Cárdenas H, Burke K A, Bigsby R M, Pope W F, Nephew K P
Department of Animal Sciences, The Ohio State University, Columbus, Ohio 43210, USA.
Biol Reprod. 2001 Jul;65(1):128-34. doi: 10.1095/biolreprod65.1.128.
Objectives were to sequence and examine the expression of the estrogen receptor beta (ERbeta) in the sheep ovary. The sequence of the ovine ERbeta (oERbeta) was determined using reverse-transcription polymerase chain reaction (RT-PCR) and cloning techniques. The reading frame of oERbeta contained 527 amino acids and exhibited high overall homology with cow (98%), rat (88%), and human (88%) ERbeta. In addition, an oERbeta isoform having a 139-base pair deletion (oERbeta1) was identified. The predicted amino acid sequence of this isoform is lacking the ligand-binding and carboxyl-terminal transactivation domains. The oERbeta protein and mRNA were determined in ovaries obtained from ewes on Days 0 (first day of estrus), 2, 6, and 10 of the estrous cycle and Day 30 of gestation. Immunohistochemistry showed that oERbeta protein was located in granulosa cells, the ovarian surface epithelium, endothelium, and Day 2 corpus luteum (CL). Weak immunostaining for ERbeta was detected in the theca interna. Relative steady-state amounts of oERbeta mRNA in the CL were determined using semiquantitative RT-PCR. Amounts of oERbeta mRNA were greater (P < 0.05) during CL formation (Day 2) than at later stages. The oERbeta to oERbeta1 mRNA ratio was lower (P < 0.05) on Day 2 than on Day 10 or Day 30 due to a decrease in amounts of oERbeta1. Results indicate that the oERbeta is a 527-amino acid protein expressed in specific cells of the ovary. Changes in relative amounts of full-length oERB and a deletion isoform in CL occurred during the estrous cycle, suggesting that these two types of ERbeta might regulate estrogen actions during early CL development in sheep.
目的是对绵羊卵巢中的雌激素受体β（ERβ）进行测序并检测其表达。使用逆转录聚合酶链反应（RT-PCR）和克隆技术确定了绵羊ERβ（oERβ）的序列。oERβ的阅读框包含527个氨基酸，与牛（98%）、大鼠（88%）和人类（88%）的ERβ具有高度的总体同源性。此外，还鉴定出一种具有139个碱基对缺失的oERβ异构体（oERβ1）。该异构体的预测氨基酸序列缺乏配体结合和羧基末端反式激活结构域。在发情周期第0天（发情第一天）、第2天、第6天和第10天以及妊娠第30天的母羊卵巢中测定了oERβ蛋白和mRNA。免疫组织化学显示，oERβ蛋白位于颗粒细胞、卵巢表面上皮、内皮细胞和第2天的黄体（CL）中。在内膜中检测到较弱的ERβ免疫染色。使用半定量RT-PCR测定CL中oERβ mRNA的相对稳态量。CL形成期间（第2天）的oERβ mRNA量比后期更多（P<0.05）。由于oERβ1量的减少，第2天的oERβ与oERβ1 mRNA比率低于第10天或第30天（P<0.05）。结果表明，oERβ是一种在卵巢特定细胞中表达的527个氨基酸的蛋白质。发情周期中CL中全长oERβ和缺失异构体的相对量发生变化，表明这两种类型的ERβ可能在绵羊CL早期发育过程中调节雌激素作用。
