Mitochondrial trafficking through Rhot1 is involved in the aggregation of germinal granule components during primordial germ cell formation in Xenopus embryos

In many animals, the germ plasm is sufficient and necessary for primordial germ cell (PGC) formation. It contains germinal granules and abundant mitochondria (germline‐Mt). However, the role of germline‐Mt in germ cell formation remains poorly understood. In Xenopus, the germ plasm is distributed as many small islands at the vegetal pole, which gradually aggregates to form a single large mass in each of the four vegetal pole cells at the early blastula stage. Polymerized microtubules and the adapter protein kinesin are required for the aggregation of germ plasm. However, it remains unknown whether germline‐Mt trafficking is important for the cytoplasmic transport of germinal granules during germ plasm aggregation. In this study, we focused on the mitochondrial small GTPase protein Rhot1 to inhibit mitochondrial trafficking during the germ plasm aggregation. Expression of Rhot1ΔC, which lacks the C‐terminal mitochondrial transmembrane domain, inhibited the aggregation of germline‐Mt during early development. In Rhot1‐inhibited embryos, germinal granule components did not aggregate during cleavage stages, which reduced the number of PGCs on the genital ridge at tail‐bud stage. These results suggest that mitochondrial trafficking is involved in the aggregation of germinal granule components, which are essential for the formation of PGCs.     

Prdm14 promotes germline fate and naive pluripotency by repressing FGF signalling and DNA methylation

Primordial germ cells (PGCs) and somatic cells originate from postimplantation epiblast cells in mice. As pluripotency is lost upon differentiation of somatic lineages, a naive epigenome and the pluripotency network are re‐established during PGC development. Here we demonstrate that Prdm14 contributes not only to PGC specification, but also to naive pluripotency in embryonic stem (ES) cells by repressing the DNA methylation machinery and fibroblast growth factor (FGF) signalling. This indicates a critical role for Prdm14 in programming PGCs and promoting pluripotency in ES cells.     

Distinct chromatin organization in the germ line founder cell of the Caenorhabditis elegans embryo

Cells belonging to the germ lineage segregate physically and molecularly from their somatic neighbors during embryogenesis. While germ line‐specific chromatin modifications have been identified at later stages in the Caenorhabditis elegans nematode, none have been found in the single P₄ germ line founder cell that arises at the beginning of gastrulation. Using light and electron microscopy, we now report that the chromatin organization in the germ line founder cell of the early C. elegans embryo is distinct from that in the neighboring somatic cells. This unique organization is characterized by a greater chromatin compaction and an expansion of the interchromatin compartment. The ultrastructure of individual chromatin domains does not differ between germ line and somatic cells, pointing to a specific organization mainly at the level of the whole nucleus. We show that this higher order reorganization of chromatin is not a consequence of the P₄ nucleus being smaller than somatic nuclei or having initiated mitosis. Imaging of living embryos expressing fluorescent markers for both chromatin and P granules revealed that the appearance of a distinct chromatin organization in the P₄ cell occurs approximately 10 min after its birth and coincides with the aggregation of P granules around the nucleus, suggesting a possible link between these two events. The higher order reorganization of chromatin that is reported here occurs during the establishment of definitive germ cell identity. The changes we have observed could therefore be a prerequisite for the programming of chromatin totipotency.     

Ultrastructural study of oogenesis in Phoronopsis harmeri (Phoronida)

Temereva, E.N., Malakhov, V.V. and Yushin, V.V. 2011. Ultrastructural study of oogenesis in Phoronopsis harmeri (Phoronida). —Acta Zoologica (Stockholm) 92 : 241–250. The successive stages of oogenesis in Phoronopsis harmeri were examined by electron microscopy methods. During the oogenesis, each oocyte is encircled by vasoperitoneal (coelomic) cells forming a follicle. The previtellogenic oocytes are small cells which accumulate ribosomes for future synthesis; their cytoplasm contains characteristic clusters of mitochondria and osmiophilic particles resembling a germ plasm of other metazoans. The cytoplasm of the vitellogenic oocytes includes numerous mitochondria, cisternae of the rough endoplasmic reticulum, Golgi bodies and annulate lamellae. The synthesis of three types of inclusions was observed: strongly osmiophilic granules (lipid droplets) as a prevalent component, distinctly larger granules surrounded by membrane (proteinaceous yolk) and numerous large vesicles with pale flocculent content. No inclusions which could be unequivocally interpreted as the cortical granules were detected. The surface of the vitellogenic oocytes is covered by microvilli which increase in number and length during development. The oogenesis in Phoronida may be interpreted as follicular because of close association of oocytes with the vasoperitoneal tissue. However, well‐developed synthetic apparatus together with a strongly developed microvillous surface and absence of endocytosis indicate a clear case of autosynthetic vitellogenesis. Thus, in phoronids, there is a combination of simply developed follicle and autosynthesis that, apparently, is plesiomorphic character.     

Heterogeneity in imprinted methylation patterns of pluripotent embryonic germ cells derived from pre-migratory mouse germ cells

Pluripotent stem cells, termed embryonic germ (EG) cells, have been generated from both human and mouse primordial germ cells (PGCs). Like embryonic stem (ES) cells, EG cells have the potential to differentiate into all germ layer derivatives and may also be important for any future clinical applications. The development of PGCs in vivo is accompanied by major epigenetic changes including DNA demethylation and imprint erasure. We have investigated the DNA methylation pattern of several imprinted genes and repetitive elements in mouse EG cell lines before and after differentiation. Analysed cell lines were derived soon after PGC specification, “early”, in comparison with EG cells derived after PGC colonisation of the genital ridge, “late” and embryonic stem (ES) cell lines, derived from the inner cell mass (ICM). Early EG cell lines showed strikingly heterogeneous DNA methylation patterns, in contrast to the uniformity of methylation pattern seen in somatic cells (control), late EG cell and ES cell lines. We also observed that all analysed XX cell lines exhibited less methylation than XY. We suggest that this heterogeneity may reflect the changes in DNA methylation taking place in the germ cell lineage soon after specification.     

Increase of cortisol levels after temperature stress activates dmrt1a causing female‐to‐male sex reversal and reduced germ cell number in medaka

In vertebrates, there is accumulating evidence that environmental factors as triggers for sex determination and genetic sex determination are not two opposing alternatives but that a continuum of mechanisms bridge those extremes. One prominent example is the model fish species Oryzias latipes which has a stable XX/XY genetic sex determination system, but still responds to environmental cues, where high temperatures lead to female‐to‐male sex reversal. However, the mechanisms behind are still unknown. We show that high temperatures increase primordial germ cells (PGC) numbers before they reach the genital ridge, which, in turn, regulates the germ cell proliferation. Complete ablation of PGCs led to XX males with germ cell less testis, whereas experimentally increased PGC numbers did not reverse XY genotypes to female. For the underlying molecular mechanism, we provide support for the explanation that activation of the dmrt1a gene by cortisol during early development of XX embryos enables this autosomal gene to take over the role of the male determining Y‐chromosomal dmrt1bY.     

A novel transforming growth factor-beta superfamily member expressed in gonadal somatic cells enhances primordial germ cell and spermatogonial proliferation in rainbow trout (Oncorhynchus mykiss)

Our understanding of the molecular mechanisms of primordial germ cell (PGC) proliferation in fish is rudimentary, but it is thought to be controlled by the surrounding somatic cells. We assumed that growth factors that are specifically involved in PGC proliferation are expressed predominantly in the surrounding genital ridge somatic cells. In order to isolate these growth factors, we compiled a complementary DNA (cDNA) subtractive library using cDNA from the genital ridges of 40-dpf rainbow trout embryos as the tester and cDNA from embryos without genital ridges as the driver. This approach identified a novel cytokine, designated gonadal soma-derived growth factor (GSDF), which is a member of the transforming growth factor (TGF)-beta superfamily. GSDF was expressed in the genital ridge somatic cells surrounding the PGCs during embryogenesis, and in both the granulosa and Sertoli cells at later stages. Inhibition of GSDF translation by antisense oligonucleotides suppressed PGC proliferation. Moreover, isolated testicular cells that were cultured with recombinant GSDF demonstrated dose-dependent proliferation of type-A spermatogonia; this effect was completely blocked by antiserum against GSDF. These results denote that GSDF, a novel member of the TGF-beta superfamily, plays an important role for proliferation of PGC and spermatogonia.     

Characterization, isolation and culture of primordial germ cells in domestic animals: recent progress and insights from the ovine species

Primordial germ cell (PGC) allocation, characterization, lineage restriction, and differentiation have been extensively studied in the mouse. Murine PGC can be easily identified using markers as alkaline phosphatase content or the expression of pluripotent markers such as Pou5f1, Nanog, Sox2, Kit, SSEA1, and SSEA4. These tools allowed us to clarify certain aspects of the complex interactions of somatic and germinal cells in the establishment of the germ cell lineage, its segregation from the neighbouring somatic tissue, and the guidance mechanisms during migration that direct most of the germ cells into the genital ridges. Few data are available from other domestic animals and here we reported our preliminary studies on the isolation, characterization, and in vitro culture of sheep PGCs. Sheep PGCs can be identified with the markers previously used in mouse, but, in some cases, these markers are not coherently expressed in the same cell depending on the grade of differentiation and on technical problems related to commercial antibodies used. Pluripotency of PGCs in culture (EGCs) from domestic animals also needs further evaluation even though the derivation of embryonic pluripotent cell lines from large mammals may be an advantage as they are more physiologically similar to the human and perhaps more relevant for clinical translation studies. Comprehensive epigenetic reprogramming of the genome in early germ cells, and derived EGCs including extensive erasure of epigenetic modifications, may be relevant for gaining insight into events that lead to reprogramming and establishment of totipotency. EGCs can differentiate in vitro in a various range of tissues, form embryonic bodies, but in many cases failed to generate tumours when transplanted into immunodeficient mice and are not able to generate germline chimeric animals after their transfer. Such incomplete information clearly indicates the urge to improve the studies on derivation of stem cells in farm animals and shows the need for a multidisciplinary investigation in order to create farm animal models to set up suitable ethical and technical systems for cell regenerative therapies in humans.     

Drosophila pericentrin‐like protein promotes the formation of primordial germ cells

Primordial germ cells (PGCs) are the precursors to the adult germline stem cells that are set aside early during embryogenesis and specified through the inheritance of the germ plasm, which contains the mRNAs and proteins that function as the germline fate determinants. In Drosophila melanogaster, formation of the PGCs requires the microtubule and actin cytoskeletal networks to actively segregate the germ plasm from the soma and physically construct the pole buds (PBs) that protrude from the posterior cortex. Of emerging importance is the central role of centrosomes in the coordination of microtubule dynamics and actin organization to promote PGC development. We previously identified a requirement for the centrosome protein Centrosomin (Cnn) in PGC formation. Cnn interacts directly with Pericentrin‐like protein (PLP) to form a centrosome scaffold structure required for pericentriolar material recruitment and organization. In this study, we identify a role for PLP at several discrete steps during PGC development. We find PLP functions in segregating the germ plasm from the soma by regulating microtubule organization and centrosome separation. These activities further contribute to promoting PB protrusion and facilitating the distribution of germ plasm in proliferating PGCs.     

Narrow H3K4me2 is required for chicken PGC formation

The development of primordial germ cells (PGCs) undergoes epigenetic modifications. The study of histone methylation in regulating PGCs is beneficial to understand the development and differentiation mechanism of germ stem cells. Notably, it provides a theoretical basis for directed induction and mass acquisition in vitro. However, little is known about the regulation of PGC formation by histone methylation. Here, we found the high enrichment of H3K4me2 in the blastoderm, genital ridges, and testis. Chromatin immunoprecipitation sequencing was performed and the results revealed that genomic H3K4me2 is dynamic in embryonic stem cells, PGCs, and spermatogonial stem cells. This trend was consistent with the H3K4me2 enrichment in the gene promoter region. Additionally, narrow region triggered PGC‐related genes (Bmp4, Wnt5a, and Tcf7l2) and signaling pathways (Wnt and transforming growth factor‐β). After knocking down histone methylase Mll2 in vitro and vivo, the level of H3K4me2 decreased, inhibiting Cvh and Blimp1 expression, then repressing the formation of PGCs. Taken together, our study revealed the whole genome map of H3K4me2 in the formation of PGCs, contributing to improve the epigenetic study in PGC formation and providing materials for bird gene editing and rescue of endangered birds.     

Analysis of localization and reorganization of germ plasm in Xenopus transgenic line with fluorescence‐labeled mitochondria

Germ plasm is found in germ‐line cells of Xenopus and thought to include the determinant of primordial germ cells (PGCs). As mitochondria is abundant in germ plasm, vital staining of mitochondria was used to analyze the movement and function of germ plasm; however, its application was limited in early cleavage embryos. We made transgenic Xenopus, harboring enhanced green fluorescent protein (EGFP) fused to the mitochondria transport signal (Dria‐line). Germ plasm with EGFP‐labeled mitochondria was clearly distinguishable from the other cytoplasm, and retained mostly during one generation of germ‐line cells in Dria‐line females. Using the Dria‐line, we show that germ plasm is reorganized from near the cell membrane to the perinuclear space at St. 9, dependent on the microtubule system.     

Sequential SDF1a and b-induced mobility guides Medaka PGC migration

Assembly and formation of the gonad primordium are the first steps toward gonad differentiation and subsequent sex differentiation. Primordial germ cells (PGCs) give rise to the gametes that are responsible for the development of a new organism in the next generation. In many organisms, following their specification the germ cells migrate toward the location of the prospective gonadal primordium. To accomplish this, the PGCs obtain directional cues from cells positioned along their migration path. One such cue, the chemokine SDF1 (stromal cell-derived factor 1) and its receptor CXCR4 have recently been found to be critical for proper PGC migration in zebrafish, chick and mouse.We have studied the mechanisms responsible for PGC migration in Medaka. In contrast to the situation observed in zebrafish, where proper PGC positioning is the result of active migration in the direction of the source of SDF1a, Medaka PGC movements are shown to be the consequence of a combination of active SDF1a and SDF1b-guided migration. In this process both SDF1 co-orthologues show only partly overlapping expression pattern and cooperate in the correct positioning of the PGCs.     

The ability of mouse nuclear transfer embryonic stem cells to differentiate into primordial germ cells

Nuclear transfer embryonic stem cells (ntESCs) show stem cell characteristics such as pluripotency but cause no immunological disorders. Although ntESCs are able to differentiate into somatic cells, the ability of ntESCs to differentiate into primordial germ cells (PGCs) has not been examined. In this work, we examined the capacity of mouse ntESCs to differentiate into PGCs in vitro. ntESCs aggregated to form embryoid bodies (EB) in EB culture medium supplemented with bone morphogenetic protein 4(BMP4) as the differentiation factor. The expression level of specific PGC genes was compared at days 4 and 8 using real time PCR. Flow cytometry and immunocytochemical staining were used to detect Mvh as a specific PGC marker. ntESCs expressed particular genes related to different stages of PGC development. Flow cytometry and immunocytochemical staining confirmed the presence of Mvh protein in a small number of cells. There were significant differences between cells that differentiated into PGCs in the group treated with Bmp4 compared to non-treated cells. These findings indicate that ntESCs can differentiate into putative PGCs. Improvement of ntESC differentiation into PGCs may be a reliable means of producing mature germ cells.     

Mitochondrial ribosomal RNA in the germinal granules in Xenopus embryos revisited

Germ cells of various animals contain a determinant that is called the germ plasm. In amphibians such as Xenopus laevis, the germ plasm is composed of mitochondria and electron dense germinal granules that are embedded in a fibrillar matrix. Previous reports indicated that one of the components of germinal granules was mitochondrial large and small ribosomal RNA (mtlrRNA and mtsrRNA). Utilizing a modified procedure for electron microscopy in situ hybridization, we investigated the distribution of these RNAs along with other components of the germ plasm in Xenopus laevis embryos. We found, that contrary to previous reports, the mtlrRNA and mtsrRNA were located in close vicinity to the germinal granules but were not major constituents of granules. The majority of the mtlrRNA and mtlsrRNAs was present inside the mitochondria and in the germ plasm matrix.