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Sahiwal cattle, known for their high milk yield, are propagated through artificial insemination (AI) using male germplasm, largely contingent on semen quality. Spermatozoa, produced in the testes, carry genetic information and molecular signals essential for successful fertilization. Seminal plasma, in addition to sperm, contains nano-sized lipid-bound extracellular vesicles (SP-EVs) that carry key biomolecules, including fertility-related miRNAs, which are essential for bull fertility. The current study focused on miRNA profiling of SP-EVs from highfertile (HF) and low-fertile (LF) Sahiwal bulls. SP-EVs were isolated using size exclusion chromatography (SEC) and characterized by dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). Western blotting detected the EVspecific protein markers TSG101 and CD63. The DLS analysis showed SP-EV sizes of 170-180 nm in HF and 130-140 nm in LF samples. The NTA revealed particle concentrations of 5.76 × 1010 to 5.86 × 1011 particles/mL in HF and 5.31 × 1010 to 2.70 × 1011 particles/mL in LF groups, with no significant differences in size and concentration between HF and LF. High-throughput miRNA sequencing identified 310 miRNAs in SP-EVs from both groups, with 61 upregulated and 119 downregulated in HF bull. Further analysis identified 41 miRNAs with significant fold changes and p-values, including bta-miR-1246, bta-miR-195, bta-miR-339b, and bta-miR-199b, which were analyzed for target gene prediction. Gene Ontology (GO) and KEGG pathway analyses indicated that these miRNAs target genes involved in transcription regulation, ubiquitindependent endoplasmic reticulum-associated degradation (ERAD) pathways, and signalling pathways. Functional exploration revealed that these genes play roles in spermatogenesis, motility, acrosome reactions, and inflammatory responses. qPCR analysis showed that bta-miR-195 had 80% higher expression in HF spermatozoa compared to LF, suggesting its association with fertility status (p < 0.05). In conclusion, this study elucidates the miRNA cargoes in SP-EVs as indicators of Sahiwal bull fertility, highlighting bta-miR-195 as a potential fertility factor among the various miRNAs identified. [ABSTRACT FROM AUTHOR]

Spermatogenesis and sperm maturation that occur in the testis and epididymis respectively are crucial for fertility. Factors secreted by the testicular and epididymal epithelial cells govern the processes of gametogenesis and maturation. Proteins encoded by the sperm‐associated antigen 11a (Spag11a) gene are implicated as having a possible role in sperm maturation. However, studies that demonstrate their definite role in fertility and sperm function using knockout models have not yet reported. In this study, Spag11a knockout mice were generated, genotyped and the reproductive parameters (fecundity, sperm count, capacitation, and acrosome reaction) and sperm proteome were determined. Litter size and sperm count were decreased in the Spag11a knockout mice when compared to the wild‐type controls. Spermatozoa from the knockout mice were able to undergo capacitation. However, acrosome reaction did not occur in sperm obtained from knockout mice. Structural abnormalities in the head and tail structures were evident in the spermatozoa of knockout mice. Perturbations in the expression of sperm proteins that are involved in gametogenesis were evident. The subfertility observed in Spag11a knockout mice could be a manifestation of lower sperm count, impaired acrosome reactions, and disturbances in the sperm proteome. The results of this study lend further support to the role of Spag11a gene in male gamete function. Summary: Analyzing the role of male reproductive tract‐specific proteins in male fertility using knockout models is an active area of investigation. Although a large number of knockout models for genes that are specific to the testis are generated and the reproductive function studied, there are very few studies that analyze the role of epididymal proteins that contribute to sperm function and fecundity. In this study, the effect of knockout of an epididymal‐specific gene, Spag11a, on male reproductive function, especially the fecundity, capacitation, acrosome reaction, and sperm proteome was analyzed. Our observations indicate subfertility and compromised sperm function associated with perturbations in sperm proteome. Using shRNA‐mediated Spag11a mRNA ablation and active immunization‐mediated SPAG11A protein ablation, we previously demonstrated the possible role of Spag11a in sperm function and fecundity. This study significantly differs from our previous studies because we used a higher level of gene ablation model, that is, the Spag11a knockout mice to provide further evidence. Furthermore, we also analyzed the molecular changes at the proteome level under conditions of Spag11a gene knockout. Thus, this study carried a lot of significance since it reiterates the observations made in our previous studies using an animal model that truly reflects the ablation of Spag11a expression. [ABSTRACT FROM AUTHOR]

Semen preservation quality affects the artificial insemination success rate, and seminal exosomes are rich in various proteins that are transferable to sperm and conducive to sperm-function preservation during storage. However, the specific effects of these proteins remain unclear. In this study, the specific effects of these proteins on semen preservation quality and fertilization capacity were investigated through a proteomic analysis of seminal exosomes from boars with high conception rates (HCRs) and low conception rates (LCRs). The results revealed significant differences in the expression of 161 proteins between the two groups, with the GPX5 level being significantly higher in the HCR group (p < 0.05). The role of GPX5 was further investigated by constructing engineered exosomes enriched with GPX5 (Exo-GPX5), which could successfully transfer GPX5 to sperm. Compared to the control group, Exo-GPX5 could significantly improve sperm motility on storage days 4 and 5 and enhance the acrosome integrity on day 5 (p < 0.05). Additionally, Exo-GPX5 increased the total antioxidant capacity (T-AOC) of sperm, reduced the malondialdehyde (MDA) level, and decreased the expression of antioxidant proteins SOD1 and CAT (p < 0.05). In simulated fertilization experiments, Exo-GPX5-treated sperm exhibited higher capacitation ability and a significant increase in the acrosome reaction rate (p < 0.05). Overall, Exo-GPX5 can improve boar semen quality under 17 °C storage conditions and enhance sperm fertilization capacity. [ABSTRACT FROM AUTHOR]

β‐nerve growth factor (βNGF) plays a crucial role in reproductive physiology and sperm quality. Enzymatic activity of seminal plasma and vaginal fluids reduces available βNGF and it has been demonstrated that chitosan microspheres could protect rrβNGF from degradation. This study examined the effects of microencapsulated rrbNGF with chitosan on rabbit sperm viability, motility and capacitation status. Results showed that 0.5 and 1 μg/mL of microencapsulated rrβNGF, as well as free rrβNGF or empty microspheres, did not adversely affect sperm viability or total motility after 2 h of incubation. However, the highest progressivity kinetic parameters were observed with 1 μg/mL free rrβNGF, while the highest curvilinear velocity (VCL) occurred with 0.5 μg/mL microencapsulated rrβNGF. Empty chitosan microspheres did not induce acrosome reaction (AR), but both concentrations of free and rrβNGFch favoured AR during in vitro incubation. The study suggests that using chitosan spheres did not show any adverse effects on sperm traits, unlike free rβNGF and rrβNGFch promoted capacitation and AR. Further research is needed to explore the potential of rrβNGFch in modifying in vitro capacitation and inducing ovulation during artificial insemination. [ABSTRACT FROM AUTHOR]

Goat production is affected by reproductive seasonality. In vitro embryo production (IVEP) could overcome this effect. This study aimed to evaluate the impact of the season of semen collection/freezing on IVEP of prepubertal goat oocytes and on sperm quality and functionality concerning capacitation. Semen from six fertile bucks was collected, pooled and cryopreserved in spring and autumn and used for IVEP of oocytes recovered during the breeding season. Oocytes were IVM in TCM‐199 with hormones, EGF and cysteamine; fertilized and cultured in BO‐IVF and BO‐IVC media (IVF Bioscience, UK). Semen samples were assessed at 0 and 3 h after culture in capacitating (BO‐IVF, CAP) and non‐capacitating conditions for sperm plasma membrane and acrosome integrity, mitochondrial membrane potential (MMP), intracellular calcium and plasma membrane lipid disorder. Blastocyst production was higher with spring sperm compared to autumn (12.0% vs. 2.1%, respectively; p <.05). After CAP, acrosome reaction and intracellular calcium were higher (p <.05) in spring than autumn sperm. No differences were found in other sperm parameters. In conclusion, seasonal variations in the IVEP of prepubertal goats could be linked to differences in sperm ability to undergo in vitro capacitation. [ABSTRACT FROM AUTHOR]

Background Materials and methods Results Discussion Conclusion Ascorbic acid 2‐glucoside (AA2G) is a stabilized form of ascorbic acid and a potent antioxidant. Ascorbic acid is present in the testes and epididymis and helps maintain the physiological integrity of reproductive organs. Its properties have been utilized to protect spermatozoa of different species from oxidative stress.Spermatozoa of C57Bl/6J and C57Bl/6N strains were frozen and analyzed, after thawing, by supplementing the capacitation medium with AA2G, both in the presence and absence of methyl‐β‐cyclodextrin (MBCD). The effect of treatment was evaluated by SCA System (Microptic) analyzing the velocity, vitality, morphology, and the DNA fragmentation. We also examined sperm capacitation (CTC), acrosome reaction (Coomassie Brillant Blue), and fertility (in vitro fertilization) of treated spermatozoa.AA2G improved sperm quality and fertility particularly in association with MBCD. We observed a significant increase of sperm motility, velocity, and vitality associated with an enhanced capacitation and acrosome reaction. These improvements resulted in a marked increase in in vitro fertilization success. Embryos obtained were cultured and reached normally the blastocyst stage.This study aimed to determine if AA2G could safeguard mouse spermatozoa during cryopreservation. We found a protective effect of AA2G that increased sperm survivability resulting in higher fertilization rate.This newly improved protocol shows potential for reanimating cryopreserved GEMMs stored in mouse biobanks and international repositories, such as the European Mouse Mutant Archive (EMMA).This can serve as a pivotal tool in fulfilling the 3Rs mission (replacement, reduction, and refinement), promoting ethical and humane research practices. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H2S) is an endogenously produced signaling molecule that belongs to the group of gasotransmitters along with nitric oxide (NO) and carbon monoxide (CO). H2S plays a pivotal role in male reproductive processes. It is produced in various tissues and cells of the male reproductive system, including testicular tissue, Leydig and Sertoli cells, epididymis, seminal plasma, prostate, penile tissues, and sperm cells. This review aims to summarize the knowledge about the presence and effects of H2S in male reproductive tissues and outline possible therapeutic strategies in pathological conditions related to male fertility, e. g. spermatogenetic disorders and erectile dysfunction (ED). For instance, H2S supports spermatogenesis by maintaining the integrity of the blood-testicular barrier (BTB), stimulating testosterone production, and providing cytoprotective effects. In spermatozoa, H2S modulates sperm motility, promotes sperm maturation, capacitation, and acrosome reaction, and has significant cytoprotective effects. Given its vasorelaxant effects, it supports the erection of penile tissue. These findings suggest the importance and therapeutic potential of H2S in male reproduction, paving the way for further research and potential clinical applications. [ABSTRACT FROM AUTHOR]

Backgroud: Before fertilization, spermatozoa undergo a crucial maturation step called capacitation, which is a unique event regulates the sperm's ability for successful fertilization. The capacitation process takes place as the spermatozoa pass through the female reproductive tract (FRT). Dihydrolipoamide dehydrogenase (DLD) protein is a post-pyruvate metabolic enzyme, exhibiting reactive oxygen species (ROS) production which causes capacitation. Additionally, other vital functions of DLD in buffalo spermatozoa are hyperactivation and acrosome reaction. DLD produces the optimum amount of ROS required to induce capacitation process in FRT. Depending on physiological or pathophysiological conditions, DLD can either enhance or attenuate the production of reactive oxygen species (ROS). Aim of this study was to investigate whether changes in the production of ROS in sperm cells can impact their ability to fertilize by triggering the capacitation and acrosome reaction. Results: In this study, abundance of DLD protein was quantified between high (n = 5) and low fertile bull (n = 5) spermatozoa. It was found that compared to high-fertile (HF) bulls, low-fertile (LF) bulls exhibited significantly (P < 0.05) higher DLD abundances. Herein, we optimised the MICA concentration to inhibit DLD function, spermatozoa were treated with MICA in time (0, 1, 2, 3, 4, and 5 h) and concentrations (1, 2.5, 5, and 10 mmol/L) dependent manner. Maximum DLD inhibition was found to be at 4 h in 10 mmol/L MICA concentration, which was used for further experimentation in HF and LF. Based on DLD inhibition it was seen that LF bull spermatozoa exhibited significantly (P < 0.05) higher ROS production and acrosome reaction in comparison to the HF bull spermatozoa. The kinematic parameters of the spermatozoa such as percent total motility, velocity parameters (VCL, VSL, and VAP) and other parameters (BCF, STR, and LIN) were also decreased in MICA treated spermatozoa in comparison to the control (capacitated) spermatozoa. Conclusions: The present study provides an initial evidence explaining the buffalo bull spermatozoa with higher DLD abundance undergo early capacitation, which subsequently reduces their capacity to fertilize. [ABSTRACT FROM AUTHOR]

Purpose: Platelet-rich plasma (PRP) is enriched with active biological components which showed proliferative and cytoprotective properties in healing different injuries in medicinal fields. This study was designed to assess the cryoprotective effects of autologous PRP on the quality of oligoasthenoteratospermia (OAT) samples during freezing and thawing procedure. Materials and Methods: The present study is an experimental research. Twenty OAT semen samples were ob- tained from individuals and prepared by discontinuous density – gradients technique DGC). The control group is sperm samples after DGC. After the procedure, the specimen was divided into four groups. The Freeze group which has no additive and the other three groups were cryopreserved with different concentrations of PRP (1×105 / µL, 0.5×105 /µL and 0.25×105 /µL). Autologous PRP was provided by each participant. After thawing, sperm pa- rameters, DNA fragmentation by sperm chromatin dispersion test (SCD), protamine deficiency by (Chromomycin A3) CMA3 staining, acrosome integrity and malondialdehyde (MDA) level were evaluated. Results: Cryopreservation resulted in a significant decrease in all factors compared to the control group. There were no significant changes in sperm count, morphology, non-progressive motility and acrosome reaction by adding PRP as a cryoprotectant in comparison with the freeze group. PRP at all three concentrations showed a significant increase in progressive motility (3.05 ± 2.01 vs. 14.05 ± 4.13, 12.35 ± 4.90 and 12.15 ± 9.65, P < 0.001) and viability (36.85 ± 10.25 vs. 47.85 ± 5.86, 51.30 ± 5.54 and 50.05 ± 5.67, P < 0.001) compared to the sperm samples without PRP. The percentage of immotile sperms decreased at all PRP concentrations compared to the freeze group. Moreover, PRP at 1×105 /µL concentration showed cryoprotective effects on DNA fragmentation, protamine deficiency and MDA level compared to the other three concentrations. Conclusion: Cryopreservation and thawing procedures may exert adverse effects on biological factors of sperm samples. Therefore, adding PRP as cryoprotectant at all three concentrations especially 1×105 /µL can be promising strategy to reduce adverse effects of cryopreservation on OAT samples. [ABSTRACT FROM AUTHOR]

Sea urchin eggs are covered with layers of extracellular matrix, namely, the vitelline layer (VL) and jelly coat (JC). It has been shown that sea urchin eggs' JC components serve as chemoattractants or ligands for the receptor on the fertilizing sperm to promote the acrosome reaction. Moreover, the egg's VL provides receptors for conspecific sperm to bind, and, to date, at least two sperm receptors have been identified on the surface of sea urchin eggs. Interestingly, however, according to our previous work, denuded sea urchin eggs devoid of the JC and VL do not fail to become fertilized by sperm. Instead, they are bound and penetratedby multiple sperm, raising the possibility that an alternative pathway independent of the VL-residing sperm receptor may be at work. In this research, we studied the roles of the JC and VL using intact and denuded eggs and the synthetic polyamine BPA-C8. BPA-C8 is known to bind to the negatively charged macromolecular complexes in the cells, such as the JC, VL, and the plasma membrane of echinoderm eggs, as well as to the actin filaments in fibroblasts. Our results showed that, when added to seawater, BPA-C8 significantly repressed the Ca2+ wave in the intact P. lividus eggs at fertilization. In eggs deprived of the VL and JC, BPA-C8 binds to the plasma membrane and increases fibrous structures connecting microvilli, thereby allowing the denuded eggs to revert towards monospermy at fertilization. However, the reduced Ca2+ signal in denuded eggs was nullified compared to the intact eggs because removing the JC and VL already decreased the Ca2+ wave. BPA-C8 does not cross the VL and the cell membrane of unfertilized sea urchin eggs to diffuse into the cytoplasm at variance with the fibroblasts. Indeed, the jasplakinolide-induced polymerization of subplasmalemmal actin filaments was inhibited in the eggs microinjected with BPA-C8, but not in the ones bath-incubated with the same dose of BPA-C8. [ABSTRACT FROM AUTHOR]

Simple Summary: Artificial insemination is nowadays a widespread practice; boar semen analysis is an essential key to guaranteeing pregnancy and high prolificacy. This analysis can include sperm motility, concentration, morphology, membrane integrity, DNA damage and seminal plasma components; a huge amount of data is available for each ejaculate and the interpretation of these results is complicated. This study aims to summarize these data, to classify ejaculates in several categories (clusters) and to investigate the potential differences among clusters on fertility and prolificacy. Two groups of Pietrain boars were studied (90 and 30 ejaculates weekly in groups 1 and 2, respectively). Computer-assisted semen analysis (CASA) and flow cytometry analysis were performed, and statistical analysis of the results was carried out using SPSS v.26 software. In both groups, variables were grouped in three principal components related to sperm velocity, linearity and DNA damage that allowed the ejaculates to be grouped into four clusters. Although an adequate description of the characteristics of the ejaculates was achieved, no differences were found among clusters for fertility or prolificacy since, with efficient quality control of semen, no relationships with fertility parameters would be expected once the minimum requirements have been met. Boar semen analysis includes sperm motility, concentration, morphology and other more complex analyses such as membrane integrity, DNA damage and seminal plasma components. This study aims to summarize these numerous data by linear combinations of them, to classify ejaculates in several categories (clusters) and to investigate the potential differences among clusters on fertility and prolificacy. Young Pietrain boars (23 ± 3.6 months) were investigated: ten boars from the Nucléus genetic line (group 1: 90 ejaculates weekly) and five boars from the Batallé genetic line (group 2: 30 ejaculates weekly). Computer-assisted semen analysis (CASA) examined motility. Sperm viability, acrosome reaction, early apoptosis, mitochondrial activity and DNA damage were studied by flow cytometry analysis. SPSS v.26 software was used to perform principal component analysis (PCA) and clustering. Three principal components (PC1: speed; PC2: linear path; PC3: DNA damage) were detected and four clusters identified in both groups. Clusters also differed significantly in several variables not included in these PCs (group 1: beat cross frequency and poly (ADP-ribose) polymerase; group 2: cathepsin B, abnormal forms, mitochondrial activity and high DNA stainability). PCA and clustering achieved adequate description of these ejaculates, but no differences among clusters were found for fertility or prolificacy, probably because the minimum sperm requirements had been met. [ABSTRACT FROM AUTHOR]

Abstract Backgroud Before fertilization, spermatozoa undergo a crucial maturation step called capacitation, which is a unique event regulates the sperm’s ability for successful fertilization. The capacitation process takes place as the spermatozoa pass through the female reproductive tract (FRT). Dihydrolipoamide dehydrogenase (DLD) protein is a post-pyruvate metabolic enzyme, exhibiting reactive oxygen species (ROS) production which causes capacitation. Additionally, other vital functions of DLD in buffalo spermatozoa are hyperactivation and acrosome reaction. DLD produces the optimum amount of ROS required to induce capacitation process in FRT. Depending on physiological or pathophysiological conditions, DLD can either enhance or attenuate the production of reactive oxygen species (ROS). Aim of this study was to investigate whether changes in the production of ROS in sperm cells can impact their ability to fertilize by triggering the capacitation and acrosome reaction. Results In this study, abundance of DLD protein was quantified between high (n = 5) and low fertile bull (n = 5) spermatozoa. It was found that compared to high-fertile (HF) bulls, low-fertile (LF) bulls exhibited significantly (P

Abstract Notch is a conserved cell-signaling pathway involved in spermatogenesis regulation. This study firstly evaluated the presence, localization patterns, acquisition origin and relation to acrosome reaction of Notch proteins in bull sperm. Western Blot analysis detected all Notch proteins in ejaculated bull sperm, and immunostaining described their specific sperm localization. Recovery of sperm from different segments showed that Notch proteins have testicular origin (NOTCH1, NOTCH2, DLL4), are sequentially acquired during sperm maturation along epididymal transit (NOTCH3, DLL3, JAGGED1-2), or post-ejaculation (DLL1, NOTCH4). Testis NOTCH2 is ubiquitously expressed in all germ-cell lines, whereas DLL4 is expressed in round and elongated spermatids during the Golgi, Cap, Acrosome and Maturation phases. In vitro spontaneous and induced sperm acrosome reaction induce consistent sperm regional relocation of NOTCH2, DLL4 and JAGGED1, and these relocation patterns are significantly associated to sperm acrosome status. NOTCH2 and JAGGED1 are relocated from the head apical to the post-equatorial regions, whereas DLL4 is lost along with the acrosome, evidencing that sperm spatial redistribution of NOTCH2 and JAGGED1 is linked to acrosome reaction onset, whereas DLL4 loss is linked to AR completion. Overall, results prompt for a relevant Notch role in bull sperm acrosome testicular development, epididymal maturation and acrosome reaction.

Abstract To acquire the ability to fertilize the egg, mammalian spermatozoa must undergo a series of changes occurring within the highly synchronized and specialized environment of the female reproductive tract, collectively known as capacitation. In an attempt to replicate this process in vitro, various culture media for mouse sperm were formulated over the past decades, sharing a similar overall composition but differing mainly in ion concentrations and metabolic substrates. The widespread use of the different media to study the mechanisms of capacitation might hinder a comprehensive understanding of this process, as the medium could become a confounding variable in the analysis. In this context, the present side-by-side study compares the influence of four commonly used culture media (FD, HTF and two TYH versions) on mouse sperm capacitation. We evaluated the induction of protein kinase A phosphorylation pathway, motility, hyperactivation and acrosome reaction. Additionally, in vitro fertilization and embryo development were also assessed. By analyzing these outcomes in two mouse colonies with different reproductive performance, our study provides critical insights to improve the global understanding of sperm function. The results obtained highlight the importance of considering variations in medium composition, and their potential implications for the future interpretation of results.

Background: While calcium is known to play a crucial role in mammalian sperm physiology, how it flows in and out of the male gamete is not completely understood. Herein, we investigated the involvement of Na+/Ca2+ exchangers (NCX) in mammalian sperm capacitation. Using the pig as an animal model, we first confirmed the presence of NCX1 and NCX2 isoforms in the sperm midpiece. Next, we partially or totally blocked Ca2+ outflux (forward transport) via NCX1/NCX2 with different concentrations of SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline; 0, 0.5, 5 and 50 µM) and Ca2+ influx (reverse transport) with SN6 (ethyl 2-[[4-[(4-nitrophenyl)methoxy]phenyl]methyl]-1,3-thiazolidine-4-carboxylate; 0, 0.3, 3 or 30 µM). Sperm were incubated under capacitating conditions for 180 min; after 120 min, progesterone was added to induce the acrosome reaction. At 0, 60, 120, 130, and 180 min, sperm motility, membrane lipid disorder, acrosome integrity, mitochondrial membrane potential (MMP), tyrosine phosphorylation of sperm proteins, and intracellular levels of Ca2+, reactive oxygen species (ROS) and superoxides were evaluated. Results: Partial and complete blockage of Ca2+ outflux and influx via NCX induced a significant reduction of sperm motility after progesterone addition. Early alterations on sperm kinematics were also observed, the effects being more obvious in totally blocked than in partially blocked samples. Decreased sperm motility and kinematics were related to both defective tyrosine phosphorylation and mitochondrial activity, the latter being associated to diminished MMP and ROS levels. As NCX blockage did not affect the lipid disorder of plasma membrane, the impaired acrosome integrity could result from reduced tyrosine phosphorylation. Conclusions: Inhibition of outflux and influx of Ca2+ triggered similar effects, thus indicating that both forward and reverse Ca2+ transport through NCX exchangers are essential for sperm capacitation. [ABSTRACT FROM AUTHOR]

Buffalo bulls are backbone of Indian dairy industry, and the quality of semen donating bulls determine the overall production efficiency of dairy farms. Seminal plasma harbor millions of lipid bilayer nanovesicles known as extracellular vesicles (EVs). These EVs carry a heterogenous cargo of essential biomolecules including fertility-associated proteins which contribute to fertilizing potential of spermatozoa. In this study, we explored size, concentration, and complete proteome profiles of SP EVs from two distinct fertility groups to uncover proteins influencing bull fertility. Through Dynamic Light Scattering (DLS) it was found that purified EVs were present in 7-14 size exclusion chromatographic (SEC) fractions with sizes ranging from 146.5 to 258.7 nm in high fertile (HF) and low fertile (LF) bulls. Nanoparticle Tracking Analysis (NTA) confirmed the size of seminal EVs up to 200 nm, and concentrations varying from 2.84 to 6.82 × 1011 and 3.57 to 7.74 × 1011 particles per ml in HF and LF bulls, respectively. No significant difference was observed in size and concentration of seminal EVs between two groups. We identified a total of 1,862 and 1,807 proteins in seminal EVs of HF and LF bulls, respectively using high throughput LC-MS/MS approach. Out of these total proteins, 1,754 proteins were common in both groups and about 87 proteins were highly abundant in HF group while 1,292 were less abundant as compared to LF bulls. Gene ontology (GO) analysis, revealed that highly abundant proteins in HF group were mainly part of the nucleus and involved in nucleosome assembly along with DNA binding. Additionally, highly abundant proteins in EVs of HF group were found to be involved in spermatogenesis, motility, acrosome reaction, capacitation, gamete fusion, and cryotolerance. Two highly abundant proteins, protein disulfide-isomerase A4 and gelsolin, are associated with sperm-oocyte fusion and acrosome reaction, respectively, and their immunolocalization on spermatozoa may indicate that these proteins are transferred through EVs. Our evidences support that proteins in EVs and subsequently their presence on sperm, are strongly associated with sperm functions. Altogether, our investigation indicates that SPEVs possess crucial protein repertoires that are essential for enhancing sperm fertilizing capacity. [ABSTRACT FROM AUTHOR]

• Extracts of Mondia whitei improve semen parameters in vitro in a dose dependent manner. • Extracts of Mondia whitei reduce the levels of sperm DNA fragmentation in vitro in a dose dependent manner. • Extracts of Mondia whitei increase the percentage of sperm with intact mitochondrial membrane potential in vitro. • Extracts of Mondia whitei increase the percentage of capacitated and acrosome reacted sperm in vitro. Mondia whitei (Mw) is a prominent African medicinal plant that is widely used as traditional treatment in the management of sexual dysfunction and male infertility. Yet, there is a paucity of scientific evidence to support its medicinal use. Therefore, the aim of this study was to investigate the in vitro effect of aqueous root extract of Mw on human semen parameters and sperm function. Semen samples were obtained from healthy donors (n = 30) and infertile patients (n = 30) for in vitro experiments. Samples were washed using human tubular fluid medium supplemented with bovine serum albumin (HTF-BSA), and incubated with increasing concentrations (0.0185, 0.185, 1.85, 18.5 and 185 μg/mL) of aqueous Mw rhizome extracts for 1 h at 37 °C. HTF-BSA washed sperm served as a control for the experiments. Sperm total and progressive motility, kinematic parameters, vitality, mitochondrial membrane potential (MMP), sperm DNA fragmentation (SDF), intracellular reactive oxygen species (ROS), capacitation and acrosome reaction were assessed following the termination of experiments. Extracts of Mw increased spermatozoa total motility, vitality, percentage of spermatozoa with intact MMP and intracellular ROS in both the donor and infertile groups. Mw increased progressive motility, SDF, capacitation and acrosome reaction in the infertile group only. Sperm kinetic motility parameters and hyperactivation were not significantly affected in any group. Although there was increased intracellular ROS generation, this did not negatively affect MMP, SDF, hyperactivation or acrosome reaction and capacitation. Mw improves functional human sperm parameters in vitro , increasing total and progressive motility, and intact MMP in a dose dependent manner. Further studies instigating the effect of Mw on male fertility are warranted. [ABSTRACT FROM AUTHOR]

Simple Summary: Research efforts over the past decade have uncovered new insights into functions of erythropoietin that point to new roles beyond erythropoiesis. Recent studies have demonstrated that erythropoietin is implicated in tissue protection, regeneration, and reproduction. In this context, the present study reveals for the first time that erythropoietin inhibits cell death by apoptosis and enhances the fertilizing capacity of bovine spermatozoa. Erythropoietin (EPO), a hormone secreted mainly by the kidney, exerts its biological function by binding to its cell-surface receptor (EpoR). The presence of EPO and EpoR in the male and female reproductive system has been verified. Therefore, some of the key properties of EPO, such as its antioxidant and antiapoptotic effects, could improve the fertilizing capacity of spermatozoa. In the present study, the effect of two different concentrations of EPO (10 mIU/μL and 100 mIU/μL) on bovine sperm-quality parameters was evaluated during a post-thawing 4-h incubation at 37 °C. EPO had a positive effect on sperm motility, viability, and total antioxidant capacity. Moreover, EPO inhibited apoptosis, as it reduced both BCL2-associated X apoptosis regulator (Bax)/B-cell lymphoma 2 (Bcl-2) ratio and cleaved cysteine-aspartic proteases (caspases) substrate levels in a dose-dependent manner. In addition, EPO induced sperm capacitation and acrosome reaction in spermatozoa incubated in capacitation conditioned medeia. These results establish a foundation for the physiological role of EPO in reproductive processes and hopefully will provide an incentive for further research in order to fully decipher the role of EPO in sperm physiology and reproduction. [ABSTRACT FROM AUTHOR]

Cryopreservation is crucial for conserving genetic diversity in endangered species including the critically endangered group of sturgeons (Acipenseridae), but it can compromise sperm quality and protein profiles. Although cryopreservation with dimethyl sulfoxide (DMSO) and methanol (MeOH) results in the recovery of good post-thaw motility, DMSO-preserved sperm show reduced fertilization ability. This study was conducted in Siberian sturgeon as a model for Acipenserid fishes to explore the effects of DMSO and MeOH on the proteome of semen using advanced proteomics methods—liquid chromatography‒mass spectrometry and two-dimensional difference gel electrophoresis. We analyzed the proteomic profiles of fresh and cryopreserved spermatozoa and their extracellular medium and showed that cryopreservation decreases motility and viability and increases reactive oxygen species levels, membrane fluidity, and acrosome damage. Despite having similar post-thaw semen motility, sperm treated with DMSO had significantly lower fertilization success (6.2%) than those treated with MeOH (51.2%). A total of 224 and 118 differentially abundant proteins were identified in spermatozoa preserved with MeOH and DMSO, respectively. MeOH-related proteins were linked to chromosomal structure and mitochondrial functionality, while DMSO-related proteins impacted fertilization by altering the acrosome reaction and binding of sperm to the zona pellucida and nuclear organization. Additionally, cryopreservation led to alterations in the proacrosin/acrosin system in both cryoprotectants. This study provides the first comprehensive proteomic characterization of Siberian sturgeon sperm after cryopreservation, offering insights into how cryoprotectants impact fertilization ability. [ABSTRACT FROM AUTHOR]

Hexokinase (HK) catalyzes the first irreversible rate-limiting step in glycolysis that converts glucose to glucose-6-phosphate. HK1 is ubiquitously expressed in the brain, erythrocytes, and other tissues where glycolysis serves as the major source of ATP production. Spermatogenic cell-specific type 1 hexokinase (HK1S) is expressed in sperm but its physiological role in male mice is still unknown. In this study, we generate Hk1s knockout mice using the CRISPR/Cas9 system to study the gene function in vivo. Hk1s mRNA is exclusively expressed in testes starting from postnatal day 18 and continuing to adulthood. HK1S protein is specifically localized in the outer surface of the sperm fibrous sheath (FS). Depletion of Hk1s leads to infertility in male mice and reduces sperm glycolytic pathway activity, yet they have normal motile parameters and ATP levels. In addition, by using in vitro fertilization (IVF), Hk1s deficient sperms are unable to fertilize cumulus-intact or cumulus-free oocytes, but can normally fertilize zona pellucida-free oocytes. Moreover, Hk1s deficiency impairs sperm migration into the oviduct, reduces acrosome reaction, and prevents capacitation-associated increases in tyrosine phosphorylation, which are probable causes of infertility. Taken together, our results reveal that HK1S plays a critical role in sperm function and male fertility in mice. Author summary: About 8%-12% of couples worldwide are infertile, and male infertility is associated with about half of these cases. Common defects of male infertility include low sperm count, reduced sperm motility, abnormal sperm morphology, and unexplained male infertility with normal sperm parameters but infertility. The cause of over 50% of male infertility cases remains unidentified. Research on male reproduction has helped to unravel male infertility. In this study, we used a knockout mouse model to study the function of HK1S in the male mouse reproductive system. We showed that depletion of Hk1s leads to infertility in male mice but with normal sperm morphology and motility. These phenotypes are similar to patients with unexplained male infertility. Furthermore, HK1S is essential for sperm glycolysis. Hk1s deficiency in mice results in decreased glycolysis activity and leads to impaired sperm migration into the oviduct, reduced acrosome reaction, and defective capacitation-associated increase in tyrosine phosphorylation. These results suggested that HK1S is necessary for male fertility in mice. Our study suggests a new avenue for the development of new therapeutic strategies for male infertility. [ABSTRACT FROM AUTHOR]

Protein glycosylation is a post-translational modification involved in wide range of biological processes. In mammalian spermatozoa this modification has been identified in numerous proteins, and membrane glycoproteins are involved in the fertilization process. The objective of the present study was to identify changes in protein glycosylation after acrosome reaction (AR) induction using the 4-Br-A23187 ionophore. Our results showed that treatment with 10 μM of 4-Br-A23187 for 20 min significantly increased the percentage of live acrosome-reacted spermatozoa compared to the control (69.8 ± 0.8 vs. 6.4 ± 0.5; mean % ± SEM, respectively). Also, we observed an increase in 32 kDa tyrosine-phosphorylated protein (p32) and a decrease in serine/threonine phosphorylation of the protein kinase A substrates (phospho-PKA-substrates) after ionophore treatment. Furthermore, changes in glycosylated proteins following AR induction were analyzed using different HRP-conjugated lectins (GNA, DSA, and SNA), revealing changes in mannose and sialic acid residues. Proteomic analysis of isolated proteins using GNA lectin revealed that 50 proteins exhibited significantly different abundance (q-value < 0.01). Subsequent analysis using Uniprot database identified 39 downregulated and 11 upregulated proteins in the presence of 4-Br-A23187. Notably, six of these proteins were classified as transmembrane proteins, namely LRRC37A/B like protein 1 C-terminal domain-containing protein, Membrane metalloendopeptidase like 1, VWFA domain-containing protein, Syndecan, Membrane spanning 4-domains A14 and Serine protease 54. This study shows a novel protocol to induce acrosome reaction in boar spermatozoa and identifies new transmembrane proteins containing mannose residues. Further work is needed to elucidate the role of these proteins in sperm-oocyte fusion. • Incubation with 4-Br-A23187 for 20 min induces acrosome reaction in boar spermatozoa. • Glycosylated protein profile changes after acrosome reaction induction. • 50 proteins containing mannose residues showed regulation after acrosome reaction. • 6 transmembrane proteins containing mannose residues were regulated. [ABSTRACT FROM AUTHOR]

Background Objectives Material and methods Results Discussion and conclusions Mammalian spermatozoa need to undergo a process named capacitation to be able to fertilize an oocyte. During their journey in the female tract, spermatozoa obtain energy while exposed to a changing environment containing a variety of metabolic substrates. The energy requirements for sperm capacitation are species‐specific. In addition, the available energy source can hinder the process of sperm capacitation and eventually the acrosome reaction.To evaluate whether the metabolic substrates available in the in vitro sperm capacitation medium allow or interfere with the pig sperm capacitation process.The effect of different metabolic substrates on sperm capacitation process was evaluated by analyzing phosphorylation in the p32 protein; the acrosome reaction and the ATP intracellular content.The presence of glucose in the in vitro capacitating medium diminishes, in a concentration‐dependent manner, parameters associated with the capacitated status: induced acrosome exocytosis, plasma membrane destabilization, and protein tyrosine phosphorylation. Conversely, sperm incubation with pyruvate or lactate, either individually or in combination, allows the attainment of the capacitated status. Unexpectedly, pig spermatozoa incubated without any extracellular energy substrates or with a non‐metabolizable substrate (l‐glucose) for 4 h displayed similar sperm viability to the control and exhibited a capacitated phenotype. The capacitation‐like phenotype observed in starved pig spermatozoa (absence of glucose, lactate, and pyruvate) was dependent on extracellular bicarbonate and calcium levels, and these spermatozoa exhibited lower intracellular ATP content compared to those not capacitated. Nevertheless, the intracellular content of calcium was not modified in comparison to the control.Our findings suggest that the metabolic substrates used to fuel pig sperm metabolism are important in achieving the capacitated status. The results of this work could be used to refine the capacitating medium employed in pig in vitro fertilization. [ABSTRACT FROM AUTHOR]

Background: Exposure of humans and animals to heavy metals is increasing day-by-day; thus, lead even today remains of significant public health concern. According to CDC, blood lead reference value (BLRV) ranges from 3.5 µg/dl to 5 μg/dl in adults. Recently, almost 2.6% decline in male fertility per year has been reported but the cause is not well established. Lead (Pb2+) affects the size of testis, semen quality, and secretory functions of prostate. But the molecular mechanism(s) of lead toxicity in sperm cells is not clear. Thus, present study was undertaken to evaluate the adverse effects of lead acetate at environmentally relevant exposure levels (0.5, 5, 10 and 20 ppm) on functional and molecular dynamics of spermatozoa of bucks following in vitro exposure for 15 min and 3 h. Results: Lead significantly decreased motility, viable count, and motion kinematic patterns of spermatozoa like curvilinear velocity, straight-line velocity, average path velocity, beat cross frequency and maximum amplitude of head lateral displacement even at 5 ppm concentration. Pb2+ modulated intracellular cAMP and Ca2+ levels in sperm cells through L-type calcium channels and induced spontaneous or premature acrosome reaction (AR) by increasing tyrosine phosphorylation of sperm proteins and downregulated mitochondrial transmembrane potential. Lead significantly increased DNA damage and apoptosis as well. Electron microscopy studies revealed Pb2+ -induced deleterious effects on plasma membrane of head and acrosome including collapsed cristae in mitochondria. Conclusions: Pb2+ not only mimics Ca2+ but also affects cellular targets involved in generation of cAMP, mitochondrial transmembrane potential, and ionic exchange. Lead seems to interact with Ca2+ channels because of charge similarity and probably enters the sperm cell through these channels and results in hyperpolarization. Our findings also indicate lead-induced TP and intracellular Ca2+ release in spermatozoa which in turn may be responsible for premature acrosome exocytosis which is essential feature of capacitation for fertilization. Thus, lead seems to reduce the fertilizing capacity of spermatozoa even at 0.5 ppm concentrations. [ABSTRACT FROM AUTHOR]

In addition to its central role in cellular metabolism, adenosine 5′-triphosphate (ATP) is an important extracellular signalling molecule involved in various physiological processes. In reproduction, extracellular ATP participates in both autocrine and paracrine paths regulating gametogenesis, gamete maturation and fertilisation. This review focusses on how extracellular ATP modulates sperm physiology with emphasis on the mammalian acrosome reaction. The presence of extracellular ATP in the reproductive tract is primarily determined by the ion channels and transporters that influence its movement within the cells comprising the tract. The main targets of extracellular ATP in spermatozoa are its own transporters, particularly species-specific sperm purinergic receptors. We also discuss notable phenotypes from knock-out mouse models and human Mendelian inheritance related to ATP release mechanisms, along with immunological, proteomic, and functional observations regarding sperm purinergic receptors and their involvement in sperm signalling. Reproductive systems express different ion channels and transporters that release ATP into their lumens, which may regulate sperm physiology. Epididymal ATP has been suggested to influence basal sperm motility. Oviductal ATP triggers a head sperm volume increase which contributes to acrosome reaction. Image by López-González, I. This article belongs to the Collection Dedication to Jim Cummins. [ABSTRACT FROM AUTHOR]

There has been emerging research linking per- and poly-fluoroalkyl substances (PFAS) to gamete viability and fertility. PFAS, prevalent in the environment and water supplies, undergo slow degradation due to their C-F bond and a long half-life (2.3–8.5 years). In females, PFAS inhibit the hypothalamic–pituitary–gonadal (HPG) axis, reducing follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels, leading to the inhibition of androgen and estradiol production. PFAS have been found to cause detrimental effects on egg quality through impairing folliculogenesis. In males, PFAS can impair sperm motility and morphology: two fundamental qualities of successful fertilization. PFAS exposure has been proven to inhibit testosterone production, sperm capacitation, and acrosomal reaction. After fertilization, the results of PFAS exposure to embryos have also been investigated, showing reduced development to the blastocyst stage. The aim of this review is to report the main findings in the literature on the impact of PFAS exposure to gamete competency and fertilization capability by highlighting key studies on both male and female fertility. We report that there is significant evidence demonstrating the negative impacts on fertility after PFAS exposure. At high doses, these environmentally abundant and widespread compounds can significantly affect human fertility. [ABSTRACT FROM AUTHOR]

This article, titled "Unraveling the Molecular Mechanisms Underlying N-formyl-L-aspartate-mediated Sperm Chemotaxis," explores the role of N-formyl-l-aspartate (NFA) in guiding sperm towards the egg during in vitro fertilization (IVF). The study investigates the chemotactic signaling, receptor identification, and the effect of NFA on sperm capacitation and acrosome reaction. The results indicate that NFA induces chemotaxis in sperm through interaction with the β-2-adrenergic receptor (β-2-AR), leading to calcium mobilization and linear swimming towards the egg. This research provides insights into the molecular mechanisms of sperm chemotaxis and suggests the potential use of NFA as a chemoattractant to improve IVF success rates. [Extracted from the article]

Glycogen synthase kinase-3β (GSK3β) not only plays a crucial role in regulating sperm maturation but also is pivotal in orchestrating the acrosome reaction. Here, we integrated single-molecule long-read and short-read sequencing to comprehensively examine GSK3β expression patterns in adult Diannan small-ear pig (DSE) testes. We identified the most important transcript ENSSSCT00000039364 of GSK3β, obtaining its full-length coding sequence (CDS) spanning 1263 bp. Gene structure analysis located GSK3β on pig chromosome 13 with 12 exons. Protein structure analysis reflected that GSK3β consisted of 420 amino acids containing PKc-like conserved domains. Phylogenetic analysis underscored the evolutionary conservation and homology of GSK3β across different mammalian species. The evaluation of the protein interaction network, KEGG, and GO pathways implied that GSK3β interacted with 50 proteins, predominantly involved in the Wnt signaling pathway, papillomavirus infection, hippo signaling pathway, hepatocellular carcinoma, gastric cancer, colorectal cancer, breast cancer, endometrial cancer, basal cell carcinoma, and Alzheimer's disease. Functional annotation identified that GSK3β was involved in thirteen GOs, including six molecular functions and seven biological processes. ceRNA network analysis suggested that DSE GSK3β was regulated by 11 miRNA targets. Furthermore, qPCR expression analysis across 15 tissues highlighted that GSK3β was highly expressed in the testis. Subcellular localization analysis indicated that the majority of the GSK3β protein was located in the cytoplasm of ST (swine testis) cells, with a small amount detected in the nucleus. Overall, our findings shed new light on GSK3β's role in DSE reproduction, providing a foundation for further functional studies of GSK3β function. [ABSTRACT FROM AUTHOR]

Background: Considering the growing therapeutic use of cannabidiol as well as the presence of cannabinoid receptors in sperm and its possible genotoxic activity, the effect of cannabidiol on sperm quality and function was examined. Methods: Thirty male NMRI mice were randomly divided into three groups: control (no injection), sham (intraperitoneal (IP) injection of DMSO daily for 34 days), and cannabidiol (IP injection of cannabidiol 30 mg/ml daily for 34 days). Following 35 days after the last injection, sperm parameters, chromatin integrity (CMA3 staining), acrosome reaction (FITC-PNA method), fertility-related genes (IZUMO1, PLCζ), and blastulation rate of the embryos obtained from the oocytes fertilized with the mentioned sperms was investigated. Results: Count, motility, and morphology of sperm were not significantly affected by cannabidiol. CMA3+ sperms (protamine deficiency) were significantly higher in the cannabidiol group compared to the control group (P = 0.03). The acrosomal reaction and fertility-related genes (IZUMO1, PLCζ) in the cannabidiol group did not differ significantly compared to the control group. Also, there was no significant difference between the cannabidiol group and the control group in the two-cell and the eight-cell stages but the rate of blastocyst formation was significantly lower in the cannabidiol group compared to other groups (P < 0.0001). Conclusions: Our results showed that cannabidiol leads to negative effects on the male reproductive system through an effect on sperm chromatin and the rate of reaching the blastocyst stage of the embryo. [ABSTRACT FROM AUTHOR]

As in most cells, intracellular pH regulation is fundamental for sperm physiology. Key sperm functions like swimming, maturation, and a unique exocytotic process, the acrosome reaction, necessary for gamete fusion, are deeply influenced by pH. Sperm pH regulation, both intracellularly and within organelles such as the acrosome, requires a coordinated interplay of various transporters and channels, ensuring that this cell is primed for fertilization. Consistent with the pivotal importance of pH regulation in mammalian sperm physiology, several of its unique transporters are dependent on cytosolic pH. Examples include the Ca2+ channel CatSper and the K+ channel Slo3. The absence of these channels leads to male infertility. This review outlines the main transport elements involved in pH regulation, including cytosolic and acrosomal pH, that participate in these complex functions. We present a glimpse of how these transporters are regulated and how distinct sets of them are orchestrated to allow sperm to fertilize the egg. Much research is needed to begin to envision the complete set of players and the choreography of how cytosolic and organellar pH are regulated in each sperm function. [ABSTRACT FROM AUTHOR]

Background: Tyrosine phosphorylation, acrosin, and acrosome reaction play an important role in fertilisation. However, cryopreservation causes changes in tyrosine phosphorylation, acrosin expression, and acrosome reaction which affect the quality of spermatozoa. Cryoprotectant media added with antioxidants is needed to protect Spermatozoa from the effects of cryopreservation so that the quality of spermatozoa can be maintained. Objectives: This research examined the effect of glutathione (GSH) supplementation in cryopreservation media on tyrosine phosphorylation, acrosin expression, and acrosome reaction. In this research, pure modified Cryoprotectant (CPA) was compared with CPA supplemented with GSH in three different concentrations. Materials and Methods: The research sample was male mus musculus albinus strain Deutchland Denken Yoken (DDY). Mice spermatozoa was cryopreserved and several parameters were measured including tyrosine phosphorylation, acrosin expression, and acrosome reaction. Results: The addition of GSH to the modified CPA increased tyrosine phosphorylation, acrosin expression, and acrosome reaction (maintaining acrosome integrity). The group with 1.00 mM GSH obtained the highest results among the other groups. Significant increases were found in tyrosine phosphorylation, acrosin expression, and acrosome reaction after the addition of 1.00 mM GSH. Conclusion: Glutathione supplementation in modified CPA can increase tyrosine phosphorylation, acrosin expression, and acrosome reaction of frozen-thawed spermatozoa. Treatment using GSH at a dose of 1.00 mM is the most effective and modification of CPA with the addition of glutathione can improve the tyrosine phosphorylation, acrosin expression and acrosome reaction in cryopreserved spermatozoa. [ABSTRACT FROM AUTHOR]

The protective action of melatonin (MLT) against the harmful effects of cadmium (Cd) on testicular activity in rats has been documented previously; however, the involved molecular mechanisms have yet to be elucidated. Herein, we investigate the involvement of the mammalian target of rapamycin (mTOR) on the ability of MLT to counteract the damage induced by Cd on the rat testicular activity. Our study confirmed that Cd has harmful effects on the testes of rats and the protective action exerted by MLT. We reported, for the first time, that the addition of rapamycin (Rapa), a specific mTOR inhibitor, to animals co‐treated with Cd and MLT completely abolished the beneficial effects exerted by MLT, indicating that the mTOR pathway partially modulates its helpful effects on Cd testicular toxicity. Interestingly, Rapa‐alone treatment, provoking mTOR inhibition, produced altered morphological parameters, increased autophagy of germ and somatic cells, and reduced serum testosterone concentration. In addition, mTOR inhibition also reduced protein levels of markers of steroidogenesis (3β‐Hydroxysteroid dehydrogenase) and blood–testis barrier integrity (occludin and connexin 43). Finally, Rapa altered sperm parameters as well as the ability of mature spermatozoa to perform a proper acrosome reaction. Although further investigation is needed to better clarify the molecular pathway involved in MLT action, we confirm that MLT alleviating Cd effects can be used as a supplement to enhance testicular function and improve male gamete quality. Research Highlights: We confirmed the Cd harmful effects and MLT protective action on rat testes.The mTOR inhibitor Rapa completely abolished the MLT beneficial effects.Results indicate that the mTOR pathway modulates MLT counteractive effects on Cd testicular toxicity. [ABSTRACT FROM AUTHOR]

Infertility affects approximately 15% of couples trying to conceive. Male-related causes account for roughly 50% of cases, with obesity emerging as a possible significant factor. Obesity, defined as a body mass index of 30.0 or higher, has become a widespread epidemic associated with numerous health issues, including a decrease of fertility. This review discusses the relationship between obesity and male infertility, particularly focusing on sperm quality and function. An overview of the literature suggests that obesity may influence the male reproductive system via disruptions in hormonal profiles, oxidative stress, and inflammation, leading to changes in sperm parameters. Several studies have discussed if obesity causes a decrease in sperm concentration, motility, and normal morphology, so far without a consensus being reached. However, available evidence suggests an impairment of sperm function in obese men, due to an increase in DNA damage and oxidative stress, impaired mitochondrial function and acrosome reaction in response to progesterone. Finally, the relationship between obesity and assisted reproductive technologies outcomes remains debatable, with conflicting evidence regarding the influence on fertilisation, pregnancy, and live birth rates. Therefore, the actual impact of obesity on human spermatozoa still needs to be clarified, due to the multiple factors potentially in play. This review focuses on the potential impact of male obesity on sperm quality and function – a long-debated topic. The adverse effects of obesity on sperm parameters can significantly impair male and couple fertility, resulting in an increased necessity for fertility assistance among obese men. Furthermore, although more definitive research is needed, these individuals often experience poorer outcomes in assisted reproductive technologies. Image by João Ramalho-Santos. This article belongs to the Collection Dedication to Jim Cummins. [ABSTRACT FROM AUTHOR]

The purpose of this study was to evaluate the differences in sperm function and embryo morphokinetics following sperm cryopreservation by ultra-rapid freezing or rapid freezing methods compared to fresh spermatozoa. Thirty samples of normozoospermia were divided equally into fresh, ultra-rapid freezing, and rapid freezing groups. In the rapid freezing, sperm suspension was placed horizontally on nitrogen vapor. In the ultra-rapid freezing, sperm suspension with a straw-in-straw system was directly immersed in liquid nitrogen. Sperm function was assessed in terms of motility, morphology, viability, mitochondrial membrane potential, sperm DNA fragmentation, and acrosome reaction status. Also, the effects of two cryopreservation methods were assessed on global DNA methylation and DNA methyltransferase activity. Moreover, 730 embryos in three groups were cultured using time-lapse imaging until day 6 for embryo morphokinetics. Progressive motility (38.80 ± 4.21 vs. 34.86 ± 4.19; p < 0.001) and viability (64.30 ± 6.24 vs. 58.10 ± 8.69; p < 0.01) in ultra-rapid freezing were significantly higher than rapid group. DNA fragmentation and acrosome reaction were significantly increased in both cryopreserved groups (p < 0.001). However, DNA fragmentation (16.30 ± 1.14 vs. 14.33 ± 2.94; p < 0.01) was significantly higher in the rapid than the ultra-rapid freezing group. No significant differences were noted in global DNA methylation (p > 0.05) and DNA methyltransferases activity (p > 0.05) in fresh compared to cryopreservation groups. The kinetic times, including tPB2, tPNa, tPNf, t2, t3, t4, t5, t6, t7, t8, and tM, showed a significant delay in cell divisions in both cryopreservation groups. Furthermore, tPNa, tPNf, and t8 occurred with a significantly higher delay in embryos fertilized by sperm from the rapid freezing compared to the ultra-rapid freezing group. In addition, blastocysts formation was similar in both cryopreservation groups. Ultra-rapid freezing preserved the sperm biological integrity and lead to better embryo morphokinetics compared to the rapid freezing method. However, both methods of sperm cryopreservation were epigenetically safe. [ABSTRACT FROM AUTHOR]

Peroxidative damage to human spermatozoa has been shown to be the primary cause of male infertility. The possible role of nitric oxide (NO) in affecting sperm motility, capacitation, and acrosome reaction has been reported, too. The overproduction of NO by the enzyme inducible nitric oxide synthase (iNOS) could be responsible as it has been implicated in the pathogenesis of many diseases. There have been many studies on regulating iNOS function in various tissues, especially by protein–protein interaction; however, no study has looked for iNOS-interacting proteins in the human testis. Here, we have reported the identification of two proteins that interact with iNOS. We initially undertook a popular yeast two-hybrid assay to screen a human testis cDNA library in yeast using an iNOS-peptide fragment (amino acids 181–335) as bait. We verified our data using the mammalian chemiluminescent co-IP method; first, employing the same peptide and, then, a full-length protein co-expressed in HEK293 cells in addition to the candidate protein. In both cases, these two protein partners of iNOS were revealed: (a) sperm acrosome-associated 7 protein and (b) retinoblastoma tumor-suppressor binding protein. [ABSTRACT FROM AUTHOR]

The phenylpyrazole insecticide fipronil has wide-ranging applications from agriculture to public health to control undesirable organisms. However, several studies have reported the residual environmental hazards of fipronil and demonstrated its harmful effects even in mammalian reproduction. Therefore, this study was conducted to demonstrate the mode of action of fipronil on mouse spermatozoa. We treated fipronil to spermatozoa and performed comprehensive function evaluations. Moreover, proteomic analyses were conducted to identify the alteration of protein expression levels in spermatozoa. Most of sperm motility and kinematic parameters and intracellular ATP levels were diminished, and the spontaneous acrosome reaction was promoted after treatment with fipronil. Proteomic analyses revealed altered expression levels of 14 proteins after treatment. These proteins have been reported to be associated with sperm-specific pathways, prominently the cytoskeleton of the sperm, "9 + 2" axoneme composition, metabolism, and fertility. Collectively, our results showed that fipronil alters sperm functional-related proteins and therefore influences male fertility. This study elucidates the possible reproductive toxic hazards associated with male infertility through aberrant suppression of sperm proteins. [ABSTRACT FROM AUTHOR]

The milk added to the semen extender media acts by binding to seminal plasma proteins, which may protect the sperm cell from undergoing the capacitation process or acrosomal reaction in advance. Among these proteins, sodium caseinate stands out, which represents 80% of all proteins present in milk. Caseins are also able to protect the sperm cell by decreasing the loss of lipids through the plasma membrane, decreasing the binding of seminal plasma proteins to the plasma membrane. The objective of this study was to determine whether the addition of sodium caseinate in different concentrations to the freezing extender medium favors sperm viability after a long period of refrigeration. For this, three concentrations of sodium caseinate were used, 0.0, 1.0, and 2.0%, in the cryopreservation medium. In addition to caseinate concentrations, three refrigeration times were tested at 5 ºC, 12 and 24 hours. So, the ejaculates were divided into nine groups 0H/0%, 0H/1%, 0H/2%, 12H/0%, 12H/1%, 12H/2%, 24H/0%, 24H/1% and 24H/2%. Ten stallions underwent reproductive evaluation, being included only those with sperm motility ≥ 70%. Fluorescent probes, hypoosmotic (HOST), and thermoresistance (TTR) tests were used to evaluate the functionality of the plasma membrane and the longevity of sperm cells, respectively. There was no stat. difference (p>0.05) between groups for membrane integrity. However, for the TTR, it was possible to observe a stat. difference (p<0.05) for the 24H/2% group with a lower response. The integrity of the plasma membrane, compared to the refrigeration time and the concentration of sodium caseinate, did not change. Therefore, it was concluded that sperm longevity was impaired with the use of a 2% concentration of sodium caseinate, and its use is not recommended for sending refrigerated semen, for insemination or freezing, that exceeds 12 hours. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Whether and how do Na+/H+ exchangers (NHEs) regulate the physiological functions of human sperm? SUMMARY ANSWER NHE-mediated flagellar intracellular pH (pHi) homeostasis facilitates the activation of the pH-sensitive, sperm-specific Ca2+ channel (CatSper) and the sperm-specific K+ channel (KSper), which subsequently modulate sperm motility, hyperactivation, flagellar tyrosine phosphorylation, and the progesterone (P4)-induced acrosome reaction. WHAT IS KNOWN ALREADY Sperm pHi alkalization is an essential prerequisite for the acquisition of sperm-fertilizing capacity. Different sperm functions are strictly controlled by particular pHi regulatory mechanisms. NHEs are suggested to modulate sperm H+ efflux. STUDY DESIGN, SIZE, DURATION This was a laboratory study that used samples from >50 sperm donors over a period of 1 year. To evaluate NHE action on human sperm function, 5-(N , N -dimethyl)-amiloride (DMA), a highly selective inhibitor of NHEs, was utilized. All experiments were repeated at least five times using different individual sperm samples or cells. PARTICIPANTS/MATERIALS, SETTING, METHODS By utilizing the pH fluorescent indicator pHrodo Red-AM, we detected alterations in single-cell pHi value in human sperm. The currents of CatSper and KSper in human sperm were recorded by the whole-cell patch-clamp technique. Changes in population and single-cell Ca2+ concentrations ([Ca2+]i) of human sperm loaded with Fluo 4-AM were measured. Membrane potential (Vm) and population pHi were quantitatively examined by a multimode plate reader after sperm were loaded with 3,3′-dipropylthiadicarbocyanine iodide and 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, respectively. Sperm motility parameters were assessed by a computer-assisted semen analysis system. Tyrosine phosphorylation was determined by immunofluorescence, and sperm acrosome reaction was evaluated by Pisum sativum agglutinin-FITC staining. MAIN RESULTS AND THE ROLE OF CHANCE DMA-induced NHEs inhibition severely acidified the human sperm flagellar pHi from 7.20 ± 0.04 to 6.38 ± 0.12 (mean ± SEM), while the effect of DMA on acrosomal pHi was less obvious (from 5.90 ± 0.13 to 5.57 ± 0.12, mean ± SEM). The whole-cell patch-clamp recordings revealed that NHE inhibition remarkably suppressed alkalization-induced activation of CatSper and KSper. As a consequence, impairment of [Ca2+]i homeostasis and Vm maintenance were detected in the presence of DMA. During the capacitation process, pre-treatment with DMA for 2 h potently decreased sperm pHi, which in turn decreased sperm motility and kinetic parameters. Sperm capacitation-associated functions, including hyperactivation, tyrosine phosphorylation, and P4-induced acrosome reaction, were also compromised by NHE inhibition. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION This was an in vitro study. Caution should be taken when extrapolating these results to in vivo applications. WIDER IMPLICATIONS OF THE FINDINGS This study revealed that NHEs are important physiological regulators for human CatSper and KSper, which are indispensable for human sperm fertility, suggesting that malfunction of NHEs could be an underlying mechanism for the pathogenesis of male infertility. FUNDING/COMPETING INTEREST(S) This work was supported by the National Natural Science Foundation of China (32271167 and 81871202 to X.Z.), Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC20211543 to X.Z.), the Social Development Project of Jiangsu Province (No. BE2022765 to X.Z.), the Society and livelihood Project of Nantong City (No. MS22022087 to X.Z.), and the Natural Science Foundation of Jiangsu Province (BK20220608 to H.K.). The authors have no competing interests to declare. [ABSTRACT FROM AUTHOR]

Before fertilization of the oocyte, the spermatozoa must undergo through a series of biochemical changes in the female reproductive tract named sperm capacitation. Spermatozoa regulates its functions by post-translational modifications, being historically the most studied protein phosphorylation. In addition to phosphorylation, recently, protein acetylation has been described as an important molecular mechanism with regulatory roles in several reproductive processes. However, its role on the mammal's sperm capacitation process remains unraveled. Sirtuins are a deacetylase protein family with 7 members that regulate protein acetylation. Here, we investigated the possible role of SIRT1 on pig sperm capacitation-related events by using YK 3-237, a commercial SIRT1 activator drug. SIRT1 is localized in the midpiece of pig spermatozoa. Protein tyrosine phosphorylation (focused at p32) is an event associated to pig sperm capacitation that increases when spermatozoa are in vitro capacitated in presence of YK 3-237. Eventually, YK 3-237 induces acrosome reaction in capacitated spermatozoa: YK 3-237 treatment tripled (3.40 ± 0.40 fold increase) the percentage of acrosome-reacted spermatozoa compared to the control. In addition, YK 3-237 induces sperm intracellular pH alkalinization and raises the intracellular calcium levels through a CatSper independent mechanism. YK 3-237 was not able to bypass sAC inhibition by LRE1. In summary, YK 3-237 promotes pig sperm capacitation by a mechanism upstream of sAC activation and independent of CatSper calcium channel. [ABSTRACT FROM AUTHOR]

Background. Extracellular vesicles (EVs) are membrane-bound vesicles containing various proteins, lipids, and nucleic acids. EVs are found in many body fluids, such as blood and urine. The release of EVs can facilitate intercellular communication through fusion with the plasma membrane or endocytosis into the recipient cell or through internalization of the contents. Recent studies have reported that EVs isolated from human endometrial epithelial cells (EECs) promote sperm fertilization ability. EVs from uterine flushing fluid more closely resemble the physiological condition of the uterus. However, it is unclear whether EVs derived directly from uterine flushing fluid have the same effect on sperm. This study aimed to research the effect of EVs from uterine flushing fluid on sperm. Methods. EVs were isolated from the uterine flushing fluid. The presence of EVs was confirmed by nanoparticle tracking analysis (NTA), Western blot, and transmission electron microscopy (TEM). EVs were incubated with human sperm for 2 h and 4 h. The effects of EVs on sperm were evaluated by analyzing acrosome reaction, sperm motility, and reactive oxygen species (ROS). Results. The EVs fractions isolated from the uterine fluid were observed in cup-shaped vesicles of different sizes by TEM. All isolated vesicles contained similar numbers of vesicles in the expected size range (30&lowbar;200 nm) by NTA.CD9and CD63 were detected in EVs by western blot. Comparing the motility of the two groups incubated sperm motility significantly differed at 4 h. The acrosome reactions were promoted by incubating with EVs significantly. ROS were increased in sperm incubated with EVs. Conclusion. Our results showed EVs present in the uterine fluid. Acrosome reactions and ROS levels increased in human sperm incubated with EVs. EVs from uterine fluid can promote the capacitation of human sperm. The increased capacitation after sperm interaction with EVs suggests a possible physiological effect during the transit of the uterus. [ABSTRACT FROM AUTHOR]

Global cannabis use has risen 23% since 2010, with 209 million reported users, most of whom are males of reproductive age. Delta-9-tetrahydrocannabinol (THC), the main psychoactive phytocannabinoid in cannabis, disrupts pro-homeostatic functions of the endocannabinoid system (ECS) within the male reproductive system. The ECS is highly involved in regulating morpho-functional and intrinsic sperm features that are required for fertilization and pre-implantation embryo development. Previous work by our group demonstrated that THC altered sperm capacitation and the transcriptome, including several fertility-associated microRNAs (miRs). Despite the prevalent use of cannabis among males of reproductive age, clinical and pre-clinical research investigating the impact of paternal cannabis on sperm function and the outcomes of artificial reproductive technologies (ARTs) remains inconclusive. Therefore, the present study investigates the impact of in vitro THC exposure on morpho-functional and intrinsic sperm functions, including contributions to embryo development following IVF. Bovine sperm were used as a translational model for human and treated with concentrations of THC that reflect plasma levels after therapeutic (0.032μM), and low (0.32μM)-high (4.8μM) recreational cannabis use. After 6-hours of treatment, THC did not alter the acrosomal reaction, but 4.8μM significantly reduced mitochondrial membrane potential (MMP) (p<0.05), primarily through agonistic interactions with CB-receptors. Fertilization of bovine oocytes with THC-treated sperm did not alter developmental rates, but blastocysts generated from sperm treated with 0.32–4.8μM THC had fewer trophoblasts (p<0.05), while blastocysts generated from sperm exposed to any concentration of THC had fewer cells in the inner cell mass (ICM), particularly within the 0.032μM group (p<0.001). Fertility associated miRs, including miR-346, miR-324, miR-33b, and miR-34c were analyzed in THC-exposed sperm and associated blastocysts generated by IVF, with lower levels of miRs-346, -324, and -33b found in sperm treated with 0.32μM THC, while miR-34c levels were higher in sperm treated with 0.032μM THC (p<0.05). Levels of miR-346 were also lower in sperm treated with 0.032μM THC, but higher in blastocysts generated from sperm exposed to 0.32μM THC (p<0.05). Our findings suggest that THC may alter key morpho-functional and epigenetic sperm factors involved in fertilization and embryo development. This is the first study to demonstrate that sperm exposed to THC in vitro negatively affects embryo quality following IVF. [ABSTRACT FROM AUTHOR]

Rabbits constitute an interesting model to understand gamete interaction and test novel Artificial Reproductive Techniques, but in vitro fertilization (IVF) is particularly problematic in this species. We have conducted a series of experiments to develop a consistent IVF technique. Initially, we checked viability, acrosome integrity, capacitation and motility in ejaculated sperm purified by a density gradient and incubated at different times in three different media: Tyrode's Albumin Lactate Pyruvate (TALP), human tubal fluid (HTF), and Brackett and Oliphant (BO). Total and progressive motility at 10–24 h and linearity from 3 h onwards was significantly higher in BO medium compared to TALP and HTF. Subsequently, cumulus-oocyte complexes (COCs) collected 10 h after induction of ovulation were incubated with sperm in TALP, HTF or BO for 18 h with or without performing sperm pre-incubation for 6 h. Pronuclear formation rate at 18 h was significantly higher in BO compared to other media (∼84 % vs. 17–22 %) and was not improved by pre-incubation. As COCs recovery rate was low at 10 h after induction of ovulation, COCs were collected at 12 h and co-incubated with sperm in BO. Pronuclear formation rate was similar than those obtained in COCs collected at 10 h (∼85 %), and when embryos were allowed to develop in vitro, the protocol yielded high cleavage and blastocyst rates (91 and 59 %, respectively). In conclusion, ejaculated rabbit sperm purified in a density gradient fertilize efficiently COCs collected at 12 h in BO medium. • Motility parameters of rabbit sperm after prolonged incubation were better in BO medium compared to TALP and HTF. • Fertilization rates were significantly higher in BO medium compared to TALP or HTF. • Sperm pre-incubation for 6 h in BO did not improved fertilization rates compared to no pre-incubation. • Rabbit sperm purified by a density gradient fertilize efficiently COCs collected at 10–12 h after ovulation induction. [ABSTRACT FROM AUTHOR]

Simple Summary: The objective was to identify candidate miRNAs and their integrated genes regulating acrosome function in capacitated sperm of high- versus low-fertility dairy bulls and to elucidate functional biological pathways using a systems biology approach featuring miRNA–mRNA cluster analyses. Based on categorized bovine miRNAs (n = 84), 19 were differentially expressed in high- compared to low-fertility capacitated sperm (p ≤ 0.05, fold regulation ≥ 2 magnitudes). mRNA expression of highly scored integrated genes of differentially expressed miRNAs was greater, ranging from 2.0 to 9.1-fold (p < 0.05) in high- compared to low-fertility sperm, with predicted pathways regulating acrosome vesicle exocytosis, acrosome reaction, and binding of sperm to zona pellucida. In conclusion, highly differentially expressed miRNAs in high-fertility bovine sperm regulating acrosome function have potential for predicting bull fertility. Bioinformatics envisage experimental data as illustrated biological networks, exploring roles of individual proteins and their interactions with other proteins in regulation of biological functions. The objective was to identify differentially expressed miRNAs and their associated genes regulating the acrosome reaction in capacitated sperm of high- compared to low-fertility dairy bulls and to elucidate biological functional pathways using a systems biology approach, featuring miRNA–mRNA cluster analysis. Categorized bovine-specific miRNAs (n = 84) were analyzed by RT-PCR; 19 were differentially expressed in high- compared to low-fertility sperm (p ≤ 0.05, fold regulation ≥ 2 magnitudes). Six miRNAs (bta-miR-129-5p, bta-miR-193a-3p, bta-miR-217, bta-mir-296-5p, bta-miR-27a, and bta-miR-320a) were highly upregulated (p < 0.05; fold regulation ≥ 5 magnitudes) in high- compared to low-fertility sperm. Highly scored integrated genes of differentially expressed miRNAs predicted associations with pathways regulating acrosome vesicle exocytosis, acrosome reaction, and sperm-oocyte binding. The mRNA expressions of genes associated with the acrosome reaction (including hub genes) were greater, ranging from 2.0 to 9.1-fold (p < 0.05) in high- compared to low-fertility capacitated bull sperm. In conclusion, differentially expressed miRNAs in high-fertility bovine sperm regulating acrosome functions have potential for predicting bull fertility. [ABSTRACT FROM AUTHOR]

Several authors have demonstrated that low levels of reactive oxygen species (ROS) are necessary for the physiological functions of sperm, such as capacitation, hyperactivation, acrosomal reaction and fertilization. However, high levels of ROS are associated with oxidative stress and detrimental effects on fertility. Consequently, deep characterization of ROS presence using different fluorescent probes could be crucial. In this sense, the study of intracellular ROS localization and the relationships between ROS and other conventional parameters could improve the characterization of sperm quality for semen preservation protocols in rams. In this work, a multiparametric study was carried out by analyzing four experimental groups of ram sperm with different initial qualities: fresh semen (from both breeding and nonbreeding seasons), frozen-thawed semen and, a positive control group treated with hydrogen peroxide (300µM) as a marker of extreme damage. Sperm analyses, including viability, apoptosis, lipid peroxidation, motility and kinetic parameters, were applied to compare several experimental groups with different sperm qualities. After that, the signals from two different ROS probes: CellROX™ Deep Red (CRDR) and Green (CRG), were examined by flow cytometry (percentage of cells that express ROS) and fluorescencemicroscopy (intracellular ROS location). Comparing conventional parameters, fresh samples from the breeding season showed the highest sperm quality, while the positive control samples showed the worst sperm quality. Concerning the ROS probes, the CRDR levels were higher in fresh samples from the breeding season than in the positive control and cryopreserved samples. Surprisingly, CRG presented its highest level (P < 0.05) in the positive control group treated with peroxide by flow cytometry. CRDR and CRG presented opposite labeling patterns that were corroborated by fluorescence microscopy, which determined that the probes localized in different parts of sperm. CRDR was found in the sperm mitochondrial region, while CRG was observed in the cell nucleus, suggesting that ROS localization is an important factor. Finally, our study indicates that CRDR is correlated with proper viability and sperm motility, and could be associated with high mitochondrial activity, while CRG is associated with sperm damage. [ABSTRACT FROM AUTHOR]

The fourth industrial revolution extensively reshapes the reality we are living in by blurring the boundaries of physical, digital and biological worlds. A good example is the previously unthinkable incursion of nanoscale waste materials, such as soot, into the technologies for assisted reproduction. Although the rapeseed oil soot may efficiently enhance the progressive motility of human spermatozoa, it is yet unknown whether this material induces undesirable oxidative stress and premature acrosome reaction, endangering the sperm-oocyte fusion and blastocyst formation. In an attempt to clarify this issue, we reveal that the three-hour incubation of human semen mixed with three main types of soot does not cause oxidative stress and spontaneous acrosome reaction of the sperm. These unique findings are attributed to synchronous elimination and stabilization of the oxidants via hydrogen bonding to the acidic groups of the soot (i.e., C=O and/or C-O-C) and electron donation by its basic chemical sites (i.e., C-OH and/or COOH). Moreover, the soot nanoparticles are electrostatically attracted by discrete positively charged areas on the sperm head, increasing its negative charge and in some cases interfering the acrosome reaction. Such novel mechanistic insights emphasize the credibility of rapeseed oil soot to confidently shift from the purely diagnostic and therapeutic phases in reproductive medicine to research dealing with the effect of carbon nanomaterials on the embryo development and implantation. [ABSTRACT FROM AUTHOR]

Abstract Purpose Since the unexplained in vitro fertilization failure occurs frequently, it is of great importance and clinical value to identify potential underlying predictors. This study aimed to explore whether the percentage of sperm with a small acrosome was correlated with unexplained in vitro fertilization failure. Methods A new acrosomal function evaluation index (the percentage of sperm with a small acrosome) was introduced into the analysis of sperm morphology. The association between the index and acrosome function by acrosin activity detection test and acrosome reaction test was investigated. In addition, the correlation with unexplained in vitro fertilization failure was further explored. Finally, the ROC curve was used to analyze the diagnostic efficacy on the failure of in vitro fertilization and the cutoff value was calculated. Results As the increasing of the percentage of sperm with a small acrosome, the value of acrosin activity, acrosome reaction rate, and in vitro fertilization rate were reduced, with a statistically significant difference (P

ABSTRACTNesfatin-1, a polypeptide hormone derived from the nucleobindin 2 (NUCB2) precursor protein, is known to regulate appetite and energy metabolism. Recent studies have also shown that NUCB2/nesfatin-1 is expressed in the reproductive organs of mice. However, the expression and potential role of NUCB2/nesfatin-1 in the mouse epididymis remain unclear. Therefore, we investigated the expression of NUCB2/nesfatin-1 in the mouse epididymis and its potential function. NUCB2/nesfatin-1 was detected in the epididymis by qRT-PCR and western blotting, and high expression levels were observed in epididymal epithelial cells by immunohistochemical staining. Pregnant mare’s serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) injections significantly increased NUCB2/nesfatin-1 expression in the epididymis. After castration, NUCB2/nesfatin-1 expression in the epididymis decreased, but was significantly increased by testosterone injection. Nesfatin-1-binding sites were found in the middle piece of testicular sperm, but were scarcely detected in the sperm head. By contrast, nesfatin-1 binding sites were identified on the sperm head within the epididymis. Furthermore, nesfatin-1 treatment inhibited the acrosome reaction in epididymal sperm. These results suggest that the nesfatin-1 protein produced in the epididymis binds to nesfatin-1 binding sites on the sperm head and plays a role in suppressing the acrosome reaction before ejaculation.

Male contraceptive options and infertility treatments are limited, and almost all innovation has been limited to updates to medically assisted reproduction protocols and methods. To accelerate the development of drugs that can either improve or inhibit fertility, we established a small molecule library as a toolbox for assay development and screening campaigns using human spermatozoa. We have profiled all compounds in the Sperm Toolbox in several automated high-throughput assays that measure stimulation or inhibition of sperm motility or the acrosome reaction. We have assayed motility under non-capacitating and capacitating conditions to distinguish between pathways operating under these different physiological states. We also assayed cell viability to ensure any effects on sperm function are specific. A key advantage of our studies is that all compounds are assayed together in the same experimental conditions, which allows quantitative comparisons of their effects in complementary functional assays. We have combined the resulting datasets to generate fingerprints of the Sperm Toolbox compounds on sperm function. The data are included in an on-line R-based app for convenient querying. [ABSTRACT FROM AUTHOR]

Background: Equine spermatozoa appear to differ from spermatozoa of other species in using oxidative phosphorylation preferentially over glycolysis. However, there is little information regarding effects of different energy sources on measured parameters in equine spermatozoa. Objective: To determine the effect of three individual energy substrates, glucose, pyruvate, and lactate, on motion characteristics, membrane integrity, and acrosomal status of stallion spermatozoa. Materials and methods: Freshly ejaculated stallion spermatozoa were incubated with combinations of glucose (5 mm), pyruvate (10 mm), and lactate (10 mm) for 0.5 to 4 h. Response to calcium ionophore A23187 (5 μm) was used to evaluate capacitation status. Motility was evaluated using computer‐assisted sperm analysis, and plasma membrane and acrosomal integrity were evaluated by flow cytometry. Results: Incubation with lactate alone for 2 h increased acrosomal sensitivity to A23187. Notably, incubation with lactate alone for 4 h induced a significant spontaneous increase in acrosome‐reacted, membrane‐intact (viable) spermatozoa, to approximately 50% of the live population, whereas no increase was seen with incubation in glucose or pyruvate alone. This acrosomal effect was observed in spermatozoa incubated at physiological pH as well as under alkaline conditions (medium pH approximately 8.5). Sperm motility declined concomitantly with the increase in acrosome‐reacted spermatozoa. Sperm motility was significantly higher in pyruvate‐only medium than in glucose or lactate. The addition of pyruvate to lactate‐containing medium increased sperm motility but reduced the proportion of live acrosome‐reacted spermatozoa in a dose‐dependent fashion. Discussion: This is the first study to demonstrate that incubation with a specific energy substrate, lactate, is associated with spontaneous acrosome reaction in spermatozoa. The proportion of live, acrosome‐reacted spermatozoa obtained is among the highest reported for equine spermatozoa. Conclusion: These findings highlight the delicate control of key sperm functions, and may serve as a basis to increase our understanding of stallion sperm physiology. [ABSTRACT FROM AUTHOR]