P-651 Oocyte secreted factors supress the expression of prostaglandin synthase-2 (PTGS2), a key maturation promoter in cumulus cells: the oocyte is not in a hurry

Abstract

Abstract Study question Does the oocyte control the expression of genes regulating the ovulatory cascade and the formation of the extracellular matrix in cumulus cells? Summary answer Oocyte secreted factors (OSFs) suppress the expression of prostaglandin synthase-2 (PTGS2) in cumulus cells, a key mediator of the maturation cascade. What is known already Oocyte quality depends on cytoplasmic and nuclear maturation synchrony. OSFs modulate maturation dynamics by inhibiting the expression of AREG (amphiregulin) and EREG (epiregulin), key mediators of the ovulatory cascade. AREG and EREG stimulate PTGS2 expression in cumulus cells, which catalyses the synthesis of prostaglandins, that amplify and translate AREG/EREG signals leading to meiotic completion and expression of genes required for cumulus expansion, among which HAS2 (hyaluronan synthase-2), essential for the synthesis of hyaluronan, the backbone of cumulus extracellular matrix, as well as PTX3 (pentraxin 3) and TNFAIP6 (tumor necrosis factor alpha-induced protein 6), genes encoding for matrix structural proteins. Study design, size, duration Relative mRNA levels of PTGS2, HAS2, PTX3 and TNFAIP6 were compared in cumulus cells from 3 treatment-groups: 1-COC: Intact cumulus-oocyte complexes subjected to in vitro maturation (IVM); 2-OOX: oocytectomized COC subjected to IVM; 3-OOX+DO: OOX subjected to IVM with the addition of denuded oocytes (1 DO/µL). Four experimental replicates including all three treatment-groups were performed. The inclusion of OOX+DO aimed to assess whether the effects of the oocyte on gene expression are mediated by OSFs. Participants/materials, setting, methods COC were aspirated from 3-8mm follicles of bovine ovaries. Oocytectomy was achieved by ooplasm aspiration with a micromanipulator. Pools of 20 COCs/OOX/OOX+DO were cultured for 22 hours in TCM199 with 100ng/mL AREG and 0.4% BSA. Subsequently, cumulus cells were separated, total RNA extracted, and mRNA abundance assessed by real-time RT-PCR with oligo-dT primers and two internal controls for relative quantification. Treatment effects were tested by ANOVA and groups were compared with the Tukey test. Main results and the role of chance Oocyctectomy drastically increased PTGS2 mRNA abundance, which was reversed by the addition of denuded oocytes (DO) to culture. Relative PTGS2 mRNA levels (mean ± EPM) were 1.02 ± 0.12, 10.40 ± 1.65 and 0.88 ± 0.08 for COC, OOX and OOX+DO, respectively (P = 0.02). Consistently, although in a lower magnitude, oocytectomy increased mRNA levels of PTX3(1.00 ± 0.04, 2.50 ± 0.18 and 0.75 ± 0.15 for COC, OOX and OOX+DO, respectively. P < 0.001) and TNFAIP6 (1.00 ± 0.07, 2.04 ± 0.17 and 0.67 ± 0.04 for COC, OOX and OOX+DO, respectively. P < 0.001), an effect again reversed by the presence of DO in culture. In contrast, oocytectomy decreased the expression of HAS2, which was neutralised in the presence of DO (1.01 ± 0.09, 0.69 ± 0.02 and 1.11 ± 0.08 for COC, OOX and OOX+DO, respectively. P = 0.005). Our data indicate that the oocyte modulates its own maturation pace by inhibiting the expression of PTGS2, which is accompanied by similar negative impacts on genes that strongly respond to prostaglandin signalling such as PTX3 and TNFAIP6. In parallel, our data suggest that OSFs exert a potent stimulatory effect on HAS2 transcription, capable to overcome their concomitant inhibitory influence via supressed AREG/EREG and PTGS2 expression. Limitations, reasons for caution Our study utilised animal and culture models that may not exactly represent human COC physiological maturation. In addition, our data are restricted to mRNA levels. Nevertheless, somatic cells predominantly transcribe poly A-mRNA that is promptly exported and translated. Accordingly, our RT-PCR strategy utilised poly-A reverse transcription with oligo-dT primers. Wider implications of the findings Our data indicate that OSFs inhibit the expression of PTGS2, a key gene for activation and amplification of the maturation cascade in cumulus cells. We speculate that this may represent an effort of the oocyte to prevent precocious meiotic resumption, thus favouring nuclear and cytoplasmic maturation synchrony and developmental competence. Trial registration number Not applicable