Recent advances in ovulation synchronization and superovulation in ...

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Abstracts. II International Symposium on Animal Biology of Reproduction, Nov. 19-22, 2008, São Paulo, SP, Brazil. Renin-angiotensin system (RAS) components are overexpressed after PMSG treatment in the rat ovary V.M. Pereira 1 , F.M. Reis 2 , K. Honorato-Sampaio 1 , S.H. Santos 1 , R.A. Santos 1 , A.M. Reis 1 Departments of 1 Physiology & Biophysics, 2 Obstetrics & Gynecology, Federal University of Minas Gerais, 31270-901, Belo Horizonte, Brazil. Introduction Many studies point to an involvement of the ovarian Renin-Angiotensin System (RAS) as a paracrine/autocrine regulator of follicular development, steroidogenesis, oocyte maturation, ovulation, and follicular atresia [1]. In spite of these studies, the role of Angiotensin II (Ang II) in the ovarian physiology was not completely clarified. Furthermore, Angiotensin-(1-7) [Ang-(1-7)], an new active peptide of the RAS has been identified in the ovary [3]. Ang-(1-7) can be generated from Ang I by neutral-endopeptidase (NEP) and prolil-endopeptidase (PEP) or from Ang II by PEP or angiotensin-converting enzyme 2 (ACE-2) [2]. Costa et al. (2003) showed that Ang-(1-7) induces estradiol and progesterone production by in vitro perfused rat ovaries, an effect blocked by A-779, a specific Ang- (1-7) antagonist. More recently, it has been demonstrated that Mas protein is a specific receptor for Ang-(1-7) [4]. The present study was undertaken to identify the cellular localization of Ang-(1-7) and the expression of Mas receptor and ECA2 in the rat ovary following gonadotropin stimulation. Materials and Methods 1) Animals: female Wistar immature rats (23-25 days old) were injected sc with PMSG (20 IU) or vehicle solution and sacrificed 48 hours later. 2) Immunohistochemistry: after anesthesia and laparotomy, rats were perfused with 4% paraformaldheyde, ovaries were removed, embedded in paraffin, sectioned at 4 μm and mounted on gelatinized slides. Tissue distribution of Ang-(1-7), Ang II and Mas receptor was evaluated by immunohistochemistry, following the avidin-biotinperoxidase method, using rabbit polyclonal specific antibodies. The immunostaining was visualized with DAB and counterstained with hematoxylin. 3) Real Time Polimerase Chain Reaction: mRNA expression levels of Mas receptor and ECA2 were evaluated by quantitative RT-PCR. Rats were sacrificed by decapitation and ovaries immediately removed, frozen in liquid nitrogen and stored at -80 o C for RNA extraction according phenol-guanidine isothiocyanate (Trizol) protocol. cDNA was synthetized from 1 μg total RNA and subsequently submmited to real time PCR analysis, conducted following manufactor’s procedures (Apllied Biosystems) using SYBR Green Master Mix. Specific primers for Mas and ECA2 were used and expression levels were normalized to the S26 housekeeping gene and calculated as 2 -ΔΔCt . Results and Discussion PMSG treatment increased Ang-(1-7), Ang II and Mas immunoreactivity in rat ovaries: strong staining for Ang-(1- 7) and Mas was observed mainly in thecal-interstitial cells, while Ang II was more evident in the granulosa layer. The expression of Mas mRNA was higher in treated (3.36 ± 0.48 UA) compared to control animals (1.66 ± 0.47 UA). Also, PMSG treatment enhanced ECA2 mRNA levels (2.15 ± 0.26 UA) compared to control (1.29 ± 0.25 UA), suggesting that this enzyme can be responsible for Ang-(1-7) synthesis in the gonadotropin-stimulated rat ovary. These data provide evidence for gonadotropin-induced changes in the ovarian expression of renin-angiotensin system components. Hence, we may suggest that the Ang-(1-7)/Mas axis, present in theca-interstitial cells, could stimulate the production of androgen precursors, which are later converted to estradiol in granulosa cells under Ang II action. References 1) Yoshimura Y. 1997. Front Neuroendocrinol, 18:247-91; 2) Santos et al. 2000. Regul Peptides, 91:45-62; 3) Costa et al. 2003. Endocrinology, 144:1942-1948; 4) Santos et al. 2003. PNAS, 100:8258-8263. E-mail: virginiamara@gmail.com Anim. Reprod., v.6, n.1, p.205, Jan./Mar. 2009 205
206 Abstracts. II International Symposium on Animal Biology of Reproduction, Nov. 19-22, 2008, São Paulo, SP, Brazil. Immunolocalization of BRG1 – SWI/SNF protein during folliculogenesis in the porcine ovary L.A. Lisboa 1 , V. Bordignon 2 , M.M. Seneda 1 1 Laboratório de Reprodução Animal DCV CCA UEL, Londrina, PR, 86051-990. 2 Department of Animal Science – McGill University, Montreal, Canada Introduction Dynamic changes in chromatin structure and gene expression occur during follicular and oocyte growth. Epigenetic mechanisms regulate these changes through biochemical reactions that modify the nucleosome structure, affecting DNA expression. Chromatin remodelers that alter DNA-histones interactions can influence transcriptional activity by facilitating or repressing DNA access. The SWI/SNF complex represents an important chromatin remodelling family, which comprises many proteins subunits including the BRG1 (brahma-related gene 1). Our aim in this study was to perform a qualitative approach of BRG1 expression patterns in different stages of follicular development. Material and Methods Ovaries (n = 10) were collected from pre-pubertal gilts after evisceration and then rinsed in cold PBS. Ovarian fragments were cut and placed into a 4% paraformaldehyde solution at 4˚C for 12 hours. Tissues were processed using standard histological protocols. For immunofluorescence, samples were deparaffinized and rehydrated through three changes of alcohol. After washing in PBS-Brij, antigen retrieval was performed by incubating the tissue sections in sodium citrate buffer for 5 min in a pressure cooker. Sections were subsequently blocked for 1 h with normal goat serum and then incubated with primary antibodies, polyclonal rabbit anti-BRG1 (1/200) or control rabbit IgG at the same concentration, overnight at 4˚C. Primary antibodies were detected using Alexa Fluor 594anti-rabbit 1/1000 diluted secondary antibody. Cells were counterstained with DAPI. We evaluated primary, secondary, tertiary and antral follicles. Results and Discussion Positive fluorescent signal for BRG1 was detected in all analyzed samples. In primary follicles, only oocytes showed BRG1. Secondary follicles expressed positive signals in the oocyte and few granulosa cells. At the tertiary stage, a strong expression was identified in the oocyte, suggesting a co-localization with euchromatin. At this stage, only granulosa cells close to theca layer were positive. On antral follicles, almost all granulosa and theca cells showed positive signals to BRG1, suggesting a possible role on proliferative and steroidogenic events. Our results suggest an important role of BRG1 in all steps of oocyte development, since primary to antral phase. Granulosa and theca cells showed positive signals especially at steroidogenic steps. Considering BRG1 a candidate gene for embryonic genome activation (1), our results suggest an important role of this chromatin remodeler since oocyte into primary follicles. The strong signal during steroidogenic phases is in accordance with BRG1 activity in estrogenic receptors (2). Finally, we consider the possibility of an important role played by BRG1 during growth and differentiation of oocytes and follicles, possibly playing activities at cellular proliferation. Primary follicle Secondary follicle Tertiary follicle Antral follicle Figure 1. Immunofluorescence of BRG1 expression (red) in primary, secondary, tertiary and antral follicles (blue, DAPI stain).O oocyte, GC granulosa cells, TC theca cells. References (1) Bultman S, Gebuhr TC, Pan H, Svoboda T, Schultz R.M, Magnuson T. 2006. Maternal BRG1 regulates zygote genome activation in the mouse. Genes Dev, 20:1744-1754; (2) Ichinose H, Garnier JM, Chambon P, Losson R. 1997. Liganddependent interaction between the estrogen receptor and the human homologues of SWI2/SNF2, Gene, 188:95-100. E-mail: mseneda@uel.br Anim. Reprod., v.6, n.1, p.206, Jan./Mar. 2009
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