The Role of Immune Cells in DKD: Mechanisms and Targeted Therapies.

Diabetic kidney disease (DKD), is a common microvascular complication and a major cause of death in patients with diabetes. Disorders of immune cells and immune cytokines can accelerate DKD development of in a number of ways. As the kidney is composed of complex and highly differentiated cells, the interactions among different cell types and immune cells play important regulatory roles in disease development. Here, we summarize the latest research into the molecular mechanisms underlying the interactions among various immune and renal cells in DKD. In addition, we discuss the most recent studies related to single cell technology and bioinformatics analysis in the field of DKD. The aims of our review were to explore immune cells as potential therapeutic targets in DKD and provide some guidance for future clinical treatments.

Effect of proanthocyanidins on blood lipids: A systematic review and meta-analysis.

Proanthocyanidins (PCs) are natural antioxidant polyphenols and their effect on the regulation of blood lipids is still controversial. This study was conducted to evaluate the effect of PCs on lipid metabolism. We searched PubMed, Embase, Web of Science, Chinese biomedical literature service system, China National Knowledge Internet, and Wanfang Data with no time restriction until March 18, 2022, using various forms of "proanthocyanidins" and "blood lipid" search terms. Randomized controlled trials investigating the relationship between PCs and lipid metabolism were included. The standard system of Cochrane Collaboration was used to assess the quality of studies. We standardized mean differences (SMDs) with 95% confidence interval (CI) using the random-effects model, Cohen approach. Seventeen studies (17 trials, N = 1138) fulfilled the eligibility criteria. PCs significantly reduced triglyceride, and increased recombinant apolipoprotein A1. Subgroup analysis showed a significant reduction in triglycerides in older adults (≥60 years) and total cholesterol for participants who were not overweight or obese (body mass index <24). An intervention duration of greater than 8 weeks reduced triglyceride and low-density lipoprotein cholesterol levels but increased high-density lipoprotein cholesterol. Different doses of PCs could regulate triglycerides, high-density lipoprotein cholesterol and total cholesterol. PCs have beneficial effects on circulating lipids and may represent a new approach for treating or preventing lipid metabolism disorders. However, more high-quality studies are needed to confirm these results.

Hyperglycemia-induced STING signaling activation leads to aortic endothelial injury in diabetes.

Hyperglycaemia-induced endothelial dysfunction is a key factor in the pathogenesis of diabetic microangiopathy and macroangiopathy. STING, which is a newly discovered regulator of innate immunity, has also been reported to play an important role in various metabolic diseases. However, the role of STING in diabetes-induced endothelial cell dysfunction is unknown. In this study, we established a diabetic macroangiopathy mouse model by streptozotocin (STZ) injection combined with high-fat diet (HFD) feeding and a glucotoxicity cell model in high glucose (HG)-treated rat aortic endothelial cells (RAECs). We found that STING expression was specifically increased in the endothelial cells of diabetic arteries, as well as in HG-treated RAECs. Moreover, genetic deletion of STING significantly ameliorated diabetes-induced endothelial cell dysfunction and apoptosis in vivo. Likewise, STING inhibition by C-176 reversed HG-induced migration dysfunction and apoptosis in RAECs, whereas STING activation by DMXAA resulted in migration dysfunction and apoptosis. Mechanistically, hyperglycaemia-induced oxidative stress promoted endothelial mitochondrial dysfunction and mtDNA release, which subsequently activated the cGAS-STING system and the cGAS-STING-dependent IRF3/NF-kB pathway, ultimately resulting in inflammation and apoptosis. In conclusion, our study identified a novel role of STING in diabetes-induced aortic endothelial cell injury and suggested that STING inhibition was a potential new therapeutic strategy for the treatment of diabetic macroangiopathy. Video Abstract.

BDH1-mediated ßOHB metabolism ameliorates diabetic kidney disease by activation of NRF2-mediated antioxidative pathway.

A ketogenic diet (KD) and ß-hydroxybutyrate (ßOHB) have been widely reported as effective therapies for metabolic diseases. ß-Hydroxybutyrate dehydrogenase 1 (BDH1) is the rate-limiting enzyme in ketone metabolism. In this study, we examined the BDH1-mediated ßOHB metabolic pathway in the pathogenesis of diabetic kidney disease (DKD). We found that BDH1 is downregulated in the kidneys in DKD mouse models, patients with diabetes, and high glucose- or palmitic acid-induced human renal tubular epithelial (HK-2) cells. BDH1 overexpression or ßOHB treatment protects HK-2 cells from glucotoxicity and lipotoxicity by inhibiting reactive oxygen species overproduction. Mechanistically, BDH1-mediated ßOHB metabolism activates NRF2 by enhancing the metabolic flux of ßOHB-acetoacetate-succinate-fumarate. Moreover, in vivo studies showed that adeno-associated virus 9-mediated BDH1 renal expression successfully reverses fibrosis, inflammation, and apoptosis in the kidneys of C57 BKS db/db mice. Either ßOHB supplementation or KD feeding could elevate the renal expression of BDH1 and reverse the progression of DKD. Our results revealed a BDH1-mediated molecular mechanism in the pathogenesis of DKD and identified BDH1 as a potential therapeutic target for DKD.

Based on network pharmacology and molecular docking to explore the molecular mechanism of Ginseng and Astragalus decoction against postmenopausal osteoporosis.

Traditional Chinese medicine suggests that Ginseng and Astragalus Decoction (GAD) may effectively treat postmenopausal osteoporosis (PMO). However, the exact mechanism of action for GAD remains unclear. This study aims to utilize network pharmacology and molecular docking technology to explore the potential mechanism of GAD in treating PMO. The main chemical components of GAD were identified by consulting literature and traditional Chinese medicine systems pharmacology database. GeneCards and online mendelian inheritance in man were used to identify PMO disease targets, and Cytoscape 3.8.2 software was used to construct a herb-disease-gene-target network. The intersection of drug targets and disease targets was introduced into the search tool for the retrieval of interacting genes platform to construct a protein-protein interaction network. Additionally, we further conducted gene ontology and Kyoto encyclopedia of genes and genomes enrichment analyses, followed by molecular docking between active ingredients and core protein targets. We have identified 59 potential targets related to the treatment of PMO by GAD, along with 33 effective components. Quercetin and kaempferol are the compounds with higher degree. In the protein-protein interaction network, IL6, AKT1, and IL1B are proteins with high degree. The enrichment analysis of gene ontology and KEEG revealed that biological processes involved in treating PMO with GAD mainly include response to hormones, positive regulation of phosphorylation, and regulation of protein homodimerization activity. The signal pathways primarily include Pathways in cancer, PI3K-Akt signaling pathway, and AGE-RAGE signaling pathway. Molecular docking results indicate that kaempferol and quercetin have a high affinity for IL6, AKT1, and IL1B. Our research predicts that IL6, AKT1, and IL1B are highly likely to be potential targets for treating PMO with GAD. PI3K/AKT pathway and AGE-ARGE pathway may play an important role in PMO.

The role of oxidative stress in diabetes mellitus-induced vascular endothelial dysfunction.

Diabetes mellitus is a metabolic disease characterized by long-term hyperglycaemia, which leads to microangiopathy and macroangiopathy and ultimately increases the mortality of diabetic patients. Endothelial dysfunction, which has been recognized as a key factor in the pathogenesis of diabetic microangiopathy and macroangiopathy, is characterized by a reduction in NO bioavailability. Oxidative stress, which is the main pathogenic factor in diabetes, is one of the major triggers of endothelial dysfunction through the reduction in NO. In this review, we summarize the four sources of ROS in the diabetic vasculature and the underlying molecular mechanisms by which the pathogenic factors hyperglycaemia, hyperlipidaemia, adipokines and insulin resistance induce oxidative stress in endothelial cells in the context of diabetes. In addition, we discuss oxidative stress-targeted interventions, including hypoglycaemic drugs, antioxidants and lifestyle interventions, and their effects on diabetes-induced endothelial dysfunction. In summary, our review provides comprehensive insight into the roles of oxidative stress in diabetes-induced endothelial dysfunction.

Lipotoxicity-induced mtDNA release promotes diabetic cardiomyopathy by activating the cGAS-STING pathway in obesity-related diabetes.

Diabetic cardiomyopathy (DCM) is characterized by lipid accumulation, mitochondrial dysfunction, and aseptic inflammatory activation. Mitochondria-derived cytosolic DNA has been reported to induce inflammation by activating cyclic GMP-AMP synthase (cGAS)/the stimulator of interferon genes (STING) pathway in the adipose, liver, and kidney tissues. However, the role of cytosolic mtDNA in the progression of DCM is unclear. In this study, with an obesity-related DCM mouse model established by feeding db/db mice with a high-fat diet (HFD), we observed increased mtDNA in the cytosol and activated cGAS-STING signaling pathway during DCM, as well as the downstream targets, IRF3, NF-κB, IL-18, and IL-1ß. In a further study with a palmitic acid (PA)-induced lipotoxic cell model established in H9C2 cells, we revealed that the cytosolic mtDNA was the result of PA-induced overproduction of mitochondrial ROS, which also led to the activation of the cGAS/STING system and its downstream targets. Notably, treatment of extracted mtDNA alone was sufficient to activate the cGAS-STING signaling pathway in cultured H9C2 cells. Besides, both knockdown of STING in PA-induced H9C2 cells and inhibition of STING by C-176 injection in the DCM mouse model could remarkably block the inflammation and apoptosis of cardiomyocytes. In conclusion, our study elucidated the critical role of cytosolic mtDNA-induced cGAS-STING activation in the pathogenesis of obesity-related DCM and provided preclinical validation for using a STING inhibitor as a new potential therapeutic strategy for the treatment of DCM.

Effects of Camellia tea and herbal tea on cardiometabolic risk in patients with type 2 diabetes mellitus: A systematic review and meta-analysis of randomized controlled trials.

Evidence for the anti-diabetic actions of camellia and herbal tea in diabetic patients has not been summarized. Several data sources were searched for randomized trials assessing the effect of different teas on cardiometabolic risk factors in T2D subjects. Two independent reviewers extracted relevant data and assessed the risk of bias. Results were summarized using mean differences (MDs) based on a random model. Sixteen studies (19 trials, N = 832) fulfilled the eligibility criteria. Mean differences were measured for body weight, body mass index, fasting blood glucose, glycosylated hemoglobin, a homeostatic model for insulin resistance, high and low-density lipoproteins, triglycerides, and systolic and diastolic blood pressure. No effects on total cholesterol and waist circumference were observed when either camellia or herbal tea was consumed. Tea produced moderate regulatory effects on adipose, glycemic control, lipid profiles, and blood pressure. In terms of efficacy, camellia and herbal teas yield different benefits in regulating metabolism. This discovery has some implications for clinical research and drug development. However, more high-quality trials are needed to improve the certainty of our estimates.

Bdh1 overexpression ameliorates hepatic injury by activation of Nrf2 in a MAFLD mouse model.

In 2020, a group of experts officially suggested metabolic dysfunction associated with fatty liver disease "MAFLD" as a more appropriate overarching term than NAFLD, indicating the key role of metabolism in fatty liver disease. Bdh1, as the rate-limiting enzyme of ketone metabolism, acts as an important metabolic regulator in liver. However, the role of Bdh1 in MAFLD is unclear. In this study, we used the transgenic db/db mice as a MAFLD mouse model and observed the downregulated expression of Bdh1 in fatty liver. In addition, expression of Bdh1 was also reduced by palmitic acid (PA) treatment in LO2 cells. Bdh1 knockdown led to ROS overproduction and ROS-induced inflammation and apoptosis in LO2 cells, while Bdh1 overexpression protected LO2 cells from lipotoxicity by inhibiting ROS overproduction. Mechanistically, Bdh1-mediated ßOHB metabolism inhibits ROS overproduction by activation of Nrf2 through enhancement of metabolic flux composed of ßOHB-AcAc-succinate-fumarate. Notably, adeno-associated virus (AAV)-mediated Bdh1 overexpression successfully reversed the hepatic function indexes, fibrosis, inflammation, and apoptosis in fatty livers from db/db mice. In conclusion, our study revealed a Bdh1-mediated molecular mechanism in pathogenesis of metabolic dysfunction related liver disease and identified Bdh1 as a novel potential therapeutic target for MAFLD.

Chronic Ethanol Consumption Induces Osteopenia via Activation of Osteoblast Necroptosis.

Chronic high-dose alcohol consumption impairs bone remodeling, reduces bone mass, and increases the risk of osteoporosis and bone fracture. However, the mechanisms underlying alcohol-induced osteoporosis are yet to be elucidated. In this study, we showed that excess intake of ethyl alcohol (EtOH) resulted in osteopenia and osteoblast necroptosis in mice that led to necrotic lesions and reduced osteogenic differentiation in bone marrow mesenchymal stem cells (BMMSCs). We found that EtOH treatment led to the activation of the RIPK1/RIPK3/MLKL signaling, resulting in increased osteoblast necroptosis and decreased osteogenic differentiation and bone formation both in vivo and in vitro. We further discovered that excessive EtOH treatment-induced osteoblast necroptosis might partly depend on reactive oxygen species (ROS) generation; concomitantly, ROS contributed to necroptosis of osteoblasts through a positive feedback loop involving RIPK1/RIPK3. In addition, blocking of the RIPK1/RIPK3/MLKL signaling by necrostatin-1 (Nec-1), a key inhibitor of RIPK1 kinase in the necroptosis pathway, or antioxidant N-acetylcysteine (NAC), an inhibitor of ROS, could decrease the activation of osteoblast necroptosis and ameliorate alcohol-induced osteopenia both in vivo and in vitro. Collectively, we demonstrated that chronic high-dose alcohol consumption induced osteopenia via osteoblast necroptosis and revealed that RIPK1 kinase may be a therapeutic target for alcohol-induced osteopenia.

G-quadruplex DNA: a novel target for drug design.

G-quadruplex (G4) DNA is a type of quadruple helix structure formed by a continuous guanine-rich DNA sequence. Emerging evidence in recent years authenticated that G4 DNA structures exist both in cell-free and cellular systems, and function in different diseases, especially in various cancers, aging, neurological diseases, and have been considered novel promising targets for drug design. In this review, we summarize the detection method and the structure of G4, highlighting some non-canonical G4 DNA structures, such as G4 with a bulge, a vacancy, or a hairpin. Subsequently, the functions of G4 DNA in physiological processes are discussed, especially their regulation of DNA replication, transcription of disease-related genes (c-MYC, BCL-2, KRAS, c-KIT et al.), telomere maintenance, and epigenetic regulation. Typical G4 ligands that target promoters and telomeres for drug design are also reviewed, including ellipticine derivatives, quinoxaline analogs, telomestatin analogs, berberine derivatives, and CX-5461, which is currently in advanced phase I/II clinical trials for patients with hematologic cancer and BRCA1/2-deficient tumors. Furthermore, since the long-term stable existence of G4 DNA structures could result in genomic instability, we summarized the G4 unfolding mechanisms emerged recently by multiple G4-specific DNA helicases, such as Pif1, RecQ family helicases, FANCJ, and DHX36. This review aims to present a general overview of the field of G-quadruplex DNA that has progressed in recent years and provides potential strategies for drug design and disease treatment.

A Toolbox for Site-Specific Labeling of RecQ Helicase With a Single Fluorophore Used in the Single-Molecule Assay.

Fluorescently labeled proteins can improve the detection sensitivity and have been widely used in a variety of biological measurements. In single-molecule assays, site-specific labeling of proteins enables the visualization of molecular interactions, conformational changes in proteins, and enzymatic activity. In this study, based on a flexible linker in the Escherichia coli RecQ helicase, we established a scheme involving a combination of fluorophore labeling and sortase A ligation to allow site-specific labeling of the HRDC domain of RecQ with a single Cy5 fluorophore, without inletting extra fluorescent domain or peptide fragment. Using single-molecule fluorescence resonance energy transfer, we visualized that Cy5-labeled HRDC could directly interact with RecA domains and could bind to both the 3' and 5' ends of the overhang DNA dynamically in vitro for the first time. The present work not only reveals the functional mechanism of the HRDC domain, but also provides a feasible method for site-specific labeling of a domain with a single fluorophore used in single-molecule assays.

Protein Phosphatase 6 Protects Prophase I-Arrested Oocytes by Safeguarding Genomic Integrity.

Mammalian oocytes are arrested at prophase of the first meiotic division in the primordial follicle pool for months, even years, after birth depending on species, and only a limited number of oocytes resume meiosis, complete maturation, and ovulate with each reproductive cycle. We recently reported that protein phosphatase 6 (PP6), a member of the PP2A-like subfamily, which accounts for cellular serine/threonine phosphatase activity, functions in completing the second meiosis. Here, we generated mutant mice with a specific deletion of Ppp6c in oocytes from the primordial follicle stage by crossing Ppp6cF/F mice with Gdf9-Cre mice and found that Ppp6cF/F; GCre+ mice are infertile. Depletion of PP6c caused folliculogenesis defects and germ cell loss independent of the traditional AKT/mTOR pathway, but due to persistent phosphorylation of H2AX (a marker of double strand breaks), increased susceptibility to DNA damage and defective DNA repair, which led to massive oocyte elimination and eventually premature ovarian failure (POF). Our findings uncover an important role for PP6 as an indispensable guardian of genomic integrity of the lengthy prophase I oocyte arrest, maintenance of primordial follicle pool, and thus female fertility.

Oocyte-specific deletion of N-WASP does not affect oocyte polarity, but causes failure of meiosis II completion.

STUDY QUESTION: There is an unexplored physiological role of N-WASP (neural Wiskott-Aldrich syndrome protein) in oocyte maturation that prevents completion of second meiosis. SUMMARY ANSWER: In mice, N-WASP deletion did not affect oocyte polarity and asymmetric meiotic division in first meiosis, but did impair midbody formation and second meiosis completion. WHAT IS KNOWN ALREADY: N-WASP regulates actin dynamics and participates in various cell activities through the RHO-GTPase-Arp2/3 (actin-related protein 2/3 complex) pathway, and specifically the Cdc42 (cell division cycle 42)-N-WASP-Arp2/3 pathway. Differences in the functions of Cdc42 have been obtained from in vitro compared to in vivo studies. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: By conditional knockout of N-WASP in mouse oocytes, we analyzed its in vivo functions by employing a variety of different methods including oocyte culture, immunofluorescent staining and live oocyte imaging. Each experiment was repeated at least three times, and data were analyzed by paired-samples t-test. MAIN RESULTS AND THE ROLE OF CHANCE: Oocyte-specific deletion of N-WASP did not affect the process of oocyte maturation including spindle formation, spindle migration, polarity establishment and maintenance, and homologous chromosome or sister chromatid segregation, but caused failure of cytokinesis completion during second meiosis (P < 0.001 compared to control). Further analysis showed that a defective midbody may be responsible for the failure of cytokinesis completion. LIMITATIONS, REASONS FOR CAUTION: The present study did not include a detailed analysis of the mechanisms underlying the results, which will require more extensive further investigations. WIDER IMPLICATIONS OF THE FINDINGS: N-WASP may play an important role in mediating and co-ordinating the activity of the spindle (midbody) and actin (contractile ring constriction) when cell division occurs. The findings are important for understanding the regulation of oocyte meiosis completion and failures in this process that affect oocyte quality. LARGE SCALE DATA: None. STUDY FUNDING AND COMPETING INTERESTS: This work was supported by the National Basic Research Program of China (No. 2012CB944404) and the National Natural Science Foundation of China (Nos 30930065, 31371451, 31272260 and 31530049). There are no potential conflicts of interests.

Geminin deletion in mouse oocytes results in impaired embryo development and reduced fertility.

Geminin controls proper centrosome duplication, cell division, and differentiation. We investigated the function of geminin in oogenesis, fertilization, and early embryo development by deleting the geminin gene in oocytes from the primordial follicle stage. Oocyte-specific disruption of geminin results in low fertility in mice. Even though there was no evident anomaly of oogenesis, oocyte meiotic maturation, natural ovulation, or fertilization, early embryo development and implantation were impaired. The fertilized eggs derived from mutant mice showed developmental delay, and many were blocked at the late zygote stage. Cdt1 protein was decreased, whereas Chk1 and H2AX phosphorylation was increased, in fertilized eggs after geminin depletion. Our results suggest that disruption of maternal geminin may decrease Cdt1 expression and cause DNA rereplication, which then activates the cell cycle checkpoint and DNA damage repair and thus impairs early embryo development.

LKB1 acts as a critical gatekeeper of ovarian primordial follicle pool.

Liver Kinase b1 (LKB1/STK11)is a tumor suppressor responsible for the Peutz-Jeghers syndrome, an autosomal-dominant, cancer-prone disorder in which patients develop neoplasms in several organs, including the oviduct, ovary, and cervix. Besides, the C allele of a SNP in the Lkb1 gene impedes the likelihood of ovulation in polycystic ovary syndrome (PCOS) in women treated with metformin, a known LKB1-AMPK activator. It is very likely that LKB1 plays roles in female fertility. To identify the physiological functions of LKB1 in the mouse ovary, we selectively disrupted LKB1 in oocytes by the Cre-LoxP conditional knockout system and found that Lkb1fl/fl; Gdf9-Cre mice were severely subfertile with significantly enlarged ovaries compared to Lkb1fl/fl mice. Interestingly, without Lkb1 expression in oocytes from the primordial follicle stage, the entire primordial follicle pool was activated but failed to mature and ovulate, subsequently causing premature ovarian failure (POF). Further investigation demonstrated that elevated mTOR signaling regulated by an AKT-independent LKB1-AMPK pathway was responsible for the excessive follicle activation and growth. Our findings reveal the role of LKB1 as an indispensable gatekeeper for the primordial follicle pool, offer new functional understanding for the tumor suppressor genes in reproductive organs, and might also provide valuable information for understanding POF and infertility.

Loss of protein phosphatase 6 in oocytes causes failure of meiosis II exit and impaired female fertility.

Dynamic protein phosphorylation and dephosphorylation, mediated by a conserved cohort of protein kinases and phosphatases, regulate cell cycle progression. Among the well-known PP2A-like protein phosphatases, protein phosphatase 6 (PP6) has been analyzed in mammalian mitosis, and Aurora A has recently been identified as its key substrate. However, the functions of PP6 in meiosis are still entirely unknown. To identify the physiological role of PP6 in female gametogenesis, Ppp6c(F/F) mice were first generated and crossed with Zp3-Cre mice to selectively disrupt Ppp6c expression in oocytes. Here, we report for the first time that PP6c is dispensable for oocyte meiotic maturation but essential for exit from meiosis II (MII) after fertilization. Depletion of PP6c caused an abnormal MII spindle and disrupted MII cytokinesis, resulting in zygotes with high risk of aneuploidy and defective early embryonic development, and thus severe subfertility. We also reveal that PP6 inactivation interferes with MII spindle formation and MII exit owing to increased Aurora A activity, and that Aurora A inhibition with MLN8237 can rescue the PP6c depletion phenotype. In conclusion, our findings uncover a hitherto unknown role for PP6 as an indispensable regulator of oocyte meiosis and female fertility.

Different fates of oocytes with DNA double-strand breaks in vitro and in vivo.

In female mice, despite the presence of slight DNA double-strand breaks (DSBs), fully grown oocytes are able to undergo meiosis resumption as indicated by germinal vesicle breakdown (GVBD); however, severe DNA DSBs do reduce and delay entry into M phase through activation of the DNA damage checkpoint. But little is known about the effect of severe DNA DSBs on the spindle assembly checkpoint (SAC) during oocyte maturation. We showed that nearly no first polar body (PB1) was extruded at 12 h of in vitro maturation (IVM) in severe DNA DSBs oocytes, and the limited number of oocytes with PB1 were actually at telophase. However, about 60% of the severe DNA DSBs oocytes which underwent GVBD at 2 h of IVM released a PB1 at 18 h of IVM and these oocytes did reach the second metaphase (MII) stage. Chromosome spread at MI and MII stages showed that chromosomes fragmented after GVBD in severe DNA DSBs oocytes. The delayed PB1 extrusion was due to the disrupted attachment of microtubules to kinetochores and activation of the SAC. At the same time, misaligned chromosome fragments became obvious at the first metaphase (MI) in severe DNA DSBs oocytes. These data implied that the inactivation of SAC during the metaphase-anaphase transition of first meiosis was independent of chromosome integrity. Next, we induced DNA DSBs in vivo, and found that the number of superovulated oocytes per mouse was significantly reduced; moreover, this treatment increased the percentage of apoptotic oocytes. These results suggest that DNA DSBs oocytes undergo apoptosis in vivo.

Scaffold subunit Aalpha of PP2A is essential for female meiosis and fertility in mice.

Ppp2r1a encodes the scaffold subunit Aalpha of protein phosphatase 2A (PP2A), which is an important and ubiquitously expressed serine threonine phosphatase family and plays a critical role in many fundamental cellular processes. To identify the physiological role of PP2A in female germ cell meiosis, we selectively disrupted Ppp2r1a expression in oocytes by using the Cre-Loxp conditional knockout system. Here we report for the first time that oocyte-specific deletion of Ppp2r1a led to severe female subfertility without affecting follicle survival, growth, and ovulation. PP2A-Aalpha was essential for regulating oocyte meiotic maturation because depletion of PP2A-Aalpha facilitated germinal vesicle breakdown, causing elongation of the MII spindle and precocious separation of sister chromatids. The resulting eggs had high risk of aneuploidy, though they could be fertilized, leading to defective embryonic development and thus subfertility. Our findings provide strong evidence that PP2A-Aalpha within the oocyte plays an indispensable role in oocyte meiotic maturation, though it is dispensable for folliculogenesis in the mouse ovary.

Specific deletion of Cdc42 does not affect meiotic spindle organization/migration and homologous chromosome segregation but disrupts polarity establishment and cytokinesis in mouse oocytes.

Mammalian oocyte maturation is distinguished by highly asymmetric meiotic divisions during which a haploid female gamete is produced and almost all the cytoplasm is maintained in the egg for embryo development. Actin-dependent meiosis I spindle positioning to the cortex induces the formation of a polarized actin cap and oocyte polarity, and it determines asymmetric divisions resulting in two polar bodies. Here we investigate the functions of Cdc42 in oocyte meiotic maturation by oocyte-specific deletion of Cdc42 through Cre-loxP conditional knockout technology. We find that Cdc42 deletion causes female infertility in mice. Cdc42 deletion has little effect on meiotic spindle organization and migration to the cortex but inhibits polar body emission, although homologous chromosome segregation occurs. The failure of cytokinesis is due to the loss of polarized Arp2/3 accumulation and actin cap formation; thus the defective contract ring. In addition, we correlate active Cdc42 dynamics with its function during polar body emission and find a relationship between Cdc42 and polarity, as well as polar body emission, in mouse oocytes.