Mechanism of apoptosis induction in amphibian spermatogonia by prolactin

本研究探讨了催乳素诱导蝾螈精原细胞凋亡的机制。我们阐明了以下几点：(1)绵羊催乳素(OPRL)仅在精原细胞的末期(从初级精原细胞产生的第7代)诱导细胞死亡，而与猪促卵泡激素(PFSH)联合给药可抑制这种细胞死亡。在睾丸碎片器官培养中，PRL通过直接作用于睾丸而诱导精原细胞死亡。(2)低温条件下移植能引起精原细胞的凋亡和血浆PRL水平的升高。低温注射抗催乳素血清后，精原细胞几乎完全恢复变性，维持3天之久。这些结果表明，低温引起了Newt血液中催乳素浓度的升高，导致了精原细胞在减数分裂前的细胞死亡。(3)蛋白质合成抑制剂放线菌酮(CHX)也诱导了精原细胞的凋亡。广谱caspase抑制剂zVAD-fmk可抑制PRL和CHX诱导的细胞凋亡。另一方面，ICE抑制剂Ac-YVAD-CHO和caspase-3抑制剂Ac-DEVD-CHO不能抑制这些参数，这表明当一些未知的caspase诱导第7代精原细胞死亡时，发生了凋亡形态的变化。(4)FSH活性对于完成最后一次有丝分裂是必不可少的，因为在体外没有pFSH的情况下，次级精原细胞在减数分裂前发生细胞死亡。这些结果表明，PRL和FSH生物活性的平衡是决定精原细胞末期的生死存亡和精细胞发生与否的关键因素。

We have investigated about the mechanism of apoptosis induction in newt spermatogonia by prolactin. We clarified the following things.(1) Ovine prolactin (oPRL) administration into newts induced apoptotic cell death only in the last stage of spermatogonia (7th generation from primary spermatogonium) and co-administration of porcine follicle-stimulating hormone (pFSH) inhibited this cell death. In organ culture of testes fragments, it was demonstrated that PRL induces spermatogonial cell death by acting directly on the testis.(2) Transfer of newts to low temperature caused spermatogonial apoptosis and elevation of plasma PRL levels. Injection of anti-PRL serum into newt at low temperature almost completely rescued spermatogonia from degeneration for as long as 3 days. These results demonstrate that low temperature caused elevation of prolactin concentration in the newt blood, which induced cell death of spermatogonia just before meiosis.(3) Cycloheximide (CHX), a protein synthesis inhibitor, also induced apoptosis. zVAD -fmk, a broad caspase inhibitor, inhibited the apoptosis induced by PRL and CHX. On the other hand, these parameters were not suppressed by ICE inhibitor Ac-YVAD-CHO or by caspase-3 inhibitor Ac-DEVD-CHO, indicating that apoptotic morphological changes occurring when the 7th generation of spermatogonia dies are induced by some unknown caspases. We have isolated cDNA for newt caspase 3.(4) FSH activity is indispensable for accomplishment of the last mitosis of newt spermatogonia, because cell death of secondary spermatogonia just before meiosis occurred without pFSH in vitro. These results indicate that balance of the biological activity of PRL and FSH is the key factor for life-or-death of the last stage of spermatogonia and for decision whether spermatocytogenesis commence or not.