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.

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“…To visualize the target protein, membranes were incubated with the ECL substrate (GE Healthcare, Chicago, IL, USA) in the dark for 5 min. After incubation, the membranes were placed into the ChemiDoc Imaging System, which automatically calculated the protein visualization time based on the light signal emitted by the membranes [ 99 ].…”