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Capacitation

Capacitation is a maturation process that mammalian spermatozoa undergo primarily in the female reproductive tract, involving a series of physiological and biochemical changes that enable them to fertilize an oocyte.^[1] These changes include alterations in membrane fluidity through cholesterol efflux, increased intracellular calcium and bicarbonate levels, activation of signaling pathways such as cAMP-protein kinase A (PKA), and protein tyrosine phosphorylation, culminating in hyperactivated motility and the capacity for the acrosome reaction.^[1]^[2] Discovered in the early 1950s, capacitation is essential for successful fertilization, as only capacitated sperm can penetrate the zona pellucida surrounding the egg.^[3]^[4] The process was first described independently by Colin Russell Austin and Min Chueh Chang in 1951 using rabbit and rat models, respectively, with Austin coining the term "capacitation" in 1952 to denote the functional maturation required for sperm to gain fertilizing capacity after deposition in the female tract.^[3]^[4] Prior to this discovery, it was assumed that ejaculated sperm were immediately competent for fertilization, but experiments showed that sperm recovered from the female tract could fertilize eggs in vitro, while those from the male tract could not.^[1] In vitro capacitation was later achieved in 1977 using boar spermatozoa, marking a key advancement that facilitated the development of assisted reproductive technologies, including the first successful human in vitro fertilization in 1978.^[5]^[6] Molecularly, capacitation is triggered by environmental factors in the female reproductive tract, such as elevated bicarbonate (HCO₃⁻) and calcium (Ca²⁺) concentrations, which activate soluble adenylyl cyclase (sAC) to elevate cAMP levels and stimulate PKA, leading to downstream events like the efflux of cholesterol via albumin and the modulation of ion channels.^[2] Key ion channels involved include CatSper, a Ca²⁺-permeable channel essential for hyperactivation and regulated by progesterone from cumulus cells; Hv1, a proton channel that regulates intracellular pH; and Slo3, a potassium channel linked to flagellar hyperactivation.^[1] Reactive oxygen species (ROS), produced at physiological levels, also play a regulatory role by promoting tyrosine phosphorylation and membrane remodeling, though excessive ROS can impair the process.^[2] These mechanisms exhibit species-specific variations, with human sperm showing distinct responses compared to rodents, underscoring the importance of model-specific research in understanding infertility and developing therapies.^[1] Capacitation's clinical significance lies in its role in male infertility, where defects in this process—often due to genetic mutations in ion channels like CatSper or environmental factors—are implicated in many cases by preventing sperm from achieving fertilizing competence.^[1] Advances in defining capacitation have improved in vitro fertilization protocols by optimizing media with bicarbonate, calcium, and cholesterol acceptors to mimic the female tract, enhancing success rates in assisted reproduction.^[2] Ongoing research continues to elucidate the precise regulation of these pathways, with implications for contraception and fertility preservation.^[1]

Definition and Biological Role

Core Definition and Process Overview

Capacitation refers to the series of physiological changes that ejaculated mammalian spermatozoa undergo to acquire the competence to fertilize an oocyte.^[7] These changes, first described in the early 1950s, enable sperm to penetrate the zona pellucida and undergo the acrosome reaction upon encountering the egg.^[8] Key aspects include the efflux of cholesterol from the sperm plasma membrane, which increases membrane fluidity and alters protein distribution, preparing the sperm for subsequent fertilization events.^[9] In vivo, capacitation typically unfolds over a period of 1 to 7 hours within the female reproductive tract, though the exact duration varies by species and individual factors.^[10] This process is reversible; if sperm are removed from the capacitating environment before completion, they can revert to an uncapacitated state, losing their fertilizing potential until re-exposed.^[11] Capacitation is distinct from epididymal maturation, which occurs prior to ejaculation as spermatozoa transit through the epididymis, where they acquire initial motility, nuclear condensation, and surface modifications necessary for viability.^[12] In contrast, capacitation represents a post-ejaculatory maturation step that can only proceed in the female reproductive tract or under specific in vitro conditions mimicking that environment.^[13] While post-ejaculatory modifications to sperm occur across many animal taxa, true capacitation—as defined by these specific mammalian membrane and preparatory changes—is absent in non-mammals such as birds, fish, and invertebrates, which rely on alternative activation mechanisms for fertilization.^[14] These alterations during capacitation ultimately lead to hyperactivated sperm motility, enhancing their ability to navigate the female tract.^[9]

Role in Mammalian Fertilization and Species Specificity

Capacitation is essential for mammalian fertilization, as it transforms immature spermatozoa into forms capable of binding to the zona pellucida, undergoing the acrosome reaction, and fusing with the oocyte plasma membrane. Without capacitation, sperm remain unable to penetrate the egg's protective layers, rendering fertilization impossible despite reaching the site of insemination. This priming process enhances sperm hyperactivated motility and membrane hyperpolarization, which facilitate zona pellucida recognition and subsequent acrosomal exocytosis, allowing enzymes to digest the zona and enable gamete fusion.^[2]^[9] From an evolutionary perspective, capacitation represents an adaptive mechanism shaped by the female reproductive tract's selective pressures, ensuring only robust sperm achieve fertilization competence at the optimal time near ovulation. This process promotes post-copulatory sperm selection by delaying full activation until sperm encounter specific tract conditions, such as bicarbonate ions and albumin, which vary across species to match reproductive strategies. The energetic cost of hyperactivation post-capacitation underscores its role in prioritizing high-quality sperm, potentially reducing polyspermy risks and enhancing offspring viability in diverse mammalian lineages.^[15]^[16] Species-specific variations in capacitation highlight its tailored evolution, with timelines and molecular requirements differing markedly; for instance, rodent sperm capacitate rapidly in minutes, while human and bovine sperm require hours, reflecting differences in tract length and fluid composition. In bovines, unique proteins like those involved in cholesterol efflux are necessary for effective capacitation, absent or functionally divergent in humans, which affects cross-species IVF compatibility. These disparities, including variations in cholesterol-to-phospholipid ratios (e.g., lower in boars at 0.20 versus ~0.40 in bovines), ensure reproductive isolation and optimize fertilization success within each species.^[1]^[2] Clinically, defects in capacitation contribute significantly to male infertility, implicated in 30-50% of cases where traditional semen analysis appears normal, often leading to reduced fertilization rates in assisted reproduction. Such impairments, detectable through capacitation assays, underscore the need for advanced diagnostics beyond motility or count assessments to identify subtle functional failures.^[17]^[18]

Physiological and Molecular Mechanisms

Membrane Dynamics and Motility Alterations

During capacitation, the sperm plasma membrane undergoes significant remodeling, primarily through the efflux of cholesterol, which is facilitated by acceptors such as bovine serum albumin (BSA) or β-cyclodextrins. This removal of cholesterol from lipid rafts reduces membrane order and increases fluidity, enabling the redistribution of membrane proteins and lipids essential for subsequent fertilization events.88109-7/fulltext) Studies in mammalian species, including humans and mice, demonstrate that cholesterol depletion correlates directly with enhanced membrane permeability and the initiation of capacitation-associated changes.^[19] Concomitant with these lipid alterations, capacitation involves dynamic ion fluxes across the sperm membrane, particularly influxes of calcium (Ca²⁺) and bicarbonate (HCO₃⁻). Ca²⁺ entry occurs primarily through the sperm-specific CatSper channel complex, a pH- and voltage-sensitive cation channel localized to the flagellar principal piece, which triggers asymmetric flagellar bending and supports hyperactivation.00499-9) Similarly, HCO₃⁻ influx, mediated by anion exchangers like SLC26A3 and SLC26A6, elevates intracellular pH (pH_i) from approximately 6.5 to 7.2-7.5, promoting soluble adenylyl cyclase activation and downstream physiological shifts. These ion movements are interdependent, with HCO₃⁻-induced alkalization sensitizing CatSper to Ca²⁺ influx, thereby amplifying membrane destabilization. These membrane and ionic changes culminate in profound alterations to sperm motility, transitioning from symmetrical, progressive thrusting to hyperactivated motility characterized by vigorous, asymmetric flagellar whipping. Hyperactivation enhances the sperm's ability to navigate the oviduct and penetrate the zona pellucida, with key computer-assisted sperm analysis (CASA) metrics including curvilinear velocity (VCL) exceeding 150 μm/s and amplitude of lateral head displacement (ALH) greater than 7 μm in human spermatozoa. In capacitated bull and mouse sperm, this motility pattern features high-velocity, star-spin trajectories, reflecting increased flagellar beat frequency and power stroke asymmetry driven by the aforementioned Ca²⁺ and pH_i elevations. Overall, these dynamics ensure sperm readiness for fertilization without compromising viability.

Biochemical Signaling and Metabolic Shifts

Capacitation in mammalian sperm is initiated by bicarbonate (HCO₃⁻) stimulation of soluble adenylyl cyclase (sAC), which catalyzes the rapid elevation of intracellular cyclic adenosine monophosphate (cAMP) levels within minutes of exposure to capacitating conditions.^[20] This cAMP surge activates protein kinase A (PKA), a key effector that phosphorylates target proteins, leading to a cascade of downstream events essential for sperm maturation.^[21] PKA-mediated signaling promotes tyrosine phosphorylation (pY) of numerous sperm proteins, including A-kinase anchoring proteins (AKAPs) such as AKAP3 and AKAP82, which localize PKA to specific compartments like the flagellum and acrosomal region, enhancing motility and acrosome reaction competence.^[21] This pY pattern is a hallmark of capacitation, with inhibitors of PKA (e.g., H89) blocking phosphorylation and capacitation progression, while cAMP analogs like dibutyryl cAMP accelerating it.^[20] A critical component of this signaling involves HCO₃⁻-induced activation of the sperm-specific CatSper calcium channel, which facilitates intracellular calcium (Ca²⁺) influx to support hyperactivated motility. The increase in intracellular Ca²⁺ concentration [ \ce{Ca^{2+}} ]_i during capacitation can be described by a basic flux model:

\frac{d[\ce{Ca^{2+}}]_i}{dt} = J_{\ce{CatSper}} - J_{\ce{clearance}}

where J_{\ce{CatSper}} represents Ca²⁺ influx through voltage-dependent CatSper channels sensitized by HCO₃⁻ via cAMP/PKA, and J_{\ce{clearance}} denotes extrusion by pumps like PMCA; this model highlights how HCO₃⁻ elevates [ \ce{Ca^{2+}} ]_i from basal levels (~50 nM) to ~400 nM, triggering flagellar waveform changes.^[22] Metabolic reprogramming accompanies these signaling events, with capacitation shifting sperm energy production toward enhanced glycolysis to meet ATP demands for hyperactivation. In mouse and human sperm, glucose uptake increases several-fold during capacitation, fueling glycolytic flux and ATP generation, while intracellular ATP levels paradoxically decline due to heightened consumption.^[23] Oxidative phosphorylation (OXPHOS) is also modulated, with elevated activity in epididymal mouse sperm derived from glycolytic intermediates like pyruvate, but ejaculated sperm exhibiting greater flexibility by utilizing alternative substrates such as citrate.^[23] These 2023 findings underscore capacitation's role in optimizing bioenergetics, as blocking glycolysis impairs pY and motility.^[23] Recent transcriptomic analyses reveal dynamic changes in the sperm mRNA interactome during capacitation, activating genes linked to fertilization. A 2025 study in bovine sperm identified 48 RNA-binding proteins (RBPs) modulating mRNA interactions, with 10 RBPs undergoing phosphorylation exclusively or differentially in capacitated states, including metabolic enzymes like hexokinase-1 that double as RBPs to regulate translation of fertilization-related transcripts.^[24] Using RNA antisense purification-mass spectrometry (RAP-MS), the research demonstrated shifts in the mRNA-RBP network, promoting selective translation of genes involved in acrosome reaction and zygote development, challenging prior views of sperm translational quiescence.^[24] Protective mechanisms within these pathways prevent premature acrosome reaction (AR), ensuring sperm reach the oocyte. Actin polymerization into F-actin during capacitation, driven by PKA inhibition of phospholipase C and activation of Fer kinase, stabilizes the acrosomal region and suppresses spontaneous AR by limiting Ca²⁺ overload via CatSper regulation.^[25] 2023 evidence shows that disrupting this polymerization (e.g., via jasplakinolide) triggers untimely AR, while PKA-dependent cortactin and Arp2/3 complex activation reinforces cytoskeletal integrity, with CaMKII further buffering Ca²⁺ signals to maintain AR timing.^[25]

Induction Processes

In Vivo Induction in the Female Tract

Capacitation of mammalian spermatozoa occurs primarily within the oviduct and uterus of the female reproductive tract, where sperm transit facilitates the removal of inhibitory factors from seminal plasma that maintain sperm in a decapacitated state.^[2] Ejaculated sperm exposed to seminal plasma undergo decapacitation, but entry into the female tract reverses this by diluting and eliminating these inhibitors, such as high zinc concentrations and prostasomes, through fluid dynamics and binding interactions.^[1]^[2] Key environmental cues in the female tract initiate capacitation through specific secretory components. Bicarbonate ions (HCO₃⁻) abundant in oviductal and uterine fluids elevate intracellular pH, activating soluble adenylyl cyclase to produce cAMP and trigger downstream signaling.^[2] Albumin present in these fluids binds and effluxes cholesterol from the sperm plasma membrane, increasing fluidity and permeability essential for the process.^[2] Additionally, uterine enzymes, including glycosidases, modify sperm surface glycoproteins by cleaving sugar residues, further altering membrane properties to support capacitation.^[2] The timing of in vivo capacitation typically spans 1-6 hours after insemination, allowing sperm to progressively mature as they ascend the tract and interact with these cues.^[2] In humans, the fallopian tube epithelium plays a pivotal role, with ciliated cells aiding sperm transport and secretions providing localized concentrations of bicarbonate, albumin, and other factors to facilitate capacitation.^[1] This process ultimately enables calcium (Ca²⁺) influx critical for hyperactivated motility.^[2]

Key Inducers and Regulatory Factors

Capacitation in mammalian spermatozoa is primarily induced by bicarbonate (HCO₃⁻), calcium ions (Ca²⁺), and albumin, which collectively trigger essential membrane and metabolic changes. HCO₃⁻ enters sperm cells via Na⁺/HCO₃⁻ cotransporters (NBC) and Cl⁻/HCO₃⁻ exchangers (SLC26), elevating intracellular pH and activating adenylyl cyclase to initiate cAMP-dependent signaling.^[2] Ca²⁺ influx, often facilitated through the sperm-specific CatSper channel, supports hyperactivated motility and acrosomal readiness by modulating ion gradients and protein phosphorylation.^[26] Albumin acts as a cholesterol acceptor, promoting cholesterol efflux from the sperm plasma membrane to enhance fluidity and facilitate HCO₃⁻-induced effects, while also regulating ATP pools independently of HCO₃⁻.^[27] Several regulators enhance capacitation by amplifying these inductive signals, particularly through protein kinase A (PKA) pathways. Progesterone binds to sperm surface receptors, triggering rapid Ca²⁺ influx via CatSper and accelerating PKA-mediated capacitation completion, which increases the proportion of spermatozoa capable of zona pellucida-induced acrosome reaction.^[28] Seminal plasma contains decapacitation factors that inhibit premature capacitation to preserve sperm fertility until reaching the female tract. These include proteins from seminal vesicles, such as seminal vesicle proteins (SVS2, SVS3, and SVS4), which bind to spermatozoa and stabilize the plasma membrane by preventing cholesterol efflux and ion perturbations, thereby reversibly blocking capacitation signals.^[29] Recent advances highlight the role of Na⁺-dependent transporters in fine-tuning capacitation through pH regulation and membrane hyperpolarization. A 2024 review emphasizes that Na⁺/H⁺ exchangers like NHE1, activated by Ca²⁺ influx during capacitation, drive intracellular alkalization to support hyperpolarization of the sperm membrane potential, which is essential for CatSper gating and hyperactivated motility.^[26] Complementary 2024 studies on secondary active transporters, including the sperm-specific Na⁺/H⁺ exchanger (sNHE) and Na⁺/Ca²⁺ exchanger (NCX1), demonstrate their involvement in reducing intracellular Na⁺ and promoting K⁺ efflux via SLO3 channels, thereby modulating hyperpolarization and preventing over-alkalinization that could impair fertility.^[30]^[31] These transporters integrate with primary inducers to ensure timed progression of capacitation events.

In Vitro Techniques

Traditional Capacitation Protocols

Traditional capacitation protocols for in vitro induction primarily involve the use of defined synthetic media that replicate key ionic and energetic components of the female reproductive tract to promote sperm hyperactivation, cholesterol efflux, and protein tyrosine phosphorylation. A widely adopted medium is Tyrode's albumin lactate pyruvate (TALP), initially formulated for bovine sperm and containing 114 mM NaCl, 3.2 mM KCl, 0.3 mM NaH₂PO₄, 0.4 mM MgSO₄·7H₂O, 21.6 mM Na-lactate, 1 mM Na-pyruvate, 25 mM NaHCO₃, 2 mM CaCl₂·2H₂O, and 3-6 mg/mL bovine serum albumin (BSA) as a cholesterol acceptor. In human IVF, human tubal fluid (HTF) medium is standard, composed of 101.6 mM NaCl, 4.69 mM KCl, 0.2 mM MgSO₄·7H₂O, 1.19 mM KH₂PO₄, 2.04 mM CaCl₂·2H₂O, 21.4 mM Na-lactate, 0.2 mM Na-pyruvate, 21.4 mM NaHCO₃, and supplemented with 3 mg/mL BSA; bicarbonate levels range from 5-25 mM, calcium at 2 mM, and BSA at 3 mg/mL across protocols to facilitate essential signaling. These components, particularly bicarbonate and calcium, are critical for activating soluble adenylyl cyclase and cAMP-dependent pathways during capacitation.^[32] Sperm preparation begins with selection of motile spermatozoa via swim-up or density gradient centrifugation using Percoll. In the swim-up method, semen is placed beneath a layer of medium (e.g., HTF), allowing progressively motile sperm to migrate upward over 30-60 minutes at 37°C, yielding a population enriched for high-quality cells.^[33] Alternatively, Percoll gradient centrifugation separates sperm by density through discontinuous layers (e.g., 45% and 90% Percoll), isolating viable sperm in the 45-90% interface after 15-20 minutes at 600g, which reduces debris and abnormal forms more effectively than swim-up in suboptimal samples.^[34] Selected sperm (typically 10-20 × 10⁶/mL) are then resuspended in capacitation medium and incubated for 3-6 hours at 37°C under 5% CO₂ in air to equilibrate pH and induce physiological changes.^[35] These protocols are routinely applied in IVF to prepare sperm for oocyte insemination, supporting fertilization rates of 50-80% in human conventional IVF cycles, though outcomes vary with oocyte quality and insemination conditions.^[36] Albumin in the media serves as a key inducer by scavenging cholesterol from the sperm membrane, as detailed in studies of regulatory factors. A primary limitation is high inter-ejaculate variability, where initial semen parameters like motility and DNA fragmentation influence capacitation efficiency and subsequent fertilization success.^[37]

Emerging Methods and Optimizations

Recent advancements in in vitro capacitation have introduced advanced sperm selection techniques to enhance the quality of spermatozoa used in assisted reproductive technologies (ART). Physiological intracytoplasmic sperm injection (PICSI) employs hyaluronan binding to select mature, competent sperm, as hyaluronan mimics the natural oocyte-cumulus matrix interactions that favor capacitated cells with intact chromatin and low DNA fragmentation.^[38] This method improves sperm maturity assessment by identifying those capable of binding hyaluronan, thereby reducing the risk of injecting immature or damaged sperm during ICSI.^[39] Similarly, magnetic-activated cell sorting (MACS) targets the removal of apoptotic sperm by using annexin V-conjugated magnetic beads to bind externalized phosphatidylserine on early apoptotic cells, isolating viable, non-apoptotic populations suitable for capacitation.^[40] MACS effectively depletes sperm with high DNA fragmentation and caspase activity, preserving a cohort with enhanced viability for downstream fertilization processes.^[41] Innovative tools have emerged to simulate physiological conditions more accurately during in vitro capacitation. Microfluidic devices, particularly those developed in 2023 studies, replicate rheological gradients of the female reproductive tract, such as structural cues mimicking the uterotubal junction, to promote directed sperm motility and reduce stress-induced damage without external stressors.^[42] These biomimetic platforms use laminar flow and rheotaxis to sort progressively motile sperm, achieving higher recovery of capacitated cells compared to traditional methods. Additionally, nanoparticle-based supplements have been explored for targeted delivery of signaling molecules to induce capacitation. Progesterone-loaded solid lipid nanoparticles, for instance, enhance acrosome reaction and hyperactivated motility in asthenozoospermic sperm by facilitating progesterone's role in calcium influx and protein tyrosine phosphorylation.^[43] Optimizations in capacitation media focus on mitigating oxidative stress and boosting energy metabolism. The addition of antioxidants like flavones has shown promise in canine sperm cryopreservation, where flavone and 3-hydroxyflavone supplementation reduces lipid peroxidation and apoptosis, maintaining membrane integrity and capacitation competence post-thaw.^[44] In 2024 studies, these flavones preserved motility and viability by scavenging reactive oxygen species, supporting sustained ATP levels during capacitation.^[45] Metabolic modulators, such as those targeting glycolytic shifts, enhance ATP production to meet the increased energy demands of hyperactivation; for example, supplementation with substrates like glucose promotes a transition from oxidative to glycolytic metabolism in murine sperm, optimizing capacitation efficiency.^[46] These emerging methods have led to measurable improvements in ART outcomes, including higher fertilization and live birth rates through better sperm selection and induction. PICSI and MACS integration has been associated with enhanced implantation and clinical pregnancy rates in ICSI cycles, particularly for patients with sperm DNA damage.^[47] Refinements in phosphotyrosine (PTyr) detection for boar sperm, as detailed in 2025 analyses, address methodological variability in assessing capacitation status, enabling more precise monitoring of tyrosine phosphorylation patterns as a marker of readiness.^[48] Overall, these optimizations yield superior embryo quality and fertilization success in species-specific protocols.

Assessment Methods

Functional Assays for Motility and Readiness

Functional assays for motility and readiness evaluate sperm capacitation through observable behavioral changes and fertilization competence, focusing on dynamic responses rather than static markers. These tests measure hyperactivation—a hallmark of capacitation involving asymmetric, high-amplitude flagellar movements that facilitate zona pellucida penetration—and overall preparedness for egg interaction. By quantifying these traits, assays provide insights into sperm fertilizing potential without altering cellular integrity. Computer-assisted sperm analysis (CASA) employs video microscopy and software algorithms to objectively track sperm trajectories, distinguishing hyperactivated motion from progressive or immotile patterns during capacitation. Key parameters often include curvilinear velocity (VCL >100–150 μm/s), indicating rapid, non-linear path speed; amplitude of lateral head displacement (ALH >5–7.5 μm), reflecting wide lateral swings; and beat-cross frequency (BCF >20 Hz), measuring flagellar beat rate in hyperactivated cells, though these thresholds vary by species and CASA system.^[49]^[50] These parameters correlate with increased sperm penetration ability in assisted reproduction. During capacitation, a subset of sperm shifts to hyperactivated motility, enhancing oviductal transport and egg binding as part of broader membrane alterations. Chlortetracycline (CTC) staining assesses capacitation via fluorescence patterns tied to intracellular calcium redistribution and membrane stability. Fresh, non-capacitated sperm exhibit an F pattern—uniform head fluorescence with a bright equatorial segment—indicating acrosome-intact, uncapacitated status. Upon capacitation, sperm transition to a B pattern, featuring a dark post-acrosomal band and bright anterior head fluorescence, signifying membrane destabilization and readiness for acrosome reaction while remaining acrosome-intact. This assay, first developed for mouse sperm, has been adapted for human and other species, with the F-to-B shift occurring within 1–2 hours in vitro.^[51] Zona pellucida binding assays test sperm adhesion to the egg's extracellular matrix, a functional endpoint of capacitation that requires hyperactivated motility and surface modifications for species-specific recognition. Capacitated sperm bind tightly to immobilized zonae from surplus oocytes, with binding ratios (sperm per zona) reflecting fertilizing capacity; low binding predicts poor outcomes. In human IVF, higher binding correlates independently with fertilization rates, outperforming basic semen parameters in cases of teratozoospermia.^[52] These assays offer advantages as non-invasive tools that preserve sperm viability for downstream use in IVF, directly linking motility dynamics and binding proficiency to clinical success rates, with studies showing positive correlations to fertilization outcomes.^[53]^[54]

Molecular Markers and Detection Techniques

One of the primary molecular markers of sperm capacitation is the increased tyrosine phosphorylation (pY) of proteins, which rises 2- to 10-fold during the process due to activation of the cAMP/PKA pathway.^[55] This marker reflects hyperactivated signaling essential for fertilization competence and is detected through phosphorylation assays such as Western blot, which quantifies pY levels in sperm extracts using anti-phosphotyrosine antibodies, or immunofluorescence microscopy to visualize phosphorylated proteins in situ.^[55] These techniques allow for the identification of specific substrates like AKAP3 and p32, which are upregulated during capacitation in species such as humans and boars.^[55] Fluorescence-based probes provide dynamic insights into capacitation-associated changes, including acrosome integrity and ion fluxes. FITC-conjugated peanut agglutinin (FITC-PNA) binds to the outer acrosomal membrane, enabling detection of acrosome reaction status via flow cytometry or microscopy, where intact acrosomes show strong fluorescence that diminishes post-reaction.^[56] Similarly, Fluo-4 AM serves as a calcium-sensitive indicator for live-cell imaging of Ca²⁺ influx, a hallmark of capacitation that triggers downstream events like hyperactivation; this probe exhibits a marked increase in fluorescence intensity upon Ca²⁺ binding, quantifiable through confocal microscopy or spectrofluorometry.^[56] Recent advances include phosphotyrosine (PTyr) flow cytometry for high-throughput analysis in boar sperm, where optimized protocols using anti-PTyr antibodies and fixation methods (e.g., formaldehyde-methanol) detect PTyr patterns with reduced variability, identifying up to 10 differentially phosphorylated proteins linked to capacitation.^[57] Transcriptomic profiling via RNA-seq has also emerged, revealing differentially expressed mRNAs and miRNAs in capacitated boar sperm, such as those in PI3K-Akt and cAMP-PKA pathways, with over 5,000 mRNAs altered to support gene activation for motility and binding.^[58] Despite these tools, challenges persist in marker detection, including inter-sample variability due to sperm handling and media composition, which can lead to inconsistent PTyr signals across populations.^[57] False positives may arise from non-specific antibody binding or over-capacitation in vitro, necessitating standardized protocols and complementary markers like Ca²⁺ levels to validate results.^[57]

Historical Development

Initial Discovery and Early Studies

The discovery of capacitation emerged from independent experiments in 1951 by Min Chueh Chang and Colin Russell Austin, who demonstrated that mammalian spermatozoa require a period of residence in the female reproductive tract to acquire fertilizing capacity. Chang's work with rabbits showed that freshly ejaculated sperm deposited directly into the fallopian tubes failed to fertilize ova if introduced simultaneously with ovulation, but achieved fertilization when deposited 6-8 hours earlier, allowing time for physiological maturation in the tract. Similarly, Austin's studies on hamster sperm revealed that insemination into the female tract was necessary for successful fertilization, as sperm transferred directly to ova in vitro or from the male tract lacked this ability. These findings established capacitation as a prerequisite step distinct from sperm motility or ejaculation, coining the term in Austin's 1952 follow-up.^[59] Key experiments further elucidated the site's specificity for capacitation. In rabbits, Chang demonstrated that epididymal or ejaculated sperm remained infertile when placed in the male reproductive tract or simple saline but gained fertilizing potential after 5-6 hours in the female uterus or fallopian tubes, even in hormone-modified conditions like ovariectomized or estrogen-treated animals. Early attempts at in vitro induction used uterine fluid extracts from estrous females, which mimicked the tract's environment and enabled partial capacitation in rabbit sperm, though full efficacy required co-culture refinements in the 1960s.^[60] These in vivo contrasts highlighted the female tract's unique role in altering sperm physiology, paving the way for understanding environmental cues. Initial research faced significant challenges, including the absence of defined culture media, which forced reliance on undefined biological fluids like blood serum or uterine secretions, complicating reproducibility and mechanistic insights. Additionally, capacitation was often conflated with the acrosome reaction—a later-identified event involving enzymatic release for zona penetration—discovered by Austin and Bishop in 1958 using phase-contrast microscopy on penetrating sperm. This overlap led to interpretive errors until the 1960s, when distinct assays separated the preparatory capacitation from the triggered acrosome response. A major milestone in the 1970s was the identification of cholesterol's role in capacitation, with studies showing that efflux of membrane cholesterol, facilitated by albumin in uterine fluids, increased membrane fluidity and initiated signaling cascades essential for sperm competence. Davis's work demonstrated that cholesterol depletion from rat and rabbit sperm plasma membranes correlated with enhanced fertilizing ability, linking lipid dynamics to the process's biochemistry.

Modern Insights and Research Advances

During the 1980s and 1990s, the cloning of key genes involved in sperm signaling advanced the understanding of capacitation mechanisms. The soluble adenylyl cyclase (sAC), encoded by the ADCY10 gene, was cloned in 1999, revealing its role as a bicarbonate-activated enzyme essential for cAMP production in sperm, which initiates downstream signaling for motility and acrosome reaction. Similarly, the CatSper1 gene, encoding a sperm-specific calcium channel, was cloned in 2001, demonstrating its necessity for hyperactivated motility during capacitation; subsequent studies through the 2010s identified additional CatSper subunits (2-4), forming a heterotetrameric channel complex critical for calcium influx. Elucidation of the protein kinase A (PKA) pathway in the 1990s and 2000s further clarified capacitation signaling. Seminal work in 1995 established that bicarbonate stimulates sAC to elevate cAMP levels, activating PKA and leading to protein tyrosine phosphorylation, a hallmark of capacitation; this pathway was confirmed across species, with PKA anchoring proteins localizing the kinase to sperm flagella for targeted phosphorylation events.^[2] Recent research from 2020 to 2025 has highlighted the role of sodium transporters in capacitation. A 2024 review detailed how hyperpolarizing Na⁺ dynamics, mediated by Na⁺/Ca²⁺ exchangers (NCX1) and Na⁺/H⁺ exchangers (NHEs), facilitate membrane hyperpolarization and calcium homeostasis essential for hyperactivation, with inhibition of these transporters blocking capacitation in mammalian models.^[26] Transcriptomic studies in 2025 revealed that capacitation induces 337 differentially expressed genes in human sperm, linking these changes to epigenetic modifications such as altered DNA methylation patterns that influence fertility competence.^[61] Metabolic rewiring investigations in 2023 demonstrated that capacitation enhances flux through glycolysis and oxidative phosphorylation in mouse and human sperm, supporting increased ATP demands for hyperactivated motility without compromising viability.^[62] Clinically, capacitation defects have been implicated in asthenozoospermia, a common cause of male infertility. A 2025 update on genetic etiologies showed that mutations in sAC and CatSper genes lead to impaired motility and capacitation, resulting in sterility; mouse models of these deficiencies mirror human phenotypes, underscoring their diagnostic relevance.^[63] In assisted reproductive technologies (ART), microfluidic optimizations have improved outcomes by selecting capacitated sperm with intact DNA; a 2025 meta-analysis reported that microfluidic sorting reduces DNA fragmentation by up to 50% compared to conventional methods, enhancing fertilization rates in intracytoplasmic sperm injection (ICSI) procedures.^[64] Looking forward, CRISPR/Cas9 gene editing has enabled precise infertility models to study capacitation. Reviews from 2022 onward highlight its use in knocking out testis-enriched genes like those in the CatSper complex, revealing dispensable versus essential loci for fertility and paving the way for therapeutic targets in idiopathic infertility.^[65] Additionally, artificial intelligence (AI) integration in computer-assisted sperm analysis (CASA) promises refined capacitation assessment; a 2025 review emphasized AI-enhanced CASA systems that accurately quantify hyperactivation and tyrosine phosphorylation via machine learning, improving diagnostic precision over traditional metrics.^[66]

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Apr 11, 2016 · Now we know that in the U.S., about 40% to 50% of infertility involves the male partner. In some other countries, the problem is even higher.
[19]
Regulation of human sperm capacitation by a cholesterol efflux ...
Capacitation has been shown to be correlated with changes in sperm plasma membrane fluidity, intracellular ion concentrations, metabolism and motility ...
[20]
Central role of soluble adenylyl cyclase and cAMP in sperm ...
This review will be centered on the role of cAMP and those enzymes involved in cAMP signaling pathways in mammalian sperm.
[21]
Role of tyrosine phosphorylation in sperm capacitation / acrosome ...
The increase in protein tyrosine phosphorylation during capacitation has been shown to be regulated by a cAMP-dependent pathway involving protein kinase A (PKA) ...Missing: adenylyl | Show results with:adenylyl
[22]
Mathematical modeling of calcium signaling during sperm ... - NIH
The major mechanism for providing Ca2+ to the flagellum, at least in mice, are CatSper channels in the plasma membrane of the principal piece of the flagellum, ...Missing: HCO3- | Show results with:HCO3-
[23]
Capacitation induces changes in metabolic pathways supporting ...
Jun 26, 2023 · Capacitation induces changes in metabolic pathways supporting motility of epididymal and ejaculated sperm.
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Systematic mRNA interactome analysis reconceptualizes ... - NIH
Oct 3, 2025 · Ejaculated mammalian sperm must undergo a series of biochemical changes called capacitation to gain fertilizing competence.Missing: transcriptomic | Show results with:transcriptomic
[25]
Mechanisms That Protect Mammalian Sperm from the Spontaneous ...
Nov 30, 2023 · Several biochemical reactions that occur during sperm capacitation, including actin polymerization, protect sperm from sAR.
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Molecular mechanisms of mammalian sperm capacitation, and its ...
Oct 16, 2024 · Capacitation is a collective term used to describe the physiological and biochemical changes necessary for spermatozoa to acquire fertilization competency.
[27]
Serum albumin and HCO3 regulate separate pools of ATP in human ...
Feb 26, 2014 · Recent findings have demonstrated that, in addition, serum albumin activates the sperm- specific Ca2+ channel CatSper (Xia and Ren, 2009).Missing: inducers | Show results with:inducers
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Time to revise: impact of methodology on boar sperm capacitation in ...
Jul 7, 2025 · Capacitation is a sperm maturation process that occurs in vivo within the female genital tract. During capacitation, the spermatozoa undergo ...<|control11|><|separator|>
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Progesterone Accelerates the Completion of Sperm Capacitation ...
Progesterone (1 μM) induced up to a 3-fold increase in the percentage of sperm undergoing the zona pellucida-induced acrosome reaction with the lowest threshold ...
[30]
Heparin-binding proteins from seminal plasma bind to bovine ...
Bovine spermatozoa that have been exposed to seminal plasma possess more binding sites for heparin than sperm from the cauda epididymis that have not been ...
[31]
Bovine In vitro fertilization: In vitro oocyte maturation and sperm ...
The binding of heparin is to a series of bovine seminal plasma proteins (BSPs), that bind to epididymal sperm at ejaculation [30]. These proteins include BSP-A1 ...
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Semen-Coagulating Protein, SVS2, in Mouse Seminal Plasma ...
Aug 9, 2025 · Mammalian seminal plasma is known to contain a decapacitation factor(s) that prevents capacitation and thus, the fertility of sperm.
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The modulation of sperm function by fertilization promoting peptide
Fertilization promoting peptide (FPP; pGlu-Glu-ProNH2) is a peptide produced by the prostate gland and then secreted into seminal plasma.Missing: decapacitation factors SVA
[34]
The sodium–proton exchangers sNHE and NHE1 control plasma ...
In this study, we examined the impact of Na+/H+ exchangers (NHEs) on mouse sperm membrane hyperpolarization during capacitation. Pharmacological inhibition of ...
[35]
The sodium-proton exchangers sNHE and NHE1 control plasma ...
Mar 6, 2024 · Therefore, upon the increase in intracellular Ca2+ associated with capacitation, NHE1 could drive pHi to a more alkaline state. In human sperm, ...
[36]
Manipulation of bicarbonate concentration in sperm capacitation ...
Mar 11, 2019 · A concentration of 15 mmol/L of HCO3− in the medium is sufficient to trigger the in vitro sperm capacitation and increase the fertilisation ...
[37]
Cumulative live birth rates after IVF/ICSI cycles with sperm prepared ...
Mar 31, 2022 · Swim-up (SU) and density gradient centrifugation (DGC) are the two most widely used sperm preparation methods for ART. Both methods are ...Missing: traditional | Show results with:traditional
[38]
Comparative auto-controlled study between swim-up and Percoll ...
This study compared swim-up and Percoll preparation of fresh semen samples for in-vitro fertilization. Sixty trials of in-vitro fertilization (IVF), ...Missing: human | Show results with:human
[39]
Preparation and incubation conditions affect the DNA integrity ... - NIH
In general, spermatozoa are usually incubated at 37°C in an atmosphere of 5% CO2 for at least 1–2 h before intracytoplasmic sperm injection or conventional ...
[40]
Fertilization rate as a novel indicator for cumulative live birth rate
In most IVF programs, on average, fertilization rates of 70%–75% are not uncommon. However, among individual cycles, a large variability in fertilization rates ...Original Article · Results · DiscussionMissing: traditional capacitation
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Limitations of Semen Analysis as a Test of Male Fertility and ... - NIH
Routine semen analysis does not measure the fertilizing potential of spermatozoa and the complex changes that occur in the female reproductive tract before ...
[42]
A Comprehensive Comparison of PICSI and ICSI Techniques ... - NIH
May 1, 2025 · PICSI is a sperm selection technique designed to facilitate a more targeted evaluation of the semen sample, thereby improving the quality of ...
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Use of Hyaluronan in the Selection of Sperm for Intracytoplasmic ...
The cohort studies indicated a significant advantage of HA-ICSI in terms of LBR, CPR, BPR, IR, FR, and good-quality embryo rates.
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Magnetic-Activated Cell Sorting (MACS): A Useful Sperm-Selection ...
Dec 8, 2020 · Magnetic-activated cell sorting (MACS) can be used to separate apoptotic sperm with high proportions of fragmented DNA from the rest, ...Missing: 2020-2025 | Show results with:2020-2025
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The Use of Non-Apoptotic Sperm Selected by Magnetic Activated ...
MACS enables the removal of apoptotic sperm from an ejaculate, thus leaving the non-apoptotic available to be microinjected.
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Sperm quality metrics were improved by a biomimetic microfluidic ...
Mar 28, 2023 · We demonstrate a 3D printed, biologically inspired microfluidic sperm selection device (MSSP) that utilizes multiple methods to simulate a sperms journey ...
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Effects of progesterone nanoparticles on the sperm capacitation and ...
Oct 5, 2021 · Findings in this study showed that progesterone solid lipid nanoparticles increase acrosomal reaction, sperm capacity and motility.
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Flavone and 3-hydroxyflavone supplementation in cryopreservation ...
Sep 15, 2024 · Our study aimed to assess the efficacy of flavone (FL) and 3-hydroxyflavone (3-OH = ) supplementation in the cryopreservation medium to protect canine sperm.
[49]
Flavone and 3-hydroxyflavone supplementation in cryopreservation ...
Sep 15, 2024 · Flavone and 3-hydroxyflavone supplementation in cryopreservation medium protects canine sperm against apoptosis and lipid peroxidation. ...
[50]
Capacitation promotes a shift in energy metabolism in murine sperm
Our results show that capacitation promotes a shift in the usage ratio of the two main metabolic pathways, from oxidative to glycolytic.
[51]
Sperm selection with hyaluronic acid improved live birth outcomes ...
Apr 23, 2022 · With ICSI, the sperm is injected directly into the egg, bypassing many of the natural barriers that would normally prevent the entry of abnormal ...
[52]
Computer‐aided sperm analysis: past, present and future - Lu - 2014
Apr 1, 2013 · Because different CASA instruments compute ALH using different algorithms, the values of ALH may usually not be comparable among CASA systems.
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Determination of the time course of capacitation in mouse ... - PubMed
Determination of the time course of capacitation in mouse spermatozoa using a chlortetracycline fluorescence assay. Dev Biol. 1984 Aug;104(2):287-96. doi ...Missing: staining | Show results with:staining
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A sperm-zona pellucida binding test and in vitro fertilization - PubMed
In conclusion, the sperm-zona pellucida binding ratio test is useful for predicting sperm fertilizing ability in vitro, particularly in men with poor sperm ...Missing: capacitation readiness success
[55]
Sperm function tests in clinical practice - PMC - NIH
To examine the predictive value of sperm zona pellucida binding assay for IVF outcomes, Results demonstrated a high predictive power of the sperm zona ...
[56]
Sperm function and assisted reproduction technology - PMC
These assays determine the sperm's capability to fertilize an oocyte. It also appears that sperm functions and semen parameters are interrelated and ...
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The 'Capacitation' of the Mammalian Sperm - Nature
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Sperm Capacitation by Uterine Fluid or Beta-Amylase in vitro - Science
Rabbit sperm developed the capacity to fertilize ova when incubated in utero or in vitro in fluids from estrous uteri. Incubation of sperm with beta-amylase ...Missing: early | Show results with:early
[63]
Sperm capacitation triggers transcriptomic changes linked to the ...
Aug 21, 2025 · Our findings revealed that sperm capacitation is associated with transcriptomic changes, characterized by 337 differential gene transcripts.Missing: mRNA interactome
[64]
Capacitation induces changes in metabolic pathways supporting ...
Jun 27, 2023 · Capacitation induces changes in metabolic pathways supporting motility of epididymal ... differences or reflect the distinct maturation states of ...
[65]
Human asthenozoospermia: Update on genetic causes, patient ...
Jan 2, 2025 · As a result, sAC deficiency in mice was shown to result in male sterility because of sperm motility and capacitation defects, precluding sperm ...
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Sperm Selection Using Microfluidic Techniques Significantly ... - NIH
Jun 30, 2025 · This meta-analysis compiled all available studies evaluating the impact of microfluidic techniques on semen parameters and reproductive outcomes ...Missing: optimizations | Show results with:optimizations
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Application of CRISPR/Cas Technology in Spermatogenesis ...
This review summarizes the applications of gene editing technologies, especially CRISPR/Cas9, in deepening our understanding of the function of spermatogenesis.
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Revolutionizing Sperm Analysis with AI: A Review of Computer ...
This review focuses on integrating AI with Computer-Aided Sperm Analysis (CASA) systems to improve assessments of sperm motility, morphology, and DNA integrity.