Transforming growth factor beta-1 decreases the yield of the second meiotic division of rat pachytene spermatocytes in vitro.

BACKGROUND: TGF beta and its receptors are present in both germ cells and somatic cells of the male gonad. However, knock-out strategies for studying spermatogenesis regulation by TGF beta have been disappointing since TGF beta-or TGF beta receptor-null mice do not survive longer than a few weeks. METHODS: In the present study, we addressed the role of TGF beta-1 on the completion of meiosis by rat pachytene spermatocytes (PS) cocultured with Sertoli cells. Identification and counting of meiotic cells were performed by cytology and cytometry. RESULTS: Under our culture conditions, some PS differentiated into round spermatids (RS). When TGF beta-1 was added to the culture medium, neither the number of PS or of secondary spermatocytes nor the half-life of RS was modified by the factor. By contrast, the number of RS and the amount of TP1 mRNA were lower in TGF beta-1-treated cultures than in control cultures. Very few metaphase I cells were ever observed both in control and TGF beta-1-treated wells. Higher numbers of metaphase II were present and their number was enhanced by TGF beta-1 treatment. A TGF beta-like bioactivity was detected in control culture media, the concentration of which increased with the time of culture. CONCLUSION: These results indicate that TGF beta-1 did not change greatly, if any, the yield of the first meiotic division but likely enhanced a bottleneck at the level of metaphase II. Taken together, our results suggest strongly that TGF beta participates in an auto/paracrine pathway of regulation of the meiotic differentiation of rat spermatocytes.

beta-Nerve growth factor participates in an auto/paracrine pathway of regulation of the meiotic differentiation of rat spermatocytes.

NGF appears to be involved in spermatogenesis. However, mice lacking NGF or TrkA genes do not survive more than a few days whereas p75(NTR) knockout mice are viable and fertile. Therefore, we addressed the effect of betaNGF on spermatogenesis by using the systems of rat germ cell culture we established previously. betaNGF did not modify the number of Sertoli cells, pachytene spermatocytes, secondary spermatocytes nor the half-life of round spermatids, but increased the number of secondary meiotic metaphases and decreased the number of round spermatids formed in vitro. These effects of betaNGF were reversible and maximal at about 4 x 10(-11) M. Conversely, K252a, a Trk-specific kinase inhibitor, enhanced the number of round spermatids above that of control cultures. The presence of betaNGF and its receptors TrkA and p75(NTR) was investigated in testis sections, in Sertoli cell and germ cell fractions, and in germ cell and Sertoli cell co-cultures. betaNGF was detected only in germ cells from pachytene spermatocytes of stages VII up to spermatids of stages IX-X. TrkA and p75(NTR) were detected in Sertoli cells and in these germ cells. Taken together, these results indicate that betaNGF should participate in an auto/paracrine pathway of regulation of the second meiotic division of rat spermatocytes in vivo.

Key role for cyclin-dependent kinases in the first and second meiotic divisions of rat spermatocytes.

In all systems examined so far, the G2/M phase transition is controlled by the M-phase promoting factor (MPF), a complex of cdc2 (CDK1) and cyclin B1. Histone H1 kinase activity and MPF components are present in pachytene spermatocytes (PS). However, it has not been demonstrated yet that direct inhibition of MPF activity prevents the G2/M transition in these cells. When roscovitine, a potent inhibitor of CDK1, CDK2, and CDK5 activities, was added to cocultures of PS with Sertoli cells, the number of both secondary spermatocytes and round spermatids formed were lower than in control cultures, despite similar cell viability. This effect of roscovitine was reversible, did not involve the Sertoli cells, and was dependent on the concentration of the inhibitor. Roscovitine did not modify the amount of MPF in these germ cells but inhibited the CDK1- or CDK2-associated histone H1 kinase activity of PS. Hence a functional relationship between cyclin-dependent kinase activity and the spontaneous processing of the first meiotic division and, for the first time, of the second meiotic division of male germ cells is shown.