353 CUMULUS CELLS RESPONSE TO HEAT SHOCK AND INSULIN-LIKE GROWTH FACTOR-I

Abstract

Heat-stress induced maternal hyperthermia has been shown to compromise the series of events associated with oocyte growth and maturation reducing oocyte competence. Such events are regulated by a variety of growth factors and dynamic communication between the oocyte and its surrounding cumulus cells. The objective of the current study was to evaluate the modulatory effects of COCs quality and IGF-I on mitochondrial membrane potential (MMP) and apoptosis in cumulus cells induced by heat shock. In this study high (≥3 layers of compact cumulus cells and homogeneous cytoplasm) and low-grade COCs (<3 layers of less compact cumulus cells and irregular cytoplasm) derived from slaughterhouse ovaries were exposed to control (CTR: 39°C) or heat shock (HS: 41°C) treatments in the presence of 0 or 100 ng mL^-1 IGF-I during the first 12 h of in vitro maturation (12 h-IVM). Immediately after 12 h-IVM COCs were denuded by repeated pipetting and cumulus cells evaluated for MMP (MitoProbe JC-1 assay kit. JC-1 is a cationic dye that exhibits potential-depend accumulation in the mitochondria) and apoptosis (Annexin V-FITC and propidium iodide) by flow cytometry (Guava EasyCyte Mini Flow Cytometry System, Millipore, Billerica, MA, USA). This factorial experiment was replicated 4 times using 75-100 COCs per treatment. Data were subjected to three-way analysis of variance using the General Linear Models procedure of SAS. Results are shown in Table 1. Exposure of high and low-grade COCs to HS reduced (P < 0.01) the percentage of cumulus cells carrying high MMP regardless of IGF-I. Even though HS caused cumulus cells mitochondrial membrane depolarization there was neither temperature nor COCs quality effect on cumulus cells apoptosis as indicated by the lack of phosphatidylserine (PS) translocation from the inner to the outer leaflet of the plasma membrane. On the other hand, addition of IGF-I to maturation medium reduced (P < 0.05) the percentage of cumulus cells labeled with Annexin V + PI regardless of COCs quality or temperature. There was no statistical interaction between COCs quality × IGF-I × temperature. In conclusion, exposure of COCs to HS during 12 h-IVM caused cumulus cells mitochondrial depolarization without inducing apoptosis. It is possible that a period longer than 12 h is required for most PS translocation to occur in cumulus cells. Moreover, IGF-I exerted protective effect reducing cumulus cells late apoptosis/necrosis events.

Table 1.  Effect of heat-shock and IGF-I on cumulus cells mitochondrial membrane potential and apoptosis. Results are least-squares means ± SEM.