Kawate N, Okuda K
Laboratory of Animal Reproduction, Faculty of Agriculture, Yamaguchi University, Japan.
Mol Reprod Dev. 1998 Sep;51(1):66-75. doi: 10.1002/(SICI)1098-2795(199809)51:1<66::AID-MRD8>3.0.CO;2-U.
We characterized splice variants for LH receptor mRNA in the bovine corpus luteum and examined the levels of expression of all the splice variants during the development of corpus luteum. Total RNA was extracted from bovine corpora lutea, and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to amplify a part of the extracellular and transmembrane domains. As a result, four distinct bands were observed on analyses with polyacrylamide gel electrophoresis, and sequencing of the products revealed that the largest form (519 bp) was the full-length A form and that the three shorter forms (438, 253, and 172 bp) were its splicing variants. Splicing patterns of the 438-, 253-, and 172-bp forms are identical to the F, B, and G forms, respectively, found in the ovine ovary. In order to examine the level of expression of all these splice variants simultaneously, semiquantitative RT-PCR was set up and performed. The relative intensities for all four forms of LH receptor mRNA significantly increased (P < 0.05) from Stage I (Days 1-4; Day 1 = day of ovulation) to IIS (Days 5-10, CL weight < 4 g), and from Stage IIS to IIL (Days 5-10, CL weight > or = 4 g). The intensities of all forms for LH receptor mRNA decreased slightly, but not significantly, from Stage IIL to III (Days 11-17). The ratios of the levels for all the forms did not change significantly during the development of the corpus luteum. Determination of the steady-state levels of LH receptor mRNA by Northern blotting showed similar changes during the development of the corpus luteum to changes of the splice variants examined by the semiquantitative RT-PCR. These results suggest that at least three splicing variants of LH receptor mRNA (i.e., F, B, and G forms) exist in the bovine corpus luteum and that the levels of all the forms of LH receptor mRNA increased in a coordinated manner during the development of bovine corpus luteum.
我们对牛黄体中促黄体生成素（LH）受体mRNA的剪接变体进行了表征，并检测了黄体发育过程中所有剪接变体的表达水平。从牛黄体中提取总RNA，进行逆转录聚合酶链反应（RT-PCR）以扩增细胞外和跨膜结构域的一部分。结果，聚丙烯酰胺凝胶电泳分析观察到四条不同的条带，产物测序表明最大的形式（519 bp）是全长A形式，而三种较短的形式（438、253和172 bp）是其剪接变体。438 bp、253 bp和172 bp形式的剪接模式分别与在绵羊卵巢中发现的F、B和G形式相同。为了同时检测所有这些剪接变体的表达水平，建立并进行了半定量RT-PCR。从I期（第1 - 4天；第1天 = 排卵日）到II期S（第5 - 10天，黄体重量 < 4 g），以及从II期S到II期L（第5 - 10天，黄体重量≥4 g），LH受体mRNA所有四种形式的相对强度均显著增加（P < 0.05）。从II期L到III期（第11 - 17天），LH受体mRNA所有形式的强度略有下降，但不显著。在黄体发育过程中，所有形式的水平比值没有显著变化。通过Northern印迹法测定LH受体mRNA的稳态水平，结果显示在黄体发育过程中的变化与通过半定量RT-PCR检测的剪接变体的变化相似。这些结果表明，牛黄体中至少存在三种LH受体mRNA的剪接变体（即F、B和G形式），并且在牛黄体发育过程中，LH受体mRNA所有形式的水平以协调的方式增加。
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