Gamete fusion is a critical event of mammalian fertilization. A random one-bead one-compound combinatorial peptide library represented synthetic human egg mimics and identified a previously unidentified ligand as Fc receptor–like 3, named MAIA after the mythological goddess intertwined with JUNO. This immunoglobulin super family receptor was expressed on human oolemma and played a major role during sperm-egg adhesion and fusion. MAIA forms a highly stable interaction with the known IZUMO1/JUNO sperm-egg complex, permitting specific gamete fusion. The complexity of the MAIA isotype may offer a cryptic sexual selection mechanism to avoid genetic incompatibility and achieve favorable fitness outcomes.

Proteins CD9 and CD81 are members of the tetraspanin superfamily and were detected in mammalian sperm, where they are suspected to form an active tetraspanin web and to participate in sperm–egg membrane fusion. The importance of these two proteins during the early stages of fertilization is supported by the complete sterility of CD9/CD81 double null female mice. In this study, the putative mechanism of CD9/CD81 involvement in tetraspanin web formation in sperm and its activity prior to fertilization was addressed. Confocal microscopy and colocalization assay was used to determine a mutual CD9/CD81 localization visualised in detail by super-resolution microscopy, and their interaction was address by co-immunoprecipitation. The species-specific traits in CD9 and CD81 distribution during sperm maturation were compared between mice and humans. A mutual position of CD9/CD81 is shown in human spermatozoa in the acrosomal cap, however in mice, CD9 and CD81 occupy a distinct area. During the acrosome reaction in human sperm, only CD9 is relocated, compared to the relocation of both proteins in mice. The structural modelling of CD9 and CD81 homologous and possibly heterologous network formation was used to propose their lateral Cis as well as Trans interactions within the sperm membrane and during sperm–egg membrane fusion.

Seminal plasma (SP) is the natural environment for spermatozoa and contains a number of components, especially proteins important for successful sperm maturation and fertilization. Nevertheless, in standard frozen stallion insemination doses production, SP is completely removed and is replaced by a semen extender. In the present study, we analyzed the effects of the selected seminal plasma protein groups that might play an important role in reducing the detrimental effects on spermatozoa during the cryopreservation process. SP proteins were separated according to their ability to bind to heparin into heparin-binding (Hep+) and heparin-non-binding (Hep−) fractions. The addition of three concentrations—125, 250, and 500 µg/mL—of each protein fraction was tested. After thawing, the following parameters were assessed: sperm motility (by CASA), plasma membrane integrity (PI staining), and acrosomal membrane integrity (PNA staining) using flow cytometry, and capacitation status (anti-phosphotyrosine antibody) using imaging-based flow cytometry. Our results showed that SP protein fractions had a significant effect on the kinematic parameters of spermatozoa and on a proportion of their subpopulations. The 125 µg/mL of Hep+ protein fraction resulted in increased linearity (LIN) and straightness (STR), moreover, with the highest values of sperm velocities (VAP, VSL), also this group contained the highest proportion of the fast sperm subpopulation. In contrast, the highest percentage of slow subpopulation was in the groups with 500 µg/mL of Hep+ fraction and 250 µg/mL of Hep− fraction. Interestingly, acrosomal membrane integrity was also highest in the groups with Hep+ fraction in concentrations of 125 µg/mL. Our results showed that the addition of protein fractions did not significantly affect the plasma membrane integrity and capacitation status of stallion spermatozoa. Moreover, our results confirmed that the effect of SP proteins on the sperm functionality is concentration-dependent, as has been reported for other species. Our study significantly contributes to the lack of studies dealing with possible use of specific stallion SP fractions in the complex puzzle of the improvement of cryopreservation protocols. It is clear that improvement in this field still needs more outputs from future studies, which should be focused on the effect of individual SP proteins on other sperm functional parameters with further implication on the success of artificial insemination in in vivo conditions.

Spermatozoa are highly specialized male cells that are characterized by a unique ability to move, which is a critical factor for successful fertilization. The relative simplicity of motility assessment, especially in livestock, has made it a widely used parameter for evaluating ejaculate quality or cryopreserved semen in the clinical field, and an advanced tool in reproductive physiology and toxicology research. Technological advances in image analysis and computational methods have substantially increased its accuracy through the use of computer-assisted sperm analysis (CASA) to minimize subjective bias in motility assessments. Nevertheless, this more objective method still presents some significant challenges, including variability in the sample preparation, imaging conditions, and analytical parameters. These issues contribute to inconsistency and impair the reproducibility and comparability of data between laboratories. The implementation of standardized protocols, combined with comprehensive training and rigorous evaluation, can serve to mitigate some of the emerging inconsistencies. In addition, the in vitro conditions under which CASA analyses are performed often differ significantly from the natural environment of the female reproductive tract in vivo. This review discusses the methodologies, critical issues, and limitations of sperm motility analyses using CASA, with a particular focus on the boar as an important agricultural and biomedical model species in which this system is widely used.

The long-term storage of boar sperm presents an ongoing challenge, and the modification of the cryoprotective compounds in semen extenders is crucial for improving cryopreservation’s success rate. The aim of our study was to reduce the percentage of glycerol in the extender by elimination or substitution with biocompatible, non-toxic polysaccharides. For boar semen extender improvement, we tested a novel modification with the polysaccharides dextran and pentaisomaltose in combination with unique in silico predictive modeling. We targeted the analysis of in vitro qualitative sperm parameters such as motility, viability, mitochondrial activity, acrosome integrity, and DNA integrity. Non-penetrating polysaccharide-based cryoprotective agents interact with sperm surface proteins such as spermadhesins, which are recognized as fertility markers of boar sperm quality. The in silico docking study showed a moderate binding affinity of dextran and pentaisomaltose toward one specific spermadhesin known as AWN, which is located in the sperm plasma membrane. Pentaisomaltose formed a hydrophobic pocket for the AWN protein, and the higher energy of this protein–ligand complex compared with dextran was calculated. In addition, the root mean square deviation (RMSD) analysis for the molecular dynamics (MD) of both polysaccharides and AWN simulation suggests their interaction was highly stable. The in silico results were supported by in vitro experiments. In the experimental groups where glycerol was partially or entirely substituted, the use of pentaisomaltose resulted in improved sperm mitochondrial activity and DNA integrity after thawing when compared with dextran. In this paper, we demonstrate that pentaisomaltose, previously used for cryopreservation in hematopoietic stem cells, represents a promising compound for the elimination or reduction of glycerol in extenders for boar semen cryopreservation. This novel approach, using in silico computer prediction and in vitro testing, represents a promising technique to help identify new cryoprotectants for use in animal breeding or genetic resource programs.

A series of biochemical and biophysical changes during sperm capacitation initiates various signaling pathways related to protein phosphorylation leading to sperm hyperactivation, simultaneously with the regulation of proteasomal activity responsible for protein degradation and turnover. Our study aimed to unveil the role of the proteasome in the regulation of boar sperm motility, hyperactivated status, tyrosine phosphorylation, and total protein ubiquitination. The proteolytic activity of the 20S proteasomal core was inhibited by MG-132 in concentrations of 10, 25, 50, and 100 μM; and monitored parameters were analyzed every hour during 3 h of in vitro capacitation (IVC). Sperm motility and kinematic parameters were analyzed by Computer Assisted Sperm Analysis (CASA) during IVC, showing a significant, negative, dose-dependent effect of MG-132 on total and progressive sperm motility (TMOT, PMOT, respectively). Furthermore, proteasomal inhibition by 50 and 100 μM MG-132 had a negative impact on velocity-based kinematic sperm parameters (VSL, VAP, and VCL). Parameters related to the progressivity of sperm movement (LIN, STR) and ALH were the most affected by the highest inhibitor concentration (100 μM). Cluster analysis revealed that the strongest proteasome-inhibiting treatment had a significant effect (p ≤ 0.05) on the hyperactivated sperm subpopulation. The flow cytometric viability results proved that reduced TMOT and PMOT were not caused by disruption of the integrity of the plasma membrane. Neither the protein tyrosine phosphorylation profile changes nor the accumulation of protein ubiquitination was observed during the course of capacitation under proteasome inhibition. In conclusion, inhibition of the proteasome reduced the ability of spermatozoa to undergo hyperactivation; however, there was no significant effect on the level of protein tyrosine phosphorylation and accumulation of ubiquitinated proteins. These effects might be due to the presence of compensatory mechanisms or the alteration of various ubiquitin-proteasome system-regulated pathways.