Search Results (21)

Microfluidic platforms have emerged as critical technologies for exploring sperm chemotaxis, providing precise gradient control, and facilitating in-depth behavioral assessment. We designed a novel, user-friendly microfluidic device that is optimized for human sperm morphology and motility. The device was validated using two well-established sperm chemoattractants, progesterone and bourgeonal, demonstrating its reliability and reproducibility. Given the key role of olfactory receptors (ORs) in mediating sperm chemotaxis, the newly developed device was employed to identify additional receptors that may contribute to sperm behavior. Using the Atlas database, we identified OR2H1 as a candidate receptor. It is enriched in testis-derived cells, particularly in early and late spermatids, and it is broadly expressed across human spermatozoa. We demonstrated that OR2H1’s ligand, methional, a sulfur-containing aldehyde naturally found in vaginal fluid and biosynthesized by Lactococcus lactis, significantly enhances sperm migration and progressive motility. Methional stimulation also triggered increased intracellular calcium levels, indicating receptor activation. Computer-assisted sperm analysis revealed that methional treatment improved sperm linearity, straightness, and wobble without affecting the average velocity, suggesting enhanced directional movement. These findings provide evidence that methional promotes sperm chemotaxis via OR2H1 and highlight the potential role of the vaginal microbiome in influencing human fertility. Full article

Bisphenol A (BPA), a typical endocrine disruptor, is known to have various adverse effects on the male reproductive system. However, the toxic effects and mechanisms of low-dose BPA have not yet been fully explored. In this study, male Kunming mice were orally administered low-dose BPA (0.03, 0.3 and 3 mg/kg/d) for ten consecutive weeks. Pathological sections of testicular tissue showed no significant morphological differences after BPA exposure. An analysis of the functional parameters of sperm revealed that exposure to low-dose BPA significantly decreased sperm motility, chemotaxis, and the acrosome reaction. An in vitro BPA exposure model combined with an omics data analysis showed that the olfactory receptor-related pathway was significantly enriched after BPA treatment. Subsequent experiments verified the reduced mRNA level of a novel olfactory receptor gene, Olfr25, in vivo and in vitro exposure models. Meanwhile, exposure to low-dose BPA reduced the intracellular calcium ion concentration and the mRNA levels of pore-forming subunits of the CatSper channel in sperm. Importantly, the knockdown of Olfr25 inhibited calcium ion levels and CatSper subunit expression in GC-2 cells. Olfr25 overexpression attenuated the BPA-induced downregulation of CatSper subunit expression in GC-2 cells. These findings indicate that Olfr25 might participate in low-dose BPA-induced sperm dysfunction by affecting the CatSper-Ca²⁺ signaling pathway. This study reveals a new mechanism underlying the effects of low-dose BPA on sperm function and provides a reference for assessing the safety of low-dose BPA exposure. Full article

Flagellar motility in sperm is activated and regulated by factors related to the eggs at fertilization. In the ascidian Ciona intestinalis, a sulfated steroid called the SAAF (sperm activating and attracting factor) induces both sperm motility activation and chemotaxis. Cyclic AMP (cAMP) is one of the most important intracellular factors in the sperm signaling pathway. Adenylyl cyclase (AC) is the key enzyme that synthesizes cAMP at the onset of the signaling pathway in all cellular functions. We previously reported that both transmembrane AC (tmAC) and soluble AC (sAC) play important roles in sperm motility in Ciona. The tmAC plays a major role in the SAAF-induced activation of sperm motility. On the other hand, sAC is involved in the regulation of flagellar beat frequency and the Ca²⁺-dependent chemotactic movement of sperm. In this study, we focused on the role of sAC in the regulation of flagellar motility in Ciona sperm chemotaxis. The immunochemical analysis revealed that several isoforms of sAC protein were expressed in Ciona sperm, as reported in mammals and sea urchins. We demonstrated that sAC inhibition caused strong and transient asymmetrization during the chemotactic turn, and then sperm failed to turn toward the SAAF. In addition, real-time Ca²⁺ imaging in sperm flagella revealed that sAC inhibition induced an excessive and prolonged Ca²⁺ influx to flagella. These results indicate that sAC plays a key role in sperm chemotaxis by regulating the clearance of [Ca²⁺]_i and by modulating Ca²⁺-dependent flagellar waveform conversion. Full article

Olfactory receptors (ORs), key components in ensuring the detection of myriad odorants, are expressed not only on the surface of olfactory neurons but also in many other tissues. In the case of ORs expressed at the sperm membrane, in vitro experiments with human and mouse spermatozoids have shown that they move toward the regions with the highest concentration of bourgeonal and lyral, respectively. However, to date, no in vivo experiment has shown any biological function of these ORs. To demonstrate a possible role in vivo of ORs in sperm chemotaxis, we overloaded the vaginal space of female mice from the prolific Swiss CD1 strain with lyral to induce competition with the supposed natural ligand and to prevent its detection. As shown, the mice that received lyral had much fewer newborns than the control mice treated with PBS, showing that lyral has a strong negative impact on procreation. This indicates that the ORs at the sperm surface are biologically active and make an important contribution to reproduction. Control experiments performed with hexanal, which does not alter sperm movement in vitro, indicate that the inhibition of reproduction observed was specific to lyral. In addition, we show that males are attracted to the smell of lyral, which acts as a pheromone, and prefer to copulate with mice marked on their back with lyral rather than with those that have not been marked. These results suggest an explanation for some cases of human infertility, which could result from an absence of recognition between the natural ligand and the ORs, either due to a mutation or a lack of expression from one of the two partners, allowing for the development of a diagnostic tests. These results might also lead to the development of a novel contraception strategy based on the use of vaginal tablets delivering an odorant or a drug that competes with the natural ligand. Full article

Abhydrolase domain containing 2-acylglycerol lipase (ABHD2) was recently claimed as the membrane receptor of progesterone (P4) in sperm cells, mediating cell processes such as sperm chemotaxis and acrosome reaction. Here, we investigated the role of membrane cholesterol (Chol) on ABHD2-mediated human sperm chemotaxis. Human sperm cells were obtained from twelve normozoospemic healthy donors. ABHD2–Chol interaction was modelled by computational molecular-modelling (MM). Sperm membrane Chol content was depleted by incubating cells with cyclodextrin (CD) or augmented by the incubation with the complex between CD and Chol (CD:Chol). Cell Chol levels were quantified by liquid chromatography-mass spectrometry. Sperm migration upon P4 gradient was evaluated through the accumulation assay in a specific migration device. Motility parameters were evaluated by sperm class analyzer, whilst intracellular calcium concentration, acrosome reaction and mitochondrial membrane potential were evaluated with calcium orange, FITC-conjugated anti-CD46 antibody and JC-1 fluorescent probes, respectively. MM analysis showed the possible stable binding Chol to ABHD2, resulting in to major impact on the protein backbone flexibility. The treatment with CD was associated with a dose-dependent increase in sperm migration in a 160 nM P4 gradient, together with increase in sperm motility parameters and levels of acrosome reaction. The treatment with CD:Chol was associated with essentially opposite effects. Chol was, thus, suggested to inhibit P4-mediated sperm function through the possible inhibition of ABHD2. Full article

In Phlebobranchiata ascidians, oocytes and spermatozoa are stored in the oviduct and spermiduct, respectively, until spawning occurs. Gametes in the gonoducts are mature and fertilizable; however, it was found that the gametes of the ascidians Phallusia philippinensis and Ciona intestinalis could not undergo fertilization in the gonoductal fluids. The body fluids of the ascidians, especially in the gonoducts, were much more acidic (pH 5.5–6.8) than seawater (pH 8.2), and the fertilization rate was low under such acidic conditions. Hence, we examined the effect of pH on gametes. Pre-incubation of gonoductal eggs at pH 8.2 prior to insemination increased fertilization rates, even when insemination was performed under low pH conditions. Furthermore, an increase in ambient pH induced an increase in the intracellular pH of the eggs. It was also found that an increase in ambient pH triggered the release of sperm attractants from the egg and is therefore necessary for sperm chemotaxis. Hence, acidic conditions in the gonoductal fluids keep the gametes, especially eggs, infertile, and the release of eggs into seawater upon spawning induces an increase in ambient pH, which enables egg fertilization. Full article

The non-orthotopic expression of olfactory receptors (ORs) includes the male reproductive system, and in particular spermatozoa; their active ligands could be essential to sperm chemotaxis and chemical sperm–oocyte communication. OR51E2 expression has been previously reported on sperm cells’ surface. It has been demonstrated in different cellular models that olfactory receptor 51E2 (OR51E2) binds volatile short-chain fatty acids (SCFAs) as specific ligands. In the present research, we make use of Western blot, confocal microscopy colocalization analysis, and the calcium-release assay to demonstrate the activation of sperm cells through OR51E2 upon SCFAs stimulus. Moreover, we perform a novel modified swim-up assay to study the involvement of OR51E2/SCFAs in sperm migration. Taking advantage of computer-assisted sperm analysis (CASA system), we determine the kinematics parameters of sperm cells migrating towards SCFAs-enriched medium, revealing that these ligands are able to promote a more linear sperm-cell orientation. Finally, we obtain SCFAs by mass spectrometry in cervico-vaginal mucus and show for the first time that a direct incubation between cervical mucus and sperm cells could promote their activation. This study can shed light on the possible function of chemosensory receptors in successful reproduction activity, laying the foundation for the development of new strategies for the treatment of infertile individuals. Full article

Fertilization requires sperm migration toward oocytes and subsequent fusion. Sperm chemotaxis, a process in which motile sperm are attracted by factors released from oocytes or associated structures, plays a key role in sperm migration to oocytes. Here, we studied sperm chemotaxis in the nematode Ascaris suum. Our data show that uterus-derived factor (UDF), the protein fraction of uterine extracts, can attract spermatozoa. UDF is heat resistant, but its activity is attenuated by certain proteinases. UDF binds to the surface of spermatozoa but not spermatids, and this process is mediated by membranous organelles that fuse with the plasma membrane. UDF induces spermatozoa to release ATP from intracellular storage sites to the extracellular milieu, and extracellular ATP modulates sperm chemotaxis. Moreover, UDF increases protein serine phosphorylation (pS) levels in sperm, which facilitates sperm chemotaxis. Taken together, we revealed that both extracellular ATP and intracellular pS signaling are involved in Ascaris sperm chemotaxis. Our data provide insights into the mechanism of sperm chemotaxis in Ascaris suum. Full article

Spermatozoa sense and respond to their environmental signals to ensure fertilization success. Reception and transduction of signals are reflected rapidly in sperm flagellar waveforms and swimming behavior. In the ascidian Ciona intestinalis (type A; also called C. robusta), an egg-derived sulfated steroid called SAAF (sperm activating and attracting factor), induces both sperm motility activation and chemotaxis. Two types of CNG (cyclic nucleotide-gated) channels, Ci-tetra KCNG (tetrameric, cyclic nucleotide-gated, K⁺-selective) and Ci-HCN (hyperpolarization-activated and cyclic nucleotide-gated), are highly expressed in Ciona testis from the comprehensive gene expression analysis. To elucidate the sperm signaling pathway to regulate flagellar motility, we focus on the role of CNG channels. In this study, the immunochemical analysis revealed that both CNG channels are expressed in Ciona sperm and localized to sperm flagella. Sperm motility analysis and Ca²⁺ imaging during chemotaxis showed that CNG channel inhibition affected the changes in flagellar waveforms and Ca²⁺ efflux needed for the chemotactic turn. These results suggest that CNG channels in Ciona sperm play a vital role in regulating sperm motility and intracellular Ca²⁺ regulation during chemotaxis. Full article

There is considerable evidence that female reproductive fluid (FRF) interacts intimately with sperm, affecting several sperm traits, including sperm motility and longevity, and ultimately fertilization success. One of the first documented interactions between FRF and sperm is the ability of FRF to attract and guide sperm towards the eggs. However, most of the evidence of FRF’s chemoattraction proprieties comes from a limited number of taxa, specifically mammals and invertebrate broadcasting spawners. In other species, small FRF volumes and/or short sperm longevity often impose methodological difficulties resulting in this gap in chemoattraction studies in non-model species. One of the outcomes of sperm chemotaxis is sperm accumulation towards high chemoattractant concentrations, which can be easily quantified by measuring sperm concentration. Here, we tested sperm accumulation towards FRF in the zebrafish, Danio rerio, using an ad hoc developed, 3D printed, device (‘sperm selection chamber’). This easy-to-use tool allows to select and collect the sperm that swim towards a chemical gradient, and accumulate in a chemoattractant-filled well thus providing putative evidence for chemoattraction. We found that sperm accumulate in FRF in zebrafish. We also found that none of the sperm quality traits we measured (sperm swimming velocity and trajectory, sperm motility, and longevity) were correlated with this response. Together with the 3D printable project, we provide a detailed protocol for using the selection chamber. The chamber is optimized for the zebrafish, but it can be easily adapted for other species. Our device lays the foundation for a standardized way to measure sperm accumulation and in general chemoattraction, stimulating future research aimed at understanding the role and the mechanisms of sperm chemoattraction by FRF. Full article

The fertilization of freshwater fish occurs in an environment that may negatively affect the gametes; therefore, the specific mechanisms triggering the encounters of gametes would be highly expedient. The egg and ovarian fluid are likely the major sources of these triggers, which we confirmed here for rainbow trout (Oncorhynchus mykiss). The ovarian fluid affected significantly spermatozoa performance: it supported high velocity for a longer period and changed the motility pattern from tumbling in water to straightforward moving in the ovarian fluid. Rainbow trout ovarian fluid induced a trapping chemotaxis-like effect on activated male gametes, and this effect depended on the properties of the activating medium. The interaction of the spermatozoa with the attracting agents was accompanied by the “turn-and-run” behavior involving asymmetric flagellar beating and Ca²⁺ concentration bursts in the bent flagellum segment, which are characteristic of the chemotactic response. Ovarian fluid created the optimal environment for rainbow trout spermatozoa performance, and the individual peculiarities of the egg (ovarian fluid)–sperm interaction reflect the specific features of the spawning process in this species. Full article

In echinoderms, sperm swims in random circles and turns in response to a chemoattractant. The chemoattractant evokes transient Ca²⁺ influx in the sperm flagellum and induces turning behavior. Recently, the molecular mechanisms and biophysical properties of this sperm response have been clarified. Based on these experimental findings, in this study, we reconstructed a sperm model in silico to demonstrate an algorithm for sperm chemotaxis. We also focused on the importance of desensitizing the chemoattractant receptor in long-range chemotaxis because sperm approach distantly located eggs, and they must sense the chemoattractant concentration over a broad range. Using parameters of the sea urchin, simulations showed that a number of sperm could reach the egg from millimeter-order distances with desensitization, indicating that we could organize a functional sperm model, and that desensitization of the receptor is essential for sperm chemotaxis. Then, we compared the model with starfish sperm, which has a different desensitization scheme and analyzed the properties of the model against various disturbances. Our approach can be applied as a novel tool in chemotaxis research. Full article

Based on the abundance of scientific publications, the polymodal sensor TRPV1 is known as one of the most studied proteins within the TRP channel family. This receptor has been found in numerous cell types from different species as well as in spermatozoa. The present review is focused on analyzing the role played by this important channel in the post-ejaculatory life of spermatozoa, where it has been described to be involved in events such as capacitation, acrosome reaction, calcium trafficking, sperm migration, and fertilization. By performing an exhaustive bibliographic search, this review gathers, for the first time, all the modulators of the TRPV1 function that, to our knowledge, were described to date in different species and cell types. Moreover, all those modulators with a relationship with the reproductive process, either found in the female tract, seminal plasma, or spermatozoa, are presented here. Since the sperm migration through the female reproductive tract is one of the most intriguing and less understood events of the fertilization process, in the present work, chemotaxis, thermotaxis, and rheotaxis guiding mechanisms and their relationship with TRPV1 receptor are deeply analyzed, hypothesizing its (in)direct participation during the sperm migration. Last, TRPV1 is presented as a pharmacological target, with a special focus on humans and some pathologies in mammals strictly related to the male reproductive system. Full article

Sperm chemotaxis, which guide sperm toward oocyte, is tightly associated with sperm capacitation, motility, and fertility. However, the molecular mechanism of sperm chemotaxis is not known. Reproductive odorant and taste receptors, belong to G-protein-coupled receptors (GPCR) super-family, cause an increase in intracellular Ca²⁺ concentration which is pre-requisite for sperm capacitation and acrosomal reaction, and result in sperm hyperpolarization and increase motility through activation of Ca²⁺-dependent Cl^¯ channels. Recently, odorant receptors (ORs) in olfactory transduction pathway were thought to be associated with post-thaw sperm motility, freeze tolerance or freezability and cryo-capacitation-like change during cryopreservation. Investigation of the roles of odorant and taste receptors (TRs) is important for our understanding of the freeze tolerance or freezability mechanism and improve the motility and fertility of post-thaw sperm. Here, we reviewed the roles, mode of action, impact of odorant and taste receptors on sperm chemotaxis and post-thaw sperm quality. Full article