FMNL2 regulates actin for endoplasmic reticulum and mitochondria distribution in oocyte meiosis.

During mammalian oocyte meiosis, spindle migration and asymmetric cytokinesis are unique steps for the successful polar body extrusion. The asymmetry defects of oocytes will lead to the failure of fertilization and embryo implantation. In present study, we reported that an actin nucleating factor Formin-like 2 (FMNL2) played critical roles in the regulation of spindle migration and organelle distribution in mouse and porcine oocytes. Our results showed that FMNL2 mainly localized at the oocyte cortex and periphery of spindle. Depletion of FMNL2 led to the failure of polar body extrusion and large polar bodies in oocytes. Live-cell imaging revealed that the spindle failed to migrate to the oocyte cortex, which caused polar body formation defects, and this might be due to the decreased polymerization of cytoplasmic actin by FMNL2 depletion in the oocytes of both mice and pigs. Furthermore, mass spectrometry analysis indicated that FMNL2 was associated with mitochondria and endoplasmic reticulum (ER)-related proteins, and FMNL2 depletion disrupted the function and distribution of mitochondria and ER, showing with decreased mitochondrial membrane potential and the occurrence of ER stress. Microinjecting Fmnl2-EGFP mRNA into FMNL2-depleted oocytes significantly rescued these defects. Thus, our results indicate that FMNL2 is essential for the actin assembly, which further involves into meiotic spindle migration and ER/mitochondria functions in mammalian oocytes.

Stevioside Ameliorates Palmitic Acid-Induced Abnormal Glucose Uptake via the PDK4/AMPK/TBC1D1 Pathway in C2C12 Myotubes.

BACKGROUND: Stevioside (SV) with minimal calories is widely used as a natural sweetener in beverages due to its high sweetness and safety. However, the effects of SV on glucose uptake and the pyruvate dehydrogenase kinase isoenzyme (PDK4) as an important protein in the regulation of glucose metabolism, remain largely unexplored. In this study, we used C2C12 skeletal muscle cells that was induced by palmitic acid (PA) to assess the effects and mechanisms of SV on glucose uptake and PDK4. METHODS: The glucose uptake of C2C12 cells was determined by 2-NBDG; expression of the Pdk4 gene was measured by quantitative real-time PCR; and expression of the proteins PDK4, p-AMPK, TBC1D1 and GLUT4 was assessed by Western blotting. RESULTS: In PA-induced C2C12 myotubes, SV could significantly promote cellular glucose uptake by decreasing PDK4 levels and increasing p-AMPK and TBC1D1 levels. SV could promote the translocation of GLUT4 from the cytoplasm to the cell membrane in cells. Moreover, in Pdk4-overexpressing C2C12 myotubes, SV decreased the level of PDK4 and increased the levels of p-AMPK and TBC1D1. CONCLUSION: SV was found to ameliorate PA-induced abnormal glucose uptake via the PDK4/AMPK/TBC1D1 pathway in C2C12 myotubes. Although these results warranted further investigation for validation, they may provide some evidence of SV as a safe natural sweetener for its use in sugar-free beverages to prevent and control T2DM.

Clinical Utility of Spectral-Domain Optical Coherence Tomography Marker Disorganization of Retinal Inner Layers in Diabetic Retinopathy.

BACKGROUND AND OBJECTIVE: Disorganization of retinal inner layers (DRIL) is a potential spectral-domain optical coherence tomography (SD-OCT) imaging biomarker with clinical utility in diabetic retinopathy (DR). PATIENTS AND METHODS: A cross-sectional study was conducted at a large academic center. The cohort was composed of 1,175 patients with type 2 diabetes with and without retinopathy on initial examination between September 2009 and January 2019 (n = 2,083 eyes). DR risk and progression factors were obtained from the medical record. Trained graders masked to patients' clinical histories evaluated SD-OCT scans for DRIL. RESULTS: Of 2,083 eyes, 28.1% (n = 585) demonstrated presence of DRIL with high interrater reliability (K = 0.88, 95% CI 0.86-0.90). DRIL was associated with worse visual acuity (VA) (P < 0.001) and DR severity (P < 0.0001). Insulin users had more severe DR (P < 0.0001). DR-related factors, race (Black, White) and sex (male) were significantly associated with DRIL (P < 0.05). CONCLUSIONS: DRIL was strongly associated with DR severity and worse VA, supporting its utility as an unfavorable prognostic indicator. [Ophthalmic Surg Lasers Imaging Retina 2023;54:692-700.].

Deep Learning Algorithm Detects Presence of Disorganization of Retinal Inner Layers (DRIL)-An Early Imaging Biomarker in Diabetic Retinopathy.

Purpose: To develop and train a deep learning-based algorithm for detecting disorganization of retinal inner layers (DRIL) on optical coherence tomography (OCT) to screen a cohort of patients with diabetic retinopathy (DR). Methods: In this cross-sectional study, subjects over age 18, with ICD-9/10 diagnoses of type 2 diabetes with and without retinopathy and Cirrus HD-OCT imaging performed between January 2009 to September 2019 were included in this study. After inclusion and exclusion criteria were applied, a final total of 664 patients (5992 B-scans from 1201 eyes) were included for analysis. Five-line horizontal raster scans from Cirrus HD-OCT were obtained from the shared electronic health record. Two trained graders evaluated scans for presence of DRIL. A third physician grader arbitrated any disagreements. Of 5992 B-scans analyzed, 1397 scans (∼30%) demonstrated presence of DRIL. Graded scans were used to label training data for the convolution neural network (CNN) development and training. Results: On a single CPU system, the best performing CNN training took ∼35 mins. Labeled data were divided 90:10 for internal training/validation and external testing purpose. With this training, our deep learning network was able to predict the presence of DRIL in new OCT scans with a high accuracy of 88.3%, specificity of 90.0%, sensitivity of 82.9%, and Matthews correlation coefficient of 0.7. Conclusions: The present study demonstrates that a deep learning-based OCT classification algorithm can be used for rapid automated identification of DRIL. This developed tool can assist in screening for DRIL in both research and clinical decision-making settings. Translational Relevance: A deep learning algorithm can detect disorganization of retinal inner layers in OCT scans.

Inheritance of perturbed methylation and metabolism caused by uterine malnutrition via oocytes.

BACKGROUND: Undernourishment in utero has deleterious effects on the metabolism of offspring, but the mechanism of the transgenerational transmission of metabolic disorders is not well known. In the present study, we found that undernourishment in utero resulted in metabolic disorders of female F1 and F2 in mouse model. RESULTS: Undernutrition in utero induced metabolic disorders of F1 females, which was transmitted to F2 females. The global methylation in oocytes of F1 exposed to undernutrition in utero was decreased compared with the control. KEGG analysis showed that genes with differential methylation regions (DMRs) in promoters were significantly enriched in metabolic pathways. The altered methylation of some DMRs in F1 oocytes located at the promoters of metabolic-related genes were partially observed in F2 tissues, and the expressions of these genes were also changed. Meanwhile, the abnormal DNA methylation of the validated DMRs in F1 oocytes was also observed in F2 oocytes. CONCLUSIONS: These results indicate that DNA methylation may mediate the transgenerational inheritance of metabolic disorders induced by undernourishment in utero via female germline.

Continuous light exposure influences luteinization and luteal function of ovary in ICR mice.

With the rapid change of people's lifestyle, more childbearing couples live with irregular schedules (i.e., staying up late) and suffer from decreased fertility and abortion, which can be caused by luteal phase defect (LPD). We used continuous light-exposed mice as a model to observe whether continuous light exposure may affect luteinization and luteal function. We showed that the level of progesterone in serum reduced (p < .001), the number of corpus luteum (CL) decreased (p < .01), and the expressions of luteinization-related genes (Lhcgr, Star, Ptgfr, and Runx2), clock genes (Clock and Per1), and Mt1 were downregulated (p < .05) in the ovaries of mice exposed to continuous light, suggesting that continuous light exposure induces defects in luteinization and luteal functions. Strikingly, injection of melatonin (3 mg/kg) could improve luteal functions in continuous light-exposed mice. Moreover, we found that, after 2 h of hCG injection, the level of pERK1/2 in the ovary decreased in the continuous light group, but increased in the melatonin administration group, suggesting that melatonin can improve LPD caused by continuous light exposure through activating the ERK1/2 pathway. In summary, our data demonstrate that continuous light exposure affects ovary luteinization and luteal function, which can be rescued by melatonin.

SQSTM1 and its MAP1LC3B-binding domain induce forced mitophagy to degrade mitochondrial carryover during mitochondrial replacement therapy.

Mitophagy is a process that selectively degrades mitochondria in cells, and it involves a series of signaling events. Our recent paper shows that the ectopic expression of SQSTM1 and its MAP1LC3B-binding domain (Binding) at the mitochondrial outer membrane, can directly cause mitophagy. To distinguish this mitophagy from others, we called it forced mitophagy. Further results show that the forced mitophagy can degrade half of the mitochondria and their DNA in HeLa cells and mouse embryos. Meanwhile, there are no apparent effects on mitochondrial membrane potential (MMP), reactive oxygen species (ROS), mitosis and embryo development. Thus, the forced mitophagy was examined to selectively degrade mitochondrial carryover in the nuclear donor embryos' mitochondria by pre-labeling with Binding before mitochondrial replacement therapy (MRT). The results show that the forced mitophagy can reduce mitochondrial carryover from an average of 4% to 0.09% compared to the controls in mouse embryos and tissues. In addition, the offspring from MRT mice show negligible effects on growth, reproduction, exercise and behavior. Furthermore, results from human tri-pronuclear embryos show that the forced mitophagy results in undetectable mitochondrial carryover in 77% of embryos following MRT. Therefore, forced mitophagy is efficient and safe for degrading mitochondrial carryover in MRT.

Private equity in ophthalmology: lessons from other specialties.

PURPOSE OF REVIEW: In the absence of long-term data of the effects of private equity in ophthalmology, this article reviews the role of private equity in other medical specialties as a guide to the future for ophthalmology. RECENT FINDINGS: Across an array of medical specialties, including anesthesiology, dermatology, emergency medicine, ophthalmology/optometry, radiology, and urology, medical practices are being consolidated into a few larger platform groups. Although there has been a short-term financial success for both private equity firms and senior medical practice partners, there exists broad skepticism from peer-reviewed publications and the national media. SUMMARY: Although the impact of private equity on ophthalmology is largely speculative, ophthalmology may follow some of the trends observed in other medical specialties. These trends include increased volume of services, increased profits, improved payer mix, increased payment per patient visit, increased use of midlevel practitioners, decreased physician autonomy, and decreased physician salaries.

Reduction of mtDNA heteroplasmy in mitochondrial replacement therapy by inducing forced mitophagy.

Mitochondrial replacement therapy (MRT) has been used to prevent maternal transmission of disease-causing mutations in mitochondrial DNA (mtDNA). However, because MRT requires nuclear transfer, it carries the risk of mtDNA carryover and hence of the reversion of mtDNA to pathogenic levels owing to selective replication and genetic drift. Here we show in HeLa cells, mouse embryos and human embryos that mtDNA heteroplasmy can be reduced by pre-labelling the mitochondrial outer membrane of a donor zygote via microinjection with an mRNA coding for a transmembrane peptide fused to an autophagy receptor, to induce the degradation of the labelled mitochondria via forced mitophagy. Forced mitophagy reduced mtDNA carryover in newly reconstructed embryos after MRT, and had negligible effects on the growth curve, reproduction, exercise capacity and other behavioural characteristics of the offspring mice. The induction of forced mitophagy to degrade undesired donor mtDNA may increase the clinical feasibility of MRT and could be extended to other nuclear transfer techniques.

A Prediction Model to Discriminate Small Choroidal Melanoma from Choroidal Nevus.

Objective: This study aimed to develop a validated machine learning model to diagnose small choroidal melanoma. Design: This is a cohort study. Subjects Participants and/or Controls: The training data included 123 patients diagnosed as small choroidal melanocytic tumor (5.0-16.0 mm in largest basal diameter and 1.0 mm-2.5 mm in height; Collaborative Ocular Melanoma Study criteria). Those diagnosed as melanoma (n = 61) had either documented growth or pathologic confirmation. Sixty-two patients with stable lesions classified as choroidal nevus were used as negative controls. The external validation dataset included 240 patients managed at a different tertiary clinic, also with small choroidal melanocytic tumor, observed for malignant growth. Methods: In the training data, lasso logistic regression was used to select variables for inclusion in the final model for the association with melanoma versus choroidal nevus. Internal and external validation was performed to assess model performance. Main Outcome Measures: The main outcome measure is the predicted probability of small choroidal melanoma. Results: Distance to optic disc ≥3 mm and drusen were associated with decreased odds of melanoma, whereas male versus female sex, increased height, subretinal fluid, and orange pigment were associated with increased odds of choroidal melanoma. The area under the receiver operating characteristic "discrimination value" for this model was 0.880. The top four variables that were most frequently selected for inclusion in the model on internal validation, implying their importance as predictors of melanoma, were subretinal fluid, height, distance to optic disc, and orange pigment. When tested against the validation data, the prediction model could distinguish between choroidal nevus and melanoma with a high discrimination of 0.861. The final prediction model was converted into an online calculator to generate predicted probability of melanoma. Conclusions: To minimize diagnostic uncertainty, a machine learning-based diagnostic prediction calculator can be readily applied for decision-making and counseling patients with small choroidal melanoma.

High-Survival Rate After Microinjection of Mouse Oocytes and Early Embryos With mRNA by Combining a Tip Pipette and Piezoelectric-Assisted Micromanipulator.

Utilizing microinjection to introduce biological molecules such as DNA, mRNA, siRNA, and proteins into the cell is well established to study oocyte maturation and early embryo development in vitro. However, microinjection is an empirical technology. The cellular survival after microinjection is mainly dependent on the operator, and an experienced operator should be trained for a long time, from several months to years. Optimizing the microinjection to be highly efficient and quickly learned should be helpful for new operators and some newly established laboratories. Here, we combined the tip pipette and piezo-assisted micromanipulator to microinject the oocyte and early embryos at different stages of mouse. The results showed that the survival rate after microinjection was more than 85% for cumulus-oocyte complex, germinal vesicle oocyte, two-cell, and four-cell embryos, and close to 100% for MII oocyte and zygotes. The high-rate survival of microinjection can save many experimental samples. Thus, it should be helpful in studying some rare animal models such as aging and conditional gene knockout mice. Furthermore, our protocol is much easier to learn for new operators, who can usually master the method proficiently after several training times. Therefore, we would like to publicly share this experience, which will help some novices master microinjection skillfully and save many laboratory animals.

Single-cell RNA sequencing analysis of mouse follicular somatic cells†.

Within the development of ovarian follicle, in addition to cell proliferation and differentiation, sophisticated cell-cell cross talks are established among follicular somatic cells such as granulosa cells (GCs) and theca cells. To systematically reveal the cell differentiation and signal transductions in follicular somatic cells, we collected the mouse follicular somatic cells from secondary to ovulatory stage, and analyzed the single cell transcriptomes. Having data filtered and screened, we found 6883 high variable genes in 4888 single cells. Then follicular somatic cells were clustered into 26 cell clusters, including 18 GC clusters, 4 theca endocrine cell (TEC) clusters, and 4 other somatic cell clusters, which include immune cells and Acta2 positive theca externa cells. From our data, we found there was metabolic reprogramming happened during GC differentiation. We also found both Cyp19a1 and Cyp11a1 could be expressed in TECs. We analyzed the expression patterns of genes associated with cell-cell interactions such as steroid hormone receptor genes, insulin signaling genes, and cytokine/transformation growth factor beta associated genes in all cell clusters. Lastly, we clustered the highly variable genes into 300 gene clusters, which could be used to search new genes involved in follicle development. These transcriptomes of follicular somatic cells provide us potential clues to reveal how mammals regulating follicle development and could help us find targets to improve oocyte quality for women with low fertility.

A hypothetical role for autophagy during the day/night rhythm-regulated melatonin synthesis in the rat pineal gland.

Melatonin is a highly conserved molecule that regulates day/night rhythms; it is associated with sleep improvement, reactive oxygen species (ROS) scavenging, anti-aging effects, and seasonal and circadian rhythms and has been a hot topic of research for decades. Using single-cell RNA sequencing, a recent study describes a single-cell transcriptome atlas for the rat pineal gland. Based on a more comprehensive analysis of the retrieved data (Mays et al., PLoS One, 2018, 13, e0205883), results from the current study unveiled the underappreciated gene regulatory network behind different cell populations in the pineal gland. More importantly, our study here characterized, for the first time, the day/night activation of autophagy flux in the rat pineal gland, indicating a potential role of autophagy in regulating melatonin synthesis in the rat pineal gland. These findings emphasized a hypothetical role of day/night autophagy in linking the biological clock with melatonin synthesis. Furthermore, ultrastructure analysis of pinealocytes provided fascinating insights into differences in their intracellular structure between daytime and nighttime. In addition, we also provide a preliminary description of cell-cell communication in the rat pineal gland. In summary, the current study unveils the day/night regulation of autophagy in the rat pineal gland, raising a potential role of autophagy in day/night-regulated melatonin synthesis.

Double-strand breaks induce short-scale DNA replication and damage amplification in the fully grown mouse oocytes.

Break-induced replication (BIR) is essential for the repair of DNA double-strand breaks (DSBs) with single ends. DSBs-induced microhomology-mediated BIR (mmBIR) and template-switching can increase the risk of complex genome rearrangement. In addition, DSBs can also induce the multi-invasion-mediated DSB amplification. The mmBIR-induced genomic rearrangement has been identified in cancer cells and patients with rare diseases. However, when and how mmBIR is initiated have not been fully and deeply studied. Furthermore, it is not well understood about the conditions for initiation of multi-invasion-mediated DSB amplification. In the G2 phase oocyte of mouse, we identified a type of short-scale BIR (ssBIR) using the DNA replication indicator 5-ethynyl-2'-deoxyuridine (EdU). These ssBIRs could only be induced in the fully grown oocytes but not the growing oocytes. If the DSB oocytes were treated with Rad51 or Chek1/2 inhibitors, both EdU signals and DSB marker γH2A.X foci would decrease. In addition, the DNA polymerase inhibitor Aphidicolin could inhibit the ssBIR and another inhibitor ddATP could reduce the number of γH2A.X foci in the DSB oocytes. In conclusion, our results showed that DNA DSBs in the fully grown oocytes can initiate ssBIR and be amplified by Rad51 or DNA replication.

Benefits and side effects of prisms in the management of diplopia in adults: a prospective study.

PURPOSE: To analyze the various factors affecting patient satisfaction with prism glasses in adults with diplopia. METHODS: In this prospective case series, the benefits and side effects of prisms and factors affecting prism satisfaction were assessed by means of a questionnaire. RESULTS: A total of 134 patients were included. Overall, 58% of patients were highly satisfied, 22% were somewhat satisfied, and 20% were either neutral, somewhat dissatisfied, or very dissatisfied with prism glasses. Prior history of prism use, amplitude of prisms, comitancy of deviation, and type of lenses worn, had no influence on satisfaction rates. The extent of resolution of diplopia was highly correlated with prism satisfaction (P < 0.001), improvement in depth perception (P < 0.001), driving (P < 0.001), and reading (P < 0.008). Up to 22% of the participants noted "bothersome" side effects, including headaches, dizziness, eye strain and pulling (22%), alteration of depth perception (16%), visual distortion (13%), halos (8%), and weight of the prisms (6%). CONCLUSIONS: In this cohort of adults with diplopia, prisms were beneficial in treating diplopia of different etiologies, incomitance, and a wide range of deviations. The side effects experienced with prism glasses themselves accounted for patient dissatisfaction in our study.

Cytokinesis During the First Division of a Mouse Embryo.

Cell division consists of nuclear division (mitosis for somatic cells and meiosis for germ cells) and cytoplasmic division (cytokinesis). Embryonic developments are highly programmed, and thus, each cellular event during early embryo development is stable. For mouse embryos, the first time of mitosis is completed about 22 h after fertilization. However, it remains unclear when the embryo completes its first cytokinesis. Here, we microinjected only one cell in the 2-cell stage mouse embryos with mRNA, which encodes green fluorescence protein (GFP). By monitoring the GFP protein transport dynamics between the two cells, we demonstrated that the first time of cytokinesis in mouse embryos is completed about 15 h after mitosis, namely 37 h after fertilization. In addition, our results indicate that the cytoplasmic protein transport between daughter cells is very effective, which relies on microtubules instead of microfilaments in 2-cell mouse embryos. These results should enrich people's understanding of the first cell division and cytoskeleton in mouse embryos and then learn more about the mechanisms of early embryo development in mammals.

Small Choroidal Melanoma: Correlation of Growth Rate with Pathology.

PURPOSE: The aim of the study was to evaluate equivalence of growth rate and pathologic confirmation in small choroidal melanoma (SCM). DESIGN: This study is a case series. SUBJECTS PARTICIPANTS AND CONTROLS: A total of 61 patients with a choroidal melanocytic tumor of size 5.0-16.0 mm in the largest basal diameter and 1.0-2.5 mm in thickness were classified into the pathology-confirmed group (n = 19), growth-confirmed group (n = 30), and with combined observations (n = 12). METHODS: Distribution of clinical variables (age, gender, laterality, tumor dimensions, tumor location, and presence of orange pigment, subretinal fluid, drusen, and retinal pigment epithelial [RPE] atrophy) between the groups was analyzed. Patient and disease characteristics were summarized as the median and interquartile range for continuous variables and the frequency and percentage for categorical variables. Comparisons were made using the Wilcoxon rank sum test for continuous variables and either Fisher's exact test or the χ2 test for categorical variables with a p value threshold of 0.05 for statistical significance. Growth rate (change in basal dimension/12 months) diagnostic of SCM was quantified. MAIN OUTCOME MEASURES: The primary aim of this study was to test the hypothesis that "growth" was diagnostic of SCM with the secondary aim of quantifying the malignant "growth rate" (growth rate of SCM). RESULTS: The clinical characteristics among all 3 groups were similar except more patients with symptoms (68 vs. 20 vs. 42%, p = 0.004) and juxtapapillary location (p = 0.03) were in the pathology group than in the growth-confirmed group. Those in the combined and growth-confirmed groups had more patients with drusen (11 vs. 60 vs. 50%, p = 0.003) and RPE atrophy (11 vs. 23 vs. 67%, p = 0.003), respectively, than in the pathology group. The median time to detect growth was 9 months (range 3-26 months). The mean growth rate in basal dimension was 1.8 mm/12 months (range, 0.0-7.4 mm; [95% CI: 1.32-2.28]). CONCLUSIONS AND RELEVANCE: Choroidal melanocytic lesions exhibiting a defined growth rate can be clinically diagnosed as SCM without a need for biopsy.

Small Choroidal Melanoma: Correlation between Clinical Characteristics and Metastatic Potential.

IMPORTANCE: Diagnosis of small choroidal melanoma is based upon clinical features and presence of factors predictive of local malignant growth. Prognostic biopsy quantifies risk of metastasis. OBJECTIVE: The aim of this study is to explore relationship between clinical characteristics and metastatic potential of a small choroidal melanoma. DESIGN: Retrospective review of 53 patients with small choroidal melanoma treated in a tertiary oncology clinic. Patients were derived from 3 cohorts, with pathologic confirmation, with growth confirmation, and those treated only on clinical basis. Based upon prognostic biopsy outcomes, each case was classified into low or high metastatic potential groups. Distribution of clinical characteristics such as age, laterality, symptoms, tumor dimensions, tumor distance from optic nerve and fovea, presence of surface orange pigment, drusen, retinal pigment epithelial atrophy, and subretinal fluid was analyzed between metastatic groups. MAIN OUTCOME MEASURES: Distribution of clinical characteristics between low or high metastatic potential groups was analyzed. RESULTS: A total of 53 patients [mean age, 61 years (range, 27-81 years); 32 (60%) men and 21 (40%) women] were classified into pathology confirmed group (n = 13), growth confirmed group (n = 26), and with clinical group (n = 14). Prognostic biopsy in the growth, pathology, and clinical groups revealed low metastatic potential in 23, 10, and 11 patients, respectively, and high metastatic potential in 3 patients in each group. Distribution of clinical characteristics between low or high metastatic potential groups was not statistically significantly different. CONCLUSION: Clinical characteristics do not identify metastatic potential of a small choroidal melanoma.

Single-cell transcriptome analysis reveals that maternal obesity affects DNA repair, histone methylation, and autophagy level in mouse embryos.

Obesity causes many reproductive dysfunctions such as reduced conception, infertility, and early pregnancy loss, and this is largely due to the negative effects of obesity on oocyte and embryo quality. In the present study, we employed single-cell RNA transcriptome sequencing to investigate the potential causes for the maternal obesity effects on mouse embryos. Our results showed that the 4-cell and morula/blastocyst rates were all significantly decreased during embryo development in obese mice. Genome-wide analysis indicated that obesity altered the expression of more than 1100 genes in 2-cell embryos, including the genes which were related to the p53 signaling pathway and apoptosis. Further analysis showed that the expression of 47 genes related to DNA damage was changed, and a positive γH2A signal and the altered expression of Rad51 and Tex15 were observed in the obese embryos. Obesity also affected histone methylation, shown by the decrease of the H3K4-me2 level. Besides this, we observed the occurrence of autophagy and apoptosis in the embryos of obese mice. There were 42 genes that were related to autophagy/apoptosis that showed aberrant expression, and the positive LC3 signal and the decrease of Clec16a, Rraga, and Atg10 level were also observed. In summary, our study suggested that obesity affected early embryonic development by inducing DNA damage, aberrant histone methylation, and autophagy levels in mice.

Mitochondrial Ca2 + Is Related to Mitochondrial Activity and Dynamic Events in Mouse Oocytes.

Mitochondrial energy insufficiency is strongly associated with oocyte activation disorders. Ca2+, especially that in the mitochondrial matrix, plays a pivotal role in mitochondrial energy supplementation, but the underlying mechanisms are still only poorly understood. An encoded mitochondrial matrix Ca2+ probe (Mt-GCaMP6s) was introduced to observe mitochondrial Ca2+ ([Ca2+]m) dynamic changes during oocyte maturation and activation. We found that active mitochondria surrounding the nucleus showed a higher [Ca2+]m than those distributed in the cortex during oocyte maturation. During oocyte partheno-activation, the patterns of Ca2+ dynamic changes were synchronous in the cytoplasm and mitochondria. Such higher concentration of mitochondrial matrix Ca2+ was closely related to the distribution of mitochondrial calcium uptake (MICU) protein. We further showed that higher [Ca2+]m mitochondria around the chromosomes in oocytes might have a potential role in stimulating mitochondrial energy for calmodulin-responsive oocyte spindle formation, while synchronizing Ca2+ functions in the cytoplasm and nuclear area are important for oocyte activation.