abortion with Wheat Germ Oil (Vitamin E)," Lancet, 2:182-183, 1931. 68 Waddell, J. and H. Steenbock, "Vitamin E in Iron Treated Dry Rations," J. Nutrition. 4:79-93, 1931. Watson, E.M. and W.P. Tew, "îftieat Germ Oil (Vitamin E) Therapy in Obstetrics," Am. J. Obst. and Gvnec.. 31:352-358, 1936. Weisner, B.P. and A.L. Bacharach, "Effect upon Sex Behaviour of a Diet Deficient in Vitamin 1," Nature. 140:972-973, 1937. Wintz, H., "A Review of the Effects of X-rays on the Testes, Ovary, and Embryo," Biol* Abstr.« 6: #322, 1932. Witschi, E., W.T. Levine, and R.T. Hill, "Endocrine Reactions of X-ray Sterilized Males," Proc. Soc. Exner. Biol, and Med.. 29:1024-1026, 1931. Young, J., "The Habitual Abortion and Stillbirth Syndrome and Late Pregnancy Toxaemia, Vitamin E and the Prolan-Progesterone Mechanism," Brit. 1. J., 1:953-957, 1937.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

The effects of alloxan on the fetal rat pancreas

PDF

Survey of the vocational activities of the Japanese in the city of Los Angeles

PDF

A comparison of the attitudes of selected groups toward the provision of dental care by the federal government with compulsory individual wage deductions, including provision for voluntary accept...

PDF

The effect of desoxycorticosterone on the height of normal thyroid epithelium

PDF

The effect of the pH of perfusates on the activity of the intact urinary bladder in the rat

PDF

Effect of certain drugs on the rate of excretion of vitamin C

PDF

Histological changes induced in the harderian gland of the mouse by transplantation in the anterior chamber of the guinea pig eye

PDF

The effect of anoxia on some aspects of the blood picture, with experiments on the albino rat and the rabbit

PDF

The effect of steroids and lecithin on the resistance of the beef erythrocyte to osmotic stress

PDF

Histological variations in the walls of the digestive tract of salamanders

PDF

The physiological effects of the biochemical properties in drinking water

PDF

A comparison of the biologic effects of several gonadotropic substances

Some effects of partial substitutions of lithium for environmental sodium on <italic>Limnoria</italic> species

PDF

Some effects of partial substitutions of lithium for environmental sodium on Limnoria species

PDF

A study of the histological distribution of glycogen in tumors of hypo-physectomized and non-hypophysectomized rats

PDF

Lateralized effects of blurring: A test of the visual spatial frequency model of cerebral hemisphere asymmetry

Cytological and genetical study of certain regions of the chromosomes in <italic>Drosophila pseudoobscura</italic>

PDF

Cytological and genetical study of certain regions of the chromosomes in Drosophila pseudoobscura

PDF

Influence of a liquid diet on water and electrolyte equilibrium and nitrogen excretion in the albino rat

PDF

The participation of the Chinese in the community life of Los Angeles

PDF

Mites parasitic in the lungs and air passages of seals, with special emphasis on their occurence in the California sea lion

PDF

Some estrogenic effects of endometrial extract

Asset Metadata

Creator Schwartz, S. C. (author)

Core Title The effect of vitamin E on the damage in the germinal epithelium in the testis of the rat caused by irradiation

School Department of Zoology

Degree Master of Arts

Degree Program Zoology

Degree Conferral Date 1939-05

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

Tag biological sciences,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Contributor Digitized by ProQuest (provenance)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c39-256637

Unique identifier UC11315018

Identifier EP67131.pdf (filename),usctheses-c39-256637 (legacy record id)

Legacy Identifier EP67131.pdf

Dmrecord 256637

Document Type Thesis

Format application/pdf (imt)

Rights Schwartz, S. C.

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA