RESUMO
The oxidation of the cellular membrane through lipid peroxidation (LPO) is linked to aging and disease. Despite the physiological importance, the chemical mechanisms underlying LPO and oxidative reactions in membranes in general remain incompletely understood, and challenges exist in translating LPO inhibitor efficacies from in vitro to in vivo. The complexity of LPO, including multiple oxidation reactions in complex membrane environments and the difficulty in quantifying reaction kinetics, underlies these difficulties. In this work, we developed a robust and straightforward method for quantifying the oxidation rate kinetics of fluorescent molecules and determined the oxidation kinetics of widely fluorophores used as indicators of membrane LPO, diphenylhexatriene (DPH), BODIPY-C11, and Liperfluo. The measurement is initiated by lipoxygenase, which provides chemical specificity and enables a straightforward interpretation of oxidation kinetics. Our results reveal that the membrane composition significantly impacts the observed kinetics oxidation in DPH and BODIPY-C11 but not Liperfluo. Reaction mechanisms for their lipid peroxide-induced oxidation are proposed. This work provides a foundation for the quantitative analysis of LPO with fluorescence and extricating the complexity of oxidation reactions within membranes.
Assuntos
Compostos de Boro , Corantes Fluorescentes , Peroxidação de Lipídeos , Oxirredução , Cinética , Corantes Fluorescentes/química , Compostos de Boro/química , Membrana Celular/metabolismo , Difenilexatrieno/química , Difenilexatrieno/análogos & derivados , Lipídeos de Membrana/metabolismo , Lipídeos de Membrana/química , Humanos , Lipoxigenase/metabolismoRESUMO
The primary constituents of honeybee venom, melittin and phospholipase A2 (PLA2), display toxin synergism in which the PLA2 activity is significantly enhanced by the presence of melittin. It has been shown previously that this is accomplished by the disruption in lipid packing, which allows PLA2 to become processive on the membrane surface. In this work, we show that melittin is capable of driving miscibility phase transition in giant unilamellar vesicles (GUVs) and that it raises the miscibility transition temperature (Tmisc) in a concentration-dependent manner. The induced phase separation enhances the processivity of PLA2, particularly at its boundaries, where a substantial difference in domain thickness creates a membrane discontinuity. The catalytic action of PLA2, in response, induces changes in the membrane, rendering it more conducive to melittin binding. This, in turn, facilitates further lipid phase separation and eventual vesicle lysis. Overall, our results show that melittin has powerful membrane-altering capabilities that activate PLA2 in various membrane contexts. More broadly, they exemplify how this biochemical system actively modulates and capitalizes on the spatial distribution of membrane lipids to efficiently achieve its objectives.
Assuntos
Venenos de Abelha , Meliteno , Meliteno/farmacologia , Lipossomas Unilamelares , Fosfolipases A2 , Lipídeos de MembranaRESUMO
Desmosterol and cholesterol are essential lipid components of the sperm plasma membrane. Cholesterol efflux is required for capacitation, a process through which sperm acquire fertilizing ability. In this study, using a transgenic mouse model overexpressing 24-dehydrocholesterol reductase (DHCR24), an enzyme in the sterol biosynthesis pathway responsible for the conversion of desmosterol to cholesterol, we show that disruption of sterol homeostasis during spermatogenesis led to defective sperm morphology characterized by incomplete mitochondrial packing in the midpiece, reduced sperm count and motility, and a decline in male fertility with increasing paternal age, without changes in body fat composition. Sperm depleted of desmosterol exhibit inefficiency in the acrosome reaction, metabolic dysfunction, and an inability to fertilize the egg. These findings provide molecular insights into sterol homeostasis for sperm capacitation and its impact on male fertility.
RESUMO
Radial spokes (RS) are T-shaped multiprotein complexes on the axonemal microtubules. Repeated RS1, RS2, and RS3 couple the central pair to modulate ciliary and flagellar motility. Despite the cell type specificity of RS3 substructures, their molecular components remain largely unknown. Here, we report that a leucine-rich repeat-containing protein, LRRC23, is an RS3 head component essential for its head assembly and flagellar motility in mammalian spermatozoa. From infertile male patients with defective sperm motility, we identified a splice site variant of LRRC23. A mutant mouse model mimicking this variant produces a truncated LRRC23 at the C-terminus that fails to localize to the sperm tail, causing male infertility due to defective sperm motility. LRRC23 was previously proposed to be an ortholog of the RS stalk protein RSP15. However, we found that purified recombinant LRRC23 interacts with an RS head protein RSPH9, which is abolished by the C-terminal truncation. Evolutionary and structural comparison also shows that LRRC34, not LRRC23, is the RSP15 ortholog. Cryo-electron tomography clearly revealed that the absence of the RS3 head and the sperm-specific RS2-RS3 bridge structure in LRRC23 mutant spermatozoa. Our study provides new insights into the structure and function of RS3 in mammalian spermatozoa and the molecular pathogenicity of LRRC23 underlying reduced sperm motility in infertile human males.
Assuntos
Infertilidade Masculina , Motilidade dos Espermatozoides , Camundongos , Animais , Masculino , Humanos , Sêmen , Axonema/metabolismo , Cauda do Espermatozoide , Proteínas/metabolismo , Espermatozoides , Infertilidade Masculina/genética , Flagelos/metabolismo , MamíferosRESUMO
A water-compatible and recyclable catalyst for nuclear magnetic resonance (NMR) hyperpolarization via signal amplification by reversible exchange (SABRE) was developed. The [Ir(COD)(IMes)Cl] catalyst was attached to a polymeric resin of bis(2-pyridyl)amine (heterogeneous SABRE catalyst, HET-SABRE catalyst), and it amplified the 1H NMR signal of pyridine up to (-) 4455-fold (43.2%) at 1.4 T in methanol and (-) 50-fold (0.5%) in water. These are the highest amplification factors ever reported among HET-SABRE catalysts and for the first time in aqueous media. Moreover, the HET-SABRE catalyst demonstrated recyclability by retaining its activity in water after more than three uses. This newly designed polymeric resin-based heterogeneous catalyst shows great promise for NMR signal amplification for biomedical NMR and MRI applications in the future.
RESUMO
Calcium signaling is critical for successful fertilization. In spermatozoa, calcium influx into the sperm flagella mediated by the sperm-specific CatSper calcium channel is necessary for hyperactivated motility and male fertility. CatSper is a macromolecular complex and is repeatedly arranged in zigzag rows within four linear nanodomains along the sperm flagella. Here, we report that the Tmem249-encoded transmembrane (TM) domain-containing protein, CATSPERθ is essential for the CatSper channel assembly during sperm tail formation. CATSPERθ facilitates the channel assembly by serving as a scaffold for a pore-forming subunit CATSPER4. CATSPERθ is specifically localized at the interface of a CatSper dimer and can self-interact, suggesting its potential role in CatSper dimer formation. Male mice lacking CATSPERθ are infertile because the sperm lack the entire CatSper channel from sperm flagella, rendering sperm unable to hyperactivate, regardless of their normal expression in the testis. In contrast, genetic abrogation of any of the other CatSper TM subunits results in loss of CATSPERθ protein in the spermatid cells during spermatogenesis. CATSPERθ might act as a checkpoint for the properly assembled CatSper channel complex to traffic to sperm flagella. This study provides insights into the CatSper channel assembly and elucidates the physiological role of CATSPERθ in sperm motility and male fertility.
Assuntos
Sêmen , Motilidade dos Espermatozoides , Animais , Masculino , Camundongos , Membrana Celular , Canais Iônicos , Proteínas de Membrana/genética , Proteínas de Plasma Seminal , Motilidade dos Espermatozoides/genética , Cauda do Espermatozoide , EspermatozoidesRESUMO
Rufinamide, possessing a triazole ring, is a new antiepileptic drug (AED) relatively well-absorbed in the lower dose range (10 mg/kg per day) and is currently being used in antiepileptic medications. Triazole derivatives can interact with various enzymes and receptors in biological systems via diverse non-covalent interactions, thus inducing versatile biological effects. Strain-promoted azide-alkyne cycloaddition (SPAAC) is a significant method for obtaining triazoles, even under physiological conditions, in the absence of a copper catalyst. To confirm the progress of chemical reactions under biological conditions, research on reaction monitoring at low concentrations is essential. This promising strategy is gaining acceptance for applications in fields such as drug development and nanoscience. We investigated the optimum Ir catalyst and magnetic field for achieving maximum proton hyperpolarization transfer in triazole derivatives. These reactions were analyzed using signal amplification by reversible exchange (SABRE) to overcome the limitations of low sensitivity in nuclear magnetic resonance spectroscopy, when monitoring copper-free click reactions in real time. Finally, a more versatile copper-catalyzed click reaction was monitored in real time, using a 60 MHz benchtop NMR system, in order to analyze the reaction mechanism.
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Climate change is a critical issue of our time, and its causes, pathways, and forecasts remain a topic of broader discussion. In this paper, we present a novel data driven pathway analysis framework to identify the key processes behind mean global temperature and sea level rise, and to forecast the magnitude of their increase from the present to 2100. Based on historical data and dynamic statistical modeling alone, we have established the causal pathways that connect increasing greenhouse gas emissions to increasing global mean temperature and sea level, with its intermediate links encompassing humidity, sea ice coverage, and glacier mass, but not for sunspot numbers. Our results indicate that if no action is taken to curb anthropogenic greenhouse gas emissions, the global average temperature would rise to an estimated 3.28 °C (2.46-4.10 °C) above its pre-industrial level while the global sea level would be an estimated 573 mm (474-671 mm) above its 2021 mean by 2100. However, if countries adhere to the greenhouse gas emission regulations outlined in the 2021 United Nations Conference on Climate Change (COP26), the rise in global temperature would lessen to an average increase of 1.88 °C (1.43-2.33 °C) above its pre-industrial level, albeit still higher than the targeted 1.5 °C, while the sea level increase would reduce to 449 mm (389-509 mm) above its 2021 mean by 2100.
RESUMO
Radial spokes (RS) are T-shaped multiprotein complexes on the axonemal microtubules. Repeated RS1, RS2, and RS3 couple the central pair to modulate ciliary and flagellar motility. Despite the cell type specificity of RS3 substructures, their molecular components remain largely unknown. Here, we report that a leucine-rich repeat-containing protein, LRRC23, is an RS3 head component essential for its head assembly and flagellar motility in mammalian spermatozoa. From infertile male patients with defective sperm motility, we identified a splice site variant of LRRC23. A mutant mouse model mimicking this variant produces a truncated LRRC23 at the C-terminus that fails to localize to the sperm tail, causing male infertility due to defective sperm motility. LRRC23 was previously proposed to be an ortholog of the RS stalk protein RSP15. However, we found that purified recombinant LRRC23 interacts with an RS head protein RSPH9, which is abolished by the C-terminal truncation. Evolutionary and structural comparison also shows that LRRC34, not LRRC23, is the RSP15 ortholog. Cryo-electron tomography clearly revealed that the absence of the RS3 head and the sperm-specific RS2-RS3 bridge structure in LRRC23 mutant spermatozoa. Our study provides new insights into the structure and function of RS3 in mammalian spermatozoa and the molecular pathogenicity of LRRC23 underlying reduced sperm motility in infertile human males.
RESUMO
Calcium signaling is critical for successful fertilization. In spermatozoa, calcium influx into the sperm flagella mediated by the sperm specific CatSper calcium channel is necessary for hyperactivated motility and male fertility. CatSper is a macromolecular complex and is repeatedly arranged in zigzag rows within four linear nanodomains along the sperm flagella. Here, we report that the Tmem249 -encoded transmembrane domain containing protein, CATSPERθ, is essential for the CatSper channel assembly during sperm tail formation. CATSPERθ facilitates the channel assembly by serving as a scaffold for a pore forming subunit CATSPER4. CATSPERθ is specifically localized at the interface of a CatSper dimer and can self-interact, suggesting its potential role in CatSper dimer formation. Male mice lacking CATSPERθ are infertile because the sperm lack the entire CatSper channel from sperm flagella, rendering sperm unable to hyperactivate, regardless of their normal expression in the testis. In contrast, genetic abrogation of any of the other CatSper transmembrane subunits results in loss of CATSPERθ protein in the spermatid cells during spermatogenesis. CATSPERθ might acts as a checkpoint for the properly assembled CatSper channel complex to traffic to sperm flagella. This study provides insights into the CatSper channel assembly and elucidates the physiological role of CATSPERθ in sperm motility and male fertility.
RESUMO
The flagellar-specific Ca2+ channel CatSper is the predominant Ca2+ entry site in mammalian sperm. CatSper-mediated Ca2+ signaling affects nearly every event that regulates sperm to acquire fertilizing capability. In this review, we summarize some of the main findings from 20 years of CatSper research and highlight recent progress and prospects.
Assuntos
Canais de Cálcio , Sêmen , Animais , Humanos , Masculino , Canais de Cálcio/metabolismo , Sêmen/metabolismo , Espermatozoides/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , MamíferosRESUMO
Argonaute 2 (AGO2) is a ubiquitously expressed protein critical for regulation of mRNA translation and vital to animal development. AGO2 protein is found in both cytoplasmic and nuclear compartments, and although its cytoplasmic role is well studied, the biological relevance of nuclear AGO2 is unclear. Here, we address this problem in vivo using spermatogenic cells as a model. We find that AGO2 transiently binds both chromatin and nucleus-specific mRNA transcripts of hundreds of genes required for sperm production during male meiosis in mice, and that germline conditional knockout (cKO) of Ago2 causes depletion of the encoded proteins. Correspondingly, Ago2 cKO males show abnormal sperm head morphology and reduced sperm count, along with reduced postnatal viability of offspring. Together, our data reveal an unexpected nuclear role for AGO2 in enhancing expression of developmentally important genes during mammalian male reproduction.
RESUMO
Thioredoxin/glutathione reductase (TXNRD3) is a selenoprotein composed of thioredoxin reductase and glutaredoxin domains. This NADPH-dependent thiol oxidoreductase evolved through gene duplication within the Txnrd family, is expressed in the testes, and can reduce both thioredoxin and glutathione in vitro; however, the function of this enzyme remains unknown. To characterize the function of TXNRD3 in vivo, we generated a strain of mice bearing deletion of Txnrd3 gene. We show that these Txnrd3 knockout mice are viable and without discernable gross phenotypes, and also that TXNRD3 deficiency leads to fertility impairment in male mice. We found that Txnrd3 knockout animals exhibited a lower fertilization rate in vitro, a sperm movement phenotype, and an altered thiol redox status in sperm cells. Proteomic analyses further revealed a broad range of substrates reduced by TXNRD3 during sperm maturation, presumably as a part of sperm quality control. Taken together, these results show that TXNRD3 plays a critical role in male reproduction via the thiol redox control of spermatogenesis.
Assuntos
Proteômica , Sêmen , Tiorredoxina Dissulfeto Redutase/metabolismo , Animais , Fertilidade , Masculino , Camundongos , Oxirredução , Selenoproteínas , Sêmen/metabolismo , Espermatogênese , Compostos de Sulfidrila , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMO
The sperm calcium channel CatSper plays a central role in successful fertilization as a primary Ca2+ gateway. Here, we applied cryo-electron tomography to visualize the higher-order organization of the native CatSper complex in intact mammalian sperm. The repeating CatSper units form long zigzag-rows along mouse and human sperm flagella. Above each tetrameric channel pore, most of the extracellular domains form a canopy that interconnects to a zigzag-shaped roof. Murine CatSper contains an additional wing-structure connected to the tetrameric channel. The intracellular domains link two neighboring channels to a diagonal array, suggesting a dimer formation. Fitting of an atomic model of isolated monomeric CatSper to the in situ map reveals supramolecular interactions and assembly of the CatSper complex. Loss of EFCAB9-CATSPERζ alters the architecture and interactions of the channels, resulting in fragmentation and misalignment of the zigzag-rows and disruption of flagellar movement in Efcab9-/- sperm. This work offers unique insights into the structural basis for understanding CatSper regulation of sperm motility.
Assuntos
Motilidade dos Espermatozoides , Cauda do Espermatozoide , Animais , Cálcio/metabolismo , Canais de Cálcio/fisiologia , Membrana Celular/metabolismo , Masculino , Mamíferos/metabolismo , Camundongos , Motilidade dos Espermatozoides/fisiologia , Cauda do Espermatozoide/metabolismo , Espermatozoides/metabolismoRESUMO
During epididymal transit, redox remodeling protects mammalian spermatozoa, preparing them for survival in the subsequent journey to fertilization. However, molecular mechanisms of redox regulation in sperm development and maturation remain largely elusive. In this study, we report that thioredoxin-glutathione reductase (TXNRD3), a thioredoxin reductase family member particularly abundant in elongating spermatids at the site of mitochondrial sheath formation, regulates redox homeostasis to support male fertility. Using Txnrd3-/- mice, our biochemical, ultrastructural, and live cell imaging analyses revealed impairments in sperm morphology and motility under conditions of TXNRD3 deficiency. We find that mitochondria develop more defined cristae during capacitation in wildtype sperm. Furthermore, we show that absence of TXNRD3 alters thiol redox status in both the head and tail during sperm maturation and capacitation, resulting in defective mitochondrial ultrastructure and activity under capacitating conditions. These findings provide insights into molecular mechanisms of redox homeostasis and bioenergetics during sperm maturation, capacitation, and fertilization.
Assuntos
Capacitação Espermática , Motilidade dos Espermatozoides , Tiorredoxina Dissulfeto Redutase/metabolismo , Animais , Epididimo , Masculino , Mamíferos , Camundongos , Mitocôndrias/metabolismo , Oxirredução , Sêmen , Capacitação Espermática/genética , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/metabolismoRESUMO
Successful fertilization depends on sperm motility adaptation. Ejaculated and activated sperm beat symmetrically in high frequency, move linearly, and swim with clockwise chirality. After capacitation, sperm beat asymmetrically with lower amplitude and a high lateral head excursion. This motility change called hyperactivation requires CatSper activation and an increase in intracellular Ca2+ . However, whether CatSper-mediated Ca2+ influx participates in controlling the swim path chirality is unknown. In this study, we show that the clockwise path chirality is preserved in mouse sperm regardless of capacitation state but is lost in the sperm either lacking the entire CatSper channel or its Ca2+ sensor EFCAB9. Pharmacological inhibition of CatSper with either mibefradil or NNC 55-0396 leads to the same loss in swim path chirality. Exposure of sperm to the recombinant N-terminal part of the zona pellucida protein 2 randomizes chirality in capacitated cells, but not in non-capacitated ones. We conclude that Ca2+ sensitive regulation of CatSper activity orchestrates clockwise swim path chirality of sperm and any substantial change, such as the physiological stimulus of zona pellucida glycoproteins, results in a loss of chirality.
Assuntos
Canais de Cálcio , Motilidade dos Espermatozoides , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Membrana Celular/metabolismo , Masculino , Camundongos , Capacitação Espermática , Espermatozoides/metabolismo , Zona Pelúcida/metabolismoRESUMO
While it is established that the topology of lipid membranes plays an important role in biochemical processes, few direct observations exist regarding how the membranes are actively restructured and its consequences on subsequent reactions. In this work, we investigated how the two major components of bee venom, melittin and phospholipase A2 (PLA2), achieve activation by such membrane remodeling. Their membrane-disrupting functions have been reported to increase when both are present, but the mechanism of this synergism had not been established. Using membrane reconstitution, we found that melittin can form large-scale membrane deformities upon which PLA2 activity is 25-fold higher. Tracking of single-molecule PLA2 revealed that its processive behavior on these deformities underlies the enhanced activity. These results show how melittin and PLA2 work synergistically to enhance the lytic effects of the bee venom. More broadly, they also demonstrate how the membrane topology may be actively altered to modulate cellular membrane-bound reactions.
Assuntos
Venenos de Abelha , Meliteno , Venenos de Abelha/química , Meliteno/farmacologia , Fosfolipases A2RESUMO
In mammalian sperm cells, regulation of spatiotemporal Ca2+ signaling relies on the quadrilinear Ca2+ signaling nanodomains in the flagellar membrane. The sperm-specific, multi-subunit CatSper Ca2+ channel, which is crucial for sperm hyperactivated motility and male fertility, organizes the nanodomains. Here, we report CatSperτ, the C2cd6-encoded membrane-associating C2 domain protein, can independently migrate to the flagella and serve as a major targeting component of the CatSper channel complex. CatSperτ loss of function in mice demonstrates that it is essential for sperm hyperactivated motility and male fertility. CatSperτ targets the CatSper channel into the quadrilinear nanodomains in the flagella of developing spermatids, whereas it is dispensable for functional channel assembly. CatSperτ interacts with ciliary trafficking machinery in a C2-dependent manner. These findings provide insights into the CatSper channel trafficking to the Ca2+ signaling nanodomains and the shared molecular mechanisms of ciliary and flagellar membrane targeting.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Flagelos/metabolismo , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/metabolismo , Animais , Masculino , Camundongos , Transporte Proteico/fisiologiaRESUMO
The lipid raft-resident protein, MAL2, has been implicated as contributing to the pathogenesis of several malignancies, including breast cancer, but the underlying mechanism for its effects on tumorigenesis is unknown. Here, we show that MAL2-mediated lipid raft formation leads to HER2 plasma membrane retention and enhanced HER2 signaling in breast cancer cells. We demonstrate physical interactions between HER2 and MAL2 in lipid rafts using proximity ligation assays. Super-resolution structured illumination microscopy imaging displays the structural organization of the HER2/Ezrin/NHERF1/PMCA2 protein complex. Formation of this protein complex maintains low intracellular calcium concentrations in the vicinity of the plasma membrane. HER2/MAL2 protein interactions in lipid rafts are enhanced in trastuzumab-resistant breast cancer cells. Our findings suggest that MAL2 is crucial for lipid raft formation, HER2 signaling, and HER2 membrane stability in breast cancer cells, suggesting MAL2 as a potential therapeutic target.
Assuntos
Neoplasias da Mama/patologia , Proteínas do Citoesqueleto/metabolismo , Resistencia a Medicamentos Antineoplásicos , Microdomínios da Membrana/metabolismo , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/metabolismo , Fosfoproteínas/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Receptor ErbB-2/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Antineoplásicos Imunológicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proliferação de Células , Proteínas do Citoesqueleto/genética , Endocitose , Feminino , Humanos , Microdomínios da Membrana/efeitos dos fármacos , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/genética , Fosfoproteínas/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Receptor ErbB-2/genética , Trocadores de Sódio-Hidrogênio/genética , Trastuzumab/farmacologia , Células Tumorais CultivadasRESUMO
Mammalian sperm cells are not capable of fertilizing an egg immediately after ejaculation; instead, they must gradually acquire the capacity to fertilize while they travel inside the female reproductive tract. Sperm cells are transported by the muscular activity of the myometrium to the utero-tubal junction (UTJ) before entering the oviduct where they undergo this physiological process, termed capacitation. Since the successful emulation of mammalian sperm capacitation in vitro, which led to the development of in vitro fertilization techniques, sperm capacitation and gamete interaction studies have been mostly carried out under in vitro conditions. Sperm cells are typically incubated in vitro for up to several hours at a concentration of more than 1 million cells per milliliter in the capacitation media inside a 37°C incubator with 5% CO2, mimicking the tubal fluid composed of serum albumin, bicarbonate, and Ca2+. The resultant sperm are functionally and molecularly heterogeneous with respect to acrosome reaction, motility, and phosphorylation. By contrast, in vivo sperm capacitation occurs in a time- and space-dependent manner, with limits on the number of capacitating sperm in the oviduct. The small number of sperm at the fertilization site in vivo are highly homogeneous and uniformly capable of fertilization. This discrepancy makes the degree of correlation between the changes observed from in vitro capacitation as a population average and the fertilizing capacity of sperm less clear. To overcome this issue, we used CLARITY tissue clearing to visualize sperm directly inside the female tract in situ and isolated sperm capacitated in vivo from the oviducts of the female mice after timed mating ( Ded et al., 2020 ). Here, we present a step-by-step protocol to collect in vivo capacitated sperm by detailing a microdissection technique and subsequent preparation steps for fluorescent imaging. The advantage of the microdissection technique over in vitro capacitation is the ability to collect physiologically segregated, homogeneous sperm populations at different stages of capacitation. Compared to CLARITY, this technique is more straightforward and compatible with a broader spectrum of antibodies for downstream imaging studies, as it allows the researcher to avoid a potentially high background from non-sperm cells in the tissue. The disadvantage of this technique is the potential contamination of the isolated sperm from different regions of the oviduct and disruption of the fine molecular structures (e.g., CatSper nanodomains) during sperm isolation, especially when the preparation is not performed swiftly. Hence, we suggest that the combination of both in situ and ex vivo isolated sperm imaging is the best way how to address the molecular features of in vivo capacitated sperm.