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.

Fertilization is the union of two gametes, spermatozoa and eggs. In the female reproductive tract, spermatozoa need to travel a long distance to reach the egg and pass through the zona pellucida (ZP), an extracellular matrix that surrounds the egg. Sperm motility plays critical roles in these processes, which is executed by the flagellum that can be divided into three parts: midpiece, principal piece, and end piece (1, 2). The central motility apparatus of the flagellum is the axoneme, a “9+2” microtubule structure, that is found in all the parts of the flagellum. In addition to the axoneme, the midpiece possesses spirally arranged mitochondria called the mitochondrial sheath. The principal piece possesses a fibrous sheath that provides elastic rigidity and a scaffold for glycolytic and signaling molecules (1). The end piece contains no accessory structures. Defects in these structures’ formation or function could lead to impaired sperm motility and male infertility (1, 2).Calcineurin is a calcium-dependent phosphatase that is evolutionarily conserved from yeasts to mammals and is comprised of two subunits, a catalytic and a regulatory subunit. In mammals, three isoforms (PPP3CA, PPP3CB, and PPP3CC) of the catalytic subunit and two isoforms (PPP3R1 and PPP3R2) of the regulatory subunit have been identified. Ppp3ca, Ppp3cb, and Ppp3r1 are expressed ubiquitously and play roles in a variety of biological processes, including immune responses and cardiac morphogenesis (3, 4). In contrast, PPP3CC and PPP3R2 compose the testis-enriched calcineurin (sperm calcineurin), the disruption of which leads to an inflexible midpiece, impaired sperm motility, and male infertility (5–7). The administration of calcineurin inhibitors such as cyclosporine A and tacrolimus (FK506), both widely used immunosuppressant drugs, to wild-type (WT) males phenocopied the Ppp3cc and Ppp3r2 knockout (KO) mice (5). These defects appear within 4 to 5 d of treatment, and male fertility recovered 1 wk after halting the drug administration, suggesting that sperm calcineurin can be a target for reversible and rapidly acting male contraceptives (5). However, it is challenging to develop molecules that specifically inhibit sperm calcineurin and not somatic calcineurin because of sequence similarities (82% amino acid identity between human PPP3CA and PPP3CC and 85% amino acid identity between human PPP3R1 and PPP3R2). Therefore, identifying proteins that interact with sperm calcineurin widens the choice of inhibitors that target the sperm calcineurin pathway.The PxIxIT motif is a conserved sequence found in calcineurin-binding proteins (8, 9). Proteins with this consensus motif can be a substrate of calcineurin. For example, NFAT, in which the PxIxIT motif was first discovered, is translocated to the nucleus and activates gene expression when it is dephosphorylated by calcineurin, which plays critical roles in immune responses (10). In addition, PxIxIT motif-containing proteins can be regulators of calcineurin. For example, RCAN1 is an endogenous inhibitor of calcineurin (11, 12), while AKAP5 anchors calcineurin to the L-type Ca²⁺ channel in the plasma membrane (13, 14). In contrast to a variety of molecules known to bind to somatic calcineurin, little is known about the proteins that interact with sperm calcineurin.Here, we searched the testis-enriched proteins that contain the PxIxIT consensus motif to identify substrates or regulators of sperm calcineurin, which may help not only reveal how sperm calcineurin works in regulating sperm motility but also discover new male contraceptive targets and understand reproductive toxicities that can be caused by calcineurin inhibitor–based immunosuppression. By analyzing the function of candidate proteins through the generation of gene-modified mice with the CRISPR/Cas9 system, we identified that SPATA33 is essential in localizing sperm calcineurin to the mitochondria and regulating sperm motility.     

Ent-kaurane-type diterpenoids from Isodonis Herba activate human hair follicle dermal papilla cells proliferation via the Akt/GSK-3β/β-catenin transduction pathway

Journal of Natural Medicines - A methanol extract from Isodonis Herba demonstrated significant proliferative effect on human hair follicle dermal papilla cells (HFDPC, % of control:...     

Correlation of early PET findings with tumor response to molecular targeted agents in patients with advanced driver-mutated non-small cell lung cancer

Recent advances in positron emission tomography with fluorine-18-fluorodeoxyglucose (FDG-PET) have facilitated not only the diagnosis and staging of lung cancer, but also the prediction of treatment outcome. The present study was designed to assess the usefulness of early FDG-PET examination for predicting subsequent tumor size reduction in response to molecular targeted agents in metastatic non-small cell lung cancer (NSCLC) with sensitive gene anomalies. I. In 29 targeted lesions of 10 NSCLC patients, changes in FDG uptake before and on day 7 after the initiation of molecular targeted therapy (gefitinib, n = 7; crizotinib, n = 3) were compared with subsequent radiographic tumor size reduction by RECIST. FDG uptake was evaluated as the maximum standardized uptake value (SUVmax) of each targeted lesion. SUVmax decreased in all lesions after therapy (mean SUVmax 8.3 ± 3.4 before to 3.7 ± 1.8 after therapy, p < 0.05). The % decrease in SUVmax of each lesion was significantly correlated with the % tumor size reduction (r = 0.44). In addition, the reduction rate of SUVmax in metastatic bone lesions after initiation of molecular targeted therapy was significantly lower than that in targeted organs (27.1 ± 27.5 vs. 51.2 ± 21.3%, respectively, p < 0.05). Early reduction in FDG-PET uptake after initiation of molecular targeted agents was able to predict subsequent tumor reduction in patients harboring EGFR-mutated or ALK-positive NSCLC. In addition, nontargeted bone metastasis may have different glucose metabolism after TKI treatment compared with other involved organs.     

Design and synthesis of the potent,orally available,brain-penetrable arylpyrazole class of neuropeptide Y5 receptor antagonists

Novel arylpyrazole derivatives were synthesized and evaluated as neuropeptide Y (NPY) Y5 receptor antagonists. Compound (-)-7, which features a novel chiral 2,3-dihydro-1H-cyclopenta[a]naphthalene moiety, showed good binding affinity and antagonistic activity for the Y5 receptor. After intracerebroventricular administration in SD rats, (-)-7 significantly inhibited food intake that was induced by the centrally administered Y5-preferring agonist, bovine pancreatic polypeptide, but had only a negligible effect on NPY-induced feeding.     

Japanese version of The Cancer Genome Atlas,JCGA, established using fresh frozen tumors obtained from 5143 cancer patients

This study aimed to establish the Japanese Cancer Genome Atlas (JCGA) using data from fresh frozen tumor tissues obtained from 5143 Japanese cancer patients, including those with colorectal cancer (31.6%), lung cancer (16.5%), gastric cancer (10.8%) and other cancers (41.1%). The results are part of a single‐center study called “High‐tech Omics‐based Patient Evaluation” or “Project HOPE” conducted at the Shizuoka Cancer Center, Japan. All DNA samples and most RNA samples were analyzed using whole‐exome sequencing, cancer gene panel sequencing, fusion gene panel sequencing and microarray gene expression profiling, and the results were annotated using an analysis pipeline termed “Shizuoka Multi‐omics Analysis Protocol” developed in‐house. Somatic driver alterations were identified in 72.2% of samples in 362 genes (average, 2.3 driver events per sample). Actionable information on drugs that is applicable in the current clinical setting was associated with 11.3% of samples. When including those drugs that are used for investigative purposes, actionable information was assigned to 55.0% of samples. Germline analysis revealed pathogenic mutations in hereditary cancer genes in 9.2% of samples, among which 12.2% were confirmed as pathogenic mutations by confirmatory test. Pathogenic mutations associated with non–cancerous hereditary diseases were detected in 0.4% of samples. Tumor mutation burden (TMB) analysis revealed 5.4% of samples as having the hypermutator phenotype (TMB ≥ 20). Clonal hematopoiesis was observed in 8.4% of samples. Thus, the JCGA dataset and the analytical procedures constitute a fundamental resource for genomic medicine for Japanese cancer patients.     

Validity of observed differences in dietary surveys by two self-administered questionnaires over a 5-year period: Concordance with self-reported change

Differences observed by comparing the responses to two surveys taken 5 years apart were compared with self-reported changes in dietary habits in the second survey to examine the construct validity of dietary change. That is, when an observed difference about a certain food was consistent with a self-reported change, these two methods seemed to show a high validity regarding dietary change. Both surveys used the same self-administered food intake frequency questionnaire, and subjective changes in food intake were evaluated at the second survey (self-reported change). Eight hundred fifty-five males and one thousand females aged 30-69 years were analyzed. Since results by both methods showed a higher frequency of increased intake of green-yellow vegetables in general, pale- colored vegetables in general, carrots, and squash, these results were thought to be of high validity. However, cabbage, lettuce, and seaweeds showed inconsistent results regarding higher frequencies of intake. Changes among quintiles of lower frequency seemed to show lower validity because the results obtained by the two methods were often inconsistent for dairy foods and some other foods. Thus, self-reported changes may reflect respondents’ attitudes toward foods, not their actual behavior.     

A case-control study of risk factors for development of type 2 diabetes: emphasis on physical activity

The aim of this case-control study was to evaluate the association between the lifestyle risk factors, especially physical activity, and the prevalence of type 2 diabetes and the comorbidity of type 2 diabetes and dyslipidemia in middle-aged Japanese urban population. Subjects (279 males and 119 females, 53.5+/-6.8 years old) were selected from one city office in Tokyo and consisted of type 2 diabetes cases (n=53), dyslipidemia cases (n=130), the comorbidity cases (n=58) and sex- and age-matched controls (n=155). A self-administered questionnaire was used to collect physical activity data using Baecke's questionnaire translated and other lifestyle data. Our results revealed that physical activity was significantly associated with the reduction of the prevalence of type 2 diabetes and the comorbidity, and the sex- and age-adjusted odds ratios of the fourth quartile to the lowest one were 0.31 (95%CI:0.12-0.81) and 0.32 (95%CI:0.13-0.81), respectively. Family history of diabetes and smoking were independent risk factors for the prevalence of type 2 diabetes and the comorbidity.     

Estimation of benchmark doses for urinary cadmium based on beta2-microglobulin excretion in cadmium-polluted regions of the Kakehashi River basin, Japan

A benchmark dose low (BMDL) is used as a replacement for the no observed adverse effect level. The threshold levels of urinary cadmium (Cd) as BMDL were estimated using data from the Kakehashi River basin. The target population (>or=50 years) comprised 3178 and 294 participants inhabiting Cd-polluted and non-polluted areas, respectively. Cut-off values for beta2-MG-uria were defined as the 84 and 95% upper limit values calculated from control subjects, and 1000 microg/l or microg/g cr of beta2-MG. Using these cut-off values, the BMDL at which the excess risk is 0.05 was determined to be 2.9 - 4.0 microg/g cr (males) and 1.5 - 3.6 microg/g cr (females). The present study demonstrated that a BMD approach is useful to estimate the threshold level of urinary Cd in Cd-exposed subjects and people living in general environment without any known Cd-pollution since a BMD approach does not need abnormality rates of urinary findings in the controls.     

Effect of the active adult learning/patient oriented clerkship on affective reaction of students ∼ from the results of student survey

We have previously reported the efficacy of the Patient Oriented Clerkship (POC) in the clinical clerkship in Showa University Hospitals, by a trial with old four-year pharmacy program students. In the unique clerkship, each student has a patient in charge, and follows his/her clinical conditions throughout the rotation. The aim of the POC is that having the students learn spontaneously (Active Learning) and actively (Adult Learning) promoted by student's commitment and responsibility by communicating with patients and health professionals in a team. As the POC requires students both Active Learning and Adult Learning, we define the POC as Active Adult Learning (AAL). Having a patient in charge for each student gives them many opportunities to participate in the medical team and foster their problem solving skills. Our previous study eventually showed positive results of the POC in the one-month short clerkship in the four-year program. On the other hand, the effect of the unique hospital clerkship in the new six-year program is not known. We conducted a student survey to clarify the learning effect in the new six-year education system which was revised and 2.5 month clinical clerkship was scheduled according to the model core clerkship curriculum. This report is the first report to show a challenge of the AAL/POC clerkship in the new six-year pharmacy education program.     

Increased Expression of trk Proto-oncogene by γ-Interferon in Human Neuroblastoma Cell Lines

Three human neuroblastoma cell lines were examined to determine the effect of recombinant γ-interferon (IFN-γ) treatment on the expression of trk proto-oncogene. Increased levels of trk proto-oncogene mRNA were observed in two neuroblastoma cell lines (KP-N-RT and KP-N-SI(FA)) after IFN-γ treatment. The levels of trk mRNA increased with growth inhibition and morphological change in a time- and dose-dependent manner. The decreased level of N- myc mRNA after IFN-γ-treatment in KP-N-RT was inversely correlated with trk mRNA. Our results suggest that IFN-γ can modulate the signal transduction of nerve growth factor in human neuroblastoma cells.