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Noda, T., Lu, Y., Fujihara, Y., Oura, S., Koyano, T., Kobayashi, S., . . . Ikawa, M. (2020). Sperm proteins SOF1, TMEM95, and SPACA6 are required for sperm-oocyte fusion in mice. Proceedings of the National Academy of Sciences of the United States of America, 117(21) doi:10.1073/pnas.1922650117, Sperm-oocyte membrane fusion is one of the most important events for fertilization. So far, IZUMO1 and Fertilization Influencing Membrane Protein (FIMP) on the sperm membrane and CD9 and JUNO (IZUMO1R/FOLR4) on the oocyte membrane have been identified as fusion-required proteins. However, the molecular mechanisms for sperm-oocyte fusion are still unclear. Here, we show that testis-enriched genes, sperm-oocyte fusion required 1 (Sof1/Llcfc1/1700034O15Rik), transmembrane protein 95 (Tmem95), and sperm acrosome associated 6 (Spaca6), encode sperm proteins required for sperm-oocyte fusion in mice. These knockout (KO) spermatozoa carry IZUMO1 but cannot fuse with the oocyte plasma membrane, leading to male sterility. Transgenic mice which expressed mouse Sof1, Tmem95, and Spaca6 rescued the sterility of Sof1, Tmem95, and Spaca6 KO males, respectively. SOF1 and SPACA6 remain in acrosome-reacted spermatozoa, and SPACA6 translocates to the equatorial segment of these spermatozoa. The coexpression of SOF1, TMEM95, and SPACA6 in IZUMO1-expressing cultured cells did not enhance their ability to adhere to the oocyte membrane or allow them to fuse with oocytes. SOF1, TMEM95, and SPACA6 may function cooperatively with IZUMO1 and/or unknown fusogens in sperm-oocyte fusion.

In mammals, more than 2000 genes are specifically or abundantly expressed in testis, but gene knockout studies revealed several are not individually essential for male fertility. Tesmin (Metallothionein-like 5; Mtl5) was originally reported as a testis-specific transcript that encodes a member of the cysteine-rich motif containing metallothionein family. Later studies showed that Tesmin has two splicing variants and both are specifically expressed in male and female germ cells. Herein, we clarified that the long (Tesmin-L) and short (Tesmin-S) transcript forms start expressing from spermatogonia and the spermatocyte stage, respectively, in testis. Furthermore, while Tesmin-deficient female mice are fertile, male mice are infertile due to arrested spermatogenesis at the pachytene stage. We were able to rescue the infertility with a Tesmin-L transgene, where we concluded that TESMIN-L is critical for meiotic completion in spermatogenesis and indispensable for male fertility., TESMIN protein, a member of metallothionein family, is essential for mouse spermatogenesis, especially in the meiotic stage.

Miyata, H., Shimada, K., Morohoshi, A., Oura, S., Matsumura, T., Xu, Z., . . . Ikawa, M. (2020). Testis-enriched kinesin KIF9 is important for progressive motility in mouse spermatozoa. FASEB Journal, 34(4), 5389-5400. doi:10.1096/fj.201902755R, Kinesin is a molecular motor that moves along microtubules. Kinesin family member 9 (KIF9) is evolutionarily conserved and expressed strongly in mouse testis. In the unicellular flagellate Chlamydomonas, KLP1 (ortholog of KIF9) is localized to the central pair microtubules of the axoneme and regulates flagellar motility. In contrast, the function of KIF9 remains unclear in mammals. Here, we mutated KIF9 in mice using the CRISPR/Cas9 system. Kif9 mutated mice exhibit impaired sperm motility and subfertility. Further analysis reveals that the flagella lacking KIF9 showed an asymmetric waveform pattern, which leads to a circular motion of spermatozoa. In spermatozoa that lack the central pair protein HYDIN, KIF9 was not detected by immunofluorescence and immunoblot analysis. These results suggest that KIF9 is associated with the central pair microtubules and regulates flagellar motility in mice.

Globozoospermia (sperm with an abnormally round head shape) and asthenozoospermia (defective sperm motility) are known causes of male infertility in human patients. Despite many studies, the molecular details of the globozoospermia etiology are still poorly understood. Serine-rich single-pass membrane protein 1 (Ssmem1) is a conserved testis-specific gene in mammals. In this study, we generated Ssmem1 knockout (KO) mice using the CRISPR/Cas9 system, demonstrated that Ssmem1 is essential for male fertility in mice, and found that SSMEM1 protein is expressed during spermatogenesis but not in mature sperm. The sterility of the Ssmem1 KO (null) mice is associated with globozoospermia and loss of sperm motility. To decipher the mechanism causing the phenotype, we analyzed testes with transmission electron microscopy and discovered that Ssmem1-disrupted spermatids have abnormal localization of Golgi at steps eight and nine of spermatid development. Immunofluorescence analysis with anti-Golgin-97 to label the trans-Golgi network, also showed delayed movement of the Golgi to the spermatid posterior region, which causes failure of sperm head shaping, disorganization of the cell organelles, and entrapped tails in the cytoplasmic droplet. In summary, SSMEM1 is crucial for intracellular Golgi movement to ensure proper spatiotemporal formation of the sperm head that is required for fertilization. These studies and the pathway in which SSMEM1 functions have implications for human male infertility and identifying potential targets for nonhormonal contraception., Absence of serine-rich single-pass membrane protein 1 (SSMEM1) leads to male infertility because of globozoospermia in mice.

The acrosome is a cap-shaped, Golgi-derived membranous organelle that is located over the anterior of the sperm nucleus and highly conserved throughout evolution. Although morphological changes during acrosome biogenesis in spermatogenesis have been well described, the molecular mechanism underlying this process is still largely unknown. Family with sequence similarity 71, member F1 and F2 (FAM71F1 and FAM71F2) are testis-enriched proteins that contain a RAB2B-binding domain, a small GTPase involved in vesicle transport and membrane trafficking. Here, by generating mutant mice for each gene, we found that Fam71f1 is essential for male fertility. In Fam71f1-mutant mice, the acrosome was abnormally expanded at the round spermatid stage, likely because of enhanced vesicle trafficking. Mass spectrometry analysis after immunoprecipitation indicated that, in testes, FAM71F1 binds not only RAB2B, but also RAB2A. Further study suggested that FAM71F1 binds to the GTP-bound active form of RAB2A/B, but not the inactive form. These results indicate that a complex of FAM71F1 and active RAB2A/B suppresses excessive vesicle trafficking during acrosome formation., Summary: FAM71F1 interacts with RAB2A/B and regulates the formation of the acrosome in mice, the misregulation of which impacts male fertility.

Significance Calcineurin is a target of immunosuppressive drugs such as cyclosporine A and tacrolimus. In the immune system, calcineurin interacts with NFAT via the PxIxIT motif to activate T cells. In contrast, little is known about the proteins that interact with a testis-enriched calcineurin that is essential for sperm motility and male fertility. Here, we discovered that calcineurin interacts with SPATA33 via a PQIIIT sequence in the testis. Further analyses reveal that SPATA33 plays critical roles in sperm motility and male fertility. Our finding sheds new light on the molecular mechanisms of sperm motility regulation and the etiology of human male fertility. Furthermore, it may help us not only understand reproductive toxicities but also develop nonhormonal male contraceptives., Calcineurin is a calcium-dependent phosphatase that plays roles in a variety of biological processes including immune responses. In spermatozoa, there is a testis-enriched calcineurin composed of PPP3CC and PPP3R2 (sperm calcineurin) that is essential for sperm motility and male fertility. Because sperm calcineurin has been proposed as a target for reversible male contraceptives, identifying proteins that interact with sperm calcineurin widens the choice for developing specific inhibitors. Here, by screening the calcineurin-interacting PxIxIT consensus motif in silico and analyzing the function of candidate proteins through the generation of gene-modified mice, we discovered that SPATA33 interacts with sperm calcineurin via a PQIIIT sequence. Spata33 knockout mice exhibit reduced sperm motility because of an inflexible midpiece, leading to impaired male fertility, which phenocopies Ppp3cc and Ppp3r2 knockout mice. Further analysis reveals that sperm calcineurin disappears from the mitochondria in the Spata33 knockout testis. In addition, immunoprecipitation analysis indicates that sperm calcineurin interacts with not only SPATA33 but also the mitochondrial protein VDAC2. These results indicate that SPATA33 localizes calcineurin to the mitochondria and regulates sperm motility.

Significance While the emergence of gene modification technologies has produced major discoveries in biomedical sciences, the recent development of the CRISPR/Cas9 system has dramatically altered the trajectory of phenotypic analysis in animal models. In this study, we identified male-specific gene clusters (Cst and Pate) and family genes (Gdpd and Lypd) and found specific members to be required for male fertility, especially for sperm fertilizing ability. Our findings support the important roles of these proteins in sperm function and could be used to develop novel infertility treatments as well as contraceptives., CRISPR/Cas9-mediated genome editing technology enables researchers to efficiently generate and analyze genetically modified animals. We have taken advantage of this game-changing technology to uncover essential factors for fertility. In this study, we generated knockouts (KOs) of multiple male reproductive organ-specific genes and performed phenotypic screening of these null mutant mice to attempt to identify proteins essential for male fertility. We focused on making large deletions (dels) within 2 gene clusters encoding cystatin (CST) and prostate and testis expressed (PATE) proteins and individual gene mutations in 2 other gene families encoding glycerophosphodiester phosphodiesterase domain (GDPD) containing and lymphocyte antigen 6 (Ly6)/Plaur domain (LYPD) containing proteins. These gene families were chosen because many of the genes demonstrate male reproductive tract-specific expression. Although Gdpd1 and Gdpd4 mutant mice were fertile, disruptions of Cst and Pate gene clusters and Lypd4 resulted in male sterility or severe fertility defects secondary to impaired sperm migration through the oviduct. While absence of the epididymal protein families CST and PATE affect the localization of the sperm membrane protein A disintegrin and metallopeptidase domain 3 (ADAM3), the sperm acrosomal membrane protein LYPD4 regulates sperm fertilizing ability via an ADAM3-independent pathway. Thus, use of CRISPR/Cas9 technologies has allowed us to quickly rule in and rule out proteins required for male fertility and expand our list of male-specific proteins that function in sperm migration through the oviduct.

The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system is a research hotspot in gene therapy. However, the widely used Streptococcus pyogenes Cas9 (WT-SpCas9) requires an NGG protospacer adjacent motif (PAM) for target recognition, thereby restricting targetable disease mutations. To address this issue, we recently reported an engineered SpCas9 nuclease variant (SpCas9-NG) recognizing NGN PAMs. Here, as a feasibility study, we report SpCas9-NG-mediated repair of the abnormally expanded CAG repeat tract in Huntington’s disease (HD). By targeting the boundary of CAG repeats with SpCas9-NG, we precisely contracted the repeat tracts in HD-mouse-derived embryonic stem (ES) cells. Further, we confirmed the recovery of phenotypic abnormalities in differentiated neurons and animals produced from repaired ES cells. Our study shows that SpCas9-NG can be a powerful tool for repairing abnormally expanded CAG repeats as well as other disease mutations that are difficult to access with WT-SpCas9., Seiya Oura and Taichi Noda et al. overcome the challenge of gene editing in CAG repeats, such as those causing Huntington’s Disease, using their recently developed SpCas9-NG variant. They demonstrate that SpCas9-NG can precisely edit and contract the CAG repeat tracks in a Huntington’s Disease mouse model, opening new avenues for research in this disease.

The process of sperm-egg fusion is critical for successful fertilization, yet the underlying mechanisms that regulate these steps have remained unclear in vertebrates. Here, we show that both mouse and zebrafish DCST1 and DCST2 are necessary in sperm to fertilize the egg, similar to their orthologs SPE-42 and SPE-49 in C. elegans and Sneaky in D. melanogaster. Mouse Dcst1 and Dcst2 single knockout (KO) spermatozoa are able to undergo the acrosome reaction and show normal relocalization of IZUMO1, an essential factor for sperm-egg fusion, to the equatorial segment. While both single KO spermatozoa can bind to the oolemma, they rarely fuse with oocytes, resulting in male sterility. Similar to mice, zebrafish dcst1 KO males are subfertile and dcst2 and dcst1/2 double KO males are sterile. Zebrafish dcst1/2 KO spermatozoa are motile and can approach the egg, but rarely bind to the oolemma. These data demonstrate that DCST1/2 are essential for male fertility in two vertebrate species, highlighting their crucial role as conserved factors in fertilization.

Meiosis is a cell division process with complex chromosome events where various molecules must work in tandem. To find meiosis-related genes, we screened evolutionarily conserved and reproductive tract-enriched genes using the CRISPR/Cas9 system and identified potassium channel tetramerization domain containing 19 (Kctd19) as an essential factor for meiosis. In prophase I, Kctd19 deficiency did not affect synapsis or the DNA damage response, and chiasma structures were also observed in metaphase I spermatocytes of Kctd19 KO mice. However, spermatocytes underwent apoptotic elimination during the metaphase-anaphase transition. We were able to rescue the Kctd19 KO phenotype with an epitope-tagged Kctd19 transgene. Immunoprecipitation-mass spectrometry identified zinc finger protein 541 (ZFP541) and histone deacetylase 1 (HDAC1) as binding partners of KCTD19, indicating that KCTD19 is involved in chromatin modification. Phenotyping of Zfp541 KO spermatocytes demonstrated XY chromosome asynapsis and recurrent DNA damage in the late pachytene stage, leading to apoptosis. In summary, our study reveals that KCTD19 associates with ZFP541 and HDAC1, and that both KCTD19 and ZFP541 were essential for meiotic exit in male mice.Author summaryMeiosis is a fundamental process that consisting of one round of genomic DNA replication and two rounds of chromosome segregation producing four haploid cells. To properly distribute their genetic material, cells need to undergo complex chromosome events such as a physical linkage of homologous chromosomes (termed synapsis) and meiotic recombination. The molecules involved in these events have not been fully characterized yet, especially in mammals. Using a CRISPR/Cas9-screening system, we identified the potassium channel tetramerization domain containing 19 (Kctd19) as an essential factor for meiosis in male mice. Further, we identified zinc finger protein 541 (ZFP541) and histone deacetylase 1 (HDAC1) as binding partners of KCTD19. By observing meiosis of Zfp541 knockout germ cells, we found that Zfp541 was also essential for meiotic completion. These results show that the KCTD19/ZFP541 complex plays a critical role and is indispensable for male meiosis and fertility.

Meiosis is a cell division process with complex chromosome events where various molecules must work in tandem. To find meiosis-related genes, we screened evolutionarily conserved and reproductive tract-enriched genes using the CRISPR/Cas9 system and identified potassium channel tetramerization domain containing 19 (Kctd19) as an essential factor for meiosis. In prophase I, Kctd19 deficiency did not affect synapsis or the DNA damage response, and chiasma structures were also observed in metaphase I spermatocytes of Kctd19 KO mice. However, spermatocytes underwent apoptotic elimination during the metaphase-anaphase transition. We were able to rescue the Kctd19 KO phenotype with an epitope-tagged Kctd19 transgene. By immunoprecipitation-mass spectrometry, we confirmed the association of KCTD19 with zinc finger protein 541 (ZFP541) and histone deacetylase 1 (HDAC1). Phenotyping of Zfp541 KO spermatocytes demonstrated XY chromosome asynapsis and recurrent DNA damage in the late pachytene stage, leading to apoptosis. In summary, our study reveals that KCTD19 associates with ZFP541 and HDAC1, and that both KCTD19 and ZFP541 are essential for meiosis in male mice., Author summary Meiosis is a fundamental process that consists of one round of genomic DNA replication and two rounds of chromosome segregation, producing four haploid cells. To properly distribute their genetic material, cells need to undergo complex chromosome events such as a physical linkage of homologous chromosomes (termed synapsis) and meiotic recombination. The molecules involved in these events have not been fully characterized yet, especially in mammals. Using a CRISPR/Cas9-screening system, we identified the potassium channel tetramerization domain containing 19 (Kctd19) as an essential factor for meiosis in male mice. Further, we confirmed the association of KCTD19 with zinc finger protein 541 (ZFP541) and histone deacetylase 1 (HDAC1). By observing meiosis of Zfp541 knockout germ cells, we found that Zfp541 was also essential for meiosis. These results show that the KCTD19/ZFP541 complex plays a critical role and is indispensable for male meiosis and fertility.

Significance Although formation of the mitochondrial sheath is a critical process in the formation of mature spermatozoa, the molecular mechanisms involved in mitochondrial sheath genesis remain unclear. Using gene-manipulated mice, we discovered that ARMC12 regulates spatiotemporal “sperm mitochondrial dynamics” during mitochondrial sheath formation through interactions with mitochondrial proteins MIC60, VDAC2, and VDAC3 as well as testis-specific proteins TBC1D21 and GK2. In addition, we demonstrated that ARMC12-interacting proteins TBC1D21 and GK2 are also essential for mitochondrial sheath formation. Our paper sheds light on the molecular mechanisms of mitochondrial sheath formation and the regulation of sperm mitochondrial dynamics, allowing us to further understand the biology of spermatogenesis and the etiology of infertility in men., The mammalian sperm midpiece has a unique double-helical structure called the mitochondrial sheath that wraps tightly around the axoneme. Despite the remarkable organization of the mitochondrial sheath, the molecular mechanisms involved in mitochondrial sheath formation are unclear. In the process of screening testis-enriched genes for functions in mice, we identified armadillo repeat-containing 12 (ARMC12) as an essential protein for mitochondrial sheath formation. Here, we engineered Armc12-null mice, FLAG-tagged Armc12 knock-in mice, and TBC1 domain family member 21 (Tbc1d21)-null mice to define the functions of ARMC12 in mitochondrial sheath formation in vivo. We discovered that absence of ARMC12 causes abnormal mitochondrial coiling along the flagellum, resulting in reduced sperm motility and male sterility. During spermiogenesis, sperm mitochondria in Armc12-null mice cannot elongate properly at the mitochondrial interlocking step which disrupts abnormal mitochondrial coiling. ARMC12 is a mitochondrial peripheral membrane protein and functions as an adherence factor between mitochondria in cultured cells. ARMC12 in testicular germ cells interacts with mitochondrial proteins MIC60, VDAC2, and VDAC3 as well as TBC1D21 and GK2, which are required for mitochondrial sheath formation. We also observed that TBC1D21 is essential for the interaction between ARMC12 and VDAC proteins in vivo. These results indicate that ARMC12 uses integral mitochondrial membrane proteins VDAC2 and VDAC3 as scaffolds to link mitochondria and works cooperatively with TBC1D21. Thus, our studies have revealed that ARMC12 regulates spatiotemporal mitochondrial dynamics to form the mitochondrial sheath through cooperative interactions with several proteins on the sperm mitochondrial surface.

The flagellum is essential for sperm motility and fertilization in vivo. The axoneme is the main component of the flagella, extending through its entire length. An axoneme is comprised of two central microtubules surrounded by nine doublets, the nexin-dynein regulatory complex, radial spokes, and dynein arms. Failure to properly assemble components of the axoneme in a sperm flagellum, leads to fertility alterations. To understand this process in detail, we have defined the function of an uncharacterized gene, Cfap97 domain containing 1 (Cfap97d1). This gene is evolutionarily conserved in mammals and multiple other species, including Chlamydomonas. We have used two independently generated Cfap97d1 knockout mouse models to study the gene function in vivo. Cfap97d1 is exclusively expressed in testes starting from post-natal day 20 and continuing throughout adulthood. Deletion of the Cfap97d1 gene in both mouse models leads to sperm motility defects (asthenozoospermia) and male subfertility. In vitro fertilization (IVF) of cumulus-intact oocytes with Cfap97d1 deficient sperm yielded few embryos whereas IVF with zona pellucida-free oocytes resulted in embryo numbers comparable to that of the control. Knockout spermatozoa showed abnormal motility characterized by frequent stalling in the anti-hook position. Uniquely, Cfap97d1 loss caused a phenotype associated with axonemal doublet heterogeneity linked with frequent loss of the fourth doublet in the sperm stored in the epididymis. This study demonstrates that Cfap97d1 is required for sperm flagellum ultra-structure maintenance, thereby playing a critical role in sperm function and male fertility in mice., Author summary Infertility is a growing issue in modern society, affecting about 15% of reproductive age couples. About 1 in 20 men of reproductive age has fertility issues. The causes of male infertility remain undefined in more than half of the cases. Approximately one-fourth of male infertility cases can be attributed to genetic factors. Currently, only a few genes involved in testis development and sperm formation have been well studied and shown to have clinical significance. A better understanding of male fertility mechanisms will help to advance infertility treatments and the development of additional contraceptive alternatives, including those targeting male sex cells. Testes uniquely express several hundreds of genes and the functions of numerous testis-specific genes are yet uncharacterized. We have identified the evolutionarily conserved Cfap97d1 gene as a testis-specific gene in a public database screen. We used two knockout mouse models to demonstrate that the absence of the Cfap97d1 gene causes reduced sperm motility (asthenozoospermia) due to destabilization of outer microtubule doublet in sperm flagellum. This research shows that Cfap97d1 is an important regulator of male fertility.

Seminal vesicle secretions (SVSs), together with spermatozoa, are ejaculated into the female reproductive tract. SVS7, also known as PATE4, is one of the major SVS proteins found in the seminal vesicle, copulatory plug, and uterine fluid after copulation. Here, we generated Pate4 knockout (−/−) mice and examined the detailed function of PATE4 on male fecundity. The morphology and weight of Pate4−/− seminal vesicles were comparable to the control. Although Pate4−/− cauda epididymal spermatozoa have no overt defects during in vitro fertilization, Pate4−/− males were subfertile. We found that the copulatory plugs were smaller in the vagina of females mated with Pate4−/− males, leading to semen leakage and a decreased sperm count in the uterus. When the females mated with Pate4−/− males were immediately re-caged with Pate4+/+ males, the females had subsequent productive matings. When the cauda epididymal spermatozoa were injected into the uterus and plugged artificially [artificial insemination (AI)], Pate4−/− spermatozoa could efficiently fertilize eggs as compared to wild-type spermatozoa. We finally examined the effect of SVSs on AI, and observed no difference in fertilization rates between Pate4+/+ and Pate4−/− SVSs. In conclusion, PATE4 is a novel factor in forming the copulatory plug that inhibits sequential matings and maintains spermatozoa in the uterus to ensure male fecundity., The copulatory plug has dual functions not only to prevent subsequent matings but also to maintain proper sperm count for fertilization in the female reproductive tract, as a winner-take-all strategy to advance male reproduction.

The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genome-editing tool. However, the widely used Streptococcus pyogenes Cas9 enzyme (SpCas9) requires an NGG protospacer adjacent motif (PAM) for target recognition, thereby restricting the targetable genomic loci. Here, we report a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize relaxed NG PAMs. The crystal structure revealed that the loss of the base-specific interaction with the third G is compensated by newly introduced non-base-specific interactions, enabling the NG PAM recognition. We showed that SpCas9-NG induces indels at endogenous target sites bearing NG PAMs in human cells. Furthermore, we found that the fusion of SpCas9-NG and the activation-induced cytidine deaminase (AID) mediates the C-to-T conversion at target sites with NG PAMs in human cells., NICHD (Grants P01HD087157, R01HD088412), NIMH (Grants 5DP1-MH100706,1R01-MH110049), NIDDK (Grant 5R01DK097768-03)

Chang Rok Kim, Taichi Noda, Hyunkyung Kim, Gibeom Kim, Seongwan Park, Yongwoo Na, Seiya Oura, Keisuke Shimada, Injin Bang, Jun-Yeong Ahn, Yong Ryoul Kim, Se Kyu Oh, Hee-Jung Choi, Jong-Seo Kim, Inkyung Jung, Ho Lee, Yuki Okada, Masahito Ikawa, Sung Hee Baek, PHF7 Modulates BRDT Stability and Histone-to-Protamine Exchange during Spermiogenesis, Cell Reports, Volume 32, Issue 4, 2020, 107950, ISSN 2211-1247, https://doi.org/10.1016/j.celrep.2020.107950., Spermatogenesis is a complex process of sperm generation, including mitosis, meiosis, and spermiogenesis. During spermiogenesis, histones in post-meiotic spermatids are removed from chromatin and replaced by protamines. Although histone-to-protamine exchange is important for sperm nuclear condensation, the underlying regulatory mechanism is still poorly understood. Here, we identify PHD finger protein 7 (PHF7) as an E3 ubiquitin ligase for histone H3K14 in post-meiotic spermatids. Generation of Phf7-deficient mice and Phf7 C160A knockin mice with impaired E3 ubiquitin ligase activity reveals defects in histone-to-protamine exchange caused by dysregulation of histone removal factor Bromodomain, testis-specific (BRDT) in early condensing spermatids. Surprisingly, E3 ubiquitin ligase activity of PHF7 on histone ubiquitination leads to stabilization of BRDT by attenuating ubiquitination of BRDT. Collectively, our findings identify PHF7 as a critical factor for sperm chromatin condensation and contribute to mechanistic understanding of fundamental phenomenon of histone-to-protamine exchange and potential for drug development for the male reproduction system. Histone-to-protamine exchange is essential for functional sperm. Kim et al. demonstrate that PHF7 is an E3 ubiquitin ligase for histone H3, and that PHF7-mediated histone ubiquitination regulates BRDT stability during histone removal. Mice with impaired Phf7 show dysfunctional sperm caused by defects in histone ubiquitination and histone-to-protamine exchange.

High-throughput transcriptomics and proteomics approaches have recently identified a large number of germ cell–specific genes with many that remain to be studied through functional genetics approaches. Serine proteases (PRSS) constitute nearly one-third of all proteases, and, in our bioinformatics screens, we identified many that are testis specific. In this study, we chose to focus on Prss44, Prss46, and Prss54, which we confirmed as testis specific in mouse and human. Based on the analysis of developmental expression in the mouse, expression of all four genes is restricted to the late stage of spermatogenesis concomitant with a potential functional role in spermiogenesis, spermiation, or sperm function. To best understand the male reproductive requirement and functional roles of these serine proteases, each gene was individually ablated by CRISPR/Cas9-mediated ES cell or zygote approach. Homozygous deletion mutants for each gene were obtained and analyzed for phenotypic changes. Analyses of testis weights, testis and epididymis histology, sperm morphology, and fertility revealed no significant differences in Prss44, Prss46, and Prss54 knockout mice in comparison to controls. Our results thereby demonstrate that these genes are not required for normal fertility in mice, although do not preclude the possibility that these genes may function in a redundant manner. Elucidating the individual functional requirement or lack thereof of these novel genes is necessary to build a better understanding of the factors underlying spermatogenesis and sperm maturation, which has implications in understanding the etiology of male infertility and the development of male contraceptives., Summary Sentence The testis-specific serine proteases Prss44, Prss46, and Prss54 are dispensable for male fertility based on phenotype analyses of knockout mice produced using the CRISPR/Cas9 system.

More than 1000 genes are predicted to be predominantly expressed in mouse testis, yet many of them remain unstudied in terms of their roles in spermatogenesis and sperm function and their essentiality in male reproduction. Since individually indispensable factors can provide important implications for the diagnosis of genetically related idiopathic male infertility and may serve as candidate targets for the development of nonhormonal male contraceptives, our laboratories continuously analyze the functions of testis-enriched genes in vivo by generating knockout mouse lines using the CRISPR/Cas9 system. The dispensability of genes in male reproduction is easily determined by examining the fecundity of knockout males. During our large-scale screening of essential factors, we knocked out 30 genes that have a strong bias of expression in the testis and are mostly conserved in mammalian species including human. Fertility tests reveal that the mutant males exhibited normal fecundity, suggesting these genes are individually dispensable for male reproduction. Since such functionally redundant genes are of diminished biological and clinical significance, we believe that it is crucial to disseminate this list of genes, along with their phenotypic information, to the scientific community to avoid unnecessary expenditure of time and research funds and duplication of efforts by other laboratories., Thirty testis-enriched genes are dispensable for male fertility based on phenotypic analyses of knockout mice produced by the CRISPR/Cas9 system.

The CRISPR/Cas9 system can efficiently introduce biallelic mutations in ES cells (ESCs), and its application with fluorescently-tagged ESCs enables phenotype analysis in chimeric mice. We have utilized ESCs that express EGFP in the cytosol and acrosome [EGR-G101 129S2 × (CAG/Acr-EGFP) B6] in previous studies; however, the EGFP signal in the sperm cytosol is weak and the signal in the acrosome is lost after the acrosome reaction, precluding analysis between wild type and ESC derived spermatozoa. In this study, we established an ESC line from RBGS (Red Body Green Sperm) transgenic mice [B6D2-Tg (CAG/Su9-DsRed2, Acr3-EGFP) RBGS002Osb] whose spermatozoa exhibit green fluorescence in the acrosome and red fluorescence in the mitochondria within the flagellar midpiece that is retained after the acrosome reaction. We utilized these new ESCs to analyze HYDIN, which is reported to function in sperm motility in humans. Analysis of Hydin-disrupted spermatozoa in mice is difficult as Hydin-mutant mice (hy3) die within 3 weeks, before sexual maturation, due to hydrocephaly. To circumvent the early lethality of the whole-body knockout, we disrupted Hydin in RBGS-ESCs and generated chimeric mice, which survived into sexual maturity. Hydin-disrupted spermatozoa obtained from the chimeric mice possessed short tails and were immotile. When we injected Hydin-disrupted spermatozoa into oocytes, heterozygous pups were obtained, which suggests that the genome of Hydin-disrupted spermatozoa can produce viable pups. Consequently, RBGS-ESCs can be a useful tool for screening and analysis of male-fertility related genes in chimeric mice.

Retinoids exert antitumor effects through the retinoic acid receptor α (RARα). In the present study, we sought to identify the factors involved in the RARα-mediated transcriptional regulation of the tumor suppressor gene and the tissue factor pathway inhibitor 2 (TFPI2) in hepatocellular carcinoma (HCC). All-trans-retinoic acid (ATRA) was used in the in vitro experiments. Cell invasiveness was measured using trans-well invasion assay. ATRA significantly increased TFPI2 expression through RARα in a human HCC cell line known as HuH7. TFPI2 was vital in the ATRA-mediated suppression of HuH7 cell invasion. The musculo-aponeurotic fibrosarcoma oncogene homolog B (MAFB) significantly enhanced the activation of the TFPI2 promoter via RARα while MAFF inhibited it. The knockdown of RARα or MAFB counteracted the ATRA-mediated suppression of HuH7 cell invasion while the knockdown of MAFF inhibited the invasion. TFPI2 expression in HCC tissues was significantly downregulated possibly due to the decreased expression of RARβ and MAFB. Patients with HCC expressing low MAFB and high MAFF levels showed the shortest disease-free survival time. These results suggest that MAFB and MAFF play critical roles in the antitumor effects of retinoids by regulating the expression of retinoid target genes such as TFPI2 and can be promising for developing therapies to combat HCC invasion.