Effect of a Single Injection of Human Chorionic Gonadotropin (hcg) on Testicular Hormones and Gonadotropins in the Thoroughbred Stallion

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1 NOTE Effect of a Single Injection of Human Chorionic Gonadotropin (hcg) on Testicular Hormones and Gonadotropins in the Thoroughbred Stallion Nobuo TSUNODA 1, Qiang WENG 2, 3, Shunichi NAGATA 4, Hiroyuki TANIYAMA 5, Gen WATANABE 2, 6 and Kazuyoshi TAYA 2, 6 * 1 Shadai Corporation, Hokkaido , 2 Laboratory of Veterinary Physiology, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Saiwai-cho, Fuchu, Tokyo , Japan, 3 Faculty of Biological Science and Technology, Beijing Forestry University, Beijing , China, 4 Research Section, Laboratory of Racing Chemistry, Tochigi , 5 Department of Veterinary Pathology, Rakuno Gakuen University, Hokkaido , 6 Department of Basic Veterinary Science, United Graduate School of Veterinary Sciences, Gifu University, Gifu , Japan The changes in testicular and pituitary functions in response to human chorionic gonadotropin (hcg) in the breeding and non-breeding season were investigated and compared in the Thoroughbred stallion. Five mature Thoroughbred stallions ranging in ages from 7 to 21 years were injected in May (breeding season) and October (non-breeding season). All animals received an intramuscular injection of 5,000 IU hcg in the experiments. Peripheral blood samples were collected in heparinized tubes from the jugular vein for hormonal assays just before the injection (Day 0) and at a daily interval for five days following the injections (Days 1, 2, 3, 4 and 5). Basal levels of immunoreactive (ir) inhibin, testosterone, estradiol-17β, FSH and LH were higher in the breeding season than in the nonbreeding season. There were significant differences in estradiol-17β, FSH and LH between the two seasons. Ir-inhibin exhibited significant increases on Day 2 after the hcg treatment in the non-breeding season, though there was no change in the breeding season. Plasma levels of testosterone showed remarkable increases after the hcg injection in both seasons. The peak levels of plasma testosterone were observed on Days 2 and 3 in the breeding and non-breeding seasons. Plasma estradiol-17β was significantly higher than the basal level on Day 3 in the non-breeding season, whereas there was no change in the breeding season. Plasma levels of FSH declined from Day 1 to Day 3, then recovered to the basal level (Day 0) on Day 4 after hcg treatment in the breeding season, whereas there was no change in the non-breeding season. Circulating LH showed a significant decrease on Day 3 compared to the based level in the breeding season, but no significant change in the non-breeding season after a treatment of hcg. A significant negative correlation was observed between testosterone and FSH in the breeding season. In conclusion, hcg treatment stimulates secretion of testicular hormones and these testicular hormones temporarily suppress secretion of gonadotropins from the pituitary gland. These results also suggested that Leydig cells of thoroughbred stallion testes have LH receptors in the non-breeding season, the same as in the breeding season. Key words: FSH, hcg, LH, stallion, testicular hormone J. Equine Sci. Vol. 18, No. 3 pp , 2007 Human chorionic gonadotropin (hcg) is a This article was accepted June 28, 2007 *Corresponding author. taya@cc.tuat.ac.jp heterodimeric protein formed by non-covalent association between the common α- and the hormone specific β-subunit [8]. Being structurally and biologically closely related to the pituitary-derived

2 108 N. TSUNODA, Q. WENG, S. NAGATA ET AL. luteinizing hormone (LH), hcg binds to the same receptor and acts as a potent LH agonist. The LH/ hcg receptor belongs to the seven-transmembrane domain, G protein-coupled receptor family [1, 18]. Stallion testes produce large amounts of testosterone, estradiol-17β and inhibin, and the secretion of these hormones is stimulated by the pituitary gonadotropins, follicle-stimulating hormone (FSH) and LH. Previous studies have demonstrated that hcg treatment is a useful method for examining testicular endocrine functions in stallions [4 6, 25]. In equine, it has been reported that testicular tissue contains a single population of LH receptors and the binding kinetics were not significantly different when hcg was compared with horse LH [6]. Some experiments in stallions involving treatment with hcg resulted in marked increases in testosterone and estrogen levels in the peripheral blood [4, 25]. In in vitro study, using testicular cells isolated from stallions, hcg made secretion of testosterone and estrogens increase more than LH in the presence of lipoprotein [5]. Also, human studies have shown that the concentrations of circulating inhibin and testosterone increase after stimulation with hcg [3, 7, 12]. Positive regulation of testicular hormone secretions by hcg treatment is possible through its effect on secretion of gonadotropins from the pituitary, and selective FSH deficiency was induced in normal men by administration of hcg [14]. However, the effect of hcg on the secretion of inhibin and gonadotropins in the stallion is unknown. Circulating concentrations of the testicular hormones and gonadotropins in the breeding season are higher than in the non-breeding season in stallions. Seasonal change of Leydig cell populations in the stallion testis has also been reported [11]. The effect of hcg treatment on testicular endocrine functions in the stallion may be different between the breeding and the non-breeding seasons. The aim of this study was to investigate the changes in plasma levels of testicular hormones and the corresponding changes in plasma levels of gonadotropins after a single hcg injection during the breeding season and the non-breeding season in the stallion. Materials and Methods Animal and treatment Five mature stallions, ranging in ages from 7 to 21 years, were used in this study. They were kept under a natural environment and engaged in breeding during March and June. The experiments were carried out in May (breeding season) and October (non-breeding season). All animals received a single intramuscular injection of 5,000 IU hcg in each season. Peripheral blood samples (10 ml) of each horse were collected in heparin tubes from the jugular vein just before the hcg injection (Day 0) and daily for five days following the injection (Days 1, 2, 3, 4 and 5). All blood samples were immediately centrifuged at 2,000 g for 10 min at 4 C and individual plasma samples were stored at 20 C until assayed. Hormones assay Plasma concentrations of ir-inhibin were measured by a double-antibody radioimmunoassay (RIA) system using rabbit antiserum against purified bovine inhibin (TNDH-1) and 125 I-labeled 32kDa bovine inhibin, as described previously [10]. The intra- and inter-assay coefficients of variation were 8.0% and 16.2%, respectively. Plasma concentrations of testosterone and estradiol-17β were determined by double-antibody RIA systems using 125 I-labeled radioligands as described previously [24, 15]. The intra- and inter-assay coefficients of variation were 6.3% and 7.2% for testosterone and 3.7% and 6.4% for estradiol-17β, respectively. Plasma concentrations of FSH and LH were measured by double-antibody RIA systems using a rabbit antiserum against human FSH (#6; provided by NIDDK NIH, Bethesda, MD, U.S.A.) and a rabbit antiserum against ovine LH (YM #18; provided by Dr Y. Mori, Laboratory of Veterinary Ethology, University of Tokyo, Tokyo, Japan) as described previously [16]. The intra- and inter- assay coefficients of variation were 9.2 % and 13.2% for FSH and 8.8 % and 13.0 % for LH, respectively. Statistical analysis Data for each hormone concentration are presented as means ± S.E.M. The effect of hcg and season were analysed by repeated measures analysis of variance (ANOVA). The difference between pre- and posttreatment values was tested by Wilcoxon s signed rank

3 TESTICULAR RESPONSE TO HCG IN STALLIONS 109 Fig. 1. Plasma concentrations of immunoreactive (ir-) inhibin (A), testosterone (B), estradiol (C), FSH (D) and LH (E) of stallions in the breeding ( ) and non-breeding seasons ( ). Each point represents the mean ± S.E.M. of 5 stallions. Asterisks indicate significant difference (p<0.05). test. Comparison within the breeding and nonbreeding seasons were analysed by the Mann-Whitney U test. Pearson s correlation coefficient was used to examine the relationships between concentrations of each hormone. All differences with values of p<0.05 were considered significant. Results stallion were higher in the breeding season than those in the non-breeding season. Plasma concentrations of estradiol-17β, FSH and LH were significantly higher in the breeding season than in the non-breeding season (Fig. 1). The hcg treatment affected the concentrations of circulating testicular hormones and gonadotropins in both seasons, except LH in the nonbreeding season (Figs. 2 and 3). These within season changes are described below. The basal levels of each hormone (Day 0) in the

4 110 N. TSUNODA, Q. WENG, S. NAGATA ET AL. Fig. 2. Changes in circulating immunoreactive (ir-) inhibin (A, D), testosterone (B, E) and estradiol-17β (C, F) from Day 0 to Day 5 after human chorionic gonadotropin (hcg; 5,000 IU) injection in the Thoroughbred stallion during the breeding (A, B, C) and the non-breeding seasons (D, E, F). Each point represents the mean ± S.E.M. of 5 stallions. Values with different superscripts are significantly different. Ir-inhibin In the breeding season, ir-inhibin levels in the plasma did not show any significant changes compared to the value on Day 0 (Fig. 2A). On the other hand, in the non-breeding season, the level of ir-inhibin was significantly increased on Day 2 after the treatment, as compared with the value on Day 0 (Fig. 2D). Testosterone Concentrations of testosterone in the plasma showed remarkable increases after treatment with hcg in both seasons. In the breeding season, the testosterone level in the plasma reached the maximum value on Day 2, and remained significantly higher than Day 0 values on Day 3 (Fig. 2B). In the non-breeding season, the

5 TESTICULAR RESPONSE TO HCG IN STALLIONS 111 Fig. 3. Changes in circulating LH (A, B) and FSH (C, D) from Day 0 to Day 5 after human chorionic gonadotropin (hcg; 5,000 IU) injection in the Thoroughbred stallion during the breeding (A, C) and the non-breeding seasons (B, D). Each point represents the mean ± S.E.M. of 5 stallions. Values with different superscripts are significantly different. plasma level of testosterone was significantly higher on Day 1, reached a peak on Day 3, and was still significantly higher than the Day 0 value on Day 4 (Fig. 2E). The peak value of the non-breeding season was higher than that of the breeding season. Estradiol-17β In the breeding season, circulating concentrations of estradiol-17β showed an increase on Day 1, and then decreased to lower levels on Day 3, though the changes were not significantly different from the Day 0 value (Fig. 2C). In the non-breeding season, the estradiol- 17β level was significantly higher than the Day 0 value on Day 3 in response to hcg treatment (Fig. 2F). FSH and LH In the breeding season, plasma concentrations of LH decreased after the hcg treatment, reached a nadir on Day 3, then slowly recovered until Day 5. There was a significant difference between the values on Days 0 and Day 3 (Fig. 3A). There was no apparent change in circulating LH in response to hcg during the nonbreeding season (Fig. 3B). In the breeding season, plasma concentrations of FSH responded to hcg treatment by beginning a decline on Day 1, reaching to a nadir on Day 3, then recovering to the pretreatment level on Day 4. However, there was no significant difference among the daily levels of FSH in response to hcg treatment in the breeding season (Fig. 3C). On the other hand, plasma concentrations of FSH responded to hcg by gradually declining until Day 5 in the non-breeding season. The FSH values on Day 4 and Day 5 were significantly lower than that on Day 0 (Fig. 3D). Correlations between testicular hormones and gonadotropins The relationships among plasma concentrations of ir-inhibin, testosterone, estradiol-17β, LH and FSH in

6 112 N. TSUNODA, Q. WENG, S. NAGATA ET AL. Fig. 4. Changes in circulating inhibin (A, D), testosterone (B, E) and estradiol-17β (C, F) from Day 0 to Day 5 after human chorionic gonadotropin (hcg; 5,000 IU) injection compared with LH in the Thoroughbred stallion during the breeding (A, B, C) and the non-breeding seasons (D, E, F). Each point represents the mean ± S.E.M. of 5 stallions. stallions after a single injection of hcg are shown in Figs. 4 and 5. A significant negative correlation was observed between testosterone and FSH in the breeding season (r= 0.315, p<0.01) (Fig. 5B). Testosterone and LH also showed negative correlation, but without statistical significance (Fig. 4B). Discussion In this study, the pre-injection value of testicular hormones and gonadotropins of the stallion in the breeding season were higher than those in the nonbreeding season. These results indicate that equine endocrine functions of the testis and the pituitary gland were active in the breeding season. In addition, the present study also demonstrated that treatment with hcg promoted testicular hormones secretion and suppressed FSH and LH levels, and suggest that Leydig cells of Thoroughbred stallion testes have LH receptors in the non-breeding season, the same as in the breeding season. The stallion is a long-day seasonal breeder showing a clear seasonal cycle of testicular activity [2, 19, 21]. There is also a clear seasonal cycle

7 TESTICULAR RESPONSE TO HCG IN STALLIONS 113 Fig. 5. Changes in circulating inhibin (A, D), testosterone (B, E) and estradiol-17β (C, F) from Day 0 to Day 5 after human chorionic gonadotropin (hcg; 5,000 IU) treatment compared with FSH in the Thoroughbred stallion during the breeding (A, B, C) and the non-breeding season (D, E, F). Each point represents the mean ± S.E.M. of 5 stallions. in the peripheral concentration of immunoreactive (ir)-inhibin, which is related to the breeding season [16, 23]. In our previous studies, annual changes in circulating ir-inhibin, testosterone, estradiol-17β, FSH and LH were observed in the stallion, and the highest levels of these hormones were noted during the breeding season [16, 17]. The effect of a single hcg treatment on the endocrine functions in the stallion was different between the breeding season and the nonbreeding season in this study. These results suggest that stallion testes may have differing sensitivities to the gonadotropin throughout the year. In a male Rhesus monkey study, hcg treatment, which markedly elevated testicular testosterone secretion, failed to increase concentrations of immunoactive inhibin [13]. In the present study, a significant elevation of the inhibin level following hcg treatment was observed in the non-breeding season, but not in the breeding

8 114 N. TSUNODA, Q. WENG, S. NAGATA ET AL. season. As the basal inhibin level in the stallion is at its highest in the breeding season, it may have been impossible to further stimulate inhibin secretion by hcg. The peak level of hcg-induced ir-inhibin in the non-breeding season was the same value as the basal levels in the breeding season, a result which provides support for our hypothesis. In our previous study, we found that inhibin α, β A and β B -subunits were localized in Leydig cells as well as Sertoli cells in the stallion testis [17]. Also, the rise in serum immunoactive inhibin levels reported in male rats following hcg stimulation raises the possibility that Leydig cells may produce inhibin [22]. Therefore, we speculate that the irinhibin increase following hcg treatment observed in the present study was mainly due to secretion from the Leydig cells. Circulating concentrations of testosterone were markedly increased following hcg treatment in both seasons, and had a clear peak around 3 days after treatment in the present study. The results are similar to those of a previous study in the stallion, which showed that testosterone concentrations were markedly elevated for five days after a single injection of hcg [4]. In rats, after hcg stimulation, the mrna level of the cytochrome P450 cholesterol side chain cleavage responded similarly in neonatal and adult male rats. In contrast, no response of aromatase cytochrome P450 mrna to hcg stimulation was found at either age [20]. Our present data show that the secretion of testosterone in response to hcg was dramatically larger than that of estradiol-17β, which is in agreement with a previous study which reported that a differential response of steroidogenic enzymes expression to hcg stimulation possibly produced differential testosterone and estradiol-17β levels, and that the secretion of testosterone was regulated differently from other testicular hormones [20]. The present study clearly demonstrates that hcg treatment dramatically induced changes in testicular hormones and gonadotropins. These results reflect the establishment of closed loop feedback regulation of the hypothalamic-pituitary-testicular axis in stallions. A previous study showed that the circulating concentrations of ir-inhibin and steroid hormones were positively correlated with each other over a period of 2 years [17]. Our present results show that inhibin, concentrations positively correlated with estradiol-17β levels after a single hcg treatment, supporting the view that estrogens and inhibin have a strongly synergistic negative feedback action on basal FSH secretion, and to a limited extent on basal LH secretion [9]. In summary, the present study provides new evidence of hormonal change after a single hcg treatment in stallions. hcg treatment increased testicular hormones levels and suppressed FSH and LH concentration. Acknowledgments We are grateful to the National Hormone and Pituitary Program, NIDDK, NIH, Torrance, CA, USA and Dr A. F. Parlow for the equine LH and FSH kits and to Dr. G. D. Niswender, Animal Reproduction and Biotechnology Laboratory, Colorado State University (Fort Collins, CO, USA) for providing antisera to testosterone and estradiol-17β. This study was supported in part by a Grant-in-Aid for Scientific Research (Basic Research B , P06445) and the Japan Thailand joint research from the Japan Society for the Promotion of Science and National Natural Science Foundation of China (NSFC) (No ) and a Grand-in-Aid from the Equine Research Institute of the Japan Racing Association. References 1. Ascoli, M., Fanelli, F., and Segaloff, D.L The lutropin/choriogonadotropin receptor, a 2002 perspective. Endocr. Rev. 23: Berndtson, W.E., Pickett, B.W., and Nett, T.M Reproductive physiology of the stallion. J. Reprod. Fertil. 39: Comhaire, F.H., Rombauts, L., Vereecken, A., and Verhoeven, G Inhibin and steroid responses to testicular stimulation in normal men. Hum. Reprod. 10: Cox, J.E., and Redhead, P.H Prolonged effect of a single injection of human chorionic gonadotrophin on plasma testosterone and oestrone sulphate concentrations in mature stallions. Equine Vet. J. 22: Eisenhauer, K.M., and Roser, J.F Effects of lipoprotein, equine luteinizing hormone, equine follicle-stimulating hormone, and equine prolactin on equine testicular steroidogenesis in vitro. J. Androl. 16: Evans, J.W., Roser, J.F., and Mikuckis, G.M Comparison of the interaction of equine LH and human chorionic gonadotrophin to equine

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