Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA

Mashiko, D., Fujihara, Y., Satouh, Y. et al. Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA. Sci Rep 3, 3355 (2013). https://doi.org/10.1038/srep0335

The Mg2+ transporter CNNM4 regulates sperm Ca2+ homeostasis and is essential for reproduction

Ca2+ influx triggers sperm capacitation; however, the underlying regulatory mechanisms remain incompletely understood. Here, we show that CNNM4, a Mg2+ transporter, is required for Ca2+ 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 Ca2+ influx during sperm capacitation. Ca2+ imaging analyses demonstrate that Ca2+ influx is perturbed in Cnnm4-deficient sperm, and forced Ca2+ entry into these sperm normalizes the level of tyrosine phosphorylation. Furthermore, we confirm the importance of CNNM4 in sperm by generating germcell- specific Cnnm4-deficient mice. These results suggest a new role of CNNM4 in sperm Ca2+ homeostasis

Prediction of pathologic node-negative clinical stage IA lung adenocarcinoma for optimal candidates undergoing sublobar resection

ObjectivePatients with pathologic node-negative early lung cancer may be optimal candidates for sublobar resection. We aimed to identify predictors of pathologic lymph node involvement in clinical stage IA lung adenocarcinoma.MethodsThe data from a multicenter database of 502 patients with completely resected clinical stage IA lung adenocarcinoma were retrospectively analyzed to determine the relationship between the lymph node metastasis status and tumor size on high-resolution computed tomography (HRCT) or maximum standardized uptake value (SUVmax) on [18F]-fluoro-2-deoxy-d-glucose positron emission tomography/computed tomography (FDG-PET/CT). Revised SUVmax was used to correct interinstitutional discrepancies.ResultsIn multivariate analyses, either a solid tumor size on HRCT (P = .001) or an SUVmax on FDG-PET/CT (P = .049) was an independent predictor of lymph node metastasis. The predictive criteria of pathologic node-negative early lung cancer were a solid tumor size of less than 0.8 cm or an SUVmax of less than 1.5. Patients who met the predictive criteria of pathologic node-negative disease had less pathologic invasiveness, such as lymphatic, vascular, or pleural invasion (P < .001), and better disease-free survival (P < .0001) than those who did not, and 86 (40.4%) of the 213 patients with T1b (2-3 cm) tumors met the predictive criteria.ConclusionsEither a solid tumor size or an SUVmax was a significant independent predictor of nodal involvement in clinical stage IA lung adenocarcinoma. The pathologic node-negative status criteria of a solid tumor size of less than 0.8 cm on HRCT or an SUVmax of less than1.5 on FDG-PET/CT may be helpful for avoiding systematic lymphadenectomy for clinical stage IA lung adenocarcinoma, even in cases of T1b (2-3 cm) tumor

CABYR is essential for fibrous sheath integrity and progressive motility in mouse spermatozoa

Ca²⁺-binding tyrosine-phosphorylation-regulated protein (CABYR) has been implicated in sperm physiological function in several in vitro studies. It has also been implicated as a potential cause of and diagnostic tool in asthenozoospermic human males. CABYR is known to be localized to the fibrous sheath, an accessory structure in the flagellar principal piece. Utilizing the CRISPR–Cas9 technology, we have knocked out this gene in mice to understand its role in male fertility. <i>Cabyr</i>-knockout male mice showed severe subfertility with a defect in sperm motility as well as a significant disorganization in the fibrous sheath. Further, abnormal configuration of doublet microtubules was observed in the Cabyr-knockout spermatozoa, suggesting that the fibrous sheath is important for the correct organization of the axoneme. Our results show that it is the role of CABYR in the formation of the fibrous sheath that is essential for male fertility

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

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.201902755

PGAP6, a GPI-specific phospholipase A2, has narrow substrate specificity against GPI-anchored proteins

Gun-Hee Lee, Morihisa Fujita, Hideki Nakanishi, Haruhiko Miyata, Masahito Ikawa, Yusuke Maeda, Yoshiko Murakami, Taroh Kinoshita, PGAP6, a GPI-specific phospholipase A2, has narrow substrate specificity against GPI-anchored proteins, Journal of Biological Chemistry, Volume 295, Issue 42, 2020, Pages 14501-14509, ISSN 0021-9258, https://doi.org/10.1074/jbc.RA120.014643

Nexin-Dynein regulatory complex component DRC7 but not FBXL13 is required for sperm flagellum formation and male fertility in mice

Nexin-Dynein regulatory complex component DRC7 but not FBXL13 is required for sperm flagellum formation and male fertility in mice. Morohoshi A, Miyata H, Shimada K, Nozawa K, Matsumura T, et al. PLOS Genetics. 2020. 16(1) doi:10.1371/journal.pgen.100858

Septins promote dendrite and axon development by negatively regulating microtubule stability via HDAC6-mediated deacetylation

Neurite growth requires two guanine nucleotide-binding protein polymers of tubulins and septins. However, whether and how those cytoskeletal systems are coordinated was unknown. Here we show that the acute knockdown or knockout of the pivotal septin subunit SEPT7 from cerebrocortical neurons impairs their interhemispheric and cerebrospinal axon projections and dendritogenesis in perinatal mice, when the microtubules are severely hyperacetylated. The resulting hyperstabilization and growth retardation of microtubules are demonstrated in vitro. The phenotypic similarity between SEPT7 depletion and the pharmacological inhibition of α-tubulin deacetylase HDAC6 reveals that HDAC6 requires SEPT7 not for its enzymatic activity, but to associate with acetylated α-tubulin. These and other findings indicate that septins provide a physical scaffold for HDAC6 to achieve efficient microtubule deacetylation, thereby negatively regulating microtubule stability to an optimal level for neuritogenesis. Our findings shed light on the mechanisms underlying the HDAC6-mediated coupling of the two ubiquitous cytoskeletal systems during neural development

RSPH6A is required for sperm flagellum formation and male fertility in mice

The flagellum is an evolutionarily conserved appendage used for sensing and locomotion. Its backbone is the axoneme and a component of the axoneme is the radial spoke (RS), a protein complex implicated in flagellar motility regulation. Numerous diseases occur if the axoneme is improperly formed, such as primary ciliary dyskinesia (PCD) and infertility. Radial spoke head 6 homolog A (RSPH6A) is an ortholog of Chlamydomonas RSP6 in the RS head and is evolutionarily conserved. While some RS head proteins have been linkedtoPCD, littleisknown about RSPH6A. Here, weshow that mouse RSPH6A is testis-enriched and localized in the flagellum. Rsph6a knockout (KO) male mice are infertile as a result of their short immotile spermatozoa. Observation of the KO testis indicates that the axoneme can elongate but is disrupted before accessory structures are formed. Manchette removal is also impaired in the KO testis. Further, RSPH9, another radial spoke protein, disappeared in the Rsph6a KO flagella. These data indicate that RSPH6A is essential for sperm flagellar assembly and male fertility in mice. This article has an associated First Person interview with the first author of the paper

CRISPR/Cas9 mediated genome editing in ES cells and its application for chimeric analysis in mice

Oji, A., Noda, T., Fujihara, Y. et al. CRISPR/Cas9 mediated genome editing in ES cells and its application for chimeric analysis in mice. Sci Rep 6, 31666 (2016). https://doi.org/10.1038/srep3166