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Melatonin and its cytoprotective effect on the maturation of mammalian oocytes

Grant number: 13/15374-3
Support type:Regular Research Grants
Duration: April 01, 2014 - March 31, 2016
Field of knowledge:Agronomical Sciences - Veterinary Medicine
Principal Investigator:Cláudia Lima Verde Leal
Grantee:Cláudia Lima Verde Leal
Home Institution: Faculdade de Zootecnia e Engenharia de Alimentos (FZEA). Universidade de São Paulo (USP). Pirassununga , SP, Brazil


Although there have been considerable advances in the field of in vitro production (IVP) of embryos in several mammalian species, embryo development is still beyond the desirable. Among the several factors that may affect IVP results, the in vitro maturation (IVM) step plays a relevant role. Several factors are involved in the control of oocyte maturation and the resulting quality and developmental competence of the oocyte. Melatonin is a hormone synthesized in the pineal gland and exerts many functions including antiapoptotic and antioxidant activity, besides affecting different cell signaling pathways. There are few studies on the role of melatonin on oocyte maturation and the mouse is a convenient model for in vivo and in vitro studies due to its fast reproduction and lower maintenance costs. In pigs there is also limited information, although it is a species of commercial and biomedical interest. Therefore, we propose to study the effect of melatonin on oocyte maturation in vivo (mouse model) and in vitro (mouse and pig models) and its role as a cytoprotective agent in mammalian female gametes. The project is divided in two blocks, where Block I is related to the mouse study and Block II to the pig study. In Block I the effect of melatonin 0, 10 and 20 mg/kg/i.p. for 2-3 days) on in vivo maturation will be evaluated. In vivo matured oocytes (OO) will be collected and separated from the cumulus cells (CC) and assessed for nuclear maturation while CC will be analysed for expression of aopoptosis (Bax and Bcl-2) and antioxidant enzymes genes (GPx and SOD). In Experiment 2, animals will be prepared as before but cumulus oocyte complexes (COCs) will be collected immature and matured in vitro for 14-16 h and the same evaluations performed. In the last experiment, COCs will be collected as in the previous experiment, but without previous melatonin treatment of the animals and COCs will be matured with or without melatonin and hydrogen peroxide (to cause apoptosis) to test the protective effect of melatonin. After treatments, OO will be evaluated for maturation rates and ATP levels and CC for gene expression. In Block II the protective effect of melatonin on in vitro maturation and in vitro parthenogenetic embryo development and cryotolerance will be tested in porcine oocytes. Melatonin will be added to maturation medium (0, 10-6 and 10-9 M) and the following assessments will be performed: Exp 1: maturation rates and cortical granule distribution at 36, 40 and 44 h IVM; Exp2: expression of antioxidant enzymes in CC (catalase, GPx and SOD); Exp 3: the best melatonin concentration selected form the previous experiments will be used to assess parthenogenetic embryo development in vitro; embryos will be analysed also for expression of apoptosis genes (Bax and Bcl-2) and number of apoptotic cells; Exp 4: embryos produced from melatonin treated or not treated oocytes will be cryopreserved by vitrification and the rates of reexpansion, hatching and apoptotic cell index in embryos will be determined. (AU)

Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
DE CASTRO, FERNANDA CAVALLARI; COELHO CRUZ, MARIA HELENA; VERDE LEAL, CLAUDIA LIMA. Role of Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 in Ovarian Function and Their Importance in Mammalian Female Fertility - A Review. ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES, v. 29, n. 8, p. 1065-1074, AUG 2016. Web of Science Citations: 15.

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