Polar Body Extrusion Rate Research Articles

Rescue ICSI Improved Cumulative Live Birth Rate for Cycles with Second Polar Body Extrusion Rate

ObjectiveTo determine the indications for early rescue intracytoplasmic sperm injection (ICSI) application. DesignA retrospective cohort study SubjectsThe study included 19,808 patients who underwent conventional in vitro fertilization (IVF) or rescue ICSI for their first cycles between February 2017 and December 2021. ExposureRescue ICSI cycles constituted the study group, where oocytes that had not extruded the second polar body 4-6 hours after insemination were rescued by ICSI. The control group consisted of conventional IVF cycles with no interventions to rescue oocytes without the second polar body. Generalized additive models (GAMs) were constructed to describe the relationship between the second polar body extrusion rate and cumulative live birth rate in conventional IVF and rescue ICSI cycles, respectively. The cutoff value of the second polar body extrusion rate guiding rescue ICSI application was determined from the intersection point of GAMs. Maternal age range applicable to rescue ICSI was further analyzed using the same method. Clinical outcomes were compared between conventional IVF and rescue ICSI cycles across different second polar body extrusion rate and maternal age subgroups. Main outcome measuresThe second polar body extrusion rate and maternal age range for rescue ICSI application, normal fertilization rate, and cumulative live birth rate. ResultsGAMs showed that the cutoff value for the second polar body extrusion rate about rescue ICSI application was 50%. When the rate <50%, normal fertilization rate and cumulative live birth rate (63.7% vs. 46.1%, OR: 1.609, 95% CI: 1.276-2.030) were significantly higher in rescue ICSI cycles than conventional IVF cycles. When the rate ≥50%, rescue ICSI cycles had similar normal fertilization rate and cumulative live birth rate compared to conventional IVF cycles. Further analysis on maternal age in cycles with second polar body extrusion rate <50% released that rescue ICSI cycles showed a higher cumulative live birth rate (67.7% vs. 48.3%, OR: 1.732, 95% CI: 1.361-2.202) than conventional IVF cycles for women aged < 38 years. ConclusionIVF cycles with second polar body extrusion rate <50% in women aged < 38 years was applicable to early rescue ICSI.

Bisphenol M inhibits mouse oocyte maturation in vitro by disrupting cytoskeleton architecture and cell cycle processes

Bisphenol M (BPM), an alternative to bisphenol A (BPA), is commonly utilized in various industrial applications. However, BPM does not represent a safe substitute for BPA due to its detrimental effects on living beings. This research aimed to assess the influence of BPM exposure on the in vitro maturation of mouse oocytes. The findings revealed that BPM exposure had a notable impact on the germinal vesicle breakdown (GVBD) rate and polar body extrusion (PBE) rate throughout the meiotic progression of mouse oocytes, ultimately resulting in meiotic arrest. Investigations demonstrated that oocytes exposure to BPM led to continued activation of spindle assembly checkpoint. Further studies revealed that securin and cyclin B1 could not be degraded in BPM-exposed oocytes, and meiosis could not realize the transition from the MI to the AI stage. Mechanistically, BPM exposure resulted in abnormal spindle assembly and disrupted chromosome alignment of oocytes. Additionally, abnormal positioning of microtubule organizing center-associated proteins implied that MTOC may be dysfunctional. Furthermore, an elevation in the acetylation level of α-tubulin in oocytes was observed after BPM treatment, leading to decreased microtubule stability. In addition to its impact on microtubules, BPM exposure led to a reduction in the expression of the actin, signifying the disruption of actin assembly. Further research indicated a heightened incidence of DNA damage in oocytes following BPM exposure. Besides, BPM exposure induced alterations in histone modifications. The outcomes of this experiment demonstrate that BPM exposure impairs oocyte quality and inhibits meiotic maturation of mouse oocytes.

Isorhamnetin Improves Oocyte Maturation by Activating the Pi3k/Akt Signaling Pathway.

Isorhamnetin is a natural flavonoid with various pharmacological activities, which can be widely and continuously ingested by humans and animals through their daily diet. The aim of this study is to explore the benefits and molecular mechanisms of isorhamnetin on oocyte maturation. Oocytes are incubated with isorhamnetin (5, 10, 20, and 30µM) for 44h. Isorhamnetin (10µM) increases the polar body extrusion rate of oocytes. Furthermore, isorhamnetin alleviates oxidative stress by inhibiting reactive oxygen species levels and stimulating SOD2 protein expression. The changes in intracellular mitochondrial autophagy and apoptosis-related proteins (Bcl-2, Bax/Bcl-2, and C-Casp3) indicate that isorhamnetin inhibits oocyte apoptosis. Isorhamnetin inhibits endoplasmic reticulum stress by reducing the protein expression of CHOP and GRP78 and improving the normal distribution rate of endoplasmic reticulum. Mechanistic studies show that isorhamnetin activates the PI3K/Akt signaling pathway. Isorhamnetin promotes oocyte maturation by inhibiting oxidative stress, mitochondrial dysregulation, apoptosis, and endoplasmic reticulum stress, which have important potential for improving oocyte quality and treating female infertility.

O-077 Depletion of MFF from oocytes impairs mitochondrial dynamics, leading to inhibited oocyte maturation and early embryonic development in mice

Abstract Study question To investigate the effect of mitochondrial fission factor (MFF) on oocyte competence and female fertility using an oocyte-specific Mff knockout mouse model. Summary answer Oocyte-specific Mff deficiency hindered oocyte maturation and early embryonic development via regulation of mitochondrial dynamics, resulting in female subfertility. What is known already Emerging studies have demonstrated that mitochondrial dynamics play a vital role in oocyte maturation and early embryo development. MFF is one of the crucial proteins regulating mitochondrial dynamics. Our previous studies have indicated that Mff is necessary for oocyte competence and fertility using a global Mff knockout mouse model. However, further investigation is needed to determine whether the deletion of Mff in oocytes could affect oocyte competence and early embryonic development by regulating mitochondrial dynamics. Study design, size, duration The oocyte-specific Mff knockout (Mfffl/fl; Gdf9-Cre) mice were generated using the Cre-LoxP conditional knockout system. The 2-month-old female mice from each group (Mfffl/fl; Gdf9-Cre or Mfffl/fl, n = 6) were mated to assess the fecundity over 10 months. The maturation and mitochondrial dynamics of germinal vesicle (GV) oocytes were determined. The spindle and chromosome morphologies of ovulated oocytes were also analyzed. Additionally, the 2-cell embryos were collected and cultured in vitro to assess early embryonic development. Participants/materials, setting, methods Female mice from each group were mated with adult males to assess the fecundity. GV oocytes, ovulated oocytes, and 2-cell embryos were collected and cultured in vitro after superovulation, as indicated. Mitochondria, spindle, and chromosome were stained and then imaged using confocal microscopy. The microscopic morphology of mitochondria was assessed using electron microscopy. Main results and the role of chance The Mfffl/fl; Gdf9-Cre mice consistently exhibited reduced fertility with a significant decrease in litter size (3.00 vs. 5.42, p &amp;lt; 0.001), number of litters per female (4.33 vs. 7.50, p &amp;lt; 0.001), and number of pups per female (13.00 vs. 40.67, p &amp;lt; 0.001) compared to the Mfffl/fl mice. The numbers of GV oocytes (29.37 vs. 39.10, p &amp;lt; 0.05), ovulated oocytes (9.81 vs. 20.91, p &amp;lt; 0.001) and 2-cell embryos (3.50 vs. 10.17, p &amp;lt; 0.05) were significantly lower in the Mfffl/fl; Gdf9-Cre mice. The blastocyst (32.32% vs. 68.44%, p &amp;lt; 0.05) embryo development rate per 2-cell embryos was also significantly lower. In vitro maturation revealed that of Mfffl/fl; Gdf9-Cre GV oocytes had significantly lower rates of GV break-down (68.89% vs. 91.57%, p &amp;lt; 0.05) and polar body extrusion (42.76% vs. 81.87%, p &amp;lt; 0.05). The ratios of aberrant spindle (71.56% vs. 23.74%, p &amp;lt; 0.001) and misaligned chromosomes (71.82% vs. 23.14%, p &amp;lt; 0.01) in ovulated oocytes from Mfffl/fl; Gdf9-Cre mice were significantly increased. In terms of mitochondrial dynamics, the mitochondrial size (0.59 µm2 vs. 0.15µm2, p &amp;lt; 0.001) and the ratio (62.50% vs. 18.67%, p &amp;lt; 0.01) of abnormal mitochondrial distribution (unevenly aggregated) in Mfffl/fl; Gdf9-Cre oocytes significantly elevated. This study found that the Mff played a crucial role in oocyte maturation and early embryonic development. Limitations, reasons for caution Further research is needed to determine the downstream targets or pathways of Mff in oocytes. Wider implications of the findings These results shed light on the important role played by MFF in ensuring proper mitochondrial dynamics in oocytes and underscore the significance of this protein in female fertility. This study provides insights into potential therapeutic targets for infertility and related diseases in humans. Trial registration number not applicable

Vinorelbine administration impedes the timely progression of meiotic maturation and induces aneuploidy in mouse oocytes

Vinorelbine is a commonly used drug to treat various malignancies, such as breast cancer, non-small cell lung cancer, and metastatic pleural mesothelioma. Its side effects include severe neutropenia, local phlebitis, gastrointestinal reactions, and neurotoxicity. In view of the scarcity of research on vinorelbine's reproductive toxicity, this study evaluated the impact of vinorelbine ditartrate, a commonly used form of vinorelbine, on oocyte maturation in vitro. Our investigation revealed that vinorelbine ditartrate had no effect on oocyte meiotic resumption. However, it did reduce the rate of first polar body extrusion, suggesting that it could significantly impede the meiotic maturation of oocytes. Vinorelbine ditartrate exposure was found to disturb the regular spindle assembly and chromosome alignment, leading to the continuous activation of the spindle assembly checkpoint (SAC) and a delayed activation of the anaphase-promoting complex/cyclosome (APC/C), ultimately causing aneuploidy in oocytes. Consequently, the administration of vinorelbine is likely to result in oocyte aneuploidy, which can be helpful in providing a drug reference and fertility guidance in a clinical context.

Expression pattern of ATG4C and its effect on early embryonic development of porcine oocytes

Autophagy is essential for oocyte maturation and preimplantation embryo development. ATG4C, a member of the ATG4 family, plays a crucial role in the autophagy process. The effect of ATG4C on the early embryonic development in pig has not been studied. In this study, the expression patterns of ATG4C were explored using qRT-PCR and immunofluorescence staining. Different concentrations of serum were added to in vitro maturation (IVM) medium to investigate its effects on oocyte maturation and embryonic development. Finally, the developmental potential of parthenogenetic embryos was detected by downregulating ATG4C in MII stage oocytes under 0 % serum condition. The results revealed that ATG4C was highly expressed in porcine oocytes matured in vitro and in parthenogenetic embryos. Compared with the 10 % serum group, the cumulus cell expansion, first polar body (PB1) extrusion rate, and subsequent developmental competence of embryos were reduced in the 0 % and 5 % serum groups. The mRNA levels of LC3, ATG5, BECLIN1, TFAM, PGC1α, and PINK1 were significantly increased (P < 0.05) in the 0 % serum group. ATG4C was significantly upregulated in the embryos at the 1-cell, 2-cell, 8-cell, and 16-cell stages in the 0 % serum group (P < 0.05). Compared with the negative control group, downregulation of ATG4C significantly decreased the 4-cell, 8-cell, and blastocyst rates (P < 0.05), and the expression of genes related to autophagy, mitochondria, and zygotic genome activation (ZGA) was significantly decreased (P < 0.05). The relative fluorescence intensity of LC3 and mitochondrial content in the ATG4C siRNA group was significantly reduced (P < 0.05). Collectively, the results indicate that ATG4C is highly expressed in porcine oocytes matured in vitro and in early embryos, and inhibition of ATG4C effects embryonic developmental competence by decreasing autophagy, mitochondrial content, and ZGA under serum-free condition.

Ursolic acid alleviates meiotic abnormalities induced by 3-nitropropionic acid in mouse oocytes

3-nitropropionic acid (3-NPA), a toxic metabolite produced by mold, is mainly found in moldy sugarcane. 3-NPA inhibits the activity of succinate dehydrogenase that can induce oxidative stress injury in cells, reduce ATP production and induce oxidative stress in mouse ovaries to cause reproductive disorders. Ursolic acid (UA) has a variety of biological activities and is a pentacyclic triterpene compound found in many plants. This experiment aimed to investigate the cytotoxicity of 3-NPA during mouse oocyte in vitro maturation and the protective effects of UA on oocytes challenged with 3-NPA. The results showed that UA could alleviate 3-NPA-induced oocyte meiotic maturation failure. Specifically, 3-NPA induced a decrease in the first polar body extrusion rate of oocytes, abnormal distribution of cortical granules, and an increase in the proportion of spindle abnormalities. In addition, 3-NPA caused mitochondrial dysfunction and induced oxidative stress, including decreases in the GSH, mitochondrial membrane potential and ATP levels, and increases in the ROS levels, and these effects led to apoptosis and autophagy. The addition of UA could significantly improve the adverse effects caused by 3-NPA. In general, our data show that 3-NPA affects the normal development of oocytes during the in vitro culture, and the addition of UA can effectively repair the damage caused by 3-NPA to oocytes.

Fibronectin 1 supports oocyte in vitro maturation in pigs

Oocyte in vitro maturation (IVM) based on the follicular fluid (FF) environment can exploit untapped resources, however, what FF factors regulate oocyte maturation remains unclear. This work demonstrated that serum and FF significantly promoted oocyte polar body extrusion (PBE) and subsequent embryo development, and FF was especially effective. Fibronectin 1 (FN1) was predicted as one potential candidate to regulate oocyte maturation by proteomics. FN1 transcription obviously decreased, and the protein expression significantly increased and migrated to plasma membrane or even outside during oocyte IVM. Treatment with 10 ng/mL FN1 significantly improved oocyte PBE rate. FN1 significantly upregulated the percentage of regular spindle morphology, downregulated the γ-H2AX level, decreased the levels of ROS and apoptosis, and increased GSH and mitochondrion contents by ameliorating the expression of corresponding genes. Moreover, FN1 significantly increased the p-PI3K level to enhance the activation of PI3K signaling pathway. In conclusion, this study discovers and confirms that FN1 is one factor in FF that significantly enhances oocyte maturation, and the underlying mechanism is that FN1 ameliorates oocyte nuclear and cytoplasmic maturation by promoting the activation of PI3K signaling pathway.

Gonadotropin supplementation improved in vitro developmental capacity of Egyptian goat oocytes by modulating mitochondrial distribution and utilization

Goat production in Mediterranean countries is economically and socially significant. The study investigated the impact of hCG supplementation on the in vitro maturation, fertilization, and mitochondrial activity of goat oocytes. In total, 2356 good cumulus-oocyte complexes (COCs) were recovered from 476 freshly collected goat ovaries. Selected oocytes were incubated in IVM medium and supplemented with hCG at varying concentrations depending on the experimental group for 24 hours at 38.5 ° C, 5% CO2 and 95% humidity. The first group (G1) was allocated as a control group, while the G2 and G3 groups were supplemented with 10 and 20 IU/mL hCG, respectively. The maturation rate was calculated using Hoechst 33342 staining in addition to cumulus expansion and the first polar body extrusion rates. The fertilization rate was evaluated. Moreover, mitochondrial activity was assessed using Mito-Tracker fluorescent staining. The results indicated a higher rate (P≤0.05) of nuclear maturation in G3 (82.5%) than in G2 (65.9%) and the control group (64.8%). Furthermore, fertilization rate was significantly improved in G3 (18.7%) compared to G2 (10.6%) and the control group (9.5%). Notably, the percentage of diffuse patterns of mitochondrial distribution increased in G3 (73.3%) compared to G2 (13.3%) and the control group (6.7%). In the same trend, the fluorescent mitochondrial intensity was higher in G3 (95%) and G2 (89.2%) compared to the control group (79.9%). In conclusion, supplementing the IVM medium with 20 IU/mL of hCG improved goat oocytes' maturation and fertilization rates by modulating cytoplasmic distribution and promoting the utilization of oocyte mitochondria.

PAK1-Dependent Regulation of Microtubule Organization and Spindle Migration Is Essential for the Metaphase I-Metaphase II Transition in Porcine Oocytes.

P21-activated kinase 1 (PAK1) is a critical downstream target that mediates the effect of small Rho GTPase on the regulation of cytoskeletal kinetics, cell proliferation, and cell migration. PAK1 has been identified as a crucial regulator of spindle assembly during the first meiotic division; however, its roles during the metaphase I (MI) to metaphase II (MII) transition in oocytes remain unclear. In the present study, the potential function of PAK1 in regulating microtubule organization and spindle positioning during the MI-MII transition was addressed in porcine oocytes. The results showed that activated PAK1 was co-localized with α-tubulin, and its expression was increased from the MI to MII stage (p < 0.001). However, inhibiting PAK1 activity with an inhibitor targeting PAK1 activation-3 (IPA-3) at the MI stage decreased the first polar body (PB1) extrusion rate (p < 0.05), with most oocytes arrested at the anaphase-telophase (ATI) stage. IPA-3-treated oocytes displayed a decrease in actin distribution in the plasma membrane (p < 0.001) and an increase in the rate of defects in MII spindle reassembly with abnormal spindle positioning (p < 0.001). Nevertheless, these adverse effects of IPA-3 on oocytes were reversed when the disulfide bond between PAK1 and IPA-3 was reduced by dithiothreitol (DTT). Co-immunoprecipitation revealed that PAK1 could recruit activated Aurora A and transform acidic coiled-coil 3 (TACC3) to regulate spindle assembly and interact with LIM kinase 1 (LIMK1) to facilitate actin filament-mediated spindle migration. Together, PAK1 is essential for microtubule organization and spindle migration during the MI-MII transition in porcine oocytes, which is associated with the activity of p-Aurora A, p-TACC3 and p-LIMK1.

Mogroside V alleviates the heat stress-induced disruption of the porcine oocyte in vitro maturation

Heat stress (HS) is a stressor that negatively affect female reproduction. Specially, oocytes are very sensitive to HS. It has been demonstrated that some active compounds can protect oocyte from HS. We previously found that Mogroside V (MV), extracted from Siraitia grosvenorii (Luo Han Guo), can protect oocyte from many kinds of stresses. However, how MV alleviates HS-induced disruption of oocyte maturation remains unknown. In this study, we treated the HS-induced porcine oocytes with MV to examine their maturation and quality. Our findings demonstrate that MV can effectively alleviate HS-induced porcine oocyte abnormal cumulus cell expansion, decrease of first polar body extrusion rate, spindle assembly and chromosome separation abnormalities, indicating MV attenuates oocyte mature defects. We further observed that MV can effectively alleviate HS-induced cortical granule distribution abnormality and decrease of blastocyst formation rate after parthenogenesis activation. In addition, MV treatment reversed mitochondrial dysfunction and lipid droplet content decrease, reduced reactive oxygen species levels, early apoptosis and DNA damage in porcine oocytes after HS. Collectively, this study suggests that MV can effectively protect porcine oocytes from HS.

High Temperature-Induced m6A Epigenetic Changes Affect Early Porcine Embryonic Developmental Competence in Pigs.

N6-methyladenosine (m6A), the most prevalent modification in eukaryotic messenger RNA (mRNA), plays a key role in various developmental processes in mammals. Three proteins that affect RNA m6A modification have been identified: methyltransferases, demethylases, and m6A-binding proteins, known as "writer," "eraser," and "reader" proteins, respectively. However, changes in the m6A modification when early porcine embryos are exposed to stress remain unclear. In this study, we exposed porcine oocytes to a high temperature (HT, 41°C) for 10 h, after which the mature oocytes were parthenogenetically activated and cultured for 7 days to the blastocyst stage. HT significantly decreased the rates of the first polar body extrusion and blastocyst formation. Further detection of m6A modification found that HT can lead to increased expression levels of "reader," YTHDF2, and "writer," METTL3, and decreased expression levels of "eraser," FTO, resulting in an increased level of m6A modification in the embryos. Additionally, heat shock protein 70 (HSP70) is upregulated under HT conditions. Our study demonstrated that HT exposure alters m6A modification levels, which further affects early porcine embryonic development.

Epigallocatechin-3-gallate improves the quality of maternally aged oocytes.

The decline in female fertility as age advances is intricately linked to the diminished developmental potential of oocytes. Despite this challenge, the strategies available to enhance the quality of aged oocytes remain limited. Epigallocatechin-3-gallate (EGCG), characterised by its anti-inflammatory, antioxidant and tissue protective properties, holds promise as a candidate for improving the quality of maternally aged oocytes. In this study, we explored the precise impact and underlying mechanisms of EGCG on aged oocytes. EGCG exhibited the capacity to enhance the quality of aged oocytes both in vitro and in vivo. Specifically, the application of EGCG in vitro resulted in noteworthy improvements, including an increased rate of first polar body extrusion, enhanced mitochondrial function, refined spindle morphology and a reduction in oxidative stress. These beneficial effects were further validated by the improved fertility observed among aged mice. In addition, our findings propose that EGCG might augment the expression of Arf6. This augmentation, in turn, contributes to the assembly of spindle-associated F-actin, which can contribute to mitigate the aneuploidy induced by the disruption of spindle F-actin within aged oocytes. This work thus contributes not only to understanding the role of EGCG in bolstering oocyte health, but also underscores its potential as a therapeutic intervention to address fertility challenges associated with advanced age.

Nuclear-cytoplasmic asynchrony in oocyte maturation caused by TUBB8 variants via impairing microtubule function: a novel pathogenic mechanism

BackgroundTUBB8, a crucial gene encoding microtubule protein, plays a pivotal role in cellular processes. Deleterious TUBB8 variants have been shown to significantly hinder oocyte maturation. In this study, we conducted an in vitro investigation using TUBB8 mutant mouse oocytes to elucidate the pathogenic mechanisms of TUBB8 variants in oocyte nuclear and cytoplasmic maturation.MethodsA mutant model was successfully established in mouse oocytes via microinjection to further investigate the effects of four novel discovered TUBB8 mutations on the nuclear and cytoplasmic maturation of mouse oocytes. Immunofluorescence and confocal microscopy were performed to observe the cortical polarity and spindle and of mutant oocytes. Active mitochondrial staining was performed to analyze mitochondrial distribution patterns. Endoplasmic reticulum and Ca2+ staining were conducted to assess ER distribution and cytoplasmic calcium ion concentration in oocytes.ResultsIn mouse oocytes, TUBB8 variants (p.A313V, p.C239W, p.R251Q, and p.G96R) resulted in a reduction of the first polar body extrusion rate, disruption of spindle assembly, and abnormal chromosome distribution. Additionally, these variants induced oocyte organelle abnormalities, including anomalies in mitochondrial redistribution and endoplasmic reticulum stress compared to the wild-type.ConclusionDeleterious TUBB8 variants could disrupt microtubule function, affecting critical processes such as spindle assembly, chromosome distribution, and organelle rearrangement during oocyte meiosis. These disruptions culminate in compromised nuclear-cytoplasmic maturation, consequently giving rise to oocyte maturation defects.

Three-dimensional glass scaffolds improve the In Vitro maturation of porcine cumulus-oocyte complexes and subsequent embryonic development after parthenogenetic activation

In vitro maturation (IVM) methods for porcine oocytes are still deficient in achieving full developmental capacity, as the currently available oocyte in vitro culture systems still have limitations. In vitro embryo production must also improve the porcine oocyte IVM system to acquire oocytes with good developmental potential. Herein, we tested a three-dimensional (3D) glass scaffold culture system for porcine oocyte maturation. After 42 h, we matured porcine cumulus-oocyte complexes (COCs) on either two-dimensional glass dishes (2D-B), two-dimensional microdrops (2D-W), or 3D glass scaffolds. The 3D glass scaffolds were tested for porcine oocyte maturation and embryonic development. Among these culture methods, the extended morphology of the 3D group maintained a 3D structure better than the 2D-B and 2D-W groups, which had flat COCs that grew close to the bottom of the culture vessel. The COCs of the 3D group had a higher cumulus expansion index and higher first polar body extrusion rate, cleavage rate, and blastocyst rate of parthenogenetic embryos than the 2D-B group. In the 3D group, the cumulus-expansion-related gene HAS2 and anti-apoptotic gene Bcl-2 were significantly upregulated (p < 0.05), while the pro-apoptotic gene Caspase3 was significantly downregulated (p < 0.05). The blastocysts of the 3D group had a higher relative expression of Bcl-2, Oct4, and Nanog than the other two groups (p < 0.05). The 3D group also had a more uniform distribution of mitochondrial membrane potential and mitochondria (p < 0.05), and its cytoplasmic active oxygen species content was much lower than that in the 2D-B group (p < 0.05). These results show that 3D glass scaffolds dramatically increased porcine oocyte maturation and embryonic development after parthenogenetic activation, providing a suitable culture model for porcine oocytes.

Decreased HAT1 expression in granulosa cells disturbs oocyte meiosis during mouse ovarian aging

BackgroundWith advanced maternal age, abnormalities during oocyte meiosis increase significantly. Aneuploidy is an important reason for the reduction in the quality of aged oocytes. However, the molecular mechanism of aneuploidy in aged oocytes is far from understood. Histone acetyltransferase 1 (HAT1) has been reported to be essential for mammalian development and genome stability, and involved in multiple organ aging. Whether HAT1 is involved in ovarian aging and the detailed mechanisms remain to be elucidated.MethodsThe level of HAT1 in aged mice ovaries was detected by immunohistochemical and immunoblotting. To explore the function of HAT1 in the process of mouse oocyte maturation, we used Anacardic Acid (AA) and small interfering RNAs (siRNA) to culture cumulus-oocyte complexes (COCs) from ICR female mice in vitro and gathered statistics of germinal vesicle breakdown (GVBD), the first polar body extrusion (PBE), meiotic defects, aneuploidy, 2-cell embryos formation, and blastocyst formation rate. Moreover, the human granulosa cell (GC)-like line KGN cells were used to investigate the mechanisms of HAT1 in this progress.ResultsHAT1 was highly expressed in ovarian granulosa cells (GCs) from young mice and the expression of HAT1 was significantly decreased in aged GCs. AA and siRNAs mediated inhibition of HAT1 in GCs decreased the PBE rate, and increased meiotic defects and aneuploidy in oocytes. Further studies showed that HAT1 could acetylate Forkhead box transcription factor O1 (FoxO1), leading to the translocation of FoxO1 into the nucleus. Resultantly, the translocation of acetylated FoxO1 increased the expression of amphiregulin (AREG) in GCs, which plays a significant role in oocyte meiosis.ConclusionThe present study suggests that decreased expression of HAT1 in GCs is a potential reason corresponding to oocyte age-related meiotic defects and provides a potential therapeutic target for clinical intervention to reduce aneuploid oocytes.

Specc1l deficiency leads to abnormal oocyte meiosis and reduced blastocyst development in mouse.

This study aimed to investigate the potential role of the mouse Specc1l (Sperm antigen with calponin homology and coiled-coil domains 1 like), a microtubule and actin cytoskeleton-associated protein during oocyte meiotic maturation and its potential effects on preimplantation development. This study shows that the transcriptional levels of Specc1l did not significantly change from the germinal vesicle (GV) stage to the metaphase II (MII) stage, but maternal transcripts rapidly and gradually degraded after fertilization. Specc1l was detected in both the cytoplasm and GV, but not in the nucleolus-like body in the GV intact oocyte. At the MII stages, Specc1l was widely distributed in the cytoplasm but did not co-localize with chromatin. Knockdown of Specc1l expression in oocytes resulted in abnormal spindle morphology and misaligned chromosomes, as well as a decrease in the rate of polar body extrusion and a reduced developmental competence of oocytes, leading to decreased blastocyst formation rate. In conclusion, this study provides evidence that Specc1l plays a critical role in mouse oocyte meiotic maturation and early embryo development, specifically in proper bipolar spindle assembly and extrusion of the first polar body.

Epifriedelanol delays the aging of porcine oocytes matured invitro

Oocyte aging directly affects the subsequent embryonic development. Epifriedelanol is the active ingredient of Aster tataricus L.F. extract, and it possesses potential anti-cancer, anti-inflammatory and antioxidant properties. In addition, epifriedelanol can slow the aging of human skin fibroblasts. To explore the effect of epifriedelanol on the aging of porcine oocytes matured in vitro, the aging model was first established, epifriedelanol was added to in vitro maturation (IVM) medium to investigate its anti-aging effects by observing oocyte maturation and embryonic development potential, and analyzing aging-related gene expression, reactive oxygen species and mitochondrial membrane potential levels. It was found that typical aging of porcine oocytes appeared from 66 h during in vitro maturation. Compared with the 44 h group, a larger perivitelline space, increased abnormality of microtubulin formation, and significantly lower blastocyst rate were observed in the 66 h and 72 h groups. Compared with the 0 μg/mL group, the first polar body extrusion, cleavage and blastocyst rates were significantly improved (P < 0.05) in 10 μg/mL group. The expression of oocyte developmental potential-related, SIRT family-related, antioxidant and anti-apoptotic-related genes was significantly up-regulated (P < 0.05), p53 and pro-apoptotic genes were significantly down-regulated (P < 0.05). In addition, the reactive oxygen species level was significantly decreased (P < 0.01), the mitochondrial membrane potential was significantly elevated (P < 0.01) in 10 μg/mL group. In conclusion, epifriedelanol delays the aging of porcine oocytes cultured in vitro by up-regulating SIRT family gene expression, enhancing the antioxidant and anti-apoptotic capacity of oocytes.

Role of FBXL5 in redox homeostasis and spindle assembly during oocyte maturation in mice.

As an E3 ubiquitin ligase, F-box and leucine-rich repeat protein 5 (FBXL5) participates in diverse biologic processes. However, the role of Fbxl5 in mouse oocyte meiotic maturation has not yet been fully elucidated. The present study revealed that mouse oocytes depleted of Fbxl5 were unable to complete meiosis, as Fbxl5 silencing led to oocyte meiotic failure with reduced rates of GVBD and polar body extrusion. In addition, Fbxl5 depletion induced aberrant mitochondrial dynamics as we noted the overproduction of reactive oxygen species (ROS) and the accumulation of phosphorylated γH2AX with Fbxl5 knockdown. We also found that Fbxl5-KD led to the abnormal accumulation of CITED2 proteins in mouse oocytes. Our in vitro ubiquitination assay showed that FBXL5 interacted with CITED2 and that it mediated the degradation of CITED2 protein through the ubiquitination-proteasome pathway. Collectively, our data revealed critical functions of FBXL5 in redox hemostasis and spindle assembly during mouse oocyte maturation.

Promising anti-ovarian aging herbal formulation He's Yangchao promotes in vitro maturation of oocytes from advanced maternal age mice

Ethnopharmacological relevanceMarveled at the discovery of artemisinin, the world's expectations for traditional Chinese medicine are rising. He's Yangchao formula (HSYC) is a traditional Chinese herbal formula with the effects of tonifying kidney and essence, and reconciling yin and yang. It has been clinically proven to have anti-ovarian aging effects. Age is the primary cause of diminished ovarian reserve and assisted reproductive failure in women, whether HSYC has the potential to improve in vitro maturation of oocytes from advanced maternal age (AMA) mice has yet to be determined. Aim of the studyThis study aims to evaluate the efficacy and possible mechanism of HSYC in promoting in vitro maturation of oocytes from AMA mice. Materials and methodsThe GV oocytes were obtained from young and aged mice. The GV oocytes from young mice were cultured in drops of M16 medium, and the GV oocytes from AMA mice were randomly divided four groups: Vehicle group (cultured in 90% M16 medium +10% blank serum), Low HSYC group (cultured in 90% M16 medium + 10% Low HSYC-medicated serum), High-HSYC group (cultured in 90% M16 medium +10% High HSYC-medicated serum), and Quercetin group (cultured in M16 medium supplemented with 10 μM quercetin). The rates of first polar body extrusion, reactive oxygen species (ROS), intracellular calcium, and mitochondrial membrane potential levels in each groups were observed. In addition, expression levels of mitochondrial function, autophagy, DNA damage, and antioxidant-related proteins were assessed. ResultsSupplementation of HSYC in vitro alleviated age-associated meiotic progression defects in maternally aged oocytes. Importantly, HSYC supplementation eliminated the age-related ROS accumulation to suppress DNA damage and autophagy during the in vitro maturation of maternally aged oocytes. Meanwhile, the mitochondrial function was improved after HSYC treatment, as manifested by higher mitochondrial membrane potential and lower Ca2+ levels. Furthermore, we found that HSYC supplementation during in vitro maturation of maternally aged oocytes upregulated the expression level of SIRT3, a crucial protein in regulating mitochondrial function. Consistently, the expression levels of the SOD2, PCG1α, and TFAM were increased, while the SOD2 acetylation level was decreased, which further proved its antioxidant function. ConclusionsHSYC supplementation promotes in vitro maturation of oocytes from AMA mice mainly via improving mitochondrial function and alleviating oxidative stress. The mechanism may be related to the regulation of SIRT3-dependent deacetylation of the SOD2 pathway.