In vitro versus cryo-induced capacitation of bovine spermatozoa, part 1: Structural, functional, and oxidative similarities and differences

Abstract

Low temperatures during cryopreservation activate a cascade of changes, which may lead into irreversible damage and reduction of the fertilization potential, including the process of premature capacitation. The aim of our study was to evaluate the range of cell damage following the cryopreservation process and possible activation of cryocapacitation in bovine spermatozoa. For the experiments semen samples were obtained from 30 sexually mature Holstein bulls. Within the analysed parameters, we focused on the functional activity, structural integrity, capacitation status and oxidative profile. The samples were divided into three experimental groups, control (CTRL), in vitro capacitated (CAP) and cryopreserved (CRYO). Based on the collected data, there was a significant decrease in the sperm motility, mitochondrial membrane potential and concentration of cyclic adenosine monophosphate in the CRYO group when compared to CAP and CTRL (P<0.0001). A significant decrease (P<0.01; P<0.0001) in the membrane and acrosome integrity as well as DNA fragmentation index and a significant increase (P<0.0001) of necrotic cells were observed in the CRYO group. Following capacitation, a significant increase (P<0.01; P<0.0001) was recorded in the number of cells which underwent the acrosome reaction in the CRYO group against CAP and CTRL. Changes in the oxidative profile of the CRYO group indicates an increase (P<0.0001) in the reactive oxygen species generation, except for the superoxide radical, which was significantly higher (P<0.0001; P<0.001) in the CAP group in comparison with CRYO and CTRL. In summary, premature capacitation may be considered a consequence of cryopreservation and the assessed parameters could serve as physical markers of cryogenic damage to bovine spermatozoa in the future.

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

“…This series of changes may be responsible for the membrane of cryopreserved spermatozoa to respond to the chlortetracycline (CTC) stain, which has been observed in our experiments. Similar CTC staining patterns were observed in both capacitated and cryopreserved sperm in bulls [ 19 , 36 ], boars [ 37 , 38 ], stallions [ 39 ] and rams [ 40 ].…”

“…The evaluation of capacitation status was performed with chlortetracycline (CTC; Sigma-Aldrich, St. Louis, MO, USA) assay, and identification of specific CTC patterns after absorption of dye by spermatozoa was observed under the Leica DMI6000 B (Leica Camera, Wetzlar, Germany) fluorescent microscope ( Figure 17 and Figure S11 ). According to cell coloration, three basic patterns were identified: fluorescence of the whole sperm head for un-capacitated spermatozoa (“F” pattern), fluorescence of the sperm head except for post-acrosomal region, which is characteristic for capacitated spermatozoa (“B” pattern) and only a bright fluorescent band in the equatorial segment stood for acrosome-reacted spermatozoa (“AR” pattern) [ 19 , 86 ]. Thirty samples from each group were assessed, and at least 300 cells were counted in each sample.…”

“…As mentioned earlier, ion channels, cotransporters and enzymes play a decisive role in the process of mammalian sperm capacitation. In our previous study, we observed that cryopreservation can induce capacitation-like changes in bovine spermatozoa [ 19 ]. Cryogenic temperature accompanied with cryocapacitation significantly decreased the quality of post-thawed bovine spermatozoa at the level of their functional activity, structural integrity and oxidative profile.…”

Molecular Markers: A New Paradigm in the Prediction of Sperm Freezability

“…More accurate results are achieved in the case of double- or triple-staining protocols. Carboxyfluorescein diacetate (CFDA) is frequently used as an indicator of an intact membrane and is usually combined with propidium iodide (PI), permeable only for necrotic cells [ 21 ]. According to Watts [ 22 ], CFDA/PI staining is more dependable than the EN technique because at <80% of membrane-intact spermatozoa, EN overestimates the percentage of intact cells.…”

“…The chlortetracycline (CTC) assay belongs to calcium-binding assays. Three basic patterns are recognized: noncapacitated spermatozoa, where the whole sperm head is CTC-positive; capacitated spermatozoa, where the sperm head is stained excluding the postacrosomal region; and acrosome-reacted spermatozoa, which are characterized by a bright fluorescent band in the equatorial segment [ 21 ]. Another study considered the double Hoechst 33258/CTC staining assay as a good predictor of male fertility in various breeds [ 29 ].…”