167 THE EFFECT OF ELEVATED TEMPERATURE ON THE HEAT SHOCK PROTEIN 70 (HSP70) EXPRESSION IN BOVINE CUMULUS–OOCYTE COMPLEXES AFTER IN VITRO MATURATION

Abstract

Elevated temperature during in vitro maturation negatively affects the oocyte's developmental competence, leading to disturbances in nuclear and cytoplasmic maturation. In previous studies, the role of cumulus granulosa cells (CGC) in cumulus-oocyte complexes' (COC) response to elevated temperature was underestimated. However, CGC play an essential role in folliculogenesis, supporting the oocyte's metabolism as well as meiotic progression. Thus, CGC may be engaged in COC response to heat shock. Heat shock protein 70 (HSP70) is the major protein engaged in cellular response to heat stress. The aim of this study was to determine the HSP70 expression in both CGC and oocytes in response to elevated temperature after COC in vitro maturation. COC were collected from bovine ovarian follicles (diameter 2–6 mm) from slaughtered cows and divided into two treatment groups: I (control) – COC were in vitro matured in control temperature (38.5°C); II (experimental) – COC were in vitro matured in elevated temperature (41°C). After in vitro maturation they were mechanically separated into CGC and oocyte. In vitro maturation was conducted in TCM199 25 mM HEPES medium supplemented with 10% FBS, 0.02 IU mL^–1 NIH-pFSH, 1 μg mL^–1 17β-oestradiol, 22 μg mL^–1 Na-pyruvate and 10 μg mL^–1 gentamicin, adjusted to pH 7.4 in 5% CO₂. The HSP70 expression in CGC and oocytes was performed by real-time PCR and normalized to S18/H2A and S18 gene expression, respectively. In addition, the immunocytofluorescent analysis of HSP70 expression in CGC and oocyte's were performed. The HSP70 was stained using primary monoclonal mouse antibodies raised against bovine HSP70 and secondary antibody raised against mouse conjugated with Alexa 488. Nuclei were stained with Hoechst 33342. Slides were analysed under laser confocal microscope (FV-500, Olympus, Center Valley, PA, USA). The HSP70 expression in CGC was measured as total optical density of HSP70 and normalized to total optical density of nuclei. Statistical analyses were performed by Portable Statgraphics 5.0 Centurion. Mean values of HSP70 expression in CGC and oocytes in real-time PCR and immunofluorescent analysis in CGC were compared using Tukey's HSD test (α = 0.01). After in vitro maturation, the expression of HSP70 in CGC was higher (0.13 ± 0.052 a.u., n = 35) in experimental temperature compared to the control (0.058 ± 0.008 a.u., n = 35) (P < 0.01). In oocytes, HSP70 expression in experimental temperature (32.5 ± 5.2 a.u., n = 12) was similar to control (29.8 ± 5.6 a.u., n = 12). The immunofluorescent analysis confirmed data from real-time PCR analysis, indicating that the HSP70 expression in CGCs in experimental temperature was higher (0.46 ± 0.07 a.u., n = 10) compared to the control (0.12 ± 0.02 a.u., n = 10; P < 0.01). After COC in vitro maturation in stress conditions, the HSP70 expression was up-regulated in CGC, but not in oocytes. That may indicate that CGCs play the major role in COC response to elevated temperature; however, the analysis of other heat shock proteins expression in CGC and oocytes need to be conducted.

Research was supported by 505-10-023300-K00169-99.