Abstract
BackgroundIn co-cultures of pachytene spermatocytes with Sertoli cells, β-NGF regulates the second meiotic division by blocking secondary spermatocytes in metaphase (metaphase II), and thereby lowers round spermatid formation. In vertebrates, mature oocytes are arrested at metaphase II until fertilization, because of the presence of cytostatic factor (CSF) in their cytoplasm. By analogy, we hypothesized the presence of CSF in male germ cells.Methodology/Principal FindingsWe show here, that Mos, Emi2, cyclin E and Cdk2, the four proteins of CSF, and their respective mRNAs, are present in male rat meiotic cells; this was assessed by using Western blotting, immunocytochemistry and reverse transcriptase PCR. We measured the relative cellular levels of Mos, Emi2, Cyclin E and Cdk2 in the meiotic cells by flow cytometry and found that the four proteins increased throughout the first meiotic prophase, reaching their highest levels in middle to late pachytene spermatocytes, then decreased following the meiotic divisions. In co-cultures of pachytene spermatocytes with Sertoli cells, β-NGF increased the number of metaphases II, while enhancing Mos and Emi2 levels in middle to late pachytene spermatocytes, pachytene spermatocytes in division and secondary spermatocytes.Conclusion/SignificanceOur results suggest that CSF is not restricted to the oocyte. In addition, they reinforce the view that NGF, by enhancing Mos in late spermatocytes, is one of the intra-testicular factors which adjusts the number of round spermatids that can be supported by Sertoli cells.
Highlights
Spermatogenesis is a complex process during which diploid spermatogonia divide mitotically to provide a population of spermatocytes that proceed through meiosis to haploid spermatids which differenciate into spermatozoa [1]
By measuring the mos or emi2 or cyclin E or cdk2 mRNA/18S RNA ratios it was found that the amount of mos mRNA was about two fold higher in round spermatid (RS) than in pachytene spermatocytes (PS), while emi2 mRNA and cyclin E mRNA amounts were roughly similar in PS and RS
The four identified constituents of cytostatic factor (CSF) are present in male rat meiotic cells For Mos, Emi2, Cyclin E and Cdk2, both the proteins and their corresponding mRNAs were highlighted in freshly elutriated PS and RS
Summary
Spermatogenesis is a complex process during which diploid spermatogonia divide mitotically to provide a population of spermatocytes that proceed through meiosis to haploid spermatids which differenciate into spermatozoa [1]. We have previously shown that b-Nerve growth factor (b-NGF) participates in the regulation of spermatocyte differentiation by blocking secondary spermatocytes (SII) in metaphase (MPII) leading to a reduction of round spermatid (RS) formation in cocultures of pachytene spermatocytes (PS) with Sertoli cells [4]. We hypothesized the presence of CSF in male germ cells, which could be, at least in part, responsible for the b-NGF induced blockage of SII in MPII observed in co-cultures of PS with Sertoli cells. That both ERK1 and ERK2 are detected in meiotic cells, from young PS to RS, present in co-culture of PS with Sertoli cells, as in freshly isolated germ cell populations Their activated (phosphorylated) forms increased during meiotic progression, suggesting a role of MAPKs in this process [12]. We hypothesized the presence of CSF in male germ cells
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