Brilliant cresyl blue (BCB) is a super vital stain that has been used to select competent oocytes in different species. The objectives of present studies were to determine mRNA abundance for select TGFβ superfamily components, SMAD2/3 and SMAD1/5 phosphorylation levels and transcript abundance for other oocyte (JY1) and cumulus cell (CTSB, CTSK, CTSS and CTSZ) markers of oocyte quality in bovine oocytes and or adjacent cumulus cells classified based on developmental potential using BCB staining. The ability of exogenous FST, JY1, or cathepsin inhibitor treatment to enhance development of embryos derived from poor quality oocytes selected based on BCB staining was also determined. Cumulus oocyte complexes (COCs) from abattoir derived ovaries were subjected to BCB staining and GV stage oocytes and cumulus cells harvested from control, BCB+ and BCB- (poor oocyte quality) groups for real time PCR or Western blot analysis. Remaining COCs underwent in vitro maturation, in vitro fertilization and embryo culture in presence or absence of above described treatments. Levels of FST, JY1, BMP15 and SMAD1, 2, 3 and 5 transcripts were higher in BCB+ oocytes whereas abundance of CTSB, CTSK, CTSS and CTSZ mRNAs was higher in cumulus cells surrounding poor quality BCB- oocytes. Western blot analysis revealed SMAD1/5 and SMAD2/3 phosphorylation were higher in BCB+ than BCB− oocytes. Embryo culture studies demonstrated that follistatin and cathepsin inhibitor treatment but not JY-1 treatment can promote developmental competence of BCB- oocytes. Results provide further understanding of molecular indices of oocyte competence.
Despite several decades since the birth of the first test tube baby and the first calf derived from an in vitro fertilized embryo, the efficiency of assisted reproductive technologies remains less than ideal. Poor oocyte competence is a major factor limiting efficiency of in vitro embryo production. Developmental competence obtained during oocyte growth and maturation establishes the foundation of successful fertilization and pre-implantation embryonic development. Regulation of molecular and cellular events during fertilization and embryo development is mediated in part by oocyte derived factors acquired during oocyte growth and maturation and programmed by factors of follicular somatic cell origin. Available evidence supports an important intrinsic role for oocyte-derived follistatin and JY-1 proteins in mediating embryo developmental progression post fertilization and suggests that paracrine and autocrine actions of oocyte-derived GDF9 and BMP15 and follicular somatic cell derived members of the fibroblast growth factor family impact oocyte competence and subsequent embryo developmental progression post fertilization. An increased understanding of molecular mechanisms mediating oocyte competence and stage specific developmental events during early embryogenesis is crucial to further improvements in assisted reproductive technologies.
Upstream stimulating factor 1 (USF1) is a basic helix loop helix transcription factor that specifically binds to E-box DNA motifs, known cis-elements of key oocyte expressed genes essential for oocyte and early embryo development. However, the functional and regulatory role of USF1 in bovine oocyte and embryo development is not understood. In this study, we demonstrated that USF1 mRNA is maternal in origin and expressed in a stage specific manner during the course of oocyte maturation and preimplantation embryonic development. Immunocytochemical analysis showed detectable USF1 protein during oocyte maturation and early embryonic development with increased abundance at 8–16 cell stage of embryo development, suggesting a potential role in embryonic genome activation. Knockdown of USF1 in germinal vesicle stage oocytes did not impact meiotic maturation or cumulus expansion, but caused significant changes in mRNA abundance for genes associated with oocyte developmental competence. Furthermore, siRNA mediated depletion of USF1 in presumptive zygote stage embryos demonstrated that USF1 is required for early embryonic development to the blastocyst stage. A similar (USF2) yet unique (TWIST2) expression pattern during oocyte and early embryonic development for related E-box binding transcription factors known to cooperatively bind USF1 suggest a potential link to USF1 action. This study demonstrates that USF1 is a maternally derived transcription factor required for bovine early embryonic development which also functions in regulation of JY-1, GDF9 and FST genes associated with oocyte competence.
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