Molecular Human Reproduction, Vol. 7, No. 12, 1107-1114,
December 2001
© 2001 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Age-dependent activin receptor expression pinpoints activin A as a physiological regulator of rat Sertoli cell proliferation
Department of Histology and Medical Embryology, University of Rome ‘La Sapienza’, Rome, Italy
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Abstract |
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It is currently believed that the fertility level of the adult mammalian testis is related to the total number of Sertoli cells, which is established in the early prepubertal life. We have previously reported that, in an in-vitro system, terminal Sertoli cell proliferation is sustained by activin A in concert with FSH. In this paper, we have addressed the question of whether this activin A effect correlates with activin receptor II (ActRII) expression pattern during early post-natal testis development. We first determined the precise developmental interval of activin proliferative effect on Sertoli cells in vitro and then analysed the expression of ActRII in purified testicular cell populations by Northern blot and in-situ hybridization. While the 3 kb ActRII isoform was widely expressed at different ages and in several testicular cells, including Sertoli cells, germ cells and myoid cells, the canonical 6 kb ActRII isoform was specifically and transiently expressed at a high rate in Sertoli cells at 7–9 days after birth, the time when these cells respond to activin A in vitro. In the light of these results, we conclude that activin A regulates terminal Sertoli cell proliferation in the rat testis and that this effect is mediated by the 6 kb isoform of ActRII.
activin/activin receptor/proliferation/Sertoli cell/testis
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Introduction |
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During mammalian testis development, Sertoli cells proliferate at a high rate during late fetal life. Such proliferative activity then gradually declines after birth and eventually ends in the early post-natal life (Steinberger and Steinberger, 1971
; Orth et al., 1988). In the adult testis, Sertoli cells are mitotically inactive and establish physical and functional interactions with germ cells within the seminiferous epithelium and these interactions are critical for maintaining spermatogenesis. Thus, it is currently accepted that the fertility of the adult is directly linked to the total Sertoli cell number, as defined during post-natal testis development (Orth et al., 1988). Both endocrine and paracrine hormones control the process of Sertoli and germ cell differentiation (Russell and Griswold, 1993). Although several candidate factors have been proposed to play direct and/or indirect roles in spermatogenesis, the delicate balance between different signals controlling somatic and germ cell proliferation and differentiation is still poorly understood.
Activins are growth factors of the TGF-ß superfamily and were originally isolated from the gonads (Ying, 1988). Subsequent studies have shown that these proteins play a number of roles in the regulation of cell survival, proliferation, differentiation and migration in a variety of tissues (Ying et al., 1997). They also exhibit a bewildering array of actions on various testicular cells (Gnessi et al., 1997; Mather et al., 1997; de Kretser et al., 2000), including opposing effects of inhibition and stimulation upon primordial germ cells (Richards et al., 1999) and gonocyte (Meehan et al., 2000) proliferation respectively. In addition, a number of observations have recently suggested that activin is involved in Sertoli cell mitotic activity during testis development. In fact, male mice carrying a deletion of activin receptor type II (ActRII ) display a significant delay in reaching puberty and reduced seminiferous tubule volume, consistent with an overall decrease in Sertoli cell number (Matzuk et al., 1995). Similar reproductive defects have also been described in mutant mice lacking activin A, but normally producing activin B (Brown et al., 2000), leading to the idea that activin(s), and/or other members of the TGF-ß family signalling through ActRII, are involved in the process of Sertoli cell number determination during post-natal testis development.
We previously described that, when activin A is administered in combination with FSH to in-vitro organ cultures of 9-day-old rat testis, proliferation of Sertoli cells is significantly stimulated, whereas that of differentiating type A spermatogonia is reduced. These effects were concentration-dependent and follistatin-sensitive (Boitani et al., 1995), pointing to the importance of activin as a local modulator of Sertoli cell proliferation during early prepubertal life. However, in-vitro studies may not represent the actual in-vivo condition. In the present study, we have overcome this difficulty by determining in detail the developmental interval of the activin proliferative effect on Sertoli cells and the expression of activin receptors in these and other testicular cells by in-situ hybridization and Northern blot, during very early post-natal rat testis development. We show that Sertoli cells specifically express the canonical 6 kb ActRII mRNA when they respond to activin A by increasing their mitotic activity, but not during other developmental periods, when they are not responsive to activin. We also show that the testis-specific 3 kb ActRII mRNA is constantly expressed in various testicular cells including Sertoli, spermatogonial and myoid cells.
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Materials and methods |
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Animals
Male Wistar rats were used in all experiments. Animals were housed in accordance with guidelines for animal care of University of Rome, La Sapienza and were killed by asphyxiation with CO2 before organ removal.
Organ cultures
In-vitro organ cultures of testis from 1 to 18-day-old rats were performed as previously described (Boitani et al., 1993, 1995). Briefly, testicular fragments were arranged on steel grids that had been previously coated with 2% Agar. Grids were then placed in organ culture dishes (Falcon, Becton Dickinson, NJ, USA). Culture medium was Eagle's minimum essential medium (MEM) with Earle's salts (GIBCO BRL, Grand Island, NJ, USA) supplemented with glutamine (2 mmol/l), HEPES (15 mmol/l), nonessential amino acids (single-strength), penicillin (100 IU/ml), streptomycin (100 mg/ml) and gentamycin (50 mg/ml).
Ovine FSH (o-FSH-17; NHPP, Torrance, CA, USA) and recombinant human (rh) activin A were added to the culture medium either alone or in combination at a final concentration of 200 ng/ml and 100 ng/ml respectively. Tissue fragments were cultured at 32°C in a humidified incubator with 5% CO2 for the times indicated in the Results.
Testis fragments were labelled with 5 µCi/ml methyl-3H-thymidine (spec.act. 20 Ci/mmol, NEN Du Pont, Milan, Italy) or 5-bromodeoxyuridine during the last 5 h of culture (Boitani et al., 1995), then washed twice with minimum essential medium and processed as needed.
Thymidine incorporation into DNA
Testis fragments labelled with 3H-thymidine were treated as previously described (Boitani et al., 1995). Briefly, fragments were incubated overnight at 55°C in the presence of 0.5 mg/ml proteinase-K (Sigma, Milan, Italy) in 50 mmol/l Tris-HCl pH 8, 100 mmol/l EDTA, 100 mmol/l NaCl and 1% sodium dodecyl sulphate (SDS). The DNA was then extracted with phenol-chloroform-isoamyl alcohol and radioactivity measured by liquid scintillation counting. The DNA content was determined by a fluorimetric assay using Hoechst 33258 (Sigma) as a fluorescent dye. To this end, aliquots of samples were added to an appropriate solution of the dye and fluorescence was immediately determined with a Perkin–Elmer fluorimeter at 365/460 nm (excitation/emission) wavelengths using salmon sperm DNA as a standard.
Bromodeoxyuridine incorporation and labelling index measurement
BrdU-labelled testis fragments were fixed in Bouin's fluid, dehydrated, embedded in Histowax (Reichert-Jung, Milan, Italy) and serial-sectioned. Sections (5 µm) were stained as previously described (Boitani et al., 1993) using an anti-BrdU monoclonal antibody diluted 1:10 (Amersham, Buchs, UK) and a peroxidase-conjugated secondary antimouse IgG antibody diluted 1:80 (Dako, Milan, Italy).
Sertoli cells were identified by nuclear morphology and location within the seminiferous cords. Numbers of labelled/unlabelled Sertoli cells were determined by analysing 40–45 cord cross-sections, selected at random, and by expressing the labelling index in terms of percentage of labelled cells.
Cell preparations
Highly purified type A spermatogonia were obtained from 9-day-old rat testis as previously described (Morena et al., 1996). Briefly, the cell suspension obtained following enzymatic digestion of testicular tissue was plated for 1 h on plastic dishes coated with Datura stramonium agglutinin (DSA) (Sigma). Cells not adhering to the lectin were fractionated on a discontinuous percoll density gradient (Pharmacia Biotech, Milan, Italy), giving a cell fraction containing at least 85% type A spermatogonia.
Sertoli cells were isolated from 9-day-old rats as described by Schlatt et al.. (Schlatt et al., 1996), and exposed to hypotonic treatment to eliminate contaminating germ cells (Galdieri et al., 1981). In our observations, Sertoli cell cultures appeared to contain <10% myoid cells, as determined by alkaline phosphatase staining.
Myoid cells (>90–95% pure) were isolated from 9-day-old rat testis using a Percoll gradient purification step (Palombi et al., 1988; Filippini et al., 1993), then rapidly frozen and stored at –80°C.
Northern blot analysis
Total RNA was extracted from testes or isolated cell populations using the guanidinium thiocyanate-phenol-chloroform method of Chomczinsky and Sacchi (Chomczyski and Sacchi, 1987). The RNA was fractionated on a denaturating formaldehyde 1.2% Agarose gel and then transferred to a nylon membrane (Hybond-N+, Amersham, Arlington Heights, IL, USA). After pre-hybridization for 6 h at 42°C in a solution containing 50% formamide, 1 mol/l NaCl, 10% dextran sulphate, 0.2% Denhardt's solution, 1% SDS, 100 µg/ml denatured salmon sperm DNA and 50 µg/ml yeast tRNA, the blots were hybridized with rat ActRII cDNA (0.8 kb fragment coding for the intracellular domain) (Feng et al., 1993) and labelled with 32P-dCTP by random priming (Gibco BRL, Grand Island, NY, USA). After hybridization at 42°C, the filters were washed with 2xSSC, 0.1% SDS at room temperature for 30 min, followed by two 15 min washes with 0.5xSSC, 0.1% SDS at 65°C. The final wash was in 0.2xSSC, 0.1% SDS at room temperature for 30 min. Filters were eventually exposed to Hyperfilms-MP (Amersham) at –80°C for the appropriate time. For standardization of different lanes, blots were re-hybridized with a ribosomal RNA cDNA probe. Amounts of ActRII RNA and rRNA were quantitatively assessed by laser densitometry. Values of ActRII RNA were normalized relative to the rRNA signal of the same lane.
In-situ hybridization
Sense and antisense (35S) UTP-labelled RNA probes (having similar specific activities) were generated from rat ActRII cDNA (Feng et al., 1993) using T7 and Sp6 RNA polymerases (Promega, Madison, WI, USA). The plasmid was linearized with Hind III and Eco RI. Details concerning tissue sections and in-situ hybridization are described by Morena et al.. (Morena et al., 1995).
Reverse transcription–polymerase chain reaction (RT–PCR)
Total RNA from testis fragments was isolated using guanidine thiocyanate–caesium chloride ultracentrifugation method (Chirgwin et al., 1979). One to 5 µg of total RNA was reverse-transcribed using SuperScriptTM II RT kit (Gibco, BRL), following the manufacturer's instructions. Amplification of cDNA was performed in a final volume of 50 µl, with 30 cycles (95°C for 45 s, 56°C for 45 s, 72°C for 1 min). Within the range of linear amplification, this cycle number allowed a linear cDNA dose response. The following oligodeoxyribonucleotides for ActRII cDNA were used: sense, 5'-GCTCTTCAGGTGCTATAC-3'; and antisense 5'-TTTGAAGTGGGCTGTGTG-3'. The PCR amplification fragment had a length of 336 bp. As an internal control for the amount of cDNA used, S16 amplification was performed using the following primers: sense 5'-AGGAGCGATTTGCTGGTGTGGA-3'; and antisense 5'-GCTACCAGGGCCTTTGAATGG-3', giving a PCR amplification fragment of 103 bp. PCR products were separated by electrophoresis on 2% agarose gels and stained with ethidium bromide. ActRII expression was quantitated by densitometry, normalized to the content in S16 and eventually expressed as arbitrary densitometric units.
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Results |
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Age-dependent effect of activin upon testicular cell proliferation and differentiation
We have previously observed the age-dependent changes in the effect of activin and FSH upon the labelling index of Sertoli cells from 3, 9 and 18-day-old rats (Boitani et al., 1995
). To define in detail the developmental interval of Sertoli cell sensitiveness to activin, we examined the effect of activin A and FSH on testicular cell proliferation by culturing in vitro testis fragments from animals of different ages (1–18 days after birth) for 3 days (Figure 1). When testis fragments were obtained from 1 to 7-day-old rats, FSH alone significantly stimulated 3H-thymidine incorporation into DNA. In contrast, the combination of activin A and FSH had an additional stimulatory effect with respect to that of FSH alone in the time interval from 7–11 days of donor animal age, but not in other periods of testis development. Activin A alone had no effect at any animal age investigated. To demonstrate the specific effect of the hormone treatments on Sertoli cell proliferation, we measured the percentage of BrdU-labelled Sertoli cells (i.e. the labelling index) in testis fragments obtained from 7, 9 and 11-day-old rats and cultured for 3 days in the presence or absence of the hormones. Results are reported in Table I. In 7-day-old, but not in older, rats, FSH caused a significant increase in Sertoli cell proliferation, whereas activin had no effect at any age when administered alone. In contrast, the percentage of BrdU-labelled Sertoli cells dramatically increased in response to the combination of activin A and FSH, compared with FSH alone, in all three ages, demonstrating that Sertoli cells respond to the factor in this phase of testis development.
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To address the question of whether activin-dependent Sertoli cell proliferation in the developmental period defined above influences germ cell differentiation, testis fragments from 9-day-old rats were cultured in vitro for periods of up to 10 days in the presence of FSH with or without activin A, then labelled with BrdU and eventually processed for histology. A good progression of germ cell differentiation up to primary spermatocytes was observed when testis fragments were cultured for 10 days in the presence of FSH (Figure 2a,b,c
). In contrast, addition of activin A together with FSH to the medium allowed Sertoli cells to maintain their mitotic activity even after 10 days of culture, and blocked germ cell differentiation through the meiotic phase (Figure 2d,e,f). These results show that activin A differentially affects Sertoli and spermatogonial cell differentiation, in line with the finding that FSH-induced mitotic activity of differentiating type A spermatogonia was markedly reduced when fragment cultures were treated for 3 days with activin (Boitani et al., 1995).
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Activin receptor expression
We next addressed the question of whether the expression of the ActRII receptor was developmentally related to the observed biological effect during early post-natal testis growth. We therefore investigated the testicular expression pattern of activin receptor type II in rats from 1–22 days after birth by Northern blot analysis (Figure 3
). Two transcripts, having a size of 6 and 3 kb respectively were identified, and the amount of these transcripts changed as a function of donor animal age. In fact the 6 kb mRNA was the most abundant form expressed during early prepubertal life, reaching a peak in the interval of 7–9 days, and decreasing afterwards. In contrast, the 3 kb mRNA was expressed at more consistent level in all infant/juvenile stages examined, but markedly increased in the testis from 40-day-old rats. In order to identify the testicular cell types expressing activin receptors at the early period of post-natal development, we performed an in-situ hybridization analysis on histological sections of 9-day-old rat testis, using sense and antisense ActRII RNA probes (Figure 4). Hybridization with sense RNA was very low, with no significant difference in grain density between seminiferous cords and interstitium (Figure 4a). With the antisense probe, a specific above-background level signal was clearly distributed over all cells in the seminiferous cords, indicating that both somatic and germ cells actually expressed the ActRII gene (Figure 4b,c). Significant labelling was also observed on peritubular cells. However, the antisense riboprobe used in this experiment detected all ActRII transcripts, making it not possible to determine whether a specific ActRII isoform was expressed in a given testicular cell. This question was therefore addressed by Northern blot analysis of total RNA derived from homogeneous cell populations from 9-day-old rat testis, including highly purified Sertoli cells, spermatogonia and myoid cells. Even though all these cells expressed ActRII (Figure 5), it appeared that the 6 and 3 kb forms were differentially expressed in somatic and germ cells. In fact at this age, the 6 kb RNA was markedly expressed only in Sertoli cells, whereas the 3 kb mRNA was the predominant transcript of spermatogonia and, at a lower level, of myoid cells.
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To determine whether activin A regulates the steady-state level of its receptor mRNA, testis fragments from 13-day-old rats were incubated for 3 days in the presence or absence of activin A, with or without FSH. ActRII mRNA was then examined by semi-quantitative RT–PCR analysis. Treatment with activin, either alone or in combination with FSH, resulted in a significant suppression (50 and 70% respectively) of ActRII mRNA at the end of the treatment compared with the control values (Figure 6
). In contrast, the FSH treatment produced a slight, but consistent, increase in ActRII mRNA expression.
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Discussion |
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In this study, we have shown that the stimulatory effect of activin A upon testicular cell proliferation is temporally related to the expression of ActRII during early pubertal development of rat gonad. We have identified a narrow window of activin effectiveness upon cell proliferation between 7 and 11 days of post-natal age, corresponding to the period of Sertoli cell terminal proliferation during which the total number of Sertoli cells populating the adult gonad is established (Gondos and Berndston, 1993
). Present observations have clearly shown that Sertoli cell proliferation takes place by two distinct phases. During the first phase, lasting from birth to 6 days of age, Sertoli cells are maximally stimulated to proliferate by FSH alone, whereas in the second phase, lasting from 7–11 days, Sertoli cell division is maintained by activin in concert with FSH. These findings are in agreement with and extend our previous observations, showing that the labelling index of Sertoli cells is increased by the combined action of activin and FSH in 9-day-old, but not in 3-day-old rats (Boitani et al., 1995). However, the possibility that the observed increase in activin effectiveness is due to a rapid decline in FSH effect cannot be ruled out. The promoting action of activin upon Sertoli cell proliferation is further documented by the present finding that Sertoli cells derived from 9-day-old rats continued to undergo mitosis even after 10 days of culture when both activin and FSH were added to the culture medium, but not when fragments were treated with FSH alone. Overall, the data obtained in this study indicate that activin prolongs the duration of Sertoli cell proliferation, eventually resulting in an increase in their number. These results are of particular interest in the context of the role that Sertoli cells play in determining the functional potential of the adult testis. Although direct evidence for the effect of activin upon Sertoli cell proliferation in vivo is still lacking, recent studies using transgenic mutant mice have pointed to the concept that the activin/follistatin system plays an important role in testicular growth and physiology in vivo. In fact male mice overexpressing follistatin, which functions as an antagonist for activins in vivo, show decreased testis size and partial seminiferous tubule degeneration with some degree of infertility (Guo et al., 1998). A similar decrease in testis size accompanied by a delay in reaching fertility has also been described in ActRII deficient mice, although in this case the decreased serum FSH levels observed in these animals might have contributed to the reduction of testicular mass (Matzuk et al., 1995). Evidence for a physiological role of FSH in regulating Sertoli cell division has been provided by several studies. In neonatal rats, an increased exposure to FSH results in testicular hypertrophy during adulthood, with a proportionate increase in the number of both Sertoli and germ cells (Meachem et al., 1996). The present finding of an age-dependent change in the biological effect of activin is consistent with changes in the expression of testicular activin ßA mRNA during post-natal testis development. In fact, Meunier et al. and recent work from our laboratory (unpublished observations) have indicated that testis levels of ßA and ßB mRNAs during the first two weeks are higher than during postpubertal life (Meunier et al.,1988), allowing one to conclude that the regulatory role of the inhibin/activin system is more important in the immature than in the mature animal. However, activin function is interwoven with inhibin function at several levels, including mRNA and protein expression, bioactivity, receptor interactions and signalling. Therefore, further investigation is required to fully clarify the activin/inhibin regulatory mechanisms in immature rat testis.
Additional evidence for the physiological relevance of the activin/inhibin family during early post-natal testicular maturation is provided by the present observation that another player of the system, the activin receptor, is expressed in a spatio–temporal specific manner that correlates with the modulating action of activin upon Sertoli cell proliferation. A number of different receptors for activin have been identified so far. Type II activin receptors (ActRII and the closely related ActRIIB) are the primary receptors that bind the ligand. The ligand-type II receptor complex then recruits the type I receptor(s), ActRI, ActRIB and ALKs (the activin receptor-like kinase), which are phosphorylated and eventually propagate the signal (Attisano et al., 1996; Massagué, 1998). Interestingly, we have found that Sertoli cells specifically express the 6 kb transcript, one of the two ActRII mRNAs expressed in the rat (Feng et al., 1993) as well as in the mouse (Mathews and Vale, 1991), between day 7 and 9 after birth in the total testis, at much greater levels compared with the 3 kb transcript. This expression of the 6 kb transcript co-ordinates in time with the profile of activin effectiveness upon Sertoli cell growth. These findings again point to the role of activin as a physiological regulator of terminal Sertoli cell proliferation (see above). Although the significance of having two transcripts of ActRII is not yet clear, the 6 kb species is distributed in a number of tissues, including brain, intestine, liver and kidney (Mathews and Vale, 1991), whereas the 3 kb transcript is only abundant in the adult testis. Consistent with that, our finding that spermatogonia from immature rats mainly express the 3 kb mRNA is in line with previous reports on the localization of this isoform in male germ cells at more differentiated stages (Feng et al., 1993). There is no evidence that the two messengers of ActRII are translated into functionally different proteins; however, several bands of different size have been observed by affinity-labelling with I125-activin (Mathews and Vale, 1993), suggesting that several isoforms of ActRII could exist and could differ in ligand binding affinity, as is the case for ActRIIB (Attisano et al., 1992). Therefore, our observation that the 6 and 3 kb isoforms are differentially expressed in Sertoli cells and spermatogonia respectively, suggests that activin may act on either/both cell type(s) to elicit different responses, including the stimulatory effect upon Sertoli cell proliferation and the inhibitory action upon germ cell differentiation. In addition, we have also shown that the stimulatory effect of activin on Sertoli cell proliferation is followed by activin receptor down-regulation, showing that the activin signalling transduction pathway is fully functional in the prepubertal rat testis at this age of testis growth. In this regard, it has been reported that 
-inhibin deficient mice display a high level of activin ßA in the blood associated with a dramatic testis-specific reduction of ActRII mRNA (Trudeau et al., 1994), suggesting that a local increase in activin concentration in vivo leads to a significant down-regulation in activin receptor expression.
In conclusion, the present study identifies Sertoli cells as a target for the regulatory action of activin A and demonstrates the spatio–temporal localization of ActRII during early development of rat testis. Our data point to the concept that discrete changes in the activin/activin receptor complex may be relevant in the local regulation of Sertoli cell differentiation.
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We are grateful to Dr Franco Mangia for discussion and critical reading of the manuscript. We thank Mrs Tiziana Menna and Stefania De Grossi for technical assistance. Activin A used in the first part of this study was generously provided by Genentech. Additional rh-activin A and ovine FSH were obtained from the NIDDK's National Hormone and Pituitary Program and NICHD. We thank Dr Ching-Ling Chen for generously providing ActRII cDNA.
This work was supported by grants: Murst co-fin 1998–1999 (to M.S.) and Murst co-fin 1999–2000 (to C.B.).
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1 To whom correspondence should be addressed at: Dept of Histology and Medical Embryology, University of Rome `La Sapienza', Via A. Scarpa 14, 00161 Roma, Italy. E-mail: carla.boitani{at}uniroma1.it
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References |
|---|
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Attisano, L., Wrana, J.L., Cheifetz, S. et al. (1992) Novel activin receptor: distinct genes and alternative mRNA splicing generate a repertoire of serine/threonine kinase receptors.Cell, 68, 97–108.[ISI][Medline]
Attisano, L., Wrana, J.L., Montalvo, E. et al. (1996) Activation of signalling by the activin receptor complex. Mol. Cell. Biol., 16, 1066–1073.[Abstract]
Boitani, C., Politi, M.G. and Menna, T. (1993) Spermatogonial cell proliferation in organ culture of immature rat testis. Biol. Reprod, 48, 761–767.[Abstract]
Boitani, C., Stefanini, M., Fragale, A. et al. (1995) Activin stimulates Sertoli cell proliferation in a defined period of rat testis development.Endocrinology, 136, 5438–5444.[Abstract]
Brown, C.W., Houston-Hawkins, D.E., Woodruff, T.K. et al. (2000) Insertion of Inhbb into the Inhba locus rescues the Inhba-null phenotype and reveals new activin functions. Nature Genet., 25, 453–457.[ISI][Medline]
Chirgwin, J.M., Przybyla, A.E.M.R.J. and Rutter, W.J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.Biochemistry, 18, 5294–5299.[Medline]
Chomczyski, P. and Sacchi, N. (1987) Single step of RNA isolation by AGPC method. Anal. Biochem., 162, 156–159.[ISI][Medline]
de Kretser, D.M., Meinhardt, A., Meehan, T. et al. (2000) The roles of inhibin and related peptides in gonadal function. Mol. Cell. Endocrinol., 161, 43–46.[ISI][Medline]
Feng, Z.M., Madigan, M.B. and Chen, C.L. (1993) Expression of type II activin receptor genes in the male and female reproductive tissues of the rat.Endocrinology, 132, 2593–2600.[Abstract]
Filippini, A., Tripiciano, A., Palombi, F. et al. (1993) Rat testicular myoid cells respond to endothelin: characterization of binding and signal transduction pathway.Endocrinology, 133, 1789–1796.[Abstract]
Galdieri, M., Palombi, F., Russo, M.A. et al. (1981) Pure Sertoli cell cultures: a new model for the study of somatic–germ cell interactions. J. Androl., 5, 249–259.
Gnessi, L., Fabbri, A. and Spera, G. (1997) Gonadal peptides as mediators of development and functional control of the testis: an integrated system with hormones and local environment. Endocr. Rev., 18, 541–609.
Gondos, B. and Berndston, W.E. (1993) Postnatal and pubertal development. In Russell, L. and Griswold, M.D. (eds) Cache River Press, Clearwater, Florida, pp. 116–153.
Guo, Q., Kumar, T.R., Woodruff, T. et al. (1998) Overexpression of mouse follistatin causes reproductive defects in transgenic mice. Mol. Endocrinol., 12, 96–106.
Massagué, J. (1998) TGF-beta signal transduction. Ann. Rev. Biochem., 67, 753–791.[ISI][Medline]
Mather, J.P., Moore, A. and Li, R.H. (1997) Activins, inhibins, and follistatins: further thoughts on a growing family of regulators. Proc. Soc. Exp. Biol. Med., 215, 209–222.[Abstract]
Mathews, L.S. and Vale, W. (1991) Expression cloning of activin receptor, a predicted transmembrane serine kinase.Cell, 65, 973–982.[ISI][Medline]
Mathews, L.S. and Vale, W.W. (1993) Characterization of type II activin receptors. Binding, processing, and phosphorylation. J. Biol. Chem., 268, 19013–19018.
Matzuk, M.M., Kumar, T.R., Vassalli, A. et al. (1995) Different phenotypes for mice deficient in either activins or activin receptor type II.Nature, 374, 356–360.[Medline]
Meachem, S.J., McLachlan, R.I., de Kretser, D.M. et al. (1996) Neonatal exposure of rats to recombinant follicle stimulating hormone increases adult Sertoli and spermatogenic cell numbers. Biol. Reprod., 54, 36–44.[Abstract]
Meehan, T., Schlatt, S., O'Bryan, M.K. et al. (2000) Regulation of germ cell and Sertoli cell development by activin, follistatin and FSH. Dev. Biol., 220, 225–237.[ISI][Medline]
Meunier, H., Rivier, C., Evans, R.M. et al. (1988) Gonadal and extragonadal expression of inhibin , ßA and ßB subunits in various tissue predicts diverse functions. Proc. Natl Acad. Sci. USA, 85, 247–251.
Morena, A.R., Boitani, C., de Grossi, S. et al. (1995) Stage and cell-specific expression of the adenosine 3',5' monophosphate-phosphodiesterase genes in the rat seminiferous epithelium.Endocrinology, 136, 687–695.[Abstract]
Morena, A.R., Boitani, C., Pesce, M. et al. (1996) Isolation of highly purified type A spermatogonia from prepubertal rat testis. J. Androl., 17, 708–717.
Orth, J.M., Gunsalus, G.L. and Lamperti, A.A. (1988) Evidence from Sertoli cell-depleted rats indicates that spermatids number in adults depends on number of Sertoli cells produced during perinatal development.Endocrinology, 122, 787–794.[Abstract]
Palombi, F., Farini, D., De Cesaris, P. and Stefanini, M. (1988) Characterization of peritubular myoid cells in highly enriched in vitro cultures. In Cooke, B.A. and Sharpe, R.M. (eds) Molecular and Cellular Endocrinology of the Testis. Raven Press, New York, pp. 311–317.
Richards, A.J., Enders, G.C. and Resnick, J.L. (1999) Activin and TGFbeta limit murine primordial germ cell proliferation. Dev. Biol., 207, 470–475.[ISI][Medline]
Russell, L.D. and Griswold, M.D. (1993) The Sertoli Cell. Cache River Press, Clearwater, Florida.
Schlatt, S., de Kretser, D.M., and Loveland, K.L. (1996) Discriminative analysis of rat Sertoli and peritubular cells and their proliferation in vitro: evidence for follicle-stimulating hormone-mediated contact inhibition of Sertoli cell mitosis. Biol. Reprod., 55, 227–235.[Abstract]
Steinberger, A. and Steinberger, E. (1971) Replication pattern of Sertoli cells in maturing rat testis in vivo and in organ culture. Biol. Reprod, 4, 84–87.[Abstract]
Trudeau, V.L., Matzuk, M.M., Hache, R.J. et al. (1994) Overexpression of activin-beta A subunit mRNA is associated with decreased activin type II receptor mRNA levels in the testes of alpha-inhibin deficient mice. Biochem. Biophys. Res. Commun., 203, 105–112.[ISI][Medline]
Ying, S.Y. (1988) Inhibins, activins, and follistatins: gonadal proteins modulating the secretion of follicle-stimulating hormone. Endocr. Rev., 9, 267–293.[Abstract]
Ying, S.Y., Zhang, Z., Furst, B. et al. (1997) Activins and activin receptors in cell growth. Proc. Soc. Exp. Biol. Med., 214, 114–122.[Abstract]
Submitted on April 20, 2001; accepted on September 21, 2001.
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