The Oxytocin Receptor in Spermatozoa May Originate From Both Spermatogenesis and Epididymal Maturation, and Regulates Capacitation.

Receptors in Spermatozoa: Are They Real?

Sperm Ion Channels: Molecular Targets for the Next Generation of Contraceptive Medicines?

The union of the mammalian spermatozoon and oocyte represents the culmination of a series of complex interactions between male and female gametes. Yet despite the importance of this highly regulated and species specific event, relatively little is known about the molecular basis of the interaction. Mammalian testicular spermatozoa, are incapable of fertilisation. Before fertilisation can occur, spermatozoa must undergo both epididymal maturation in the male reproductive tract and capacitation in the female tract. Only sperm that have traversed the epididymis attain the functional endpoints of capacitation, the ability to acrosome react and fertilise an egg. Capacitation is correlated with an increase in the level of tyrosine phosphorylation of a number of proteins, several of which become exposed on the cell surface. Previous analysis of the surface phosphoproteome of capacitated sperm demonstrated that the molecular chaperone HSPD1 is exposed on the plasma membrane overlying the acrosome, an ideal position for interaction with the zona pellucida. Although HSPD1 is not directly involved in zona binding, it has been proposed that during capacitation, intracellular tyrosine phosphorylation activates HSPD1 inside the cell, orchestrating the assembly of a zona binding complex and its subsequent exposure on the outside of sperm. To investigate the role of HSPD1 in fertilization, a proteomics-based approach was employed to identify chaperone associated proteins in capacitated sperm. HSPD1 was immunoprecipitated from capacitated sperm and associated proteins identified by liquid chromatography tandem mass spectrometry LC-MS/MS. analysis. Protein interactions were confirmed by reciprocal co-immunoprecipitation followed by Western blotting. The expression and localisation of the identified proteins during sperm maturation and fertilisation related events were investigated using indirect immunofluorescence and flow cytometry. To this end we have identified a number of proteins including an aldose reductase, another chaperone HSPE1, citron kinase and the proacrosin binding protein. These proteins co-localise with HSPD1 to the acrosomal region. However, this expression pattern is lost once sperm have undergone calcium ionophore A23187 induced acrosome reaction, as would be expected of molecules potentially involved in sperm-egg interactions. Based on these data we hypothesize that during epididymal transit, proteins important for fertilisation are deposited on sperm. During capacitation these proteins are assembled into functional protein complexes by chaperones including HSPD1, and chaperoned to sites including the cell surface where they affect the functional competence of sperm. The characterization of these HSPD1-interacting proteins is the subject of several chapters in this thesis. In addition, a proteomic analysis of the surface proteome of functionally mature mouse sperm was performed. This work was designed to complement and reinforce the work identifying chaperone-associated proteins in sperm, and to establish a reference proteome for ongoing and future comparative proteome studies. Protocols were developed and optimized to label sperm surface proteins with a number of different biotinylation reagents. Isolated proteins were subsequently separated by SDS-PAGE and proteins identified by LC-MS/MS. The broad goals of the work were to investigate the molecular basis of sperm-egg recognition with a view to identifying the key proteins that orchestrate the process.

Melatonin is a ubiquitous molecule, present in a wide range of organisms, and involved in multiple functions. Melatonin relays the information about the photoperiod to the tissues that express melatonin-binding sites in both central and peripheral nervous systems. This hormone has a complex mechanism of action. It can cross the cell plasma membrane and exert its actions in all cells of the body. Certain melatonin actions are mediated by receptors that belong to the superfamily of G-protein-coupled receptors (GPCRs), the MT1 and MT2 membrane. Melatonin can also bind to calmodulin as well as to nuclear receptors of the retinoic acid receptor family, RORα1, RORα2 and RZRβ. The purpose of this review is to report on recent developments in the physiological role of melatonin and its receptors. Specific issues concerning the biological function of melatonin in mammalian seasonal reproduction and spermatozoa are considered. The significance of the continuous presence of melatonin in seminal plasma with a fairly constant concentration is also discussed.

Study question Do EVs from human follicular fluid (FF-EVs), cervicovaginal fluid (CVF-EVs), and endometrial stromal cells spent medium (ESCs-EVs) induce sperm capacitation and enhance sperm motility? Summary answer In humans, EVs from different female reproductive tracts actively contribute to sperm maturation by inducing capacitation and enhancing sperm progressive motility. What is known already In the human body, EVs represent crucial players in cell-to-cell communication. EVs presence has been detected also in the female and male reproductive systems, specifically in seminal, follicular, oviductal, endometrial and vaginal fluid, as well as in embryo secretions. Moreover, EVs have been shown to be of pivotal importance in the fertilization process, as they support the maturation of spermatozoa and oocytes, as well as embryos in their development and endometrial implantation. The majority of these studies, though, have been conducted in animal models, underscoring the necessity to replicate analogous investigations also in humans. Study design, size, duration Follicular fluid (FF, n = 8), cervicovaginal fluid (CVF, n = 20) and endometrial tissue (n = 8) samples were collected from women undergoing assisted reproductive technology (ART) cycles at San Raffaele Hospital Fertility Centre (Milan). CVF were collected 2 and 7 days after the LH peak (CVF LH + 2 and CVF LH + 7, respectively). ESCs-EVs were isolated from both decidualized (dESCs-EVs) and not decidualized (eESCs-EVs) endometrial cells. Semen samples (n = 15) were collected from normozoospermic men. Participants/materials, setting, methods EVs were isolated using differential centrifugations and characterized using Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA), and western blotting (WB). EVs capture by spermatozoa was assessed using flow cytometry. EVs impact on sperm progressive motility was evaluated following WHO guidelines. EVs potential to induce sperm capacitation was examined through sperm acrosome reaction (AR) induction using a Ca2+ ionophore, followed by Coomassie blue staining. All experiments were performed using EVs at 50 and 100 μg/mL. Main results and the role of chance Isolated EVs displayed comparable size, with mean diameters ranging from 147,9±11,6 nm to 168,1±6,2 nm (mean ± SEM) and average concentrations from 9,53E+10 ± 8,11E+09 to 2,70E+11 ± 3,49E+10 particles/ml (mean ± SEM). TEM analysis confirmed their round-shaped morphology and structural integrity, while WB confirmed their positivity for EVs markers (Alix, TSG101, CD63, CD9). Spermatozoa efficiency in capturing these fluorescently-labeled EVs, measured as the percentage of fluorescence-positive spermatozoa, reached up to 3,6±1,1% for FF-EVs, 12,1±3,7% for CVF-EVs and 9,4±1,9% for ESCs-EVs (mean ± SEM). Sperm cells co-incubated with any EVs group maintained unaffected vitality. Following a 4-hour incubation with EVs, a rise in the proportion of progressively motile spermatozoa was observed, with percentages of progressively motile spermatozoa ranging from a minimum of 45,4±3,8% for CVF-EVs LH + 7 to a maximum of 52,7±2,7% for dESCs-EVs (mean ± SEM). This increase resulted to be statistically significant (p < 0.05 and p < 0.01, respectively) compared to the untreated (UT) condition, which exhibited 26,2±0,7% of progressively motile sperm cells. Spermatozoa that completed the AR, indicating capacitation, after 4-hour incubation with EVs resulted to be up to 25,8±3,2% for FF-EVs, 35,0±6,6% for CVF-EVs and 26,9±3,0% for ESCs-EVs, compared to an average of 13% in the UT control. Limitations, reasons for caution This study constitutes a pilot investigation characterized by a limited sample size, necessitating larger cohorts to validate results. Wider implications of the findings Studied EVs may exert beneficial effects on capacitation and progressive motility also on asthenozoospermic (AS) men’s spermatozoa. Therefore, they could be used in ART cycles to prime spermatozoa from AS men and increase the percentage of sperm cells displaying progressive motility, potentially favouring fertilization via classical IVF instead of ICSI. Trial registration number not applicable

Molecular biology of human male infertility: links with aging, mitochondrial genetics, and oxidative stress?

Mammalian sperm capacitation and fertilization in vitro: A critique of methodology

Objective To evaluate the effects of different concentrations of parecoxib sodium on the rat sperm motility,capacitation and acrosome reaction in vitro.Methods The sperm samples from Sprague-Dawley rat epididymis were collected by Klinefelter diffusion method and randomly divided into 4 groups (n =18 each) using a random number table:control group (group C),and parecoxib sodium 100,500,1 000 μmol/L groups (P1-3 groups).Parecoxib sodium with the final concentrations of 100,500 and 1 000 μmol/L was added to the culture medium.The samples were then incubated for 5 h in an airtight container filled with 5 ％ CO2 at 37 ℃.Then sperm motility was examined in vitro at 37 ℃ and analyzed by the computer-assisted sperm analysis,including the sperm motility ((a + b)％),average path velocity (VAP),straight line velocity (VSL),curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH).The capacitation effect was assessed by using the chlortetracycline staining and phase-contract microscopy.The acrosome reaction was evaluated by coomassie brilliant blue staining.Results The VAP,VSL,VCL and capacitation ability of the sperm were gradually decreased in C and P1-3 groups (P ＜ 0.05).Compared with group C,(a + b)％ in P2,3 groups and ALH in P2 group were significantly decreased (P ＜ 0.05).There was no significant difference in the acrosome reaction between groups (P ＞ 0.05).Conclusion Parecoxib sodium has significant inhibitory effects on the rat sperm motility and capacitation in a dose-dependent manner,while has no effect on the acrosome reaction in vitro. Key words: Cyclooxygenase inhibitors; Sperm motility; Sperm capacitation; Acrosome reaction

Very long-chain (C24 to C34) polyunsaturated fatty acids (VLCPUFA) are important constituents of sphingomyelin (SM) and ceramide (Cer) in testicular germ cells. In the present paper we focused on the SM and Cer and their fatty acids in spermatozoa and their main regions, heads and tails. In bull and ram spermatozoa, SM was the third most abundant phospholipid and VLCPUFA were the major acyl groups ( approximately 70%) of SM and Cer. In rat epididymal spermatozoa the SM/Cer ratio was low in the absence of and could be maintained high in the presence of the cation chelator EDTA, added to the medium used for sperm isolation. This fact points to the occurrence of an active divalent cation-dependent sphingomyelinase. Bull and rat sperm had an uneven head-tail distribution of phospholipid, with virtually all the VLCPUFA-rich SM located at the head, the lower SM content in the rat being determined by the lower sperm head/tail size ratio. Most of the SM from bull sperm heads was readily solubilized with 1% Triton X-100 at 4 degrees C. The detergent-soluble SM fraction was richer in VLCPUFA than the nonsoluble fraction and richer in saturated fatty acids. Cer was produced at the expense of SM, thus decreasing severalfold the SM/Cer ratio in rat spermatozoa incubated for 2 h in presence of the sperm-capacitating agents, calcium, bicarbonate, and albumin. The generation of Cer from SM in the sperm head surface may be an early step among the biochemical and biophysical changes known to take place in the spermatozoon in the physiological events preceding fertilization.

Infertility, affecting one in six couples worldwide, poses significant health and social challenges. While both male and female factors contribute to infertility, male infertility causes remain underexplored, with about 34% of cases classified as unexplained. A few studies focus on the role of lipids in male fertility, and some lipids are rising as key players in spermatozoa. This review highlights the importance of lipids, particularly phospholipids, neutral lipids, and glycolipids, in spermatozoa during capacitation and the acrosome reaction (AR). The dynamic lipid profile of human spermatozoa is crucial for their development, maturation, and fertilization capability. During epididymal maturation, sperm undergo crucial biochemical changes, including increased production of phosphatidylcholine and sphingomyelin, which enhance membrane integrity and mobility. Increased levels of ceramide affect membrane fluidity and signalling necessary for sperm function. As spermatozoa enter the female reproductive tract, they adjust their lipid content for capacitation and fertilization. Lipid signalling is crucial for human spermatozoa, influencing their viability and fertilization potential during transit through the female reproductive tract. Lysophosphatidic acid, abundant in seminal plasma, enhances sperm motility, facilitates the AR by promoting glycolysis and calcium influx, and is important for maintaining sperm viability. The remodelling of lipid rafts, enriched in cholesterol and sphingolipids, is essential for signal transduction and capacitation. Sphingolipids, particularly sphingosine 1-phosphate and ceramide, play significant roles in sperm capacitation and AR by promoting reactive oxygen species production and calcium signalling, respectively. Understanding these lipid dynamics will increase our knowledge of the complexity of sperm metabolism.

Nurture vs nature: how can we optimize sperm quality?

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. 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). 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. 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.

Sperm cryosurvival rates are not optimal for most species. Therefore, new cryopreservation strategies are needed with the objective of increasing the number of surviving sperm and the quality of those sperm after thawing. Cholesterol plays important roles in many sperm functions, including effects on membrane properties. One of these effects is to stabilize membranes at low temperatures. Thus, species that produce sperm which possess high membrane cholesterol : phospholipid ratios are more resistant to cold shock than sperm with low cholesterol : phospholipid ratios. Therefore, increasing the cholesterol content of sperm membranes may be a strategy that can improve sperm quality after freeze-thawing. In this review, information is presented related to using cyclodextrins pre-loaded with cholesterol for cryopreserving sperm from different species. The topics discussed include both in vitro and in vivo assessments of sperm quality after cryopreservation, as well as how increasing sperm cholesterol content affects other sperm functions.

Oxytocin is present in the male reproductive tract and has been shown to increase contractility in the epididymis and to modulate steroidogenesis. This study investigated the effects of oxytocin in the testis in vivo, and the presence and cellular localization of oxytocin receptors in the reproductive tract of rams. During the breeding season, mature rams underwent efferent duct ligation before injection of either oxytocin (20 microg) or oxytocin plus an oxytocin antagonist (20 microg) into the testicular artery; the contralateral testicular artery received saline. Injection of oxytocin caused a significant increase (P < 0.05) in the concentration of spermatozoa collected from the rete testis. This effect was not observed after treatment with the oxytocin antagonist plus oxytocin. Western blot analysis performed using a specific oxytocin receptor antibody (020) identified a single immunoreactive band of 66 kDa in testicular and epididymal tissue. This band was present in uterine tissue but not in liver or muscle. Immunocytochemistry identified oxytocin receptors on Leydig and Sertoli cells of the testis, on epithelial cells throughout the epididymis, on peritubular smooth muscle cells in the cauda epididymidis, and on the epithelial cells and circular smooth muscle layer of the ductus deferens. These findings indicate that oxytocin can modulate sperm transport in the ram testis. A role for oxytocin in promoting sperm transit is supported by the localization of oxytocin receptors in the cauda epididymis and ductus deferens, and the presence of receptors on Leydig, Sertoli and epididymal epithelial cells provides further evidence that oxytocin may be involved in the local regulation of steroidogenesis.

Does adjudin disrupt chloride ion (Cl⁻) ion transport function in human sperm and impede sperm capacitation and fertilizing ability in vitro? In this study the results indicate that adjudin is a potent blocker of Cl⁻ channels: disrupting Cl⁻ ion transport function results in a decline in sperm capacitation and fertilizing ability in humans in vitro. Although our previous studies have demonstrated that adjudin exerts its effect by disrupting sertoli-germ cell adhesion junctions, most notably apical ectoplasmic specialization by targeting testin and actin filament bundles that disrupts the actin-based cytoskeleton in sertoli cells, it remains unclear whether adjudin impedes Cl⁻ ion transport function in the human sperm. Semen samples were obtained from 45 fertile men (aged 25-32). Spermatozoa were isolated from the semen in the human tube fluid (HTF) medium by centrifugation through a discontinuous Percoll gradient, and incubated with adjudin at 10 nM-10 µM and/or other reagents under capacitating conditions for 0-5 h. We evaluated the effect of adjudin and different reagents on sperm functions with which they were incubated at 37 °C. Sperm motility and hyperactivation were analyzed by a computer-assisted sperm analysis (CASA) system. Sperm capacitation and the acrosome reaction were assessed by chlortetracycline fluorescence staining. Sperm fertilizing ability was evaluated by sperm penetration of zona-free hamster egg assay, and cellular cAMP levels in spermatozoa were quantified by the EIA kit. The proteins tyrosine, serine and threonine phosphorylation in the presence or absence of adjudin were analyzed by means of a immunodetection of spermatozoa, especially, compared the effect of adjudin on sperm hyperactivation and capacitation in the complete HTF medium with the Cl⁻-deficient HTF medium as well as the various Cl⁻ channel blockers. Adjudin significantly inhibited sperm hyperactivation but not sperm motility. Adjudin-induced inhibition of sperm capacitation was reversible, and it was found to block the rhuZP₃β- and progesterone-induced acrosome reaction in a dose-dependent manner. Adjudin also blocked sperm penetration of zona-free hamster eggs, and significantly inhibited both forskolin-activated transmembrane adenylyl cyclase and soluble adenylyl cyclase activities leading to a significant decline in the cellular cAMP levels in human spermatozoa. Adjudin failed to reduce sperm protein tyrosine phosphorylation but it did prevent sperm serine and threonine protein phosphorylation. Interestingly, adjudin was found to exert its inhibitory effects on sperm capacitation and capacitation-associated events only in the complete Cl⁻-HTF medium but not Cl⁻-deficient medium, illustrating the likely involvement of Cl⁻. Adjudin inhibits the fertility capacity of human sperm is mediated by disrupting chloride ion and its transport function. This study has examined the effect of adjudin only on human sperm capacitation and fertilizing ability in vitro and thus has some limitations. Further investigations in vivo are needed to confirm adjudin is a potent male contraceptive. Our studies demonstrated that adjudin inhibition of capacitation is reversible and its toxicity is low, opening the door for the examination of adjudin as a mediator of male fertility control. Adjudin may be a safe, efficient and reversible male antifertility agent and applicable to initial clinical trials of adjudin as a male antifertility agent in humans. STUDING FUNDING/COMPETING INTEREST(S): This work was supported by the National Basic Research Program of China (2006CB504002), the Nature Science Foundation of China (Nos. 81000244 and 81170554), Zhejiang Project of Science and Technology (2011C23046), the Nature Science Fund of Zhejiang province (Nos.Y2100058 and Y2090236), the key Science and Technology Innovation Team of Zhejiang Province (No.2012R10048-07) and the National Institutes of Health (NICHD U54 HD029990 project 5), USA. The authors declare no conflict of interest.