Gene Expression In Oocytes Research Articles

Transcriptomic signatures of WNT-driven pathways and granulosa cell-oocyte interactions during primordial follicle activation.

Primordial follicle activation (PFA) is a pivotal event in female reproductive biology, coordinating the transition from quiescent to growing follicles. This study employed comprehensive single-cell RNA sequencing to gain insights into the detailed regulatory mechanisms governing the synchronized dormancy and activation between granulosa cells (GCs) and oocytes with the progression of the PFA process. Wntless (Wls) conditional knockout (cKO) mice served as a unique model, suppressing the transition from pre-GCs to GCs, and disrupting somatic cell-derived WNT signaling in the ovary. Our data revealed immediate transcriptomic changes in GCs post-PFA in Wls cKO mice, leading to a divergent trajectory, while oocytes exhibited modest transcriptomic alterations. Subpopulation analysis identified the molecular pathways affected by WNT signaling on GC maturation, along with specific gene signatures linked to dormant and activated oocytes. Despite minimal evidence of continuous up-regulation of dormancy-related genes in oocytes, the loss of WNT signaling in (pre-)GCs impacted gene expression in oocytes even before PFA, subsequently influencing them globally. The infertility observed in Wls cKO mice was attributed to compromised GC-oocyte molecular crosstalk and the microenvironment for oocytes. Our study highlights the pivotal role of the WNT-signaling pathway and its molecular signature, emphasizing the importance of intercellular crosstalk between (pre-)GCs and oocytes in orchestrating folliculogenesis.

The dose-dependent dual effects of alpha-ketoglutarate (AKG) on cumulus oocyte complexes during in vitro maturation

In this study, we reported for the first time the dose-dependent dual effects of Alpha-Ketoglutarate (AKG) on cumulus oocyte complexes (COCs) during in vitro maturation (IVM). AKG at appropriate concentration (30 µM) has beneficial effects on IVM. This includes improved cumulus expansion, oocyte quality, and embryo development. These effects are mediated through multiple underlying mechanisms. AKG reduced the excessive accumulation of reactive oxygen species (ROS) in cumulus cells, reduced the consumption of GSH and NADPH. Cumulus GSH and NADPH were transported to oocytes via gap junctions, thereby reducing the oxidative stress, apoptosis and maintaining the redox balance in oocytes. In addition, AKG improved the mitochondrial function by regulating the mitochondrial complex 1 related gene expression in oocytes to maintain mitochondrial membrane potential and ATP production. On the other hand, oocyte generated GDF9 could also be transported to cumulus cells to promote cumulus expansion. Conversely, a high concentration of AKG (750 µM) exerted adverse effects on IVM and suppressed the cumulus expansion as well as reduced the oocyte quality. The suppression of the cumulus expansion caused by high concentration of AKG could be rescued with GDF9 supplementation in COCs, indicating the critical role of GDF9 in IVM. The results provide valuable information on the variable effects of AKG at different concentrations on reproductive physiology.Graphical abstract

Single-nucleus and spatial transcriptomics of paediatric ovary: Molecular insights into the dysregulated signalling pathways underlying premature ovarian insufficiency in classic galactosemia.

Classic galactosemia (CG) is an inborn error of galactose metabolism caused by mutations in the GALT gene. Premature ovarian insufficiency (POI) is a later complication that affects 80% of women with CG due to a significant decline in ovarian reserve (primordial follicle pool). The definite mechanisms underlying the early onset of POI in CG patients are not fully understood. In this study, we performed single-nucleus RNA sequencing (snRNA-seq) and spatial transcriptomics on ovary tissue biopsies from prepubertal girls diagnosed with CG to investigate dynamic changes in gene expression and altered signalling pathways in granulosa cells, oocytes, and stromal cells. We generated single-nucleus and spatial transcriptomics atlas of human ovaries from prepubertal girls diagnosed with and without CG. snRNA-seq profiling of the paediatric ovary revealed a diverse ovarian microenvironment with seven distinct major cell types. Our transcriptomic analysis revealed an increase in the expression of several endoplasmic reticulum stress and oxidative stress associated genes, which can promote apoptosis of granulosa cells in CG. PTEN/PI3K/AKT signalling, which is crucial for primordial follicle activation and survival was dysregulated as supported by upregulated PTEN transcripts and a significant reduction in phospho-AKT levels in the granulosa cells and oocytes. We also found a marked increase in expression of phospho-H2A.X, LC3A/B and CASP9 in the primordial follicles of CG ovaries suggesting DNA damage, autophagy, and accelerated follicular atresia. Furthermore, we noticed genes participating in extracellular matrix organisation, integrin and gap junction signalling, essential for structural support of the ovarian stroma were profoundly altered. Our findings provide molecular insights into the dysregulated cellular signalling pathways essential for primordial follicle growth and survival that can explain the etiology of POI in CG patients. This study has implications in the development of future therapeutic interventions to preserve ovarian function and promote femalereproductive health. Created a comprehensive single-nucleus transcriptomic atlas and spatial landscape of paediatric ovary tissue from prepubertal girls diagnosed with classic galactosemia (CG). Our transcriptomic analysis revealed activation of genes associated with ER-stress signalling, oxidative stress response and ATM signalling/DNA damage response as shown by significant increase in expression of p-EIF2A, p-H2A.X and LC3A/B in the primordial follicles of CG ovary. PTEN/PI3K/AKT signalling pathways was dysregulated evidenced by a significant reduction in phospho-AKT expression in the primordial follicles of CG ovary, suggesting impaired follicle activation and survival.

Dynamic changes in gene expression of growing nonhuman primate antral follicles.

The growth of the ovarian antral follicle is a complex process that is difficult to study, especially in human and nonhuman primates. Understanding the antral stage of development is key to new approaches to regulating reproduction. This study analyzed cohorts of three sizes of developing antral follicles obtained from adult rhesus macaque females using RNA sequencing of oocytes and cumulus and granulosa cells. The overall objective of this study was to identify key developmental changes in gene expression in oocytes, granulosa, and cumulus cells, as nonhuman primate antral stage follicles transition through progressively larger sizes in the absence of exogenous hormonal stimulation. Only a relatively small number of genes displayed altered mRNA expression levels in any of the three cell types during this period. Most of the identified differentially expressed genes (DEGs) decreased in the granulosa cells or increased in the cumulus cells. Although the number of DEGs observed was small, these DEGs indicate predicted effects on distinct upstream regulators in the cumulus and granulosa cells. This study is particularly important because it shows for the first time the gene expression changes during antral follicle growth in a medically relevant model.NEW & NOTEWORTHY Changes in gene expression in oocytes, granulosa, and cumulus cells were determined in nonhuman primate antral stage ovarian follicles transitioning through progressively larger sizes without exogenous hormonal stimulation. Only a small number of genes displayed altered mRNA expression levels in any of the three cell types. Most of the differentially expressed genes (DEGs) decreased in granulosa cells or increased in cumulus cells. These results identified upstream regulators of antral follicle development.

Depletion of placental brain-derived neurotrophic factor (BDNF) is attributed to premature ovarian insufficiency (POI) in mice offspring

IntroductionPremature ovarian insufficiency (POI) is one of the causes of female infertility. Unexplained POI is increasingly affecting women in their reproductive years. However, the etiology of POI is diverse and remains elusive. We and others have shown that brain-derived neurotrophic factor (BDNF) plays an important role in adult ovarian function. Here, we report on a novel role of BDNF in the Developmental Origins of POI.MethodsPlacental BDNF knockout mice were created using CRISPR/CAS9. Homozygous knockout (cKO(HO)) mice didn’t survive, while heterozygous knockout (cKO(HE)) mice did. BDNF reduction in cKO(HE) mice was confirmed via immunohistochemistry and Western blots. Ovaries were collected from cKO(HE) mice at various ages, analyzing ovarian metrics, FSH expression, and litter sizes. In one-month-old mice, oocyte numbers were assessed using super-ovulation, and oocyte gene expression was analyzed with smart RNAseq. Ovaries of P7 mice were studied with SEM, and gene expression was confirmed with RT-qPCR. Alkaline phosphatase staining at E11.5 and immunofluorescence for cyclinD1 assessed germ cell number and cell proliferation.ResultscKO(HE) mice had decreased ovarian function and litter size in adulthood. They were insensitive to ovulation induction drugs manifested by lower oocyte release after superovulation in one-month-old cKO(HE) mice. The transcriptome and SEM results indicate that mitochondria-mediated cell death or aging might occur in cKO(HE) ovaries. Decreased placental BDNF led to diminished primordial germ cell proliferation at E11.5 and ovarian reserve which may underlie POI in adulthood.ConclusionThe current results showed decreased placental BDNF diminished primordial germ cell proliferation in female fetuses during pregnancy and POI in adulthood. Our findings can provide insights into understanding the underlying mechanisms of POI.

P-197 Current status and hotspots of in vitro oocyte maturation: a bibliometric study of the past two decades

Abstract Study question What is the current research status and hotspot of in vitro maturation? And how could we improve the efficiency of IVM technique? Summary answer Five hotspots in IVM field were found, including culturing system, supplementation, cooperation in the ovarian follicle, gene expression, and cryopreservation of oocytes. What is known already In vitro maturation (IVM) of oocytes is a promising technique among assisted reproductive technologies. Because ovarian stimulation is minimal or absent in an IVM cycle, IVM technique reduces the risk of ovarian hyperstimulation syndrome (OHSS) and captures the reproductive potential of immature oocytes. However, the developmental competence of IVM oocytes is generally lower than the oocytes matured in vivo. A recent study showed the cumulative live birth rate in IVM is still inferior to standard IVF. The protocol for IVM still needs to be improved in some details. Study design, size, duration The articles and reviews related to IVM in the Web of Science Core Collection (WoSCC) were retrieved on January 16th, 2023. Three bibliometric tools including VOSviewer 1.6.18, CiteSpace 6.1.R6, and Bibliometrix R package 4.1.0 were used to generate network maps and explore frontier knowledge and trends. Participants/materials, setting, methods The data was retrieved and downloaded from WoSCC using the search formula: TS = (“in vitro maturation” OR “IVM”) AND TS = (follic* OR oocyte*) NOT TS=“ivermectin”. The timespan was set from 2003 to 2022. The language was limited to English, and publication types to article or review. VOSviewer detected indicators, including country, institution, journal, and author. CiteSpace analyzed reference co-citation and identified emerging keywords. Bibliometrix explored trending topics in IVM field. Main results and the role of chance A total of 5133 publications about IVM were retrieved from WoSCC, including 4706 articles and 427 reviews. The number of publications in IVM has increased steadily. China and University of Adelaide were the most prolific country and institution in IVM. Theriogenology was the most published and cited journal. Thompson, Jeremy G was the most productive author in the field of IVM, and Eppig, JJ’s papers were cited most. Furthermore, five hotspots were summarized and introduced, including culturing system, supplementation, cooperation in the ovarian follicle, gene expression, and cryopreservation of oocytes. This study comprehensively and visually analyzed the hotspots and frontiers of IVM in the past two decades. In our conclusion, further study could concentrate on: (1) the mechanism of oocytes matured in vivo and in vitro, especially in energy metabolism and intercellular communications; (2) establishment of IVM culture system, including standardized biphasic IVM culturing system and standardized supplementation; (3) the genetic differences between oocytes matured in vivo and in vitro; (4) the mechanism of cryopreservation-inflicted damage and the solution. Limitations, reasons for caution Due to the limitations of the software, the data we used for bibliometric analysis was only retrieved from WoSCC. Also, while bibliometric analysis can help us understand a field visually, it cannot completely replace system retrieval. Wider implications of the findings In this review, we explored and analyzed the field of IVM from 2003 to 2022 comprehensively and visually. Possible trends and hotspots in future were predicted, which may pave the way for future study directions and developments. Trial registration number not applicable

P-542 Bidirectional signaling between oocytes and ovarian support cells (OSCs) during in vitro maturation results in improved oocyte gene expression

Abstract Study question Are the gene expression profiles of ovarian support cells (OSCs) and cumulus-free oocytes bidirectionally influenced by co-culture during in vitro maturation (IVM)? Summary answer OSC gene expression is modulated by oocyte presence and OSC-IVM matured oocytes display transcriptomic resemblance to in vivo matured oocytes potentially due to OSC-oocyte crosstalk. What is known already The application of IVM holds a significant interest to improve infertility treatment. We previously showed the development of human OSCs generated from human induced pluripotent stem cells (hiPSCs) with ability to improve the maturation rates of immature human cumulus oocyte complexes (COCs) and embryo formation rates from minimal ovarian stimulation cycles (Piechota et al. 2023). We recently demonstrated that OSC-IVM can also be used to improve the nuclear maturation rate of human denuded oocytes from conventional stimulation cycles. Study design, size, duration We aimed to determine how gene expression changed in oocytes and OSCs by OSC-IVM co-culture. Immature fresh denuded oocytes (n = 141) were collected and randomized for IVM without OSCs (Media-IVM) (MediCult; n = 59) or with OSCs (OSC-IVM) (OSCs+MediCult; n = 82) for 24-28h. Subject enrollment ranged from 12/01/2023-06/30/2023. OSCs were collected for analysis pre-IVM (time 0h; n = 3), and post-IVM with (n = 3) and without (n = 3) immature denuded oocyte co-culture. Participants/materials, setting, methods 47 fertility patients undergoing conventional ovarian stimulation donated immature denuded oocytes (GV or MI oocytes) for research, which were allocated between either the Media-IVM or OSC-IVM conditions. In vivo matured oocytes (IVF-MII) were used as controls. Single oocyte RNA sequencing (RNA-seq) was performed after IVM culture, including both mature oocytes or stalled immature oocytes, alongside IVF-MII oocytes. Bulk RNA-seq was performed on OSCs and primary cumulus cells were used as a reference control (n = 7). Main results and the role of chance RNA-seq analysis demonstrated in the absence of oocytes, OSCs retain their global transcriptomic signature during IVM, maintaining or upregulating expression of CYP19A1, MDK, NR2F2, FOXL2, and CD82 and downregulating FSHR. When co-cultured with oocytes, OSCs shifted their global transcriptomic profile to resemble primary cumulus cells. In OSCs, we identified upregulation of IGF2, EGF, and IGFBP4, and downregulation of AMHR2, EGFR and IGFBP2 in the presence of oocytes, indicating oocyte-directed remodeling of OSC gene expression. Differential gene expression analysis in oocytes comparing MIIs and GVs post-IVM identified 31 genes differentially enriched and 353 genes differentially depleted in MIIs after co-culture with OSCs compared to 37 and 230 genes respectively without OSCs. IVF MII gene expression did not differ from OSC-IVM MII oocytes but did significantly differ from Media-IVM MII gene expression (p = 0.0008). Pathway enrichment analysis demonstrated broad similarity between IVF and OSC-IVM MIIs for Unfolded Protein Response, Myc Targets, and Oxidative Phosphorylation, while Media-IVM uniquely showed enrichment in DNA Damage and Spindle Assembly pathways. Media-IVM produced MIIs enriched uniquely for genes involved in electron transport chain (ETC) and oxidative phosphorylation (OXPHOS), which were not found in OSC-IVM or IVF MIIs. Limitations, reasons for caution The number of oocytes utilized in this study was limited. Additionally, as this study used rescue IVM as a model, it is likely that approaches using cumulus enclosed oocytes and traditional IVM may differ. Wider implications of the findings Bidirectional signaling between OSCs and oocytes may contribute to an improved in vitro environment that supports oocyte nuclear and cytoplasmic maturation. Therefore, oocytes matured in the presence of OSCs are more similar to oocytes matured in vivo and display a pathway enrichment profile associated with a higher oocyte developmental competence. Trial registration number not applicable

Single-cell profiling reveals transcriptome dynamics during bovine oocyte growth

BackgroundMammalian follicle development is characterized by extensive changes in morphology, endocrine responsiveness, and function, providing the optimum environment for oocyte growth, development, and resumption of meiosis. In cattle, the first signs of transcription activation in the oocyte are observed in the secondary follicle, later than during mouse and human oogenesis. While many studies have generated extensive datasets characterizing gene expression in bovine oocytes, they are mostly limited to the analysis of fully grown and matured oocytes. The aim of the present study was to apply single-cell RNA sequencing to interrogate the transcriptome of the growing bovine oocyte from the secondary follicle stage through to the mid-antral follicle stage.ResultsSingle-cell RNA-seq libraries were generated from oocytes of known diameters (< 60 to > 120 μm), and datasets were binned into non-overlapping size groups for downstream analysis. Combining the results of weighted gene co-expression network and Trendy analyses, and differently expressed genes (DEGs) between size groups, we identified a decrease in oxidative phosphorylation and an increase in maternal -genes and transcription regulators across the bovine oocyte growth phase. In addition, around 5,000 genes did not change in expression, revealing a cohort of stable genes. An interesting switch in gene expression profile was noted in oocytes greater than 100 μm in diameter, when the expression of genes related to cytoplasmic activities was replaced by genes related to nuclear activities (e.g., chromosome segregation). The highest number of DEGs were detected in the comparison of oocytes 100–109 versus 110–119 μm in diameter, revealing a profound change in the molecular profile of oocytes at the end of their growth phase.ConclusionsThe current study provides a unique dataset of the key genes and pathways characteristic of each stage of oocyte development, contributing an important resource for a greater understanding of bovine oogenesis.

Characterization of preovulatory follicular fluid secretome and its effects on equine oocytes during in vitro maturation

In vitro maturation (IVM) of oocytes is clinically used in horses to produce blastocysts but current conditions used for horses are suboptimal. We analyzed the composition of equine preovulatory follicular fluid (FF) secretome and tested its effects on meiotic competence and gene expression in oocytes subjected to IVM. Preovulatory FF was obtained, concentrated using ultrafiltration with cut-off of 10 kDa, and stored at −80 °C. The metabolic and proteomic composition was analyzed, and its ultrastructural composition was assessed by cryo-transmission microscopy. Oocytes obtained post-mortem or by ovum pick up (OPU) were subjected to IVM in the absence (control) or presence of 20 or 40 μg/ml (S20 or S40) of secretome. Oocytes were then analyzed for chromatin configuration or snap frozen for gene expression analysis. Proteomic analysis detected 255 proteins in the Equus caballus database, mostly related to the complement cascade and cholesterol metabolism. Metabolomic analysis yielded 14 metabolites and cryo-transmission electron microscopy analysis revealed the presence of extracellular vesicles (EVs). No significant differences were detected in maturation rates among treatments. However, the expression of GDF9 and BMP15 significantly increased in OPU-derived oocytes compared to post-mortem oocytes (fold increase ± SEM: 9.4 ± 0.1 vs. 1 ± 0.5 for BMP15 and 9.9 ± 0.3 vs. 1 ± 0.5 for GDF9, respectively; p < 0.05). Secretome addition increased the expression of TNFAIP6 in S40 regardless of the oocyte source. Further research is necessary to fully understand whether secretome addition influences the developmental competence of equine oocytes.

Single-cell RNA-seq and single-cell bisulfite-sequencing reveal insights into yak preimplantation embryogenesis

Extensive epigenetic reprogramming occurs during preimplantation embryonic development. However, the impact of DNA methylation in plateau yak preimplantation embryos and how epigenetic reprogramming contributes to transcriptional regulatory networks are unclear. In this study, we quantified gene expression and DNA methylation in oocytes and a series of yak embryos at different developmental stages and at single-cell resolution using single-cell bisulfite-sequencing and RNA-seq. We characterized embryonic genome activation and maternal transcript degradation and mapped epigenetic reprogramming events critical for embryonic development. Through cross-species transcriptome analysis, we identified 31 conserved maternal hub genes and 39 conserved zygotic hub genes, including SIN3A, PRC1, HDAC1/2, and HSPD1. Notably, by combining single-cell DNA methylation and transcriptome analysis, we identified 43 candidate methylation driver genes, such as AURKA, NUSAP1, CENPF, and PLK1, that may be associated with embryonic development. Finally, using functional approaches, we further determined that the epigenetic modifications associated with the histone deacetylases HDAC1/2 are essential for embryonic development and that the deubiquitinating enzyme USP7 may affect embryonic development by regulating DNA methylation. Our data represent an extensive resource on the transcriptional dynamics of yak embryonic development and DNA methylation remodeling, and provide new insights into strategies for the conservation of germplasm resources, as well as a better understanding of mammalian early embryonic development that can be applied to investigate the causes of early developmental disorders.

201 The presence of corpus luteum affects gene expression in oocytes and cumulus cells after in vitro maturation in mares

201 The presence of corpus luteum affects gene expression in oocytes and cumulus cells after in vitro maturation in mares

Transcriptome Dynamics and Cell Dialogs Between Oocytes and Granulosa Cells in Mouse Follicle Development.

The development and maturation of follicles is a sophisticated and multistage process. The dynamic gene expression of oocytes and their surrounding somatic cells and the dialogs between these cells are critical to this process. In this study, we accurately classified the oocyte and follicle development into nine stages and profiled the gene expression of mouse oocytes and their surrounding granulosa cells and cumulus cells. The clustering of the transcriptomes showed the trajectories of two distinct development courses of oocytes and their surrounding somatic cells. Gene expression changes precipitously increased at Type 4 stage and drastically dropped afterward within both oocytes and granulosa cells. Moreover, the number of differentially expressed genes between oocytes and granulosa cells dramatically increased at Type 4 stage, most of which persistently passed on to the later stages. Strikingly, cell communications within and between oocytes and granulosa cells became active from Type 4 stage onward. Cell dialogs connected oocytes and granulosa cells in both unidirectional and bidirectional manners. TGFB2/3, TGFBR2/3, INHBA/B, and ACVR1/1B/2B of TGF-β signaling pathway functioned in the follicle development. NOTCH signaling pathway regulated the development of granulosa cells. Additionally, many maternally DNA methylation- or H3K27me3-imprinted genes remained active in granulosa cells but silent in oocytes during oogenesis. Collectively, Type 4 stage is the key turning point when significant transcription changes diverge the fate of oocytes and granulosa cells, and the cell dialogs become active to assure follicle development. These findings shed new insights on the transcriptome dynamics and cell dialogs facilitating the development and maturation of oocytes and follicles.

The effect of Coenzyme Q10 on mitochondrial biogenesis in mouse ovarian follicles during in vitro culture.

The aim of this research was to investigate the effect of Coenzyme Q10 (CoQ10) on the expression of the Transcription Factor A Mitochondrial (Tfam) gene and mtDNA copy number in preantral follicles (PFs) of mice during in vitro culture. To conduct this experimental study, PFs were isolated from 14-day-old National Medical Research Institute mice and cultured in the presence of 50 µm CoQ10 for 12 days. On the 12th day, human chorionic gonadotropin was added to stimulate ovulation. The fundamental parameters, including preantral follicle developmental rate and oocyte maturation, were evaluated. Additionally, the Tfam gene expression and mtDNA copy number of granulosa cells and oocytes were assessed using the real-time polymerase chain reaction. The results revealed that CoQ10 significantly increased the diameter of PFs, survival rate, antrum formation, and metaphase II (MII) oocytes (P < 0.05). Moreover, in the CoQ10-treated groups, the Tfam gene expression in granulosa cells and oocytes increased considerably compared with the control group. The mtDNA copy number of granulosa cells and oocytes cultured in the presence of CoQ10 was substantially higher compared with the control groups (P < 0.05). The addition of CoQ10 to the culture medium enhances the developmental competence of PFs during in vitro culture by upregulating Tfam gene expression and increasing mtDNA copy number in oocyte and granulosa cells.

Cytoplasmic granules in bovine oocytes do not affect embryonic or fetal development.

Oocyte cytoplasmic evaluation is based on homogeneity and granular appearance. Our study investigated if a granular cytoplasm, highly heterogeneous, would affect oocyte competence in bovine. In two experiments, bovine cumulus-oocyte complexes (COCs) with homogeneous cytoplasm (control, CC) and granulated cytoplasm (granular, GC) were selected from a regular pool of COCs. Experiment 1 was performed with slaughterhouse ovaries, and Experiment 2 was carried out in Girolando COCs obtained from ovum pick-up. Granular oocytes had higher caspase 3 levels (66.17 ± 11.61 vs 172.08 ± 16.95, P < 0.01) and similar GAP junction activity (5.64 ± 0.45 vs 6.29 ± 0.29). ZAR1 relative mRNA amount was lower in granular oocytes (178.27 ± 151.63 vs 0.89 ± 0.89, P = 0.01) and no effect was detected for MATER, PPP2R1A, ENY2, IGF2R, and BMP15 genes. Despite molecular differences, no detrimental effect was detected on oocyte competence in GC oocytes. Cleavage (Experiment 1: 59.52 ± 7.21% vs 59.79 ± 6.10% and Experiment 2: 68.88 ± 4.82 vs 74.41 ± 5.89%) and blastocyst (Experiment 1: 29.28 ± 4.14% vs 23.15 ± 2.96% and Experiment 2: 21.11 ± 3.28% vs 21.02 ± 6.08%) rates were similar between CC and GC (Experiments 1 and 2, respectively). Post-transfer embryo development revealed that pregnancy (CC: 24.27 ± 9.70% vs GC: 26.31 ± 7.23%) and calving (23.68% vs 33.33%) rates and fetal growth were not affected by the presence of cytoplasmic granules. Our results demonstrated that oocytes with granular cytoplasm present equivalent efficiency for IVF and calf production compared with homogenous cytoplasm oocytes. This could be observed through similar cleavage, blastocyst rates, and fetal growth development. In addition to differences in oocyte gene expression related to oocyte quality, it seems not to affect oocyte developmental competence.

Cumulus Cell and Oocyte Gene Expression in Prepubertal Gilts and Sows Identifies Cumulus Cells as a Prime Informative Parameter of Oocyte Quality.

Cumulus cells (CCs) are pivotal during oocyte development. This study aimed to identify novel marker genes for porcine oocyte quality by examining the expression of selected genes in CCs and oocytes, employing the model of oocytes from prepubertal animals being of reduced quality compared to those from adult animals. Total RNA was extracted either directly after follicle aspiration or after in vitro maturation, followed by RT-qPCR. Immature gilt CCs accumulated BBOX1 transcripts, involved in L-carnitine biosynthesis, to a 14.8-fold higher level (p < 0.05) relative to sows, while for CPT2, participating in fatty acid oxidation, the level was 0.48 (p < 0.05). While showing no differences between gilt and sow CCs after maturation, CPT2 and BBOX1 levels in oocytes were higher in gilts at both time points. The apparent delayed lipid metabolism and reduced accumulation of ALDOA and G6PD transcripts in gilt CCs after maturation, implying downregulation of glycolysis and the pentose phosphate pathway, suggest gilt cumulus-oocyte complexes have inadequate ATP stores and oxidative stress balance compared to sows at the end of maturation. Reduced expression of BBOX1 and higher expression of CPT2 in CCs before maturation and higher expression of G6PD and ALDOA after maturation are new potential markers of oocyte quality.

A DNA Methylation Perspective on Infertility.

Infertility affects a significant number of couples worldwide and its incidence is increasing. While assisted reproductive technologies (ART) have revolutionized the treatment landscape of infertility, a significant number of couples present with an idiopathic cause for their infertility, hindering effective management. Profiling the genome and transcriptome of infertile men and women has revealed abnormal gene expression. Epigenetic modifications, which comprise dynamic processes that can transduce environmental signals into gene expression changes, may explain these findings. Indeed, aberrant DNA methylation has been widely characterized as a cause of abnormal sperm and oocyte gene expression with potentially deleterious consequences on fertilization and pregnancy outcomes. This review aims to provide a concise overview of male and female infertility through the lens of DNA methylation alterations.

The effect of L-carnitine on oocyte mitochondrial health and biomarkers on cyclophosphamide chemotherapy drug in mice

Improving oocyte competence during chemotherapy is widely known as a contributing factor to increasing the probability of fertility. Additionally, the role of cumulus cells in oocyte quality is of utmost importance. Therefore, this study was designed to simultaneously probe into the relative gene expression of oocytes and cumulus cells as biomarkers of oocyte quality with cyclophosphamide and L-carnitine treatment. A total of 60 adult NMRI mice were divided into four groups: control, L-carnitine (LC), cyclophosphamide (CP), and cyclophosphamide+L-carnitine (CP+LC). The relative mRNA expression levels of oocyte quality genes including growth differentiation factor 9 (Gdf9), hyaluronan synthase 2 (Has2), and mitochondrial sirtuin 3 (Sirt3) in oocytes, and genes involved in bilateral communication between cumulus cells and between the oocyte and its neighboring cumulus cells including connexin 37 (Cx37) and connexin 43 (Cx43) were detected by Real-time-PCR. DCFH-DA staining analyzed the level of intracellular ROS in oocytes. Under the influence of L-carnitine, Gdf9, Has2, Cx43, and Cx37 were significantly up-regulated (p ≤ 0.05). However, cyclophosphamide considerably reduced the expression of all these genes (p ≤ 0.05). The expression of the Sirt3 gene in the CP group increased significantly compared to the other groups (p ≤ 0.05). Analysis of fluorescent images revealed that the level of intracellular ROS in the cyclophosphamide group was significantly increased compared to the other groups (p ≤ 0.05), while it plummeted in the L-carnitine group (p ≤ 0.05). L-carnitine as an antioxidant can reduce the destructive effects of cyclophosphamide and enhance bilateral communications between oocytes and cumulus cells, and it may ultimately lead to an increase in the fertility rate.

Lipid modulation during IVM increases the metabolism and improves the cryosurvival of cat oocytes

This study investigated the time course of lipid accumulation during IVM and assessed the role of lipid modulators added during IVM on lipid content, nuclear maturation, oxidative stress, mitochondrial activity, gene expression, and cryosurvival of cat oocytes. First, the lipid content of immature COCs was compared to those subjected to different IVM duration times (24, 28, and 32 h). Then, the lipid content was investigated after the use of different lipid modulators [conjugated linoleic acid (CLA), forskolin (FSK), l-carnitine (LC)]. Subsequently, both the CONTROL group and MIX 18 (CLA+FSK+LC) were compared regarding nuclear maturation, mitochondrial activity, reactive oxygen 19 species (ROS), and glutathione (GSH) levels, to the expression of SDHA, GDF9, BMP15, ZAR-1, 20 PRDX1, SIRT1, and SIRT3 genes (normalized by ACTB and YWHAZ genes); and to vitrification and 21 post-warming viability assessment. When not using any lipid modulator, an increase (P < 0.05) in lipid content could be observed after 28 h of IVM. The MIX group showed the greatest (P < 0.05) reduction in oocyte lipid content after 28 h of IVM. No difference (P > 0.05) was observed in the MII rate in the CONTROL (45%) and MIX (41%) groups and in mitochondrial activity ((1.00 ± 0.35 A U vs 1.19 ± 0.14 A U). Although ROS and GSH levels were higher (P < 0.05) in MIX than in CONTROL, the redox balance (ROS/GSH) was greater (P < 0.05) in the latter (C:1.00 ± 0.20b vs M:0.26 ± 0.06 a A.U). The GDF9, HSP70, PRDX1, and SIRT1 transcripts were downregulated (P < 0.05) in MIX-oocytes, compared to the CONTROL. After vitrification, MIX (74%) presented a higher (P < 0.05) viability compared to control (53%). In conclusion, MIX can reduce the total lipid content and improve viability after cryopreservation, however, it seems to affect the oocyte metabolism in a way that still needs to be better understood in the cat biological model.

O-105 Chromatin accessibility of oocytes contributes to PCOS transgenerational inheritance

Abstract Study question What is the underlying mechanism contributing to the transgenerational defects of oocytes and embryos of polycystic ovary syndrome (PCOS)? Summary answer The transgenerational inheritance of abnormal chromatin accessibility in the PCOS oocytes is responsible for the defect of oocytes and embryos in transgeneration. What is known already Studies have provided evidence of compromised oocyte quality and related adverse impacts. Observed PCOS animal-model oocyte defects include meiotic abnormalities, mitochondrial dysfunction, and increased oxidative stress. Elevated androgen levels are associated with reduced fertilization rates and lower cleavage rates during in vitro fertilization (IVF) treatment. PCOS could be transgenerationally transmitted, and unique altered gene expression has been observed in metaphase II (MII) oocytes from all offspring in PCOS mice. Study design, size, duration We profiled the transcriptome and chromatin accessibility of oocytes and preimplantation embryos in F0-F2 offspring of PCOS mice, to explored whether the inheritance of chromatin accessibility defects in PCOS oocytes was responsible for the abnormalities of oocytes and embryos in PCOS offspring. Besides, oocytes from 13 healthy women and 8 women with PCOS, 24 blood samples from 24 women with/without PCOS were analyzed to validate the molecular mechanisms of PCOS inheritance. Duration: 2019.5∼2022.12. Participants/materials, setting, methods PCOS mice models were established by means of DHEA-exposure. Smart-seq2 and DNase-seq were utilized to investigate the transcriptome and chromatin accessibility of oocytes and preimplantation embryos in F0-F2 offspring of PCOS mice. Bioinformatic analysis was applied to explore the differential gene expression and chromatin accessibility of oocytes and embryos in PCOS mice and offspring comparing to controls. Common gene signatures in the tissues of PCOS women and PCOS daughters are analyzed compared to control samples. Main results and the role of chance In PCOS mice, we observed transcriptional alteration in oocytes and 2-cell embryos compared to normal mice. There are 4229 and 1197 differentially expressed genes (DEGs) in PCOS oocytes and 2-cell embryos. The DEGs includes both maternal factors and zygotic genome activation (ZGA) genes. Interestingly, in PCOS 2-cell embryos, we observed hundreds of pre-activated genes which are silence in normal 2-cell embryo but expressed in PCOS 2-cell embryos. Notably, many DEGs in the oocytes of PCOS mice were also differentially expressed in the oocytes of F2 PCOS mice compared to control mice, supporting the transgenerational inheritance of transcriptional alteration in PCOS oocytes. Mechanismly, the changes of chromatin accessibility were associated with the DEGs in PCOS oocytes and embryos. In particular, chromatin accessibility of the pre-activated gene promoters is elevated in PCOS oocytes compared to that in normal oocytes. Furthermore, many of the abnormal chromatin accessibility signals detected in F0 PCOS oocytes were also observed in F2 PCOS oocytes. Finally, our data showed that some DEGs in the oocytes of PCOS mice were also differentially expressed in human PCOS oocytes compared to the control oocytes. Common genes identified in PCOS offspring may be used as potential predictors of PCOS phenotype. Limitations, reasons for caution Which factors contribute to the abnormalities of chromatin accessibility in PCOS oocytes are still unknown. Wider implications of the findings The candidate genes may be used as potential predictors of PCOS phenotype and provide the clues for PCOS offspring . Trial registration number not applicable

Vitrification with Dimethyl Sulfoxide Induces Transcriptomic Alteration of Gene and Transposable Element Expression in Immature Human Oocytes.

Despite substantial advancements in the field of cryobiology, oocyte and embryo cryopreservation still compromise developmental competence. Furthermore, dimethyl sulfoxide (DMSO), one of the most commonly used cryoprotectants, has been found to exert potent effects on the epigenetic landscape of cultured human cells, as well as mouse oocytes and embryos. Little is known about its impact on human oocytes. Additionally, few studies investigate the effects of DMSO on transposable elements (TE), the control of which is essential for the maintenance of genomic instability. The objective of this study was to investigate the impact of vitrification with DMSO-containing cryoprotectant on the transcriptome, including on TEs, of human oocytes. Twenty-four oocytes at the GV stage were donated by four healthy women undergoing elective oocyte cryopreservation. Oocytes were paired such that half from each patient were vitrified with DMSO-containing cryoprotectant (Vitrified Cohort), while the other half were snap frozen in phosphate buffer, unexposed to DMSO (Non-Vitrified Cohort). All oocytes underwent RNA sequencing via a method with high fidelity for single cell analysis, and which allows for the analysis of TE expression through Switching Mechanism at the 5'-end of the RNA Transcript sequencing 2 (SMARTseq2), followed by functional enrichment analysis. Of the 27,837 genes identified by SMARTseq2, 7331 (26.3%) were differentially expressed (p < 0.05). There was a significant dysregulation of genes involved in chromatin and histone modification. Mitochondrial function, as well as the Wnt, insulin, mTOR, HIPPO, and MAPK signaling pathways were also altered. The expression of TEs was positively correlated with the expression of PIWIL2, DNMT3A, and DNMT3B, and negatively correlated with age. These findings suggest that the current standard process of oocyte vitrification, involving DMSO-containing cryoprotectant, induces significant transcriptome changes, including those involving TEs.