Mammalian Germ Cells Research Articles

STRA8 Shuttles between Nucleus and Cytoplasm and Displays Transcriptional Activity

Stra8 (stimulated by retinoic acid 8) encodes a protein crucial for mammalian germ cells entering into premeiotic stages. Here, to elucidate the still unknown STRA8 molecular functions, we studied the cellular localization of the protein in several cell types, including premeiotic mouse germ cells and stem cell lines. We reported distinct STRA8 localization in germ and stem cell types and a heterogeneous protein distribution in the cytoplasm and nucleus of such cells suggesting that the protein can shuttle between these two compartments. Moreover, we identified specific protein motifs determining its nuclear import/export. Furthermore, we demonstrated that in transfected cell lines the nuclear import of STRA8 is an active process depending on an N-terminal basic nuclear localization signal. Moreover, its nuclear export is mainly mediated by the Exportin1 (XPO1) recognition of a nuclear export signal. Significantly, we also demonstrated that STRA8 associates with DNA and possesses transcriptional activity. These observations strongly suggest that STRA8 can exert important functions in the nucleus rather than in the cytoplasm as believed previously, likely depending on the cell type and regulated by its nuclear-cytoplasmic shuttling.

05-P009 Role of replacement histone H3.3 in mammalian germ cell development

05-P009 Role of replacement histone H3.3 in mammalian germ cell development

07-P009 Doublesex variants in the honey bee, Apis mellifera: Lack of sex-specific expression and characterization of novel transcripts

embryo and differentiated adult tissues, with particular focus on a cancer associated variant that excludes the 4th exon (DE4), but retains replication function. Using RT-PCR, qPCR and immunohistochemistry, we find that, although Ciz1 is present in most cells throughout development, high levels are restricted to adult testes, with temporal regulation within the developing germ cell lineage. Over 40% of adult testicular transcripts are alternatively spliced, with some variants unique to this tissue. The regulated induction and alternative splicing of Ciz1 coincides with activation of the spermatogenic cycle. The protein is dynamically regulated in germ cells at discreet stages of the differentiation process, characterised most notably by loss of Ciz1 from post-replicative cells and subsequent re-activation at greatly enhanced levels later in the differentiation process. Our data suggests that following initial replicative phases in spermatogonia and pre-leptotene spermatocytes ‘old’ Ciz1 is released or degraded. ‘New’ Ciz1 is then produced in copious amounts, indicating that Ciz1 has a novel post-replicative role in the mammalian germ cell differentiation process.

Control of KIT signalling in male germ cells: what can we learn from other systems?

The KIT ligand (KITL)/KIT-signalling system is among several pathways known to be essential for fertility. In the postnatal testis, the KIT/KITL interaction is crucial for spermatogonial proliferation, differentiation, survival and subsequent entry into meiosis. Hence, identification of endogenous factors that regulate KIT synthesis is important for understanding the triggers driving germ cell maturation. Although limited information is available regarding local factors in the testicular microenvironment that modulate KIT synthesis at the onset of spermatogenesis, knowledge from other systems could be used as a basis for identifying how KIT function is regulated in germ cells. This review describes the known regulators of KIT, including transcription factors implicated in KIT promoter regulation. In addition, specific downstream outcomes in biological processes that KIT orchestrates are addressed. These are discussed in relationship to current knowledge of mammalian germ cell development.

Dazl Functions in Maintenance of Pluripotency and Genetic and Epigenetic Programs of Differentiation in Mouse Primordial Germ Cells In Vivo and In Vitro

BackgroundMammalian germ cells progress through a unique developmental program that encompasses proliferation and migration of the nascent primordial germ cell (PGC) population, reprogramming of nuclear DNA to reset imprinted gene expression, and differentiation of mature gametes. Little is known of the genes that regulate quantitative and qualitative aspects of early mammalian germ cell development both in vivo, and during differentiation of germ cells from mouse embryonic stem cells (mESCs) in vitro.Methodology and Principal FindingsWe used a transgenic mouse system that enabled isolation of small numbers of Oct4ΔPE:GFP-positive germ cells in vivo, and following differentiation from mESCs in vitro, to uncover quantitate and qualitative phenotypes associated with the disruption of a single translational regulator, Dazl. We demonstrate that disruption of Dazl results in a post-migratory, pre-meiotic reduction in PGC number accompanied by aberrant expression of pluripotency genes and failure to erase and re-establish genomic imprints in isolated male and female PGCs, as well as subsequent defect in progression through meiosis. Moreover, the phenotypes observed in vivo were mirrored by those in vitro, with inability of isolated mutant PGCs to establish pluripotent EG (embryonic germ) cell lines and few residual Oct-4-expressing cells remaining after somatic differentiation of mESCs carrying a Dazl null mutation. Finally, we observed that even within undifferentiated mESCs, a nascent germ cell subpopulation exists that was effectively eliminated with ablation of Dazl.Conclusions and SignificanceThis report establishes the translational regulator Dazl as a component of pluripotency, genetic, and epigenetic programs at multiple time points of germ cell development in vivo and in vitro, and validates use of the ESC system to model and explore germ cell biology.

A Signaling Principle for the Specification of the Germ Cell Lineage in Mice

Specification of the germ cell lineage is vital to development and heredity. In mice, the germ cell fate is induced in pluripotent epiblast cells by signaling molecules, yet the underlying mechanism remains unknown. Here we demonstrate that germ cell fate in the epiblast is a direct consequence of Bmp4 signaling from the extraembryonic ectoderm (ExE), which is antagonized by the anterior visceral endoderm (AVE). Strikingly, Bmp8b from the ExE restricts AVE development, thereby contributing to Bmp4 signaling. Furthermore, Wnt3 in the epiblast ensures its responsiveness to Bmp4. Serum-free, defined cultures revealed that, in response to Bmp4, competent epiblast cells uniformly expressed key transcriptional regulators Blimp1 and Prdm14 and acquired germ-cell properties, including genome-wide epigenetic reprogramming, in an orderly fashion. Notably, the induced cells contributed to both spermatogenesis and fertility of offspring. By identifying a signaling principle in germ cell specification, our study establishes a robust strategy for reconstituting the mammalian germ cell lineage in vitro.

Fluorescence activated cell sorting of live female germ cells and somatic cells of the mouse fetal gonad based on forward and side scattering

Analysis of female mammalian germ cells has been hindered by difficulties in isolating high purity germ cell populations from embryonic and fetal gonads. Meiotic prophase stage oocytes are particularly difficult to isolate due to the lack of suitable surface markers. Oct4 promoter driven GFP expression has been used to distinguish germ cells/oocytes (GFP positive) from somatic cells (GFP negative), however, the requirement for transgenic animals has limited the use of this technique. We analyzed the side- and forward scattering properties of living cell populations obtained from fetal ovaries of Oct4-GFP transgenic and wild-type mice. On the basis of these measurements, we defined criteria that allow the discrimination and identification of germ cells and somatic cells within cell suspensions of nontransgenic female fetal gonads. The described method is suitable for the isolation of populations of germ cells and somatic cells of higher than 90% purity. We also demonstrated that the sorted cells can be used in downstream immunofluorescence and RT-PCR applications. Hence, we conclude that side and forward scattering based sorting of female germ cells is a valuable tool that will benefit the understanding of female gametogenesis.

Flavouring Group Evaluation 220: alpha, beta‐Unsaturated ketones and precursors from chemical subgroup 4.4 of FGE.19: 3(2H)‐Furanones

Flavouring Group Evaluation 220: alpha, beta‐Unsaturated ketones and precursors from chemical subgroup 4.4 of FGE.19: 3(2H)‐Furanones

Foetal germ cells: striking the balance between pluripotency and differentiation

The germ line is unique in that highly specialised cells must be formed while retaining genomic totipotency. In mice, germ cells are derived from the pluripotent epiblast cells under the influence of inductive signals. This specification event involves reactivation or maintenance of core regulators of pluripotency, activation of germ line specific genetic programs, silencing of somatic cell programs and strict epigenetic management. These programs are thought to allow germ line specification while at the same time protecting underlying genomic totipotency. At this stage germ cells do not exhibit overt pluripotency but are able to establish pluripotent embryonic germ cells in culture. The primordial germ cells migrate to and populate the gonads at around embryonic day 10.5 and then undergo further epigenetic reprogramming and sex determination. Under the influence of the gonadal somatic cells the germ cells commit to male development and enter mitotic arrest or commit to female development and enter meiosis. This process involves down regulation of the core regulators of pluripotency, further differentiation of the germ cells, tight cell cycle regulation and loss of the germ cells ability to form pluripotent cells in culture. If this differentiation process is disrupted, germ cells can regain pluripotency and form tumours. This review focuses on the formation and early differentiation of germ cells in mammals with particular emphasis on the balance between germ cell differentiation and the maintenance of underlying genomic totipotency.

Prion protein and RNA: a view from the cytoplasm

Since it was posited that a cytoplasmic isoform of PrP may be involved in prion diseases, controversies about the isoform's biogenesis and function have emerged in the literature. While the existence of cytoplasmic PrP in vivo and in different cell cultures systems has now been well-established, whether it has specific activity remains unknown. This review outlines recent evidence about the molecular activity of cytoplasmic PrP. Cytoplasmic PrP inhibits a normal cellular stress response by preventing the assembly of protective mRNA stress granules and the synthesis of heat-shock protein 70 following environmental stress. Interference with the stress response correlates with the coalescence of mRNAs in a large cytoplasmic ribonucleoprotein particle. This particle shares similarities with the chromatoid body, a particle that organizes and controls RNA processing in mammalian germ cells as well as in neurons and stem cells from planarians with high regenerative abilities.

Germ Cell-Intrinsic and -Extrinsic Factors Govern Meiotic Initiation in Mouse Embryos

Retinoic acid (RA) is an essential extrinsic inducer of meiotic initiation in mammalian germ cells. However, RA acts too widely in mammalian development to account, by itself, for the cell-type and temporal specificity of meiotic initiation. We considered parallels to yeast, in which extrinsic and intrinsic factors combine to restrict meiotic initiation. We demonstrate that, in mouse embryos, extrinsic and intrinsic factors together regulate meiotic initiation. The mouse RNA-binding protein DAZL, which is expressed by postmigratory germ cells, is a key intrinsic factor, enabling those cells to initiate meiosis in response to RA. Within a brief developmental window, Dazl-expressing germ cells in both XX and XY embryos actively acquire the ability to interpret RA as a meiosis-inducing signal.

Progress in gene transfer by germ cells in mammals

Use of germ cells as vectors for transgenesis in mammals has been well developed and offers exciting prospects for experimental and applied biology, agricultural and medical sciences. Such approach is referred to as either male germ cell mediated gene transfer (MGCMGT) or female germ cell mediated gene transfer (FGCMGT) technique. Sperm-mediated gene transfer (SMGT), including its alternative method, testis-mediated gene transfer (TMGT), becomes an established and reliable method for transgenesis. They have been extensively used for producing transgenic animals. The newly developed approach of FGCMGT, ovary-mediated gene transfer (OMGT) is also a novel and useful tool for efficient transgenesis. This review highlights an overview of the recent progress in germ cell mediated gene transfer techniques, methods developed and mechanisms of nucleic acid uptake by germ cells.

FSHR and Premeiotic Marker STRA8 Expression of Adult Female Bone Marrow Stem Cells Induced by Retinoic Acid

One of the most devastating adverse effects of cancer treatments including bone marrow transplantation is damage to the reproductive system, which in young girls and women less than 40 years old is frequently associated with premature menopause and infertility, although adult bone marrow stem cells (BMSCs) are able to generate cells of all three germ layers under appropriate experimental conditions. These outcomes seem to be a result of cytotoxic effects on oocytes housed within the ovaries. Recent studies showed that female BMSCs (FBMSC) could differentiate into granulosa cells (GCs) and oocyte and form primordial follicles in ovary after FBMSCs transplantation and might have repaired chemotherapy-induced damage to the niche or the ovarian stroma. The formation of new primordial follicles in adult mammal ovaries is a controversial issue because some new studies have concluded that all offspring were derived from the recipient germ line after FBMSCs transplantation. To explore the effects of FBMSCs on female reproductive function, the experiment is to investigate whether adult female BMSCs are able to differentiate into Follicle-stimulating hormone receptor (FSHR, a GCs specific marker in the ovary cells) positive GCs and stra8 (stimulated by retinoic acid gene 8, a specific expression gene in mammalian germ cell's transition from mitosis to meiosis) expression oogonia in vitro induced by all-trans retinoic acid (RA).Methods: FBMSCs were separated from adult female SD rat bone marrow. FBMSCs were cultured in DMEM supplemented with 10% fetal bovine serum. All experiments were performed using FBMSCs from the 2nd passage adherent cell fraction. FBMSCs were cultured above medium with 10−6 M RA or without RA(control group). RT-PCR was performed to detect the expression of Stra8 mRNA and FSHR mRNA, Western blot and immunocytochemistry staining was utilized to examine the expression of FSHR in FBMSCs cultured with RA.Results:Some of the FBMSCs were large and round and had a oocyte-like cell appearance 7 days after induction culture.Some FBMSCs were FSHR positive by immunocytochemistry 3 days after induction differentiation, the FSHR+ cells were small or middle and round and were found in small clusters. Following longer in culture(from day 5 to day 7), the most of FSHR+ cells gradually became larger. The FBMSCs also expressed FSHR mRNA by RT-PCR and FSHR protein by Western Blotting 7days after induction differentiation. In control group, the FBMSC still kept undifferentiated relatively elongated or spindle-shaped cells that they were FSHR negative.The FBMSCs expressed Stra8 mRNA by RT-PCR 7days after induction differentiation, the RA-regulated gene specifically expressed in premeiotic germ cells and a marker of the stem cells transdifferentiated into germ cells.In control group, the FBMSCs were stra8 negative. Recent reports indicated that Stra8 was expressed in embryonic germ cells of ovary in mice and was not expressed after birth in ovary, it implied that there was not oocyte production in postnatal ovary. Our results is not support the view and suggest that adult FBMSCs could be able to generate oocytes.Conclusions: These results suggest that RA could induce FBMSC to differentiate into GC-like cells and oogonia-like cells in vitro. Stra8 is not only expressed in embryonic germ cells of ovary, but also can be expressed in adult FBMSC induced by RA. Adult FBMSCs contain pluripotent stem cells that could differentiate into germ cells and gonadal somatic cells. It is a point to investigate the function of GC-like cells derived from FBMSCs and the differentiation capable of oogonia-like cells derived from FBMSCs in vitro in a further step of the research work.

CTF18 plays an important role in mammalian gametogenesis and fertility

OBJECTIVE: CTF18 encodes an evolutionarily conserved protein that is crucial for germline development in the fly and that is essential for faithful transmission of chromosomes during DNA replication in yeast. Recently we demonstrated that CTF18, termed Chtf18 in mice, is expressed throughout the male and female germline of the mouse. The objective of our studies is to elucidate the role of CTF18/Chtf18 in mammalian germ cell development.DESIGN: We generated a mouse model that lacks Chtf18 to directly determine the role of Chtf18 in vivo.MATERIALS AND METHODS: We employed gene targeting to derive mice that lack Chtf18 function. We utilized Cre/loxP technology to derive Chtf18-null and conditional alleles. The phenotypic consequences of Chtf18 deletion were assessed by gross, histological, chromosomal, and immunofluorescence examination of Chtf18-deficient and wild-type gonads.RESULTS: Adult Chtf18-null male and female mouse gonads are much smaller and morphologically abnormal compared to those of their wild-type adult littermates. Fertility is significantly impaired in both male and female mice. Seminiferous tubules from Chtf18-null males demonstrate an overall deficiency of spermatocytes and a significant paucity of spermatids and spermatozoa. Some areas of seminiferous tubules are almost devoid of spermatogenic cells. The tubules also contain large multi-nucleated and aberrant-appearing spermatogenic cells. Ovaries from Chtf18-null females are smaller and contain fewer follicles than wild-type females. All stages of follicle development are seen, but many of the follicles appear to be degenerating and contain aberrant-appearing cells. Surface spread nuclei from Chtf18-null mouse gonads reveal a clear chromosomal defect during meiosis. In addition, the morphological phenotype of Chtf18loxP/loxP; TNAP Cre mice, in which Chtf18 is deleted only in germ cells, is indistinguishable from that of Chtf18-null gonads, indicating that Chtf18 is required in the germ line.CONCLUSIONS: CTF18/Chtf18 plays a significant role in mammalian gametogenesis and fertility through a function in meiosis. OBJECTIVE: CTF18 encodes an evolutionarily conserved protein that is crucial for germline development in the fly and that is essential for faithful transmission of chromosomes during DNA replication in yeast. Recently we demonstrated that CTF18, termed Chtf18 in mice, is expressed throughout the male and female germline of the mouse. The objective of our studies is to elucidate the role of CTF18/Chtf18 in mammalian germ cell development. DESIGN: We generated a mouse model that lacks Chtf18 to directly determine the role of Chtf18 in vivo. MATERIALS AND METHODS: We employed gene targeting to derive mice that lack Chtf18 function. We utilized Cre/loxP technology to derive Chtf18-null and conditional alleles. The phenotypic consequences of Chtf18 deletion were assessed by gross, histological, chromosomal, and immunofluorescence examination of Chtf18-deficient and wild-type gonads. RESULTS: Adult Chtf18-null male and female mouse gonads are much smaller and morphologically abnormal compared to those of their wild-type adult littermates. Fertility is significantly impaired in both male and female mice. Seminiferous tubules from Chtf18-null males demonstrate an overall deficiency of spermatocytes and a significant paucity of spermatids and spermatozoa. Some areas of seminiferous tubules are almost devoid of spermatogenic cells. The tubules also contain large multi-nucleated and aberrant-appearing spermatogenic cells. Ovaries from Chtf18-null females are smaller and contain fewer follicles than wild-type females. All stages of follicle development are seen, but many of the follicles appear to be degenerating and contain aberrant-appearing cells. Surface spread nuclei from Chtf18-null mouse gonads reveal a clear chromosomal defect during meiosis. In addition, the morphological phenotype of Chtf18loxP/loxP; TNAP Cre mice, in which Chtf18 is deleted only in germ cells, is indistinguishable from that of Chtf18-null gonads, indicating that Chtf18 is required in the germ line. CONCLUSIONS: CTF18/Chtf18 plays a significant role in mammalian gametogenesis and fertility through a function in meiosis.

Modelling germ cell development in vitro

Germ cells have a critical role in mediating the generation of genetic diversity and transmitting this information across generations. Furthermore, gametogenesis is unique as a developmental process in that it generates highly-specialized haploid gametes from diploid precursor stem cells through meiosis. Despite the importance of this process, progress in elucidating the molecular mechanisms underpinning mammalian germ cell development has been retarded by the lack of an efficient and reproducible system of in vitro culture for the expansion and trans-meiotic differentiation of germline cells. The dearth of such a culture system has rendered the study of germ cell biology refractory to the application of new high-throughput technologies such as RNA interference, leaving in vivo gene-targeting approaches as the only option to determine the function of genes believed to be involved in gametogenesis. Recent reports detailing the derivation of gametes in vitro from stem cells may provide the first steps in developing new tools to solve this problem. This review considers the developments made in modelling germ cell development using stem cells, and some of the challenges that need to be overcome to make this a useful tool for studying gametogenesis and to realize any future clinical application.

Critical function of Prdm14 for the establishment of the germ cell lineage in mice

Specification of germ cell fate is fundamental in development and heredity. Recent evidence indicates that in mice, specification of primordial germ cells (PGCs), the common source of both oocytes and spermatozoa, occurs through the integration of three key events: repression of the somatic program, reacquisition of potential pluripotency and ensuing genome-wide epigenetic reprogramming. Here we provide genetic evidence that Prdm14, a PR domain-containing transcriptional regulator with exclusive expression in the germ cell lineage and pluripotent cell lines, is critical in two of these events, the reacquisition of potential pluripotency and successful epigenetic reprogramming. In Prdm14 mutants, the failure of these two events manifests even in the presence of Prdm1 (also known as Blimp1), a key transcriptional regulator for PGC specification. Our combined evidence demonstrates that Prdm14 defines a previously unknown genetic pathway, initiating independently from Prdm1, for ensuring the launching of the mammalian germ cell lineage.

Recent Advances in the Derivation of Germ Cells from the Embryonic Stem Cells

In recent years, considerable progress has been made in the establishment and differentiation of human embryonic stem (ES) cell lines. The primordial germ cells (PGCs) and embryonic germ (EG) cells derived from them share many of their properties with ES cells. ES cell lines have now been derived from different stages of germ cell development and they have differentiated into gametes and shown embryonic development in mice, including the production of live pups. Conversely, germ cells can also be derived from ES cells. It has been demonstrated that murine (m) ES cells can differentiate into PGCs and subsequently into early gametes (oocytes and sperms) and blastocysts. Recently, immature sperm cells derived from mES cells in culture have produced live offspring. Preliminary research has indicated that human (h) ES cells probably have the potential to differentiate into germ cells. Adult stem cells have been reported to differentiate into mature germ cells in vitro. Therefore, stem cells may offer a valuable in vitro model for the investigation of germ cell development and the early stages of human gametogenesis, including epigenetic modifications of the germ line. This review discusses recent developments in the derivation and specification of mammalian germ cells from ES cells and describes some of the mechanisms of germ cell development.

Small RNA guides for de novo DNA methylation in mammalian germ cells: Figure 1.

Germline genomic methylation is essential for gamete identity and integrity in mammals. The study by Kuramochi-Miyagawa and colleagues (908-917) in the previous issue of Genes & Development links the process of DNA methylation-dependent repression of retrotranspons with the presence of piwi-interacting RNAs (piRNAs) in fetal male germ cells undergoing de novo methylation.

Epigenetic link between DNA methylation and histone modifications

Genetic imprinting, found in flowering plants and placental mammals, uses DNA methylation to yield parent‐of‐origin‐dependent gene expression. DNA methyltransferase 3a (Dnmt3a) and its regulatory factor, DNA methyltransferase 3‐like protein (Dnmt3L), are both required for de novo DNA methylation of imprinted genes in mammalian germ cells. Dnmt3L interacts specifically with unmethylated lysine 4 of histone H3 through its N‐terminal Cys‐rich domain that has strong structural similarities to the PHD‐like domain of BHC80, a component of LSD1‐mediated histone lysine demethylase complex. The C‐terminal domain of Dnmt3L is shown by crystallography to interact with the catalytic domain of Dnmt3a, demonstrating that Dnmt3L has dual functions of binding of unmethylated histone tail and activating DNA methyltransferase. The complexed C‐terminal domains of Dnmt3a and 3L showed further dimerization through Dnmt3a‐3a interaction, forming a tetrameric complex with two active sites. Substitution of key non‐catalytic residues in the Dnmt3a‐3L interface or Dnmt3a‐3a interface eliminated enzymatic activity. Molecular modeling of a DNA‐Dnmt3a dimer suggested that the two active sites are separated by approximately one DNA helical turn. Studies were supported partially by NIH grants (GM049245 and GM068680) and Georgia Research Alliance.

Quality control in an unreliable world

A cell and its world of molecular machines and organelles is complex—and imperfect, full of small errors and looming catastrophes. It is challenged by stresses, environmental insults and general entropy, but works remarkably well. One reason for this is the existence of quality control. Quality control acts at the level of the important macromolecules, proteins, RNA and DNA, as well as at the organelle and whole‐cell level. It serves to edit mistakes and generally maintain functionality. Biological processes are allowed to be a bit messy and slightly unreliable as long as quality control exists. This focus in The EMBO Journal presents eight reviews, which relate to quality control in molecular and cellular biology. Some reviews are directly about quality control mechanisms, while others are more tangentially connected to it. Some of the processes discussed are clearly appreciated as ‘quality control’ and the term is frequently used, for example in ER quality control. Other quality control processes have other names, but they have in common that they serve to check for correctness and functionality of already produced macromolecules or organelles. For the quite diverse types of quality control, there are general questions that allow some comparison. First, what is being detected? Does the cell detect the healthy and correct molecule/complex/organelle and allow its persistence? Or is the unhealthy and incorrect version detected and removed? This dichotomy is related to whether degradation or persistence is the default behavior. Second, how is correctness versus incorrectness detected? Is it qualitatively or quantitatively different? Third, how are the mistakes dealt with? Are they corrected/remodeled or are they removed/degraded? And finally, what happens if quality control does not function well? Several of the reviews explore the relationship between quality control and human disease conditions. The purpose of bringing together reviews from experts in seemingly unrelated areas, …