Abstract
Substantially less development to the blastocyst stage occurs in vitro than in vivo and this may be due to deficiencies in oocyte competence. Although a large proportion of bovine oocytes undergo spontaneous nuclear maturation, less is known about requirements for proper cytoplasmic maturation. Commonly, supraphysiological concentrations of FSH and LH are added to maturation media to improve cumulus expansion, fertilization and embryonic development. Therefore, various concentrations of porcine FSH (pFSH) and recombinant human FSH (rhFSH) were investigated for their effect on bovine cumulus expansion in vitro. Expression of FSHr, LHr and Cx43 mRNAs was determined in cumulus-oocyte complexes to determine whether they would be useful markers of oocyte competence. In serum-free media, only 1000 ng/ml pFSH induced marked cumulus expansion, but the effect of 100 ng/ml pFSH was amplified in the presence of 10% serum. In contrast, cumulus expansion occurred with 1 ng/ml rhFSH in the absence of serum. FSHr mRNA was highest at 0–6 h of maturation, then abundance decreased. Similarly, Cx43 mRNA expression was highest from 0–6 h but decreased by 24 h of maturation. However, the relative abundance of LHr mRNA did not change from 6–24 h of maturation. Decreased levels of FSHr, LHr and Cx43 mRNAs were detected in COCs of poorer quality. In conclusion, expansion of bovine cumulus occurred at low doses of rhFSH in serum-free media. In summary, FSHr, LHr and Cx43 mRNA abundance reflects COC quality and FSHr and Cx43 mRNA expression changes during in vitro maturation; these genes may be useful markers of oocyte developmental competence.
Highlights
Cumulus-oocyte complexes (COCs) are collected from 2–8 mm antral follicles from unstimulated bovine ovaries collected from an abattoir for in vitro fertilization procedures
Bovine cumulus expansion was obtained with 1000 ng/ml porcine Follicle stimulating hormone (FSH) (pFSH) in serum-free TCM-199
Cumulus expansion was induced with only 1 ng/ml recombinant human FSH (rhFSH) in serum-free media, which is in contrast to the high concentration pFSH required for cumulus expansion
Summary
Cumulus-oocyte complexes (COCs) are collected from 2–8 mm antral follicles from unstimulated bovine ovaries collected from an abattoir for in vitro fertilization procedures. These follicles are several days from reaching pre-ovulatory size and may not have received sufficient exposure to hormones and growth factors in vivo to have the resulting accumulation of (page number not for citation purposes). Recent evidence has shown that maturation condition (oocytes matured in vivo or in vitro) has a significant influence on the numbers of embryos developing to the blastocyst stage [1] This suggests improvements in maturation media and protocols still could be made that would improve oocyte competence and developmental rate. Recombinant gonadotropins have become commercially available; these are very pure sources of hormone that can allow the individual roles of FSH and LH to be investigated without the hormone cross-contamination of pituitary, serum or urinary preparations
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