FSH and LH induce progesterone production and progesterone receptor synthesis in cumulus cells: a requirement for meiotic resumption in porcine oocytes

Masayuki Shimada 0 Takato Terada 0 0 Faculty of Applied Biological Science, Hiroshima University , Higashi-Hiroshima, Hiroshima, 739-8528, Japan The aim of this study was to investigate the role of progesterone in the meiotic resumption of porcine oocytes. Progesterone production and progesterone receptor (PR) immunoreactivity in cumulus cells were not detected in porcine cumulus-oocyte complexes (COC) when observations were made either just after collection from the follicles or after 28 h cultivation without LH and FSH. However, the addition of LH and FSH induced PR expression in cumulus cells, concomitant with increased progesterone production. To assess the role of progesterone in the COC, an inhibitor of progesterone production, aminoglutethimide (AGT), was administered. The addition of AGT to the medium with LH and FSH significantly suppressed progesterone production in a dose-dependent fashion. When COC were cultured with LH, FSH and 0.5 10-3 mol/l AGT, almost complete inhibition of progesterone production and of germinal vesicle breakdown (GVBD) was seen. However, this inhibitory effect on GVBD was overcome by additional progesterone. Moreover, 0.5 10-3 mol/l AGT also suppressed the reduction in connexin43, a gap junctional protein, in cumulus cells after 28 h cultivation, and increased the level of cyclic AMP in oocytes. These results support the hypothesis that the binding of progesterone, which was secreted by LH- and FSH-stimulated cumulus cells, to its newly synthesized receptor induces GVBD in porcine oocytes, possibly through a reduction of connexin43 in cumulus cells. - During in-vitro meiotic maturation of cumulusoocyte complexes (COC), progesterone is produced by the cumulus cells, and the level of progesterone is increased by stimulation with LH, FSH or forskolin in humans (Chian et al., 1999) and pigs (Racowsky, 1985). It has been reported that a high concentration of progesterone in follicle fluid induces meiotic maturation of rhesus monkey oocytes (Morgan et al., 1990). In porcine and bovine oocytes, the addition of progesterone into a maturation medium stimulates meiotic re-initiation, regardless of the presence or absence of gonadal hormones (Sirotkin, 1992; Eroglu, 1993). However, other reports have shown no statistically significant stimulation of meiotic resumption by progesterone in the case of porcine oocytes (McGaughey, 1977). Moreover, progesterone has been reported to suppress spontaneous meiotic maturation in mouse oocytes (Barrett and Powers, 1993). Thus, it is unclear whether or not progesterone is required for in-vitro meiotic resumption in mammalian oocytes. Many of the biological activities of progesterone are mediated by an intracellular receptor, a hormonally regulated DNA-binding protein that belongs to a superfamily of ligand-activated transcription factors (Beato et al., 1989; OMalley, 1990). The expression of progesterone receptor (PR) mRNA can be induced by either LH or FSH through a cAMP-mediated pathway in rat pre-ovulatory follicles (Natraj and Richards, 1993; Park-Sarge and Mayo, 1994). In the human ovary, PR mRNA expression in antral follicles is significantly higher than that in pre-antral follicles (Revelli et al., 1996). Recently, it was reported (Slomczynska et al., 2000) that PR are not detected in porcine granulosa cells of small antral follicles, however, treatment of the small follicles with LH or FSH can induce the expression of the PR protein. Although it is known that in-vivo expression of PR protein in follicle cells is regulated by both LH and FSH, there is no description of the expression of the PR protein in cumulus cells surrounding oocytes during in-vitro meiotic maturation of COC in primates and pig. Some of the meiosis inhibitory factors transported into oocytes via numerous gap junctions are synthesized by the cumulus and granulosa cells that are attached to oocytes (Downs and Eppig, 1984; Downs et al., 1986; Isobe et al., 1996). A loss of the cumulus-to-cumulus cell gap junction preceding the induction of meiotic resumption has been reported in rat COC (Larsen et al., 1986, 1987). In pigs, a significantly positive correlation has been observed between the proportion of oocytes undergoing germinal vesicle breakdown (GVBD) and that of COC exhibiting a loss of the gap junctional communication between the cumulus cells of outer layers (Isobe et al., 1998; Isobe and Terada, 2001). In our previous study (Shimada et al., 2001), a disruption of gap junctional communication in the outer layers of cumulus cells resulted in the depletion of connexin43 expression in those layers. Furthermore, connexin43 expression in the rat endometrium is regulated by progesterone during early pregnancy (Grummer et al., 1994). It has also been shown that progesterone down-regulates the expression of the connexin43 gene in human myometrial cells (Zhao et al., 1996). These studies support the idea that progesterone induces a reduction in connexin43 expression via the PR-mediated transcription pathway in the outer layers of cumulus cells, resulting in meiotic resumption in porcine oocytes. In order to investigate the role of progesterone and the PR in cumulus cells during meiotic resumption in porcine oocytes, the present study examined the effects of gonadotrophins and/or aminoglutethimide (AGT) on the production of progesterone, the PR, and connexin43 in cumulus cells, as well as their effects on cAMP levels and the induction of meiotic resumption in oocytes. Materials and methods Isolation and culture of porcine COC Porcine ovaries were collected from 57 month old pre-pubertal gilts at a local slaughterhouse and were transported within 1.5 h to the laboratory in 0.85% (w/v) NaCl containing 0.1 mg/ml kanamycin (Meiji Seika, Tokyo, Japan) at ~30C. The surfaces of intact healthy antral follicles measuring 38 mm in diameter were cut with a razor blade and oocytes were collected with a surgical blade used to scrape the inner surface of the follicle walls. The oocytes collected were placed in pre-warmed phosphate-buffered saline (PBS) (pH 7.4) supplemented with 0.1% (w/v) polyvinylpyrrolidone (Sigma Chemical Co., St Louis, MO, USA). Oocytes with evenly granulated cytoplasm and at least four layers of unexpanded cumulus oophorus cells (COC) were selected under a stereomicroscope and were washed three times with maturation medium. Twenty COC were cultured for 28 h in 500 l of maturation medium, i.e. basic medium supplemented with 0.6 g/ml porcine FSH (Sigma) and 1.3 g/ml equine LH (Sigma), at 39C in a humidified atmosphere of 5% CO2 in air. The basic medium was modified NCSU37 (Petters and Reed, 1991) containing 10% (v/v) fetal calf serum (Gibco BRL, Grand Island, NY, USA), 7 mmol/l taurine (Sigma), 2% (v/v) essential amino acids (Gibco), and 1% (v/v) non-essential amino acids (Gibco). Treatment of COC with progesterone Some COC were cultured for 28 h in the basic medium supplemented with 0, 10, 100 and 1000 ng/ml proges (...truncated)