【作者】 王少兵；

【导师】 徐银学；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2009， 博士

【摘要】 卵巢是重要的雌性生殖器官,决定了动物的繁殖潜能。尽管哺乳动物卵巢含有的卵泡甚多,但其一生能成熟排卵的卵泡数很少,绝大多数(约有99.9%)的卵泡在腔前卵泡阶段闭锁、退化,这无疑是极大的资源浪费。现有的研究已表明,Wnt/p-catenin通路与卵泡发育和卵巢机能有密切关联。本研究以动情周期小鼠为研究对象,通过real-time Q-PCR和阴道涂片方法确定了小鼠的动情周期；利用real-time Q-PCR技术检测了Frizzled家族基因mRNA在小鼠动情周期卵巢组织中的表达规律；运用ISH、IHC和Western blotting方法深入研究了小鼠动情周期卵巢组织中Frizzled 2和3的时空表达模式。本研究有助于阐明雌性生殖细胞的发育中Wnt/Frizzled信号通路的具体功能和机制,对提高雌性动物繁殖力、更合理有效地开发利用卵巢内的卵泡资源,以及探究繁殖疾病病因和治疗方法,都具有重要的理论和现实意义。1.小鼠动情周期的判断与证实为了选择合理的动情周期和准确的动情时期,采用阴道涂片观察和判断动情周期阴道内的细胞学变化,并通过检测动情周期子宫湿重和组蛋白H3.2的mRNA表达水平变化来进一步证实。结果显示,小鼠动情周期划分为4个时期(动情前期、动情期、动情后期、间情期),在整个动情周期中,子宫湿重是变化的,动情前期和动情期的子宫最重(P<0.05)；组蛋白H3.2水平变化类似于子宫湿重,并与子宫细胞增殖变化一致(P<0.05)。这表明本研究中实验材料小鼠的动情周期划分是合理和准确的,从而为后续研究奠定基础。2. Frizzled家族基因mRNA在小鼠动情周期卵巢组织中的表达水平哺乳动物中已确定了10种Frizzled蛋白,其能作为受体被一类糖脂蛋白Wnt配体激活,从而影响细胞命运,包括卵巢的胚胎期发育,但这些受体分子在成年动物卵泡发育期间的表达和具体作用知之甚少。故本研究运用real-time Q-PCR方法分析研究了Frizzled 1-10 mRNA在小鼠动情周期卵巢中的相对表达水平,结果表明,10个Frizzled家族基因中,除Frizzled 8外,其他都有不同水平的表达(A-I)。Frizzled 1和Frizzled 2动情前期表达水平显著高于其他三个时期水平,都约是间情期水平的4倍(P<0.05)(A、B); Frizzled 3动情前期和间情期表达水平都显著高于另两个时期的1.5倍(P<0.05)(C)；Frizzled 4在动情期显著高于其他三时期的2～3倍(P<0.05)(D)；间情期Frizzled 5表达显著高于动情前期和动情期,约是动情期水平的5倍(P<0.05)(E), Frizzled 7表达类似于Frizzled 5,约是动情前期水平的4倍(P<0.05)(G)；Frizzled 10的表达在动情前期显著高于动情期和间情期,约是动情期的3倍多9(P<0.05)(I); Frizzled 6和Frizzled 9在四个时期的表达没显著差别(P<0.05)(F、H)。这些结果提示Frizzled 1-4可能在动情周期卵泡发育过程中具有更重要作用,Frizzled 1和Frizzled 4已被研究证实,但有关表达水平较高的Frizzled 2和Frizzled 3在卵巢中的研究报道甚少。因此,后面研究将深入探明Frizzled 2和Frizzled 3在卵巢组织中的时空表达模式。3. Frizzled-2的mRNA和蛋白质在小鼠动情周期卵巢中的时空表达模式Wnt/p-catenin通路是保守的信号通路,其调节基因表达和控制细胞命运规律、细胞增殖和分化等多种发育过程。当前,有关该通路在成年动物卵巢中作用研究报道几乎没有。本研究通过实时定量PCR、原位杂交、Western印迹杂交和免疫组织化学等方法,检测了Frizzled 2在动情周期的小鼠卵巢中的表达情况。研究发现,动情前期的Frizzled 2 mRNA和蛋白水平最高,自动情期到间情期快速减弱。原位杂交结果表明：动情前期卵母细胞和基质中Frizzled 2信号强；而后三个时期的卵母细胞、颗粒细胞、基质和黄体中具有中等或微弱信号。Frizzled 2蛋白的表达模式大部分与mRNA的一致,但是,强的表达信号存在动情前期和动情期的颗粒细胞和卵母细胞膜上。以上结果表明,Frizzled 2可能调控动情周期的卵泡生长、卵母细胞成熟。4. Frizzled-3的mRNA和蛋白质在小鼠动情周期卵巢中的表达与定位动物早期和成年期间,Frizzled基因调节胚胎发育、组织和细胞极性、神经突触形成、增殖等各种生长发育过程。当前,有关Frizzled 3在成年动物卵巢中的研究报道几乎没有。所以,运用实时定量PCR、原位杂交和免疫组织化学方法检测了Frizzled3的mRNA和蛋白在动情周期的小鼠卵巢中的表达规律。结果发现,动情前期和间情期Frizzled 3相对表达水平最高,动情期和动情后期出现显著降低(P<0.05)。原位杂交结果表明：动情前期,Frizzled 3 mRNA在各期卵泡的颗粒细胞和基质上高表达；黄体上弱表达。动情期,基质中信号强,但颗粒细胞和黄体上水平弱的几乎看不见。动情后期,基质中表达中等水平,黄体细胞表达水平弱,各级卵泡几乎不表达。间情期,基质中高水平表达,颗粒细胞和黄体中水平很弱。整体来看,动情前期和间情期杂交信号最强,其他二时期次之,这基本与real time Q-PCR的结果一致。Frizzled 3蛋白的表达模式大部分与mRNA的一致,基质上的信号最强,但是,动情期和间情期的颗粒细胞存在中等强度的表达信号,以及动情前期和动情期的卵母细胞膜上低水平表达。以上结果表明,Frizzled 3可能参与调控动情周期的卵泡生长和卵母细胞成熟。更多还原

【Abstract】 Ovary, an important female reproductive organ, determines animal breeding potential. Although many follicles is in the mammalian ovary, few can develop the ovulatory follicles in life and the vast majority (about 99.9%) are atresia or degradation in preantral phase among them. This is a great waste. Existing research has shown that Wnt/β-catenin pathway closely relative to follicular development and ovarian function. In this study, firstly, we confirmed vaginal cytology-based estrous cycle stage classification through the real-time Q-PCR method, then detected mRNA expression of the Frizzled family genes in the mice ovary during estrous cycle by real-time Q-PCR, at last, using ISH, IHC and Western blotting methods, studied the temporal and spatial expression patterns of Frizzled 2 and 3 in the estrous ovary in mice. These works should have great theoretical and practical significance, help to clarify specific function and mechanism of Wnt/Frizzled signaling pathway during the development of female germ cells, to improve the fertility of female animals, a more reasonable utilization of follicles in the ovary, as well as to study the etiology and treatment of the reproductive disease.1. Estrous cycle stage classification and validation in miceIn order to choose a reasonable and accurate estrous cycle, the author observed vaginal cytology changes within estrous cycle by vaginal smears and dividing into four periods (proestrus, estrus, metestrus, diestrus). To confirm vaginal cytology-based estrous cycle stage classification, uterine wet weights and histone H3.2 mRNA expression were analyzed. Uterine weight varies with the estrous cycle. Uteri obtained from mice are the heaviest in the proestrus and estrus (p< 0.05). The relative expression level of H3.2 mRNA parallels wet weight and consistent with known changes in uterine cellular proliferation (p< 0.05). This suggest that sampled mice hav the reasonable and accurate estrous cycle in this study and lay the foundation for our follow-up study. 2. The relative expression levels of Frizzled family genes mRNA in the estrous ovary in miceTen Frizzled genes encoding Frizzleds have been identified in mammals. They can be activated as a receptor binding to Wnt ligands (a class of glycoproteins) to affect cell fate, including the development of embryonic ovary, but little known about the expression and function of the receptor molecules in the adult ovary. Therefore, we used real-time Q-PCR to analyze the relative expression levels of Frizzled 1-10 mRNAs in estrous ovary in mice. The results showed that The others had a different expression level except Frizzled 8(A-I). Expressions of Frizzled 1 and 2 were highest in proestrus and both were about 4 times higher than that in diestrus (P<0.05) (A, B); Frizzled 3 was 1.5 fold higher in proestrus and diestrus than those in the other two periods (P<0.05) (C); Frizzled 4 was found significantly high expression in estrus, which 2-3 fold than ones of the other three stages (P <0.05) (D); Frizzled 5 had marked higher signal in diestrus than those in proestrus and estrus, being approximately 5 times higher than that in estrus (P<0.05) (E), Frizzled 7 was similar to Frizzled 5, about 4 fold high than the level of proestrus (P<0.05) (G); Frizzled 10 expression in proestrus was significantly higher than those in estrus and diestrus and about more than 3 times higher than that in estrus(P<0.05) (I); but Frizzled 6 and 9 were no significant difference during estrous cycle(P<0.05) (F、H). These results suggested that Frizzled 1-4 may play an more important role in the development of follicle during estrous cycle stage. As previously studied, Frizzled 1 and 4 have been confirmed, but Frizzled 2 and 3 with higher expression levels have been little reported in the adult ovary. Therefore, the next work is that study the temporal and spatial expression patterns of Frizzled 2 and 3 in the estrus mouse ovary.3. The temporal and spatial expression patterns of Frizzled 2 mRNA and protein in estrous ovary in miceThe Wnt/β-catenin pathway is a conserved signalling pathway that regulates gene expression and controls diverse developmental processes such as cell fate specification, cell proliferation and cell differentiation. To our knowledge, the potential role of this pathway in the adult ovary has been poorly addressed. To this end, we investigated the expression pattern of Frizzled 2 in the mouse ovary during estrous cycle by real-time Q-PCR, in situ hybridization, Western blotting and immunohistochemistry. In this study, We found that during estrous cycle, Frizzled 2 mRNA and protein exhibited the highest level in the proestrus stage and rapidly decreased from estrus to diestrus. In situ hybridization results showed that the positive signals for Frizzled 2 were highly detected in the oocyte and stroma in proestrus stage, while moderate or weak Frizzled 2 mRNAs were localized in the oocyte, granulose cells, stroma and corpus luteum from estrus to diestrus. The localization pattern of Frizzled 2 protein was mostly consistent with its mRNA, but stronger Frizzled 2 proteins were present in the granulosa cells and membrane of oocyte in proestrus and estrus. Our data suggested that Frizzled 2 may be involved in regulating follicle growth, oocyte maturation during estrous cycle.4. Expression and localization of Frizzled 3 in the mice ovary during estrous cycleDuring the early period and adult, Frizzled genes regulate embryonic development, tissue and cell polarity, the formation of neural synapses, the cell proliferation, etc. To date, Frizzled 3 have not been investigated in adult ovary. Here, we detected the expression and localization of Frizzled 3 mRNA and protein in the mice ovary during estrous cycle through real-time Q-PCR, in situ hybridization and immunohistochemistry. It was found that the relative expression level of Frizzled 3 was highest in proestrus and diestrus, significantly decreased in estrus and metestrus (P<0.05). In situ hybridization results showed that in proestrus, high expression signals were found in the granulosa cell and stroma, weak level in corpus luteum. In estrous, stroma displayed high level, but granulosa cells and corpus luteum were revealed weak almost invisible levels. In metestrus, the moderate expression level was found in stroma, luteal cells were weak and almost no expression signals at all follicles. In diestrus, high expression level was in stroma, weak in granulosa cells and corpus luteum. Overall, the strongest hybridization signals were in proestrus and diestrus, second level in the other stages. This was mostly consistent with real-time Q-PCR. Expression patterns of Frizzled 3 protein were almost in accord with mRNA, the strongest signal was in the stroma, but moderate level were displayed in granulosa cells of estrus and diestrus, as well as low expression level in the oocyte cell membrane during proestrus and estrus. These results show that Frizzled 3 may regulate the developement of follicle, oocyte maturation in estrous cycle.更多还原