Cholesterol depletion affects the biophysical state of oocyte membranes disturbing

BUSCHIAZZO, J.; ALONSO, T. S.; BONINI, I.C; ANTOLLINI, S.S.

Rosario

Congreso; 49th Annual Meeting of the Society of Cryobiology; 2012

CHOLESTEROL DEPLETION AFFECTS THE BIOPHYSICAL STATE OF OOCYTE MEMBRANES DISTURBING AMPHIBIAN FERTILIZATION Buschiazzo J*, Alonso TS, Bonini IC and Antollini SS Instituto de Investigaciones Bioquímicas de Bahía Blanca, Camino La Carrindanga km. 7, C.C. 857, B8000FWB Bahía Blanca, Argentina. The steroid hormone progesterone induces the resumption of meiosis of full-grown ovarian oocytes arrested at the first meiotic division. Apart from protein-mediated signaling, progesterone-induced maturation involves enzyme activations that modify membrane fluidity and release a cascade of lipid messengers. Once the oocyte is mature, it enters meiosis II and spontaneously arrests in metaphase until fertilization, in which drastic membrane reorganization occurs when amphibian sperm fuses with the oocyte membrane and exocytosis of cortical granules takes place. The predominant lipids in most membranes are phospholipids and cholesterol. Cholesterol stabilizes membranes at low temperatures and it is an important constituent of membrane rafts. In this study, the cholesterol-binding drug methyl-β-cyclodextrin (MβCD) was used for cholesterol modulation in order to assess membrane raft involvement in amphibian fertilization. The generalized polarization (GP) of the fluorescent probe Laurdan was used to study the effect of cholesterol depletion and fertilization on the biophysical properties of membranes from Rhinella arenarum oocytes evaluated in temperatures ranging from 5°C to 40°C. For comparative reasons, cholesterol removal of ovarian oocytes and progesterone-induced maturation were also analyzed. As it was expected for native membranes of complex lipid composition, GP values gradually decreased when temperature increased. Light (L) and Heavy (H) membranes, enriched in rafts and non-raft plasma membrane, respectively, showed two different thermotropic profiles of GP. GP values were higher in L fraction with respect to H indicating a higher membrane lipid order in agreement with the enrichment in cholesterol of these low-density membranes. Cholesterol depletion of ovarian oocytes, mediated by MβCD, caused a decrease in the GP value of L membranes which corresponds to a greater penetration of water molecules and indicates a decrease in membrane lipid order. In contrast, GP values of H membranes increased after MβCD treatment. As a result, both plasma membrane fractions become more similar in their biophysical states. On the other hand, progesterone modified the biophysical properties of the plasma membrane increasing the lipid order of both fractions. Interestingly, different to ovarian oocytes, membranes isolated from ovulated oocytes showed an important lipid order resistance to variations in temperature. In these oocytes, MβCD treatment also decreased lipid order of L membranes but at a lesser extent than ovarian oocytes. In addition, fertilization increased lipid order of non-raft membranes. As we have previously shown for progesterone-induce maturation, functional studies revealed that MβCD treatment inhibited fertilization in a dose-dependent manner. Cholesterol repletion performed using MβCD pre-loaded with cholesterol induced a recovery of the fertilization rate. These results indicate that fertilization competence of amphibian oocytes is sensitive to the cholesterol content of both plasma membrane fractions. Taking into account that the plasma membrane is a major site of cryoinjury during cryopreservation of mammalian oocytes, these results open a discussion on sterol modulation to be able to obtain a stable source of oocytes available for in vitro fertilization and other related biotechnologies. This study was supported by grants from the National Council of Scientific and Technical Research, CONICET to T.S.A. and to S.S.A.