Germ cells of the mammalian female: A limited or renewable resource? Hainaut M et al. (2021) In many non-mammalian organisms, a population of germ-line stem cells supports continuing production of gametes during most or all the life of the individual, and germ-line stem cells are also present and functional in male mammals. Traditionally, however, they have been thought not to exist in female mammals, who instead generate all their germ cells during fetal life. Over the last several years, this dogma has been challenged by several reports, while supported by others. We describe and compare these conflicting studies with the aim of understanding how they came to opposing conclusions. We first consider studies that, by examining marker-gene expression, the fate of genetically marked cells, and consequences of depleting the oocyte population, addressed whether ovaries of post-natal females contain oogonial stem cells (OSC) that give rise to new oocytes. We next discuss whether ovaries contain cells that, even if inactive under physiological conditions, nonetheless possess OSC properties that can be revealed through cell-culture. We then examine studies of whether cells harvested after long-term culture of cells obtained from ovaries can, following transplantation into ovaries of recipient females, give rise to oocytes and offspring. Finally, we note studies where somatic cells have been re-programmed to acquire a female germ-cell fate. We conclude that the weight of evidence strongly supports the traditional interpretation that germ-line stem cells do not exist post-natally in female mammals. However, the ability to generate germ cells from somatic cells in vitro establishes a method to generate new gametes from cells of post-natal mammalian females.//////////////////Toyooka Y, et al 2000 reported expression and intracellular localization of mouse
Vasa-homologue protein during germ cell development.
To demonstrate the cellular and subcellular localization of mouse vasa
homologue protein during germ cell development, specific antibody was raised
against the full-length MVH protein. The immunohistochemical analyses
demonstrated that MVH protein was exclusively expressed in primordial germ
cells just after their colonization of embryonic gonads and in germ cells
undergoing gametogenic processes until the post-meiotic stage in both males
and females.
NCBI Summary:
DEAD box proteins, characterized by the conserved motif Asp-Glu-Ala-Asp (DEAD), are putative RNA helicases. They are implicated in a number of cellular processes involving alteration of RNA secondary structure such as translation initiation, nuclear and mitochondrial splicing, and ribosome and spliceosome assembly. Based on their distribution patterns, some members of this family are believed to be involved in embryogenesis, spermatogenesis, and cellular growth and division. This gene encodes a DEAD box protein, which is a homolog of VASA proteins in Drosophila and several other species. The gene is specifically expressed in the germ cell lineage in both sexes and functions in germ cell development. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Oct 2009]
General function
RNA processing
Comment
Stem Cells. 2017 Jan;35(1):236-247. doi: 10.1002/stem.2504. Epub 2016 Nov 7.
Regulation of Injury-Induced Ovarian Regeneration by Activation of Oogonial Stem Cells.
Isolation of a DEAD-family protein gene that encodes a murine homolog of Drosophila vasa and its specific expression in germ cell lineage. Fujiwara Y et al. In an effort to study the molecular basis of the determination processes of the mammalian germ cell lineage, we have tried to isolate a mouse gene homolog to vasa, which plays an essential role as a maternal determining factor for the formation of Drosophila germ cell precursors. By reverse transcriptase PCRs of mouse primordial germ cell cDNAs using family-specific primers, we obtained a gene (Mvh) encoding a DEAD-family protein that showed a much higher degree of similarity with the product of the Drosophila vasa gene (vas) than previously reported mouse genes. In adult tissues, Mvh transcripts were exclusively detected in testicular germ cells, in which Mvh protein was found to be localized in cytoplasm of spermatocytes and round spermatids including a perinuclear granule. The protein was also expressed in germ cells colonized in embryonic gonads but was not detected in pluripotential embryonic cells such as stem cells and germ cells. These results suggest the possibility that the Mvh protein may play an important role in the determination events of mouse germ cells as in the case of Drosophila vasa.
Cellular localization
Cytoplasmic, Plasma membrane
Comment
In mouse oocytes the mitochondrion-originated germinal body-like structures accumulate mouse Vasa homologue (MVH) protein. Reunov AA 2014 et al.
Summary Mouse Vasa homologue (MVH) antibodies were applied to mouse Graafian oocytes to clarify if mitochondrion-originated germinal body-like structures, described previously by conventional electron microscopy, were associated with the germ plasm. It was found that both the mitochondrion-like structures with cristae and the germinal body-like structures that lacked any signs of cristae were labelled specifically by the anti-MVH antibody. Moreover, some granules were MVH-positive ultrastructural hybrids of the mitochondria and germinal body-like structures, the presence of which clearly supported the idea of a mitochondrial origin for the germinal body-like structures. This finding is the first evidence that mitochondrion-originated germinal body-like granules represent mouse germ plasm.
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Experimental evidence showing that no mitotically active female germline progenitors exist in postnatal mouse ovaries. Zhang H et al. It has been generally accepted for more than half a century that, in most mammalian species, oocytes cannot renew themselves in postnatal or adult life, and that the number of oocytes is already fixed in fetal or neonatal ovaries. This assumption, however, has been challenged over the past decade. In this study, we have taken an endogenous genetic approach to this question and generated a multiple fluorescent Rosa26(rbw/+);Ddx4-Cre germline reporter mouse model for in vivo and in vitro tracing of the development of female germline cell lineage. Through live cell imaging and de novo folliculogenesis experiments, we show that the Ddx4-expressing cells from postnatal mouse ovaries did not enter mitosis, nor did they contribute to oocytes during de novo folliculogenesis. Our results provide evidence that supports the traditional view that no postnatal follicular renewal occurs in mammals, and no mitotically active Ddx4-expressing female germline progenitors exist in postnatal mouse ovaries.
Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women. White YA et al. Germline stem cells that produce oocytes in vitro and fertilization-competent eggs in vivo have been identified in and isolated from adult mouse ovaries. Here we describe and validate a fluorescence-activated cell sorting-based protocol that can be used with adult mouse ovaries and human ovarian cortical tissue to purify rare mitotically active cells that have a gene expression profile that is consistent with primitive germ cells. Once established in vitro, these cells can be expanded for months and can spontaneously generate 35- to 50-?m oocytes, as determined by morphology, gene expression and haploid (1n) status. Injection of the human germline cells, engineered to stably express GFP, into human ovarian cortical biopsies leads to formation of follicles containing GFP-positive oocytes 1-2 weeks after xenotransplantation into immunodeficient female mice. Thus, ovaries of reproductive-age women, similar to adult mice, possess rare mitotically active germ cells that can be propagated in vitro as well as generate oocytes in vitro and in vivo.
Production of offspring from a germline stem cell line derived from neonatal ovaries. Zou K et al. The idea that females of most mammalian species have lost the capacity for oocyte production at birth has been challenged recently by the finding that juvenile and adult mouse ovaries possess mitotically active germ cells. However, the existence of female germline stem cells (FGSCs) in postnatal mammalian ovaries still remains a controversial issue among reproductive biologists and stem cell researchers. We have now established a neonatal mouse FGSC line, with normal karyotype and high telomerase activity, by immunomagnetic isolation and culture for more than 15 months. FGSCs from adult mice were isolated and cultured for more than 6 months. These FGSCs were infected with GFP virus and transplanted into ovaries of infertile mice. Transplanted cells underwent oogenesis and the mice produced offspring that had the GFP transgene. These findings contribute to basic research into oogenesis and stem cell self-renewal and open up new possibilities for use of FGSCs in biotechnology and medicine.
Differentiation potential of germ line stem cells derived from the postnatal mouse ovary. Pacchiarotti J et al. General belief in reproductive biology is that in most mammals female germ line stem cells are differentiated to primary oocytes during fetal development and oogenesis starts from a pool of primordial follicles after birth. This idea has been challenged previously by using follicle kinetics studies and demonstration of mitotically active germ cells in the postnatal mouse ovary (Johnson et al., 2004; Kerr et al., 2006; Zhang et al., 2008). However, the existence of a population of self-renewing ovarian germ line stem cells in postnatal mammals is still controversial (Eggan et al., 2006; Telfer et al., 2005; Gosden, 2004). Recently, production of offspring from a germ line stem cell line derived from the neonatal mouse ovary was reported (Zou et al., 2009). This report strongly supports the existence of germ line stem cells and their ability to expand in vitro. Recently, using a transgenic mouse model in which GFP is expressed under a germ cell-specific Oct-4 promoter, we isolated and generated multipotent cell lines from male germ line stem cells (Izadyar et al., 2008). Using the same strategy we isolated and derived cell lines from postnatal mouse ovary. Interestingly, ovarian germ line stem cells expanded in the same culture conditions as the male suggesting that they have similar requirements for their self-renewal. After 1 year of culture and many passages, ovarian germ line stem cells maintained their characteristics and telomerase activity, expressed germ cell and stem cell markers and revealed normal karyotype. As standard protocol for differentiation induction, these cells were aggregated and their ability to form embryoid bodies (EBs) was investigated. EBs generated in the presence of growth factors showed classical morphology and expressed specific markers for three germ layers. However, in the absence of growth promoting factors EBs were smaller and large cells with the morphological and molecular characteristics of oocytes were formed. This study shows the existence of a population of germ line stem cell in postnatal mouse ovary with multipotent characteristics.
Generation of Functional Oocytes and Spermatids from Fetal Primordial Germ Cells after Ectopic Transplantation in Adult Mice. Matoba S et al. Primordial germ cells (PGCs) are undifferentiated germ cells in developing fetuses. As they give rise to definitive oocytes and spermatozoa that contribute to new life in the next generation, their development must be under strict control regarding genetic and epigenetic aspects. However, we do not know to what extent their development depends on the specific milieu. In this study, we transplanted mouse PGCs collected from male and female gonads at 12.5 days post coitum (dpc) under the kidney capsule of adult mice, together with gonadal somatic cells. The transplanted cells constructed testis-like and ovary-like tissues, respectively, under the kidney capsule within 4 wk. Normal-looking round spermatids and fully grown germinal vesicle (GV) oocytes developed within these tissues. Ectopic spermatogenesis continued thereafter, while oogenesis consisted of only a single wave. The injection of these round spermatids directly into mature in vivo-derived oocytes led to the birth at term of normal pups. PGC-derived GV oocytes were isolated, induced to mature in vitro, and injected with normal spermatozoa. The injected oocytes were successfully fertilized and developed into normal pups. Our findings demonstrate the remarkable flexibility of PGC development, which can proceed up to functional gametes under a spatially and temporally non-innate condition. This transplantation system may provide a unique technical basis for the induction of the development of early germ cells of exogenous origins, such as those from embryonic stem cells.
Expression regulated by
Comment
Ovarian localization
Primordial Germ Cell, Oocyte
Comment
Tsunekawa N, et al isolated a chicken homolog (Cvh) to vasa gene (vas), which
plays
an essential role in germline formation in Drosophila. They demonstrate the
germline-specific expression of CVH protein throughout all stages of
development. Immunohistochemical analyses using specific antibody raised
against CVH protein indicated that CVH protein was localized in cytoplasm of
germ cells ranging from presumptive primordial germ cells (PGCs) in
uterine-stage embryos to spermatids and oocytes in adult gonads. In the oocytes, CVH protein was predominantly localized in
granulofibrillar structures surrounding the mitochondrial cloud and spectrin
protein-enriched structure, indicating that the CVH-containing cytoplasmic
structure is the precursory germ plasm in the chicken, These results
strongly
suggest that the chicken germline is determined by maternally inherited
factors in the germ plasm.
Castrillon DH, et al 2000 reported that the human VASA gene is specifically expressed in the germ cell
lineage.
Northern analysis of fetal
and adult tissues showed that expression of the human VASA gene is restricted to the
ovary and testis and is undetectable in somatic tissues. The VASA protein is cytoplasmic
and expressed in migratory primordial germ cells in the region of the gonadal
ridge. VASA protein is present in fetal and adult gonadal germ cells in both
males and females and is most abundant in spermatocytes and mature oocytes,
The gene we have isolated is thus a highly specific marker of germ cells and
should be useful for studies of human germ cell determination and function.
Expression of pluripotent stem cell markers in the human fetal ovary. Kerr CL et al. BACKGROUND Human primordial germ cells (PGCs) can give rise to pluripotent stem cells such as embryonal carcinoma cells (ECCs) and embryonic germ cells (EGCs). METHODS In order to determine whether PGCs express markers associated with pluripotency in EGCs and ECCs, the following study cross examines the expression patterns of multiple pluripotent markers in the human fetal ovary, 5.5-15 weeks post-fertilizaton (pF) and relates this expression with the ability to derive pluripotent EGCs in vitro. RESULTS Specific subpopulations were identified which included OCT4(+)/Nanog(+)/cKIT(+)/VASA(+) PGCs and oogonia. Interestingly, these cells also expressed SSEA1 and alkaline phosphatase (AP) and SSEA4 expression occurred throughout the entire gonad. Isolation of SSEA1(+) cells from the gonad resulted in AP(+) EGC colony formation. The number of OCT4(+) or Nanog(+) expressing cells peaked by week 8 and then diminished after week 9 pF, as oogonia enter meiosis. In addition, the efficiency of EGC derivation was associated with the number of OCT4(+) cells. TRA-1-60 and TRA-1-81 were only detected in the lining of the mesonephric ducts and occasionally in the gonad. CONCLUSIONS These results demonstrate that PGCs, a unipotent cell, express most, but not all, of the markers associated with pluripotent cells in the human fetal ovary.
Phenotypes
Mutations
2 mutations
Species: mouse
Mutation name: type: None fertility: fertile Comment: Geography of Follicle Formation in the Embryonic Mouse Ovary Impacts Activation Pattern during the First Wave of Folliculogenesis. Cordeiro MH et al. (2015) During embryonic development mouse female germ cells enter meiosis in an anterior to posterior wave believed to be driven by retinoic acid. It has been proposed that ovarian follicle formation and activation follows the same general wave of meiotic progression, however the precise anatomical specification of these processes has not been delineated. Here, we created a mouse line using Mvh, Gdf9 and Zp3 promoters to drive distinct temporal expression of three fluorescent proteins in the oocytes and to identify where the first follicle cohort develops. The fluorescent profile revealed that the first growing follicles consistently appear in a specific region of the ovary, the anterior-dorsal region, which led us to analyze if meiotic onset occurred earlier in the dorsal ovarian region. Surprisingly, in addition to the anterior to posterior wave, we observed an early meiotic entry in the ventral region of the ovary. This additional anatomical stratification of meiosis contrasts with the localization of the initial follicle formation and activation in the dorsal region of the ovary. Therefore, our study suggests that the specification of cortical and medullar areas in the ventral and dorsal regions on the ovary, rather than the onset of meiosis, impacts where the first follicular activation event occurs.//////////////////
Species: C. elegans
Mutation name: type: null mutation fertility: None Comment: Using a combination of proteomics, cytology, and functional analysis in C. elegans, Chu et al. (2006) reduced 1,099 proteins copurified with spermatogenic chromatin to 132 proteins for functional analysis. This strategy to find fertility factors conserved from C. elegans to mammals achieved its goal: of mouse gene knockouts corresponding to nematode proteins, 37% (7 of 19) cause male sterility. This list includes PPP1CC (176914), H2AX (601772), SON (182465), TOP1 (126420), DDX4, DBY (400010), and CENPC (117141).