摘要
采用免疫组化技术检测催乳素受体蛋白(PRLR)在绵羊卵巢中的表达定位,建立绵羊卵泡颗粒细胞体外培养体系,将颗粒细胞分成4个处理组,添加不同浓度催乳素(PRL):对照组(不添加PRL)、试验Ⅰ组(0.05 mg/L PRL)、试验Ⅱ组(0.50 mg/L PRL)、试验Ⅲ组(5.00 mg/L PRL),用ELISA法检测各组颗粒细胞雌激素(E2)和孕酮(P4)的分泌水平,荧光定量RT-PCR检测FSHR、LHR、PRLR、StAR、CYP11和CYP19等基因相对表达量。结果表明:在绵羊卵巢中,PRLR蛋白主要在卵泡的颗粒细胞和黄体细胞中表达;添加PRL抑制了E2的分泌、促进了P4分泌,其中Ⅱ组和Ⅲ组E2分泌水平显著低于对照组和Ⅰ组(P<0.05);P4随着PRL添加浓度的升高而升高,各试验组P4分泌水平均显著高于对照组(P<0.05)。各试验组颗粒细胞FSHR、LHR、PRLR、CYP11、StAR的mRNA表达量均极显著高于对照组(P<0.01),而试验组CYP19的表达量极显著低于对照组(P<0.01)。综上,PRL通过上调FSHR、LHR、PRLR、CYP11和StAR mRNA表达,下调CYP19 mRNA的表达,抑制绵羊卵巢颗粒细胞E2的分泌、促进P4的分泌。通过研究添加PRL对绵羊卵巢颗粒细胞E2和P4分泌及相关基因表达的影响,为探讨PRL对卵泡发育的调控机制奠定基础。
The aim of this study was to investigate the effects of treatment with prolactin(PRL)on the secretion of estrogen(E2),progesterone(P4)and expressions of related genes in sheep granulosa cells(GCs).The role of PRL on follicular development laid the foundation for research.Immunohistochemistry was used to detect localization of PRLR expression in sheep ovary.Cell culture and microscopic observation techniques were used to establish an in vitro culture system of sheep follicular GCs.GCs were divided into four treatment groups according to the concentration of PRL added:control group(no PRL added),test group Ⅰ(0.05 mg/L PRL),test group Ⅱ(0.50 mg/L PRL),test group Ⅲ(5.00 mg/L PRL),the concentrations of E2 and P4 were tested by ELISA.The mRNA levels of related-genes(FSHR,LHR,PRLR,StAR,CYP11 and CYP19)were analyzed by quantitative real-time PCR(qRT-PCR).In sheep ovary,PRLR protein is mainly expressed in follicular granular cell layers and luteal cells.The results showed that the addition of PRL inhibited the secretion of E2.The secretion of E2 in the test group Ⅱ and the test group Ⅲ was significantly lower than that in the control group and the experimental groupⅠ(P<0.05).P4 increased with the increase of PRL concentration,the secretion of P4 in each experimental group was significantly higher than that in the control group(P<0.05).The mRNA expression levels of FSHR,LHR,PRLR,CYP11 and StAR in GCs of the experimental groups were significantly higher than those in the control group(P<0.01).The expression of CYP19 in each experimental group was significantly decreased compared with the control group(P<0.01),and gradually decreased with the increase of PRL concentration.In summary,PRL plays a key role in regulating the function of GCs in sheep,which can affect the secretion of steroid hormones and follicular development and ovulationrelated gene expression.
作者
何海迎
王泳
段春辉
杨若晨
王康
张英杰
刘月琴
HE Hai-ying;WANG Yong;DU AN Chun-hui;YANG Ruo-chen;WANG Kang;ZHANG Ying-jie;LIU Yue-qin(College of Animal Science and Technology,Hebei Agricultural University,Baoding,Hebei 071000,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2020年第11期2226-2233,共8页
Chinese Journal of Veterinary Science
基金
国家绒毛用羊产业技术体系资助项目(CARS39-23)
河北省高等学校青年拔尖人才资助项目(8042018/1081034)
河北省自然科学基金资助项目(C2019204357)
河北省重点研发计划资助项目(18226602D)。
关键词
催乳素
雌激素
孕酮
绵羊
颗粒细胞
基因表达
prolactin
estrogen
progesterone
sheep
granulosa cells
gene expression
