Acute exposure of triclocarban affects early embryo development in mouse through disrupting maternal-to-zygotic transition and epigenetic modifications.

Triclocarban (TCC) is a broad-spectrum antibacterial agent used globally, and high concentrations of this harmful chemical exist in the environment. The human body is directly exposed to TCC through skin contact. Moreover, TCC is also absorbed through diet and inhaled through breathing, which results in its accumulation in the body. The safety profile of TCC and its potential impact on human health are still not completely clear; therefore, it becomes imperative to evaluate the reproductive toxicity of TCC. Here, we explored the effect of TCC on the early embryonic development of mice and its associated mechanisms. We found that acute exposure of TCC affected the early embryonic development of mice in a dose-dependent manner. Approximately 7600 differentially expressed genes (DEGs) were obtained by sequencing the transcriptome of 2-cell mouse embryos; of these, 3157 genes were upregulated and 4443 genes were downregulated in the TCC-treated embryos. GO and KEGG analysis revealed that the enriched genes were mainly involved in redox processes, RNA synthesis, DNA damage, apoptosis, mitochondria, endoplasmic reticulum, Golgi apparatus, cytoskeleton, peroxisome, RNA polymerase, and other components or processes. Moreover, the Venn analysis showed that the zygotic genome activation (ZGA) was affected and the degradation of maternal effector genes was inhibited. TCC induced changes in the epigenetic modification of 2-cell embryos. The level of DNA methylation increased significantly. Further, the levels of H3K27ac, H3K9ac, and H3K27me3 histone modifications decreased significantly, whereas those of H3K4me3 and H3K9me3 modifications increased significantly. Additionally, TCC induced oxidative stress and DNA damage in the 2-cell embryos. In conclusion, acute exposure of TCC affected early embryo development, destroyed early embryo gene expression, interfered with ZGA and maternal gene degradation, induced changes in epigenetic modification of early embryos, and led to oxidative stress and DNA damage in mouse early embryos.

Curcumin alleviates bisphenol AF-induced oxidative stress and apoptosis in caprine endometrial epithelial cells via the Nrf2 signaling pathway.

Bisphenol AF (BPAF), a BPA-substitute, has been widely used in industrial compounds throughout the world. Several studies have shown that BPAF has endocrine interference and reproductive toxicity. However, the toxic effects of BPAF on pregnancy and placenta of goats are still unclear. Therefore, the objective of this study was to reveal the toxic effect of BPAF by using an in vitro culture model of caprine endometrial epithelial cells (EECs) and further attempted to alleviate the toxicity by curcumin pretreatment. The results showed that BPAF induces significant effects on EECs, including decreased cell viability and mitochondrial membrane potential (△ψm), elevating intracellular reactive oxygen species (ROS), promoting cell apoptosis through upregulating the expression of Bax, Cytochrome c, and downregulating the expression of Bcl-2. Meanwhile, BPAF induced dysregulation of oxidative stress by increasing the levels of malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) but decreasing the activities of superoxide dismutase (SOD). However, curcumin pretreatment could significantly attenuate BPAF-induced toxic effects in EECs. Further study revealed that BPAF treatment could activate mitogen-activated protein kinase (MAPK) pathway and nuclear factor-erythroid 2-related factor 2 (Nrf2) expression, but curcumin pretreatment significantly inhibited the activation of MAPK signal pathway and Nrf2 expression induced by BPAF. Overall, this study indicated that curcumin could prevent BPAF-induced EECs cytotoxicity, which provides a potential therapeutic strategy for female infertility associated with BPAF exposure.

High-dose synthetic phenolic antioxidant propyl gallate impairs mouse oocyte meiotic maturation through inducing mitochondrial dysfunction and DNA damage.

Propyl gallate (PG) is one of the most widely used antioxidants in food products, cosmetics and pharmaceutical industries. Increased research has suggested that exposure to PG influences reproductive health in humans and animals. However, until now, it has not yet been confirmed whether PG would impact oocyte quality. In this study, the hazardous effects of PG on oocyte meiotic maturation were investigated in mice. The findings showed that PG exposure compromises oocyte meiosis by inducing mitochondrial stress which activates apoptosis to trigger oocyte demise. Moreover, DNA damage was significantly induced in PG-treated oocytes, which might be another cause of oocyte developmental arrest and degeneration. Besides, the level of histone methylation (H3K27me2 and H3K27me3) in oocyte was also significantly increased by PG exposure. Furthermore, PG-induced oxidative stress was validated by the increased level of reactive oxygen species (ROS), which might be the underlying reason for these abnormities. In conclusion, the foregoing findings suggested that PG exposure impaired oocyte meiotic maturation by yielding mitochondrial stress to activate apoptosis, inducing DNA damage and oxidative stress, and altering histone methylation level.

Zinc pyrithione exposure compromises oocyte maturation through involving in spindle assembly and zinc accumulation.

Zinc Pyrithione (ZPT), a Food and Drug Administration (FDA) approved chemical, is widely used for topical antimicrobials and cosmetic consumer products, including anti-dandruff shampoos. ZPT and its degraded byproducts have detected in large quantities in the environment, and identified to pose healthy risks on aquatic organisms and human. However, so far, knowledge about ZPT effects on female reproduction, particularly oocyte maturation and quality, is limited. Herein, we investigated the adverse impact of ZPT on mouse oocyte maturation and quality in vitro and found exposure to ZPT significantly compromises oocyte maturation. The results revealed that ZPT disturbed the meiotic cell cycle by impairing cytoskeletal dynamics, kinetochore-microtubule attachment (K-MT), and causing spindle assembly checkpoints (SAC) continuous activation. Further, we observed the microtubule-organizing centers (MTOCs) associated proteins p-MAPK and Aurora-A were disrupted in ZPT-treated oocytes, signified by decreased expression and abnormal localization, responsible for the severe cytoskeletal defects. In addition, ZPT exposure induced a significant increase in the levels of H3K9me2, H3K9me3, H3K27me1, and H3K27me3, suggesting the alterations of epigenetic modifications. Moreover, the accumulation of zinc ions (Zn2+) was observed in ZPT-treated oocytes, which was detrimental because overmuch intracellular Zn2+ disrupted oocyte meiosis. Finally, these above alterations impaired spindle organization and chromosome alignment in metaphase-II (MII) oocytes, indicative of damaged oocytes quality. In conclusion, ZPT exposure influenced oocyte maturation and quality via involvement in MTOCs-associated proteins mediated spindle defects, altered epigenetic modifications and zinc accumulation.

Bisphenol F exposure affects mouse oocyte in vitro maturation through inducing oxidative stress and DNA damage.

Bisphenol F (BPF), a substitute for bisphenol A (BPA), is progressively used to manufacture various consumer products. Despite the established reproductive toxicity of BPF, the underlying mechanisms remain to elucidate. This in-vitro study deep in sighted the BPF toxicity on mouse oocyte meiotic maturation and quality. After treating oocytes with BPF (300 µM), the oocyte meiotic progression was blocked, accentuated by a reduced rate in the first polar body extrusion (PBE). Next, we illustrated that BPF induced α-tubulin hyper-acetylation disrupted the spindle assembly and chromosome alignment. Concurrently, BPF resulted in severe oxidative stress and DNA damage, which triggered the early apoptosis in mouse oocytes. Further, altered epigenetic modifications following BPF exposure were proved by increased H3K27me3 levels. Concerning the toxic effects on spindle structure, oxidative stress, and DNA damage in mouse oocytes, BPF toxicity was less severe to oocyte maturation and spindle structure than BPA and induced low oxidative stress. However, compared with BPA, oocytes treated with BPF were more prone to DNA damage, indicating not less intense or even more severe toxic effects of BPF than BPA on some aspects of oocytes maturation. In brief, the present study established that like wise to BPA, BPF could inhibit meiotic maturation and reduce oocyte quality, suggesting it is not a safe substitute for BPA.

Benzophenone-3 breaches mouse Sertoli cell barrier and alters F-actin organization without evoking apoptosis.

Benzophenone-3 (BP-3), one of the most commonly utilized ultraviolet filters in personal care products, has aroused public concern in recent years for its high chances of human exposure. Previous studies have found that BP-3 can impair testes development and spermatogenesis, but the targets of BP-3 are still unknown. In this study, primary Sertoli cells from 20-day-old mice were treated in vitro with 0-100 µM BP-3 for 24 h to identify its toxicity on Sertoli cells and Sertoli cell barrier. Results demonstrated that BP-3 could induce a notable change in cell morphology and impair Sertoli cell viability. The analysis of transepithelial electrical resistance showed that the integrity of the Sertoli cell barrier was destroyed by BP-3 (100 µM). Some structural proteins of the barrier including ZO-1, Occludin, and Connexin43 were lower expressed and the localization of basal ectoplasmic specializations protein ß-catenin was altered because of BP-3 treatment. Further exploration suggested that BP-3 led to Sertoli cell F-actin disorganization by affecting the expression of Rictor, a key component of the mTORC2 complex. Moreover, although increased DNA damage marker γH2A.X was observed in the treatment group, the cell apoptosis rate was changeless which was further confirmed by increased BAX and stable Bcl-2 (two primary apoptosis regulating proteins). In conclusion, this study revealed that BP-3 had the potential to perturb the Sertoli cell barrier through altered junction proteins and disorganized F-actin, but it could hardly evoke Sertoli cell apoptosis.

WDR62 is a novel participator in spindle migration and asymmetric cytokinesis during mouse oocyte meiotic maturation.

In female meiosis, oocyte meiotic maturation is a form of asymmetric cell division, producing the first polar body and a large oocyte, in which the asymmetry of oocyte meiotic division depends on spindle migration and positioning, and cortical polarization. In this study, we conclude that WDR62 (WD40-repeat protein 62) plays an important role in asymmetric meiotic division during mouse oocyte maturation. Our initial study demonstrated that WDR62 mainly co-localized with chromosomes during mouse oocyte meiotic maturation. Interference of Wdr62 by siRNA microinjection did not affect germinal vesicle breakdown (GVBD) but compromised the first polar body extrusion (PBE) with the large polar bodies generated, which is coupled with a higher incidence of spindle abnormality and chromosome misalignment. Further analysis concluded that loss of WDR62 blocked asymmetric spindle positioning and actin cap formation, which should be responsible for large polar body extrusion. Moreover, WDR62 decline intervened with the Arp2/3 complex, an upstream regulator for the cortical actin. Besides for p-MAPK, a critical regulator for the asymmetric division of oocyte, WDR62-depleted oocytes showed perturbation only in localization pattern but not expression level. In summary, our study defines WDR62 as an essential cytoskeletal regulator of spindle migration and asymmetric division during mouse oocyte meiotic maturation.

Ribonucleic Acid Export 1 Is a Kinetochore-Associated Protein That Participates in Chromosome Alignment in Mouse Oocytes.

Ribonucleic acid export 1 (Rae1) is an important nucleoporin that participates in mRNA export during the interphase of higher eukaryotes and regulates the mitotic cell cycle. In this study, small RNA interference technology was used to knockdown Rae1, and immunofluorescence, immunoblotting, and chromosome spreading were used to study the role of Rae1 in mouse oocyte meiotic maturation. We found that Rae1 is a crucial regulator of meiotic maturation of mouse oocytes. After the resumption of meiosis (GVBD), Rae1 was concentrated on the kinetochore structure. The knockdown of Rae1 by a specific siRNA inhibited GVBD progression at 2 h, finally leading to a decreased 14 h polar body extrusion (PBE) rate. However, a comparable 14 h PBE rate was found in the control, and the Rae1 knockdown groups that had already undergone GVBD. Furthermore, we found elevated PBE after 9.5 h in the Rae1 knockdown oocytes. Further analysis revealed that Rae1 depletion significantly decreased the protein level of securin. In addition, we detected weakened kinetochore-microtubule (K-MT) attachments, misaligned chromosomes, and an increased incidence of aneuploidy in the Rae1 knockdown oocytes. Collectively, we propose that Rae1 modulates securin protein levels, which contribute to chromosome alignment, K-MT attachments, and aneuploidy in meiosis.

Progressive Degenerative Myopathy and Myosteatosis in ASNSD1-Deficient Mice.

Mice with an inactivating mutation in the gene encoding asparagine synthetase domain containing 1 (ASNSD1) develop a progressive degenerative myopathy that results in severe sarcopenia and myosteatosis. ASNSD1 is conserved across many species, and whole body gene expression surveys show maximal expression levels of ASNSD1 in skeletal muscle. However, potential functions of this protein have not been previously reported. Asnsd1-/- mice demonstrated severe muscle weakness, and their normalized body fat percentage on both normal chow and high fat diets was greater than 2 SD above the mean for 3651 chow-fed and 2463 high-fat-diet-fed knockout (KO) lines tested. Histologic lesions were essentially limited to the muscle and were characterized by a progressive degenerative myopathy with extensive transdifferentiation and replacement of muscle by well-differentiated adipose tissue. There was minimal inflammation, fibrosis, and muscle regeneration associated with this myopathy. In addition, the absence of any signs of lipotoxicity in Asnsd1-/- mice despite their extremely elevated body fat percentage and low muscle mass suggests a role for metabolic dysfunctions in the development of this phenotype. Asnsd1-/- mice provide the first insight into the function of this protein, and this mouse model could prove useful in elucidating fundamental metabolic interactions between skeletal muscle and adipose tissue.

Nucleoporin35 is a novel microtubule associated protein functioning in oocyte meiotic spindle architecture.

Nucleoporins (Nups) are a large and diverse family of proteins that mediate nucleocytoplasmic transport at interphase of vertebrate cells. Nups also function in mitosis progression. However, whether Nups are involved in oocyte meiosis progression is still rarely known. In this study, we delineated the roles and regulatory mechanisms of Nucleoporin35 (Nup35) during oocyte meiotic maturation. The immunofluorescent signal of Nup35 was localized in the nuclear membrane at germinal vesicle (GV) stage, the microtubules and spindle at pro-metaphase I (pro-MI), metaphase I (MI), and metaphase II (MII), but to the spindle poles at anaphase I (AI) and telophase I (TI). The dynamic localization pattern of Nup35 during oocyte meiotic maturation implied its specific roles. We also found that Nup35 existed as a putatively phosphorylated form after resumption of meiosis (GVBD), but not at GV stage, implying its functional switch from nuclear membrane to meiotic progression. Further study uncovered that knockdown of Nup35 by specific siRNA significantly compromised the extrusion of first polar body (PBE), but not GVBD, with defects of spindle assembly and chromosome alignment and dissociated some localization signal of p-ERK1/2 from spindle poles to cytoplasm. A defective kinetochore - microtubule attachment (K-MT) was also identified in oocytes after knockdown of Nup35, which activates spindle assembly checkpoint. In conclusion, our results suggest that Nup35 is putatively phosphorylated and released to the cytoplasm after resumption of meiosis, and regulates spindle assembly and chromosome alignment.

LX2761, a Sodium/Glucose Cotransporter 1 Inhibitor Restricted to the Intestine, Improves Glycemic Control in Mice.

LX2761 is a potent sodium/glucose cotransporter 1 inhibitor restricted to the intestinal lumen after oral administration. Studies presented here evaluated the effect of orally administered LX2761 on glycemic control in preclinical models. In healthy mice and rats treated with LX2761, blood glucose excursions were lower and plasma total glucagon-like peptide-1 (GLP-1) levels higher after an oral glucose challenge; these decreased glucose excursions persisted even when the glucose challenge occurred 15 hours after LX2761 dosing in ad lib-fed mice. Further, treating mice with LX2761 and the dipeptidyl-peptidase 4 inhibitor sitagliptin synergistically increased active GLP-1 levels, suggesting increased LX2761-mediated release of GLP-1 into the portal circulation. LX2761 also lowered postprandial glucose, fasting glucose, and hemoglobin A1C, and increased plasma total GLP-1, during long-term treatment of mice with either early- or late-onset streptozotocin-diabetes; in the late-onset cohort, LX2761 treatment improved survival. Mice and rats treated with LX2761 occasionally had diarrhea; this dose-dependent side effect decreased in severity and frequency over time, and LX2761 doses were identified that decreased postprandial glucose excursions without causing diarrhea. Further, the frequency of LX2761-associated diarrhea was greatly decreased in mice either by gradual dose escalation or by pretreatment with resistant starch 4, which is slowly digested to glucose in the colon, a process that primes the colon for glucose metabolism by selecting for glucose-fermenting bacterial species. These data suggest that clinical trials are warranted to determine if LX2761 doses and dosing strategies exist that provide improved glycemic control combined with adequate gastrointestinal tolerability in people living with diabetes.

Effect of LX4211 on glucose homeostasis and body composition in preclinical models.

Treatments that lower blood glucose levels and body weight should benefit patients with type 2 diabetes mellitus (T2DM). We developed LX4211 [(2S,3R,4R,5S,6R)-2-(4-chloro-3-(4-ethoxybenzyl)phenyl)-6-(methylthio)tetrahydro-2H-pyran-3,4,5-triol], an orally available small molecule that decreases postprandial glucose excursions by inhibiting intestinal sodium/glucose cotransporter 1 (SGLT1) and increases urinary glucose excretion (UGE) by inhibiting renal SGLT2. In clinical studies of patients with T2DM, LX4211 appears to act through dual SGLT1/SGLT2 inhibition to improve glycemic control and promote weight loss. Here, we present preclinical studies that explored the ability of LX4211 to improve glycemic control and promote weight loss. We found that 1) LX4211 inhibited in vitro glucose transport mediated by mouse, rat, and dog SGLT1 and SGLT2; 2) a single daily LX4211 dose markedly increased UGE for >24 hours in mice, rats, and dogs; and 3) in the KK.Cg-Ay/J heterozygous (KKA(y)) mouse model of T2DM, LX4211 lowered A1C and postprandial glucose concentrations while increasing postprandial glucagon-like peptide 1 concentrations. Also, long-term LX4211 treatment 1) decreased oral glucose tolerance test (OGTT) glucose excursions, increased OGTT 30-minute insulin concentrations and increased pancreatic insulin content in KKA(y) mice; and 2) decreased weight gain in dogs and rats but not in KKA(y) mice while increasing food consumption in dogs, rats, and KKA(y) mice; in these KKA(y) mice, calories lost through UGE were completely offset by calories gained through hyperphagia. These findings suggest that LX4211 improves glycemic control by dual SGLT1/SGLT2 inhibition in mice as in humans, and that the LX4211-mediated weight loss observed in patients with T2DM may be attenuated by LX4211-mediated hyperphagia in some of these individuals.

Propyl gallate exposure affects the mouse 2-cell stage embryonic development through inducing oxidative stress and autophagy.

Propyl gallate (PG), owing to its exceptional antioxidant properties, is extensively used in industries such as food processing. The potential harmful impacts of PG have sparked concern among people. It has been reported that exposure of PG has certain reproductive toxicity, which can affect the maturation of mouse oocytes and induce testicular dysfunction. However, its impact on early embryonic development is still unclear. In this study, we explored the toxic effects and potential mechanisms of PG on mouse 2-cell stage embryonic development. The results showed that exposure of PG can decrease the development of 2-cell stage embryos and repress the development of 4-cell stage embryos. Further study found that PG could induce intracellular oxidative stress and the accumulation of DNA damage in 2-cell stage embryos. Moreover, exposure of PG impaired the function of mitochondria and lysosomes in 2-cell stage embryos, thereby triggering the occurrence of autophagy. In addition, exposure of PG altered the epigenetic modification of 2-cell stage embryos, displaying a decreased level of DNA methylation and an increased level of H3K4me3. In summary, our results indicated that exposure of PG can damage the development of mouse 2-cell stage embryos by inducing oxidative stress, DNA damage, and autophagy, and altering epigenetic modification.

Improved glycemic control in mice lacking Sglt1 and Sglt2.

Sodium-glucose cotransporter 2 (SGLT2) is the major, and SGLT1 the minor, transporter responsible for renal glucose reabsorption. Increasing urinary glucose excretion (UGE) by selectively inhibiting SGLT2 improves glycemic control in diabetic patients. We generated Sglt1 and Sglt2 knockout (KO) mice, Sglt1/Sglt2 double-KO (DKO) mice, and wild-type (WT) littermates to study their relative glycemic control and to determine contributions of SGLT1 and SGLT2 to UGE. Relative to WTs, Sglt2 KOs had improved oral glucose tolerance and were resistant to streptozotocin-induced diabetes. Sglt1 KOs fed glucose-free high-fat diet (G-free HFD) had improved oral glucose tolerance accompanied by delayed intestinal glucose absorption and increased circulating glucagon-like peptide-1 (GLP-1), but had normal intraperitoneal glucose tolerance. On G-free HFD, Sglt2 KOs had 30%, Sglt1 KOs 2%, and WTs <1% of the UGE of DKOs. Consistent with their increased UGE, DKOs had lower fasting blood glucose and improved intraperitoneal glucose tolerance than Sglt2 KOs. In conclusion, 1) Sglt2 is the major renal glucose transporter, but Sglt1 reabsorbs 70% of filtered glucose if Sglt2 is absent; 2) mice lacking Sglt2 display improved glucose tolerance despite UGE that is 30% of maximum; 3) Sglt1 KO mice respond to oral glucose with increased circulating GLP-1; and 4) DKO mice have improved glycemic control over mice lacking Sglt2 alone. These data suggest that, in patients with type 2 diabetes, combining pharmacological SGLT2 inhibition with complete renal and/or partial intestinal SGLT1 inhibition may improve glycemic control over that achieved by SGLT2 inhibition alone.

LX4211 increases serum glucagon-like peptide 1 and peptide YY levels by reducing sodium/glucose cotransporter 1 (SGLT1)-mediated absorption of intestinal glucose.

LX4211 [(2S,3R,4R,5S,6R)-2-(4-chloro-3-(4-ethoxybenzyl)phenyl)-6-(methylthio)tetrahydro-2H-pyran-3,4,5-triol], a dual sodium/glucose cotransporter 1 (SGLT1) and SGLT2 inhibitor, is thought to decrease both renal glucose reabsorption by inhibiting SGLT2 and intestinal glucose absorption by inhibiting SGLT1. In clinical trials in patients with type 2 diabetes mellitus (T2DM), LX4211 treatment improved glycemic control while increasing circulating levels of glucagon-like peptide 1 (GLP-1) and peptide YY (PYY). To better understand how LX4211 increases GLP-1 and PYY levels, we challenged SGLT1 knockout (-/-) mice, SGLT2-/- mice, and LX4211-treated mice with oral glucose. LX4211-treated mice and SGLT1-/- mice had increased levels of plasma GLP-1, plasma PYY, and intestinal glucose during the 6 hours after a glucose-containing meal, as reflected by area under the curve (AUC) values, whereas SGLT2-/- mice showed no response. LX4211-treated mice and SGLT1-/- mice also had increased GLP-1 AUC values, decreased glucose-dependent insulinotropic polypeptide (GIP) AUC values, and decreased blood glucose excursions during the 6 hours after a challenge with oral glucose alone. However, GLP-1 and GIP levels were not increased in LX4211-treated mice and were decreased in SGLT1-/- mice, 5 minutes after oral glucose, consistent with studies linking decreased intestinal SGLT1 activity with reduced GLP-1 and GIP levels 5 minutes after oral glucose. These data suggest that LX4211 reduces intestinal glucose absorption by inhibiting SGLT1, resulting in net increases in GLP-1 and PYY release and decreases in GIP release and blood glucose excursions. The ability to inhibit both intestinal SGLT1 and renal SGLT2 provides LX4211 with a novel dual mechanism of action for improving glycemic control in patients with T2DM.

Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 plays a critical role in the lipolytic processing of chylomicrons.

The triglycerides in chylomicrons are hydrolyzed by lipoprotein lipase (LpL) along the luminal surface of the capillaries. However, the endothelial cell molecule that facilitates chylomicron processing by LpL has not yet been defined. Here, we show that glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) plays a critical role in the lipolytic processing of chylomicrons. Gpihbp1-deficient mice exhibit a striking accumulation of chylomicrons in the plasma, even on a low-fat diet, resulting in milky plasma and plasma triglyceride levels as high as 5000 mg/dl. Normally, Gpihbp1 is expressed highly in heart and adipose tissue, the same tissues that express high levels of LpL. In these tissues, GPIHBP1 is located on the luminal face of the capillary endothelium. Expression of GPIHBP1 in cultured cells confers the ability to bind both LpL and chylomicrons. These studies strongly suggest that GPIHBP1 is an important platform for the LpL-mediated processing of chylomicrons in capillaries.

WDR62 regulates mouse oocyte meiotic maturation related to p-JNK and H3K9 trimethylation.

WDR62 (WD40-repeat protein 62) participates in diverse biological process, especially mitotic spindle organization via regulating centriole biogenesis and the function of centriole-associated protein. However, the role of WDR62 exerts in spindle assembly and meiotic progression control in oocytes lacking typical centrosomes remains obscure. In a previous study, we reported that WDR62 is involved in spindle migration and asymmetric cytokinesis in mouse oocyte meiosis. In the current study, another novel function of WDR62 regulating cell cycle progression through meiotic spindle formation during oocyte meiotic maturation was found. Knockdown of WDR62 through siRNA microinjection disrupted the meiotic cell cycle and induced metaphase-I (MI) arrest coupled with severe spindle abnormality, chromosome misalignment, and aneuploid generation. Moreover, WDR62 depletion induced defective kinetochore-microtubule attachments (K-MT) and activated spindle assembly checkpoint (SAC), which could trigger the arrest of meiotic progression. Further study demonstrated that depletion of WDR62 was associated with an aberrant location of p-JNK and reduced its expression level; concomitantly, status of H3K9 trimethylation was also altered. In addition, phenotypes similar to WDR62 depletion were observed during the function-loss analysis of p-JNK using a specific inhibitor (SP600125), which signifies that WDR62 is important for spindle organization and meiotic progression, and this function might be via its regulation of p-JNK. In conclusion, this study revealed that WDR62 functions in multiple ways during oocyte meiotic maturation, which could be related to p-JNK and H3K9 trimethylation.

Bromoacetic acid impairs mouse oocyte in vitro maturation through affecting cytoskeleton architecture and epigenetic modification.

As a major public health achievement, disinfection of drinking water significantly decreases outbreaks of waterborne disease, but produces drinking water disinfection by-products (DBPs) unfortunately. The haloacetic acids (HAAs) including bromoacetic acid (BAA), the second major class of DBPs, are considered as a global public health concern. BAA has been identified as cytotoxic, genotoxic, mutagenic, carcinogenic, and teratogenic in somatic cells. However, the toxic effects of BAA on oocyte maturation remain obscure. Herein, we documented that exposure to BAA compromised mouse oocyte maturation in vitro, causing blocked polar body extrusion (PBE). Meiotic progression analysis demonstrated that exposure to BAA induced the activated spindle assembly checkpoint (SAC) mediated metaphase I (MI) arrest in oocytes. Further study revealed that exposure to BAA resulted in the hyperacetylation of α-tubulin, disrupting spindle assembly and chromosome alignment, which is responsible for the activation of SAC. Besides, the organization of actin, the other major component of cytoskeleton in oocytes, was disturbed after BAA exposure. In addition, exposure to BAA altered the status of histone H3 methylation and 5 mC, indicative of the damaged epigenetic modifications. Moreover, we found that exposure to BAA induced DNA damage in a dose-dependent manner in oocytes. Collectively, our study evidenced that exposure to BAA intervened mouse oocyte maturation via disrupting cytoskeletal dynamics, damaging epigenetic modifications and inducing accumulation of DNA damage.

Gossypol exposure induces mitochondrial dysfunction and oxidative stress during mouse oocyte in vitro maturation.

Gossypol is a yellow natural polyphenolic compound extracted from the seeds, leaves, stems, and flower buds of the cotton plant. Several studies have shown that exposure to gossypol impacts reproductive health in both humans and animals. However, whether gossypol exposure would influence oocyte quality has not yet been determined. Here, we studied the effects of gossypol on the meiotic maturation of mouse oocytes in vitro. The results revealed that gossypol exposure did not affect germinal vesicle breakdown (GVBD) but significantly reduced polar body extrusion (PBE) rates. Moreover, we observed meiotic spindle organization and chromosome alignment were entirely disturbed after gossypol exposure. Further, gossypol exposure also caused mitochondrial dysfunction and abruptly decreased the levels of cellular ATP, and diminished the mitochondrial membrane potential (MMP). Accordingly, gossypol-induced oxidative stress was confirmed through an increased level of reactive oxygen species (ROS). Early apoptosis incidence also increased as identified by positive Annexin-V signaling. Collectively, the above findings provide evidence that gossypol exposure impaired oocyte meiotic maturation, disturbed spindle structure and chromosome dynamics, disrupted mitochondrial function, induced oxidative stress, and triggered early apoptosis. These findings emphasize gossypol's adverse effects on oocyte maturation and thus on female fertility.

High-Throughput Screening of Mouse Gene Knockouts Identifies Established and Novel High Body Fat Phenotypes.

PURPOSE: Obesity is a major public health problem. Understanding which genes contribute to obesity may better predict individual risk and allow development of new therapies. Because obesity of a mouse gene knockout (KO) line predicts an association of the orthologous human gene with obesity, we reviewed data from the Lexicon Genome5000TM high throughput phenotypic screen (HTS) of mouse gene KOs to identify KO lines with high body fat. MATERIALS AND METHODS: KO lines were generated using homologous recombination or gene trapping technologies. HTS body composition analyses were performed on adult wild-type and homozygous KO littermate mice from 3758 druggable mouse genes having a human ortholog. Body composition was measured by either DXA or QMR on chow-fed cohorts from all 3758 KO lines and was measured by QMR on independent high fat diet-fed cohorts from 2488 of these KO lines. Where possible, comparisons were made to HTS data from the International Mouse Phenotyping Consortium (IMPC). RESULTS: Body fat data are presented for 75 KO lines. Of 46 KO lines where independent external published and/or IMPC KO lines are reported as obese, 43 had increased body fat. For the remaining 29 novel high body fat KO lines, Ksr2 and G2e3 are supported by data from additional independent KO cohorts, 6 (Asnsd1, Srpk2, Dpp8, Cxxc4, Tenm3 and Kiss1) are supported by data from additional internal cohorts, and the remaining 21 including Tle4, Ak5, Ntm, Tusc3, Ankk1, Mfap3l, Prok2 and Prokr2 were studied with HTS cohorts only. CONCLUSION: These data support the finding of high body fat in 43 independent external published and/or IMPC KO lines. A novel obese phenotype was identified in 29 additional KO lines, with 27 still lacking the external confirmation now provided for Ksr2 and G2e3 KO mice. Undoubtedly, many mammalian obesity genes remain to be identified and characterized.