Human Oogenesis Research Articles

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.

GametesOmics: A Comprehensive Multi-omics Database for Exploring the Gametogenesis in Humans and Mice.

Gametogenesis plays an important role in the reproduction and evolution of species. The transcriptomic and epigenetic alterations in this process can influence the reproductive capacity, fertilization, and embryonic development. The rapidly increasing single-cell studies have provided valuable multi-omics resources. However, data from different layers and sequencing platforms have not been uniformed and integrated, which greatly limits their use for exploring the molecular mechanisms that underlie oogenesis and spermatogenesis. Here, we develop GametesOmics, a comprehensive database that integrates the data of gene expression, DNA methylation, and chromatin accessibility during oogenesis and spermatogenesis in humans and mice. GametesOmics provides a user-friendly website and various tools, including Search and Advanced Search for querying the expression and epigenetic modification(s) of each gene; Tools with Differentially expressed gene (DEG) analysis for identifying DEGs, Correlation analysis for demonstrating the genetic and epigenetic changes, Visualization for displaying single-cell clusters and screening marker genes as well as master transcription factors (TFs), and MethylView for studying the genomic distribution of epigenetic modifications. GametesOmics also provides Genome Browser and Ortholog for tracking and comparing gene expression, DNA methylation, and chromatin accessibility between humans and mice. GametesOmics offers a comprehensive resource for biologists and clinicians to decipher the cell fate transition in germ cell development, and can be accessed at http://gametesomics.cn/.

Interleukin-6 Concentration in Single-Embryo Medium Is Associated with Blastocyst Formation.

IL-6 plays an important role in oogenesis in humans. However, at the preimplantation stage, IL-6 production and the role in embryo development remain unclear. In this study, IL-6 concentrations in single-embryo media were analyzed. In addition, the association between IL-6 production and blastocyst formation was investigated. Single-embryo culture media from 194 embryos were collected on day 6 after fertilization and divided into four groups according to the developmental stage of the corresponding embryo, as follows: cleavage stage group, morula-early blastocyst group, unavailable full blastocyst group, and available full blastocyst group. IL-6 concentrations were significantly lower in the cleavage stage group than in the morula-early blastocyst group (p = 0.009), in the unavailable full blastocyst group (p = 0.003), and in the available full blastocyst group (p < 0.001). Logistic regression analysis showed that IL-6 concentration in single-embryo medium was significantly associated with blastocyst formation (odds ratios β1 = 1.876, 95% CI 1.433 to 2.644, p < 0.0001). Therefore, IL-6 was produced by human preimplantation embryos throughout the preimplantation stage and may play a role in embryo development.

From in vivo to in vitro: exploring the key molecular and cellular aspects of human female gametogenesis.

Human oogenesis is a highly complex and not yet fully understood process due to ethical and technological barriers that limit studies in the field. In this context, replicating female gametogenesis in vitro would not only provide a solution for some infertility problems, but also be an excellent study model to better understand the biological mechanisms that determine the formation of the female germline. In this review, we explore the main cellular and molecular aspects involved in human oogenesis and folliculogenesis in vivo, from the specification of primordial germ cells (PGCs) to the formation of the mature oocyte. We also sought to describe the important bidirectional relationship between the germ cell and the follicular somatic cells. Finally, we address the main advances and different methodologies used in the search for obtaining cells of the female germline in vitro.

Induction of fetal meiotic oocytes from embryonic stem cells in cynomolgus monkeys.

Human in vitro oogenesis provides a framework for clarifying the mechanism of human oogenesis. To create its benchmark, it is vital to promote in vitro oogenesis using a model physiologically close to humans. Here, we establish a foundation for in vitro oogenesis in cynomolgus (cy) monkeys (Macaca fascicularis): cy female embryonic stem cells harboring one active and one inactive X chromosome (Xa and Xi, respectively) differentiate robustly into primordial germ cell-like cells, which in xenogeneic reconstituted ovaries develop efficiently into oogonia and, remarkably, further into meiotic oocytes at the zygotene stage. This differentiation entails comprehensive epigenetic reprogramming, including Xi reprogramming, yet Xa and Xi remain epigenetically asymmetric with, as partly observed in vivo, incomplete Xi reactivation. In humans and monkeys, the Xi epigenome in pluripotent stem cells functions as an Xi-reprogramming determinant. We further show that developmental pathway over-activations with suboptimal up-regulation of relevant meiotic genes impede in vitro meiotic progression. Cy in vitro oogenesis exhibits critical homology with the human system, including with respect to bottlenecks, providing a salient model for advancing human in vitro oogenesis.

Complementarity between Early Embryogenesis and Uterine Receptivity: Toward Integrative Approach to Female Infertility Management. Editorial to the Special Issue “Molecular Mechanisms of Human Oogenesis and Early Embryogenesis”

Two highly relevant papers were recently been published in the Special Issue of Molecular Sciences entitled "Molecular Mechanisms of Human Oogenesis and Early Embryogenesis" [...].

P-531 Influence of tandemly repetitive pericentromeric DNA transcripts inactivation on the formation of membraneless RNP structures at the end of human oocytes maturation

Abstract Study question Does inactivation of tandemly repetitive (TR) pericentromeric DNA transcription affect the size and the number of DDX4-containing RNPs at the end of human oocytes maturation? Summary answer Antisense oligonucleotides (AO) can inactivate the transcription of HS2/HS3. Inactivation leads to disaggregation of the DDX4-based membraneless RNP structures. What is known already Earlier we had described for the first time the transcription of the pericentromeric TR DNA of HS2/HS3 families in late human oogenesis, during the transition of oocytes from the GV stage to the MI stage. Both HS2/HS3 strands were transcribed. The presence of HS2/HS3 transcripts was demonstrated by bioinformatic analysis, qPCR and RNA-DNA-FISH. HS2/HS3 RNAs were found in RNPs which contained DDX5 and DDX4 RNA helicases, located near mitochondria. We suggested that the RNPs could be the site of spatial sequestration of RNA and proteins at the end of human oocytes maturation. Study design, size, duration For studying the inactivation of HS2/HS3 transcription, oocytes were subjected to cytoplasmic microinjection using a micromanipulator. Before the experiment, oocytes were divided into 3 groups (n = 11). Group 1 (control, n = 4) got a dose of saline, group 2 (n = 3) – 8.5 fmol AO, group 3 (n = 4) – 12.75 fmol AO. AO or saline was injected into oocytes, the cells were then placed in a CO2 incubator, and after 48 hours, they were fixed and examined. Participants/materials, setting, methods GV and MI human oocytes are not used for the bank of donor oocytes and were transferred for research. The informed consent of the donors, as well as the permission of the ethical committee of the clinic, was obtained. The distribution of HS2/HS3 transcripts and DDX4 helicase was studied using the immuno-FISH. Fluorescent signals were analyzed using the ImageJ program. Statistical processing of the count data was carried out using GraphPad Prism 9 software. Main results and the role of chance Statistically significant inactivation of TR RNA HS2/HS3 (p = 0.01) in group 3 (AO, 12.75 fmol) was observed. The fluorescence of the HS2/HS3 RNA hybridization signal dropped to 25.68±1.777 as compared to the control group 1 (35.50±2.938). In group 2, this value (34.47 ± 1.984) did not change significantly as compared to the control group. Also, in the 3rd group, there was a tendency (p = 0.16) of an increase in the fluorescence of the antibodies signal against DDX4 up to 26.23 ± 2.223 in comparison with group 1 (19.57 ± 2.496). In group 2, this indicator (21.63 ± 3.095) changed to a lesser extent as compared to the control group. At the same time, in the oocytes of the 3rd group, there was a tendency (p = 0.19) to an increase in the number of individual cytoplasmic foci stained with the antibody against DDX4 up to 76±18.56 as compared with group 1 (44±8.994 signals). In group 2 this value (26±5.508 signals) changed less compared to control group 1. We assume, after the inactivation of HS2/HS3 transcription, DDX4-containing RNPs dissociate since an increase in the number of individual signals of the corresponding DDX4 was revealed. Limitations, reasons for caution The small sample size did not allow us to obtain statistically reliable data on the fluorescence of DDX 4. Data accumulation will be continued in future studies. Wider implications of the findings The data presented confirm the existence of DDX4-based RNPs in human preovulatory oocytes. These membraneless structures are involved in the processes of oocyte maturation. The suggested approach of transcripts inactivation can be used for oocytes without removing zona pellucidae increasing their survival after inactivation. Trial registration number not applicable

Ovarian recovery via autologous platelet-rich plasma: New benchmarks for condensed cytokine applications to reverse reproductive aging.

Health and life expectancy gains have pushed the overall number of menopausal patients to record levels. Because maternal age at first pregnancy also continues to rise, it is unsurprising that reduced birth rates are consistently reported across many populations. Both trends severely strain national demographics and present a socioeconomic challenge for which no satisfactory solution currently exists. Symptomatic menopause and infertility/miscarriage are met with standard therapies like hormone replacement therapy (HRT) and in vitro fertilization, respectively. Although these accepted interventions do supply some cover, both are expensive, low yield, and not without controversy. Meanwhile, ovarian steroid output and competent oocyte availability approach unrecoverable loss beyond age ~35 years, irrespective of treatment. Received wisdom holds that postnatal oogenesis in humans is impossible, a tenet which until recently encountered little serious confrontation. Reassessing this paradigm is overdue given proof‐of‐concept work on native sex steroid rejuvenation, de novo euploid oogenesis, ovulation, blastocyst development, fetal growth, and healthy term livebirths—all apparently possible with intraovarian insertion of platelet‐rich plasma (PRP). Discrete functional analysis of the full platelet‐derived cytokine array carried with PRP unfortunately for now, is incomplete. Here, selected platelet releasate constituents and measured effects are framed to address advances in wellness and women’s health. Emphasis is on cytokines best positioned to enable recovery of senescent ovarian function sufficient to suspend synthetic HRT dependency and/or permit egg retrieval and pregnancy. Whereas the chronicle of progress in other clinical fields does invite generalization of fresh platelet applications to reproductive endocrinology, basic mechanistic questions remain open.

Starvation May Change the Type of Paternal but Not Maternal Inheritance of the Telomere Length Throughout Generations

Our aim was to evaluate the effect of starvation on the paternal and maternal inheritance of the leucocyte telomere length (LTL) in three birth cohorts: before, during, and after the Chuvashian famine of 1922–1923. This retrospective cohort study comprised native Chuvash men (n= 678) and women (n=647) born between 1909–1980. Data were gathered during three expeditions: 1994, 1999, 2002, enabling us to treat age and birth year as independent variables. LTL was measured by a quantitative polymerase chain reaction technique. A significant difference in LTL values (χ2 d.f=2=79.04, p&amp;lt;0.0001) was observed between the sexes. In a pedigree-based variance component analysis, ~67.4% of the LTL variation was explained by an additive genetic component. In the entire sample, a significant correlation was observed between the LTL of the parents and offsprings. Fathers born between 1924–1928 demonstrated a significantly stronger LTL correlation with their offsprings. The LTL correlation between mothers and offspring in this group and between parents and offspring in those born after 1928 did not differ from the entire population. We believe that the increased correlation between the LTL in the father-offspring pairs born after starvation in Chuvashia can be explained by differences in the processes of oogenesis and spermatogenesis in humans.

Transcriptional progression during meiotic prophase I reveals sex-specific features and X chromosome dynamics in human fetal female germline.

During gametogenesis in mammals, meiosis ensures the production of haploid gametes. The timing and length of meiosis to produce female and male gametes differ considerably. In contrast to males, meiotic prophase I in females initiates during development. Hence, the knowledge regarding progression through meiotic prophase I is mainly focused on human male spermatogenesis and female oocyte maturation during adulthood. Therefore, it remains unclear how the different stages of meiotic prophase I between human oogenesis and spermatogenesis compare. Analysis of single-cell transcriptomics data from human fetal germ cells (FGC) allowed us to identify the molecular signatures of female meiotic prophase I stages leptotene, zygotene, pachytene and diplotene. We have compared those between male and female germ cells in similar stages of meiotic prophase I and revealed conserved and specific features between sexes. We identified not only key players involved in the process of meiosis, but also highlighted the molecular components that could be responsible for changes in cellular morphology that occur during this developmental period, when the female FGC acquire their typical (sex-specific) oocyte shape as well as sex-differences in the regulation of DNA methylation. Analysis of X-linked expression between sexes during meiotic prophase I suggested a transient X-linked enrichment during female pachytene, that contrasts with the meiotic sex chromosome inactivation in males. Our study of the events that take place during meiotic prophase I provide a better understanding not only of female meiosis during development, but also highlights biomarkers that can be used to study infertility and offers insights in germline sex dimorphism in humans.

Identification of calcineurin as a predictor of oocyte quality and fertilization competence based on microarray data

ObjectiveThe aim of our study was to detect a biomarker for selection of competent oocytes with acceptable fertilization potential. Calcium ion fluctuation play the most critical role of modulating intercellular signaling pathways in oocyte maturation, egg activation and the egg-to-embryo transition. Since, the stimulatory action of calcium ion is mediated by binding to certain proteins, the calcium/calmodulin-binding genes (CBGs), as the main calcium binding group, was analyzed in detail. MethodsIn this work, bioinformatics analysis was conducted on the CBGs of human cumulus cells (CCs) to elucidate a reliable biomarker for fertile oocyte selection. Calcineurin (CaN) or protein phosphatase 3 (PPP3) was selected which consists of a catalytic subunit A with PPP3CA (Aα), PPP3CB (Aβ), and PPP3CC (Aγ) isoforms and a regulatory subunit B. Whereas CaN A regulates calcium ion function, our study gives insights to probable role of related isoforms within human oogenesis process. The presence of CaN A in CCs surrounding growing and mature oocytes was confirmed by western blotting and the expression patterns of related isoforms were examined by reverse transcription-quantitative PCR (RT-qPCR). ResultsOur results indicated the increased expression of the catalytic subunit of CaN protein in the CCs of metaphase (M) II oocytes. The expression level of PPP3CB was significantly elevated in CCs of fertile MII compared with those in the germinal vesicle (GV), MI and unfertilized MII oocytes (P ≤ 0.05). ConclusionElevated level of PPP3CB isoform in the CCs of fertile MII oocyte could be a reliable indication of oocyte fertilization potential. However, further researches are required to introduce CaN Aβ as an appropriate biomarker for oocyte selection in assisted reproduction technique (ART) programs.

Failure to recombine is a common feature of human oogenesis

Failure of homologous chromosomes to recombine is arguably the most important cause of human meiotic nondisjunction, having been linked to numerous autosomal and sex chromosome trisomies of maternal origin. However, almost all information on these "exchangeless" homologs has come from genetic mapping studies of trisomic conceptuses, so the incidence of this defect and its impact on gametogenesis are not clear. If oocytes containing exchangeless homologs are selected against during meiosis, the incidence may be much higher in developing germ cells than in zygotes. To address this, we initiated studies of exchangeless chromosomes in fetal ovarian samples from elective terminations of pregnancy. In total, we examined more than 7,000 oocytes from 160 tissue samples, scoring for the number of foci per cell of the crossover-associated protein MLH1. We identified a surprisingly high level of recombination failure, with more than 7% of oocytes containing at least one chromosome pair that lacked an MLH1 focus. Detailed analyses indicate striking chromosome-specific differences, with a preponderance of MLH1-less homologs involving chromosomes 21 or 22. Further, the effect was linked to the overall level of recombination in the cell, with the presence of one or two exchangeless chromosomes in a cell associated with a 10%-20% reduction in the total number of crossovers. This suggests individuals with lower rates of meiotic recombination are at an increased risk of producing aneuploid offspring.

Do prenatal exposures pose a real threat to ovarian function? Bisphenol A as a case study.

Fetal development represents a time of potential vulnerability due to rapid cell division, organ development and limited fetal kidney/liver activity for detoxification and metabolism of exposures. Health effects of prenatal toxicant exposure have previously been described, but there is little cohesive evidence surrounding effects on ovarian function. Using bisphenol A (BPA) as a case study, we seek to examine whether a prominent prenatal environmental exposure can pose a real threat to human ovarian function. To do so, we broadly review human oogenesis and menstrual cycle biology. We then present available literature addressing prenatal bisphenol A and diverse outcomes at the level of the ovary. We highlight relevant human cohorts and mammalian models to review the existing data on prenatal exposures and ovarian disruption. Doing so suggests that while current exposures to BPA have not shown marked or consistent results, there is data sufficient to raise concerns regarding ovarian function. Challenges in the examination of this question suggest the need for additional models and pathways by which to expand these examinations in humans.

Both male and female gamete generating cells produce processed pseudogenes in the human genome

The human genome contains an unusually large number of processed pseudogenes. The fact that processed pseudogenes are roughly 33% more abundant in our X chromosome than in our autosomes suggests that this overabundance is the result of the fact that human oogenesis is much longer than that of non-mammalian species. Here, we analyze the origins of the processed pseudogenes found on the human Y chromosome to determine whether human spermatogenesis also contribute to this overabundance.Our results show that human processed pseudogenes not only retrotranspose to the Y chromosome, but are also produced by genes on the Y chromosome. Furthermore, the fact that X chromosomes are three times more abundant than Y chromosomes likely explains why the euchromatic density of processed pseudogenes is three times higher in the X chromosome than in the Y chromosome.The large number of processed pseudogenes found in our genome is therefore due to the low substrate specificity of the L1 reverse transcriptase responsible for the reverse transcription of germline mRNA molecules into processed pseudogenes, as well as the life-long production of both male and female gametes.

Molecular genetic analysis reveals atypical confined placental mosaicism with a small supernumerary marker chromosome derived from chromosome 18: A clinical report of discordant results from three prenatal tests

We present a case with discordant results in three prenatal screening methods, with additional genetic analyses. Non-invasive prenatal testing (NIPT) was performed on a 41-year-old Japanese woman at 10 weeks of gestation, and the result was positive for trisomy 18 with high accuracy. Amniocentesis was performed at 16 weeks of gestation. However, the result showed 47,XX,+mar[16]/47,XX,+18[2]. Fetal examination by ultrasound revealed no malformations. After termination of the pregnancy, we performed additional genetic analyses, and confirmed the presence of confined placental mosaicism (CPM). Furthermore, a small supernumerary marker chromosome (sSMC) was detected in fetal cells, which was derived de novo from the centromere of chromosome 18. Single nucleotide polymorphism array analysis revealed that fetal chromosome 18 was inherited with maternal uniparental disomy, with a relatively large copy-neutral loss of heterozygosity, including its centromere. Our genetic analyses strongly indicated the cause of result discrepancy in prenatal testing as incomplete trisomy 18 rescue leading to atypical CPM with a sSMC. These findings also offer insight into the mechanisms by which chromosomal aberrations form during human oogenesis and embryogenesis.

Quantitative Proteomic Profiling of the Human Ovary from Early to Mid-Gestation Reveals Protein Expression Dynamics of Oogenesis and Folliculogenesis.

The in vivo gene networks involved in coordinating human fetal ovarian development remain obscure. In this study, quantitative mass spectrometry was conducted on ovarian tissue collected at key stages during the first two trimesters of human gestational development, confirming the expression profiling data using immunofluorescence, as well as in vitro modeling with human oogonial stem cells (OSCs) and human embryonic stem cells (ESCs). A total of 3,837 proteins were identified in samples spanning developmental days 47-137. Bioinformatics clustering and Ingenuity Pathway Analysis identified DNA mismatch repair and base excision repair as major pathways upregulated during this time. In addition, MAEL and TEX11, two key meiosis-related proteins, were identified as highly expressed during the developmental window associated with fetal oogenesis. These findings were confirmed and extended using in vitro differentiation of OSCs into in vitro derived oocytes and of ESCs into primordial germ cell-like cells and oocyte-like cells, as models. In conclusion, the global protein expression profiling data generated by this study have provided novel insights into human fetal ovarian development in vivo and will serve as a valuable new resource for future studies of the signaling pathways used to orchestrate human oogenesis and folliculogenesis.

In vitro differentiation of human embryonic stem cells into ovarian follicle-like cells

Understanding the unique mechanisms of human oogenesis necessitates the development of an in vitro system of stem cell differentiation into oocytes. Specialized cell types and organoids have been derived from human pluripotent stem cells in vitro, but generating a human ovarian follicle remains a challenge. Here we report that human embryonic stem cells can be induced to differentiate into ovarian follicle-like cells (FLCs) in vitro. First, we find that two RNA-binding proteins specifically expressed in germ cells, DAZL and BOULE, regulate the exit from pluripotency and entry into meiosis. By expressing DAZL and BOULE with recombinant human GDF9 and BMP15, these meiotic germ cells are further induced to form ovarian FLCs, including oocytes and granulosa cells. This robust in vitro differentiation system will allow the study of the unique molecular mechanisms underlying human pluripotent stem cell differentiation into late primordial germ cells, meiotic germ cells and ovarian follicles.

Conserved roles of mouse DUX and human DUX4 in activating cleavage-stage genes and MERVL/HERVL retrotransposons.

To better understand transcriptional regulation during human oogenesis and pre-implantation development, we defined stage-specific transcription, which revealed the cleavage stage as highly distinctive. Here, we present multiple lines of evidence that a eutherian-specific, multi-copy retrogene, DUX4, encodes a transcription factor which activates hundreds of endogenous genes (e.g. ZSCAN4, ZFP352, KDM4E) and retroviral elements (MERVL/HERVL-family) that defines the cleavage-specific transcriptional programs in mouse and human. Remarkably, mouse Dux expression is both necessary and sufficient to convert mouse embryonic stem cells into two-cell embryo-like (‘2C-like’) cells, measured here by the reactivation of ‘2C’ genes and repeat elements, the loss of POU5F1 protein and chromocenters, and by the conversion of the chromatin landscape (assessed by ATAC-seq) to a state strongly resembling mouse two-cell embryos. Taken together, we propose mouse DUX and human DUX4 as major drivers of the cleavage/‘2C’ state.

RNA-binding proteins in human oogenesis: Balancing differentiation and self-renewal in the female fetal germline

Primordial germ cells undergo three significant processes on their path to becoming primary oocytes: the initiation of meiosis, the formation and breakdown of germ cell nests, and the assembly of single oocytes into primordial follicles. However at the onset of meiosis, the germ cell becomes transcriptionally silenced. Consequently translational control of pre-stored mRNAs plays a central role in coordinating gene expression throughout the remainder of oogenesis; RNA binding proteins are key to this regulation. In this review we examine the role of exemplars of such proteins, namely LIN28, DAZL, BOLL and FMRP, and highlight how their roles during germ cell development are critical to oogenesis and the establishment of the primordial follicle pool.

Why have Scientists Accepted Zuckerman's Opinion that no Human Oogenesis after Birth?

Why have Scientists Accepted Zuckerman's Opinion that no Human Oogenesis after Birth?