实验海洋生物学重点实验室知识库

胎生是自然界中广泛存在的一种繁殖方式，目前报道的有多种软骨鱼类、硬骨鱼类、两栖动物、爬行动物和大多数哺乳动物都营胎生繁殖。胎生这种繁殖方式为胚胎提供了稳定的营养、保护等发育条件，降低了外界条件对胚胎发育的不利影响，极大地提高了后代的成活率。胎生的一个重要特征是卵子在体内受精并且受精卵在雌性体内完成发育。在哺乳动物中，母体与胚胎之间通过胎盘完成营养、气体等物质交换，因此对胎生的硬骨鱼来说，卵巢在形态和生理上的适应性变化有着重要意义。在鲉科中，平鲉属、无鳔鮋属、眶棘鲉属和菖鲉属均包含有胎生鱼种，许氏平鲉营胎生繁殖，是我国一种重要的海洋经济鱼种，其特殊的生殖方式一直受到国内外学者的广泛关注。本文通过分析许氏平鲉卵子发生成熟及胚胎发育过程中卵母细胞周围滤泡结构的变化、激素分泌差异以及相关基因表达模式的差异，研究了许氏平鲉滤泡胎盘结构鉴定及发生过程，主要结果如下：

1、许氏平鲉卵子发生成熟过程中，最明显的特点是形成了许多独立的卵泡。卵泡由生殖上皮包绕住卵母细胞形成，最早出现在III期。雄鱼和雌鱼交配后，精子散落的分布在雌鱼卵巢腔内。独立的卵泡通过茎状结构分别与卵巢基质相连，使得每个卵泡均可以与精子接触，并且卵泡能通过滤泡茎以及包绕在滤泡层上的血管中直接获得营养，减少了卵子之间的竞争，从而保证受精和营养吸收效率。独立的卵泡可能是滤泡胎盘形成的基础。在受精过程中，卵巢间质中的血管和结缔组织迅速增生，卵母细胞的滤泡层结构破裂，基底膜和颗粒层消失，膜细胞层迅速增殖并侵入周围的间质中，形成类似滤泡胎盘的结构。这一结构极为发达，表面广泛折叠形成大片的褶皱并且高度血管化，在从受精后发育到囊胚期的过程中进一步膨大且较为致密；在之后的妊娠过程中，随着胚胎的发育，滤泡胎盘逐渐变得疏松。

2、在与卵巢发育和妊娠密切相关基因中，cyp17-I是滤泡层膜细胞层的标记基因，在妊娠期的滤泡胎盘上有强烈的表达信号。在受精前，cyp17-I在卵母细胞的膜细胞层表达，并且在V期时，卵母细胞周围的间质细胞也出现了表达信号，说明许氏平鲉滤泡胎盘的形成可能与膜细胞层的增殖，迁移，扩张有关，并且在受精之前就开始出现扩张趋势。HLA-E和lama4在哺乳动物中，对降低母体对胚胎的免疫排斥、促进滋养层入侵具有重要作用，在妊娠期的滤泡胎盘以及受精前的卵母细胞的膜细胞层上均能够检测到强烈的表达信号，说明滤泡胎盘可能来源于膜细胞层并且执行与哺乳动物胎盘相似的功能。cyp19a1a和 fshr作为颗粒细胞中特异性表达的基因，仅在受精前的卵母细胞的颗粒细胞层上检测到了信号，在妊娠期的滤泡胎盘上没有检测到信号，说明颗粒细胞可能并不参与滤泡胎盘的形成。这些结果证明了许氏平鲉滤泡胎盘可能是由膜细胞层的增殖，迁移，扩张形成的，并且可能执行与哺乳动物胎盘相似的功能。

3、在许氏平鲉体内，睾酮含量在卵子发生的过程中一直处于较低水平，受精之后逐渐升高，在囊胚期达到峰值，并在整个妊娠期保持较高水平。雌二醇水平在卵子成熟过程中显著升高，在IV期达到峰值，然后从V期到囊胚期急剧下降，但是妊娠中后期雌二醇也保持较高水平，并在原肠期再次达到高峰。在卵子发生过程中，孕酮水平较低，但受精后孕酮的水平在急速上升，在妊娠期间一直处于高位。对比大菱鲆，许氏平鲉从V期到囊胚期体内睾酮含量上升，雌二醇含量下降，导致体内雌二醇与睾酮的比率逐渐下降，在囊胚期达到最小值，这可能是促进膜细胞层和滤泡胎盘向外扩张的驱动因素。并且在妊娠期，睾酮、雌二醇和孕酮都处于较高水平，可能起到维持妊娠的作用。

Viviparous is a reproductive method that exists widely in nature. it occurrs in most vertebrates, including most cartilaginous fishes, several clades of bony fishes, amphibians, reptiles and most mammals. Viviparous reproduction provides stable nutrition, protection and other development conditions for embryos, reduces the adverse effects of external conditions on embryonic development, and greatly improves the survival rate of offspring. An important feature of viviparous birth is that the egg is fertilized in the female's body and the fertilized egg completes development in the female's body. In mammals, the placenta completes the exchange of nutrients and gas between the mother and the embryo. Therefore, for the viviparous bony fish, the morphological and physiological adaptations of the ovaries have great significance. In Scorpaenidae, four genera (Sebastes, Sebasticus, Helicolenus and Hozukius) also belong to the marine viviparity. Black rockfish (Sebastes schlegelii) belongs to viviparous fish and farmed as an important marine economic fish  in China. It is widely inhabits in the waters of China, Japan and South Korea, and its special reproductive method has been widely concerned by scholars. In this paper, by analyzing the differences in follicular structure, hormone secretion and related gene expression patterns around oocytes during the oocyte maturation and embryonic development of black rockfish, the identification of the follicular placenta structure and the occurrence process of black rockfish were studied. The main results are as follows

1. The most obvious feature of the egg maturation process of black rockfish is the formation of many independent follicles. Follicles are formed by reproductive epithelium surrounding oocytes, and they first appear in stage III. After mate, the sperm are scattered in the ovarian cavity of the female fish. The independent follicles are connected to the ovarian stroma through the stem-like structure, so that each follicle can be in contact with sperm, and the follicle can directly obtain nutrition through the follicular stem and the blood vessels surrounding the follicular layer, reducing the competition of eggs to ensure the efficiency of fertilization and nutrient absorption. Independent follicles may be the basis of follicular placenta formation. During fertilization, the blood vessels and connective tissues in the interstitium of the ovary proliferate rapidly, the follicular layer structure of the oocytes is broken, the basement membrane and granular layer disappear, and the theca cell layer rapidly proliferates and invades the surrounding interstitium, forming a follicular placenta-like structure. The structure is extremely developed, the surface is widely folded to form large folds and is highly vascularized. It is further enlarged and denser during the development from fertilization to the blastocyst stage; in the subsequent pregnancy process, as the embryo develops, it gradually becomes loose.

2. Among the genes closely related to ovarian development and pregnancy, cyp17-I is a marker gene of the theca cell layer, and it has a strong expression signal on the follicular placenta during pregnancy. Before fertilization, cyp17-I was expressed in the theca cell layer of the oocyte, and at stage V, the stroma cells around the oocyte also showed expression signals, indicating that the formation of the follicular placenta of blackrock fish may be related to the theca cells and the expansion trend begins before fertilization. In mammals, HLA-E and lama4 play an important role in reducing the mother’s immune rejection of embryos and promoting trophoblast invasion. The signal be detected on the follicular placenta during pregnancy and the theca cell layer of oocytes before fertilization. The strong expression signal indicates that the follicular placenta may originate from the theca cell layer and perform similar functions to the mammalian placenta. cyp19a1a and fshr are genes specifically expressed in granulosa cells. Signals are only detected on the granulosa cell layer of oocytes before fertilization. No signal can be detected on the follicular placenta during pregnancy, indicating that granulosa cells may not be involved in the formation of follicular placenta. These results prove that the follicular placenta of black rockfish may be formed by the proliferation, migration, and expansion of the theca cell layer, and may perform similar functions to the mammalian placenta. 

3. In black rockfish, the 11-ketotestosterone has been at a low level during the process of oogenesis, and gradually increased after fertilization, reaching a peak in the blastula stage, and maintaining a high level throughout the pregnancy. The level of 17-βestradiol increases significantly during egg maturation, peaks at stage IV, and then drops sharply from stage V to blastula stage, but 17-βestradiol also maintains a high level in the middle and late stages of pregnancy, and reaches its peak again in the gastrula stage . During the process of oogenesis, the level of progesterone is low, but the level of progesterone rises rapidly after fertilization and remains high during pregnancy. Compared with turbot, the level of 11-ketotestosterone in black rockfish increases from stage V to the blastula stage, and the level of 17-βestradiol decreases, resulting in a gradual decrease in the ratio of 17-βestradiol to 11-ketotestosterone in the body, reaching a minimum in the blastocyst stage. This may be due to the driving factor that promotes the outward expansion of the theca cell layer and follicular placenta. And during pregnancy, 11-ketotestosterone, 17-βestradiol and progesterone are all at high levels, which may help maintaining pregnancy.