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

More than 1200 genes have been shown in the database to be expressed predominantly in the mouse testes. Advances in genome editing technologies such as the CRISPR/Cas9 system have made it possible to create genetically engineered mice more rapidly and efficiently than with conventional methods, which can be utilized to screen genes essential for male fertility by knocking out testis-enriched genes. Finding such genes related to male fertility would not only help us understand the etiology of human infertility but also lead to the development of male contraceptives. In this study, we generated knockout mice for 12 genes (Acrv1, Adgrf3, Atp8b5, Cfap90, Cfap276, Fbxw5, Gm17266, Lrrd1, Mroh7, Nemp1, Spata45, and Trim36) that are expressed predominantly in the testis and examined the appearance and histological morphology of testes, sperm motility, and male fertility. Mating tests revealed that none of these genes is essential for male fertility at least individually. Notably, knockout mice for Gm17266 showed smaller testis size than the wild-type but did not exhibit reduced male fertility. Since 12 genes were not individually essential for male fertilization, it is unlikely that these genes could be the cause of infertility or contraceptive targets. It is better to focus on other essential genes because complementary genes to these 12 genes may exist.

The luciferase-based in vivo protein-protein interaction assay revealed that CHK1 promotes PP2A and PME-1 interaction.

Protein phosphatase 2A (PP2A) is an essential serine/threonine protein phosphatase, and its dysfunction is involved in the onset of cancer and neurodegenerative disorders. PP2A functions as a trimeric holoenzyme whose composition is regulated by the methyl-esterification (methylation) of the PP2A catalytic subunit (PP2Ac). Protein phosphatase methylesterase-1 (PME-1) is the sole PP2Ac methylesterase, and the higher PME-1 expression is observed in various cancer and neurodegenerative diseases. Apart from serving as a methylesterase, PME-1 acts as a PP2A inhibitory protein, binding directly to PP2Ac and suppressing its activity. The intricate function of PME-1 hinders drug development by targeting the PME-1/PP2Ac axis. This study applied the NanoBiT system, a bioluminescence-based protein interaction assay, to elucidate the molecular mechanism that modulates unknown PME-1/PP2Ac protein-protein interaction (PPI). Compound screening identified that the CHK1 inhibitors inhibited PME-1/PP2Ac association without affecting PP2Ac methylation levels. CHK1 directly phosphorylates PP2Ac to promote PME-1 association. Phospho-mass spectrometry identified multiple phospho-sites on PP2Ac, including the Thr219, that affect PME-1 interaction. An anti-phospho-Thr219 PP2Ac antibody was generated and showed that CHK1 regulates the phosphorylation levels of this site in cells. On the contrary, in vitro phosphatase assay showed that CHK1 is the substrate of PP2A, and PME-1 hindered PP2A-mediated dephosphorylation of CHK1. Our data provides novel insights into the molecular mechanisms governing the PME-1/PP2Ac PPI and the triad relationship between PP2A, PME-1, and CHK1.

Exploring Cell-Penetrating Peptides as Penetration Enhancers in Eye Drop Formulations Using a Reconstructed Human Corneal Epithelial Model.

Ocular tissues function as biological barriers that hinder drug delivery, depending on the target tissue and route of administration, and must be overcome to achieve the desired therapeutic effect. Penetration enhancers have long been investigated to improve corneal drug penetration via eye drop instillation; however, further development is warranted owing to potential safety concerns. In the present study, we focused on cell-penetrating peptides (CPPs) as a penetration enhancer to address the requirements and explored CPP candidates suitable for corneal drug delivery. Using a reconstructed human corneal epithelial tissue model, LabCyte CORNEA-MODEL24 as an alternative to animal testing that is expected to have higher reproducibility than extracted eyeballs and octa-arginine (R8) as a representative model CPP with simple structure, we investigated the enhancement of 6-carboxyfluorescein (6-FAM) uptake by fluorescence imaging and the potential of eye irritation by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Also, surface plasmon resonance (SPR) evaluated the interaction between R8 and model compounds, suggesting that the stronger interaction could facilitate the corneal uptake of compounds. A comparative screening study of corneal uptake using various CPPs showed that the CPPs other than R8 also have the potential to enhance the corneal uptake of 6-FAM. In particular, penetratin (PNT) showed stronger fluorescence intensity. Through these findings, this manuscript provides beneficial information for the development of a novel corneal penetration enhancer with CPPs. In the future, it is expected that the basic findings with R8 will be verified to be applicable to other CPPs for development as penetration enhancers for eye drop formulation.

IRGC1, a testis-enriched immunity related GTPase, is important for fibrous sheath integrity and sperm motility in mice.

Immunity-related GTPases (IRGs), also known as p47 GTPases, are a family of interferon-inducible proteins that play roles in immunity defense against intracellular pathogens. Although the molecular functions of IRGs have been well studied, the function of the family member, IRGC1, remains unclear. IRGC1 is unique among IRGs because its expression is not induced by interferon and it is expressed predominantly in the testis. Further, IRGC1 is well conserved in mammals unlike other IRGs. Here, we knocked out (KO) Irgc1 in mice using the CRISPR/Cas9 system and found that the fertility of Irgc1 KO males was severely impaired because of abnormal sperm motility. Further analyses with a transmission electron microscope revealed that the fibrous sheath (FS), an accessory structure of the sperm tail, was disorganized in Irgc1 KO mice. In addition, IRGC1 was detected in the sperm tail and fractionated with FS proteins. These results suggest that IRGC1 is a component of the FS and is involved in the correct formation of the FS.

TULP2 deletion mice exhibit abnormal outer dense fiber structure and male infertility.

Purpose: Tulp2 (tubby-like protein 2) is a member of the tubby protein family and expressed predominantly in mouse testis. Recently, it was reported that Tulp2 knockout (KO) mice exhibited disrupted sperm tail morphology; however, it remains to be determined how TULP2 deletion causes abnormal tail formation. Methods: The authors analyzed male fertility, sperm morphology, and motility of two Tulp2 KO mouse lines that were generated using the conventional method that utilizes homologous recombination in embryonic stem (ES) cells as well as the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. Furthermore, the authors observed the spermatogenesis of Tulp2 KO mice in more detail using scanning and transmission electron microscopy (SEM and TEM). Results: Both mouse lines of Tulp2 KO exhibited male infertility, abnormal tail morphology, and impaired sperm motility. No overt abnormalities were found in the formation of the mitochondrial sheath in Tulp2 KO mice using the freeze-fracture method with SEM. In contrast, abnormal outer dense fiber (ODF) structure was observed in Tulp2 KO testis with TEM. Conclusions: TULP2 may play roles in the correct formation and/or maintenance of ODF, which may lead to abnormal tail morphology, impaired sperm motility, and male infertility.

A Compressed Collagen Construct for Studying Endothelial-Smooth Muscle Cell Interaction Under High Shear Stress.

The coculture of vascular endothelial cells (ECs) on collagen gels containing smooth muscle cells (SMCs) has been carried out to investigate cellular interactions associated with blood vessel pathophysiology under wall shear stress (WSS) conditions. However, due to a lack of gel stiffness, the previous collagen gel coculture constructs are difficult to use for pathologic higher WSS conditions. Here, we newly constructed a coculture model with centrifugally compressed cell-collagen combined construct (C6), which withstands higher WSS conditions. The elastic modulus of C6 was approximately 6 times higher than that of the uncompressed collagen construct. The level of α-smooth muscle actin, a contractile SMC phenotype marker observed in healthy arteries, was elevated in C6 compared with that of the uncompressed construct, and further increased by exposure to a physiological level WSS of 2 Pa, but not by a pathological level of 20 Pa. WSS conditions of 2 and 20 Pa also induced different expression ratios of matrix metalloproteinases and their inhibitors in the C6 coculture model but did not in monocultured ECs and SMCs. The C6 coculture model will be a powerful tool to investigate interactions between ECs and SMCs under pathologically high WSS conditions.

Survey with Innovations to Increase Response Rate Reveals Low Compliance with Guidelines among Youth Baseball Coaches - Including a Survey of Differences between Cities.

Background: Compliance rates of youth baseball team coaches with guidelines regarding pitch count limits have been reported, but response rates from previous surveys have not been high, which may introduce substantial non-response bias. In addition, differences between cities in guideline compliance rates have remained unclear. Purpose: The aim of the present study was to obtain data on coach compliance with guidelines for pitch count limits with a high survey response rate. Secondary aims were to determine compliance with guidelines other than pitch count limits, and to determine whether differences in compliance exists between cities. Methods: A questionnaire was developed for coaches of youth baseball teams in Gunma to assess knowledge of and compliance with the Japan Softball Baseball Association's recommendations for preventing injuries. In the preparation, distribution, and collection of the questionnaire, four strategies were applied to increase the response rate. The questionnaire surveyed basic descriptive information about the team and coach and coaches compliance with guidelines. Survey items were compared between compliant and non-compliant groups for pitch count limits, and by city. Results: Valid responses to the questionnaire were obtained from coaches of 58 of 62 teams surveyed for a response rate of 93.5%. Despite the fact that almost all coaches were aware of the recommendations regarding pitch count limits and felt these limits were needed, only 15.5% were compliant. For guidelines other than the pitch count limits, the recommended values were exceeded for practice time on holidays. Differences between cities were evident in the compliance rate with the pitch count limit, but no differences between cities in other items were observed. Conclusion: The results of this research revealed that compliance with pitch count limits in this sample of youth baseball coaches was much lower than previously reported. Differences between cities were identified in rates of compliance with pitch count limits. These results suggest a need to increase compliance rates with guidelines for pitch count limits and to address differences between cities. Level of Evidence: Cross-sectional survey study, 3b.

CRISPR/Cas9-mediated genome editing reveals 12 testis-enriched genes dispensable for male fertility in mice.

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 motor domain of testis-enriched kinesin KIF9 is essential for its localization in the mouse flagellum.

Kinesin is a molecular motor that moves along microtubules. Testis-enriched kinesin KIF9 (Kinesin family member 9) is localized in the mouse sperm flagellum and is important for normal sperm motility and male fertility; however, it is unclear if the motor domain of KIF9 is involved in these processes. In this study, we substituted threonine of the ATP binding motif in the KIF9 motor domain to asparagine (T100N) in mice using the CRISPR/Cas9 system, which is known to impair kinesin motor activity. T100N mutant mice exhibit reduced sperm motility and male fertility consistent with Kif9 knockout mice. Further, KIF9 was depleted in the spermatozoa of T100N mutant mice although the amounts of KIF9 were comparable between wild-type and T100N mutant testes. These results indicate that the motor domain of KIF9 is essential for its localization in the sperm flagellum.

FAM71F1 binds to RAB2A and RAB2B and is essential for acrosome formation and male fertility 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.

SPATA33 localizes calcineurin to the mitochondria and regulates sperm motility in mice.

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.

Testis-enriched kinesin KIF9 is important for progressive motility in mouse spermatozoa.

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.

Localization of mechanosensitive channel TRPV4 in mouse skin.

A transient receptor potential (TRP) family, TRPV4, is a calcium-permeable swell-activated channel, playing a role in cutaneous mechanosensation. To elucidate the localization in the mechanosensitive endings, we found with immunohistochemistry in mice that TRPV4 was expressed both by small (low threshold) and large (high threshold) dorsal root ganglia neurons. In addition to free nerve endings, TRPV4 was specifically located at cutaneous mechanosensory terminals co-localized with neurofilament 200, including Meissner, Merkel, penicillate and intraepidermal terminals but not including hair follicle palisades. The distribution suggests that the sensation of pressure by mechanosensitive TRPV4 channel is transmitted through A- as well as C-fiber.