Co-activator p120 is increased by gonadotropins in the rat ovary and enhances progesterone receptor activity

Abstract

Background: Ovarian follicular development is primarily dependent on pituitary gonadotropins. Identification of gonadotropin-inducible genes in the ovary is one of the effective approaches for the study of follicular development. In this study we identify rat homologue of p120, a nuclear transcription co-activator, as one of the FSH inducible genes in the rat granulosa cells.

Methods: A full-length cDNA encoding rat p120 was cloned, and expression of the gene in the ovary was examined by Northern blotting. Tissue localization of p120 was examined by in situ hybridization. Cellular functions of p120 were studied by co-transfection of rat p120 gene together with estrogen receptor (ER)-alpha, ER-beta, androgen receptor (AR), or progesterone receptor (PR) genes.

Results: A full-length cDNA encoding rat p120 was characterized as a protein with 957 amino acid residues. Rat p120 was expressed ubiquitously, but strongly in the ovary and the testis. Expression of p120 mRNA was also induced in vivo by PMSG or PMSG/hCG treatment. Strong expression of p120 mRNA was observed in the granulosa cells of pre-ovulatory large antral follicles. Progesterone receptor was co-localized with p120 in the large antral follicles. Co-transfection experiments revealed that rat p120 activated AR, ER-alpha, ER-beta, and PR in the presence of their respective ligands.

Conclusion: These observations suggest that rat p120 is strongly induced in the ovarian granulosa cells, and may work together with PR in the granulosa cells of ovulatory follicles to promote the ovulation process.

Figure 1

Tissue Distribution of rat p120 RT- PCR was performed using p120 specific primers. Total RNA was isolated from various tissues of adult rats. Ten micrograms of total, extracted RNA were reverse-transcribed, and a portion (1/100) was subjected to the PCR reaction for the specific amplification of p120 and GAPDH, respectively. The reaction mixtures were separated 1.5 % agarose gel and visualized with EtBr staining. The samples are brain (lane 1), pituitary (lane 2), liver (lane 3), adrenal (lane 4), uterus (lane 5), testis (lane 6), kidney (lane 7), spleen (lane 8), intestine (lane 9), stomach (lane 10), untreated ovary (lane 11), ovary treated with PMSG for 48 h (lane 12).

Figure 2

Structure of p120. (a), Schematic illustrations of the structures of rat and human p120. LXXLL motif (L, leucine; X, any amino acid). (b), A deduced amino acid sequence of rat p120. The amino acids are numbered starting from the initiation codon. The proline-rich domain between 223 and 288 amino acid residues from the N-terminus is underlined, and acidic amino acid-rich domain between 602 and 731 is shown by bold type. The bromo-domain between 809 and 894 is boxed. The putative NR box (LXXLL motif) is boxed by colored background.

Figure 3

Induction of rat p120 gene expression in the ovary. Northern blot analysis of rat p120 mRNA in cultured granulosa cells treated with FSH (a), in immature rat ovary primed with PMSG (b), and in immature rat ovary primed with PMSG followed by hCG (c). Total RNA was isolated at the indicated times after the treatments. All samples contained 10 micrograms of total RNA. The blots were hybridized with each specific probe of rat p120, ER-alpha, ER-beta, or PR, respectively. The blots were striped and rehybridized with a probe specific for GAPDH or 36B4 to normalize for equivalent loading of RNA.

Figure 4

Induction of rat p120 gene expression in the ovary. Real-time PCR was performed using each specific primer of rat p120, ER-beta, or PR, respectively. Total RNA was isolated from cultured granulosa cells treated with FSH for 48 hr (a) or with FSH for 48 h followed by hCG (b). Each value represents the mean ± SEM of 4 independent Real-time PCR experiments. Different letters for the same graph indicate statistically significant differences (P < 0.05; Duncan multiple comparison test).

Figure 5

Activation of nuclear hormone receptors with p120. CV-1 cells were transfected as described in Materials and Methods with cGREx2/pGL3-TK and hAR (hAR/pSG5), or with cGREx2/pGL3-TK and hPR (hPR/pcDNA3) in the presence or absence of human p120 (a), ERE-TATA/pGL2 and rER-alpha (rER-alpha/pSV2), or ERE-TATA/pGL2 and rER-beta (rER-beta/pSG5) in the presence or absence of rat p120 (b). Ligands for each nuclear receptor (10^-8M testosterone propionate, 10^-8M progesterone, 10^-8M estradiol, and 10^-7M estradiol) were also added to the cultures. Each value represents the mean ± SEM of 4 independent transfection experiments. *P < 0.01 by Student's t test.

Figure 6

In situ hybridization of p120 mRNA in the immature rat ovary. Ovaries from 21-day-old immature rats were dissected, sectioned and hybridized with ³⁵S-labeled cRNA probes. All sections were shown by both dark-field illumination and bright-field photomicrographs of hematoxylin-staining. (A) Sections from untreated ovaries hybridized with p120 (a, b) or ER-beta (c, d) antisense probes, (B) PMSG treatment for 6 h hybridized with p120 (e, f) or ER-beta (h, i) antisense probes. Sections were also hybridized with sense probes (g, j), (C) PMSG treatment for 24 h hybridized with p120 (k, l) or ER-beta (m, n), (D) hCG treatment for 4 h after PMSG treatment hybridized with p120 (o, p) or PR (r, s) or sense probes (q, t). Original magnification was × 100. Scale bars, 0.1 mm.