Response gene to complement 32 expression is induced by the luteinizing hormone (LH) surge and regulated by LH-induced mediators in the rodent ovary

Abstract

Response gene to complement 32 (Rgc32) has recently been suggested to be expressed in the ovary and regulated by RUNX1, a transcription factor in periovulatory follicles. In the present study, we determined the expression profile of the Rgc32 gene in the rodent ovary throughout the reproductive cycle and the regulatory mechanism(s) involved in Rgc32 expression during the periovulatory period. Northern blot and in situ hybridization analyses revealed the up-regulation of Rgc32 expression in periovulatory follicles. Rgc32 mRNA was also localized to newly forming corpora lutea (CL) and CL from previous estrous cycles. Further studies using hormonally induced luteal and luteolysis models revealed a transient increase in levels of Rgc32 mRNA at the time of functional regression of the CL. Next, the regulation of Rgc32 expression was investigated in vitro using rat preovulatory granulosa cells. The effect of human chorionic gonadotropin on Rgc32 expression was mimicked by forskolin, but not phorbol 12-myristate 13-acetate, and was mediated by the activation of progesterone receptors and the epidermal growth factor-signaling pathway. The mechanism by which RUNX1 regulates Rgc32 expression was investigated using chromatin immunoprecipitation and Rgc32 promoter-luciferase reporter assays. Data from these assays revealed direct binding of RUNX1 in the Rgc32 promoter region in vivo as well as the involvement of RUNX binding sites in the transactivation of the Rgc32 promoter in vitro. In summary, the present study demonstrated the spatial/temporal-specific expression of Rgc32 in the ovary, and provided evidence of LH-initiated and RUNX1-mediated expression of Rgc32 gene in luteinizing granulosa cells.

Figure 1

Profile of Rgc32 expression in gonadotropin-stimulated rodent ovaries during the periovulatory and luteal period. Induction of Rgc32 expression during the hCG-induced periovulatory period in rat ovaries (A), mouse ovaries (B), rat COCs (C), and during the hCG-induced luteal period in rat ovaries (D). A, B, and D, Autoradiograph of a representative Northern blot analysis shows Rgc32 mRNA and ribosomal protein L32 mRNA in gonadotropin-stimulated rodent ovaries collected before or at selected hours and days after hCG injection. Relative levels of Rgc32 mRNA were normalized to the L32 band in each sample (mean ± sem; n = 3–4 independent animals). C, The levels of Rgc32 mRNA in COCs were measured using a real-time PCR as described in Materials and Methods. Bars with no common superscripts are significantly different (P < 0.05).

Figure 2

Serum progesterone (A) and Rgc32 mRNA levels (B) in the PRL ablation-replacement model of induced luteal regression. Group treatments are indicated across the abscissa; ovaries collected on PSP4 were fully functional, ovaries collected on PSP7 were functionally normal or functionally regressed (without or with bromocriptine treatment, respectively), and ovaries collected on PSP10 were functionally regressed (bromocriptine without PRL replacement) or functionally and structurally regressed (bromocriptine with PRL replacement). The levels of progesterone concentrations were measured in serum obtained from each animal (mean ± sem; n = 5 animals per time point). Relative levels of mRNA for Rgc32 were normalized to the L32 band in each sample (mean ± sem; n = 5 independent animals). Bars with no common superscripts are significantly different (P < 0.05).

Figure 3

In situ hybridization analysis of Rgc32 mRNA during the periovulatory period in ovaries obtained from naturally cycling rats. Representative bright-field (A, C, E, and G) and corresponding dark-field (B, D, F, and H) photomicrographs are depicted. Ovaries were collected at 0 (at the peak of the LH surge), 4, and 12 h after the LH surge. Panels G and H were hybridized with sense probes (negative control). Arrows in D indicate periovulatory follicles (F) expressing Rgc32 mRNA. Arrowheads in F indicate newly forming CL (nCL). Asterisks in B indicate CL generated during previous estrous cycles. Original magnification of all slides is ×40. pCL, Corpus luteum from previous cycles; PF, preovulatory follicle.

Figure 4

Stimulation of Rgc32 expression by hCG in rat granulosa cells in vitro. Autoradiograph of a representative Northern blot analysis shows Rgc32 mRNA and ribosomal protein L32 mRNA in granulosa cells obtained from rat preovulatory ovaries (48 h after PMSG) and cultured in medium alone (control), or with hCG (1 IU/ml) for 0, 3, 6, 12, or 24 h. Relative levels of Rgc32 mRNA were normalized to the L32 band in each sample (mean ± sem; n = 4 independent culture experiments). Bars with no common superscripts are significantly different (P < 0.05).

Figure 5

Regulation of Rgc32 expression by activators of intracellular signaling pathways in granulosa cells in vitro. Autoradiograph of a representative Northern blot analysis shows Rgc32 mRNA and ribosomal protein L32 in granulosa cells from rat preovulatory ovaries (48 h after PMSG) cultured in medium alone (Cont), or with hCG (1 IU/ml), FSK (10 μm), PMA (20 nm), or FSK plus PMA for 24 h. Relative levels of Rgc32 mRNA were normalized to the L32 band in each sample (mean ± sem; n = 4 independent culture experiments). Bars with no common superscripts are significantly different (P < 0.05).

Figure 6

Hormonal regulation of hCG-induced Rgc32 expression in cultured rat granulosa cells. A, Inhibition of Rgc32 expression by AG (EGF receptor tyrosine kinase-selective inhibitor, 1 μm). The cells were cultured in medium alone (Cont), hCG (1 IU/ml), AG, AREG (EGF-related peptide, 100 nm/ml), hCG plus AG, or AREG plus AG for 24 h. B, Inhibition of Rgc32 expression by RU486. Granulosa cells were cultured in medium alone (Cont), hCG (1 IU/ml), RU486 (PGR antagonist, 10 μm), or hCG plus RU486 for 24 h. Autoradiograph of a representative Northern blot analysis shows Rgc32 mRNA and ribosomal protein L32 mRNA in granulosa cells from rat preovulatory ovaries (48 h after PMSG). Relative levels of Rgc32 mRNA were normalized to the L32 band in each sample (mean ± sem; n = 6 independent culture experiments). Bars with no common superscripts are significantly different (P < 0.05).

Figure 7

Nucleotide sequences of rat Rgc32 promoter regions. The rat Rgc32 promoter sequence was analyzed using a genomic library. Nucleotide sequences are numbered from TSS at +1. Putative transcription factor binding sites (boxed sequence) are predicted by TFSEARCH. TATA box is underlined. Translation initiation site is the black shaded box (ATG).

Figure 8

Evidence of RUNX1 binding in the Rgc32 promoter region in vivo. A, Schematic map of the rat Rgc32 promoter region. The locations of primers used to amplify DNA fragments spanning RUNX transcription binding sites were designated in the rat Rgc32 promoter region. B, ChIP detection of RUNX1 transcription factor binding to the rat Rgc32 promoter region in luteinizing granulosa cells. ChIP assays were performed using DNA extracted from granulosa cells obtained at 10 h after hCG injection. One tenth of the chromatin was kept as input DNA control (Input) before immunoprecipitation. Immunoprecipitations were performed with RUNX1 antibody, or normal rabbit IgG served as a negative control. DNAs were analyzed by PCR using primers indicated previously. RUNX⁻¹⁹⁵ (203 bp) and RUNX⁻⁵⁶⁰ (304 bp) DNA fragments containing RUNX1 transcription factor binding sites were enriched in chromatin samples immunoprecipitated with RUNX1 antibody as well as input DNA. Experiments were repeated at least five times, each with different granulosa cell samples.

Figure 9

Regulation of the Rgc32 promoter in luteinizing granulosa cells. A, Stimulation of Rgc32 promoter activity by FSK and FSK plus PMA. Rat granulosa cells isolated from gonadotropin-primed immature rats (48 h after PMSG) were transiently transfected with empty, −1226/+91 bp, or −780/+91 bp Rgc32-luciferase reporter constructs, stimulated with FSK, PMA, or FSK plus PMA and cultured for 12 h. B, RUNX binding sites contribute to the transactivation of the Rgc32 promoter. The granulosa cells were transiently transfected with the Rgc32 promoter (−780/+91 bp) constructs containing deletions in RUNX binding sites (Δ−514/−565 bp and Δ−514/−565 bp + Δ−183/−200 bp) or mutation in the RUNX binding site (ψ−195/−200 bp). Black boxes represent the consensus RUNX binding site. Shadowed boxes represent the putative RUNX binding site (89% match). Firefly luciferase activities were normalized by Renilla luciferase activities, and each experiment was performed in triplicate at least three times. A significant effect of interaction between treatments and constructs was observed. Bars with no common superscripts in each panel and among vector constructs are significantly different (P < 0.05).