Intracellular distribution of hydrogen sulfide releasing enzymes in maturing porcine oocytes

The study of the molecular mechanisms regulating reproductive processes is crucial for increasing the efficiency of reproductive biotechnologies that are often used in animal breeding. A key part of many biotechnological methods is the cultivation of oocytes, which simulates natural conditions in the ovaries. However this simulation is still imperfect and presents a limiting factor for successful formation of mammalian oocytes.capable of fertilization. Meiotic maturation is influenced by many factors including for example gasotransmiter hydrogen sulfide (H2S). Until recently, hydrogen sulfide was known only as toxic gas polluting the environment. It was found that the hydrogen sulfide is at low concentrations important signaling molecule with a lot of physiological functions. Hydrogen sulfide is produced by enzymes CBS, CTH and 3-MPST in various tissues of the body, including the reproductive system. We hypothesised that enzymes responsible for the endogenous production of hydrogen sulfide, CBS, CTH and 3-MPST, are present in porcine oocytes and their presence varies during meiotic maturation in cellular organelles. The goal of this study was to prove this hypothesis by cultivating porcine oocytes, detection of mitochondria, endoplasmic reticulum and hydrogen sulfide-releasing enzymes using imunocytochemistry and colocalization analysis. Our results showed that enzymes releasing hydrogen sulfide, CBS, CTH and 3-MPST, are present in GV porcine oocytes and also in the oocytes matured to the MII stage, after 48 hours in vitro cultivation. We found that the distribution of these enzymes in cellular organelles in oocyte changed during meiotic maturation. There were significant changes in the distribution of enzymes 3-MPST and CBS in the mitochondria, levels of colocalization coefficients for enzyme 3-MPST increased, these coefficients for enzyme CBS decreased during meiotic maturation. In endoplasmic reticulum we found significant changes in the distribution of all three hydrogen sulfide-releasing enzymes. Levels of colocalitazion coefficients for CBS and 3-MPST increased, while levels of these coefficients for enzyme CTH decreased. Results of our experiments on porcine oocytes can help to clarify the role of gasotransmitter hydrogen sulfide during meiotic maturaion mammals of oocytes and are highly valueable because their possible application in breeding of farm animals and in human assisted reproduction