催乳素和抑制素互作促进鹅卵泡发育和产蛋的细胞和分子机理研究

Prolactin has been considered to be a negative regulating hormone in avian reproduction, for it inhibits pituitary LH secretion and results in reproductive regression. Inhibin, by reducing FSH secretion, has also been regarded as a reproduction repressing hormone. However, recently we had observed that both Prolactin and inhibin are required for normal development and function of avian gonades, that immunoneutralization of both hormones brought about reductions in development and function of the testis and egg laying rate in both chickens and geese. To unravel the underlying molecular and cellular mechanisms, this study planes to investigate the following: (1)Changes in egg laying rate, follicular developmental speed, gonadal and pituitary hormone secretions, and gene expression mRNA levels of LHR and steroid hormone synthases folloowing immunizations against PRL and inhibin in geese. These work will demonstrate at what level of ovarian development and what molecules whereby prolactin and inhibin play pivotal roles. (2) Effects by prolactin, inhibin and anti-prolactin and anti-inhibin antibodies on functions of ovarian tissues (cells) cocultured with or without pituitary gland, in terms of gonadal and pituitary hormone secretions, LHR and steroid hormone synthases expressions. These will help to demonstrate prolactin and inhibin's roles in gonadal steroid hormone production, and reveal how inhibin mediates the stimulation by FSH of androgen production at absence and presence of prolactin. (3) Identificication of DNA sequences in LHR and steroid synthase genes that will respond to prolactin or inhibin so to drive transcription of these genes. This work will identify prolactin and inhibin response elements in LHR or steroid synthase gene promoter regions, to illustrate the molecular mechanisms at DNA level whereby prolactin and inhibin stimulates expressions of genes instrumental in gonadal function and development. (4) Identification of transcription factors that bind to prolactin or inhibin response elements in stimulation of gene expressions of LHR and steroid synthase genes, and the gene network involved in. This work will further demonstrate and verify the cellular and molecular mechanisms mediating the stimulation of gonadal development and function by these hormones.

我们发现催乳素和抑制素在性腺水平的互作具有促进家禽繁殖活动的调控作用，即两激素的同时存在是睾丸和卵泡发育及产蛋所必需的。本研究拟开展两激素互作的细胞和分子调控机理，包括：（1）对种鹅免疫催乳素和抑制素，观察对产蛋、卵泡发育、性腺和垂体激素分泌和LHR和类固醇激素合成酶基因表达的影响，以确定两激素的作用环节和分子靶标；（2）体外培养卵巢组织，并加入催乳素和抑制素或两者抗体，观察对激素分泌、LHR和雄激素合成酶基因表达的影响，证明FSH通过抑制素促进雄激素分泌及与催乳素互作促进繁殖活动的新理论；（3）在明确催乳素和抑制素促进LHR或雄激素合成酶分子表达后，确定这些分子基因启动子区域对催乳素和抑制素的反应元件；（4）在催乳素或抑制素作用下，与以上启动子相结合的转录因子种类或其表达差异和所涉及的调控基因网络。

该项目拟研究催乳素（PRL）和抑制素（INH）协同促进鹅卵泡发育及产蛋的调控机理，然而由于在实际工作中难以观察到免疫PRL和INH对鹅产蛋的协同调控作用，因此调整为研究两激素对公鸡睾丸发育的协同性调控机理。. 第一部分工作非常好地揭示了PRL和INH对后备小公鸡睾丸发育的协同性促进作用，如免疫INH促进睾丸发育、免疫PRL显著抑制睾丸发育、而联合免疫PRL和INH则极显著抑制睾丸发育和睾酮分泌。免疫PRL显著下调睾丸组织中PRLR信号传导蛋白JAK2-STAT5磷酸化，下调LHR、睾酮合成酶StAR、P450scc和3β-HSD等基因表达。利用体外培养的睾丸间质细胞，验证了PRL通过调控JAK2-STAT信号通路，调控上述基因的表达，特别是在3β-HSD基因的5’调控区存在PRL反应元件（TTC xxx GAA），由此揭示了低浓度PRL具有促进睾酮分泌及睾丸发育的分子和细胞调控机理。细胞培养工作同时也揭示了高浓度PRL通过抑制PRLR-JAK2-STAT信号传导，从而抑制睾酮分泌的调控作用，初步验证项目的研究假设。. 第二部分工作对后备公鹅的免疫INH，则显著上调垂体FSHβ和LHβ基因的表达，同时上调睾丸中FSHR和SOX9、Wt1、Dhh和DAZL等生精基因的表达水平，促进睾丸发育。. 第三部分研究了不同时长和波长的光照，对鹅繁殖活动及产蛋性能的影响及机理。研究结果揭示光照通过影响下丘脑Opsin5的基因表达，调控下丘脑TSH、Dio2基因表达，从而影响下丘脑甲状腺激素T3的生成，然后调控下丘脑GnRH、GnIH、VIP等基因的表达变化，调控垂体FSHβ和LHβ的表达及鹅的繁殖活动。. 第四部分研究了鹅PRL的测定方法，制备了具有生物活性的鹅催乳素、其单克隆和多克隆抗体，建立了能够很好检测鹅血液PRL浓度的ELISA测定方法, 也建立了基于PRLR-STAT5信号传导系统的PRL生物测定技术。. 这些工作揭示了催乳素和抑制素的促进性腺活动的分子机理，补充了动物繁殖传统内分泌调控理论的局限。. 项目共发表SCI论文5篇，其他论文3篇，专著1部，申报发明专利1件。