Altered histone abundance as a mode of ovotoxicity during 7,12-dimethylbenz[a]anthracene exposure with additive influence of obesity†.

Histones are slowly evolving chromatin components and chromatin remodeling can incorporate histone variants differing from canonical histones as an epigenetic modification. Several identified histone variants are involved with the environmental stress-induced DNA damage response (DDR). Mechanisms of DDR in transcriptionally inactive, prophase-arrested oocytes and epigenetic regulation are under-explored in ovarian toxicology. The study objective was to identify ovarian proteomic and histone modifications induced by DMBA exposure and an influence of obesity. Post-pubertal wildtype (KK.Cg-a/a; lean) and agouti (KK.Cg-Ay/J; obese) female mice, were exposed to either corn oil (control; CT) or DMBA (1 mg/kg) for 7d via intraperitoneal injection (n = 10/treatment). Ovarian proteome analysis (LC-MS/MS) determined that obesity altered 225 proteins (P < 0.05) with histone 3 being the second least abundant (FC = -5.98, P < 0.05). Histone 4 decreased by 3.33-fold, histone variant H3.3 decreased by 3.05-fold, and H1.2, H1.4 and H1.1(alpha) variants increased by 1.59, 1.90 and 2.01-fold, respectively (P < 0.05). DMBA exposure altered 48 proteins in lean mice with no observed alterations in histones or histone variants. In obese mice, DMBA exposure altered 120 proteins and histone 2B abundance increased by 0.30-fold (P < 0.05). In DMBA-exposed mice, obesity altered the abundance of 634 proteins. Histones 4, 3 and 2A type 1-F decreased by 4.03, 3.71, 0.43-fold, respectively, whereas histone variant H1.2 and linker histone, H15 increased by 2.72- and 3.07-fold, respectively (P < 0.05). Thus, DMBA exposure alters histones and histone variants, and responsivity is more pronounced during obesity, potentially altering ovarian transcriptional regulation.

Trajectory of primordial follicle depletion is accelerated in obese mice in response to 7,12-dimethylbenz[a]anthracene exposure.

Both obesity and exposure to environmental genotoxicants, such as 7,12-dimethylbenz[a]anthracene (DMBA), negatively impair female reproductive health. Hyperphagic lean KK.Cg-a/a (n = 8) and obese KK.Cg-Ay/J (n = 10) mice were exposed to corn oil as vehicle control (CT) or DMBA (1 mg/kg/day) for 7d intraperitoneally, followed by a recovery period. Obesity increased liver and spleen weight (P < 0.05), and DMBA exposure decreased uterine weight (P < 0.05) in obese mice. Primordial follicle loss (P < 0.05) caused by DMBA exposure was observed in obese mice only. Primary (lean P < 0.1; obese P < 0.05) and secondary (lean P < 0.05, obese P < 0.1) follicle loss initiated by DMBA exposure continued across recovery. Reduced pre-antral follicle number in lean mice (P < 0.05), regardless of DMBA exposure, was evident with no effect on antral follicles or corpora lutea number. Immunofluorescence staining of DNA damage marker, γH2AX, did not indicate ongoing DNA damage but TRP53 abundance was decreased in follicles (P < 0.05) of DMBA-exposed obese mice. In contrast, increased (P < 0.05) superoxide dismutase was observed in the corpora lutea of DMBA-exposed obese mice and reduced (P < 0.05) TRP53 abundance was noted in preantral and antral follicles of DMBA-exposed obese mice. This study indicates that obesity influences ovotoxicity caused by a genotoxicant, potentially involving accelerated primordial follicle activation and hampering normal follicular dynamics.

Biomarker-based human and animal sperm phenotyping: the good, the bad and the ugly†.

Conventional, brightfield-microscopic semen analysis provides important baseline information about sperm quality of an individual; however, it falls short of identifying subtle subcellular and molecular defects in cohorts of "bad," defective human and animal spermatozoa with seemingly normal phenotypes. To bridge this gap, it is desirable to increase the precision of andrological evaluation in humans and livestock animals by pursuing advanced biomarker-based imaging methods. This review, spiced up with occasional classic movie references but seriously scholastic at the same time, focuses mainly on the biomarkers of altered male germ cell proteostasis resulting in post-testicular carryovers of proteins associated with ubiquitin-proteasome system. Also addressed are sperm redox homeostasis, epididymal sperm maturation, sperm-seminal plasma interactions, and sperm surface glycosylation. Zinc ion homeostasis-associated biomarkers and sperm-borne components, including the elements of neurodegenerative pathways such as Huntington and Alzheimer disease, are discussed. Such spectrum of biomarkers, imaged by highly specific vital fluorescent molecular probes, lectins, and antibodies, reveals both obvious and subtle defects of sperm chromatin, deoxyribonucleic acid, and accessory structures of the sperm head and tail. Introduction of next-generation image-based flow cytometry into research and clinical andrology will soon enable the incorporation of machine and deep learning algorithms with the end point of developing simple, label-free methods for clinical diagnostics and high-throughput phenotyping of spermatozoa in humans and economically important livestock animals.

Metabolic Shift in Porcine Spermatozoa during Sperm Capacitation-Induced Zinc Flux.

Mammalian spermatozoa rely on glycolysis and mitochondrial oxidative phosphorylation for energy leading up to fertilization. Sperm capacitation involves a series of well-regulated biochemical steps that are necessary to give spermatozoa the ability to fertilize the oocyte. Additionally, zinc ion (Zn2+) fluxes have recently been shown to occur during mammalian sperm capacitation. Semen from seven commercial boars was collected and analyzed using image-based flow cytometry before, after, and with the inclusion of 2 mM Zn2+ containing in vitro capacitation (IVC) media. Metabolites were extracted and analyzed via Gas Chromatography-Mass Spectrometry (GC-MS), identifying 175 metabolites, with 79 differentially abundant across treatments (p < 0.05). Non-capacitated samples showed high levels of respiration-associated metabolites including glucose, fructose, citric acid, and pyruvic acid. After 4 h IVC, these metabolites significantly decreased, while phosphate, lactic acid, and glucitol increased (p < 0.05). With zinc inclusion, we observed an increase in metabolites such as lactic acid, glucitol, glucose, fructose, myo-inositol, citric acid, and succinic acid, while saturated fatty acids including palmitic, dodecanoic, and myristic acid decreased compared to 4 h IVC, indicating regulatory shifts in metabolic pathways and fatty acid composition during capacitation. These findings underscore the importance of metabolic changes in improving artificial insemination and fertility treatments in livestock and humans.

Obesity partially potentiates dimethylbenz[a]anthracene-exposed ovotoxicity by altering the DNA damage repair response in mice†.

Obesity adversely affects reproduction, impairing oocyte quality, fecundity, conception, and implantation. The ovotoxicant, dimethylbenz[a]anthracene, is biotransformed into a genotoxic metabolite to which the ovary responds by activating the ataxia telangiectasia mutated DNA repair pathway. Basal ovarian DNA damage coupled with a blunted response to genotoxicant exposure occurs in obese females, leading to the hypothesis that obesity potentiates ovotoxicity through ineffective DNA damage repair. Female KK.Cg-a/a (lean) and KK.Cg-Ay/J (obese) mice received corn oil or dimethylbenz[a]anthracene (1 mg/kg) at 9 weeks of age for 7 days via intraperitoneal injection (n = 10/treatment). Obesity increased liver weight (P < 0.001) and reduced (P < 0.05) primary, preantral, and corpora lutea number. In lean mice, dimethylbenz[a]anthracene exposure tended (P < 0.1) to increase proestrus duration and reduced (P = 0.07) primordial follicle number. Dimethylbenz[a]anthracene exposure decreased (P < 0.05) uterine weight and increased (P < 0.05) primary follicle number in obese mice. Total ovarian abundance of BRCA1, γH2AX, H3K4me, H4K5ac, H4K12ac, and H4K16ac (P > 0.05) was unchanged by obesity or dimethylbenz[a]anthracene exposure. Immunofluorescence staining demonstrated decreased (P < 0.05) abundance of γH2AX foci in antral follicles of obese mice. In primary follicle oocytes, BRCA1 protein was reduced (P < 0.05) by dimethylbenz[a]anthracene exposure in lean mice. Obesity also decreased (P < 0.05) BRCA1 protein in primary follicle oocytes. These findings support both a follicle stage-specific ovarian response to dimethylbenz[a]anthracene exposure and an impact of obesity on this ovarian response.

A re-appraisal of mesenchymal-epithelial transition (MET) in endometrial epithelial remodeling.

Mesenchymal-epithelial transition (MET) is a mechanism of endometrial epithelial regeneration. It is also implicated in adenocarcinoma and endometriosis. Little is known about this process in normal uterine physiology. Previously, using pregnancy and menses-like mouse models, MET occurred only as an epithelial damage/repair mechanism. Here, we hypothesized that MET also occurs in other physiological endometrial remodeling events, outside of damage/repair, such as during the estrous cycle and adenogenesis (gland development). To investigate this, Amhr2-Cre-YFP/GFP mesenchyme-specific reporter mice were used to track the fate of mesenchymal-derived (MD) cells. Using EpCAM (epithelial marker), EpCAM+YFP+ MD-epithelial cells were identified in all stages of the estrous cycle except diestrus, in both postpartum and virgin mice. EpCAM+YFP+ MD-epithelial cells comprised up to 80% of the epithelia during estrogen-dominant proestrus and significantly declined to indistinguishable from control uteri in diestrus, suggesting MET is hormonally regulated. MD-epithelial cells were also identified during postnatal epithelial remodeling. MET occurred immediately after birth at postnatal day (P) 0.5 with EpCAM+GFP+ cells ranging from negligible (0.21%) to 82% of the epithelia. EpCAM+GFP+ MD-epithelial cells declined during initiation of adenogenesis (P8, avg. 1.75%) and then increased during gland morphogenesis (P14, avg. 10%). MD-epithelial cells expressed markers in common with non-MD-epithelial cells (e.g., EpCAM, FOXA2, ESR1, PGR). However, MD-epithelial cells were differentially regulated postnatally and in adults, suggesting a functional distinction in the two populations. We conclude that MET occurs not only as an epithelial damage/repair mechanism but also during other epithelial remodeling events, which to our knowledge has not been demonstrated in other tissues.

Sperm capacitation as a predictor of boar fertility.

Prediction of a boar's fertility level has great economic importance for sow herds. After standard sperm morphology and motility metrics are met, approximately 25% of boars have less than 80% conception rates. Due in part to the many factors involved in the fertilization process, a multifactorial model incorporating multiple relevant sperm physiology factors will likely lead to increased understanding of boar fertility. Here we review the current literature on boar sperm capacitation as a predictor of boar fertility. While limited, several studies have provided correlations between the percentage of sperm in an ejaculate that are capable of undergoing sperm capacitation in a chemically defined media and artificial insemination field fertility as well as proteome and other methods. Work summarized here underscores the need for further understanding of boar fertility.

Spermatozoan Metabolism as a Non-Traditional Model for the Study of Huntington's Disease.

Huntington's Disease (HD) is a fatal autosomal dominant neurodegenerative disease manifested through motor dysfunction and cognitive deficits. Decreased fertility is also observed in HD animal models and HD male patients, due to altered spermatogenesis and sperm function, thus resulting in reduced fertilization potential. Although some pharmaceuticals are currently utilized to mitigate HD symptoms, an effective treatment that remedies the pathogenesis of the disease is yet to be approved by the FDA. Identification of genes and relevant diagnostic biomarkers and therapeutic target pathways including glycolysis and mitochondrial complex-I-dependent respiration may be advantageous for early diagnosis, management, and treatment of the disease. This review addresses the HD pathway in neuronal and sperm metabolism, including relevant gene and protein expression in both neurons and spermatozoa, indicated in the pathogenesis of HD. Furthermore, zinc-containing and zinc-interacting proteins regulate and/or are regulated by zinc ion homeostasis in both neurons and spermatozoa. Therefore, this review also aims to explore the comparative role of zinc in both neuronal and sperm function. Ongoing studies aim to characterize the products of genes implicated in HD pathogenesis that are expressed in both neurons and spermatozoa to facilitate studies of future treatment avenues in HD and HD-related male infertility. The emerging link between zinc homeostasis and the HD pathway could lead to new treatments and diagnostic methods linking genetic sperm defects with somatic comorbidities.

Progesterone induces porcine sperm release from oviduct glycans in a proteasome-dependent manner.

In mammals, the oviduct retains sperm, forming a reservoir from which they are released in synchrony with ovulation. However, the mechanisms underlying sperm release are unclear. Herein, we first examined in greater detail the release of sperm from the oviduct reservoir by sex steroids, and secondly, if the ubiquitin-proteasome system (UPS) mediates this release in vitro. Sperm were allowed to bind to oviductal cells or immobilized oviduct glycans, either bi-SiaLN or a suLeX, and channeled with steroids in the presence or absence of proteasome inhibitors. Previously, we have demonstrated progesterone-induced sperm release from oviduct cells and immobilized glycans in a steroid-specific manner. Herein, we found that the release of sperm from an immobilized oviduct glycan, a six-sialylated branched structure, and from immobilized fibronectin was inhibited by the CatSper blocker NNC 055-0396, akin to the previously reported ability of NNC 055-0396 to inhibit sperm release from another oviduct glycan, sulfated Lewis-X trisaccharide. Thus, CatSper may be required for release of sperm from a variety of adhesion systems. One possible mechanism for sperm release is that glycan receptors on sperm are degraded by proteasomes or shed from the sperm surface by proteasomal degradation. Accordingly, the inhibition of proteasomal degradation blocked sperm release from oviduct cell aggregates both immobilized oviduct glycans as well as fibronectin. In summary, progesterone-induced sperm release requires both active CatSper channels and proteasomal degradation, suggesting that hyperactivation and proteolysis are vital parts of the mechanism by which sperm move from the oviduct reservoir to the site of fertilization.

Reciprocal surface expression of arylsulfatase A and ubiquitin in normal and defective mammalian spermatozoa.

Defective mammalian spermatozoa are marked on their surface by proteolytic chaperone ubiquitin. To identify potential ubiquitinated substrates in the defective spermatozoa, we resolved bull sperm protein extracts on a two-dimensional gel and isolated a 64-65-kDa spot (p64) corresponding to one of the major ubiquitin-immunoreactive bands observed in the one-dimensional Western blots. Immune serum raised against this protein recognized a prominent, possibly glycosylated band/spot in the range of 55-68 kDa, consistent with the original spot used for immunization. Internal sequences obtained by Edman degradation of this spot matched the sequence of arylsulfatase A (ARSA), the sperm acrosomal enzyme thought to be important for fertility. By immunofluorescence, a prominent signal was detected on the acrosomal surface (boar and bull) and on the sperm tail principal piece (bull). A second immune serum raised against a synthetic peptide corresponding to an immunogenic internal sequence (GTGKSPRRTL) of the porcine ARSA also labeled sperm acrosome and principal piece. Both sera showed diminished immunoreactivity in the defective bull spermatozoa co-labeled with an anti-ubiquitin antibody. Western blotting and image-based flow cytometry (IBFC) confirmed a reduced ARSA immunoreactivity in the immotile sperm fraction rich in ubiquitinated spermatozoa. Larger than expected ARSA-immunoreactive bands were found in sperm protein extracts immunoprecipitated with anti-ubiquitin antibodies and affinity purified with matrix-bound, recombinant ubiquitin-binding UBA domain. These bands did not show the typical pattern of ARSA glycosylation but overlapped with bands preferentially binding the Lens culinaris agglutinin (LCA) lectin. By both epifluorescence microscopy and IBFC, the LCA binding was increased in the ubiquitinated spermatozoa with diminished ARSA immunoreactivity. ARSA was also found in the epididymal fluid suggesting that in addition to intrinsic ARSA expression in the testis, epididymal spermatozoa take up ARSA on their surface during the epididymal passage. We conclude that sperm surface ARSA is one of the ubiquitinated sperm surface glycoproteins in defective bull spermatozoa. Defective sperm surface thus differs from normal sperm surface by increased ubiquitination, reduced ARSA binding, and altered glycosylation.

Porcine model for the study of sperm capacitation, fertilization and male fertility.

Mammalian fertilization remains a poorly understood event with the vast majority of studies done in the mouse model. The purpose of this review is to revise the current knowledge about semen deposition, sperm transport, sperm capacitation, gamete interactions and early embryonic development with a focus on the porcine model as a relevant, alternative model organism to humans. The review provides a thorough overview of post-ejaculation events inside the sow's reproductive tract including comparisons with humans and implications for human fertilization and assisted reproductive therapy (ART). Porcine methodology for sperm handling, preservation, in vitro capacitation, oocyte in vitro maturation, in vitro fertilization and intra-cytoplasmic sperm injection that are routinely used in pig research laboratories can be successfully translated into ART to treat human infertility. Last, but not least, new knowledge about mitochondrial inheritance in the pig can provide an insight into human mitochondrial diseases and new knowledge on polyspermy defense mechanisms could contribute to the development of new male contraceptives.

Sperm Cohort-Specific Zinc Signature Acquisition and Capacitation-Induced Zinc Flux Regulate Sperm-Oviduct and Sperm-Zona Pellucida Interactions.

Building on our recent discovery of the zinc signature phenomenon present in boar, bull, and human spermatozoa, we have further characterized the role of zinc ions in the spermatozoa's pathway to fertilization. In boar, the zinc signature differed between the three major boar ejaculate fractions, the initial pre-rich, the sperm-rich, and the post-sperm-rich fraction. These differences set in the sperm ejaculatory sequence establish two major sperm cohorts with marked differences in their sperm capacitation progress. On the subcellular level, we show that the capacitation-induced Zn-ion efflux allows for sperm release from oviductal glycans as analyzed with the oviductal epithelium mimicking glycan binding assay. Sperm zinc efflux also activates zinc-containing enzymes and proteases involved in sperm penetration of the zona pellucida, such as the inner acrosomal membrane matrix metalloproteinase 2 (MMP2). Both MMP2 and the 26S proteasome showed severely reduced activity in the presence of zinc ions, through studies using by gel zymography and the fluorogenic substrates, respectively. In the context of the fertilization-induced oocyte zinc spark and the ensuing oocyte-issued polyspermy-blocking zinc shield, the inhibitory effect of zinc on sperm-borne enzymes may contribute to the fast block of polyspermy. Altogether, our findings establish a new paradigm on the role of zinc ions in sperm function and pave the way for the optimization of animal semen analysis, artificial insemination (AI), and human male-factor infertility diagnostics.

Ubiquitin-proteasome system participates in the de-aggregation of spermadhesins and DQH protein during boar sperm capacitation.

We studied the participation of the ubiquitin-proteasome system (UPS) in spermadhesin release during in vitro capacitation (IVC) of domestic boar spermatozoa. At ejaculation, boar spermatozoa acquire low molecular weight (8-16 kDa) seminal plasma proteins, predominantly spermadhesins, aggregated on the sperm surface. Due to their arrangement, such aggregates are relatively inaccessible to antibody labeling. As a result of de-aggregation and release of the outer layers of spermadhesins from the sperm surface during IVC, antibody labeling becomes feasible in the capacitated spermatozoa. In vivo, the capacitation-induced shedding of spermadhesins from the sperm surface is associated with the release of spermatozoa from the oviductal sperm reservoir. We took advantage of this property to perform image-based flow cytometry to study de-aggregation and shedding of boar spermadhesins (AQN, AWN, PSP protein families) and boar DQH (BSP1) sperm surface protein which induces higher fluorescent intensity in capacitated vs ejaculated spermatozoa. Addition of a proteasomal inhibitor (100 µM MG132) during IVC significantly reduced fluorescence intensity of all studied proteins (P < 0.05) compared to vehicle control IVC. Western blot detection of spermadhesins did not support their retention during IVC with proteasomal inhibition (P > 0.99) but showed the accumulation of DQH (P = 0.03) during IVC, compared to vehicle control IVC. Our results thus demonstrate that UPS participates in the de-aggregation of spermadhesins and DQH protein from the sperm surface during capacitation, with a possible involvement in sperm detachment from the oviductal sperm reservoir and/or sperm-zona pellucida interactions. The activity of sperm UPS modulates de-aggregation of boar spermadhesins and DQH sperm surface protein/binder of sperm1 (BSP1) during the sperm capacitation.

Modifications of the 26S proteasome during boar sperm capacitation.

Protein ubiquitination is a stable, reversible post-translational modification, targeting proteins for degradation/recycling by the 26S proteasome in a well-characterized enzymatic cascade. Studies have revealed the role of UPS in the regulation of fertilization, including sperm-zona pellucida interactions and the early event of sperm capacitation. The present study investigates the changes in proteasome compartmentalization, subunit composition and post-translational modifications during in vitro capacitation of fresh boar spermatozoa. We observed capacitation-dependent shedding of both 20S core and 19S regulatory particles from the acrosome that was associated with decreased plasma membrane integrity, independent of proteasomal inhibition. Subunits PSMA1-7 of the 20S core did not appear to undergo post-translational modifications during capacitation, based on invariant molecular masses before and after capacitation; however, we observed multiple PSMD4 forms of 19S regulatory particles (50, 53, 70, 115-140, 160 and >176 kDa) sequentially released from spermatozoa. PSMD4 subunit was found to be post-translationally modified during the course of capacitation, resulting in changes of apparent molecular mass, some of which were dependent on proteasomal inhibition. These results show that the sperm proteasomes are being modified during sperm capacitation. Additional studies of individual 26S proteasome subunits will be required to elucidate these modifications and to understand how UPS modulates sperm capacitation.

Zinc: A Necessary Ion for Mammalian Sperm Fertilization Competency.

The importance of zinc for male fertility only emerged recently, being propelled in part by consumer interest in nutritional supplements containing ionic trace minerals. Here, we review the properties, biological roles and cellular mechanisms that are relevant to zinc function in the male reproductive system, survey available peer-reviewed data on nutritional zinc supplementation for fertility improvement in livestock animals and infertility therapy in men, and discuss the recently discovered signaling pathways involving zinc in sperm maturation and fertilization. Emphasis is on the zinc-interacting sperm proteome and its involvement in the regulation of sperm structure and function, from spermatogenesis and epididymal sperm maturation to sperm interactions with the female reproductive tract, capacitation, fertilization, and embryo development. Merits of dietary zinc supplementation and zinc inclusion into semen processing media are considered with livestock artificial insemination (AI) and human assisted reproductive therapy (ART) in mind. Collectively, the currently available data underline the importance of zinc ions for male fertility, which could be harnessed to improve human reproductive health and reproductive efficiency in agriculturally important livestock species. Further research will advance the field of sperm and fertilization biology, provide new research tools, and ultimately optimize semen processing procedures for human infertility therapy and livestock AI.

Regulation of Sperm Capacitation by the 26S Proteasome: An Emerging New Paradigm in Spermatology.

The ubiquitin proteasome system (UPS) participates in many biological processes ranging from cell cycle and antigen processing to cellular defense and signaling. Work of the last decade has made it evident that the UPS is involved in many sperm-related processes leading up to and as part of fertilization. The current knowledge of UPS involvement and changes during sperm capacitation are reviewed together with a list of known proteasome-associated sperm proteins and a discussion of the relationships between these proteins and the proteasome. Proteasomal inhibitors such as MG-132 and epoxomicin significantly alter capacitation and prevent acrosome reaction. The 26S proteasome degrades AKAP3, an A-kinase anchoring protein, partially regulating the release of protein-kinase A (PKA), a vital component necessary for the steps leading up to capacitation. Further, changes occur in 20S core subunit localization and abundance throughout capacitation. Proteasome-interacting valosine-containing protein (VCP) undergoes tyrosine phosphorylation; however, its physiological roles in capacitation and fertilization remain unknown. The E1-type ubiquitin-activating enzyme (UBA1) inhibitor PYR-41 also alters acrosomal membrane remodeling during capacitation. Furthermore, after capacitation, the acrosomal proteasomes facilitate the degradation of zona pellucida glycoproteins leading up to fertilization. Methods to modulate the sperm proteasome activity during sperm storage and capacitation may translate to increased reproductive efficiency in livestock animals. Human male infertility diagnostics may benefit from incorporation of research outcomes built upon relationships between UPS and capacitation. Altogether, the studies reviewed here support the involvement of UPS in sperm capacitation and present opportunities for new discoveries.

The Ubiquitin-Proteasome System Participates in Sperm Surface Subproteome Remodeling during Boar Sperm Capacitation.

Sperm capacitation is a complex process endowing biological and biochemical changes to a spermatozoon for a successful encounter with an oocyte. The present study focused on the role of the ubiquitin-proteasome system (UPS) in the remodeling of the sperm surface subproteome. The sperm surface subproteome from non-capacitated and in vitro capacitated (IVC) porcine spermatozoa, with and without proteasomal inhibition, was selectively isolated. The purified sperm surface subproteome was analyzed using high-resolution, quantitative liquid chromatography-mass spectrometry (LC-MS) in four replicates. We identified 1680 HUGO annotated proteins, out of which we found 91 to be at least 1.5× less abundant (p < 0.05) and 141 to be at least 1.5× more abundant (p < 0.05) on the surface of IVC spermatozoa. These proteins were associated with sperm capacitation, hyperactivation, metabolism, acrosomal exocytosis, and fertilization. Abundances of 14 proteins were found to be significantly different (p < 0.05), exceeding a 1.5-fold abundance between the proteasomally inhibited (100 µM MG132) and vehicle control (0.2% ethanol) groups. The proteins NIF3L1, CSE1L, NDUFB7, PGLS, PPP4C, STK39, and TPRG1L were found to be more abundant; while BPHL, GSN, GSPT1, PFDN4, STYXL1, TIMM10, and UBXN4 were found to be less abundant in proteasomally inhibited IVC spermatozoa. Despite the UPS having a narrow range of targets, it modulated sperm metabolism and binding by regulating susceptible surface proteins. Changes in CSE1L, PFDN4, and STK39 during in vitro capacitation were confirmed using immunocytochemistry, image-based flow cytometry, and Western blotting. The results confirmed the active participation of the UPS in the extensive sperm surface proteome remodeling that occurs during boar sperm capacitation. This work will help us to identify new pharmacological mechanisms to positively or negatively modulate sperm fertilizing ability in food animals and humans.

Semen quality of Nellore and Angus bulls classified by fertility indices and relations with field fertility in fixed-time artificial insemination.

Despite being the most important form of biotechnology in animal reproduction, artificial insemination was used in about 23% of Brazilian bovine herds in 2021. This is due to the variability of results caused by varying bull fertility and body condition of the cows. This study aimed to correlate the fertility indices of bulls with qualitative attributes of the semen. Semen samples from 28 bulls (Nellore and Angus) were used to evaluate postthaw sperm morphology and kinetics using conventional analysis, image-based flow cytometry (IBFC) and computer-assisted semen analysis (CASA). The fertility index was effective in separating bulls into 4 different fertility classes (P < 0.001), and fertility rates in timed artificial insemination (TAI) remained constant between the cows' fertility categories (P < 0.001) and in the different grades of female body condition (P < 0.005). After partial least squares regression (PLS) analysis, four models were proposed with different variables. The coefficients of determination for the conventional analysis, CASA, and IBCFC models were 0.154, 0.380, and 0.259, respectively. The composite model, including select IBFC and CASA parameters, showed a greater R2 (0.481) with progressive motility, average speed (VAP, µm/s), membrane integrity, and mitochondrial potential, showing a positive effect. Linear speed (VSL, µm/s) and acrosomal integrity had a negative effect on bull fertility indices. Bulls classified by the fertility index attained dispersed pregnancy rates in different cow body condition score (BCS) classes, and the sperm quality pattern was consistent with this classification. In conclusion, this novel composite model including CASA and IBFC parameters improves the prediction of bull fertility used in TAI.