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Endocrinology Vol. 145, No. 7 3532-3541
Copyright © 2004 by The Endocrine Society

Changes in Circulating and Testicular Levels of Inhibin A and B and Activin A During Postnatal Development in the Rat

Jeremy J. Buzzard, Kate L. Loveland, Moira K. O’Bryan, Anne E. O’Connor, Marilyn Bakker, Tetsuo Hayashi, Nigel G. Wreford, John R. Morrison and David M. de Kretser

Monash Institute of Reproduction and Development (J.J.B., K.L.L., M.K.O., A.E.O., M.B., T.H., J.R.M., D.M.d.K.), Australian Research Council Centre of Excellence in Biotechnology and Development (K.L.L., M.K.O., D.M.d.K.), and Department of Anatomy and Cell Biology (J.J.B., N.G.W.), Monash University, Clayton, 3168 Melbourne, Australia

Address all correspondence and requests for reprints to: David M. de Kretser, Monash Institute of Reproduction and Development, 27-31 Wright Street, Clayton, Victoria 3168, Australia. E-mail: david.de.kretser{at}med.monash.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study describes the testicular levels of inhibin/activin subunits by Northern analysis and in situ hybridization and serum and testicular levels of inhibins A and B and activin A by enzyme linked immunosorbent assays (ELISA) during postnatal development in the rat. We show that serum inhibin A levels are less than 4 pg/ml throughout postnatal life. Serum inhibin B levels peak at 572 ± 119 pg/ml (mean ± SE) at d 40 post partum (pp) before falling to 182 ± 35 pg/ml in mature males. Serum activin A decreases from 294 ± 29 pg/ml at d 6 to 132 ± 27 pg/ml at maturity. Within the testis, inhibin A levels fall from 0.330 ± 0.108 ng/g at d 15 to less than 0.004 ng/g at maturity. Inhibin B levels peak at 43.9 ± 4.2 ng/g at d 6 before falling to 1.6 ± 0.13 ng/g at maturity. Testicular activin A levels fall from 18.6 ± 2.2 ng/g at d 6 to 0.094 ± 0.013 ng/g at maturity. Northern profiles of testicular inhibin/activin subunits correlate with immunoreactive levels demonstrated by ELISA. In situ hybridization suggests that ßA and ßB subunit expression is largely restricted to the seminiferous tubule, particularly Sertoli cells, spermatogonia, and primary spermatocytes. These data support the view that inhibin B is the major inhibin in the male rat and that levels relate to Sertoli cell number and activity. Furthermore, the demonstration of high local concentrations of activin A during the period of Sertoli cell proliferation and the onset of spermatogenesis support its proposed role because a modulator of testicular development and function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ACTIVINS AND INHIBINS are disulfide linked dimeric glycoproteins that have the capacity to modulate FSH secretion by the pituitary (1, 2). Both protein families are formed from common components termed {alpha}- and ß-subunits. Although five distinct ß-subunits have been isolated (termed ßA to ßE), biological activities have been demonstrated for only ßA and ßB. Inhibins are heterodimers of one {alpha}-subunit and a ß-subunit forming inhibin A ({alpha}ßA) and inhibin B ({alpha}ßB). Activins are dimers of two ß-subunits, forming activin A (ßAßA), activin AB (ßAßB), and activin B (ßBßB) (3).

Despite their shared ß-subunits, the biological activities of activins and inhibins are opposed. In normal postnatal male animals, inhibin secretion is restricted primarily to the Sertoli cells in the testis (4, 5, 6) and act on pituitary gonadotropes to suppress FSH secretion (7). Furthermore, inhibin appears to have local effects within the testis to suppress spermatogonial numbers (8). Recent data have indicated that, in the human male, inhibin B is the major circulating inhibin (9), and its levels in circulation are directly related to Sertoli cell number (10).

In contrast, the activins are pleiotropic, affecting a range of processes from early embryonic development through to adulthood (11, 12). Although activin is clearly secreted by the adult testis, castration does not lead to a decline in its circulating levels, indicating the testis contribution to the circulating activin A pool is insignificant (13). The activins oppose the actions of inhibin by stimulating both basal and GnRH-induced pituitary FSH secretion (11, 14). Furthermore, activin has a complex local role in the regulation of gonadal development; activin A can modulate DNA synthesis in the fetal gonad, promoting mitosis in the female and suppressing it in the male (although the cell types affected are not clear) (15). Postnatally, activin A has age-dependent effects on Sertoli cell division in the rat testis (16, 17) and affects the development of germ cells in the male (18, 19, 20). Activin A has been particularly implicated in the modulation of inflammation in the testis, an immune privileged site (21). Collectively, these data highlight the potential significance of the changes in both local and circulating levels of activin A and the inhibins.

Previous studies by this group and others have examined levels of inhibin using a bioassay to measure testicular bioactive inhibin (22) and RIAs to measure both testicular and serum immunoreactive inhibin (23, 24, 25). Many of the inhibin RIAs that have been used for previous studies measure not only dimeric inhibin but also {alpha}-subunit products of unclear biological significance including free {alpha}-subunit and pro-{alpha}C (26). Of similar concern, the inhibin bioassay measures the suppressive effect of fluids or tissue extracts on FSH secretion by rat pituitary cells, and hence the presence of activin or its binding protein follistatin can cause inaccurate biopotencies because activin stimulates FSH and follistatin suppresses it (27, 28). Given these caveats, results from both inhibin RIA and bioassay are potentially misleading.

Recently specific ELISAs have been developed for each of inhibin A (29), inhibin B (30), and activin A (31), which exhibit minimal cross-reactivity with each other or their precursor proteins. These assays allow us to study the secretion of activin A and inhibins without the limited specificity imposed by other assays such as the inhibin RIA (32) or bioassay (27). Unfortunately, no ELISA is yet available for activin B, whereas activin AB levels in the male rat have been previously reported to be below the detection limit of the ELISA (33). Furthermore, despite the highly conserved nature of follistatin between species, it has not been possible to date to measure rat follistatin in serum or culture medium by RIAs that are used to measure concentrations in humans and sheep (34, 35). In the present study, we used the best available techniques and established standards to describe the changes in serum and testicular levels of the inhibins and activin A during sexual maturation of the male rat, the model of choice for studying regulation of testicular development and spermatogenesis. These are evaluated in conjunction with mRNA analyses that localize each subunit mRNA in the testis. The data provide new insights into the role of these proteins in the biology of the testis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male Sprague Dawley rats were obtained from the Monash Central Animal House, and were kept in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (1997, National Health and Medical Research Council, Australia). The study was approved by the Monash Medical Centre Animal Ethics Committee. For ELISAs, rats at d 3, 6, 8 and 10 post partum (pp) were killed by anesthesia on ice followed by decapitation. Rats at day 15, 20, 25, 30, 40, 50, and 90 pp were killed by a lethal ip dose of sodium pentobarbital. Blood was removed by cardiac puncture and left overnight at 4 C before being centrifuged for 30 min at 300 x g; serum was removed and stored at –20 C until required. For Northern analysis, rats at d 3 and 10 were killed by decapitation and rats at d 20 and between 60–90 d were killed by carbon dioxide anesthesia followed by decapitation. For both Northern analysis and ELISA measurements, the testes were removed, weighed, decapsulated, reweighed to account for loss of tissue during decapsulation, snap frozen in liquid nitrogen, and stored at –75 C until required. For in situ hybridization, testes were perfusion fixed in Bouin’s fixative, dehydrated in graded ethanol, and embedded in paraffin. Five-micrometer sections were floated on diethyl pyrocarbonate-treated deionized water and dried onto slides (Superfrost Plus; Biolabs Scientific, Melbourne, Australia). In some experiments, enriched preparations of primary spermatocytes and round spermatids were isolated from the testes of adult rats using techniques previously described and the purity of the germ cell preparations were assessed by morphological assessments using light microscopy of thin sections of resin embedded cell preparations (36).

RNA isolations and Northern analysis
RNA was prepared from d 3, 10, 30, and 90 pp rat testes, enriched elutriation fractions of primary spermatocytes (at least 86% pure) and round spermatids (at least 90% pure), and adult rat ovary using the acid phenol extraction method (37). Thirty micrograms of total RNA per lane was separated on a 1.2% agarose formaldehyde gel and transferred by capillary action onto a Nytran SuperCharge membrane (Schleicher and Schuell, Keene, NH) according to the manufacturer’s instructions.

Random primed, radiolabeled cDNA probes were prepared with 32P-dCTP using the Rediprime II kit (Amersham Biosciences, Buckinghamshire, UK). Probes were prepared from the following cDNA templates: {alpha}-subunit (corresponding to bp 198–496 of the inhibin {alpha}-subunit, accession number: NM_012590.1), ßA-subunit [a 370-bp cDNA specific for the rat inhibin ßA-subunit (38)], ßB-subunit (a 240-bp cDNA corresponding to bp 17–257 of the mouse inhibin ßB-subunit, accession no. X69620.1).

Northern hybridization of membranes with radiolabeled probes were performed at 42 C for 16 h in Ultrahyb hybridization solution (Ambion, Austin, TX) according to the manufacturer’s instructions. The membranes were washed to high stringency in 0.1% saline sodium citrate buffer (SSC), 0.1% sodium dodecyl sulfate at 57–58 C before exposure to x-ray film. Northern assays were performed twice, with quantitation performed on one set of samples for each probe as described below. Quantitative loading normalization was achieved using a cDNA probe directed toward message encoding the S26 ribosomal protein (accession no. X02414). The signal was collected for quantitation on a Molecular Dynamics PhosphorImage analyzer (FLA2000, Fujifilm, Tokyo, Japan) and analyzed using Image Gauge version 3.46.

In situ hybridizations
In situ hybridization using digoxigenin (DIG)-labeled cRNA was used to localize mRNAs encoding the activin/inhibin ßA- and ßB-subunits in adult rat testis sections. Plasmids containing the same cDNAs as used for Northern analysis (shown above) were linearized and used to produce DIG-labeled cRNAs using previously described techniques (39). RNA polymerases T7 and SP6 (Promega, Madison, WI) and DIG-labeled deoxyuridine 5-triphosphate (Roche, Stockholm, Sweden) were used to produce antisense and sense (negative control) cRNAs for each target sequence using protocols and reagents supplied with the DIG-labeling kit. In every experiment the sense cRNA was included in parallel with the antisense cRNA as a negative control. The in situ hybridization procedure was performed as previously described (19).

Serum and testicular extracts
To have sufficient serum to assay, some samples were pooled: d 3 pp and d 6 pp serum was pooled into two aliquots, each containing equal quantities of serum from six rats. Day 8 pp serum was pooled into four aliquots, each containing serum from three rats. Day 10 and 15 pp serum was pooled into six aliquots, each containing serum from two rats. Serum from d 20, 25, 30, 40, 50, and 90 pp rats was isolated from six rats individually at each age. To have sufficient testicular tissue extract to assay, some testes were pooled: 12 rats were used to prepare six isolates from each of d 3, 6, 8, 10, and 15 pp (i.e. four testes per isolate). Six single-rat isolates (i.e. two testes per isolate) were prepared from each of d 20, 25, 30, 40, 50, and 90 pp rats.

Decapsulated testis pools less than 100 mg were homogenized in 100 µl PBS using a handheld plastic homogenizer in a microfuge tube. Testis pools greater than 100 mg were homogenized in an equal volume of PBS in a dounce homogenizer (i.e. 800 mg homogenized in 800 µl). Homogenates were centrifuged at 4 C twice for 30 min at 4000 x g each time and supernatant was frozen at –75 C until required for analysis.

ELISAs
Inhibin A and B concentrations were measured using specific ELISAs (29, 30) according to the manufacturer’s instructions (Oxford Bio-Innovations, Oxfordshire, UK) with the following modifications: standard (inhibin A, World Health Organization 91/624, inhibin B, World Health Organization 96/784) and samples were diluted in 5% BSA in 0.1 M PBS and treated as per the manufacturer’s protocol. Duplicate samples were added to the plates and incubated overnight at room temperature. The plates were washed and the detection antibody (Q1 coupled to alkaline phosphatase) was added for 3 h at room temperature. After washing, the alkaline phosphatase activity was detected using an amplification kit (ELISA amplification system; Invitrogen Ltd., Carlsbad, CA) according to the manufacturer’s instructions; substrate was incubated with the sample for 2 h at room temperature before measurement. For inhibin A assays, the average intraplate coefficient of variation (CV) was 4.2%, the average interplate CV was 8.2% (n = 3) and the limit of detection for the assay was 4 pg/ml. For inhibin B, the intraplate and interplate CVs were 4.1 and 6.6%, respectively (n = 7), and the limit of detection was 5 pg/ml.

Activin A concentrations were measured using a specific ELISA (31) according to the manufacturer’s instructions (Oxford Bio-Innovations) with the following modifications. The standard used was recombinant human activin A as previously described (40). Standards and samples were diluted in 5% BSA in 0.1 M PBS; 6% sodium dodecylsulfate solution was added (3% final concentration) followed by boiling for 3 min to dissociate follistatin-bound activin A. The samples were allowed to cool before the addition of H2O2 (2% final concentration) and a subsequent 30 min incubation. Then 25 µl 5% BSA/0.1 M Tris/5% Triton X-100/0.9% NaCl/0.1% NaN3 was added to each well before the addition of the treated samples. Duplicates were added to the E4 (anti-ßA subunit) monoclonal antibody-coated plate and incubated overnight at room temperature. The plates were washed and the detection antibody (biotinylated-E4) was added for 2 h at room temperature. After washing, alkaline phosphatase linked to streptavidin was added to the wells and incubated at room temperature for 1 h. After further washes, the alkaline phosphatase activity was detected using an amplification kit according to the manufacturer’s instructions; the substrate was incubated for 1 h at room temperature. The intraplate CV was 5.0%, the interplate CV was 10.9% (n = 4), and the limit of detection for the assay was 10 pg/ml.

Previous studies demonstrated low levels of cross-reactivity among these ELISAs. The activin A ELISA has been reported to cross-react with inhibin A (0.5% cross-reactivity), whereas cross reactivity with inhibin B, inhibin pro-{alpha}-C subunit, and follistatin are all less than 0.1% (31). The inhibin B ELISA cross-reacts with inhibin A (0.5% cross-reactivity), whereas cross-reactivity with activin A, inhibin pro-{alpha}-C, and follistatin are all less than 0.1% (30). The inhibin A ELISA does not cross-react (i.e. <0.1%) with inhibin B or free {alpha}-subunits (41).

Sertoli cell number approximations
Numbers of Sertoli cells present in d 10, 15, 20, 25, 30, and 40 pp testes were determined from previously published stereological data from the same colony of Sprague Dawley rats (42). Numbers of Sertoli cells present in d 3, 6, 8, 50, and 90 pp testes were interpolated from numbers present at d 1, 5, 10, 48, and 70 pp: cell cycle duration was determined using the equation, tcell cycle = ({Delta}t)/[log2 (n2/n1)], where {Delta}t is the time between counts (e.g. 4 d between d 1 and d 5 pp), n1 is the cell number at the time of first count, and n2 is the cell number at the time of second count. It was assumed that the cell cycle duration between two cell counts was constant. Sertoli cell number was then approximated from the equation, napprox = n1 x 2x, where n1 is the known cell number (at time t1), and x = (tapprox – t1)/tcell cycle.

Statistics
ELISA data were analyzed using one-way ANOVA combined with post hoc tests for linear trend and Bartlett’s tests for equal variances to test variation from linearity. When concentrations were below the limit of detection for the assay, for statistical purposes data were analyzed as whether the average concentration was the limit of detection (i.e. 4 pg/ml for inhibin A, 5 pg/ml for inhibin B, and 10 pg/ml for activin A) and the SE was equal to half the limit of detection.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Testis weights and Sertoli cell number approximations
Testis weights increased progressively from d 3 (6.87 mg) until d 90 (1.9 g). Sertoli cell numbers were interpolated from previously published data (42) to estimate the average amount of inhibin produced per Sertoli cell. Sertoli cell numbers increase from 2.97 million per testis at d 3 pp to plateau at 38.4 million per testis at d 15. Testis weights and approximate Sertoli cell numbers are shown in Table 1Go.


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  		TABLE 1. Left testis weights and approximated Sertoli cell numbers

 
Testicular activin/inhibin subunit mRNA expression
A 1.5-kb band corresponding to the inhibin {alpha}-subunit mRNA was highly expressed at d 3 pp (at similar levels to adult ovary), decreasing progressively to a relatively low level at d 90 pp (Fig. 1AGo). A 6-kb band corresponding to the activin/inhibin ßA-subunit mRNA was highly expressed at d 3 pp (at similar levels to adult ovary), becoming barely detectable at d 10 pp, and undetectable at d 30 and 90 pp (Fig. 1BGo). Two bands (at 4.2 and 3.5 kb) corresponding to activin/inhibin ßB subunit were detected. The 4.2-kb band was most abundant in d 3 pp testis, decreasing to barely detectable levels at d 90 pp. The 3.5-kb band was expressed at low levels at d 3 pp, increasing markedly at d 10 pp, and then decreasing progressively at d 30 and 90 pp (Fig. 1CGo).



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  		FIG. 1. Northern blot analysis of inhibin/activin subunit mRNAs during postnatal rat development. RNA prepared from whole testes illustrates changing levels of inhibin {alpha}-subunit (A), ßA-subunit (B), and ßB-subunit (C) mRNAs in d 2, 10, and 30 and adult (90 pp) testis, adult ovary (as a positive control), and preparations of round spermatids (R s’tids) and primary spermatocytes (Prim. s’cytes; D) when examined by Northern analysis. Transcript sizes are indicated by arrows. Quantitative loading normalization using a probe for mRNA encoding the S26 ribosomal protein is illustrated below A–C, with the mRNA signal level indexed relative to adult testis value. D, Lane loading is illustrated by the Northern image for mRNA encoding the S26 ribosomal protein. The different-sized transcripts of mRNA encoding S26 ribosomal protein in d 20 testis, adult testis, and round spermatid and primary spermatocyte fractions are thought to be due to haploid cell-specific splice variation and polyadenylation patterns as noted in previous studies (77 ). This phenomenon is more clearly demonstrated in D because these gels were separated to a higher resolution than gels depicted in A–C.

 
In Northern analyses performed on enriched fractions of germ cells, ßB-subunit mRNA was found in both preparations of primary spermatocytes and round spermatids (Fig. 1CGo). Both the 3.5- and 4.2-kb transcripts were present. Of particular interest, inhibin {alpha}-subunit was clearly detectable (albeit at low levels) in preparations of primary spermatocytes, which were 86% pure.

Cellular localization of the activin/inhibin subunits by in situ hybridizations
In this study, nonradioactive in situ hybridization was used to examine the expression of activin/inhibin ßA- and ßB-subunits mRNAs within the adult testis. The activin ßA-subunit mRNA was readily evident in vascular endothelial cells (Fig. 2AGo), whereas the signal within the seminiferous epithelium was comparatively weak, especially within the cytoplasm of germ cells (Fig. 2Go, A and C). Peritubular cells had a moderate staining level, whereas most other interstitial cells had a comparatively weaker signal. A strong, stage-specific staining was observed in vacuoles at the base of the seminiferous epithelium at late stage IX through to stage XI, which correspond to the size, position, and timing of residual bodies undergoing phagocytosis by Sertoli cells. No signal was evident in spermatogonia, whereas the weak signal detected in Sertoli cells, spermatocytes, and spermatids was evident only after a relatively prolonged period of color development.



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  		FIG. 2. In situ hybridization localization of activin/inhibin subunit mRNAs in adult rat testis. In situ hybridization was used to localize mRNA encoding the activin/inhibin ßA-subunit (A–C) and the activin/inhibin ßB-subunit (D and E). A, Hybridization of ßA-antisense riboprobe to an adult testis section. Note the strong staining of vascular endothelium and the heterogeneous intensity of staining between tubules. B, Hybridization of a ßA-sense riboprobe to an adult testis section (i.e. negative control). C, Higher-power detail of hybridization of ßA-antisense riboprobe. D, Hybridization of a ßB-antisense riboprobe to an adult testis section. Note the very strong staining of Sertoli cells and early germ cells. E, Hybridization of a ßB sense riboprobe to an adult testis section (i.e. negative control).

 
Expression of ßB-subunit mRNA was noted in type B spermatogonia, preleptotene, leptotene, and zygotene spermatocytes (weak) and pachytene and diplotene spermatocytes commencing at stages IV-V and peaking at stages XII-XIV (moderate). Spermatids from step 6 to 13 expressed ßB-subunit mRNA, peaking at step 11 (strong). Expression of ßB-subunit mRNA in Sertoli cells occurred throughout the cycle but peaked at stages XI-XIII (moderate). Peritubular cells expressed ßB-subunit mRNA and some Leydig cells exhibited expression (weak) (Fig. 2EGo).

In situ hybridization (43, 44) and immunohistochemistry (4, 5, 6) have previously indicated that inhibin {alpha}-subunit mRNA expression is restricted to Sertoli cells within the adult testis.

Serum and testicular inhibin concentrations
Serum inhibin A levels were below the detection limit of the assay (<4 pg/ml) at all ages. In contrast, serum inhibin B levels increased significantly (P < 0.0001) in a linear manner from 165 ± 28 pg/ml (mean ± SEM) at d 3 pp to a peak of 572 ± 119 pg/ml at d 40 pp before decreasing to levels of 182 ± 35 pg/ml at d 90 pp (Fig. 3AGo).



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  		FIG. 3. Serum levels of inhibin B and activin A during postnatal rat development. Serum concentrations of inhibin B and activin A were measured by specific ELISA. Inhibin A concentrations were below the limit of detection for the assay (4 pg/ml) at all ages. A, Serum concentrations of inhibin B and previously published levels of FSH [as determined by RIA (22 ), dotted line]. Serum inhibin B concentrations significantly followed a linear trend (P < 0.0001) and significantly varied from linearity (P < 0.0001). B, Serum concentrations of activin A. Values for inhibin B and activin A are presented as mean ± SEM. Serum activin A concentrations significantly followed a linear trend (P < 0.0001) and significantly varied from linearity (P < 0.05).

 
Concentrations of each of the proteins studied were presented in terms of amount per gram of testis and amount per whole testis (whereas inhibin B concentrations were also presented in terms of amount per Sertoli cell). The. amount of protein per gram of testis is important because it demonstrates the local concentrations of these hormones. The amount of protein per whole testis is important in determining the total production of these hormones (i.e. how much is available to contribute to the general circulation) but has very little bearing on the local concentrations, given that the testis weight/body weight ratio is changing dramatically during this period. Inhibin B levels were additionally presented in terms of amount per Sertoli cell because within the testis, inhibin B is widely believed to be exclusively a product of Sertoli cells. Inhibin A levels were not presented in this manner because they were below the detection limit of the assay in d 3, 6, 8, 10, 50, and 90 pp.

Testicular inhibin A concentrations were highest at d 15 pp (0.330 ± 0.108 ng/g testis) and decreased rapidly (P < 0.0001) until they fell below the detection limit of the assay (0.004 ng/g testis) at d 50–90 pp (Fig. 4Go).



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  		FIG. 4. Testicular levels of inhibin A during postnatal rat development. Inhibin A concentrations in rat testis homogenates were measured by specific ELISA. Levels were not determined in d 3, 6, 8, or 10 pp testes due to the small size of testes and relatively low level expression of inhibin A. Levels fell below the detection limit of the assay (4 pg/ml) at d 50 and 90 pp. A, Local inhibin A levels expressed as amount per gram of testis. Local inhibin A levels significantly followed a linear trend (P < 0.0001) and significantly varied from linearity (P < 0.0001). B, Local inhibin A levels expressed as total amount per testis.

 
Testicular concentrations of inhibin B increased to a peak of 43.9 ± 4.16 ng/g testis at d 6 pp before decreasing (P < 0.0001) to basal levels of 1.56 ± 0.128 ng/g testis at d 90 pp (Fig. 5AGo). These concentrations of inhibin B were 100- to 1000-fold higher than concentrations of inhibin A at equivalent times. Total testicular inhibin B content increased from 0.2 ng per testis at d 3 pp to between 0.45 and 0.58 ng per testis between d 6 pp and d 25 pp. Thereafter, the total testicular inhibin B content again increased steadily to 3.02 ± 0.25 ng/g per testis at d 90 pp (Fig. 5BGo).



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  		FIG. 5. Testicular levels of inhibin B during postnatal rat development. Inhibin B concentrations in rat testis homogenates were measured by specific ELISA. A, Local inhibin B levels expressed as amount per gram of testis. Local inhibin B levels significantly followed a linear trend (P < 0.0001) and significantly varied from linearity (P < 0.0001). B, Local inhibin B levels expressed as total amount per testis. C, Assuming that inhibin B is produced by only the Sertoli cells, average inhibin B content per Sertoli cell.

 
Serum and testicular activin A
Serum activin A levels ranged between 259 and 307 pg/ml from d 3 until d 10 pp before slowly decreasing (P < 0.0001) to 132 ± 27 pg/ml at d 90 pp (Fig. 3BGo).

Concentrations of activin A in testis homogenates were highest at d 6 pp (18.6 ± 2.21 ng/g testis) before decreasing (P < 0.0001) significantly to 0.295 ± 0.017 ng/g testis at d 20 pp. The levels then slowly declined to 0.094 ± 0.013 ng/g testis at d 90 pp (Fig. 6AGo). The total testicular activin A content per testis peaked at 0.202 ng/testis at d 6 pp before decreasing sharply to 0.031–0.036 ng/testis between d 15 and 25 pp. Levels then increased moderately to reach a steady state of 0.167–0.181 ng/testis at d 50 and 90 pp (Fig. 6BGo).



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  		FIG. 6. Testicular levels of activin A during postnatal rat development. Activin A concentrations in rat testis homogenates were measured by specific ELISA. A, Local activin A levels expressed as amount per gram of testis. Local activin A levels significantly followed a linear trend (P < 0.0001) and significantly varied from linearity (P < 0.0001). B, Local activin A levels expressed as total amount per testis.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study provides measurements of circulating and testicular levels of inhibins A and B and activin A, using specific assays, during rat testicular development. Taken together with the results of Northern analysis and the in situ hybridization localization of {alpha}-, ßA-, and ßB-subunits, these studies have identified the sites of cellular production, thereby facilitating an understanding of the actions of these proteins in the testis.

Inhibin secretion
The use of specific assays for inhibin A and inhibin B have enabled us to more clearly delineate the secretory patterns of these two proteins. This study and that of Sharpe et al. (10) demonstrate that, in serum, inhibin B levels rise progressively from early postnatal life to reach a peak (at 40 d in this study and at 20 d in the study by Sharpe et al.), both demonstrating a progressive decline beyond 40 d. This decline parallels and correlates with declining FSH concentrations. Our data showing that serum inhibin A concentrations are less than 4 pg/ml throughout postnatal life confirm the view that inhibin B is the major circulating form in the male rat, similar to observations in other species (9).

Given earlier data that the testis is the principal source of inhibin in the circulation (32), we examined the testicular inhibin content during development. Our data demonstrate that both inhibin B and A are produced in the testis in the first 20 d pp, with the peak inhibin B concentration preceding that of inhibin A by 5–10 d. Although testicular inhibin A concentrations appear to reach a peak at d 15 pp, their levels are around 100-fold lower than the concentrations of inhibin B, indicating that inhibin B is the predominant form produced by the testis.

The elevated levels of inhibin B and A in the immediate postnatal period parallel the period of Sertoli cell activity in male infants as shown by elevated inhibin B levels during the first year of human life (45). The stimulus for this period of Sertoli cell activity may result from the perinatal elevation of FSH noted in man and identified in the rat by earlier studies from our laboratory (22, 46). The physiological role of the perinatal increase in testicular inhibin production remains unclear and the specific role of each inhibin remains to be established.

Although the testicular concentrations of inhibin B decline dramatically (due to dilution by the germ cell populations, which are believed not produce inhibin), the total testicular content of inhibin B rises progressively from d 25 in concert with rising FSH levels, plateauing at 50 d. This is in keeping with the existing data that FSH is the principal stimulus for inhibin secretion by Sertoli cells (47).

Given that within the testis, inhibin B is believed to be produced by Sertoli cells exclusively (4, 5, 6), we used previously published Sertoli cell number data to estimate the average amount of inhibin B produced per Sertoli cell. When expressed as the amount secreted per Sertoli cell, inhibin B levels fall sharply from 68.9 ± 13.1 ag per Sertoli cell at d 3 pp to 14.9 ± 15.4 ag per Sertoli cell at d 15–25 pp. Levels of inhibin B remain low until d 30 pp, when they begin to increase to 79.4 ± 6.5 ag per Sertoli cell at d 90 pp (Fig. 5CGo). This pattern parallels the two distinct phases of rat Sertoli cell activity; the first phase may be associated with elevated circulating FSH levels (46), which induce Sertoli cell proliferation. The rate of postnatal Sertoli cell proliferation decreases steadily until d 15 pp, after which no further division occurs (42, 48). The second phase is associated with the increasing levels of FSH that are present during pubertal maturation in the rat (10, 22, 46). This action of FSH on inhibin is well documented (49) but may be augmented by local interactions between germ cells and Sertoli cells. In this context, the appearance of spermatids in the seminiferous epithelium at d 25–30 pp (50) may be responsible for part of the stimulation of Sertoli cell inhibin expression, despite serum inhibin and FSH levels declining from peaks at 30–40 d in adult rats [(10, 22, 46) and illustrated in Fig. 3AGo]. This hypothesis is supported by a number of recent studies that indicate that germ cells are capable of modulating Sertoli cell inhibin B expression both in vitro (51, 52) and in vivo (53, 54). The expression of mRNA encoding the inhibin/activin subunits correlates well with the protein levels described in this paper. Inhibin {alpha}-subunit mRNA decreases progressively with age, which correlates with the progressive decrease of testicular concentrations of both inhibin A and inhibin B proteins. However, this observation is due to the relative decrease in Sertoli cell mass, the principal source of the {alpha}-subunit in comparison with germ cell mass, and the overall testicular expression of the {alpha}-subunit results in increasing total testicular content and serum levels of inhibin B (Figs. 3Go and 5Go).

Previous attempts to detect {alpha}-subunit in the testis by immunohistochemistry (4, 5, 6) and in situ hybridization (43, 44) failed to demonstrate signal in germ cells; however, these techniques would be insensitive to low-level expression in the context of high-level expression from the Sertoli cells. Notably, when we probed RNA from isolated fractions of germ cells for inhibin {alpha}-subunit, we detected a low level but distinct signal in RNA from primary spermatocytes. Because these preparations were 86% pure, it is impossible, based on this evidence alone, to conclude that {alpha}-subunit is produced by germ cells. The data would be in keeping with the observation of Tena Sempere et al. (44), who noted that the levels of {alpha}-subunit decrease from d 0 to d 30 but then plateau to d 60 despite a continuing increase in testis weight due to germ cell multiplication. This observation raises the possibilities that the Sertoli cells are producing more {alpha}-subunit mRNA associated with the known increase in FSH stimulation or germ cells may be contributing. Our data do raise the possibility that primary spermatocytes may produce {alpha}-subunit, which [combined with the data shown here and elsewhere (55) that ß-subunits are produced in germ cells] raises the possibility that germ cells may be capable of producing both the inhibins and activins. Further studies using such techniques as laser capture dissection will be required to resolve these issues.

Inhibin/activin ßA-subunit decreases rapidly to undetectable levels at d 30 pp, which correlates with the decrease to undetectable levels of inhibin A protein by d 50 pp and the dramatic decrease in activin A levels toward d 15–20 pp. The progressive decrease in both isoforms of inhibin/activin ßB-subunit mRNA toward adulthood correlates with the similar decrease in inhibin B protein. The results of in situ hybridization studies indicate that Sertoli cells express both the ßA- and ßB-subunits, which, taken together with prior studies showing a subunit expression in these cells, indicates the capacity of Sertoli cells to produce inhibins A and B and activins A and B. Our data using specific ELISAs demonstrate that the testis contains predominantly inhibin B in the adult and lesser levels in the early postnatal period, a time at which inhibin A levels are at their highest (albeit still less than one tenth of inhibin B levels).

Activin secretion
The testicular concentrations of activin A are maximal at d 6 pp (18.6 ± 2.2 ng/g) and decline to levels of 0.094 ± 0.013 ng/g at d 90 pp. The early peak in concentrations coincides with the highest circulating levels of activin A, raising the possibility that the testis may contribute to the circulating pool in the immediate postnatal period in contrast to our earlier studies that showed that the testis does not significantly contribute to circulating activin A levels in the adult (13).

The principal source of activin A in the testis is difficult to determine because our in situ data indicate the presence of ßA-subunit mRNA in Sertoli cells, peritubular cells, endothelial cells, and also germ cells. Historically, testicular activin has been considered to be a product of Sertoli cells (55, 56, 57). Recent findings, however, have indicated that during early postnatal testis development, the peritubular (17, 58) and germ cells (19) may also be major sources of activin A. The demonstration in this study that the ßB-subunit mRNA and to a lesser extent the ßA-subunit mRNA is localized in germ cells supports the report of ßB-protein in germ cells in post-vitamin A-deficient rat testes (59) and the human testis (55). The cellular localization to spermatogonia, primary spermatocytes, and round spermatids is similar in both species and suggests that the products are potentially activin B and to a lesser extent activin A [although Andersson et al. (55) did not demonstrate ßA-immunolocalization in the human]. Taken together, these data suggest that the activins play autocrine or paracrine roles in the testis. This view is further supported by the identification of type IIA activin receptor in primary spermatocytes, early round spermatids, Sertoli cells, and Leydig cells and activin type IIB receptors in spermatogonia (60, 61). The capacity of these receptors to function has been demonstrated by the binding of iodinated activin A to primary spermatocytes and round spermatids (62). Additionally, modulation of the actions of activin is possible locally by the presence of follistatin in Sertoli cells, spermatogonia, primary spermatocytes, and round spermatids (39, 63, 64).

The peak concentrations of testicular activin A at d 6 coincide with the time at which Sertoli cell proliferation becomes sensitive to the mitogenic effects of activin A (16, 17), and the levels decline dramatically at d 15 pp, correlating with the cessation of the Sertoli cell proliferative phase. This role of activin, together with FSH, may represent the major factors controlling the size of the total Sertoli cell population, which ultimately determines total sperm output (65, 66, 67, 68). This concept is supported by the observation of a decline in total Sertoli cell numbers in the testis in studies in which targeted gene inactivation of the ß-subunit of FSH or the activin type IIA receptor was performed in mice (69, 70).

Given that inhibin A is undetectable by ELISA in the adult rat testis and ßA-subunit is present in Sertoli cells, it is possible that in the adult, these cells produce activin A, a conclusion that is consistent with the tissue localization of the ßA-subunit mRNA in previous studies (61, 71). Unfortunately, due to the lack of an assay for activin B, it is not possible to determine whether ßB-mRNA expression leads predominantly to inhibin B or activin B secretion.

The function of germ cell localization of activin subunits in the adult testis is less clear. Deletion of the ßB-subunit by gene targeting in mice does not result in a testicular phenotype (72), but it is possible that due to its high degree of homology, the ßA-subunit is compensating. However, disruption of activin action by targeted disruption of the activin type IIA receptor resulted in smaller but still fertile testes (11, 70). The phenotype has, however, been interpreted to result from the decreased FSH levels in these mice, causing lower Sertoli cell numbers (69, 70). The data in this and other studies now clearly raise the probability that the absence of activin action on Sertoli cell proliferation in the neonatal period, together with lower FSH levels, combined to decrease Sertoli cell number.

Overexpression of the ßA-subunit in the testis of mice led to a severe disruption of spermatogenesis (73). Equally, overexpression of follistatin, presumably through its ability to neutralize the action of activin, also led to disruption of spermatogenesis in some lines (74). Furthermore, targeted disruption of either the ßA-gene or follistatin result in neonatal death, preventing the determination of a testicular phenotype (75). The directed expression of the coding region of the ßB-subunit into the ßA-subunit gene locus prevented neonatal death and led to a delay in the onset of spermatogenesis, presumably due to the less potent action of the ßB-subunit (76). Furthermore, in these mice, there was a 50% decrease in testis weight, compared with controls, supporting the importance of the ßA-subunit, probably acting through the formation of activin A, in the development of the testis (75).

Together, the data presented in this paper represent the best currently available description of the intratesticular and circulating levels of activin A, inhibin A, and inhibin B. It is hoped that an ELISA is generated for activin B, which is similar to the ELISAs used for this study. The data would be further improved by the development of an assay that is capable of measuring follistatin in the rat. The data presented here will greatly aid the dissection of the activin/inhibin endocrine pathways during development in the rat, the animal model of choice for studying testis development.


   Footnotes
 
This work was supported by grants from the Australian Research Council (Grant A09927208) and the National Health and Medical Research Council of Australia (Grants 973218, 143781, and 143792).

Abbreviations: CV, Coefficient of variation; DIG, digoxigenin; pp, post partum.

Received August 11, 2003.

Accepted for publication March 29, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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