Molecular interactions between gametes and somatic cells in the in vitro model of fertilization

Author: Konstantine Buchukuri
Keywords: spermatozoa, capacitation, oxysterols
Annotation:

Fertilization is a complex multi-step process that involves the fusion of female and male gametes and the formation of a zygote, which gives birth to a new life. In mammals, fertilization takes place in the reproductive tract of the female organism, where the sperm moves towards the egg as well as undergoes physiological and biochemical changes, as a result of which, it acquires the fertilization ability. This series of transformations is called capacitation, which is stimulated by the environment created in the female reproductive tract. However, capacitation can be achieved in an in vitro environment as well, that is an integral part of Assisted Reproductive Technologies. Capacitation strongly depends on the formation of reactive oxygen species (ROS), which oxidize cholesterol in the plasma membrane of spermatozoa and convert it into oxysterols. This serves to one of the most important event - the outflow of cholesterol from the membrane, which increases the permeability of the latter. On the other hand, reactive oxygen species may induce apoptosis in spermatozoa. Since their excessive production causes oxidative stress, various apoptosis-inducing events may occur, such as mitochondrial dysfunction, DNA damage, lipid peroxidation, or direct activation of the apoptotic signaling system. Therefore, in order to promote in vitro fertilization-related technologies there is a need to prevent this phenomenon harmful to fertilization and to develop protective mechanisms for spermatozoa against oxidative stress. The aim of the present study was to identify the oxysterols involved in capacitation and understand their biochemistry. The research object - boar spermatozoa - were capacitated in vitro, using the validated protocol for different time duration (0, 2, 4, hours). After that, we isolated and quantitatively measured oxysterols by ultra-high-performance liquid chromatography in tandem with mass spectrometry. The following compound was isolated from the samples: cholesterol, desmosterol, 7α-hydroxycholesterol, β-hydroxycholesterol, and 24-hydroxycholesterol. From those the presence of cholesterol and desmosterol and their high levels was expected, since they are naturally present in the plasma membrane. As for 7α-hydroxycholesterol, β-hydroxycholesterol, and 24-hydroxycholesterol, these are oxidized forms of the above-mentioned compounds, which are involved in the capacitation process. Thus, the study identified different forms of oxysterols formed during the capacitation process. The obtained data will expand the knowledge about the molecular mechanisms of capacitation and will contribute to the improvement of in-vitro methods of inducing capacitation, which will increase the efficiency of fertilization technologies.