Oocyte Meiotic Maturation Research Articles

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.

Mammalian oocytes store mRNAs in a mitochondria-associated membraneless compartment.

Full-grown oocytes are transcriptionally silent and must stably maintain the messenger RNAs (mRNAs) needed for oocyte meiotic maturation and early embryonic development. However, where and how mammalian oocytes store maternal mRNAs is unclear. Here, we report that mammalian oocytes accumulate mRNAs in a mitochondria-associated ribonucleoprotein domain (MARDO). MARDO assembly around mitochondria was promoted by the RNA-binding protein ZAR1 and directed by an increase in mitochondrial membrane potential during oocyte growth. MARDO foci coalesced into hydrogel-like matrices that clustered mitochondria. Maternal mRNAs stored in the MARDO were translationally repressed. Loss of ZAR1 disrupted the MARDO, dispersed mitochondria, and caused a premature loss of MARDO-localized mRNAs. Thus, a mitochondria-associated membraneless compartment controls mitochondrial distribution and regulates maternal mRNA storage, translation, and decay to ensure fertility in mammals.

Optimizing swine in vitro embryo production with growth factor and antioxidant supplementation during oocyte maturation

Porcine in vitro fertilization often results in low embryo development rates compared to other livestock species, which is often associated with either a low fertilization rate or high incidence of polyspermy. Since the quality of oocyte maturation is known to play a significant role in oocyte competence, we investigated the impact of supplementing in vitro maturation (IVM) medium containing porcine follicular fluid (pFF) with the growth factors FGF2, LIF and IGF1 (FLI), along with different combinations of cysteine, melatonin and ITS, on cumulus cell expansion, oocyte meiotic maturation, fertilization outcome, embryo development and blastocyst cell numbers. Maturation medium containing pFF yielded the greatest cumulus expansion. Compared to pFF and FLI individually, using pFF and FLI together resulted in the best embryo development rates over total oocyte placed in IVF (12.5% vs. 15.0% vs. 26.6%, respectively). Supplementation of IVM medium containing pFF and FLI with either cysteine, melatonin or insulin-transferrin-selenium, revealed that cysteine was essential to improve embryo development, while melatonin and ITS had a limited impact on improving blastocyst rates. Finally, we observed that pig oocytes matured in medium supplemented with pFF, FLI, cysteine and melatonin had a high proportion of monospermic zygotes (68.2%) and low proportion of polyspermic zygotes (15.9%) following IVF and yielded superior cleavage (78.2%) and blastocyst (32.0%) rates.

A field guide to Aurora kinase inhibitors: an oocyte perspective.

The Aurora protein kinases have critical functions in controlling oocyte meiotic maturation. In this study, we describe an assay for examining their activation state in oocytes and establish the best working doses of three commonly used inhibitors. Several small molecule inhibitors exist for targeting Aurora kinase proteins in somatic cells. From this point of view, we evaluate the specificity of these inhibitors in mouse oocytes, and we demonstrate that MLN 8237 and AZD 1152 are specific for Aurora kinase A and Aurora kinase C, respectively, only when used at low concentrations.

Transient inhibition of CDK2 activity prevents oocyte meiosis I completion and egg activation in mouse.

Mammalian oocytes are arrested at the diplotene stage of prophase I during fetal or postnatal development. It was reported that cyclin-dependent kinases(CDK1) was the sole CDK to drive the resumption of meiosis and CDK2 was dispensable for meiosis progression in mouse oocytes according to the conditional knockout studies. However, a recent study showed that CDK2 activity is essential for meiotic division and gametogenesis by means of gene-directed mutagenesis, which avoids the compensatory activation of other CDKs. Taken the compensatory effect between CDKs after gene knockout, the physiological function of CDK2 activity in oocyte maturation remains unclear. To address this issue, we applied a specific small-molecule inhibitor to restrain CDK2 activity transiently during oocyte meiotic maturation. Surprisingly, transient inhibition of CDK2 activity severely prevented the meiosis I completion although the meiotic resumption was not affected. Then we found that CDK2 activity was required for establishment of normal spindle and chromosome dynamics. Notably, CDK2 inhibition interrupted the anaphase-promoting complex/cyclosome (APC/C)-dependent degradation pathway by maintaining the activation of spindle assembly checkpoint (SAC). Interestingly, CDK2 inhibition prevented the egg activation as well. Overall, our data demonstrate that CDK2 kinase activity is required for proper dynamics of spindle and chromosomes, whose disturbance induces the continuous SAC activation and subsequent inactivation of APC/C activity in oocyte meiosis.

The Science of IVM

Oocyte in vitro maturation (IVM) makes use of oocytes from patients that have received minimal or no gonadotrophin stimulation. Whilst this brings many advantages to patients, this typically means oocytes are collected from small-medium sized (4-12 mm) antral follicles. These oocytes are under-developed, and yet removal of these from the follicle and culture in vitro leads to spontaneous oocyte meiotic maturation, without the benefit of the physiological signals that naturally induce oocyte maturation at ovulation. Using animals models, over the past decade seminal advances have been made in our understanding of the fundamental mechanisms regulating oocyte maturation in vivo. Most IVM systems, as practised clinically today, typically do not recapitulate these key cellular processes, possibly accounting for the lower efficiency of IVM compared to IVF. A key objective of modern approaches to IVM is to restore in vitro, as far as possible, the natural processes that occur during oocyte maturation in vivo. One strategy to achieve this in IVM is to: 1) prevent spontaneous meiotic resumption at oocyte collection using phosphodiesterase inhibitors, then 2) artificially maintain or elevate cumulus-oocyte complex (COC) cAMP levels, and finally to 3) induce oocyte meiotic resumption using EGF-like peptides. This typically requires the use of biphasic or 2-step IVM systems. One such biphasic IVM system is called capacitation-IVM (CAPA-IVM), as the oocyte is “capacitated” for development in vitro. CAPA-IVM uses the follicle’s natural oocyte meiotic inhibitor, c-type natriuretic peptide (CNP), in the pre-IVM phase, and amphiregulin as the oocyte meiotic inducer in the second IVM phase. Such biphasic-IVM systems typically lead to subsequent improvements in embryo yield compared to standard IVM, and indeed this is the case for CAPA-IVM. Several pilot RCTs and a large RCT of CAPA-IVM vs conventional IVF have been completed, and CAPA-IVM is now practiced in a few centres globally and is commencing in Sydney. The development and clinical application of modern IVM systems, built on decades of animal scientific research, is a bench-to-bedside success story of biomedical research. IVM is now a viable minimally invasive procedure for the treatment of infertility and for fertility preservation in female cancer patients.

Supplementation of porcine in vitro maturation medium with FGF2, LIF, and IGF1 enhances cytoplasmic maturation in prepubertal gilts oocytes and improves embryo quality

In porcine in vitro production (IVP) systems, the use of oocytes derived from prepubertal gilts, whilst being commercially attractive, remains challenging due to their poor developmental competence following in vitro maturation (IVM). Follicular fluid contains important growth factors and plays a key role during oocyte maturation; therefore, it is a common supplementation for porcine IVM medium. However, follicular fluid contains many poorly characterized components, is batch variable, and its use raises biosecurity concerns. In an effort to design a defined IVM system, growth factors such as cytokines have been previously tested. These include leukaemia inhibitory factor (LIF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1 (IGF1), the combination of which is termed 'FLI'. Here, using abattoir-derived oocytes in a well established porcine IVP system, we compared follicular fluid and FLI supplementation during both IVM and embryo culture to test the hypothesis that FLI can substitute for follicular fluid without compromising oocyte nuclear and cytoplasmic maturation. We demonstrate that in oocytes derived from prepubertal gilts, FLI supplementation enhances oocyte meiotic maturation and has a positive effect on the quality and developmental competence of embryos. Moreover, for the first time, we studied the effects of follicular fluid and FLI combined showing no synergistic effects.

Octocrylene exposure impairs mouse oocyte quality by inducing spindle defects and mitochondria dysfunction

One of organic ultraviolet (UV) filters, Octocrylene (OCL), is mainly used in various cosmetic products, which is being frequently detected in soil, sediment, aquatic systems and food chain. There is evidence confirmed the reproductive toxicity of OCL in Japanese medaka. However, less was known about the effects of OCL exposure on oocyte quality. Here, we investigated the impacts of OCL on mouse oocyte maturation and quality by exposing oocytes to OCL in vitro at concentrations of 8, 22, 30, 40 and 50 nM. The results showed that OCL markedly reduced mouse oocyte germinal vesicle breakdown (GVBD) at 50 nM and polar body extrusion (PBE) rates at 40 and 50 nM. OCL exposure further disrupted spindle assembly and chromosome alignment, finally inducing aneuploid. Mitochondrial function was also damaged by OCL exposure, leading to ROS overproduction and apoptosis in oocytes. Moreover, OCL treatment impaired the distribution of cortical granules and sperm binding ability of oocytes. In summary, these data demonstrated that OCL could disturb the oocyte meiotic maturation and reduce oocyte quality.

Ovarian transcriptome analysis of diploid and triploid rainbow trout revealed new pathways related to gonadal development and fertility

Triploidisation represents several advantages (e.g. sterility) and therefore is routinely applied in aquaculture of several commercially important fish species, including rainbow trout. The comparative transcriptomic analysis of ovaries of triploid (3N) and diploid (2N) female rainbow trout revealed a total of 9 075 differentially expressed genes (DEGs; 4 105 genes upregulated in 2N and 4 970 genes upregulated in 3N ovaries, respectively). Identified clusters for DEGs upregulated in 3N and 2N ovaries were different, including carbohydrate and lipid metabolic process and transport, protein modification, signalling (related to folliculogenesis) and response to stimulus for DEGs upregulated in 2N, and developmental process, signalling (related to apoptosis, cellular senescence and adherence junctions) and regulation of RNA metabolic process for DEGs upregulated in 3N. The enrichment of processes involved in carbohydrate and lipid metabolism in 2N ovaries indicated high metabolism of ovarian tissue and the energy reservoir generation indispensable during the earliest stages of development. Our results highlight the importance of oocyte hydration along with oestrogen, insulin, leptin, fibroblast growth factor, and Notch signalling and pathways related to the regulation of cyclic adenosine monophosphate (cAMP) levels in proper oocyte meiotic maturation prior to ovulation in 2N ovaries. Conversely, triploidisation may lead to an increase in ovarian cellular senescence and apoptosis, which in turn can result in abnormal gonadal morphology and fibrosis. The downregulation of genes responsible for the precise regulation of meiosis and proper chromosome segregation during meiosis probably affects meiotic maturation via irregular meiotic division of chromosomes. The induction of triploidy of the rainbow trout genome resulted in enhanced expression of male-specific genes, genes responsible for re-establishing the transcriptional balance after genome reorganisation and genes involved in regulatory mechanisms, including gene silencing and DNA methylation. To the best of our knowledge, this is the first genome-wide investigation providing in-depth comprehensive and comparative gene expression patterns in the ovary from 2N and 3N rainbow trout females helping in elucidating the molecular mechanisms leading to impaired gonadal development and sterility of female triploids.

Glyphosate exposure deteriorates oocyte meiotic maturation via induction of organelle dysfunctions in pigs

BackgroundRecently, defects in mammalian oocytes maturation induced by environmental pollution results in the decreasing animal reproduction. Animal exposed to glyphosate is largely unavoidable because glyphosate is one of the most widely used herbicide worldwide due to its high-efficiency and broad-spectrum effects, which causes glyphosate an environmental contaminant found in soil, water and food. During the last few years, the growing and wider use of glyphosate has raised great concerns about its effects of reproductive toxicity. In this study, using porcine models, we investigated effects of glyphosate on organelle functions during oocyte meiosis.ResultsThe results showed glyphosate exposure disrupted porcine oocyte maturation. Expression levels of cumulus expansion-related genes were interfered, further indicating the meiotic defects. The damaging effects were mediated by destruction of mitochondrial distribution and functions, which induced ROS accumulation and oxidative stress, also indicated by the decreased mRNA expression of related antioxidant enzyme genes. We also found an interference of endoplasmic reticulum (ER) distribution, disturbance of Ca2+ homeostasis, as well as fluctuation of ER stress, showing with the reduced ER stress-related mRNA or protein expression, which could indicate the dysfunction of ER for protein processing and signal transduction in glyphosate-exposed oocytes. Moreover, glyphosate exposure induced the disruption of lysosome function for autophagy, showing with the decrease of LAMP2 expression and autophagy-related genes mRNA expression. Additionally, our data showed the distribution of Golgi apparatus and the functions of ribosome were disturbed after glyphosate exposure, which might affect protein synthesis and transport.ConclusionsCollectively, our study showed that exposed to glyphosate could affect animal reproduction by compromising the quality of oocytes through its wide toxic effects on organelle functions.

P-222 Differential regulation of active translation in mouse oocytes and zygotes upon maturation in vitro and in vivo

Abstract Study question What are the differences in the translation of specific transcripts between in vitro matured versus in vivo oocytes and IVF made versus in vivo zygotes Summary answer Our RNA-seq data show a selection of actively translated trancripts up and down regulated respective to total transcript among in vitro and in vivo conditions What is known already Our project wants to address whether there is room for an improvement towards the more physiological aspects of in vitro maturation technology. As the oocyte is transcriptionally silent during meiotic maturation, we want to approach this goal by addressing changes on global translational level. There is not much known about the employment of transcripts during meiotic maturation. Some get degraded, some silent and some recruited to be translated. To the current knowledge, nobody has so far focused on describing changes in active translation on the polysomal level between in vitro and in vivo culture conditions in mouse oocytes. Study design, size, duration Study performed on a mouse model. The following samples in quadruplicates have been analyzed and compared: in vitro meiotically matured denuded MII oocytes, in vitro meiotically matured cumulus-oocyte complexes supplemented with rFSH Gonal and hCG, in vivo meiotically matured MII oocytes, in vitro meiotically matured and IVF fertilized pronuclear zygotes, in vivo matured and fertilized pronuclear zygotes. Culture conditions were optimized according to the conventional clinical IVM technology. Participants/materials, setting, methods Mouse oocytes and zygotes were input material. Collected samples were subjected to a scarce sample profiling (polysomal fractionation on a sucrose gradient, total RNA isolation and qPCR). From respective quadruplicate fractionated samples, cDNA library was prepared and NGS deep RNA-sequencing performed. Raw data were analysed by Illumina DRAGEN software and differential gene expression was performed on Qiagen CLC Genomics. Candidate validation process is undergoing at the moment. Main results and the role of chance Results show a list of differentially regulated actively translated trancripts with implications for in vitro matured oocyte's ability to become fertilized. Similar pattern regarding developmental competence can be seen between in vitro and in vivo matured zygotes. Some candidate genes are in accordance with the latest publications regarding in vitro maturation. However due to ongoing validation and unpublished work we prefer not to disclose further and more concrete results. Limitations, reasons for caution First of all, it is a mouse model which is a kind of approximation regarding human oocyte and embryo development. RNA-seq results have to be cautiously interpreted, especially regarding the interpretation and translation of findings for further validation in clinical settings. Wider implications of the findings Place findings in context with the importance of correct translational regulation during oocyte meiotic maturation and present preliminary data for further validation in clinical assisted reproduction practice. Moreover we want to show key regulatory areas affected by in vitro maturation technology and suggest improvements for particular clinical applications. Trial registration number GA19-13491S

P-705 Influence of ovulation induction strategy in oocyte meiotic maturation: hCG versus agonist

Abstract Study question What is the influence of the ovulation induction strategy on the meiotic status of collected oocytes? Summary answer Non-invasive imaging of meiotic spindles during ICSI showed that ovulation induction with a hCG improved meiotic maturation and subsequent blastocyst formation rate. What is known already Owing to the shorter half-life of GnRH, this strategy has gained attention by significantly reducing risk of ovarian hyperstimulation syndrome. Reduced exposure to LH often requires luteal support and has been associated with a lower live birth rate, a lower ongoing pregnancy rate (pregnancy beyond 12 weeks) and a higher rate of early miscarriage (less than 12 weeks) in fresh autologous cycles, nevertheless. Mid-cycle LH surge triggers molecular activity in follicle cells initiating resumption of meiosis I for competition of the first meiotic division and formation of a MII oocyte inferring competence for subsequent fertilization and further development. Study design, size, duration A retrospective observational study was carried in a cohort of 25 - > 36 years old women including 85 patients. Analysis was carried in two groups: 1: GnRHa trigger and 2: hCG trigger and later stratified for patient age. Participants/materials, setting, methods Women between 25 and > 36 years old, non PCOS, with an AMH > 1.2 ng/ml and stimulated under a short scheme GnRH-antagonist for downregulation, FSH and ovulation induction with GnRHa or hCG. Oocytes allocated to (intracytoplasmic sperm injection) ICSI treatment were cultured for 3-4 hours striped from cumulus cells 2 hours after retrieval and placed in G-MOPS PLUS in aglass bottom dish and with an Octax PolarAide system. Main results and the role of chance A spindle was present in 39.3% (218/554) of all morphologically mature (MII) oocytes alone. Group 1 (GnRHa trigger) and group 2 (hCG trigger) had 56% (309/554) and 44% (245/554) of oocytes at MII stage from which 38% (120/309) and 39% (96/245) showed spindles. A better meiotic maturation stage (scores 3, 4) at ICSI was found when ovulation induction was performed with hCG (group 2) (81% (98/120) versus 88% (85/96), p = 0,0031), nevertheless. Fertilization rate was no different between groups (60% and 58%, respectively), while significantly more blastocysts (at culture day 4 and 5) were generated from group 2 MII oocytes presenting a spindle and a good score (36% (35/98) versus 62% (53/85), p = 0.039). Good blastocyst quality (≥3BB) did not differ between groups. When stratifying for age, women between 25 – 30 presented no differences in presence or meiotic maturation (score 2, 3) in MII oocytes, while fertilization (42% versus 77%, p = 0.022) and blastocyst formation (32% versus 45%, p = 0.012) were better in group 2. Women between > 36 years old presented no differences in presence of meiotic maturation, however, fertilization (40% versus 54%, p = 0.042) and blastocyst formation (25% versus 57%, p = 0.007) were best in group 2. Limitations, reasons for caution Thorough multivariable analysis in a larger patient cohort is required to endorse these results, nevertheless. Wider implications of the findings Prediction of ovulation induction efficacy in different patient cohorts could help fine tuning cycle management. Trial registration number not applicable

An interpretable and versatile machine learning approach for oocyte phenotyping.

ABSTRACTMeiotic maturation is a crucial step of oocyte formation, allowing its potential fertilization and embryo development. Elucidating this process is important for both fundamental research and assisted reproductive technology. However, few computational tools based on non-invasive measurements are available to characterize oocyte meiotic maturation. Here, we develop a computational framework to phenotype oocytes based on images acquired in transmitted light. We trained neural networks to segment the contour of oocytes and their zona pellucida using oocytes from diverse species. We defined a comprehensive set of morphological features to describe an oocyte. These steps were implemented in an open-source Fiji plugin. We present a feature-based machine learning pipeline to recognize oocyte populations and determine morphological differences between them. We first demonstrate its potential to screen oocytes from different strains and automatically identify their morphological characteristics. Its second application is to predict and characterize the maturation potential of oocytes. We identify the texture of the zona pellucida and cytoplasmic particle size as features to assess mouse oocyte maturation potential and tested whether these features were applicable to the developmental potential of human oocytes. This article has an associated First Person interview with the first author of the paper.

Paclitaxel is evidence to reduce growing ovarian follicle growth in mice model study

We aimed to investigate the effects of paclitaxel on ovarian follicle development and oocyte meiotic competence. Early secondary follicles were cultured individually with or without paclitaxel 2.5 × 10−10, 2.5 × 10−9, and 2.5 × 10−8 M for 12 days. Thereafter, the follicles were treated with human chorionic gonadotropin (hCG) and epidermal growth factor (EGF). Follicle morphology, oocytes meiotic maturation, immunofluorescence for α-tubulin of the oocytes, mRNA expression of growth differentiation factor 9 (GDF9), B-cell lymphoma 2 (Bcl2), and Bcl2-associated X (Bax) were examined every 4, 8, and 12 days of the culture period. We demonstrated that high dose paclitaxel treatment decreased follicle survival (p ≤ 0.05), while lower dose (2.5 × 10−9 M) reduced the survival compared to control after 12 days of culture. The number of oocytes at MII stage was not significantly different between control and paclitaxel groups (p > 0.05). Paclitaxel increased GDF9 expression in oocytes after 4 days of the culture (p ≤ 0.05). Bcl2 declined significantly compared to control after 8 days (p ≤ 0.05 for all groups), while Bax expression tended to be consistent (p ≥ 0.05 for all groups). To conclude, high concentration paclitaxel reduces follicle preantral follicle growth, while in lower concentration it decreases more growing follicle growth and survival.

TPX2 deficiency leads to spindle abnormity and meiotic impairment in porcine oocytes

The targeting protein for Xklp2 (TPX2) is a spindle assembly factor, that can stimulate microtubule formation and promote spindle completion during mitosis. However, the role of TPX2 in mammalian oocyte meiotic maturation is still not fully understood. This study was conducted to address the dynamic distribution and potential roles of TPX2 in microtubule nucleation during meiotic maturation in porcine oocytes by microinjecting specific siRNAs. Western blotting results revealed that the expression of TPX2 displayed a lower level from 0 to 22 h of culture, while its expression exhibited a higher level after 28 h of culture. Immunofluorescence staining demonstrated that TPX2 was distributed along the microtubules and enriched in the poles after meiotic spindle formation at the 28 and 44 h of culture. From immunoprecipitation, TPX2 can interact with the microtubule-associated proteins aurora kinase A (AURKA) and transforming acidic coiled-coil 3 (TACC3). Meanwhile, the dynamic changes in the expression and localization of AURKA and TACC3 were highly consistent with TPX2 during meiotic maturation. After knocking down TPX2 by siRNA injection, the proportion of oocytes with aberrant spindles and scattered cytoplasmic actin filaments was significantly increased. In addition, TPX2 depletion markedly downregulated the expression of AURKA and TACC3. Thus, these results suggested that TPX2 is essential for meiotic spindle formation in the porcine oocyte, and that this regulation is related to its effect on AURKA and TACC3 expression.

Analysis of A-kinase Anchoring Protein 13 (AKAP13) in Peri-Ovulatory Oocyte Maturation [A328

INTRODUCTION: Arrest of oocytes at prophase I of meiosis is crucial for fertility and is maintained by Protein Kinase A (PKA). PKA activity is regulated by A-kinase anchoring proteins (AKAPs) that bind the regulatory subunit of PKA to control enzymatic activity. AKAP13 is expressed in oocytes and granulosa cells of humans and mice. Female mice haploinsufficient for Akap13 showed reduced litter sizes. To determine if AKAP13 in oocytes contributes to female reproductive function, we used an oocyte-specific conditional knockout (cKO) to delete Akap13 in the final stages of oocyte growth. METHODS: Oocytes were collected from mice harboring an oocyte-specific conditional knockout of Akap13 mice, with Cre recombinase expression driven by the Zp3 promoter (Zp3-Cre+, Akap13flox/flox). Akap13 DNA and mRNA were assessed by polymerase chain reaction (PCR) and quantitative PCR (qPCR), respectively. Controls were oocytes harvested from Akap13+/flox mice lacking Cre. Oocytes from control or cKO mice were examined for morphology, meiotic resumption, spindle organization, and spindle positioning at metaphase II. RESULTS: Cre-mediated excision of Akap13 DNA in oocytes was confirmed using PCR. Oocyte mRNA levels of Akap13 in cKO mice were reduced by 90% compared to controls. Live-cell imaging revealed no differences in germinal vessel breakdown, polar body emission, or completion of meiosis I in cKO oocytes as compared to controls. Staining of fixed oocytes revealed that metaphase II spindles and chromosome alignment appeared normal in in vitro-matured cKO oocytes. CONCLUSION: These studies suggest that impairment of female fertility in Akap13 haploinsufficient mice is not caused by impairment of oocyte meiotic maturation.

MicroRNA-342-3p regulates yak oocyte meiotic maturation by targeting DNA methyltransferase 1.

MicroRNAs (miRNAs) play vital roles in the development of oocytes and ovarian follicles. We have previously shown differential expression of miR-342-3p during yak oocyte maturation. In this study, we investigated the role of miR-342-3p in meiotic maturation of yak oocytes and the underlying mechanism. The profile of ovarian DNA methyltransferase 1 (DNMT1) expression was investigated in yak by RT-qPCR and western blot analyses. The pattern of Dnmt1 expression in various meiotic stages (GV stage, MI stage and MII stage) of yak oocyte maturation was then measured by immunofluorescence staining. The interaction between Dnmt1 and miR-342-3p was verified by dual-luciferase reporter assay. Finally, miR-342-3p inhibitors were microinjected into yak cumulus-oocyte complex to evaluate the effects on oocyte maturation. MiR-342-3p expression was upregulated in oocytes during meiotic maturation, with significantly higher levels in the MII stage compared with the GV- and MI stages (p<.05), whereas the opposite pattern of Dnmt1 expression was detected. In the period to sexual maturity (3-year-old), DNMT1 showed an age-related pattern of ovarian expression at both the gene and protein levels. Immunohistochemistry analysis also indicated maturation-stage-related differences in DNMT1 expression in the ovarian follicles and corpus luteum, with expression predominantly detected in cumulus cells and oocytes. MiR-342-3p inhibitors effectively upregulated Dnmt1 expression and significantly inhibited oocyte meiotic maturation. Taken together, our results indicate that miR-342-3p plays a vital role in the meiotic maturation of yak oocytes by targeting the 3'-untranslated regions (UTR) of Dnmt1 and provide a new perspective on the mechanism of this process.

Perfluorooctanoic acid alters the developmental trajectory of female germ cells and embryos in rodents and its potential mechanism

The epidemiological studies regarding perfluorooctanoic acid (PFOA) suggests that its exposure causes reproductive health issues, the underlying mechanisms of which are still in its infancy. Here, we report that PFOA deteriorates female reproduction at multiple development stages. Oocyte meiosis and preimplantation development are severely impaired by PFOA with oxidative stress being a contributor. Supplementing with antioxidant melatonin partially rescues oocyte meiotic maturation and non-apoptotic demise. The attenuation in ovarian follicle development however can be improved by metformin but not melatonin. Importantly, metformin blunts PFOA-induced fetal growth retardation (FGR) and such protective effect could be recapitulated by transplantation of fecal material and pharmacological activation of AMPK. Mechanistically, PFOA causes gut microbiota dysbiosis, which might thereby rewire host metabolism of L-phenylalanine, histamine and L-palmitoylcarnitine that triggers hyperphenylalaninaemia, inflammation and ferroptosis to initiate FGR. Deregulated serine metabolism by the gut microbe constitutes an alternative mechanism underlying PFOA-induced FGR in that modulation of serine in dam’s diet phenocopied the FGR. Our study expands the understanding of risk factors that impair human reproductive health, and proposes restoration of gut microbiota diversity and intervention of metabolism as therapeutics mitigating health risks predisposed by environmental perturbation.

Impact of obesity on medically assisted reproductive treatments.

Increasing evidence has demonstrated that obesity impairs female fertility and negatively affects human reproductive outcome following medically assisted reproduction (MAR) treatment. In the United States, 36.5% of women of reproductive age are obese. Obesity results not only in metabolic disorders including type II diabetes and cardiovascular disease, but might also be responsible for chronic inflammation and oxidative stress. Several studies have demonstrated that inflammation and reactive oxygen species (ROS) in the ovary modify steroidogenesis and might induce anovulation, as well as affecting oocyte meiotic maturation, leading to impaired oocyte quality and embryo developmental competence. Although the adverse effect of female obesity on human reproduction has been an object of debate in the past, there is growing evidence showing a link between female obesity and increased risk of infertility. However, further studies need to clarify some gaps in knowledge. We reviewed the recent evidence on the association between female obesity and infertility. In particular, we highlight the association between fat distribution and reproductive outcome, and how the inflammation and oxidative stress mechanisms might reduce ovarian function and oocyte quality. Finally, we evaluate the connection between female obesity and endometrial receptivity.

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.