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

Abstract

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.

Figure 1

Changes in the number of germ cells of different ploidy in cocultures of Sertoli cells and PS (4C cells) maintained in the absence (control) or presence of 10 ng/ml TGFβ1 (TGFβ1). Results are the mean ± SEM of seven experiments. ** p < 0.01 vs control; * p < 0.05 vs control.

Figure 2

Effect of TGFβ1 on the TP1 mRNA/16S rRNA ratio on day 14 of cocultures of Sertoli cells and PS. PS were cocultured with Sertoli cells for 14 days either in the absence (control) or presence of 10 ng/ml TGFβ1. On day 14 of coculture, total RNA was extracted and TP1 mRNA and 16S rRNA were coamplified by RT-PCR, as described in the Materials and Methods section, and their ratio were calculated. Each point is the mean ± SEM of four experiments. * p < 0.05 vs control.

Figure 3

Staining of metaphase I (left) and metaphase II (right) by a Ser 10 phosphorylated histone H₃ antibody in cocultures of PS and Sertoli cells (day one of coculture; bars represent 10 μm).