Dual role of valosin-containing protein (VCP/p97) in mouse sperm during capacitation.

Valosin-containing protein (VCP; aka p97), a member of the AAA (ATPases Associated with various cellular Activities) family, has been associated with a wide range of cellular functions. While previous evidence has shown its presence in mammalian sperm, our study unveils its function in mouse sperm. Notably, we found that mouse VCP does not undergo tyrosine phosphorylation during capacitation and exhibits distinct localization patterns. In the sperm head, it resides within the equatorial segment and, following acrosomal exocytosis, it is released and cleaved. In the flagellum, VCP is observed in the principal and midpiece. Furthermore, our research highlights a unique role for VCP in the cAMP/PKA pathway during capacitation. Pharmacological inhibition of sperm VCP led to reduced intracellular cAMP levels that resulted in decreased phosphorylation in PKA substrates and tyrosine residues and diminished fertilization competence. Our results show that in mouse sperm, VCP plays a pivotal role in regulating cAMP production, probably by the modulation of soluble adenylyl cyclase activity.

Membrane potential hyperpolarization: a critical factor in acrosomal exocytosis and fertilization in sperm within the female reproductive tract.

Hyperpolarization of the membrane potential (Em), a phenomenon regulated by SLO3 channels, stands as a central feature in sperm capacitation-a crucial process conferring upon sperm the ability to fertilize the oocyte. In vitro studies demonstrated that Em hyperpolarization plays a pivotal role in facilitating the mechanisms necessary for the development of hyperactivated motility (HA) and acrosomal exocytosis (AE) occurrence. Nevertheless, the physiological significance of sperm Em within the female reproductive tract remains unexplored. As an approach to this question, we studied sperm migration and AE incidence within the oviduct in the absence of Em hyperpolarization using a novel mouse model established by crossbreeding of SLO3 knock-out (KO) mice with EGFP/DsRed2 mice. Sperm from this model displays impaired HA and AE in vitro. Interestingly, examination of the female reproductive tract shows that SLO3 KO sperm can reach the ampulla, mirroring the quantity of sperm observed in wild-type (WT) counterparts, supporting that the HA needed to reach the fertilization site is not affected. However, a noteworthy distinction emerges-unlike WT sperm, the majority of SLO3 KO sperm arrive at the ampulla with their acrosomes still intact. Of the few SLO3 KO sperm that do manage to reach the oocytes within this location, fertilization does not occur, as indicated by the absence of sperm pronuclei in the MII-oocytes recovered post-mating. In vitro, SLO3 KO sperm fail to penetrate the ZP and fuse with the oocytes. Collectively, these results underscore the vital role of Em hyperpolarization in AE and fertilization within their physiological context, while also revealing that Em is not a prerequisite for the development of the HA motility, essential for sperm migration through the female tract to the ampulla.

A versatile kinase mobility shift assay (KiMSA) for PKA analysis and cyclic AMP detection in sperm physiology (and beyond).

The cAMP-dependent protein kinase (PKA) is one of the most extensively distributed kinases among intracellular signal cascades, with a pivotal role in the regulation of various processes, including the capacitation of sperm cells. Traditional assessments of PKA activity relies on the utilization of [γ-32P] ATP and the Kemptide substrate. This methodology presents several major drawbacks, including high-costs and health risks derived from the manipulation of radioactive isotopes. In this work we introduce an enhanced non-radioactive assay for quantifying PKA activity, termed KiMSA which relies on the use of a fluorescent-labeled Kemptide (Kemptide-FITC). Once the kinase reaction is terminated, the products can be easily resolved through electrophoresis on an agarose gel and quantified by fluorescence densitometry. We show that the KiMSA assay is suitable for purified PKA, and also to address both basal and capacitation induced PKA activity in mouse sperm cells. Furthermore, the assay enables monitoring the inhibition of PKA with inhibitors such as sPKI and H-89 in live cells. Therefore, the experimental and optimal assay conditions are set so that the KiMSA assay can be used to either assess in vitro as well as in vivo PKA activity in sperm cells. Finally, this method allows for measurement of cAMP concentrations, rendering a versatile technique for the study of cAMP/PKA pathways.

The sodium-proton exchangers sNHE and NHE1 control plasma membrane hyperpolarization in mouse sperm.

Sperm capacitation, crucial for fertilization, occurs in the female reproductive tract and can be replicated in vitro using a medium rich in bicarbonate, calcium, and albumin. These components trigger the cAMP-PKA signaling cascade, proposed to promote hyperpolarization of the mouse sperm plasma membrane through activation of SLO3 K+ channel. Hyperpolarization is a hallmark of capacitation: proper membrane hyperpolarization renders higher in vitro fertilizing ability, while Slo3 KO mice are infertile. However, the precise regulation of SLO3 opening remains elusive. Our study challenges the involvement of PKA in this event and reveals the role of Na+/H+ exchangers. During capacitation, calcium increase through CatSper channels activates NHE1, while cAMP directly stimulates the sperm-specific NHE, collectively promoting the alkalinization threshold needed for SLO3 opening. Hyperpolarization then feeds back Na+/H+ activity. Our work is supported by pharmacology, and a plethora of KO mouse models, and proposes a novel pathway leading to hyperpolarization.

High-throughput screening method for discovering CatSper inhibitors using membrane depolarization caused by external calcium chelation and fluorescent cell barcoding.

The exclusive expression of CatSper in sperm and its critical role in sperm function makes this channel an attractive target for contraception. The strategy of blocking CatSper as a male, non-hormonal contraceptive has not been fully explored due to the lack of robust screening methods to discover novel and specific inhibitors. The reason for this lack of appropriate methodology is the structural and functional complexity of this channel. We have developed a high-throughput method to screen drugs with the capacity to block CatSper in mammalian sperm. The assay is based on removing external free divalent cations by chelation, inducing CatSper to efficiently conduct monovalent cations. Since Na+ is highly concentrated in the extracellular milieu, a sudden influx depolarizes the cell. Using CatSper1 KO sperm we demonstrated that this depolarization depends on CatSper function. A membrane potential (Em) assay was combined with fluorescent cell barcoding (FCB), enabling higher throughput flow cytometry based on unique fluorescent signatures of different sperm samples. These differentially labeled samples incubated in distinct experimental conditions can be combined into one tube for simultaneous acquisition. In this way, acquisition times are highly reduced, which is essential to perform larger screening experiments for drug discovery using live cells. Altogether, a simple strategy for assessing CatSper was validated, and this assay was used to develop a high-throughput drug screening for new CatSper blockers.

Extending resolution within a single imaging frame.

The resolution of fluorescence microscopy images is limited by the physical properties of light. In the last decade, numerous super-resolution microscopy (SRM) approaches have been proposed to deal with such hindrance. Here we present Mean-Shift Super Resolution (MSSR), a new SRM algorithm based on the Mean Shift theory, which extends spatial resolution of single fluorescence images beyond the diffraction limit of light. MSSR works on low and high fluorophore densities, is not limited by the architecture of the optical setup and is applicable to single images as well as temporal series. The theoretical limit of spatial resolution, based on optimized real-world imaging conditions and analysis of temporal image stacks, has been measured to be 40 nm. Furthermore, MSSR has denoising capabilities that outperform other SRM approaches. Along with its wide accessibility, MSSR is a powerful, flexible, and generic tool for multidimensional and live cell imaging applications.

The early molecular events leading to COFILIN phosphorylation during mouse sperm capacitation are essential for acrosomal exocytosis.

The actin cytoskeleton reorganization during sperm capacitation is essential for the occurrence of acrosomal exocytosis (AR) in several mammalian species. Here, we demonstrate that in mouse sperm, within the first minutes of exposure upon capacitating conditions, the activity of RHOA/C and RAC1 is essential for LIMK1 and COFILIN phosphorylation. However, we observed that the signaling pathway involving RAC1 and PAK4 is the main player in controlling actin polymerization in the sperm head necessary for the occurrence of AR. Moreover, we show that the transient phosphorylation of COFILIN is also influenced by the Slingshot family of protein phosphatases (SSH1). The activity of SSH1 is regulated by the dual action of two pathways. On one hand, RHOA/C and RAC1 activity promotes SSH1 phosphorylation (inactivation). On the other hand, the activating dephosphorylation is driven by okadaic acid-sensitive phosphatases. This regulatory mechanism is independent of the commonly observed activating mechanisms involving PP2B and emerges as a new finely tuned modulation that is, so far, exclusively observed in mouse sperm. However, persistent phosphorylation of COFILIN by SSH1 inhibition or okadaic acid did not altered actin polymerization and the AR. Altogether, our results highlight the role of small GTPases in modulating actin dynamics required for AR.

Human Sperm Remain Motile After a Temporary Energy Restriction but do Not Undergo Capacitation-Related Events.

To acquire fertilization competence, mammalian sperm must undergo several biochemical and physiological modifications known as capacitation. Despite its relevance, the metabolic pathways that regulate the capacitation-related events, including the development of hyperactivated motility, are still poorly described. Previous studies from our group have shown that temporary energy restriction in mouse sperm enhanced hyperactivation, in vitro fertilization, early embryo development and pregnancy rates after embryo transfer, and it improved intracytoplasmic sperm injection results in the bovine model. However, the effects of starvation and energy recovery protocols on human sperm function have not yet been established. In the present work, human sperm were incubated for different periods of time in medium containing glucose, pyruvate and lactate (NUTR) or devoid of nutrients for the starving condition (STRV). Sperm maintained in STRV displayed reduced percentages of motility and kinematic parameters compared to cells incubated in NUTR medium. Moreover, they did not undergo hyperactivation and showed reduced levels of ATP, cAMP and protein tyrosine phosphorylation. Similar to our results with mouse sperm, starvation induced increased intracellular Ca2+ concentrations. Starved human sperm were capable to continue moving for more than 27 h, but the incubation with a mitochondrial uncoupler or inhibitors of oxidative phosphorylation led to a complete motility loss. When exogenous nutrients were added back (sperm energy recovery (SER) treatment), hyperactivated motility was rescued and there was a rise in sperm ATP and cAMP levels in 1 min, with a decrease in intracellular Ca2+ concentration and no changes in sperm protein tyrosine phosphorylation. The finding that human sperm can remain motile for several hours under starvation due to mitochondrial use of endogenous metabolites implies that other metabolic pathways may play a role in sperm energy production. In addition, full recovery of motility and other capacitation parameters of human sperm after SER suggests that this treatment might be used to modulate human sperm fertilizing ability in vitro.

Dissecting EPPIN protease inhibitor domains in sperm motility and fertilizing ability: repercussions for male contraceptive development.

EPPIN (epididymal protease inhibitor) is a mammalian conserved sperm-binding protein displaying an N-terminal WFDC (whey-acidic protein four-disulfide core) and a C-terminal Kunitz protease inhibitor domains. EPPIN plays a key role in regulating sperm motility after ejaculation via interaction with the seminal plasma protein SEMG1 (semenogelin-1). EPPIN ligands targeting the SEMG1 binding site in the Kunitz domain are under development as male contraceptive drugs. Nevertheless, the relative contributions of EPPIN WFDC and Kunitz domains to sperm function remain obscure. Here, we evaluated the effects of antibodies targeting specific epitopes in EPPIN's WFDC (Q20E antibody, Gln20-Glu39 epitope) and Kunitz (S21C and F21C antibodies, Ser103-Cys123 and Phe90-C110 epitopes, respectively) domains on mouse sperm motility and fertilizing ability. Computer-assisted sperm analysis showed that sperm co-incubation with S21C antibody (but not F21C antibody) lowered progressive and hyperactivated motilities and impaired kinematic parameters describing progressive (straight-line velocity; VSL, average path velocity; VAP and straightness; STR) and vigorous sperm movements (curvilinear velocity; VCL, amplitude of lateral head movement; ALH, and linearity; LIN) compared with control. Conversely, Q20E antibody-induced milder inhibition of progressive motility and kinematic parameters (VAP, VCL and ALH). Sperm co-incubation with S21C or Q20E antibodies affected in vitro fertilization as revealed by reduced cleavage rates, albeit without changes in capacitation-induced tyrosine phosphorylation. In conclusion, we show that targeting specific epitopes in EPPIN Kunitz and WFDC domains inhibits sperm motility and capacitation-associated events, which decrease their fertilizing ability; nevertheless, similar observations in vivo remain to be demonstrated. Simultaneously targeting residues in S21C and Q20E epitopes is a promising approach for the rational design of EPPIN-based ligands with spermostatic activity.

Cdc42 localized in the CatSper signaling complex regulates cAMP-dependent pathways in mouse sperm.

Sperm acquire the ability to fertilize in a process called capacitation and undergo hyperactivation, a change in the motility pattern, which depends on Ca2+ transport by CatSper channels. CatSper is essential for fertilization and it is subjected to a complex regulation that is not fully understood. Here, we report that similar to CatSper, Cdc42 distribution in the principal piece is confined to four linear domains and this localization is disrupted in CatSper1-null sperm. Cdc42 inhibition impaired CatSper activity and other Ca2+ -dependent downstream events resulting in a severe compromise of the sperm fertilizing potential. We also demonstrate that Cdc42 is essential for CatSper function by modulating cAMP production by soluble adenylate cyclase (sAC), providing a new regulatory mechanism for the stimulation of CatSper by the cAMP-dependent pathway. These results reveal a broad mechanistic insight into the regulation of Ca2+ in mammalian sperm, a matter of critical importance in male infertility as well as in contraception.

Membrane hyperpolarization abolishes calcium oscillations that prevent induced acrosomal exocytosis in human sperm.

Sperm capacitation is essential to gain fertilizing capacity. During this process, a series of biochemical and physiological modifications occur that allow sperm to undergo acrosomal exocytosis (AE). At the molecular level, hyperpolarization of the sperm membrane potential (Em) takes place during capacitation. This study shows that human sperm incubated under conditions that do not support capacitation (NC) can become ready for an agonist stimulated AE by pharmacologically inducing Em hyperpolarization with Valinomycin or Amiloride. To investigate how Em hyperpolarization promotes human sperm's ability to undergo AE, live single-cell imaging experiments were performed to simultaneously monitor changes in [Ca2+ ]i and the occurrence of AE. Em hyperpolarization turned [Ca2+ ]i dynamics in NC sperm from spontaneously oscillating into a sustained slow [Ca2+ ]i increase. The addition of progesterone (P4) or K+ to Valinomycin-treated sperm promoted that a significant number of cells displayed a transitory rise in [Ca2+ ]i which then underwent AE. Altogether, our results demonstrate that Em hyperpolarization is necessary and sufficient to prepare human sperm for the AE. Furthermore, this Em change decreased Ca2+ oscillations that block the occurrence of AE, providing strong experimental evidence of the molecular mechanism that drives the acquisition of acrosomal responsiveness.

Seeing is believing: Current methods to observe sperm acrosomal exocytosis in real time.

Acrosomal exocytosis (AR) is a critical process that sperm need to undergo to fertilize an egg. The evaluation of the presence or absence of the acrosome is usually performed by using lectins or dyes in fixed cells. With this approach, it is neither possible to monitor the dynamic process of exocytosis and related molecular events while discriminating between live and dead cells, nor to evaluate the acrosomal status while sperm reside in the female reproductive tract. However, over the last two decades, several new methodologies have been used to assess the occurrence of AR in living cells allowing different groups to obtain information that was not possible in the past. These techniques have revolutionized the whole study of this process. This review summarizes current methods available to analyze AR in living cells as well as the important information that emerged from studies using these approaches.

Everything you ever wanted to know about PKA regulation and its involvement in mammalian sperm capacitation.

The 3', 5'-cyclic adenosine monophosphate (cAMP) dependent protein kinase (PKA) is a tetrameric holoenzyme comprising a set of two regulatory subunits (PKA-R) and two catalytic (PKA-C) subunits. The PKA-R subunits act as sensors of cAMP and allow PKA-C activity. One of the first signaling events observed during mammalian sperm capacitation is PKA activation. Thus, understanding how PKA activity is restricted in space and time is crucial to decipher the critical steps of sperm capacitation. It is widely accepted that PKA specificity depends on several levels of regulation. Anchoring proteins play a pivotal role in achieving proper localization signaling, subcellular targeting and cAMP microdomains. These multi-factorial regulation steps are necessary for a precise spatio-temporal activation of PKA. Here we discuss recent understanding of regulatory mechanisms of PKA in mammalian sperm, such as post-translational modifications, in the context of its role as the master orchestrator of molecular events conducive to capacitation.

Reactive oxygen species are involved in the signaling of equine sperm chemotaxis.

Sperm chemotaxis may facilitate the finding of the oocyte. Only capacitated spermatozoa can orient their movement by chemotaxis, which as well as capacitation, is regulated in part by the cAMP-PKA pathway. Reactive oxygen species (ROS) are produced during sperm capacitation which is closely related to chemotaxis. Then, the ROS participation in the chemotactic signaling can be expected. Here we studied the role of ROS in the chemotaxis signaling of equine spermatozoa which produce high quantities of ROS because of their energy metabolism. The level of capacitated and chemotactic spermatozoa was increased with 0.1 and 0.2 mM hydrogen peroxide (H2O2), which was involved in the chemotactic signaling. By combining a concentration gradient of H2O2 with inhibitors/chelators of some of the signaling pathway elements, we showed that the activation of NOX (membrane NADPH oxidase) increases the intracellular ROS which activate the chemotaxis AMPc-PKA pathway. Our results provide evidence about the participation of ROS in the chemotactic signaling mediated by progesterone (P).

Determination of a Robust Assay for Human Sperm Membrane Potential Analysis.

Mammalian sperm must undergo a complex process called capacitation in order to fertilize the egg. During this process, hyperpolarization of the sperm plasma membrane has been mostly studied in mouse, and associated to its importance in the preparation to undergo the acrosome reaction (AR). However, despite the increasing evidence of membrane hyperpolarization in human sperm capacitation, no reliable techniques have been set up for its determination. In this report we describe human sperm membrane potential (Em) measurements by a fluorimetric population assay, establishing optimal conditions for Em determination. In addition, we have conducted parallel measurements of the same human sperm samples by flow cytometry and population fluorimetry, before and after capacitation, to conclusively address their reliability. This integrative analysis sets the basis for the study of Em in human sperm allowing future work aiming to understand its role in human sperm capacitation.

Membrane Potential Assessment by Fluorimetry as a Predictor Tool of Human Sperm Fertilizing Capacity.

Mammalian sperm acquire the ability to fertilize eggs by undergoing a process known as capacitation. Capacitation is triggered as the sperm travels through the female reproductive tract. This process involves specific physiological changes such as rearrangement of the cell plasma membrane, post-translational modifications of certain proteins, and changes in the cellular permeability to ions - with the subsequent impact on the plasma membrane potential (Em). Capacitation-associated Em hyperpolarization has been well studied in mouse sperm, and shown to be both necessary and sufficient to promote the acrosome reaction (AR) and fertilize the egg. However, the relevance of the sperm Em upon capacitation on human fertility has not been thoroughly characterized. Here, we performed an extensive study of the Em change during capacitation in human sperm samples using a potentiometric dye in a fluorimetric assay. Normospermic donors showed significant Em hyperpolarization after capacitation. Em values from capacitated samples correlated significantly with the sperm ability to undergo induced AR, highlighting the role of hyperpolarization in acrosomal responsiveness, and with successful in vitro fertilization (IVF) rates. These results show that Em hyperpolarization could be an indicator of human sperm fertilizing capacity, setting the basis for the use of Em values as a robust predictor of the success rate of IVF.

Low level laser therapy (LLLT) modulates ovarian function in mature female mice.

It is known that LLLT has beneficial effects on several pathological conditions including wound healing, pain and inflammation. LLLT modulates biological processes, including cell proliferation, apoptosis and angiogenesis. In the present study, we examined the effect of local application of LLLT on follicular dynamics, ovarian reserve, AMH expression, progesterone levels, apoptosis, angiogenesis, and reproductive outcome in adult mice. LLLT (200 J/cm2) increased the percentage of primary and preantral follicles, whilst decreasing the percentage of corpora lutea compared to control ovaries. LLLT-treated ovaries did not exhibit any changes regarding the number of primordial follicles. We observed a higher percentage of AMH-positive follicles (in early stages of development) in LLLT-treated ovaries compared to control ovaries. LLLT reduced the P4 concentration and the apoptosis in early antral follicles compared to control ones. LLLT caused a reduction in the endothelial cell area and an increase in the periendothelial cell area in the ovary. Additionally, LLLT was able to improve oocyte quality. Our findings suggest that local application of LLLT modulates follicular dynamics by regulating apoptosis and the vascular stability in mouse ovary. In conclusion, these data indicate that LLLT might become a novel and useful tool in the treatment of several pathologies, including female reproductive disorders.

Super-resolution imaging of live sperm reveals dynamic changes of the actin cytoskeleton during acrosomal exocytosis.

Filamentous actin (F-actin) is a key factor in exocytosis in many cell types. In mammalian sperm, acrosomal exocytosis (denoted the acrosome reaction or AR), a special type of controlled secretion, is regulated by multiple signaling pathways and the actin cytoskeleton. However, the dynamic changes of the actin cytoskeleton in live sperm are largely not understood. Here, we used the powerful properties of SiR-actin to examine actin dynamics in live mouse sperm at the onset of the AR. By using a combination of super-resolution microscopy techniques to image sperm loaded with SiR-actin or sperm from transgenic mice containing Lifeact-EGFP, six regions containing F-actin within the sperm head were revealed. The proportion of sperm possessing these structures changed upon capacitation. By performing live-cell imaging experiments, we report that dynamic changes of F-actin during the AR occur in specific regions of the sperm head. While certain F-actin regions undergo depolymerization prior to the initiation of the AR, others remain unaltered or are lost after exocytosis occurs. Our work emphasizes the utility of live-cell nanoscopy, which will undoubtedly impact the search for mechanisms that underlie basic sperm functions.This article has an associated First Person interview with the first author of the paper.

Lysine acetylation modulates mouse sperm capacitation.

Mammalian sperm are unable to fertilize the egg immediately after ejaculation. To gain fertilization competence, they need to undergo a series of modifications inside the female reproductive tract, known as capacitation. Capacitation involves several molecular events such as phosphorylation cascades, hyperpolarization of the plasma membrane and intracellular Ca2+ changes, which prepare the sperm to develop two essential features for fertilization competence: hyperactivation and acrosome reaction. Since sperm cells lack new protein biosynthesis, post-translational modification of existing proteins plays a crucial role to obtain full functionality. Here, we show the presence of acetylated proteins in murine sperm, which increase during capacitation. Pharmacological hyperacetylation of lysine residues in non-capacitated sperm induces activation of PKA, hyperpolarization of the sperm plasma membrane, CatSper opening and Ca2+ influx, all capacitation-associated molecular events. Furthermore, hyperacetylation of non-capacitated sperm promotes hyperactivation and prepares the sperm to undergo acrosome reaction. Together, these results indicate that acetylation could be involved in the acquisition of fertilization competence of mammalian sperm.