Homologous and heterologous regulation of gonadotropin-releasing hormone receptor gene expression in preovulatory rat granulosa cells.

ABSTRACT

The reverse transcription-polymerase chain reaction was used to evaluate the influence of gonadotropins and GnRH on GnRH receptor gene expression in cultured preovulatory rat granulosa cells. Cells were obtained from immature female rats 48 h after priming with 10 IU PMSG, sc, and cultured in medium containing LH, FSH, or GnRH after a 24-h preincubation period. After culture, cells were lysed, total RNA was extracted, and culture medium was assayed for its content of progesterone, and estradiol by RIA. Subsequent to reverse transcription, complementary DNA was subjected to polymerase chain reaction, after which the products formed were transferred to nylon filters and hybridized with a 416-basepair internal rat GnRH receptor complementary DNA probe. As an amplification control, Southern hybridization of glyceraldehyde-3-phosphate dehydrogenase messenger RNA (mRNA) was performed. Treatment with LH increased both progesterone and estradiol output, whereas GnRH receptor mRNA levels were markedly suppressed in a dose-related fashion, with maximal inhibition seen at 100-1000 ng (P < 0.05). In contrast, FSH in concentrations between 1-1000 ng was without effect on GnRH receptor gene expression. Time-course analysis revealed that GnRH receptor gene expression was not affected by LH (1000 ng) until 12 h, when transcript levels fell to 24% of those seen at 0 h (P < 0.05). This pronounced decrease in GnRH receptor mRNA abundance is, however, transient, because after 48 h, levels returned to those seen before LH treatment. As GnRH has been postulated to directly influence its own receptor synthesis in the pituitary, the effect of GnRH treatment of granulosa cells during a 24-h culture period was also evaluated. In GnRH-treated cells, a dose-dependent increase in GnRH receptor mRNA levels was seen, with maximal effects (2.4-fold; P < 0.05) at 10(-6) M. The demonstration of agonist-specific up- and down-regulation of GnRH receptor gene expression in preovulatory granulosa cells adds further support to the extrapituitary actions of GnRH as an important autocrine/paracrine factor involved in the regulatory events during the periovulatory period.