Sperm functionality is differentially regulated by porcine oviductal extracellular vesicles from the distinct phases of the estrous cycle.

Context Extracellular vesicles (EVs) derived from the oviductal fluid (oEVs) play a critical role in various reproductive processes, including sperm capacitation, fertilisation, and early embryo development. Aims To characterise porcine oEVs (poEVs) from different stages of the estrous cycle (late follicular, LF; early luteal, EL; mid luteal, ML; late luteal, LL) and investigate their impact on sperm functionality. Methods poEVs were isolated, characterised, and labelled to assess their binding to boar spermatozoa. The effects of poEVs on sperm motility, viability, acrosomal status, protein kinase A phosphorylation (pPKAs), tyrosine phosphorylation (Tyr-P), and in in vitro fertility were analysed. Key results poEVs were observed as round or cup-shaped membrane-surrounded vesicles. Statistical analysis showed that poEVs did not significantly differ in size, quantity, or protein concentration among phases of the estrous cycle. However, LF poEVs demonstrated a higher affinity for binding to sperm. Treatment with EL, ML, and LL poEVs resulted in a decrease in sperm progressive motility and total motility. Moreover, pPKA levels were reduced in presence of LF, EL, and ML poEVs, while Tyr-P levels did not differ between groups. LF poEVs also reduced sperm penetration rate and the number of spermatozoa per penetrated oocyte (P Conclusions poEVs from different stages of the estrous cycle play a modulatory role in sperm functionality by interacting with spermatozoa, affecting motility and capacitation, and participating in sperm-oocyte interaction. Implications The differential effects of LF and LL poEVs suggest the potential use of poEVs as additives in IVF systems to regulate sperm-oocyte interaction.

Exosomes from uterine fluid promote capacitation of human sperm.

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 (30-200 nm) by NTA. CD9 and 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.

Comparative Proteomics Elucidates the Potential Mechanism of Sperm Capacitation of Chinese Mitten Crabs (Eriocheir sinensis).

Sperm capacitation is broadly defined as a suite of biochemical and biophysical changes resulting from the acquisition of fertilization ability. To gain insights into the regulation mechanism of crustacean sperm capacitation, 4D label-free quantitative proteomics was first applied to analyze the changes of sperm in Eriocheir sinensis under three sequential physiological conditions: seminal vesicles (X2), hatched with the seminal receptacle content (X3), and incubated with egg water (X5). In total, 1536 proteins were identified, among which 880 proteins were quantified, with 82 and 224 proteins significantly altered after incubation with the seminal receptacle contents and egg water. Most differentially expressed proteins were attributed to biological processes by Gene Ontology annotation analysis. As the fundamental bioenergetic metabolism of sperm, the oxidative phosphorylation, glycolysis, and the pentose phosphate pathway presented significant changes under the treatment of seminal receptacle contents, indicating intensive regulation for sperm in the seminal receptacle. Additionally, the seminal receptacle contents also significantly increased the oxidation level of sperm, whereas the enhancement of abundance in superoxide dismutase, peroxiredoxin 1, and glutathione S-transferase after incubation with egg water significantly improved the resistance against oxidation. These results provided a new perspective for reproduction studies in crustaceans.

Extracellular cAMP and MRP4 activity influence in vitro capacitation and fertilizing ability of pig spermatozoa.

cAMP has been reported to be an essential driver of sperm capacitation. In bovine sperm cAMP efflux through multidrug resistance-associated protein 4 (MRP4) has been suggested to maintain intracellular cAMP homeostasis and generate extracellular signaling able to regulate capacitation. The aim of this work was to determine whether extracellular cAMP may influence in vitro pig sperm capacitation and acquisition of fertilizing ability and to evaluate the role of MRP4. In vitro sperm capacitation and gamete coincubation were performed in Brackett and Oliphant's medium (BO) in presence of caffeine (Ctr+) or in BO without caffeine (Ctr-) supplemented with 0, 8, 9, 10 mM cAMP. Despite the percentage of capacitated sperm, assayed by immunolocalization of tyrosine-phosphorylated proteins, was significantly lower in Ctr- compared to Ctr+, it increased supplementing 10 mM cAMP to Ctr- reaching values similar to Ctr+. The absence of caffeine during gamete coincubation reduced the fertilization rate compared to Ctr+, while 10 mM cAMP supplementation to Ctr- increased the fertilization rate reaching values similar to Ctr + . The presence of MRP4 in pig spermatozoa was detected for the first time by western blot and immunohistochemistry assays. To evaluate MRP4 role on pig sperm capacitation, in vitro capacitation and gamete coincubation were performed in Ctr + in presence of MK571, a MRP4 selective inhibitor. MK571 reduced the percentage of capacitated cells and the fertilization rate, while cAMP addition fully reversed MRP4 blockade consequences. Present findings suggest that, under our in vitro conditions, extracellular cAMP and MRP4 activity influence pig sperm capacitating events.

Deletion of the Sodium Glucose Cotransporter 1 (Sglt-1) impairs mouse sperm movement.

The Sodium Glucose Cotransporter Isoform 1 (Sglt-1) is a symporter that moves Na+ and glucose into the cell. While most studies have focused on the role of Sglt-1 in the small intestine and kidney, little is known about this transporter's expression and function in other tissues. We have previously shown that Sglt-1 is expressed in the mouse sperm flagellum and that its inhibition interferes with sperm metabolism and function. Here, we further investigated the importance of Sglt-1 in sperm, using a Sglt-1 knockout mouse (Sglt-1 KO). RNA, immunocytochemistry, and glucose uptake analysis confirmed the ablation of Sglt-1 in sperm. Sglt-1 KO male mice are fertile and exhibit normal sperm counts and morphology. However, Sglt-1 null sperm displayed a significant reduction in total, progressive and other parameters of sperm motility compared to wild type (WT) sperm. The reduction in motility was exacerbated when sperm were challenged to swim in media with higher viscosity. Parameters of capacitation, namely protein tyrosine phosphorylation and acrosomal reaction, were similar in Sglt-1 KO and WT sperm. However, Sglt-1 KO sperm displayed a significant decrease in hyperactivation. The impaired motility of Sglt-1 null sperm was observed in media containing glucose as the only energy substrate. Interestingly, the addition of pyruvate and lactate to the media partially recovered sperm motility of Sglt-1 KO sperm, both in the low and high viscosity media. Altogether, these results support an important role for Sglt-1 in sperm energetics and function, providing sperm with a higher capacity for glucose uptake.

The compound YK 3-237 promotes pig sperm capacitation-related events.

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.

Existence and importance of Na+K+-ATPase in the plasma membrane of boar spermatozoa.

Sodium-potassium-ATPase (Na+K+-ATPase), a target to treat congestive heart failure, is the only known receptor for cardiac glycosides implicated in intracellular signaling and additionally functions enzymatically in ion transport. Spermatozoa need transmembrane ion transport and signaling to fertilize, and Na+K+-ATPase is identified here for the first time in boar spermatozoa. Head plasma membrane (HPM) isolated from boar spermatozoa was confirmed pure by marker enzymes acid and alkaline phosphatase (218 ± 23% and 245 ± 38% enrichment, respectively, versus whole spermatozoa). Western immunoblotting detected α and ß subunits (isoforms α1, α3, ß1, ß2, and ß3) in different concentrations in whole spermatozoa and HPM. Immunofluorescence of intact sperm only detected α3 on the post-equatorial exterior membrane; methanol-permeabilized sperm also had α3 post-equatorially and other isoforms on the acrosomal ridge and cap. Mass spectrometry confirmed the presence of all isoforms in HPM. Incubating boar sperm in capacitating media to induce the physiological changes preceding fertilization significantly increased the percentage of capacitated sperm compared to 0 h control (33.0 ± 2.6% vs. 19.2 ± 2.6% capacitated sperm, respectively; p = 0.014) and altered the ß2 immunofluorescence pattern. These results demonstrate the presence of Na+K+-ATPase in boar sperm HPM and that it changes during capacitation.

Lipidomics profiles of human spermatozoa: insights into capacitation and acrosome reaction using UPLC-MS-based approach.

Introduction: Lipidomics elucidates the roles of lipids in both physiological and pathological processes, intersecting with many diseases and cellular functions. The maintenance of lipid homeostasis, essential for cell health, significantly influences the survival, maturation, and functionality of sperm during fertilization. While capacitation and the acrosome reaction, key processes before fertilization, involve substantial lipidomic alterations, a comprehensive understanding of the changes in human spermatozoa's lipidomic profiles during these processes remains unknown. This study aims to explicate global lipidomic changes during capacitation and the acrosome reaction in human sperm, employing an untargeted lipidomic strategy using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Methods: Twelve semen specimens, exceeding the WHO reference values for semen parameters, were collected. After discontinuous density gradient separation, sperm concentration was adjusted to 2 x 106 cells/ml and divided into three groups: uncapacitated, capacitated, and acrosome-reacted. UPLC-MS analysis was performed after lipid extraction from these groups. Spectral peak alignment and statistical analysis, using unsupervised principal component analysis (PCA), bidirectional orthogonal partial least squares discriminant analysis (O2PLS-DA) analysis, and supervised partial least-squares-latent structure discriminate analysis (PLS-DA), were employed to identify the most discriminative lipids. Results: The 1176 lipid peaks overlapped across the twelve individuals in the uncapacitated, capacitated, and acrosome-reacted groups: 1180 peaks between the uncapacitated and capacitated groups, 1184 peaks between the uncapacitated and acrosome-reacted groups, and 1178 peaks between the capacitated and acrosome-reacted groups. The count of overlapping peaks varied among individuals, ranging from 739 to 963 across sperm samples. Moreover, 137 lipids had VIP values > 1.0 and twenty-two lipids had VIP > 1.5, based on the O2PLS-DA model. Furthermore, the identified twelve lipids encompassed increases in PI 44:10, LPS 20:4, LPA 20:5, and LPE 20:4, and decreases in 16-phenyl-tetranor-PGE2, PC 40:6, PS 35:4, PA 29:1, 20-carboxy-LTB4, and 2-oxo-4-methylthio-butanoic acid. Discussion: This study has been the first time to investigate the lipidomics profiles associated with acrosome reaction and capacitation in human sperm, utilizing UPLC-MS in conjunction with multivariate data analysis. These findings corroborate earlier discoveries on lipids during the acrosome reaction and unveil new metabolites. Furthermore, this research highlights the effective utility of UPLC-MS-based lipidomics for exploring diverse physiological states in sperm. This study offers novel insights into lipidomic changes associated with capacitation and the acrosome reaction in human sperm, which are closely related to male reproduction.

Sperm Capacitation and Kinematics in Phodopus Hamsters.

This study was designed to analyze changes in the spermatozoa of three species of Phodopus hamsters incubated under different conditions. Cauda epididymal sperm were incubated for 4 h in modified Tyrode's medium containing albumin, lactate, pyruvate, and Hepes (mTALP-H), in the same medium with the addition of bicarbonate (mTALP-BH), or with bicarbonate and 20 ng/mL of progesterone (mTALP-BH+P4). Media with bicarbonate are believed to promote capacitation in rodent species. Sperm motility, viability, capacitation patterns, and kinematics were assessed at different times. Capacitation in live cells was quantified after staining with Hoechst 33258 and chlortetracycline. Patterns believed to correspond to non-capacitated cells (F pattern), capacitated, acrosome-intact cells (B pattern), and acrosome-reacted cells (AR pattern) were recognized. Kinematics were examined via computer-assisted sperm analysis (CASA). The results showed a decrease in total motility in all three species in different media, with a sharp decrease in progressive motility in bicarbonate-containing media (without or with progesterone), suggesting hyperactivated motion. However, none of the other signs of hyperactivation described in rodents (i.e., decrease in STR or LIN, together with an increase in ALH) were observed. F pattern cells diminished with time in all media and were generally lower in P. roborovskii and higher in P. campbelli. B pattern cells increased in mTALP-BH media in all species. Progesterone did not enhance the percentage of B pattern cells. Finally, AR pattern cells increased in all species incubated in different media, showing the highest percentage in P. roborovskii and the lowest in P. campbelli. Comparisons between media revealed that there were higher percentages of F pattern cells and lower percentages of B pattern cells over time in medium without bicarbonate (mTALP-H) in comparison to media containing bicarbonate (mTALP-BH; mTALP-BH+P4). Overall, changes consistent with the acquisition of capacitation and development of hyperactivated motility were found; however, further studies are required to better characterize media necessary to support the pathways involved in these processes in Phodopus species.

Cryopreservation, in addition to protein tyrosine phosphorylation, alters the distribution of phosphatidyl inositol bisphosphate and the localization of cytoskeletal and signaling proteins (gelsolin, tyrosine kinase c-SRC and phospholipase C-ζ) in the perinuclear theca of boar sperm.

Cryopreservation of boar spermatozoa affects the perinuclear theca (PT) and involves several proteins and molecules that play important roles during capacitation and the acrosomal reaction. The objective of the present study was to evaluate whether the deleterious effects of cryopreservation in addition to protein tyrosine phosphorylation are accompanied by changes in the distribution of phosphatidyl inositol bisphosphate (PIP2) and the localization of cytoskeletal and signaling proteins in the perinuclear theca of cryopreserved boar spermatozoa. For this purpose, by immunocytochemistry (IC) the changes in localization of phosphorylated proteins in tyrosine residues, gelsolin, c-SRC kinase and PLC-ζ, as well as in the distribution of phosphatidyl inositol bisphosphate were analyzed in thawed spermatozoa (T) non capacitated (NC), capacitated (C) and in those with acrosomal reaction (AR) and compared with fresh spermatozoa (F) under the same physiological status. Western blotting (WB) and co-immunoprecipitation were performed to confirm the presence of these proteins in PT and to determine the interaction between these molecules. IC showed that immunostaining for phosphorylated proteins significantly increased in the acrosomal region and flagellum in TNC spermatozoa (p < 0.05). The proportion of cells displaying immunolabeling for gelsolin in the acrosomal region decreased after capacitation in cryopreserved spermatozoa; the same change was found (p < 0.05) in the proportion of spermatozoa immunoreactive to PIP2 in the sperm head. c-SRC was observed in the equatorial segment and acrosomal region, subdomains that coincide with the site where phosphorylated proteins were detected. PLC-ζ immunolocalization in fresh spermatozoa underwent changes after capacitation and acrosomal reaction, with a significant increase in the equatorial segment and post-acrosomal region in cryopreserved spermatozoa (p < 0.05). WB analysis indicated the presence of gelsolin, c-SRC and PLC-ζ in PT; besides, we confirmed that gelsolin co-immunoprecipitated with c-SRC and PLC-ζ, which changes according to the physiological state of spermatozoa. As a conclusion, cryopreservation together with increased immunodetection of tyrosine phosphorylated proteins decreases the detection of PIP2 and alters the immunolocalization patterns of gelsolin, c-SRC and PLC-ζ in the PT in boar spermatozoa.

The other side of capacitation: role of mouse male molecules in the regulation of time and place of capacitation.

In brief: Capacitation is regulated by decapacitation factors secreted by male ducts and accessory sex glands. This revision is focused on targets and events regulated by decapacitation factors in Mus musculus and their potential use for fertility control. Abstract: Sperm capacitation is a necessary process for mammalian spermatozoa to acquire fertilization capability. This process occurs when the sperm enters the female's reproductive duct, involving a vital interplay with the uterine and oviductal environment, leading to morphological, physiological, and biochemical modifications in the male gamete. Besides, for a successful sperm capacitation, molecules are incorporated onto the sperm's surface during its passage through the male reproductive tract followed by their subsequent removal. These molecules, referred to as decapacitation factors (DFs), also regulate capacitation, preventing this process from occurring in the wrong site or at the wrong time. While decapacitation factors have been extensively studied in recent decades in species such as Mus musculus, there is no comprehensive report consolidating information on all the identified decapacitation factors and the molecular basis of their function. The aim of this review is to summarize the data related to decapacitation factors discovered and characterized in Mus musculus. Concurrently, this review aims to elucidate the implications of different decapacitation factors throughout the fertilization process (i.e. capacitation, acrosomal reaction, and fertilization), as well as the methodologies employed for their investigation. Given that mice (Mus musculus) have served as a valuable model in reproductive research due to their genetic similarity to humans, this review contributes to our understanding of the role of decapacitation factors in male fertility.

Exposure to Cumulus Cell Secretome Improves Sperm Function: New Perspectives for Sperm Selection In Vitro.

In the literature, there is a well-known correlation between poor semen quality and DNA sperm integrity, which can turn into negative outcomes in terms of embryo development and clinical pregnancy. Sperm selection plays a pivotal role in clinical practice, and the most widely used methods are mainly based on sperm motility and morphology. The cumulus oophorus complex (COC) during natural fertilization represents a barrier that spermatozoa must overcome to reach the zona pellucida and fertilize the oocyte. Spermatozoa that can pass through the COC have better structural and metabolic characteristics as well as enhanced acrosome reaction (AR). The present study aimed to evaluate the exposure of sperm to cumulus cell secretome during swim-up treatment (SUC) compared with the routinely used swim-up method (SU). To determine the effectiveness of this method, biological factors critical for the ability of sperm to fertilize an oocyte, including capacitation, AR, tyrosine phosphorylation signature, DNA integrity, and mitochondrial functionality, were assessed. The SUC selection assures recovery of high-quality spermatozoa, with enhanced mitochondrial functionality and motility compared with both SU-selected and unselected (U) sperm. Furthermore, using this modified swim-up procedure, significantly reduced sperm DNA damage (p < 0.05) was detected. In conclusion, the SUC approach is a more physiological and integrated method for sperm selection that deserves further investigation for its translation into clinical practice.

Boar fertility is controlled through systematic changes of mitochondrial protein expression during sperm capacitation.

Vigorous activation of mitochondria in spermatozoa during capacitation induces the biological and morphological changes of spermatozoa to acquire fertilizing ability. To in-depth understand the dynamic roles of mitochondrial and male fertility, this study was to identify how the mitochondrial proteins are changed during sperm capacitation and regulate male fertility using boar spermatozoa. The mitochondrial proteins were differentially changed during sperm capacitation according to fertility status, i.e., superior litter size (SL) and normal litter size (NL). Following sperm capacitation, ubiquitin-cytochrome c reductase core protein (UQCRC1) and ATP synthase F1 (ATP5F1) increased in NL, while cytochrome c oxidase subunit 5B (COX5B), and cytochrome c1 (CYC1) proteins decreased. In contrast, only and ubiquinone oxidoreductase core subunit 8 (NDUFS8) protein was increased in SL following capacitation. The protein expression difference value of CYC1, COX5B, and NDUFS8 following sperm capacitation was lower in NL than SL boars. Based on these complicated changes during sperm capacitation, the accuracy for predicting male fertility of NDUFS8 was increased to 87 %. Overall, considering the systematic orchestration of mitochondrial protein expression according to sperm capacitation status, it will be possible to better understand male fertility.

Boar sperm motility is modulated by CCK at a low concentration of bicarbonate under capacitation conditions.

In a previous study, our group detected the cholecystokinin (CCK) protein in the porcine oviduct. This fact, together with the involvement of CCK in the regulation of sperm protein tyrosine phosphorylation by the modulation of HCO3 - uptake (in mice and humans) suggests a role for CCK during sperm capacitation. Therefore, on the one hand, the expression of CCK receptors (CCK1R and CCK2R) on boar testes has been investigated and probed; on the other hand, boar spermatozoa (from seminal doses of 1-day and 5-day storage) were exposed to different concentrations of CCK (0-control, 25 or 50 µM) in a medium supporting capacitation supplemented with 0, 5 or 25 mmol/L of HCO3 - for 1 h at 38.5°C. Sperm motion (total and progressive motility), kinetic parameters, viability, acrosome status, and mitochondrial activity were determined. No differences between groups (0, 25 or 50 µM of CCK) were observed when HCO3 - was absent in the media (p > .05). However, the results showed that when the media was supplemented with 5 mmol/L HCO3 - in 1-day seminal dose storage, the linearity index (LIN, %), straightness index (STR, %) and oscillation index (WOB, %) (sperm kinetics parameters) increased in the presence of CCK regardless the concentration (p < .05). Nevertheless, CCK in sperm from 5-day storage only increased the WOB parameter in comparison to the control (p < .05). Furthermore, the average amplitude of the lateral displacement of the sperm head (ALH, µm) and curvilinear velocity (VCL, µm/s) decreased when CCK was present, depending on its concentration and sperm aging (1-day vs. 5-days) (p < .05). In the case of the media supporting capacitation supplemented with 25 mmol/L HCO3 - , any differences were observed except for sperm viability in the 5-day seminal doses, which increased in the 50 µM-CCK group compared to the control (p < .05). In conclusion, these data suggest an implication of CCK protein during sperm capacitation under low bicarbonate concentration increasing the sperm linear trajectory.

Sperm capacitation as a predictor of boar fertility.

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

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

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

Effect of arginine-induced motility and capacitation on RNA population in goat spermatozoa.

INTRODUCTION: In vitro capacitation is essential in assisted reproductive technologies (ART) for embryo production. Recently, arginine has been proven to enhance capacitation in mammalian spermatozoa. However, the detailed mechanism of action of arginine remains elusive. AIM: This study investigated the effect of arginine-induced capacitation and motility enhancement on the spermatozoal RNA (spRNA) population in goats. MATERIAL AND METHODS: Goat spermatozoa were treated with arginine for up to six hours and compared with non-treated or PHE (penicillamine, hypotaurine, and epinephrine)-treated spermatozoa at different intervals (0, 1, 2, 4, and 6 hours). Sperm parameters, including viability, individual motility, capacitation, acrosome reaction, and ROS production, were evaluated. The spRNA population was analyzed by short-read RNA sequencing (RNA-seq). RESULTS: The percentage of capacitated (73.21 ± 4.22%) and acrosome reacted (18.35 ± 0.56%) spermatozoa was highest in arginine treatment, while PHE treatment showed the highest percentage (79.82 ± 4.31%) of motile spermatozoa from 0 to 4 hours of incubation. RNA-seq analysis identified 1,321 differentially expressed genes (DEGs) in arginine-treated spermatozoa compared to the control. The PGK2, RNASE10, ODF1, and ROPN1L genes involved in sperm motility and ACR, DKKL1, KCNJ11, and PRND genes involved in the capacitation process were upregulated in arginine-treated spermatozoa. The DEGs regulate sperm capacitation-related cAMP-PKA, PI3-Akt, calcium, and MAPK signaling pathways. CONCLUSION: The arginine-induced capacitation and enhanced sperm motility were associated with the upregulation of several genes involved in sperm motility and capacitation pathways. The comparative study also suggests that arginine may be used in lieu of PHE for motility enhancement and in vitro capacitation of goat spermatozoa.

Understanding the impact of varicocele on sperm capacitation.

OBJECTIVE: To study the relationship between the seminal sample quality of men with varicocele and sperm capacitation. DESIGN: Cross-sectional observational study. SETTING: Academic hospital. PATIENT(S): Seventy-six men (19 control and 57 with varicocele) were analyzed. INTERVENTION(S): Semen samples were submitted to a discontinuous density gradient for sperm selection. Sperm capacitation was induced using a human tubal fluid medium supplemented with bovine serum albumin. MAIN OUTCOME MEASURE(S): After capacitation induction, the sperm were assessed by capacitation state, computer-assisted sperm motility, mitochondrial activity, membrane integrity, acrosome reaction, and intracellular oxidative stress. RESULT(S): The capacitation period increased sperm motility, showing an increase in the average path velocity and a decrease in the straightness compared with sperm before capacitation (paired analysis). After capacitation, the rate of capacitated sperm, motility, and mitochondrial activity showed differences between groups (control and varicocele). The varicocele group showed lower mitochondrial activity and capacitation than the control group. On the other hand, no significant differences were observed in the other variables evaluated. CONCLUSION(S): Varicocele men showed less viable sperm and mitochondrial activity than control men after capacitation sperm. The induction of capacitation altered motility by increasing path velocity and decreasing straightness in all of the studied groups, evidencing the occurrence of hyperactivation.

The Role of Sperm Membrane Potential and Ion Channels in Regulating Sperm Function.

During the last seventy years, studies on mammalian sperm cells have demonstrated the essential role of capacitation, hyperactivation and the acrosome reaction in the acquisition of fertilization ability. These studies revealed the important biochemical and physiological changes that sperm undergo in their travel throughout the female genital tract, including changes in membrane fluidity, the activation of soluble adenylate cyclase, increases in intracellular pH and Ca2+ and the development of motility. Sperm are highly polarized cells, with a resting membrane potential of about -40 mV, which must rapidly adapt to the ionic changes occurring through the sperm membrane. This review summarizes the current knowledge about the relationship between variations in the sperm potential membrane, including depolarization and hyperpolarization, and their correlation with changes in sperm motility and capacitation to further lead to the acrosome reaction, a calcium-dependent exocytosis process. We also review the functionality of different ion channels that are present in spermatozoa in order to understand their association with human infertility.

Analyzing the effect of heparin on in vitro capacitation and spermatozoal RNA population in goats.

Heparin is a glycosaminoglycan polymer that is commonly used as an anticoagulant. Heparin also induces in vitro capacitation in spermatozoa, although its molecular mechanism is elusive. This study investigated the effect of heparin on in vitro capacitation and spermatozoal RNA (spRNA) population in goats. Goat spermatozoa were treated with 20 µM heparin for 0-6 h and evaluated for motility, capacitation, acrosome reaction, and spRNA population by RNA sequencing (RNA-seq). It was observed that heparin enhanced sperm motility up to 6 h of incubation (p < 0.05). Heparin also induced capacitation and acrosome reaction within 4 h. RNA-seq identified 1254 differentially expressed genes (DEGs) between heparin-treated and control spermatozoa. Most DEGs (1251 nos.) were upregulated and included 1090 protein-coding genes. A few genes (PRND, ITPR1, LLCFC1, and CHRM2) showed >5-fold increased expression in heparin-treated spermatozoa compared to the control. The upregulated genes were found to be involved in cAMP-PKA, PI3-Akt, calcium, MAPK signaling, and oxidative stress pathways. DCFDA staining confirmed the increased oxidative stress in heparin-treated spermatozoa compared to the control (p < 0.05). In conclusion, the results of the present study suggest that heparin enhances sperm motility and induces capacitation by upregulation of the spRNA population and oxidative stress pathway.