湖羊PGR基因对卵泡颗粒细胞体外增殖与凋亡的影响

旨在探究孕酮受体(progesterone receptor,PGR)基因对湖羊卵泡颗粒细胞体外增殖与凋亡的影响。利用RT-PCR技术扩增和克隆获得PGR基因编码序列(CDS),通过生物信息学软件对其氨基酸序列及同源性进行比对;用所获得序列构建过表达载体和干扰siRNA,分别转染湖羊颗粒细胞,并用CCK8技术检测颗粒细胞的细胞活力;采用RT-qPCR和Western blot技术,检测细胞周期和凋亡的相关基因或蛋白表达水平。结果显示,羊PGR基因的CDS区全长2736 bp,编码911个氨基酸;与其他物种氨基酸序列的同源性为39.66%~95.44%。干扰PGR基因通过下调CDK4、Bcl-2和上调Caspas3、Caspase8、BAX基因mRNA的表达(P<0.05),进而抑制颗粒细胞的增殖,但对CyclinD1未产生影响(P>0.05);过表达PGR基因通过上调CDK4、CyclinD1、Bcl-2和下调Caspase3、Caspase8、BAX基因mRNA的表达(P<0.05),进而促进颗粒细胞的增殖;BAX蛋白表达变化与对应mRNA的表达趋势一致(P<0.05);过表达PGR基因显著上调PCNA基因表达,而干扰PGR基因则下调PCNA蛋白表达(P<0.05)。研究表明,PGR基因通过调控细胞周期和凋亡关键基因的表达,影响湖羊颗粒细胞的增殖与凋亡,进而调节湖羊卵泡发育。     

RNA干扰水牛MTPN基因对颗粒细胞增殖、凋亡及激素分泌的影响

本研究旨在探讨敲低MTPN基因对体外培养水牛颗粒细胞增殖、凋亡及雌激素、孕酮分泌的影响。应用RNAi技术敲低颗粒细胞中MTPN基因的表达水平,通过实时荧光定量PCR方法检测体外培养水牛颗粒细胞中MTPN基因及增殖和周期相关基因的表达情况,CCK-8法检测细胞增殖,借助流式细胞仪检测细胞周期的分布,采用ELISA试剂盒检测细胞培养液中雌激素与孕酮含量。结果显示,经siRNA(si-MTPN)转染颗粒细胞后,MTPN基因相对表达量下降60%(P<0.01);细胞增殖受到显著抑制(P<0.05),G1期细胞数量下降,S期细胞数量上升,G2期细胞数量极显著上升(P<0.01),细胞被阻滞在G2期;增殖与周期相关基因Cyclin D2、Cytochrom C表达量显著上升(P<0.05),Caspase9、Fas基因表达量极显著上升(P<0.01);ELISA检测雌激素和孕酮分泌水平均显著下降(P<0.05)。综上表明,敲低MTPN基因能通过调控相关基因的表达抑制体外培养水牛颗粒细胞的增殖及雌激素、孕酮的分泌水平,为阐明MTPN基因参与家畜卵泡发生的分子机制提供参考。     

不同浓度GnIH对鸭颗粒细胞周期和增殖的影响

为了研究不同浓度GnIH对鸭颗粒细胞周期、增殖及相关基因表达的影响。本研究分别用不同浓度GnIH(0、0.1、1、10和100 ng·mL~(-1))处理体外培养的鸭颗粒细胞24 h(n=3),观察细胞的生长状态,通过流式细胞术和EdU方法检测细胞周期和细胞增殖,并用qRT-PCR方法检测增殖相关基因CDK6、CyclinD1、IGF-2、IGFBP-2、p27~(kip1)的表达。结果显示,各浓度GnIH处理组的细胞生长状态良好,形态正常,细胞轮廓清晰,组间死亡细胞数差异不显著(P0.05);在0.1和1 ng·mL~(-1) GnIH处理组,细胞周期阻滞在G2期的比例显著上升(P0.05);随着GnIH处理浓度的增加,EdU阳性细胞数所占的百分比降低;在0.1和1 ng·mL~(-1) GnIH处理组,颗粒细胞中CDK6、CyclinD1、IGF-2、IGFBP-2、p27~(kip1)基因的相对表达量均下降,在10和100 ng·mL~(-1) GnIH处理组中这些基因的相对表达量则有所上升。研究表明,在体外培养的鸭颗粒细胞中,GnIH能使细胞周期阻滞在G2期,并降低EdU阳性细胞所占百分比和下调增殖相关基因的表达水平,从而抑制颗粒细胞增殖来影响动物繁殖性能。     

RNA干扰水牛MTPN基因对颗粒细胞增殖、凋亡及激素分泌的影响

本研究旨在探讨敲低MTPN基因对体外培养水牛颗粒细胞增殖、凋亡及雌激素、孕酮分泌的影响。应用RNAi技术敲低颗粒细胞中MTPN基因的表达水平,通过实时荧光定量PCR方法检测体外培养水牛颗粒细胞中MTPN基因及增殖和周期相关基因的表达情况,CCK-8法检测细胞增殖,借助流式细胞仪检测细胞周期的分布,采用ELISA试剂盒检测细胞培养液中雌激素与孕酮含量。结果显示,经siRNA(si-MTPN)转染颗粒细胞后,MTPN基因相对表达量下降60%(P0.01);细胞增殖受到显著抑制(P0.05),G1期细胞数量下降,S期细胞数量上升,G2期细胞数量极显著上升(P0.01),细胞被阻滞在G2期;增殖与周期相关基因Cyclin D2、Cytochrom C表达量显著上升(P0.05),Caspase9、Fas基因表达量极显著上升(P0.01);ELISA检测雌激素和孕酮分泌水平均显著下降(P0.05)。综上表明,敲低MTPN基因能通过调控相关基因的表达抑制体外培养水牛颗粒细胞的增殖及雌激素、孕酮的分泌水平,为阐明MTPN基因参与家畜卵泡发生的分子机制提供参考。     

siRNA干扰牦牛Bmal1基因对颗粒细胞激素分泌的影响

本研究旨在探讨敲低Bmal1基因对体外培养牦牛颗粒细胞雌二醇(E2)和孕酮(P4)分泌的影响。应用siRNA技术下调颗粒细胞中Bmal1基因的表达,利用ELISA试剂盒检测细胞培养液中E2与P4含量,再通过实时荧光定量PCR(qRT-PCR)和蛋白印迹(Western Blot)检测细胞激素分泌相关基因的表达情况。结果显示:经si-Bmal1转染颗粒细胞后,E2分泌量显著下降,P4分泌量极显著下降;在mRNA检测水平上,激素合成相关基因Cyp19a1、Star的表达量下降;在蛋白检测水平上,Cyp19a1、Star的结果与mRNA表达基本一致。综上表明,敲低Bmal1基因能通过调控相关基因的表达抑制体外培养牦牛颗粒细胞E2和P4的分泌水平,Bmal1基因参与牦牛颗粒细胞激素分泌的调控。     

抑制BMP/Smad信号通路对水牛卵巢颗粒细胞生长和类固醇激素合成的影响

【目的】探究BMP/Smad信号通路对水牛卵巢颗粒细胞生长和类固醇激素合成的影响。【方法】利用脂质体转染的方法将合成的Smad4基因的3对siRNAs(Smad4-siRNA1、Smad4-siRNA2和Smad4-siRNA3)和NC-siRNA(对照组)分别转染水牛颗粒细胞,通过实时荧光定量PCR检测Smad4基因的表达水平,比较不同siRNA的干扰效率。然后利用筛选的干扰效率最高的siRNA,转染水牛卵巢颗粒细胞,通过CCK-8法检测对照组和干扰组细胞的增殖情况,实时荧光定量PCR检测上述两组细胞凋亡相关基因Bax和Bcl2,细胞周期相关调控基因CyclinD2和CDK4,以及类固醇激素合成相关基因Cyp19A1和Cyp11A1的表达水平,并通过ELISA检测雌二醇(E₂)和孕酮(P₄)的含量。【结果】基因干扰试验结果表明,3对siRNAs对Smad4基因的表达均具有极显著的干扰作用(P<0.01),其中Smad4-siRNA1的干扰效率最高,达到了64%。CCK-8检测结果显示,与对照组相比,干扰组细胞的增殖效率显著降低(P&l...     

FOXO3基因过表达对鹅卵泡颗粒细胞凋亡和自噬相关基因mRNA表达水平的影响

本研究首先克隆鹅FOXO3基因编码区(CDS)序列,构建FOXO3基因的真核表达载体,然后将其转染至鹅卵泡颗粒细胞中,利用荧光定量PCR方法检测FOXO3基因对细胞凋亡和自噬相关基因mRNA表达水平的影响。结果显示,本研究成功克隆了鹅FOXO3基因CDS序列,并由此构建了真核表达载体pcDNA3.1-FOXO3。鹅卵泡颗粒细胞FOXO3基因过表达实验发现,过表达组凋亡相关基因Bax、Bcl-2、Caspase3、Fasl的mRNA表达水平较对照组均升高(P<0.05),而自噬相关基因LC3和Beclin1的mRNA表达水平较对照组均降低(P<0.05)。本研究结果表明,FOXO3基因可能影响鹅卵泡颗粒细胞中凋亡及自噬相关基因的表达,进而调控颗粒细胞的凋亡和自噬。     

猪RPL36A基因对PK15细胞增殖过程的影响

【目的】探究核糖体蛋白L36A(ribosomal protein L36A,RPL36A)基因对PK15细胞增殖过程的影响,为解析马身猪和大白猪生长速度差异的生理机制奠定基础。【方法】采用脂质体法将RPL36A基因干扰和过表达载体转染至PK15细胞中,通过实时荧光定量PCR和Western blotting技术检测RPL36A基因表达效率及细胞增殖标志基因(PCNA、Ki67、Cyclin B、CDK4)的表达变化,并通过划痕试验、CCK-8和EdU法检测细胞增殖情况。【结果】过表达RPL36A基因后,PK15细胞中PCNA、Ki67、CDK4基因mRNA表达量均极显著升高(P<0.01),Cyclin B基因mRNA表达量显著升高(P<0.05);PCNA蛋白表达量显著升高(P<0.05);PK15细胞在48 h的细胞数量极显著高于空载组(P<0.01),细胞增殖速度升高;阳性细胞数极显著升高(P<0.01)。干扰RPL36A基因后,PK15细胞中PCNA、Cyclin B基因mRNA表达量均极显著降低(P<0.01),Ki67、CDK4基因mR...     

miR-24-3p对猪颗粒细胞雌二醇合成的作用

旨在探究miR-24-3p对猪颗粒细胞雌二醇合成的影响。本试验收集180日龄健康母猪的卵巢组织,每次试验取20对卵巢进行颗粒细胞的分离培养,将miR-24-3p的mimics及inhibitor转染进颗粒细胞,通过ELISA、RT-qPCR、Western blot、双荧光素酶报告试验等技术探究miR-24-3p对猪颗粒细胞雌二醇合成的作用。结果表明,过表达miR-24-3p可显著促进雌二醇的合成(P<0.01),并加快StAR、CYP19A1和CYP11A1的转录和翻译(P<0.05);而干扰miR-24-3p则显著抑制雌二醇的合成(P<0.05),并显著下调CYP11A1、CYP19A1的mRNA和蛋白水平(P<0.05)。进一步研究发现,TOP1是miR-24-3p的直接靶基因,过表达miR-24-3p可显著抑制TOP1的表达(P<0.05),干扰miR-24-3p可显著上调TOP1的表达(P<0.05)。而过表达TOP1则可减弱miR-24-3p对颗粒细胞雌二醇合成的促进作用(P<0.05)。综上所述,miR-24-3p通过靶向TOP1...     

基于RNA-Seq分析食欲素A对绵羊卵巢黄体化颗粒细胞基因表达的影响

食欲素A(orexin A)作为一种重要的神经肽类激素,通过与G蛋白耦联受体结合影响生殖功能。为了研究orexin A对绵羊黄体化颗粒细胞基因表达的影响及其信号网络,培养绵羊卵巢黄体化颗粒细胞进行鉴定,提取黄体化颗粒细胞组和添加Orexin A的黄体化颗粒细胞组的RNA,采用lllumina测序技术进行测序,利用生物信息学方法对转录组数据进行分析,在测序数据质量控制的基础上,对差异表达的基因进行筛选、功能注释和富集分析。结果表明,黄体化颗粒细胞组的孕酮分泌量显著高于未黄体化颗粒细胞组;转录组测序共获得了50个差异基因,其中上调表达20个,下调30个;发现多个与细胞周期调控、繁殖及代谢相关的高表达基因,如PRRT2、ID1、RRM2、ATP6、ATP8、KIRREL、SOX4、TBX3等基因,结合GO分析和KEGG通路分析表明,差异基因主要参与代谢途径、氧化磷酸化、癌症信号通路、GnRH信号通路、cGMP-PKG信号通路、Rap1信号通路、Wnt信号通路以及Ca离子信号通路等。本试验为进一步阐明orexin A影响绵羊的生殖调控提供了依据。     

miR-495-3p对山羊卵巢颗粒细胞功能的影响

旨在探究miR-495-3p对山羊卵巢颗粒细胞功能的影响及作用机制.本研究选取健康的3～4月龄大足黑山羊母羊,收集卵巢颗粒细胞,利用miR-495-3p模拟物(mimics)和抑制物(inhibitor)构建过表达和抑制模型,通过流式细胞术检测细胞凋亡和周期,ELISA分析颗粒细胞的雌二醇(E2)和孕酮(P4)分泌,采...     

干扰和过表达BAMBI基因对猪卵泡颗粒细胞表型和功能的影响

为研究转化生长因子-β（TGF-β）信号通路中的骨形态发生蛋白和激活素膜结合抑制剂（bone morphogenetic protein and activin membrane-bound inhibitor,BAMBI）基因对猪卵泡颗粒细胞的调控作用,本研究设计构建BAMBI干扰和过表达载体,并将构建好的干扰（pSIREN-BAMBI-sh1、pSIREN-BAMBI-sh2、pSIREN-BAMBI-sh3）和过表达（pcDNA3.1-BAMBI）重组质粒转染猪卵泡颗粒细胞,通过实时荧光定量PCR技术进行有效片段的筛选,并对TGF-β信号通路下游基因（TGF-βRⅠ、TGF-βRⅡ、SMAD1、SMAD2、SMAD3、SMAD4、SMAD5）和细胞凋亡基因（Bax、Bcl-2）mRNA的表达水平进行检测。最后,用MTT法和流式细胞术检测BAMBI干扰和过表达对卵泡颗粒细胞增殖及凋亡的影响。结果表明,BAMBI干扰和过表达载体成功构建,pSIREN-BAMBI-sh2抑制BAMBI表达的效果最好,干扰效率最高。干扰BAMBI时,TGF-βRⅡ表达量显著上升（P<0.05）,使得SMAD2、SMAD3的表达量显著上升（P<0.05）;过表达BAMBI时,TGF-βRⅡ表达显著下降（P<0.05）,使得SMAD2、SMAD3的表达量显著下降（P<0.05）;上调BAMBI可显著抑制猪卵泡颗粒细胞增殖,极显著促进猪卵泡颗粒细胞凋亡（P<0.01）。研究结果表明,BAMBI基因显著影响猪卵泡颗粒细胞的生长发育,可能通过调节TGF-β信号通路间接影响猪的繁殖性能。     

可变剪接体WNT4-β对山羊卵泡颗粒细胞增殖和激素分泌的影响

旨在研究WNT4的一个可变剪接体（WNT4-β）对山羊卵泡颗粒细胞增殖的影响。本研究选取4~6月龄健康母羊20只,采集双侧卵巢,体外分离卵泡颗粒细胞进行培养。通过免疫荧光染色技术确定WNT4-β的表达位置;在山羊颗粒细胞中过表达或干扰WNT4-β后,利用RT-qPCR、Western blot检测WNT4-β和WNT信号通路中关键标记因子ROA1、RHOA及颗粒细胞增殖标记基因cyclin-D2、CDK4的表达变化;CCK-8技术检测颗粒细胞增殖情况;并通过ELISA分析颗粒细胞中生殖激素水平的变化。免疫荧光染色结果显示,WNT4-β只在山羊卵泡颗粒细胞中表达,在卵母细胞不表达;过表达WNT4-β后,WNT4-β和颗粒细胞增殖因子cyclin-D2、CDK4的mRNA相对表达量极显著增加（P<0.01）,蛋白表达水平显著增加（P<0.05）;WNT信号通路标记因子ROA1、RHOA mRNA表达水平显著增加（P<0.05）,β-catenin蛋白表达水平显著增加（P<0.05）;干扰WNT4-β后,WNT4-β、cyclin-D2、CDK4、ROA1和RHOA 的mRNA表达显著降低（P<0.05）,WNT4-β、cyclin-D2、CDK4及β-catenin蛋白表达显著降低（P<0.05）。CCK-8结果显示,过表达WNT4-β促进颗粒细胞增殖（P<0.05）;ELISA结果显示,过表达WNT4-β后,颗粒细胞中雌二醇（estradiol,E₂）水平显著增加（P<0.05）,孕酮（progesterone,P₄）水平升高但不显著（P>0.05）;干扰WNT4-β后则结果相反,颗粒细胞增殖受到抑制（P<0.05）,E₂和P₄的水平显著降低（P<0.05）。综上所述,WNT4可变剪接体WNT4-β通过调控WNT信号通路促进山羊卵泡颗粒细胞增殖及类固醇激素分泌,本研究为解析WNT4调控山羊颗粒细胞增殖的潜在分子机制提供理论基础。     

SMAD7对山羊卵泡颗粒细胞增殖、凋亡的影响

旨在探究SMAD7对山羊卵泡颗粒细胞增殖和凋亡的影响。本试验收集3~4月龄大足黑山羊母羊的卵泡颗粒细胞,通过过表达或siRNA干扰、ELISA、qRT-PCR、Western blot及流式细胞术等技术与方法探究SMAD7对颗粒细胞增殖、凋亡及类固醇激素分泌的影响。结果发现,SMAD7过表达显著下调颗粒细胞增殖活力并促进细胞凋亡,抑制PCNA表达（P<0.05）,下调BCL2/BAX的比值（P<0.01）;同时,SMAD7干扰显著上调颗粒细胞增殖活力,显著上调PCNA表达（P<0.05）与BCL2/BAX表达量比值（P<0.05）。SMAD7过表达极显著上调颗粒细胞的孕酮分泌,下调雌二醇表达水平（P<0.01）;同时SMAD7干扰极显著下调孕酮分泌,上调雌二醇分泌（P<0.01）。进一步研究发现,SMAD7过表达显著抑制SMAD2、SMAD3的mRNA和蛋白表达（P<0.05）;SMAD7干扰则显著促进SMAD2、SMAD3的mRNA和蛋白表达（P<0.05）。结果表明,SMAD7抑制山羊卵泡颗粒细胞的增殖和雌二醇分泌,促进凋亡和孕酮的合成,并且抑制SMAD2、SMAD3的表达,进而调节卵泡的发育与闭锁。     

Quantification of insulin-like growth factor binding protein mRNA using real-time PCR in bovine granulosa and theca cells: effect of estradiol, insulin, and gonadotropins

The effects of estradiol, insulin, and gonadotropins on levels of insulin-like growth factor binding protein (IGFBP)-2, -3, -4, and -5 mRNA levels in bovine granulosa and theca cells were evaluated in vitro using serum-free medium containing various hormone treatments arranged in four different experiments. Amounts of IGFBP-2, -3, -4 and -5 mRNA were quantitated using fluorescent quantitative real-time RT-PCR. In small-follicle (1-5 mm) granulosa cells, follicle-stimulating hormone (FSH) in the presence or absence of insulin increased (P<0.05) IGFBP-3 mRNA but did not change IGFBP-2, -4, or -5 mRNA levels; estradiol was without effect on IGFBP-2, -3, -4, or -5 mRNA levels in the absence of insulin but increased (P<0.05) IGFBP-2 mRNA levels in the presence of insulin. Luteinizing hormone (LH) in the absence (but not presence) of insulin increased (P<0.05) small-follicle granulosa cell IGFBP-3 mRNA levels. In large-follicle (>7.9 mm) granulosa cells, insulin alone increased (P<0.05) IGFBP-2 gene expression while LH, FSH, and estradiol were without effect (P>0.10). Estradiol (3 and 300 ng/ml) decreased (P<0.05) IGFBP-5 mRNA levels in large-follicle granulosa cells. In theca cells, insulin decreased (P<0.05) IGFBP-4 expression, but had no effect (P>0.10) on IGFBP-2, -3, or -5 mRNA levels. Estradiol decreased (P<0.05) IGFBP-2, -3, and -4 mRNA levels but had no effect on IGFBP-5 mRNA levels in theca cells. LH had no effect on levels of IGFBP-2, -3, -4, or -5 mRNA in theca cells. These results indicate that expression of IGFBP-2, -3, -4, and -5 mRNA by granulosa and theca cells are differentially regulated by estradiol, insulin and gonadotropins, therefore discretely modulating the amount of bioavailable IGFs to these cells depending upon the specific hormonal stimuli. In particular, these studies are the first in cattle to show that estradiol selectively inhibits IGFBP-2, -3, and -4 gene expression in theca cells, inhibits IGFBP-5 gene expression in large-follicle granulosa cells, and stimulates IGFBP-2 gene expression in small-follicle granulosa cells.     

Prostaglandin F2α Stimulates Endothelial Nitric Oxide Synthase Depending on the Existence of Bovine Granulosa Cells: Analysis by Co‐culture System of Endothelial Cells,Smooth Muscle Cells and Granulosa Cells

Prostaglandin F_2α (PGF_2α) induces luteolysis in the mid but not in the early luteal phase; despite this, both the early and the mid corpus luteum (CL) have PGF_2α receptor (FPr). We previously indicated that the luteal blood flow surrounding the CL drastically increases prior to a decrease of progesterone (P) in the cows, suggesting that an acute increase of luteal blood flow may be an early sign of luteolysis in response to PGF_2α and that this may be induced by a vasorelaxant nitric oxide (NO). The aim of this study was to investigate the luteal stage‐dependent and the site‐restricted effect of PGF_2α and NO on the mRNA expressions and P secretion. To mimic the local luteal region both of peripheral and central areas of the CL, we utilized co‐cultures using bovine aorta endothelial cells (EC), smooth muscle cells (SMC) and luteinizing granulosa cells (GC) or fully‐luteinized GC. PGF_2α stimulated the expression of endothelial NO synthase (eNOS) mRNA at 0.5 h in mix‐cultures of EC and SMC with fully‐luteinized GC but not with luteinizing GC. The expression of eNOS mRNA in EC was increased by PGF_2α at 1 h only when EC was cultured together with fully‐luteinized GC but not with luteinizing GC. In all co‐cultures, PGF_2α did not affect the mRNA expression of FPr. Treatment of NO donor inhibited P secretion at 0.5 h. In conclusion, the present study suggests that the coexistence of the mature luteal cells (fully‐luteinized GC) with EC/SMC may be crucial for acquiring functional NO synthesis induced by PGF_2α.     

The Adverse Effect of Phytoestrogens on the Synthesis and Secretion of Ovarian Oxytocin in Cattle

The current investigations were undertaken to study the mechanism of the adverse effect of phytoestrogens on the function of bovine granulosa (follicles >1< cm in diameter) and luteal cells from day 1–5, 6–10, 11–15, 16–19 of the oestrous cycle. The cells were incubated with genistein, daidzein or coumestrol (each at the dose of 1 × 10^?6 m ). The viability and secretion of estradiol (E2), progesterone (P4) and oxytocin (OT) were measured after 72 h of incubation. Moreover, the expression of mRNA for neurophysin‐I/OT (NP‐I/OT; precursor of OT) and peptidyl‐glycine‐α‐amidating monooxygenase (PGA, an enzyme responsible for post‐translational OT synthesis) was determined after 8 h of treatment. None of the phytoestrogens used affected the viability of cells except for coumestrol. The increased secretion of E2 and P4 was only obtained by coumestrol (p < 0.05) from granulosa cells from follicles <1 cm in diameter and decreased from luteal cells on days 11–15 of the oestrous cycle, respectively. All three phytoestrogens stimulated (p < 0.05) OT secretion from granulosa and luteal cells in all stages of the oestrous cycle and the expression of NP‐I/OT mRNA in the both types of cells. The expression of mRNA for PGA was stimulated (p < 0.05) by daidzein and coumestrol in granulosa cells, and by genistein and coumestrol in luteal cells. In conclusion, our results demonstrate that these phytoestrogens can impair the ovary function in cattle by adversely affecting the synthesis of OT in follicles and in corpus luteum. However, their influence on the ovarian steroids secretion was less evident.     

Association Between Leptin, LH and its Receptor and Luteinization and Progesterone Accumulation (P4) in Bovine Granulosa Cell In Vitro

A full understanding of the cellular events that occur during in vitro luteinization of bovine granulosa cells, stimulated by LH and by leptin, is a complex goal that has not been completely achieved. The aim of this work was to study the effects of leptin, LH and leptin + LH on progesterone accumulation (P4) and on the expression of LH receptors (LHR) in bovine granulosa cells in culture. The results confirm that this in vitro model is representative of functional and morphological luteinization/differentiation. The pattern of expression of LHR with time of incubation was an important marker of in vitro luteinization, with 50–90% of cells expressing LHR by 96 h in culture. Cytoplasmic lipidic droplets were highly abundant in granulosa cells, suggesting a sufficient source of precursors for steroid hormone synthesis: P4 accumulation ranged between 40 and 550 ng/ml. In addition, a positive correlation ( r  = 0.58, p < 0.05) between the expression of LHR and accumulation of P4 throughout the time of incubation was observed. The expression of LHR was inhibited by LH and leptin + LH treatment. In conclusion, we found an inverse modulation between the expression of LHR and the concentration of LH, and the expression of LHR could be regulated by P4 produced by the luteinized granulosa cells. These findings are contributing to elucidate further the panoply of interactions during the differentiation of granulosa cells into luteal cells in vitro .