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Spermatozoa need to undergo an exocytotic event called the acrosome reaction before fusing with eggs. Although calcium ion (Ca 2+ ) is essential for the acrosome reaction, its molecular mechanism remains unknown. Ferlin is a single transmembrane protein with multiple Ca 2+ -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 Ca 2+ ionophore cannot induce the acrosome reaction in Fer1l5 mutant spermatozoa. These results suggest that FER1L5 is the missing link between Ca 2+ and the acrosome reaction.

Spermatozoa become mature and competent for fertilization during transit from the caput epididymis to the cauda epididymis. However, detailed molecular mechanisms of epididymal sperm maturation are still unclear. Here, we focused on multiple epididymis-enriched genes: lipocalin family genes (Lcn5, Lcn6, Lcn8, Lcn9, and Lcn10) and Ly6 family genes (Ly6g5b and Ly6g5c). These genes are evolutionarily conserved in mammals and form clusters on chromosomes 2 and 17 in the mouse, respectively.To clarify whether these genes are required for epididymal sperm maturation and acquisition of fertilizing ability, we generated knockout (KO) mice using the CRISPR/Cas9 system and analyzed their phenotype.We generated four lines of KO mice: Lcn9 single KO, the lipocalin family quadruple KO (Lcn5, Lcn6, Lcn8, and Lcn10), quintuple KO (Lcn5, Lcn6, Lcn8, Lcn10, and Lcn9), and double KO of Ly6 family genes (Ly6g5b and Ly6g5c).Although the Lcn9 single KO did not affect male fertility, the quadruple KO and quintuple KO male mice were subfertile and mostly infertile, respectively, with a reduced amount of ADAM3, an essential protein for sperm binding to the zona pellucida. Further analysis revealed that the quintuple KO spermatozoa lack the CMTM2A/B that are required for ADAM3 maturation. Intriguingly, Ly6g5b and Ly6g5c double KO male mice also showed subfertility with reduced sperm ADAM3.These results suggest epididymal secretory proteins are involved in ADAM3 maturation and acquisition of sperm fertilizing ability.

A safe, effective, and reversible nonhormonal male contraceptive drug is greatly needed for male contraception as well as for circumventing the side effects of female hormonal contraceptives. Phosducin-like 2 (PDCL2) is a testis-specific phosphoprotein in mice and humans. We recently found that male PDCL2 knockout mice are sterile due to globozoospermia caused by impaired sperm head formation, indicating that PDCL2 is a potential target for male contraception. Herein, our study for the first time developed a biophysical assay for PDCL2 allowing us to screen a series of small molecules, to study structure-activity relationships, and to discover two PDCL2 binders with novel chemical structure.To identify a PDCL2 ligand for therapeutic male contraception, we performed DNA-encoded chemical library (DECL) screening and off-DNA hit validation using a unique affinity selection mass spectrometry (ASMS) biophysical profiling strategy.We employed the screening process of DECL, which contains billions of chemically unique DNA-barcoded compounds generated through individual sequences of reactions and different combinations of functionalized building blocks. The structures of the PDCL2 binders are proposed based on the sequencing analysis of the DNA barcode attached to each individual DECL compound. The proposed structure is synthesized through multistep reactions. To confirm and determine binding affinity between the DECL identified molecules and PDCL2, we developed an ASMS assay that incorporates liquid chromatography with tandem mass spectrometry (LC-MS/MS).After a screening process of PDCL2 with DECLs containing 440 billion compounds, we identified a series of hits. The selected compounds were synthesized as off-DNA small molecules, characterized by spectroscopy data, and subjected to our ASMS/LC-MS/MS binding assay. By this assay, we discovered two novel compounds, which showed good binding affinity for PDCL2 in comparison to other molecules generated in our laboratory and which were further confirmed by a thermal shift assay.With the ASMS/LC-MS/MS assay developed in this paper, we successfully discovered a PDCL2 ligand that warrants further development as a male contraceptive.

Each year, infertility affects 15% of couples worldwide, with 50% of cases attributed to men. Globozoospermia is an uncommon cause of male factor infertility, characterized by defects in sperm acrosome formation, leading to round-headed spermatozoa.We generated Pdcl2 knockout mice to investigate the essential roles of PDCL2 in mammalian reproduction.We used reverse transcription-polymerase chain reaction to demonstrate that PDCL2 was expressed exclusively in the male reproductive tract in mice and humans. We created Pdcl2 knockout mice using the CRISPR-Cas9 system and analyzed their fertility. Pdcl2 null spermatozoa underwent further evaluation using computer-assisted sperm analysis, light microscopy, and ultrastructural microscopy. We used immunoblot analysis and immunofluorescence to elucidate relationships between PDCL2 and other acrosomal proteins.The PDC family is highly conserved in eukaryotes. Mouse and human PDCL2 are testis enriched and localized to the testicular endoplasmic reticulum. Loss of the protein causes sterility because of abnormal acrosome biogenesis during spermiogenesis and immotility. Furthermore, Pdcl2 null spermatozoa have rounded heads, similar to globozoospermia in humans. Observation of the knockout testis shows a lack of acrosomal cap formation, aberrant localization of mitochondria in the sperm head, and misshapen nuclei.PDCL2 is essential for sperm acrosome development and male fertility in mice and is a putative contraceptive target in men.

Local control of sperm maturation Newly produced spermatozoa within the testis do not have fertilizing ability but become fully functional when they mature in the epididymis. The development of the epididymis itself is dependent on testicular factors arriving via luminal flow. Improper signaling between the testis and epididymis is hypothesized to result in male infertility. Kiyozumi et al. identified NELL2 as a testicular luminal protein that binds to its receptor, ROS1, on the luminal epididymis surface and induces epididymal differentiation (see the Perspective by Lord and Oatley). In turn, differentiated epididymis secretes a fertility-essential protease, ovochymase-2, to make spermatozoa fully mature and functional. Thus, testis-epididymis interorgan communication by this “lumicrine” regulation ensures mammalian reproduction. Science , this issue p. 1132 ; see also p. 1053

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.

As the world population continues to increase to unsustainable levels, the importance of birth control and the development of new contraceptives are emerging. To date, male contraceptive options have been lagging behind those available to women, and those few options available are not satisfactory to everyone. To solve this problem, we have been searching for new candidate target proteins for non-hormonal contraceptives. Testis-specific proteins are appealing targets for male contraceptives because they are more likely to be involved in male reproduction and their targeting by small molecules is predicted to have no on-target harmful effects on other organs. Using in silico analysis, we identified Erich2, Glt6d1, Prss58, Slfnl1, Sppl2c, Stpg3, Tex33, and Tex36 as testis-abundant genes in both mouse and human. The genes, 4930402F06Rik and 4930568D16Rik, are testis-abundant paralogs of Glt6d1 that we also discovered in mice but not in human, and were also included in our studies to eliminate the potential compensation. We generated knockout (KO) mouse lines of all listed genes using the CRISPR/Cas9 system. Analysis of all of the individual KO mouse lines as well as Glt6d1/4930402F06Rik/4930568D16Rik TKO mouse lines revealed that they are male fertile with no observable defects in reproductive organs, suggesting that these 10 genes are not required for male fertility nor play redundant roles in the case of the 3 Glt6D1 paralogs. Further studies are needed to uncover protein function(s), but in vivo functional screening using the CRISPR/Cas9 system is a fast and accurate way to find genes essential for male fertility, which may apply to studies of genes expressed elsewhere. In this study, although we could not find any potential protein targets for non-hormonal male contraceptives, our findings help to streamline efforts to find and focus on only the essential genes., Ten testis-enriched genes are dispensable for male fertility, as determined by phenotypic analyses of knockout mice.

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.

Fujihara, Y., Lu, Y., Noda, T., Oji, A., Larasati, T., Kojima-Kita, K., . . . Ikawa, M. (2020). Spermatozoa lacking fertilization influencing membrane protein (FIMP) fail to fuse with oocytes in mice. Proceedings of the National Academy of Sciences of the United States of America, 117(17), 9393-9400. doi:10.1073/pnas.1917060117, Sperm-oocyte fusion is a critical event in mammalian fertilization, categorized by three indispensable proteins. Sperm membrane protein IZUMO1 and its counterpart oocyte membrane protein JUNO make a protein complex allowing sperm to interact with the oocyte, and subsequent sperm-oocyte fusion. Oocyte tetraspanin protein CD9 also contributes to sperm-oocyte fusion. However, the fusion process cannot be explained solely by these three essential factors. In this study, we focused on analyzing a testis-specific gene 4930451I11Rik and generated mutant mice using the CRISPR/Cas9 system. Although IZUMO1 remained in 4930451I11Rik knockout (KO) spermatozoa, the KO spermatozoa were unable to fuse with oocytes and the KO males were severely subfertile. 4930451I11Rik encodes two isoforms: a transmembrane (TM) form and a secreted form. Both CRISPR/Cas9-mediated TM deletion and transgenic (Tg) rescue with the TM form revealed that only the TM form plays a critical role in sperm-oocyte fusion. Thus, we renamed this TM form Fertilization Influencing Membrane Protein (FIMP). The mCherry-tagged FIMP TM form was localized to the sperm equatorial segment where the sperm-oocyte fusion event occurs. Thus, FIMP is a sperm-specific transmembrane protein that is necessary for the sperm-oocyte fusion process.

Spermatozoa are produced in the testis but gain their fertilizing ability during epididymal migration. This necessary step in sperm maturation includes posttranslational modification of sperm membrane proteins that includes protein processing by proteases. However, the molecular mechanism underpinning this epididymal sperm maturation remains unknown. In this study, we focused on transmembrane serine protease 12 (Tmprss12). Based on multi-tissue expression analysis by PCR, Tmprss12 was specifically expressed in the testis, and its expression started on day 10 postpartum, corresponding to the stage of zygotene spermatocytes. TMPRSS12 was detected in the acrosomal region of spermatozoa by immunostaining. To reveal the physiological function of TMPRSS12, we generated two knockout (KO) mouse lines using the CRISPR/Cas9 system. Both indel and large deletion lines were male sterile showing that TMPRSS12 is essential for male fertility. Although KO males exhibited normal spermatogenesis and sperm morphology, ejaculated spermatozoa failed to migrate from the uterus to the oviduct. Further analysis revealed that a disintegrin and metalloprotease 3 (ADAM3), an essential protein on the sperm membrane surface that is required for sperm migration, was disrupted in KO spermatozoa. Moreover, we found that KO spermatozoa showed reduced sperm motility via computer-assisted sperm analysis, resulting in a low fertilization rate in vitro. Taken together, these data indicate that TMPRSS12 has dual functions in regulating sperm motility and ADAM3-related sperm migration to the oviduct. Because Tmprss12 is conserved among mammals, including humans, our results may explain some genetic cases of idiopathic male infertility, and TMPRSS12 and its downstream cascade may be novel targets for contraception., TMPRSS12 has dual functions for uterotubal junction migration ability by affecting ADAM3 processing and sperm motility.

Background Ubiquitination is a post-translational modification required for a number of physiological functions regulating protein homeostasis, such as protein degradation. The endoplasmic reticulum (ER) quality control system recognizes and degrades proteins no longer needed in the ER through the ubiquitin–proteasome pathway. E2 and E3 enzymes containing a transmembrane domain have been shown to function in ER quality control. The ER transmembrane protein UBE2J1 is a E2 ubiquitin-conjugating enzyme reported to be essential for spermiogenesis at the elongating spermatid stage. Spermatids from Ube2j1 KO male mice are believed to have defects in the dislocation step of ER quality control. However, associated E3 ubiquitin-protein ligases that function during spermatogenesis remain unknown. Results We identified four evolutionarily conserved testis-specific E3 ubiquitin-protein ligases [RING finger protein 133 (Rnf133); RING finger protein 148 (Rnf148); RING finger protein 151 (Rnf151); and Zinc finger SWIM-type containing 2 (Zswim2)]. Using the CRISPR/Cas9 system, we generated and analyzed the fertility of mutant mice with null alleles for each of these E3-encoding genes, as well as double and triple knockout (KO) mice. Male fertility, male reproductive organ, and sperm-associated parameters were analyzed in detail. Fecundity remained largely unaffected in Rnf148, Rnf151, and Zswim2 KO males; however, Rnf133 KO males displayed severe subfertility. Additionally, Rnf133 KO sperm exhibited abnormal morphology and reduced motility. Ultrastructural analysis demonstrated that cytoplasmic droplets were retained in Rnf133 KO spermatozoa. Although Rnf133 and Rnf148 encode paralogous genes that are chromosomally linked and encode putative ER transmembrane E3 ubiquitin-protein ligases based on their protein structures, there was limited functional redundancy of these proteins. In addition, we identified UBE2J1 as an E2 ubiquitin-conjugating protein that interacts with RNF133. Conclusions Our studies reveal that RNF133 is a testis-expressed E3 ubiquitin-protein ligase that plays a critical role for sperm function during spermiogenesis. Based on the presence of a transmembrane domain in RNF133 and its interaction with the ER containing E2 protein UBE2J1, we hypothesize that these ubiquitin-regulatory proteins function together in ER quality control during spermatogenesis.

Glycosylphosphatidylinositol (GPI) is a posttranslational glycolipid modification of proteins that anchors proteins in lipid rafts on the cell surface. Although some GPI-anchored proteins (GPI-APs), including the prion protein PrP

Fertilization is the fundamental process that initiates the development of a new individual in all sexually reproducing species. Despite its importance, our understanding of the molecular players that govern mammalian sperm-egg interaction is incomplete, partly because many of the essential factors found in nonmammalian species do not have obvious mammalian homologs. We have recently identified the lymphocyte antigen-6 (Ly6)/urokinase-type plasminogen activator receptor (uPAR) protein Bouncer as an essential fertilization factor in zebrafish [S. Herberg, K. R. Gert, A. Schleiffer, A. Pauli, Science 361, 1029-1033 (2018)]. Here, we show that Bouncer's homolog in mammals, Sperm Acrosome Associated 4 (SPACA4), is also required for efficient fertilization in mice. In contrast to fish, in which Bouncer is expressed specifically in the egg, SPACA4 is expressed exclusively in the sperm. Male knockout mice are severely subfertile, and sperm lacking SPACA4 fail to fertilize wild-type eggs in vitro. Interestingly, removal of the zona pellucida rescues the fertilization defect of Spaca4-deficient sperm in vitro, indicating that SPACA4 is not required for the interaction of sperm and the oolemma but rather of sperm and the zona pellucida. Our work identifies SPACA4 as an important sperm protein necessary for zona pellucida penetration during mammalian fertilization.

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.

Irie and colleagues identified a "toxic conformer", which possesses a turn structure at positions 22-23, among various conformations of Aβ and have been reporting its potent oligomeric capacity and neurotoxicity. This toxic conformer was detected in the brains of AD patients and AD model mice (Tg2576 line), and passive immunization targeting this conformer ameliorated the cognitive dysfunction in an AD model. In this study, we developed a novel AD mouse model (AppNL-P-F/NL-P-F) with Swedish mutation (NL), Iberian mutation (F), and mutation (P) overproducing E22P-Aβ, a mimic of the toxic conformer, utilizing the knock-in technique that well recapitulates the Aβ pathology of AD patients in mice and avoids the artificial phenotype observed in transgenic-type model mice. We confirmed that AppNL-P-F/NL-P-F mice produce Aβ by ELISA and accumulate senile plaques by immunohistochemistry at eight months of age. In WB, we observed a potential trimer band and high molecular-weight oligomer bands without a monomeric band in the TBS-soluble fraction of AppNL-P-F/NL-P-F mice at six months of age. In the novel object recognition test, cognitive impairment was observed at six months of age in these mice. These findings suggest that the toxic conformer of Aβ induces cognitive dysfunction mediated by its oligomer formation in this mouse brain. AppNL-P-F/NL-P-F mice may be a useful model for evaluating Aβ oligomer-induced cognitive impairment in AD and will aid in exploring therapeutic targets for AD pathology.

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.

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) sperm 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 sperm can bind to the oolemma, they show the fusion defect, resulting that Dcst1 KO males become almost sterile and Dcst2 KO males become sterile. Similar to mice, zebrafish dcst1 KO males are subfertile and dcst2 and dcst1/2 double KO males are sterile. Zebrafish dcst1/2 KO sperm are motile and can approach the egg, but are defective in binding to the oolemma. Furthermore, we find that DCST1 and DCST2 interact with each other and are interdependent. These data demonstrate that DCST1/2 are essential for male fertility in two vertebrate species, highlighting their crucial role as conserved factors in fertilization.

SUMMARYFertilization is the fundamental process that initiates the development of a new individual in all sexually reproducing species. Despite its importance, our understanding of the molecular players that govern mammalian sperm-egg interaction is incomplete, partly because many of the essential factors found in non-mammalian species do not have obvious mammalian homologs. We have recently identified the Ly6/uPAR protein Bouncer as a new, essential fertilization factor in zebrafish (Herberg et al., 2018). Here, we show that Bouncer’s homolog in mammals, SPACA4, is also required for efficient fertilization in mice. In contrast to fish, where Bouncer is expressed specifically in the egg, SPACA4 is expressed exclusively in the sperm. Male knockout mice are severely sub-fertile, and sperm lacking SPACA4 fail to fertilize wild-type eggs in vitro. Interestingly, removal of the zona pellucida rescues the fertilization defect of Spaca4-deficient sperm in vitro, indicating that SPACA4 is not required for the interaction of sperm and the oolemma but rather of sperm and zona pellucida. Our work identifies SPACA4 as an important sperm protein necessary for zona pellucida penetration during mammalian fertilization.

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.

Kim, H., Takegahara, N., Walsh, M. C., Ueda, J., Fujihara, Y., Ikawa, M., & Choi, Y. (2020). Protocadherin-7 contributes to maintenance of bone homeostasis through regulation of osteoclast multinucleation. BMB Reports, 53(9), 472-477. doi:10.5483/BMBRep.2020.53.9.050, Osteoclasts are hematopoietic-derived cells that resorb bone. They are required to maintain proper bone homeostasis and skeletal strength. Although osteoclast differentiation depends on receptor activator of NF-κB ligand (RANKL) stimulation, additional molecules further contribute to osteoclast maturation. Here, we demonstrate that protocadherin-7 (Pcdh7) regulates formation of multinucleated osteoclasts and contributes to maintenance of bone homeostasis. We found that Pcdh7 expression is induced by RANKL stimulation, and that RNAi-mediated knockdown of Pcdh7 resulted in impaired formation of osteoclasts. We generated Pcdh7-deficient mice and found increased bone mass due to decreased bone resorption but without any defect in bone formation. Using an in vitro culture system, it was revealed that formation of multinucleated osteoclasts is impaired in Pcdh7-deficient cultures, while no apparent defects were observed in differentiation and function of Pcdh7-deficient osteoblasts. Taken together, these results reveal an osteoclast cell-intrinsic role for Pcdh7 in maintaining bone homeostasis.

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.

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.

Aldosterone excess is a cardiovascular risk factor. Aldosterone can directly stimulate an electrical remodeling of cardiomyocytes leading to cardiac arrhythmia and hypertrophy. L-type and T-type voltage-gated calcium (Ca2+) channels expression are increased by aldosterone in cardiomyocytes. To further understand the regulation of these channels expression, we studied the role of a transcriptional repressor, the inhibitor of differentiation/DNA binding protein 2 (Id2). We found that aldosterone inhibited the expression of Id2 in neonatal rat cardiomyocytes and in the heart of adult mice. When Id2 was overexpressed in cardiomyocytes, we observed a reduction in the spontaneous action potentials rate and an arrest in aldosterone-stimulated rate increase. Accordingly, Id2 siRNA knockdown increased this rate. We also observed that CaV1.2 (L-type Ca2+ channel) or CaV3.1, and CaV3.2 (T-type Ca2+ channels) mRNA expression levels and Ca2+ currents were affected by Id2 presence. These observations were further corroborated in a heart specific Id2- transgenic mice. Taken together, our results suggest that Id2 functions as a transcriptional repressor for L- and T-type Ca2+ channels, particularly CaV3.1, in cardiomyocytes and its expression is controlled by aldosterone. We propose that Id2 might contributes to a protective mechanism in cardiomyocytes preventing the presence of channels associated with a pathological state.

©2016 Gun-Hee Lee et al. Originally published in Journal of Cell Biology. https://doi.org/10.1083/jcb.201605121, Glycosylphosphatidylinositol-anchored proteins (GPI-APs) can be shed from the cell membrane by GPI cleavage. In this study, we report a novel GPI-processing enzyme, termed post-glycosylphosphatidylinositol attachment to proteins 6 (PGAP6), which is a GPI-specific phospholipase A2 mainly localized at the cell surface. CRI PTO, a GPI-AP, which plays critical roles in early embryonic development by acting as a Nodal coreceptor, is a highly sensitive substrate of PGAP6, whereas CRY PTIC, a close homologue of CRI PTO, is not sensitive. CRI PTO processed by PGAP6 was released as a lysophosphatidylinositol-bearing form, which is further cleaved by phospholipase D. CRI PTO shed by PGAP6 was active as a coreceptor in Nodal signaling, whereas cell-associated CRI PTO activity was reduced when PGAP6 was expressed. Homozygous Pgap6 knockout mice showed defects in early embryonic development, particularly in the formation of the anterior-posterior axis, which are common features with Cripto knockout embryos. These results suggest PGAP6 plays a critical role in Nodal signaling modulation through CRI PTO shedding.

Chromosome 16p13.11 microduplication is a risk factor associated with various neurodevelopmental disorders such as attention-deficit/hyperactivity disorder, intellectual disabilities, developmental delay and autistic spectrum disorder. The underlying molecular mechanism of this genetic variation remained unknown, but its core genetic locus-conserved across mice and humans-contains seven genes. Here, we generated bacterial artificial chromosome-transgenic mice carrying a human 16p13.11 locus, and these mice showed the behavioral hyperactivity phenotype. We identified miR-484 as the responsible gene using a combination of expression and functional analyses. Mature miR-484 was expressed during active cortical neurogenesis, and overexpression of miR-484 decreased proliferation and increased neural progenitor differentiation in vivo. Luciferase screening identified the 3'-untranslated region of protocadherin-19 (Pcdh19) as a target of miR-484. The effect of miR-484 on neurogenesis was rescued by ectopic PCDH19 expression. These results demonstrate that miR-484 promotes neurogenesis by inhibiting PCDH19. Dysregulation of neurogenesis by imbalanced miR-484/PCDH19 expression contributes to the pathogenesis of 16p13.11 microduplication syndrome.

Gene-expression analysis studies from Schultz et al. estimate that more than 2,300 genes in the mouse genome are expressed predominantly in the male germ line. As of their 2003 publication [Schultz N, Hamra FK, Garbers DL (2003) Proc Natl Acad Sci USA 100(21):12201–12206], the functions of the majority of these testis-enriched genes during spermatogenesis and fertilization were largely unknown. Since the study by Schultz et al., functional analysis of hundreds of reproductive-tract–enriched genes have been performed, but there remain many testis-enriched genes for which their relevance to reproduction remain unexplored or unreported. Historically, a gene knockout is the “gold standard” to determine whether a gene’s function is essential in vivo. Although knockout mice without apparent phenotypes are rarely published, these knockout mouse lines and their phenotypic information need to be shared to prevent redundant experiments. Herein, we used bioinformatic and experimental approaches to uncover mouse testis-enriched genes that are evolutionarily conserved in humans. We then used gene-disruption approaches, including Knockout Mouse Project resources (targeting vectors and mice) and CRISPR/Cas9, to mutate and quickly analyze the fertility of these mutant mice. We discovered that 54 mutant mouse lines were fertile. Thus, despite evolutionary conservation of these genes in vertebrates and in some cases in all eukaryotes, our results indicate that these genes are not individually essential for male mouse fertility. Our phenotypic data are highly relevant in this fiscally tight funding period and postgenomic age when large numbers of genomes are being analyzed for disease association, and will prevent unnecessary expenditures and duplications of effort by others.

Ca(2+) influx triggers sperm capacitation; however, the underlying regulatory mechanisms remain incompletely understood. Here, we show that CNNM4, a Mg(2+) transporter, is required for Ca(2+) influx during capacitation. We find that Cnnm4-deficient male mice are almost infertile because of sperm dysfunction. Motion analyses show that hyperactivation, a qualitative change in the mode of sperm motility during capacitation, is abrogated in Cnnm4-deficient sperm. In contrast, tyrosine phosphorylation of flagellar proteins, a hallmark of capacitation, is excessively augmented. These seemingly paradoxical phenotypes of Cnnm4-deficient sperm are very similar to those of sperm lacking a functional cation channel of sperm (CatSper) channel, which plays an essential role in Ca(2+) influx during sperm capacitation. Ca(2+) imaging analyses demonstrate that Ca(2+) influx is perturbed in Cnnm4-deficient sperm, and forced Ca(2+) entry into these sperm normalizes the level of tyrosine phosphorylation. Furthermore, we confirm the importance of CNNM4 in sperm by generating germ-cell-specific Cnnm4-deficient mice. These results suggest a new role of CNNM4 in sperm Ca(2+) homeostasis.

Tsumagari, R., Kakizawa, S., Kikunaga, S., Fujihara, Y., Ueda, S., Yamanoue, M., . . . Shirai, Y. (2020). Dgkg knock-out mice show impairments in cerebellar motor coordination, ltd, and the dendritic development of purkinje cells through the activation of pkcg. ENeuro, 7(2) doi:10.1523/ENEURO.0319-19.2020, Diacylglycerol kinase g (DGKg) regulates protein kinase C (PKC) activity by converting DG to phosphatidic acid (PA). DGKg directly interacts with PKCg and is phosphorylated by PKCg, resulting in the upregulation of lipid kinase activity. PKC dysfunction impairs motor coordination, indicating that the regulation of PKC activity is important for motor coordination. DGKg and PKC are abundantly expressed in cerebellar Purkinje cells. However, the physiological role of DGKg has not been elucidated. Therefore, we developed DGKg knock-out (KO) mice and tested their cerebellar motor coordination. In DGKg KO mice, cerebellar motor coordination and long-term depression (LTD) were impaired, and the dendrites of Purkinje cells from DGKg KO mice were significantly retracted. Interestingly, treatment with the cPKC inhibitor Gö6976 (Gö) rescued the dendritic re-traction of primary cultured Purkinje cells from DGKg KO mice. In contrast, treatment with the PKC activator 12-o-tetradecanoylphorbol 13-acetate (TPA) reduced morphologic alterations in the dendrites of Purkinje cells from wild-type (WT) mice. In addition, we confirmed the upregulation of PKCg activity in the cerebellum of DGKg KO mice and rescued impaired LTD in DGKg KO mice with a PKCg-specific inhibitor. Furthermore, im-pairment of motor coordination observed in DGKg KO mice was rescued in tm1c mice with DGKg reexpres-sion induced by the FLP-flippase recognition target (FRT) recombination system. These results indicate that DGKg is involved in cerebellar LTD and the dendritic development of Purkinje cells through the regulation of PKCg activity, and thus contributes to cerebellar motor coordination.

Kim, H., Takegahara, N., Walsh, M.C. et al. IgSF11 regulates osteoclast differentiation through association with the scaffold protein PSD-95. Bone Res 8, 5 (2020). https://doi.org/10.1038/s41413-019-0080-9, Osteoclasts are multinucleated, giant cells derived from myeloid progenitors. While receptor activator of NF-κB ligand (RANKL) stimulation is the primary driver of osteoclast differentiation, additional signaling further contributes to osteoclast maturation. Here, we demonstrate that immunoglobulin superfamily member 11 (IgSF11), whose expression increases during osteoclast differentiation, regulates osteoclast differentiation through interaction with postsynaptic density protein 95 (PSD-95), a scaffold protein with multiple protein interaction domains. IgSF11 deficiency in vivo results in impaired osteoclast differentiation and bone resorption but no observed defect in bone formation. Consequently, IgSF11-deficient mice exhibit increased bone mass. Using in vitro osteoclast culture systems, we show that IgSF11 functions through homophilic interactions. Additionally, we demonstrate that impaired osteoclast differentiation in IgSF11-deficient cells is rescued by full-length IgSF11 and that the IgSF11-PSD-95 interaction requires the 75 C-terminal amino acids of IgSF11. Our findings reveal a critical role for IgSF11 during osteoclast differentiation and suggest a role for IgSF11 in a receptor- and signal transduction molecule-containing protein complex.

Formin is one of the two major classes of actin binding proteins (ABPs) with nucleation and polymerization activity. However, despite advances in our understanding of its biochemical activity, whether and how formins generate specific architecture of the actin cytoskeleton and function in a physiological context in vivo remain largely obscure. It is also unknown how actin filaments generated by formins interact with other ABPs in the cell. Here, we combine genetic manipulation of formins mammalian diaphanous homolog1 (mDia1) and 3 (mDia3) with superresolution microscopy and single-molecule imaging, and show that the formins mDia1 and mDia3 are dominantly expressed in Sertoli cells of mouse seminiferous tubule and together generate a highly dynamic cortical filamentous actin (F-actin) meshwork that is continuous with the contractile actomyosin bundles. Loss of mDia1/3 impaired these F-actin architectures, induced ectopic noncontractile espin1-containing F-actin bundles, and disrupted Sertoli cell–germ cell interaction, resulting in impaired spermatogenesis. These results together demonstrate the previously unsuspected mDia-dependent regulatory mechanism of cortical F-actin that is indispensable for mammalian sperm development and male fertility., Author summary Paternal genetic information is transmitted to the offspring via sperm. The unique cell morphology of the sperm plays essential roles in sperm transport through the female reproductive tract and in fertilization with oocytes. Sertoli cells are somatic cells located in the seminiferous tubules of the testis and are known to contribute to the development of sperm. While many studies have analyzed sperm development, the mechanisms underlying its morphogenesis remain obscure. In this work, we showed that the interaction between developing sperm and Sertoli cells is critical for sperm morphogenesis. We further unraveled that this interaction is strongly dependent on the cortical F-actin meshwork and contractile actomyosin bundles of Sertoli cells, and that two actin polymerization and nucleation factors of the formin family, mDia1 and mDia3, are involved in the generation of both actin-based structures. Loss of these formins in mice result in disrupted Sertoli cell actin structures, abnormal sperm morphology, and male infertility. We conclude that mDia1 and mDia3 play a role in sperm development through the regulation of the actin cytoskeletal architecture of Sertoli cells and that defects in these proteins might contribute to male infertility.

Masanaga Muto, Yoshitaka Fujihara, Tomohiro Tobita, Daiji Kiyozumi, Masahito Ikawa, Lentiviral Vector-Mediated Complementation Restored Fetal Viability but Not Placental Hyperplasia in Plac1-Deficient Mice, Biology of Reproduction, Volume 94, Issue 1, 1 January 2016, 6, 1–9, https://doi.org/10.1095/biolreprod.115.133454, The X-linked Plac1 gene is maternally expressed in trophoblast cells during placentation, and its disruption causes placental hyperplasia and intrauterine growth restriction. In contrast, Plac1 is also reported to be one of the upregulated genes in the hyperplastic placenta generated by nuclear transfer. However, the effect of overexpressed Plac1 on placental formation and function remained unaddressed. We complemented the Plac1 knockout placental dysfunction by lentiviral vector-mediated, placenta-specific Plac1 transgene expression. Whereas fetal development and the morphology of maternal blood sinuses in the labyrinth zone improved, placental hyperplasia remained, with an expanded the junctional zone that migrated and encroached into the labyrinth zone. Further experiments revealed that wild-type placenta with transgenically expressed Plac1 resulted in placental hyperplasia without the encroaching of the junctional zone. Our findings suggest that Plac1 is involved in trophoblast cell proliferation, differentiation, and migration. Its proper expression is required for normal placentation and fetal development.

Summary Global DNA demethylation and subsequent de novo DNA methylation take place in mammalian male embryonic germ cells [1–3]. P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs), which are germline-specific small RNAs, have been postulated to be critically important for de novo DNA methylation of retrotransposon genes, and many proteins, including PIWI family proteins, play pivotal roles in this process [4–6]. In the embryonic mouse testis, two mouse PIWI proteins, mouse PIWI-like (MILI) and mouse PIWI2 (MIWI2), are involved in the biogenesis of piRNAs through the so-called ping-pong amplification cycle [7–10], and long single-stranded RNAs transcribed from the gene regions of piRNA clusters have been proposed to be the initial material [11–16]. However, it remains unclear whether transcription from the piRNA clusters is required for the biogenesis of piRNAs. To answer this question, we developed a novel artificial piRNA production system by simple expression of sense and antisense EGFP mRNAs in embryonic male germ cells in the piRNA biogenesis phase. EGFP expression was silenced by piRNA-dependent DNA methylation, indicating that concomitant expression of sense and antisense RNA transcripts is necessary and sufficient for piRNA production and subsequent piRNA-dependent gene silencing. In addition, we demonstrated that this artificial piRNA induction paradigm could be applied to an endogenous gene essential for spermatogenesis, DNMT3L [3, 17, 18]. This study not only provides novel insights into the molecular mechanisms of piRNA production, but also presents an innovative strategy for inducing epigenetic modification in germ cells.

Significance Immune cell activation is accompanied by dynamic changes in expression of genes related to inflammation. Concomitantly, immune reactions are tightly controlled to prevent harmful pathologies due to sustained inflammation. Gene expression is controlled at multiple checkpoints. Among these, the post-transcriptional regulation of the balance between Arid5a and Regnase-1 is important for the induction of inflammation. Our findings provide important insight into the role of the regulation of nucleocytoplasmic localization of Arid5a in the development of inflammation through the induction of a change in the ratio of Arid5a to Regnase-1., Adenine-thymine (AT)-rich interactive domain 5a (Arid5a) is an RNA-binding protein found in the cytoplasm and nucleus of normally growing cells. Although Arid5a is known to play an important role in immune regulation, whether and how Arid5a subcellular localization impacts immune regulation has remained unclear. In this study, we generated Arid5a transgenic (TG) mice to address this question. While ectopic Arid5a overexpression did not affect expression of inflammatory cytokines under unstimulated conditions, significantly higher levels of inflammatory cytokines, such as IL-6, were produced in response to lipopolysaccharide (LPS) stimulation. Consistent with this, TG mice were more sensitive to LPS treatment than wild-type mice. We also found that Arid5a is imported into the nucleus via a classical importin-α/β1–mediated pathway. On stimulation, nuclear Arid5a levels were decreased, while there was a concomitant increase in cytoplasmic Arid5a. Arid5a is associated with up-frameshift protein 1, and its nuclear export is regulated by a nuclear export receptor, chromosomal region maintenance 1. Taken together, these data indicate that Arid5a is a dynamic protein that translocates to the cytoplasm from the nucleus so as to properly exert its dual function in mRNA stabilization and transcriptional regulation during inflammatory conditions.