RESUMO
The structure of the sperm flagellar axoneme is highly conserved across species and serves the essential function of generating motility to facilitate the meeting of spermatozoa with the egg. During spermiogenesis, the axoneme elongates from the centrosome, and subsequently the centrosome docks onto the nuclear envelope to continue tail biogenesis. Mycbpap is expressed predominantly in mouse and human testes and conserved in Chlamydomonas as FAP147. A previous cryo-electron microscopy analysis has revealed the localization of FAP147 to the central apparatus of the axoneme. Here, we generated Mycbpap knockout mice and demonstrated the essential role of Mycbpap in male fertility. Deletion of Mycbpap led to disrupted centrosome-nuclear envelope docking and abnormal flagellar biogenesis. Furthermore, we generated transgenic mice with tagged MYCBPAP, which restored the fertility of Mycbpap knockout males. Interactome analyses of MYCBPAP using Mycbpap transgenic mice unveiled binding partners of MYCBPAP including central apparatus proteins such as CFAP65 and CFAP70 that constitute the C2a projection and centrosome-associated proteins such as CCP110. These findings provide insights into a MYCBPAP-dependent regulation of the centrosome-nuclear envelope docking and sperm tail biogenesis.
RESUMO
Ribonucleoprotein (RNP) granules are membraneless electron-dense structures rich in RNAs and proteins, and involved in various cellular processes. Two RNP granules in male germ cells, intermitochondrial cement and the chromatoid body (CB), are associated with PIWI-interacting RNAs (piRNAs) and are required for transposon silencing and spermatogenesis. Other RNP granules in male germ cells, the reticulated body and CB remnants, are also essential for spermiogenesis. In this study, we disrupted FBXO24, a testis-enriched F-box protein, in mice and found numerous membraneless electron-dense granules accumulated in sperm flagella. Fbxo24 knockout (KO) mice exhibited malformed flagellar structures, impaired sperm motility, and male infertility, likely due to the accumulation of abnormal granules. The amount and localization of known RNP granule-related proteins were not disrupted in Fbxo24 KO mice, suggesting that the accumulated granules were distinct from known RNP granules. Further studies revealed that RNAs and two importins, IPO5 and KPNB1, abnormally accumulated in Fbxo24 KO spermatozoa and that FBXO24 could ubiquitinate IPO5. In addition, IPO5 and KPNB1 were recruited to stress granules, RNP complexes, when cells were treated with oxidative stress or a proteasome inhibitor. These results suggest that FBXO24 is involved in the degradation of IPO5, disruption of which may lead to the accumulation of abnormal RNP granules in sperm flagella.
Assuntos
Proteínas F-Box , Infertilidade Masculina , Camundongos Knockout , Cauda do Espermatozoide , Masculino , Animais , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Camundongos , Cauda do Espermatozoide/metabolismo , Proteínas F-Box/metabolismo , Proteínas F-Box/genética , Grânulos Citoplasmáticos/metabolismo , Espermatozoides/metabolismoRESUMO
Infertility is a global health problem affecting one in six couples, with 50% of cases attributed to male infertility. Spermatozoa are male gametes, specialized cells that can be divided into two parts: the head and the flagellum. The head contains a vesicle called the acrosome that undergoes exocytosis and the flagellum is a motility apparatus that propels the spermatozoa forward and can be divided into two components, axonemes and accessory structures. For spermatozoa to fertilize oocytes, the acrosome and flagellum must be formed correctly. In this Review, we describe comprehensively how functional spermatozoa develop in mammals during spermiogenesis, including the formation of acrosomes, axonemes and accessory structures by focusing on analyses of mouse models.
Assuntos
Acrossomo , Espermatogênese , Espermatozoides , Animais , Masculino , Espermatogênese/fisiologia , Espermatozoides/fisiologia , Espermatozoides/metabolismo , Acrossomo/metabolismo , Acrossomo/fisiologia , Humanos , Mamíferos/fisiologia , Camundongos , Axonema/metabolismo , Flagelos/fisiologia , Flagelos/metabolismoRESUMO
Sperm heads contain not only the nucleus but also the acrosome which is a distinctive cap-like structure located anterior to the nucleus and is derived from the Golgi apparatus. The Golgi Associated RAB2 Interactors (GARINs; also known as FAM71) protein family shows predominant expression in the testis and all possess a RAB2-binding domain which confers binding affinity to RAB2, a small GTPase that is responsible for membrane transport and vesicle trafficking. Our previous study showed that GARIN1A and GARIN1B are important for acrosome biogenesis and that GARIN1B is indispensable for male fertility in mice. Here, we generated KO mice of other Garins, namely Garin2, Garin3, Garin4, Garin5a, and Garin5b (Garin2-5b). Using computer-assisted morphological analysis, we found that the loss of each Garin2-5b resulted in aberrant sperm head morphogenesis. While the fertilities of Garin2-/- and Garin4-/- males are normal, Garin5a-/- and Garin5b-/- males are subfertile, and Garin3-/- males are infertile. Further analysis revealed that Garin3-/- males exhibited abnormal acrosomal morphology, but not as severely as Garin1b-/- males; instead, the amounts of membrane proteins, particularly ADAM family proteins, decreased in Garin3 KO spermatozoa. Moreover, only Garin4 KO mice exhibit vacuoles in the sperm head. These results indicate that GARINs assure correct head morphogenesis and some members of the GARIN family function distinctively in male fertility.
Assuntos
Fertilidade , Infertilidade Masculina , Camundongos Knockout , Cabeça do Espermatozoide , Animais , Masculino , Camundongos , Acrossomo/metabolismo , Fertilidade/genética , Complexo de Golgi/metabolismo , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Morfogênese/genética , Proteína rab2 de Ligação ao GTP/metabolismo , Proteína rab2 de Ligação ao GTP/genética , Cabeça do Espermatozoide/metabolismo , Espermatozoides/metabolismo , Testículo/metabolismo , Testículo/crescimento & desenvolvimentoRESUMO
More than 1200 genes have been shown in the database to be expressed predominantly in the mouse testes. Advances in genome editing technologies such as the CRISPR/Cas9 system have made it possible to create genetically engineered mice more rapidly and efficiently than with conventional methods, which can be utilized to screen genes essential for male fertility by knocking out testis-enriched genes. Finding such genes related to male fertility would not only help us understand the etiology of human infertility but also lead to the development of male contraceptives. In this study, we generated knockout mice for 12 genes (Acrv1, Adgrf3, Atp8b5, Cfap90, Cfap276, Fbxw5, Gm17266, Lrrd1, Mroh7, Nemp1, Spata45, and Trim36) that are expressed predominantly in the testis and examined the appearance and histological morphology of testes, sperm motility, and male fertility. Mating tests revealed that none of these genes is essential for male fertility at least individually. Notably, knockout mice for Gm17266 showed smaller testis size than the wild-type but did not exhibit reduced male fertility. Since 12 genes were not individually essential for male fertilization, it is unlikely that these genes could be the cause of infertility or contraceptive targets. It is better to focus on other essential genes because complementary genes to these 12 genes may exist.
Assuntos
Sistemas CRISPR-Cas , Fertilidade , Infertilidade Masculina , Camundongos Knockout , Motilidade dos Espermatozoides , Testículo , Animais , Masculino , Testículo/patologia , Testículo/metabolismo , Camundongos , Fertilidade/genética , Infertilidade Masculina/genética , Motilidade dos Espermatozoides/genética , Feminino , Edição de Genes , Humanos , Camundongos Endogâmicos C57BLRESUMO
The Ras homology (Rho) family of GTPases serves various functions, including promotion of cell migration, adhesion, and transcription, through activation of effector molecule targets. One such pair of effectors, the Rho-associated coiled-coil kinases (ROCK1 and ROCK2), induce reorganization of actin cytoskeleton and focal adhesion through substrate phosphorylation. Studies on ROCK knockout mice have confirmed that ROCK proteins are essential for embryonic development, but their physiological functions in adult mice remain unknown. In this study, we aimed to examine the roles of ROCK1 and ROCK2 proteins in normal adult mice. Tamoxifen (TAM)-inducible ROCK1 and ROCK2 single and double knockout mice (ROCK1flox/flox and/or ROCK2flox/flox;Ubc-CreERT2) were generated and administered a 5-day course of TAM. No deaths occurred in either of the single knockout strains, whereas all of the ROCK1/ROCK2 double conditional knockout mice (DcKO) had died by Day 11 following the TAM course. DcKO mice exhibited increased lung tissue vascular permeability, thickening of alveolar walls, and a decrease in percutaneous oxygen saturation compared with noninducible ROCK1/ROCK2 double-floxed control mice. On Day 3 post-TAM, there was a decrease in phalloidin staining in the lungs in DcKO mice. On Day 5 post-TAM, immunohistochemical analysis also revealed reduced staining for vascular endothelial (VE)-cadherin, ß-catenin, and p120-catenin at cell-cell contact sites in vascular endothelial cells in DcKO mice. Additionally, VE-cadherin/ß-catenin complexes were decreased in DcKO mice, indicating that ROCK proteins play a crucial role in maintaining lung function by regulating cell-cell adhesion.
Assuntos
Células Endoteliais , Camundongos Knockout , Quinases Associadas a rho , Animais , Quinases Associadas a rho/metabolismo , Quinases Associadas a rho/genética , Camundongos , Células Endoteliais/metabolismo , Junções Intercelulares/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Caderinas/metabolismo , Caderinas/genética , beta Catenina/metabolismo , beta Catenina/genética , Masculino , Antígenos CDRESUMO
Each year, infertility affects 15% of couples worldwide, with 50% of cases attributed to men. It is assumed that sperm head shape is important for sperm-zona pellucida (ZP) penetration but research has yet to elucidate why. We generated testis expressed 46 (Tex46) knockout mice to investigate the essential roles of TEX46 in mammalian reproduction. We used RT-PCR to demonstrate that Tex46 was expressed exclusively in the male reproductive tract in mice and humans. We created Tex46-/- mice using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system and analyzed their fertility. Tex46 null spermatozoa underwent further evaluation using computer-assisted sperm analysis, light microscopy, and ultrastructural microscopy. We used immunoblot analysis to elucidate relationships between TEX46 and other acrosome biogenesis-related proteins. Mouse and human TEX46 are testis-enriched and encode a transmembrane protein which is conserved from amphibians to mammals. Loss of the mouse TEX46 protein causes male sterility primarily due to abnormal sperm head formation and secondary effects on sperm motility. Tex46 null spermatozoa morphologically lack the typical hooked sperm head appearance and fail to penetrate through the ZP. Electron microscopy of the testicular germ cells reveals malformation of the acrosomal cap, with misshapen sperm head tips and the appearance of a gap between the acrosome head and the nucleus. TEX46 is essential for sperm head formation, sperm penetration through the ZP, and male fertility in mice, and is a putative contraceptive target in men.
RESUMO
In mammals, females undergo reproductive cessation with age, whereas male fertility gradually declines but persists almost throughout life. However, the detailed effects of ageing on germ cells during and after spermatogenesis, in the testis and epididymis, respectively, remain unclear. Here we comprehensively examined the in vivo male fertility and the overall organization of the testis and epididymis with age, focusing on spermatogenesis, and sperm function and fertility, in mice. We first found that in vivo male fertility decreased with age, which is independent of mating behaviors and testosterone levels. Second, overall sperm production in aged testes was decreased; about 20% of seminiferous tubules showed abnormalities such as germ cell depletion, sperm release failure, and perturbed germ cell associations, and the remaining 80% of tubules contained lower number of germ cells because of decreased proliferation of spermatogonia. Further, the spermatozoa in aged epididymides exhibited decreased total cell numbers, abnormal morphology/structure, decreased motility, and DNA damage, resulting in low fertilizing and developmental rates. We conclude that these multiple ageing effects on germ cells lead to decreased in vivo male fertility. Our present findings are useful to better understand the basic mechanism behind the ageing effect on male fertility in mammals including humans.
Assuntos
Epididimo , Testículo , Animais , Masculino , Camundongos , Envelhecimento , Fertilidade , Mamíferos , Sêmen , EspermatogôniasRESUMO
Ribonucleoprotein (RNP) granules are membraneless electron-dense structures rich in RNAs and proteins, and involved in various cellular processes. Two RNP granules in male germ cells, intermitochondrial cement and the chromatoid body (CB), are associated with PIWI-interacting RNAs (piRNAs) and are required for transposon silencing and spermatogenesis. Other RNP granules in male germ cells, the reticulated body and CB remnants, are also essential for spermiogenesis. In this study, we disrupted FBXO24, a testis-enriched F-box protein, in mice and found numerous membraneless electron-dense granules accumulated in sperm flagella. Fbxo24 knockout (KO) mice exhibited malformed flagellar structures, impaired sperm motility, and male infertility, likely due to the accumulation of abnormal granules. The amount and localization of known RNP granule-related proteins were not disrupted in Fbxo24 KO mice, suggesting that the accumulated granules were distinct from known RNP granules. Further studies revealed that RNAs and two importins, IPO5 and KPNB1, abnormally accumulated in Fbxo24 KO spermatozoa. In addition, IPO5 and KPNB1 were recruited to stress granules, RNP complexes, when cells were treated with oxidative stress or a proteasome inhibitor. These results suggest that FBXO24 plays a critical role in preventing the accumulation of importins and RNP granules in sperm flagella.
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
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
Celastrol, a bioactive molecule extracted from the Tripterygium wilfordii plant, has been shown to exhibit anti-inflammatory properties. However, its mechanism of action has not been fully elucidated. Here, we show that celastrol suppresses humoral immune responses and autoimmunity by disabling a protein complex consisting of copper metabolism MURR1 domain-containing (COMMD) 3 and COMMD8 (COMMD3/8 complex), a signaling adaptor for chemoattractant receptors. Having demonstrated the involvement of the COMMD3/8 complex in a mouse model of rheumatoid arthritis, we identified celastrol as a compound that covalently bound to and dissociated the COMMD3/8 complex. Celastrol inhibited B cell migration, reduced antibody responses, and blocked arthritis progression, recapitulating deficiency of the COMMD3/8 complex. These effects of celastrol were abolished in mice expressing a celastrol-resistant mutant of the COMMD3/8 complex. These findings establish that celastrol exerts immunosuppressive activity by targeting the COMMD3/8 complex. Our study suggests that the COMMD3/8 complex is a potentially druggable target in autoimmune diseases and points to celastrol as a lead pharmacologic candidate in this capacity.
Assuntos
Doenças Autoimunes , Imunidade Humoral , Camundongos , Animais , Autoimunidade , Triterpenos PentacíclicosRESUMO
BACKGROUND: TSN (translin), also called testis brain RNA-binding protein, binds to TSNAX (translin-associated factor X) and is suggested to play diverse roles, such as RNA metabolism and DNA damage response. TSNAXIP1 (Translin-associated factor X-interacting protein 1) was identified as a TSNAX-interacting protein using a yeast two-hybrid system, but its function in vivo was unknown. OBJECTIVE: To reveal the function of TSNAXIP1 in vivo in mice. MATERIALS AND METHODS: We generated Tsnaxip1 knockout mice using the CRISPR/Cas9 system and analyzed their fertility and sperm motility. Further, we generated 1700010I14Rik knockout mice, because 1700010I14RIK is also predominantly expressed in testes and contains the same Pfam (protein families) domain as TSNAXIP1. RESULTS: Reduced male fertility and impaired sperm motility with asymmetric flagellar waveforms were observed in not only Tsnaxip1 but also 1700010I14Rik knockout mice. Unlike Tsn knockout mice, no abnormalities were found in testicular sections of either Tsnaxip1 or 1700010I14Rik knockout mice. Furthermore, TSNAXIP1 was detected in the sperm tail and fractionated with axonemal proteins. DISCUSSION AND CONCLUSION: Unlike the TSN-TSNAX complex, whose disruption causes abnormal vacuoles in mouse testes, TSNAXIP1 and 1700010I14RIK may play roles in regulating sperm flagellar beating patterns.
Assuntos
Motilidade dos Espermatozoides , Testículo , Animais , Masculino , Camundongos , Fator X/metabolismo , Fertilidade , Camundongos Knockout , Proteínas/metabolismo , Sêmen , Motilidade dos Espermatozoides/genética , Espermatozoides/metabolismo , Testículo/metabolismoRESUMO
Spermatozoa need to undergo an exocytotic event called the acrosome reaction before fusing with eggs. Although calcium ion (Ca2+) is essential for the acrosome reaction, its molecular mechanism remains unknown. Ferlin is a single transmembrane protein with multiple Ca2+-binding C2 domains, and there are six ferlins, dysferlin (DYSF), otoferlin (OTOF), myoferlin (MYOF), fer-1-like 4 (FER1L4), FER1L5, and FER1L6, in mammals. Dysf, Otof, and Myof knockout mice have been generated, and each knockout mouse line exhibited membrane fusion disorders such as muscular dystrophy in Dysf, deafness in Otof, and abnormal myogenesis in Myof. Here, by generating mutant mice of Fer1l4, Fer1l5, and Fer1l6, we found that only Fer1l5 is required for male fertility. Fer1l5 mutant spermatozoa could migrate in the female reproductive tract and reach eggs, but no acrosome reaction took place. Even a Ca2+ ionophore cannot induce the acrosome reaction in Fer1l5 mutant spermatozoa. These results suggest that FER1L5 is the missing link between Ca2+ and the acrosome reaction.
Assuntos
Proteínas Musculares , Testículo , Masculino , Feminino , Animais , Camundongos , Membrana Celular/metabolismo , Proteínas Musculares/metabolismo , Testículo/metabolismo , Fusão de Membrana , Fertilidade , Espermatozoides/metabolismo , Mamíferos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismoRESUMO
BACKGROUND: Lactate dehydrogenase C (LDHC) is specifically expressed in male germ cells and plays critical roles in glycolysis. Glycolysis is required to supply energy for sperm motility. Previous studies showed that Ldhc knock-out mice exhibit impaired sperm motility. OBJECTIVES: We established human LDHC knock-in (hLDHC KI) mice and examined whether hLDHC KI mice can be used to assess LDHC-targeting drugs. MATERIAL AND METHODS: HLDHC was knocked-in to the mouse Ldhc (mLdhc) allele using the CRISPR/Cas9 system. Mating tests, sperm motility examinations with a computer-assisted sperm analysis (CASA) system, and in vitro fertilization (IVF) were performed. Furthermore, the effect of an LDH inhibitor was analyzed with CASA and IVF. RESULTS: HLDHC was detected at the protein level in hLDHC KI spermatozoa. hLDHC KI mice exhibited comparable sperm motility and male fertility to wild-type (WT) mice. When we performed IVF using the LDH inhibitor more specific to hLDHC than mLDHC, fertilization rates were reduced in hLDHC KI mice but not in WT mice. DISCUSSION AND CONCLUSION: Our results reveal that hLDHC can rescue the absence of mLDHC. Differences in the effect of the LDH inhibitor between WT and hLDHC KI mice indicate that hLDHC KI mice can be a good model to assess hLDHC inhibitors for preclinical contraceptive studies.
Assuntos
Sêmen , Motilidade dos Espermatozoides , Humanos , Masculino , Camundongos , Animais , Espermatozoides/metabolismo , Anticoncepcionais , Camundongos KnockoutRESUMO
Purpose: Tulp2 (tubby-like protein 2) is a member of the tubby protein family and expressed predominantly in mouse testis. Recently, it was reported that Tulp2 knockout (KO) mice exhibited disrupted sperm tail morphology; however, it remains to be determined how TULP2 deletion causes abnormal tail formation. Methods: The authors analyzed male fertility, sperm morphology, and motility of two Tulp2 KO mouse lines that were generated using the conventional method that utilizes homologous recombination in embryonic stem (ES) cells as well as the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. Furthermore, the authors observed the spermatogenesis of Tulp2 KO mice in more detail using scanning and transmission electron microscopy (SEM and TEM). Results: Both mouse lines of Tulp2 KO exhibited male infertility, abnormal tail morphology, and impaired sperm motility. No overt abnormalities were found in the formation of the mitochondrial sheath in Tulp2 KO mice using the freeze-fracture method with SEM. In contrast, abnormal outer dense fiber (ODF) structure was observed in Tulp2 KO testis with TEM. Conclusions: TULP2 may play roles in the correct formation and/or maintenance of ODF, which may lead to abnormal tail morphology, impaired sperm motility, and male infertility.
RESUMO
Immunity-related GTPases (IRGs), also known as p47 GTPases, are a family of interferon-inducible proteins that play roles in immunity defense against intracellular pathogens. Although the molecular functions of IRGs have been well studied, the function of the family member, IRGC1, remains unclear. IRGC1 is unique among IRGs because its expression is not induced by interferon and it is expressed predominantly in the testis. Further, IRGC1 is well conserved in mammals unlike other IRGs. Here, we knocked out (KO) Irgc1 in mice using the CRISPR/Cas9 system and found that the fertility of Irgc1 KO males was severely impaired because of abnormal sperm motility. Further analyses with a transmission electron microscope revealed that the fibrous sheath (FS), an accessory structure of the sperm tail, was disorganized in Irgc1 KO mice. In addition, IRGC1 was detected in the sperm tail and fractionated with FS proteins. These results suggest that IRGC1 is a component of the FS and is involved in the correct formation of the FS.
Assuntos
Motilidade dos Espermatozoides , Testículo , Animais , Masculino , Camundongos , GTP Fosfo-Hidrolases/metabolismo , Interferons/metabolismo , Mamíferos , Camundongos Knockout , Proteínas/metabolismo , Cauda do Espermatozoide/metabolismo , Espermatozoides/metabolismo , Testículo/metabolismoRESUMO
Although several long noncoding RNAs (lncRNAs) have recently been shown to encode small polypeptides, those in testis remain largely uncharacterized. Here we identify two sperm-specific polypeptides, Kastor and Polluks, encoded by a single mouse locus (Gm9999) previously annotated as encoding a lncRNA. Both Kastor and Polluks are inserted in the outer mitochondrial membrane and directly interact with voltage-dependent anion channel (VDAC), despite their different amino acid sequences. Male VDAC3-deficient mice are infertile as a result of reduced sperm motility due to an abnormal mitochondrial sheath in spermatozoa, and deficiency of both Kastor and Polluks also severely impaired male fertility in association with formation of a similarly abnormal mitochondrial sheath. Spermatozoa lacking either Kastor or Polluks partially recapitulate the phenotype of those lacking both. Cooperative function of Kastor and Polluks in regulation of VDAC3 may thus be essential for mitochondrial sheath formation in spermatozoa and for male fertility.
Assuntos
Motilidade dos Espermatozoides , Canais de Ânion Dependentes de Voltagem , Animais , Masculino , Camundongos , Peptídeos/genética , Peptídeos/metabolismo , Espermatogênese/genética , Espermatozoides/metabolismo , Canais de Ânion Dependentes de Voltagem/genética , Canais de Ânion Dependentes de Voltagem/metabolismoRESUMO
Kinesin is a molecular motor that moves along microtubules. Testis-enriched kinesin KIF9 (Kinesin family member 9) is localized in the mouse sperm flagellum and is important for normal sperm motility and male fertility; however, it is unclear if the motor domain of KIF9 is involved in these processes. In this study, we substituted threonine of the ATP binding motif in the KIF9 motor domain to asparagine (T100N) in mice using the CRISPR/Cas9 system, which is known to impair kinesin motor activity. T100N mutant mice exhibit reduced sperm motility and male fertility consistent with Kif9 knockout mice. Further, KIF9 was depleted in the spermatozoa of T100N mutant mice although the amounts of KIF9 were comparable between wild-type and T100N mutant testes. These results indicate that the motor domain of KIF9 is essential for its localization in the sperm flagellum.
Assuntos
Cinesinas , Testículo , Animais , Fertilidade/genética , Flagelos , Cinesinas/genética , Masculino , Camundongos , Camundongos Knockout , Motilidade dos Espermatozoides , EspermatozoidesRESUMO
Gene expression analyses suggest that more than 1000-2000 genes are expressed predominantly in mouse and human testes. Although functional analyses of hundreds of these genes have been performed, there are still many testis-enriched genes whose functions remain unexplored. Analyzing gene function using knockout (KO) mice is a powerful tool to discern if the gene of interest is essential for sperm formation, function, and male fertility in vivo. In this study, we generated KO mice for 12 testis-enriched genes, 1700057G04Rik, 4921539E11Rik, 4930558C23Rik, Cby2, Ldhal6b, Rasef, Slc25a2, Slc25a41, Smim8, Smim9, Tmem210, and Tomm20l, using the clustered regularly interspaced short palindromic repeats /CRISPR-associated protein 9 (CRISPR/Cas9) system. We designed two gRNAs for each gene to excise almost all the protein-coding regions to ensure that the deletions in these genes result in a null mutation. Mating tests of KO mice reveal that these 12 genes are not essential for male fertility, at least when individually ablated, and not together with other potentially compensatory paralogous genes. Our results could prevent other laboratories from expending duplicative effort generating KO mice, for which no apparent phenotype exists.