Molecular Human Reproduction, Vol. 6, No. 10, 877-881,
October 2000
© 2000 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
The effect of FSH on male germ cell survival and differentiation in vitro is mimicked by pentoxifylline but not insulin
1 Laboratoire d'Eylau, 55 rue Saint-Didier, 75116 Paris, France, 2 MAR&Gen, Molecular Assisted Reproduction and Genetics, Gracia 36, 18002 Granada, Spain, 3 University of Granada, Campus Universitario Fuentenueva, 18004 Granada, Spain and 4 European Hospital, Via Portuense 700, Rome, Italy
Abstract
High concentrations of FSH have been shown to boost in-vitro differentiation of germ cells from men with normal spermatogenesis and from some patients with in-vivo maturation arrest. This study shows that the differentiation-promoting effect of FSH is connected to protection against germ cell apoptosis and that both effects can be mimicked by the intracellular cyclic AMP (cAMP)-elevating drug pentoxifylline. On the other hand, a high concentration of insulin, supposed to act at the insulin-like growth factor I receptor, did not exert any effect either on differentiation or apoptosis of germ cells in vitro. These data show that the in-vitro effects of supraphysiological concentrations of FSH on human spermatogenesis are mediated by the classical FSH signal transduction pathway involving cAMP as a second messenger. Pentoxifylline may thus be useful as an alternative means for intracellular cAMP elevation in men with high circulating FSH concentrations leading to desensitization of the FSH receptor.
FSH/in-vitro spermatogenesis/insulin/male germ cell/pentoxifylline
Introduction
Recent studies have shown that certain phases of human spermatogenesis can be substantially accelerated by in-vitro culture of seminiferous tubule segments recovered from men with normal spermatogenesis (Tesarik et al., 1998a
,b). When the same in-vitro culture system was used in cases of maturation arrest, meiotic and post-meiotic germ cells from some patients overcame the in-vivo block and resumed differentiation, leading to the first pregnancies and births in cases of complete spermiogenesis failure and in a case of maturation arrest at the primary spermatocyte stage (Tesarik et al., 1999a,b). In the former indication, in-vitro culture is supposed to boost cytoplasmic maturation of spermatids (Tesarik et al., 1998a) and to facilitate the distinction between healthy spermatids and those carrying irreversible DNA damage due to incipient apoptosis (Tesarik et al., 1999c).
In spite of these encouraging data, in-vitro culture fails to overcome the in-vivo maturation arrest in 50–75% of cases, depending on the in-vivo blocking stage and serum FSH concentration (Tesarik et al., 2000). The observation that the success of in-vitro culture is lower in men with very high serum FSH concentrations (>20 IU/l), compared with those with normal or slightly elevated FSH (Tesarik et al., 2000) has suggested that the failures may be at least partly due to desensitization of the FSH receptor on Sertoli cells within the cultured seminiferous tubule segments which are thus unable to respond to FSH present in culture medium by creating a spermatogenesis-promoting microenvironment. If this is the case, the problem might be resolved by acting directly at the signal transduction pathway downstream of the FSH receptor or by using alternative hormones and growth factors known to promote survival and differentiation of cultured cells.
The effect of FSH on Sertoli cells is mediated by cyclic AMP (cAMP) (Parvinen, 1982) whose action is modulated by endogenous cAMP phosphodiesterase (Morena et al., 1995; Naro et al., 1996). Insulin and insulin-like growth factors I and II (IGF-I and IGF-II) can also promote germ cell in-vitro differentiation by acting at Sertoli cells (Borland et al., 1984; Mita et al., 1985) or directly through receptors located on germ cells (Tres et al., 1986; Vannelli et al., 1988).
In this study we examined whether the beneficial effect of FSH on human male germ cell in-vitro survival and differentiation is mimicked by pentoxifylline, a phosphodiesterase inhibitor increasing cAMP concentration in cells, and insulin added at a high concentration at which it is known to act through the IGF-I receptor (Sara and Hall, 1990; Francis et al., 1993). The two substances were chosen with regard to the ease of their eventual future clinical application for promoting in-vitro differentiation of germ cells from men with maturation arrest, since both are currently used in other therapeutic indications in human medicine. This study compares the effects of FSH, pentoxifylline and insulin on the appearance of apoptotic DNA damage, on morphological differentiation and on cytoplasmic maturation of cultured germ cells recovered from men with obstructive azoospermia and with ongoing complete in-vivo spermatogenesis as a prelude to future studies with samples from men with maturation arrest.
Materials and methods
Testicular tissue source and handling
Ten patients with obstructive azoospermia undergoing testicular biopsy aimed at the extraction of spermatozoa for assisted reproduction gave their informed consent for part of the testicular tissue recovered to be used in the present experiments. A small piece of testicular tissue was obtained from each patient by open testicular biopsy and placed in Gamete-100 medium (Scandinavian IVF Science, Gothenborg, Sweden). Samples were disintegrated mechanically by using sterile glass slides. After isolation of sufficient numbers of spermatozoa to be used for assisted reproduction, the rest of the disintegrated testicular tissue from each patient, consisting of individual cells, cell clusters and intact segments of the seminiferous tubules, was brought in a homogenous suspension and distributed into five equal aliquots. One aliquot was used immediately for the evaluation of germ cell DNA damage and differentiation activity. The remaining four aliquots were assigned to in-vitro culture in the presence of different medium supplements. Each aliquot was thus centrifuged at 200 g for 10 min and then resuspended in Gamete-100 medium with the corresponding supplement.
In-vitro culture
In-vitro culture of four aliquots of each testicular tissue sample, prepared as described above, was carried out at 30°C in cellculture tubes containing 2 ml of medium. Three aliquots of each testicular biopsy sample were cultured in Gamete-100 medium (including testosterone; see Discussion) supplemented with 50 IU/l human recombinant FSH (Puregon, Organon, Oss, The Netherlands), 1 mg/ml pentoxifylline (Sigma, St Louis, MO, USA), or 10 µg/ml insulin (Sigma) respectively. The fourth aliquot was cultured in unsupplemented Gamete-100 medium to serve as control. After 24 h of culture, cells from each aliquot were harvested by centrifugation and distributed into two subaliquots that were analysed for DNA damage and for differentiation activity respectively.
Evaluation of germ cell DNA damage
Segments of the seminiferous tubules and cell clusters were dissociated by incubation for 1 h in Gamete-100 medium with the addition of collagenase I (1000 IU/ml) and elastase (10 IU/ml) (both purchased from Sigma). The resulting cell suspensions were smeared on microscope slides, left to air-dry and fixed for 15 min with 5% glutaraldehyde in 0.05 mol/l cacodylate buffer (pH 7.4). Fixed smears were processed by a modified terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) procedure (Tesarik et al., 1998c) using the apoptosis detection system, fluorescein kit (Promega, Charbonnières, France) and 4D4 monoclonal antibody, reacting with proacrosin in human germ cells from the pachytene stage onwards (Escalier et al., 1991) as specific germline marker. Primary spermatocytes (n = 200) and round spermatids (n = 200) were assessed for each aliquot.
Evaluation of germ cell differentiation activity
Aliquots assigned to this analysis were first treated with collagenase I and elastase as described in the previous section. The resulting cell suspensions were smeared onto four microscope slides, allowed to air-dry and fixed for 10 min with 100% ethanol. For each aliquot, two slides were stained with the use of Papanicolaou method (World Health Organization, 1992), and the other two were processed for immunocytochemistry with 4D4 monoclonal antibody (Tesarik et al., 1998a). The percentages of normal and atypical forms of elongating and elongated spermatids were determined in Papanicolaou-stained preparations by using the previously described staging criteria and terminology (de Kretser and Kerr, 1988; Tesarik et al., 1998a,b). Briefly, Sa, Sb, Sc and Sd stages represent normal forms of round, early elongating, late elongating and elongated spermatids respectively (de Kretser and Kerr, 1988), whereas Saf, Sbp and Scp refer to abnormal spermatid forms (Tesarik et al., 1998a,b) characterized by a round shape, non-condensed nucleus and a flagellum (Saf), a round shape, condensed and protruding nucleus and the absence of flagellum (Sbp), and a round shape, condensed and protruding nucleus and a flagellum (Scp).
Germ cell cytoplasmic maturation was evaluated by examining the status of acrosome development in 4D4-immunoreactive germ cells as described (Tesarik et al., 1998a). Briefly, three sequential developmental stages of acrosome assembly, reticular, vesicular and cap stage, were distinguished, and the percentage of germ cells showing each of them was determined. All analyses performed with the Papanicolaou-stained smears and the immunocytochemical preparations were based on the evaluation of 200 germ cells in each aliquot.
Statistical analysis
Statistical significance of differences in the proportion of germ cells with damaged DNA and in the percentages of germ cells at different stages of morphological differentiation and cytoplasmic maturation between individual treatment groups were evaluated by using 
2 and Kruskal–Wallis tests.
Results
Effects of FSH, pentoxifylline and insulin on the preservation of germ cell DNA integrity during in-vitro culture
Before the beginning of in-vitro culture, the percentages of primary spermatocytes and of round spermatids showing TUNEL-positive nuclei (indicative of DNA fragmentation) was 19 ± 2 and 22 ± 3% respectively. After 24 h of culture in unsupplemented Gamete-100 medium, the corresponding values for primary spermatocytes and round spermatids were significantly elevated (P < 0.01) and were 78 ± 8 and 64 ± 7% respectively. However, medium supplementation with either 50 IU/l FSH or 1 mg/ml pentoxifylline resulted in a significant (P < 0.01) reduction of the percentage of TUNEL-positive primary spermatocytes (Figure 1A) and round spermatids (Figure 1B), compared with unsupplemented medium. In contrast, no difference was observed between samples cultured in unsupplemented medium and in medium supplemented with 10 µg/ml insulin (Figure 1).
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Effects of FSH, pentoxifylline and insulin on germ cell morphological differentiation and cytoplasmic maturation
After 24 h of in-vitro culture in unsupplemented medium, the percentages of the major stages of post-meiotic germ cells were similar to freshly obtained samples (Table I
). After culture in medium supplemented with FSH, the percentage of round spermatids (Sa stage) was reduced, and the percentages of early and late elongating spermatids (Sb and Sc stages) was increased as compared to the control incubation in unsupplemented medium, and this effect was mimicked in medium supplemented with pentoxifylline (Table I). In contrast, the percentages of individual stages of germ cells after culture in medium supplemented with insulin did not differ from control incubations in unsupplemented medium (Table I).
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In-vitro culture in the presence of FSH or pentoxifylline also led to a significant acceleration of germ cell cytoplasmic maturation, as evidenced by a shift in the proportion of cells showing different stages of acrosome assembly in favour of more mature forms (vesicular and cap-like), accompanied by a decrease in the less mature, reticular form (Figure 2
). Insulin had no measurable effect on this process when compared with unsupplemented medium (Figure 2).
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Discussion
The results of this study provide the first direct demonstration that artificial augmentation of intracellular cAMP concentration, produced by inhibition of phosphodiesterase with pentoxifylline, mimics the effects of FSH on in-vitro survival and differentiation of human germ cells cultured in a system retaining, at least partly, the original Sertoli-germ cell associations. In this culture system, FSH has previously been shown to accelerate meiotic and post-meiotic germ cell maturation (Tesarik et al., 1998a,b) and to overcome in-vivo maturation arrest in some testiculopathies (Tesarik et al., 1999a,b). Because of the unusual speed of the in-vitro differentiation events and of the need for supraphysiological hormone concentrations to achieve these effects, the mechanism of this FSH action was questioned. The present data indicate that the signal transduction pathway implying cAMP, known to mediate the physiological FSH effects on spermatogenesis (Parvinen, 1982), is also involved in these in-vitro effects.
Since germ cells are believed to lack a functional FSH receptor (Böckers et al., 1994), these effects are likely to be dependent on the presence of Sertoli cells in the culture system. Moreover, cAMP also appears to play important regulatory roles within germ cells, since post-meiotic germ cell differentiation is disrupted in mice carrying mutations of the cAMP-responsive element modulator (CREM) gene (Blendy et al., 1996; Nantel et al., 1996). In our culture system, in which intact original Sertoli-germ cell associations were largely retained, cAMP produced by Sertoli cells in response to FSH may have been entering germ cells via specialized cell–cell junctions and thus mediate the observed stimulatory effect of FSH on post-meiotic differentiation. With regard to the ongoing debate about the role of cAMP in post-meiotic differentiation (Daniel and Habener, 2000), our data support the hypothesis that cAMP is important for the activity of CREM that accumulates in post-meiotic male germ cells of mice (Delmas et al., 1993), hamsters (Foulkes et al., 1993), rats (Walker and Habener, 1996) and men with normal spermatogenesis (Weinbauer et al., 1998), although recent evidence suggests the existence of alternative pathways of CREM activation in male germ cells (Fimia et al., 1999). Interestingly, CREM expression is reduced in round spermatids from men with post-meiotic maturation arrest (Weinbauer et al., 1998; Steger et al., 1999). Our data also suggest that cAMP, rather than FSH itself, is an important regulatory factor in mammalian spermatogenesis, since the in-vivo effect of FSH on intracellular cAMP accumulation can be mimicked, in the testis, by locally produced pituitary adenylate cyclase-activating polypeptide (Daniel and Habener, 2000) whose action may explain the sporadical observations of complete spermatogenesis in men with inactivating mutations of the FSH receptor (Tapanainen et al., 1997).
The present observations also indicate that, in addition to stimulation of germ cell differentiation, FSH also protects in-vitro cultured germ cells against apoptosis. This is in agreement with a previous study conducted in the rat (Henriksen et al., 1996). The anti-apoptotic effect of FSH added to culture medium is likely to be related to the improved developmental potential of in-vitro cultured spermatids (Tesarik et al., 1999a,b), since high frequencies of apoptotic spermatids have been reported both in an animal model of primary testicular failure and in patients suffering from post-meiotic maturation arrest (Jurisicova et al., 1999; Tesarik et al., 1998c). In this study, this FSH effect also could be mimicked by pentoxifylline. The anti-apoptotic action of FSH in the rat seminiferous epithelium is mediated partially through the stem cell factor/c-kit pathway (Yan et al., 2000). The production of stem cell factor by isolated and cultured rat seminiferous tubules has been shown to be much more dependent on FSH compared with the in-vivo condition (Yan et al., 1999). It remains to be determined whether stem cell factor, when added directly to culture medium, would also mimick the anti-apoptotic effect of FSH on human germ cells.
Unlike pentoxifylline, the presence of insulin in culture medium did not mimick any of the differentiation-promoting and anti-apoptotic effects of FSH. Because the concentration of insulin used (10 µg/ml) was high enough to stimulate the IGF-I receptor, IGFs do not appear to be involved in the regulation of human germ cell in-vitro survival and differentiation. This is in contrast with the in-vitro culture of human ovarian follicles where both FSH and high-dose insulin show similar differentiation-promoting and anti-apoptotic effects (Wright et al., 1999). However, this difference may also be due to the fact that different culture systems were used in both studies. For instance, the high concentration of testosterone, that was added to all culture groups in the present study in order to protect Sertoli cells against apoptosis (Tesarik et al., 1998b), may have up-regulated the production of IGFs in some of the cultured cells, by analogy with the effects of androgen in cultured primate ovarian follicles (Vendola et al., 1999).
One of the objectives of this study was the search for novel medium supplements with which germ cell maturation blocks refractory to in-vitro treatments using media supplemented with FSH and testosterone only (Tesarik et al., 1998b) might be overcome. The results of this study suggest that medium supplementation with insulin is not likely to be effective in this respect. On the other hand, pentoxifylline, added either instead of, or together with, FSH, might be useful in some of these cases, particularly in those in which the circulating concentration of FSH is highly elevated, leading to desensitization of the FSH receptor on Sertoli cells.
Circulating FSH concentrations are usually elevated as a result of reduced secretion of inhibin B by Sertoli cells (Illingworth et al., 1996), which has been shown, in animal models, to be a consequence of reduced numbers of post-meiotic germ cells in the testis (Pineau et al., 1990; Allenby et al., 1991). The persisting post-meiotic cells may thus be arrested at the round spermatid stage and undergo apoptosis because of the failure of Sertoli cells to respond to FSH by secreting the necessary differentiation-promoting and anti-apoptotic factors. This possibility is corroborated by the observation that germ cells from men with highly elevated serum FSH concentrations (>20 IU/l) are less likely to resume differentiation during in-vitro culture, in comparison with men with only moderately elevated FSH concentrations (Tesarik et al., 2000). In contrast, the use of pentoxifylline appears to be less justified in cases in which maturation arrest at the round spermatid stage is caused by a reduced or absent expression of CREM in round spermatids. Studies are underway to test the usefulness of pentoxifylline for in-vitro spermatogenesis of germ cells from patients with highly elevated FSH concentrations.
Notes
5 To whom correspondence should be addressed at: Laboratoire d'Eylau, 55 rue Saint-Didier, 75116 Paris. 
References
Allenby, G., Foster, P.M.D. and Sharpe, R.M. (1991) Evidence that secretion of immunoreactive inhibin by seminiferous tubules from the adult rat testis is regulated by specific germ cell types: correlation between in vivo and in vitro studies. Endocrinology, 128, 467–476.[Abstract]
Blendy, J.A., Kaestner, K.H., Weinbauer, G.F. et al. (1996) Severe impairment of spermatogenesis in mice lacking the CREM gene. Nature, 380, 162–165.[Medline]
Böckers, T.M., Nieschlag, E., Kreutz, M.R. and Bergmann, M. (1994) Localization of follicle-stimulating hormone (FSH) immunoreactivity and hormone receptor mRNA in testicular tissue of infertile men. Cell Tiss. Res., 278, 595–600.[ISI][Medline]
Borland, K., Mita, M., Oppenheimer, C.L. et al. (1984) The actions of insulin-like growth factors I and II on cultured Sertoli cells. Endocrinology, 114, 240–246.[Abstract]
Daniel, P.B. and Habener, J.F. (2000) Pituitary adenylate cyclase-activating polypeptide gene expression regulated by a testis-specific promoter in germ cells during spermatogenesis. Endocrinology, 141, 1218–1227.
de Kretser, D.M. and Kerr, J.B. (1988) The cytology of the testis. In Knobil, E. and Neill, J. (eds), The Physiology of Reproduction. Raven Press, New York, USA, pp. 837–932.
Delmas, V., van der Hoorn, F., Mellstrom, B. et al. (1993) Induction of CREM activator proteins in spermatids: down-stream targets and implications for haploid germ cell differentiation. Mol. Endocrinol., 7, 1502–1514.[Abstract]
Escalier, D., Gallo, J.-M., Albert, M. et al. (1991) Human acrosome biogenesis: immunodetection of proacrosin in primary spermatocytes and of its partitioning pattern during meiosis. Development, 113, 779–788.[Abstract]
Fimia, G.M., De Cesare, D. and Sassone-Corsi, P. (1999) CBP-independent activation of CREM and CREB by the LIM-only protein ACT. Nature, 398, 165–169.[Medline]
Foulkes, N.S., Schloter, F., Pevet, P. and Sassone-Corsi, P. (1993) Pituitary hormone FSH directs the CREM functional switch during spermatogenesis. Nature, 362, 264–267.[Medline]
Francis, G.L., Aplin, S.E., Milner, S.J. et al. (1993) Insulin-like growth factor (IGF)-II binding to IGF-I binding proteins and IGF receptors is modified by deletion of the N-terminal hexapeptide or substitution of arginine for glutamate-6 in IGF-II. Biochem. J., 293, 713–719.
Henriksen, K., Kangasniemi, M., Parvinen, M. et al. (1996) In vitro, follicle-stimulating hormone prevents apoptosis and stimulates deoxyribonucleic acid synthesis in the rat seminiferous epithelium in a stage-specific fashion. Endocrinology, 137, 2141–2149.[Abstract]
Illingworth, P.J., Groome, N.P., Byrd, W. et al. (1996) Inhibin B: a likely candidate for the physiologically important form of inhibin in men. J. Clin. Endocrinol. Metab., 81, 1321–1325.[Abstract]
Jurisicova, A., Lopes, S., Meriano, J. et al. (1999) DNA damage in round spermatids of mice with a targeted disruption of the Pp1c
gene and in testicular biopsies of patients with non-obstructive azoospermia. Mol. Hum. Reprod., 5, 323–330.
Mita, M., Borland, K., Price, J.M. and Hall, P.F. (1985) The influence of insulin and insulin-like growth factor-I on hexose transport by Sertoli cells. Endocrinology, 116, 987–992.[Abstract]
Morena, A.R., Boitani, C., de Grossi, S. et al. (1995) Stage and cell-specific expression of the adenosine 3',5' monophosphate-diesterase genes in the rat seminierous epithelium. Endocrinology, 136, 687–695.[Abstract]
Nantel, F., Monaco, L., Foulkes, N.S. et al. (1996) Spermiogenesis deficiency and germ-cell apoptosis in CREM-mutant mice. Nature, 380, 159–162.[Medline]
Naro, F., Zhang, R. and Conti, M. (1996) Developmental regulation of unique adenosine 3',5'-monophosphate-specific phosphodiesterase variants during rat spermatogenesis. Endocrinology, 137, 2464–2472.[Abstract]
Parvinen, M. (1982) Regulation of the seminiferous epithelium. Endocr. Rev., 3, 404–417.[ISI][Medline]
Pineau, C., Sharpe, R.M., Saunders, P.T.K. et al. (1990) Regulation of Sertoli cell inhibin production and of inhibin 
-subunit mRNA levels by specific germ cell types. Mol. Cell. Endocrinol., 72, 13–22.[ISI][Medline]
Sara, V.R. and Hall, K. (1990) Insulin-like growth factors and their binding proteins. Physiol. Rev., 70, 591–641.
Steger, K., Klonisch, T., Gavenis, K. et al. (1999) Round spermatids show normal testis-specific H1t but reduced cAMP-responsive element modulator and transition protein 1 expression in men with round-spermatid maturation arrest. J. Androl., 20, 747–754.
Tapanainen, J.S., Aittomaki, K., Min, J. et al. (1997) Men homozygous for an inactivating mutation of the follicle-stimulating hormone (FSH) receptor gene present variable suppression of spermatogenesis and fertility. Nature Genet., 15, 205–206.[ISI][Medline]
Tesarik, J., Greco, E., Rienzi L. et al. (1998a) Differentiation of spermatogenic cells during in-vitro culture of testicular biopsy samples from patients with obstructive azoospermia: effect of recombinant follicle stimulating hormone. Hum. Reprod., 13, 2772–2781.
Tesarik, J., Guido, M., Mendoza, C. et al. (1998b) Human spermatogenesis in vitro: respective effects of follicle-stimulating hormone and testosterone on meiosis, spermiogenesis, and Sertoli cell apoptosis. J. Clin. Endocrinol. Metab., 83, 4467–4473.
Tesarik, J., Greco, E., Cohen-Bacrie, P. and Mendoza, C. (1998c) Germ cell apoptosis in men with complete and incomplete spermiogenesis failure. Mol. Hum. Reprod., 4, 757–762.
Tesarik, J., Bahceci, M., Özcan, C. et al. (1999a) Restoration of fertility by in-vitro spermatogenesis. Lancet, 353, 555–556.[ISI][Medline]
Tesarik, J., Bahceci, M., Özcan, C. et al. (1999b) In-vitro spermatogenesis. Lancet, 353, 1708.[ISI][Medline]
Tesarik, J., Mendoza, C. and Greco, E. (1999c) In vitro culture facilitates the selection of healthy spermatids for assisted reproduction. Fertil. Steril., 72, 809–813.[ISI][Medline]
Tesarik, J., Balaban, B., Isiklar, A. et al. (2000) In-vitro spermatogenesis resumption in men with maturation arrest: relationship with in-vivo blocking stage and serum FSH. Hum. Reprod., 15, 1350–1354.
Tres, L.L., Smith, E.P., Van Wyk, J.J. and Kierszenbaum, A.L. (1986) Immunoreactive sites and accumulation of somatomedin-C in rat Sertoli-spermatogenic cell co-cultures. Exp. Cell Res., 162, 33–50.[ISI][Medline]
Vannelli, B.G., Barni, T., Orlando, C. et al. (1988) Insulin-like growth factor-I (IGF-I) and IGF-I receptor in human testis: an immunohisto- chemical study. Fertil. Steril., 49, 666–669.[ISI][Medline]
Vendola, K., Zhou, J., Wang, J. et al. (1999) Androgens promote oocyte insulin-like growth factor I expression and initiation of follicle development in the primate ovary. Biol. Reprod., 61, 353–357.
Walker, H.W. and Habener, J.F. (1996) Role of transcription factors CREB and CREM in cAMP-regulated transcription during spermatogenesis. Trends Endocrinol. Metab., 7, 133–138.
Weinbauer, G.F., Behr, R., Bergmann, M. and Nieschlag, E. (1998) Testicular cAMP responsive element modulator (CREM) protein is expressed in round spermatids but is absent or reduced in men with round spermatid maturation arrest. Mol. Hum. Reprod., 4, 9–15.
World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and Sperm–Cervical Mucus Interaction. 3rd edn. Cambridge University Press, Cambridge, UK.
Wright, C.S., Hovatta, O., Margara, R. et al. (1999) Effects of follicle-stimulating hormone and serum substitution on the in-vitro growth of human ovarian follicles. Hum. Reprod., 14, 1555–1562.
Yan, W., Lindenborg, J., Suominen, J. and Toppari, J. (1999) Stage-specific regulation of stem cell factor gene expression in the rat seminiferous epithelium. Endocrinology, 140, 1499–1504.
Yan, W., Suominen, J. and Toppari, J. (2000) Stem cell factor protects germ cells from apoptosis in vitro. J. Cell Sci., 113, 161–168.[Abstract]
Submitted on May 19, 2000; accepted on July 24, 2000.
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