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In Vitro versus Cryo-Induced Capacitation of Bovine Spermatozoa, Part 2: Changes in the Expression Patterns of Selected Transmembrane Channels and Protein Kinase A.

ABSTRACT: Since the molecular similarities and differences among physiological capacitation and cryocapacitation have not been studied in detail, this study was designed to assess the gene and protein expression levels of the Cation channel of sperm (CatSper) 1 and 2, sodium bicarbonate (Na+/HCO3−) cotransporter (NBC) and protein kinase A (PKA) in un-capacitated (control), in vitro capacitated (CAP) and cryopreserved (CRYO) bovine spermatozoa. All samples were subjected to motility evaluation using the computer assisted sperm analysis and chlortetracycline (CTC) assay for the assessment of the capacitation patterns. Furthermore, quantitative reverse transcription PCR (qRT-PCR) and Western blots were used to monitor the expression patterns of the selected capacitation markers. The results showed a significant reduction in the gene and protein expression levels of CatSper1 and 2 in the CRYO group when compared to the CAP group (p < 0.0001). In the case of NBC, the results were not significantly different or were inconclusive. While a non-significant down-regulation of PKA was found in the CRYO group, a significant reduction in the expression of the PKA protein was found in frozen-thawed spermatozoa in comparison to the CAP group (p < 0.05). In conclusion, we may hypothesize that while in vitro capacitated and cryopreserved spermatozoa exhibit CTC-patterns consistent with capacitation events, the molecular machinery underlying CTC-positivity may be different.

SUBMITTER: Benko F

PROVIDER: S-EPMC9739406 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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In Vitro versus Cryo-Induced Capacitation of Bovine Spermatozoa, Part 2: Changes in the Expression Patterns of Selected Transmembrane Channels and Protein Kinase A.

Benko Filip F Fialková Veronika V Žiarovská Jana J Ďuračka Michal M Lukáč Norbert N Tvrdá Eva E

International journal of molecular sciences 20221124 23

Since the molecular similarities and differences among physiological capacitation and cryocapacitation have not been studied in detail, this study was designed to assess the gene and protein expression levels of the Cation channel of sperm (CatSper) 1 and 2, sodium bicarbonate (Na+/HCO3−) cotransporter (NBC) and protein kinase A (PKA) in un-capacitated (control), in vitro capacitated (CAP) and cryopreserved (CRYO) bovine spermatozoa. All samples were subjected to motility evaluation using the co ...[more]

PMID: 36498971

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Project description:Epicatechin (EPC) is a flavonoid belonging to the family of catechins; it has been described as a powerful scavenger of a wide spectrum of reactive oxygen species (ROS) and a modulator of ex vivo sperm vitality. In this study, we assessed the potential protective abilities of EPC on cryopreserved bovine spermatozoa. We focused on conventional quality parameters, as well as the oxidative profile of spermatozoa alongside capacitation patterns, and expression profiles of proteins involved in the process of capacitation. Semen samples were cryopreserved in the presence of 25, 50 or 100 μmol/L EPC and compared to native semen (negative control) as well as ejaculates frozen in the absence of EPC (positive control). A dose-dependent improvement of conventional sperm quality parameters was observed following EPC administration, particularly in case of the sperm motility, membrane, acrosome and DNA integrity in comparison to the positive control. Experimental groups exposed to all EPC doses presented with a significantly lower proportion of capacitated spermatozoa as opposed to the positive control. While no significant effects of EPC were observed in cases of superoxide production, a significant decrease in the levels of hydrogen peroxide and hydroxyl radical were recorded particularly in the experimental groups supplemented with 50 and 100 μmol/L EPC. Western blot analysis revealed that supplementation of particularly 100 μmol/L EPC to the semen extender prevented the loss of the cation channel of sperm (CatSper) isoforms 1 and 2, sodium bicarbonate cotransporter (NBC) and protein kinase A (PKA), which play important roles in the process of sperm capacitation. In summary, we may hypothesize that EPC is particularly effective in the stabilization of the sperm membrane during the freeze–thaw process through its ability to quench ROS involved in damage to the membrane lipids and to prevent the loss of membrane channels crucial to initiate the process of sperm capacitation. These attributes of EPC provide an additional layer of protection to spermatozoa exposed to low temperatures, which may be translated into a higher post-thaw structural integrity and functional activity of male gametes.

Project description:BACKGROUND: There has been an ongoing debate in the reproductive field about whether mammalian spermatozoa rely on glycolysis, oxidative phosphorylation or both for their energy production. Recent studies have proposed that human spermatozoa depend mainly on glucose for motility and fertilization but the mechanism behind an efficient glycolysis in human spermatozoa is not well understood. Here, we demonstrate how human spermatozoa utilize exogenous pyruvate to enhance glycolytic ATP production, motility, hyperactivation and capacitation, events that are crucial for male fertility. METHODS: Purified human spermatozoa from healthy donors were incubated under capacitating conditions (including albumin, bicarbonate and glucose) and tested for changes in ATP levels, motility, hyperactivation and tyrosine phosphorylation after treatment with pyruvate. The experiments were repeated in the presence of sodium cyanide in order to assess the contribution from mitochondrial respiration. The metabolism of (13)C labeled glucose and pyruvate was traced by a combination of liquid chromatography and mass spectrometry. RESULTS: The treatment of human spermatozoa with exogenous pyruvate increased intracellular ATP levels, progressive motility and hyperactivation by 56, 21 and 130%, respectively. In addition, added pyruvate induced a significant increase in tyrosine phosphorylation levels. Blocking of the electron transport chain did not markedly affect the results, indicating that the mechanism is independent of oxidative phosphorylation. However, the observed effects could be counteracted by oxamate, an inhibitor of lactate dehydrogenase (LDH). Metabolic tracing experiments revealed that the observed rise in ATP concentration resulted from an enhanced glycolytic flux, which was increased by more than 50% in the presence of exogenous pyruvate. Moreover, all consumed (13)C labeled pyruvate added was converted to lactate rather than oxidized in the tricarboxylic acid cycle. CONCLUSIONS: Human spermatozoa seem to rely mainly, if not entirely, on glycolysis as the source of ATP fueling the energy-demanding processes of motility and capacitation. The efficient glycolysis is dependent on exogenous pyruvate, which indirectly feeds the accelerated glycolysis with NAD(+) through the LDH-mediated conversion of pyruvate to lactate. Pyruvate is present in the human female reproductive tract at concentrations in accordance with our results. As seen in other mammals, the motility and fertility of human spermatozoa seem to be dictated by the available energy substrates present in the conspecific female.

Dataset Information

In Vitro versus Cryo-Induced Capacitation of Bovine Spermatozoa, Part 2: Changes in the Expression Patterns of Selected Transmembrane Channels and Protein Kinase A.

Publications

In Vitro versus Cryo-Induced Capacitation of Bovine Spermatozoa, Part 2: Changes in the Expression Patterns of Selected Transmembrane Channels and Protein Kinase A.

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