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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

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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.

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A Bonnet, R Dalbiès-Tran and M A Sirard

Ovarian oogenesis and folliculogenesis are complex and coordinated biological processes which require a series of events that induce morphological and functional changes within the follicle, leading to cell differentiation and oocyte development. In this context, the challenge of the researchers is to describe the dynamics of gene expression in the different compartments and their interactions during the follicular programme. In recent years, high-throughput arrays have become a powerful tool with which to compare the whole population of transcripts in a single experiment. Here, we review the challenges of applying genomics to this model in farm animal species. The first limitation lies in limited the availability of biological material, which makes the study of the follicle compartments (oocyte, granulosa cells and thecal cells) or early embryo much more difficult. The concept of observing all transcripts at once is very attractive but despite progress in sequencing, the genome annotation remains very incomplete in non-model species. Particularly, oogenesis and early embryo development relate to the high proportion of unknown expressed sequence tags. Then, it is important to consider post-transcriptional and translational regulation to understand the role of these genes. Ultimately, these new inferred insights will still have to be validated by functional approaches. In addition to in vitro or ex vivo functional approaches, both ‘natural mutant’ ewe models and RNA interference represent, at the moment, the best hope for functional genomics. Advances in our understanding of reproductive physiology should be facilitated by gene expression data exchange and translation into a better understanding of the underlying biological phenomena.

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M. A. Sirard, R. D. Lambert, D. P. Ménard and M. Bedoya

Summary. Cow embryos fertilized in vitro (1–8-cell) (n = 113) were transferred surgically to the ligated oviduct of pseudopregnant female rabbits (31 ± 4 h after 75 i.u. hCG). Rabbits were killed 99 ± 5 h later and 67 embryos were recovered: 45 (67%) had cleaved at least once, 15 had reached the morula stage and 2 blastocysts were obtained. Transfer of cleaved embryos (2–8-cell) led to a high recovery rate (84%) compared to 39% for one-cell embryos. Of the embryos incubated for more than 99 h in the rabbit oviduct, 41% were at the morula stage. Embryos incubated in vivo (n = 21) (8-cell to blastocyst) were transferred to the uterus of 14 synchronized recipient heifers by a surgical (N = 5) or a non-surgical (N = 9) procedure: 6 pregnancies resulted (4 from the non-surgical procedures). In addition, 27 (2–8-cell) cow embryos developed in vitro were transferred to the oviduct of synchronized heifers by a paralumbar surgical approach on a standing animal, but no pregnancies resulted. It is therefore concluded that (1) the rabbit oviduct can be used to obtain cow eggs at an embryonic stage sufficiently advanced to permit transfer to the uterus of a synchronized recipient; and (2) the pregnancy rate after in-vitro fertilization and incubation in the rabbit oviduct are similar to results with fertilization in vivo.

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F C F Dias, M I R Khan, M A Sirard, G P Adams and J Singh

Microarray analysis was used to compare the gene expression of granulosa cells from dominant follicles with that of those after superstimulatory treatment. Cows were allocated randomly to two groups (superstimulation and control, n=6/group). A new follicular wave was induced by ablation of follicles ≥5 mm in diameter, and a progesterone-releasing device controlled internal drug release (CIDR) was placed in the vagina. The superstimulation group was given eight doses of 25 mg FSH at 12-h intervals starting from the day of wave emergence (day 0), whereas the control group was not given FSH treatment. Both groups were given prostaglandin F2 α twice, 12 h apart, on day 3 and the CIDR was removed at the second injection; 25 mg porcine luteinizing hormone (pLH) was given 24 h after CIDR removal, and cows were ovariectomized 24 h later. Granulosa cells were collected for RNA extraction, amplification, and microarray hybridization. A total of 190 genes were downregulated and 280 genes were upregulated. To validate the microarray results, five genes were selected for real-time PCR (NTS, FOS, THBS1, FN1, and IGF2). Expression of four genes increased significantly in the three different animals tested (NTS, FOS, THBS1, and FN1). The upregulated genes are related to matrix remodeling (i.e. tissue proliferation), disturbance of angiogenesis, apoptosis, and oxidative stress response. We conclude that superstimulation treatment i) results in granulosa cells that lag behind in maturation and differentiation (most of the upregulated genes are markers of the follicular growth stage), ii) activates genes involved with the NFE2L2 oxidative stress response and endoplasmic reticulum stress response, and iii) disturbs angiogenesis.

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P. Leclerc, J. Langlais, R. D. Lambert, M. A. Sirard and J. G. Chafouleas

Summary. A 125I-labelled calmodulin gel overlay procedure in the presence and the absence of Ca2+ was used to evaluate bull spermatozoa calmodulin-binding proteins. Frozen spermatozoa were thawed, washed and incubated for 6 h before being processed for SDS polyacrylamide gel electrophoresis and the 125I-labelled calmodulin gel overlay procedure. In non-incubated spermatozoa, up to 14 binding proteins were detected. Some exhibited greater calmodulin binding in the presence of Ca2+ while others exhibited greater binding when Ca2+ was absent. When heparin (2 μg/ml) was present in the incubation medium, a decrease in the calmodulin binding to the proteins of M r 28 000 and 30 000 was detected in the presence of Ca2+ and EGTA. This effect of heparin was time- and dose-dependent and was increased by the presence of the acrosin inhibitor benzamidine. Sperm capacitation could thus be related to a decrease in the binding of calmodulin to these proteins.

Keywords: bull; spermatozoa; calmodulin; calmodulin-binding proteins; heparin; capacitation

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P. Leclerc, M. A. Sirard, J. G. Chafouleas and R. D. Lambert

Summary. Concentrations of the intracellular Ca2+-mediator calmodulin (CaM), were measured by radioimmunoassay during heparin-induced capacitation of bull spermatozoa. Heparin reduced sperm CaM concentrations in a dose-dependent manner corresponding with an increase in in-vitro fertilization rates. Such reductions were observed after heparin treatment for 4–6 h, which is in agreement with the length of the capacitation period in bulls and was concomitant with an increase in CaM concentration in the incubation medium, suggesting translocation of CaM from the spermatozoa to the surrounding milieu. This CaM translocation was inhibited partly by the protease inhibitor benzamidine, suggesting a role for the sperm protease in this process.

Keywords: calmodulin; spermatozoa; capacitation; heparin; cow