Search Results

You are looking at 1 - 2 of 2 items for

  • Author: S. Bilodeau x
Clear All Modify Search
Free access

M. A. Sirard and S. Bilodeau

Summary. This study was undertaken to create an in-vitro model using granulosa cell monolayers to replace the role of the follicle in the maturation of bovine oocytes. Cumulus–oocyte complexes were co-incubated with fresh or 7-day granulosa cell cultures (with new or conditioned medium) or with conditioned medium alone, in the presence or absence of IBMX (isobutylmethylxanthine), adenosine or heparin. Progression to the metaphase-II stage was significantly affected by the co-culture of oocytes with bovine granulosa cell monolayers and to a lesser degree when cultured with supernatant alone (conditioned medium). The oocytes attached rapidly to the monolayer, suggesting that the intimate contact between the granulosa cells and the cumulus–oocyte complexes is an important signal for the maintenance of meiotic arrest. Heparin did not prevent maturation itself, but prevented attachment of cumulus–oocyte complexes to monolayers, thereby reducing their inhibitory effect. Adenosine prevented cumulus expansion and reduced maturation and IBMX was an effective inhibitor only in the presence of additional granulosa cells.

Keywords: oocyte; meiosis; granulosa cells; in vitro; cattle

Free access

S. Bilodeau, M. A. Fortier and M. A. Sirard

The effect of adenylate cyclase stimulation via the components of the enzyme on nuclear maturation in bovine cumulus-enclosed and zona-free oocytes was examined. The stimulating agents were cholera toxin, pertussis toxin, forskolin, sodium fluoride and prostaglandin E2. Cyclic AMP contents were measured in cumulus-oocyte complexes, cumulus-enclosed oocytes and in zona-free oocytes after stimulation, to establish the relationship between cumulus cell and oocyte cAMP concentrations and the meiotic status of the oocyte. In cumulus-enclosed oocytes, forskolin alone and 3-isobutyl-1-methylxanthine (IBMX), at 0.5 mmol l−1, inhibited the resumption of meiosis after 8 h of culture; the other agents were without effect. After 24 h of culture, IBMX at 0.5 mmol l−1 was without effect, but at 2 mmol l−1 reduced the percentage of oocytes at the mature stage (51 versus 82% in control medium). Forskolin alone reduced the proportion of oocytes at the mature stage from 82 to 58%. Forskolin plus IBMX at 2 mmol l−1 and sodium fluoride plus IBMX at 2 mmol l−1 significantly diminished the maturation rate (6 and 17% mature oocytes, respectively). Cholera toxin (with IBMX) and forskolin (alone or with IBMX) stimulated the synthesis of high amounts of cAMP in complexes, but only forskolin had a significant effect on the cAMP contents of oocytes derived from complexes. Forskolin was more effective in zona-free oocytes than in cumulusenclosed oocytes in inhibiting nuclear maturation (24% mature oocytes versus 73% in control medium) even after 24 h of culture; its effect was potentiated by IBMX; forskolin also stimulated cAMP synthesis. IBMX was as effective as forskolin in delaying nuclear maturation, but did not cause an accumulation of cAMP above the control value. The other agents were without effect on meiosis and cAMP concentrations in zona-free oocytes. These results suggest that increases in cAMP concentration in denuded oocytes inhibit maturation; but, when cAMP concentrations are high in cumulus cells, a maturation signal can be generated that bypasses the inhibitory effect of cAMP in the oocyte. Bovine oocytes can synthesize high amounts of cAMP, but its adenylate cyclase may not be coupled to G proteins sensitive to cholera or pertussis toxin.