Proliferative Phase Sertoli Cells Display a Developmentally Regulated Response to Activin in Vitro
Abstractx}-z:3, 百拇医药
We have used cultures of highly purified, proliferating rat Sertoli cells collected from d 3, 6, and 9 rat pups to investigate the role of activin A on Sertoli cell division. These studies demonstrate that activin A acts directly on d 6 and 9, but not d 3, Sertoli cells to induce proliferation, both alone and synergistically with FSH. In addition to stimulating proliferation, activin A induces secretion of inhibins A and B as determined by specific ELISAs. We demonstrate that the synergy between activin A and FSH is not due to local actions of secreted inhibin or follistatin. We have used real-time fluorometric RT-PCR to demonstrate that activin regulates expression of activin receptor and follistatin mRNA by Sertoli cells. Saturation binding studies using 125I-activin A indicate that synergy between activin and FSH may be due to increased numbers of activin receptors on the Sertoli cell. Finally, we show that activin A was secreted at high levels by cultured peritubular cells but was undetectable in high purity proliferating Sertoli cell cultures, suggesting that activin A functions as a paracrine factor during postnatal testis development.
Introductiond(9o, 百拇医药
SERTOLI CELLS, WHICH form the somatic component of the seminiferous tubules, are a major determinant of sperm output by the testis (1). In the rat, Sertoli cells divide in the fetal and early postnatal periods before differentiating into nonproliferative cells at around d 15 post partum (pp) (2, 3). FSH appears to be the primary Sertoli cell mitogen (4), whereas exposure to thyroid hormone leads to the premature cessation of mitosis and Sertoli cell differentiation (5, 6, 7). Recent studies have provided evidence that activin (a homodimer of inhibin ß subunits) may also be involved in Sertoli cell division (8, 9); however, elucidating the actual nature of its role has proven difficult.d(9o, 百拇医药
In the female, activin has been demonstrated to have mitogenic effects on granulosa cells (the female equivalent of Sertoli cells) in the ovary, especially in combination with FSH (10, 11). In cultured testis fragments obtained from d 9 rats, treatment with activin A and FSH resulted in a synergistic increase in Sertoli cell proliferation (compared with untreated fragments or fragments treated with FSH alone) as determined by 5-bromo-2'deoxyuridine incorporation (8). No significant effect was observed in fragments isolated from d 3 or d 18 rats. Further investigation of the window of activin responsiveness led to the demonstration that in testis fragments, Sertoli cells become significantly responsive to activin at some time between d 5 and 7 pp and become unresponsive to activin at some time between d 11 and 13 pp (12). Contrasting with these findings, treatment of d 3 testis fragment cultures with activin A resulted in a significant decrease in Sertoli cell proliferation in both the presence and absence of FSH (9). Treatment of these d 3 cultures with the activin binding-protein follistatin also suppressed Sertoli cell proliferation, suggesting a role for endogenous activin or bone morphogenetic proteins (which are also antagonized by follistatin) (9). Of note, treatment of d 3 testis fragments with activin was found to induce proliferation of gonocytes (9), and at later stages of development, activin has been demonstrated to promote spermatogonial proliferation (13), and to maintain mitochondrial differentiation in spermatocytes (14), suggesting a multifaceted role for activin during testis development.
A direct role for activin A in the regulation of Sertoli cell development would require evidence of the receptor to be present in, and the ligand available to Sertoli cells. Immunohistochemical analysis demonstrates expression of the ßA and ßB subunits of activin/inhibin in both the Sertoli cells and isolated interstitial cells in d 12 rats (15), whereas medium conditioned by cultured primary Sertoli cells isolated from d 21 rats or Sertoli derived TM-4 cells contains activin-like activity (16). These findings led to a widespread assumption that, within the seminiferous tubule, activins are produced by Sertoli cells as autocrine factors. However, peritubular myoid cells have also been demonstrated to express high levels of activin (17). Furthermore, most primary Sertoli cell cultures contain significant degrees of contamination by peritubular cells (18, 19), whereas the TM-4 Sertoli cell line is known to be highly mutated (20) and hence is unlikely to be representative of normal Sertoli cells. It is also quite likely that the profile of activin expression in the testis may change significantly after Sertoli cells differentiate and during postnatal development.
The activin receptors are divided into two subtypes, type II receptors (ActRIIa and ActRIIb) are ligand binding receptors, which then recruit type I receptors that initiate the signal transduction cascade. Within the testis, there is a relatively widespread expression of activin receptors. ActRIIa message has been demonstrated by Northern blot in isolated pachytene spermatocytes, round spermatids, Sertoli cells, and at low levels in isolated Leydig cells (21). In situ hybridization has confirmed the expression of ActRIIa message in spermatocytes, spermatids, and Sertoli cells, but not Leydig cells (22). ActRIIb message on the other hand has been demonstrated by in situ hybridization in primary spermatocytes and Sertoli cells (23). A transient up-regulation of ActRIIa has been reported in Sertoli cells between d 7 and 9 pp (12). No studies appear to have addressed the specific expression of activin type I receptors in the testis.i*cjesv, 百拇医药
While testis fragment cultures are very effective for investigating the action of exogenous factors on testis development, it is difficult to elucidate precise local actions. Since testis fragments contain Sertoli cells, peritubular cells, Leydig cells, testicular macrophages, blood vessels, gonocytes, and spermatogonia, it is possible that a hormone or growth factor applied to such a culture system will exert effects on many different cell types, in some cases inducing expression of further paracrine factors that may affect Sertoli cells. Furthermore, because activin is produced by the testis, it is difficult to delineate the effects of endogenous activin from activin applied to the fragment (16, 17, 24).
To investigate the role of activin A on Sertoli cell development in the absence of other cell types, we have used a high purity, proliferative phase Sertoli cell culture method that has been recently developed in our laboratory. These methods clearly demonstrate that activin A is capable of directly inducing mitosis in Sertoli cells isolated from d 6 and d 9 rat pups. Sertoli cells isolated from d 3 rat pups are initially unresponsive to activin A but become responsive after 8 d in culture. A functional role for activin on Sertoli cells is further supported by the finding that activin A induces secretion of both inhibin A and B by the Sertoli cell. Furthermore, we show that Sertoli cells isolated from d 6 rats produce undetectable amounts of activin A, whereas peritubular cells from the same age produce significant quantities, suggesting that peritubular cells are a major source of activin during early postnatal testicular development.q@, 百拇医药
Materials and Methodsq@, 百拇医药
Cell culture
Male Sprague Dawley rats were obtained from the Monash University (Victoria, Australia) 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. In most experiments, Sertoli cells were isolated from d 6 pp rats; where indicated in the text, cells were also isolated from d 3 or 9 pp rats.(7u, 百拇医药
Sertoli cells were isolated according to previously described methods (25). Briefly, testes were removed, decapsulated, and diced. The resultant fragments of seminiferous cords were rinsed in DMEM/F12 medium (no. 12400-024, Life Technologies, Inc., Rockville, MD), then digested with 3 x 105 U/liter collagenase type II (no. C6885, Sigma) in DMEM/F12 at 22 C for 1 h, washed and then treated with 0.05% wt/vol hyaluronidase (no. H2126, Sigma) in DMEM/F12 for 1 h at 22 C. The seminiferous cords were then rinsed and plated down onto culture dishes coated with 1 µg/cm2 laminin (no. L2020, Sigma) in DMEM/F12 containing insulin, transferrin and selenium (ITS concentrate no. 41400-045, Life Technologies, Inc.), and penicillin/streptomycin (Life Technologies, Inc.). After 24 h of culture, medium was changed and some cultures were exposed to media supplemented with recombinant human (rh) activin A (R&D Systems, Minneapolis, MN; 0–100 ng/ml), rhFSH (Organon, 0–390 IU/liter), rh inhibin A (0–100 ng/ml) (26), and/or bovine follistatin (0–200 ng/ml) (27) alone or in combinations.
Suspended peritubular cells were recovered from the supernatant following collagenase digestion of d 6 pp rat seminiferous cords as previously described (28). Suspended cells were centrifuged at 2000 x g and rinsed once before being seeded onto culture vessels in DMEM/F12 with penicillin, streptomycin, and 10% fetal calf serum. Cells were grown to confluence and passaged once before use.[[{2)@, 百拇医药
When required, cells were passaged by treatment with a volume of trypsin/versene solution containing 0.1% wt/vol trypsin (T2021, Sigma), 0.5 mM tetrasodium EDTA (E6511, Sigma) in PBS for 5 min at 37 C followed by 2 vol 0.05% wt/vol soybean trypsin inhibitor (no. T6522, Sigma) in DMEM/F12. Recovered cells were counted on a hemocytometer using trypan blue exclusion to determine viability. Viable cells were rinsed and replated at 4 x 104 cells/cm2.[[{2)@, 百拇医药
Proliferation assay[[{2)@, 百拇医药
[Methyl-3H]-thymidine incorporation was used as a measure of proliferative activity. [Methyl-3H]-thymidine (2.0 Ci/mmol, 3 x 106cpm/ml media, Amersham Biosciences, Castle Hill, New South Wales, Australia) was added to the culture medium for 6 h. Cells were harvested using a Micromate 196 cell harvester (Packard Instruments, Meriden, CT), and incorporated radionucleotide counted using a liquid scintillation counter. All [3H]-thymidine incorporation assays were performed in quadruplicate in at least three separate experiments.
Inhibin and activin assaysz?}0:, http://www.100md.com
Inhibin A and B concentrations were measured using specific ELISAs (29, 30) according to the manufacturer’s instructions (Oxford Bio-Innovations, Oxfordshire, UK) with some modifications. Standard (inhibin A, WHO 91/784; inhibin B, WHO 96/78) and samples were diluted in unconditioned culture media containing the same additives as were used in the culture. Samples were then 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 alkaline phosphatase-conjugated inhibin detection antibody was added for 3 h at room temperature. After washing, the alkaline phosphatase activity was detected using an amplification kit (ELISA Amplification System, Life Technologies, Inc.) as per the manufacturer’s instructions; the substrate was then incubated for 2 h at room temperature. Inhibin A assays were all performed on the same plate; the intraplate % coefficient of variation (%CV) was 9% and the limit of detection for the assay was 4 pg/ml. Inhibin B assays were performed in three plate; the intraplate %CV was 8%, the interplate %CV was 9% and the limit of detection was 5 pg/ml. Samples were diluted out in a dose-dependent manner and were parallel to the standard curve in both assays (data not shown).
Activin A concentrations were measured using a specific ELISA (31) according to the manufacturer’s instructions (Oxford Bio-Innovations) with some modifications. The standard used was rh activin A as previously described (32). Standards and samples were diluted in unconditioned culture media containing the same additives as were used in the culture. Six percent sodium dodecyl sulfate in 0.05 M PBS solution was added (3% final concentration) followed by boiling for 3 min to denature proteins and remove the interference of follistatin and other known sources of error. The samples were allowed to cool before the addition of H2O2 (2% final concentration) and subsequent 30 min incubation. A total of 25 µl 20% 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 plate was 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 (ELISA Amplification System, Life Technologies, Inc.) as per the manufacturer’s instructions; the substrate was then incubated for 1 h at room temperature. All samples were measured in a single plate, the intraplate %CV was 7%, and the limit of detection for the assay was 0.01 ng/ml. Media samples were diluted out in a dose-dependent manner and were parallel to the standard. One hundred percent recoveries were obtained from spiked samples (Okuma, Y., D. M. de Kretser, and M. P. Hedger, manuscript in preparation).
RNA isolationm-, http://www.100md.com
Cells for RNA isolation were cultured in laminin coated six-well culture dishes (Falcon, North Ridge, New South Wales, Australia). Cells were harvested by treatment with trypsin/versene for 5 min at 37 C, counted on a hemocytometer, and 2 x 106 cells pelleted at 500 x g. Poly A mRNA was isolated using oligo-(deoxythymidine)25 magnetic dynabeads (DynAl, Lake Success, CA) according to the manufacturer’s instructions. mRNA was eluted into 10 mM Tris-HCl (pH 7.5) and stored at -70 C.m-, http://www.100md.com
Reverse transcription (RT)m-, http://www.100md.com
RT was performed using Superscript II (Life Technologies, Inc.) according to the manufacturer’s instructions, primed with oligo-(deoxythymidine)10–15 (Pharmacia, Uppsala, Sweden). For every sample, a no RT control was performed in which all incubations and buffers were identical but no Superscript enzyme was added. This control verified the absence of contaminating genomic DNA in PCRs (data not shown).
PCR primers2tr], 百拇医药
PCR primers (sequences shown in Table 1) were designed using primer3 (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) to previously described sequences for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, accession no. NM_017008.1), ß-actin (NM031144.1), activin receptor type I (ActRI; L19341.1), activin receptor type IIa (ActRIIa; L10639.1), activin receptor type IIb (ActRIIb; M87067.1), follistatin (NM012561.1), and FSH receptor (FSHr; L02842.1). Primers were synthesized by SigmaGenosys Australia (Castle Hill, New South Wales, Australia). For each primer set, annealing temperature was optimized by performing PCR on identical replicates in a gradient block thermocycler (PCR express with 96-well gradient block, Hybaid, Ashford, UK). PCR was performed in a 25-µl solution containing 0.5 U of Taq polymerase (Pharmacia), 10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, 0.2 mM deoxynucleotide triphosphates (Biotech, Roseville, New South Wales, Australia), 1 mM forward and reverse primers, and 1 µl of cDNA from the RT reaction. The initial denaturation step was at 94 C for 3 min, followed by 35 cycles of denaturation for 30 sec at 94 C, annealing for 30 sec at 54–66 C (described below), and extension for 1–1.5 min (depending upon product size) at 72 C. The reaction was subjected to a final extension for 3 min at 72 C before being electrophoresed at 90 V on a 1% agarose gel stained with ethidium bromide. The annealing temperature used was the highest temperature at which a large amount of specific PCR product could be seen when run on an agarose gel. For all primer sets tested, PCR at this temperature yielded no products of size other than that expected.
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Table 1. Primer sequences and PCR conditions for real-time RT-PCR+h+0?, http://www.100md.com
To verify the absence of genomic DNA contamination in RT reactions, a PCR was performed for ß-actin on each RT sample and its equivalent no RT control. PCR was performed as above; however, annealing temperature was constant at 59 C, and 45 cycles of PCR were performed revealing no genomic DNA contamination in any samples (data not shown).+h+0?, http://www.100md.com
Real-time PCR+h+0?, http://www.100md.com
For quantitative PCR, a real-time fluorometric capillary based thermocycler was used (LightCycler, Roche Diagnostics, Basel, Switzerland). PCR was performed in a 10-µl solution containing SYBR green PCR master mix (1x, Roche), MgCl2 (concentrations determined empirically; final concentrations used shown in Table 1), 0.1 mM primers, and 0.2 µl of cDNA from the RT reaction. To minimize pipetting error, solutions were diluted such that 4 µl was the smallest volume pipetted at any time. PCR was performed as follows: 10 min initial denaturation at 95 C, followed by 45 cycles of 95 C denaturation for 10 sec, annealing at 59–63 C (see Table 1) for 5 sec and extension at 72 C for 5–20 sec (see Table 1). DNA content was measured by fluorometry at the end of each extension step. At the end of the PCR, melting curve analysis was performed to verify product specificity by increasing temperature from 66–95 C at 0.1 C per second, measuring fluorescence constantly. In each reaction, a standard curve was prepared from serial dilutions of an arbitrary standard.
PCR products were quantified using the LightCycler software. These values were controlled for RT efficiency and cDNA loading by normalizing with an endogenous control (GAPDH). Normalized values were then calibrated against values for untreated control Sertoli cells to give a rational value.2vmhv, 百拇医药
Radiolabeled activin A binding2vmhv, 百拇医药
Sertoli cells were plated at 0.25, 0.5, 1, and 2 x 106 cells/well in 24-well plates and maintained as described above in the presence or absence of FSH and/or activin A for 3 d before assay.2vmhv, 百拇医药
Recombinant human carrier-free activin A (1 µg, R&D Systems) was iodinated by a chloramine-T method (33) using Na125I (ICN Australia, Seven Hills, New South Wales, Australia), yielding [125I]activin of specific activity 72 µCi/µg. Cell cultures were washed twice with binding assay buffer [(DMEM/F12 medium supplemented with 50 mM HEPES, 0.1% BSA, 0.4 mM EDTA, and 50 ng/ml phenylmethylsulfonylfluoride (Sigma)]. Monolayers of Sertoli cells were exposed to activin tracer at 32,000 cpm per 0.25 ml assay buffer/well (~ 40 pM), in the absence or presence of unlabeled activin (0.003–10 nM) for 4 h at 26 C, with rotary mixing at 30 cycles/min. Nonspecific binding to duplicate Sertoli cell cultures that had received each hormone treatment was determined using an excess of unlabeled activin (10 nM), and was subtracted from total binding data to assess specific binding. After removal of the assay mixture, cultures were placed on ice and washed three times with ice-cold PBS, then each monolayer was dissolved in 0.10 ml of 0.1% Triton X-100 in PBS, and bound [125I]activin in the recovered lysate and a single rinse was pooled for subsequent {gamma} radioactivity determination. Binding data for Sertoli cells plated at different cell densities were corrected for cell number, determined by counting cells in duplicate cultures that received the same treatments in parallel.
Statistical analysesit'\, 百拇医药
All statistical analyses involved one-way ANOVA followed by Tukey’s post hoc test. Differences between groups were deemed significant if P < 0.05.it'\, 百拇医药
Resultsit'\, 百拇医药
Activin A stimulates Sertoli cell proliferationit'\, 百拇医药
Treatment of d 6 rat Sertoli cells with rhFSH induced a significant 3.4-fold increase in thymidine uptake compared with untreated cells. Treatment with activin A in the absence of FSH induced a 2.8-fold increase (P < 0.05) in thymidine uptake (Fig. 1A). In the presence of a maximal stimulating dose of FSH, activin A synergized with FSH in a dose-dependent manner by inducing Sertoli cell division to reach a plateau at 50 ng/ml representing a 24-fold increase in thymidine uptake compared with cells treated with FSH alone (Fig. 1B). The effects of activin were blocked by addition of the activin antagonist, follistatin.it'\, 百拇医药
fig.ommitteed
Figure 1. Activin A influences proliferation of d 6 rat Sertoli cells in the presence and absence of FSH. A, Sertoli cells were treated with activin (100 ng/ml), rhFSH (390 IU/liter), follistatin (200 ng/ml), or combinations as indicated for 3 d before being pulsed with 3H-thymidine for 6 h and incorporated radionucleotide counted in a liquid scintillation counter. B, Sertoli cells were treated with FSH (390 IU/liter) and activin and follistatin for 3 d before being pulsed with 3H-thymidine for 6 h. Activin and follistatin concentrations are in nanograms per milliliter. n = 4 independent samples per treatment group; shared superscripts indicate no significant difference (at the P < 0.05 level).r, 百拇医药
Activin A stimulates inhibin secretionr, 百拇医药
Secretion of inhibin B in the culture medium was approximately 100-fold greater than inhibin A across all groups. FSH or activin A alone had no significant effect on inhibin A production; however, the combination of activin A and FSH resulted in a significant 11-fold increase (Fig. 2A). Inhibin B secretion was significantly stimulated (P < 0.001) by both FSH (1.5-fold) and activin A (2.3-fold) independently, whereas the combination of these factors resulted in a 13-fold increase (Fig. 2B). After 3 d of culture, there were no significant differences in the numbers of Sertoli cells per well between treatment groups (data not shown).
fig.ommitteed6hig, 百拇医药
Figure 2. FSH and activin A induce inhibin secretion by d 6 rat Sertoli cells. Sertoli cells were treated with FSH (390 IU/liter) and/or activin (100 ng/ml) for 2 d before the vessel was rinsed and media was replaced. Cells were cultured for a final 24 h and media was assayed for inhibin A and B by ELISA. Following removal of media for assay, Sertoli cells were stripped from the plate and counted on a hemocytometer. A, Expression of inhibin A from cultured Sertoli cells; B, expression of inhibin B from cultured Sertoli cells. n = 3 independent samples; shared superscripts indicate no significant difference (at the P < 0.05 level).6hig, 百拇医药
Inhibin and follistatin do not influence Sertoli cell proliferation6hig, 百拇医药
To verify that increased inhibin secretion in activin A- and FSH-treated cultures was not responsible for influencing proliferation, we assessed the effect of various doses of inhibin A on proliferation of Sertoli cells isolated from d 6 rats after 3 d in culture. The addition of inhibin A (1–100 ng/ml) to cultures did not significantly affect Sertoli cell thymidine uptake in the presence or absence of FSH (Fig. 3A).
fig.ommitteed4$.?dhk, http://www.100md.com
Figure 3. Inhibin and follistatin do not affect d 6 rat Sertoli cell proliferation. Sertoli cells were cultured in unsupplemented medium or treated with FSH (390 IU/liter) and/or inhibin or follistatin for 3 d before being pulsed with 3H-thymidine for 6 h. Activin and follistatin concentrations are in ng/ml. A, Sertoli cells treated with inhibin A; B, Sertoli cells treated with follistatin. n = 4 independent samples; shared superscripts indicate no significant difference (at the P < 0.05 level).4$.?dhk, http://www.100md.com
Treatment of Sertoli cells with activin A significantly stimulated expression of follistatin mRNA [Fig. 4E; follistatin protein could not be determined because the follistatin RIA (34) does not detect rat follistatin]. To verify that follistatin was not responsible for the increased Sertoli cell proliferation in activin A treated cultures, we assessed the influence of various doses of follistatin on thymidine incorporation by Sertoli cells. Follistatin did not significantly affect Sertoli cell thymidine incorporation over a wide range of concentrations (10–200 ng/ml) in either the presence and absence of FSH (Fig. 3B).
fig.ommitteed|u8cz7e, 百拇医药
Figure 4. Synergy between FSH and activin A in d 6 rat Sertoli cells is not due to changes in FSH receptor (FSHr), activin receptor, or follistatin mRNA expression. Sertoli cells were cultured with FSH (390 IU/liter), activin (100 ng/ml) and/or follistatin (200 ng/ml) for 3 d before mRNA was extracted and reverse transcribed. Real-time fluorometric RT-PCR was used to determine the relative expression levels of mRNA encoding ActRI (A), ActRIIa (B), ActRIIb (C), FSHR (D), and follistatin (E). n = 4; shared superscripts indicate no significant difference (at the P < 0.05 level).|u8cz7e, 百拇医药
Synergy between activin A and FSH is not explained by changes in activin and FSH receptor mRNA expression|u8cz7e, 百拇医药
We hypothesized that the synergy between activin and FSH was due to changes in activin or FSH receptor expression or decreased expression of follistatin. To test this, we performed real-time PCR to quantify expression of ActRI, ActRIIa, ActRIIb, FSH receptor (FSHr) and follistatin mRNA in response to treatment with FSH, activin A, and/or follistatin.
ActRI expression was significantly increased in response to combined FSH and activin A and this increase could be inhibited by follistatin. No significant changes in ActRI expression were observed in any other treatment groups (Fig. 4A). ActRIIa expression was not changed by FSH or activin A alone, but was significantly suppressed in response to FSH and activin A in combination. This action of FSH and activin A was completely reversed by the addition of follistatin to the culture. In contrast, ActRIIa expression increased significantly in response to the combination of activin A and follistatin, but when FSH was added, this increase was suppressed (Fig. 4B).l, 百拇医药
ActRIIb expression was significantly suppressed in response to FSH and activin A in combination, and the addition of follistatin reversed this change (Fig. 4C). FSH receptor expression was not significantly altered by any treatment (Fig. 4D). Follistatin expression was significantly increased in response to activin A (P < 0.001) and the response to FSH and activin A in combination was significantly (P < 0.001) greater than activin A alone. Again, the addition of follistatin to the various activin A treated cultures suppressed the increases (Fig. 4E).
Sertoli cells express activin receptors, and expression is increased by treatment with FSH and activin Aw-j, 百拇医药
Binding of [125I]activin A to Sertoli cells (1 x 106 cell/well) was determined after 3 d of culture in the presence of FSH. Scatchard analysis of the binding data revealed a population of high affinity binding sites (Kd of 100 pM; 280 sites/cell) and a population of low affinity binding sites (Kd of 32 nM; 11,000 sites/cell; Fig. 5A).w-j, 百拇医药
fig.ommitteedw-j, 百拇医药
Figure 5. FSH and activin A synergistically increase the number of activin binding sites on d 6 rat Sertoli cells. A, Activin saturation binding curve and Scatchard analysis (inset). Sertoli cells were seeded at 1 x 106 cells/well and cultured for 3 d. Cells were incubated with radiolabeled activin A (40 pM, equivalent to 1 ng/ml) and various concentrations of unlabeled activin A (0–250 ng/ml, equivalent to 0–10 nM) and bound radionucleotide detected with a {gamma} counter. B, Sertoli cells were seeded at 0.25, 0.5, 1, and 2 x 106 cells/well and cultured for 3 d in media alone or supplemented with activin A and/or FSH. Cells were then incubated with radiolabeled activin A (40 pM) in the absence (total binding) or presence (nonspecific binding) of 250 ng/ml (10 nM) unlabeled activin A. Nonspecific binding was subtracted from total binding to yield a value for specific binding. The derived specific binding data were corrected for differences in cell proliferation arising from the hormonal treatments using cell numbers assessed in duplicate parallel cultures. Exposure of untreated cultures to activin for 2 h before the binding assay did not reduce the level of specific binding relative to control cultures, discounting the possibility that residual activin bound during the treatment phase interfered with the estimate of activin binding.
Specific binding of activin A to Sertoli cells was increased marginally by 3 d pretreatment of the cells with FSH compared with untreated Sertoli cells (after correcting the results for treatment-induced changes in cell number; Fig. 5B). Treatment of cultures with activin A also caused a small but insignificant increase in the specific binding of activin, but the combination of FSH with activin increased binding 8-fold above untreated Sertoli cells (P < 0.01; Fig. 5B).5!6jcx, http://www.100md.com
Responsiveness of Sertoli cells to activin A is age related5!6jcx, http://www.100md.com
To investigate the previously reported age-dependent activin response (8), we isolated Sertoli cells from d 3, 6, and 9 pp rats and cultured them with FSH and/or activin A for 2 or 8 d before measuring thymidine incorporation and mRNA expression. When treated with FSH, Sertoli cells collected from d 3 pups and cultured for 2 d showed a significant increase in thymidine uptake. Activin A, alone or in combination with FSH, did not affect thymidine uptake in these cultures (Fig. 6A). In contrast, when cultured for 8 d, these d 3 Sertoli cells had acquired responsiveness to activin A, and showed an augmented response to treatment with activin and FSH together, becoming indistinguishable in their response from Sertoli cells collected from d 6 pups. Sertoli cells collected from d 6 (Fig. 6B) and d 9 (Fig. 6C) rats were responsive to activin A after both 2 and 8 d in culture.
fig.ommitteedy%!, 百拇医药
Figure 6. Proliferative response of Sertoli cells to activin is related to age of the donor animal and duration in culture. Sertoli cells were isolated from d 3 (A), d 6 (B), and d 9 (C) rats and treated with FSH and/or activin for 2 or 8 d before being pulsed with 3H-thymidine for 6 h. n = 4; shared superscripts indicate no significant difference (at the P < 0.05 level).y%!, 百拇医药
Of interest, there were no significant differences in the expression of mRNA encoding either ActRI, ActRIIa or ActRIIb between Sertoli cells isolated from d 3 rats and (which were unresponsive to activin) and Sertoli cells isolated from d 6 rats (which did respond to activin) when cultured for 2 d (Fig. 7, A–C). When cultured for a 8 d, d 3 Sertoli cells, which acquire activin A responsiveness over this period, had significant increases in both ActRIIa and ActRIIb expression compared with the more moderate increases in expression seen by d 6 Sertoli cells over this culture period (Fig. 7, B and C). Day 6 Sertoli cells did also show a significant increase in ActRI expression over this longer culture period (Fig. 7A).
fig.ommitteedz@+f, http://www.100md.com
Figure 7. Expression of mRNA encoding activin receptors is not affected by donor age but is affected by duration in culture. Sertoli cells were isolated from d 3 and 6 rats and treated with FSH and activin A for 2 or 8 d before mRNA was extracted and reverse transcribed. Real-time fluorometric RT-PCR was used to determine the relative expression levels of mRNA encoding ActRI (A), ActRIIa (B), and ActRIIb (C). n = 4; shared superscripts indicate no significant difference (at the P < 0.05 level).z@+f, http://www.100md.com
Peritubular cells in vitro express activin Az@+f, http://www.100md.com
To elucidate whether activin A is primarily an autocrine or paracrine factor affecting Sertoli cells, we measured the amount of activin A secreted in 24 h by peritubular cells and Sertoli cells isolated from d 6 rats and cultured for 3 d by specific ELISA. Secretion of activin A by cultured Sertoli cells was below the detection limit of the assay (0.01 ng/ml), whereas secretion by peritubular cells reached 2.3 ng/ml in the media (Fig. 8).
fig.ommitteednu;1/q, http://www.100md.com
Figure 8. Peritubular cells express activin A in vitro. Cells were isolated and Sertoli cells cultured in DMEM/F12 with 390 IU/liter FSH, whereas peritubular cells were cultured in DMEM/F12 + 10% fetal calf serum. Cells were cultured for 4 d before being rinsed, trypsinized, counted, and reseeded onto 1 µg/cm2 laminin-coated plates (at 4 x 104 cells/cm2) in DMEM/F12 with FSH. Cells were cultured for an additional 2 d before media was replaced and incubated for a further 24 h. Media were removed and assayed for activin A content by ELISA.nu;1/q, http://www.100md.com
Discussionnu;1/q, http://www.100md.com
This study represents the first comprehensive analysis of the effects of activin A on Sertoli cell proliferation in vitro. The results largely explain the contrasting observations of Boitani and co-workers (8, 12) and Meehan et al. (9), who showed, in testis fragment cultures, that the responses to activin A differed with age. This study demonstrates that highly purified Sertoli cells isolated from d 6 or d 9 rats can respond directly to activin A; however, cells isolated from d 3 rats initially cannot. Our data show that Sertoli cells are capable of maturing in vitro so that d 3 cells gain activin responsiveness when maintained in culture for 8 d. This maturation did not involve increased expression of mRNA encoding the activin receptors.
Our initial experiments indicated that activin A alone was capable of stimulating d 6 Sertoli cell proliferation to a degree similar to that observed for the classical Sertoli cell mitogen FSH. In combination, activin A and FSH synergistically stimulated Sertoli cell proliferation, as has been previously reported in testis fragment cultures by Boitani and co-workers (8, 12). We demonstrate that synergy between activin A and FSH is not due to inhibin or follistatin exerting a mitogenic effect on Sertoli cells. When we investigated the expression of mRNA encoding follistatin and receptors for activin and FSH, it appeared that the changes in expression observed would resist the synergy between activin and FSH rather than reinforcing it. Direct measurement of activin receptors using binding of radiolabeled activin; however, demonstrated that treatment with activin and FSH caused an increase in the number of activin receptors per cell. Taken together, this data suggests that treatment with activin and FSH cause activin receptor complexes to be stabilized rather than inducing increased expression. This effect could account, at least in part, for the dramatic increase in Sertoli cell mitosis induced by FSH and activin together.
Further to the stimulation of Sertoli cell proliferation, activin A showed a capacity to induce expression of inhibins (Fig. 2), especially in the presence of FSH. In humans, Sertoli cell proliferation is marked in the late fetal stages and during the first 2 yr of life (35), which correlates well with circulating inhibin B levels (36). Following birth, there is a significant elevation of serum inhibin B, which relates to increased gonadotropin levels; in hypogonadotropic patients, in whom Sertoli cell division is suppressed, inhibin B levels remain low, but rise in response to treatment with exogenous FSH (37, 38). Taken together, previous data indicate that inhibin B is a good marker for the Sertoli cells response to the mitogenic activity of FSH (39). The data presented in this paper indicate that activin A is similarly capable of inducing both proliferation, and inhibin B (which in vivo would lead to decreased FSH secretion by the pituitary). Activin A can thus contribute both to the generation of Sertoli cells, and the feedback loop that keeps FSH secretion in check.
Given the concentrations of activin A required to elicit a response in Sertoli cells, we hypothesized (as others have) that activin A may be primarily a paracrine or autocrine factor. To investigate this, we measured the secretion of activin A by both Sertoli and peritubular cells in vitro. We found that Sertoli cell secretion of activin A was undetectable, whereas peritubular cells secreted relatively large quantities (Fig. 8), suggesting a primarily paracrine (rather than autocrine) role during testicular development. This finding could explain the discrepancies between the findings of Fragale et al. (12) and the present study; Fragale et al. (12) showed that, in testis fragment cultures, exogenous activin only elicits a Sertoli cell proliferative response when applied in combination with FSH. In this paper, we show that isolated Sertoli cells are capable of proliferating in response to activin alone, albeit at lower levels than in combination with FSH. If peritubular cells are a major source of activin A in the testis, then testis fragments necessarily secrete endogenous activin A, which is likely to impact upon Sertoli cell proliferation even in the absence of exogenous factors. It is likely, therefore, that Sertoli cells in testis fragments are not significantly responsive to exogenous activin A because they are already stimulated by endogenous activin A.
Previous studies have demonstrated that Sertoli cells contain activin receptors, that the testis produces activin and that activin may affect Sertoli cell development. Our data provide strong evidence that activin A is capable of directly inducing Sertoli cell development. Given the observed synergy between activin A and FSH, the age-dependent responsiveness of Sertoli cells to activin and the high levels of activin A secreted by peritubular cells, we suggest that in vivo, activin A provides a means for peritubular cells to exert paracrine control over seminiferous epithelium development at the end of the Sertoli cell proliferative phase. This hypothesis is consistent with the notion that activin promotes proliferation and suppresses differentiation of gonocytes during postnatal testis development (9). Together, these mechanisms would allow coordinated development of gonocytes and Sertoli cells, preventing premature gonocyte differentiation.f#rh, 百拇医药
During the window of activin responsiveness, minor changes to the rate of Sertoli cell proliferation could have major consequences in defining the number of Sertoli cells present in the adult. Furthermore, because activin A is also capable of inducing expression of inhibin by Sertoli cells, it may contribute to the pituitary-gonadal feedback loop in a similar manner to FSH. Because Sertoli cell number is a key regulator of sperm production and fertility, this mechanism could provide a further level of control over testis growth and subsequent fertility.
Acknowledgmentso99, 百拇医药
The authors gratefully acknowledge the efforts of Lynda Foulds for preparing inhibin A and follistatin. We thank Professor Gail Risbridger and Drs. Kate Loveland and Mark Hedger for reading the manuscript.o99, 百拇医药
Received June 6, 2002.o99, 百拇医药
Accepted for publication October 4, 2002.o99, 百拇医药
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Tso JY, Sun XH, Kao TH, Reece KS, Wu R 1985 Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucleic Acids Res 13:1485–1502(Jeremy J. Buzzard Paul G. Farnworth David M. de Kretser Anne E. O’Connor Nigel G. Wreford and John )
We have used cultures of highly purified, proliferating rat Sertoli cells collected from d 3, 6, and 9 rat pups to investigate the role of activin A on Sertoli cell division. These studies demonstrate that activin A acts directly on d 6 and 9, but not d 3, Sertoli cells to induce proliferation, both alone and synergistically with FSH. In addition to stimulating proliferation, activin A induces secretion of inhibins A and B as determined by specific ELISAs. We demonstrate that the synergy between activin A and FSH is not due to local actions of secreted inhibin or follistatin. We have used real-time fluorometric RT-PCR to demonstrate that activin regulates expression of activin receptor and follistatin mRNA by Sertoli cells. Saturation binding studies using 125I-activin A indicate that synergy between activin and FSH may be due to increased numbers of activin receptors on the Sertoli cell. Finally, we show that activin A was secreted at high levels by cultured peritubular cells but was undetectable in high purity proliferating Sertoli cell cultures, suggesting that activin A functions as a paracrine factor during postnatal testis development.
Introductiond(9o, 百拇医药
SERTOLI CELLS, WHICH form the somatic component of the seminiferous tubules, are a major determinant of sperm output by the testis (1). In the rat, Sertoli cells divide in the fetal and early postnatal periods before differentiating into nonproliferative cells at around d 15 post partum (pp) (2, 3). FSH appears to be the primary Sertoli cell mitogen (4), whereas exposure to thyroid hormone leads to the premature cessation of mitosis and Sertoli cell differentiation (5, 6, 7). Recent studies have provided evidence that activin (a homodimer of inhibin ß subunits) may also be involved in Sertoli cell division (8, 9); however, elucidating the actual nature of its role has proven difficult.d(9o, 百拇医药
In the female, activin has been demonstrated to have mitogenic effects on granulosa cells (the female equivalent of Sertoli cells) in the ovary, especially in combination with FSH (10, 11). In cultured testis fragments obtained from d 9 rats, treatment with activin A and FSH resulted in a synergistic increase in Sertoli cell proliferation (compared with untreated fragments or fragments treated with FSH alone) as determined by 5-bromo-2'deoxyuridine incorporation (8). No significant effect was observed in fragments isolated from d 3 or d 18 rats. Further investigation of the window of activin responsiveness led to the demonstration that in testis fragments, Sertoli cells become significantly responsive to activin at some time between d 5 and 7 pp and become unresponsive to activin at some time between d 11 and 13 pp (12). Contrasting with these findings, treatment of d 3 testis fragment cultures with activin A resulted in a significant decrease in Sertoli cell proliferation in both the presence and absence of FSH (9). Treatment of these d 3 cultures with the activin binding-protein follistatin also suppressed Sertoli cell proliferation, suggesting a role for endogenous activin or bone morphogenetic proteins (which are also antagonized by follistatin) (9). Of note, treatment of d 3 testis fragments with activin was found to induce proliferation of gonocytes (9), and at later stages of development, activin has been demonstrated to promote spermatogonial proliferation (13), and to maintain mitochondrial differentiation in spermatocytes (14), suggesting a multifaceted role for activin during testis development.
A direct role for activin A in the regulation of Sertoli cell development would require evidence of the receptor to be present in, and the ligand available to Sertoli cells. Immunohistochemical analysis demonstrates expression of the ßA and ßB subunits of activin/inhibin in both the Sertoli cells and isolated interstitial cells in d 12 rats (15), whereas medium conditioned by cultured primary Sertoli cells isolated from d 21 rats or Sertoli derived TM-4 cells contains activin-like activity (16). These findings led to a widespread assumption that, within the seminiferous tubule, activins are produced by Sertoli cells as autocrine factors. However, peritubular myoid cells have also been demonstrated to express high levels of activin (17). Furthermore, most primary Sertoli cell cultures contain significant degrees of contamination by peritubular cells (18, 19), whereas the TM-4 Sertoli cell line is known to be highly mutated (20) and hence is unlikely to be representative of normal Sertoli cells. It is also quite likely that the profile of activin expression in the testis may change significantly after Sertoli cells differentiate and during postnatal development.
The activin receptors are divided into two subtypes, type II receptors (ActRIIa and ActRIIb) are ligand binding receptors, which then recruit type I receptors that initiate the signal transduction cascade. Within the testis, there is a relatively widespread expression of activin receptors. ActRIIa message has been demonstrated by Northern blot in isolated pachytene spermatocytes, round spermatids, Sertoli cells, and at low levels in isolated Leydig cells (21). In situ hybridization has confirmed the expression of ActRIIa message in spermatocytes, spermatids, and Sertoli cells, but not Leydig cells (22). ActRIIb message on the other hand has been demonstrated by in situ hybridization in primary spermatocytes and Sertoli cells (23). A transient up-regulation of ActRIIa has been reported in Sertoli cells between d 7 and 9 pp (12). No studies appear to have addressed the specific expression of activin type I receptors in the testis.i*cjesv, 百拇医药
While testis fragment cultures are very effective for investigating the action of exogenous factors on testis development, it is difficult to elucidate precise local actions. Since testis fragments contain Sertoli cells, peritubular cells, Leydig cells, testicular macrophages, blood vessels, gonocytes, and spermatogonia, it is possible that a hormone or growth factor applied to such a culture system will exert effects on many different cell types, in some cases inducing expression of further paracrine factors that may affect Sertoli cells. Furthermore, because activin is produced by the testis, it is difficult to delineate the effects of endogenous activin from activin applied to the fragment (16, 17, 24).
To investigate the role of activin A on Sertoli cell development in the absence of other cell types, we have used a high purity, proliferative phase Sertoli cell culture method that has been recently developed in our laboratory. These methods clearly demonstrate that activin A is capable of directly inducing mitosis in Sertoli cells isolated from d 6 and d 9 rat pups. Sertoli cells isolated from d 3 rat pups are initially unresponsive to activin A but become responsive after 8 d in culture. A functional role for activin on Sertoli cells is further supported by the finding that activin A induces secretion of both inhibin A and B by the Sertoli cell. Furthermore, we show that Sertoli cells isolated from d 6 rats produce undetectable amounts of activin A, whereas peritubular cells from the same age produce significant quantities, suggesting that peritubular cells are a major source of activin during early postnatal testicular development.q@, 百拇医药
Materials and Methodsq@, 百拇医药
Cell culture
Male Sprague Dawley rats were obtained from the Monash University (Victoria, Australia) 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. In most experiments, Sertoli cells were isolated from d 6 pp rats; where indicated in the text, cells were also isolated from d 3 or 9 pp rats.(7u, 百拇医药
Sertoli cells were isolated according to previously described methods (25). Briefly, testes were removed, decapsulated, and diced. The resultant fragments of seminiferous cords were rinsed in DMEM/F12 medium (no. 12400-024, Life Technologies, Inc., Rockville, MD), then digested with 3 x 105 U/liter collagenase type II (no. C6885, Sigma) in DMEM/F12 at 22 C for 1 h, washed and then treated with 0.05% wt/vol hyaluronidase (no. H2126, Sigma) in DMEM/F12 for 1 h at 22 C. The seminiferous cords were then rinsed and plated down onto culture dishes coated with 1 µg/cm2 laminin (no. L2020, Sigma) in DMEM/F12 containing insulin, transferrin and selenium (ITS concentrate no. 41400-045, Life Technologies, Inc.), and penicillin/streptomycin (Life Technologies, Inc.). After 24 h of culture, medium was changed and some cultures were exposed to media supplemented with recombinant human (rh) activin A (R&D Systems, Minneapolis, MN; 0–100 ng/ml), rhFSH (Organon, 0–390 IU/liter), rh inhibin A (0–100 ng/ml) (26), and/or bovine follistatin (0–200 ng/ml) (27) alone or in combinations.
Suspended peritubular cells were recovered from the supernatant following collagenase digestion of d 6 pp rat seminiferous cords as previously described (28). Suspended cells were centrifuged at 2000 x g and rinsed once before being seeded onto culture vessels in DMEM/F12 with penicillin, streptomycin, and 10% fetal calf serum. Cells were grown to confluence and passaged once before use.[[{2)@, 百拇医药
When required, cells were passaged by treatment with a volume of trypsin/versene solution containing 0.1% wt/vol trypsin (T2021, Sigma), 0.5 mM tetrasodium EDTA (E6511, Sigma) in PBS for 5 min at 37 C followed by 2 vol 0.05% wt/vol soybean trypsin inhibitor (no. T6522, Sigma) in DMEM/F12. Recovered cells were counted on a hemocytometer using trypan blue exclusion to determine viability. Viable cells were rinsed and replated at 4 x 104 cells/cm2.[[{2)@, 百拇医药
Proliferation assay[[{2)@, 百拇医药
[Methyl-3H]-thymidine incorporation was used as a measure of proliferative activity. [Methyl-3H]-thymidine (2.0 Ci/mmol, 3 x 106cpm/ml media, Amersham Biosciences, Castle Hill, New South Wales, Australia) was added to the culture medium for 6 h. Cells were harvested using a Micromate 196 cell harvester (Packard Instruments, Meriden, CT), and incorporated radionucleotide counted using a liquid scintillation counter. All [3H]-thymidine incorporation assays were performed in quadruplicate in at least three separate experiments.
Inhibin and activin assaysz?}0:, http://www.100md.com
Inhibin A and B concentrations were measured using specific ELISAs (29, 30) according to the manufacturer’s instructions (Oxford Bio-Innovations, Oxfordshire, UK) with some modifications. Standard (inhibin A, WHO 91/784; inhibin B, WHO 96/78) and samples were diluted in unconditioned culture media containing the same additives as were used in the culture. Samples were then 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 alkaline phosphatase-conjugated inhibin detection antibody was added for 3 h at room temperature. After washing, the alkaline phosphatase activity was detected using an amplification kit (ELISA Amplification System, Life Technologies, Inc.) as per the manufacturer’s instructions; the substrate was then incubated for 2 h at room temperature. Inhibin A assays were all performed on the same plate; the intraplate % coefficient of variation (%CV) was 9% and the limit of detection for the assay was 4 pg/ml. Inhibin B assays were performed in three plate; the intraplate %CV was 8%, the interplate %CV was 9% and the limit of detection was 5 pg/ml. Samples were diluted out in a dose-dependent manner and were parallel to the standard curve in both assays (data not shown).
Activin A concentrations were measured using a specific ELISA (31) according to the manufacturer’s instructions (Oxford Bio-Innovations) with some modifications. The standard used was rh activin A as previously described (32). Standards and samples were diluted in unconditioned culture media containing the same additives as were used in the culture. Six percent sodium dodecyl sulfate in 0.05 M PBS solution was added (3% final concentration) followed by boiling for 3 min to denature proteins and remove the interference of follistatin and other known sources of error. The samples were allowed to cool before the addition of H2O2 (2% final concentration) and subsequent 30 min incubation. A total of 25 µl 20% 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 plate was 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 (ELISA Amplification System, Life Technologies, Inc.) as per the manufacturer’s instructions; the substrate was then incubated for 1 h at room temperature. All samples were measured in a single plate, the intraplate %CV was 7%, and the limit of detection for the assay was 0.01 ng/ml. Media samples were diluted out in a dose-dependent manner and were parallel to the standard. One hundred percent recoveries were obtained from spiked samples (Okuma, Y., D. M. de Kretser, and M. P. Hedger, manuscript in preparation).
RNA isolationm-, http://www.100md.com
Cells for RNA isolation were cultured in laminin coated six-well culture dishes (Falcon, North Ridge, New South Wales, Australia). Cells were harvested by treatment with trypsin/versene for 5 min at 37 C, counted on a hemocytometer, and 2 x 106 cells pelleted at 500 x g. Poly A mRNA was isolated using oligo-(deoxythymidine)25 magnetic dynabeads (DynAl, Lake Success, CA) according to the manufacturer’s instructions. mRNA was eluted into 10 mM Tris-HCl (pH 7.5) and stored at -70 C.m-, http://www.100md.com
Reverse transcription (RT)m-, http://www.100md.com
RT was performed using Superscript II (Life Technologies, Inc.) according to the manufacturer’s instructions, primed with oligo-(deoxythymidine)10–15 (Pharmacia, Uppsala, Sweden). For every sample, a no RT control was performed in which all incubations and buffers were identical but no Superscript enzyme was added. This control verified the absence of contaminating genomic DNA in PCRs (data not shown).
PCR primers2tr], 百拇医药
PCR primers (sequences shown in Table 1) were designed using primer3 (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) to previously described sequences for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, accession no. NM_017008.1), ß-actin (NM031144.1), activin receptor type I (ActRI; L19341.1), activin receptor type IIa (ActRIIa; L10639.1), activin receptor type IIb (ActRIIb; M87067.1), follistatin (NM012561.1), and FSH receptor (FSHr; L02842.1). Primers were synthesized by SigmaGenosys Australia (Castle Hill, New South Wales, Australia). For each primer set, annealing temperature was optimized by performing PCR on identical replicates in a gradient block thermocycler (PCR express with 96-well gradient block, Hybaid, Ashford, UK). PCR was performed in a 25-µl solution containing 0.5 U of Taq polymerase (Pharmacia), 10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, 0.2 mM deoxynucleotide triphosphates (Biotech, Roseville, New South Wales, Australia), 1 mM forward and reverse primers, and 1 µl of cDNA from the RT reaction. The initial denaturation step was at 94 C for 3 min, followed by 35 cycles of denaturation for 30 sec at 94 C, annealing for 30 sec at 54–66 C (described below), and extension for 1–1.5 min (depending upon product size) at 72 C. The reaction was subjected to a final extension for 3 min at 72 C before being electrophoresed at 90 V on a 1% agarose gel stained with ethidium bromide. The annealing temperature used was the highest temperature at which a large amount of specific PCR product could be seen when run on an agarose gel. For all primer sets tested, PCR at this temperature yielded no products of size other than that expected.
fig.ommitteed+h+0?, http://www.100md.com
Table 1. Primer sequences and PCR conditions for real-time RT-PCR+h+0?, http://www.100md.com
To verify the absence of genomic DNA contamination in RT reactions, a PCR was performed for ß-actin on each RT sample and its equivalent no RT control. PCR was performed as above; however, annealing temperature was constant at 59 C, and 45 cycles of PCR were performed revealing no genomic DNA contamination in any samples (data not shown).+h+0?, http://www.100md.com
Real-time PCR+h+0?, http://www.100md.com
For quantitative PCR, a real-time fluorometric capillary based thermocycler was used (LightCycler, Roche Diagnostics, Basel, Switzerland). PCR was performed in a 10-µl solution containing SYBR green PCR master mix (1x, Roche), MgCl2 (concentrations determined empirically; final concentrations used shown in Table 1), 0.1 mM primers, and 0.2 µl of cDNA from the RT reaction. To minimize pipetting error, solutions were diluted such that 4 µl was the smallest volume pipetted at any time. PCR was performed as follows: 10 min initial denaturation at 95 C, followed by 45 cycles of 95 C denaturation for 10 sec, annealing at 59–63 C (see Table 1) for 5 sec and extension at 72 C for 5–20 sec (see Table 1). DNA content was measured by fluorometry at the end of each extension step. At the end of the PCR, melting curve analysis was performed to verify product specificity by increasing temperature from 66–95 C at 0.1 C per second, measuring fluorescence constantly. In each reaction, a standard curve was prepared from serial dilutions of an arbitrary standard.
PCR products were quantified using the LightCycler software. These values were controlled for RT efficiency and cDNA loading by normalizing with an endogenous control (GAPDH). Normalized values were then calibrated against values for untreated control Sertoli cells to give a rational value.2vmhv, 百拇医药
Radiolabeled activin A binding2vmhv, 百拇医药
Sertoli cells were plated at 0.25, 0.5, 1, and 2 x 106 cells/well in 24-well plates and maintained as described above in the presence or absence of FSH and/or activin A for 3 d before assay.2vmhv, 百拇医药
Recombinant human carrier-free activin A (1 µg, R&D Systems) was iodinated by a chloramine-T method (33) using Na125I (ICN Australia, Seven Hills, New South Wales, Australia), yielding [125I]activin of specific activity 72 µCi/µg. Cell cultures were washed twice with binding assay buffer [(DMEM/F12 medium supplemented with 50 mM HEPES, 0.1% BSA, 0.4 mM EDTA, and 50 ng/ml phenylmethylsulfonylfluoride (Sigma)]. Monolayers of Sertoli cells were exposed to activin tracer at 32,000 cpm per 0.25 ml assay buffer/well (~ 40 pM), in the absence or presence of unlabeled activin (0.003–10 nM) for 4 h at 26 C, with rotary mixing at 30 cycles/min. Nonspecific binding to duplicate Sertoli cell cultures that had received each hormone treatment was determined using an excess of unlabeled activin (10 nM), and was subtracted from total binding data to assess specific binding. After removal of the assay mixture, cultures were placed on ice and washed three times with ice-cold PBS, then each monolayer was dissolved in 0.10 ml of 0.1% Triton X-100 in PBS, and bound [125I]activin in the recovered lysate and a single rinse was pooled for subsequent {gamma} radioactivity determination. Binding data for Sertoli cells plated at different cell densities were corrected for cell number, determined by counting cells in duplicate cultures that received the same treatments in parallel.
Statistical analysesit'\, 百拇医药
All statistical analyses involved one-way ANOVA followed by Tukey’s post hoc test. Differences between groups were deemed significant if P < 0.05.it'\, 百拇医药
Resultsit'\, 百拇医药
Activin A stimulates Sertoli cell proliferationit'\, 百拇医药
Treatment of d 6 rat Sertoli cells with rhFSH induced a significant 3.4-fold increase in thymidine uptake compared with untreated cells. Treatment with activin A in the absence of FSH induced a 2.8-fold increase (P < 0.05) in thymidine uptake (Fig. 1A). In the presence of a maximal stimulating dose of FSH, activin A synergized with FSH in a dose-dependent manner by inducing Sertoli cell division to reach a plateau at 50 ng/ml representing a 24-fold increase in thymidine uptake compared with cells treated with FSH alone (Fig. 1B). The effects of activin were blocked by addition of the activin antagonist, follistatin.it'\, 百拇医药
fig.ommitteed
Figure 1. Activin A influences proliferation of d 6 rat Sertoli cells in the presence and absence of FSH. A, Sertoli cells were treated with activin (100 ng/ml), rhFSH (390 IU/liter), follistatin (200 ng/ml), or combinations as indicated for 3 d before being pulsed with 3H-thymidine for 6 h and incorporated radionucleotide counted in a liquid scintillation counter. B, Sertoli cells were treated with FSH (390 IU/liter) and activin and follistatin for 3 d before being pulsed with 3H-thymidine for 6 h. Activin and follistatin concentrations are in nanograms per milliliter. n = 4 independent samples per treatment group; shared superscripts indicate no significant difference (at the P < 0.05 level).r, 百拇医药
Activin A stimulates inhibin secretionr, 百拇医药
Secretion of inhibin B in the culture medium was approximately 100-fold greater than inhibin A across all groups. FSH or activin A alone had no significant effect on inhibin A production; however, the combination of activin A and FSH resulted in a significant 11-fold increase (Fig. 2A). Inhibin B secretion was significantly stimulated (P < 0.001) by both FSH (1.5-fold) and activin A (2.3-fold) independently, whereas the combination of these factors resulted in a 13-fold increase (Fig. 2B). After 3 d of culture, there were no significant differences in the numbers of Sertoli cells per well between treatment groups (data not shown).
fig.ommitteed6hig, 百拇医药
Figure 2. FSH and activin A induce inhibin secretion by d 6 rat Sertoli cells. Sertoli cells were treated with FSH (390 IU/liter) and/or activin (100 ng/ml) for 2 d before the vessel was rinsed and media was replaced. Cells were cultured for a final 24 h and media was assayed for inhibin A and B by ELISA. Following removal of media for assay, Sertoli cells were stripped from the plate and counted on a hemocytometer. A, Expression of inhibin A from cultured Sertoli cells; B, expression of inhibin B from cultured Sertoli cells. n = 3 independent samples; shared superscripts indicate no significant difference (at the P < 0.05 level).6hig, 百拇医药
Inhibin and follistatin do not influence Sertoli cell proliferation6hig, 百拇医药
To verify that increased inhibin secretion in activin A- and FSH-treated cultures was not responsible for influencing proliferation, we assessed the effect of various doses of inhibin A on proliferation of Sertoli cells isolated from d 6 rats after 3 d in culture. The addition of inhibin A (1–100 ng/ml) to cultures did not significantly affect Sertoli cell thymidine uptake in the presence or absence of FSH (Fig. 3A).
fig.ommitteed4$.?dhk, http://www.100md.com
Figure 3. Inhibin and follistatin do not affect d 6 rat Sertoli cell proliferation. Sertoli cells were cultured in unsupplemented medium or treated with FSH (390 IU/liter) and/or inhibin or follistatin for 3 d before being pulsed with 3H-thymidine for 6 h. Activin and follistatin concentrations are in ng/ml. A, Sertoli cells treated with inhibin A; B, Sertoli cells treated with follistatin. n = 4 independent samples; shared superscripts indicate no significant difference (at the P < 0.05 level).4$.?dhk, http://www.100md.com
Treatment of Sertoli cells with activin A significantly stimulated expression of follistatin mRNA [Fig. 4E; follistatin protein could not be determined because the follistatin RIA (34) does not detect rat follistatin]. To verify that follistatin was not responsible for the increased Sertoli cell proliferation in activin A treated cultures, we assessed the influence of various doses of follistatin on thymidine incorporation by Sertoli cells. Follistatin did not significantly affect Sertoli cell thymidine incorporation over a wide range of concentrations (10–200 ng/ml) in either the presence and absence of FSH (Fig. 3B).
fig.ommitteed|u8cz7e, 百拇医药
Figure 4. Synergy between FSH and activin A in d 6 rat Sertoli cells is not due to changes in FSH receptor (FSHr), activin receptor, or follistatin mRNA expression. Sertoli cells were cultured with FSH (390 IU/liter), activin (100 ng/ml) and/or follistatin (200 ng/ml) for 3 d before mRNA was extracted and reverse transcribed. Real-time fluorometric RT-PCR was used to determine the relative expression levels of mRNA encoding ActRI (A), ActRIIa (B), ActRIIb (C), FSHR (D), and follistatin (E). n = 4; shared superscripts indicate no significant difference (at the P < 0.05 level).|u8cz7e, 百拇医药
Synergy between activin A and FSH is not explained by changes in activin and FSH receptor mRNA expression|u8cz7e, 百拇医药
We hypothesized that the synergy between activin and FSH was due to changes in activin or FSH receptor expression or decreased expression of follistatin. To test this, we performed real-time PCR to quantify expression of ActRI, ActRIIa, ActRIIb, FSH receptor (FSHr) and follistatin mRNA in response to treatment with FSH, activin A, and/or follistatin.
ActRI expression was significantly increased in response to combined FSH and activin A and this increase could be inhibited by follistatin. No significant changes in ActRI expression were observed in any other treatment groups (Fig. 4A). ActRIIa expression was not changed by FSH or activin A alone, but was significantly suppressed in response to FSH and activin A in combination. This action of FSH and activin A was completely reversed by the addition of follistatin to the culture. In contrast, ActRIIa expression increased significantly in response to the combination of activin A and follistatin, but when FSH was added, this increase was suppressed (Fig. 4B).l, 百拇医药
ActRIIb expression was significantly suppressed in response to FSH and activin A in combination, and the addition of follistatin reversed this change (Fig. 4C). FSH receptor expression was not significantly altered by any treatment (Fig. 4D). Follistatin expression was significantly increased in response to activin A (P < 0.001) and the response to FSH and activin A in combination was significantly (P < 0.001) greater than activin A alone. Again, the addition of follistatin to the various activin A treated cultures suppressed the increases (Fig. 4E).
Sertoli cells express activin receptors, and expression is increased by treatment with FSH and activin Aw-j, 百拇医药
Binding of [125I]activin A to Sertoli cells (1 x 106 cell/well) was determined after 3 d of culture in the presence of FSH. Scatchard analysis of the binding data revealed a population of high affinity binding sites (Kd of 100 pM; 280 sites/cell) and a population of low affinity binding sites (Kd of 32 nM; 11,000 sites/cell; Fig. 5A).w-j, 百拇医药
fig.ommitteedw-j, 百拇医药
Figure 5. FSH and activin A synergistically increase the number of activin binding sites on d 6 rat Sertoli cells. A, Activin saturation binding curve and Scatchard analysis (inset). Sertoli cells were seeded at 1 x 106 cells/well and cultured for 3 d. Cells were incubated with radiolabeled activin A (40 pM, equivalent to 1 ng/ml) and various concentrations of unlabeled activin A (0–250 ng/ml, equivalent to 0–10 nM) and bound radionucleotide detected with a {gamma} counter. B, Sertoli cells were seeded at 0.25, 0.5, 1, and 2 x 106 cells/well and cultured for 3 d in media alone or supplemented with activin A and/or FSH. Cells were then incubated with radiolabeled activin A (40 pM) in the absence (total binding) or presence (nonspecific binding) of 250 ng/ml (10 nM) unlabeled activin A. Nonspecific binding was subtracted from total binding to yield a value for specific binding. The derived specific binding data were corrected for differences in cell proliferation arising from the hormonal treatments using cell numbers assessed in duplicate parallel cultures. Exposure of untreated cultures to activin for 2 h before the binding assay did not reduce the level of specific binding relative to control cultures, discounting the possibility that residual activin bound during the treatment phase interfered with the estimate of activin binding.
Specific binding of activin A to Sertoli cells was increased marginally by 3 d pretreatment of the cells with FSH compared with untreated Sertoli cells (after correcting the results for treatment-induced changes in cell number; Fig. 5B). Treatment of cultures with activin A also caused a small but insignificant increase in the specific binding of activin, but the combination of FSH with activin increased binding 8-fold above untreated Sertoli cells (P < 0.01; Fig. 5B).5!6jcx, http://www.100md.com
Responsiveness of Sertoli cells to activin A is age related5!6jcx, http://www.100md.com
To investigate the previously reported age-dependent activin response (8), we isolated Sertoli cells from d 3, 6, and 9 pp rats and cultured them with FSH and/or activin A for 2 or 8 d before measuring thymidine incorporation and mRNA expression. When treated with FSH, Sertoli cells collected from d 3 pups and cultured for 2 d showed a significant increase in thymidine uptake. Activin A, alone or in combination with FSH, did not affect thymidine uptake in these cultures (Fig. 6A). In contrast, when cultured for 8 d, these d 3 Sertoli cells had acquired responsiveness to activin A, and showed an augmented response to treatment with activin and FSH together, becoming indistinguishable in their response from Sertoli cells collected from d 6 pups. Sertoli cells collected from d 6 (Fig. 6B) and d 9 (Fig. 6C) rats were responsive to activin A after both 2 and 8 d in culture.
fig.ommitteedy%!, 百拇医药
Figure 6. Proliferative response of Sertoli cells to activin is related to age of the donor animal and duration in culture. Sertoli cells were isolated from d 3 (A), d 6 (B), and d 9 (C) rats and treated with FSH and/or activin for 2 or 8 d before being pulsed with 3H-thymidine for 6 h. n = 4; shared superscripts indicate no significant difference (at the P < 0.05 level).y%!, 百拇医药
Of interest, there were no significant differences in the expression of mRNA encoding either ActRI, ActRIIa or ActRIIb between Sertoli cells isolated from d 3 rats and (which were unresponsive to activin) and Sertoli cells isolated from d 6 rats (which did respond to activin) when cultured for 2 d (Fig. 7, A–C). When cultured for a 8 d, d 3 Sertoli cells, which acquire activin A responsiveness over this period, had significant increases in both ActRIIa and ActRIIb expression compared with the more moderate increases in expression seen by d 6 Sertoli cells over this culture period (Fig. 7, B and C). Day 6 Sertoli cells did also show a significant increase in ActRI expression over this longer culture period (Fig. 7A).
fig.ommitteedz@+f, http://www.100md.com
Figure 7. Expression of mRNA encoding activin receptors is not affected by donor age but is affected by duration in culture. Sertoli cells were isolated from d 3 and 6 rats and treated with FSH and activin A for 2 or 8 d before mRNA was extracted and reverse transcribed. Real-time fluorometric RT-PCR was used to determine the relative expression levels of mRNA encoding ActRI (A), ActRIIa (B), and ActRIIb (C). n = 4; shared superscripts indicate no significant difference (at the P < 0.05 level).z@+f, http://www.100md.com
Peritubular cells in vitro express activin Az@+f, http://www.100md.com
To elucidate whether activin A is primarily an autocrine or paracrine factor affecting Sertoli cells, we measured the amount of activin A secreted in 24 h by peritubular cells and Sertoli cells isolated from d 6 rats and cultured for 3 d by specific ELISA. Secretion of activin A by cultured Sertoli cells was below the detection limit of the assay (0.01 ng/ml), whereas secretion by peritubular cells reached 2.3 ng/ml in the media (Fig. 8).
fig.ommitteednu;1/q, http://www.100md.com
Figure 8. Peritubular cells express activin A in vitro. Cells were isolated and Sertoli cells cultured in DMEM/F12 with 390 IU/liter FSH, whereas peritubular cells were cultured in DMEM/F12 + 10% fetal calf serum. Cells were cultured for 4 d before being rinsed, trypsinized, counted, and reseeded onto 1 µg/cm2 laminin-coated plates (at 4 x 104 cells/cm2) in DMEM/F12 with FSH. Cells were cultured for an additional 2 d before media was replaced and incubated for a further 24 h. Media were removed and assayed for activin A content by ELISA.nu;1/q, http://www.100md.com
Discussionnu;1/q, http://www.100md.com
This study represents the first comprehensive analysis of the effects of activin A on Sertoli cell proliferation in vitro. The results largely explain the contrasting observations of Boitani and co-workers (8, 12) and Meehan et al. (9), who showed, in testis fragment cultures, that the responses to activin A differed with age. This study demonstrates that highly purified Sertoli cells isolated from d 6 or d 9 rats can respond directly to activin A; however, cells isolated from d 3 rats initially cannot. Our data show that Sertoli cells are capable of maturing in vitro so that d 3 cells gain activin responsiveness when maintained in culture for 8 d. This maturation did not involve increased expression of mRNA encoding the activin receptors.
Our initial experiments indicated that activin A alone was capable of stimulating d 6 Sertoli cell proliferation to a degree similar to that observed for the classical Sertoli cell mitogen FSH. In combination, activin A and FSH synergistically stimulated Sertoli cell proliferation, as has been previously reported in testis fragment cultures by Boitani and co-workers (8, 12). We demonstrate that synergy between activin A and FSH is not due to inhibin or follistatin exerting a mitogenic effect on Sertoli cells. When we investigated the expression of mRNA encoding follistatin and receptors for activin and FSH, it appeared that the changes in expression observed would resist the synergy between activin and FSH rather than reinforcing it. Direct measurement of activin receptors using binding of radiolabeled activin; however, demonstrated that treatment with activin and FSH caused an increase in the number of activin receptors per cell. Taken together, this data suggests that treatment with activin and FSH cause activin receptor complexes to be stabilized rather than inducing increased expression. This effect could account, at least in part, for the dramatic increase in Sertoli cell mitosis induced by FSH and activin together.
Further to the stimulation of Sertoli cell proliferation, activin A showed a capacity to induce expression of inhibins (Fig. 2), especially in the presence of FSH. In humans, Sertoli cell proliferation is marked in the late fetal stages and during the first 2 yr of life (35), which correlates well with circulating inhibin B levels (36). Following birth, there is a significant elevation of serum inhibin B, which relates to increased gonadotropin levels; in hypogonadotropic patients, in whom Sertoli cell division is suppressed, inhibin B levels remain low, but rise in response to treatment with exogenous FSH (37, 38). Taken together, previous data indicate that inhibin B is a good marker for the Sertoli cells response to the mitogenic activity of FSH (39). The data presented in this paper indicate that activin A is similarly capable of inducing both proliferation, and inhibin B (which in vivo would lead to decreased FSH secretion by the pituitary). Activin A can thus contribute both to the generation of Sertoli cells, and the feedback loop that keeps FSH secretion in check.
Given the concentrations of activin A required to elicit a response in Sertoli cells, we hypothesized (as others have) that activin A may be primarily a paracrine or autocrine factor. To investigate this, we measured the secretion of activin A by both Sertoli and peritubular cells in vitro. We found that Sertoli cell secretion of activin A was undetectable, whereas peritubular cells secreted relatively large quantities (Fig. 8), suggesting a primarily paracrine (rather than autocrine) role during testicular development. This finding could explain the discrepancies between the findings of Fragale et al. (12) and the present study; Fragale et al. (12) showed that, in testis fragment cultures, exogenous activin only elicits a Sertoli cell proliferative response when applied in combination with FSH. In this paper, we show that isolated Sertoli cells are capable of proliferating in response to activin alone, albeit at lower levels than in combination with FSH. If peritubular cells are a major source of activin A in the testis, then testis fragments necessarily secrete endogenous activin A, which is likely to impact upon Sertoli cell proliferation even in the absence of exogenous factors. It is likely, therefore, that Sertoli cells in testis fragments are not significantly responsive to exogenous activin A because they are already stimulated by endogenous activin A.
Previous studies have demonstrated that Sertoli cells contain activin receptors, that the testis produces activin and that activin may affect Sertoli cell development. Our data provide strong evidence that activin A is capable of directly inducing Sertoli cell development. Given the observed synergy between activin A and FSH, the age-dependent responsiveness of Sertoli cells to activin and the high levels of activin A secreted by peritubular cells, we suggest that in vivo, activin A provides a means for peritubular cells to exert paracrine control over seminiferous epithelium development at the end of the Sertoli cell proliferative phase. This hypothesis is consistent with the notion that activin promotes proliferation and suppresses differentiation of gonocytes during postnatal testis development (9). Together, these mechanisms would allow coordinated development of gonocytes and Sertoli cells, preventing premature gonocyte differentiation.f#rh, 百拇医药
During the window of activin responsiveness, minor changes to the rate of Sertoli cell proliferation could have major consequences in defining the number of Sertoli cells present in the adult. Furthermore, because activin A is also capable of inducing expression of inhibin by Sertoli cells, it may contribute to the pituitary-gonadal feedback loop in a similar manner to FSH. Because Sertoli cell number is a key regulator of sperm production and fertility, this mechanism could provide a further level of control over testis growth and subsequent fertility.
Acknowledgmentso99, 百拇医药
The authors gratefully acknowledge the efforts of Lynda Foulds for preparing inhibin A and follistatin. We thank Professor Gail Risbridger and Drs. Kate Loveland and Mark Hedger for reading the manuscript.o99, 百拇医药
Received June 6, 2002.o99, 百拇医药
Accepted for publication October 4, 2002.o99, 百拇医药
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