-
Nature May 2024Epigenetic reprogramming resets parental epigenetic memories and differentiates primordial germ cells (PGCs) into mitotic pro-spermatogonia or oogonia. This process...
Epigenetic reprogramming resets parental epigenetic memories and differentiates primordial germ cells (PGCs) into mitotic pro-spermatogonia or oogonia. This process ensures sexually dimorphic germ cell development for totipotency. In vitro reconstitution of epigenetic reprogramming in humans remains a fundamental challenge. Here we establish a strategy for inducing epigenetic reprogramming and differentiation of pluripotent stem-cell-derived human PGC-like cells (hPGCLCs) into mitotic pro-spermatogonia or oogonia, coupled with their extensive amplification (about >10-fold). Bone morphogenetic protein (BMP) signalling is a key driver of these processes. BMP-driven hPGCLC differentiation involves attenuation of the MAPK (ERK) pathway and both de novo and maintenance DNA methyltransferase activities, which probably promote replication-coupled, passive DNA demethylation. hPGCLCs deficient in TET1, an active DNA demethylase abundant in human germ cells, differentiate into extraembryonic cells, including amnion, with de-repression of key genes that bear bivalent promoters. These cells fail to fully activate genes vital for spermatogenesis and oogenesis, and their promoters remain methylated. Our study provides a framework for epigenetic reprogramming in humans and an important advance in human biology. Through the generation of abundant mitotic pro-spermatogonia and oogonia-like cells, our results also represent a milestone for human in vitro gametogenesis research and its potential translation into reproductive medicine.
PubMed: 38768632
DOI: 10.1038/s41586-024-07526-6 -
Fertility and Sterility Jan 2024The oocyte, a long-lived, postmitotic cell, is the locus of reproductive aging in women. Female germ cells replicate only during fetal life and age throughout... (Review)
Review
The oocyte, a long-lived, postmitotic cell, is the locus of reproductive aging in women. Female germ cells replicate only during fetal life and age throughout reproductive life. Mechanisms of oocyte aging include the accumulation of oxidative damage, mitochondrial dysfunction, and disruption of proteins, including cohesion. Nobel Laureate Bob Edwards also discovered a "production line" during oogonial replication in the mouse, wherein the last oocytes to ovulate in the adult-derived from the last oogonia to exit mitotic replication in the fetus. On the basis of this, we proposed a two-hit "telomere theory of reproductive aging" to integrate the myriad features of oocyte aging. The first hit was that oocytes remaining in older women traversed more cell cycles during fetal oogenesis. The second hit was that oocytes accumulated more environmental and endogenous oxidative damage throughout the life of the woman. Telomeres (Ts) could mediate both of these aspects of oocyte aging. Telomeres provide a "mitotic clock," with T attrition an inevitable consequence of cell division because of the end replication problem. Telomere's guanine-rich sequence renders them especially sensitive to oxidative damage, even in postmitotic cells. Telomerase, the reverse transcriptase that restores Ts, is better at maintaining than elongating T. Moreover, telomerase remains inactive during much of oogenesis and early development. Oocytes are left with short Ts, on the brink of viability. In support of this theory, mice with induced T attrition and women with naturally occurring telomeropathy suffer diminished ovarian reserve, abnormal embryo development, and infertility. In contrast, sperm are produced throughout the life of the male by a telomerase-active progenitor, spermatogonia, resulting in the longest Ts in the body. In mice, cleavage-stage embryos elongate Ts via "alternative lengthening of telomeres," a recombination-based mechanism rarely encountered outside of telomerase-deficient cancers. Many questions about Ts and reproduction are raised by these findings: does the "normal" T attrition observed in human oocytes contribute to their extraordinarily high rate of meiotic nondisjunction? Does recombination-based T elongation render embryos susceptible to mitotic nondisjunction (and mosaicism)? Can some features of Ts serve as markers of oocyte quality?
Topics: Male; Female; Humans; Mice; Animals; Aged; Telomerase; Semen; Reproduction; Aging; Oocytes; Telomere
PubMed: 37993053
DOI: 10.1016/j.fertnstert.2023.11.012 -
Biology of Reproduction Jul 2023Follicular development is a critical process in reproductive biology that determines the number of oocytes and interacts with various cells within the follicle (such as...
Follicular development is a critical process in reproductive biology that determines the number of oocytes and interacts with various cells within the follicle (such as oocytes, granulosa cells, cumulus cells and theca cells, etc.), and plays a vital role in fertility and reproductive health due to the dogma of a limited number of oogonia. Dysregulation of follicular development can lead to infertility problems and other reproductive disorders. To explore the physiological and pathological mechanisms of follicular development, immunology-based methods, microarrays, and next-generation sequencing have traditionally been used for characterization at the tissue level. However, with the proliferation of single-cell sequencing techniques, research has uncovered unique molecular mechanisms in individual cells that have been masked by previous holistic analyses. In this review, we briefly summarize the achievements and limitations of traditional methods in the study of follicular development. Simultaneously, we focus on how to understand the physiological process of follicular development at the single-cell level and reveal the relevant mechanisms leading to the pathology of follicular development and intervention targets. Moreover, we also summarize the limitations and application prospects of single cell sequencing in follicular development research.
PubMed: 37504504
DOI: 10.1093/biolre/ioad080 -
Theriogenology Jan 2023The aim of this study was to test whether vitrification of sterlet Acipenser ruthenus and Russian sturgeon Acipenser gueldenstaedtii ovarian tissue through...
The aim of this study was to test whether vitrification of sterlet Acipenser ruthenus and Russian sturgeon Acipenser gueldenstaedtii ovarian tissue through needle-immersed vitrification (NIV) is an efficient strategy for the preservation of oogonia (OOG) in order to supplement the current conservation efforts for these endangered fish species. Histological analyses of the gonads displayed that the ovaries of both species were immature and contained predominantly OOG and primary oocytes. The germline origin of these cells was verified by localization of the vasa protein through immunocytochemistry. NIV protocol was optimized by testing different equilibration (ES) and vitrification solutions (VS) containing various concentrations of dimethyl sulfoxide (MeSO), propylene glycol (PG) or methanol (MeOH). In sterlet, the highest average viability (55.7 ± 11.5%) was obtained by using a combination of 1.5 M PG and 1.5 M MeSO in the ES, and 1.5 M MeOH and 5.5 M MeSO in the VS. In Russian sturgeon, the highest average viability (49.4 ± 17.1%) was obtained by using a combination of 1.5 M MeOH and 1.5 M MeSO in the ES, and 3 M PG and 3 M MeSO in the VS. To test whether vitrified/warmed OOG are functional, we have conducted an intra-specific transplantation assay to verify whether transplanted sterlet OOG will colonize the gonads of recipient fish. Fluorescently labelled cells were detected within recipient gonads at 2 and 3 months post-fertilization (mpf). Colonization rates of vitrified/warmed OOG (70% at 2 mpf and 61% at 3 mpf) were similar to those of fresh OOG (80% at 2 mpf and 70% at 3 mpf). This study has demonstrated that vitrification of ovarian tissue is an effective method for the preservation of OOG, and that the vitrified/warmed cells are functional and are able to colonize recipient gonads after transplantation similarly to the fresh cells. Since the vitrification procedure displayed in this study is simple and does not require complex and expensive laboratory equipment, it can be readily applied in field conditions, and therefore it can be invaluable for the conservation efforts of the critically endangered sturgeon species. However, care needs to be taken that despite the research conducted so far, donor-derived progeny was not yet obtained in sturgeons.
Topics: Animals; Vitrification; Fishes; Ovary
PubMed: 36375212
DOI: 10.1016/j.theriogenology.2022.11.009 -
The EMBO Journal May 2023Human 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...
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.
Topics: Animals; Female; Humans; Macaca fascicularis; Oocytes; Oogenesis; Ovary; Embryonic Stem Cells
PubMed: 36929479
DOI: 10.15252/embj.2022112962 -
Sexual Development : Genetics,... 2022Whether to produce sperm or eggs is the most basic and important choice from the perspective of germ cell development and differentiation. However, the induction... (Review)
Review
BACKGROUND
Whether to produce sperm or eggs is the most basic and important choice from the perspective of germ cell development and differentiation. However, the induction mechanism has not received much attention until relatively recently. This is because the issue of sexual differentiation has generally been considered a theme of somatic cells to make a testis or ovary. Basically, the sex of individual somatic cells and germ cells matches. Therefore, the sex of germ cells is thought to follow the sex of somatic cells once determined. However, researchers realized that a big, open question remained: What somatic cell signals actually induce the sexual differentiation of germ cells and what is the sex determinant in germ cells?
SUMMARY
In vitro experiments demonstrated that 2 somatic signals (BMP and RA) act directly on germ cells to induce oogonia. Therefore, these 2 signals may be referred to as oogonia inducers. From the viewpoint of germ cells, an independent experiment identified SMAD4 and STRA8, which are directly downstream of BMP and RA, respectively, acting in germ cells as female determinants. However, what about male? If these factors are female determinants, their absence may result in the induction of spermatogonia. This may be true in vivo because germ cells enter a male pathway if they do not receive these signals even in the ovary. However, this has not been confirmed in an in vitro culture system. There should be signals required for germ cells to enter a male pathway.
KEY MESSAGES
The important message is that although testis-specific factors secreted from the testis are considered to include male-inducing factors for germ cells, this may not be the case, and the male-inducing factor, if it exists, also exists in the ovary.
PubMed: 35263749
DOI: 10.1159/000520976 -
Plant Disease May 2022Sanqi (Panax notoginseng (Burk.) F. H. Chen) is a precious traditional Chinese herbal medicine. During April of 2021, a root rot disease with approximate 15% incidence...
Sanqi (Panax notoginseng (Burk.) F. H. Chen) is a precious traditional Chinese herbal medicine. During April of 2021, a root rot disease with approximate 15% incidence was observed on 2-year-old Sanqi plants in a field of Zhouning (27º12' N, 119°33' E), Fujian Province of China. The disease symptoms included severe stunting, leaf chlorosis, root rotting and necrosis, as the disease progressed, the whole plant gradually wilted and died. To recover the causal agent, symptomatic roots were excised, surface sterilized in 75% alcohol for 1.5 min, rinsed in sterilized water three times, dried, and placed on PARP selective medium (Jeffers and Martin 1986), and incubated at 20°C in dark. After 5 days, total of 26 Pythium-like isolates were obtained, and one representative isolate Py21-6 (available from the Institute of Plant Protection, Fujian Academy of Agricultural Sciences) was selected for further identification. Colonies of Py21-6 on PARP plate were white with dense, cottony, aerial, and transparent mycelia. Sporangia were terminal or intercalary, non-papillate, spherical, pyriform or ovoid, measuring 21.7 ± 2.8 × 19.3 ± 2.3 μm (n = 30). Zoospores were saucer-like, released out of sporangium after maturation, and dispersed quickly by swimming. Oogonia were spherical, terminal or occasionally intercalary. Oospores were globose, smooth and aplerotic. The dimensions of zoospores, oogonia, and oospores were 6.8 ± 0.7 μm, 21.6 ± 2.2 μm and 18.2 ± 2.7 μm (n = 30), respectively. Antheridia were bell-shaped or irregular, terminal, monoclinous, and usually one per oogonium. According to the morphological characteristics the isolate was initially identified as Pythium spp. (Van der Plaats-Niterink 1981, Yong et al. 2016). For further identification, DNA extracted from Py21-6, the cytochrome c oxidase subunit I (COI) gene and internal transcribed spacer (ITS) region were amplified and sequenced with primers FM55/FM52R (Long et al. 2012) and ITS1 /ITS4 (White et al. 1990), respectively. BLAST analysis of 680-bp COI (OM688194) and 728-bp ITS (OM663703) sequences revealed 99.86% and 99.99% similarity to Pythium vexans in GenBank (HQ708995 [COI], GU133572 [ITS]). Therefore, the pathogen was identified as P. vexans. In order to fulfill Koch's postulates, isolate Py21-6 was grown on Martin's liquid medium (Martin 1992) for 72 h to produce a spore suspensions of 106 oospores/ml, and the pathogenicity test was conducted by root-dip method. Three groups of 2-year-old Sanqi (15 plants per group) with root soaked for 20 min in oospore suspension were used for pathogenicity, and the other three groups (15 plants per group) with root dipped in sterilized water as control. All treated plants were replanted in (15-cm-diameter) pots (2 plants/pot) filled with mixture of sterilized soil: vermiculite: pearlite (2:1:1, v/v), maintained in greenhouse under 60% black shade cloth at 20 to 26°C with 80% relative humidity, and watered once every three days. After 21days, all inoculated plants showed the same symptoms observed on the original diseased plants in the field, whereas, the control plants remained symptomless. The same pathogen was successfully re-isolated from the inoculated plants, and identical to those of the originals based on morphological and sequence data. To our knowledge, this is the first report of P. vexans causing root rot on Sanqi in China (Farr and Rossman 2022). Root rot is one of the destructive diseases in Sanqi production, identification of the pathogen will be useful to develop effective field management strategies to control this disease.
PubMed: 35581918
DOI: 10.1094/PDIS-04-22-0781-PDN -
Stem Cells International 2021Germ cells are capable of maintaining species continuity through passing genetic and epigenetic information across generations. Female germ cells mainly develop during... (Review)
Review
Germ cells are capable of maintaining species continuity through passing genetic and epigenetic information across generations. Female germ cells mainly develop during the embryonic stage and pass through subsequent developmental stages including primordial germ cells, oogonia, and oocyte. However, due to the limitation of using early human embryos as research model, research models are needed to reveal the early developmental process and related mechanisms of female germ cells. After birth, the number of follicles gradually decreases with age. Various conditions which damage ovarian functions would cause premature ovarian failure. Alternative treatments to solve these problems need to be investigated. Germ cell differentiation from pluripotent stem cells can simulate early embryonic development of female germ cells and clarify unresolved issues during the development process. In addition, pluripotent stem cells could potentially provide promising applications for female fertility preservation after proper differentiation. Mouse female germ cells have been successfully reconstructed and delivered to live offspring. However, the derivation of functional human female germ cells has not been fully achieved due to technical limitations and ethical issues. To provide an updated and comprehensive information, this review centers on the major studies on the differentiation of mouse and human female germ cells from pluripotent stem cells and provides references to further studies of developmental mechanisms and potential therapeutic applications of female germ cells.
PubMed: 33510796
DOI: 10.1155/2021/8849230 -
International Journal of Molecular... Apr 2023It is a well-known fact that the reproductive organs in women, especially oocytes, are exposed to numerous regulatory pathways and environmental stimuli. The maternal... (Review)
Review
It is a well-known fact that the reproductive organs in women, especially oocytes, are exposed to numerous regulatory pathways and environmental stimuli. The maternal age is one cornerstone that influences the process of oocyte fertilization. More precisely, the longer a given oocyte is in the waiting-line to be ovulated from menarche to menopause, the longer the duration from oogenesis to fertilization, and therefore, the lower the chances of success to form a viable embryo. The age of menarche in girls ranges from 10 to 16 years, and the age of menopause in women ranges from approximately 45 to 55 years. Researchers are paying attention to the regulatory pathways that are impacting the oocyte at the very beginning during oogenesis in fetal life to discover genes and proteins that could be crucial for the oocyte's lifespan. Due to the general trend in industrialized countries in the last three decades, women are giving birth to their first child in their thirties. Therefore, maternal age has become an important factor impacting oocytes developmental competence, since the higher a woman's age, the higher the chances of miscarriage due to several causes, such as aneuploidy. Meiotic failures during oogenesis, such as, for instance, chromosome segregation failures or chromosomal non-disjunction, are influencing the latter-mentioned aging-related phenomenon too. These errors early in life of women can lead to sub- or infertility. It cannot be neglected that oogenesis is a precisely orchestrated process, during which the oogonia and primary oocytes are formed, and RNA synthesis takes place. These RNAs are crucial for oocyte growth and maturation. In this review, we intend to describe the relevance of regulatory pathways during the oogenesis in women. Furthermore, we focus on molecular pathways of oocyte developmental competence with regard to maternal effects during embryogenesis. On the background of transcriptional mechanisms that enable the transition from a silenced oocyte to a transcriptionally active embryo, we will briefly discuss the potential of induced pluripotent stem cells.
Topics: Pregnancy; Female; Humans; Maternal Age; Oogenesis; Oocytes; Ovulation; Stem Cells
PubMed: 37047809
DOI: 10.3390/ijms24076837