Oocyte Growth Research Articles

Metformin improves epigenetic modification involved in oocyte growth and embryo development in polycystic ovary syndrome mice model.

The possible relationship between dehydroepiandrosterone (DHEA)-induced polycystic ovary syndrome (PCOS) and epigenetic changes (ECs) leading to the impaired oocyte quality, has not been investigated yet. So, this study aimed to provide an insight into the relationship of the impaired oocyte quality with ECs in a mice DHEA-induced PCOS model and to further reveal the effect of metformin treatment. For this purpose, 80 female BALB/C mice were randomly divided into four equal groups, named as the control, sham, (DHEA) and DHEA + Metformin groups. The alterations in acetylation of H4K5 and H4K16, and in methylation of DNA (5MeC) and H3K9 were evaluated using immunocytochemical. Moreover, the expression of Hdac1, Hdac2, Dnmt1, and Dnmt3a genes involved in ECs were analyzed using reverse-transcription polymerase chain reaction. As well, the levels of mitochondrial membrane potential (MMP), oxidative stress (OS), embryo development, ovarian morphology, sexual hormone, ovulatory function, and AMPKα phosphorylation activity were compared in all the studied groups. Metformin attenuated the damages induced by DHEA as indicated by the normalized the estrous cycle, the improved ovarian morphology, the decreased sexual hormone and OS levels, and the increased MMP and AMPKα phosphorylation levels. In the metformin group, the Dnmt1, Dnmt3a, and Hdac2 genes have significantly upregulated compared to the DHEA group. However, metformin was found to have no effect on the expression level of Hdac1. In this regard, significant decrease and increase were observed in both the acetylated H4K16 and methylated H3K9 within MII oocytes in the DHEA + Metformin group compared with the DHEA group. Our results show that metformin could enhance the developmental competence of PCOS oocytes via reducing OS and ECs.

Metabolome and Transcriptome Analysis of Liver and Oocytes of Schizothorax o'connori Raised in Captivity.

Schizothorax o’connori (S. o’connori) is a representative tetraploid species in the subfamily Schizothoracinae and an important endemic fish in the Qinghai-Tibet Plateau. However, the domestication of S. o’connori remains challenging due to the lack of basic research. Here, we investigated the effects of artificial feeding on the oocytes and liver of S. o’connori by comparing the histological, metabolomic, and transcriptomic data. Histological results showed that the oocytes and liver of captive-reared S. o’connori had abnormal cell morphology. After comparison with the self-built database, a total of 233 metabolites were annotated. In oocytes, a total of 37 differentially accumulated metabolites (DAMs) were detected and two pathways were significantly enriched. There were obvious differences in the metabolites related to ovarian development, including pregnenolone and arachidonic acid. In liver, a total of 70 DAMs were detected and five pathways were significantly enriched. Based on the transcriptomic data, a total of 159 differentially expressed genes (DEGs) were significantly related with cell growth and death pathway in oocytes, while a total of 2841 DEGs were significantly related with 102 pathways in liver. Comparing the metabolomic and transcriptomic data showed that there were three common significant enrichment pathways in liver, including biosynthesis of unsaturated fatty acids, starch and sucrose metabolism, and fatty acid biosynthesis. These results showed that special attention should be given to the composition and intake of fatty acids during the artificial breeding of S. o’connori. In addition, many of metabolite-gene pairs were related to adenosine 5′-diphosphate, adenosine monophosphate, and pregnenolone. In summary, these data provide an overview of global metabolic and transcriptomic resources and broaden our understanding of captive-reared S. o’connori.

Association of Atorvastatin with Increased Growth and Quality of Immature Mouse Oocytes In-Vitro

Association of Atorvastatin with Increased Growth and Quality of Immature Mouse Oocytes In-Vitro

Mechanical mapping of mammalian follicle development using Brillouin microscopy

In early mammalian development, the maturation of follicles containing the immature oocytes is an important biological process as the functional oocytes provide the bulk genetic and cytoplasmic materials for successful reproduction. Despite recent work demonstrating the regulatory role of mechanical stress in oocyte growth, quantitative studies of ovarian mechanical properties remain lacking both in vivo and ex vivo. In this work, we quantify the material properties of ooplasm, follicles and connective tissues in intact mouse ovaries at distinct stages of follicle development using Brillouin microscopy, a non-invasive tool to probe mechanics in three-dimensional (3D) tissues. We find that the ovarian cortex and its interior stroma have distinct material properties associated with extracellular matrix deposition, and that intra-follicular mechanical compartments emerge during follicle maturation. Our work provides an alternative approach to study the role of mechanics in follicle morphogenesis and might pave the way for future understanding of mechanotransduction in reproductive biology, with potential implications for infertility diagnosis and treatment.

The Function of Cumulus Cells in Oocyte Growth and Maturation and in Subsequent Ovulation and Fertilization.

Cumulus cells (CCs) originating from undifferentiated granulosa cells (GCs) differentiate in mural granulosa cells (MGCs) and CCs during antrum formation in the follicle by the distribution of location. CCs are supporting cells of the oocyte that protect the oocyte from the microenvironment, which helps oocyte growth and maturation in the follicles. Bi-directional communications between an oocyte and CCs are necessary for the oocyte for the acquisition of maturation and early embryonic developmental competence following fertilization. Follicle-stimulation hormone (FSH) and luteinizing hormone (LH) surges lead to the synthesis of an extracellular matrix in CCs, and CCs undergo expansion to assist meiotic resumption of the oocyte. The function of CCs is involved in the completion of oocyte meiotic maturation and ovulation, fertilization, and subsequent early embryo development. Therefore, understanding the function of CCs during follicular development may be helpful for predicting oocyte quality and subsequent embryonic development competence, as well as pregnancy outcomes in the field of reproductive medicine and assisted reproductive technology (ART) for infertility treatment.

Characterization of the foxl2 gene involved in the vtg expression in mud crab (Scylla paramamosain).

Forkhead box protein L2 (Foxl2) is involved in multiple physiological processes, such as ovarian development, granulosa cell differentiation, ovarian follicle development, and oocyte growth. In this study, a Spfoxl2 gene encoded 530 amino acid protein with characteristic forkhead (FH) domain was identified from transcriptome data of mud crab Scylla paramamosain and validated the accuracy by PCR technology. Meanwhile, the orthologues of the Spfoxl2 gene in other 14 crustacean species were identified with the same method. Further multiple sequence alignment analysis revealed the Foxl2 was highly conserved, especially in the FH domain, even completely identical in several species. Besides, the semi-quantitative PCR (Sq-PCR) result showed Spfoxl2 gene was mainly expressed in the gonad (testis and ovary). Further quantitative real-time PCR (qRT-PCR) result demonstrated its expression level in the testis was significantly higher than that in the ovary (p<0.01). In addition, the qRT-PCR result showed that in zoea V, megalopa, and larval I, the expression level of Spfoxl2 in megalopa is the highest. In addition, a putative Foxl2 binding site was identified on the promoter region of Spvtg, and knockdown of Spfoxl2 mediated by RNAi technology increased the expression of Spvtg in the ovary, suggesting Spfoxl2 might be the upstream negative regulator of Spvtg. Overall, this study provided new insights into the role of Spfoxl2 in ovary development through regulating Spvtg expression in S. paramamosain.

Probiotic Administration Mitigates Bisphenol A Reproductive Toxicity in Zebrafish.

Although the use of bisphenol A (BPA) has been banned in a number of countries, its presence in the environment still creates health issues both for humans and wildlife. So far, BPA toxicity has been largely investigated on different biological processes, from reproduction to development, immune system, and metabolism. In zebrafish, Danio rerio, previous studies revealed the ability of environmentally relevant concentrations of this contaminant to significantly impair fertility via epigenetic modification. In addition, several studies demonstrated the ability of different probiotic strains to improve organism health. This study provides information on the role of the probiotic mixture SLAb51 to counteract adverse BPA effects on reproduction. A 28-day trial was set up with different experimental groups: BPA, exposed to 10 µg/L BPA; P, receiving a dietary supplementation of SLAb51 at a final concentration of 109 CFU/g; BPA+P exposed to 10 µg/L BPA and receiving SLAb51 at a final concentration of 109 CFU/g and a C group. Since oocyte growth and maturation represent key aspects for fertility in females, studies were performed on isolated class III (vitellogenic) and IV (in maturation) follicles and liver, with emphasis on the modulation of the different vitellogenin isoforms. In males, key signals regulating spermatogenesis were investigated. Results demonstrated that in fish exposed to the combination of BPA and probiotic, most of the transcripts were closer to C or P levels, supporting the hypothesis of SLAb51 to antagonize BPA toxicity. This study represents the first evidence related to the use of SLAb51 to improve reproduction and open new fields of investigation regarding its use to reduce endocrine disrupting compound impacts on health.

Comprehensive CircRNA Profiling and Selection of Key CircRNAs Reveal the Potential Regulatory Roles of CircRNAs throughout Ovarian Development and Maturation in Cynoglossus semilaevis.

Simple SummaryCircRNAs: as molecules involved in gene regulation, have become a new research hotspot in the non-coding RNA field. CircRNAs show tissue- or developmental stage-specific patterns of expression and can influence the expression levels of their parental genes. Recent studies have documented the potential biological roles of circRNAs in the growth, development, reproduction and health of humans and animals. Tongue sole (Cynoglossus semilaevis) is a marine flatfish that is an economically important farmed species in China. The commercial aquaculture of tongue sole has developed in the last few years because wild resources have decreased. Reproduction is regulated by brain-pituitary-gonad-liver axis which limits the development of artificial tongue sole culture. However, the roles of circRNAs in the ovarian development and maturation of tongue sole has never been reported. The identification of the potential functions of circRNAs provides a foundation for understanding the genetic mechanisms that regulate oocyte growth and maturation, which will allow the efficiency of tongue sole reproduction to be improved. Moreover, our findings extend the knowledge about a new type of endogenous RNA involved in regulating the ovarian development and maturation of tongue sole.CircRNAs are novel endogenous non-coding small RNAs involved in the regulation of multiple biological processes. However, little is known regarding circRNAs in ovarian development and maturation of fish. Our study, for the first time, provides the genome-wide overview of the types and relative abundances of circRNAs in tongue sole tissues during three ovarian developmental stages. We detected 6790 circRNAs in the brain, 5712 in the pituitary gland, 4937 in the ovary and 4160 in the liver. Some circRNAs exhibit tissue-specific expression, and qRT-PCR largely confirmed 6 differentially expressed (DE) circRNAs. Gene Ontology and KEGG pathway analyses of DE mRNAs were performed. Some DE circRNA parental genes were closely associated with biological processes in key signalling pathways and may play essential roles in ovarian development and maturation. We found that the selected circRNAs were involved in 10 pathways. RNase R digestion experiment and Sanger sequencing verified that the circRNA had a ring structure and was RNase R resistant. qRT-PCR results largely confirmed differential circRNA expression patterns from the RNA-seq data. These findings indicate that circRNAs are widespread in terms of present in production-related tissues of tongue sole with potentially important regulatory roles in ovarian development and maturation.

What defines the maternal transcriptome?

In somatic cells, RNA polymerase II (Pol II) transcription initiation starts by the binding of the general transcription factor TFIID, containing the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs), to core promoters. However, in growing oocytes active Pol II transcription is TFIID/TBP-independent, as during oocyte growth TBP is replaced by its vertebrate-specific paralog TBPL2. TBPL2 does not interact with TAFs, but stably associates with TFIIA. The maternal transcriptome is the population of mRNAs produced and stored in the cytoplasm of growing oocytes. After fertilization, maternal mRNAs are inherited by the zygote from the oocyte. As transcription becomes silent after oocyte growth, these mRNAs are the sole source for active protein translation. They will participate to complete the protein pool required for oocyte terminal differentiation, fertilization and initiation of early development, until reactivation of transcription in the embryo, called zygotic genome activation (ZGA). All these events are controlled by an important reshaping of the maternal transcriptome. This procedure combines cytoplasmic readenylation of stored transcripts, allowing their translation, and different waves of mRNA degradation by deadenylation coupled to decapping, to eliminate transcripts coding for proteins that are no longer required. The reshaping ends after ZGA with an almost total clearance of the maternal transcripts. In the past, the murine maternal transcriptome has received little attention but recent progresses have brought new insights into the regulation of maternal mRNA dynamics in the mouse. This review will address past and recent data on the mechanisms associated with maternal transcriptome dynamic in the mouse.

Relationship between chromatin configuration and in vitro maturation ability in guinea pig oocytes.

BackgroundGerminal vesicle (GV) chromatin configurations of oocytes are proposed to be related to oocyte competence and may reflect the quality of oocyte. Currently, a limited number of published studies investigated the GV chromatin configurations of guinea pig oocytes.ObjectiveIn this study on the in vitro maturation (IVM) of guinea pig oocytes, we examined the changes in their GV chromatin configurations during meiotic progression.MethodsBased on the degree of chromatin compaction, the GV chromatin configurations of guinea pig oocytes could be divided into three categories depending on whether the nucleolus‐like body (NLB) was surrounded or partly surrounded by compacted chromatin, namely the uncondensed (NSN), the intermediate type (SN‐1) and the compacted type (SN‐2).ResultsThe percentage of cells displaying the SN‐2 configuration increased with the growth of guinea pig oocytes, suggesting that this configuration presents the potential for maturation in oocytes. Oocytes derived from larger follicle exhibited increased meiotic potential. Serum starvation affected the GV chromatin configurations of guinea pig oocytes.ConclusionsCollectively, these results suggest that the SN‐2 type might be a more mature form of configuration in guinea pig oocyte, whose proportion was associated with the follicle size and susceptible to the environment (e.g. serum concentration).

The birth of a healthy child in the assisted reproductive technologies program after autologous co-culture of embryo with cumulus cells and a new CAT transfer technology. Case report

Cumulus cells are essential during oocytes growth and development, as well as during their maturation and fertilization. Research results have shown that embryo co-cultivation with autologous cumulus cells increases the frequency of blastocyst formation, and also improves the effectiveness of ART programs. Embryo transfer in such programs is recommended to be carried out using the CAT technology (Cumulus-Aided embryo Transfer), which includes embryo cultivation on a layer of cumulus cells and embryo transfer with a certain amount of diluted cumulus cells. Patient G., 38 years old, came to the department with infertility for 15 years and recurrent implantation failure in history. The patient had ART program with autologous co-cultivation of embryos with cumulus cells and a new CAT transfer technology. The patient fell pregnant and gave birth to a healthy child. Autologous cumulus cells can be a source of biologically active substances and improve embryological parameters and implantation rate in ART programs. Embryo co-cultivation with cumulus cells is especially important for patients with recurrent implantation failure. This technique can become an alternative for optimizing human embryos culturing.

P–458 A computational biology approach to improve in-vitro folliculogenesis

Abstract Study question Considering the complexity of mechanisms involved in mammalian ovarian folliculogenesis, how about improving the current in-vitro folliculogenesis (ivF) protocols to prolong individual reproductive chance? Summary answer Computational modelling approach based on network theory was used to manage complexity, improve ivF knowledge and discover new molecules to be targeted for innovating assisted-reproductive-technologies. What is known already: Over the past decades, based on the large ovarian-pool of immature-gametes availability, ivF systems were developed in several mammalian species to support oocyte growth in order to preserve human-fertility and contrast endangered species extinction. Only mouse live-births were obtained when primordial/primary follicles were cultured in-vitro, instead the oocyte differentiation is extremely slow in medium-sized mammals. Moreover, the degree of meiotic-competence is quite incomplete if compared to mice, because oocytes must proceed until late antral-follicle stage to acquire a complete developmental competence. These observations denote the importance to adopt further investigations for establishing a complete ivF protocol in translational mammal model. Study design, size, duration Two researchers expert on reproductive biology generated the Web of Science-Mammals-Made in-vitro folliculogenesis (WoS_MMivF) database including 1111 manuscripts published in peer-reviewed international papers indexed selected in Advanced Search of WoS “Core-collection” by carrying out an independent analysis. Two additional researchers verified the correctness of the records. Participants/materials, setting, methods WoS_MMivF network was built up using Cytoscape 2.6.3 software. The network was analyzed for topological parameters (closeness-centrality, betweenness-centrality and edge count) and to identify key controllers (Hub.BN). Bidimensional-kernel-density-estimation (2D KDE) identifies Hub.BN controllers; Search-Tool-for-the-Retrieval-of-Interacting-Genes/Proteins (STRING) were used to enrich the network with new proteins. Main results and the role of chance The analysis of topological parameters demonstrated that the network is scale-free according to Barabási-Albert-model with a high-degree of robustness-against-random-damage, great controllability and navigability. The network reproduces a coherent framework identifying cross-talking molecules playing a key role in the inter-follicular/intra (somatic and germinal compartment) dialogue. The network allows to organize signalling transduction events/molecules by stratifying them in three layers: input-layer recognizes molecules generating the information flux working as systemic endocrine (pituitary/chorion/enteric-related endocrine hormones) and local paracrine-factors (TGFbeta-superfamily-members and growth-factors) exerting either intrafollicular control or remote feedback on reproductive-cycle. Processing-layer presents molecules able to elaborate/amplify the endocrine/paracrine controllers of ovarian functions, including components of codified intracellular-signaling-pathways like PI3K, KIT and MAPK and second messengers cAMP and Ca2+. These cascades are necessary to promote in-vitro reproducible follicular functions and modulate steroidogenesis, representing molecular events stratified in the output-layer. STRING analysis allowed to extend the regulatory flow of information towards two major biological action contexts: metabolic-control (paracrine-factors and signal-transduction) and angiogenesis. Metabolic-control mediated by mTOR and its interactor cognates FOXO1, FOXO3/SIRT1 plays a key role for ivF, representing the energy sensors of the reproductive cells in hypothalamic-pituitary-ovarian-axis first regulating the status of follicle quiescence/activation and then fate of the structure (specialization or apoptosis). Limitations, reasons for caution - Wider implications of the findings: STRING identified mTOR as key pathway of folliculogenesis, which might act as a molecular-switch to be pharmacologically targeted for potential new in-vitro strategies modulating follicular fate. These results suggest that computational approach in biology might offer perspective in identifying unknown signals, implementing research questions and innovative protocols to face female-fertility. Trial registration number Not applicable

The reproductive system of Ceracis cornifer (Mellié) and first description of sperm structure in a minute tree-fungus beetle (Tenebrionoidea: Ciidae)

Studies on the spermatogenesis of Tenebrionidae beetles (Tenebrionoidea) have shown an unusual organization of spermatozoa, in which they are arranged antiparallelly within the testicular cysts. Despite such works, many taxa of Tenebrionoidea remain to be studied, including the minute tree-fungus beetles (Ciidae). Among the challenges in the study of the internal morphology of ciids is their small body size (about 2 mm or less), which makes dissections and comparisons extremely arduous. Here, we provide, for the first time, an anatomical and histological study of the reproductive system (female and male) and a description of sperm structure of Ceracis cornifer Mellié (Ciidae), under light and transmission electron microscopes. We pointed out the growth of a single oocyte at a time in females, aside of evidences toward a constant copulatory activity that can provide a continuous supply of sperm in their small spermatheca. In males, sperm have an antiparallel organization within the cysts, a condition observed so far only in members of Tenebrionoidea. Furthermore, we observed sperm dimorphism in C. cornifer. We described, for the first time, this characteristic in a Tenebrionoidea taxon, and discussed the possible relationship between sperm dimorphism and antiparallel organization of sperm.

Single Molecule RNA Localization and Translation in the Mammalian Oocyte and Embryo

During oocyte growth the cell accumulates RNAs to contribute to oocyte and embryo development which progresses with ceased transcription. To investigate the subcellular distribution of specific RNAs and their translation we developed a technique revealing several instances of localized translation with distinctive regulatory implications. We analyzed the localization and expression of candidate non-coding and mRNAs in the mouse oocyte and embryo. Furthermore, we established simultaneous visualization of mRNA and in situ translation events validated with polysomal occupancy. We discovered that translationally dormant and abundant mRNAs CyclinB1 and Mos are localized in the cytoplasm of the fully grown GV oocyte forming cloud-like structures with consequent abundant translation at the center of the MII oocyte. Coupling detection of the localization of specific single mRNA molecules with their translation at the subcellular context is a valuable tool to quantitatively study temporal and spatial translation of specific target mRNAs to understand molecular processes in the developing cell.

Effect of docosahexaenoic acid on in vitro growth of bovine oocytes.

PurposeThe present study investigated the effects of docosahexaenoic acid (DHA) on the growth of bovine oocytes.MethodsOocytes and granulosa cell complexes (OGCs) were collected from early antral follicles (0.4‐0.7 mm) on the surface of ovaries harvested from a slaughterhouse. The OGCs were cultured with 0, 1, and 10 μmol/L docosahexanoic acid (DHA) for 16 days.ResultsAntrum formation of the OGCs and the number of granulosa cells (GCs) surrounding the oocytes were comparable among groups, whereas supplementation of 0.1 μmol/L of DHA significantly improved oocyte growth. Oocytes grown with DHA had a higher fertilization rate, acetylation levels of H4K12, and ATP contents, as well as a lower lipid content compared with those grown without DHA. In addition, GCs surrounding OGCs grown with DHA had low lipid content compared with vehicle counterparts. Furthermore, when GCs were cultured in vitro, DHA increased ATP production, mitochondrial membrane potential, and reduced lipid content and levels of reactive oxygen species. RNA‐seq of GCs revealed that DHA increased CPT1A expression levels and affect genes associated with focal adhesion, oxidative phosphorylation, and PI3K‐AKT etcConclusionThe results suggest that DHA supplementation affects granulosa cell characteristics and supports oocyte growth in vitro.

PADI6 Regulates Trophoblast Cell Migration-Invasion Through the Hippo/YAP1 Pathway in Hydatidiform Moles.

PurposePeptidyl arginine deiminase, type VI (PADI6), a member of the subcortical maternal complex, plays an important role in oocyte growth and the development of fertilized oocytes. Human patients with PADI6 mutations can suffer from multiple reproductive deficiencies including hydatidiform moles and miscarriages. Recent studies have demonstrated that the Hippo signaling pathway plays a central role in the specification of the first cell fates and the maintenance of the human placental trophoblast epithelium. The present study aimed to verify the hypothesis that PADI6 regulates the biological functions of trophoblast cells by targeting YAP1 and to explore the mechanism by which PADI6 accomplishes this in trophoblast cells.MethodsVilli from HMs and human trophoblast cell lines were used to identify the localization of PADI6 and YAP1 by immunohistochemistry and immunocytochemistry. PADI6 overexpression and knockdown were induced in human trophoblast cells. Co-immunoprecipitation was used to explore the interaction between PADI6 and YAP1. Wound healing, Transwell and EdU staining assays were used to detect migration, invasion and proliferation. Flow cytometric analysis was used to analyze the cell cycle and apoptosis. β-Tubulin and F-actin levels were determined by Western blot, quantitative real-time PCR and phalloidin staining.ResultsThe results showed that PADI6 and YAP1 had the same expression pattern in villi and colocalized in the cytotrophoblast. An interaction between PADI6 and YAP1 was also confirmed in human trophoblast cell lines. We found that PADI6 positively regulated the expression of YAP1. Functionally, overexpression of PADI6 promoted cell cycle progression and enhanced migration, invasion, proliferation and apoptosis, whereas downregulation of PADI6 showed the opposite effects.ConclusionThis study demonstrates that YAP1 is a novel target of PADI6 that serves as an important regulator of trophoblast dysfunction. The crosstalk between the Hippo/YAP1 pathway and the SCMC might be a new topic to explore to uncover the pathological mechanisms of HMs.

Oxygen concentration affects de novo DNA methylation and transcription in in vitro cultured oocytes

BackgroundReproductive biology methods rely on in vitro follicle cultures from mature follicles obtained by hormonal stimulation for generating metaphase II oocytes to be fertilised and developed into a healthy embryo. Such techniques are used routinely in both rodent and human species. DNA methylation is a dynamic process that plays a role in epigenetic regulation of gametogenesis and development. In mammalian oocytes, DNA methylation establishment regulates gene expression in the embryos. This regulation is particularly important for a class of genes, imprinted genes, whose expression patterns are crucial for the next generation. The aim of this work was to establish an in vitro culture system for immature mouse oocytes that will allow manipulation of specific factors for a deeper analysis of regulatory mechanisms for establishing transcription regulation-associated methylation patterns.ResultsAn in vitro culture system was developed from immature mouse oocytes that were grown to germinal vesicles (GV) under two different conditions: normoxia (20% oxygen, 20% O2) and hypoxia (5% oxygen, 5% O2). The cultured oocytes were sorted based on their sizes. Reduced representative bisulphite sequencing (RRBS) and RNA-seq libraries were generated from cultured and compared to in vivo-grown oocytes. In the in vitro cultured oocytes, global and CpG-island (CGI) methylation increased gradually along with oocyte growth, and methylation of the imprinted genes was similar to in vivo-grown oocytes. Transcriptomes of the oocytes grown in normoxia revealed chromatin reorganisation and enriched expression of female reproductive genes, whereas in the 5% O2 condition, transcripts were biased towards cellular stress responses. To further confirm the results, we developed a functional assay based on our model for characterising oocyte methylation using drugs that reduce methylation and transcription. When histone methylation and transcription processes were reduced, DNA methylation at CGIs from gene bodies of grown oocytes presented a lower methylation profile.ConclusionsOur observations reveal changes in DNA methylation and transcripts between oocytes cultured in vitro with different oxygen concentrations and in vivo-grown murine oocytes. Oocytes grown under 20% O2 had a higher correlation with in vivo oocytes for DNA methylation and transcription demonstrating that higher oxygen concentration is beneficial for the oocyte maturation in ex vivo culture condition. Our results shed light on epigenetic mechanisms for the development of oocytes from an immature to GV oocyte in an in vitro culture model.

Immature Follicular Origins and Disrupted Oocyte Growth Pathways Contribute to Decreased Gamete Quality During Reproductive Juvenescence in Mice.

Egg quality dictates fertility outcomes, and although there is a well-documented decline with advanced reproductive age, how it changes during puberty is less understood. Such knowledge is critical, since advances in Assisted Reproductive Technologies are enabling pre- and peri-pubertal patients to preserve fertility in the medical setting. Therefore, we investigated egg quality parameters in a mouse model of the pubertal transition or juvenescence (postnatal day; PND 11–40). Animal weight, vaginal opening, serum inhibin B levels, oocyte yield, oocyte diameter, and zona pellucida thickness increased with age. After PND 15, there was an age-associated ability of oocytes to resume meiosis and reach metaphase of meiosis II (MII) following in vitro maturation (IVM). However, eggs from the younger cohort (PND 16–20) had significantly more chromosome configuration abnormalities relative to the older cohorts and many were at telophase I instead of MII, indicative of a cell cycle delay. Oocytes from the youngest mouse cohorts originated from the smallest antral follicles with the fewest cumulus layers per oocyte, suggesting a more developmentally immature state. RNA Seq analysis of oocytes from mice at distinct ages revealed that the genes involved in cellular growth signaling pathways (PI3K, mTOR, and Hippo) were consistently repressed with meiotic competence, whereas genes involved in cellular communication were upregulated in oocytes with age. Taken together, these data demonstrate that gametes harvested during the pubertal transition have low meiotic maturation potential and derive from immature follicular origins.

Small body size of pseudoscorpions and a distinct architecture of the ovary: A step to miniaturization?

Chelicerata, the second largest subphylum of Arthropoda, includes invertebrates with a wide range of body size. Pseudoscorpions are among small or miniature chelicerates which exhibit several morphological, anatomical, and developmental features related to miniaturization, e.g., replacement of book lungs by tracheae, unpaired gonads, and matrotrophic development of the embryos outside the female body, in the brood sac. In this paper, we show the ovary structure of two pseudoscorpion species, Cheiridium museorum and Apocheiridium ferum (Cheiridiidae). Both cheiridiids are one of the smallest pseudoscorpions. The results of our observations conducted in light, transmission electron, and confocal microscopy demonstrate that the ovary of C.museorum and A.ferum, displays a significant structural difference that is unusual for chelicerates. The difference concerns the spatially restricted position of the germarium. We show that such ovary architecture results in a significantly reduced number of growing oocytes and in consequence a reduced number of deposited eggs. A centrally located germarium implies also a modified pattern of ovary development during oocyte growth due to long distance migration of the germline and the accompanying somatic cells. Herein, we postulate that such an ovary structure is related to the pseudoscorpion's small body size and it is a step towards miniaturization in the smaller pseudoscorpions species.

Cyclophosphamide Exposure Causes Long-Term Detrimental Effect of Oocytes Developmental Competence Through Affecting the Epigenetic Modification and Maternal Factors' Transcription During Oocyte Growth.

Cyclophosphamide (CTX) is widely used in various cancer therapies and in immunosuppression, and patients can still have babies after CTX chemotherapy. CTX directly causes primordial follicle loss with overactivation and DNA damage-induced apoptosis. Previous studies have shown that maternal exposure to CTX before conception increases the incidence of birth abnormalities and alters the methylation of genes in the oocytes of offspring. Mice were treated with a single dose of CTX (100 mg/kg) at post-natal day 21 and sacrificed 47 days later when primordial follicles surviving chemotherapy developed to the antral stage. Acute DNA damage and acceleration of the activation of primordial follicles after CTX treatment were repaired within several days, but the remaining follicle numbers remarkably decrease. Although partial surviving primordial follicle were developed to mature oocyte, oocyte quality hemostasis was impaired exhibiting aberrant meiosis progression, abnormal spindle and aneuploidy, mitochondrial dysfunction and increased endoplasmic reticulum stress. Thereafter, embryo development competency significantly decreased with fewer blastocyst formation after CTX exposure. CTX treatment resulted in alteration of DNA methylations and histone modifications in fully grown GV oocytes. Single-cell RNA-seq revealed CTX treatment suppressed multiple maternal genes’ transcription including many methyltransferases and maternal factor YAP1, which probably accounts for low quality of CTX-repaired oocyte. In vitro addition of lysophosphatidic acid (LPA) to embryo culture media to promote YAP1 nuclear localization improved CTX-repaired embryo developmental competence. This study provides evidence for the consistent toxic effect of CTX exposure during follicle development, and provide a new mechanism and new insights into future clinical interventions for fertility preservation.