Do the main psychoactive phytocannabinoid delta-9-tetrahydrocannabinol (THC) and its non-psychoactive analog cannabidiol (CBD) affect human sperm function?

THC and CBD affect the sperm-specific Ca2+ channel CatSper, suppress activation of the channel by progesterone (P4) and prostaglandin E1 (PGE1), and THC also alters human sperm function in vitro.

Marijuana (Cannabis sativa) is one of the most commonly used recreational drugs worldwide. Although the effects of phytocannabinoids on semen parameters have been studied, there is no evidence of a direct impact of THC and CBD on human sperm.

We investigated the effects of the major psychoactive phytocannabinoid, THC, its non-psychoactive analog, CBD, and their major metabolites on Ca2+ influx via CatSper in human spermatozoa. THC and CBD were selected to further evaluate their action on P4-, PGE1-, and pH-induced activation of CatSper. The effects of THC and CBD on sperm motility, penetration into viscous media, and acrosome reaction (AR) were also assessed.

The effects of phytocannabinoids on CatSper activity were investigated on semen samples from healthy volunteers and men with homozygous deletion of the CATSPER2 gene using kinetic Ca2+ fluorimetry and patch-clamp recordings. Motility was assessed by computer-assisted sperm analysis (CASA). Sperm penetration into viscous media was assessed using a modified Kremer test. The AR was evaluated by flow cytometry using Pisum sativum agglutinin-stained spermatozoa.

Both THC and CBD increased the intracellular calcium concentration with CBD inducing a greater increase compared to THC. These Ca2+ signals were abolished in men with homozygous deletion of the CATSPER2 gene demonstrating that they are mediated through CatSper. THC suppressed the P4- and the PGE1-induced Ca2+ increase with a half-maximal inhibitory concentration (IC50) of 1.88 ± 1.15 µM and 0.98 ± 1.10, respectively. CBD also suppressed the P4- and PGE1-induced Ca2+ signal with an IC50 of 2.47 ± 1.12 µM and 6.14 ± 1.08 µM, respectively. The P4 and PGE1 responses were also suppressed by THC and CBD metabolites, yet with greatly reduced potency and/or efficacy. THC and CBD were found to inhibit the Ca2+ influx evoked by intracellular alkalization via NH4Cl, with THC featuring a higher potency compared to CBD. In conclusion, THC and CBD inhibit both the ligand-dependent and -independent activation of CatSper in a dose-dependent manner. This indicates that these phytocannabinoids are genuine CatSper inhibitors rather than P4 and PGE1 antagonists. Finally, THC, but not CBD, impaired sperm hyperactivation and penetration into viscous media and induced a small increase in AR.

Future studies are needed to assess whether cannabis consumption can affect fertility since this study was in vitro.

The action of THC and CBD on CatSper in human sperm may interfere with the fertilization process, but the impact on fertility remains to be elucidated. THC inhibits the P4 and the PGE1 response more potently than CBD and most previously described CatSper inhibitors. THC can be used as a starting point for the development of non-hormonal contraceptives targeting CatSper.

This work was supported by the Swiss Center for Applied Human Toxicology (SCAHT), the Département de l'Instruction Publique (DIP) of the State of Geneva and the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation). The authors declare that no conflicts of interest have been identified that might affect the impartiality of the research reported.

N/A.

The female reproductive environment modulates sperm fertilization potential and may exert numerous selective forces on sperm during fertilization. Exploring cetacean (whales, dolphins, and porpoises) sperm responses to variable extracellular salinity and pH, either from seawater exposure or from the vaginal environment, offers insight into the selective pressures exerted on sperm in aquatically-mating mammals. This study examined common bottlenose dolphin (Tursiops truncatus) sperm motility, plasma membrane integrity, morphology, and acrosome integrity upon semen exposure to biologically relevant extracellular salinities (0-40 ppt) and in situ measured dolphin vaginal pHs (5-7.4). Sperm motility and kinematics were assessed by computer-aided sperm analysis (CASA). Sperm plasma membrane integrity, morphology, and acrosome integrity were assessed post-hoc. Dolphin sperm motility and plasma membrane integrity significantly decreased when exposed to solutions ≥ average oceanic salinity (35 ppt). Vaginal pHs were highly variable among individuals (pH 5.04-7.4). Sperm motility appeared optimal at pH 7 and sperm viability decreased significantly at pHs < 6.5. There was more inter-individual variation in sperm motility and kinematics when exposed to pHs < 7 compared to any salinity. Optimal extracellular salinity (8 - 15 ppt) and pH (pH 7) ranges that improve sperm motility and viability can inform dolphin semen processing methods for cryopreservation.

The flagellar calcium channel CatSper is essential for male fertility, as it regulates calcium influx to trigger the hyperactive motility required for sperm to fertilize the egg. Precise activation of CatSper is critical, as premature activation can impair sperm function. While optimal temperature is known to influence fertilization, its effect on CatSper remains unknown. By directly recording from mouse spermatozoa, we reveal that CatSper functions as a temperature-gated ion channel, with a thermal threshold of 33.5 °C and a temperature coefficient Q10 of 5.1. Additionally, we show that physiological levels of spermine reversibly inhibit CatSper's temperature gating, protecting against premature activation. Our findings highlight for the first time the presence of the temperature-gating modality of CatSper and reveal the protective role of spermine, a major component of seminal plasma. These results emphasize the need to maintain testes below 34 °C for optimal fertility and advance understanding of CatSper regulation in male fertility.

Serotonin (5-HT) induces sperm hyper-motility in bivalves. This process has been suggested to be associated with K+ efflux due to higher concentrations of K+ ions in testicular fluid compared to that of seawater. This hypothesis was supported by inhibition of 5-HT-induced sperm hyper-motility in artificial seawater (ASW) containing high extracellular K+ ions or in the presence of a voltage-dependent K+ channel blocker (4-AP). Here, we studied changes of sperm membrane potential to elucidate 5-HT-induced sperm hyper-motility signaling in Pacific oyster (Crassostrea gigas). Sperm motility was partially initiated (48.34 ± 7.80%) in ASW, and decreased at 5 min post-activation (p < 0.05). In the presence of 10-5 M 5-HT, sperm motility was recorded 81.63 ± 3.55%, which remained unchanged within 60 min post-activation. After sperm activation in ASW with or without 5-HT, fluorescence intensity of membrane potential-sensitive fluorescent (DiSC3(5)) was decreased to lower than that of the resting stage, indicating membrane hyperpolarization. Induction of membrane hyperpolarization, using valinomycin or in K+-free ASW (KF-ASW) could not trigger sperm hyper-motility, suggesting that hyperpolarization itself did not induce sperm hyper-motility. Next, we showed that membrane hyperpolarization was due to K+ efflux. The fluorescence intensity of DiSC3(5) was increased in ASW or KF-ASW containing 4-AP, suggesting membrane depolarization due to inhibition of K+ efflux. The valinomycin-induced membrane hyperpolarization was changed to depolarization by subsequent additions of KCl, suggesting that changes in the electrochemical gradient of K+ ions resulted in the retention of intracellular K+ ions. Observed membrane depolarization in the presence of 4-AP or high K+ ions was associated with inhibition of 5-HT-induced sperm hyper-motility. Taken together, this study shows that 5-HT-induced sperm hyper-motility was associated with membrane hyperpolarization due to K+ efflux.

Bisphenol A (BPA) is a widely used chemical that is slowly being phased out due to its toxic properties. The industry is therefore looking for alternatives in the form of BPA analogs. However, studies have shown that BPA analogs can have comparable or even stronger endocrine and toxic effects than BPA. This review describes various mechanisms and interactions of BPA analogs with individual nuclear receptors. They interfere with downstream signaling pathways not only by binding to the nuclear receptors, but also by various alternative mechanisms, such as altering receptor expression, affecting co-receptors, altering signal transduction pathways, and even epigenetic changes. Further studies are needed to fully investigate the potential synergistic and additive effects that may result. In the search for a less harmful alternative to BPA, affinity to the nuclear receptor may not be the decisive factor. We therefore recommend a different study approach to assess their effects on the endocrine system before new BPA analogs are introduced to the market to protect public health and the environment.

Androgens, such as 5α-dihydrotestosterone (5α-DHT), regulate numerous functions by binding to nuclear androgen receptors (ARs) and potential unknown membrane receptors. Here, we report that the androgen 5α-DHT activates membrane receptor GPR133 in muscle cells, thereby increasing intracellular cyclic AMP (cAMP) levels and enhancing muscle strength. Further cryoelectron microscopy (cryo-EM) structural analysis of GPR133-Gs in complex with 5α-DHT or its derivative methenolone (MET) reveals the structural basis for androgen recognition. Notably, the presence of the "Φ(F/L)2.64-F3.40-W6.53" and the "F7.42××N/D7.46" motifs, which recognize the hydrophobic steroid core and polar groups, respectively, are common in adhesion GPCRs (aGPCRs), suggesting that many aGPCRs may recognize different steroid hormones. Finally, we exploited in silico screening methods to identify a small molecule, AP503, which activates GPR133 and separates the beneficial muscle-strengthening effects from side effects mediated by AR. Thus, GPR133 represents an androgen membrane receptor that contributes to normal androgen physiology and has important therapeutic potentials.

After ejaculation, mammalian spermatozoa are not capable of fertilizing a metaphase II-arrested egg. They require to undergo a series of biochemical and physiological processes collectively known as capacitation. In all these processes, the regulation of calcium ions fluxes plays essential roles and involves participation of many channels and transporters localized in the plasma membrane as well as in the membrane of intracellular organelles. In mammalian sperm, a fraction of these molecules has been proposed to contribute to mature sperm function. However, in many cases, the evidence for the presence of a given protein is based on the use of agonists and antagonists with more than one target. In this review, we will critically analyze the published evidence supporting the presence of these molecules in mammalian sperm with special emphasis to methods involving tandem mass spectrometry identification, electrophysiological evidence and controlled immunoassays.

Most animals have male and female, whereas flowering plants are hermaphrodites. Exceptionally, a small population of invertebrates, including ascidians and nematodes, has hermaphrodite in reproductive strategies. Several ascidians exhibit strict self-sterility (or self-incompatibility), similar to flowering plants. Such a self-incompatibility mechanism in ascidian has been revealed to be very similar to those of flowering plants. Here, we describe the mechanisms of ascidian fertilization shared with invertebrates and mammals, as well as with plants. In the nematode Caenorhabditis elegans, having self-fertile hermaphrodite and male, several genes responsible for fertilization are homologous to those of mammals. Thus, novel proteins responsible for fertilization will be easily disclosed by the analyses of sterile mutants. In this review, we focus on the same or similar reproductive strategies by shedding lights on the common mechanisms of fertilization, particularly in hermaphrodites.

Calcium enters human sperm through the "Cation Channel of Sperm" (CatSper), while potassium ions exit via the sperm potassium channel (KSper). These two channels regulate intracellular calcium concentration ([Ca2+]i) and membrane potential. Our study aims to investigate and compare the contributions of these channels in capacitated sperm function.

NNC and quinidine significantly decreased progressive sperm motility (P=0.001) and reduced sperm kinematics (P=0.001). NNC but not quinidine significantly decreased sperm survival (P=0.001), reduced [Ca2+]i in live spermatozoa (P=0.05), and induced the acrosomal reaction (P=0.012).

Inhibition of KSper without effect on [Ca2+]i can inhibit sperm motility and increase mortality rate. It seems that the function of KSper is as vital as CatSper in human sperm physiology.

Progesterone (P4) secreted from the cumulus cells mediates nongenomic signalling in sperm cells and induces capacitation and acrosome reaction in the oviduct. The induction of these events in bull spermatozoa is not fully understood. Recently, we reported the presence of membrane nongenomic P4 receptors in bull spermatozoa and the involvement of P4-nongenomic signalling through the involvement of complex signalling pathways with cannabinoid receptors in the regulation of bull sperm capacitation and acrosome reaction. Here in this study, using pharmacological agents, we report the involvement of cyclic adenosine 3'5' monophosphate-protein kinase A (cAMP-PKA) and calcium channels (sperm cation channel CatSper-and L-type) in the regulation of P4-dependent nongenomic induction of bull sperm capacitation and acrosome reaction. A total of 24 semen ejaculates were used in the study, and 1 picomolar (pM) and 1 micromolar (μM) P4 concentrations were used for capacitation and acrosome reaction induction, respectively in bull spermatozoa. Further, the P4-receptor blocker mifepristone (20 μM) and several other selective blockers KH7 (cAMP, 3 μM), P9115 (PKA, 10 μM), Nifedipine (L-type, 3 μM) and NNC 55-0396 (CatSper, 3 μM) were used for the exploration of downstream P4-signalling in the regulation of bull sperm capacitation and acrosome reaction. The sperm cells showed 50%-70% inhibition and reduction of P4-response in inducing capacitation and acrosome reaction after selective inhibition of these molecules during the study. Our results from the in vitro experiments in bull sperm cells suggested the involvement of cAMP-PKA, CatSper and L-type calcium channels during P4-induced bull sperm capacitation and acrosome reaction. These results also provided new insights into P4-nongenomic signalling in bull spermatozoa in the regulation of capacitation and acrosome reaction, further strengthening our understanding of these complex events in the oviduct.

Unlocking the secrets of reproductive success in domestic animals requires a deep understanding of the molecular biology and immunology of spermatozoa, capacitation, fertilization, and conception. This review highlights the complex processes involved in spermatogenesis and sperm capacitation, including changes in membrane properties, signaling pathways, and the crucial acrosome reaction. The interaction with the zona pellucida in species-specific gamete recognition and binding is emphasized. The implications of fertilization defects for infertility and assisted reproduction are discussed, underscoring the challenges faced in breeding programs. The future directions for research in this field involve advancements in molecular techniques, understanding the immune regulation of spermatozoa, investigating environmental factors' impact, and integrating multi-omics approaches to enhance assisted reproduction techniques in domestic animals. This review contributes to our understanding of the intricate mechanisms underlying successful reproduction and provides insights into potential strategies for improving fertility outcomes in domestic animals.

Calcium signaling is a critical regulator of sperm activation and function during the processes of capacitation and fertilization. Here, we describe a combined method for calcium imaging of single, live human sperm in response to stimuli administered with a precisely targeted delivery technique. This protocol is an adaptation of techniques developed for studies of murine sperm [1, 2], and enables real-time monitoring of human sperm calcium dynamics with high spatiotemporal resolution and concurrent detection of acrosome exocytosis (AE), a functional endpoint of sperm capacitation and requirement for physiological fertilization.The described imaging technique provides a valuable tool for exploration of calcium regulation in human sperm, which is essential to answer important questions and knowledge gaps regarding the link between calcium dynamics, AE, and fertilization. The versatility of this technique can be amplified through use of various indicator dyes or integration with pharmacological strategies such as pre-treating sperm with inhibitors or activators targeting specific receptors, channels, or intracellular signaling pathways of interest. Beyond fundamental inquiries into sperm physiology, this method can also be applied to assess the impact of potential contraceptive compounds on calcium signaling, AE, and membrane integrity.

The mechanisms enabling sperm to locate unfertilized eggs within the fallopian tubes remain a subject of debate in reproductive biology. Previous studies using polytocous mammals observed a 1:1 sperm-egg ratio within the ampulla at the time of fertilization. From these observations, it is hypothesized that this mechanism could be linked to sperm-egg fusion, such that unfertilized eggs may attract sperm until fusion occurs, whereupon the attraction ceases.

To test this, fertile male mice were mated with infertile homozygous Cd9Null females, whose eggs cannot fuse with sperm, leading to the accumulation of supernumerary sperm in the perivitelline space. Fertile heterozygous Cd9Het females, were used as controls.

The results revealed that both Cd9Null and Cd9Het females ovulated similar numbers of eggs (6.53 ± 0.61 vs. 5.50 ± 0.53 eggs/ampulla). The majority of eggs produced by Cd9Het females were fertilized by one single sperm, without any additional sperm found bound to the zona or within the perivitelline space. In contrast, most of the eggs ovulated by Cd9Null females either showed an accumulation of supernumerary sperm within in the perivitelline space or showed no sperm bound to the zona nor present within the perivitelline space.

These findings indicate that genetic ablation of Cd9 leads to an imbalance in the 1:1 sperm-egg ratio observed within the ampulla. This information may set the foundation for future studies with the aim to identify the specific mechanisms that sperm use to locate unfertilized eggs and whether they become ineffective when gamete fusion is prevented.

A strong link between antipsychotic drug use and reduced human sperm quality has been reported. Trifluoperazine (TFP), a commonly used antipsychotic, is now being explored for anticancer applications. Although there are hints that TFP might affect the male reproductive system, its impact on human sperm quality remains uncertain. Using a human sperm and TFP in vitro coculture system, we examined the effect of TFP (12.5, 25, 50 and 100 μM) on human sperm function and physiological parameters. The results showed that 50 μM and 100 μM TFP induced the accumulation of reactive oxygen species (ROS) and a decrease in the mitochondrial membrane potential (MMP) of human sperm, leading to decreased sperm viability, while 25 μM TFP inhibited only the penetration ability, total sperm motility, and progressive motility. Although 12.5 μM and 25 μM TFP increased [Ca2+]i in human sperm, they did not affect capacitation or the acrosome reaction. These results may be explained by the observation that 12.5 μM and 25 μM TFP did not increase tyrosine phosphorylation in human sperm, although TFP increased [Ca2+]i in a time-course traces similar to that of progesterone. Our results indicated that TFP could cause male reproductive toxicity by inducing the accumulation of ROS and a decrease in the MMP in human sperm.

Sperm-specific potassium channel (KSper) comprised of pore-forming subunit SLO3 and auxiliary subunit LRRC52 is of importance for sperm fertility. The deficiency of KSper in both mice and humans resulted in severe impairments of sperm functions including sperm hyperactivity and acrosome reaction. Previous reports suggested that mouse KSper modulated sperm function possibly by affecting sperm intracellular pH (pHi). However, the precise signaling mechanism of human KSper (hKSper) on the regulation of sperm functions was largely unclear.

To explore the regulatory role of hKSper on sperm flagellar pHi.

More than 50 sperm donors were recruited during a period of 1 year. As reported in our previous work, we quantitatively measured flagellar pHi by employing a single-cell pH fluorescent recording on human spermatozoa loaded with pH indicator pHrodo Red. Three different hKSper antagonists including clofilium, quinidine, and a polyclonal antibody of LRRC52 (LID1) were utilized to evaluate the effect of hKSper inhibition on sperm flagellar pHi.

Given the predominant role of hKSper on the regulation of membrane potential (Em), we first detected a considerable depolarization (about 25-30 mV) of Em evoked by clofilium and quinidine. Subsequently, it was shown that flagellar pHi values of human spermatozoa were significantly decreased by the treatment of clofilium (50 µM, from 7.13 ± 0.11 to 6.43 ± 0.12), quinidine (500 µM, from 7.00 ± 0.11 to 6.64 ± 0.08) and LID1 (20 µg/mL, from 6.98 ± 0.16 to 6.67 ± 0.22). Moreover, we found that when human spermatozoa were pre-incubated with a high K+ solution (135 mM), both the depolarization of Em and the acidification of flagellar pHi evoked by clofilium and quinidine were abolished. In addition, we found that extracellular substitution of N-methyl-D-glucamine for Na+ abolished pHi acidification induced by hKSper inhibition.

Our results demonstrate that hKSper inhibition evokes flagellar pHi acidification of human spermatozoa, suggesting that flagellar pHi maintenance is an important signaling mechanism of hKSper on the regulation of sperm functions.

Long-chain polyunsaturated fatty acids (LC-PUFA) like arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3), and docosahexaenoic acid (DHA, 22:6n-3) constitute one-third to half of fish sperm lipids. Fish sperm is rich in phospholipid (PL)-primarily phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin. DHA is generally the most abundant LC-PUFA in each PL class, followed by competition between ARA and EPA. While the total n-6: n-3 PUFA ratio does not correlate significantly with sperm biomechanics, LC-PUFA do. DHA positively influences sperm biomechanics, while ARA and EPA may be negatively associated. Fish sperm maintains lower (≤1) total n-6 PUFA per unit of n-3 PUFA but keep a higher (>1) ARA per unit EPA. A weak dietary influence on sperm EPA and DHA exists but not on ARA. The DHA: EPA ratio in fish sperm is often >1, though values <1 occur. Certain species cannot fortify DHA sufficiently during spermatogenesis, diverging through whole genome duplications. Fish sperm can show ARA: EPA ratios greater or less than 1, due to shifts in prostaglandin pathways in different evolutionary eras. DHA-rich PL bilayers provide unique packing and fusogenic properties, with ARA/EPA-derived eicosanoids guiding sperm rheotaxis/chemotaxis, modulated by DHA-derived resolvins. Docosapentaenoic acid (DPA, 22:5n-3) sometimes substitutes for DHA in fish sperm.

Cryopreservation of porcine spermatozoa is detrimental due to their high sensitivity to cold shock, leading to changes akin to capacitation, known as cryocapacitation. These changes, including the acrosomal reaction, hypermotility induction, and protein phosphorylation, might be influenced by the presence of progesterone in seminal plasma and egg yolk, used in most freezing extenders. We tested the effect of various progesterone concentrations added to the freezing extenders (1, 10, and 100 μg/mL). At 100 μg/mL, progesterone decreased the proportion of straightness and tended to reduce viability and the proportion of progressive motility (p < 0.1). At 10 μg/mL, it increased reacted acrosomes in dead sperm (p < 0.05), protein phosphorylation rate (p < 0.05), and tended (p < 0.1) to enhance linear movement compared to the control. To counteract the capacitating effect of progesterone, we examined the effect of antiprogesterone mifepristone (RU 486) at concentrations of 5, 10, 20, 50, 100, and 200 μM, and co-incubated 10 μM of RU 486 with 10 μg/mL of progesterone. RU 486 maintained capacitation levels and motility parameters similar to the control, although high concentrations (100 μM) tended (p = 0.152) to increase protein phosphorylation. Co-incubation reduced the acrosome reaction in dead sperm, and RU 486 appeared to prevent hypermotility stabilizing motility and viability parameters compared to samples with progesterone alone. Protein phosphorylation increased and RU 486 could not restore capacitation to control levels due to its competitive antagonism for progesterone receptors, having less affinity than progesterone, which displaces RU 486 at high concentrations, allowing normal sperm capacitation.

Mammalian sperm are characterized as specialized cells, as their transcriptional and translational processes are largely inactive. Emerging researches indicate that Ca2+ serves as a crucial second messenger in the modulation of various sperm physiological processes, such as capacitation, hyperactivation, and the acrosome reaction. Specifically, sperm-specific calcium channels, including CatSper, voltage-gated calcium channels (VGCCs), store-operated calcium channels (SOCCs), and cyclic nucleotide-gated (CNG) channels, are implicated in the regulation of calcium signaling in mammalian sperm. Calcium stores located in the sperm acrosomes, along with the IP3 receptors in the neck of the redundant nuclear envelope and the mitochondria in the tail, play significant roles in modulating intracellular Ca2+ levels in sperm. However, the functions and mechanisms of these calcium channels in modulating mammalian sperm physiological functions have not yet been well elucidated. Therefore, by focusing on humans and mice, this study aims to provide a comprehensive review of the current advancements in research regarding the roles of calcium signaling and associated calcium channels in regulating sperm function. This endeavor seeks to enhance the understanding of calcium signaling in sperm regulation and to facilitate the development of drugs for the treatment of infertility or as non-hormonal male contraceptives.

It has long been thought that exocytosis was driven exclusively by well-studied fusion proteins. Some decades ago, the role of lipids became evident and escalated interest in the field. Our laboratory chose a particular cell to face this issue: the human sperm. What makes this cell special? Sperm, as terminal cells, are characterized by their scarcity of organelles and the complete absence of transcriptional and translational activities. They are specialized for a singular membrane fusion occurrence: the exocytosis of the acrosome. This unique trait makes them invaluable for the study of exocytosis in isolation. We will discuss the lipids' role in human sperm acrosome exocytosis from various perspectives, with a primary emphasis on our contributions to the field. Sperm cells have a unique lipid composition, very rare and not observed in many cell types, comprising a high content of plasmalogens, long-chain, and very-long-chain polyunsaturated fatty acids that are particular constituents of some sphingolipids. This review endeavors to unravel the impact of membrane lipid composition on the proper functioning of the exocytic pathway in human sperm and how this lipid dynamic influences its fertilizing capability. Evidence from our and other laboratories allowed unveiling the role and importance of multiple lipids that drive exocytosis. This review highlights the role of cholesterol, diacylglycerol, and particular phospholipids like phosphatidic acid, phosphatidylinositol 4,5-bisphosphate, and sphingolipids in driving sperm acrosome exocytosis. Furthermore, we provide a comprehensive overview of the factors and enzymes that regulate lipid turnover during the exocytic course. A more thorough grasp of the role played by lipids transferred from sperm can provide insights into certain causes of male infertility. It may lead to enhancements in diagnosing infertility and techniques like assisted reproductive technology (ART).

To locate and fertilize the egg, sperm probe the varying microenvironment prevailing at different stages during their journey across the female genital tract. To this end, they are equipped with a unique repertoire of mostly sperm-specific proteins. In particular, the flagellar Ca2+ channel CatSper has come into focus as a polymodal sensor used by human sperm to register ligands released into the female genital tract. Here, we provide the first comprehensive study on the pharmacology of the sperm-specific human Slo3 channel, shedding light on its modulation by reproductive fluids and their constituents. We show that seminal fluid and contained prostaglandins and Zn2+ do not affect the channel, whereas human Slo3 is inhibited in a non-genomic fashion by diverse steroids as well as by albumin, which are released into the oviduct along with the egg. This indicates that not only CatSper but also Slo3 harbours promiscuous ligand-binding sites that can accommodate structurally diverse molecules, suggesting that Slo3 is involved in chemosensory signalling in human sperm.

Progesterone (P4) acts as a key conserved signalling molecule in vertebrate reproduction. P4 is especially important for mature sperm physiology and subsequent reproductive success. "CatSpermasome", a multi-unit molecular complex, has been suggested to be the main if not the only P4-responsive atypical Ca2+-ion channel present in mature sperm. Altogether, here we analyse the protein sequences of CatSper1-4 from more than 500 vertebrates ranging from early fishes to humans. CatSper1 becomes longer in mammals due to sequence gain mainly at the N-terminus. Overall the conservation of full-length CatSper1-4 as well as the individual TM regions remain low. The lipid-water-interface residues (i.e. a 5 amino acid stretch sequence present on both sides of each TM region) also remain highly diverged. No specific patterns of amino acid distributions were observed. The total frequency of positively charged, negatively charged or their ratios do not follow in any specific pattern. Similarly, the frequency of total hydrophobic, total hydrophilic residues or even their ratios remain random and do not follow any specific pattern. We noted that the CatSper1-4 genes are missing in amphibians and the CatSper1 gene is missing in birds. The high variability of CatSper1-4 and gene-loss in certain clades indicate that the "CatSpermasome" is not the only P4-responsive ion channel. Data indicate that the molecular evolution of CatSper is mostly guided by diverse hydrophobic ligands rather than only P4. The comparative data also suggest possibilities of other Ca2+-channel/s in vertebrate sperm that can also respond to P4.

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.

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.

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.

The viscoelastic properties of the female reproductive tract influence sperm swimming behavior, but the exact role of these rheological changes in regulating sperm energetics remains unknown. Using high-speed dark-field microscopy, the flagellar dynamics of free-swimming sperm across a physiologically relevant range of viscosities is resolved. A transition from 3D to 2D slither swimming under an increased viscous loading is revealed, in the absence of any geometrical or chemical stimuli. This transition is species-specific, aligning with viscosity variations within each species' reproductive tract. Despite substantial drag increase, 2D slithering sperm maintain a steady swimming speed across a wide viscosity range (20-250 and 75-1000 mPa s for bull and human sperm) by dissipating over sixfold more energy into the fluid without elevating metabolic activity, potentially by altering the mechanisms of dynein motor activity. This energy-efficient motility mode is ideally suited for the viscous environment of the female reproductive tract.

Infertility is a growing health concern among many couples worldwide. Men account for half of infertility cases. CatSper, a sperm-specific Ca2+ channel, is expressed on the cell membrane of mammalian sperm. CatSper plays an important role in male fertility because it facilitates the entry of Ca2+ necessary for the rapid change in sperm motility, thereby allowing it to navigate the hurdles of the female reproductive tract and successfully locate the egg. Many pollutants present in the environment have been shown to affect the functions of CatSper and sperm, which is a matter of capital importance to understanding and solving male infertility issues. Environmental pollutants can act as partial agonists or inhibitors of CatSper or exhibit a synergistic effect. In this article, we briefly describe the structure, functions, and regulatory mechanisms of CatSper, and discuss the body of literature covering the effects of environmental pollutants on CatSper.

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.

Successful male fertilization requires the main processes such as normal spermatogenesis, sperm capacitation, hyperactivation, and acrosome reaction. The progress of these processes depends on some endogenous and exogenous factors. So, the optimal level of ions and essential and rare elements such as selenium, zinc, copper, iron, manganese, calcium, and so on in various types of cells of the reproductive system could affect conception and male fertility rates. The function of trace elements in the male reproductive system could be exerted through some cellular and molecular processes, such as the management of active oxygen species, involvement in the action of membrane channels, regulation of enzyme activity, regulation of gene expression and hormone levels, and modulation of signaling cascades. In this review, we aim to summarize the available evidence on the role of trace elements in improving male reproductive performance. Also, special attention is paid to the cellular aspects and the involved molecular signaling cascades.

In female tract, mammalian sperm develop hyperactivated motility which is a key physiological event for sperm to fertilize eggs. This motility change is triggered by Ca2+ influx via the sperm-specific Ca2+ channel, CatSper. Although previous studies in human and mice largely contributed to understanding CatSper and Ca2+ signaling for sperm hyperactivation, the differences on their activation mechanisms are not well understood yet. There are several studies to examine expression and significance of the CatSper channel in non-human and non-mouse models, such as domestic animals. In this review, I summarize key knowledge for the CatSper channel from previous studies and propose future aspects for CatSper study using sperm from domestic animals.

Ca2+ is a key secondary messenger that determines sperm motility patterns. Mammalian sperm undergo capacitation, a process to acquire fertilizing ability, in the female reproductive tract. Capacitated sperm change their flagellar waveform to develop hyperactivated motility, which is crucial for successful sperm navigation to the eggs and fertilization. The sperm-specific channel, CATSPER, and an ATPase transporter, PMCA4, serve as major paths for Ca2+ influx and efflux, respectively, in sperm. The ionic paths coordinate Ca2+ homeostasis in the sperm, and their loss-of-function impairs sperm motility, to cause male infertility. In this review, we summarize the physiological significance of these two Ca2+ gates and suggest their potential applications in novel male contraceptives.

Progesterone (P) and 17β-estradiol (Eβ) form the well-known hormone pair that regulates sperm capacitation. Here, we examined the regulatory effects of P and Eβ on sperm hyperactivation in mice and evaluated the in vitro fertilization (IVF) success. Although P enhanced hyperactivation, Eβ dose-dependently suppressed the P-enhanced hyperactivation. Moreover, P increased IVF success, whereas Eβ suppressed the P-induced increase in IVF success in a dose-dependent manner. Thus, P and Eβ competitively regulate hyperactivation and IVF success in mice. Since P and Eβ concentrations generally change during the estrous cycle, sperm are speculated to capacitate in response to the oviductal environment and fertilize the oocyte.

Whether and how do Na+/H+ exchangers (NHEs) regulate the physiological functions of human sperm?

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.

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.

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.

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.

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.

N/A.

This was an in vitro study. Caution should be taken when extrapolating these results to in vivo applications.

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.

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.

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.

Exposure to bisphenols has been found to have adverse effects on male reproductive function in animals. Human exposure to bisphenols is widespread. Bisphenol A (BPA) and its analogues, including bisphenol S (BPS), bisphenol F (BPF), and bisphenol AF (BPAF) are utilized in various consumer products such as food contact materials and dental resins. The effects of these compounds on male fertility and spermatogenesis are unclear and findings from human studies are inconsistent. In this cross-sectional study, we evaluated the influence of BPA, BPS, BPF, BPAF (BPs) measured in semen on number of spermatozoa, total motility, progressive motility, morphology, and DNA fragmentation. We also examined the association of bisphenols (BPs) exposure with patients' occupation. A total of 358 patients aged 17-62 years with BMI 18-42 were included in the study from 2019 to 2021. BPs were extracted using solvent extraction followed by preconcentration step and determined by high-performance liquid chromatography and tandem mass spectrometry (LC/MSMS). Bisphenols were detected in 343 from 349 analysed samples (98.3% of all the samples). In 6 samples, the concentration of all BPs was under the limit of detection and in 20 samples under the limit of quantification. We did not find a statistically significant relationship between occupation and BPs. However, we observed significant correlations between the concentration of BPA and a lower motility and normal morphology. For BPS, a significant correlation with a lower ejaculate volume and a lower total sperm count was found. BPF and BPAF were detected only in 14.3% and 23.9% of samples, respectively. For BPF and BPAF, no significant correlations with spermiogram parameters were observed. Our results show that BPs are widespread in the male population (more than 90% of analysed samples), independently of an occupation and in case of BPA and BPS having a negative impact on spermiogram parameters.

Hormones mediate long-range cell communication and play vital roles in physiology, metabolism, and health. Traditionally, endocrinologists have focused on one hormone or organ system at a time. Yet, hormone signaling by its very nature connects cells of different organs and involves crosstalk of different hormones. Here, we leverage the organism-wide single cell transcriptional atlas of a non-human primate, the mouse lemur (Microcebus murinus), to systematically map source and target cells for 84 classes of hormones. This work uncovers previously-uncharacterized sites of hormone regulation, and shows that the hormonal signaling network is densely connected, decentralized, and rich in feedback loops. Evolutionary comparisons of hormonal genes and their expression patterns show that mouse lemur better models human hormonal signaling than mouse, at both the genomic and transcriptomic levels, and reveal primate-specific rewiring of hormone-producing/target cells. This work complements the scale and resolution of classical endocrine studies and sheds light on primate hormone regulation.

Uterine leiomyomas (ULs) are the most common benign smooth muscle cell steroid-dependent tumors that occur in women of reproductive age. Progesterone (P4) is a major hormone that promotes the ULs development and growth. P4 action in ULs is mediated mainly by its nuclear progesterone receptors (PGRs), although rapid non-genomic responses have also been observed. Data on the membrane progesterone receptors (mPRs) regulated signaling pathways in ULs in the available literature is still very limited. One of the essential characteristics of ULs is the excessive production of extracellular matrix (ECM). P4 has been shown to stimulate ECM production and collagen synthesis in ULs. Recent research demonstrated that, despite their benign nature, ULs may present with abnormal vasculature. P4 has been shown to regulate angiogenesis in ULs through the upregulation of vascular endothelial growth factor (VEGF) and by controlling the secretion of permeability factors. This review summarizes the key findings regarding the role of PGRs and mPRs in ULs, especially highlighting the potential ECM and angiogenesis modulation by P4. An increased understanding of this mechanistic role of nuclear and specifically mPRs in the biology of P4-modulated ECM and angiogenesis in the growth of ULs could turn out to be fundamental for developing effective targeted therapies for ULs.

Intracellular calcium homeostasis plays a crucial role in spermatozoa by regulating physiological functions associated with sperm quality and male fertility potential. Intracellular calcium fine balance in the sperm cytoplasm is strictly dependent on sperm surface channels including the CatSper channel. CatSpers' role is to ensure the influx of extracellular calcium, while intracellular pH alkalinization serves as a stimulus for the activation of several channels, including CatSper. Overall, the generation of intracellular calcium spikes through CatSper is essential for fertilization-related processes, such as sperm hyperactivation, acrosome reaction, egg chemotaxis, and zona pellucida penetration. Multiple lines of evidence suggest that disruption in the close interaction among ions, pH, and CatSper could impair male fertility potential. In contemporary times, the growing reliance on Medically Assisted Reproduction procedures underscores the impact of cryopreservation on gametes. In fact, a large body of literature raises concerns about the cryo-damages provoked by the freeze-thawing processes, that can affect the plasma membrane integrity, thus the structure of pivotal ion channels, and the fine regulation of both intracellular calcium and pH. This review aims to provide an overview of the importance of the CatSper channel in sperm quality and further fertilization potential. Additionally, it addresses the emerging issue of cryopreservation's impact on the functionality of this sperm channel.

Ca2+ is a key secondary messenger that modulates sperm motility by tuning flagellar movement in various species. The sperm-specific Ca2+ channel, CatSper, is a primary Ca2+ gate that is essential for male fertility in mammals. CatSper-mediated Ca2+ signaling enables sperm to develop hyperactivated motility and fertilize the eggs in the female tract. Therefore, altered CatSper function compromises the entry of Ca2+ into the sperm, followed by impairing hyperactivation and male fertility. However, methods to evaluate the function of the CatSper channel are limited to patch clamping and functional imaging using Ca2+ dye. Previous studies have revealed that various parameters for sperm motility are highly correlated with intracellular Ca2+ levels in mouse. Here, I cover a step-by-step protocol to analyze the change in Ca2+-mediated sperm motility by using computer-assisted semen analysis (CASA) to evaluate the functional normality of the CatSper channel in sperm. This approach analyzes sperm motility parameters during intracellular Ca2+ chelation followed by in vitro capacitation to recover intracellular Ca2+ via the activated CatSper channel. Thus, this Ca2+-handling method is handy and could be broadly applied in reproductive biology labs and clinics that have CASA equipment to examine the functional normality of the CatSper channel.

The field of aptamer-based sensing has evolved considerably over the past three decades. The aptamer sensor-based detection of small-molecule targets in body fluids is designed for real-time or rapid, low-cost, non- or minimally invasive tracking and diagnosis of human health status. It can be achieved by specifically monitoring biomarkers or metabolites excreted from various body fluids, including blood, urine, cerebrospinal fluid, saliva, ect. This article reviews a comprehensive collection of aptamer-based sensors for detecting small-molecule in various body fluids. A comparative analysis of aptamer features, emerging chemistry, advanced sensing materials, transduction techniques, and detection performance is conducted, and the strengths and pitfalls of each approach are discussed. Finally, the development process and application challenges of aptamer-based sensors in the detection of small-molecule in body fluids are presented and discussed.

Type 2 taste receptors (TAS2Rs), traditionally known for their role in bitter taste perception, are present in diverse reproductive tissues of both sexes. This review explores our current understanding of TAS2R functions with a particular focus on reproductive health. In males, TAS2Rs are believed to play potential roles in processes such as sperm chemotaxis and male fertility. Genetic insights from mouse models and human polymorphism studies provide some evidence for their contribution to male infertility. In female reproduction, it is speculated that TAS2Rs influence the ovarian milieu, shaping the functions of granulosa and cumulus cells and their interactions with oocytes. In the uterus, TAS2Rs contribute to uterine relaxation and hold potential as therapeutic targets for preventing preterm birth. In the placenta, they are proposed to function as vigilant sentinels, responding to infection and potentially modulating mechanisms of fetal protection. In the cervix and vagina, their analogous functions to those in other extraoral tissues suggest a potential role in infection defense. In addition, TAS2Rs exhibit altered expression patterns that profoundly affect cancer cell proliferation and apoptosis in reproductive cancers. Notably, TAS2R agonists show promise in inducing apoptosis and overcoming chemoresistance in these malignancies. Despite these advances, challenges remain, including a lack of genetic and functional studies. The application of techniques such as single-cell RNA sequencing and clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated endonuclease 9 gene editing could provide deeper insights into TAS2Rs in reproduction, paving the way for novel therapeutic strategies for reproductive disorders.

Embryonic development and adult physiology are dependent on the action of steroid hormones. In particular, the reproductive system is reliant on hormonal signaling to promote gonadal function and to ensure fertility. Here we will describe hormone receptor functions and their impacts on testicular function, focusing on a specific group of essential hormones: androgens, estrogens, progesterone, cortisol, and aldosterone. In addition to focusing on hormone receptor function and localization within the testis, we will highlight the effects of altered receptor signaling, including the consequences of reduced and excess signaling activity. These hormones act through various cellular pathways and receptor types, emphasizing the need for a multifaceted research approach to understand their critical roles in testicular function. Hormones exhibit intricate interactions with each other, as evidenced, for example, by the antagonistic effects of progesterone on mineralocorticoid receptors and cortisol's impact on androgens. In light of research findings in the field demonstrating an intricate interplay between hormones, a systems biology approach is crucial for a nuanced understanding of this complex hormonal network. This review can serve as a resource for further investigation into hormonal support of male reproductive health.

The infertility of many couples rests on an enigmatic dysfunction of the man's sperm. To gain insight into the underlying pathomechanisms, we assessed the function of the sperm-specific multisubunit CatSper-channel complex in the sperm of almost 2,300 men undergoing a fertility workup, using a simple motility-based test. We identified a group of men with normal semen parameters but defective CatSper function. These men or couples failed to conceive naturally and upon medically assisted reproduction via intrauterine insemination and in vitro fertilization. Intracytoplasmic sperm injection (ICSI) was, ultimately, required to conceive a child. We revealed that the defective CatSper function was caused by variations in CATSPER genes. Moreover, we unveiled that CatSper-deficient human sperm were unable to undergo hyperactive motility and, therefore, failed to penetrate the egg coat. Thus, our study provides the experimental evidence that sperm hyperactivation is required for human fertilization, explaining the infertility of CatSper-deficient men and the need of ICSI for medically assisted reproduction. Finally, our study also revealed that defective CatSper function and ensuing failure to hyperactivate represents the most common cause of unexplained male infertility known thus far and that this sperm channelopathy can readily be diagnosed, enabling future evidence-based treatment of affected couples.

The progesterone (P4) secreted by cumulus cells has gained attention for its role as a possible physiological inducer of sperm acrosome exocytosis. In mammals, it is generally accepted that fertilization rates of oocytes without cumulus are markedly low. This study assessed the integrity of capacitated bovine sperm acrosome when exposed to increasing concentrations of P4, and evaluated whether exogenous P4 during in vitro fertilization (IVF) increases the developmental competence of partially cumulus-denuded oocytes in serum-free conditions. After a 4-h capacitation induction, sperm were incubated with increasing concentrations of P4 (0, 0.1, 10 and 100 μM) and evaluated for viability, caspase activation and acrosome status at three different times (4, 5, and 22 h), including the capacitation induction period. Progesterone induced sperm acrosomal exocytosis without compromising sperm viability or activating sperm caspases. Sperm undergoing acrosome reaction exhibited three differential Concanavalin A patterns, corresponding to early, intermediate and late acrosomal exocytosis. The percentage of these patterns significantly increased over time, regardless of P4 concentration, except for those spermatozoa with late acrosomal exocytosis, which only showed an increase at 22 h of incubation. After incubation for 1 h with 100 μM P4, spermatozoa showing intermediate acrosomal exocytosis significantly increased. At 22 h of incubation, the pattern corresponding to early acrosomal exocytosis evidenced a dose-dependent increase. However, prematurely high levels of acrosome reaction induced by 100 μM P4 led to inefficient IVF outcomes (P < 0.05). Therefore, IVF trials with partially cumulus-denuded oocytes were carried out with lower P4 concentrations (0, 0.1, 1, 5, 10 μM). Cleavage rate significantly increased at 1 μM P4, which translated to increased total embryo production after 7 days of in vitro culture (P < 0.05). Significantly higher percentages of expanded blastocysts were observed at both 1 μM and 10 μM P4 as compared to the other experimental conditions. In conclusion, the different patterns of acrosomal exocytosis identified over time by incubation of live sperm with a fluorescent lectin revealed the existence of sperm subpopulations heterogeneous in their physiological states. Moreover, exogenous P4 at 1 μM during IVF improved the developmental competence of partially cumulus-denuded oocytes in serum-free conditions.

Progesterone (P) is a well-known enhancer of hyperactivation which is associated with the success of in vitro fertilization (IVF). In this study, we examined whether P-enhanced hyperactivation affected IVF success in rats. When rat spermatozoa were exposed to 10, 20, and 40 ng/ml P, 20 ng/ml P enhanced hyperactivation via the membrane progesterone receptor. In addition, the enhancement of hyperactivation by 20 ng/ml P was regulated by phospholipase C, transmembrane adenylate cyclase, and protein kinase A. However, 20 ng/ml P did not affect IVF success. These results suggest that 20 ng/ml P enhances rat spermatozoal hyperactivation through non-genomic pathways. Because the concentration of P changes during the estrous cycle, it seems that rat spermatozoa are hyperactivated in response to the oviductal environment. However, the effect of 20 ng/ml P does not seem to fully capacitate spermatozoa.

Na+/H+ exchangers (NHEs) are known to be important regulators of pH in multiple intracellular compartments of eukaryotic cells. Sperm function is especially dependent on changes in pH and thus it has been postulated that NHEs play important roles in regulating the intracellular pH of these cells. For example, in order to achieve fertilization, mature sperm must maintain a basal pH in the male reproductive tract and then alkalize in response to specific signals in the female reproductive tract during the capacitation process. Eight NHE isoforms are expressed in mammalian testis/sperm: NHE1, NHE3, NHE5, NHE8, NHA1, NHA2, NHE10, and NHE11. These NHE isoforms are expressed at varying times during spermatogenesis and localize to different subcellular structures in developing and mature sperm where they contribute to multiple aspects of sperm physiology and male fertility including proper sperm development/morphogenesis, motility, capacitation, and the acrosome reaction. Previous work has provided evidence for NHE3, NHE8, NHA1, NHA2, and NHE10 being critical for male fertility in mice and NHE10 has recently been shown to be essential for male fertility in humans. In this article we review what is known about each NHE isoform expressed in mammalian sperm and discuss the physiological significance of each NHE isoform with respect to male fertility.

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.