USF2 and TFEB compete in regulating lysosomal and autophagy genes.

Autophagy, a highly conserved self-digestion process crucial for cellular homeostasis, is triggered by various environmental signals, including nutrient scarcity. The regulation of lysosomal and autophagy-related processes is pivotal to maintaining cellular homeostasis and basal metabolism. The consequences of disrupting or diminishing lysosomal and autophagy systems have been investigated; however, information on the implications of hyperactivating lysosomal and autophagy genes on homeostasis is limited. Here, we present a mechanism of transcriptional repression involving upstream stimulatory factor 2 (USF2), which inhibits lysosomal and autophagy genes under nutrient-rich conditions. We find that USF2, together with HDAC1, binds to the CLEAR motif within lysosomal genes, thereby diminishing histone H3K27 acetylation, restricting chromatin accessibility, and downregulating lysosomal gene expression. Under starvation, USF2 competes with transcription factor EB (TFEB), a master transcriptional activator of lysosomal and autophagy genes, to bind to target gene promoters in a phosphorylation-dependent manner. The GSK3ß-mediated phosphorylation of the USF2 S155 site governs USF2 DNA-binding activity, which is involved in lysosomal gene repression. These findings have potential applications in the treatment of protein aggregation-associated diseases, including α1-antitrypsin deficiency. Notably, USF2 repression is a promising therapeutic strategy for lysosomal and autophagy-related diseases.

Loss of CCDC188 causes male infertility with defects in the sperm head-neck connection in mice.

Acephalic spermatozoa syndrome (ASS) represents a rare genetic and reproductive disease, which is defined as semen composed of mostly headless spermatozoa. The connecting piece in the neck region, also known as the head-to-tail coupling apparatus (HTCA), plays a crucial role in the tight linkage between the sperm head and tail. Dysfunction of this structure can lead to separation of sperm heads and tails, and male infertility. Using the mouse as an experimental model, several proteins have been identified as associated with the HTCA and disruption of these proteins causes acephalic spermatozoa. However, the molecular mechanism underlying this morphologic anomaly and HTCA remains elusive. In this study, we focused on coiled-coil domain containing 188 (Ccdc188), which shows testis-enriched expression. To elucidate the physiological role of CCDC188, we generated a knockout (KO) mouse line using the CRISPR/Cas9 system. Ccdc188 KO male mice were sterile, indicating that CCDC188 is indispensable for male fertility. Most Ccdc188-null spermatozoa were acephalic. Transmission electron microscopy revealed that while the sperm HTCA could assemble properly without CCDC188, the HTCA failed to attach to the nucleus during spermiogenesis, leading to sperm head and neck separation. In addition, we found almost all of the spermatozoa in the cauda epididymis lacked a mitochondrial sheath. Taken together, we demonstrated that CCDC188 plays a crucial role in forming a tight sperm head-neck junction.

Comprehensive posttranslational modifications in the testis-specific histone variant H3t protein validated in tagged knock-in mice.

During the development of multicellular organisms and cell differentiation, the chromatin structure in the cell nucleus undergoes extensive changes, and the nucleosome structure is formed by a combination of various histone variants. Histone variants with diverse posttranslational modifications are known to play crucial roles in different regulatory functions. We have previously reported that H3t, a testis-specific histone variant, is essential for spermatogenesis. To elucidate the function of this chromatin molecule in vivo, we generated knock-in mice with a FLAG tag attached to the carboxyl terminus of H3t. In the present study, we evaluated the utility of the generated knock-in mice and comprehensively analyzed posttranslational modifications of canonical H3 and H3t using mass spectrometry. Herein, we found that H3t-FLAG was incorporated into spermatogonia and meiotic cells in the testes, as evidenced by immunostaining of testicular tissue. According to the mass spectrometry analysis, the overall pattern of H3t-FLAG posttranslational modification was comparable to that of the control H3, while the relative abundances of certain specific modifications differed between H3t-FLAG and the control bulk H3. The generated knock-in mice could be valuable for analyzing the function of histone variants in vivo.

Multiple lines of evidence for disruption of nuclear lamina and nucleoporins in FUS amyotrophic lateral sclerosis.

Advanced pathological and genetic approaches have revealed that mutations in fused in sarcoma/translated in liposarcoma (FUS/TLS), which is pivotal for DNA repair, alternative splicing, translation and RNA transport, cause familial amyotrophic lateral sclerosis (ALS). The generation of suitable animal models for ALS is essential for understanding its pathogenesis and developing therapies. Therefore, we used CRISPR-Cas9 to generate FUS-ALS mutation in the non-classical nuclear localization signal (NLS), H517D (mouse position: H509D) and genome-edited mice. Fus WT/H509D mice showed progressive motor impairment (accelerating rotarod and DigiGait system) with age, which was associated with the loss of motor neurons and disruption of the nuclear lamina and nucleoporins and DNA damage in spinal cord motor neurons. We confirmed the validity of our model by showing that nuclear lamina and nucleoporin disruption were observed in lower motor neurons differentiated from patient-derived human induced pluripotent stem cells (hiPSC-LMNs) with FUS-H517D and in the post-mortem spinal cord of patients with ALS. RNA sequence analysis revealed that most nuclear lamina and nucleoporin-linking genes were significantly decreased in FUS-H517D hiPSC-LMNs. This evidence suggests that disruption of the nuclear lamina and nucleoporins is crucial for ALS pathomechanisms. Combined with patient-derived hiPSC-LMNs and autopsy samples, this mouse model might provide a more reliable understanding of ALS pathogenesis and might aid in the development of therapeutic strategies.

Gelsolin alleviates rheumatoid arthritis by negatively regulating NLRP3 inflammasome activation.

Despite numerous biomarkers being proposed for rheumatoid arthritis (RA), a gap remains in our understanding of their mechanisms of action. In this study, we discovered a novel role for gelsolin (GSN), an actin-binding protein whose levels are notably reduced in the plasma of RA patients. We elucidated that GSN is a key regulator of NLRP3 inflammasome activation in macrophages, providing a plausible explanation for the decreased secretion of GSN in RA patients. We found that GSN interacts with NLRP3 in LPS-primed macrophages, hence modulating the formation of the NLRP3 inflammasome complex. Reducing GSN expression significantly enhanced NLRP3 inflammasome activation. GSN impeded NLRP3 translocation to the mitochondria; it contributed to the maintenance of intracellular calcium equilibrium and mitochondrial stability. This maintenance is crucial for controlling the inflammatory response associated with RA. Furthermore, the exacerbation of arthritic symptoms in GSN-deficient mice indicates the potential of GSN as both a diagnostic biomarker and a therapeutic target. Moreover, not limited to RA models, GSN has demonstrated a protective function in diverse disease models associated with the NLRP3 inflammasome. Myeloid cell-specific GSN-knockout mice exhibited aggravated inflammatory responses in models of MSU-induced peritonitis, folic acid-induced acute tubular necrosis, and LPS-induced sepsis. These findings suggest novel therapeutic approaches that modulate GSN activity, offering promise for more effective management of RA and a broader spectrum of inflammatory conditions.

The significance of electrical signals in maturing spermatozoa for phosphoinositide regulation through voltage-sensing phosphatase.

Voltage-sensing phosphatase (VSP) exhibits voltage-dependent phosphatase activity toward phosphoinositides. VSP generates a specialized phosphoinositide environment in mammalian sperm flagellum. However, the voltage-sensing mechanism of VSP in spermatozoa is not yet characterized. Here, we found that VSP is activated during sperm maturation, indicating that electric signals in immature spermatozoa are essential. Using a heterologous expression system, we show the voltage-sensing property of mouse VSP (mVSP). The voltage-sensing threshold of mVSP is approximately -30 mV, which is sensitive enough to activate mVSP in immature spermatozoa. We also report several knock-in mice in which we manipulate the voltage-sensitivity or electrochemical coupling of mVSP. Notably, the V312R mutant, with a minor voltage-sensitivity change, exhibits abnormal sperm motility after, but not before, capacitation. Additionally, the V312R mutant shows a significant change in the acyl-chain profile of phosphoinositide. Our findings suggest that electrical signals during sperm maturation are crucial for establishing the optimal phosphoinositide environment in spermatozoa.

MYCBPAP is a central apparatus protein required for centrosome-nuclear envelope docking and sperm tail biogenesis in mice.

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, which 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.

Eighteen genes primarily expressed in the testis are not required for male fertility in mice.

There are approximately 20,000 protein-coding genes in humans and mice. More than 1000 of these genes are predominantly expressed in the testis or are testis-specific and thought to play an important role in male reproduction. Through the production of gene knockout mouse models and phenotypic evaluations, many genes essential for spermatogenesis, sperm maturation, and fertilization have been discovered, greatly contributing to the elucidation of their molecular mechanisms. On the other hand, there are many cases in which single-gene knockout models do not affect fertility, indicating that tissue-specific genes are not always critical. Here, we selected 18 genes whose mRNA expression is restricted to the testis or higher than in other tissues, but whose function in male reproduction is unknown. We then created single-gene KO mouse models using the CRISPR/Cas9 system. The established KO males were subjected to mating tests and screened for effects on fecundity, revealing that these genes were not essential for spermatogenesis and male fertility. This knowledge will contribute to understanding the functions of genes characteristic of the testis and identify the cause of male infertility.

FBXO24 deletion causes abnormal accumulation of membraneless electron-dense granules in sperm flagella and male infertility.

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.

Kdm4d mutant mice show impaired sperm motility and subfertility.

Regulation of gene expression through histone modifications underlies cell homeostasis and differentiation. Kdm4d and Kdm4dl exhibit a high degree of similarity and demethylate H3K9me3. However, the physiological functions of these proteins remain unclear. In this study, we generated Kdm4dl mutant mice and found that Kdm4dl was dispensable for mouse development. However, through the generation of Kdm4d mutant mice, we unexpectedly found that Kdm4d mutant male mice were subfertile because of impaired sperm motility. The absence of Kdm4d was associated with an altered distribution of H3K9me3 in round spermatids, suggesting that the Kdm4d-mediated adjustment of H3K9me3 levels is required to generate motile sperm. Further analysis revealed that the absence of Kdm4d did not affect the functionality of sperm nuclei in generating offspring. As KDM4D is specifically expressed in the human testes, our results suggest that changes in KDM4D expression or its activity may be a risk factor for human infertility.

Development of functional spermatozoa in mammalian spermiogenesis.

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.

Semaphorin 6D tunes amygdalar circuits for emotional, metabolic, and inflammatory outputs.

Regulated neural-metabolic-inflammatory responses are essential for maintaining physiological homeostasis. However, the molecular machinery that coordinates neural, metabolic, and inflammatory responses is largely unknown. Here, we show that semaphorin 6D (SEMA6D) coordinates anxiogenic, metabolic, and inflammatory outputs from the amygdala by maintaining synaptic homeostasis. Using genome-wide approaches, we identify SEMA6D as a pleiotropic gene for both psychiatric and metabolic traits in human. Sema6d deficiency increases anxiety in mice. When fed a high-fat diet, Sema6d-/- mice display attenuated obesity and enhanced myelopoiesis compared with control mice due to higher sympathetic activity via the ß3-adrenergic receptor. Genetic manipulation and spatial and single-nucleus transcriptomics reveal that SEMA6D in amygdalar interneurons is responsible for regulating anxiogenic and autonomic responses. Mechanistically, SEMA6D is required for synaptic maturation and γ-aminobutyric acid transmission. These results demonstrate that SEMA6D is important for the normal functioning of the neural circuits in the amygdala, coupling emotional, metabolic, and inflammatory responses.

Golgi associated RAB2 interactor protein family contributes to murine male fertility to various extents by assuring correct morphogenesis of sperm heads.

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.

Gene-deficient mouse model established by CRISPR/Cas9 system reveals 15 reproductive organ-enriched genes dispensable for male fertility.

Since the advent of gene-targeting technology in embryonic stem cells, mice have become a primary model organism for investigating human gene function due to the striking genomic similarities between the two species. With the introduction of the CRISPR/Cas9 system for genome editing in mice, the pace of loss-of-function analysis has accelerated significantly. This has led to the identification of numerous genes that play crucial roles in male reproductive processes, including meiosis, chromatin condensation, flagellum formation in the testis, sperm maturation in the epididymis, and fertilization in the oviduct. Despite the advancements, the functions of many genes, particularly those enriched in male reproductive tissues, remain largely unknown. In our study, we focused on 15 genes and generated 13 gene-deficient mice [4933411K16Rik, Adam triple (Adam20, Adam25, and Adam39), BC048671, Cfap68, Gm4846, Gm4984, Gm13570, Nt5c1b, Ppp1r42, Saxo4, Sh3d21, Spz1, and Tektl1] to elucidate their roles in male fertility. Surprisingly, all 13 gene-deficient mice exhibited normal fertility in natural breeding experiments, indicating that these genes are not essential for male fertility. These findings have important implications as they may help prevent other research laboratories from duplicating efforts to generate knockout mice for genes that do not demonstrate an apparent phenotype related to male fertility. By shedding light on the dispensability of these genes, our study contributes to a more efficient allocation of research resources in the exploration of male reproductive biology.

Individual disruption of 12 testis-enriched genes via the CRISPR/Cas9 system does not affect the fertility of male mice.

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.

Thirteen Ovary-Enriched Genes Are Individually Not Essential for Female Fertility in Mice.

Infertility is considered a global health issue as it currently affects one in every six couples, with female factors reckoned to contribute to partly or solely 50% of all infertility cases. Over a thousand genes are predicted to be highly expressed in the female reproductive system and around 150 genes in the ovary. However, some of their functions in fertility remain to be elucidated. In this study, 13 ovary and/or oocyte-enriched genes (Ccdc58, D930020B18Rik, Elobl, Fbxw15, Oas1h, Nlrp2, Pramel34, Pramel47, Pkd1l2, Sting1, Tspan4, Tubal3, Zar1l) were individually knocked out by the CRISPR/Cas9 system. Mating tests showed that these 13 mutant mouse lines were capable of producing offspring. In addition, we observed the histology section of ovaries and performed in vitro fertilization in five mutant mouse lines. We found no significant anomalies in terms of ovarian development and fertilization ability. In this study, 13 different mutant mouse lines generated by CRISPR/Cas9 genome editing technology revealed that these 13 genes are individually not essential for female fertility in mice.

Generation and characterization of cerebellar granule neurons specific knockout mice of Golli-MBP.

Golli-myelin basic proteins, encoded by the myelin basic protein gene, are widely expressed in neurons and oligodendrocytes in the central nervous system. Further, prior research has shown that Golli-myelin basic protein is necessary for myelination and neuronal maturation during central nervous system development. In this study, we established Golli-myelin basic protein-floxed mice to elucidate the cell-type-specific effects of Golli-myelin basic protein knockout through the generation of conditional knockout mice (Golli-myelin basic proteinsfl/fl; E3CreN), in which Golli-myelin basic proteins were specifically deleted in cerebellar granule neurons, where Golli-myelin basic proteins are expressed abundantly in wild-type mice. To investigate the role of Golli-myelin basic proteins in cerebellar granule neurons, we further performed histopathological analyses of these mice, with results indicating no morphological changes or degeneration of the major cellular components of the cerebellum. Furthermore, behavioral analysis showed that Golli-myelin basic proteinsfl/fl; E3CreN mice were healthy and did not display any abnormal behavior. These results suggest that the loss of Golli-myelin basic proteins in cerebellar granule neurons does not lead to cerebellar perturbations or behavioral abnormalities. This mouse model could therefore be employed to analyze the effect of Golli-myelin basic protein deletion in specific cell types of the central nervous system, such as other neuronal cells and oligodendrocytes, or in lymphocytes of the immune system.

Conditional deficiency of Rho-associated kinases disrupts endothelial cell junctions and impairs respiratory function in adult mice.

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.

Threonine phosphorylation of STAT1 restricts interferon signaling and promotes innate inflammatory responses.

Since its discovery over three decades ago, signal transducer and activator of transcription 1 (STAT1) has been extensively studied as a central mediator for interferons (IFNs) signaling and antiviral defense. Here, using genetic and biochemical assays, we unveil Thr748 as a conserved IFN-independent phosphorylation switch in Stat1, which restricts IFN signaling and promotes innate inflammatory responses following the recognition of the bacterial-derived toxin lipopolysaccharide (LPS). Genetically engineered mice expressing phospho-deficient threonine748-to-alanine (T748A) mutant Stat1 are resistant to LPS-induced lethality. Of note, T748A mice exhibited undisturbed IFN signaling, as well as total expression of Stat1. Further, the T748A point mutation of Stat1 recapitulates the safeguard effect of the genetic ablation of Stat1 following LPS-induced lethality, indicating that the Thr748 phosphorylation contributes inflammatory functionalities of Stat1. Mechanistically, LPS-induced Toll-like receptor 4 endocytosis activates a cell-intrinsic IκB kinase-mediated Thr748 phosphorylation of Stat1, which promotes macrophage inflammatory response while restricting the IFN and anti-inflammatory responses. Depletion of macrophages restores the sensitivity of the T748A mice to LPS-induced lethality. Together, our study indicates a phosphorylation-dependent modular functionality of Stat1 in innate immune responses: IFN phospho-tyrosine dependent and inflammatory phospho-threonine dependent. Better understanding of the Thr748 phosphorylation of Stat1 may uncover advanced pharmacologically targetable molecules and offer better treatment modalities for sepsis, a disease that claims millions of lives annually.

Tex46 knockout male mice are sterile secondary to sperm head malformations and failure to penetrate through the zona pellucida.

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.