【作者】 李宇；

【导师】 刘月琴；

【作者基本信息】 河北农业大学 ， 畜牧学， 2022， 硕士

【摘要】 本文旨在研究黄体期不同阶段注射前列腺激素(PGF2α)对育成绵羊生殖激素分泌、免疫细胞因子、前列腺素受体基因及程序性坏死通路基因表达的影响。选择健康、体况良好、体重相近、发情周期正常的湖羊育成母羊60只,用“孕酮栓+PMSG”法进行发情周期同步化处理后,选择发情基本同步的48只母羊随机均分为6组。发情当天记为第0d,黄体前期试验组、黄体中期试验组和黄体末期试验组绵羊分别在第6d、11d、16d注射1 mLPGF2α(0.1 mg),黄体前期对照组、中期对照组和末期对照组绵羊分别在第6 d、11 d、16 d注射1 mL生理盐水。每次注射后第0.5、1、2、3 h采血,用于血液指标检测,第3 h采集黄体组织,用于基因表达检测及HE组织染色检测黄体组织形态变化。结果表明:在黄体期不同阶段注射PGF2α对育成绵羊血清生殖激素及免疫指标的影响:①各试验组和对照组在注射PGF2α后的0.5、1、2、3 h间血清中FSH、LH、PRL、P4、E2水平以及TNF-α、IL-1β、IL-6、IFN-β水平无差异(P>0.05)。②试验组注射 PGF2α后 0.5、1、2、3 h血清中 FSH、LH、PRL及 IL-1β、IFN-β与对照组差异不显著(P>0.05);前期试验组注射PGF2α后第3 h的P4水平显著低于前期对照组,E2和IL-6水平显著高于前期对照组(P<0.05);前期试验组注射PGF2α后第2和3 h TNF-α水平显著高于前期对照组(P<0.05);中期试验组注射PGF2α后第1 h P4水平显著低于中期对照组(P<0.05)。③黄体前期注射PGF2α后,前期试验组0.5～3 h内FSH、E2、TNF-α、IL-6整体水平显著高于前期对照组(P<0.05),P4整体水平显著低于前期对照组(P<0.05),对LH、PRL、IL-1 β和IFN—β无影响(P>0.05);黄体中期注射PGF2α后,中期试验组0.5～3h内E2整体水平显著高于中期对照组(P<0.05),P4整体水平显著低于中期对照组(P<0.05),对 FSH、LH、PRL、TNF-α、IL-1β、IL-6 和 IFN-β无影响(P>0.05);黄体末期注射PGF2α对生殖激素与相关细胞因子没有显著性影响(P>0.05)。在黄体期不同阶段注射PGF2α对育成绵羊程序性坏死基因表达及卵巢形态的影响:①未注射PGF2α的对照组绵羊,黄体前期、中期FPA mRNA表达水平显著高于黄体末期(P<0.05);黄体前期FPB表达水平显著高于黄体末期(P<0.05),与黄体中期无差异(P>0.05);黄体中期TNFR1表达水平显著高于黄体前期和末期(P<0.05);黄体中期RIPK3表达水平显著高于黄体末期(P<0.05),黄体中、末期与黄体前期无差异(P>0.05);TNF-α、RIPK1和MLKL表达水平在整个黄体期没有显著变化(P>0.05)。②注射PGF2α后试验羊黄体组织形态发生了变化,出现炎症细胞浸润与细胞凋亡,其中黄体中期退化程度最高。③黄体前期注射PGF2α显著降低FPA表达水平(P<0.05),显著提高FPB、RIPK1、MLKL表达量(P<0.05),对TNF-α、TNFR1、RIPK3基因表达无影响(P>0.05);黄体中期注射PGF2α显著上调了FPB、TNF-α、RIPK1与MLKL表达量(P<0.05),对FPA、TNFR1和RIPK3表达没有影响(P>0.05);黄体末期注射PGF2α显著上调了RIPK1表达(P<0.05),对 FPA、FPB、TNF-α、TNFR1、RIPK3和MLKL 表达没有影响(P>0.05)。④FPA与RIPK3和TNFR1 mRNA表达呈正相关(P<0.05),FPB与MLKL和TNF-αmRNA表达呈正相关(P<0.05)。综上,PGF2α对黄体的作用存在阶段性差异,黄体前期对PGF2α的短期应答反应强于黄体中末期。PGF2α在黄体中期诱导黄体退化效果较好,PGF2α可能通过TNF-α/TNFR1通路启动程序性坏死,FPB在介导PGF2α诱导黄体退化和程序性坏死中发挥主要作用。更多还原

【Abstract】 The aim of this study was to investigate the effects of Prostaglandin F2α(PGF2α)injection at different phases of luteal on on endocrine regulationexpression of PGF2αreceptors and programmed necrosis-associated genes.Sixty healthy sheep in good condition with normal estrous cycle were used in this study.Forty eight of sixty sheep with basically synchronous estrus were randomly divided into 6 groups after synchronization of estrus cycle with " MAP+PMSG" method.The day of oestrus is recorded as day 0.The experiment groups of early-,mid-and late-luteal phase were injected with 1mL PGF2α(0.1mg)on day 6,day 11 and day 16,respectively.The control groups of different luteal phases were injected with ImL normal saline on day 6,11 and 16,respectively.Blood was collected at 0.5,1,2,and 3 hours after each injection for the determination of blood index.The corpus luteum tissue was collected at 3 h after each injection for gene expression detection and the morphological changes of corpus luteum.The results showed as follows:Effects of PGF2α on reproductive hormones and immune indices at different phases of the luteal phase in sheep.① There were no differences in levels of FSH,LH,PRL,P4,E2,TNF-α,IL-1β,IL-6 and IFN-β in experimental group and control group at 0.5,1,2,3h after injection(P>0.05).② There were no significant differences in levels of FSH,LH,PRL,IL-1β and IFN-β between experimental group and control group at 0.5,1,2 and 3 h after injection(P>0.05).At 3h after injection of PGF2α,P4 level was significantly lower,and E2 and IL-6 levels were significantly higher in the early-luteal group than the control group(P<0.05).The level of TNF-α in the early-luteal group was significantly higher than the control group at the 2h and 3h after injection of PGF2α(P<0.05),and the level of P4 in the mid-luteal group was significantly lower than that in the control group at 1h after injection of PGF2α(P<0.05).③ After injection of PGF2α in the early-luteal phase,the overall levels of FSH,E2,TNF-α,and IL-6 in the early-luteal group were significantly higher than the control group within 3 h(P<0.05),and the overall level of P4 was significantly lower than control group(P<0.05).There were no effects on LH,PRL,IL-1β and IFN-β(P>0.05).After injection of PGF2α in the mid-luteal phase,the overall level of E2 in the mid-luteal group was significantly higher than the control group(P<0.05),and P4 was significantly lower than the control group(P<0.05).There were no effects on FSH,LH,PRL,TNF-α,IL-1β,IL-6 and IFN-β(P>0.05).Effects of PGF2α on luteal tissue morphology and expression of necroptosisassociated genes at different phases of the luteal phase in sheep.① The results showed that,for sheep of the control group that were not injected with PGF2α,the mRNA expression level of FPA in the corpus luteum was significantly higher in the early-luteal and midluteal phases than in the late-luteal phase(P<0.05);the FPB expression level in the corpus luteum was significantly higher than the late-luteal phase(P<0.05),and there was no difference from the mid-luteal phase(P>0.05);the expression level of TNFR1 in the mid-luteal phase was significantly higher than that in the early-luteal and lateluteal phases(P<0.05);The expression level of RIPK3 in the mid-luteal phase was significantly higher than that in the late-luteal phase(P<0.05),and both of them was no significant difference to the early-luteal phase(P>0.05);TNF-α,RIPK1 and MLKL expression levels did not change significantly throughout the luteal phase(P>0.05).②After the injection of PGF2α,the morphology of corpus luteum was changed,with inflammatory cell infiltration and apoptosis,especially in the mid-luteal phase,with a higher degree of degeneration.③ In the early-luteal phase,injection of PGF2αsignificantly reduced the expression of FPA(P<0.05),and significantly increased the expression of FPB,RIPK1,MLKL(P<0.05),and had no effect on the expression of TNFα,TNFR1,and RIPK3(P>0.05).In the mid-luteal phase,injection of PGF2α can significantly up-regulate the expression of FPB,TNF-α,RIPK1 and MLKL(P<0.05),but has no effect on the expression of FPA,TNFR1 and RIPK3(P>0.05);In the lateluteal phase,injection of PGF2α can significantly up-regulate the expression of RIPK1(P<0.05)),has no effect on the expression of FPA,FPB,TNF-α,TNFR1,RIPK3 and MLKL(P>0.05).④The mRNA expression level of FPA was positively correlated with RIPK3 and TNFR1(P<0.05),and the mRNA expression level of FPB was positively correlated with MLKL and TNF-α(P<0.05).In conclusion,there are stage differences of the effect of PGF2α on corpus luteum.The short-term response to PGF2α was stronger in early-luteal phase than in mid-and late-luteal in sheep;PGF2α has a better effect on inducing luteal degeneration in the midluteal phase.PGF2α may initiate necroptosis through the TNF-α/TNFR1 pathway,and FPB plays a major role in mediating PGF2α-induced luteal degeneration and necroptosis.更多还原