With all advances on embryo in vitro production (IVP) on several domestic species, embryo yield and quality are still beyond the desireable. Among several factors interferring with success, the in vitro maturation stage (IVM) is certainly fundamental. Many factors are involved in the control of oocyte maturation and developmental competence. Melatoni is a pineal hormone showing varied functions including antioxidant and antiapoptotic activities, besides influencing many cell signaling pathways. Melatonin has been detected in follicular fluid and its receptors found in oocytes and granulosa cells. In vitro studies have shown positive effects on oocyte maturation and embryo development. There are few studies on the effects of melatonin on oocyte maturation and the mouse, due to its fast reproduction and lower cost for maintencance, is an interesting model, widely used for in vitro studies, but also in vivo, which are much slower and expensive in domestic animals. Therefore, we propose to study the effects of melatonin on in vivo and in vitro maturation and its cytoprotective action (antioxidant/antiapoptotic)in murine cumulus-oocyte complexes (COCs). The effect of different doses of melatonin injections 0, 10 and 20 ng/kg/i.p. for 2-3 days)for in vivo treatment of mouse females will be studied. Treatment will begin with 5 IU eCG for follicular development. To assess the effect on in vivo maturation, animals receiving melatonin injections will receive 5IU hCG to induce maturation and ovulation animais que receberam melatonina, 48 h after the first injection (eCG). Sixteen hours later in vivo matured COCs will be collected. Oocytes(OO) wil be separated from their cumulus cells (CC) and will be analyzed for maturation rates (1st polar body extrusion - 1PB). CC will be analyzed by PCR for expression of apoptosis (Bax and Bcl2) and antioxidant enzymes (SODs and GPx). On the second experiment, effects of in vivo treatment on in vitro maturation will be evaluated. Similar treatment will be given to animals as described in the first one, except the hCG will not be given, but insted, immature COCs will be collected from the ovaries and matured in vitro with FSH. The same analyses in OO and CC will be done at the end of maturation. On the third experiment, different concentrations of melatonin during IVM. OO and CC will be analyzed as previoulsy. On the fourth experiment, oocytes will be in vitro matured with H2O2 to induce oxidative stress and another group with the association of melatonin to evaluate its cytoprotective effects. OO and CC will be assessed and described previously, and OO will be additionally evaluated for ATP prodution.
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