IN A PREVIOUS communication 5 from this laboratory, a study of the lipid

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1 The Sertoli Cell as Related to Age of Man and Experimental Alteration of the Pituitary Gonad Axis in the Animal With Consideration of Its Role in Spermatogenesis Kenneth M. Lynch, Jr., M.D.,* and William Wallace Scott, Ph.D., M.D. t IN A PREVIOUS communication 5 from this laboratory, a study of the lipid content of the Leydig cell and Sertoli cell in the human testis as related to age, benign prostatic hyperplasia, and prostatic cancer in a series of 168 cases was presented. Our particular interest at that time was the determination of a possible variation in the lipid pattern in individuals with benign prostatic hyperplasia, individuals with prostatic cancer, and in individuals who clinically or at autopsy had neither. Accepting at that time the hypotheses that the Leydig cell produces androgen,6 the Sertoli cell estrogen, 5 6 and that the lipid contained in these cells might represent these steroids, 6 it was felt that evidence for or against the androgen theory of the production of prostatic cancer and the estrogen theory in the origin of benign prostatic hyperplasia might be obtained. We were unable to demonstrate, however, any significant variation in the three groups subjected to study. This work was done wholly under a Grant-in-Aid from the American Cancer Society upon the recommendation of the Committee on Growth of the National Research Council. From the Department of Urology, Medical College of the State of South Carolina, Charleston, South Carolina; formerly in the James Buchanan Brady Urological Institute, Johns Hopkins Hospital, Baltimore, Maryland. f From the James Buchanan Brady Urological Institute, Johns Hopkins Hospital. Read before the seventh annual meeting of the American Society for the Study of Sterility, June 9, 1951, Atlantic City, N. J. 35

2 36 LYNCH & SCOTT [Fertility & Sterility Conclusive evidence was afforded, nevertheless, that there is a definite pattern of Leydig cell lipid and Sertoli cell lipid in relation to age. Although not commented on in our previous reports, this pattern seems to be closely related to the presence and degree of spermatogenesis. In order to obtain experimental data which might assist in the interpretation of these findings, groups of young adult 200-Gm. albino rats were subjected to various manipulations of the pituitary-gonad system. 7 It is our purpose to present in this report an analysis of these two groups of studies with particular reference to spermatogenesis. METHODS Testicular tissue was obtained from 197 human males ranging in age from 2 months premature to 84 years. Most of the specimens were obtained from cases of accidental death; those from patients having benign prostatic hyperplasia and prostatic cancer were obtained by biopsy during the preoperative work-up. The animal tissue was obtained from young adult 200-Gm. albino rats of the Sprague-Dawley and Wistar strains. These animals were subjected to various manipulations, hormonal and otherwise, the technic and details of which we have described elsewhere. 7 The animal and human testis tissue was fixed in formalin, then infiltrated and embedded in gelatin. The gelatin blocks were fixed in formalin and frozen sections cut from these blocks were stained with Oil-Red-O using acid hematoxylin counterstain. This variation of preparing testis tissue for frozen section has been outlined in a previous communication. 5 RESULTS Human Studies Figure 1, A, B, and C shows the estimated Leydig cell lipid and Sertoli cell lipid plotted against age in the postpubertal group. The pattern in the prepubertal group is elsewhere described and discussed and is not related to spermatogenesis.5 Each point on the curves represents one specimen; each circle represents the average of the points in each five-year period. These graphs were prepared from our first 168 cases. In the complete series, Leydig cell lipid appeared at 12 years as shown in Fig. 2, A and Sertoli cell lipid appeared at 15 years as shown in Fig. 2, B. Spermatogenesis was not present until lipid was demonstrable in the Sertoli cells. Spermato-

3 Vol. 3, No. 1, 1952] THE SERTOLI CELL 37 genesis was maximum during the ages from 17 to 35 years during which period the Leydig cell lipid was estimated as 4 plus and the Sertoli cell LEYDIG CELL LIPID ++++ '? Y.\. o, I.... /.o..._/... o,.'b-\' FIGURE AG IN YEARS Estimated Leydig cell lipid and Sertoli cell lipid plotted against age in postpubertal group. A. (From J. Urol. 64: , 1950.) SERTOLI CELL LIPID Ht +t +..-.o-...,... oy.haii I.~/. r...-(. ;: " AGE fn YEARS FIGURE 1 (Continued). Estimated Leydig cell lipid and Sertoli cell lipid plotted against age in post-pubertal group. B. (From J. Urol. 64: , 1950.) lipid as 1 to 2 plus, as illustrated in Fig. 3, A. In the higher age groups, the depth of the germinal epithelium gradually diminished although spermatogenesis was still present in 75 per cent of the 71- to 84-year-old

4 38 LYNCH & SCOTT [Fertility & Sterility age group, as exemplified in Fig. 3, B. With increasing age and decreasing spermatogenesis, there was a decline in the amount of Leydig cell lipid and an increase in the amount of Sertoli cell lipid so that in old age there was a 4:1 ratio in favor of the Sertoli cell lipid whereas in the 20-year-old age group this ratio was reversed. In the series of 197 cases there were 34 specimens in which spermatogenesis was absent. In 17 of these, either thickening of the tubular tunica.. z w.. z.... <.. e ~ w ::1 ++ w + /cr SERTOLI CELLS t- 10 ~» ~ " ~ ~ ~ AGE IN YEARS FIGURE 1 (Continued). Estimated Leydig cell lipid and Sertoli cell lipid plotted against age in post-pubertal group. C. (From J. Urol. 64: , 1950.) propria or interstitial fibrosis was present, indicating abnormal testes. In 3 cases the degeneration was due to prolonged. administration of estrogen given for prostatic canc~r (Fig. 4), in another there was severe cirrhosis of the liver with presumable failure of hepatic inactivation of circulating estrogen, and another was a cryptorchid testis. Since 17 -ketosteroid and pituitary gonadotropin determinations were not made, the cause of the testicular degeneration in the remaining 29 cases is not known. It is felt that they probably fall into the various groups recently classified by Howard and his associates. 4 Animal Studies NoRMAL TESTIS. There was a consistent and characteristic lipid pattern in the unmanipulated testis of the adult albino rat. There were various stages of maturation of sperm in the seminiferous tubules. Tubules

5 Vol. 3, No. 1, 1952] THE SERTOLl CELL 39 2A 2B 3A FIGURE 2. (From J. Ural. 64: , 1950.) A, Specimen 125, aged 12 years, showing lipid in the Leydig cells only and no spermatogenesis (X 135). B, Specimen 168, aged 15 years, showing 3 plus Leydig cell lipid, very small amount of Sertoli cell lipid, and active spermatogenesis (X 270). FIGURE 3. (From J. Ural. 64:773, 1950.) A, Specimen 142, aged 17 years, showing 4 plus Leydig cell lipid, one plus Sertoli cell lipid, and active spermatogenesis (X 70). B, Specimen 20, aged 76 years, showing 4 plus Sertoli cell lipid, absent Leydig cell lipid, and minimal spermatogenesis (X 70). 3B

6 40 LYNCH & SCOTT [Fertility & Sterility in the "resting phase" with no spermatozoa present contained moderatesized droplets of lipid at the basement membrane, presumably in the Sertoli cells. As the sperm matured, these droplets became smaller and seemed to migrate toward the tubular lumen, finally occupying a position adjacent to the sperm heads where they formed a halo or rosette. These droplets disappeared when the sperm were released into the lumen of the tubule and then reappeared at the basement membrane as the cycle continued. These phases are represented in adjacent tubules in Fig. 5, A. FIGURE 4. Specimen 1, 59-year-old patient with prostatic cancer and on estrogen therapy for 9 months. There is thickening of the tunica propria and marked accumulation of lipid in the Sertoli cells which are the only cells remaining in the tubules. EFFECT OF HYPOPHYSECTOMY. Hypophysectomy resulted in degeneration of the germinal epithelium, complete absence of spermatogenesis, and accumulation of large drops of lipid in the Sertoli cells which, in many of the tubules, were the only cells remaining, as seen in Fig. 5, B. Spermatogenesis and the normal lipid pattern could be maintained by injections of pituitary gonadotropins." EFFECT OF EsTROGEN. Injections of estradiol benzoatet resulted in varying degrees of impairment of spermatogenesis and the normal lipid pattern, the 0 Provided by Dr. W. E. Hambourger of G. D. Searle and Company in the form of desiccated gonadotropic factor of sheep anterior pituitary gland labeled Gonadophysin. t Estradiole benzoate and testosterone propionate were provided by Dr. Edward Henderson of The Schering Corporation.

7 Vol. 3, No. 1, 1952] THE SERTOLI CELL 41 most marked being similar to the changes following hypophysectomy, and the least marked showing merely an increase in the numbers of tubules in the "resting" or aspermatogenic phase. EFFECT OF ANDROGEN. The testes of animals injected with testosterone propionate"' showed tubular changes similar to the minimal changes following estrogen injection, but, in addition, there was atrophy of the Leydig SA 58 FIGURE 5. A, Testis of control young adult 200-Gm. albino rat showing, in adjacent tubules, the "resting," intermediate, and mature phases of spermatogenesis. B, Testis of hypophysectomized littermate showing degeneration of the germinal cells and large drops of lipid in the Sertoli cells. cells with an accumulation of lipid in an occasional interstitial cell nest. At no time was lipid demonstrated in the normally active Leydig cell of the albino rat, although it was present in copious quantities in a number of wild rats examined. EFFECT OF INANITION. Subjection of the animals to a loss of 50 per cent body weight resulted in impairment of spermatogenesis and accumulation of lipid in the Sertoli cells. Estradiole benzoate and testosterone propionate were provided by Dr. Edward Henderson of The &!hering Corporation.

8 42 LYNCH & SCOTT [Fertility & Sterility EFFECT OF CRYPTOHCHIDISM. Animals rendered artificially cryptorchid showed degeneration of the germinal cells and accumulation of lipid in the Sertoli cells. EFFECT OF VAs DEFERENS LIGATION. In the pilot experiment the vasa were ligated transabdominally and on autopsy the testes showed the same changes as did the cryptorchid testes. It occurred to us that we might be inadvertently causing cryptorchidism by our surgical approach, for which reason a second group of animals was subjected to vas ligation through scrotal incisions. The testes of these animals were entirely normal on examination one month subsequently. We conclude that degeneration of the germinal cells following vas ligation as reported in a standard text on endocrinology may have its basis in accidental cryptorchidism. DISCUSSION From these studies of the human and animal testis we have deduced the following information regarding the lipid produced in the Sertoli cell. The lipid does not appear until puberty and then only after the appearance of lipid in the Leydig cells. This supports the belief that both androgen from the Leydig cells and follicle stimulating hormone from the pituitary are required for proper function of the seminiferous tubules. Mature spermatogenesis appears simultaneously with demonstrable Sertoli cell lipid indicating that the lipid is necessary for the production of spermatozoa. The lipid is released into the lumina of the tubules with the mature sperm, but retained within the Sertoli cells in the absence of spermatogenesis. This is compatible with the hypothesis that the Sertoli cells have a nutritive function and suggests that the lipid may well be the nutritive material. These studies indicate, in addition, that the Sertoli cell lipid fails to be utilized and accumulates in the cell, probably because it is not utilized, when spermatogenesis is impaired or absent; i.e., following hypophysectomy, estrogen therapy, androgen therapy, and in inanition and cryptorchidism. We have been able to learn little about the nature of the Sertoli cell lipid; extraction and biologic assay of the lipid in this laboratory has not demonstrated estrogenic activity. Histochemical studies reported by Perlman have shown that it consists largely of cholesterol and cholesterol esters. We do not feel, however, that these findings contradict the hypothesis that the tubules produce a "hormone X" which inhibits the pituitary-as sug-

9 Vol. 3, No. 1, 1952] THE SERTOll CEll 43 gested by Howard and his associates 3 -or the established fact that the estrogen has been isolated from the animal testis by Courrier and by Beall. REFERENCES 1. Beall, D.: Biochem. J. 34:1293, ComTier, R.: Compt. rend. Soc. de Bioi. 117:1117, Howard, R. P., Sniffen, R. C., Simmons, F. A., and Albright, F.: J. Clin. Endocrinol. 10:121, Howard, R. P., Simmons, F. A., and Sniffen, R. C.: Fertil. & Steril. 2:95, Lynch, K. M., Jr., and Scott, W. W.: J. Ural. 64:767, Lynch, K. M., Jr., and Scott, W. W.: In Surgical Forum, American College of Surgeons. Philadelphia, Saunders, 1951, pp Lynch, K. M., Jr., and Scott, W. W.: Submitted to Endocrinology. 8. Scott, W. W., and Lynch, K. M., Jr.: In Proceedings of Conference on Egg and Ovum, Sperm and Testis. (In press.) 9. Perlman, P. L.: Endocrinology 46:347, DISCUSSION l\f. L. BRODNY, Boston, Massachusetts: The identification and localization of the intratesticular hormonal mechanisms and their correlation with endocrine dvsfunctions have long been a challenge to medicine. The Leydig cell was discove;ed in 1850 and the Sertoli cell in 1865, but their biochemical composition and the nature of their physiologic processes remained for decades a scientific enigma. Dr. Lynch and Dr. Scott are to be congratulated for having ingeniously unraveled many of the mysteries concerning the biochemistry of these cells, and it is their basic research which may furnish the answers to many pertinent questions in testicular physiology. Their previous reports dealt with the lipid content of these cells in prostatic hyperplasia, prostatic carcinoma, and testicular neoplasms. Today's report correlates the function of the Sertoli cell and spermatogenesis and is of special interest to this Society. The Fertility Clinic of which I am a member is in the Beth Israel Hospital, Boston, and there at the Yamins Laboratory for Surgical Research, investigations are also being carried on in this field. In 1949, a method for demonstrating the carbonyl group in the lipids of formalin fixed tissues was developed."' The technic was applied to organs which were known to contain ketosteroids such as the adrenal glands and corpus luteum of the dog and the placenta and testes of the rat. Supplementary studies have shown the carbonyl group to be ketonic in character rather than aldehydic. The authors stated, 'Whenever lipoidal ketones are demonstrated in tissue known to contain ketosteroid, it is presumed although not conclusively proved that ketosteroids are responsible for the reaction." Variations of the hormonal patterns were noted in different species and also seasonally. Lipid and ketosteroids which were absent in the Sertoli cells of the rat's testes were present in abundance in those of the pig's testes. The Sertoli cells Ash bel, R., and Seligman, A. M.: A New Reagent for the Histochemical Demonstration of Active Carbonyl Groups. A New Method for Staining Ketonic Steroids.

10 44 LYNCH & SCOTT [Fertility & Sterility of the testes of the cryptorchid dog contained an abundance of ketosteroid as contrasted with the Sertoli cell of the testes of normal dogs. This observation merits investigation in the human. Drs. Ashbel, Cohen, and Seligman have now in print the results of studies which localize and identify the ketosteroids in normal and neoplastic testes in human and various species." Estrogenic steroids are alkali soluble and androgenic steroids are not. Ashbel, Cohen, and Seligman found that alkaline extraction did not effect the ketosteroid reaction in three interstitial cell tumors but completely blocked it in three Sertoli cell tumors indicating that the ketosteroid in interstitial cells is for the most part alkali insoluble androgens and in Sertoli cells alkali soluble estrones. Drs. Lynch and Scott's presentation today will open new fields in the investigation and understanding of male fertility. DR. R. PALMER HowARD, Oklahoma City, Oklahoma: The authors are to be congratulated not only for their detailed examinations of the human testis, but also for their fundamental comparative studies on the changes in the rat testis under controlled experimental conditions. I would like to illustrate a few points in my discussion by the use of slides. Huggins and Russell (Endocrinology 39:1, 1946) did not note an accumulation of lipid in the Sertoli cells of testes in hypophysectomized dogs. However, the lipid accumulation which Dr. Lynch demonstrated in the testes of hypophysectomized rats does occur in adult humans suffering from severe hypopituitarism. The first slide shows a testes of a 64-year-old male, who had severe hypopihlitarism of two-years' duration with radiologic evidence of erosion of the sella turcica, very low urinary 17-ketosteroids, and urinary gonadotropins (FSH) of less than 3m. u. per day. The tubular degeneration and the accumulation of large lipid droplets in the Sertoli cells are comparable to the changes demonstrated in the hypophysectomized rat by the authors. The slide I have shown you was prepared from a specimen stained by routine hematoxylin and eosin stain, but we have used fat stains on frozen sections to demonstrate that these droplets are truly sudanophilic. This second slide is from a higher magnification of the same specimen (Fig. 1). You will note the marked thickening of the tunica propria, almost complete absence of spermatogenesis, considerable lipid in the center of the tubules, and the absence of Leydig cells in the interstitial spaces. In similar cases we have never seen a normal number or a normal appearance of the Leydig cells. In exceptional instances there are rare atrophic cells, which probably represent degenerating Leydig cells. These changes in the interstitial cells, which occurred in all patients with evidence of organic lesions affecting the pituitary gland, appeared to us to be a more sensitive indication of interference with pituitary function than changes in the tubules. It is our presumption that lesions similar to those illustrated in the last two slides indicate absence both of the follicle stimulating hormone ( FSH) and the luteinizing hormone ( LH, ICSH). Patients suffering from more recent or incomplete destructive lesions of the Ashbel, R., Cohen, R. B., and Seligman, A. M.: Histochemical Demonstration of Ketosteroids in 1\ormal and Neoplastic Testes.

11 Vol. 3, No. 1, 1952] ihe SERTOLI CELL 45 pituitary may show less evidence of damage to the testes. Indeed patients with acromegaly in its early stages frequently have normal testes. \Ve have seen a few patients with hypopituitarism due to organic lesions, who had impotence, FIGURE 1. Section of a tubule in the testis showing advanced changes due to injury of the pituitary. These changes consist of an accumulation of sudanophilic material in the Sertoli cytoplasm (A), thickening of the tunica propria (B), and suppression of spermatogenesis. The Leydig cells have disappeared. A multinucleated spermatogonium is shown at C. Hematoxylin and eosin stain, X 500. (Case 26 of Howard, Sniffen, Simmons, and Albright: J. Clin. Endocrinol. 10:121, 1950.) FIGURE 2. An area in the testis of a patient with injury to the hypophysis. Spermatogenesis is active, but the Leydig cells have disappeared. Hematoxylin and eosin stain, X 120. (Case 32 in J. Clin. Endocrinol. 10:121, Photoprints for Figs. 1 and 2 were prepared by Dr. R. C. Sniffen.) low urinary 17-ketosteroids (approximately 3 mg. per day, or less), but normal values for the excretion of gonadotropins ( FSH). The following slide illustrates the testis of one of these patients (Fig. 2). It will be noted that spermatogenesis 2

12 46 LYNCH & SCOTT [Fertility & Sterility is essentially normal in the majority of the tubules but the interstitial spaces show an absence of Leydig cells. It is felt that this indicates a persistence of FSH but a virtual absence of LH ( ICSH). Some support for this concept appears to come from the recent paper of Simpson, Li, and Evans (Endocrinology 48:370, 1951). Those authors showed that low doses of an electrophoretically homogeneous preparation of sheep's FSH injected into hypophysectomized rats resulted in the maturation of tubules up to a state of spermatogenesis without the appearance of recognizable Leydig cells. In Fig. 4 of the present paper the speakers demonstrated the effect of prolonged courses of estrogen therapy on the human testis. This effect is probably the result of suppression of the hypophyseal secretion of both FSH and LH, since there is absence of the Leydig cells in association with degenerative changes in the tubules. As a practical question to the speakers and to the audience I would like to inquire whether anyone has observed recovery of the human testis after a period of estrogen therapy, during which severe testicular damage was demonstrated? Concerning the significance of lipid accumulation in the Sertoli cells in various types of testicular deficiencies, the speakers' suggestion that this represents an unutilized accumulation of nutritional substrates seems preferable to ours. The previous suggestion of our group that the lipid represents unreleased "X" hormone appears less likely. Problems as to whether the Sertoli cells form a second testicular hormone, and as to the identity of the testicular cells responsible for the production of estrogen are not answered by the work presented this afternoon. However, Maddock and Nelson gave evidence at the Thirty-third Meeting of the Endocrine Society that the Leydig cells are the source of estrogen elaborated by the testis. Dr. Brodny has just mentioned the Sertoli cell tumor of the dog in which estrogen is apparently formed. However, that is a special condition and the evidence of Maddock and Nelson in the human is not directly comparable to it. DR. WARREN NELSON, Iowa City, Iowa: As usual, the papers that come from Dr. Scott and Dr. Lynch's laboratories have a very fine story to bring us and this paper is no exception. I would like to speak about the lipid picture in the fetal testis. It carries the story back a few months from the point where Dr. Lynch began it. By the fourteenth week one can see lipid in the interstitial cells of the fetal testis and this lipid continues to be present throughout the remainder of fetal life. A very extensive Leydig cell development characterizes the human fetal testis and disappears within a month or six weeks, as Dr. Lynch has said, to appear again only when the boy begins to enter puberty. We can reiterate that the lipid that is seen in the Sertoli cells of those species, which have been studied, including man, appears to be inversely proportional to the activity of the germinal epithelium. Thus a picture such as he has described is seen not only after hypophysectomy, estrogen treatment and cryptorchidism, but also in X-irradiated testes, testes of animals receiving androgen in which spermatogenesis is interfered with and in the testes of

13 Vol. 3, No. 1, 1952] THE SERTOLI CELL 47 animals receiving nitrofuranes. We have seen it as well in the testes of men who had suffered severe febrile episodes that caused spermatogenesis to cease. For example, after pneumonia there is a cessation of spermatogenesis that continues for some time. In such conditions Sertoli cell lipid appears to accumulate and to be related to its lack of utilization. We have been interested in the site of estrogen production in the testis and have studied the testes of stallions since, surprising to say, stallions produce tremendous amounts of estrogenic hormone. Large quantities can be obtained not only from the urine of these animals but also by extraction from the testes. Cytochemical studies of horse testes have shown much the same picture that has been seen in other animals: lipid in the Sertoli cells which appears to be related to spermatogenesis, and lipid in the Leydig cells. We are not satisfied with any of the cytochemical methods, including the Ashbel and Seligman method, as certifying with any reasonable certainty the presence of steroid hormones. DR. CHARLES W. CHARNY, Philadelphia, Pennsylvania: I would like to answer Dr. Howard's question about the permanency of estrogen effect on spermatogenesis. I have had 3 cases, 1 of which was reported a few years ago with Dr. Charles Dunn. This was the case of a man who had been arrested on four occasions, charged with criminal rape. He was given 5 mg. of estrogen daily for about :30 days. At the end of that time semen examination showed no spermatozoa and testicular biopsy revealed complete degeneration of the seminiferous tubules with only Sertoli cells remaining. Another biopsy done about six or eight weeks later revealed complete regeneration of the tubules. The 2 other instances were cases of infertility which I thought might be caused by hyperandrogenism. One patient received 2 mg. and the other 3 mg. daily. In both instances there was a disappearance of spermatozoa from the semen and treatment was promptly stopped. However, reexamination of the semen several weeks later showed a return of spermatozoa not only to the original level but an increase which was definitely above the original starting point. I mention this to suggest the possibility that the rebound phenomenon reported by Drs. Heckel and McDonald might possibly be observed after the administration of substances other than androgens. DR. LYNCH, closing: I am very sorry that Dr. Scott is not here to discuss this, but he is in England as Exchange Professor of Urology at Guy's Hospital in London. There are several observations that I should like to make. We are certainly not pioneers in this study, as Dr. Brodny was kind enough to indicate. Many others have preceded us in the study of testicular lipids. Dr. Perlman has done some excellent work, and Dr. Ashbel and Dr. Seligman. In regard to their stain, we have a great deal of difficulty in controlling the ph. After we add the absolute alcohol, the ph is unstable and we can keep it between 7 and 7.2 for only about half an hour; that is the reason we did not routinely run the Seligman stain on all our sections. I think that the Seligman stain is specific for the carbonyl group. This has been demonstrated chemically. But it is not specific

14 48 LYNCH & SCOTT (Fertility & Sterility for 17-ketosteroids because one gets a dense blue stain in the cerebellum. We always run a piece of cerebellum as a control to demonstrate that our stain has worked. We have had a lot of difficulty in interpreting the blue color with the Ashbel Seligman stain. We often get the purple color that was demonstrated in some of the slides that Dr. Brodny showed. I don't know whether it is a positive reaction or a negative one. So far we have been accepting it as positive only if it is bright blue. Dr. Harlan Firminger has done some beautiful studies with ultraviolet spectramicroscopy and has demonstrated that the lipid droplets in the interstitial cells of the mouse are testosterone. He, by the way, did these lipase stains for us, which I should like to acknowledge. I made very little comment on the interstitial cell lipid in the rat because there is no lipid in the interstitial cells in the albino rat unless the pituitary is inhibited. I did not mention this, but the lipid does appear in the interstitial cells in androgen-treated rats. Apparently the lipid is normally rapidly liberated or utilized, just as rapidly as it is formed, and it accumulates only when the pituitary is suppressed. On the other hand, the wild rat interstitial cells are loaded with lipid. ' It has been well known for a long time that one can differentially extract estrogen and androgen by separating the phenolic and nonphenolic fractions. The test that Dr. Brodny has described seems to do this histochemically. In regard to Drs. Huggins' and Moulder's cryptorchid dog that had no lipid in its Sertoli cells, I wonder if the dog had not been cryptorchid for a long time and had complete atrophy, even of the Sertoli cells. We have examined the testes from a patient who died of cirrhosis of the liver. There was complete fibrosis of the interstitial areas and hyalinization of the tubules with lipid being demonstrated in an occasional lumen only. Dr. Howard brings up an important question: whether the testes of animals and humans will recover after atrophy of the testes caused by estrogen and androgen. We are planning to initiate studies to determine whether recovery occurs after estrogen therapy and whether it is complete. Dr. Nelson, I think, has very effectively demonstrated already that there is complete recovery and actually a rebound after androgen therapy. Dr. Nelson's comments on the fetal testis are certainly very interesting. We studied two premature infants and they showed no lipid in the testis. Following birth, there was lipid in the interstitial cells. There may be suppression of the fetal pituitary (this is pure speculation) by circulating estrogens from the mother which are able to cross placental membranes. Gonadotropins have been demonstrated not to cross the placental membranes.