The effect of FSH on male germ cell survival and differentiation in vitro is mimicked by pentoxifylline but not insulin

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1 Molecular Human Reproduction vol.6 no.10 pp , 2000 The effect of FSH on male germ cell survival and differentiation in vitro is mimicked by pentoxifylline but not insulin Jan Tesarik 1,2,5, Carmen Mendoza 2,3 and Ermanno Greco 4 1 Laboratoire d Eylau, 55 rue Saint-Didier, Paris, France, 2 MAR&Gen, Molecular Assisted Reproduction and Genetics, Gracia 36, Granada, Spain, 3 University of Granada, Campus Universitario Fuentenueva, Granada, Spain and 4 European Hospital, Via Portuense 700, Rome, Italy 5 To whom correspondence should be addressed at: Laboratoire d Eylau, 55 rue Saint-Didier, Paris. 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. Key words: FSH/in-vitro spermatogenesis/insulin/male germ cell/pentoxifylline Introduction or by using alternative hormones and growth factors Recent studies have shown that certain phases of human known to promote survival and differentiation of cultured spermatogenesis can be substantially accelerated by in-vitro cells. culture of seminiferous tubule segments recovered from men The effect of FSH on Sertoli cells is mediated by cyclic with normal spermatogenesis (Tesarik et al., 1998a,b). When AMP (camp) (Parvinen, 1982) whose action is modulated the same in-vitro culture system was used in cases of maturation by endogenous camp phosphodiesterase (Morena et al., arrest, meiotic and post-meiotic germ cells from some patients 1995; Naro et al., 1996). Insulin and insulin-like growth overcame the in-vivo block and resumed differentiation, leadcell factors I and II (IGF-I and IGF-II) can also promote germ ing to the first pregnancies and births in cases of complete in-vitro differentiation by acting at Sertoli cells (Borland spermiogenesis failure and in a case of maturation arrest at et al., 1984; Mita et al., 1985) or directly through receptors the primary spermatocyte stage (Tesarik et al., 1999a,b). In located on germ cells (Tres et al., 1986; Vannelli et al., the former indication, in-vitro culture is supposed to boost 1988). cytoplasmic maturation of spermatids (Tesarik et al., 1998a) In this study we examined whether the beneficial effect and to facilitate the distinction between healthy spermatids of FSH on human male germ cell in-vitro survival and and those carrying irreversible DNA damage due to incipient differentiation is mimicked by pentoxifylline, a phospho- apoptosis (Tesarik et al., 1999c). diesterase inhibitor increasing camp concentration in cells, In spite of these encouraging data, in-vitro culture fails to and insulin added at a high concentration at which it is known overcome the in-vivo maturation arrest in 50 75% of cases, to act through the IGF-I receptor (Sara and Hall, 1990; Francis depending on the in-vivo blocking stage and serum FSH et al., 1993). The two substances were chosen with regard to concentration (Tesarik et al., 2000). The observation that the the ease of their eventual future clinical application for success of in-vitro culture is lower in men with very high promoting in-vitro differentiation of germ cells from men with serum FSH concentrations ( 20 IU/l), compared with those maturation arrest, since both are currently used in other with normal or slightly elevated FSH (Tesarik et al., 2000) therapeutic indications in human medicine. This study com- has suggested that the failures may be at least partly due to pares the effects of FSH, pentoxifylline and insulin on the desensitization of the FSH receptor on Sertoli cells within the appearance of apoptotic DNA damage, on morphological cultured seminiferous tubule segments which are thus unable differentiation and on cytoplasmic maturation of cultured germ to respond to FSH present in culture medium by creating a cells recovered from men with obstructive azoospermia and spermatogenesis-promoting microenvironment. If this is the with ongoing complete in-vivo spermatogenesis as a prelude case, the problem might be resolved by acting directly at the signal transduction pathway downstream of the FSH receptor to future studies with samples from men with maturation arrest. European Society of Human Reproduction and Embryology 877

2 J.Tesarik, C.Mendoza and E.Greco Materials and methods terminology (de Kretser and Kerr, 1988; Tesarik et al., 1998a,b). Briefly, Sa, Sb, Sc and Sd stages represent normal forms of round, Testicular tissue source and handling early elongating, late elongating and elongated spermatids respectively Ten patients with obstructive azoospermia undergoing testicular (de Kretser and Kerr, 1988), whereas Saf, Sbp and Scp refer to biopsy aimed at the extraction of spermatozoa for assisted reproduction abnormal spermatid forms (Tesarik et al., 1998a,b) characterized by gave their informed consent for part of the testicular tissue recovered a round shape, non-condensed nucleus and a flagellum (Saf), a round to be used in the present experiments. A small piece of testicular shape, condensed and protruding nucleus and the absence of flagellum tissue was obtained from each patient by open testicular biopsy (Sbp), and a round shape, condensed and protruding nucleus and a and placed in Gamete-100 medium (Scandinavian IVF Science, flagellum (Scp). Gothenborg, Sweden). Samples were disintegrated mechanically by Germ cell cytoplasmic maturation was evaluated by examining the using sterile glass slides. After isolation of sufficient numbers of status of acrosome development in 4D4-immunoreactive germ cells spermatozoa to be used for assisted reproduction, the rest of the as described (Tesarik et al., 1998a). Briefly, three sequential developdisintegrated testicular tissue from each patient, consisting of indivimental stages of acrosome assembly, reticular, vesicular and cap dual cells, cell clusters and intact segments of the seminiferous stage, were distinguished, and the percentage of germ cells showing tubules, was brought in a homogenous suspension and distributed each of them was determined. All analyses performed with the into five equal aliquots. One aliquot was used immediately for the Papanicolaou-stained smears and the immunocytochemical preparaevaluation of germ cell DNA damage and differentiation activity. tions were based on the evaluation of 200 germ cells in each aliquot. 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- Statistical analysis 100 medium with the corresponding supplement. Statistical significance of differences in the proportion of germ cells with damaged DNA and in the percentages of germ cells at different In-vitro culture stages of morphological differentiation and cytoplasmic maturation In-vitro culture of four aliquots of each testicular tissue sample, between individual treatment groups were evaluated by using χ 2 and prepared as described above, was carried out at 30 C in cellculture Kruskal Wallis tests. 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 Results human recombinant FSH (Puregon, Organon, Oss, The Netherlands), 1 mg/ml pentoxifylline (Sigma, St Louis, MO, USA), or 10 µg/ml Effects of FSH, pentoxifylline and insulin on the insulin (Sigma) respectively. The fourth aliquot was cultured in preservation of germ cell DNA integrity during in- unsupplemented Gamete-100 medium to serve as control. After 24 h vitro culture of culture, cells from each aliquot were harvested by centrifugation Before the beginning of in-vitro culture, the percentages and distributed into two subaliquots that were analysed for DNA of primary spermatocytes and of round spermatids showing damage and for differentiation activity respectively. TUNEL-positive nuclei (indicative of DNA fragmentation) was 19 2 and 22 3% respectively. After 24 h of culture Evaluation of germ cell DNA damage in unsupplemented Gamete-100 medium, the corresponding Segments of the seminiferous tubules and cell clusters were dissociated values for primary spermatocytes and round spermatids were by incubation for 1hinGamete-100 medium with the addition of collagenase I (1000 IU/ml) and elastase (10 IU/ml) (both purchased significantly elevated (P 0.01) and were 78 8 and 64 from Sigma). The resulting cell suspensions were smeared on microeither 50 IU/l FSH or 1 mg/ml pentoxifylline resulted in a 7% respectively. However, medium supplementation with scope 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 significant (P 0.01) reduction of the percentage of TUNEL- by a modified terminal deoxyribonucleotidyl transferase-mediated positive primary spermatocytes (Figure 1A) and round deoxyuridine triphosphate nick-end labelling (TUNEL) procedure spermatids (Figure 1B), compared with unsupplemented (Tesarik et al., 1998c) using the apoptosis detection system, fluorescein medium. In contrast, no difference was observed between kit (Promega, Charbonnières, France) and 4D4 monoclonal antibody, samples cultured in unsupplemented medium and in medium reacting with proacrosin in human germ cells from the pachytene supplemented with 10 µg/ml insulin (Figure 1). stage onwards (Escalier et al., 1991) as specific germline marker. Primary spermatocytes (n 200) and round spermatids (n 200) were assessed for each aliquot. Effects of FSH, pentoxifylline and insulin on germ cell morphological differentiation and cytoplasmic Evaluation of germ cell differentiation activity maturation Aliquots assigned to this analysis were first treated with collagenase After 24 h of in-vitro culture in unsupplemented medium, the I and elastase as described in the previous section. The resulting cell percentages of the major stages of post-meiotic germ cells suspensions were smeared onto four microscope slides, allowed to were similar to freshly obtained samples (Table I). After air-dry and fixed for 10 min with 100% ethanol. For each aliquot, culture in medium supplemented with FSH, the percentage of two slides were stained with the use of Papanicolaou method (World round spermatids (Sa stage) was reduced, and the percentages Health Organization, 1992), and the other two were processed for immunocytochemistry with 4D4 monoclonal antibody (Tesarik et al., of early and late elongating spermatids (Sb and Sc stages) 1998a). The percentages of normal and atypical forms of elongating was increased as compared to the control incubation in and elongated spermatids were determined in Papanicolaou-stained unsupplemented medium, and this effect was mimicked in preparations by using the previously described staging criteria and medium supplemented with pentoxifylline (Table I). In contrast, 878

3 Pentoxifylline mimics FSH in germ cell culture Figure 2. Proportions of spermatids in different phases of acrosomal development after 24 h of culture in unsupplemented medium or supplemented medium. Data are presented as means SEM. 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 Figure 1. Proportion of TUNEL-positive cells among (A) primary survival and differentiation of human germ cells cultured in a spermatocytes and (B) round spermatids after 24 h of culture in unsupplemented medium or supplemented medium. Data are system retaining, at least partly, the original Sertoli-germ cell presented as means SEM. 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 Table I. Effects of FSH, pentoxifylline and insulin on germ cell in-vitro morphological differentiation as reflected by changes in the proportion of concentrations to achieve these effects, the mechanism of this round (Sa), early elongating (Sb), late elongating (Sc) and elongated (Sd) FSH action was questioned. The present data indicate that the spermatids a signal transduction pathway implying camp, known to mediate Treatment group Post-meiotic germ cells (%) the physiological FSH effects on spermatogenesis (Parvinen, 1982), is also involved in these in-vitro effects. Sa Sb b Sc c Sd Since germ cells are believed to lack a functional FSH Before culture receptor (Böckers et al., 1994), these effects are likely to be Cultured, unsupplemented dependent on the presence of Sertoli cells in the culture system. Cultured, FSH 11 2 d 16 2 d 18 2 d 55 6 Moreover, camp also appears to play important regulatory Cultured, pentoxifylline 13 2 d 15 2 d 16 2 d 56 6 Cultured, insulin roles within germ cells, since post-meiotic germ cell differentiation is disrupted in mice carrying mutations of the campa Normal and abnormal spermatid forms are denoted using the classifications responsive element modulator (CREM) gene (Blendy et al., by de Kretser and Kerr (1988) and by Tesarik et al. (1998a) respectively. Including the atypical form Sbp. 1996; Nantel et al., 1996). In our culture system, in which c Including the atypical form Scp. intact original Sertoli-germ cell associations were largely Significantly different from culture in unsupplemented medium (P 0.01). 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 the percentages of individual stages of germ cells after culture debate about the role of camp in post-meiotic differentiation in medium supplemented with insulin did not differ from (Daniel and Habener, 2000), our data support the hypothesis control incubations in unsupplemented medium (Table I). that camp is important for the activity of CREM that In-vitro culture in the presence of FSH or pentoxifylline accumulates in post-meiotic male germ cells of mice (Delmas also led to a significant acceleration of germ cell cytoplasmic et al., 1993), hamsters (Foulkes et al., 1993), rats (Walker maturation, as evidenced by a shift in the proportion of cells and Habener, 1996) and men with normal spermatogenesis showing different stages of acrosome assembly in favour of (Weinbauer et al., 1998), although recent evidence suggests more mature forms (vesicular and cap-like), accompanied by the existence of alternative pathways of CREM activation in a decrease in the less mature, reticular form (Figure 2). Insulin male germ cells (Fimia et al., 1999). Interestingly, CREM had no measurable effect on this process when compared with expression is reduced in round spermatids from men with postmeiotic unsupplemented medium (Figure 2). maturation arrest (Weinbauer et al., 1998; Steger et al., 879

4 J.Tesarik, C.Mendoza and E.Greco 1999). Our data also suggest that camp, rather than FSH concentration of FSH is highly elevated, leading to desensitization itself, is an important regulatory factor in mammalian spermatogenesis, of the FSH receptor on Sertoli cells. since the in-vivo effect of FSH on intracellular camp Circulating FSH concentrations are usually elevated as a accumulation can be mimicked, in the testis, by locally result of reduced secretion of inhibin B by Sertoli cells produced pituitary adenylate cyclase-activating polypeptide (Illingworth et al., 1996), which has been shown, in animal (Daniel and Habener, 2000) whose action may explain the models, to be a consequence of reduced numbers of postsporadical observations of complete spermatogenesis in men meiotic germ cells in the testis (Pineau et al., 1990; Allenby with inactivating mutations of the FSH receptor (Tapanainen et al., 1991). The persisting post-meiotic cells may thus be et al., 1997). arrested at the round spermatid stage and undergo apoptosis The present observations also indicate that, in addition to because of the failure of Sertoli cells to respond to FSH by stimulation of germ cell differentiation, FSH also protects inapoptotic secreting the necessary differentiation-promoting and anti- vitro cultured germ cells against apoptosis. This is in agreement factors. This possibility is corroborated by the with a previous study conducted in the rat (Henriksen et al., observation that germ cells from men with highly elevated 1996). The anti-apoptotic effect of FSH added to culture serum FSH concentrations ( 20 IU/l) are less likely to resume medium is likely to be related to the improved developmental differentiation during in-vitro culture, in comparison with men potential of in-vitro cultured spermatids (Tesarik et al., with only moderately elevated FSH concentrations (Tesarik 1999a,b), since high frequencies of apoptotic spermatids have et al., 2000). In contrast, the use of pentoxifylline appears to been reported both in an animal model of primary testicular be less justified in cases in which maturation arrest at the failure and in patients suffering from post-meiotic maturation round spermatid stage is caused by a reduced or absent arrest (Jurisicova et al., 1999; Tesarik et al., 1998c). In this expression of CREM in round spermatids. Studies are underway study, this FSH effect also could be mimicked by pentoxifylline. to test the usefulness of pentoxifylline for in-vitro spermato- The anti-apoptotic action of FSH in the rat seminiferous genesis of germ cells from patients with highly elevated FSH epithelium is mediated partially through the stem cell factor/ concentrations. c-kit pathway (Yan et al., 2000). The production of stem cell factor by isolated and cultured rat seminiferous tubules has References been shown to be much more dependent on FSH compared 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 with the in-vivo condition (Yan et al., 1999). It remains to be is regulated by specific germ cell types: correlation between in vivo and determined whether stem cell factor, when added directly to in vitro studies. 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