J Sirois

Université du Québec à Montréal, Montréal, Quebec, Canada

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Publications (89)305.61 Total impact

  • Khampoun Sayasith, Jean Sirois
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    ABSTRACT: The interaction between stromal cell-derived factor-1 (SDF1) and chemokine CXC motif receptor 4 (CXCR4) has been implicated in leukocyte attraction, tissue remodeling and angiogenesis. The objective of the present study was to characterize the expression and regulation of SDF1 and CXCR4 in equine follicles during the ovulatory process. Equine preovulatory follicles were isolated during estrus 0-39 h after hCG treatment. Follicle wall preparations (theca interna with attached granulosa cells) and isolated preparations of granulosa cells and theca interna were obtained, and total RNA extracts were analyzed by RT-PCR/Southern blot. Results showed that levels of CXCR4 transcripts were induced by hCG in follicles at 36 h post-hCG (P < 0.05 vs 0 h), with the induction observed in both granulosa and theca cells. Immunoblotting and immunohistochemical analyses confirmed an increase in CXCR4 protein in follicles after hCG treatment. In contrast, levels of SDF1 transcripts were very low in granulosa cells but high in theca interna cells throughout most of the ovulatory period. Studies in vivo performed with bovine preovulatory follicles collected 0-24 h post-hCG revealed a marked and significant up-regulation of CXCR4 transcripts after hCG (P < 0.05), as observed in equine follicles. A similar pattern of CXCR4 mRNA up-regulation was observed in cultures of bovine granulosa cells treated with forskolin (P < 0.05). This forskolin-dependent induction of CXCR4 mRNA was suppressed by co-treatment with inhibitors of PKA, ERK1/2 and EGFR, and by the progesterone receptor antagonist RU486 (P < 0.05), underscoring the contribution of multiple signaling pathways. In complementary studies, treatment of bovine granulosa cells with EGF or the hypoxia mimetic cobalt chloride significantly increased CXCR4 transcript levels, whereas co-treatment with forskolin and a CXCR4 antagonist repressed the expression of several ovulation-related genes. Collectively, this study describes for the first time the gonadotropin-dependent up-regulation of CXCR4 transcript in ovarian follicles of large monoovulatory species, provides some insights into the regulation of CXCR4 gene expression in granulosa cells, and identifies a potential link between follicular SDF1/CXCR4 activation and the regulation of ovulation-related genes.
    Molecular and Cellular Endocrinology 01/2014; · 4.04 Impact Factor
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    ABSTRACT: Vanin-2 (VNN2) is known to be involved in inflammation and leukocyte migration, but its regulation in follicles remains unknown. The objectives of this work were to study the regulation of VNN2 transcripts in bovine follicles prior to ovulation, and to characterize the control of its expression in bovine granulosa cells. VNN2 expression was studied using total RNA extracted from granulosa cells of small follicles (2-4 mm in diameter), dominant follicles obtained on day 5 of the estrous cycle, ovulatory follicles obtained 0-24 h after human chorionic gonadotropin (hCG), and corpora lutea on day 5 of the cycle. Results from RT-PCR analyses showed that levels of VNN2 mRNA were high in ovulatory follicles 24 h post-hCG but low in other tissues. In ovulatory follicles, levels of VNN2 mRNA were low at 0 h but significantly upregulated 12-24 h post-hCG. To determine factors controlling VNN2 gene expression, established primary cultures of granulosa cells isolated from bovine dominant follicles were used. Treatment with forskolin elevated VNN2 mRNA expression as observed in vivo. Mutation studies identified the minimal region conferring basal and forskolin-stimulated VNN2 promoter activities, which were dependent on chicken ovalbumin upstream promoter-transcription factor (COUP-TF), GATA and Ebox cis-elements. EMSAs identified COUP-TF, GATA4, and upstream stimulating factor (USF) proteins as key factors interacting with these elements. Chromatin immunoprecipitation assays confirmed basal and forskolin-induced interactions between these proteins and the VNN2 promoter in bovine granulosa cell cultures. VNN2 promoter activity and mRNA expression were markedly stimulated by forskolin and overexpression of the catalytic subunit of PKA, but inhibited by PKA and ERK1/2 inhibitors. Collectively, findings from this study describe for the first time the gonadotropin/forskolin-dependent up-regulation of VNN2 transcripts in granulosa cells of preovulatory follicles, and provide insights into some of the molecular basis of VNN2 gene expression in follicular cells.
    Biology of Reproduction 09/2013; · 4.03 Impact Factor
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    ABSTRACT: The ovulatory process involves a complex remodeling of the extracellular matrix during which a desintegrin and metalloproteinase with thrombospondin motif 1 (ADAMTS1) is thought to play a key role, but its transcriptional regulation in bovine follicles remains largely unknown. The objectives of this study were to characterize the regulation of ADAMTS1 in bovine follicles before ovulation and to determine its transcriptional control in bovine granulosa cells. Regulation of ADAMTS1 was assessed using total RNA isolated from bovine preovulatory follicles obtained at various times after human chorionic gonadotropin treatment. Results from RT-PCR analyses showed that levels of ADAMTS1 mRNA were very low at 0 hours but increased at 6- to 24 hours after human chorionic gonadotropin in granulosa cells. To determine the regulatory mechanisms controlling ADAMTS1 gene expression in vitro, primary cultures of bovine granulosa cells were established, and treatment with forskolin up-regulated ADAMTS1 mRNA levels. Promoter activity assays, 5` deletion, and site-directed mutagenesis identified a minimal region conferring full-length basal and forskolin-stimulated ADAMTS1 promoter activities, with both being dependent on Ebox cis-acting elements. EMSAs revealed upstream stimulating factor (USF) proteins as key trans-activating factors interacting with Ebox. Chromatin immunoprecipitation assays confirmed such interactions between USF and Ebox in vivo, and USF binding to Ebox elements was increased by forskolin treatment. ADAMTS1 promoter activity and mRNA expression were increased by forskolin and overexpression of the catalytic subunit of protein kinase A, but not by cotreatment with inhibitors of protein kinase A, ERK1/2, and epidermal growth factor receptor signaling pathways. Furthermore, treatment with a soluble epidermal growth factor induced ADAMTS1 mRNA expression in granulosa cells. Collectively, results from this study describe the gonadotropin/forskolin-dependent up-regulation of ADAMTS1 mRNA in granulosa cells of bovine preovulatory follicles in vivo and in vitro and identify for the first time some of the molecular mechanisms responsible for ADAMTS1 promoter activation in follicular cells of a large monoovulatory species.
    Endocrinology 06/2013; · 4.72 Impact Factor
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    ABSTRACT: Little is know about the expression and regulation of epiregulin (EREG) and amphiregulin (AREG) in ovarian follicles of large monoovulatory animal species. To characterize the gonadotropin-dependent regulation of EREG and AREG mRNAs in equine follicles prior to ovulation, extracts were prepared from equine follicles collected during estrus between 0 and 39 h post-hCG and corpora lutea obtained on day 8 of the estrous cycle (day 0 = day of ovulation). Results from RT-PCR/Southern blot analyses showed that levels of EREG and AREG mRNAs were very low in follicles obtained at 0 h but increased thereafter (P < 0.05), with maximal levels observed 33 to 39 h post-hCG. This significant increase was observed in both granulosa and theca cells. Immunohistochemistry and immunoblot analyses confirmed the hCG-dependent induction of EREG protein in both cell types. RT-PCR/Southern blot analyses of ADAM17, which encodes an enzyme that cleaves and releases soluble bioactive EREG and AREG, showed that levels of its transcript were high and remained constant throughout the period studied. Studies on the hCG-dependent regulation of EREG and AREG in bovine preovulatory follicles in vivo showed that the induction of both transcripts was transient, observed predominantly at 6 h post-hCG and localized only in granulosa cells. To characterize the effect of epidermal growth factor receptor (EGFR) activation on the expression of ovulation-related genes in granulosa cells of a large monoovulatory animal species, primary cultures of bovine granulosa cells were established. Results from RT-PCR analyses revealed that EREG and AREG mRNAs were induced by forskolin treatment in vitro; but the EGFR inhibitor PD153035 suppressed the forskolin-dependent induction of several ovulation-related transcripts, including PTGS2, PTGER2, TNFAIP6, PGR, MMP1, VEGFA, and CTSL2 mRNAs. Moreover, these transcripts were induced in granulosa cell cultures by EGF, an analogue of EREG and AREG. Collectively, this study identifies differences in the temporal and cellular localization of EREG and AREG expression in equine and bovine preovulatory follicles, and underscores the potential role of follicular EGFR activation in the regulation of ovulation-regulated genes in large monoovulatory species.
    General and Comparative Endocrinology 11/2012; · 2.82 Impact Factor
  • K Sayasith, J Sirois, M Doré
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    ABSTRACT: Cyclooxygenase-2 (COX-2), the rate-limiting enzyme in the biosynthesis of prostaglandins, plays an important role in inflammation and tumorigenesis. COX-2 primary structure has been characterized in many species and its expression demonstrated in a variety of cancers in humans and dogs, including mammary cancer. In contrast, there is currently little information on the structure of feline COX-2. Also, information on COX-2 expression in feline mammary cancer is limited and conflicting. The objectives of this study were therefore to characterize the molecular structure of feline COX-2 and to evaluate by immunohistochemistry its expression in mammary carcinomas. Our results show that the predicted coding region of feline COX-2 encodes a 604-amino acid protein, which is identical in length to several COX-2 homologs. Feline COX-2 amino acid sequence is highly similar to other mammalian COX-2 homologs. Immunohistochemical analysis of 40 mammary carcinomas showed that the majority of tumors studied (35/40; 87%) expressed COX-2 at a level varying from low (20/40; 50%) to intermediate (13/40; 32%) and high (2/40; 5%). These results provide the first molecular characterization of feline COX-2 and demonstrate that COX-2 is expressed in the majority of feline mammary carcinomas.
    Veterinary Pathology 02/2009; 46(3):423-9. · 1.93 Impact Factor
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    ABSTRACT: To study the regulation of bovine TNFalpha-induced protein 6 (TNFAIP6) prior to ovulation, preovulatory follicles obtained after the treatment with human chorionic gonadotropin (hCG) were used. RT-PCR analyses showed that levels of TNFAIP6 mRNA were low before hCG but significantly increased after hCG treatment in follicles. Further analyses and immunohistochemistry indicated that this increase in transcript and protein levels occurred in theca and granulosa cells. To investigate molecular mechanisms involved in TNFAIP6 transactivation, the activity of bovine TNFAIP6 promoter was studied in granulosa cell cultures. Mutant studies identified the minimal region conferring full-length promoter activity, in which activator protein-1 (AP1) and cAMP response element (CRE) elements were required for promoter activity. Overexpression of dominant-negative AP1 and activating transcription factor/cAMP response element-binding protein (CREB) inhibited forskolin-inducible promoter activity. DNA binding assays demonstrated the importance of AP1 and CRE for activity and identified JunD, FosB, Fra2, CREB1, and CREB2 as being part of the AP1 complex, and FosB, Fra2, and CREB1 for the CRE complex. Chromatin immunoprecipitation assays confirmed binding of these proteins with endogenous TNFAIP6 promoter. Treatment with forskolin, prostaglandin E2, and catalytic subunit protein kinase (cPKA) stimulated, but H89, PKA inhibitor peptide, and indomethacin inhibited, TNFAIP6 promoter activity and gene expression in granulosa cells. Collectively, this study is the first to describe that the ovulatory process in cows is associated with a gonadotropin-dependent induction of TNFAIP6 in ovarian follicles and provide the molecular basis through which AP1 and CRE sites and PKA activation played important roles in the regulation of TNFAIP6 in granulosa cells.
    Endocrinology 09/2008; 149(12):6213-25. · 4.72 Impact Factor
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    ABSTRACT: The objectives of the study were to clone the primary structure of the prostaglandin E2 receptor subtype 2 (PTGER2) cDNA and to characterize its regulation in equine follicles during gonadotropin-induced ovulation. Results from DNA isolation indicated that the equine PTGER2 cDNA encodes a predicted 353-amino acid protein, which is highly similar (76-85%) to known mammalian homologues. The regulation of PTGER2 was studied by semi-quantitative RT-PCR/Southern blot using preparations of theca interna and mural granulosa cells isolated from equine follicles 0-39 hr post-treatment with human chorionic gonadotropin (hCG). Results indicated that a significant increase of PTGER2 mRNA occurred at 24 and 39 hr post-hCG in granulosa cells, and 30 and 33 hr post-hCG in theca cells (P < 0.05). Immunohistochemical staining and immunoblotting performed on equine follicular samples showed a corresponding increase of PTGER2 protein in both cell types after treatment with hCG. Levels of PTGER2 mRNA were also high in uterus, thymus and spleen, but moderate to low in other tested tissues. In the ovary, the expression of PTGER4 mRNA was observed and predominantly occurred in granulosa cells, with highest abundance of transcripts observed at 12 and 39 hr post-hCG. Thus, this study reports for the first time in mares that the ovulatory process is accompanied by the gonadotropin-dependent up-regulation of PTGER2 and PTGER4, which may in turn regulate PGE2-mediated preovulatory effects.
    Molecular Reproduction and Development 06/2008; 76(2):191-201. · 2.81 Impact Factor
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    ABSTRACT: Vacuolar system-associated protein-60 (VASAP-60) constitutes the bovine ortholog of the human "protein kinase C substrate 80K-H" (PRKCSH or 80K-H). We characterized the bovine VASAP-60/PRKCSH gene structure and promoter, identified cis-acting elements controlling VASAP-60 expression, searched for mRNA splice variants, and analyzed mRNA expression in ovarian follicles. Expression of VASAP-60 mRNA showed a 2.4-fold increase (P<0.0001) in granulosa cells of dominant follicles compared to small follicles (2-4 mm) or ovulatory follicles, and no mRNA splice variant was identified. The bovine VASAP-60 gene encompasses 12.5 kb and is composed of 18 exons and 17 introns. Primer extension analysis revealed a single transcription initiation site, and the promoter lacks a TATA box. Promoter activity assays were performed with a series of deletion constructs in different bovine cell lines (endometrial epithelial glandular, kidney epithelial and aortic endothelial) to identify cis-acting elements. The -53/+16 bp fragment (+1 = transcription start site) conferred minimal promoter activity whereas activator and repressor elements were located in the -200/-53 bp and -653/-200 bp fragments, respectively. Analysis of cis-acting elements in the -200/-53 bp activation domain revealed by gel shift assays and chromatin immunoprecipitation assay that transcription factor YY1 binds to VASAP-60 promoter. This study is the first to report that VASAP-60 is up-regulated in granulosa cells of dominant follicles, to document the primary structure of the bovine VASAP-60 gene and promoter, and to demonstrate that YY1 binds to the VASAP-60 proximal promoter and may act as a positive transcriptional regulator.
    Gene 05/2007; 391(1-2):63-75. · 2.20 Impact Factor
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    ABSTRACT: The type 1 form of 17beta-hydroxysteroid dehydrogenase (17betaHSD1) was the first isoform to be identified and is capable of converting estrone to 17beta-estradiol. This study was aimed at characterizing the molecular structure of the equine 17betaHSD1 gene and cDNA, as well as its molecular regulation during human chorionic gonadotropin (hCG)-induced follicular luteinization/ovulation in vivo. The equine 17betaHSD1 gene was cloned from an equine genomic library and shown to have a conserved genomic structure composed of six exons. Its cDNA sequence was also identified and coded for a 308 amino acid protein, 72 x 1-74 x 5% homologous to other mammalian orthologs. RT-PCR/Southern blot analyses were performed to study the regulation of the 17betaHSD1 transcript in equine preovulatory follicles isolated between 0 and 39 h after hCG treatment. Results demonstrated the presence of high 17betaHSD1 mRNA expression prior to hCG treatment with a marked decrease observed 12 h after hCG (P<0 x 05). Analyses on isolated preparations of granulosa and theca interna cells identified the granulosa cell layer as the site of 17betaHSD1 transcript expression and downregulation (P<0 x 05). A 1412 bp fragment of the equine 17betaHSD1 proximal promoter was sequenced and shown to contain many putative transcription factor binding sites. Electromobility shift assays (EMSA) using a fragment of the proximal promoter (-230/-30) and nuclear extracts prepared from granulosa cells isolated prior to hCG (0 h post-hCG) revealed the presence of a major complex, and results from competition assays suggest that steroidogenic factor-1 (SF-1), NFkappaB, GATA, and Sp1 cis-elements are involved. Supershift assays using an antibody against the p65 subunit of NFkappaB led to the displacement of the binding nuclear proteins to the DNA probe, whereas the use of an anti-equine SF-1 antibody demonstrated the clear formation of a DNA-protein-antibody complex, confirming the potential role of these transcription factors in EMSA results. Interestingly, a notable decrease in DNA binding was observed when granulosa cell nuclear extracts isolated 30 h post-hCG were used, which paralleled the decrease in 17betaHSD1 transcript after hCG treatment. Thus, this study is the first to report the gonadotropin-dependent downregulation of 17betaHSD1 transcript expression in a monoovulatory species, the presence and regulation of protein/DNA interactions in the proximal region of the 17betaHSD1 promoter during gonadotropin treatment, and the characterization of the primary structure of equine 17betaHSD1 cDNA and gene.
    Journal of Molecular Endocrinology 03/2007; 38(1-2):67-78. · 3.58 Impact Factor
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    ABSTRACT: To study the regulation of bovine pituitary adenylate cyclase-activating polypeptide (PACAP) in preovulatory follicles prior to ovulation, PACAP cDNA was isolated by RT-PCR. Its open reading frame (ORF) is composed of 531 bp, and encodes for a 176-amino acid protein that bears 76-90% identity with other PACAP homologs. Using bovine preovulatory follicles obtained between 0 and 24 h after human chorionic gonadotropin (hCG) and semiquantitative RT-PCR/Southern blot, we demonstrate that levels of PACAP mRNA were low at 0 h, markedly increased at 6 and 12 h (P<0.05), and declined 18 and 24 h after hCG. Levels of PACAP mRNA were high in the bovine pituitary, testis, intestine and uterus, but moderate to low in other tissues. Analyses performed on isolated preparations of granulosa and theca cells showed a significant increase of PACAP transcripts in both cell types after hCG, whereas primary granulosa cell cultures revealed high levels of PACAP as well as its receptors PAC-1 and VPAC-2 mRNA after forskolin treatment. Overexpression of the catalytic subunit of protein kinase A (PKA) in granulosa cells stimulated, but treatment with H89 or PKA inhibitor protein inhibited PACAP mRNA expression, whereas PACAP overexpression stimulated an increase in abundance of transcripts for PGHS-2, PGES, EP2 receptor, progesterone receptor, and ADAMTS-1, but not for P450-side chain cleavage and P450 aromatase. Thus, this study demonstrates the gonadotropin-dependent regulation of PACAP mRNA in bovine preovulatory follicles, the importance of PKA activation in the expression of PACAP in granulosa cells, and stimulating effect of PACAP on gene expression during the ovulatory process.
    Reproduction (Cambridge, England) 02/2007; 133(2):441-53. · 3.56 Impact Factor
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    ABSTRACT: The mammalian ovulatory process is accompanied by a gonadotropin-dependent increase in follicular levels of prostaglandin E2 (PGE2) and PGF2alpha, which are metabolized by 15-hydroxy prostaglandin dehydrogenase (PGDH). Little is known about ovarian PGDH regulation in non-primate species. The objectives of this study were to characterize the structure of equine PGDH and its regulation in follicles during human chorionic gonadotropin (hCG)-induced ovulation. The full-length equine PGDH was obtained by RT-PCR, 5'- and 3'-rapid amplification of cDNA ends (RACE). Its open reading frame encodes a 266-amino acid protein that is 72-95% homologous to other species. Semi-quantitative RT-PCR/Southern blot were used to study PGDH regulation in follicles isolated 0-39 h post-hCG. Results showed that PGDH mRNA expression was low in follicles obtained at 0 h, increased at 12 and 24 h (P < 0.05), and decreased at 36-h post-hCG. This induction of expression was biphasic, with elevated abundance of transcripts at 12 and 33 h post-hCG (P < 0.05) in mural granulosa and theca cells. Immunohistochemistry and immunoblotting confirmed regulated expression of PGHD protein in both cell types of preovulatory follicles after hCG. High levels of PGDH mRNA were observed in corpus luteum and other non-ovarian tissues tested, except kidney, muscle, brain, and heart. Thus, this study is the first to report the gonadotropin-dependent regulation of PGDH during ovulation in a non-primate species. PGDH induction was biphasic in theca and mural granulosa cells differing from primates in which this induction was monophasic and limited to granulosa cells, suggesting species-specific differences in follicular control of PGDH expression during ovulation.
    Reproduction (Cambridge, England) 02/2007; 133(2):455-66. · 3.56 Impact Factor
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    ABSTRACT: The preovulatory rise in gonadotropins causes an expansion of the cumulus-oocyte complex, a process requiring the induction of several genes. The objectives of this study were to clone the equine tumor necrosis factor alpha-induced protein 6 (TNFAIP6), and investigate its regulation in equine follicles during human chorionic gonadotropin (hCG)-induced ovulation. The isolation of the equine TNFAIP6 cDNA revealed that it contains an open reading frame of 834 bp (including the stop codon), encoding a predicted 277 amino acid protein that is highly similar (91-93% identity) to known mammalian homologs. The regulation of TNFAIP6 mRNA was studied in equine follicles isolated during estrus between 0 and 39 h post-hCG and in corpora lutea (CL) obtained on day 8 of the estrous cycle. Results from semi-quantitative RT-PCR/Southern blot showed that levels of TNFAIP6 mRNA were low in follicles obtained at 0 h, increased at 12 h, returned to basal levels at 24 h, and increased again at 36 h post-hCG (P<0.05). Levels of TNFAIP6 transcripts were relatively moderate in CL, but low in non-ovarian tissues tested. Analyses performed with isolated preparations of theca and granulosa cells indicated that TNFAIP6 mRNA was regulated in both layers, with a maximal induction obtained 33-36 h post-hCG (P<0.05). Immunohistochemical staining of sections of equine follicles isolated at 0 and 33 h post-hCG confirmed the induction of TNFAIP6 protein in both cell types after hCG treatment. Thus, the present study describes for the first time the gonadotropin-dependent regulation of follicular TNFAIP6 during the ovulation in a monoovulatory species. The biphasic induction of TNFAIP6 in equine theca and granulosa cells differs from the pattern observed in rodents, suggesting a distinct control of gene expression in this monoovulatory species.
    Reproduction (Cambridge, England) 02/2007; 133(1):135-45. · 3.56 Impact Factor
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    ABSTRACT: Members of the organic anion transporting polypeptide (SLCO/OATP) superfamily are capable of importing anionic compounds across the lipid bilayer in a sodium-independent manner. Member 2B1 has been shown to transport few substrates, two of which are dihydroepiandrosterone-3-sulfate (DHEA-S) and estrone-3-sulfate. Steroid sulfatase (STS) catalyses the hydrolysis of these steroids into their unconjugated counterparts. The objective of this study was to investigate the regulation of SLCO2B1 and STS mRNAs during human chorionic gonadotropin (hCG)-induced ovulation/luteinization. The equine SLCO2B1 cDNA was cloned and shown to encode a 709-amino acid protein (OATP2B1) that is highly conserved when compared to mammalian orthologs. RT-PCR/Southern blot analyses were performed to study the regulation of SLCO2B1 and STS transcripts in equine preovulatory follicles isolated between 0 and 39h after hCG treatment. Results showed high levels of SLCO2B1 mRNA expression before hCG, with a marked decrease observed in follicles obtained 24-39h post-hCG (P<0.05). Analyses of isolated granulosa and theca interna cells identified high mRNA expression in both cell types prior to hCG treatment, with granulosa cells showing a more rapid SLCO2B1 mRNA down-regulation. No significant change in STS mRNA was observed in intact follicle walls. However, when both cell types were isolated, a significant decrease in STS mRNA was observed in granulosa cells 24-39h post-hCG. Collectively, these results demonstrate that the hCG-dependent induction of follicular luteinization is accompanied by the down-regulation of SLCO2B1 and STS transcripts. Considering that OATP2B1 can import sulfoconjugated DHEA and estrogens, and that STS can remove the sulfonate moiety from these steroids, their down-regulation in luteinizing preovulatory follicles may provide an additional biochemical basis for the decrease in ovarian 17beta-estradiol biosynthesis after the LH surge.
    The Journal of Steroid Biochemistry and Molecular Biology 02/2007; 103(1):10-9. · 3.98 Impact Factor
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    ABSTRACT: Caveolins are implicated in endocytosis, cholesterol trafficking and signal transduction. A cDNA fragment corresponding to caveolin-1 (CAV1) was identified in a mRNA profiling expression study in bovine granulosa cells (GC) following human chorionic gonadotropin (hCG)-induced ovulation. Thus, we have characterized CAV1 cDNA and studied its spatio-temporal expression pattern in bovine ovarian follicles. The full-length bovine alphaCAV1 cDNA was cloned and encodes a putative 22 kDa protein. Expression of alphaCAV1 was studied in bovine GC obtained from follicles at different developmental stages: small follicles (SF: 2-4 mm), dominant follicles (DF), ovulatory follicles (OF: 24 hr post-hCG), and corpus luteum (CL). Semiquantitative RT-PCR analysis showed a 6.5-fold increase in alphaCAV1 mRNA in GC of OF versus DF (P < 0.0001), whereas CAV2 mRNA was increased by only twofold (P < 0.0007). Temporal expression of alphaCAV1 mRNA from OF recovered at 0, 6, 12, 18, and 24 hr after hCG injection showed an 8.5-fold increase of alphaCAV1 mRNA after 24 hr compared to 0 hr (P < 0.0018) whereas no significant variation was detected for CAV2. Immunoblot demonstrated an initial increase in alphaCAV1 protein level 12 hr post-hCG, reaching a maximum at 24 hr. Immunohistochemical localization of CAV1 was observed in GC of OF isolated 18 and 24 hr after hCG injection, whereas no signal was detected in GC of DF and SF. The induction of alphaCAV1 in GC of OF suggests that alphaCAV1 likely contributes to control the increase in membrane signaling that occurs at the time of ovulation and luteinization.
    Molecular Reproduction and Development 11/2006; 73(11):1353-60. · 2.81 Impact Factor
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    ABSTRACT: Early growth response factor-1 (EGR-1) is a transcription factor that is involved in the transactivation of several genes. The objective of this study was to characterize gonadotropin-dependent regulation of bovine EGR-1 in preovulatory follicles prior to ovulation. Bovine EGR-1 cDNA was obtained by RT-PCR, 5'- and 3'-RACE, its open reading frame composed of 1623 bp, and its coding region encodes a 540-amino acid protein that displays 62-93% identity to known mammalian homologs. The regulation of EGR-1 mRNA was studied in bovine preovulatory follicles which were isolated 0-24 h post-hCG using semiquantitative RT-PCR/Southern blot. Results revealed that the levels of EGR-1 mRNA were very low in follicles at 0 h, markedly increased at 6 h (P < 0.05) when compared to 0 h, and decreased between 12 and 24 h post-hCG. High levels of the EGR-1 mRNA were also observed in corpus luteum, uterus, kidney, pituitary, and spleen, moderate and low in other bovine tissues tested. Analyses performed on isolated preparations of granulosa and theca cells indicated that EGR-1 mRNA was regulated in both cell types, with a predominant expression in granulosa cells. Immunohistochemistry on sections of preovulatory follicles isolated before and after hCG confirmed its protein expression in granulosa cells, 24 h post-hCG. Studies of EGR-1 regulation in primary granulosa cells cultured with forskolin showed that levels of EGR-1 mRNA were low at 0 h, highly increased at 6 h post-forskolin (P < 0.05), and declined to steady state thereafter. Immunoblotting confirmed forskolin-induced EGR-1 protein in cultures. Interestingly, overexpression of EGR-1 increased the levels of mRNA for prostaglandin (PG) G/H synthase-2 (PGHS-2), PG E synthase (PGES), PG E2 receptor (EP2), LH receptor (LH-R), but not for cytochrome P450-side chain cleavage (P450scc), and cytochrome P450 aromatase (P450arom) in granulosa cultures. Thus, this study reports for the first time, a gonadotropin-dependent induction of follicular EGR-1 prior to ovulation in large monoovulatory species and its stimulating effect on the expression of genes known to be involved in prostaglandin biosynthesis pathway, thereby suggesting its potential involvement in the regulation of preovulatory events in cattle.
    Journal of Molecular Endocrinology 11/2006; 37(2):239-50. · 3.58 Impact Factor
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    ABSTRACT: Estrogen sulfotransferase (EST) is responsible for the sulfoconjugation of estrogens, thereby changing their physical properties and preventing their action via the estrogen receptors. These sulfoconjugated steroids no longer diffuse freely across the lipid bilayer; instead, they are exported by members of the ATP-binding cassette family, such as ABCC1. The objective of this study was to investigate the regulation of EST and ABCC1 during human chorionic gonadotropin (hCG)-induced ovulation/luteinization. The transcripts for EST and ABCC1 were cloned by RT-PCR, and the regulation of their mRNAs was studied in preovulatory follicles obtained during estrus at 0, 12, 24, 30, 33, 36, and 39 h after hCG. Results obtained from RT-PCR/Southern blot analyses showed significant changes in steady-state levels of both EST and ABCC1 mRNA after hCG treatment (P < 0.05). In granulosa cells, a significant increase in EST transcript was observed 30-39 h after hCG. Similarly, ABCC1 transcript levels were induced in granulosa cells 12-39 h after hCG. In contrast, no significant changes in either EST or ABCC1 were detected in theca interna samples after hCG. The increase in EST and ABCC1 transcripts observed in granulosa cells was reflected in preparations of intact follicle walls, suggesting that the granulosa cell layer contributes the majority of EST and ABCC1 expression in preovulatory follicles. The present study demonstrates that follicular luteinization is accompanied not only by a decrease in 17 beta-estradiol biosynthesis but also by an increase in expression of genes responsible for estrogen inactivation and elimination from granulosa cells, such as EST and ABCC1, respectively.
    Endocrinology 10/2006; 147(9):4222-33. · 4.72 Impact Factor
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    ABSTRACT: Mammary cancer is the most common cancer in female dogs. Induction of cyclooxygenase-2 (COX-2), a key enzyme in prostaglandins (PGs) biosynthesis, has been demonstrated in various cancers in humans and dogs, including mammary cancer. The objective of this study was to investigate the expression and regulation of COX-2 in canine mammary epithelial cells. Cell lines derived from normal and neoplastic canine mammary glands were cultured in the absence or presence of phorbol 12-myristate 13-acetate (PMA), and immunoblots, immunocytochemistry, radioimmunoassays, and a cell proliferation assay were used to study COX-2 expression and PGs production. Results showed that the neoplastic cell line CMT12 constitutively overexpressed COX-2 protein whereas other mammary cell lines expressed low to undetectable basal levels of COX-2 protein. Basal PGE(2) production was significantly higher (P < .05) in CMT12 compared to other cell lines. Levels of COX-2 protein in CMT12 decreased in a time-dependent manner with serum starvation, and PMA stimulation induced a strong time-dependent increase in COX-2 protein. Treatment of CMT12 cells with NS-398 (a specific COX-2 inhibitor) significantly blocked PGE(2) synthesis and reduced cell proliferation (P < .05). These results indicate that some neoplastic canine mammary cell lines constitutively overexpress COX-2, and that COX-2 inhibition decreases PGE(2) production and cell proliferation, supporting a role for COX-2 and PGs in canine mammary oncogenesis.
    Veterinary Pathology 09/2006; 43(5):656-66. · 1.93 Impact Factor
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    ABSTRACT: The progressive rise in gonadotropins prior to ovulation triggers a marked increase in intrafollicular levels of prostaglandin F(2alpha)(PGF(2alpha)), which is known to interact with PGF(2alpha) receptor (FP). Little is known about the regulation of FP during ovulation. This study was undertaken to characterize the equine FP and its gonadotropin-dependent regulation in preovulatory follicles prior to ovulation. The full-length equine FP encodes a 366-amino acid protein that is 82-93% homologous to other species. Using semi-quantitative RT-PCR/Southern blot, we showed that FP mRNA expression was low in follicles obtained before hCG treatment (0h) and at 24, but increased at 12 and 36h post-hCG (P<0.05). This expression was regulated in both follicular cells, with high levels of the transcript at 33 and 36h post-hCG in granulosa cells, and at 12, 30 and 33h post-hCG in theca cells (P<0.05). Immunohistochemistry confirmed the induction of FP protein in both follicular cells after hCG, and immunoblotting revealed the increase of FP protein in preovulatory follicles 36h post-hCG. High levels of FP mRNA were detected in the corpora lutea and heart, but very low or undetectable in other tissues. This study reports for the first time the expression of FP and its up-regulation by hCG in preovulatory follicles prior to ovulation. FP regulation was occurred in different pattern than that observed in other species, suggesting a distinct and species-specific follicular control of FP expression during ovulation, and a potential involvement of PGF(2alpha), acting on granulosa and theca cells, in the ovulatory process.
    Prostaglandins & other lipid mediators 07/2006; 80(1-2):81-92. · 2.42 Impact Factor
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    ABSTRACT: Prostaglandins are required for the ovulatory process, and their biosynthesis depends on the initial release of arachidonic acid from membrane phospholipids. We hypothesized that phospholipase A2 group IVA (PLA2G4A) expression is upregulated in granulosa cells (GC) at ovulation. We have characterized bovine PLA2G4A cDNA, and investigated its spatiotemporal regulation at the mRNA and protein levels in hCG-induced ovulatory follicles and in vitro, using forskolin-stimulated GC. Regulation of PLA2G4A mRNA expression was studied in GC obtained from bovine follicles collected at different developmental stages: small follicles (2-4 mm), dominant follicles at Day 5 (D5) of the estrous cycle, ovulatory follicles 24 h following injection of hCG, and corpus luteum at D5. PLA2G4A mRNA increased by 14-fold in GC of hCG-stimulated versus dominant follicles (P < 0.0001). Follicular walls obtained from ovulatory follicles recovered at 0, 6, 12, 18, and 24 h post-hCG injection showed an initial 16-fold increase in PLA2G4A transcript at 12 h that reached a 45-fold increase at 24 h, as compared to 0 h (P < 0.0001). Immunoblots of GC extracts showed an initial induction of the PLA2G4A protein at 18 h post-hCG, reaching a maximum at 24 h. Immunohistochemistry observations showed that PLA2G4A signal was mainly observed in mural GC compared to antral GC in hCG-stimulated follicles. Stimulation of cultured bovine GC with 10 microM of forskolin caused an increase in PLA2G4A mRNA and protein. Ovulation is associated with an LH/hCG-dependent induction of PLA2G4A in GC via the adenylyl cyclase/cAMP pathway.
    Biology of Reproduction 07/2006; 74(6):1096-103. · 4.03 Impact Factor
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    ABSTRACT: Aldo-keto reductases (AKRs) are multifunctional enzymes capable of acting on a wide variety of substrates, including sex steroids. AKRs having 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) activity can reduce progesterone to 20alpha-hydroxy-4-pregnen-3-one (20alpha-DHP), a metabolite with lower affinity for the progesterone receptor. The objective of this study was to investigate the regulation of equine AKR1C23 during human chorionic gonadotropin (hCG)-induced ovulation/luteinization. The equine AKR1C23 cDNA was cloned and shown to encode a 322 amino acid protein that is conserved (71-81% identity) when compared with mammalian orthologs. RT-PCR/Southern blotting analyses were performed to study the regulation of AKR1C23 transcripts in equine preovulatory follicles isolated between 0 and 39 h after hCG treatment (ovulation occurring 39-42 h post-hCG). Results showed the presence of low AKR1C23 expression before hCG treatment, but a marked increase was observed in follicles obtained 12 h after hCG (P<0.05). Analyses of isolated preparations of granulosa and theca interna cells identified low mRNA expression in both cell types prior to hCG treatment, with granulosa cells clearly being the predominant site of follicular AKR1C23 mRNA induction. A specific polyclonal antibody was raised against a fragment of the equine protein and immunoblotting analyses showed an increase in AKR1C23 protein in granulosa cell extracts when comparing follicles isolated at 36 h post-hCG vs those collected prior to treatment, in keeping with mRNA results. Immunohistochemical data confirmed the induction of the enzyme in follicular cells after hCG treatment. The enzyme was tested for 20alpha-HSD activity and was shown to exhibit a K(M) of 3.12 microM, and a V(max) of 0.86 pmol/min per 10 microg protein towards progesterone. The levels of 20alpha-DHP measured in follicular fluid reflected this activity. Collectively, these results demonstrate for the first time that the gonadotropin-dependent induction of follicular luteinization is accompanied by an increase in AKR1C23 expression. Considering the 20alpha-HSD activity of AKR1C23, its regulated expression in luteinizing preovulatory follicles may provide a biochemical basis for the increase in ovarian 20alpha-DHP observed during gonadotropin-induced luteinization/ovulation. (The nucleotide sequence reported in this paper has been submitted to GenBank with accession number AY955082.).
    Journal of Molecular Endocrinology 06/2006; 36(3):449-61. · 3.58 Impact Factor

Publication Stats

2k Citations
305.61 Total Impact Points

Institutions

  • 1987–2014
    • Université du Québec à Montréal
      Montréal, Quebec, Canada
  • 1996–2008
    • Université de Montréal
      Montréal, Quebec, Canada
  • 1998
    • Hôpital Maisonneuve-Rosemont
      Montréal, Quebec, Canada
  • 1988–1995
    • Cornell University
      • College of Veterinary Medicine
      Ithaca, NY, United States