发情和乏情初产母猪下丘脑-垂体-卵巢轴差异表达基因筛选及分析

Screening and Analysis of Differentially Expressed Genes in Hypothalamus-Pituitary-Ovary Axis of Estrous and Anestrous Primiparous Sows

研究旨在分析发情和乏情初产母猪下丘脑-垂体-卵巢轴基因表达的差异,探讨母猪乏情调控的分子机制。选用6头健康的断奶初产母猪,分为发情组(3头)和乏情组(3头),屠宰后分别采集下丘脑、垂体、卵巢进行转录组测序(RNA-Seq)、GO功能富集、KEGG通路及相关蛋白的Western blotting分析。结果发现,发情组下丘脑、垂体、卵巢分别获得52432800、52573730和52209252条clean reads,与猪参考基因组(Sus scrofa 11.1)的比对率分别为78.69%、81.49%和79.26%;乏情组下丘脑、垂体、卵巢分别获得52516724,52476820和52195962条clean reads,与猪参考基因组的比对率分别为82.38%、83.05%和80.20%。与发情组母猪相比,乏情组母猪下丘脑中共有710个差异表达基因,其中上调基因392个,下调基因318个;垂体中共有707个差异表达基因,其中上调基因283个,下调基因424个;卵巢中共有956个差异表达基因,其中上调基因635个,下调基因321个。3种组织中的共有差异表达基因为36个。与母猪情期调控相关的亲吻素-1(KISS1)、G-蛋白偶联受体54(GPR54)、速激肽3(TAC3)、速激肽3受体(TACR3)、17-α-羟化酶/17,20碳链裂解酶(CYP17A1)、芳香化酶(CYP19A1)、类固醇激素合成急性调节蛋白(STAR)、促性腺激素释放激素受体(GnRHR)和雌激素受体1(ESR1)基因在乏情组母猪体内显著下调(P<0.05;P<0.01)。Western blotting分析结果显示,TAC3、TAC3R、KISS1和GPR54相关蛋白在乏情组母猪下丘脑中也显著下调(P<0.05)。GO和KEGG通路分析发现,差异表达基因显著富集于正调控胆固醇酯化反应、卵巢类固醇生成、GnRH信号通路、FoxO信号等一些与类固醇激素生成和卵泡发育相关的信号通路。本研究结果表明,发情和乏情初产母猪下丘脑-垂体-卵巢轴上与情期调控相关的基因存在差异表达,尤其是Kisspeptin/GPR54和TAC3/TACR3两大系统的mRNA及蛋白表达水平在乏情组母猪下丘脑中均显著下调,推测可能是Kisspeptin/GPR54和TAC3/TACR表达受损导致了下丘脑调节功能障碍,进而导致了初产母猪乏情。该研究为揭示初产母猪乏情分子调控机制提供了重要支持,为今后利用分子技术改善母猪乏情问题提供了重要理论依据。

In this study,gene expression differences in hypothalamus-pituitary-ovary axis of estrous and anestrous primiparous sows was analyzed and molecular mechanisms for the regulation of sow estrus was explored.6 healthy primiparous sows were divided into 2 groups:The estrous group(3 sows)and anestrous group(3 sows),and then all sows were slaughtered to collect their hypothalamuses,pituitaries and ovaries for RNA-Seq analysis,GO classification,KEGG enrichment analysis and Western blotting analysis of related protein.It was found that 52432800,52573730 and 52209252 clean reads were obtained from hypothalamuses,pituitaries and ovaries of estrous sows,respectively.And the ratio of these reads to porcine reference genome(Sus scrofa 11.1)were 78.69%,81.49%and 79.26%,respectively.52516724,52476820 and 52195962 clean reads were obtained from hypothalamuses,pituitaries and ovaries of anestrous sows,respectively.And the ratio of these reads to porcine reference genome(Sus scrofa 11.1)were 82.38%,83.05%and 80.20%,respectively.Compared with estrous sows,there were 710 differentially expressed genes in the hypothalamus of anestrous sows,including 392 up-regulated and 318 down-regulated genes.There were 707 differentially expressed genes in the pituitary of anestrous sows,including 283 up-regulated and 424 down-regulated genes.There were 956 differentially expressed genes in the ovary of anestrous sows,including 635 up-regulated and 321 down-regulated genes.36 differentially expressed genes were shared in 3 kinds of tissues.This study found KISS1,GPR54,TAC3,TACR3,CYP17A1,CYP19A1,STAR,GnRHR and ESR 1 genes were all significantly down-regulated in anestrous sow(P<0.05;P<0.01),which were related to regulation of sow estrus.Western blotting analysis indicated TAC3,TAC3R,KISS1 and GPR54 proteins were also all significantly down-regulated in anestrous sow.Analysis of GO and KEGG pathway revealed the differently expressed genes were significantly enriched in some signaling pathways related to steroid hormone production and follicular development,such as positive regulation cholesterol esterification,ovarian steriodogenesis,GnRH signaling pathway and FoxO signaling pathway.This study showed that genes related to the regulation of sow estrus were differentially expressed in hypothalamus-pituitary-ovary axis of estrous and anestrous primiparous sows and especially the mRNA and protein expression levels of Kisspeptin/GPR54 and TAC3/TACR3 systems were all significantly down-regulated in hypothalamus of anestrous primiparous sows.It was speculated that impaired expression of Kisspeptin/GPR54 and TAC3/TACR3 systems led to hypothalamic dysfunction,and in turn led to anestrus in primiparous sows.This study provided important support for revealing the molecular regulation mechanism of anestrus in primiparous sows and a important theoretical basis for the use of molecular technology to improve sow anestrus in the future.