Gustavo Zamberlam

Université de Montréal, Montréal, Quebec, Canada

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Publications (7)18.89 Total impact

  • Gustavo Zamberlam, Fatiha Sahmi, Christopher A Price
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    ABSTRACT: In rabbits and rodents, nitric oxide (NO) is generally considered to be critical for ovulation. In monovulatory species, however, the importance of NO has not been determined, nor is it clear where in the preovulatory cascade NO may act. The objectives of the present study were (1) to determine if nitric oxide synthase (NOS) enzymes are regulated by luteinizing hormone (LH) and (2) to determine if and where endogenous NO is critical for expression of genes essential for the ovulatory cascade in bovine granulosa cells in serum-free culture. Time- and dose-response experiments demonstrated that LH had a significant stimulatory effect on endothelial NOS (NOS3) mRNA abundance but in a prostaglandin-dependent manner. NO production was stimulated by LH before a detectable increase in NOS3 mRNA levels was observed. Pretreatment of cells with the NOS inhibitor, L-NAME, blocked the effect of LH on the epidermal growth factor (EGF)-like ligands epiregulin and amphiregulin, as well as prostaglandin-endoperoxide synthase-2 (PTGS2) mRNA abundance and protein levels. Similarly, EGF treatment increased mRNA encoding epiregulin, amphiregulin and the early response gene EGR1, and this was inhibited by pretreatment with L-NAME. Interestingly, pretreatment with L-NAME had no effect on either ERK1/2 or AKT activation. Taken together, these results suggest that endogenous NOS activity is critical for LH-induced ovulatory cascade in granulosa cells of a monotocous species and acts downstream of EGF receptor activation but upstream of the EGF-like ligands.
    Free radical biology & medicine. 06/2014;
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    ABSTRACT: The ovarian promoter of the primate and rodent genes encoding cytochrome P450 aromatase (CYP19A1) are robustly responsive to forskolin in luteinized cell models, whereas the ruminant ovarian promoter is minimally active. We explored this discrepancy by investigating the activity of the bovine ovarian promoter in two bovine granulosa cell models, luteinizing and non-luteinizing cells in vitro. In non-luteinizing cells, both FSH and IGF1 increased abundance of transcripts derived from the ovarian promoter. Comparison of the activity of promoters of several species in response to transcription factors (forskolin, NR5A2, FOXL2) in luteinizing cells demonstrated that a rat ovarian promoter-luciferase reporter was regulated mainly by forskolin (18-fold increase over basal expression) and addition of NR5A2 or FOXL2 had no further effect. Activity of a human promoter was significantly increased by NR5A2 plus forskolin (153-fold) compared with forskolin alone (71-fold over basal); addition of FOXL2 did not significantly increase promoter activity. Forskolin alone provoked minor activation of caprine and bovine promoter reporters (3-fold over basal), and addition of NR5A2 increased activity (7 to 11-fold). When forskolin, NR5A2 and FOXL2 treatments were combined, the activity of the caprine and bovine promoters increased to 20- and 34-fold, respectively. These data suggest that a major reason why CYP19A1 is not expressed in luteinized cells (and the corpus luteum) of ruminants may be the stimulatory effect of FOXL2, which does not appear to be the case in the human and rat.
    General and Comparative Endocrinology 01/2014; · 2.82 Impact Factor
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    ABSTRACT: Fibroblast growth factors (FGFs) are involved in the paracrine modulation of ovarian function, and typically exert mitogenic effects by inhibiting apoptosis and increasing cell proliferation. However, in a previous study with bovine granulosa cells we observed one FGF had the opposite effect; addition of FGF18 increased caspase-3 activation and increased the proportion of apoptotic cells, and FGF18 mRNA levels were higher in atretic follicles compared with healthy follicles. The objectives of the present study were (1) determine if culture conditions that increase apoptosis also increase FGF18 mRNA levels, and (2) to identify the mediators of FGF18-induced apoptosis in granulosa cells. Granulosa cells were collected from 2-5 mm diameter follicles and cultured in serum-free medium supplemented with insulin. At the medium change on day 2, FSH (1 ng/ml) was added to the cells. Treatments were applied on day 4 and cells harvested on day 6. In the first experiment, cells were treated with insulin alone, IGF alone or neither (to reduce cell viability). FGF18 mRNA levels were significantly upregulated by the withdrawal of IGF/insulin support, and this was associated with an increase in abundance of mRNA encoding the pro-apoptotic factor FasL, and a decrease in CYP19A1 mRNA levels. Estradiol is mitogenic in granulosa cells, therefore we manipulated estradiol levels by withdrawal of FSH or the replacement of the aromatase substrate androstenedione with the non-aromatizable androgen DHT; both conditions increased FGF18 mRNA levels, and addition of exogenous estradiol failed to reduce FGF18 mRNA levels to those of control. To identify mediators of FGF18 action, cells were treated with FGF18 from day 2 of culture. FGF18 increased apoptosis, as measured by cell cycle analysis and immunofluorescence for cleaved caspase-3, and this effect was abrogated by the addition of estradiol. In contrast, addition of FGF2 did not increase granulosa cell apoptosis, even though both FGFs inhibited estradiol secretion. We then assessed the effect of FGF18 on an apoptotic pathway known to be influenced by estradiol, the MDM2-p53 pathway. Addition of FGF18 inhibited abundance of MDM2 mRNA and this was reversed by estradiol. FGF18 also inhibited abundance of mRNA encoding the MDM2 binding protein, MTBP, and this was again reversed by estradiol. Two p53 target genes are BBC3 and BAX; FGF18 did not alter BAX mRNA levels but significantly increased BBC3 mRNA levels, and this was reversed by estradiol. Finally, to assess the effect of FGF18 on follicle growth, we used an established model of intrafollicular injection in cattle in vivo. Injection of the growing dominant follicle caused follicle regression within 24 h of injection, whereas saline-injected control follicles continued to grow for 72 h post-injection. We conclude that increased FGF18 expression may be part of an atretic pathway in bovine follicles, and that FGF18 act through an estrogen-sentitive MDM2 pathway. This work was supported by CNPq, Brazil and NSERC, Canada.
    Society for Study of Reproduction, SSR; 07/2013
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    ABSTRACT: Angiotensin II (AGT-2) induces follicular prostaglandin release in a number of species and ovulation in rabbits. Conversely, AGT-2 antagonists block ovulation in cattle. To determine the mechanism of action of AGT-2, we used a bovine granulosa cell model in which luteinizing hormone (LH) increased the expression of genes essential for ovulation in a time- and dose-dependent manner. The addition of AGT-2 to LH-stimulated cells significantly increased abundance of prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA and protein, whereas AGT-2 alone had no effect. Upstream of PTGS2, AGT-2 increased abundance of mRNA encoding the epidermal growth factor-like ligands amphiregulin (AREG) and epiregulin (EREG) at 6 h posttreatment and abundance of a disintegrin and metalloprotease 17 (ADAM17), a sheddase, within 3 h of treatment. Inhibiting sheddase activity abolished the stimulatory effect of AGT-2 on AREG, EREG, and PTGS2 mRNA. The addition of selective AGT-2 antagonists to cells stimulated with LH plus AGT-2 demonstrated that AGT-2 did not act through the type 1 receptor and did not increase mitogen-activated protein kinase 3/1 phosphorylation. Combined with previous data from studies in vitro, we conclude that AGT-2 is an essential cofactor for LH in the early increase of ADAM expression/activity that induces the cascade of events leading to ovulation.
    Biology of Reproduction 08/2011; 85(6):1167-74. · 4.03 Impact Factor
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    ABSTRACT: Nitric oxide (NO) is a potential regulator of ovarian follicle growth, and ovarian granulosa cells reportedly generate NO in response to gonadotrophins, suggesting that the regulated form of nitric oxide synthase (iNOS) is present. The objectives of the present study were to gain insight into the expression and role of iNOS in the follicle. Messenger RNA encoding iNOS was detected in granulosa cells, and abundance was higher in growing dominant follicles compared to subordinate follicles (P<0.01). FSH (P<0.05) and IGF1 (P<0.01) stimulated oestradiol secretion and iNOS mRNA abundance in granulosa cells in vitro, whereas FGF2 (P<0.05) and EGF (P<0.01) decreased oestradiol secretion and iNOS expression. The addition of an anti-oestrogen prevented FSH-induced iNOS mRNA accumulation. Inhibition of endogenous NO production did not affect steroidogenesis in granulosa cells, but increased FasL mRNA abundance, caspase-3 activation and the incidence of apoptotic cell death (P<0.05). These results demonstrate that iNOS is expressed in ruminant granulosa cells and is regulated by gonadotrophins and oestradiol. Physiological levels of NO may contribute to the survival of granulosa cells.
    Molecular and Cellular Endocrinology 01/2011; 335(2):189-94. · 4.04 Impact Factor
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    ABSTRACT: Fibroblast growth factors (FGF) are involved in paracrine signaling between cell types in the ovarian follicle. FGF8, for example, is secreted by oocytes and controls cumulus cell metabolism. The closely related FGF18 is also expressed in oocytes in mice. The objective of this study was to assess the potential role of FGF18 in follicle growth in a monovulatory species, the cow. Messenger RNA encoding FGF18 was detected primarily in theca cells, and in contrast to the mouse, FGF18 was not detected in bovine oocytes. Addition of FGF18 protein to granulosa cell cultures inhibited estradiol and progesterone secretion as well as the abundance of mRNA encoding steroidogenic enzymes and the follicle-stimulating hormone receptor. In vivo, onset of atresia of the subordinate follicle was associated with increased thecal FGF18 mRNA levels and FGF18 protein in follicular fluid. In vitro, FGF18 altered cell cycle progression as measured by flow cytometry, resulting in increased numbers of dead cells (sub-G1 peak) and decreased cells in S phase. This was accompanied by decreased levels of mRNA encoding the cell cycle checkpoint regulator GADD45B. Collectively, these data point to a unique role for this FGF in signaling from theca cells to granulosa cells and suggest that FGF18 influences the process of atresia in ovarian follicles.
    Biology of Reproduction 09/2010; 83(3):339-46. · 4.03 Impact Factor
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    ABSTRACT: To determine if initial cell plating density alters steroidogenesis and the E(2):P ratio in granulosa cells in long-term serum-free culture. Experimental study. Academic institution. Cattle of slaughterhouse origin. Culture of granulosa cells in vitro at different cell plating density. Steroid secretion was measured by RIA, mRNA levels were measured by real-time polymerase chain reaction, and cell death was assessed by flow cytometry. Low plating density favored E(2) secretion and mRNA encoding estrogenic enzymes, whereas higher density inhibited E(2) secretion and enhanced P secretion and levels of mRNA encoding progestagenic enzymes. Increasing plating density decreased the E(2):P ratio and cell health. Lower cell density favors an estrogenic granulosa cell phenotype, whereas higher density favors luteinization. Serum-free culture systems should be optimized with this in mind.
    Fertility and sterility 04/2009; 93(6):2050-5. · 3.97 Impact Factor