Cyclin B1 expression in meiotically competent and incompetent goat oocytes

Abstract

To start determining the nature of meiotic incompetence in goat oocytes, we have examined the expression of one of the potential pre‐MPF subunits: the cyclin B1. We have been isolating a small DNA probe encoding the goat cyclin B1 box to analyze the expression of the cyclin B1 gene in competent and incompetent goat oocytes. This probe was easily obtained by polymerase‐chain‐reaction (PCR) on reverse‐transcribed mRNA from granulosa cells, using cyclin B specific primers derived from a bovine cDNA. The transcript corresponding to cyclin B1 in goat granulosa cells is 1.8 kb. In situ hybridization analysis indicated that competent and incompetent oocytes contained cyclin B1 mRNA, but also that active cyclin B1 mRNA synthesis occured at the end of the growth phase, e.g., when oocytes progressed in the acquisition of meoitic competence. Western blot analysis, performed with a monoclonal anticyclin B1 antibody, revealed in competent and incompetent oocytes a polypeptide of 65kDa corresponding to the goat cyclin B1 protein. This pattern of cyclin B1 expression further suggested that meiotic incompetence in goat oocytes could not be primarily correlated with a lack of cyclin B1 protein as potential pre‐MPF subunit, but to a limiting amount of this protein. Mol. Reprod. Dev. 47:222–228, 1997. © 1997 Wiley‐Liss, Inc.

Mitochondria directly influence fertilisation outcome in the pig

“…Similar results were obtained from analysis of cyclin B mRNA transcripts (Fig. 1B), which would be expected to persist in the oocyte cytoplasm (Hue et al 1997). Interestingly, the oocytes used in these studies were obtained from a commercial dam line of pure white pigs.…”

Quantification of Cyclin B1 and p34cdc2 in Bovine Cumulus-Oocyte Complexes and Expression Mapping of Genes Involved in the Cell Cycle by Complementary DNA Macroarrays1

“…The pelleted RNA was washed in 70% ethanol and dried prior to reverse transcription. First-strand cDNA was synthesized using a SuperScript Preamplification System for First Strand cDNA Synthesis kit (Life Technologies) and 100 ng of oligo(dT) [12][13][14][15][16][17][18] primer in a total reaction volume of 20 l. A cDNA aliquot of 2 l was subjected to quantitative polymerase chain reaction (PCR) amplification using a Light Cycler apparatus (Roche Diagnostics, Laval, PQ, Canada). Products were detected with SYBR Green (Roche Diagnostics).…”

“…In the mouse, the acquisition of meiotic competence is partly associated with the synthesis of p34 cdc2 [14][15][16][17], suggesting that a lack of one of the MPF components keeps the oocyte in meiotic arrest. Similarly, although both cyclin B1 mRNA and protein have been detected in meiotically competent and incompetent goat oocytes [18], only p34 cdc2 mRNA is present in meiotically incompetent oocytes [19]. In contrast, both components of MPF are present in the pig oocytes, suggesting that meiotic arrest in the pig is likely controlled by another pathway that involves protein phosphate transfer (kinase and/or phosphatase) [20].…”

Brilliant Cresyl Blue stain selects largest oocytes with highest mitochondrial activity, maturation-promoting factor activity and embryo developmental competence in prepubertal sheep

“…MPF activity appears just before germinal vesicle breakdown (GVBD) increasing until metaphase I; its activity is decreased in anaphase-telophase while its maximum level is reached at metaphase II (MII). It has been shown that incompetent goat oocytes have a limited amount of cyclin B1 (Hue et al 1997) and p34 cdc2 . MPF activity in calf and lamb oocytes were significantly lower than in cow and ewe oocytes (Ledda et al 2001, Salamone et al 2001, whereas (Han et al 2010) showed in mice that the MPF activity of prepubertal oocytes was significantly higher than that of adult oocytes.…”