Oocyte Meiotic Progression Research Articles

IP3R1 is required for meiotic progression and embryonic development by regulating mitochondrial calcium and oxidative damage

Calcium ions (Ca2+) regulate cell proliferation and differentiation and participate in various physiological activities of cells. The calcium transfer protein inositol 1,4,5-triphosphate receptor (IP3R), located between the endoplasmic reticulum (ER) and mitochondria, plays an important role in regulating Ca2+ levels. However, the mechanism by which IP3R1 affects porcine meiotic progression and embryonic development remains unclear. We established a model in porcine oocytes using siRNA-mediated knockdown of IP3R1 to investigate the effects of IP3R1 on porcine oocyte meiotic progression and embryonic development. The results indicated that a decrease in IP3R1 expression significantly enhanced the interaction between the ER and mitochondria. Additionally, the interaction between the ER and the mitochondrial Ca2+ ([Ca2+]m) transport network protein IP3R1-GRP75-VDAC1 was disrupted. The results of the Duolink II in situ proximity ligation assay (PLA) revealed a weakened pairwise interaction between IP3R1-GRP75 and VDAC1 and a significantly increased interaction between GRP75 and VDAC1 after IP3R1 interference, resulting in the accumulation of large amounts of [Ca2+]m. These changes led to mitochondrial oxidative stress, increased the levels of reactive oxygen species (ROS) and reduced ATP production, which hindered the maturation and late development of porcine oocytes and induced apoptosis. Nevertheless, after treat with [Ca2+]m chelating agent ruthenium red (RR) or ROS scavenger N-acetylcysteine (NAC), the oocytes developmental abnormalities, oxidative stress and apoptosis caused by Ca2+ overload were improved. In conclusion, our results indicated IP3R1 is required for meiotic progression and embryonic development by regulating mitochondrial calcium and oxidative damage.

Melatonin improves the viability and ultrastructure of bovine oocyte-granulosa complexes of invitro cultured early antral follicles.

This study aims to investigate the effects of melatonin on follicular growth, viability and ultrastructure, as well as on the levels of mRNA for antioxidant enzymes, reactive oxygen species (ROS) and meiotic progression in oocytes from invitro cultured bovine early antral follicles. To this end, isolated early antral follicles (500-600 μm) were cultured in TCM-199+ alone or supplemented with 10-6 , 10-7 or 10-8 M melatonin at 38.5°C with 5% CO2 for 8 days. Follicle diameters were evaluated at days 0, 4 and 8 of culture. At the end of culture, ultrastructure, chromatin configuration, viability (calcein-AM and ethidium homodimer-1 staining), and the levels of ROS and mRNA for catalase (CAT), superoxide dismutase (SOD) and peroxiredoxin 6 (PRDX6) and glutathione peroxidase (GPx) were investigated in oocyte-granulosa cell complexes (OGCs). The results showed that early antral follicles cultured with 10-6 and 10-8 M melatonin had a progressive and significant increase in their diameters throughout the culture period (p < .05). Additionally, oocytes from follicles cultured with 10-7 or 10-8 M melatonin had increased fluorescence for calcein-AM, while those cultured with 10-6 or 10-7 M had reduced fluorescence for ethidium homodimer-1. Different from follicles cultured in other treatments, those cultured with 10-8 M melatonin had well-preserved ultrastructure of oocyte and granulosa cells. Melatonin, however, did not influence the levels of ROS, the mitochondrial activity, oocyte meiotic resumption and expression mRNA for SOD, CAT, GPX1 and PRDX6. In conclusion, the presence of 10-8 M melatonin in culture medium improves viability and preserves the ultrastructure of oocyte and granulosa cells of early antral follicles cultured invitro.

Core fucosylation regulates the ovarian response via FSH receptor during follicular development

IntroductionOvarian low response to follicle-stimulating hormone (FSH) causes infertility featuring hypergonadotropic hypogonadism, ovarian failure, and/or defective ovarian response. ObjectivesN-glycosylation is essential for FSH receptor (FSHR). Core fucosylation catalyzed by fucosyltransferase 8 (FUT8) is the most common N-glycosylation. Core fucosylation level changes between individuals and plays important roles in multiple physiological and pathological conditions. This study aims to elucidate the significance of FUT8 to modulate FSHR function in female fertility. MethodsSamples from patients classified as poor ovary responders (PORs) were detected with lectin blot and real-time PCR. Fut8 gene knockout (Fut8−/−) mice and FUT8-knockdown human granulosa cell line (KGN-KD) were established and in vitro fertilization (IVF) assay, western blot, molecular interaction, immunofluorescence and immunoprecipitation were applied. ResultsCore fucosylation is indispensable for oocyte and follicular development. FSHR is a highly core-fucosylated glycoprotein. Loss of core fucosylation suppressed binding of FSHR to FSH, and attenuated FSHR downstream signaling in granulosa cells. Transcriptomic analysis revealed the downregulation of several transcripts crucial for oocyte meiotic progression and preimplantation development in Fut8−/− mice and in POR patients. Furthermore, loss of FUT8 inhibited the interaction between granulosa cells and oocytes, reduced transzonal projection (TZP) formation and caused poor developmental competence of oocytes after fertilization in vitro. While L­fucose administration increased the core fucosylation of FSHR, and its sensitivity to FSH. ConclusionThis study first reveals a significant presence of core fucosylation in female fertility control. Decreased fucosylation on FSHR reduces the interaction of FSH-FSHR and subsequent signaling, which is a feature of the POR patients. Our results suggest that core fucosylation controls oocyte and follicular development via the FSH/FSHR pathway and is essential for female fertility in mammals.

The ERβ-cAMP signaling pathway regulates estradiol-induced ovine oocyte meiotic arrest

Although 17β-estradiol (E2) and its receptors (ERs) are reported to play important roles in regulating oocyte maturation, the specific mechanism remains unclear. First, we performed immunohistochemistry analyses to determine the expression of the ERα and ERβ proteins in ovine ovarian tissue. Second, E2 (0.5 ng/mL and 1 μg/mL) were added to pre-IVM medium for 0 h, 1 h and 2 h. The effects of E2 (0.5 ng/mL and 1 μg/mL) on cyclic adenosine monophosphate (cAMP) level in cumulus-oocyte complexes (COCs) and on oocyte meiotic progression were evaluated by ELISA and DAPI staining respectively. Third, the effects of E2 on the gene and protein expression of ERα and ERβ in COCs were investigated by Western blotting and real-time PCR. Afterward, ERβ and cAMP regulators were added to the 2-h pretreatment medium with or without E2 (0.5 ng/mL) to explore the possible interactions among E2, cAMP and ERβ. The results showed that both ERα and ERβ proteins were expressed in ovine cumulus layers and oocytes. E2 significantly increased intra-COC cAMP levels, maintained oocyte meiotic arrest, and promoted ERβ transcript and protein expression. E2 treatment increased the cAMP concentration, which was enhanced by ERβ agonist treatment and remarkably attenuated by ERβ inhibitor treatment. Forskolin plus IBMX treatment increased ERβ protein expression in COCs (P < 0.05), and this was attenuated by Rp-cAMP treatment. In conclusion, E2 (0.5 ng/mL) increased intra-COC cAMP levels by promoting ERβ expression, thereby maintaining oocyte meiotic arrest. cAMP in COCs has a positive feedback effect on ERβ expression, which provides a novel explanation for the positive role of E2 in regulating ovine follicle development and oocyte maturation.

Maternal NAT10 orchestrates oocyte meiotic cell-cycle progression and maturation in mice

In mammals, the production of mature oocytes necessitates rigorous regulation of the discontinuous meiotic cell-cycle progression at both the transcriptional and post-transcriptional levels. However, the factors underlying this sophisticated but explicit process remain largely unclear. Here we characterize the function of N-acetyltransferase 10 (Nat10), a writer for N4-acetylcytidine (ac4C) on RNA molecules, in mouse oocyte development. We provide genetic evidence that Nat10 is essential for oocyte meiotic prophase I progression, oocyte growth and maturation by sculpting the maternal transcriptome through timely degradation of poly(A) tail mRNAs. This is achieved through the ac4C deposition on the key CCR4-NOT complex transcripts. Importantly, we devise a method for examining the poly(A) tail length (PAT), termed Hairpin Adaptor-poly(A) tail length (HA-PAT), which outperforms conventional methods in terms of cost, sensitivity, and efficiency. In summary, these findings provide genetic evidence that unveils the indispensable role of maternal Nat10 in oocyte development.

Decreased LONP1 expression contributes to DNA damage and meiotic defects in oocytes.

Meiotic defects in oocytes are the primary reason for decreased female fertility with advanced maternal age. In this study, we revealed that decreased expression of ATP-dependent Lon peptidase 1 (LONP1) in aged oocytes and oocyte-specific depletion of LONP1 disrupt oocyte meiotic progression accompanying with mitochondrial dysfunction. In addition, LONP1 downregulation increased oocyte DNA damage. Moreover, we demonstrated that splicing factor proline and glutamine rich directly interacts with LONP1 and mediate the effect of LONP1 depletion on meiotic progression in oocytes. In summary, our data suggest that decreased expression of LONP1 is involved in advanced maternal age-related meiosis defects and that LONP1 represents a new therapeutic target to improve aged oocyte quality.

In vitro effect of zearalenone on sperm parameters, oocyte maturation and embryonic development in buffalo

The negative impact of zearalenone (ZEN; potent estrogenic mycotoxin) exposure on buffalo embryo production has not yet been determined. In the current study, buffalo sperm and oocytes were exposed to ZEN at different concentrations during maturation. Sperms (with and without ZEN exposure) were incubated for 2h and evaluated for motility, viability, acrosome integrity, normality, and ultrastructure. Matured oocytes exposed to ZEN were stained to determine their nuclear maturation. Further, their developmental ability was evaluated after in vitro fertilization. Our results showed the toxic effects of ZEN at high concentrations (2000ng/mL) on different buffalo sperm parameters. The number of acrosome-intact sperm was reduced at 0h after exposure to a concentration of ≥100ng/mL. Furthermore, the maturation rate of buffalo oocytes (telophase I + metaphase II) was significantly decreased in ZEN-treated oocytes with a higher degeneration rate. Oocytes matured in 1000ng/mL ZEN and subsequently exhibited considerable reduction in cleavage rate and blastocyst formation compared with control oocytes (2.6% vs. 13.1%). Moreover, the morula rate was decreased (p<0.001) in ZEN-treated oocytes at concentrations of ≥10ng/mL. Overall, the adverse effects of in vitro ZEN exposure on buffalo sperm parameters and oocyte meiotic progression with a notable reduction in cleavage, morula, and blastocyst rates were defined by these results. Altogether, buffaloes should be considered sensitive to ZEN exposure with respect to their reproductive function.

Effects of N-acetylcysteine on growth, viability and reactive oxygen species levels in small antral follicles cultured in vitro

Objective: To investigate the effects of different concentrations of N-acetylcysteine on follicular growth and morphology, as well as on viability, levels of reactive oxygen species (ROS) and meiotic progression of oocytes from in vitro cultured bovine early antral follicles. Methods: Isolated early antral follicles (about 500 μm) were cultured in TCM-199+ alone or supplemented with 1.0, 5.0 or 25.0 mM N-acetylcysteine at 38.5 °C with 5% CO2 for 8 days. Follicle diameters were evaluated at day 0, 4 and 8 of culture. At the end of culture, the levels of ROS, chromatin configuration and viability (calcein-AM and ethidium homodimer-1 staining) were investigated in the cumulus-oocyte complexes. Comparisons of follicle diameters between treatments were performed. Data on percentages of morphologically normal follicles, growth rates and chromatin configuration in different treatments were compared. Results: An increase in follicular diameters after culture in all treatments was observed, except for follicles cultured with 25.0 mM N-acetylcysteine. Fluorescence microscopy showed that oocytes cultured in all treatments were stained positively with calcein-AM, and that 5.0 mM N-acetylcysteine reduced fluorescence for ethidium homodimer-1. Intracellular levels of ROS in oocytes from follicles cultured with 1.0 mM N-acetylcysteine showed a significant reduction compared to other treatments. The presence of N-acetylcysteine in culture medium did not influence the rates of oocyte at the germinal vesicle stage. Conclusions: N-acetylcysteine at concentrations of 1.0 and 5.0 mM reduces ROS levels and staining for ethidium homodimer-1 in in vitro cultured follicles, respectively, while 25.0 mM N-acetylcysteine decreases follicular growth and the percentages of continuously growing follicles.

Cyclic adenosine 3’5’monophosphate (cAMP) regulates meiotic maturation in goldfish oocytes Carassius auratus (Teleostei: Cyprinidae)

A good understanding of the mechanism of oocyte development and maturation, and the regulatory factors involved therein are important for the success of a breeding program especially of species used in aquaculture. It is crucial in a breeding program for females to supply high quality mature oocytes, which is generally achieved by fine-tuning oocyte meiotic arrest and resumption progression. Therefore, knowledge of the oocyte maturation process is fundamental for the development of methodologies to increase the success of fish production in aquaculture. TheFigure regulations of oocyte maturation still are not totally understood. The hypothesis whether elevated levels or a drop in intraoocyte cAMP triggers resumption of meiotic maturation was investigated in denuded goldfish oocytes. In the presence of agents that elevate cAMP like forskolin, dibutyryl cAMP, isobutyl methylxanthine, maturation of denuded oocytes was inhibited. Whereas 2’deoxyadenosine-3-monophosphate, an inhibitor of adenylate cyclase promoted oocyte maturation. Forskolin decreased spontaneous maturation as well as hormone-induced maturation of goldfish oocytes. At 10 µM forskolin added up to 4 h after hormonal stimulation completely inhibited oocyte maturation. Beyond 4 h, however, when oocyte maturation had already started, forskolin can no longer inhibit maturation. Intraoocyte cAMP assayed in denuded oocytes with or without hormonal stimulation decreased immediately within 30 minutes. With forskolin, it did not cause a significant decrease in cAMP not until at 4 h. The results were consistent with the findings that elevated levels of cAMP maintain meiotic arrest while a decrease in cAMP is necessary and sufficient to trigger resumption of meiotic maturation.

Mitochondrial E3 ubiquitin ligase MARCH5 is required for mouse oocyte meiotic maturation†.

As the most abundant organelles in oocytes, mitochondria play an important role in maintaining oocyte quality. Here, we report that March5, encoding a mitochondrial ubiquitin ligase that promotes mitochondrial elongation, plays a critical role in mouse oocyte meiotic maturation via regulating mitochondrial function. The subcellular localization of MARCH5 was similar to the mitochondrial distribution during mouse oocyte meiotic progression. Knockdown of March5 caused decreased ratios of the first polar body extrusion. March5-siRNA injection resulted in oocyte mitochondrial dysfunctions, manifested by increased reactive oxygen species, decreased ATP content as well as decreased mitochondrial membrane potential, leading to reduced ability of spindle formation and an increased ratio of kinetochore-microtubule detachment. Further study showed that the continuous activation of the spindle assembly checkpoint and the failure of Cyclin B1 degradation caused MI arrest and first polar body (PB1) extrusion failure in March5 knockdown oocytes. Taken together, our results demonstrated that March5 plays an essential role in mouse oocyte meiotic maturation, possibly via regulation of mitochondrial function and/or ubiquitination of microtubule dynamics- or cell cycle-regulating proteins.

Septin 9 controls CCNB1 stabilization via APC/CCDC20 during meiotic metaphase I/anaphase I transition in mouse oocytes.

The anaphase promoting complex/cyclosome (APC/C) and its cofactors CDH1 and CDC20 regulate the accumulation/degradation of CCNB1 during mouse oocyte meiotic maturation. Generally, the CCNB1 degradation mediated by APC/CCDC20 activity is essential for the transition from metaphase to anaphase. Here, by using siRNA and mRNA microinjection, as well as time-lapse live imaging, we showed that Septin 9, which mediates the binding of septins to microtubules, is critical for oocyte meiotic cell cycle progression. The oocytes were arrested at the MI stage and the connection between chromosome kinetochores and spindle microtubules was disrupted after Septin 9 depletion. As it is well known that spindle assembly checkpoint (SAC) is an important regulator of the MI-AI transition, we thus detected the SAC activity and the expression of CDC20 and CCNB1 which were the downstream proteins of SAC during this critical period. The signals of Mad1 and BubR1 still remained on the kinetochores of chromosomes in Septin 9 siRNA oocytes at 9.5h of in vitro culture when most control oocytes entered anaphase I. The expression of CCNB1 did not decrease and the expression of CDC20 did not increase at 9.5h in Septin 9 siRNA oocytes. Microinjection of mRNA encoding Septin 9 or CDC20 could partially rescue MI arrest caused by Septin 9 siRNA. These results suggest that Septin 9 is required for meiotic MI-AI transition by regulating the kinetochore-microtubule connection and SAC protein localization on kinetochores, whose effects are transmitted to APC/CCDC20 activity and CCNB1 degradation in mouse oocytes.

Toxic effects of AZD1208 on mouse oocytes and its possible mechanisms.

AZD1208, a pan-inhibitor that can effectively inhibit PIM kinase, is used for the treatment of advanced solid tumors and malignant lymphomas. Numerous studies have proved its curative effects while its potential cellular toxicity on reproduction was still little known. In this study, we investigated the toxic effects of AZD1208 on mouse oocytes. The results showed that AZD1208 treatment did not affect meiotic resumption, but postponed oocyte maturation as indicated by delayed first polar body extrusion. Further mechanistic study showed that AZD1208 treatment delayed spindle assembly. In addition, we found that oocytes treated with AZD1208 showed mitochondrial dysfunction. Abnormal mitochondrial clusters with decreased mitochondrial membrane potential were observed in oocytes during incubation in vitro. Moreover, increased oxidative stress was observed by testing the level of reactive oxygen species. In summary, our results suggest that AZD1208 treatment influences oocyte meiotic progression by causing mitochondrial dysfunctions and subsequent delayed spindle assembly.

P-216 Low meiotic progression rates in oocytes from older women are due to impaired mitochondrial metabolism

Abstract Study question Could a decreased mitochondrial metabolism explain the lower oocyte meiotic progression competence observed in advanced maternal age (AMA) oocytes? Summary answer Inhibition of mitochondrial activity in young oocytes mirrors the phenotype of AMA oocytes, pointing to a causative link between effective mitochondrial metabolism and meiotic competence What is known already Oocyte meiotic maturation is a fundamental and finely regulated process required to form haploid gametes, and errors during this process invariably cause loss of oocyte competence. Similarly, mitochondria metabolism is required for the acquisition of oocyte competence and support of embryo development. In oocytes from AMA women, both meiotic competence and mitochondrial activity are impaired; in mice, it has been suggested the two processes might be functionally linked. However, functional studies in human oocytes evaluating whether alterations in mitochondrial metabolism cause reduced meiotic competence are lacking Study design, size, duration A total of 216 oocytes from young (age ≤30) and AMA (age &amp;gt;37) women were included. Oocytes at the germinal vesicle (GV) stage were matured in vitro (in G2plus medium for 30 h; maturation was assessed by polar body (PB) extrusion. For loss of function studies on mitochondrial metabolism, 64 GVs from young women were treated with 1uM of the mitochondria inhibitor Trifluoromethoxy-carbonylcyanide-phenylhydrazone (FCCP) for 30 min and cultured for 30 h in G2plus Participants/materials, setting, methods The proteins Dihydrolipoamide S-Acetyltransferase (D-LAT) and Translocase of outer mitochondrial membrane (TOMM20) were analyzed by immunofluorescence in young and AMA oocytes to assess mitochondrial activity and localization, respectively. Fluorescent mean intensities were quantified with ImageJ and compared with t-test. For D-LAT, we assessed an area of 30 um approx. inward from the oocyte cortex, whereas for TOMM20, the whole oocyte diameter was used. Maturation rates were compared between groups by Chi-squared test Main results and the role of chance Mitochondrial staining of the two proteins showed a common pattern in the two age groups, with a uniform localization of mitochondria in the whole ooplasm observed by TOMM20, and a subcortical localization of active organelles detected by D-LAT. Quantification of the fluorescent signal showed no differences in term of total mitochondrial presence (intensity of 61674±24322 AU in young, 32186±33414 AU in AMA, p = 0.195), whereas a significant decrease in activity (D-LAT) was measured in the AMA group (intensity of 78614±58534 AU in young, 12517±10187 AU in AMA, p = 0.003). Young oocytes also matured at a higher rate (86.3%; 63/73) than AMA oocytes (62.3%; 38/61; p = 0.002). Young GV oocytes treated with FCCP presented a phenotype similar to AMA oocytes, with a significant decrease in of DLAT (intensity of 78614±58534 AU in non-treated, 11554±16131 AU in treated, p = 0.019), indicating a decrease in mitochondria activity, and a concomitant drop in maturation rate, with only 39.5% (17/43) of the treated oocytes able to extrude the PB Limitations, reasons for caution Maturation rates have been assessed by PB extrusion and some variability in spindle assembly may have been overlooked. We assessed one mitochondria activity inhibitor, FCCP, so indirect effects of this molecule on the observed phenotype cannot be ruled out Wider implications of the findings We show a functional link between decreased mitochondrial metabolism and impaired meiotic progression. The metabolic inhibition with FCCP in young oocytes might serve as tool to further investigate the molecular mechanisms driving the loss of quality observed in AMA oocytes Trial registration number NA

HIF1α is dispensable for oocyte development and female fertility in mice.

BackgroundIt has been thought that oocyte may develop in a low oxygen environment, as changes in follicle structure and formation of a fluid-filled antrum. The survival of hypoxic tissues is controlled by hypoxia-inducible factors (HIFs) that are activated in a low oxygen state. HIF1α is expressed in mature mouse oocytes and continues to be expressed after fertilization, from the 2-cell to blastocyst stage. However, the physiological roles of HIF pathway during oogenesis and embryogenesis have still not been elucidated in detail.MethodsMutant mice with oocyte-specific HIF1α deletion were generated by crossing Hif1αfl/fl mice with transgenic mice expressing Gdf9-promoter-mediated Cre recombinase. Breeding assay was carried out to detect female fertility. In vitro fertilization and embryo culture were used to assess early embryo development. Oocyte meiotic progression was also examined. Quantitative RT-PCR was used for analyzing of candidate genes expression.ResultsWe successfully generated mutant mice with oocyte-specific deletion of HIF1α. Oocytes loss of HIF1α did not affect female fertility, ovulation and early embryo development. Moreover, oocytes can mature in vitro, and form well-organized spindle in the absence of HIF1α. In addition, pronounced differences in Hif2α and Hif3α mRNA expression were not observed in HIF1α-deleted oocytes. These results revealed that HIF pathway in oocytes is not essential for female fertility.

Dynamic zinc fluxes regulate meiotic progression in Caenorhabditis elegans†.

Zinc influx and efflux events are essential for meiotic progression in oocytes of several mammalian and amphibian species, but it is less clear whether this evolutionary conservation of zinc signals is also important in late-stage germline development in invertebrates. Using quantitative, single cell elemental mapping methods, we find that Caenorhabditis elegans oocytes undergo significant stage-dependent fluctuations in total zinc content, rising by over sevenfold from Prophase I through the beginning of mitotic divisions in the embryo. Live imaging of the rapid cell cycle progression in C. elegans enables us to follow changes in labile zinc pools across meiosis and mitosis in single embryo. We find a dynamic increase in labile zinc prior to fertilization that then decreases from Anaphase II through pronuclear fusion and relocalizes to the eggshell. Disruption of these zinc fluxes blocks extrusion of the second polar body, leading to a range of mitotic defects. We conclude that spatial temporal zinc fluxes are necessary for meiotic progression in C. elegans and are a conserved feature of germ cell development in a broad cross section of metazoa.

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.

Insights into the NAD+ biosynthesis pathways involved during meiotic maturation and spindle formation in porcine oocytes.

Treatments that elevate NAD+ levels have been found to improve oocyte quality in mice, cattle, and pigs, suggesting that NAD+ is vital during oocyte maturation. This study aimed to examine the influence of different NAD+ biosynthetic pathways on oocyte quality by inhibiting key enzymes. Porcine oocytes from small antral follicles were matured for 44 h in a defined maturation system supplemented with 2-hydroxynicotinic acid [2-HNA, nicotinic acid phosphoribosyltransferase (NAPRT) inhibitor], FK866 [nicotinamide phosphoribosyltransferase (NAMPT) inhibitor], or gallotannin [nicotinamide mononucleotide adenylyltransferase (NMNAT) inhibitor] and their respective NAD+ pathway modulators (nicotinic acid, nicotinamide, and nicotinamide mononucleotide, respectively). Cumulus expansion was assessed after 22 h of maturation. At 44 h, maturation rates were determined and mature oocytes were fixed and stained to assess spindle formation. Each enzyme inhibitor reduced oocyte maturation rate and adversely affected spindle formation, indicating that NAD+ is required for meiotic spindle assembly. Furthermore, NAMPT and NMNAT inhibition reduced cumulus expansion, whereas NAPRT inhibition affected chromosomal segregation. Treating oocytes with gallotannin and nicotinamide mononucleotide together showed improvements in spindle width, while treating oocytes with 2-HNA and nicotinic acid combined showed an improvement in both spindle length and width. These results indicate that the salvage pathway plays a vital role in promoting oocyte meiotic progression, while the Preiss-Handler pathway is essential for spindle assembly.

Cell Division Cycle 5-Like Regulates Metaphase-to-Anaphase Transition in Meiotic Oocyte.

The quality of oocytes is a vital factor for embryo development. Meiotic progression through metaphase I usually takes a relatively long time to ensure correct chromosome separation, a process that is critical for determining oocyte quality. Here, we report that cell division cycle 5-like (Cdc5L) plays a critical role in regulating metaphase-to-anaphase I transition during mouse oocyte meiotic maturation. Knockdown of Cdc5L by small interfering RNA injection did not affect spindle assembly but caused metaphase I arrest and subsequent reduced first polar body extrusion due to insufficient anaphase-promoting complex/cyclosome activity. We further showed that Cdc5L could also directly interact with securin, and Cdc5L knockdown led to a continuous high expression level of securin, causing severely compromised meiotic progression. The metaphase-to-anaphase I arrest caused by Cdc5L knockdown could be rescued by knockdown of endogenous securin. In summary, we reveal a novel role for Cdc5L in regulating mouse oocyte meiotic progression by interacting with securin.

Luteolin Orchestrates Porcine Oocyte Meiotic Progression by Maintaining Organelle Dynamics Under Oxidative Stress.

Increasing evidence has demonstrated that oxidative stress impairs oocyte maturation, but the underlying mechanisms remain largely unknown. Here, for the first time, we examined the antioxidant role of luteolin in meiotic progression and the underlying mechanisms. Supplementation of 5 μM luteolin increased the rates of first polar body extrusion and blastocyst formation after parthenogenetic activation, and the expression levels of oocyte competence (BMP15 and GDF9)-, mitogen-activated protein kinase (MOS)-, and maturation promoting factor (CDK1 and Cyclin B)-related genes were also improved. Luteolin supplementation decreased intracellular reactive oxygen species levels and increased the expression levels of oxidative stress-related genes (SOD1, SOD2, and CAT). Interestingly, luteolin alleviated defects in cell organelles, including actin filaments, the spindle, mitochondria, the endoplasmic reticulum, and cortical granules, caused by H2O2 exposure. Moreover, luteolin significantly improved the developmental competence of in vitro-fertilized embryos in terms of the cleavage rate, blastocyst formation rate, cell number, cellular survival rate, and gene expression and markedly restored the competencies decreased by H2O2 treatment. These findings revealed that luteolin supplementation during in vitro maturation improves porcine meiotic progression and subsequent embryonic development by protecting various organelle dynamics against oxidative stress, potentially increasing our understanding of the underlying mechanisms governing the relationship between oxidative stress and the meiotic events required for successful oocyte maturation.

Checkpoint kinases are required for oocyte meiotic progression by the maintenance of normal spindle structure and chromosome condensation

Checkpoint kinases (Chk) 1/2 are known for DNA damage checkpoint and cell cycle control in somatic cells. According to recent findings, the involvement of Chk1 in oocyte meiotic resumption and Chk2 is regarded as an essential regulator for progression at the post metaphase I stage (MI). In this study, AZD7762 (Chk1/2 inhibitor) and SB218078 (Chk1 inhibitor) were used to uncover the joint roles of Chk1/2 and differentiate the importance of Chk1 and Chk2 during oocyte meiotic maturation. Inhibition of Chk1/2 or Chk1 alone had no significant effect on germinal vesicle breakdown (GVBD) but significantly inhibited the first polar body (PB1). Interestingly, inhibition of Chk1 alone could not increase or completely block the extrusion of PB1 like Chk1/2 inhibition. Also, Chk1/2 inhibition resulted in defective meiotic spindle organization and chromosome condensation both in MI and metaphase II (MII) stages of oocytes. The location of γ-tubulin and Securin were abnormal or missing, while P38 MAPK was activated by Chk1/2 inhibition. Meanwhile, Chk1/2 inhibition reduced the percentage of the second polar body extrusion and pronuclear formation. In conclusion, our results further understand the functions and regulatory mechanism of Chk1/2 during oocyte meiotic maturation.