【作者】 刘欣；

【导师】 张贵学；

【作者基本信息】 东北农业大学 ， 动物遗传育种与繁殖， 2014， 硕士

【摘要】 牛的发情周期按卵巢状态分为两个时期,卵泡期和黄体期,生殖内分泌调节着发情周期的过程,导致卵泡形成、成熟、排鸾及黄体形成等一系列的变化,同时,卵巢产生的类固醇激素等对副性器官作用,使生殖系统产生相应的变化,完成相应的生理功能,如,卵子成熟、受精、着床及妊娠的。雌激素、孕酮在发情周期或妊娠过程中发挥重要的作用,故本实验以不同生理状态牛的生殖器官为研究对象,探讨雌激素、孕酮在发情周期激素受体的大致分布变化,为充实繁殖理论和提高繁殖率提供理论和实验依据。同时,制备体外培养输卵管细胞,为进一步研究牛的生殖生理提供体外细胞研究素材。试验分为两部分：一是分别选取4头卵巢上有明显卵泡的卵泡期母牛和黄体期母牛的生殖器官冷冻保存。实验3个部位(卵巢、输卵管、子宫体)的横切取样,利用RT-PCR的方法,检测雌激素受体、孕激素受体的分布变化,为进一步研究发情周期过程中,激素的作用变化提供依据；二是分离输卵管上皮细胞,鉴定后进行培养和冷冻保存,建立输卵管上皮细胞库,为今后输卵管体外生殖生理研究储备材料。主要研究结果如下：1、ERα、ER β和PR分布依生殖系统部位不同：在卵泡期,ERα、ER β和PR在卵巢中最高,其次是输卵管,子宫含量最低(P<0.05)。在黄体期,子宫中ER a最高,其次是输卵管,卵巢含量最低(P<0.05)；输卵管中ER β最高,其次是卵巢,子宫含量最低(P<0.05)；PR表达量卵巢最高,输卵管其次,子宫最低,差异显著(P<0.05)。2、ERα、ER β和PR生理状态不同处于动态的变化中。卵泡期卵巢、输卵管的ER a明显高于黄体期(P<0.05),而在子宫中,两个时期中ER a没有变化(P>0.05)；卵巢ER β含量卵泡期明显高于黄体期,在子宫中的表达量黄体期却高于卵泡期(P<0.05)而在输卵管中的含量无差异(P>0.05)；PR在在卵巢、输卵管的含量卵泡期高于黄体期(P<0.05),在子宫中,卵泡期和黄体期的含量没有变化(P>0.05)。3、各不同生理状态和部位,Erα的表达量显著高于Erβ的表达量,即：Er a型为主要雌激素受体。4、建立了输卵管上皮细胞库,细胞纯度90%以上,冷冻保存解冻后活率在78%以上。输卵管上皮细胞初次培养贴壁速度较慢,悬浮时间为5-7天,容易成团存在。上皮细胞贴壁后先形成细胞集落,一周时间可铺满瓶底。第二代以后贴壁时间缩短为1-1.5天时间,可传7代,第二代以后随着传代数的增加,传代代次间隔延长。更多还原

【Abstract】 Bovine estrous cycle is divided by ovarian state into two periods, the follicular phase and the luteal phase. Reproductive Endocrinology regulates the process of the estrous cycle, leading to a series of changes of follicle formation, maturation, ovulation and corpus luteum formation. Meanwhile, steroid hormones produced by the ovaries affect the gonads, makes reproductive system to produce a corresponding change and to complete the appropriate physiological functions, such as, egg maturation, fertilization, implantation and pregnancy. Estrogen and progesterone play an important role in the estrous cycle or pregnancy process. Therefore, this study experiment on cow reproductive organs with different physiological states in order to investigate estrogen and progesterone receptors distribution change in different physiological states, providing theoretical and experimental evidence for enriching reproduction theory and improving reproductive rate. Meanwhile, oviduct cells have been already cultured in vitro, in vitro materials have been provided for further cow reproductive physiology research.Test is divided into two parts:firstly selected four sets of cow reproductive organs in a clear follicular or luteal phase then frozen. Use the RT-PCR method to evaluate distribution of estrogen and progesterone receptor change in experiment of3position (the ovary, fallopian tube and uterus) crosscut samples, providing a basis for further study of hormonal changes in the course of the estrous cycle. Secondly, oviduct epithelial cells were isolated, cultured and identified in vitro. Cells were cryopreservated and established cell bank of oviduct cells, providing materials in vitro for the future research of oviduct productive physiology.Main results are as follows:1Receptors of ER α, ERβ and PR distribute differently according to different parts of the reproductive system.In the follicular phase, ERa, ERβ and PR levels all in ovarian highest, followed by the fallopian tubes, in uterus lowest, different significantly (P<0.05). During the luteal phase, ERa is highest in the uterus, followed by the fallopian tubes, ovaries are lowest, different significantly (P <0.05);in oviduct ER(3level is highest, followed by ovarian, lowest in uterine, different significantly (P<0.05); PR is highest in the ovary, secondly fallopian tube, uterus lowest, different significantly (P<0.05).2ERa, ERβ and PR are in a dynamic change in different physiological statesOvaries and fallopian tubes in follicular phase, ERa was significantly higher than the luteal phase (P<0.05), while in the uterus, two periods of ERa unchanged (P>0.05), Ovaries in follicular phase ERβ was significantly higher than the luteal phase(P<0.05),while ERβin the uterus of luteal phase was significantly higher than follicular phase; and has no difference in the two states of oviduct (P>0.05).PR in ovaries and fallopian tubes of follicular phase was significantly higher than the luteal phase (P<0.05); in uterus, the content of PR in the follicular phase and the luteal phase did not change (P>0.05).3All parts and the physiological state, ERα expression level was significantly higher than the expression of ER β, that means:ER a is the main type of estrogen receptor.4Established cell bank of oviduct cells, cell purity more than90%,the motility was above78%after thaw.First isolated epithelial cells takes a long time to adhere, suspension time is5-7days, like to stay in a group. After adherent cells first form to colony, one week can cover the bottom. From the second generation the attaching time changes to1-1.5days, epithelial cells could be passaged to the7th generation. After the second generation, with the increase of the number of passages, the cell passaging interval extended.更多还原