126 Subcortical maternal complex (SCMC) expression during folliculogenesis is affected by oocyte donor age in sheep
Authors: Bebbere D. , Abazari-Kia A. , Ariu F. , Bogliolo L. , Ledda S. 
Published: December 2019

Age-associated decline in female fertility is largely attributable to decrease in oocyte quality. However, the molecular mechanisms that shape oocyte developmental competence, and that may be involved in reproductive aging, are yet to be elucidated. The subcortical maternal complex (SCMC) is a multiprotein complex located in the subcortex of oocytes that is essential for early embryogenesis and female fertility. It appears to be functionally conserved across mammals; aberrant expression of its members was observed in several animal models of differential competence, and mutations in human SCMC genes were associated with certain human reproductive disorders. At least seven proteins contribute to the complex: KH domain-containing 3 like (KHDC3/FILIA), NLR family pyrin domain-containing 2 (NLRP2), NLRP5 (MATER), oocyte expressed protein (OOEP), peptidyl arginine deiminase 6 (PADI6), transducin-like enhancer of split 6 (TLE6), and zinc finger BED-type-containing 3 (ZBED3), all encoded by maternal effect genes (MEGs). The aim of the present work was to evaluate expression dynamics of the SCMC components during folliculogenesis in relation to maternal age in sheep. Total RNA was isolated and reverse-transcribed from pools of denuded growing oocytes (GO) of different diameters (70-90μm (small, S), 90-110μm (medium, M), or 110-130μm (large, L)) derived from nonhormonally treated prepubertal (Pr, age 40 days), adult (Ad, age &lt;4 years), or aged (Aged, age &gt;6 years) animals (5 pools of 30 oocytes per experimental group). The SCMC expression was assessed by real-time PCR (PCR efficiency of 90-110% and correlation coefficient r2&gt;0.99). Data were normalized against oocyte number and an exogenous spike-in mRNA, Luciferase, as reference gene. Expression dynamics were analyzed within each age group (general linear model ANOVA). Strikingly, patterns specifically associated with donor age were observed during folliculogenesis for six of the seven SCMC components. The Pr group showed active transcription of all mRNA, except ZBED3, during the entire window of oocyte growth (P&lt;0.05). On the contrary, the similar abundance of NLRP2, NLRP5, PADI6, and ZBED3 in Ad S, M, and L GO suggests earlier storage during folliculogenesis; FILIA, OOEP, and TLE6 showed an increase between Ad S and M GO (P&lt;0.05), indicating that the synthesis of these transcripts is complete at this stage (M GOs). Notably, oocytes derived from Aged donors showed a completely inverse expression pattern, with a decrease in abundance of NLRP2, TLE6, FILIA, and PADI6 mRNAs during the last stage of oocyte growth (L GO; P&lt;0.05). Interestingly, MATER showed very high variability in expression (standard error (SE) ranging from 0.79 to 1.13 quantitation cycles (Cq)) in Aged GO, compared to Ad GO (SE 0.16-0.24 Cq) or Pr GO (SE 0.16-0.26 Cq), suggesting large inter-oocyte differences. In conclusion, age affects the storage of the MEGs encoding the SCMC during folliculogenesis. The observed depletion in SCMC transcripts in GO of aged donors is likely to be involved in the age-related decline in oocyte quality.