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Biology of Reproduction 65, 209-215 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Production and Endocrine Role of Inhibin During the Early Development of Bull Calves1

H. Kaneko2,a, J. Noguchia, K. Kikuchia, S. Akagi3,b, A. Shimadaa, K. Tayac, G. Watanabec, and Y. Hasegawad

a Department of Genetic Resources II, National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305-8602, Japan b Department of Animal Production, Kyushu National Agricultural Experiment Station, Nishigoshi, Kumamoto 861-1192, Japan c Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan d Laboratory Animal Science, Kitasato University, School of Veterinary Medicine and Animal Science, Towada, Aomori 034-8628, Japan

ABSTRACT

This study investigated the ontogeny of control of FSH secretion by inhibin during early prepubertal development of bulls by 1) measurements of circulating levels of inhibin and FSH from 1 to 13 wk of age, and 2) immunoneutralization of endogenous inhibin at 7, 21, 60, and 120 days of age. In addition, production and localization of inhibin in testes were examined by immunohistochemistry and Western blots at 7, 21, 60, and 120 days of age. Plasma immunoreactive inhibin levels were relatively low between 1 and 3 wk of age and then showed a tendency to rise (P < 0.1) from 4 wk of age. Circulating concentrations of FSH were low during 3 wk after birth and increased at 5 wk, remained high (P < 0.05) until 16 wk of age. Treatment with inhibin antiserum resulted in a significant (P < 0.05) increase in plasma FSH at 7, 21, 60, and 120 days of age compared to those following injection of control serum; however, the magnitude of the FSH rise after inhibin immunization was greater as bulls aged. There were no significant changes in plasma LH after inhibin immunization. An intense staining of inhibin {alpha} subunits was found in Sertoli cells within the solid seminiferous cords from 7 to 120 days of age, while no specific immune reaction was found in interstitial cells. Western blot analysis of testicular homogenates isolated from bulls 7–120 days of age revealed presence of a 28.5-kDa molecule that cross-reacted with inhibin {alpha} subunit and ßB subunit-specific antibodies. In this study, before 13 wk of age in bull calves, there was no inverse relationship between plasma concentrations of immunoreactive inhibin and FSH. However, the present immunization study clearly indicates that inhibin participates in the regulation of FSH secretion from infancy to early prepubertal stage, although the endocrine significance of inhibin becomes greater in older bulls. The results also indicate that the major production site of inhibin in the testis is Sertoli cells and that these cells produce inhibin that exerts a negative feedback effect on FSH secretion from early stages of development.

follicle-stimulating hormone, inhibin, mechanisms of hormone action, Sertoli cells, testis

INTRODUCTION

Circulating levels of gonadotropins in young bulls are low in the immediate postnatal period, show a transient increase prior to 20 wk of age, and then decrease from 22 to 26 wk of age [1–3]. The early rise in gonadotropin secretion precedes differentiation of gonocytes into spermatogonia and more mature germ cells, pre-Sertoli cells to Sertoli cells, and lumen formation in testicular tubules [4–6]. These observations lead to the hypothesis that this early rise in concentrations of gonadotropins initiates testicular growth and development. The early rise in gonadotropin secretion is associated with an increase in GnRH secretion [7]. Opioids and testosterone modulate LH secretion during the early lives of bulls [1, 2, 8], but opioidergic control of FSH secretion appears to be much less than that for LH [1]. Also, testosterone is only involved in FSH regulation after 24 wk of age [2]. Although steroid control of FSH secretion is not evident before 24 wk of age, castration of bull calves younger than 24 wk of age (7.5–20 wk) resulted in increased FSH secretion [9, 10], indicating that the testes exert inhibitory effects on FSH secretion at this time.

Inhibin is a glycoprotein of gonadal origin that suppresses FSH secretion in the male [11, 12]. In bulls at 1–2 yr of age, circulating FSH levels were decreased following injection of charcoal-extracted bovine follicular fluid, indicating that inhibin has the ability to suppress FSH secretion in bulls [13]. Active immunization of bulls against fragments of inhibin {alpha} subunit commenced between 2 and 14 wk of age, and increased FSH secretion was detected around the pubertal period [14–16]. Passive immunoneutralization of endogenous inhibin produced a marked increase in FSH levels in 6-mo-old bulls [17]. The above findings indicate that inhibin is involved in the regulation of FSH secretion in bulls after later stages of prepubertal development. However, little is known about the control of FSH secretion during the early lives of bull calves.

Therefore, the objectives of this study were to 1) examine the role of inhibin in the control of FSH secretion during early stages of prepubertal development, and 2) determine the cellular source of inhibin production in the testes.

MATERIALS AND METHODS

Hormonal Profile During Early Developmental Stage

Weekly blood samples were taken from five Holstein bull calves from 1 wk after birth until 13 wk of age. Plasma was recovered following centrifugation and stored at -30°C.

Preparation of Antisera

Inhibin antiserum (GB) used in the immunization study was raised against bovine 32-kDa inhibin A in a male castrated goat [18]. This antiserum neutralizes the FSH-suppressing activity of follicular fluid [19]. Inhibin antiserum was purified by an affinity column packed with 1 ml of N-hydroxysuccinimide-activated Sepharose (HiTrap NHS-activated; Amersham Pharmacia Biotech, Buckinghamshire, UK) to which bovine inhibin A was attached. Bovine inhibin A (1 mg/ml) was coupled to N-hydroxysuccinimide-activated Sepharose according to the manufacturer's recommendations. Coupling efficiency of inhibin A to N-hydroxysuccinimide-activated Sepharose was 80%. The antiserum was diluted with an equal volume of PBS (0.05 M, pH 7.5, 0.15 M NaCl), and saturated ammonium sulfate (40% of final volume) was added. After incubation for 1 h at 4 °C, the mixture was centrifugated at 1000 x g for 30 min at 4°C. The precipitate was dissolved in Tris-buffered saline (TBS; 0.05 M Tris/HCl, pH 7.5, 0.15 M NaCl) containing 0.1 % (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS; Sigma, St. Louis, MO) and applied onto the inhibin A-coupled column. After incubation for 1 h at room temperature, the column was washed twice with TBS containing 0.1% CHAPS for five column volumes. The antiserum was eluted with five column volumes of 0.1 M glycine-HCl (pH 2.0). Based on immunoblotting, the purified antiserum recognizes the {alpha} subunits of various molecular weight forms of inhibin including the 30- to 32-kDa form based on immunoblotting [20] (see Results). The {alpha} subunit-specific antibody was used for immunohistochemistry, Western blots, and affinity purification of inhibin from testicular homogenates in the present study.

Antiserum to the ßB subunit (G542) was raised in a castrated male goat against amino acids 84–99 of the ßB subunit of bovine inhibin conjugated to hemocyanin. The antiserum was purified using an affinity column packed with 1 ml of N-hydroxysuccinimide-activated Sepharose (HiTrap NHS-activated; Amersham Pharmacia Biotech) to which bovine inhibin ßB (84–99) was attached. Bovine inhibin ßB (84–99) (2.5 mg/ml) was coupled to N-hydroxysuccinimide-activated Sepharose according to the manufacturer's recommendations. Coupling efficiency of inhibin ßB (84–99) to N-hydroxysuccinimide-activated Sepharose was 70%. Cross reaction of the purified ßB subunit antibody with the ßA and ßB subunits was tested by immunoblotting. Activin A (ßA-ßA dimer) and B (ßB-ßB dimer) were purified from bovine follicular fluid (bFF) by the methods of Hasegawa et al. [21]. Serial doses of activin A (50–800 ng/lane) and B (12.5–200 ng/lane) were subjected to 15% SDS-PAGE [22], and the proteins on the gel were transferred onto polyvinylidene difluoride membrane (Immobilon-P; Millipore, Bedford, MA) at 1 mA/cm2 for 2 h using a transblot apparatus (Horize blot, ATTO, Tokyo, Japan). The membrane was incubated overnight with the ßB subunit antibody at a concentration of 4 µg protein/ml (determined by absorbance at 280 nm) at room temperature, then with alkaline phosphatase-labeled anti-goat IgG (Sigma) for 1 h at room temperature. Five-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium (Sigma Fast BCIP/NBT; Sigma) was used for visualizing the immune reaction. The purified ßB subunit antibody cross-reacted slightly with activin A (ßA-ßA) compared to activin B (ßB-ßB) (Fig. 1) and thus was used as a ßB subunit-specific antibody in Western blot analyses.



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  		FIG. 1. Western blot of activin A (ßA-ßA dimer) and B (ßB-ßB dimer) with an affinity-purified antiserum to inhibin ßB subunit (G542). Note that the ßB subunit antibody is highly specific for activin B

Immunoneutralization of Endogenous Inhibin

Twenty-nine clinically normal Holstein bull calves were used. Mean body weights for the calves at 7, 21, 60, and 120 days of age were 42.2 ± 2.5 kg (mean ± SEM, n = 7), 58.1 ± 5.6 kg (n = 10), 82.5 ± 5.2 kg (n = 6), and 122.0 ± 11.2 kg (n = 6), respectively. All animals in each group were given a single i.v. injection of 40 ml inhibin antiserum (GB). The volume of inhibin antiserum (40 ml) was estimated to be sufficient to immunoneutralize circulating inhibin in the calves used in this study, because injection of the same antiserum, at a dose of 50 ml, caused a significant elevation of serum FSH levels in bulls weighing about 170 kg [17]. Forty-eight hours before anti-inhibin serum injection, the animals received 40 ml castrated goat serum (control serum). Endocrine changes during the period from injection of control serum to injection of inhibin antiserum served as controls. Control serum contained no inhibin immunoreactivity determined by RIA. The time of injection of inhibin antiserum was defined as 0 h. Blood samples were collected from a jugular vein of each calf at -48 (time of injection of control serum), -46, -44, -42, -36, -24, -12, 0 (time of injection of inhibin antiserum), 2, 4, 6, 12, 24, 36, 48, 72, and 96 h. Plasma was recovered after centrifugation and stored at -30°C.

Immunohistochemistry

Testes were obtained from two calves at 7, 21, 60, and 120 days of age and were fixed with Methacarn solution (methanol-chloroform-acetic acid, 6:3:1, by volume) and embedded in paraffin. Immunohistochemical procedure was based on the methods of Noguchi et al. [23]. Briefly, 4-µm sections were immersed in 6 M urea for 30 min at room temperature and treated with 0.5 % (w/v) periodic acid to remove endogenous peroxidase activity. The sections were then incubated overnight at 4°C with the inhibin {alpha} subunit-specific antibody (0.43 µg protein/ml). Antigen-antibody complexes were visualized using avidin-biotin-peroxidase complex technique (Elite ABC kit, Vector Laboratories, Burlingame, CA) with diaminobenzidine. To test the specificity of immunereaction, sections obtained from a 60-day-old bull were incubated with the purified antiserum that had been preabsorbed with excess bovine inhibin described as follows. Bovine inhibin was purified from bFF using an affinity column coupled with {alpha}-specific monoclonal antibody (256H) [24]. The affinity-purified inhibin preparation was mixed with the {alpha} subunit-specific antibody to a final concentration of 0.375 µg/ml and incubated overnight at 4°C. After centrifugation at 10 000 x g for 30 min at 4°C, supernatant was used as the preabsorbed antibody for immuhistochemical procedures.

Immunoblotting

Testes were obtained from two calves at 7, 21, 60, and 120 days of age and were homogenized in 10-fold weight of TBS containing 0.1% CHAPS. Inhibin was purified from testicular homogenates using an affinity column packed with 1 ml of N-hydroxysuccinimide-activated Sepharose (HiTrap NHS-activated; Amersham Pharmacia Biotech) to which the purified {alpha} subunit-specific antibody was attached. The {alpha} subunit-specific antibody (5 mg/ml) was coupled to N-hydroxysuccinimide-activated Sepharose according to the manufacture's recommendations. Coupling efficiency of the antibody to N-hydroxysuccinimide-activated Sepharose was 80%. Testicular homogenate was applied to the immunoaffinity column and incubated for 1 h at room temperature. The column was washed twice with TBS containing 0.1% CHAPS for five column volumes. The inhibin fraction was eluted with five column volumes of 0.1 M glycine-HCl (pH 2.0). The eluate fraction was concentrated with centrifugal filter devices (Centricon, cut-off 10 000; Millipore) and applied to 12.5% SDS-PAGE [22].

Rainbow-colored molecular weight markers (14.3 to 220 kDa; Amersham Pharmacia Biotech) were used to estimate molecular size of proteins. The amount of immunoreactive inhibin subjected to SDS-PAGE, as determined by RIA, was 132–180 ng per lane for subsequent blots probed with the {alpha} subunit-specific antibody, and 330–465 ng per lane for blots probed with the ßB subunit-specific antibody. For positive control, 20 ng of bovine inhibin A was also subjected to SDS-PAGE and immunoblotting. Proteins on the gel were transferred onto polyvinylidine difluoride membrane (Immobilon-P; Millipore) at 1 mA/cm2 for 2.5 h using the transblot apparatus (Horize blot; ATTO). The membrane was incubated overnight with the {alpha} subunit-specific antibody (4.3 µg protein/ml) or the ßB subunit antibody (4 µg/ml) at room temperature and then with alkaline phosphatase-labeled anti-goat IgG (Sigma) for 1 h at room temperature. Sigma Fast BCIP/NBT (Sigma) was used to visualize the immune reaction.

Radioimmunoassays

Plasma concentrations of FSH were measured by RIA [25] using anti-bovine FSHß subunit serum (U.S. Department of Agriculture [USDA]-5-pool) USDA-bFSH-12 for radioiodination, and USDA-bFSH-I2 as a reference standard. Plasma concentrations of LH were measured by RIA [26] using anti-ovine LH serum (USDA-309-684P), USDA-bLH-I-1 for radioiodination, USDA-bLH-B5 as reference standard (RIA materials were provided by USDA Animal Hormone Program, Germplasm and Gamete Physiology Laboratory, Beltsville Agricultural Research Center, Beltsville, MD). Sensitivities of the assays for LH and FSH, based on a 95% confidence limit of the zero standard, were 0.01 ng/tube (0.1 ng/ml) and 5 pg/tube (25 pg/ml), respectively. The intra- and interassay coefficients of variation, calculated according to the method of Rodbard [27], were 9.5% and 11.5% for LH, and 5.3% and 9.8% for FSH, respectively.

Concentrations of immunoreactive inhibin were measured by a homologous double-antibody RIA [28] using anti-bovine inhibin serum (TNDH-1) and bovine 32-kDa inhibin A for radioiodination, and bovine 32-kDa inhibin A as a reference standard. This assay system cross-reacts with inhibin A and pro-{alpha}C, but cross-reaction with pro-{alpha}C in the inhibin RIA was 35% compared to that with bovine 32-kDa inhibin (100%) [29]. The sensitivity of the assay was 7.8 pg/tube (0.078 ng/ml). The intra- and interassay coefficients of variation were 6.0% and 9.8%.

Inhibin Antibody Titer Determination

Changes in the titer of inhibin antibodies was determined by measuring the binding of 125I-labeled bovine 32-kDa inhibin to a 1:50 dilution of plasma, as described previously [18]. Results were expressed as a percentage of the total counts added.

Statistics

Follicle-stimulating hormone and immunoreactive inhibin profiles during the infantile period were subjected to analysis of variance (ANOVA) for repeated measures [30]. Results obtained from inhibin immunization were analyzed as follows. Hormonal profiles after injection of control (-48 to 0 h) or anti-inhibin serum (0 to 96 h) were subjected to repeated-measures ANOVA. Effect of immunization treatment was determined by comparing data during the first 48 h after injection of inhibin antiserum to data during the first 48 h after control serum. The data were subjected to repeated-measures two-way ANOVA [30]. When a significant effect was obtained with ANOVA, the significance of the difference between two means was tested by Student t-test. When more than two means were compared, the significance of the difference between means was determined by Duncan multiple range test. All data were analyzed using the General Linear Model Procedure of the Statistical Analysis Systems [31]. A value of P < 0.05 was considered to be significant.

RESULTS

Hormonal Profiles During Early Development (Fig. 2)

Plasma FSH concentrations were low between 1 and 3 wk of age and then increased (P < 0.05) and remained high from 5 to 13 wk of age (Fig. 2a). Immunoreactive-inhibin concentrations were around 1.5 ng/ml between 1 and 3 wk of age and then showed a tendency (P < 0.1) to rise with age (Fig. 2b).



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  		FIG. 2. Concentrations of a) FSH and b) immunoreactive inhibin in bulls from 1 to 13 wk of age. Values are means ± SEM for five bull calves

Immunoneutralization of Endogenous Inhibin (Figs. 3 and 4)

At each different age, titer of inhibin antibodies was the highest 6 h after injection of inhibin antiserum and then decreased gradually thereafter (Fig. 3). Regardless of the age of bulls, concentrations of plasma FSH showed no significant changes during the 48 h following injection of control serum (Fig. 4). In contrast, as bulls aged, the injection of inhibin antiserum resulted in a significant (P < 0.05) increase in plasma FSH concentrations compared with those after injection of control serum. Specifically FSH levels increased 5-fold (P < 0.001) after an injection of inhibin antiserum for bulls at 60 and 120 days of age, but only 1.5–2-fold (P < 0.01) for younger bulls. Plasma LH concentrations were unaltered following passive immunization against inhibin, although pulsatile discharge of LH was seen in several bulls 60 and 120 days of age.



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  		FIG. 3. Changes in titer of inhibin antibodies after passive immunization of bulls at 7 (open circles; n = 6 bulls), 21 (closed circles; n = 10), 60 (triangles; n = 6), and 120 days (open squares; n = 6) of age. The time of injection of inhibin antiserum is defined as 0 h. Values are means ± SEM



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  		FIG. 4. Changes in FSH concentrations after injection of inhibin antiserum to bulls at a) 7 (n = 7), b) 21 (n = 10), c) 60 (n = 6), and d) 120 days (n = 6) of age. All calves in each group were given inhibin antiserum 48 h following injection of castrated goat serum (control serum). Time of injection of inhibin antiserum was defined as 0 h. Arrows indicate the time of injection of inhibin antiserum (A/INH) or castrated goat serum (CGS). Values are means ± SEM

Immunohistochemistry (Fig. 5)

Preabsorption of the {alpha} subunit-specific antibody with affinity-purified bFF abolished immunostaining (Fig. 5A). Intense immunostaining for the {alpha} subunit-specific antibody was found in the cytoplasm of Sertoli cells within solid seminiferous tubules of bull calves at 7, 21, 60, and 120 days of age (Fig. 5, B–E). No specific inhibin {alpha} subunit immunostaining was observed in the interstitial cells and germ cells.



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  		FIG. 5. Immunohistochemical localization of inhibin {alpha} subunit in the testis of B) 7-, C) 21-, D) 60-, and E) 120-day-old bull calves. A) The absorption control for inhibin {alpha} subunit. Counterstaining with hematoxylin was also performed. Black lines in the bottom right corner of each panel represent 50 µm. Note the intense immunopositive staining within the solid seminiferous cords at 7, 21, 60, and 120 days of age

Immunoblot Analysis of Testicular Homogenate (Fig. 6)

Immunoblot analysis using the {alpha} subunit antibody detected the presence of a 25.5-kDa band and a 28.5-kDa band in testicular homogenates from bulls of all ages. The ßB subunit-specific antibody detected primarily the 28.5-kDa band.



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  		FIG. 6. Western blot of testicular homogenates from bull calves at 7, 21, 60, and 120 days of age using the {alpha} subunit (lanes 1–5) or ßB subunit-specific antibody (lanes 6–9) as probe. Lane 1, purified bovine inhibin A; lanes 2 and 6, samples at 7 days of age; lanes 3 and 7, 21 days of age; lanes 4 and 8, 60 days of age; lanes 5 and 9, 120 days of age. Note the presence of the 28.5-kDa band cross-reacted with both the {alpha} and ßB subunit-specific antibodies.

DISCUSSION

The main findings of our study demonstrated that 1) there was no direct inverse correlation between plasma immunoreactive inhibin and FSH from 4 to 13 wk of age; 2) passive immunoneutralization of bull calves against inhibin at 7, 21, 60, and 120 days of age resulted in a significant increase in plasma FSH, although the greatest increase in FSH was observed in 60- and 120-day-old bull calves; 3) inhibin {alpha} subunits were found exclusively in Sertoli cells; and 4) inhibin B was present in testes of bull calves throughout early development.

Concentrations of plasma FSH are similar to results previously published [1–3]. Immunoreactive-inhibin levels showed a tendency to rise in parallel with increasing FSH: this suggests little contribution of inhibin to the regulation of FSH during early development of bull calves. However, injection of inhibin antiserum at 7, 21, 60, and 120 days of age produced a significant increase in plasma FSH, without alteration of LH concentrations. The results provide clear evidence that inhibin has a physiological role in the regulation of FSH secretion during the early stages of development. The above results coupled with previous findings that immunization of bulls against inhibin increased FSH secretion from later prepubertal to pubertal periods [14–17] indicate that inhibin functions as a regulator for FSH secretion from infancy to adulthood. However, androgens also have a negative feedback role in regulation of FSH secretion in bulls after 24 wk of age [2].

The FSH response to inhibin immunization was the greatest at 60 and 120 days compared to those at 7 and 21 days, although antiserum was effective in neutralizing endogenous inhibin at each age based on data about inhibin antibody titer in the circulation. This result suggests either that the inhibin system has not fully matured yet, probably due to low production of dimeric inhibin or low pituitary sensitivity to inhibin, or that amounts of releasable FSH in the pituitary are small, owing to low stimulatory input from the hypothalamus. With regard to the former aspect, it is necessary to develop a sensitive assay for dimeric inhibin of cattle. Around 6–12 wk of age, the number of GnRH pulses [7] and GnRH receptors [32] increases in bulls. The FSH response to GnRH administration at 1 mo of age is smaller than in other older animals [3]. These previous findings coupled with the present results that endogenous inhibin should have been neutralized support the latter suggestion.

Fetal [33] and prepubertal [34] bovine testes contain inhibin bioactivity. In the present study, Western blot analysis using {alpha} subunit-specific antibody revealed the presence of 28.5-kDa and 25.5-kDa bands in the testicular homogenates isolated from bulls at 7, 21, 60, and 120 days of age. The ßB subunit-specific antibody recognized the 28.5-kDa band but not the 25.5-kDa band. The 28.5-kDa protein is therefore likely to be inhibin B, with a molecular weight similar to that reported previously in the rat and human follicular fluid [20]. In bovine fetal testis, {alpha} and ßA subunit mRNA was detected [33] and a 30-kDa protein with FSH-suppressing activity was identified [35]. In this study, the presence of 30- to 32-kDa inhibin A was not examined because of the absence of a sensitive ßA subunit-specific antibody. The 26-kDa protein, detected in testicular homogenates in the present study is most likely to be pro-{alpha}C, which is also in bFF [36–38]. Based on our immunoblot results, coupled with immunohistochemical detection of inhibin {alpha} subunits in Sertoli cells, it appears that Sertoli cells have the ability to produce inhibin B and pro-{alpha}C throughout early prepubertal development.

Castration of bull calves resulted in increased FSH concentrations in early prepubertal stages [9, 10, 39], indicating that testes exert inhibitory effects on FSH secretion at this time. Together with the present results that inhibin immunization increased FSH secretion and that inhibin B was detected in testes using Western blot and immuhistochemical analyses, inhibin of testicular origin acts as a long loop feedback system controlling FSH secretion throughout early prepubertal development. However, there are data that indicate that inhibin {alpha} and ßB proteins are expressed in rat pituitary [40] and may locally modify FSH secretion in vitro [41]. Neutralization of pituitary inhibin also may be involved in the rise in FSH secretion in the present study.

The physiological role of FSH during prepubertal development of bulls is unclear. Proliferation of pre-Sertoli cells is associated with the initial increase in FSH during early prepubertal development, and pre-Sertoli cells differentiate into Sertoli cells just after the early FSH rise [4, 5]. In rats, just before the onset of maximal Sertoli cell proliferation in the fetal period, administration of FSH antiserum or removal of the hypothalamus-pituitary system by decapitation attenuated the activity of Sertoli cell proliferation [42]. Further, addition of FSH to cultures of testes after decapitation restored the proliferation activity of Sertoli cells, suggesting that FSH is important in establishing the number of Sertoli cells in rats. In daily cows, the number of Sertoli cells is highly correlated with daily sperm production [43, 44]. Taken together, it is likely that inhibin acts as a regulator of Sertoli cell differentiation and population via controlling FSH secretion during early prepubertal development, which in turn influences sperm production in mature bulls. Recent evidence also supports an autocrine and paracrine action of inhibin in the regulation of spermatogenesis. Intratesticular injection of bFF reduced the number of spermatogonia in the treated testis of adult mice and hamsters with no effect on the contralateral testis [45]. Inhibin treatment of rat seminiferous tuble segments reduced DNA synthesis in spermatogonia [46]. These findings raise the possibility that inhibin may have direct effects on developing testis in bulls.

In summary, the present results demonstrated that passive immunoneutralization of bulls against inhibin during the early prepubertal period resulted in a significant increase in plasma FSH and that testes have the ability to have produce inhibin B throughout early development. Testicular inhibin is involved in controlling FSH secretion during early prepubertal development and may act as a regulator of Sertoli cell differentiation and population.

ACKNOWLEDGMENTS

We are grateful to Dr. J.J. Ireland, Department of Animal Science, Michigan State University, for reading the original manuscript and for valuable suggestions. We are grateful to the USDA Animal Hormone Program, Germplasm and Gamete Physiology Laboratory, Beltsville Agricultural Research Center, Beltsville, MD, for providing RIA materials for bovine FSH and LH; Dr. K. Wakabayashi, Hormone Assay Center, Institute of Endocrinology, Gunma University, Maebashi, Japan, for providing antiserum to rabbit {gamma}-globulin.

FOOTNOTES

First decision: 21 November 2000.

1 This work was supported by the Ministry of Agriculture, Forestry and Fisheries. Back

2 Correspondence: Hiroyuki Kaneko, Department of Genetic Resources II, National Institute of Agrobiological Resources, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan. FAX: 81 298 38 7408; kaneko{at}abr.affrc.go.jp Back

3 Current address: National Institute of Animal Industry, Inashikigun, Ibaraki 305-0901, Japan. Back

Accepted: February 28, 2001.

Received: October 23, 2000.

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