REPRODUCTIVE TRACT OF ANIMALS FED A DIET DEFICIENT IN VITAMIN A ALCOHOL BUT CON TAINING VITAMIN A ACID. I. THE MALE RAT

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1 HISTOLOGY OF THE LESIONS PRODUCED IN THE REPRODUCTIVE TRACT OF ANIMALS FED A DIET DEFICIENT IN VITAMIN A ALCOHOL BUT CON TAINING VITAMIN A ACID. I. THE MALE RAT J. McC. HOWELL, J. N. THOMPSON and G. A. J. PITT Departments of Veterinary Pathology and Biochemistry, University of Liverpool {Received 20th August 1962) Summary. Male rats maintained on a diet in which the vitamin A alcohol had been replaced by vitamin A acid developed lesions in the reproductive tract. The testicular changes were a sloughing of the cells of the germinal epithelium followed an by obliteration of the lumen of the tubule by Sertoli cells. Testicular regeneration was produced by the administration ofvitamin A alcohol. The lesions are comparable to those of vitamin A deficiency as described by Mason (1933) but are un complicated by the secondary manifestations of vitamin A deficiency. The present paper also describes for the first time lesions in the testes of healthy growing prepubertal rats fed the vitamin A acid diet. INTRODUCTION Vitamin A acid, the carboxylic acid corresponding to the primary alcohol, vitamin A, was thought to replace vitamin A alcohol in all its known functions except that it could not provide vitamin A aldehyde for the prosthetic group of the visual pigments. Thus Dowling & Wald (1960) a using diet deficient in vitamin A alcohol, but supplemented with vitamin A acid, found that rats grew well and appeared normal, except that they were blind. It has been suggested that vitamin A acid is the form of the vitamin active systemically, i.e. concerned with growth and general tissue maintenance (Moore, 1953; Dowling & Wald, 1960). However, Thompson, Howell & Pitt (in preparation) have shown that rats maintained on a diet deficient in vitamin A alcohol, but containing vitamin A acid, showed normal mating behaviour but failed to reproduce. Lesions were present in the reproductive tract of male and female rats. The purpose of this communication is to give a more detailed report of the histological changes in the male reproductive tract, and to discuss these changes in relation to those of orthodox vitamin A deficiency as described by Mason (1933). 159

2 160 J. McC. Howell, J. N. Thompson and G. A. J. Pitt MATERIALS AND METHODS ANIMALS The animals used in our experiments were hooded rats from the closed colony maintained in the Biochemistry Department of the University of Liverpool. They could be divided into two types according to their vitamin A reserves at weaning {see Table 1). Type 1 rats were born of stock females which had been maintained on a diet containing a generous amount of vitamin A (Diet No. 4, British Extracting Co. Bromborough). The Type 1 rats were fed from weaning the basic diet (see Table 1 parentage and dietary regime of the rats used in this work Dam Diet Vitamin A stores at weaning Type 1 rat Stock rat Basic diet from weaning Present Control to Type 1 Stock rat Basic diet plus vitamin A alcohol given in oil by a weekly dose Type 2 rat Type 1 rat given doses of 0-5 to 2-0 Mg of vitamin A acetate in oil by mouth every day during preg nancy Control to Type 2 Type 1 rat given doses of 0-5 to 2-0 v-g of vitamin A acetate in oil by mouth every day during preg nancy mouth as of 140 or 200 ug ofvitamin A acetate OR the basal diet without vitamin A acid Basic diet available from birth Basic diet plus vitamin A alcohol given in oil by mouth as a weekly dose of 140 or 200 Mg of vitamin A acetate Present Absent Absent below), i.e. a diet containing vitamin A acid but not vitamin A alcohol. They had stores of vitamin A alcohol in the liver which lasted from 4 to 5 weeks after weaning. The control rats were fed the basic diet with modifications as indicated in Table 1 and in the text. Type 2 rats were born of Type 1 females which were maintained on the basic the minimal amount of vitamin A alcohol diet and given during pregnancy (given as the acetate) required for the successful delivery of living young (Thompson, Howell & Pitt, in preparation). The administration of vitamin A alcohol ceased as soon as the young were born. At weaning the Type 2 rats were continued on the basic diet. Any stores of vitamin A alcohol in the Type 2 rats were exhausted before these animals were weaned. Control rats, born under this regimen, were fed the basic diet with modifications as indicated in Table 1 and in the text.

3 Vitamin A alcohol deficiency in the rat 161 DIET The basic diet (Thompson, Howell & Pitt, in preparation) consisted of: sucrose 65%, casein 18%, yeast 8%, edible ground-nut oil 5%, and minerals 4 %. Supplements were added containing adequate amounts of all known vitamins, with the exception of vitamin A alcohol. In the supplements vitamin A acid was substituted for vitamin A alcohol. The vitamin A acid was mixed into the food as the methyl ester, the dose being 10 pg/g of diet. A new supply of diet was mixed at least once a week and usually more frequently. Certain animals received higher levels of vitamin A acid given as 1 mg of vitamin A acid methyl ester by mouth every day in 0 1 ml of ground-nut oil or mixed with the diet at a level of up to 0-5 mg/g of diet. The basic diet contained 0-15 g //-oc-tocopheryl acetate per kg of diet. EXPERIMENTAL TECHNIQUES In our regeneration experiments, Type 1 rats were divided into pairs matched for body weight. Under ether anaesthesia the right testis of these rats was re moved and fixed in Bouin's fluid. The animals were retained on the basic diet but one of the pair also received vitamin A alcohol as a dietary supplement, i.e. in addition to vitamin A acid. The vitamin A was given by mouth in oil as a weekly dose of 200 µg of vitamin A acetate. All rats were killed by chloroform inhalation. Testes and seminal vesicles were removed and weighed prior to fixation. Material for histological examina tion was fixed in Bouin's fluid as soon as possible after death. The tissue was embedded in paraffin wax and sections were cut at 4 µ and stained with haematoxylin and eosin; selected sections were also stained by the periodic acid-schiff technique. Where appropriate, sections were cut on the freezing microtome and stained with Sudan IV or by the pas technique. Type 1 rats RESULTS HISTOLOGY OF THE TESTIS AND EPIDIDYMIS The rats were weaned on to the basic diet when they were 3 to 4 weeks old. accumulated a store of vitamin A alcohol. At this time they had already Such rats, given the basic diet from which the vitamin A acid was omitted, showed the classical symptoms of vitamin A deficiency (failure to maintain a gain in weight, xerophthalmia) after 4 to 5 weeks. The first lesion in the male reproductive tract of the rats given the basic diet was seen after 5 weeks. This first lesion was an increase in the number of At 5 weeks cells, probably spermatids, in the ductus epididymis (PL 1, Fig. 1). the testes weighed less than those of an animal given this diet supplemented with vitamin A alcohol, but little difference could be seen in the histological structure. As the length of time on the basic diet increased, the number of sloughed cells in the ductus epididymis became more numerous, being replaced by degenerate cells, which finally disappeared leaving only eosinophilic material.

4 162 J. McC. Howell, J. N. Thompson and G. A. J. Pitt The number of spermatozoa in the ductus epididymis decreased and they were absent from the ductus epididymis of rats killed when 20 weeks old. They were, however, seen to persist in the proximal part of the vas deferens and a few were found in this site in an animal killed when 11 months old. The first lesion to be seen in the testis was a loss of spermatids. This was apparent after about 6 to 8 weeks on the basic diet. This lesion occurred at about the same time in animals completely deficient in vitamin A, i.e. animals fed the basic diet without the vitamin A acid. This early lesion was followed by a patchy loss of spermatocytes from the seminiferous tubules. At this time some tubules contained healthy spermatocytes together with a few cells having dark structureless nuclei and homogeneous darkly eosinophilic cytoplasm. These were thought to be degenerating spermatocytes. Although many spermatocytes had disappeared from the testes of rats examined after a period of 20 weeks on the diet, isolated spermatocytes persisted and were seen in the testes of one animal killed when 1 year old (PL 1, Fig. 2). During the time that the spermatocytes were disappearing, vacuolation was seen in the cytoplasm of the Sertoli and spermatogonia cell layers (PL 1, Fig. 3). An examination of frozen sections revealed that these vacuoles con tained neither sudanophilic nor pas positive material. After the disappearance of the spermatocytes and spermatogonia, the lumen of the tubule became obliterated by a mass of Sertoli cells (PL 1, Fig. 4). These lesions were patchily distributed throughout the degenerating testis. Thus in any one section of the testis the following changes could be seen: tubules completely filled with Sertoli cells, and tubules with a lumen, lined by Sertoli cells and spermatogonia. Many of the latter showed vacuolation and some contained spermatocytes (PL 1, Fig. 5). Type 2 rats These rats were given the basic diet as soon as they could eat. They were born with negligible stores of vitamin A which would not last until weaning. Thus they were deficient in vitamin A alcohol from the start of the experiment. Type 2 rats fed on the basic diet never formed spermatids (PL 1, Fig. 6). A lumen was formed in the seminiferous tubule, only to be closed later by Sertoli cells. Healthy dividing spermatocytes and degenerating spermatocytes were seen in the same tubule (PL 2, Fig. 7) and in the early stages spermatocytes were numerous (PL 1, Fig. 6). They persisted in reduced numbers for a con siderable period of time and some spermatocytes were still present in the tubules of a rat killed when 231 days old. Vacuolation was seen in the cytoplasm of Ser toli cells. Loss of spermatocytes and spermatogonia was followed by what would seem to be a proliferation of Sertoli cells to fill the seminiferous tubules. This was seen in animals only 61 days old (PL 2, Fig. 8) but filling of the tubule was still only to be seen in isolated tubules in the testes of rats aged 80 days. The majority of tubules were filled when the rats were 130 days old, but even in a rat killed when 231 days old this lesion was not present in all the tubules (PL 2, Fig. 9). Varying stages in the development of the lesion could be seen in any one section of the testis. The ductus epididymis of these rats at no time contained spermatozoa

5 Vitamin A alcohol deficiency in the rat 163 (PL 2, Fig. 10). In a few rats one or two degenerating cells were observed but the usual picture was of a ductus that was empty or which contained only eosinophilic material (PL 2, Fig. 11). REGENERATION OF THE TUBULAR EPITHELIUM These experiments were performed on Type 1 rats. The right testis was re moved after 2 to 4 months on the basic diet. This was done in order to observe regeneration from different stages in the development of the lesion. All stages in the development of the lesion were seen, ranging from a testis with a normal histological structure but with an increase in the number of cells in the epi didymis, to a testis having almost all its tubules filled with Sertoli cells. The period allowed for regeneration varied from 49 days to 99 days. There was only slight evidence of regeneration in the left testis examined after 49 days, but definite evidence of regeneration was seen after longer periods on the basic diet supplemented with vitamin A alcohol (PL 2, Fig. 12 and PL 3, Fig. 13). Sections prepared from the testis removed at operation from one such animal, shown in PL 2, Fig. 12, contained no spermatids, but after 69 days regeneration spermatids were present in about half the tubules of the remaining testis and there were some immature spermatozoa (Leblond & Cleremont, 1952). Our results indicated that regeneration could be produced in severely degenerate testes if spermatocytes or spermatogonia were present in the tubules. In all cases the testes of the animals maintained on the basic diet alone showed a more severe degree of degeneration at the end of the experiment than did the testis that had previously been removed. PREVENTION OF DEVELOPMENT OF THE LESION Type 1 rats, whose diet was supplemented with vitamin A alcohol at or up 3 weeks after weaning, did to not show degeneration of the germinal epithelium. Normal testicular development was seen even in the Type 2 rats on such a regimen. High dietary levels of vitamin A acid, even those sufficient to produce signs of hypervitaminosis A (Thompson & Pitt, 1960) in no way altered the develop ment of the lesion. Animals on high levels of vitamin A acid who also received a supplement of vitamin A alcohol had normal testes. MACROSCOPIC APPEARANCE OF THE TESTIS In Type 1 rats the first macroscopic sign of testicular abnormality was a change in colour. There was a change from the normal white to a slightly pink appear ance. This was followed by a reduction in size and weight; the degenerate testes had a greyish pink translucent appearance and they were markedly oedematous. Even when the synthetic diet was supplemented with a high level of vitamin A acid, the testes weighed less than those of similar rats whose diet was supplemented with vitamin A alcohol (Thompson, Howell & Pitt, in preparation). The testes of the Type 2 rats were small and slightly pink but of a relatively firm consistency (PL 3, Fig. 14), and they were not markedly oedematous.

6 164 J. McC. Howell, J. TV. Thompson and G. A. J. Pitt ACCESSORY SEXUAL ORGANS and Type 2 rats fed on the basic diet were The seminal vesicles of Type 1 smaller and weighed less than those of equivalent animals receiving this diet supplemented with vitamin A alcohol. In the regeneration experiments the rats whose diet had been supplemented with vitamin A alcohol had larger seminal vesicles at the end of the experiment than those rats which had been maintained on the basic diet alone (Thompson, Howell & Pitt, in preparation). No marked histological changes were seen in either the prostatic acini or the seminal vesicles. Foci of squamous metaplasia and keratinization were not seen in any of the rats given the basic diet. Such lesions were frequently present in the prostate and seminal vesicles of rats fed this diet without vitamin A acid. DISCUSSION Dowling & Wald (1960) concluded that "... the only function vitamin A may perform directly in the rat is to supply the prosthetic group of its visual pig ments. All other functions growth, general tissue maintenance are served equally well by vitamin A acid". Our results have indicated that this statement must be modified (Thompson, Howell & Pitt, in preparation). Rats main tained on such a diet, the basic diet, show severe degenerative changes in the testes. These changes are similar to those described by Mason (1933) in rats deficient in vitamin A, although Mason's results can only be compared with those obtained in our Type 1 rats. Mason observed the first changes in the reproductive organs at about the time xerophthalmia developed, and he illustrates this lesion in a rat that had been on the deficient diet for about 50 days. This lesion was essentially a sloughing of the cells of the germinal epithelium. The first cells to be sloughed were the spermatids. At this time many cells were seen in the ductus epididy mis. He noted: "This sloughing process was often observed in sections of the ductuli efferentes and ductus epididymis before any other pathological change could be noted in the tubules themselves". He further stated: "Often, small numbers of deeply staining pycnotic cells were observed throughout the epithelium of the seminiferous tubules". These were in all probability the cells that we consider to be degenerating spermatocytes. In the advanced stages of vitamin A deficiency few germ cells were left. Mason recorded a reduction in tubule size throughout the course of the development of the lesion, but he did not mention vacuole formation although vacuole-like structures can be seen in Figs. 11 and 51 of his paper. Neither does Mason report that the lesion of vitamin A deficiency had a patchy distribution throughout the testes, but some evidence of a patchy distribution of the lesions can be seen in Figs. 8, 11, 26, 27 and 51 of his paper. These are essentially the same lesions observed in our animals fed on a diet deficient in vitamin A alcohol but supplemented with vitamin A acid. They are the characteristic lesions of vitamin A deficiency as observed in the testes. However, Mason did not report Sertoli cells filling the seminiferous tubules, and this lesion was not seen in any of our control animals which were given at

7 Vitamin A alcohol deficiency in the rat 165 weaning a diet completely deficient in vitamin A, i.e. the basic diet without vitamin A acid. The proliferation of Sertoli cells was first seen in Type 1 rats fed from weaning the basic diet, when they were 11 weeks old, and the lesion was seen in one or two tubules only. However, Beaver (1961) working on vitamin A deficiency in germ-free rats does show Sertoli cells filling the semi niferous tubules {seefig. 25 of his paper) ; three of the germ-free rats in his experi ment lived for 109 days and the average survival time in eight animals allowed to die was 95 days. One would expect vitamin A deficient rats kept under normal laboratory conditions to die when about 9 weeks old. It is thought that our results only differ from those of Mason in that our rats lived longer and did not suffer from inanition. Had Mason's deficient rats lived longer, presumably they too would have shown tubules full of Sertoli cells. The results of our experiments in Type 1 2 rats indicate that the and Type basic lesion of vitamin A deficiency in the testes is to be found at a stage in spermatocytogenesis before the meiotic division of the spermatocytes to form spermatids. Spermatids were never formed in our Type 2 rats. Degenerating spermatocytes were seen for a considerable period of time and during this time healthy and even dividing spermatocytes might be seen in the same tubule. Eventually all the spermatocytes and probably all the spermatogonia disappear leaving a tubule filled with Sertoli cells. The mechanism for these changes remains obscure. In the Type 2 rats we have demonstrated for the first time, the importance of vitamin A in the development and maturation of the germinal epithelium of the testes. Mason considered that one of the main differences between testicular damage due to vitamin A deficiency and that due to vitamin E deficiency was that regeneration could be produced in the former but not in the latter. Striking regeneration was produced in our rats by the administration ofvitamin A alcohol. Evidence has been cited by Mason (1939) that "variable degrees of gross atrophy and the occasional appearance of foci of epithelial keratinization, have been observed in the epididymis, vas deferens, seminal vesicle, prostate and preputial glands of the A deficient rat". He went on to say: "That (the atrophy) of the prostate and seminal vesicle appears to be more closely related to the body growth and nutritive state of the animal than to the degree of A de ficiency." This cannot relate to our findings with vitamin A acid for in our experiments Type 1 rats had a body weight equal to that of the control rats on the same diet but supplemented with vitamin A alcohol, but the seminal vesicles of the former were smaller. It is interesting to note that in the re generation experiment the rats receiving the vitamin A alcohol postoperatively seminal vesicles than the controls maintained on vitamin A acid had bigger (Thompson, Howell & Pitt, in preparation). These results indicate that vitamin A alcohol has an effect on the seminal and nutritive state vesicles which is independent of its effect on the growth of the animal. Fiske (1941) has shown that pituitary, seminal vesicle and testes weights of

8 166 J. McC. Howell, J. jv. Thompson and G. A. J. Pitt in rats rats kept in the dark were smaller than the weights of these organs kept under normal laboratory conditions. However, there was little histolo gical difference between the testes of these two groups. In the Type 1 rats the testes were markedly degenerate and the seminal vesicles were reduced in size before one would have expected the rats to be blind (Dowling & Wald, 1960). Regeneration of the retina may have in fluenced the seminal vesicle weights of the rats receiving vitamin A alcohol in our regeneration experiment. One would expect a much earlier development of the retinal lesion in the Type 2 rats. If this is so, blindness could be a contributory factor in the develop ment of the lesions in the reproductive tract of these animals. This aspect of the problem is now being investigated. ACKNOWLEDGMENTS We wish to thank Professor D. L. Hughes and Professor R. A. Morton, f.r.s. for their encouragement and advice, Miss M. W. Royston, a.i.m.l.t. and Mr J. Camp for technical assistance, Mr G. Weston, f.i.m.l.t. for the photographs, and Dr O. Isler of F. Hoffmann-La Roche & Co, Basel for supplies of vitamin A acid. This work was supported in part by a grant from the Agricultural Re search Council. REFERENCES Beaver, D. L. (1961) Vitamin A deficiency in the germ-free rat. Amer. J. Path. 38, 335. Dowling, J. E. & Wald, G. (1960) The biological function of vitamin A acid. Proc. nat Acad. Sci., Wash. 46, 587. Fiske, V. M. (1941) Effect of light on sexual maturation, oestrous cycles, and anterior pituitary of the rat. Endocrinology, 29, 187. Leblond, C. P. & Cleremont, Y. (1952) Spermiogenesis of rat, mouse, hamster and guinea-pig as revealed by the 'periodic acid-fuchsin sulfurous acid' technique. Amer. J. Anat. 90, 167. Mason, K. E. (1933) Differences in testes injury and repair after vitamin A deficiency, vitamin E deficiency and inanition. Amer. J. Anat. 52, 153. Mason, K. E. (1939) Relation of the vitamins to the sex glands. Sex and Internal Secretions, 2nd edn. Eds. E. Allen, C. H. Danforth and E. A. Doisy. Williams & Wilkins, Baltimore. Moore, T. (1953) Vitamin A in the normal individual. Symposium on Nutrition. Ed. R. M. Herriott. Johns Hopkins Press, Baltimore. Thompson, J. N. & Pitt, G. A. J. (1960) Vitamin A acid and hypervitaminosis A. Nature, Lond. 188, 672. EXPLANATION OF PLATES PLATE 1 Fig. 1. Spermatozoa and sloughed cells in the ductus epididymis. Type 1 rat fed on the basic diet for 6 weeks, i.e. a diet containing vitamin A acid but not vitamin A alcohol. The histological structure of the testis was normal. Haematoxylin and eosin Fig. 2. Isolated spermatocytes present in the seminiferous tubule. One-year-old-Type 1 rat. Haematoxylin and eosin Fig. 3. Vacuolation in the cytoplasm of the cells lining the tubules. Type 1 rat fed the basic diet for 11 weeks. Haematoxylin and eosin Fig. 4. Tubules filled with Sertoli cells. Eosinophilic oedema fluid surrounds these tubules. Type 1 rat fed the basic diet for 15 weeks. Haematoxylin and eosin. X 189. Fig. 5. Some tubules are filled with Sertoli cells, others have a lumen. Vacuolation and spermatocytes can be seen in the latter. Type 1 rat fed the basic diet for 15 weeks. Haematoxylin and eosin Fig. 6. Seminiferous tubule containing numerous spermatocytes but no spermatids. Fifty-six-day-old Type 2 rat. Periodic acid Schiff reagent. x375.

9 PLATE 1 (Facing p. 166)

10 PLATE 2

11 PLATE 3 (Facing p. 167)

12 Vitamin A alcohol deficiency in the rat PLATE 2 Fig. 7. Tubule from the testis shown in Fig. 6. Healthy spermatocytes and degenerating cells are present. Periodic acid-schiff reagent, 500. Fig. 8. The tubule in the centre of the field is filled with Sertoli cells. Sixty-one-day-old Type 2 rat. Haematoxylin and eosin Fig. 9. Testes. Some of the tubules still have a lumen. Two hundred and thirty-oneday-old Type 2 rat. Haematoxylin and eosin Fig. 10. The ductus epididymis is empty. Fifty-six-day-old Type 2 rat (see Fig. 6). Haematoxylin and eosin Fig. 11. The ductus epididymis is either empty or contains eosinophilic material. Two hundred and thirty-one-day-old Type 2 rat (see Fig. 9). Haematoxylin and eosin. X52-5. Fig. 12. Right testis showing severe degeneration. Tubule in the centre of the field con tains spermatocytes. Eighteen-week-old Type 1 rat. This testis was removed surgically during a regeneration experiment. The animal had been fed on the basic diet. Haema toxylin and eosin. 70. PLATE 3 Fig. 13. Left testis removed 69 days after the surgical removal of the right testis which is shown in Fig. 12. Postoperatively the rat had been fed on the basic diet supplemented with a weekly dose of vitamin A alcohol, given in oil, by mouth as 200 Mg of vitamin A acetate. Spermatids are present. Many of these are immature spermatozoa. Twentyeight-weeks-old Type 1 rat. Haematoxylin and eosin. X 70. Fig. 14. Testes and seminal vesicles of a 67-day-old Type 2 rat and of a 67-day-old control animal. The control animal was born on the same regimen as the Type 2 rat but from 40 days old it had been fed the basic diet supplemented with a weekly dose of vitamin A alcohol. The testes and seminal vesicles of this animal, shown at the top of the figure, are larger than those of the Type 2 rat.