The aim of our in vitro studies was to understand the role of leptin in controlling proliferation, apoptosis, and protein kinase A (PKA) in human ovarian cells. We analyzed the in vitro effects of leptin (0, 1, 10 or 100 ng/ml) on the accumulation of proliferation-related peptides (PCNA, cyclin B1), apoptosis-associated peptide (Bax) and the intracellular signaling molecule PKA in cultured human granulosa cells using immunocytochemistry and Western immunoblotting. It was observed that leptin stimulated in a dose-dependent manner the accumulation of PCNA (at doses 1-100 ng/ml), cyclin B1 (at doses 10 or 100 ng/ml), Bax (at doses 10 or 100 ng/ml) and PKA (at doses 1-100 ng/ml) in cultured human ovarian cells. These observations suggest the ability of leptin to control directly human ovarian cell functions: proliferation, apoptosis, and intracellular messenger PKA., A. V. Sirotkin, M. Mlynček, A. V. Makarevich, I. Florkovičová, L. Hetényi., and Obsahuje bibliografii a bibliografické odkazy
The aim of our study was to test the in fluence of short exposure (6 h) of preimplantation rabbit embryos to elevated temperatures (41.5 ºC or 42.5 ºC) in vitro on their developmental capacity. Fertilized eggs recovered from female oviducts at the pronuclear stage (19 hpc) were cultured at standard temperature (37.5 ºC) until the morula stage (72 hpc). Afterwards, the embryos were divided into two groups, cultured for 6 h either at hyperthermic (41.5 ºC or 42.5 ºC) or standard temperature (control 37.5 ºC), post-incubated overnight (16-20 h) at 37.5 ºC and then evaluated for developmental stages, apoptosis (TUNEL), proliferation (cell number), actin cytoskeleton and presence of heat-shock proteins Hsp70. It was observed that hyperthermia at 41.5 ºC did not alter progression of embryos to higher preimplantation stages (expanded and hatching/hatched blastocysts), rate of apoptosis, total cell number of blastocysts and structure of actin filament compared to 37.5 ºC. We stern-blotting revealed the presence of heat stress-induced 72 kDa fraction of Hsp70 proteins in granulosa cells (exposed to 41 ºC) and embryos (exposed to 41.5 ºC). Following the elevation of temperature to 42.5 ºC embryo development was dramati cally compromised. The embryos were arrested at the morula or early blastocyst stage, showed an increased rate of apoptosis and decreased total cell number compared to control. The structure of actin filaments in most of blastomeres was damaged and such blastomeres often contained apoptotic nuclei. In this group a presence of heat-stress-induced fraction of Hsp70 proteins had not been confirmed. This is the first report demonstrating a threshold of thermotolerance of rabbit preimplantation embryos to hyperthermic exposure in vitro. A detrimental effect of higher temperature on the embryo is probably associated with the loss of their ability to produce Hsp70 de novo, which leads to cytoskeleton alterations and enhanced apoptosis., A. V. Makarevich, L. Olexiková, P. Chrenek, E. Kubovičová, K. Fréharová, J. Pivko., and Obsahuje bibliografii a bibliografické odkazy