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BOR - Papers in Press, published online ahead of print December 27, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.012856
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BIOLOGY OF REPRODUCTION 68, 1918–1925 (2003)
DOI: 10.1095/biolreprod.102.012856
© 2003 by the Society for the Study of Reproduction, Inc.

Testis

Molecular Weight Forms of Inhibin A and Inhibin B in the Bovine Testis Change with Age1

H. Kaneko2,3, J. Noguchi3, K. Kikuchi3, and Y. Hasegawa4

Genetic Diversity Department,3 National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan Laboratory Animal Science,4 School of Veterinary Medicine and Animal Science, Kitasato University, Towada, Aomori 034-8628, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To investigate alterations in the molecular weight forms of inhibin in bull testis from the infantile (4–5 wk of age) to postpubertal (49–56 wk of age) periods, testicular homogenates were obtained from animals of various ages and fractionated by a combination of immunoaffinity chromatography and SDS-PAGE. Subsequently, the fractions eluted from the SDS gels were assayed for total inhibin, inhibin A, and inhibin B by fluoroimmunoassay or immunofluorometric assays (IFMAs) and for inhibin bioactivity by an in vitro bioassay. The molecular mass patterns of inhibin A and inhibin B in the testis, as determined by the dimer-specific IFMAs, showed the presence of a peak of approximate 47 kDa until 21–26 wk of age. However, the peak disappeared after 31–32 wk of age. As bulls aged, especially after 31–32 wk of age, inhibin A and inhibin B levels increased in the molecular mass region of 27–34 kDa. Total inhibin showed two peaks, of between 20 and 26 kDa and at approximately 47 kDa, until 21–26 wk of age and a single peak between 20 and 30 kDa after 31–32 wk of age. The eluted fractions corresponding to 29, 31, or 47 kDa gave a dose-response curve that was parallel to the curve generated with 32-kDa inhibin A or 29-kDa inhibin B standard in the IFMA for inhibin A or inhibin B. The fractions corresponding to 29 and 31 kDa suppressed basal release of FSH from rat pituitary cells, but the 47-kDa fraction had a lower FSH-suppressing activity. In the testes of older bulls, immunoblot analysis revealed the presence of a 29-kDa band cross-reacting with inhibin {alpha} and inhibin ßB antibodies and of a 31-kDa band cross-reacting with inhibin {alpha} and inhibin ßA antibodies. The 47-kDa band was recognized by the {alpha}, ßA, and ßB antibodies. Immunohistochemisty of the testis at each age showed that inhibin {alpha} subunits were found exclusively in Sertoli cells, but the intensity of immunostaining diminished in older bulls, in parallel with the decrease in the testicular concentrations of total inhibin. We conclude that 1) bovine Sertoli cells produce both inhibin A and inhibin B, 2) inhibin production in Sertoli cells during the prepubertal period is characterized by the 47 kDa inhibin-related material that contains precursor forms of inhibin A and inhibin B, and 3) the proportion of the mature forms of inhibin A and inhibin B increases as bulls age, although total inhibin production in Setroli cells decreases.

inhibin, Sertoli cells, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inhibin is a dimeric protein composed of an {alpha} subunit and either one of two ß subunits (ßA or ßB = inhibin A or inhibin B, respectively). Inhibin is synthesized as two precursor chains that are linked by disulfide bonds, and the largest precursor form (105 kDa) is processed successively to form the lowest molecular mass form (29–32 kDa) through the smaller 55-, 65-, and 95-kDa forms [1, 2]. Several forms of the free {alpha} subunit ({alpha}N{alpha}C, pro-{alpha}C, and {alpha}C) have been identified [3–5] in follicular fluid. The most well-established role of inhibin in male animals is that of an endocrine hormone that suppresses secretion of FSH. In bulls, immunization studies revealed that inhibin functions as a negative regulator of FSH secretion from the early prepubertal period [6], but the endocrine significance of inhibin becomes greater as bulls age [6–10]. In addition to its endocrine role, inhibin may also act as a paracrine/autocrine regulator of testicular functions. For example, inhibin reversed the action of activin on testosterone production in primary cultures of Leydig cells [11]. Intratesticular treatment with bovine follicular fluid (bFF) or inhibin preparation reduced the number of spermatogonia in mice and hamsters [12] and the number of round spermatids in rats [13]. In vitro, inhibin treatment of rat seminiferous tubule segments reduced DNA synthesis in spermatogonia [14].

Clarification of the molecular weight profile of inhibin is important for further investigation of the role of inhibin in gonadal functions. The molecular weight distribution of inhibin changes in relation to various physiological conditions in human follicular fluid [15] and in human female plasma [16–18], suggesting that various inhibin forms are regulators or markers of gonadal functions. In contrast to studies in females [1–5, 19], only a limited number of studies have investigated the molecular weight forms of inhibin present in the testis [20]. Inhibin B forms of 26–36 kDa and 110 kDa were found in the circulation of the neonatal to adult male rhesus monkey [21]. However, little is known about whether the molecular weight profile of inhibin in the testis alters in response to testicular development. Inhibin B is the major type of dimeric inhibin produced in the testis in humans [22], primates [23], rats [24], hamsters [25], and pigs [26], but it is not known whether bovine Sertoli cells produce inhibin A or/and inhibin B.

The aim of this study was to clarify whether the molecular weight forms of dimeric inhibin(s) change during testicular development in bulls. We used immunoaffinity chromatography and SDS-PAGE to isolate various molecular weight forms of inhibin from testes. We then identified inhibin molecules by specific immunoassays, immunoblotting, and bioassay.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Samples

Protocols for the use of animals in the present study were approved by the Animal Care Committee of National Institute of Biological Sciences. To study alterations in production and the molecular nature of inhibin during postnatal development, testes were surgically obtained from 10–15 Japanese black cattle at each stage of maturation (4–5, 7–9, 15, 21–25, 31–32, 39–42, and 49–56 wk of age) under tranquilization with xylazine (Celactal; Bayer Japan Co., Tokyo, Japan). Testes were homogenized in a 10-fold concentration of Tris-buffered saline (TBS; 0.05 M Tris-HCl, pH 7.5, 0.15 M NaCl) containing 5 mM EDTA, 0.1% (w/v) 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS; Sigma, St. Louis, MO), and 0.05% NaN3 and then stored at -80°C. For immunohistochemistry, testes were obtained from two bulls at 5, 9, 15, 22, 32, 40, and 50 wk of age, fixed with Bouin solution, and embedded in paraffin.

Isolation of Different Molecular Weight Forms of Inhibin from Testis

Procedure Inhibin was isolated from pooled testicular homogenates, which were prepared from 10–15 bulls of each age, by using two-cycle immunoaffinity chromatography with a modification of the methods of Miyamoto et al. [27]. The globulins were obtained from inhibin {alpha} antiserum (GB, [28]) by precipitation with saturated ammonium sulfate (40%, w/v) and were coupled (~25 mg protein/ml gel) to an agarose gel (Affi-Gel 10; Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's recommendations. Testicular homogenates (300 ml/trial) were applied to a column packed with the coupled gel (75 ml) at a flow rate of 0.3–0.4 ml/min at 4°C. The column was washed twice with five column volumes of cold TBS containing 5 mM EDTA and 0.1% CHAPS. Inhibin was eluted with five column volumes of cold 0.1 M glycine-HCl (pH 2.0) containing 0.1 mM PMSF, and the eluate was immediately neutralized with 2.0 M Tris-HCl (pH 8.5) and stored at -80°C. The pooled eluate for each age group was concentrated to 2–3 ml by ultrafiltration (cutoff 10 000, Ultrafiltration Membrane; Millipore Corp., Bedford, MA) at 4°C and then applied to a column packed with 1 ml of N-hydroxysuccinimide-activated Sepharose (HiTrap NHS-activated; Amersham Biosciences, Piscataway, NJ) to which 1 mg of purified {alpha} subunit antibody (GB) had been attached [6]. The final eluate was concentrated with a centrifugal filter device (cutoff 10 000, Ultrafree; Millipore) and was subjected to 12.5% SDS-PAGE. Rainbow-colored molecular mass markers (14.3–220 kDa; Amersham) were used to estimate the sizes of proteins. The gel was cut into 2.0-mm slices, and inhibin was extracted from each gel slice with 2 ml of TBS containing EDTA and CHAPS under gentle shaking overnight at 4°C. The gel eluates were stored at -80°C for subsequent inhibin immunoassays, bioassay, or immunoblotting. For immunoassays, aliquots of the immunoaffinity extracts containing 10 µg of total inhibin as determined by fluoroimmunoassay (FIA) were subjected to SDS-PAGE. Aliquots of the extracts containing 200 µg of total inhibin at 4–5 and 50 wk of age were applied to SDS-PAGE for subsequent immunoblotting and bioassay.

Validation To examine whether modifications in the molecular weight profile of inhibin occur during the steps of immunoaffinity chromatography, homogenates of testes at 5 and 50 wk of age were applied directly to SDS-PAGE. The molecular weight patterns of inhibin in the resulting eluted fractions were compared with those in the eluted fractions obtained after the application of the immunoaffinity testicular extracts to SDS-PAGE. Recovery of inhibin from 50-wk-old testes was also estimated by the Tr-FIA for total inhibin or by spectrophotometry (DV 7500; Beckman Instruments, Palo Alto, CA; optical density = 280 nm) during the fractionation procedures.

FIA for Total Inhibin

Concentrations of total inhibin were determined by a competitive immunoassay using europium (Eu)-labeled inhibin A as a probe [29]. In the FIA of total inhibin, anti-bovine inhibin serum (TNDH-1 [30]) was used as a primary antibody. Bovine 32-kDa inhibin A was used for Eu labeling and as a reference standard (anti-inhibin serum was provided by Dr. Taya, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan). The detection limit of the FIA was 0.078 ng/ml. The intra- and interassay coefficients of variation (CVs) were 11.0% and 15.5%, respectively.

Immunofluorometric Assay for Bovine Inhibin A

Concentrations of inhibin A were determined by an immunofluorometric assay (IFMA) using purified polyclonal antibodies to the {alpha} and ßA subunits of bovine inhibin A as described previously [31]. Bovine 32-kDa inhibin A purified from bFF [32] was used as a reference standard. The sensitivity of the inhibin A IFMA was 3.3 pg/ml. The intra- and interassay CVs were 7.5% and 10.2%, respectively.

IFMA for Bovine Inhibin B

Assay procedure Concentrations of inhibin B were determined by an IFMA using purified polyclonal antibodies to the {alpha} and ßB subunits of inhibin by a modification of the inhibin A IFMA [31]. Porcine 29-kDa inhibin B purified from follicular fluid by the methods described previously [32] was used as a reference standard. Antibody against the ßB subunit (G544) was purified from a goat antiserum against amino acids 84–99 of the ßB subunit of porcine inhibin B by two-cycle immunoaffinity chromatography. The affinity column used in the present study was a 5-ml prepacked column of N-hydroxysuccinimide-activated Sepharose (HiTrap NHS-activated; Amersham) to which 5 mg of amino acids 84–99 of the ßB subunit were attached. The purified ßB antibody was adsorbed onto wells of a 96-microwell plate (FluoroNunc Modules; Nalge Nunc International, Rochester, NY) by incubating each well with 100 µl of the antibody preparation diluted to a concentration of 10 µg/ml with coating buffer (0.05 M K2HPO4 containing 0.15 M NaCl and 0.05% (w/v) NaN3, pH 8.9). Triplicate aliquots (100 µl) of standards (0.78–100 ng/ml) and unknown samples were pipetted into ßB antibody-coated wells. The final volume for each well was adjusted to 200 µl with assay buffer (TBS containing 0.05% (w/v) BSA, 0.1% (w/v) bovine {gamma}-globulin, 0.05% (w/v) NaN3, 0.01% (v/v) Tween 40, 0.0015% (w/v) Phenol Red, and 0.02 M diethylenetriaminepentaacetic acid). After incubation, the wells were washed 12 times with wash buffer (TBS containing 0.1% (w/v) Tween 20 and 0.05% (w/v) NaN3). The {alpha} antibody, labeled with the Eu chelate of N1-(p-isothiocyanatobenzyl)-diethylenetriamine-N1,N2,N3,N3-tetraacetic acid (Eu-Labelling reagent; Wallac Oy, Turku, Finland) [31] and diluted in assay buffer (2 x 106 counts/sec per 100 µl), was added to each well. Thereafter, wells were incubated for 3 h at 25°C. After rinsing (12 times), 100 µl of enhancement solution was added to each well, and the wells were shaken for 5 min. Fluorescence was measured with a fluorometer (1234 DELFIA fluorometer; Wallac Oy).

Validation Specificity of the inhibin B IFMA was assessed by comparing the fluorescence intensity obtained in the presence of various inhibin-related proteins. The IFMA recognized porcine inhibin B from a concentration of 0.39 ng/ml to >100 ng/ml, whereas inhibin A, activin A, activin AB, activin B, and pro-{alpha}C, which were purified from bFF [32, 33], showed low cross-reactivity (<2%) (Fig. 1). The intra- and interassay CVs were 6.5% and 9.4%, respectively.



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  		FIG. 1. Dose-response curves of various inhibin-related proteins in the inhibin B IFMA. Materials tested were 29-kDa inhibin B (•) purified from porcine follicular fluid and inhibin A ({circ}), activin A ({triangleup}), activin AB ({blacktriangledown}), activin B ({square}), and pro-{alpha}C ({blacktriangleup}) purified from bFF

Immunoblotting

Immunoaffinity extracts of testes before fractionation by SDS-PAGE and eluted fractions after SDS-PAGE were subjected to immunoblotting as described previously [6]. The amount of total inhibin subjected to SDS-PAGE, as determined by the FIA, was approximately 150 ng/lane for blots of the immunoaffinity extracts before fractionation probed with the inhibin {alpha} antibody [6]. For the eluted fractions, 5 ng/lane was used when probed with the {alpha} antibody, 60–90 ng/lane when probed with the inhibin ßA antibody (R105 [31]), and 200–300 ng/lane when probed with the inhibin ßB antibody (R205) raised against amino acids 84–99 of the ßB subunit of porcine inhibin B in a castrated rabbit. The ßA antibody was highly specific for activin A (ßA-ßA dimer) and the ßB antibody was highly specific for activin B (ßB-ßB dimer) (Fig. 2).



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  		FIG. 2. Immunoblotting of bovine activin A and activin B with the purified ßA antibody (R105) or ßB antibody (R205). Note the specificity of the antibodies

Inhibin Bioassay

Inhibin bioactivity was determined with an in vitro bioassay for suppression of spontaneous FSH release from cultured rat anterior pituitary cells, as reported previously [34]. Aliquots of gel fractions containing 300 ng of total inhibin were concentrated to approximately 100 µl with a centrifugal filter device (cutoff 10 000, Centricon; Millipore) and then dialyzed against 0.85% (w/v) NaCl for 24 h at 4°C to remove SDS. The gel fractions were made up to 1 ml with culture medium. Samples were diluted with culture medium, and aliquots (100 µl) of the diluted samples were added to the dispersed pituitary cells. The final concentration of total inhibin in the samples was 9.4–150 ng/ml. Bovine inhibin A was used as a reference standard. After 3 days of incubation, 100-µl aliquots of the resulting incubation medium were subjected to FIA for rat FSH using anti-rFSH-S-11 as a primary antibody, rFSH-I-9 for Eu labeling, and rFSH-RP-2 as a reference standard. (Rat FSH RIA kit was provided by Dr. A.F. Parlow, National Hormone and Peptide Program, Harbor-UCLA Medical Center, Torrance, CA.)

Immunohistochemistry

For antigen retrieval, the sections were autoclaved in 0.01 M sodium citrate buffer (pH 6.0) at 121°C for 15 min. The other immunohistochemical procedures using the purified {alpha} antibody (GB) have been described elsewhere [6, 35]. As a negative control, sections obtained from a 32-wk-old bull were incubated with the {alpha} antibody, which had been preabsorbed with excessive inhibin, and were subjected to staining procedures as described previously [6].

Statistics

Changes in testicular concentrations of total inhibin were subjected to one-way ANOVA. When a significant effect was obtained with the ANOVA, the significance of the difference between means was determined by Duncan multiple range test. All data were analyzed with the general linear models procedure of the Statistical Analysis System (SAS Inc., Cary, NC) [36]. Differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Validation of the Purification Procedure

The molecular weight patterns of inhibin in the testis obtained after the direct application of testicular homogenate to SDS-PAGE were similar to those obtained after the application of immunoaffinity testicular extracts to SDS-PAGE (data not shown). After the first cycle of immunoaffinity chromatography, a total of 383 µg of total inhibin was obtained from testicular homogenates from animals at 50 wk of age (899 µg total inhibin/9 L homogenate). A total of 240 µg total inhibin was extracted by the rechromatography. The ratio of total inhibin:total protein was 0.21% after the first immunoaffinity chromatography and increased to 27.4% after the rechromatography.

Testicular Inhibin Concentration

Testicular concentrations (mean ± SEM) of total inhibin were high at 4–5 wk of age (13.9 ± 0.7 µg/g wet tissue) but were significantly (P < 0.05) decreased at 15 wk of age (5.4 ± 1.2 µg/g wet tissue). The levels of total inhibin further declined to 2.8 ± 0.04 µg/g at 21–25 wk of age.

Molecular Weight Distribution of Inhibins in the Testes of Developing Bulls

At each age, a similar amount of total inhibin (6.4–7.1 µg) was eluted from each SDS gel after overnight shaking. In bull testes at 4–5 wk of age, high levels of total inhibin were observed between 20 and 26 kDa and at approximately 47 kDa (Fig. 3a). At this age, both inhibin A and inhibin B IFMAs detected the 47-kDa peak, whereas inhibin A IFMA also recognized the 23- and 31-kDa peaks. The 47-kDa peak was not detected by the three different assays at 31–32 wk of age (Fig. 3b). At 49–56 wk of age, inhibin A and inhibin B IFMAs identified 31- and 29-kDa peaks, respectively (Fig. 3c). Figure 4 shows sequential changes in the proportion of inhibin A and inhibin B in the defined molecular mass regions (27–34 and 40–50 kDa) compared with the overall inhibin A and inhibin B recovered from the gels. The proportion of inhibin A and inhibin B between 40 and 50 kDa was constant until 21–25 wk of age but decreased at 31–32 wk. In contrast, the proportion of inhibin A and inhibin B in the region of 27–34 kDa increased as bulls aged.



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  		FIG. 3. Molecular weight distribution of total inhibin, inhibin A, and inhibin B in bull testes at 4–5 (a), 31–32 (b), and 49–56 (c) wk of age. Testicular homogenates were processed by a combination of immunoaffinity chromatography and SDS-PAGE. The vertical broken lines represent molecular weights and positions of marker proteins used to calibrate the gel.



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  		FIG. 4. Changes in the proportion of inhibin A (a) and inhibin B (b) in the defined molecular mass regions (27–34 kDa and 40–50 kDa) in bull testes at each age of maturation. Testicular homogenates were processed by a combination of immunoaffinity chromatography and SDS-PAGE

Dose-Response Curves of Eluted Fractions

Serial dilution of the eluted fractions corresponding to 29, 31, and 47 kDa resulted in dose-response curves that were parallel to the standard curve in the IFMA for inhibin A or inhibin B (Fig. 5, a and b).



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  		FIG. 5. Dose-response curves of reference standards (bovine inhibin A, •; porcine inhibin B, {block}) and eluted fractions corresponding to 29 kDa ({square}), 31 kDa ({triangleup}), and 47 kDa ({diamond}) in the IFMAs for inhibin A (a) and inhibin B (b)

Inhibin Bioassay

The eluted fractions corresponding to 29 or 31 kDa showed a dose-dependent suppression of FSH secretion, and the response curves were parallel to the curve generated with bovine 32-kDa inhibin A (Fig. 6). The eluted fractions of 47 kDa showed a lower FSH-suppressing activity.



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  		FIG. 6. Suppression curves for inhibin preparations in the in vitro bioassay. Materials tested were bovine 32 kDa inhibin A (•) and eluted fractions corresponding to 29 kDa ({square}), 31 kDa ({triangleup}), and 47 kDa ({diamond})

Immunoblotting

Immunoblotting using the {alpha} antibody detected the presence of clear bands of 20, 21, 23, 26, 29, 31, 39, 43, and 47 kDa in the extracts obtained from testes with the two-cycle immunoaffinity chromatography (Fig. 7). The 39-, 43-, and 47-kDa bands were prominent until 21–25 wk of age but disappeared at 39–42 wk of age. The 29- and 31-kDa bands became prominent as the bulls aged. The 20-, 23-, and 26-kDa bands were detected consistently throughout the study. The ßA and ßB antibodies recognized the 31- and 29-kDa bands, respectively (Fig. 8a), and the 47-kDa band also showed a positive reaction to the ßA and ßB antibodies (Fig. 8b).



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  		FIG. 7. Immunoblotting of immunoaffinity extracts of bull testes (before fractionation) at various ages using the purified inhibin {alpha} antibody. Lane 1: 4–5 wk of age; lane 2: 7–9 wk of age; lane 3: 21–25 wk of age; lane 4: 31–32 wk of age; lane 5: 39–42 wk of age; lane 6: 49–56 wk of age



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  		FIG. 8. Immunoblotting of eluted fractions from the SDS gels corresponding to 29–31 kDa (a) and 47 kDa (b). As a probe, purified inhibin {alpha}, ßA, or ßB antibodies (A/{alpha}, A/ßA, or A/ßB) were used. a) Lanes 1 and 4: 31-kDa fraction at 4–5 wk of age; lanes 2 and 5: 31-kDa fraction at 49–56 wk of age; lanes 3 and 6: 29-kDa fraction at 49–56 wk of age. b) Lanes 1–3: 47-kDa fraction at 4–5 wk of age

Immunohistochemistry

Clear immunostaining for the {alpha} subunits was found in the cytoplasm of Sertoli cells between 5 (Fig. 9a) and 15 wk of age. The immune reaction became weak between 22 and 32 wk of age (Fig. 9b), but Sertoli cells remained positive to the {alpha} antibody at 50 wk of age (Fig. 9c). No specific inhibin {alpha} immunostaining was observed in the cytoplasm of interstitial cells and germ cells. Preabsorption of the {alpha} antibody abolished immunostaining (Fig. 9d).



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  		FIG. 9. Immunohistochemical localization of inhibin {alpha} in the testis at 5 wk (a, bar = 25 µm), 32 wk (b), and 50 wk (c) of age, and the absorption control for inhibin {alpha} (32-wk-old testis) (d). Counterstaining with hematoxylin was also performed. Arrows indicate Sertoli cells at 50 wk of age


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The significant findings of our study are that 1) inhibin A and inhibin B were present in bull testis from the infantile to postpubertal periods, 2) infantile bull testis contained a significant amount of the precursor form of inhibin-related material, which contained inhibin A and inhibin B, 3) a marked increase in the proportion of the mature form of inhibin A and inhibin B occurred in the testes of adult bulls, and 4) inhibin {alpha} was exclusively found in Sertoli cells until the postpubertal period, although the immunoreaction diminished as the bulls aged, consistent with a decrease in total production of inhibin.

The decrease in testicular concentration of total inhibin with age is consistent with the previous finding of Matsuzaki et al. [37]. However, the present study is the first to demonstrate that the molecular weight distribution of inhibin changes during testis development. The 29- and 31-kDa forms, which were prominent in adult testis, are similar in size to 30-kDa inhibin purified from bovine fetal testes [20]. Analysis by specific IFMAs and immunoblotting in the present study clearly indicates that the 29-kDa form is inhibin B and the 31-kDa form is inhibin A. This designation was confirmed by the results of the in vitro bioassay showing that eluted fractions corresponding to 29 and 31 kDa had FSH-suppressing activity. These materials are likely to correspond to the mature forms of inhibin previously described in bovine [38] and human [39] ovaries. The presence of inhibin A and inhibin B in bull testis is consistent with the previous report that both ßA and ßB subunits can be detected immunologically in seminiferous tubules in adult bulls [40]. In contrast to the 29- to 31-kDa fraction, the 47-kDa fraction has low inhibin bioactivity. A precursor form of the free {alpha} subunit that is of similar size (45 kDa; pro-{alpha}N{alpha}C) but is devoid of biological activity has been isolated from bovine fetal testis [20] and bFF [3]. However, the 47-kDa material was recognized by both inhibin A and inhibin B IFMAs and cross-reacted with antibodies against {alpha}, ßA, and ßB subunits by immunoblotting, which indicate that the 47-kDa material contains dimeric inhibin A and inhibin B. The most likely explanation is that the 47-kDa material, which was specifically found in the testis until 21–25 wk of age, is a combination of precursor forms of inhibin A, inhibin B, and the free {alpha} subunit.

The structure of the 47-kDa dimeric form is unclear. Variation in glycosylation of the {alpha} subunit produces inhibin variants of various sizes [2, 41], which suggests the possibility that the 47-kDa dimeric form is deglycosylated 55-kDa precursor form. Generation of the 47-kDa form may be attributed to deletion of the terminal sequence of the N-terminal region of the {alpha} subunit. Good et al. [19] identified a material of similar size (49 kDa) that cross-reacts with {alpha} and ßA subunits in bFF. In contrast to the shift in the molecular masses of dimeric inhibin, 19.9- to 26-kDa forms except for the 20.7-kDa form were consistently present during testis development. These materials are similar in size to the 20-kDa {alpha}C, 23-kDa {alpha}C, and 26-kDa pro{alpha}C described by others [2, 3–5].

The age-related changes in the intensity of the immune reaction of inhibin {alpha} in Sertoli cells were parallel to changes in testicular concentrations of total inhibin, which indicates that these cells are the major production site of inhibin in the bull testis from the infantile to postpubertal periods. These results extend the previous findings that the {alpha} subunits are localized in prepubertal Sertoli cells [6, 37, 40, 42]. In contrast to the findings that the testes of most species, such as humans [22], nonhuman primates [23], rats [24], hamsters [25], and pigs [26], largely produce inhibin B, the present results indicate that bovine Sertoli cells produce both inhibin A and inhibin B. In bulls, morphologically undifferentiated pre-Sertoli cells are dominant in seminiferous tubules until 24 wk of age, whereas a significant number of matured Sertoli cells are found after 24–28 wk of age, concomitant with formation of the tubule lumen [43, 44]. The present finding that the molecular weight profile of inhibin in bull testis progresses in an age-dependent manner suggests that the differentiation processes of bovine Sertoli cells include changes in the molecular weight forms of dimeric inhibins that they produce. An age-related change in the processing of the {alpha} subunit is most likely to be involved in this change.

The physiological roles of the various molecular weight forms of dimeric inhibin are not clear. With respect to the endocrine role of inhibin in bulls, inhibin immunization raised FSH secretion between 1 and 3 wk of age, but the FSH response after immunization was much smaller than that in more developed animals [6]. This finding suggests that the inhibin system controlling FSH secretion is not fully functional during infancy. The present results indicate that the infantile testis contains high levels of total inhibin, but the proportion of the mature forms of dimeric inhibins was much lower than that in older bulls. It is unclear whether inhibin forms in the circulation are similar to those in the testis; however, the low proportion of the mature inhibin may be responsible in part for the deficiency in FSH regulation by inhibin. Although total inhibin production in the testis became much lower after the pubertal period, inhibin plays a significant role in FSH regulation [7, 8, 10], which is consistent with the increased proportion of mature inhibin A and inhibin B. Until 28 wk of age, gonocytes transfer to the basement membrane of the tubules and differentiate to spermatogonia [43]. 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 [12]. Inhibin treatment of rat seminiferous tubule segments reduced DNA synthesis in spermatogonia [14]. These findings suggest that inhibin may have direct inhibitory effects on spermatogenesis in bulls. A low proportion of bioactive inhibin may favor the differentiation of gonocytes.

To test the possibility that artifacts appearing during the isolation process may have been responsible for the changes in molecular weight distribution of inhibin in developing testis observed in this study, we fractionated testicular homogenates at 5 and 50 wk of age directly through SDS-PAGE. The molecular weight patterns of inhibin obtained after direct fractionation of testicular homogenates were similar to those obtained after application of immunoaffinity extracts to SDS-PAGE. Therefore, the observed changes in inhibin were probably not artifactual.

The combination of immunoaffinity chromatography and SDS-PAGE was used to isolate inhibin from the testes of very young to adult bulls. Subsequent specific IFMAs for inhibin A and inhibin B and immunoblot analysis revealed that bull testis produces inhibin A and inhibin B and that the molecular weights forms of these dimeric inhibins change during testicular development.


   ACKNOWLEDGMENTS
 
We thank Dr. A.F. Parlow (Science Director of National Hormone and Peptide Program of National Institute of Diabetes and Digestive and Kidney Diseases, Harbor-UCLA Medical Center, Torrance, CA) for providing the rat FSH RIA kit. We also thank Dr. K. Taya (Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan) for providing anti-bovine inhibin serum (TNDH-1) for Tr-FIA. We thank Ms. T. Aoki and Ms. E. Yamauchi for technical assistance.


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

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

Received: 29 October 2002.

First decision: 14 November 2002.

Accepted: 16 December 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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