Subcortical maternal complex (SCMC) expression during folliculogenesis is affected by oocyte donor age in sheep.

PURPOSE: The age-associated decline in female fertility is largely ascribable to the decrease in oocyte quality. The subcortical maternal complex (SCMC) is a multiprotein complex essential for early embryogenesis and female fertility and functionally conserved across mammals. The present work evaluated expression dynamics of its components during folliculogenesis in relation to maternal age in sheep. METHODS: The expression of the SCMC components (KHDC3/FILIA, NLRP2, NLRP5/MATER, OOEP/FLOPED, PADI6, TLE6 and ZBED3) was analyzed by real-time PCR in pools of growing oocytes (GO) of different diameters (70-90 µm (S), 90-110 µm (M), or 110-130 µm (L)) derived from non-hormonally treated adult (Ad; age < 4 years), prepubertal (Pr; age 40 days), or aged ewes (age > 6 years). RESULTS: Specific expression patterns associated with donor age were observed during folliculogenesis for all genes, except ZBED3. In oocytes of adult donors, the synthesis of NLRP2, NLRP5, PADI6, and ZBED3 mRNAs was complete in S GO, while FILIA, TLE6, and OOEP were actively transcribed at this stage. Conversely, Pr GO showed active transcription of all mRNAs, except for ZBED3, during the entire window of oocyte growth. Notably, aged GO showed a completely inverse pattern, with a decrease of NLRP2, TLE6, FILIA, and PADI6 mRNA abundance during the latest stage of oocyte growth (L GO). Interestingly, MATER showed high expression variability, suggesting large inter-oocyte differences. CONCLUSION: Our study describes the SCMC expression dynamics during sheep oogenesis and reports age-specific patterns that are likely involved in the age-related decline of oocyte quality.

Simple, fast, and efficient method of manual oocyte enucleation using a pulled Pasteur pipette.

Cloning mammals by somatic cell nuclear transfer entails the replacement of oocyte chromosomes with the nucleus of a somatic cell. A major step in this technique is to efficiently produce large batches of enucleated oocytes, a process that requires considerable micromanipulation skills and expensive equipments. Here, a simple, fast, and efficient method of manual oocyte enucleation was introduced that can be adopted in every laboratory with the minimum equipments. Common laboratory glass pipettes were pulled on the flame of a burner and then used for manual bisection or enucleation of sheep and goat zona-free oocytes by passing them through the discontinuous cutting border of culture medium and mineral oil. The described techniques showed a certain efficiency to conveniently bisect or enucleate large batches of sheep, and goat oocytes being pre-treated with demecolcine. The method may be straightforward for simple manipulation of oocytes of other species and for development of automated cloning methods as well.