The Effects of Selective Withdrawal of FSH or LH. on Spermatogenesis in the Immature Rat

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1 BIOLOGY OF REPRODUCTION 14, (1976) The Effects of Selective Withdrawal of FSH or LH on Spermatogenesis in the Immature Rat H. G. MADHWA RAJ and MARTIN DYM Departments of Obstetrics and Gynaecology and Physiology and Department of Anatomy, Laboratory of Human Reproduction and Reproductive Biology, Harvard Medical School, Boston, Massachusetts ABSTRACT Neutralizing antisera to rat follicle stimulating hormone (FSH) and sheep luteinizing hormone (LH) have been prepared and made monospecific by suitable absorption. The antiserum to rat FSH is devoid of contaminating antibodies to LII, binds only to 1 25 FSH, and neutralizes the biological activity of rat FSH in the HCG augmentation assay. The anti-sheep LH serum cross-reacts with rat LII and causes abortion when injected into 8-day pregnant rats. Administration of anti-lh serum to 20 day male rats, over a 14 day period, resulted in an 80% reduction in testes weight, accompanied by a marked reduction in the weights of the epididymides, ventral prostate and seminal vesicles. The size of the Leydig cells, the diameter of the seminiferous tubules, and the number of germ cells were all reduced. In this anti-lh group serum testosterone levels were 113 ± 6 pg/mi compared to control values of 616 ± 138 pg/mi. Treatment with anti-fsh serum decreased the weight of the testes by 50% without any reduction in the weights of the accessory organs or in the levels of serum testosterone (532 ± 75 pg/mi). The Leydig cells appeared normal; however, the tubule diameter and the number of spermatids and spermatocytes were also reduced when compared to the controls. Thus, it appears that selective withdrawal of FSH interferes with spermatogenesis in the immature rat, without alterations in the serum testosterone levels. INTRODUCTION Hypophysectomy and replacement therapy with follicle stimulating hormone (FSH) and/or luteinizing hormone (LH) have been extensively utilized to delineate the role of gonadotropins in spermatogenesis and androgen production (Steinberger and Steinberger, 1975). In recent years availability of purified gonadotropin preparations led to the production of neutralizing antisera to FSH and LH, and their use as immunological tools to selectively inhibit the biological activity of a single hormone without resorting to hypophysectomy (Moudgal, 1974; Eshkol and Lunenfeld, 1975). In the adult male rabbit and rat, administration of anti-ovine LH serum interrupted male reproductive function and eventually resulted in cryptorchidism and loss of libido (Hayashida, 1963; Talaat and Lawrence, 1971). However, active immunization with ovine FSH had no effect on these parameters, thus leaving the role of FSH in the male in an inconclusive state (Talaat and Lawrence, 1969). Furthermore, the effects of passive immunization with FSH antiserum have Accepted January 13, Received December 3, not been thoroughly investigated. Since the FSH antiserum used in the previous studies was raised against ovine FSH, the degree of cross reaction with the endogenous FSH of the rat would determine its neutralizing ability. Therefore, we decided to prepare antisera to rat FSH. In the present investigation we report the effects of selective withdrawal of either FSH or LH on spermatogenesis in the young rat using specific antisera. MATERIALS AND METHODS Antisera to purified ovine LH (2.7 X NIH-LH-S1) and rat FSH (NIAMDDFSHB1; 3.7 X NIHFSWS1) were produced by immunizing rabbits according to the method of Madhwa Raj and Moudgal (1970). The antiserum to rat FSH was absorbed with hypophysectomized rat serum and rat LH (1 Mg/mI). The antiserum to ovine LH was absorbed with normal lamb serum. The absence of any antibodies to LH in the anti-fsh serum was verified by the highly sensitive radiolabeled hormone binding technique. Completion of absorption and absence of antibodies to contaminating hormones in the anti-lh serum were verified using the agar-gel double diffusion test (Ouchterlony, 1958). The ability of the absorbed FSH antiserum to neutralize rat FSH activity was tested by the HCG augmentation assay as modified by Johnson and Naqui (1970) using 24 day old female rats. Fifty Hi of human chorionic gonadotropin (HCG) and various doses of rat FSH were mixed and injected subcutan- 489

2 490 MADHWA RAJ AND DYM TABLE labeled hormone binding study to demonstrate the specificity of the antiserum to rat FSH. % ne t bindinga using Antiserum (0.1 ml of! log dilution) 1125 rat LHb I 25 rat FSI1C Unabsorbed antiserum to rat FSH Absorbed antiserum to rat FSHd 0 26 NIAMD antiserum to rat LH aafter subtracting the non-specific binding of 2.6%. b,cniamd rat LH-I-1 ;NIAMD rat FSH-I-1. dabsorbed with hypophysectomized rat serum and rat LH. eously while the FSH antiserum was injected intraperitoneally. The animals were autopsied 72 h later and the ovaries were weighed. The capability of the anti-ovine LH serum to cross-react and neutralize rat LH was tested by its ability to terminate pregnancy on the 8th day in rats. Twenty day old male rats were divided into 3 groups and injected daily, by subcutaneous route, with antiserum to rat FSH (0.5 ml), antiserum to ovine LII (0.5 ml) or normal rabbit serum (0.5 ml) for 14 days. At autopsy on the 34th day blood was collected under light ether anesthesia. The testes, epididymides, ventral prostate and seminal vesicles were weighed. Several testes from each group were perfused with 5 percent glutaraldehyde according to a technique previously described by Vitale et al. (1973); the tissues were postfixed in 2 percent 0504, embedded in epon and examined with the light microscope. Serum testosterone levels were determined by radioimmunoassay as described by Bartke et al. (1973); however, Sephadex-LH-20 column chromatography was omitted since rat serum has been shown to have negligible amounts of dihydro-testosterone (Falvo and Nalbandov, 1974). RESULTS Characterization of the Antisera The presence of specific antibodies in the FSH antiserum was confirmed by the observation that it retained its ability to bind I 25 labeled rat FSH (Table 1). The unabsorbed FSH antiserum bound both 1125 FSH and, to a lesser extent, 1125 LII. Upon absorption with rat serum and LI-I the FSH antiserum completely lost its ability to bind 1125 LII; however, it retained most of its ability to specifically bind the 1125 rat FSH (Table 1). The reduction in binding with 1125 FSH observed after absorption with LH may be due to the loss of some antibodies to the a subunit, common to both FSH and LH. Table 2 summarizes the ability of the FSH antiserum to neutralize the biological activity of exogenously administered rat FSH in the HCG augmentation assay. It can be readily seen that 0.3 ml of the antiserum administered at a separate site is capable of inhibiting the ovarian weight increase caused by 40 jig rat FSH. This inhibition was due to a reduction in the number and size of the Graafian follicles. The absorbed anti-lh serum showed a single precipitin band against ovine LH in the agar-gel double diffusion test. This precipitin band partially cross-reacted with the rat pituitary TABLE 2. Inhibition of FSH induced ovarian weight gain by antiserum to rat FSH in the HCG augmentation assaya. Group (5 rats per group) HCG (IU) Rat FSH (Mg) NIAMD-FSH-Bl Antiserum (ml) Ovarian weight (mg) Mean ± S.E ± ± ± ± 59* ± 8.7 atwenty4our day Sprague-Dawley rats were used for the bioassay. P<0.01, compared with group 3.

3 RESPONSE TESTIS ANTIGONADOTROPIC SE RA 491 FSH or anti-lh sera on the weights of the testes and accessory organs. It can be seen that following treatment with anti-lh serum both the testes and the accessory organ weights have been drastically reduced. The reduction in epididymides, seminal vesicles and ventral prostate weights indicates an inhibition of testosterone production. In the anti-fsh treated group a 50 percent reduction in the testes weight was noted. However, the weights of the FIG. 1. Immunologic reactions of ovine LII antiserum in agar gel. Central well contains antiserum to sheep LH. Peripheral wells contain, clock-wise from top, sheep LH (Papkoff, 10 Mg) rat pituitary extract (1 pituitary equivalent), normal sheep serum, sheep FSH (NIH-FSI-I-S9, 20 Mg) sheep prolactin (NIH-P- SlO, 20 Mg) and normal sheep serum. extract line, indicating the ability of the ovine antiserum to combine with rat LH (Fig. 1). The absorbed LH antiserum did not show any bands against ovine FSH, prolactin, liver extract or serum. With this antiserum 0.3 ml is capable of terminating pregnancy when given on the 8th day in the rat, indicating its ability to neutralize the biological activity of endogenous LH of the rat. Effects of Antiserum on Male Rats Administration of anti-rat FSH or anti-ovine LII sera to groups of 20 day old male rats, over a period of 14 days, had no apparent ill effects on the general health of these animals. However, the anti-lh treated animals were cryptorchid with no scrotal development visible. The anti-fsh treated rats had a well-developed scrotum but the testes were smaller and less firm than the controls receiving normal rabbit serum. Table 3 demonstrates the effect of anti- epididymides, seminal vesicles and ventral prostate were similar to those of controls (Table 3) suggesting that serum testosterone levels were normal in the group treated with antiserum to FSH. In the 34 day old normal rabbit serum (NRS) treated group the Leydig cells were very well-developed, possessing large, spherical nuclei and abundant cytoplasm filled with a variety of organelles. Following anti-lh treatment for 14 days (Days 20-34) the size of the Leydig cells was dramatically reduced. The cytoplasm was frequently not evident in light microscopic examination and the nucleus appeared smaller and irregularly shaped, when compared to the controls. However, in the anti- FSH treated group the Leydig cells were perfectly normal. Analyses of serum testosterone values confirmed our morphological observations. Following treatment with anti-lh serum the serum testosterone levels were reduced by more than 80 percent, compared to the normal controls. However, serum testosterone levels remained normal in the anti-fsh treated group (Table 4). The most advanced germ cells in the normal 34 day old rats (NRS group) were round spermatids at stage VIII of the cycle of the seminiferous epithelium. The seminiferous tubules possessed large, well developed lumina and were 233 ± 4.6 micra in diameter. The tubules of the anti-lh group were much smaller in TABLE 3. Effect of antigonadotro pie sera on organ weights. Organ weights (mg) mean ± S.E. Treatment Body weight (g) Mean ± S.E. Testes Epididymides Seminal vesicles Ventral prostate Normal rabbit serum (12 rats) Antiserum to LI-I (10 rats) Antiserum to FSH(9 rats) 132 ± ± ± ± ± 12* 563 ± 21* 156 ± 5 42 ± 4* 161 ± 4** 75 ± 7 19 ± 2 81 ± ± 4 12 ± 1 76 ± 5*S *P<0.01, compared to NRS group. s snot significant, compared to NRS group (P>O.2).

4 492 MADHWA RAJ AND DYM TABLE 4. Serum testosterone levels after treatment with antigonadotropic sera. Testosterone (pg/mi) Treatmenta Mean ± SE. Normal rabbit serum 616 ± 138 Antiserum to LH 113 ± 6 Antiserum to FSH 532 ± 755* a5 rats per group. P<0.01, compared to NRS group. 5Not significant, compared to NRS group (P>0.2). diameter (139.5 ± 3.5 micra) and the lumina were either very small or absent. Only rarely were spermatids present. The number of pachytene spermatocytes (corrected per 100 Sertoli cell nucleoli) was reduced by 50 percent, compared to the control values, although spermatogonia and their proliferation appeared normal. Many degenerating spermatocytes were present. The size of the individual germ cells was similar to that observed in the NRS group. However, the height of the Sertoli cells was significantly reduced in the anti-li-i group and numerous lipid droplets accumulated in the basal cytoplasm. Following 14 days of anti-fsh treatment the tubule diameter was about 20 percent smaller than control values (196.8 ± 3 micra). The tubula lumen appeared well developed, although somewhat smaller than controls. The number of pachytene spermatocytes was reduced to 65 percent of control values and the number of spermatids was reduced to 33 percent of controls. Many of the spermatids that were present appeared to be in various phases of degeneration. Binucleate and other abnormal forms were observed. Another interesting feature was that a small number of the spermatids appeared to develop more rapidly and therefore some elongated forms were also apparent. A more detailed quantitative and ultrastructural examination will be presented in a subsequent paper (Dym and Mad hwa Raj, in preparation). DISCUSSION Pharmacological (estrogen or testosterone) or surgical blockade of pituitary function, coupled with replacement therapy, have been used in the past to study the role of gonadotropins in male reproductive function (Steinberger and Steinberger, 1975; Chemes et al., 1976). Chronic hypophysectomy results in a very sick animal since all the metabolic processes are seriously altered. The weight loss alone of hypophysectomized adult rats is more than 33 percent within two weeks. Estrogens may have a direct effect on the seminiferous tubules. The use of monospecific antisera permits an evaluation of the effects of withdrawal of a single hormone without affecting the other metabolic processes. The absorbed antiserum to rat FSH retained its ability to specifically bind only radiolabeled FSH, as well as to neutralize the biological activity of exogenous FSH in vivo. Therefore, the use of such an antiserum to neutralize endogenous FSH in the male rat appears justified. The ability of the ovine LH antiserum to crossreact with rat LII and neutralize its biological activity is well known (Schwartz, 1974). The observed reduction in serum testosterone levels and accessory organ weights in the anti-lh treated rats correlated very well with the morphological appearance of the Leydig cells. The small diameter of the seminiferous tubules, the lack of a tubule lumen and the inhibition of spermatogenesis appear to mimic the effects of hypophysectomy. Indeed, the presence of a residual spermatogenesis that we find after anti-lh treatment in the immature rat has been previously reported following hypophysectomy in adult animals (Clermont and Morgentaler, 1955). A significant feature of our results is that we have demonstrated, for the first time, that selective withdrawal of FSH is deleterious for normal spermatogenesis in the intact immature rat. That the effects of the anti-fsh serum on the seminiferous tubules are specific and not due to contaminating LH antibodies are borne out by the following three observations: 1) Leydig cell morphology is similar to controls; 2) serum testosterone levels are not altered; 3) accessory organ weights are not reduced. Thus, the effects on the seminiferous epithelium following treatment with anti-fsh serum must be attributed to specific neutralization of endogenous FSH. The role of FSH in spermatogenesis remains an enigma despite the earlier work of Greep et al. (1936) which suggested that FSH regulates germ cell maturation. Numerous more recent reports have appeared demonstrating the important actions of FSH on

5 RESPONSE TESTIS ANTIGONADOTROPIC SERA 493 the seminiferous tubules at the biochemical level (Means, 1975; Steinberger et al., 1975; Dorrington and Fritz, 1974). However, it has proved difficult to assign a definitive requirement for this hormone in normal spermatogenesis in the immature rat. It is well established that when FSH is added to seminiferous tubules obtained from immature animals the following events occur: 1) a binding of FSH to receptors on the plasma membrane of Sertoli cells; 2) an increase in cyclic AMP levels and 3) the activation of cyclic AMP dependent protein kinases and an eventual increase in general protein synthesis. It has also been demonstrated that androgen binding protein (ABP) is produced by the Sertoli cells, possibly under dual control of FSH and testosterone (Hansson et a!., 1974; Means et al., 1976). All of these reports indicate that the Sertoli cell is the primary target for FSH in the testis. Although our results do demonstrate that FSH is required for spermatogenesis in the young rat it should be noted that following selective withdrawal of LH (and testosterone) the effects on the seminiferous tubules and on the Sertoli cells are more dramatic than when FSH is withdrawn. In normal rats the Sertoli cell is a tall columnar cell which extends from the base of the seminiferous epithelium to the tubule lumen (Dym and Fawcett, 1970). The height of the Sertoli cell was much smaller after treatment with anti-lh serum compared to anti-fsh treatment. Germ cell numbers, tubule diameter and tubule lumen diameter were all lower in the anti-lh treated group. In the immature testis a tubule lumen develops concomitantly with the onset of fluid production by the Sertoli cells (Vitale et al., 1973). This lumen serves to channel the fluid into the rete testis. The presence of a tubule lumen may be used as an indicator of active fluid secretion. After treatment with anti-lh serum the luminal diameter is markedly reduced or absent, when compared to controls, suggesting that the important function of fluid production by the Sertoli cell has been turned off. After anti-fsh treatment the lumina of the seminiferous tubules are somewhat smaller than controls, implying that the Sertoli cells are still producing fluid but at a reduced rate. Preliminary results carried out in collaboration with Des. Nicholas Kotite and Frank French indicate that androgen binding protein levels decrease in both the testes and epididymides following anti-fsh or anti-lh treatment, although the effects were more dramatic following LH withdrawal. This confirms that both FSH and testosterone (produced under the influence of LH) are involved in the control of the production of this protein. In view of the various above mentioned effects of FSH antiserum on the testes we conclude that FSH is essential for the normal spermatogenesis in the immature rat. ACKNOWLEDGMENTS Supported in part by grant HD from the National Institute of Child Health and Human Development, and by The Bing Fund. The authors thank NIAMDD and Dr. A. F. Parlow for the gifts of sheep and rat FSH, LH and prolactin preparations, Dr. H. Papkoff for the purified ovine LH, and Dr. B. V. Caldwell for the testosterone antiserum. REFERENCES Bartke, A., Steele, R. E., Musto, N. and Caldwell, B. V. (1973). Fluctuations in plasma testosterone levels in adult male rats and mice. Endocrinology 92, Clermont, V. and Morgentaler, A. (1955). Quantitative study of spermatogenesis in the hypophysectomized rat. Endocrinology 57, 369. Chemes, H. E., Podesta, E. and Rivarola, M. A. (1976). Action of testosterone, dihydrotestosterone and 5cs androstane 3o, 17(3 diol on the spermatogenesis of immature rats. Biol. Reprod. In press. Dorrington, J. H. and Fritz, E. B. (1974). Cell types influenced by FSH in the rat testis. In Gonadotropins and gonadal functions. (N. It Moudgal, Ed.). Academic Press, N. V., p Dym, M. and Fawcett, D. W. (1970). The bloodtestis barrier in the rat and the physiological compartmentation of the seminiferous epithelium. Biol. Reprod. 3, 308. Eshkol, A. and Lunenfeld, B. (1975). Use of antisera to gonadotropins in reproduction research. In Immunication with hormones in reproduction research. (E. Nieschlag, Ed.). North Holland Publishing Company. Amsterdam, p. 55. Greep, R. 0., Fevold, H. L and Hisaw, F. L (1936). Effect of two hypophyseal gonadotropic hormones on the reproductive system of the male rat. Anat. Rec. 65, 261. Hansson, V., French, F. S., Weddington, W. C., Nayfeh, S. H. and Ritzen, M. E. (1974). FSH stimulation of testicular androgen binding protein (ABP). In Hormone Binding and Target Cell Activation in the Testis. (M. Dufau and K. Catt, Eds.). pp Plenum Press, N. V. Hayashida, T. (1963). Inhibition of spermiogenesis, prostate and seminal vesicle development in normal animals with antigonadotropic hormone serum. J. Endocrinol. 26, 75. Johnson, D. C. and Naqui, It H. (1970). A simplified augmented ovarian weight assay for FSI{ Proc. Soc. Exp. Biol. Med. 133, 536. Madhwa Raj, H. G. and Moudgal, N. B. (1970). Hor-

6 494 MADHWA RAJ AND DYM monal control of gestation in the intact rat. Endocrinology 86, 874. Means, A. It (1975). Biochemical effect of follicle stimulating hormone on the testis. In Handbook of Physiology: Male Reproductive System. (D. W. Hamilton and Greep, It 0., Eds.). Vol. V, Section 7, p Means, A. It, Fakunding, J. L, Huckins, C., Tindall, D. J. and Vitale, R. (1976). Follicle stimulating hormone, the Sertoli cell, and spermatogenesis. Recent Progress in Hormone Research. In press. Moudgal, N. B. (1974). Hormone antibodies - an appraisal of their use in reproductive physiology. (It 0. Greep, Ed.). Series 1, Vol. 8, p. 33. Butterworths and University Park Press. London, Baltimore. Falvo, It E. and Nalbandov, A. V. (1974). Radioimmunoassay of peripheral plasma testosterone in males from eight species using a specific antibody without chromatography. Endocrinology 95, Ouchterlony, 0. (1958). Diffusion-in-gel methods for immunological analysis. Progress in Allergy 5, 1. Schwartz, N. B. (1974). The role of FSH, LH and their antibodies on follicle growth and ovulation. Biol. Reprod. 10, 236. Steinberger, A., Lindsey, J. N. and Heidel, J. J. (1975). Direct effect of follicle stimulating hormone (FSH) on isolated Sertoli cells cultured in vitro. Abstracts of the 57th Annual Meeting of the Endocrine Society. June, p Abstract No Steinberger, E. and Steinberger, A. (1975). Hormonal control of testicular function in mammals. In Handbook of Physiology, (E. Knobil and W. H. Sawyer, Eds.). Vol. IV, Part 2, p American Physiological Society. Washington, D. C. Talaat, M. and Laurence, K. A. (1969). Effects of active immunization with ovine FSH on the reproductive capacity of female rats and rabbits. Endocrinology 84, 185. Talaat, M. and Laurence, K. A. (1971). Impairment of spermatogenesis and libido through antibodies to leutinizing hormone. Fertil. Steril. 22, 118. Vitale-Calpe, R. D., Fawcett, D. W. and Dym, M. (1973). The normal development of the bloodtestis barrier and the effects of clomiphene and estrogen treatment. Anat. Rec. 176, 333. RECOMMENDED REVIEWS Eshkol, A. and Lunenfeld, B. (1975). Use of antisera to gonadotropins in reproduction research. In Immunization with hormones in reproduction research. (E. Nieschlag, Ed.). North-Holland Publishing Company. Amsterdam, p. 55. Moudgal, N. B. (1974). Hormone antibodies - an appraisal of their use in reproductive physiology. In Reproductive Physiology. (B. 0. Greep, Ed.). Series 1, Vol. 8, p. 33. Butterworths and University Park Press, London, Baltimore.