Capacitation is a crucial sperm maturation process occurring in vivo in the female reproductive tract, enabling spermatozoa to fertilize the oocyte. In vitro, capacitation can be induced using defined capacitation media (CM), with further assessment of protein tyrosine phosphorylation (PTyr) patterns widely used as a marker to evaluate sperm capacitation. This review critically examines the factors influencing PTyr detection in boar spermatozoa variability introduced by different methodological approaches. Discrepancies in PTyr patterns may be a result of different sperm handling, including preservation methods, selection techniques, and capacitation protocols. Semen extenders, which may contain unknown components, can affect the variability in capacitation status. Selection techniques commonly employed to isolate viable spermatozoa may initiate different capacitation regulatory pathways, resulting in variability in analyzed sperm subpopulations and inconsistencies in PTyr detection. Similarly, the lack of standardization in CM composition significantly impacts capacitation outcomes. Fixation protocols further increase variability in PTyr pattern detection, as aldehydic fixatives potentially alter protein structures, while alcohol-based fixatives cause protein aggregation and plasma membrane disruption. While PTyr immunofluorescence remains a valuable tool for capacitation assessment, its reliability is limited by methodological variability. Mimicking in vivo conditions is crucial, and even minor modifications in the sperm capacitation process may provide inconsistent results in PTyr patterns across studies. This review offers valuable insights into often-disregarded methodological details and highlights the need for improved for better standardization of capacitation protocols. The uniform methodological approach improves reproducibility and reliability in capacitation studies and stimulates further investigation leading to the discovery of alternative additional markers to determine the capacitation status in mammalian spermatozoa.

• Klíčová slova
• Capacitation media, Immunofluorescence, Protein phosphorylation, Reproduction, Signaling pathway, Sperm fixation,
• MeSH
• analýza spermatu veterinární   MeSH
• fosforylace MeSH
• fosfotyrosin * metabolismus   MeSH
• kapacitace spermií * fyziologie   MeSH
• prasata fyziologie   MeSH
• spermie * fyziologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• fosfotyrosin * MeSH

In the last two decades, a school of thought emerged that perceives male reproductive health, testicular function, and sperm output as a sentry for general, somatic health. Large-scale epidemiologic studies have already linked the reduced sperm count to increased risk of chronic somatic disease (e.g., cancer, cardiovascular, neurological and bone diseases), yet most of these studies have not taken full advantage of advanced andrological analysis. Altered proteostasis, i.e., the disbalance between protein synthesis and turnover, is a common denominator of many diseases, including but not limited to cancer and neurodegenerative diseases. This chapter introduces the concept of cellular proteostasis as a measure of sperm structural and functional integrity and an endpoint of varied impacts on spermiogenesis and sperm maturation, including heritability, general health, lifestyle, and occupational and environmental reprotoxic exposure. Special consideration is given to small molecule protein modifiers, sperm-binding seminal plasma proteins, zinc-interacting proteins, and redox proteins responsible for the maintenance of protein structure and the protection of spermatozoa from oxidative damage. While the main focus is on human male infertility, serious consideration is given to relevant animal models, and in particular to male food animals with extensive records of fertility from artificial insemination services. Altogether, the proteostatic biomarker discovery and validation studies set the stage for the integration of proteomics of sperm proteostasis with genomic and high throughput phenomic approaches to benefit both human and animal reproductive medicine.

• Klíčová slova
• Biomarker, Infertility, Omics, Proteasome, Proteostasis, Seminal plasma, Sperm, Thioredoxin, Ubiquitin,
• MeSH
• fertilita * fyziologie   MeSH
• homeostáze proteinů * fyziologie   MeSH
• lidé MeSH
• mužská infertilita * metabolismus   genetika   patologie   patofyziologie   MeSH
• spermatogeneze * MeSH
• spermie * metabolismus   patologie   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Capacitation is an essential post-testicular maturation event endowing spermatozoa with fertilizing capacity within the female reproductive tract, significant for fertility, reproductive health, and contraception. By using a human-relevant large animal model, the domestic boar, this study focuses on furthering our understanding of the involvement of the ubiquitin-proteasome system (UPS) in sperm capacitation. The UPS is a universal, evolutionarily conserved, cellular proteome-wide degradation and recycling machinery, that has been shown to play a significant role in reproduction during the past two decades. Herein, we have used a bottom-up proteomic approach to (i) monitor the capacitation-related changes in the sperm protein levels, and (ii) identify the targets of UPS regulation during sperm capacitation. Spermatozoa were capacitated under proteasomal activity-permissive and inhibiting conditions and extracted sperm proteins were subjected to high-resolution mass spectrometry. We report that 401 individual proteins differed at least two-fold in abundance (P < 0.05) after in vitro capacitation (IVC) and 13 proteins were found significantly different (P < 0.05) between capacitated spermatozoa with proteasomal inhibition compared to the vehicle control. These proteins were associated with biological processes including sperm capacitation, sperm motility, metabolism, binding to zona pellucida, and proteasome-mediated catabolism. Changes in RAB2A, CFAP161, and TTR during IVC were phenotyped by immunocytochemistry, image-based flow cytometry, and Western blotting. We conclude that (i) the sperm proteome is subjected to extensive remodeling during sperm capacitation, and (ii) the UPS has a narrow range of distinct protein substrates during capacitation. This knowledge highlights the importance of the UPS in sperm capacitation and offers opportunities to identify novel pharmacological targets to modulate sperm fertilizing ability for the benefit of human reproductive health, assisted reproductive therapy, and contraception, as well as reproductive management in food animal agriculture.

• Klíčová slova
• Pig, Sperm capacitation, Sperm proteomics, Ubiquitin-proteasome system,
• MeSH
• kapacitace spermií * fyziologie   MeSH
• prasata MeSH
• proteasomový endopeptidasový komplex * metabolismus   MeSH
• proteom metabolismus   MeSH
• proteomika * metody   MeSH
• spermie * metabolismus   fyziologie   MeSH
• ubikvitin * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteasomový endopeptidasový komplex * MeSH
• proteom MeSH
• ubikvitin * MeSH

Sperm-zona pellucida (ZP) interaction, involving the binding of sperm surface ligands to complementary carbohydrates of ZP, is the first direct gamete contact event crucial for subsequent gamete fusion and successful fertilization in mammals. It is a complex process mediated by the coordinated engagement of multiple ZP receptors forming high-molecular-weight (HMW) protein complexes at the acrosomal region of the sperm surface. The present article aims to review the current understanding of sperm-ZP binding in the four most studied mammalian models, i.e., murine, porcine, bovine, and human, and summarizes the candidate ZP receptors with established ZP affinity, including their origins and the mechanisms of ZP binding. Further, it compares and contrasts the ZP structure and carbohydrate composition in the aforementioned model organisms. The comprehensive understanding of sperm-ZP interaction mechanisms is critical for the diagnosis of infertility and thus becomes an integral part of assisted reproductive therapies/technologies.

• Klíčová slova
• ZP-ligands, gamete interaction, sperm-ZP receptors, spermatozoa, zona pellucida,
• MeSH
• lidé MeSH
• ligandy MeSH
• membránové glykoproteiny metabolismus   MeSH
• mezibuněčná komunikace * MeSH
• receptory buněčného povrchu metabolismus   MeSH
• savci metabolismus   MeSH
• spermie cytologie   metabolismus   MeSH
• zona pellucida metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• ligandy MeSH
• membránové glykoproteiny MeSH
• receptory buněčného povrchu MeSH

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.

• Klíčová slova
• artificial insemination, biotechnology, cryobiology, phosphorylation, spermatozoa,
• MeSH
• koně MeSH
• kryoprezervace veterinární   MeSH
• lidský sérový albumin metabolismus   MeSH
• proteiny semenné plazmy metabolismus   MeSH
• sérové globuliny metabolismus   MeSH
• spermie fyziologie   MeSH
• uchování spermatu metody   veterinární   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• lidský sérový albumin MeSH
• plasma protein fraction MeSH Prohlížeč
• proteiny semenné plazmy MeSH
• sérové globuliny MeSH