Personne : Richard, François J.
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Université Laval. Département des sciences animales
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- PublicationRestreintEffects of follicular cells on oocyte maturation. I : Effects of follicular hemisections on bovine oocyte maturation in vitro(Society for the Study of Reproduction, 1996-01-01) Richard, François J.; Sirard, Marc-AndréThis study was designed to evaluate the role of follicular cells in the maintenance of meiotic arrest (germinal vesicle [GV] stage). Bovine ovaries were obtained from a slaughterhouse. Ten oocytes were selected and cocultured for 24 h with two follicular hemisections in a 50-microliters drop of TCM-199 supplemented with 10% fetal calf serum. Follicular hemisections were separated into cellular components--granulosa, theca interna, and theca externa. When the oocytes were cocultured in physical contact with granulosa cells, 36% were maintained in the GV stage. However, the percentage of oocytes in the GV stage was significantly increased (p < 0.05) when the theca interna layer associated with (86%) or not with (78%) granulosa cells was used rather than the hemisection consisting of all three follicular cell layers (57%). When we cocultured oocytes without direct contact, using theca interna associated with granulosa cells, only 35% of the oocytes were maintained in the GV stage. Conditioned medium obtained after culture with two follicular hemisections also maintained a high percentage of oocytes in the GV stage when it was replenished every 4 h. An additional culture of 12 h with fresh unconditioned medium resulted in 95% meiotic resumption. In conclusion, these results suggest that the maintenance of bovine oocytes in the GV stage is influenced by theca cells, independent of contact, and that the oocytes can be maintained by conditioned medium from follicular hemisections.
- PublicationRestreintEffects of follicular cells on oocyte maturation. II : Theca cell inhibition of bovine oocyte maturation in vitro(Society for the Study of Reproduction, 1996-01-01) Richard, François J.; Sirard, Marc-AndréThis study was undertaken to assess the role of follicular cells in the maintenance of meiotic arrest (germinal vesicle [GV] stage). Bovine cumulus-oocyte complexes (COC) were obtained by puncture of ovaries collected at a slaughterhouse. Different monolayers of follicular cells--granulosa cells, theca interna, theca externa, or both types of theca cells together--were cultured in 24-well plates with 1 ml of TCM-199 supplemented with 10% fetal calf serum. Theca cells were obtained by digesting theca layers with collagenase. The medium was renewed 48 h before coculturing selected COC with confluent follicular cell monolayers. Oocytes were maintained in GV stage when cocultured for 12 h directly on monolayers of theca cells. The percentage of oocytes in GV stage was not significantly different between treatments using different types of theca cells (51-66%). Whether COC were cocultured in contact or not in contact with theca cells, the percentage of GV stage was similar (61%). The reversibility of this inhibition was high (85%). However, granulosa cells did not exert meiotic arrest (10%). When oocytes were denuded of their cumulus cells and cocultured with theca cells, only 3% were maintained in GV stage; 59% were maintained in GV stage when COC were not removed before culture. This data provided evidence of the essential role of cumulus cells in maintaining GV stage. In conclusion, we have demonstrated that theca cells, in vitro, maintained bovine oocytes in meiotic arrest. The inhibitory factor(s) produced by theca cells is soluble in the medium and acts through the cumulus cells.
- PublicationRestreintCumulus cell transcripts transit to the bovine oocyte in preparation for maturation(Society for the Study of Reproduction., 2015-11-13) Gilbert, Isabelle; Ettaoumi, Sara; Bastien, Alexandre; Gagné, Dominic; Ashkar, Fazl; Khandjian, Edward William; Macaulay, Angus; Shojaei Saadi, Habib Allah; Richard, François J.; Robert, Claude; Fournier, Éric; Sirard, Marc-André; Hyttel, P.So far, the characteristics of a good quality egg have been elusive, similar to the nature of the physiological, cellular, and molecular cues leading to its production both in vivo and in vitro. Current understanding highlights a strong and complex interdependence between the follicular cells and the gamete. Secreted factors induce cellular responses in the follicular cells, and direct exchange of small molecules from the cumulus cells to the oocyte through gap junctions controls meiotic arrest. Studying the interconnection between the cumulus cells and the oocyte, we previously demonstrated that the somatic cells also contribute transcripts to the gamete. Here, we show that these transcripts can be visualized moving down the transzonal projections (TZPs) to the oocyte, and that a time course analysis revealed progressive RNA accumulation in the TZPs, indicating that RNA transfer occurs before the initiation of meiosis resumption under a timetable fitting with the acquisition of developmental competence. A comparison of the identity of the nascent transcripts trafficking in the TZPs, with those in the oocyte increasing in abundance during maturation, and that are present on the oocyte's polyribosomes, revealed transcripts common to all three fractions, suggesting the use of transferred transcripts for translation. Furthermore, the removal of potential RNA trafficking by stripping the cumulus cells caused a significant reduction in maturation rates, indicating the need for the cumulus cell RNA transfer to the oocyte. These results offer a new perspective to the determinants of oocyte quality and female fertility, as well as provide insight that may eventually be used to improve in vitro maturation conditions.
- PublicationRestreintActive 3'-5' cyclic nucleotide phosphodiesterases are present in detergent-resistant membranes of mural granulosa cells(CSIRO, 2016-01-04) Bergeron, Annick; Richard, François J.; Guillemette, Christine; Sirard, Marc-AndréLipids rafts are specialised membrane microdomains involved in cell signalling that can be isolated as detergent-resistant membranes (DRMs). The second messenger cyclic AMP (cAMP) has a central role in cell signalling in the ovary and its degradation is carried out by the phosphodiesterase (PDE) enzyme family. We hypothesised that PDEs could be functionally present in the lipid rafts of porcine mural granulosa cell membranes. PDE6C, PDE8A and PDE11A were detected by dot blot in the DRMs and the Triton-soluble fraction of the mural granulosa cells membrane and the cytosol. As shown by immunocytochemistry, PDEs showed clear immunostaining in mural granulosa cell membranes and the cytosol. Interestingly, cAMP-PDE activity was 18 times higher in the DRMs than in the Triton-soluble fraction of cell membranes and was 7.7 times higher in the cytosol than in the DRMs. cAMP-PDE activity in mural granulosa cells was mainly contributed by the PDE8 and PDE11 families. This study shows that PDEs from the PDE8 and PDE11 families are present in mural granulosa cells and that the cAMP-PDE activity is mainly contributed by the cytosol. In the cell membrane, the cAMP-PDE activity is mainly contributed by the DRMs. In addition, receptors for prostaglandin E2 and LH, two G-protein-coupled receptors, are present in lipid rafts and absent from the non-raft fraction of the granulosa cell membrane. These results suggest that in these cells, the lipid rafts exist as a cell-signalling platform and PDEs are one of the key enzyme families present in the raft.
- PublicationRestreintCharacterization of FSH signalling networks in bovine cumulus cells : a perspective on oocyte competence acquisition(Oxford University Press, 2015-06-24) Khan, Daulat Raheem; Richard, François J.; Guillemette, Christine; Sirard, Marc-AndréUnderstanding the mechanisms regulating oocyte developmental competence is essential to enhance the clinical efficiency of assisted reproduction. FSH orchestrates the acquisition of oocyte competence, both in vivo and in vitro. Multiple pathways are implicated in FSH signalling; however, their precise coordination remains unresolved. A robust system to investigate FSH signalling is oocyte in vitro maturation (IVM) and we have previously demonstrated better bovine embryo development after FSH addition for the first 6 h during IVM. Using this model, we investigated FSH signalling in cumulus through transcriptomic and pharmacological tools. We demonstrate modulation of cumulus transcriptome by FSH mainly through protein kinase A (PKA) and epidermal growth factor (EGF) pathways. Differentially expressed transcripts were implicated in cumulus expansion, steroidogenesis, cell metabolism and oocyte competence. FSH required rouse-sarcoma oncogene (SRC) for EGF receptor transactivation. PKA and EGF pathway crosstalk was investigated using extracellular signal-regulated kinases (ERK1/2) phosphorylation as the functional end-point. FSH enhanced ERK1/2 activation by the EGF pathway with a simultaneous diminution through PKA. More specifically, FSH increased dual specific phosphatase (DUSP1) transcripts via PKA although DUSP1 protein did not change since EGF was required to prevent degradation. Our findings implicate FSH in PKA and EGF pathway activation, which interact to maintain appropriate levels of ERK1/2 phosphorylation and eventually cumulus expansion, metabolism and steroidogenesis. Moreover, considering the implication of the EGF pathway in GDF9 and BMP15 actions, our findings suggest that FSH may have a role in modulation of the cumulus response to oocyte-secreted factors. This information has implications for improvement of IVM and hence oocyte developmental competence.
- PublicationRestreintEffects of intramuscular administration of folic acid and vitamin B 12 on granulosa cells gene expression in postpartum dairy cows(Elsevier, 2015-11-01) Khan, Daulat Raheem; Laforest, Jean-Paul; Girard, Christiane; Gagnon, Annie; Richard, François J.; Sirard, Marc-AndréThe fertility of dairy cows is challenged during early lactation, and better nutritional strategies need to be developed to address this issue. Combined supplementation of folic acid and vitamin B12 improve energy metabolism in the dairy cow during early lactation. Therefore, the present study was undertaken to explore the effects of this supplement on gene expression in granulosa cells from the dominant follicle during the postpartum period. Multiparous Holstein cows received weekly intramuscular injection of 320 mg of folic acid and 10 mg of vitamin B12 (treated group) beginning 24 (standard deviation = 4) d before calving until 56 d after calving, whereas the control group received saline. The urea plasma concentration was significantly decreased during the precalving period, and the concentration of both folate and vitamin B12 were increased in treated animals. Milk production and dry matter intake were not significantly different between the 2 groups. Plasma concentrations of folates and vitamin B12 were increased in treated animals. Daily dry matter intake was not significantly different between the 2 groups before [13.5 kg; standard error (SE) = 0.5] and after (23.6 kg; SE = 0.9) calving. Average energy-corrected milk tended to be greater in vitamin-treated cows, 39.7 (SE = 1.4) and 38.1 (SE = 1.3) kg/d for treated and control cows, respectively. After calving, average plasma concentration of β-hydroxybutyrate tended to be lower in cows injected with the vitamin supplement, 0.47 (SE = 0.04) versus 0.55 (SE = 0.03) for treated and control cows, respectively. The ovarian follicle ≥12 mm in diameter was collected by ovum pick-up after estrus synchronization. Recovered follicular fluid volumes were greater in the vitamin-treated group. A microarray platform was used to investigate the effect of treatment on gene expression of granulosa cells. Lower expression of genes involved in the cell cycle and higher expression of genes associated with granulosa cell differentiation before ovulation were observed. Selected candidate genes were analyzed by reverse transcription quantitative PCR. Although the effects of intramuscular injections of folic acid and vitamin B12 on lactational performance and metabolic status of animals were limited, ingenuity pathway analysis of gene expression in granulosa cells suggests a stimulation of cell differentiation in vitamin-treated cows, which may be the result of an increase in LH secretion.
- PublicationRestreintOocyte maturation and quality : role of cyclic nucleotides(Society for Reproduction and Fertility, 2016-11-01) Gilchrist, Robert B.; Richard, François J.; Luciano, Alberto Maria; Richani, Dulama; Zeng, Hai-tao; Wang, X.; De Vos, Michel; Sugimura, Satoshi; Smitz, Johan; Thompson, Jeremy G.The cyclic nucleotides, cAMP and cGMP, are the key molecules controlling mammalian oocyte meiosis. Their roles in oocyte biology have been at the forefront of oocyte research for decades, and many of the long-standing controversies in relation to the regulation of oocyte meiotic maturation are now resolved. It is now clear that the follicle prevents meiotic resumption through the actions of natriuretic peptides and cGMP – inhibiting the hydrolysis of intra-oocyte cAMP – and that the pre-ovulatory gonadotrophin surge reverses these processes. The gonadotrophin surge also leads to a transient spike in cAMP in the somatic compartment of the follicle. Research over the past two decades has conclusively demonstrated that this surge in cAMP is important for the subsequent developmental capacity of the oocyte. This is important, as oocyte in vitro maturation (IVM) systems practised clinically do not recapitulate this cAMP surge in vitro, possibly accounting for the lower efficiency of IVM compared with clinical IVF. This review particularly focuses on this latter aspect – the role of cAMP/cGMP in the regulation of oocyte quality. We conclude that clinical practice of IVM should reflect this new understanding of the role of cyclic nucleotides, thereby creating a new generation of ART and fertility treatment options.
- PublicationAccès libreMitochondrial sub-cellular localization of cAMP-specific phosphodiesterase 8A in ovarian follicular cells(Nature Publishing Group, 2019-08-28) Tremblay, Marie-Ève; Vernoux, Nathalie; Lounas, Amel; Richard, François J.; Germain, MarcCyclic adenosine monophosphate (cAMP) is a ubiquitous secondary messenger that plays a central role in endocrine tissue function, particularly in the synthesis of steroid hormones. The intracellular concentration of cAMP is regulated through its synthesis by cyclases and its degradation by cyclic nucleotide phosphodiesterases (PDEs). Although the expression and activity of PDEs impact the specificity and the amplitude of the cAMP response, it is becoming increasingly clear that the sub-cellular localization of PDE emphasizes the spatial regulation of the cell signalling processes that are essential for normal cellular function. We first examined the expression of PDE8A in porcine ovarian cells. PDE8A is expressed in granulosa cells, cumulus cells and oocytes. Second, we assessed the mitochondrial sub-cellular localization of PDE8A. Using western blotting with isolated mitochondrial fractions from granulosa cells and cumulus-oocyte complexes revealed immuno-reactive bands. PDE assay of isolated mitochondrial fractions from granulosa cells measured specific PDE8 cAMP-PDE activity as PF-04957325-sensitive. The immune-reactive PDE8A signal and MitoTracker labelling co-localized supporting mitochondrial sub-cellular localization of PDE8A, which was confirmed using immuno-electron microscopy. Finally, the effect of PDE8 on progesterone production was assessed during the in-vitro maturation of cumulus-oocyte complexes. Using PF-04957325, we observed a significant increase (P < 0.05) in progesterone secretion with follicle-stimulating hormone (FSH). Active mitochondria stained with MitoTracker orange CMTMRos were also increased by the specific PDE8 inhibitor supporting its functional regulation. In conclusion, we propose the occurrence of mitochondrial sub-cellular localization of PDE8A in porcine granulosa cells and cumulus cells. This suggests that there is potential for new strategies for ovarian stimulation and artificial reproductive technologies, as well as the possibility for using new media to improve the quality of oocytes.
- PublicationRestreintThe use of adenosine to inhibit oocyte meiotic resumption in Bos taurus during pre-IVM and its potential to improve oocyte competence(Elsevier Inc., 2019-10-07) Vigneault, Christian; Caballero, Julieta; Richard, François J.; Sirard, Marc-André; Blondin, Patrick.One of the major challenges of artificial reproductive technologies is to develop new methods for pro-ducing greater numbers of embryos. An oocyte fosters the ability to develop into an embryo beforeoocyte meiotic resumption. The aim of the present study was to assess the effect of adenosine (ADO), apurine nucleoside found in follicularfluid, on the inhibition of oocyte meiotic resumption and theproduction of blastocysts. The results showed the efficacy of ADO to inhibit oocyte meiotic resumption.The use of ADO (3 mM) during a pre-in vitro maturation (pre-IVM) culture period of 6 h resulted in asignificant increase (p<0.05) of blastocysts compared to control conditions with no pre-IVM cultureperiod. No effect on the percentage of cleavage was observed. The effect of adenosine on blastocyst yieldwas time- and concentration-dependent with an optimum effect at 3 mM for 6 h. Supplementing theADO pre-IVM culture medium with estradiol, follicle-stimulating hormone, progesterone, epidermalgrowth factor, insulin-like growth factor-2 or reelin did not improve the blastocyst yield. Transcriptionalanalyses of ADO-treated cumulus cells revealed that NRP1, RELN, MAN1A1, THRA and GATM were up-regulated. Finally, bioinformatic analysis identified mitochondrial function as the top canonicalpathway affected by ADO. This opens up new opportunities for further investigations.
- PublicationRestreintCumulus cell gene expression associated with pre-ovulatory acquisition of developmental competence in bovine oocytes(Commonwealth Scientific and Industrial Research Organization, 2013-07-05) Vigneault, Christian; Bunel, Audrey; Nivet, Anne-Laure; Richard, François J.; Sirard, Marc-André; Blondin, Patrick.The final days before ovulation impact significantly on follicular function and oocyte quality. This study investigated the cumulus cell (CC) transcriptomic changes during the oocyte developmental competence acquisition period. Six dairy cows were used for 24 oocyte collections and received FSH twice daily over 3 days, followed by FSH withdrawal for 20, 44, 68 and 92 h in four different oestrous cycles for each of the six cows. Half of the cumulus–oocyte complexes were subjected to in vitro maturation, fertilisation and culture to assess blastocyst rate. The other half of the CC underwent microarray analysis (n = 3 cows, 12 oocyte collections) and qRT-PCR (n = 3 other cows, 12 oocyte collections). According to blastocyst rates, 20 h of FSH withdrawal led to under-differentiated follicles (49%), 44 and 68 h to the most competent follicles (71% and 61%) and 92 h to over-differentiated ones (51%). Ten genes, from the gene lists corresponding to the three different follicular states, were subjected to qRT-PCR. Interestingly, CYP11A1 and NSDHL gene expression profiles reflected the blastocyst rate. However most genes were associated with the over-differentiated status: GATM, MAN1A1, VNN1 and NRP1. The early period of FSH withdrawal has a minimal effect on cumulus gene expression, whereas the longest period has a very significant one and indicates the beginning of the atresia process.
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