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许氏平鮋滤泡胎盘结构及母源营养供应的研究
杜腾飞
Subtype博士
Thesis Advisor李军
2022-05-21
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Keyword许氏平鮋 滤泡胎盘 母源营养 卵黄蛋白原
Abstract

鱼类生殖方式分为卵生和胎生,许氏平鲉是我国北方海区胎生鱼类的代表种,长期以来无论是在优质蛋白供给还是在近岸生态环境修复和增养殖方面都扮演着重要角色。近年来,学者们认为许氏平鲉胚胎发育的营养来源可能是母体和卵黄两个,但仅见于生理生化的少量研究。迄今没有直接证据证明许氏平鮋胚胎发育过程中存在着母源营养。本研究以胚胎发育期许氏平鮋为研究对象,通过解剖学、组织学和电镜技术对母胎连接结构进行观察;通过液相色谱和气相色谱技术,对血液中的营养成分进行测定;通过荧光定量和蛋白质免疫印迹法对胚胎发育过程中肝脏卵黄蛋白原合成进行研究;并通过亮氨酸稳定同位素标记和FITC卵黄蛋白示踪标记寻找母源营养的直接证据,以期证明许氏平鮋胚胎发育过程中存在母源营养。主要研究结果如下:

许氏平鮋胚胎发育过程中有胎盘样结构出现,存在母源营养的结构基础。许氏平鮋的母胎连接结构,形态上类似于滤泡胎盘,由内外两层组成,靠近胚胎的内层由上皮滤泡细胞组成,外层由高度血管化的疏松结缔组织组成。高度血管化是许氏平鮋滤泡胎盘最显著的特征,离胚胎最近的血管存在于滤泡层,母体通过血液运输来的营养物质可直接转移到胚胎中。许氏平鮋的类胎盘结构,从受精后开始增生,直接表现为结缔组织绒毛状结构的增多。胚胎发育初期,滤泡胎盘与胚胎之间的联系比较紧密,滤泡层细胞紧贴胚胎外膜;随着胚胎的发育和卵巢液的增多,滤泡胎盘和胚胎之间的联系变得疏松;超微观察发现,孵化前期的胚胎和滤泡胎盘之间存在空隙,包裹胚胎的卵膜有一部分裸露在胎盘外部,薄的卵膜上有大量的小孔和嵴状突起。这些结构的变化加上雌鱼卵巢内初孵仔鱼的表面鳞状上皮细胞的存在,均为母胎之间的营养传递提供了可能性。

许氏平鮋的血液中含有丰富的营养物质,包括16种水解氨基酸(9种必需氨基酸),20种脂肪酸,葡萄糖和果糖,氨基酸在血液中的含量最高3.44-4.58 g/100g,大约是脂肪酸的3-5倍,是糖的44-155倍。雌鱼血液中含量较高的营养素比较固定,含量较高的氨基酸有脯氨酸>亮氨酸>谷氨酸>缬氨酸>丙氨酸;含量较高的脂肪酸有DHA>棕榈酸> EPA>油酸>芥酸。同种营养素随胚胎发育也发生变化,大多数营养素含量(13种氨基酸、15种脂肪酸和葡萄糖)呈现胚胎发育前期增加后期减少的趋势。同位素标记结果显示,许氏平鮋卵细胞、原肠期胚胎和器官形成期胚胎中未检测到15N亮氨酸,却在对照组和实验组的卵细胞和胚胎中均检测到了高丰度的14N亮氨酸(常规),表明亮氨酸作为水产动物的必需氨基酸,在配子发育和胚胎发育过程中起着重要的作用。血液和胚胎的卵巢绒毛中检测到15N标记的亮氨酸丰度而未在胚胎中检测到。这表明在卵细胞及胚胎中后期发育阶段(原肠期和器官形成期),母体血液中游离丰富的亮氨酸没有被卵细胞和胚胎吸收利用,说明卵细胞和胚胎对营养物质的吸收具有一定的自主选择性,优先主动运输一些功能性的大分子营养物质(如卵黄蛋白原),即复合型的大分子,可以同时满足卵细胞和胚胎对多种营养物质的需求。

卵黄蛋白原作为复合型大分子营养物质,在许氏平鲉胚胎发育过程中在母体雌二醇的作用下,持续表达提供胚胎营养。肝脏荧光定量和Western blottingWB)结果均显示在胚胎发育过程中肝脏持续合成VtgAaVtgAbVtgC,且都显示了相同的表达规律,囊胚期表达量最高,随着胚胎的发育表达量逐渐下降。卵巢WB实验,发现胚胎发育过程中,胚胎体内卵黄蛋白原含量在胚胎发育前期变化不明显,到器官形成期和分娩前,胚胎内的卵黄明显减少,推测胚胎发育前期更多的是营养物质的储备,胚胎发育后期更多的是营养物质的利用。通过雌激素人工诱导、分离、纯化许氏平鮋卵黄蛋白原, FITC体外标记和注射FITC-Vtg的方法,追踪胚胎是否从母体吸收Vtg。结果显示,胚胎发育过程中母体的卵黄蛋白原可通过血液,经各级毛细血管,进入滤泡胎盘及间质中,并在体节期胚胎内部以及器官形成期的卵黄囊内均检测到FITC-Vtg。因此我们认为许氏平鲉胚胎发育期,肝脏可继续合成Vtg,运输至卵巢,Vtg可持续被胚胎吸收和利用,即胚胎发育过程中存在母源营养。

Other Abstract

Fish reproduction is divided into oviparous and viviparous. Sebastes schlegelii is a representative species of viviparous fish in the northern sea area of China. For a long time, it has played an important role in both high-quality protein supply and coastal ecological environment restoration and aquaculture. In recent years, scholars believe that the nutritional sources of embryonic development of S. schlegelii may be maternal and yolk, but it is only found in a small number of physiological and biochemical studies. So far, there is no direct evidence to prove that maternal nutrition exists in the process of embryonic development. In this study, the structure of maternal fetal connection was observed by anatomy, histology and electron microscope; The nutritional components in blood were determined by liquid chromatography and gas chromatography; The synthesis of hepatic vitellogenin during embryonic development was studied by fluorescence quantification and Western blotting; The direct evidence of maternal nutrition was found by leucine stable isotope labeling and FITC yolk protein tracer labeling, in order to prove that maternal nutrition exists in the process of embryonic development of Sebastes schlegelii Sebastes schlegelii. The main results are as follows:

There are placental like structures during embryonic development and maternal nutritional structure. The maternal fetal connection structure of S. schlegelii is morphologically similar to follicular pseudoplacenta, which is composed of inner and outer layers. The inner layer near the embryo is composed of epithelial follicular cells, and the outer layer is composed of highly vascularized loose connective tissue. High vascularization is the most significant feature of follicular placenta of Sebastes schlegelii. The blood vessel closest to the embryo exists in the follicular layer, and the nutrients transported by the mother through blood can be directly transferred to the embryo. The placental like structure of S. schlegelii begins to proliferate after fertilization, which is directly manifested in the increase of villous structure of connective tissue. In the early stage of embryo development, the follicles are closely connected with the outer membrane of the embryo; With the development of embryo and the increase of ovarian fluid, the connection between follicular placenta and embryo becomes loose; Ultrastructural observation showed that there was a gap between the embryo and follicular placenta in the early hatching stage. Part of the egg membrane surrounding the embryo was exposed outside the placenta, and there were a large number of small holes and cristae on the thin egg membrane. These structural changes and the presence of squamous epithelial cells on the surface of newly hatched larvae in the female ovary provide the possibility of nutrient transmission between mother and fetus.

S. schlegelii blood is rich in nutrients, including 16 hydrolyzed amino acids (9 essential amino acids), 20 fatty acids, glucose and fructose. The highest content of amino acids in the blood is 3.44-4.58 g/100g, about 3-5 times that of fatty acids and 44-155 times that of sugar. The higher content of nutrients in the blood of female fish is relatively fixed, and the higher content of amino acids are proline > leucine > glutamate > valine > alanine; The fatty acids with high content are DHA > palmitic acid > EPA > oleic acid > erucic acid. The same nutrients also changed with the development of embryos. Most nutrients (13 amino acids, 15 fatty acids and glucose) showed a trend of increasing in the early stage of embryonic development and decreasing in the later stage. The results of isotope labeling showed that 15N leucine was not detected in eggs, gastrula embryos and organogenesis embryos, but high abundance of 14N leucine (conventional) was detected in eggs and embryos of control and experimental groups, indicating that leucine, as an essential amino acid of aquatic animals, plays an important role in gamete development and embryo development. The abundance of 15N labeled leucine was detected in the blood and ovarian villi of embryos, but not in embryos, which indicates that the free and abundant leucine in the maternal blood in the middle and late development stages of eggs and embryos (gastrula stage and organogenesis stage) was not absorbed and utilized by eggs and embryos, indicating that eggs and embryos have a certain degree of autonomy and selectivity in the absorption of nutrients, give priority to the active transport of some functional macromolecular nutrients (such as Vitellogenin), that is, complex macromolecules, which can meet the needs of eggs and embryos for a variety of nutrients at the same time.

Vitellogenin, as a complex macromolecular nutrient, was continuously expressed during the embryonic development of S. schlegelii under the action of maternal estradiol to provide embryonic nutrition. The results of liver fluorescence quantification and Western blotting (WB) showed that the liver continuously synthesized VtgAa, VtgAb and VtgC during embryonic development, and all showed the same expression law. The highest expression amount was at the blastocyst stage, which gradually decreased with the development of embryos. Ovarian WB experiment found that during embryonic development, the content of vitellogenin in the embryo did not change significantly in the early stage of embryonic development, and the yolk in the embryo decreased significantly during organ formation and before delivery. It is speculated that the early stage of embryonic development is more the reserve of nutrients, and the later stage of embryonic development is more the utilization of nutrients. The vitellogenin was artificially induced, isolated and purified by estrogen, labeled by FITC in vitro and injected with FITC-Vtg to track whether the embryo absorbed Vtg from the mother. The results showed that during embryonic development, maternal vitellogenin could enter the follicular placenta and stroma through blood and capillaries at all levels, and FITC-Vtg was detected in the interior of somite embryos and yolk sac during organogenesis. Therefore, we believed that during embryonic development, the liver could continue to synthesize VTG and transported it to the ovary. Vtg could be continuously absorbed and utilized by embryos. There was maternal nutrition during embryonic development.

Language中文
Table of Contents

第一章 前言... 1

1.1 许氏平鮋... 1

1.1.1 许氏平鮋分类地位... 1

1.1.2 许氏平鮋形态学特征... 1

1.1.3 许氏平鮋的地理分布... 2

1.1.4 许氏平鮋的繁殖习性... 2

1.1.5 许氏平鮋的经济价值和生态价值... 3

1.1.6 许氏平鮋的研究现状... 3

1.2 脊椎动物的繁殖策略... 3

1.2.1 脊椎动物的生殖方式... 4

1.2.2 脊椎动物胚胎发育的营养方式... 4

1.3 卵生鱼类的卵黄营养... 5

1.3.1 卵黄蛋白原的生成... 5

1.3.2 卵黄蛋白原的转运... 6

1.3.3 卵黄的生物学作用... 7

1.4 胎生鱼类的母源营养... 8

1.4.1 母源营养的类型... 8

1.4.2 母源营养研究途径... 9

1.5 鱼类氨基酸营养和卵黄蛋白原营养的研究进展... 10

1.5.1 鱼类氨基酸营养的研究... 10

1.5.2 鱼类卵黄蛋白营养的研究... 12

1.5.3 三种追踪物质转运的研究方法... 13

1.6 本研究的目的与意义... 14

第二章 许氏平鮋滤泡胎盘结构研究... 17

2.1 研究背景... 17

2.2 材料和方法... 18

2.2.1 实验材料... 18

2.2.2 主要试剂和仪器... 18

2.2.3 实验方法... 18

2.3 实验结果... 20

2.3.1 许氏平鮋滤泡胎盘结构的解剖学观察... 20

2.3.2 许氏平鮋滤泡胎盘结构的组织学观察... 23

2.3.3 许氏平鮋滤泡胎盘结构的超微观察... 30

2.4 讨论... 33

2.5 小结... 35

第三章 许氏平鮋血液营养组分分析及亮氨酸同位素示踪研究... 37

3.1 研究背景... 37

3.2 材料和方法... 38

3.2.1 实验材料... 38

3.2.2 主要试剂和仪器... 38

3.2.3 实验方法... 38

3.3 实验结果... 42

3.3.1 许氏平鮋雌鱼血液中氨基酸含量变化... 43

3.3.2 许氏平鮋雌鱼血液中脂肪酸含量变化... 46

3.3.3 许氏平鮋雌鱼血液中葡萄糖和果糖含量变化... 50

3.3.4 亮氨酸稳定同位素示踪... 50

3.4 讨论... 55

3.5 小结... 57

第四章 许氏平鮋卵黄蛋白原表达特征及母胎运输研究... 59

4.1 研究背景... 59

4.2 材料和方法... 60

4.2.1 实验材料... 60

4.2.2 主要试剂和仪器... 60

4.2.3 实验方法... 61

4.3 实验结果... 72

4.3.1 许氏平鮋三种卵黄蛋白原氨基酸序列以及蛋白结构分析... 72

4.3.2 卵黄蛋白原VtgAb抗体制备... 80

4.3.3 许氏平鮋胚胎发育过程中雌二醇及卵黄蛋白原表达量的变化... 85

4.3.4 人工激素诱导Vtg合成及FITC标记示踪... 90

4.4 讨论... 95

4.5 小结... 97

第五章 结论与展望... 99

5.1 结论... 99

5.2 展望... 101

参考文献... 103

... 123

作者简历及攻读学位期间发表的学术论文与研究成果... 127

Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/178277
Collection实验海洋生物学重点实验室
Recommended Citation
GB/T 7714
杜腾飞. 许氏平鮋滤泡胎盘结构及母源营养供应的研究[D]. 中国科学院海洋研究所. 中国科学院大学,2022.
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