Cumulus-oocyte expansion in rhesus macaques: A critical role for prostaglandin E2 (PGE2) and the PGE2 receptor subtype-2 (PTGER2)

PELUFFO, MC; MURPHY, MJ; ZELINSKI, MB; LINDENTHAL, B; STOUFFER, RL; HENNEBOLD, JD

Portland, OR

Congreso; 44th SSR Annual Meeting; 2011

Society for the Study of Reproduction (SSR)

Ovulation is a complex process wherein a fully-developed follicle ruptures in response to the actions of the midcycle gonadotropin surge, releasing the cumulus-oocyte complex (COC) for passage into the reproductive tract and possible fertilization of the oocyte. Prior to and following ovulation, the cumulus cells that surround the oocyte undergo a transformation whereby they lose their cell-to-cell contacts and synthesize various extracellular matrix components including hyaluronic acid (HA), which in turn results in an “expanded” cumulus cell layer (termed cumulus-oocyte expansion, or C-OE). Recent data from nonprimate species indicate that C-OE is critical for allowing fertilization and involves prostaglandin E2 (PGE2) synthesis and signaling, while the role of PGE2 and gonadotropins in primate C-OE is unknown. Thus, the aim of this study was to determine the involvement of PGE2 signaling in rhesus macaque C-OE in vitro, as well as to compare its effectiveness with that of gonadotropins. COCs were obtained from adult female monkeys that underwent controlled ovarian stimulation cycles without an ovulatory hCG stimulus. COCs (n=3-6/per treatment; each experiment repeated 3-4 times) were cultured in TALP media containing (a) monkey serum (MS) alone (negative control); (b) MS plus FSH and LH (100ng/ml, each); or (c) MS plus PGE2 (500ng/ml) and assessed for expansion at 0, 24 and 48 hr post-treatment by microscopy. MS alone typically resulted in few expanded COCs, while equivocal C-OE was observed in the gonadotropin-treated group as determined by an increase in area. In contrast, PGE2 treatment of macaque COCs resulted in a consistent expansion that appeared similar to what occurs in vivo. To serve as a molecular marker of C-OE, we developed a fluorescence-based technique using HA binding protein to assess the presence of HA in the extracellular matrix. COCs were fixed in 4% paraformaldehyde after 30 hr of culture under the same conditions mentioned above along with two additional groups that included MS plus each gonadotropin alone (FSH or LH, 100 ng/ml). In the MS control group, HA staining was minimal. Gonadotropins (FSH, LH and FSH plus LH) did not lead to uniform HA expression throughout the cumulus mass, wherein staining was observed primarily in the periphery of the COC. In contrast, PGE2 consistently induced HA levels throughout the whole complex. To determine if PGE2 signaling in primate COCs is mediated via the PGE2 receptor subtype 2 (PTGER2), as reported in mice, a specific antagonist (ZK888, 15 µM) was added to the culture media in the presence of PGE2. Levels of cAMP in the culture media were measured after 30 hr of culture to further validate inhibition of the receptor signaling, as PTGER2 binding leads to cAMP synthesis. ZK888 treatment of macaque COCs in vitro blocked PGE2 dependent-synthesis of HA and cAMP (control and ZK888 + PGE2, undetectable cAMP; PGE2 alone, 4.1 ± 1.4 pg/ml). These data demonstrate, for the first time in primates, that gonadotropins minimally impact C-OE; whereas PGE2 consistently induces C-OE and stimulates the secretion of HA in cumulus cells through PTGER2. These findings suggest a critical role for PGE2 in the primate C-OE process. Support: RR000163, U54 HD55744 and a Lalor Foundation Postdoctoral Fellowship (MCP).