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ABSTRACT:The present study is focused on the methodology of fluorescence activated cell sorting (FACS) of spermatozoa stained by the antibody against extracellular surface marker ubiquitin (eUb) and subsequent protocol for their long term storage in liquid nitrogen (LN). High level of spermatozoa surface ubiquitination has been previously discussed as a negative quality marker. From a general point of view, any other outer membrane antigen would be compatible with our approach. Regarding our experimental design we found that only those insemination doses with at least 40% of motile spermatozoa after freezing and thawing (F/T) in the egg-yolk medium with lactose are suitable for the subsequent antibody staining and FACS. The sorting rate was sufficient for the preparation of up to 20 spermatozoa aliquots for intracytoplasmic sperm injections (ICSI). Two significantly different groups with good freezability were prepared and stored in LN (0.73% contamination of spermatozoa with high eUb level in non-ubiquitinated group and reversely 6.65% spermatozoa without eUb in highly ubiquitinated group). Sperm viability after FACS varied from 11 to 28% regardless of the used media (P = 0.15). Required viability of F/T sorted spermatozoa was obtained by using Solusem ® extender as a load and collection medium. In this case 12% of viable spermatozoa with progressive motility in low eUb level group and 7% in high eUb level group (P < 0.05) were detected. Our approach allows obtaining sufficient number of viable spermatozoa for subsequent artificial fertilization by ICSI. This procedure could be used for a wide variety of spermatozoa sorting based on different surface markers.

BackgroundHydrogen sulfide has been shown to improve the quality of oocytes destined for in vitro fertilization. Although hydrogen sulfide is capable of modulating ion channel activity in somatic cells, the role of hydrogen sulfide in gametes and embryos remains unknown. Our observations confirmed the hypothesis that the KATP and L-type Ca2+ ion channels play roles in porcine oocyte ageing and revealed a plausible contribution of hydrogen sulfide to the modulation of ion channel activity.ResultsWe confirmed the benefits of the activation and suppression of the KATP and L-type Ca2+ ion channels, respectively, for the preservation of oocyte quality.ConclusionsOur experiments identified hydrogen sulfide as promoting the desired ion channel activity, with the capacity to protect porcine oocytes against cell death. Further experiments are needed to determine the exact mechanism of hydrogen sulfide in gametes and embryos.

The 5-hydroxytryptamine (serotonin) receptor type 7 (5-HT7R) is a G protein-coupled receptor (GPCR) present primarily in the nervous system and gastrointestinal tract, where it regulates mood, cognition, digestion, and vasoconstriction. 5-HT7R has previously been shown to bind to its cognate stimulatory Gs protein in the inactive state. This phenomenon, termed inverse coupling, is thought to counteract the atypically high intrinsic activity of 5-HT7R. However, it is not clear how active and inactive 5-HT7 receptors affect the mobility of the Gs protein in the plasma membrane. Here, we used single-molecule imaging of the Gs protein and 5-HT7R to evaluate Gs mobility in the membrane in the presence of 5-HT7R and its mutants. We show that expression of 5-HT7R dramatically reduces the diffusion rate of Gs. Expression of the constitutively active mutant 5-HT7R (L173A) is less effective at slowing Gs diffusion presumably due to the reduced ability to form long-lasting inactive complexes. An inactive 5-HT7R (N380K) mutant slows down Gs to the same extent as the wild-type receptor. We conclude that inactive 5-HT7R profoundly affects Gs mobility, which could lead to Gs redistribution in the plasma membrane and alter its availability to other GPCRs and effectors.