IHEM   20887
INSTITUTO DE HISTOLOGIA Y EMBRIOLOGIA DE MENDOZA DR. MARIO H. BURGOS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Calcium oscillations pattern in in vitro and in vivo matured oocytes
Autor/es:
WETTEN, P; CAPPA AI; CAPPA AI; GARRIDO F; GARRIDO F; MICHAUT MA; MICHAUT MA; WETTEN, P
Reunión:
Congreso; XXXVI Reunión Científica Anual Sociedad de Biología de Cuyo; 2018
Resumen:
Before fertilization, mammalian eggs are arrested at the metaphase stage of the second meiosis (MII). Sperm entry triggers a series of increases in the intracellular free-Ca2+ concentration, termed calcium oscillations, which enable exit from the MII and induce egg activation. In vitro maturation has become an important tool in assisted reproductive technology in humans and animals, but it is unknown how it affects oocyte activation and embryo development. One of the first steps in oocyte activation is the cortical reaction, which consists in the exocytosis of small vesicles called ?cortical granules?. This exocytosis is activated by the first arise of intracellular calcium after the first sperm entry and is responsible for blocking polyspermy. We have developed a method that allows the analysis of cortical reaction in live oocytes activated parthenogenetically with SrCl2, which produces calcium oscillations mimicking the sperm entry. Using this method we have shown that in vitro maturation alters cortical reaction in mouse MII oocytes. Based on this observation, we hypothesized that the altered cortical reaction is due to altered calcium oscillations in in vitro matured oocytes. In consequence, the aims of this work were: 1) to set up the standard method to observe and analyze calcium oscillations in live mouse oocytes and 2) to compare calcium oscillations in in vivo and in vitro matured oocytes. Ovulated oocytes (in vivo matured) were obtained from superovulated CF-1 females (8-12 weeks). In vitro matured oocytes were collected 45 h after PMSG injection and in vitro matured overnight in G-IVF medium. In vivo and in vitro oocytes were loaded with Fura2-AM and activated parthenogenetically with SrCl2. The increase of intracellular free-Ca2+ concentration in live oocytes was registered every 10 seconds during 1 h in a thermostatic chamber by confocal microscopy. Results showed that in vivo matured oocytes had 3-4 calcium oscillations during the first hour; however, in vitro matured oocytes showed 0-1 calcium oscillations during the same time. In conclusion, our results showed that in vitro maturation affects the calcium oscillations pattern in mouse MII oocytes.