Sperm mitochondria dysfunction in response to testicular cancer.

Testicular cancer is the most common form of cancer in young men of reproductive age and its incidence is increasing globally. With the currently successful treatment and 95% survival rate, there is a need for deeper understanding of testicular cancer-related infertility. Most patients with testicular cancer experience semen abnormalities prior to cancer therapy. However, the exact mechanism of the effect of testicular cancer on sperm anomalies is not known. Mitochondria are organelles that play a crucial role in both tumorigenesis and spermatogenesis and their malfunction may be an important factor resulting in sperm abnormalities in testicular cancer patients. Within the scope of this review, we will discuss current knowledge of testicular cancer-related alterations in the ATP production pathway, a possible pathophysiological switch from oxidative phosphorylation (OXPHOS) to glycolysis, as well as the role of oxidative stress promoting sperm dysfunction. In this regard, the review provides a summary of the impact of testicular cancer on sperm quality as a possible consequence of impaired mitochondrial function including the energy metabolic pathways that are known to be altered in the sperm of testicular cancer patients.

Fertilization, but Not Post-Implantation Development, Can Occur in the Absence of Sperm and Oocyte Beta1 Integrin in Mice.

Fertilization is a complex process that requires successive stages and culminates in the adhesion/fusion of gamete membranes. If the question of the involvement of oocyte integrins has been swept away by deletion experiments, that of the involvement of sperm integrins remains to be further characterized. In the present study, we addressed the question of the feasibility of sperm-oocyte adhesion/fusion and early implantation in the absence of sperm ß1 integrin. Males and females with ß1 integrin-depleted sperm and oocytes were mated, and fertilization outcome was monitored by a gestational ultrasound analysis. Results suggest that although the sperm ß1 integrin participates in gamete adhesion/fusion, it is dispensable for fertilization in mice. However, sperm- and/or oocyte-originated integrin ß1 is essential for post-implantation development. Redundancy phenomena could be at the origin of a compensatory expression or alternative dimerization pattern.

Timing of ICSI with Respect to Meiotic Spindle Status.

The aim of this study was to evaluate the efficiency of using meiotic spindle (MS) visibility and relative position to the polar body (PB) as indicators of oocyte maturation in order to optimize intracytoplasmic sperm injection (ICSI) timing. This was a cohort study of patients younger than 40 years with planned ICSI, the timing of which was determined by MS status, compared with those without MS evaluation. The angle between PB and MS and MS visibility were evaluated by optical microscope with polarizing filter. Oocytes with MS evaluation were fertilized according to MS status either 5-6 h after ovum pick-up (OPU) or 7-8 h after OPU. Oocytes without MS evaluation were all fertilized 5-6 h after OPU. For patients over 35 years visualization of MS influenced pregnancy rate (PR): 182 patients with MS visualization had 32% PR (58/182); while 195 patients without MS visualization had 24% PR (47/195). For patients under 35 years, visualization of MS did not influence PR: 140 patients with MS visualization had 41% PR (58/140), while 162 patients without MS visualization had 41% PR (66/162). Visualization of MS therefore appears to be a useful parameter for assessment of oocyte maturity and ICSI timing for patients older than 35.

H4K5 Butyrylation Coexist with Acetylation during Human Spermiogenesis and Are Retained in the Mature Sperm Chromatin.

Male germ cells experience a drastic chromatin remodeling through the nucleo-histone to nucleo-protamine (NH-NP) transition necessary for proper sperm functionality. Post-translational modifications (PTMs) of H4 Lys5, such as acetylation (H4K5ac), play a crucial role in epigenetic control of nucleosome disassembly facilitating protamine incorporation into paternal DNA. It has been shown that butyrylation on the same residue (H4K5bu) participates in temporal regulation of NH-NP transition in mice, delaying the bromodomain testis specific protein (BRDT)-dependent nucleosome disassembly and potentially marking retained nucleosomes. However, no information was available so far on this modification in human sperm. Here, we report a dual behavior of H4K5bu and H4K5ac in human normal spermatogenesis, suggesting a specific role of H4K5bu during spermatid elongation, coexisting with H4K5ac although with different starting points. This pattern is stable under different testicular pathologies, suggesting a highly conserved function of these modifications. Despite a drastic decrease of both PTMs in condensed spermatids, they are retained in ejaculated sperm, with 30% of non-colocalizing nucleosome clusters, which could reflect differential paternal genome retention. Whereas no apparent effect of these PTMs was observed associated with sperm quality, their presence in mature sperm could entail a potential role in the zygote.

Kinetic Study of 17α-Estradiol Mechanism during Rat Sperm Capacitation.

17α-Estradiol (αE2) is a natural diastereoisomer of 17ß-estradiol (E2). It is well known that αE2 can bind to estrogen receptors. However, its biological activity is less than that of E2 and is species and tissue specific. The goal of our study was to propose the mechanism of αE2 hormonal response in rat sperm during their capacitation in vitro and compare it with a previously studied mouse model. Concentration changes in externally added αE2 during capacitation of rat sperm were monitored by the high-performance liquid chromatographic method with tandem mass spectrometric detection (HPLC-MS/MS). The calculated values of relative concentrations Bt were subjected to kinetic analysis. The findings indicated that αE2 in rat sperm did not trigger autocatalytic reaction, in contrast to the mouse sperm, and that the initiation of the hormone penetration through the sperm plasma membrane was substantially faster in rats.

Important parameters affecting quality of vitrified donor oocytes.

For group of 281 oocytes obtained from 43 stimulated donors and cryopreserved by vitrification protocol using Cryotop and Kitazato medium we determined important parameters of oocytes collection and vitrification processes which strongly affect the probability that warmed oocytes will produce high-quality embryos for transfer. The probability to obtain high-quality embryos for transfer from vitrified and warmed oocytes was highest when two conditions were fulfilled: 1. oocytes were incubated before vitrification for 7-10 h and 2. stimulated ovaries of donors in one cycle produced a smaller number of oocytes (<7 oocytes from one donor per stimulated cycle). The probable reasons for these observations were: 1. early vitrification (less than 7 h) before final oocyte metaphase II maturation negatively affected the crucial process of post-warm remodelling of spindles and chromosomes, which reduced the fertilization and utilization rates, 2. the evaluated vitrification protocol amplifies negative impact of membrane defects of oocytes of those cohorts containing more than 6 oocytes - freezing places great demands on the integrity and elasticity of the cell membranes. The fact that cryopreservation influences a complex state of oocytes was confirmed by confocal microscopy.

Role of Integrins in Sperm Activation and Fertilization.

In mammals, integrins are heterodimeric transmembrane glycoproteins that represent a large group of cell adhesion receptors involved in cell-cell, cell-extracellular matrix, and cell-pathogen interactions. Integrin receptors are an important part of signalization pathways and have an ability to transmit signals into and out of cells and participate in cell activation. In addition to somatic cells, integrins have also been detected on germ cells and are known to play a crucial role in complex gamete-specific physiological events, resulting in sperm-oocyte fusion. The main aim of this review is to summarize the current knowledge on integrins in reproduction and deliver novel perspectives and graphical interpretations presenting integrin subunits localization and their dynamic relocation during sperm maturation in comparison to the oocyte. A significant part of this review is devoted to discussing the existing view of the role of integrins during sperm migration through the female reproductive tract; oviductal reservoir formation; sperm maturation processes ensuing capacitation and the acrosome reaction, and their direct and indirect involvement in gamete membrane adhesion and fusion leading to fertilization.

αV Integrin Expression and Localization in Male Germ Cells.

Integrins are transmembrane receptors that facilitate cell adhesion and cell-extracellular matrix communication. They are involved in the sperm maturation including capacitation and gamete interaction, resulting in successful fertilization. αV integrin belongs to the integrin glycoprotein superfamily, and it is indispensable for physiological spermiogenesis and testosterone production. We targeted the gene and protein expression of the αV integrin subunit and described its membrane localization in sperm. Firstly, in mouse, we traced αV integrin gene expression during spermatogenesis in testicular fraction separated by elutriation, and we detected gene activity in spermatogonia, spermatocytes, and round spermatids. Secondly, we specified αV integrin membrane localization in acrosome-intact and acrosome-reacted sperm and compared its pattern between mouse, pig, and human. Using immunodetection and structured illumination microscopy (SIM), the αV integrin localization was confined to the plasma membrane covering the acrosomal cap area and also to the inner acrosomal membrane of acrosome-intact sperm of all selected species. During the acrosome reaction, which was induced on capacitated sperm, the αV integrin relocated and was detected over the whole sperm head. Knowledge of the integrin pattern in mature sperm prepares the ground for further investigation into the pathologies and related fertility issues in human medicine and veterinary science.

Specific Inhibition of VanZ-Mediated Resistance to Lipoglycopeptide Antibiotics.

Teicoplanin is a natural lipoglycopeptide antibiotic with a similar activity spectrum as vancomycin; however, it has with the added benefit to the patient of low cytotoxicity. Both teicoplanin and vancomycin antibiotics are actively used in medical practice in the prophylaxis and treatment of severe life-threatening infections caused by gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, Enterococcus faecium and Clostridium difficile. The expression of vancomycin Z (vanZ), encoded either in the vancomycin A (vanA) glycopeptide antibiotic resistance gene cluster or in the genomes of E. faecium, as well as Streptococcus pneumoniae and C. difficile, was shown to specifically compromise the antibiotic efficiency through the inhibition of teicoplanin binding to the bacterial surface. However, the exact mechanisms of this action and protein structure remain unknown. In this study, the three-dimensional structure of VanZ from E. faecium EnGen0191 was predicted by using the I-TASSER web server. Based on the VanZ structure, a benzimidazole based ligand was predicted to bind to the VanZ by molecular docking. Importantly, this new ligand, named G3K, was further confirmed to specifically inhibit VanZ-mediated resistance to teicoplanin in vivo.

In Silico Identification and Validation of Organic Triazole Based Ligands as Potential Inhibitory Drug Compounds of SARS-CoV-2 Main Protease.

The SARS-CoV-2 virus is highly contagious to humans and has caused a pandemic of global proportions. Despite worldwide research efforts, efficient targeted therapies against the virus are still lacking. With the ready availability of the macromolecular structures of coronavirus and its known variants, the search for anti-SARS-CoV-2 therapeutics through in silico analysis has become a highly promising field of research. In this study, we investigate the inhibiting potentialities of triazole-based compounds against the SARS-CoV-2 main protease (Mpro). The SARS-CoV-2 main protease (Mpro) is known to play a prominent role in the processing of polyproteins that are translated from the viral RNA. Compounds were pre-screened from 171 candidates (collected from the DrugBank database). The results showed that four candidates (Bemcentinib, Bisoctrizole, PYIITM, and NIPFC) had high binding affinity values and had the potential to interrupt the main protease (Mpro) activities of the SARS-CoV-2 virus. The pharmacokinetic parameters of these candidates were assessed and through molecular dynamic (MD) simulation their stability, interaction, and conformation were analyzed. In summary, this study identified the most suitable compounds for targeting Mpro, and we recommend using these compounds as potential drug molecules against SARS-CoV-2 after follow up studies.

Tetraspanins in mammalian reproduction: spermatozoa, oocytes and embryos.

It is known that tetraspanin proteins are involved in many physiological somatic cell mechanisms. Additionally, research has indicated they also have a role in various infectious diseases and cancers. This review focuses on the molecular interactions underlying the tetraspanin web formation in gametes. Primarily, tetraspanins act in the reproductive tract as organizers of membrane complexes, which include the proteins involved in the contact and association of sperm and oocyte membranes. In addition, recent data shows that tetraspanins are likely to be involved in these processes in a complex way. In mammalian fertilization, an important role is attributed to CD molecules belonging to the tetraspanin superfamily, particularly CD9, CD81, CD151, and also CD63; mostly as part of extracellular vesicles, the significance of which and their potential in reproduction is being intensively investigated. In this article, we reviewed the existing knowledge regarding the expression of tetraspanins CD9, CD81, CD151, and CD63 in mammalian spermatozoa, oocytes, and embryos and their involvement in reproductive processes, including pathological events.

Correction to: Tetraspanins in mammalian reproduction: spermatozoa, oocytes and embryos.

The original article can be found online.

The Role of the LINC Complex in Sperm Development and Function.

The LINC (LInker of Nucleoskeleton and Cytoskeleton) complex is localized within the nuclear envelope and consists of SUN (Sad1/UNc84 homology domain-containing) proteins located in the inner nuclear membrane and KASH (Klarsicht/Anc1/Syne1 homology domain-containing) proteins located in the outer nuclear membrane, hence linking nuclear with cytoplasmic structures. While the nucleoplasm-facing side acts as a key player for correct pairing of homolog chromosomes and rapid chromosome movements during meiosis, the cytoplasm-facing side plays a pivotal role for sperm head development and proper acrosome formation during spermiogenesis. A further complex present in spermatozoa is involved in head-to-tail coupling. An intact LINC complex is crucial for the production of fertile sperm, as mutations in genes encoding for complex proteins are known to be associated with male subfertility in both mice and men. The present review provides a comprehensive overview on our current knowledge of LINC complex subtypes present in germ cells and its central role for male reproduction. Future studies on distinct LINC complex components are an absolute requirement to improve the diagnosis of idiopathic male factor infertility and the outcome of assisted reproduction.

The Role of Taste Receptor mTAS1R3 in Chemical Communication of Gametes.

Fertilization is a multiple step process leading to the fusion of female and male gametes and the formation of a zygote. Besides direct gamete membrane interaction via binding receptors localized on both oocyte and sperm surface, fertilization also involves gamete communication via chemical molecules triggering various signaling pathways. This work focuses on a mouse taste receptor, mTAS1R3, encoded by the Tas1r3 gene, as a potential receptor mediating chemical communication between gametes using the C57BL/6J lab mouse strain. In order to specify the role of mTAS1R3, we aimed to characterize its precise localization in testis and sperm using super resolution microscopy. The testis cryo-section, acrosome-intact sperm released from cauda epididymis and sperm which underwent the acrosome reaction (AR) were evaluated. The mTAS1R3 receptor was detected in late spermatids where the acrosome was being formed and in the acrosomal cap of acrosome intact sperm. AR is triggered in mice during sperm maturation in the female reproductive tract and by passing through the egg surroundings such as cumulus oophorus cells. This AR onset is independent of the extracellular matrix of the oocyte called zona pellucida. After AR, the relocation of mTAS1R3 to the equatorial segment was observed and the receptor remained exposed to the outer surroundings of the female reproductive tract, where its physiological ligand, the amino acid L-glutamate, naturally occurs. Therefore, we targeted the possible interaction in vitro between the mTAS1R3 and L-glutamate as a part of chemical communication between sperm and egg and used an anti-mTAS1R3-specific antibody to block it. We detected that the acrosome reacted spermatozoa showed a chemotactic response in the presence of L-glutamate during and after the AR, and it is likely that mTAS1R3 acted as its mediator.

Mitochondrial Function in Modulating Human Granulosa Cell Steroidogenesis and Female Fertility.

Ovarian follicle steroidogenesis associated with embryo quality results in a successful pregnancy. Each follicle consists of an oocyte surrounded by granulosa cells, which secrete several steroid and peptide hormones. Follicles harvested from women who conceived after assisted reproductive therapy (ART) had significantly higher estradiol levels in follicular fluids than the follicles from women who failed to conceive after ART. The higher follicular estradiol levels correlate well with successful fertilization following ART. Mitochondria are the central sites for steroid hormone biosynthesis. The first and rate-limiting step in the biosynthesis of steroid hormones occurs in the mitochondria of granulosa cells. In the present study, we hypothesized that the mitochondria in granulosa cells are critical for maintaining oocyte quality and fertility capacity. This study aims to clarify the relationship between mitochondrial function and granulosa cell steroidogenesis, and the relationship between hormone levels and fertility capacity. Sera, follicular fluids and granulosa cells were obtained from individuals undergoing IVF-ET treatment. The oocyte numbers, oocyte quality, fertilization rate, and pregnancy rate were also recorded. The patients who provided the granulosa cells were further classified into four groups: endometriosis, ovarian endometrioma, endometriosis without ovarian endometrioma, and polycystic ovary syndrome (PCOS); patients with other female factor infertility and male factor infertility were used as controls. We measured the levels of estradiol (E2) by radioimmunoassay. Concurrently, we analyzed the mitochondrial mass and membrane potential, and apoptosis by flow cytometry using nonyl acridine orange, TMRE, Annexin V-FITC and PI. Mitochondrial morphology was visualized by transfection with pLV-mitoDsRed. In addition, we assessed the protein levels of steroidogenic enzymes, steroidogenic acute regulatory protein (StAR) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) by Western blot. The results showed significantly decreased serum E2 and follicular E2 levels, and decreased IVF outcomes, in the patients with endometriosis. Reduced mitochondrial mass and decreased mitochondrial membrane potential were correlated with lower E2. Furthermore, a significant decrease in StAR and 3ß-HSD was found in patients with ovarian endometrioma. The enzyme levels of StAR and 3ß-HSD were highly correlated with E2 levels. Finally, elevated cumulus cell apoptosis was found in the patient group with ovarian endometrioma and PCOS. In conclusion, mitochondrial dysfunction of human granulosa cells may contribute to the decline of steroidogenesis, decreased fertilization rate, oocyte maturation rate, and oocyte quality, and it can ultimately jeopardize fertility.

Fluorescent analysis of boar sperm capacitation process in vitro.

BACKGROUND: Capacitation involves physiological changes that spermatozoa must undergo in the female reproductive tract or in vitro to obtain the ability to bind, penetrate and fertilize the egg. Up to date, several methods have been developed to characterize this complex biological process. The goal of the presented study is to mutually compare several fluorescent techniques, check their ability to detect changes in molecular processes during the capacitation progress and determine their ability to predict the percentage of acrosome reacted (AR) sperm after the exposure to solubilized zona pellucida (ZP). The capacitation process was analyzed using four fluorescent techniques: 1. chlortetracycline (CTC) staining, 2. anti-acrosin antibody (ACR.2) assay, 3. anti-phosphotyrosine (pY) antibody assay, 4. fluorescein isothiocyanate-conjugated phalloidin (FITC-phall) assay. All these methods were tested using fluorescent microscopy and flow cytometry. RESULTS: All selected methods are capable to detect the capacitation progress of boar sperm in vitro, but there are significant differences in their outcome when using fluorescent microscopy or flow cytometry experimental arrangements and subsequent statistical analysis (KW-ANOVA). Also, the ability to predict the absolute numbers of sperm which will undergo ZP-induced AR differ significantly (CTC and ACR.2 gave the best predictions). CONCLUSIONS: Our study compared four largely used methods used to characterize capacitation process, highlighted their differences and showed that all are able to detect capacitation progress, CTC and ACR.2 are furthermore able to accurately predict the percentage of AR sperm after ZP-induced AR.

The correlation between human seminal plasma sialoproteins and ejaculate parameters.

Sialic acids are negatively charged carbohydrates that are components of saccharide chains covalently linked to macromolecules. Sialylated glycoproteins are important for most biological processes, including reproduction, where they are associated with spermatogenesis, sperm motility, immune responses, and fertilization. Changes in the glycoprotein profile or sialylation in glycoproteins are likely to affect the quality of ejaculate. The aim of this study was to determine differences in the degree of sialylation between normozoospermic ejaculates and ejaculates with a pathological spermiogram using two lectins, Sambucus nigra (SNA) and Maackia amurensis (MAL II/MAA) recognizing α-2,6 or α-2,3 linkage of Sia to galactosyl residues. Our results show a close relationship between seminal plasma (SP) sialoproteins and the presence of anti-sperm antibodies in the ejaculate, apoptotic spermatozoa, and ejaculate quality. Using mass spectrometry, we identified SP sialoproteins such as, semenogelins, glycodelin, prolactin-inducible protein, lactotransferrin, and clusterin that are associated with spermatozoa and contribute to the modulation of the immune response and sperm apoptosis. Our findings suggest a correlation between the degree of SP glycoprotein sialylation and the existence of possible pathological states of spermatozoa and reproductive organs. Glycoproteins sialylation represents a potential parameter reflecting the overall quality of ejaculate and could potentially be utilised in diagnostics.

Protamine 2 Deficiency Results In Septin 12 Abnormalities.

There is a well-established link between abnormal sperm chromatin states and poor motility, however, how these two processes are interdependent is unknown. Here, we identified a possible mechanistic insight by showing that Protamine 2, a nuclear DNA packaging protein in sperm, directly interacts with cytoskeletal protein Septin 12, which is associated with sperm motility. Septin 12 has several isoforms, and we show, that in the Prm2 -/- sperm, the short one (Mw 36 kDa) is mislocalized, while two long isoforms (Mw 40 and 41 kDa) are unexpectedly lost in Prm2 -/- sperm chromatin-bound protein fractions. Septin 12 co-immunoprecipitated with Protamine 2 in the testicular cell lysate of WT mice and with Lamin B1/B2/B3 in co-transfected HEK cells despite we did not observe changes in Lamin B2/B3 protein or SUN4 expression in Prm2 -/- testes. Furthermore, the Prm2 -/- sperm have on average a smaller sperm nucleus and aberrant acrosome biogenesis. In humans, patients with low sperm motility (asthenozoospermia) have imbalanced histone- protamine 1/2 ratio and modified levels of cytoskeletal proteins. We detected retained Septin 12 isoforms (Mw 40 and 41 kDa) in the sperm membrane, chromatin-bound and tubulin/mitochondria protein fractions, which was not true for healthy normozoospermic men. In conclusion, our findings expand the current knowledge regarding the connection between Protamine 2 and Septin 12 expression and localization, resulting in low sperm motility and morphological abnormalities.

Proteomic analysis of the mouse sperm acrosome - towards an understanding of an organelle with diverse functionality.

The acrosome located within the mammalian sperm head is essential for successful fertilization, as it enables the sperm to penetrate the extracellular layers of the oocyte and fuse with oolemma. However, the mammalian acrosomal vesicle is no longer considered to contain only hydrolytic enzymes. Using label-free nano-scale liquid chromatography tandem mass spectrometry (nLC-MS/MS) proteomics, we identified a total of 885 proteins in the acrosome isolated from spermatozoa obtained from cauda epididymis of free-living house mice Mus musculus musculus contains a total of 885 proteins. Among these, 334 proteins were significantly enriched in the acrosome thus representing 27.3% of the whole proteome of the intact sperm. Importantly, we have detected a total of nine calycins while eight of them belong to the lipocalin protein family. In mice, lipocalins are involved in multi-level chemical communication between individuals including pheromone transport and odor perception. Using an indirect immunofluorescence assay, we demonstrated that lipocalin 5 (LCN5) is expressed in the mouse germ cells, and after completing spermatogenesis, it remains localized in the sperm acrosome until the last step of the extratesticular maturation, the acrosome reaction. The presence of lipocalins in the acrosome and acrosome-reacted sperm suggests their original role as chelators of organic and potentially toxic compounds resulting from ongoing spermiogenesis. Along with this evidence, detected mitochondrial (e.g., a subunit of the cytochrome c oxidase MTCO1) and proteasomal proteins (subunits of both 20 S core proteasome [PSMA2, PSMBs] and 19 S regulatory particle [PSMDs]) in acrosomes provide further evidence that acrosomes could also function as `waste baskets` after testicular sperm maturation.

Modulatory effect of MG-132 proteasomal inhibition on boar sperm motility during in vitro capacitation.

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.