Transforming growth factor β (TGFβ) signaling pathways are integral for a plethora of biological processes. SMAD2 and SMAD3 are the principal transcriptional effectors of TGFβ superfamily ligands, yet quantitative, genome-wide mapping of their DNA-associated complexes under physiological contexts has remained limited due to the lack of specific, robust models. Here, we generated two versatile epitope-tagged mouse models in which endogenous SMAD2 and SMAD3 proteins are globally tagged with hemagglutinin (HA) and podoplanin (PA) sequences, respectively, enabling high-fidelity profiling of SMAD2 and SMAD3 binding across tissues. To demonstrate the broad application of our models, we exemplified the usage of our lines in ovarian biology, where we defined the transcriptional programs downstream of GDF9, a key oocyte-derived ligand in folliculogenesis from the TGFβ superfamily. By integrating genomic and transcriptomic analyses, we identified direct genes induced by the GDF9-SMAD2/3 axis and identified gene sets suppressed by this signaling cascade, highlighting a previously underappreciated role of GDF9 in attenuating competing pathways to ensure proper ovarian granulosa cell fate transitions. Short-term GDF9 stimulation shifts SMAD2/3 cofactor recruitment toward lineage- and differentiation-associated transcription factors, without significant global changes in H3K27ac landscapes, indicating that GDF9 signals through targeted SMAD recruitment to preacetylated chromatin regions. Network analyses further demonstrated that GDF9-SMAD2/3 direct targets align with luteinizing hormone-driven preovulatory signaling. Together, our study generated epitope-tagged mouse models that provide extensive and applicable in vivo genetic toolkits for tissue-specific dissection of TGFβ family signaling and reveal a comprehensive, direct transcriptional network through which GDF9 coordinates granulosa cell differentiation and follicular maturation.

Spermatogenesis is a highly ordered developmental process that occurs in the seminiferous tubules and involves mitotic proliferation of spermatogonial stem cells, meiotic division of spermatocytes, and post-meiotic spermiogenesis, ultimately producing mature spermatozoa. Defects at any stage of this process can lead to male infertility. Large-scale transcriptomic and proteomic studies have identified thousands of testis-enriched genes in humans and mice, supporting the use of mouse models to uncover genetic regulators of male fertility. In this study, we focused on transmembrane channel-like protein 7 (TMC7), which is highly expressed in the testis. To investigate its physiological function, we generated a Tmc7 knockout (KO) mouse line using the CRISPR/Cas9 system. Tmc7 KO male mice were sterile, and no spermatozoa were observed in the epididymis. Instead, multinucleated giant cells containing multiple elongating spermatids were detected in the lumens of seminiferous tubules. We found that these abnormalities were associated with defective intercellular bridge (ICB) stabilization. Transmission electron microscopy further revealed that spermatid nuclei passed through gaps within the ICBs, leading to the formation of multinucleated giant cells. Given the Golgi localization of TMC7, ICB destabilization may occur secondarily due to blood-testis barrier disruption caused by alterations in the testicular microenvironment. Importantly, ectopic expression of TMC7 in Tmc7 KO mice rescued the defective spermiogenesis phenotype. Taken together, these findings demonstrate that TMC7 plays a critical role in spermiogenesis and indirectly contributes to the maintenance of ICB integrity.

Fertilization depends on the proper formation and function of the acrosome, a specialized organelle essential for sperm-oocyte interaction. Defects in acrosome biogenesis impair fertilization and cause severe male infertility, including globozoospermia. However, the molecular mechanisms underlying acrosome formation and maintenance remain poorly understood. Here, we investigated the roles of the testis-enriched proteins LRRC37 and LRRC37A using knockout (KO) mouse models. While Lrrc37 KO males were fertile with only mild sperm head abnormalities, Lrrc37a KO males were completely infertile and produced round-headed spermatozoa characteristic of globozoospermia. LRRC37A localized to the acrosome, and its loss resulted in abnormal acrosome enlargement accompanied by the accumulation of Golgi- and vesicle-like structures. Acrosome formation in Lrrc37a KO mice appeared normal during early spermiogenesis but became abnormal at later stages, indicating a requirement for LRRC37A in acrosomal structural integrity. These defects were associated with impaired sperm head shaping during spermiogenesis, leading to globozoospermia. Together, these findings establish LRRC37A as an essential factor in maintaining acrosome integrity and sperm head morphogenesis, highlighting its distinct function from LRRC37. This work provides new insight into the molecular basis of acrosome-related infertility and has important implications for understanding the etiology of globozoospermia.

Acephalic spermatozoa syndrome 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, 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 head-to-tail coupling apparatus and disruption of these proteins causes acephalic spermatozoa. However, the molecular mechanism underlying this morphologic anomaly and head-to-tail coupling apparatus 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 mouse line using the CRISPR/Cas9 system. Ccdc188 knockout 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 head-to-tail coupling apparatus could assemble properly without CCDC188, the head-to-tail coupling apparatus 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.

It is known that various testis-specific mitochondrial proteins are associated with energy metabolism and male meiosis. PDHA2 is a testis-specific mitochondrial protein, and its encoding gene is speculated to be an autosomal retrogene of the progenitor X-linked Pdha1. Here, we show that Pdha2 knockout (KO) mice exhibit azoospermia due to failure at the late pachytene-diplotene transition. We found that PDHA2 interacts with PDHB and PDHA1. PDHA2 absence leads to decreased PDHB amounts and ATP levels in male germ cells. ATP reduction impairs the function of the ATPase recombination proteins RAD51 and DMC1, causing crossover formation deficiency, further resulting in double-strand break repair failure at the pachytene stage. Pdha1 expression by transgenes in Pdha2 KO germ cells rescues fertility and PDHB expression in Pdha2 KO males, confirming the functional equivalence of PDHA1 and PDHA2. Because X-linked Pdha1 expression is silenced during meiotic sex chromosome inactivation, our findings also support the hypothesis that Pdha2 was transposed from Pdha1. In summary, PDHA2 compensates for silenced PDHA1 in male germ cells, and plays a crucial role in maintaining efficient double-strand break repair for proper meiotic progression.

Sperm morphogenesis is a tightly regulated differentiation process, disruption of which leads to sperm malfunction and male infertility. Here, we show that Tex38 knockout (KO) male mice are infertile. Tex38 KO spermatids exhibit excess retention of residual cytoplasm around the head, resulting in abnormal sperm morphology with backward head bending. TEX38 interacts and colocalizes with ZDHHC19, a testis-enriched acyltransferase catalyzing protein S-palmitoylation, at the plasma membrane of spermatids. ZDHHC19 and TEX38 are each downregulated in mouse testes lacking the other protein. TEX38 stabilizes and localizes ZDHHC19 to the plasma membrane of cultured cells and vice versa, consolidating their interdependence. Mice deficient in ZDHHC19 or harboring a C142S mutation that disables the palmitoyltransferase activity of ZDHHC19 display phenotypes resembling those of Tex38 KO mice. Strikingly, ZDHHC19 palmitoylates ARRDC5, an arrestin family protein regulating sperm differentiation. Overall, our findings indicate that TEX38 forms a stable complex with ZDHHC19 at the plasma membrane of spermatids, which governs downstream S-palmitoylation of proteins essential for morphological transformation of spermatids.

Radial spokes (RSs) are conserved multimolecular structures attached to the axonemal microtubule doublets and are essential for the motility control of both cilia and sperm flagella. CFAP91, an RS3 protein, is implicated in human male infertility, yet its molecular function remains poorly understood. Here, we demonstrate that Cfap91 knockout (KO) mice exhibit impaired sperm flagellum formation and male infertility. Using a transgenic rescue model expressing FLAG- and BioID2-tagged CFAP91, we reveal that CFAP91 immunoprecipitates with RS3 proteins CFAP251 and LRRC23, whose localization is disrupted in Cfap91 KO sperm flagella. In addition, proximity labeling in mature spermatozoa identifies EFCAB5 as a sperm-specific CFAP91-proximal component. We show that Efcab5 KO males exhibit reduced sperm motility and fertility. Our findings establish CFAP91 as an essential scaffolder of RS3 assembly and EFCAB5 as a sperm-specialized movement regulator, advancing understanding of axonemal specialization in mammalian spermatozoa and its relevance to male infertility.

BACKGROUND:RIBC1 (RIB43A domain with coiled-coils 1) and RIBC2 (RIB43A domain with coiled-coils 2) are homolog proteins of RIB43a which is localized to microtubules in the cilia and flagella of unicellular organisms. Cryo-electron microscopy and artificial intelligence studies showed that RIBC1 and RIBC2 are microtubule inner proteins (MIPs) localized in the inner lumen of the doublet microtubules (DMTs) in mouse sperm flagella. However, the function of RIBC1 and RIBC2 in mammalian reproduction and sperm flagella is still unknown. OBJECTIVE:To clarify the function of RIBC1 and RIBC2 in mouse spermatozoa. MATERIALS AND METHODS:We generated Ribc1 knockout (KO), Ribc2 KO, and Ribc1 and Ribc2 double-knockout (Ribc1/2 DKO) mice using the CRISPR/Cas9 system and analyzed their phenotypes. RESULTS:We revealed that the loss of either RIBC1 or RIBC2 alone did not affect male fertility, but the absence of both caused a decrease in pup numbers. Sperm motility analysis showed that Ribc1 KO spermatozoa had reduced velocity, but Ribc2 KO sperm velocities were comparable with WT mice. However, Ribc1/2 DKO sperm velocities were significantly lower than those from Ribc1 KO mice. No structural abnormalities in the axonemal structure at the transmission electron microscope (TEM) level and no abnormalities in the flagellar waveform pattern were observed in Ribc1/2 DKO spermatozoa. DISCUSSION AND CONCLUSION:Both RIBC1 and RIBC2 are not significant for maintaining the axonemal structure in mouse spermatozoa, but both proteins function cooperatively in sperm motility. This result may indicate that minor structural changes due to RIBC protein absence are not detected at the TEM level, and RIBC2 function depends on RIBC1 in sperm motility. We think that reduced litter size in Ribc1/2 DKO mice is caused by reduced sperm motility due to minor structural abnormalities caused by the loss of two RIBC proteins.

Background More than 1000 genes have been identified as predominantly expressed in the human testis. Advances in gene editing technologies have enabled the rapid and efficient generation of genetically engineered mice. This approach facilitates the screening of genes essential for spermatogenesis by analyzing knockout mouse models.Objectives This study aimed to elucidate the essential genes in male reproductive function by generating knockout mouse models.Materials and Methods We selected 11 target genes that may have potential roles in the male reproductive system based on a public database. Knockout mouse lines of these target genes were generated using the CRISPR/Cas9 system to elucidate their functions in male reproduction. Also, we conducted natural mating tests to elucidate fecundity and analyzed the phenotype of the knockout males.Results Natural mating tests revealed that all 11 gene-deficient mouse lines maintained normal male fertility. The phenotypic analysis, including testis appearance and weight, histology of testis and epididymis, and sperm motility and morphology, showed no apparent abnormalities.Discussion and Conclusion These results suggest that each gene is not essential for male reproductive function.

BackgroundMale infertility is an intricate multifactorial disease involving the interplay between genetic and environmental factors. Genetic anomalies account for more than 15% of all male infertility cases; however, diagnosing them exhibits enormous challenges due to variable symptomatic presentations and limited knowledge of gene functions. Therefore, a thorough investigation into gene regulatory networks underlying male reproduction is demanded to improve patient counseling and infertility treatment.ObjectiveIn this study, we aimed to identify testis-expressed genes essential for male fertility.MethodsWe searched public databases, such as the National Center for Biotechnology Information (NCBI), Ensembl genome browser, the Human Protein Atlas (HPA), and the Mammalian Reproductive Genetics Database V2 (MRGDv2), to identify genes predominantly expressed in male reproductive tissues. Genetically engineered mouse lines lacking individual genes of interest were generated using either targeted gene replacement or the CRISPR/Cas9 system. To determine the gene functions, we analyzed fertility, testis weight, testis and epididymis histology, and sperm motility and morphology in adult knockout (KO) male mice.ResultsThrough the in silico screen, we identified 18 testis-expressed genes, including coiled-coil domain containing 182 (Ccdc182), EF-hand calcium-binding domain 15 (Efcab15), family with sequence similarity 187, member B (Fam187b), family with sequence similarity 24, member A (Fam24a), family with sequence similarity 24, member B (Fam24b), glial cell line derived neurotrophic factor family receptor alpha 2 (Gfra2), GLI pathogenesis-related 1 like 1, 2, and 3 (Glipr1l1-3), interleukin 3 (Il3), IZUMO family member 4 (Izumo4), peptidyl-prolyl cis/trans isomerase, NIMA-interacting 1, retrogene 1 (Pin1rt1), solute carrier family 22 (organic cation transporter), member 16 (Slc22a16), sperm microtubule inner protein 2 (Spmip2), testis expressed 51 (Tex51), transmembrane and coiled-coil domains 2 (Tmco2), and tripartite motif family-like 1 and 2 (Triml1/2). The KO males displayed no obvious health problems, and normal mating behavior, fecundity, testis and epididymis histology, and sperm morphology and motility.Discussion and ConclusionOur findings indicate that these 18 testis-expressed genes are individually dispensable for male reproduction in mice. Disseminating such genes would promote our understanding of male reproduction and expedite the discovery of novel key male factors. Although we anticipate that mutations in these genes may not impair fertility in men, their enrichment in male germ cells makes them potential biomarkers for sperm count, quality, and morphological anomalies.

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.

Mammalian sperm flagellum contains the midpiece characterized by a mitochondrial sheath that packs tightly around the axoneme and outer dense fibers. Mitochondria are known as the "powerhouse" of the cell, and produce ATP through the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS). However, the contribution of the TCA cycle and OXPHOS to sperm motility and male fertility is less clear. Cytochrome c oxidase (COX) is an oligomeric complex localized within the mitochondrial inner membrane, and the terminal enzyme of the mitochondrial electron transport chain in eukaryotes. Both COX6B2 and COX8C are testis-enriched COX subunits whose functions in vivo are poorly studied. Here, we generated Cox6b2 and Cox8c knockout (KO) mice using the CRISPR/Cas9 system. We examined their fertility and sperm mitochondrial function to determine the significance of testis-enriched COX subunits in male fertility. The mating test revealed that disrupting COX6B2 induces male subfertility, while disrupting COX8C does not affect male fertility. Cox6b2 KO spermatozoa showed low sperm motility, but mitochondrial function was normal according to oxygen consumption rates. Therefore, low sperm motility seems to cause subfertility in Cox6b2 KO male mice. These results also indicate that testis-enriched COX, COX6B2 and COX8C, are not essential for OXPHOS in mouse spermatozoa.

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.

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.

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.

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.

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.

Endometrial decidualization, a prerequisite for successful pregnancies, relies on transcriptional reprogramming driven by progesterone receptor (PR) and bone morphogenetic protein (BMP)-SMAD1/SMAD5 signaling pathways. Despite their critical roles in early pregnancy, how these pathways intersect in reprogramming the endometrium into a receptive state remains unclear. To define how SMAD1 and/or SMAD5 integrate BMP signaling in the uterus during early pregnancy, we generated two novel transgenic mouse lines with affinity tags inserted into the endogenous SMAD1 and SMAD5 loci (Smad1HA/HA and Smad5PA/PA). By profiling the genome-wide distribution of SMAD1, SMAD5, and PR in the mouse uterus, we demonstrated the unique and shared roles of SMAD1 and SMAD5 during the window of implantation. We also showed the presence of a conserved SMAD1, SMAD5, and PR genomic binding signature in the uterus during early pregnancy. To functionally characterize the translational aspects of our findings, we demonstrated that SMAD1/5 knockdown in human endometrial stromal cells suppressed expressions of canonical decidual markers (IGFBP1, PRL, FOXO1) and PR-responsive genes (RORB, KLF15). Here, our studies provide novel tools to study BMP signaling pathways and highlight the fundamental roles of SMAD1/5 in mediating both BMP signaling pathways and the transcriptional response to progesterone (P4) during early pregnancy.