Effects of PRL and FSH on LH Binding and Number of Leydig Cells in Hypophysectomized Mice

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1 Endocrinol. Japon. 1990, 37 (2), Effects of PRL and FSH on LH Binding and Number of Leydig Cells in Hypophysectomized Mice MINORU TAKASE õ, KAZUYOSHI TSUTSUI AND SEIICHIRO KAWASHIMA* Zoological Institute, Faculty of Science, Hiroshima University, Hiroshima 730 and *Zoological Institute, Faculty of Science, University of Tokyo, Tokyo 113, Japan Abstract The effects of PRL and FSH on testicular LH receptors were studied in hypophysectomized (hypox) mice. From the eleventh day after hypophysectomy, they were given 100ƒÊg ovine (o) PRL and/or 2ƒÊg ofsh in two injections per day for 10 days. Hypophysectomy reduced the weight of testis, epididymides and seminal vesicles, and the LH binding to the testis. Treatment,with oprl and/or ofsh in hypox mice resulted in an increase in the weight of testis and epididymides, and the LH binding per testis. There was no :difference between the testicular LH binding in ofsh- and oprl-treated mice. Histological examination showed that oprl and/or ofsh treatment in hypox mice restored normal spermatogenesis. Administration of ofsh to hypox mice led to an increase in the number of typical Leydig cells, whereas oprl was not effective. These results suggest that either PRL or FSH stimulates the LH binding to the testis, but that the action of PRL and FSH on the increase in testicular LH binding is different. Contradictory results have been obtained on the effects of PRL on LH binding to the testis. In adult rats, Morris and Saxena (1980) reported that moderate doses of PRL increased the LH binding, while Ota et al. (1983) reported a direct inhibitory action of PRL on the LH binding by heterologous down-regulation. In contrast, when the PRL concentration greatly increased in rats by transplanting pituitary grafts under the kidney capsule, testicular LH binding increased (Bartke et al., 1977: McNeilly et Received September 29, 1989 Biology, Faculty of Science, Hiroshima University Naka-ku, Hiroshima 730 Japan *To whom reprint requests should be addressed. al., 1978: Sharpe et al., 1980). However, a supranormal concentration of PRL in the mouse induced by transplanting pituitary grafts induced a decrease in LH binding (Klemcke and Bartke, 1981). In hereditary dwarf mice, which are deficient in plasma PRL and GH, the administration of PRL increased testicular LH binding (Bohnet and Friesen, 1976). Our unpublished data indicated that the reduction in plasma PRL by bromocryptine treatment in intact adult mice was accompanied by a decrease in plasma FSH and testicular LH binding and that the administration of PRL to bromocryptine-treated mice increased plasma FSH and restored testicular LH binding. In order to understand the effects of

2 TAKASE et al. Endocrinol. April 1990 Japon. PRL on LH receptors, its action through changes in plasma LH and FSH levels cannot be omitted, if experiments are carried out in intact animals. In the present study, the direct effect of PRL on the testicular LH binding was tested in hypophysectomized (hypox) mice. Although hypox rats have already been used for the study of PRL effects on testicular LH binding (Zipf et al., 1978: Purvis et al., 1979a), no experiments on hypox mice had been carried out. In addition to the effect of PRL on LH binding in hypox mice, we examined whether exogenous administration of PRL and FSH has different effects on Leydig cells. Animals and treatments Materials and Methods Male mice of the C57BL/6NCrj strain were hypophysectomized at 90 days of age by the external auditory canal method (Tanaka, 1955). The success of hypophysectomy was verified at autopsy and by very low levels of plasma FSH. Ovine (o) PRL was dissolved in 0.03M NaHCO3 containing 0.15M NaC1 (ph 9.5). ofsh was dissolved in saline. oprl had a contamination level of 0.002% NIADDK-oFSH- RP-1, 0.02% NIADDK-oLH-23, and 0.03% NIADDK-oGH-I-3 by weight. ofsh was contaminated with less than 0.1% PRL, 0.04 times NIH-LH-SI, and less than 0.1% GH by weight. From the eleventh day after hypophysectomy, the operated mice received subcutaneous injections of 100ƒÊg oprl and/or 2ƒÊg ofsh in two injections per day for 10 days in a volume of 0.05 ml per injection. The dose of oprl was chosen on the basis of our unpublished experiments, and was effective in increasing LH binding to the intact mouse testis, and the dose of ofsh was determined from the report indicating down-regulation of FSH receptors (Tsutsui et al., 1985). Sham-operated mice and hypox mice given vehicle only served as controls. Sample preparations The testes, epididymides, and seminal vesicles were taken out, and each testis and the accessory organs were weighed. The left testis was homogenized in 1.5 ml 0.04M Tris-HCl buffer (ph 7.4) containing 5mM MgSO4 at about 0 Ž. The homogenates were centrifuged at 11,000 ~g for 20 min at 4 Ž. The resulting pellets were stored at -80 Ž after instantaneous freezing on dry ice-ethanol until radioreceptor assay (RRA) was performed. RRA of LH Highly purified LH (NIADDK-rat LH-I-6) was iodinated with 131I by the lactoperoxidase method (Tsutsui and Ishii, 1978). The specific radioactivity of labeled LH was about 1.11 MBq/ genates were quickly thawed and suspended in cold 0.04M Tris-HC1 buffer (ph 7.4) containing 5mM MgSO4 and 0.1% BSA, which was used as the assay buffer for RRA. Re:eptor preparation (100ƒÊl) and 131I-LH (50ƒÊl; ng) were incubated at 35 Žfor 2 h with or without unlabeled LH (50ƒÊl: 2ƒÊg NIH-oLH-24) in a reaction tube precoated with 5% BSA to reduce hormone adsorption to the tube wall. At the end of incubation, bound and free 131I-LH were separated by centrifugation at 11,000 ~g for 3min after adding 1ml assay buffer to each tube. The washing by 1ml assay bufer was repeated three times. The radioactivity of the resulting pellets was counted in an autowell r counter. Specific binding was found by subtracting non-specific binding from total binding. Histological examination The right testis was fixed in Bouin's solution and embedded in paraplast. Cross sections of the medial part of the testis were serially cut into 6ƒÊm thicknesses and stained with periodic acid-schiff (PAS) reagent and hematoxylin. Typical Leydig cells were identified by pale-staining of the spherical to oval nucleus with a rim of heterochromatin (Fawcett, 1986: Teerds et al., 1989). The number of typical Leydig cells was estimated by the method described in previous reports (Abercrombie, 1946: Takasugi et al., 1985) as follows. In five sections taken randomly from the middle part of the testis, the mean number of Leydig cells per section was calculated by the following formula: TC = CC ~Si (S + D); TC, mean true counts of Leydig cells per cross section: CC, mean counts of all identifiable Leydig cell nuclei per section: S, thickness of section (6ƒÊm): D, mean diameter of nucleus of

3 counted Leydig cells. The total number of Leydig cells per testis was calculated by the calculated as a rotation ellipsoid, where a is longitudinal length of the testis, and b is testicular diameter perpendicular to a). Statistical analysis Comparisons of change in the weight of testis, epididymides and seminal vesicles, and the LH binding in sham-operated and hypox mice were made by Student's t-test. Differences in the weight of testis, epididymides and seminal vesicles, LH binding, and the number of Leydig cells in the testis among groups after oprl and/ or ofsh administration to hypox mice were analyzed by Duncan's multiple range test. Results Changes in body weight, weight of testis and accessory organs, and LH binding after hypophysectomy Body weight was not affected by hypophysectomy (Fig. 1A). Testicular weight significantly reduced by hypophysectomy (Fig.1B). Hypophysectomy also induced a significant reduction in the weight of epididymides and seminal vesicles 10 days after the operation, but further reduction was not noted when observed at 20 days postoperation (Fig.1C, D). The binding of LH per 4 mg equivalent of testicular tissue 10 days after hypophysectomy showed a reduction to 44% of that in matched control mice, but no further reduction was detected at 20 days postoperation (Fig. 2A). Specific LH binding per testis was calculated from the specific LH binding per unit testicular weight. LH binding per testis in hypox mice was 32% of that in sham-operated mice at 10 days, and further reduction was observed at 20 days, due to the decrease in testicular weight (Fig.2B). Fig.1. Effects of hypophysectomy (hypox) on the weight of body (A), testis (B), epididymides (C) and seminal vesicles (D) in mice. Hypophysectomy was performed at 90 days of age. Each point represents the mean }SE. The number of mice is indicated in parenthesis. Difference from matched sham-operated controls : š, p<0.05. Effects of PRL and/or FSH on body weight, and weight of testis and accessory organs in hypox mice PRL and/or FSH did not affect the body weight (Fig.3A). Either separate or combined injections of PRL and FSH increased

4 testicular weight, compared with that of hypox mice (Fig.3B). The influence of FSH on testicular weight was more marked than that of PRL, and no additive effect of PRL and FSH was observed. The weight of epididymides was significantly increased by FSH treatment (Fig. 3C, vs. hypox). PRL tended to increase the epididymal Fig.2. Effects of hypophysectomy (hypox)on the specific LH binding per 4 mg testis (A) and per testis (B). Hypophysectomy was performed at 90 days of age. Each point represents the mean mice is indicated in parenthesis. Difference from matched sham-operated controls: *, p<0.05. Table 1. Effects of PRL and/or FSH on the number of Leydig cells and the LH binding in hypophysectomized mice Hypophysectomy (Hypox) was performed at 90 days of age. *: Mean }SE (n=4). Significance of difference: a, p<0.05 (vs. Sham) ; b, p<0.05 (vs. Hypox) ; c, p<0.05 (vs. Hypox+PRL).

5 weight, but the increase was not statistically significant. Combined treatment with PRL and FSH was most effective among treatments in increasing epididymal weight. The weight of seminal vesicles was not influenced by PRL and/or FSH treatment (Fig.3D). Effects of PRL and/or FSH on LH binding in hypox mice Neither PRL nor FSH treatment significantly affected the LH binding per testicular weight (Table 1). However, on a per gland basis the LH binding was enhanced by PRL and/or FSH treatment (Table 1). There were no significant differences in the LH binding per 4 mg or per gland among the three treatment groups, showing the lack of an additive effect of PRL and FSH on the binding. Fig.3. Effects of PRL and/or FSH treatment on the weight of body (A), testis (B), epididymides (C) and seminal vesicles (D) in hypophysectomized mice. Hypophysectomy (H) and sham-operation (S) were performed at 90 days of age. From 10 days after the operation, hypophysectomized mice were given 100 ofsh-17 in two injections per day for IU days. Hypophysectomized control mice received vehicle only. Each column and vertical line represent the mean }SE. The number of mice is indicated in parenthesis. Significance of difference: a, p<0.05 (vs. S); b, p<0.05 (vs. H); c, p<0.05 (vs.h }PRL). Histological changes The seminiferous tubules in shamoperated mice consisted of cells at all stages of spermatogenesis, and the interstitial space was well-developed, containing typical Leydig cells with abundant cytoplasm and a pale-stained round nucleus with a rim of heterochromatin (Fig. 4, a and b). Following hypophysectomy the testis underwent degeneration, the seminiferous tubules containing few spermatids and sperms and the interstitial cells mostly consisting of dark-staining cells (Fig. 4, c and d). In PRL-treated mice, active spermatogenesis and enlarged interstitial space were observed, but the treatment was not effective in restoring the histology of interstitial tissue to its normal appearance (Fig. 4, e and f). In FSH-treated hypox mice the histology of the testis was similar to that of sham-operated mice (Fig.4, g and h). The frequency of typical Leydig cells was greater in FSH-treated hypox mice (Fig.4, g and h) than in control hypox mice (Fig. 4, c and d). Simultaneous treatment of

6 198 TAKASE et al. a b C d e f g h i j Endocrinol. April 1990 Japon.

7 hypox mice with PRL and FSH yielded testicular histology similar to that in shamoperated mice and FSH-treated hypox mice (Fig. 4, i and j). No mitotic figures of Leydig cells were encountered in any of the five groups. Table 1 shows the changes in the number of typical Leydig cells after hypophysectomy and after PRL and/or FSH treatment in hypox mice. Hypophysectomy led to a reduction in the number of typical Leydig cells per testis. Although PRL had no effect on the number of Leydig cells, either FSH treatment or combined treatment with FSH and PRL increased the number of typical Leydig cells. Compared with the LH binding per 1.0 ~106 typical Leydig cells in hypox mice, PRL treatment induced a significant increase, but FSH treatment was not effective. Discussion Several investigators have indicated that PRL stimulates testicular function in dwarf mice (Bohnet and Friesen, 197 ; Bartke et al., 1977a), rats (Aragona et al., 1977; Zipf et al., 1978; Purvis et al., 1979a; Belanger et al., 1979; McNeilly et al., 1979; Morris and Saxena, 1980), and hamsters (Bex and Bartke, 1977; Bex et al., 1978; Fig.4. Histological appearance of a part of the testis from a sham-operated mouse (a, b), hypophysectomized mice given vehicle (c, d), 100ƒÊg oprl (e, f), 2ƒÊg ofsh (g, h), and 100ƒÊg oprl plus 2ƒÊg ofsh (i, j) in two injections per day for 10 days. A number of typical Leydig cells which have round palestaining nuclei with a rim of heterochromatin are visible in the interstitial tissue of the sham-operated control mouse (arrow, Fig. b). Note the cells with a dark-staining nucleus in the hypophysectomized mouse (arrow heads, g. d). Bar: 100ƒÊm, applicable to left panel; 20ƒÊm applicable to right panel. Matthews et al., 1978). FSH is also known to stimulate the increase in the number of LH receptors and LH-stimulated steroidogenesis in rats (El Safoury and Bartke, 1974 ; van Beurden et al., 1976; Chen et al., 1976; Chen et al., 1977; McNeilly et al., 1979; Closset and Hennen, 1989), and pig (Tabone et al., 1984; Benahmed et al., 1985a, b). In mice, several authors reported the effects of PRL on testosterone production and 3ƒÀ-hydroxysteroid dehydrogenase (HSD) activity (Bliriska, 1983 ; Odell and Larsen, 1984). However, there were no reports indicating whether PRL increases the testicular LH binding in hypox mice. Concerning the effect of FSH, Tsutsui et al. (1985) showed that there was a tendency toward an increase in testicular LH binding following FSH administration to adult hypox mice, but the increase was not significant. The present study showed that either PRL or FSH induced a significant increase in LH binding to the testis of hypox mice, and FSH induced an increase in the number of typical Leydig cells in hypox mice. On the other hand, PRL had no significant effect on the number of typical Leydig cells. Accordingly, it may be concluded that PRL augmented testicular LH binding through the increase in LH binding sites per Leydig cell, while FSH stimulated LH binding through the increase in the number of Leydig cells. In earlier reports it was found that PRL stimulated LH binding per unit testicular weight, per testis and per 3ƒÀ-HSD positive cells (Zipf et al., 1978; McNeilly et al., 1979 ; Purvis et al., 1979a; Morris and Saxena, 1980). FSH also enhanced the LH binding per testis, but it was ineffective on the bases of unit weight or 3ƒÀ-HSD positive cells (Chen et al., 1976 ; Thanki and Steinberger, 1976 ; McNeilly et al., 1979; Purvis et al., 1979b ; Kerr and Sharpe, 1985b). In immature hypox rats, FSH treatment increased the volume of interstitial cells per testis (Kerr and Sharpe, 1985b). With

8 cells might be due to the difference in the cell types bearing specific receptors for each hormone. Specific binding sites for PRL and LH were localized on the Leydig cell surface in rats (Aragona et al., 1977 ; Wahlstrom et al., 1983). Similarly, in the mouse testis the accumulation of labeled PRL was detected on Leydig cells (Vanha- Perttula et al., 1973). Of course, FSH binding sites were located on Sertoli cells in the seminiferous tubules (Fritz et al., 1975; Setchell et al., 1976; Shimizu et al., 1987). It has been reported that unknown factor(s) secreted from Sertoli cells following FSH treatment augmented the responsiveness of Leydig cells to LH and increased the LH binding to cultured porcine testicular cells (Benahmed et al., 1985 a, b). Kerr and Sharpe (1985b) showed that FSH induced maturation of primitive mesenchymal cells to Leydig cells. Therefore, it is probable that PRL acted only on surviving Leydig cells in hypox mice, and that FSH stimulated Sertoli cells to secrete unknown factor(s) which was effective in the maturation of interstitial cells, resulting in the increase in the number of typical Leydig cells in the present study. Furthermore, because PRL binding is greater in other organs, such as liver and prostates, than in testis (Vanha-Perttula et al., 1973 ; Aragona and Friesen, 1975), the influence of these organs might also be very important. In Figure 4, a number of cells bearing small and dark-staining polyhedral nuclei (shrunk cells) are visible in the interstitial space of hypox mice. The testis of FSHtreated mice contained more numerous typical Leydig cells and fewer shrunk cells than that of control hypox mice. On the these studies as a background the present other hand, PRL appeared to have no effect on the ratio of the two cell types in between the effects of PRL and FSH on the interstitial tissue. From these observations, it is speculated that FSH enhanced LH receptors. The difference between PRL and FSH the maturation of shrunk cells to typical effects on the number of typical Leydig Ledyig cells. However, it is unclear whether the shrunk cells are the atrophied Leydig cells, the precursor Leydig cells in the process of development from the mesenchymal cells, or other cell types not belonging to the category of Leydig cells. In the present study, the LH binding per 106 Leydig cells increased in hypox PRL-treated mice but was unchanged in hypox FSH-treated mice (Table 1). However, until more exact methods for the identification of Leydig cells are established, no firm conclusion may be drawn on the changes per Leydig cell. Bambino et al. (1980) reported the additive effect of PRL and FSH on the testicular LH binding in immature hypox rats. Because PRL and FSH had different effects on the increase in testicular LH binding, additive effects of these two hormones were expected in the present study. However, this failed to occur in the combination of PRL and FSH doses used. Whether the failure to detect the additive effects was specific to adult mice or not was not clarified in the present study. Histological observations in the present study showed that the testis of hypox mice was almost totally devoid of sperm but following PRL and/or FSH treatment sperm became visible. It is well known that androgen is required for spermatogenesis (Lostroh, 1976). In the present study, though circulating testosterone levels in hypox mice with and without treatment with PRL and/or FSH could be expected to be very low because of the lower weight of the seminal vesicles, spermatogenesis was observed in hypox mice treated with either hormone. The positive regulatoryeffect on Sertoli cells was detected following the administration of PRL to hypox

9 mice (our unpublished data). Because specific binding sites for PRL are restricted to Leydig cells (Venha-Perttula et al., 1973 Wahlstroni et al., 1983), it could be presumed that some factor(s) secreted from Leydig cells due to the stimulation by PRL acted on Sertoli cells. Therefore, indirect and direct effects of PRL and FSH, respectively, on Sertoli cells may be the reason for stimulatory effects on spermatogenesis in hypox mice given injections of either hormone. experimentally-induced chronic hyperprolactinemia on testosterone and gonadotropin levels in male rats and mice. Endocrinology 100, Belanger, A., C. Anclair, C. Seguin, P. A. Kelly and F. Labrie (1979). Down-regulation of testicular androgen biosynthesis and LH receptor levels by an LHRH agonist : role of prolactin. Mol. Cell. Endocrinol. 13, Benahmed, M., C. Grenot, E. Tabone, P. Sanchez and A. M. Morera (1985a). FSH regulates cultured Leydig cell function via Sertoli cell proteins : an in vitro study. Biochem. Biophys. Res. Commun. 132, Benahmed, M., J. Reventos, E. Tabone and J. M. Saez (1985b). Cultured Sertoli cell-mediated Acknowledgements This work was supported in part by a Grantin-Aid from the Ministry of Education, Science and Culture, Japan (to S. K.). We are grateful to Dr. S. Raiti, the National Hormone and Pituitary Program, University of Maryland School of Medicine, the National Institute of Arthritis, Diabetes, and Digestive and Kidney Disease (NIADDK) and Dr. A. F. Parlow, Pituitary HormonejAntisera Center, Harbor-UCLA Medical Center for the supply of pituitary hormones and antisera. References Abercrombie, M.(1946). Estimation of nuclear population from microtome sections. Anat. Rec. 94, Aragona, C., H. G. Bohnet and H. G. Friesen (1977). Localization of prolactin binding in prostate and testis : the role of serum prolactin concentration Acta Endocr.(Copenh) 84, Aragona, C. and H. G. Friesen (1975). on the testicular LH receptor. Specific prolactin binding sites in the prostate and testis of rats. Endocrinology 97, Bambino, T. H., J. R. Schreiber and A. J. W. Hsueh (1980). Gonodotropin-releasing hormone and its agonist inhibit testicular luteinizing hormone receptor and steroidogenesis in immature and adult hypophysectomized rats. Endocrinology 107, Bartke, A., M. S. Smith, S. D. Michael, F. G. Perou and S. Dalterio (1977). Effects of FSH stimulatory effect on Leydig cell steroidogenesis. Am. J. Physiol. 248, E Bex, F., A. Bartke, B. D. Goldman and S. Dalterio (1978). Prolactin, growth hormone, luteinizing hormone receptors, and seasonal changes in testicular activity in the golden hamster. Endocrinology 103, Bex, F. J. and A. Bartke (1977). Testicular LH binding in the hamster : modification by photoperiod and prolactin. Endocrinology 100, Bilinska, B.(1983). Effect of in vitro administration of LH, prolactin separately and LH and prolactin in mixture, on cultured Leydig cells from mouse testes : I. Changes of 45, 3ƒÀ-hydroxysteroid dehydrogenase during postnatal life. Folia Histochem. Cytochem. 21, Bohnet, H. G. and H. G. Friesen (1976). Effect of prolactin and growth hormone on prolactin and LH receptors in the dwarf mouse. J. Reprod. Fertil. 48, Chen, Y. -D. I., A. H. Payne and R. P. Kelch (1976). FSH stimulation of Leydig cell function in the hypophysectomized immature rat. Prc. Soc. Exp. Biol. Med. 153, Chen, Y. -D. I., M. J. Shaw and A. H. Payne (1977). Steroid and FSH action on LH receptors and LH-sensitive testicular responsiveness during sexual maturation of the rat. Mol. Cell. Endocrinol. 8, Closset, J. and G. Hennen (1989). Biopotency of highly purified porcine FSH and human LH on gonadal function. J. Endocrinol. 120, El Safoury, S. and A. Bartke (1974). Effects of follicle-stimulating hormone and luteinizing

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