The present study evaluated the effects of melatonin (MEL) during IVM of bovine cumulus-oocyte complexes (COC). The COC were cultured in droplets (25–30/100 µL) under mineral oil at 38.5°C and 5% CO2 in air. Medium (TCM199 + 0.1% polyvinyl alcohol, 0.25 mM sodium pyruvate, and 25 µg mL–1 of gentamycin) was supplemented with FSH (0.5 µg mL–1), MEL (10–9 and 10–6 M), or no hormones (control). In experiment 1, oocytes were assessed for nuclear maturation rates (6, 12, 18, and 24 h of IVM). In experiment 2, relative abundance of antioxidant enzymes copper, zinc superoxide dismutases (CuZnSOD), manganese superoxide dismutases (MnSOD), and glutathione peroxidase (GPx) was evaluated in oocytes and cumulus cells (0 and 24 h) by real time PCR. The immature group was the reference and endogenous controls were actin β and glyceraldehyde 3-phosphate dehydrogenase. In experiment 3, nuclear fragmentation in cumulus was assessed by TUNEL and flow cytometry (24 h). In experiment 4, embryo development after in vitro fertilization and culture was analysed (cleavage rates D2, blastocysts Day 8, and hatching Day 10). The control group was matured in complete IVM medium (10% FCS, 0.5 µg mL–1 of FSH, 50 µg mL–1 of LH, and 1 µg mL–1 of oestradiol). The MEL and FSH groups were the same. Data (3–4 replicates) were analysed by Chi-square (experiment 1; GraphPad Prism) or ANOVA and Tukey test (SAS, 1995) and 5% significance. At 6 h of IVM, all oocytes were at germinal vesicle stage. At 12 h, hormone groups had similar metaphase I rates (71–81%, P > 0.05) and were superior to control (54%, P < 0.05). At 18 h, metaphase II (MII) rates were similar (57–74%, P > 0.05). After 24 h, MEL (10–9 and 10–6 M) was similar to control (51, 67, and 69% MII, respectively, P > 0.05) and FSH had the highest MII rates (90%, P > 0.05). The 10–6 M MEL was similar to FSH (P > 0.05). Antioxidant enzymes were unaffected in oocytes (P > 0.05%). The CuZnSOD transcripts increased in cumulus after IVM with 10–6 M MEL compared to immature cells (3.5 and 1.0, respectively, P < 0.05). Control, FSH, and 10–9 M MEL did not differ (2.1–2.5, P > 0.05) or increased relative to immature cells (P < 0.05), but were inferior to 10–6 M MEL (P < 0.05). The MnSOD relative abundance was similar for immature, control, and 10–9 M MEL (1.0–3.0, P > 0.05). The 10–6 M MEL increased MnSOD relative to immature cells (3.0 and 1.0, respectively, P < 0.05), but was similar to control and 10–9 M MEL (P > 0.05). The FSH showed the highest levels (9.1, P < 0.05); GPx4 transcripts were not affected (P > 0.05). Nuclear fragmentation in cumulus was not influenced (33.4–41.5/10 000 cells; P > 0.05). Embryo development rates were similar for all groups (cleavage: 82–87%, blastocysts: 49–54, hatching: 91–96%, P > 0.05). In conclusion, MEL during IVM stimulates meiosis resumption at rates similar to FSH and embryo development similar to FSH and complete IVM medium. The MEL increased CuZnSOD expression in cumulus, but no parallel effect was observed on nuclear fragmentation.

No abstract

Ao meu grande amor Josiel Valerino da Cunha, pelo incentivo constante na realização desse sonho. Por todo amor, carinho, paciência e providão dedicado a mim durante a realização do mesmo. Aos meus queridos pais, Antônio Carlos Rodrigues e Maria Zeni Cardozo Rodrigues, por todo amor, carinho e sabedoria presente em nosso lar, cujos valores e virtudes contribuíram para as escolhas que fiz, sobretudo,acreditar na educação, quem me trouxe até aqui. Às minhas queridas irmãs, Lenita Rodrigues e Beatriz Rodrigues, que tanto amo. Quem sempre torceu pela realização desse sonho. AGRADECIMENTOS À Deus, Quem viu em meu coração o desejo de fazer ciência e me abençoou na realização desse sonho. Guardou-me e me capacitou ao ingresso da Universidade, e diante das dificuldades enfrentadas na pesquisa, me abraçou com seu amor, tornando-me forte para enfrentá-las com mérito. À Faculdade de Zootecnia e Engenharia de Alimentos, por proporcionar a realização do curso de Mestrado. À minha família, pelo apoio, incentivo e auxílio desde os primeiros momentos em que esse sonho começara a nascer em mim. Ao meu amado esposo, meu grande companheiro, por sustentar essa minha dedicação à pesquisa, e acreditar nos frutos que colheremos juntos através desse tempo em que se fez presente na minha vida acadêmica. Em você encontro a força que me inspira permanecer caminhando na busca da realização de tantos outros sonhos. À Profa. Dra. Claudia Lima Verde Leal, minha orientadora, quem acreditou em mim e abriu as portas para eu fazer ciência. A quem eu devo toda gratidão pelos seus ensinamentos, conselhos, apoio constante, disponibilidade, amizade e dedicação ao meu projeto de pesquisa. A quem eu sempre me espelharei como exemplo de profissionalismo, competência e grande amor a área acadêmica. À Fernanda Cavallari Castro, minha irmãzinha nas rotinas do laboratório. Minha imensa gratidão por ter me acolhido de uma forma tão especial a casa em que passou a ser nossa durante esses três anos. Agradeço por compartilhar comigo o que o ser humano guarda de mais precioso em seu coração, o conhecimento... Conhecimentos básicos e ao mesmo tempo fundamentais, a mim necessários para que ao longo dessa jornada, eu pudesse caminhar com meus próprios passos e assim crescer, adquirir maturidade, ter a liberdade de decidir, optar por escolhas, enfrentar os muitos espinhos... e hoje poder dizer que os venci. Meus sinceros agradecimentos pela atenção, paciência e disponibilidade dedicada a mim. À Ligia Garcia Mesquita, pela atenção, paciência e disposição em me auxiliar. Agradeço de coração pela oportunidade de trabalhar contigo e reconhecer em você o exemplo de inteligência, competência, profissionalismo, dedicação à pesquisa, e acima de tudo, ser dona de um precioso coração disposto a me aconselhar, me acalmar, me fazer perseverar e crer no sonho que sonhei pra mim. Minha eterna gratidão por ter enfrentado ao meu lado os desafios da Biologia Molecular. À minha querida amiga Gabriella Mamede, pela linda amizade construída no decorrer desses três anos. Sou muito grata p...

Intracellular levels of cyclic adenosine monophosphate modulators (cAMP) and cGMP, in adipocytes, are important for the regulation of the lipolysis rate. The phosphodiesterases (PDE) control cGMP and cAMP levels by degradation. Different PDE isoforms are expressed in bovine oocytes and cumulus cells. Previously, we found that using an inhibitor of PDE5A (sildenafil, SILD) increased cGMP levels in bovine oocytes during in vitro maturation (IVM). In the current study we investigated if inhibition of PDE5A during maturation reduces the lipid content in IVF embryos. For this, oocytes were cultured for 24 h in maturation medium with 10% FCS and 10–7 M SILD (treatment I), 10% FCS (treatment II) and 0.4% BSA (control; N ± 160 COC/groups submit to IVF). After COC were in vitro fertilized, cleavage (Day 4) and blastocyst rates (Day 7) were measured. Blastocysts were stained with Nile Red (1 μg mL–1) for lipid content quantification, by mean fluorescence intensity per μm2, measured in the ImageJ program (fluorescence intensity, f.i.). Four replicates were transformed to log10 and subjected to statistical analysis using the SAS system (SAS Institute Inc., Cary, NC, USA) by ANOVA followed by Tukey test with a significance level of 5%. No difference in cleavage (Day 4) and blastocyst (Day 7) rates were observed in all groups (82 and 41.9%, respectively), showing that presence of FCS, SILD, or both in IVM medium did not affect embryo development. Treatment I had higher lipid content (40.35 f.i.) than treatment II (31.12 f.i.), which in turn was also superior to control (22.31 f.i.). According to the results, the presence of FCS in IVM media generates embryos with higher lipid content, and association of FCS and SIL further increased lipid content. Although inhibition of PDE5 increases cGMP levels and leads to higher lipolysis, such an effect was not observed when SIL was used as the PDE5 inhibitor. Reasons for such findings are still unclear, but a possibility would be the activation of a negative feedback mechanism by the increased cGMP generated by SIL, because this nucleotide activates PKG, which in turn inhibits cGMP synthesis by guanylate cyclase. During development the lower cGMP levels could reduce lipolysis, resulting in increased lipid accumulation in embryos. Further studies are needed to address this possibility.