Mutation of the Thap4 gene causes dwarfism and testicular anomalies in rats and mice.

The petit (pet) locus is associated with dwarfism, testicular anomalies, severe thymic hypoplasia, and high postnatal lethality, which are inherited in autosomal recessive mode of inheritance in rats with a Wistar strain genetic background. Linkage analysis localized the pet locus between 98.7 Mb and 101.2 Mb on rat chromosome 9. Nucleotide sequence analysis identified 2 bp deletion in exon 2 of the Thap4 gene as the causative mutation for pet. This deletion causes a frameshift and premature termination codon, resulting in a truncated THAP4 protein lacking approximately two-thirds of the C-terminal side. Thap4 is expressed in various organs, including the testis and thymus in rats. To elucidate the biological function of THAP4 in other species, we generated Thap4 knockout mice lacking exon 2 of the Thap4 gene through genome editing. Thap4 knockout mice also exhibited dwarfism and small testis but did not show high postnatal lethality. Thymus weights of adult Thap4 knockout male mice were significantly higher compared to wild-type male mice. Although Thap4 knockout male mice were fertile, their testis contained seminiferous tubules with spermatogenesis and degenerative seminiferous tubules lacking germ cells. Additionally, we observed vacuoles in seminiferous tubules, and clusters of cells in the lumen in seminiferous tubules in Thap4 knockout male mice. These results demonstrate that spontaneous mutation of Thap4 gene in rats and knockout of Thap4 gene in mice both cause dwarfism and testicular anomalies. Thap4 gene in rats and mice is essential for normal testicular development, maintaining spermatogenesis throughout the entire region of seminiferous tubules.

Effectiveness of intravenous methylprednisolone pulse in patients with severe microscopic polyangiitis and granulomatosis with polyangiitis.

OBJECTIVES: To evaluate the effectiveness and safety of two different intravenous methylprednisolone (IVMP) pulse doses in patients with severe microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA). METHODS: We emulated a target trial using observational data from the nationwide registry in Japan. Patients with severe glomerulonephritis or diffuse alveolar haemorrhage were selected and pseudo-randomised into three groups using propensity score-based overlap weighting as follows: non-IVMP, IVMP 0.5 g/day, and IVMP 1.0 g/day. The primary outcome was all-cause death, and the secondary outcomes were composite all-cause death and kidney failure, severe relapse, and serious infection from 2 to 48 weeks after treatment initiation. To estimate the treatment effects, the Cox proportional hazard model and Fine-Gray subdistribution hazard model were used. RESULTS: In this emulated target trial, of 201 eligible patients (MPA, 175; GPA, 26), 6 (2.8%) died, 4 (2.0%) had kidney failure, 11 (5.3%) had severe relapse, and 40 (19.8%) had severe infections. Hazard ratios (HR) for IVMP 0.5 g/day and IVMP 1.0 g/day pulse groups compared with non-IVMP pulse were as follows: all-cause death = 0.46 (95% confidence interval [95%CI]: 0.07-2.81) and 0.07 (95%CI: 0.01-0.41); all-cause death/kidney failure = 1.18 (95%CI: 0.26-5.31) and 0.59 (95%CI: 0.08-4.52); subdistribution HRs for severe relapse = 1.26 (95%CI: 0.12-13.70) and 3.36 (95%CI: 0.49-23.29); and serious infection = 1.88 (95%CI: 0.76-4.65) and 0.94 (95%CI: 0.28-3.13). CONCLUSIONS: IVMP 1.0 g/day pulse may improve 48-week mortality in patients with severe MPA/GPA.

Dynamics of extracellular vesicle uptake by mural granulosa cells in mice.

Extracellular vesicles (EVs) play an important role in the development and function of mammalian ovarian follicles. However, the mechanisms by which they are taken up by the follicular granulosa cells remain unclear. In addition, while oocytes play a pivotal role in follicular development, the possible interactions between oocyte-derived paracrine factors (ODPFs) and EV signals are unknown. Therefore, this study aimed to elucidate the mechanism of EV uptake and the effects of ODPFs on EV uptake by follicular somatic mural granulosa cells in mice. Fluorescence-labeled transferrin (TRF) and cholera toxin B (CTB), substrates for clathrin- and caveolae-mediated endocytosis, respectively, were taken up by mural granulosa cells in vitro. Their uptake was inhibited by Pitstop 2 and genistein, inhibitors of clathrin and caveolae pathways, respectively. Mural granulosa cells took up EVs, and this uptake was suppressed by Pitstop 2 and genistein. Moreover, ODPFs promoted the uptake of EVs and CTB, but not TRF, by mural granulosa cells. These results suggest that mural granulosa cells take up EVs through both clathrin- and caveolae-mediated endocytosis and that oocytes may promote caveolae-mediated endocytosis to facilitate the uptake of EVs.

Correlation between attitude towards dental care for older adults and ageism in dental students and clinical trainees: Cross-sectional study.

INTRODUCTION: Since the needs for dental care among older adults in Japan are expected to increase in the future, this study aimed to examine the influence of ageism among dental students and clinical trainees on the attitude towards the older adults in dental care. MATERIALS AND METHODS: In 2022, we conducted a questionnaire survey of 9th-, 11th-semester dental students and first-year clinical trainees enrolled at Kyushu Dental University, using the Japanese version of Ageism Scale for Dental Students (ASDS-J), Japanese version of Fraboni Scale of Ageism (FSA-J). Questions on intention to provide dental care to the older adults, relationships with the older adults and living with the older adults were also surveyed. RESULTS: There were 162 participants (84 males and 78 females), and the response rate was 71.7%. The participants were divided into the positive group for dental care to older adults (68 respondents) and the negative group (94 respondents). The ASDS-J score of the negative group was significantly higher than that of the positive group [49 (36-66) vs. 52 (34-88), p < .001]. The binomial logistic regression analysis revealed that the odds ratio of being negative attitude was 1.1 per one point of ASDS-J (95% confidence interval: 1.0-1.2) in the model adjusted for student grade, interaction with the older adults, gender, experience living with the older adults and FSA-J. CONCLUSION: These findings suggest that ageism specific to dental students and clinical trainees (as assessed using the ASDS-J) was independently correlated with negative attitudes towards dental care for older adults.

Periosteum-derived podoplanin-expressing stromal cells regulate nascent vascularization during epiphyseal marrow development.

Bone marrow development and endochondral bone formation occur simultaneously. During endochondral ossification, periosteal vasculatures and stromal progenitors invade the primary avascular cartilaginous anlage, which induces primitive marrow development. We previously determined that bone marrow podoplanin (PDPN)-expressing stromal cells exist in the perivascular microenvironment and promote megakaryopoiesis and erythropoiesis. In this study, we aimed to examine the involvement of PDPN-expressing stromal cells in postnatal bone marrow generation. Using histological analysis, we observed that periosteum-derived PDPN-expressing stromal cells infiltrated the cartilaginous anlage of the postnatal epiphysis and populated on the primitive vasculature of secondary ossification center. Furthermore, immunophenotyping and cellular characteristic analyses indicated that the PDPN-expressing stromal cells constituted a subpopulation of the skeletal stem cell lineage. In vitro xenovascular model cocultured with human umbilical vein endothelial cells and PDPN-expressing skeletal stem cell progenies showed that PDPN-expressing stromal cells maintained vascular integrity via the release of angiogenic factors and vascular basement membrane-related extracellular matrices. We show that in this process, Notch signal activation committed the PDPN-expressing stromal cells into a dominant state with basement membrane-related extracellular matrices, especially type IV collagens. Our findings suggest that the PDPN-expressing stromal cells regulate the integrity of the primitive vasculatures in the epiphyseal nascent marrow. To the best of our knowledge, this is the first study to comprehensively examine how PDPN-expressing stromal cells contribute to marrow development and homeostasis.

The UFMylated ribosome-recognition protein SAYSD1 is predominantly expressed in spermatids but is dispensable for fertility in mice.

SAYSVFN domain-containing protein 1 (SAYSD1) is an evolutionarily conserved membrane protein that has recently been identified as a ubiquitin-fold modifier 1 (UFM1)-conjugated ribosome-recognition protein that plays a critical role in translocation-associated quality control (TAQC). However, its expression and roles in mammals in vivo remain largely unknown. We found that SAYSD1 is predominantly expressed in round and elongating spermatids and localizes in the endoplasmic reticulum (ER) of mouse testes, but not in differentiated spermatozoa. Mice deficient in Saysd1 developed normally post-partum. Furthermore, Saysd1-deficient mice were fertile, with no apparent differences in sperm morphology or motility compared with wild-type sperm, although the cauda epididymis contained slightly less sperm. Expression of the ER stress markers spliced X-box binding protein 1s (XBP1s) and CCAAT/enhancer binding protein (C/EBP)-homologous protein (CHOP) in the testes was comparable between Saysd1-deficient and wild-type mice. These results suggested that SAYSD1 is involved in sperm production in mice but is dispensable for their development and fertility.

Association between early immunophenotypic changes and therapeutic response of belimumab in patients with systemic lupus erythematosus.

Belimumab is a therapeutic medication that inhibits the B-cell-activating factor (BAFF) used for systemic lupus erythematosus (SLE); however, the response sometimes varies among individuals, even when patients are stratified based on general clinical characteristics. Therefore, we focused on immunological phenotypic changes with belimumab, investigated their association with subsequent clinical courses, and sought to identify relevant immunological indicators to stratify patients who would benefit from belimumab. We assessed changes in B and T cell phenotypes, as well as BAFF-related factors, such as levels of BAFF and a proliferation-inducing ligand, and expression of three BAFF receptors: BAFF receptor (BAFF-R), B-cell maturation antigen (BCMA), transmembrane activator and cyclophilin ligand interactor (TACI), in 19 patients with SLE who were treated with belimumab before and 3 months after treatment. First, to visualize patterns in complex and diverse data, we summarized B cell changes such as subsets and BAFF receptor expressions into two axes, the first and second principal components (PC1 and PC2), and characterized broad phenotypic changes by cluster analysis. Next, we evaluated whether the B cell changes represented by PC1 and PC2 were associated with other concurrent phenotypic changes, baseline factors, and treatment response at 6 months. We found that lower PC2, indicating increased BAFF-R expression and decreased percentage of naïve B cells, was associated with a subsequent therapeutic response at 6 months (odds ratio 5.3, 95% confidence interval 1.2-24, p = .031). Furthermore, higher percentages of effector memory CD3+CD4+ T cells at baseline were associated with lower PC2 and therapeutic response. Further analysis revealed that increased PC1, as reflected by increased BCMA and TACI expression and an increase in the percentage of class-switched memory B cells, was associated with both T and B cell activation. Although belimumab is a B-cell targeted therapy, it can also influence T-cell phenotypes. Thus, early B cell changes could be used to predict treatment response, and their changes could be predicted from baseline T cell phenotypes, indicating the importance of B and T cell interactions.

Ascorbic acid enhances chondrocyte differentiation of ATDC5 by accelerating insulin receptor signaling.

Chondrogenesis is strictly regulated by several factors, including cytokines, hormones, and extracellular matrix proteins. Mouse teratocarcinoma-derived lineage cells, differentiate into chondrocytes in the presence of insulin. Although ascorbic acid promotes chondrogenic differentiation, the detailed regulative mechanisms underlying its role in chondrogenesis remain unclear. Therefore, in this study, we evaluated the effects of ascorbic acid on insulin-induced chondrogenic differentiation of ATDC5 cells and the underlying intracellular signaling. The results revealed that insulin-stimulated collagen deposition, matrix formation, calcification, and expression of chondrogenic differentiation marker genes in ATDC5 cells. This enhancement by insulin was amplified with the addition of ascorbic acid. Molecular analysis revealed that the activation of insulin-induced phosphoinositide 3-kinase (PI3K)/Akt signaling was enhanced in the presence of ascorbic acid. In contrast, Wnt/ß-catenin signaling was suppressed during chondrocyte differentiation via upregulation of the Wnt agonist, secreted Frizzled-related protein 1 (sFRP-1) and 3 (sFRP-3). Notably, ascorbic acid upregulated the expression of insulin receptors and their substrates (IRS-1 and IRS-2). Furthermore, ascorbic acid reversed the suppression of IRS-1 and IRS-2 protein by insulin. These results indicate that ascorbic acid positively regulates the chondrogenic differentiation of ATDC5 cells via enhancement of insulin signaling. Our findings provide a substantial basis for further elucidation of the regulatory mechanisms of chondrocyte differentiation and the pathophysiology of OA, thus aiding in development of effective treatment strategies.

L-PGDS Attenuates Acute Lung Injury by Prostaglandin D2 in Both Dependent and Independent Ways.

Lipocalin-type PG D synthase (L-PGDS) has two roles: it can be a PGD synthase, or it can be a carrier protein of hydrophobic small molecules. In this study, we investigated the dual roles of L-PGDS in acute lung injury by using L-PGDS-deficient and point-mutated mice, which lack PGD2 producibility but maintain lipocalin ability. Hydrochloride (HCl) administration (0.1 M intratracheally for 6 h) caused hemorrhage and dysfunction in the wild-type (WT) mouse lung. These symptoms were accompanied by an increase in PGD2 production. Both deficiency and point mutation of L-PGDS aggravated the HCl-induced hemorrhage and dysfunction. Although both the gene modifications decreased PGD2 production, only L-PGDS-deficient mice, but not point mutation mice, lacked protein expressions of L-PGDS in the lungs. In the WT mice, HCl administration caused pulmonary edema, indexed as an increase in lung water content and protein leakage in bronchoalveolar lavage fluid. L-PGDS deficiency and point mutation similarly aggravated edema formation. HCl administration also stimulated mucin production and bronchoalveolar lavage fluid leukocyte infiltration in the WT mouse lungs. Of interest, L-PGDS deficiency, but not point mutation, exacerbated these manifestations. Consistently, only L-PGDS deficiency increased the mRNA expression of IL-33, which stimulates mucin production in the inflamed lung. These results show that L-PGDS attenuated HCl-induced acute lung injury progresses in two different ways: L-PGDS produced PGD2, which inhibited pulmonary edema formation, whereas its lipocalin ability decreased mucin formation and inflammatory cell infiltration in the inflamed lung.

The effect of ACVR1B/TGFBR1/ACVR1C signaling inhibition on oocyte and granulosa cell development during in vitro growth culture.

The signals of the transforming growth factor ß (TGF-ß) superfamily play a critical role in follicular development in mammals. ACVR1B/TGFBR1/ACVR1C receptors mediate the signaling of several TGF-ß superfamily ligands in granulosa cells. Although the requirement for ACVR1B/TGFBR1/ACVR1C receptor signaling in follicular development has been confirmed using mutant mouse models, the detailed roles of the signaling in granulosa cell and oocyte development have not been clearly defined. In the present study, we examined the requirement for ACVR1B/TGFBR1/ACVR1C receptor signaling in granulosa cells using an in vitro growth culture of oocyte-granulosa cell complexes (OGCs) and SB431542, a potent inhibitor of the receptor signaling. Although cumulus-oocyte complexes isolated from the control OGCs were able to undergo cumulus expansion, those isolated from OGCs grown with the inhibitor were not competent, even in the presence of in vivo-grown oocytes. The diameter of the oocytes in the SB431542-treated OGCs was comparable with that of the control; however, these oocytes were not competent for complete meiotic maturation or preimplantation development. Therefore, ACVR1B/TGFBR1/ACVR1C receptor signaling is not required for oocytes to increase their volume but is essential for the normal development of cumulus cells and oocyte developmental competence.

Accumulation of senescent cells in the stroma of aged mouse ovary.

Senescent cells play a detrimental role in age-associated pathogenesis by producing factors involved in senescence-associated secretory phenotype (SASP). The present study was conducted to examine the possibility that senescent cells are present in aged ovaries and, if so, to determine the tissue region where senescent cells accumulate using a mouse model. Female mice at 2-4 and 8-10 months were used as reproductively young and aged models, respectively; the latter included mice with and without reproductive experience. Cells positive for senescence-associated ß-galactosidase (SA-ß-Gal) staining, one of the markers of cellular senescence, were detected in the stromal region of aged, but not young, ovaries regardless of reproductive experience. Likewise, the localization of cells expressing CDKN2A (cyclin dependent kinase inhibitor 2A), another senescence marker, in the stromal region of aged ovaries was detected with immunohistochemistry. CDKN2A expression detected by western blotting was significantly higher in the ovaries of aged mice with reproductive experience than in those without the experience. Moreover, cells positive for both γH2AX (a senescence marker) and fluorescent SA-ß-Gal staining were present in those isolated from aged ovaries. In addition, the transcript levels of several SASP factors were significantly increased in aged ovaries. These results suggest that senescent cells accumulate in the ovarian stroma and may affect ovarian function in aged mice. Additionally, reproductive experience may promote accumulation.

Mineralocorticoid Receptor Antagonists for Preventing Chronic Kidney Disease Progression: Current Evidence and Future Challenges.

Regulation and action of the mineralocorticoid receptor (MR) have been the focus of intensive research over the past 80 years. Genetic and physiological/biochemical analysis revealed how MR and the steroid hormone aldosterone integrate the responses of distinct tubular cells in the face of environmental perturbations and how their dysregulation compromises fluid homeostasis. In addition to these roles, the accumulation of data also provided unequivocal evidence that MR is involved in the pathophysiology of kidney diseases. Experimental studies delineated the diverse pathological consequences of MR overactivity and uncovered the multiple mechanisms that result in enhanced MR signaling. In parallel, clinical studies consistently demonstrated that MR blockade reduces albuminuria in patients with chronic kidney disease. Moreover, recent large-scale clinical studies using finerenone have provided evidence that the non-steroidal MR antagonist can retard the kidney disease progression in diabetic patients. In this article, we review experimental data demonstrating the critical importance of MR in mediating renal injury as well as clinical studies providing evidence on the renoprotective effects of MR blockade. We also discuss areas of future investigation, which include the benefit of non-steroidal MR antagonists in non-diabetic kidney disease patients, the identification of surrogate markers for MR signaling in the kidney, and the search for key downstream mediators whereby MR blockade confers renoprotection. Insights into these questions would help maximize the benefit of MR blockade in subjects with kidney diseases.

Factors affecting the number of bacteria in saliva and oral care methods for the recovery of bacteria in contaminated saliva after brushing: a randomized controlled trial.

BACKGROUND: Oral care is important in preventing aspiration pneumonia in older adults. However, it is not clear what kind of oral care can reduce the number of bacteria in saliva. The purposes of this study are to clarify whether there is a relationship between plaque amounts and salivary bacterial counts, and how bacteria dispersed into the oral cavity by brushing can be reduced. METHODS: First, saliva samples were collected from 10 healthy adult volunteers after 30 h of unbrushing and after thorough brushing, and the total bacterial count was determined by real-time PCR. Next, 40 older adults attending an outpatient dental clinic were randomly assigned into two groups: a wiping group (20 patients) and a mouthwashing group (20 patients). Saliva was collected before and after brushing, and after wiping in the wiping group and after mouthwashing in the mouthwashing group, and the total bacterial count was quantified by real-time PCR. RESULTS: In a study of volunteers, there was no association between plaque amounts and salivary bacterial counts. In a study of older adult patients, salivary bacterial counts were significantly higher in patients with higher oral hygiene index and fewer remaining teeth. Brushing increased salivary bacterial counts. Wiping did not significantly reduce the number of bacteria, while mouthwash returned the increased number of bacteria after brushing to the pre-brushing level. CONCLUSIONS: There is no direct relationship between the amount of plaque and the number of bacteria in saliva. Brushing disperses bacteria into the oral cavity, resulting in a marked increase in the number of bacteria in saliva. Wiping does not collect the dispersed bacteria, and it seems essential to rinse the mouth after brushing. TRIAL REGISTRATION: UMIN000045854.

Optimal Dose of Intravenous Cyclophosphamide during remission induction therapy in ANCA-associated vasculitis: a retrospective cohort study of J-CANVAS.

OBJECTIVES: To identify the optimal dose of intravenous cyclophosphamide (IVCY) for induction therapy for anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV). METHODS: We retrospectively assessed patients with AAV who received IVCY every 2-3 weeks during the remission induction phase. The associations of the IVCY dose with infection-free survival and relapse-free survival were analysed using a Cox regression model. We compared patients in three categories: very low-dose (VLD), low-dose (LD), and conventional dose (CD) (<7.5 mg/kg, 7.5-12.5 mg/kg, and >12.5 mg/kg, respectively). The non-linear association between IVCY dose and the outcomes were also evaluated. RESULTS: Of the 80 patients (median age 72 years), 12, 42, and 26 underwent the VLD, LD, and CD regimens, respectively, of whom 4, 3, and 7 developed infection or died. The adjusted hazard ratios for infection or death were 4.3 (95% confidence interval (CI) 0.94-19.8) for VLD and 5.1 (95% CI 1.21-21.3) for CD, compared with LD. We found the hazard ratio for infection or death increased when the initial IVCY dose exceeded 9 mg/kg. Relapse-free survival did not differ clearly. CONCLUSION: Low-dose IVCY (7.5-12.5 mg/kg) may result in fewer infections and similar relapse rates compared with the conventional regimen (>12.5 mg/kg).

Two acquired mouse Y chromosome-linked genes, Prssly and Teyorf1, are dispensable for male fertility‡.

Prssly (Protease, serine-like, Chr Y) and Teyorf1 (Testis expressed, chromosome Y open reading frame 1) are two acquired single-copy genes located on the distal tip of the non-pairing short arm of the mouse Y chromosome adjacent to telomeric sequence. Both genes lack X chromosome-linked homologues and are expressed in testicular germ cells. We first performed analysis of Prssly and Teyorf1 genomic sequences and demonstrated that previously reported Prssly sequence is erroneous and the true Prssly sequence is longer and encodes a larger protein than previously estimated. We also confirmed that both genes encode pseudogenes that are not expressed in testes. Next, using CRISPR/Cas9 genome targeting, we generated Prssly and Teyorf1 knockout (KO) mice and characterized their phenotype. To create Prssly KO mice, we targeted the conserved exon 5 encoding a trypsin domain typical for serine proteases. The targeting was successful and resulted in a frame shift mutation that introduced a premature stop codon, with the Prssly KO males retaining only residual transcript expression in testes. The Teyorf1 targeting removed the entire open reading frame of the gene, which resulted in no transcript expression in KO males. Both Prssly KO and Teyorf1 KO males were fertile and had normal testis size and normal sperm number, motility, and morphology. Our findings show that Prssly and Teyorf1 transcripts with potential to encode proteins are dispensable for male fertility.

Effect of fibroblast growth factor signaling on cumulus expansion in mice in vitro.

The expansion of cumulus cells associated with oocytes is an essential phenomenon in normal mammalian ovulation. Indeed, attenuated expression of cumulus expansion-related genes, including Has2, Ptgs2, Ptx3, and Tnfaip6, results in ovulation failure, leading to female subfertility or infertility. Moreover, emerging evidence suggests that proteins of the fibroblast growth factor (FGF) family, produced within ovarian follicles, regulate the development and function of cumulus cells; however, the effects of FGF signaling on cumulus expansion have not been investigated extensively. Herein, we investigate the effects of FGF signaling, particularly those of FGF8 secreted by oocytes, on epidermal growth factor-induced cumulus expansion in mice. The phosphorylation level of MAPK3/1, an intracellular mediator of FGF signaling, was significantly decreased in cumulus-oocyte complexes (COCs) following treatment with NVP-BGJ398, an FGF receptor inhibitor. Moreover, even though NVP-BGJ398 treatment did not affect cumulus cell expansion, it significantly upregulated the expression of Ptgs2 and Ptx3. In contrast, treatment with recombinant FGF8 did not affect the degree of cumulus expansion or the expression of expansion-related genes in COCs or oocytectomized cumulus cell complexes. Collectively, these results suggest that FGFs, other than FGF8, exert suppressive effects on the cumulus expansion process in mice.

Expression and regulation of estrogen receptor 2 and its coregulators in mouse granulosa cells.

The cooperative effects of estrogen and oocyte-derived paracrine factors (ODPFs) play critical roles in the normal development of ovarian follicles; however, the mechanism underlying this cooperation has not been well studied. The present study aimed to determine whether ODPFs affect estrogen signaling by regulating the expression of estrogen receptor (ESR) and its coregulators in mouse granulosa cells. Some transcripts encoding ESR coregulators were differentially expressed between cumulus and mural granulosa cells (MGCs). The transcript levels of ESR coregulators, including nuclear receptor corepressor 1 and activator 2, in cumulus cells were significantly suppressed by ODPFs; however, they increased when cumulus cell-oocyte complexes were treated with the transforming growth factor beta receptor I inhibitor, SB431542. Moreover, MGCs exhibited significantly higher ESR2 protein and transcript levels than those in cumulus cells. ODPFs promoted Esr2 expression in cumulus cells but had no effect on that in MGCs. Overall, regulation of the expression of ESR2 and its coregulators in cumulus cells by oocytes seems to be one of the mechanisms underlying estrogen-oocyte cooperation in well-developed antral follicles in mice.

Mouse embryonic stem cells maintain differentiation potency into somatic lineage despite alternation of ploidy.

Vertebrates, including mammals, are considered to have evolved by whole genome duplications. Although some fish have been reported to be polyploids that have undergone additional genome duplication, there have been no reports of polyploid mammals due to abnormal development after implantation. Furthermore, as the number of physiologically existing tetraploid somatic cells is small, details of the functions of these ploidy-altered cells are not fully understood. In this present study, we aimed to clarify the details of the differentiation potency of tetraploids using tetraploid embryonic stem cells. To clarify the differentiation potency, we used mouse tetraploid embryonic stem cells derived from tetraploid embryos. We presented tetraploid embryonic stem cells differentiated into neural and osteocyte lineage in vitro and tetraploid cells that contributed to various tissues of chimeric embryos ubiquitously in vivo. These results revealed that mouse embryonic stem cells maintain differentiation potency after altering the ploidy. Our results provide an important basis for the differentiation dynamics of germ layers in mammalian polyploid embryogenesis.

Insulin promotes sodium transport but suppresses gluconeogenesis via distinct cellular pathways in human and rat renal proximal tubules.

Insulin is known to promote sodium transport and regulate gluconeogenesis in renal proximal tubules. Although protein kinase B (also known as Akt) and mammalian target of rapamycin complexes (mTORC) have been established as key regulators in the insulin signaling pathway, their roles in proximal tubules are poorly understood. To help define this, we examined the components of insulin signaling in sodium transport and gluconeogenesis in isolated human and rat proximal tubules, and also investigated the role of insulin in sodium handling and mTORC1 in insulin signaling in vivo. In isolated human and rat proximal tubules, Akt and mTORC1/2 inhibition suppressed insulin-stimulated sodium-bicarbonate co-transporter 1 (NBCe1) activity, whereas mTORC1 inhibition had no effect. Akt2 and mTORC2 gene silencing largely inhibited insulin-stimulated NBCe1 activity, whereas silencing of Akt1 and mTORC1 had no effect. Furthermore, insulin decreased sodium excretion, and this effect depended on phosphoinositide 3 kinase in vivo. Moreover, insulin reduced glucose production in rat proximal tubules and the expression of gluconeogenic genes in human and rat proximal tubules. Akt and mTORC1 inhibition largely abolished the observed insulin-mediated inhibitory effects. Gene silencing of insulin receptor substrate 1 (IRS1), Akt2, mTORC1, and mTORC2 also abolished insulin-mediated inhibition of gluconeogenesis. Additionally, in vivo, mTORC1 inhibition abolished insulin-mediated inhibitory effects in rat proximal tubules, although not in liver. These results indicate that insulin-stimulated proximal tubule sodium transport is mediated via the Akt2/mTORC2 pathway, whereas insulin-suppressed proximal tubule gluconeogenesis is mediated via the IRS1/Akt2/mTORC1/2 pathway. Thus, distinct pathways may play important roles in hypertension and hyperglycemia in metabolic syndrome and diabetes.

Oocytes suppress FOXL2 expression in cumulus cells in mice†.

Cumulus cells and mural granulosa cells (MGCs) play distinct roles during follicular development, and normal development of these cell lineages is critical for the female fertility. Transcriptomic diversification between the two cell lineages is obviously a critical mechanism for their functional diversification; however, the transcriptional regulators responsible for this event have not been fully defined. In this study, we sought to identify key transcriptional regulators responsible for the differential gene expression between the two cell lineages. In silico analysis of transcriptomic comparison between cumulus cells and MGCs identified several candidate regulators responsible for the diversification of the two cell lineages. Among them, we herein focused on forkhead box L2 (FOXL2) and showed that expressions of FOXL2 as well as its target transcripts were differentially regulated between cumulus cells and MGCs. The lower expression of FOXL2 in cumulus cells seemed to be due to the suppression by oocyte-derived paracrine signals. These results suggest that FOXL2 is one of the critical transcription factors that determine cumulus cell and MGC lineages under the control of oocytes.