OOCYTE SECRETED FACTORS REGULATE THE EXPRESSION OF INDUCERS OF THE OVULATORY CASCADE IN CUMULUS CELLS

Abstract

Considering that oocyte secreted factors (OSFs) seem to slow the passe of oocyte nuclear maturation by stimulating the production and delivery of cyclic guanosine monophosphate (cGMP) to the oocyte via increased expression of natriuretic peptide C (NPPC) in cumulus cells, we hypothesized that, as part of the same regulatory effort, OSFs would also inhibit the expression of the main inducers of the ovulatory cascade in cumulus cells, namely amphiregulin (AREG) and epiregulin (EREG), as well as of a metalloproteinase crucial for autocrine signal amplification (disintegrin and metalloprotease 17; ADAM17). The relative mRNA abundance of AREG, EREG and ADAM17 in cumulus cell was compared among 3 treatment-groups: (1) Intact COC subjected to in vitro maturation (IVM); (2) Oocytectomized COC (OOX) subjected to IVM and (3) OOX subjected to IVM with the addition of denuded oocytes (OOX+DO; 1 DO/μL). Four culture replicates including all three treatment-groups were performedCOC were aspirated from 3-8mm follicles of bovine ovaries. Oocytectomy was achieved by aspiration of the ooplasm with a micromanipulator while preserving the structure of the COC. COC and OOX underwent IVM in pools of 20 for 22 hours. IVM was performed in 100 μL of TCM199 with Earl’s salts supplemented with 1μg/mL FSH, 0.4% BSA, 22 μg/mL sodium pyruvate, 75 μg/mL of amikacin at 38,5°C and 5.5% CO2 in humid atmosphere. Abundance of mRNA encoding the target genes in cumulus cells was assessed by real-time RT-PCR using Power SybrGreen (LifeTech®) and RPL15 (ribosomal protein 15) as the reference gene. Effects of treatments were tested by ANOVA and groups were compared with the Tukey test. Statistically significant differences were those with p<0.05. Oocyctectomy increased AREG, EREG and ADAM17 mRNA levels (p<0.02), while the addition of secreting DO reversed the effects of oocytectomy on all genes. Relative mRNA levels (target gene/RPL15 mRNA abundance; mean ± SEM) in COC, OOX and OOX+DO, respectively, were: 0.91 ± 0.14, 7.53 ± 3.36 and 1.53 ± 0.64 for AREG; 0.87 ± 0.13, 3.15 ± 1.03 and 1.14 ± 0,08 for EREG; 1.22 ± 0.08, 1.64 ± 0.06 and 1,01 ± 0,12 for ADAM17. Oocyte secreted factors appear to reduce the expression of genes triggering the ovulatory cascade and final oocyte maturation. We speculate that this is part of an effort of the oocyte to prevent precocious meiotic resumption thus favouring nuclear and cytoplasmic maturation synchrony and developmental competence.