The steroid inhibitor trilostane decreases gonadotropin-, but not prostaglandin e2-, stimulated haluronic acid expression by macaque cumulus-oocyte-complexes during culture

Abstract

To determine whether the inhibition of steroid synthesis with trilostane (TRL) affects synthesis of the cumulus expansion-associated glycosaminoglycan, haluronic acid (HA), in macaque culumus-oocyte-complexes (COCs) in the absence or presence of gonadotropins or prostaglandin E2 (PGE2). Nonhuman primate model, 3x2 factorial design, randomized, controlled study. Healthy macaque (n=5) COCs (n=3-6/group/animal) from 0.5-1.5mm small antral follicles during spontaneous menstrual cycles were cultured with or without TRL (250ng/ml) in 1) TALP media with 5% charcoal-stripped monkey serum (CON), 2) media plus gonadotropins (FSH, LH; 75 mIU/ml each), or 3) media plus PGE2 (5ng/ml) for 30 hours in 5% CO2. Following culture, COCs were fixed, immunolabeled with HA binding protein, phalloidin (actin) and Hoechst 33342 (DNA), and imaged by confocal microscopy. Non-cultured COCs were used as negative controls for HA staining. HA staining intensity for each COC was assigned a score (1 to 3) by 3 blind observers based on the percentage (%) of extracellular space occupied by HA; “1”: less than 25%, “2”: 25-75%, “3”-over 75%. Statistical significance (p≤0.05) was determined using the SAS GLM method. Non-cultured COCs exhibited no HA staining. In CON and CON+TRL groups, HA staining remained low and unchanged. Gonadotropins (p<0.05) as well as PGE2 (p<0.05) increased HA staining in cultured COCs in comparison to that of the CON. HA localized to the extracellular matrix between neighboring cumulus cells and sometimes concentrated in the outermost area of expanded COCs, resulting in a ring-like appearance surrounding the COC. However, addition of TRL reduced (p<0.01) HA staining in the FSH+LH+TRL group compared to FSH+LH alone. Interestingly, TRL did not alter HA staining (p=0.66) in the PGE2+TRL group in comparison to COCs treated with PGE2 alone. In macaque COCs, cumulus expansion and HA expression appears to be steroid-dependent during gonadotropin stimulation, but either independent of steroid hormones or downstream of the steroid hormone signaling pathway during PGE2 induction of cumulus expansion. This study supports an important role of steroid hormones in the intrafollicular environment for cumulus expansion during oocyte maturation in primates, and has implications for improving in vitro fertilization techniques and fertility preservation in humans. Ongoing studies include hormone replacement to determine which steroid hormone is required for HA synthesis.