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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 (Zn 2+ ) 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 ZnCl 2 . 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). During sperm capacitation physiological changes occur that are required for fertilization. Here, the authors describe four sperm zinc signatures during invitro capacitation that are indicative of sperm quality and capacity to fertilize.

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.

Maternal inheritance of mitochondria and mtDNA is a universal principle in human and animal development, guided by selective ubiquitin-dependent degradation of the sperm-borne mitochondria after fertilization. However, it is not clear how the 26S proteasome, the ubiquitin-dependent protease that is only capable of degrading one protein molecule at a time, can dispose of a whole sperm mitochondrial sheath. We hypothesized that the canonical ubiquitin-like autophagy receptors [sequestosome 1 (SQSTM1), microtubule-associated protein 1 light chain 3 (LC3), gamma-aminobutyric acid receptor-associated protein (GABARAP)] and the nontraditional mitophagy pathways involving ubiquitin-proteasome system and the ubiquitin-binding protein dislocase, valosin-containing protein (VCP), may act in concert during mammalian sperm mitophagy. We found that the SQSTM1, but not GABARAP or LC3, associated with sperm mitochondria after fertilization in pig and rhesus monkey zygotes. Three sperm mitochondrial proteins copurified with the recombinant, ubiquitin-associated domain of SQSTM1. The accumulation of GABARAP-containing protein aggregates was observed in the vicinity of sperm mitochondrial sheaths in the zygotes and increased in the embryos treated with proteasomal inhibitor MG132, in which intact sperm mitochondrial sheaths were observed. Pharmacological inhibition of VCP significantly delayed the process of sperm mitophagy and completely prevented it when combined with microinjection of autophagy-targeting antibodies specific to SQSTM1 and/or GABARAP. Sperm mitophagy in higher mammals thus relies on a combined action of SQSTM1-dependent autophagy and VCP-mediated dislocation and presentation of ubiquitinated sperm mitochondrial proteins to the 26S proteasome, explaining how the whole sperm mitochondria are degraded inside the fertilized mammalian oocytes by a protein recycling system involved in degradation of single protein molecules.

Artificial insemination of livestock has been a staple technology for producers worldwide for over sixty years. This reproductive technology has allowed for the rapid improvement of livestock genetics, most notably in dairy cattle and pigs. This field has experienced continuous improvements over the last six decades. Though much work has been carried out to improve the efficiency of AI, there are still many areas which continue to experience improvement, including semen analysis procedures, sperm selection techniques, sperm sexing technologies, and semen storage methods. Additionally, the use of AI continues to grow in beef cattle, horses, and small ruminants as the technology continues to become more efficient and yield higher pregnancy rates. In this review, AI trends in the various livestock species as well as cutting edge improvements in the aforementioned areas will be discussed at length. Future work will continue to refine the protocols which are used for AI and continue to increase pregnancy rates within all livestock species.

Fertilisation is an orchestrated, stepwise process during which the participating male and female gametes undergo irreversible changes, losing some of their structural components while contributing others to the resultant zygote. Following sperm penetration through the egg coat, the sperm plasma membrane fuses with its oocyte counterpart, the oolemma. At least two plasma membrane proteins essential for sperm–oolemma fusion – IZUMO and CD9 on the male and female gametes, respectively – have been identified recently by classical cell biology approaches and confirmed by gene deletion. Oolemma-associated tetraspanin CD81, closely related to CD9, also appears to have an essential role in fusion. Additional proteins that may have nonessential yet still facilitating roles in sperm–oolemma adhesion and fusion include oolemma-anchored integrins and oocyte-expressed retroviral envelope proteins, sperm disintegrins, and sperm-borne proteins of epididymal origin such as CRISP1 and CRISP2. This review discusses these components of the gamete fusion mechanism within the framework of gamete structure, membrane biology, cell signalling and cytoskeletal dynamics, and revisits the topic of antipolyspermy defence at the oolemma level. Harnessing the mechanisms of sperm–egg fusion is of importance to animal biotechnology and to human assisted fertilisation, wherein male patients with reduced sperm fusibility have been identified.

Fertility is one of the most economically important traits in both beef and dairy cattle production; however, only female fertility is typically subjected to selection. Male and female fertility have only a small positive genetic correlation which is likely due to the existence of a relatively small number of genetic variants within each breed that cause embryonic and developmental losses. Genomic tools have been developed that allow the identification of lethal recessive loci based upon marker haplotypes. Selection against haplotypes harbouring lethal alleles in conjunction with selection to improve female fertility will result in an improvement in male fertility. Genomic selection has resulted in a two to fourfold increase in the rate of genetic improvement of most dairy traits in US Holstein cattle, including female fertility. Considering the rapidly increasing rate of adoption of high-throughput single nucleotide polymorphism genotyping in both the US dairy and beef industries, genomic selection should be the most effective of all currently available approaches to improve male fertility. However, male fertility phenotypes are not routinely recorded in natural service mating systems and when artificial insemination is used, semen doses may be titrated to lower post-thaw progressively motile sperm numbers for high-merit and high-demand bulls. Standardization of sperm dosages across bull studs for semen distributed from young bulls would allow the capture of sire conception rate phenotypes for young bulls that could be used to generate predictions of genetic merit for male fertility in both males and females. These data would allow genomic selection to be implemented for male fertility in addition to female fertility within the US dairy industry. While the rate of use of artificial insemination is much lower within the US beef industry, the adoption of sexed semen in the dairy industry has allowed dairy herds to select cows from which heifer replacements are produced and cows that are used to produce terminal crossbred bull calves sired by beef breed bulls. Capture of sire conception rate phenotypes in dairy herds utilizing sexed semen will contribute data enabling genomic selection for male fertility in beef cattle breeds. As the commercial sector of the beef industry increasingly adopts fixed-time artificial insemination, sire conception rate phenotypes can be captured to facilitate the development of estimates of genetic merit for male fertility within US beef breeds.

Biomarker-based sperm analysis elevates the treatment of human infertility and ameliorates reproductive performance in livestock. The negative biomarker-based approach focuses on proteins and ligands unique to defective spermatozoa, regardless of their morphological phenotype, lending itself to analysis by flow cytometry （FC）. A prime example is the spermatid specific thioredoxin SPTRX3/TXNDC8, retained in the nuclear vacuoles and superfluous cytoplasm of defective human spermatozoa. Infertile couples with high semen SPTRX3 are less likely to conceive by assisted reproductive therapies （ART） and more prone to recurrent miscarriage while low SPTRX3 has been associated with multiple ART births. Ubiquitin, a small, proteolysis-promoting covalent posttranslational protein modifier is found on the surface of defective posttesticular spermatozoa and in the damaged protein aggregates, the aggresomes of spermiogenic origin. Semen ubiquitin content correlates negatively with fertility and conventional semen parameters, and with sperm binding of lectins LCA （Lens culinaris agglutinin; reveals altered sperm surface） and PNA （Arachis hypogaealpeanut agglutinin; reveals acrosomal malformation or damage）. The Postacrosomal Sheath WWl Domain Binding Protein （PAWP）, implicated in oocyte activation during fertilization, is ectopic or absent from defective human and animal spermatozoa. Consequently, FC-parameters of PAWP correlate with ART outcomes in infertile couples and with fertility in bulls. Assays based on the above biomarkers have been combined into multiplex FC semen screening protocols, and the surface expression of lectins and ubiquitin has been utilized to develop nanoparticle-based bull semen purification method validated by field artificial insemination trials. These advances go hand-in-hand with the innovation of FC-technology and genomics/proteomics-based biomarker discovery.

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.

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.

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.