McDonald Emily A, Smith Jacqueline E, Cederberg Rebecca A, White Brett R
Laboratory of Reproductive Biology, Department of Animal Science, Institute of Agriculture and Natural Resources, University of Nebraska-Lincoln, Lincoln, NE, USA.
Present address: Center for International Health Research, Rhode Island Hospital, Providence, RI, USA.
Reprod Biol Endocrinol. 2016 Jun 29;14(1):36. doi: 10.1186/s12958-016-0170-0.
Binding of gonadotropin-releasing hormone (GnRH) to its receptor (GnRHR) on gonadotropes within the anterior pituitary gland is essential to reproduction. In pigs, the GnRHR gene is also located near a genetic marker for ovulation rate, a primary determinant of prolificacy. We hypothesized that pituitary expression of the GnRHR gene is alternatively regulated in genetic strains with elevated ovulation rates (Chinese Meishan and Nebraska Index) vs. standard white crossbred swine (Control).
Luciferase reporter vectors containing 5118 bp of GnRHR gene promoter from either the Control, Index or Meishan swine lines were generated. Transient transfection of line-specific, full length, deletion and mutation constructs into gonadotrope-derived αT3-1 cells were performed to compare promoter activity and identify regions necessary for divergent regulation of the porcine GnRHR gene. Additionally, transcription factors that bind the GnRHR promoter from each line were identified with electrophoretic mobility shift assays (EMSA).
Dramatic differences in luciferase activity among Control, Index and Meishan promoters (19-, 27- and 49-fold over promoterless control, respectively; P < 0.05) were established. A single bp substitution (-1690) within a previously identified upstream enhancer (-1779/-1667) bound GATA-4 in the Meishan promoter and the p52/p65 subunits of nuclear factor (NF)-κB in the homologous Control/Index promoters. Transient transfection of vectors containing block replacement mutations of either the GATA-4 or NF-κB binding sites within the context of their native promoters resulted in a 50 and 60 % reduction of luciferase activity, respectively (P < 0.05). Furthermore, two single-bp substitutions in the Meishan compared to Control/Index promoters resulted in binding of the p52 and p65 subunits of NF-κB and a specificity protein 1 (SP1)-like factor (-1235) as well as GATA-4 (-845). Vectors containing the full-length Meishan promoter harboring individual mutations spanning these regions reduced luciferase activity by 25 and 20 %, respectively, compared to native sequence (P < 0.05).
Elevated activity of the Meishan GnRHR gene promoter over Control/Index promoters in αT3-1 cells is partially due to three single nucleotide polymorphisms resulting in the unique binding of GATA-4 (-1690), the p52/p65 subunits of NF-kB in combination with a SP1-like factor (-1235), and GATA-4 (-845).
促性腺激素释放激素（GnRH）与其在前脑垂体促性腺细胞上的受体（GnRHR）结合对繁殖至关重要。在猪中，GnRHR基因也位于排卵率的遗传标记附近，排卵率是繁殖力的主要决定因素。我们假设，与标准白色杂交猪（对照）相比，排卵率升高的遗传品系（中国梅山猪和内布拉斯加指数猪）中GnRHR基因的垂体表达受到不同调控。
构建了含有来自对照、指数或梅山猪品系的5118 bp GnRHR基因启动子的荧光素酶报告载体。将品系特异性、全长、缺失和突变构建体瞬时转染到促性腺细胞来源的αT3-1细胞中，以比较启动子活性并确定猪GnRHR基因差异调控所需的区域。此外，通过电泳迁移率变动分析（EMSA）鉴定了与每个品系的GnRHR启动子结合的转录因子。
确定了对照、指数和梅山启动子之间荧光素酶活性的显著差异（分别比无启动子对照高19倍、27倍和49倍；P<0.05）。在先前鉴定的上游增强子（-1779/-1667）内的一个单碱基替换（-1690）在梅山启动子中与GATA-4结合，在同源的对照/指数启动子中与核因子（NF）-κB的p52/p65亚基结合。在其天然启动子背景下瞬时转染含有GATA-4或NF-κB结合位点的阻断替换突变的载体，分别导致荧光素酶活性降低50%和60%（P<0.05）。此外，与对照/指数启动子相比，梅山启动子中的两个单碱基替换导致NF-κB的p52和p65亚基以及一个特异性蛋白因子1（SP-1）样因子（-1235）以及GATA-4（-845）结合。与天然序列相比，含有跨越这些区域的单个突变的全长梅山启动子的载体分别使荧光素酶活性降低25%和20%（P<0.05）。
在αT3-1细胞中，梅山GnRHR基因启动子的活性高于对照/指数启动子，部分原因是三个单核苷酸多态性导致GATA-4（-1690）、NF-κB的p52/p65亚基与SP-1样因子（-1235）以及GATA-4（-845）的独特结合。
