Testicular Inhibin in the Stallion: Cellular Source and Seasonal Changes in Its Secretion¹

^a Laboratory of Racing Chemistry, Tokyo 158-0098, Japan ^b Shadai Corporation, Hokkaido 059-1432, Japan ^c Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan ^d Rakuno Gakuen University, Hokkaido 069-0836, Japan ^e Japan Racing Association, Equine Research Institute, Tochigo 320-0856, Japan

	ABSTRACT

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The cellular localization of inhibin , ß_A, and ß_B subunits, 3ß-hydroxysteroid dehydrogenase (3ß-HSD), and cytochrome P450 aromatase (aromatase) in stallion testes was investigated. In addition, detailed seasonal changes in circulating immunoreactive (ir)-inhibin were investigated in correlation with testosterone, estradiol, LH, and FSH. Inhibin subunit-positive staining was observed in Sertoli cells, and more clearly positive staining was noted in Leydig cells. Inhibin ß_A and ß_B subunits were also stained in both types of cells. Immunoreactivity of 3ß-HSD and aromatase was confined to the Leydig cells. There was no seasonal effect on the percentage of the areas within seminiferous tubules and interstitial tissues that stained positive for the inhibin subunit. The highest plasma concentrations of ir-inhibin were observed in the breeding season, and the lowest levels were noted during the nonbreeding season. The circulating concentrations of ir-inhibin, steroid hormones, and gonadotropins were positively correlated with each other throughout the 2 years studied. The presence of the inhibin and ß subunits in Leydig cells and Sertoli cells in the equine testis suggests that these cells may secrete dimetric (bioactive) inhibin in circulation of stallions, and that the circulating ir-inhibin may be a useful indicator of the testicular function of stallions.

	INTRODUCTION

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The stallion is a long-day seasonal breeder showing a clear seasonal cycle of testicular activity [1–3]. There is also a clear seasonal cycle in the peripheral concentrations of immunoreactive (ir)-inhibin, which has a relationship with the breeding season [4, 5]. Stallion testes secrete a large amount of immunoreactive and bioactive inhibin [4, 6] in circulation. These results suggest that the circulating levels of inhibin may reflect testicular functions in the stallion. However, the correlation among secretion of ir-inhibin and other testicular hormones is not clear in the stallion. Also, the cellular source of inhibin in the equine testis has not yet been determined.

In the present study, to determine the cellular source of testicular inhibin, immunolocalization of the inhibin , ß_A, and ß_B subunits in the stallion testis was investigated during the breeding season and the nonbreeding season, and these results were compared to findings for two key steroidogenic enzymes, 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and cytochrome P450 aromatase (aromatase). Further, detailed annual changes in the plasma concentrations of ir-inhibin were investigated and compared to changes in two testicular steroids, testosterone and estradiol, and two pituitary gonadotropins, FSH and LH, in adult stallions.

	MATERIALS AND METHODS

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Animals and Blood Samples

Five mature Thoroughbred stallions, ranging from 7 to 21 yr of age, were used for evaluating the annual change in plasma concentrations of ir-inhibin, testosterone, estradiol, LH, and FSH. They were kept in grass fields daily in all seasons and given supplementary feeding of hay in the winter. These stallions engaged in breeding during March and June. All stallions were investigated for two annual cycles, and their blood samples (10 ml) were collected by venipuncture from the jugular vein at 1-mo intervals over 2 yr (except for August of the first year and July of the second year). Individual plasma samples were stored at -20°C until required for assay of ir-inhibin, testosterone, estradiol, LH, and FSH.

Testes Samples

For immunohistochemical localization of three inhibin subunits and two steroidogenic enzymes, testes were collected from 10 Thoroughbred mature stallions (4–9 yr old). They were castrated during both the breeding (n = 5, March to June) and the nonbreeding (n = 5, September to December) season. Castrations were performed under general anesthesia. A part of the testicular parenchyma (0.5–1 cm³) was taken from the center of the testes. These samples were immediately fixed in 4% paraformaldehyde (Sigma Chemical Co., St. Louis, MO) in 0.01 M PBS, pH 7.4, and embedded in paraffin. The paraffin-embedded testes were serially sectioned at 6-µm thickness and placed on silane-coated slides (Dako Japan Co., Kyoto, Japan).

RIA of Inhibin, FSH, LH, Testosterone, and Estradiol

Concentrations of ir-inhibin in plasma were measured using a rabbit antiserum against purified bovine inhibin (TNDH 1) and ¹²⁵I-labeled 32-kDa bovine inhibin, as described previously [6]. The results were expressed in terms of 32-kDa bovine inhibin. The sensitivity of the assay was 7.8 pg/tube (78 pg/ml). The intra- and interassay coefficients of variation were 8.0% and 16.2%, respectively.

Plasma FSH concentrations were measured using a rabbit antiserum against human FSH (#6; provided by NIDDK NIH, Bethesda, MD) as described previously by Hines et al. [7]. Highly purified equine FSH (E219B; kindly provided by Dr. H. Papkoff, Hormone Research Laboratory, University of California, San Francisco, CA) was used as a standard and for iodination. Dilutions of stallion, mare, and gelding plasma were parallel to the standard curve after logit-log transformation, as assessed from identity of slope values. Cross-reactivity of the antiserum with highly purified equine LH (E98A; provided by Dr. H. Papkoff) was less than 0.2%. The sensitivity of the assay was 312.5 pg/tube (1560 pg/ml). The intra- and interassay coefficients of variation were 9.2% and 13.2%, respectively.

Plasma LH concentrations were measured using 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 by Nambo et al. [8]. Highly purified equine LH (E98A; provided by Dr. H. Papkoff) was used as a standard and for iodination. Dilutions of stallion, mare, and gelding plasma were parallel to the standard curve after logit-log transformation, as assessed from identity of slope values. Cross-reactivity of the antiserum with highly purified equine FSH (E219B; provided by Dr. H. Papkoff) was less than 0.5%. The sensitivity of the assay was 31.2 pg/tube (300 pg/ml). The intra- and interassay coefficients of variation were 8.8% and 13.0%, respectively.

Plasma concentrations of testosterone and estradiol were determined by double-antibody RIA systems using ¹²⁵I-labeled radioligands as described previously [9, 10]. Antisera against testosterone (GDN 250; [11]) and estradiol-17ß (GDN 244; [12]), kindly supplied by Dr. G.D. Niswender (Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO), were used in each RIA. The intra- and interassay coefficients of variation were 6.3% and 7.2% for testosterone and 3.7% and 6.4% for estradiol, respectively.

Immunohistochemistry

Inhibin , ß_A, and ß_B subunits. After the sections were deparaffinized with xylene, they were subjected to antigen retrieval by autoclaving in 0.01 M sodium citrate buffer (pH 6.0) at 121°C for 15 min [13]. The sections were then incubated in 3% H₂O₂ in methanol at room temperature for 30 min followed by 0.5% casein-Tris saline (0.05 M Tris-HCl with 0.15 M NaCl, pH 7.6) (CTS) at 37°C for 1 h to quench nonspecific staining. A section was incubated for 8–12 h at 4°C with polyclonal antibody against an inhibin subunit at a dilution of 1:2000–4000 in CTS. The antibody against each inhibin subunit was anti-[Tyr30]-inhibin -chain (1-30)-NH₂ conjugated to rabbit serum albumin (kindly provided by Dr. N. Ling, Neuroendocrine Inc., San Diego, CA), anti-cyclic inhibin ß_A (81-113)-NH₂ (#305-24-D; kindly provided by Dr. W. Vale, the Salk Institute for Biological Studies, La Jolla, CA), and anti-cyclic inhibin ß_B (80-112)-NH₂ (#305-25-D; provided by Dr. W. Vale). After this incubation, the sections were treated with 0.5% biotinylated goat anti-rabbit secondary antibody (ABC kit Elite; Vector Labs, Burlingame, CA) in CTS for 1 h and were subsequently incubated with 2% avidin-biotin complex (ABC kit Elite) in CTS for 30 min at 37°C. The antibody bound to the sections was visualized by treating with 0.05% 3.3'-diaminobenzidine tetrachloride (Sigma) in 10 mM Tris-buffered saline containing 0.01% H₂O₂ for 3 min. Specificity of the antibody against inhibin subunit was examined using normal rabbit serum and neutralized antibody (the primary antibody had been preincubated with 5 µg/ml purified bovine inhibin overnight at 4°C) instead of primary antibody. In order to identify the cell types in stallion testis, a serial section of each testis sample was also stained with hematoxylin and eosin.

3ß-HSD and aromatase. Sections were also stained with antiserum against 3ß-HSD or aromatase in the same manner as described for inhibin. 3ß-HSD antiserum used in the present experiment was polyclonal antiserum against human placental 3ß-HSD raised in a rabbit (diluted 1:1000, kindly provided by Dr. J.I. Mason [14], Cecil H. and Ida Green Center for Reproductive Science, University of Texas, Southern Medical Center, Dallas, TX). Aromatase antiserum used in the present experiment was polyclonal antiserum against human placental P450 aromatase (R-8-1, diluted 1:2000) raised in a rabbit (kindly provided by Dr. Y. Osawa [15], the Medical Foundation of Buffalo, Buffalo, NY). These antisera have been used in the horse tissue [16, 17].

Evaluation of Immunostaining Sites

The percentages of area in the seminiferous tubule and in the interstitial tissue that stained positive for the inhibin subunit were determined by analyzing sections of stallion testes during the breeding (n = 5) and the nonbreeding seasons (n = 5). Ten seminiferous tubules and ten fields within the interstitial tissue in each section were evaluated. The images were captured in a personal computer (NEC, Tokyo, Japan) using a Nikon photomicroscope with a x40 objective (Nikon, Tokyo, Japan), and the areas were determined using an image analysis system with a Graphics Digitizer (PIAS, Osaka, Japan). The repeatability of measurements, expressed as the coefficient of variation for ten measurements of a positive area, was 1.94% at 10 µm² and 1.62% at 100 µm², respectively.

Statistical Analysis

All data are presented as means ± SEM. When there was heterogeneity of variance and the standard deviations were proportional to the means, logarithmic transformation was carried out before analysis of variance. The significance of annual changes in the concentrations of each hormone was examined by a two-way ANOVA with the animal and month as factors. Pearson's correlation coefficient was used to examine the relationship between concentrations of each hormone throughout 2 years. Student's t-test was used to compare the evaluations of immunostaining-positive areas between the breeding and the nonbreeding season. All differences with values of p < 0.05 were considered significant.

	RESULTS

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Immunohistochemical Localization of Inhibin , ß_A, and ß_B Subunits, 3ß-HSD, and Aromatase

The hematoxylin- and eosin-stained sections showed many seminiferous tubules including a large amount of sperm, germ cells, and Sertoli cells. They were surrounded by interstitial tissue filled with large cells that had a spherical nucleus and an eosinophilic cytoplasm (Fig. 1a).

View larger version (103K): [in this window] [in a new window]   FIG. 1. Demonstration of inhibin subunit staining specificity in the adult stallion testis. A section was stained with hematoxylin-eosin (a). The seminiferous tubules are surrounded by interstitial tissue that is filled with Leydig cells. The serial sections were incubated with inhibin subunit antiserum (b), with polyclonal inhibin subunit antiserum neutralized with bovine 32-kDa inhibin (c), and with normal rabbit serum (d). Immunostaining of the inhibin subunit is seen in Sertoli cells within the seminiferous tubules and also Leydig cells (b), but no staining was demonstrable in c and d. Bar = 30 µm.FIG. 2. Immunohistochemical localization of inhibin ßA and ßB subunits in the adult stallion testis. Immunostaining of the inhibin ßA subunit (a) and ßB subunit (b) is observed in both Sertoli cells and Leydig cells. Bar = 30 µm.FIG. 3. Immunohistochemical localizations of 3ß-HSD (a) and aromatase (b) in the testis of an adult stallion. Only Leydig cells are immunopositive to 3ß-HSD and aromatase. Bar = 30 µm.

Preparations stained with antibody to the inhibin subunit, with neutralized antibody, and with normal rabbit serum are shown in Figure 1, b, c, and d, respectively. Inhibin subunit positive-staining cells were observed in the seminiferous tubules and in the interstitial tissue (Fig. 1b). Neither neutralized antibody nor normal rabbit serum showed any immunostaining cells (Fig. 1, c and d). Therefore, these results showed that this polyclonal antibody specifically stained inhibin subunit-containing cells. Immunohistochemical localization of inhibin ß_A and ß_B subunits was observed in the same cells as that of inhibin subunit (Fig. 2, a and b). In the seminiferous tubules, the germ cells were completely negative, and a positive-staining site was observed around the germ cells near the seminiferous epithelium. Consequently, the inhibin , ß_A, and ß_B subunit-positive cells in the seminiferous tubules were judged to be Sertoli cells. The interstitial inhibin subunit-positive cells were also confirmed for the colocalization of 3ß-HSD and aromatase (Fig. 3, a and b), indicating that those cells could be judged Leydig cells. Immunohistochemical localization of 3ß-HSD and aromatase was observed only in Leydig cells in interstitial tissue, not in Sertoli cells and germ cells.

Evaluation of Inhibin Subunit Immunostaining Sites

The percentages of area in the seminiferous tubules and interstitial tissue that stained positive for inhibin subunit are shown in Figure 4. There was no significant difference in the percentage of area in the testis that stained positive between the breeding season and the nonbreeding season. Variations in the inhibin subunit-positive area among individual seminiferous tubules were observed in all testes. The coefficients of variation per testis were 10.1–27.8% in the breeding season (n = 5) and 11.2–31.2% in the nonbreeding season (n = 5). All Leydig cells were stained by antiserum to the inhibin subunit, and the intensity for individual cells was similar.

View larger version (13K): [in this window] [in a new window]   FIG. 4. Percentages of area that immunostained positive for the inhibin subunit within the seminiferous tubules (a) and interstitial tissues (b) in an adult stallion testis. Ten testes of adult stallions were collected in both the breeding season (n = 5) and the nonbreeding season (n = 5), and the means of percentages for immunopositive area within the seminiferous tubules and the interstitial tissue are represented (means ± SE).

Annual Changes in Plasma Concentrations of ir-Inhibin, Testosterone, Estradiol, LH, and FSH

The results from 5 stallions are summarized in Figure 5. There were significant (p < 0.05) changes in plasma concentrations of ir-inhibin, testosterone, estradiol, FSH, and LH throughout the 2 years. All hormones showed the highest concentrations during the period of the breeding season of these animals (from March to June) and the lowest during the oppositional season (from September to December). The circulating concentrations of these hormones throughout the 2 years were positively correlated with each other (p < 0.01). Concentrations of ir-inhibin exhibited a particularly high correlation with estradiol (Table 1).

View larger version (36K): [in this window] [in a new window]   FIG. 5. Annual changes in plasma concentrations of ir-inhibin (a), testosterone (b), estradiol (c), FSH (d), and LH (e) in stallions. The plasma samples were collected monthly for 2 years (except for August of the first year and July of the second year). Values are expressed as the mean ± SEM for five animals. The shaded areas in each panel represent the period of the breeding season of these stallions.

	DISCUSSION

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The present study demonstrated that a clear annual change in three circulating testicular hormones and two gonadotropins occurred in stallions and that the highest levels of these hormones were noted during the breeding season. These results for seasonal changes in ir-inhibin agree with results from a previous study in this species [4]. Other seasonal breeders, such as the ram [18] and the Japanese monkey [19], showed a similar seasonal change in circulating ir-inhibin concentration in circulation in accordance with the testicular activity. In our previous study, the stallion testis contained a large amount of immunoreactive and bioactive inhibin, and ir-inhibin in plasma vanished as a result of castration [5]. These results clearly indicated that the testis is the major source of ir-inhibin in stallion plasma. The annual change in circulating ir-inhibin in stallions showed a positive correlation with two other testicular hormones and gonadotropins, and an especially high positive correlation was observed between ir-inhibin and estradiol. In addition, the results of immunohistochemistry demonstrate that the inhibin and ß subunits are clearly localized in the Leydig cells as compared to the Sertoli cells. To the authors' knowledge, there has been no report of localization of the inhibin subunit with such strong immunostaining in adult-type Leydig cells in any other species.

In a previous study in the rat, the majority of the inhibin subunit and ß_B subunits were immunolocalizated in fetal-type Leydig cells during fetal life, but the immunostaining was reduced in adult-type Leydig cells after birth [20, 21]. In the present study, the immunopositive areas of inhibin subunits in Leydig cells were obviously larger than those in the Sertoli cells in the stallion testis, indicating that a major part of the circulating ir-inhibin of the stallion may be secreted from the Leydig cells. Recently, in the rat testis, colocalization of the inhibin subunit and 3ß-HSD in the same cells was demonstrated in fetal-type Leydig cells [21]. In the present study, colocalization of the inhibin and ß subunits in the Leydig cells of the adult stallion indicated that the Leydig cells may secrete dimetric and bioactive inhibin. Immunohistochemical results also demonstrated colocalization of the inhibin and ß subunits with 3ß-HSD and aromatase in the same Leydig cells of adult stallion testes, indicating that Leydig cells of the adult stallion have the ability to secrete three hormones—testosterone, estradiol, and inhibin. In a recent study of the adult mouse, aromatase was localized in germ cells and sperm in the testis, and it was suggested that germ cells are a site of estrogen synthesis in the adult mouse testis [22, 23]. In the present study, however, aromatase and 3ß-HSD were localized only in the Leydig cells, not in Sertoli cells and germ cells. Previous studies in the stallion [17] and other species, such as rats [24] and boars [25], have also shown that aromatase is localized only in Leydig cells. It has been reported that the aromatase activity in the testis of adult rats is regulated by LH [26]. On the other hand, in vitro studies in the adult rat [27] and the boar [28] indicated that aromatase activity in Leydig cells was stimulated by coculture with Sertoli cells. It has also been reported that inhibin produced by Sertoli cells stimulates steroidogenesis in Leydig cells by paracrine regulation [29].

Because previous reports indicated that seasonal changes occur in the number of Sertoli cells [30, 31] and Leydig cells [32] in the testis of stallions, it was of interest to examine whether or not the localization of inhibin subunit changed from the breeding season to the nonbreeding season. In the present study, however, no significant effect of season on the area of localization of immunoreactivity of inhibin in either interstitial tissue or seminiferous tubules was observed. There has been no report concerning season-related changes in testicular inhibin localization in any other species. It has been demonstrated that inhibin secretion from Sertoli cells of the rat testis is different among specific germ cell types [33]. In the present study, the ratio of inhibin subunit-positive area in the seminiferous tubules seemed to be contrary to the number of germ cells in each seminiferous tubule. Although the difference was not significant, the percentage of area positive for the inhibin subunit in seminiferous tubules tended to be higher in the nonbreeding season than in the breeding season.

Annual changes in plasma concentrations of ir-inhibin showed a high positive correlation with estradiol concentrations. Previous study of other seasonal breeders, such as the golden hamster, suggested that testicular ir-inhibin secretion may not be directly and immediately influenced by circulating FSH levels [34]. In addition, studies from the rat [35] and the human [36] also suggested that LH plays a stimulatory role in the regulation of adult testicular inhibin _A mRNA. Drummond et al. [37] demonstrated that hCG treatment caused a significant and dose-dependent increase in the serum ir-inhibin level in adult male rats and also indicated that the Leydig cells are involved in the regulation of ir-inhibin secretion. Therefore, LH may have an important role in the secretion of ir-inhibin from Leydig cells of the testis in the stallion.

A recent study of human males showed that the circulating inhibin forms in the adult man were inhibin B and the inhibin subunit precursor, pro-C, and the physiologically important form was inhibin B [38]. However, the circulating inhibin forms in other male animals, including the stallion, are not known.

In conclusion, we have shown that the inhibin , ß_A, and ß_B subunits are colocalized within Leydig cells as well as Sertoli cells in stallion testes. In addition, these inhibin subunits are colocalized with aromatase and 3ß-HSD within Leydig cells, and season-related change in circulating ir-inhibin shows a high positive correlation with estradiol. These results suggest that inhibin may be involved in steroidogenesis in Leydig cells. The presence of the inhibin and ß subunits in Leydig cells and Sertoli cells in the testis suggests that these cells may secrete a dimetric inhibin in circulation and that circulating ir-inhibin may be a useful indicator of the testicular function of stallions.

	ACKNOWLEDGMENTS

 
We are grateful to National Hormone and Pituitary Program, NIDDK, NIH, Bethesda, MD, for antiserum to human FSH (#6); Dr. G.D. Niswender, Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO, for providing antisera to estradiol-17ß (GDN 244) and testosterone (GDN 250); Dr. H. Papkoff, Hormone Research Laboratory, University of California, San Francisco, CA, for providing purified equine FSH (E265B) and LH (E263B); Dr. Y. Mori, Laboratory of Veterinary Ethology, University of Tokyo, Tokyo, Japan, for providing antiserum to ovine LH (YM#18); Dr. N. Ling, Neuroendocrine Inc., San Diego, CA, for providing [Tyr30]-inhibin (1-30); Dr. W. Vale, the Salk Institute for Biological Studies, La Jolla, CA, for providing anti-cyclic inhibin ßA(81-113) (#305-24-D) and anti-cyclic inhibin ßB(80-112) (#305-25-D); Dr. J.I. Mason, Cecil H. and Ida Green Center for Reproductive Science, University of Texas, Southern Medical Center, Dallas, TX, for providing antiserum against 3ß-HSD; and Dr. Y. Osawa of the Medical Foundation of Buffalo, Buffalo, NY, for providing polyclonal antibody against human placental P450 aromatase (R-8-1).

	FOOTNOTES

 
¹ This work was supported in part by a grant-in-aid for Co-operative Research from the Ministry of Education of Japan (08306015).

² Correspondence: Shun-ichi Nagata, Laboratory of Racing Chemistry, 3-47-6 Kamiyoga Setagaya-ku, Tokyo 158-0098, Japan. FAX: 81-3-3429-4402; BXD01742{at}niftyserve.or.jp

Accepted: February 18, 1998.

Received: September 5, 1997.

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