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.

Ejaculatory Abstinence Affects the Sperm Quality in Normozoospermic Men—How Does the Seminal Bacteriome Respond?

“…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 ].…”

Sperm freezing damage: the role of regulated cell death

“…An increase in intracellular Ca 2+ flux, mediated by CatSper, can induce sperm hyperactivation, acrosome reaction, oocyte chemotaxis, and zona pellucida penetration during the fertilization process [ 191 ]. However, the expression of CatSper is reduced during sperm cryopreservation [ 192 , 193 ], suggesting that the elevation of Ca 2+ flux in cryopreserved sperm is independent of CatSper. Intriguingly, this may be associated with the development of some types of RCD.…”

Kaempferol as an Alternative Cryosupplement for Bovine Spermatozoa: Cytoprotective and Membrane-Stabilizing Effects

“…Unlike other cell types, spermatozoa are small cells with a large surface [5] which will affect the intracellular viscosity and transition temperature [2]. Hence, if the cryopreservation procedure is not carried out properly, or if cryoprotective agents are not present in the extender, ice crystals and thermal and osmotic shock may lead to structural sperm damage [1,4], cell death by apoptosis or necrosis [6,7] and reactive oxygen species (ROS) overgeneration, which is considered as the prime mechanism of the loss of sperm function following the freeze-thaw process [7,8]. Accordingly, a high proportion of spermatozoa with peroxidized membranes, oxidative damage to the proteins and DNA [6,9] and high levels of peroxyl radicals and lipid hydroperoxides [8] have been found in frozen-thawed semen samples.…”

“…Under physiological circumstances, capacitation is initiated by the activation of the transmembrane channels including the cation channels of sperm (CatSper) and the sodium bicarbonate cotransporter (NBC), leading to an influx of calcium (Ca 2+ ) and bicarbonate (HCO 3 − ) into the sperm intracellular compartments, followed by a rise in pH and synthesis of cyclic adenosine monophosphate (cAMP). This will then lead to the activation of protein kinase A (PKA), which will trigger tyrosine kinases, leading to sperm hyperactivation and changes in the architecture of the sperm plasma membrane [7,10]. In the meantime, protein kinase C (PKC) will play essential roles in the acquisition of hyperactive motility as a serine/threonine kinase and a mediator in Ca 2+ mobilization for the signaling pathways leading to the acrosome reaction [10].…”

“…In the meantime, protein kinase C (PKC) will play essential roles in the acquisition of hyperactive motility as a serine/threonine kinase and a mediator in Ca 2+ mobilization for the signaling pathways leading to the acrosome reaction [10]. As opposed to the physiological process, cryocapacitation is initiated by the loss of Ca 2+ and HCO 3 − transmembrane channels responsible for the initiation of sperm activation [7,11]. Prematurely capacitated spermatozoa are characterized by low cAMP production [12] and the loss of cholesterol and phospholipids from the plasma membrane leading to cells with compromised membranes that will exhibit chlortetracycline hydrochloride (CTC) positivity typical for physiologically capacitated spermatozoa [6,11,13].…”