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

Flow cytometry becomes a common method for analysis of spermatozoa quality. Standard sperm characteristics such as viability, acrosome and chromatin integrity, oxidative damage (ROS) etc. can be easily assess in any animal semen samples. Moreover, several fertility-related markers were observed in humans and some other mammals. However, these fertility biomarkers have not been previously studied in ram. The aim of this study was to optimize the flow-cytometric analysis of these standard and novel markers in ram semen. Ram semen samples from Slovak native sheep breeds were analyzed using CASA system for motility and concentration and were subsequently stained with several fluorescent dyes or specific antibodies to evaluate sperm viability (SYBR-14), apoptosis (Annexin V, YO-PRO-1, FLICA, Caspases 3/7), acrosome status (PNA, LCA, GAPDHS), capacitation (merocyanine 540, FLUO-4 AM), mitochondrial activity (MitoTracker Green, rhodamine 123, JC-1), ROS (CM-H2DCFDA, DHE, MitoSOX Red, BODIPY), chromatin (acridine orange), leukocyte content, ubiquitination and aggresome formation, and overexpression of negative biomarkers (MKRN1, SPTRX-3, PAWP, H3K4me2). Analyzed semen samples were divided into two groups according to viability as indicators of semen quality: Group 1 (viability over 60%) and Group 2 (viability under 60%). Significant (p < 0.05) differences were found between these groups in sperm motility and concentration, apoptosis, acrosome integrity (only PNA), mitochondrial activity, ROS production (except for DHE), leukocyte and aggresome content, and high PAWP expression. In conclusion, several standard and novel fluorescent probes have been confirmed to be suitable for multiplex ram semen analysis by flow cytometry as well as several antibodies have been validated for the specific detection of ubiquitin, PAWP and H3K4me2 in ram spermatozoa.

• Klíčová slova
• H3K4me2, MKRN1, PAWP, SPTRX-3, biomarkers, flow cytometry, native breeds, ram, semen, ubiquitin,
• MeSH
• analýza spermatu MeSH
• biologické markery MeSH
• chromatin MeSH
• fertilita MeSH
• kryoprezervace metody   MeSH
• motilita spermií * MeSH
• ovce MeSH
• průtoková cytometrie MeSH
• reaktivní formy kyslíku MeSH
• savci MeSH
• spermie MeSH
• uchování spermatu * metody   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• biologické markery MeSH
• chromatin MeSH
• reaktivní formy kyslíku MeSH

Sperm capacitation, one of the key events during successful fertilization, is associated with extensive structural and functional sperm remodeling, beginning with the modification of protein composition within the sperm plasma membrane. The ubiquitin-proteasome system (UPS), a multiprotein complex responsible for protein degradation and turnover, participates in capacitation events. Previous studies showed that capacitation-induced shedding of the seminal plasma proteins such as SPINK2, AQN1, and DQH from the sperm surface is regulated by UPS. Alterations in the sperm surface protein composition also relate to the porcine β-microseminoprotein (MSMB/PSP94), seminal plasma protein known as immunoglobulin-binding factor, and motility inhibitor. MSMB was detected in the acrosomal region as well as the flagellum of ejaculated boar spermatozoa, while the signal disappeared from the acrosomal region after in vitro capacitation (IVC). The involvement of UPS in the MSMB degradation during sperm IVC was studied using proteasomal interference and ubiquitin-activating enzyme (E1) inhibiting conditions by image-based flow cytometry and Western blot detection. Our results showed no accumulation of porcine MSMB either under proteasomal inhibition or under E1 inhibiting conditions. In addition, the immunoprecipitation study did not detect any ubiquitination of sperm MSMB nor was MSMB detected in the affinity-purified fraction containing ubiquitinated sperm proteins. Based on our results, we conclude that UPS does not appear to be the regulatory mechanism in the case of MSMB and opening new questions for further studies. Thus, the capacitation-induced processing of seminal plasma proteins on the sperm surface may be more complex than previously thought, employing multiple proteolytic systems in a non-redundant manner.

• Klíčová slova
• MSMB, PSP94, boar, capacitation, spermatozoa, ubiquitin-proteasome system, β-microseminoprotein,
• MeSH
• kapacitace spermií fyziologie   MeSH
• prasata MeSH
• proteasomový endopeptidasový komplex metabolismus   MeSH
• sekreční proteiny prostaty metabolismus   MeSH
• spermie metabolismus   fyziologie   MeSH
• ubikvitin metabolismus   MeSH
• ubikvitinace MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• beta-microseminoprotein MeSH Prohlížeč
• proteasomový endopeptidasový komplex MeSH
• sekreční proteiny prostaty MeSH
• ubikvitin MeSH

Sperm capacitation, the ultimate maturation event preparing mammalian spermatozoa for fertilization, was first described in 1951, yet its regulatory mechanisms remain poorly understood. The capacitation process encompasses an influx of bicarbonate and calcium ions, removal of decapacitating factors, changes of pH and sperm proteasomal activities, and the increased protein tyrosine phosphorylation. Here, we document a novel biological phenomenon of a unique zinc (Zn2+) ion redistribution associated with mammalian sperm in vitro capacitation (IVC). Using image-based flow cytometry (IBFC), we identified four distinct types of sperm zinc ion distribution patterns (further zinc signature) and their changes during IVC. The zinc signature was altered after sperm capacitation, reduced by proteasomal inhibitors, removed by zinc chelators, and maintained with addition of external ZnCl2. These findings represent a fundamental shift in the understanding of mammalian fertilization, paving the way for improved semen analysis, in vitro fertilization (IVF), and artificial insemination (AI).

• MeSH
• analýza spermatu MeSH
• chelátory farmakologie   MeSH
• chloridy farmakologie   MeSH
• kapacitace spermií účinky léků   fyziologie   MeSH
• kationty dvojmocné metabolismus   MeSH
• prasata MeSH
• průtoková cytometrie metody   MeSH
• sloučeniny zinku farmakologie   MeSH
• spermie metabolismus   MeSH
• zinek metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• chelátory MeSH
• chloridy MeSH
• kationty dvojmocné MeSH
• sloučeniny zinku MeSH
• zinc chloride MeSH Prohlížeč
• zinek MeSH