雄激素（11-KT）和孕激素（DHP）被认为是促进鱼类精子发生和成熟的关键因素，但雄激素合成酶抑制剂处理，或DHP受体基因突变未影响雄性育性。我们发现松弛素（Relaxin3，RLN3）缺失导致精子发生不能进入减数第二次分裂，不能形成精子细胞，雄性不育，促性腺激素和雄激素合成水平下降，表明RLN3可能是鱼类精子发生过程的关键调控因子。本项目拟鉴定RLN3及其受体在脑和精巢的表达模式，建立RLN3及其受体的敲除模型，分别用RLN3转基因、hCG、11-KT对基因敲除个体进行回救，深入研究RLN3对精子发生的影响。通过RNA-seq研究基因敲除对lncRNAs和生殖轴关键基因表达水平的影响，检测DNA甲基化修饰状态的变化。通过ChIP-seq筛选RLN3的靶基因，qPCR和原位杂交等检测靶基因的表达。本项目旨在揭示RLN3调控精子发生的分子机制和表观遗传机制。

Fish androgen (11-KT) and progestin hormone (17α, 20β-dihydroxy-4-pregnen-3-one, DHP) were considered as the key hormones in spermatogenesis and sperm maturation. However, treatment by steroidogenic enzyme inhibitor of 11-KT, or mutation of DHP receptor gene had no effects on fertility of male fish. Intriguingly, we found that mutation of RLN3 resulted in failure of second meiotic division, absence of spermatids and infertility. Meanwhile, RLN3 gene deficiency led to the decrease of androgen and gonadotropin production. Therefore, fish spermatogenesis might be tightly controlled by RLN3 peptide hormone. In this proposal, the expression pattern of RLN3 and its receptor in testis and brain will be characterized. Knockout of RLN3a, RLN3b and their receptor and rescue by each RLN3 transgene, 11-KT, hCG will be carried out to further clarify the possible roles of RLN3 in fish spermatogenesis. To understand the epigenetic modification in RLN3-regulated spermatogenesis, lncRNA sequencing, DNA methylation modification of the key genes of reproductive axis will be examined between control and RLN3 knockout fish in the brain and testis. Finally, ChIP-seq will be carried out to clarify the possible target genes of RLN3, and Real-time PCR and in situ hybridization will be used to characterize the expression level of potential RLN3 target. The final goal of this project is to bring insights into the molecular and epigenetic mechanisms of RLN3 in spermatogenesis regulation in teleosts.