173 THE IMPACT OF ELEVATED TEMPERATURE ON HSP70 EXPRESSION IN CATTLE MURAL GRANULOSA CELLS

Abstract

Elevated temperature has an adverse impact on cattle fertility, causing disorders in ovarian functions and follicle development. Heat shock caused by elevated temperature leads to disruption in the cytoskeleton structure and the nuclear maturation of oocytes. Furthermore, it has an impact on mural granulosa cells (MGC), which are responsible for maintaining an appropriate microenvironment for oocyte development and signal transmission through the ovarian follicle. Heat-shock protein 70 is considered as a fundamental marker of cellular defence mechanisms related to heat shock. It protects other proteins from denaturation by forming complexes and stabilisation of their structure. HSP70 has also an ability to repair damaged proteins and allows them to return to their native structure. Furthermore, members of the HSP70 subfamily participate in the folding of newly synthesised proteins and their transport to different cell compartments. The aim of this study was to determine the impact of elevated temperature on HSP70 expression in mural granulosa cells. MGCs were obtained postmortem from mural layers of cattle ovarian follicles with diameters greater than 15 mm and randomly assigned to one of 5 variants: I (control) – MGCs after isolation; II – MGCs cultured in medium with LH at 38.5°C; III – MGCs cultured in medium with LH at elevated temperature, 41°C; IV – MGCs cultured in medium without LH at 38.5°C; and V – MGCs cultured in medium without LH at elevated temperature, 41°C. HSP70 expression was determined using real-time PCR method, and was normalised to s18/h2a expression. Statistical analysis was made in Statgraphics (Statpoint Technologies Inc., Warrenton, VA, USA; P = 0.01) using one-way ANOVA to compare expression of hsp70 between experimental variants and multifactor ANOVA to determine the influence of temperature and LH stimulation on hsp70 expression. Mean values of hsp70 expression in real-time PCR were compared using Tukey's test (a = 0.05). The significant increase of hsp70 expression was observed in MGCs cultured at 41°C (groups III and V) in comparison to MGCs cultured at 38.5°C (groups II and IV) and the control group. Simultaneously, there is no significant impact of LH stimulation on hsp70 expression. In conclusion, mural granulosa cells are susceptible to elevated temperature, which induces activation of cellular defence mechanisms performed by increased hsp70 expression. This may lead to disorders in the function of MGCs followed by changes in follicular fluid composition and signal transmission through the follicle.

Research was supported by 505-10-023300-L00171-99.