IF SEXUALLY MATURE male rats are subjected to experimental cryptorchidism,

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1 Cryptorchidism Its Pre- and Postpubertal Effects on the Hypophysis of the Rat JAMES R. MOREHEAD, PH.D.,* and CHARLES F. MORGAN, Pn.D:r IF SEXUALLY MATURE male rats are subjected to experimental cryptorchidism, gonadotrophs of the anterior lobe of the hypophysis undergo characteristic alterations. Nelson 13,14 noted an increase in the number of basophilic cells in the anterior lobe of the hypophysis after a cryptorchid period of 75 days. Vacuolation of some of these cells was observed at 140 days. The morphologic changes in the gonadotrophs of cryptorchid animals appear identical to those observed by Addison following castration in rats. These particular cells are designated "castration cells" or "signet-ring gonadotrophs" due to the appearance of large vacuoles in the cytoplasm. During a prolonged cryptorchid state of up to 496 days in the rat, Moore 9 reported an increase in the weight of the hypophysis; however, he did not examine the organ histologically. Both Moore and Nelson attributed the morphologic and weight changes in the hypophysis to the reduced levels of androgen in the cryptorchid and castrate animals. Several other methods of investigation indicate that the cryptorchid testis is incapable of maintaining normal androgen production. Using weights of the male accessory reproductive organs, conflicting results have been reported. Over a period of 3 years, Nelson 15 observed a gradual decrease in organ weights beginning 240 days postoperation and continuing to a level comparable to that of castrate animals. Moore,9 using the same time span, found an initial loss of accessory reproductive organ weights followed by a return to normal levels. Clegg 2 reported a loss in weight of accessory reproductive organs within From the Department of Anatomy, University of Minnesota Medical School, Minneapolis, Minn. Supported by Grant GM IH. from the U. S. Health Service. A portion of this paper was submitted to the Graduate School of the University of Minnesota in a thesis in partial fulfillment of the requirements for the Ph.D. degree. "Present address: Department of Anatomy, Tufts University School of Medicine, Boston, Mass. tdeceased, Mar. 10,

2 VOL. 18, No.4, 1967 CRYPTORCHIDISM days after induction of cryptorchidism. Subsequently, a rise to normal was noted, followed by a decrease to 90% normal weight. Citric acid and fructose levels were observed to follow the same pattern of fluctuation as organ weights. Clegg3 also correlated quantitive changes in the numbers of interstitial cells of Leydig with the functional state of the accessory reproductive organs. Martins and Rocha-using parabiotic, cryptorchid male and normal female rats-observed long, 42-day estrous periods in the female. This experiment suggested an increased production of gonadotrophic hormones induced by the decreased production of androgen in the cryptorchid testis of the male partner. Morehead and Morgan, using autoradiographic technics, observed a reduction in the radioactive index of both the seminal vesicle and coagulating gland epithelia following a cryptorchid period of 28 days. In the cryptorchid rat testis, a reduction in enzymes involved in the synthesis of androgen has been reported by Kormano et al., and by Lluarado and Dominguez. It may be concluded that the mature cryptorchid rat testis is incapable of producing androgen in normal amounts. Also, it is apparent that in the sexually mature animal, the cryptorchid condition produces alterations within the anterior lobe of the hypophysis. In humans, the cryptorchid condition is frequently treated conservatively with the thought that the testis may descend at the time of puberty. The prime consideration in these cases is focused on the problem of sterility as a result of the cryptorchid condition extending into the period of sexual maturity. This approach, however, does not take into consideration the effect of cryptorchidism upon related organs such as the pituitary and accessory sex organs. Therefore, it seemed of interest to examine the pituitary in animals made experimentally cryptorchid before they reached sexual maturity. MATERIALS AND METHODS At the age of 4 weeks, male rats (Sprague-Dawley strain) were made experimentally cryptorchid as previously described by Morehead and Morgan. The animals were sacrificed following intervals of experimental cryptorchidism: Group I, 6 days; Group II, 2 weeks; Group III, 4 weeks; and Group IV, 26 weeks. Controls were animals of comparable ages with scrotal testes. Each group consisted of 5 animals. After weighing to the nearest milligram, the pituitaries were fixed in Zenker-formalin solution. The tissues, embedded in Paraplast, * were sec- *Fisher Scientific Co., Boston, Mass.

3 532 MOREHEAD & MORGAN FERTILITY & STERILITY tioned serially at 5 JL. The sections were stained with either PAS-hematoxylin or Mallory's azan itain. Using the classification for pituitary cell types of Purves and Griesbach,16.17 cell counts were made by counting 1000 gonadotrophs in each organ. The cell counts were expressed as the percentage of signet-ring cells in the total number of gonadotrophs. Pituitary weights were presented as the ratio of the organ weight, in milligrams, to 100 gm. of body weight. Pituitary Weights RESULTS As noted in Table 1 and Fig. 1, the values for the ratio of pituitary weight to body weight, lie on 2 lines that intersect at a point which coincides closely with the age of sexual maturity. As judged by the development of the accessory reproductive organs, sexual maturity was attained by these animals during the 2- to 4- week cryptorchid interval (6-8 weeks of age). It may be noted that before 6 weeks of age, the pituitary weights for the control animals are significantly greater than those of the cryptorchid animals; whereas, in the postpubertal stages, the cryptorchid animals have significantly heavier pituitaries than do the controls. Pituitary Cell Types In this investigation, no signet-ring cells were observed in the pituitaries of prepubertal animals. Following puberty, the cryptorchid animals had a larger number of signet-ring gonadotrophs than did the controls. As recorded in Table 2, Group III cryptorchid animals had 6 times as many signet-ring gonadotrophs as the controls. In the oldest age group examined, Group IV, one-half of the gonadotrophs present in the cryptorchid animals were signet-ring cells (Fig. 2); the controls, by contrast, had only 16% signet-ring cells (Fig. 3). TABLE 1. Relative Weight of Pituitary at Varying Time Intervals Mean pituitary wt. (mg./l00 gm. body wt.) Cryptorchid Group A ge when sacrificed intervals (wk.) Cryptorchid Controls p I 4 wk., 6 days 6 (days) 1.4 ± ±.5 < 0.01 II 6 wk ± ±.2 < 0.05 III 8 wk ± ±.3 < 0.02 IV 30 wk ± ±.1 < 0.001

4 VOL. 18, No.4, 1967 CRYPTORCHIDISM 533 DISCUSSION The primary clinical manifestation associated with cryptorchidism is sterility. The failure of the cryptorchid testis to produce normal spermatozoa has been attributed by Fukui,4.5 and later by Moore and his coworkers10 11 to the higher temperature of the body cavity as compared with the scrotal temperature. Few references in the literature, however, deal with the effects of this condition upon target organs such as the accessory reproductive organs in the male or the anterior lobe of the pituitary. Within the past 5 years, several new approaches have been used to study this problem. Using biochemical assays, Kormano et al., and Lluarado and Dominguez reported that the cryptorchid testis does not produce androgen in normal quantities. These studies have shown a reduction in the secretory products of the accessory organs in cryptorchid animals, as well as a reduction in enzymes directly associated with the production of androgen within the interstitial cells of the cryptorchid testis, itself. Morehead and Fig. 1. Influence of cryptorchid state on weight of hypophysis; 0, cryptorchid; X, controls. 0.4 o L--2~-4LI --~L-~\'~\--~26- CRYPTORCHID TIME (WK.l TABLE 2. Percentage of Signet-Ring Cells among Total Number of Gonadotrophs* Signet-ring cells Crypwrchid Group interval Crypwrchid animals (%) Controls (%) I 6 days 0 0 II 2 wk. 0 0 III 4 wk IV 26 wk * Mean values for each group of 5 animals.

5 534 :\10REHEAD & MORGAN FERTILITY & STERILITY Morgan, using autoradiographic technics, observed that the radioactive index of the seminal vesicle and coagulating gland was diminished in cryptorchid animals as compared with controls. Following castration, gonadotrophs in the anterior lobe of the pituitary Fig. 2. Section of anterior pituitary from Group IV cryptorchid animal. Signetring cells comprise approximately 50% of total number of gonadotrophs in this field. (Mallory azan stain, X 225) Fig. 3. Section of anterior pituitary from Group IV control animal. Gonadotrophs adjacent to sinusoids with only 1 signet-ring cell. (Mallory azan stain, X 225) had been observed by Addison to undergo vacuolation. These particular cells are designated "castration cells" or "signet-ring gonadotrophs" owing to their characteristic morphology. Although this cell type was associated, initially, with the loss of androgen due to castration, a similar morphologic alteration in the pituitary as the result of experimental cryptorchidism, was observed by Nelson U sing sexually mature rats, Nelson observed an increase in the number of gonadotrophs after a cryptorchid period of 75 days. These same cells

6 VOL. 18, No.4, 1967 CRYPTORCHIDISM 535 underwent vacuolation to form signet-ring gonadotrophs following an experimental cryptorchid period of 140 days in the mature rat. Under the condition used in this experiment-induction of experimental cryptorchidism at 4 weeks of age-signet-ring gonadotrophs became apparent in the anterior lobe of the hypophysis at the time of sexual maturity. Normal, basophil gonadotrophs were present in the pituitaries of both control and cryptorchid animals prior to puberty; however, vacuolation of these cells did not occur in cryptorchid animals until the time of puberty. Moreover, as the animals-still in the cryptorchid state-passed into adulthood, the number of signet-ring cells increased to 50% of the total number of gonadotrophs. The variation in experimental results between Nelson's studies and the present investigation may be due to the fact that the animals in the present experiment were rendered cryptorchid before puberty, while the animals in Nelson's study were sexually mature before induction of the cryptorchid state. The present investigation would seem to indicate that the gonadotrophs of the anterior lobe of the pituitary are more sensitive to levels of circulating androgen at the time of puberty than they are at a later stage in sexual maturation. As a correlate to the development of signet-ring cells, the hypophysis also displays an increase in both absolute and relative organ weight. While studying the effect of cryptorchidism on the accessory reproductive organs in the male rat, Moore9 noted "... a predominating tendency for increase in pituitary weight in the cryptorchid males... " However, he did not describe the histologic state of the organ. With regard to organ weight, the results of the present investigation agree with Moore's findings in the case of the postpubertal animals; however, before sexual maturity, the pituitaries of the cryptorchid groups are significantly lighter in weight than are those of the controls. Histologically, the anterior pituitaries of the prepubertal experimental and control groups appear identical. At the present time, then, there is no explanation for the phenomenon. The 2 observations in this investigation-( 1) appearance of signet-ring gonadotrophs and (2) increase in weight of the pituitary in sexually rna.. ture, cryptorchid rats-would implicate a reduction in the level of circulating androgen. These observations are in agreement with the results of the studies using biochemical assays. Whereas, Nelson did not observe alterations in the accessory reproductive organs and pituitaries until many months following the induction of experimental cryptorchidism in sexually mature rats, the reports based on biochemical studies show a reduction of androgen within several days following elevation of the testes into the

7 536 MOREHEAD & MORGAN FERTILITY & STERILITY abdominal cavity. The present investigation would be in close agreement with the biochemical studies in that the reduction in circulating androgen -as reflected by alterations in anterior pituitary cellular morphology and organ weight-becomes apparent "immediately" with the onset of puberty. Our results warrant the conclusion that, morphologically, the cryptorchid condition has no effect on the anterior lobe of the pituitary in prepubertal animals; however, at the time of sexual maturation, the gonadotrophs respond to the reduction in androgen by undergoing vacuolation with concomitant increases in weight of the entire hypophysis. SUMMARY Four-week-old male rats were made experimentally cryptorchid. Following intervals of 6 days, and 2, 4, and 26 weeks, the hypophyses were examined with respect to organ weight and histologic appearance. Before the animal reached puberty, the organ weights of the hypophyses were significantly higher in the control animals than in the cryptorchid groups. After puberty, the values were reversed; the hypophyses from cryptorchid animals were significantly heavier than those from normal controls. Signet-ring gonadotrophs were not present in prepubertal animals in either the control or cryptorchid groups; however, normal gonadotrophs were present in both of these groups. Following puberty, there were greater numbers of signet-ring gonadotrophs in the cryptorchid groups as compared with normal controls; the differences were statistically significant. Thus, it would seem that with respect to gonadotrophs, the cryptorchid condition has little effect upon the prepubertal hypophysis. In sexually mature animals, however, the anterior pituitary does respond to the cryptorchid condition. This response appears to be indicative of a reduction of androgen production by the cryptorchid testis. Department of Anatomy Tufts University School of Medicine Boston, Mass REFERENCES ADDISON, W. H. F. The cell-changes in the hypophysis of the albino rat after castration. ] Comp Neurol28:441, CLEGG, E. J. Some effects of artificial cryptorchidism on the accessory reproductive organs of the rat. ] Endocr 20:2lO, CLEGG, E. J. Further studies on artificial cryptorchidism: Quantitative changes in the interstitial cells of the rat testis. I Endocr 21:433, FUKUI, N. On a hitherto unknown action of heat rayon testicles. lap Med World 3:27, 1923.

8 VOL. 18, No.4, 1967 CRYPTORCHIDISM FUKUI, N. Action of body temperature on the testicle. Jap Med World 3:160, KORMANO, M., HARKONEN, M., and KONTINEN, E. Effect of experimental cryptorchidism on the histochemically demonstrable dehydrogenase of the rat testis. Endocrinology 74:44, LLUARADO, J. G., and DOMINGUEZ, O. V. Effect of cryptorchidism on testicular enzymes involved in androgen synthesis. Endocrinology 72:292, MARTINS, T., and ROCHA, A. The regulation of the hypophysis by the testicle, and some problems of sexual dynamics. Endocrinology 15:421, MOORE, C. R Hormone secretion by experimental cryptorchid testes. Yale J BioI Med 17:203, MOORE, C. R, and OSLUND, R. Experiments on the sheep testis-cryptorchidism, vasectomy, and scrotal insulation. Arner J Physiol 67:595, MOORE, C. R, and QUICK, W. J. The scrotum as a temperature regulator for the testes. Arner ] Physioi 68:70, MOREHEAD, J. R, and MORGAN, C. F. Hormone production by experimental cryptorchid rat testes as indicated by radioautographic studies of the seminal vesicles and coagulating glands. Fertil SteriI18:232, NELSON, W. O. A study of the anterior hypophysis and sex accessory glands of castrated and experimental cryptorchid rats. Anat Rec 58 (Supp!.) :30, NELSON, W. O. Effect of gonadotropic hormone injections upon hypophyses and sex accessories of experimental cryptorchid rats. Proc Soc Exp BioI Med 31:1192, NELSON, W. O. Some factors involved in the control of gametogenic and endocrine functions of the testis. Syrnpos Quant Bioi 5:123, PURVES, H. D., and GRIESBACH, W. E. The site of thyrotrophin and gonadotrophin production in the rat pituitary studied by McManus-Hotchkiss staining for glycoprotein. Endocrinology 49:244, PURVES, H. D., and GRIESBACH, W. E. The site of follicle stimulating and luteinizing hormone production in the rat pituitary. Endocrinology 55:785, Pacific Coast Fertility Society Meeting The Pacific Coast Fertility Society will hold its Fifteenth Annual Meeting Sept. 28-0ct. 1, 1967, at Mountain Shadows Resort and Country Club, Scottsdale, Ariz. A stimulating and outstanding program in the broad field of reproductive physiology will be offered. Dr. M. C. Shelesnyak, Weizmann Institute of Science, Rehovogh, Israel, will be one of the distinguished Guest Lecturers. Among other special presentations will be a panel, "Current Trends in Contraception," and a workshop, "Current Laboratory Tests Concerning Infertility." Additional features of the 1967 program will be announced later. The meeting is open to physicians, interns, residents, fellows, and nurses. For further information, please write to the office of the Pacific Coast Fertility Society, 5410 Wilshire Blvd., Los Angeles, Calif