黑色素细胞影响乌骨鸡产蛋性能的分子机制研究

Cross generation records showed that the egg production performance of black bone chicken is 20% lower than non black bone chicken in Dongxiang blue-shelled population. After bred the black bone and non black bone chickens in two strains separately, the difference of egg production was more obvious. Thinking about the two lines originated from the same population, and raised in the same environmen, the difference of their egg production should be related to the black bone traits. It is presumed that the distribution of melanocytes may influence the reproduction trait in black bone chicken. Section examination showed that melanocytes were not seen in the hypothalamus and pituitary tissues, but big amount of melanocytes were observed in the ovary tissues, mainly distributed in the granulosa cell layer and theca of ovary. Melanocytes belong to dendritic cells, which are rich in synaptic structure, can contact with neighboring cells, and secrete exocrine, and transfer melanocytes molecules to adjacent cells, affecting the function of neighboring cells. This study plan to work from the perspective of interaction of melanocytes and ovarian granulosa cells and theca cell, by differential gene expression screening of black bone chicken and non black bone chicken in ovarian tissues, and co-culture of melanocytes and ovarian granulosa cells or theca cells, and with exosomes transfection method, we want to explore the interaction between melanocytes and granulosa cells or theca cell, to understand the molecular mechanism of lower egg production performance in black bone chicken.

东乡黑羽绿壳蛋鸡多个世代的记录显示，群体中的乌骨鸡产蛋量显著低于非乌骨鸡，全年产蛋量相差20%。将该群体中乌骨鸡与非乌骨鸡单独纯繁，形成乌骨系和非乌骨系，其产蛋差异更加明显。乌骨系与非乌骨系源于同一群体，在相同的环境下饲养，其产蛋量的差异显然应与乌骨性状相关，推测受黑色素细胞分布的影响。切片检测显示，乌骨鸡的下丘脑、垂体组织未见黑色素细胞，但卵巢组织存在大量的黑色素细胞，在主要的功能细胞层卵巢颗粒细胞层和卵泡膜层都有分布。黑色素细胞属于树突状细胞，拥有丰富的突触结构，可与相邻细胞接触，并能够分泌外泌体，将黑色素细胞的分子向相邻细胞传递，影响相邻细胞的功能。本研究拟从黑色素细胞与卵巢颗粒细胞和卵泡膜细胞互作的角度，筛选乌骨鸡与非乌骨鸡卵巢细胞的差异表达基因，利用与黑色素细胞共培养和黑色素细胞外泌体转染等方法，探究黑色素细胞与卵巢颗粒细胞和卵泡膜细胞之间的互作，解析乌骨鸡产蛋性能低的分子机制。

中国特色地方鸡品种乌骨鸡体内广泛分布大量的黑色素细胞，因而呈现乌皮、乌骨、乌肉的特征。尤其乌骨鸡部分内脏组织也呈现乌色，如卵巢和卵泡。乌骨鸡具有很高的经济价值，但是，乌骨鸡的低产蛋量极大地限制了其生产效率。本课题组前期研究发现乌骨鸡产蛋量显著低于非乌骨鸡，推测其乌质性状与产蛋性能有关。本研究在乌骨鸡卵巢组织和卵泡颗粒细胞和膜细胞层发现了大量的黑色素细胞，黑色素细胞是否影响卵泡生殖细胞的生物学功能是本研究的重点。. 本研究发现在卵泡发育各重要阶段，乌骨鸡卵泡数量均显著低于非乌骨鸡。卵泡储备量不足可能是导致乌骨鸡产蛋连续性差，产蛋量低的生殖发育原因。卵泡颗粒细胞的增殖和分化对卵泡的发育以及排卵十分重要，因而我们进行了卵泡颗粒细胞体外培养，发现乌骨鸡颗粒细胞增殖能力和性腺激素合成与分泌能力均显著低于非乌骨鸡，激素不足是乌骨鸡卵泡发育受限的生理原因。为探究乌骨鸡和非乌骨鸡颗粒细胞及膜细胞基因表达差异，进行了转录组测序。在卵泡发育不同阶段乌骨鸡颗粒层和膜层的细胞增殖和性腺激素合成相关基因表达量显著低于非乌骨鸡，这可能是导致乌骨鸡产蛋率低的分子调节原因。基于转录组测序结果，我们针对关键差异基因EDN3，该基因是黑色素细胞增殖基因，是乌骨鸡乌质表型的原因基因，构建了过表达载体。在非乌骨鸡卵泡颗粒细胞中过表达后，发现EDN3基因具有抑制颗粒细胞促黄体生成素受体表达的作用，说明乌骨鸡EDN3基因的变异产生了对卵泡发育抑制的一因多效作用，从遗传上解释了乌骨鸡低产蛋量的原因。.为研究黑色素细胞对乌骨鸡卵泡发育的影响，本研究构建了乌骨鸡卵巢黑色素细胞及其分泌的外泌体与非乌骨鸡颗粒细胞的共培养体系，共培养后发现黑色素细胞的存在对颗粒细胞的生物学功能具有抑制作用，并且外泌体是其与颗粒细胞互作的主要作用方式。本研究证明乌骨鸡黑色素细胞能够影响卵泡生殖细胞的生物学功能，导致乌骨鸡产蛋量降低。