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Scientific Reports Oct 2022Bucky ball was identified as germ plasm organizer in zebrafish and has proven crucial for Balbiani body condensation. A synteny comparison identified an uncharacterized...
Bucky ball was identified as germ plasm organizer in zebrafish and has proven crucial for Balbiani body condensation. A synteny comparison identified an uncharacterized gene locus in the chicken genome as predicted avian counterpart. Here, we present experimental evidence that this gene locus indeed encodes a 'Bucky ball' equivalent in matured oocytes and early embryos of chicken. Heterologous expression of Bucky ball fusion proteins both from zebrafish and chicken with a fluorescent reporter revealed unique patterns indicative for liquid-liquid phase separation of intrinsically disordered proteins. Immuno-labeling detected Bucky ball from oocytes to blastoderms with diffuse distribution in matured oocytes, aggregation in first cleavage furrows, and co-localization to the chicken vasa homolog (CVH). Later, Bucky ball translocated to the cytoplasm of first established cells, and showed nuclear translocation during the major zygotic activation together with CVH. Remarkably, during the phase of area pellucida formation, Bucky ball translocated back into the cytoplasm at stage EGK VI, whereas CVH remained within the nuclei. The condensation of Bucky ball and co-localization with CVH in cleavage furrows and nuclei of the centrally located cells strongly suggests chicken Bucky ball as a germ plasm organizer in birds, and indicate a special importance of the major zygotic activation for germline specification.
Topics: Animals; Chickens; Cytoplasm; Germ Cells; Intrinsically Disordered Proteins; Oocytes; Zebrafish
PubMed: 36207377
DOI: 10.1038/s41598-022-21239-8 -
Current Biology : CB Mar 2009In many animals, gamete formation during embryogenesis is specified by maternal cytoplasmic determinants termed germ plasm. During oogenesis, germ plasm forms a distinct...
In many animals, gamete formation during embryogenesis is specified by maternal cytoplasmic determinants termed germ plasm. During oogenesis, germ plasm forms a distinct cellular structure such as pole plasm in Drosophila or the Balbiani body, an aggregate of organelles also found in mammals. However, in vertebrates, the key regulators of germ plasm assembly are largely unknown. Here, we show that, at the beginning of zebrafish oogenesis, the germ plasm defect in bucky ball (buc) mutants precedes the loss of polarity, indicating that Buc primarily controls Balbiani body formation. Moreover, we molecularly identify the buc gene, which is exclusively expressed in the ovary with a novel, dynamic mRNA localization pattern first detectable within the Balbiani body. We find that a Buc-GFP fusion localizes to the Balbiani body during oogenesis and with the germ plasm during early embryogenesis, consistent with a role in germ plasm formation. Interestingly, overexpression of buc seems to generate ectopic germ cells in the zebrafish embryo. Because we discovered buc homologs in many vertebrate genomes, including mammals, these results identify buc as the first gene necessary and sufficient for germ plasm organization in vertebrates.
Topics: Amino Acid Sequence; Animals; Cell Polarity; Female; Humans; Male; Molecular Sequence Data; Oocytes; Oogenesis; Ovary; Phylogeny; Recombinant Fusion Proteins; Sequence Alignment; Zebrafish; Zebrafish Proteins
PubMed: 19249209
DOI: 10.1016/j.cub.2009.01.038 -
Developmental Dynamics : An Official... Jan 2016Animal germ cells have specific organelles that are similar to ribonucleoprotein complex, called germ plasm, which is accumulated in eggs. Germ plasm is essential for...
BACKGROUND
Animal germ cells have specific organelles that are similar to ribonucleoprotein complex, called germ plasm, which is accumulated in eggs. Germ plasm is essential for inherited mechanism of germ line segregation in early embryogenesis. Sea urchins have early germ line segregation in early embryogenesis. Nevertheless, organization of germ plasm-related organelles and their molecular composition are still unclear. Another issue is whether maternally accumulated germ plasm exists in the sea urchin eggs.
RESULTS
I analyzed intracellular localization of germ plasm during oogenesis in sea urchin Strongylocentrotus intermedius by using morphological approach and immunocytochemical detection of Vasa, a germ plasm marker. All ovarian germ cells have germ plasm-related organelles in the form of germ granules, Balbiani bodies, and perinuclear nuage found previously in germ cells in other animals. Maternal germ plasm is accumulated in late oogenesis at the cell periphery. Cytoskeletal drug treatment showed an association of Vasa-positive granules with actin filaments in the egg cortex.
CONCLUSIONS
All female germ cells of sea urchins have germ plasm-related organelles. Eggs have a maternally accumulated germ plasm associated with cortical cytoskeleton. These findings correlate with early segregation of germ line in sea urchins.
Topics: Animals; Cytoplasm; Female; Germ Cells; Oocytes; Oogenesis; Organelles; Sea Urchins
PubMed: 26385846
DOI: 10.1002/dvdy.24348 -
Poultry Science Dec 1996Little is known about the growth and differentiation of the primary oocyte in the sexually mature chicken or turkey hen. In this study, primary oocytes from turkey hens...
Little is known about the growth and differentiation of the primary oocyte in the sexually mature chicken or turkey hen. In this study, primary oocytes from turkey hens in egg production were examined by light and electron microscopy. Based on oocyte and germinal vesicle (GV) diameters and organelle morphology and distribution, the sequential development of the primary oocyte was divided into five stages. No Balbiani body was observed in Stage I oocytes (< 80 microns in diameter). Pleomorphic mitochondria were localized around the GV and multivesicular bodies were scattered in the ooplasm. By Stage II (81 to 150 microns), the Balbiani body was observed adjacent to the GV. Pleomorphic mitochondria, macrobodies, and smooth endoplasmic reticulum (SER) were associated with the Balbiani body. Lipid droplets were predominantly localized to the periphery of the oocyte. The Balbiani body was partially dispersed by Stage III (151 to 350 microns) and associated organelles appeared in clusters in the ooplasm. Golgi and SER were observed immediately subjacent to the oolemma. Stage IV oocytes (351 to 500 microns) were characterized by the absence of the Balbiani body, a more centrally located GV, and the redistribution of the mitochondria to the periphery of the oocyte. Throughout the ooplasm was vesicular SER. By Stage V (501 to 800 microns), zonation of the organelles was completed with the mitochondrial ring immediately subjacent to the oolemma and a concentric layer of lipid droplets subjacent to the mitochondrial ring. The GV was in the periphery of the oocyte. Organelle and inclusion redistribution and organelle pleomorphism were presumed to be reflective of increasing metabolic and transport requirements of the growing oocyte in the mature turkey hen.
Topics: Animals; Chickens; Cytoplasmic Granules; Egg Yolk; Eggs; Endoplasmic Reticulum, Smooth; Female; Golgi Apparatus; Microscopy, Electron; Mitochondria; Oocytes; Organelles; Oviposition; Sexual Maturation; Turkeys
PubMed: 9000285
DOI: 10.3382/ps.0751569 -
Journal of Ovarian Research Mar 2019Normal pubertal ovary displays all stages of follicular development and a biased BAX/BCL2 protein ratio in favor of pro-apoptotic BAX protein comparable to the adult...
BACKGROUND
Normal pubertal ovary displays all stages of follicular development and a biased BAX/BCL2 protein ratio in favor of pro-apoptotic BAX protein comparable to the adult ovary. However, adolescents suffering malignant extra-gonadal disease show a limited follicle development after cytotoxic drug treatment and a reduced capacity of in vitro follicle growth. We evaluated the expression of pro- and anti-apoptotic members of the BCL2 gene family, the FAS/FAS-L proteins from the extrinsic apoptosis pathway, the germ-cell-specific marker VASA, the pluripotency marker OCT3/4, and markers of early and late apoptosis in the ovary of pubertal patients with malignant extra-gonadal disease, which received or not pre-surgery chemotherapy, entering a cryopreservation program.
RESULTS
Ovarian biopsies from 12 adolescent girls were screened for follicle count and expression of VASA, OCT3/4, BAX, BCL2, MCL1L and S, cleaved-BID, FAS/FAS-L and CASPASE 3 through immunohistochemistry, western blot and RT-PCR. All stages of folliculogenesis, from primordial to antral follicle, were present in all 12 patients analyzed. VASA and most of the screened apoptosis-related genes showed a pattern of immune-expression comparable to that previously reported. OCT3/4 showed a cytoplasmic localization in the great majority of the primordial follicles; however, in some cases the localization was nuclear. In addition, OCT3/4B showed a significant reduction compared to OCT3/4A. Unexpectedly, BCL2 was detected at all stages of folliculogenesis, associated to the Balbiani's body in the primordial follicles, regardless of whether patients had or had not received chemotherapy, ruling out the possibility that its expression is a protective response to chemotherapy.
CONCLUSIONS
These findings reveal new information on the morphological status of the follicular reserve and the expression of apoptosis-related genes in histologically normal adolescent ovary from patients undergoing extragonadal cancer. The unexpected expression of apoptosis-inhibiting BCL2 protein, both in patients that had or had not received chemotherapy, opens a new avenue for thorough investigations. Moreover, the nuclear localization of OCT3/4 protein in primordial follicle-enclosed oocytes suggests a possible increased activity of ovarian stem cells in response to chemotherapy and/or extragonadal cancer. This new information can be essential for a better managing of in vitro culture of follicles that can be removed by filtration from preserved ovarian tissue, especially in girls that entered a cryopreservation program.
Topics: Adolescent; Apoptosis Regulatory Proteins; Cell Nucleus; Child; Cryopreservation; Cytoplasm; Female; Gene Expression; Humans; Neoplasms, Germ Cell and Embryonal; Ovarian Follicle; Ovarian Reserve; Ovary; RNA, Messenger; Young Adult
PubMed: 30857552
DOI: 10.1186/s13048-019-0496-2 -
International Journal of Cell Biology 2020Mitochondria change their morphology and distribution depending on the metabolism and functional state of a cell. Here, we analyzed the mitochondria and selected...
Mitochondria change their morphology and distribution depending on the metabolism and functional state of a cell. Here, we analyzed the mitochondria and selected structures in female germ-line cysts in a representative of clitellate annelids - the white worm in which each germ cell has one cytoplasmic bridge that connects it to a common cytoplasmic mass. Using serial block-face scanning electron microscopy (SBEM), we prepared three-dimensional ultrastructural reconstructions of the entire selected compartments of a cyst at the advanced stage of oogenesis, i.e. the nurse cell, cytophore, and cytoplasmic bridges of all 16 cells (15 nurse cells and oocyte). We revealed extensive mitochondrial networks in the nurse cells, cytophore and mitochondria that pass through the cytoplasmic bridges, which indicates that a mitochondrial network can extend throughout the entire cyst. The dynamic hyperfusion state was suggested for such mitochondrial aggregations. We measured the mitochondria distribution and revealed their polarized distribution in the nurse cells and more abundant accumulation within the cytophore compared to the nurse cell. A close association of mitochondrial networks with dispersed nuage material, which seems to be the structural equivalent of a Balbiani body, not described in clitellate annelids so far, was also revealed.
PubMed: 32395131
DOI: 10.1155/2020/7483467 -
BMC Evolutionary Biology Oct 2016The primordial germ cells (PGCs) giving rise to gametes are determined by two different mechanisms in vertebrates. While the germ cell fate in mammals and salamanders is...
BACKGROUND
The primordial germ cells (PGCs) giving rise to gametes are determined by two different mechanisms in vertebrates. While the germ cell fate in mammals and salamanders is induced by zygotic signals, maternally delivered germ cell determinants specify the PGCs in birds, frogs and teleost fish. Assembly of the germ plasm in the oocyte is organized by the single Buc in zebrafish, named Velo1 in Xenopus, and by Oskar in Drosophila. Secondary loss of oskar in several insect lineages coincides with changes in germline determination strategies, while the presence of buc in mammals suggests functions not associated with germline formation.
RESULTS
To clarify the evolutionary history of buc we searched for the gene in genomes available from various chordates. No buc sequence was found in lamprey and chordate invertebrates, while the gene was identified in a conserved syntenic region in elephant shark, spotted gar, teleosts, Comoran coelacanth and most tetrapods. Rodents have probably lost the buc gene, while a premature translation stop was found in primates and in Mexican axolotl lacking germ plasm. In contrast, several buc and buc-like (bucL) paralogs were identified in the teleosts examined, including zebrafish, and the tetraploid genome of Atlantic salmon harbors seven buc and bucL genes. Maternal salmon buc1a, buc2a and buc2b mRNAs were abundant in unfertilized eggs together with dnd and vasa mRNAs. Immunostained salmon Buc1a was restricted to cleavage furrows in 4-cell stage embryos similar to a fluorescent zebrafish Buc construct injected in salmon embryos. Salmon Buc1a and Buc2a localized together with DnD, Vasa and Dazl within the Balbiani body of early oocytes.
CONCLUSIONS
Buc probably originated more than 400 million years ago and might have played an ancestral role in assembling germ plasm. Functional redundancy or subfunctionalization of salmon Buc paralogs in germline formation is suggested by the maternally inherited mRNAs of three salmon buc genes, the localized Buc1a in the cleavage furrows and the distribution of Buc1a and Buc2a in the Balbiani body during oogenesis. The extra-ovarian expression of salmon buc genes and the presence of a second zebrafish bucL gene suggest additional functions not related to germ cell specification.
Topics: Ambystoma mexicanum; Animals; Evolution, Molecular; Female; Fish Proteins; Gene Dosage; Oocytes; Oogenesis; Primates; RNA, Messenger; Rodentia; Salmo salar
PubMed: 27784263
DOI: 10.1186/s12862-016-0809-7 -
PLoS Biology Jan 2016The source of symmetry breaking in vertebrate oocytes is unknown. Animal-vegetal oocyte polarity is established by the Balbiani body (Bb), a conserved structure found in...
The source of symmetry breaking in vertebrate oocytes is unknown. Animal-vegetal oocyte polarity is established by the Balbiani body (Bb), a conserved structure found in all animals examined that contains an aggregate of specific mRNAs, proteins, and organelles. The Bb specifies the oocyte vegetal pole, which is key to forming the embryonic body axes as well as the germline in most vertebrates. How Bb formation is regulated and how its asymmetric position is established are unknown. Using quantitative image analysis, we trace oocyte symmetry breaking in zebrafish to a nuclear asymmetry at the onset of meiosis called the chromosomal bouquet. The bouquet is a universal feature of meiosis where all telomeres cluster to one pole on the nuclear envelope, facilitating chromosomal pairing and meiotic recombination. We show that Bb precursor components first localize with the centrosome to the cytoplasm adjacent to the telomere cluster of the bouquet. They then aggregate around the centrosome in a specialized nuclear cleft that we identified, assembling the early Bb. We show that the bouquet nuclear events and the cytoplasmic Bb precursor localization are mechanistically coordinated by microtubules. Thus the animal-vegetal axis of the oocyte is aligned to the nuclear axis of the bouquet. We further show that the symmetry breaking events lay upstream to the only known regulator of Bb formation, the Bucky ball protein. Our findings link two universal features of oogenesis, the Bb and the chromosomal bouquet, to oocyte polarization. We propose that a meiotic-vegetal center couples meiosis and oocyte patterning. Our findings reveal a novel mode of cellular polarization in meiotic cells whereby cellular and nuclear polarity are aligned. We further reveal that in zygotene nests, intercellular cytoplasmic bridges remain between oocytes and that the position of the cytoplasmic bridge coincides with the location of the centrosome meiotic-vegetal organizing center. These results suggest that centrosome positioning is set by the last mitotic oogonial division plane. Thus, oocytes are polarized in two steps: first, mitotic divisions preset the centrosome with no obvious polarization yet, then the meiotic-vegetal center forms at zygotene bouquet stages, when symmetry is, in effect, broken.
Topics: Animals; Cell Polarity; Centrosome; Chromosomes; Meiosis; Microtubules; Oocytes; Zebrafish
PubMed: 26741740
DOI: 10.1371/journal.pbio.1002335 -
Development (Cambridge, England) Mar 2024Gametogenesis is the process through which germ cells differentiate into sexually dimorphic gametes, eggs and sperm. In the teleost fish medaka (Oryzias latipes), a germ...
Gametogenesis is the process through which germ cells differentiate into sexually dimorphic gametes, eggs and sperm. In the teleost fish medaka (Oryzias latipes), a germ cell-intrinsic sex determinant, foxl3, triggers germline feminization by activating two genetic pathways that regulate folliculogenesis and meiosis. Here, we identified a pathway involving a dome-shaped microtubule structure that may be the basis of oocyte polarity. This structure was first established in primordial germ cells in both sexes, but was maintained only during oogenesis and was destabilized in differentiating spermatogonia under the influence of Sertoli cells expressing dmrt1. Although foxl3 was dispensable for this pathway, dazl was involved in the persistence of the microtubule dome at the time of gonocyte development. In addition, disruption of the microtubule dome caused dispersal of bucky ball RNA, suggesting the structure may be prerequisite for the Balbiani body. Collectively, the present findings provide mechanistic insight into the establishment of sex-specific polarity through the formation of a microtubule structure in germ cells, as well as clarifying the genetic pathways implementing oocyte-specific characteristics.
Topics: Animals; Female; Male; Oryzias; Semen; Germ Cells; Gametogenesis; Oogenesis
PubMed: 38471539
DOI: 10.1242/dev.201840 -
Journal of Developmental Biology Mar 2016The germ cell lineage in is specified by the inheritance of germ plasm that assembles within the mitochondrial cloud or Balbiani body in stage I oocytes. Specific RNAs,...
The germ cell lineage in is specified by the inheritance of germ plasm that assembles within the mitochondrial cloud or Balbiani body in stage I oocytes. Specific RNAs, such as , localize to the germ plasm. has the essential germline function of blocking somatic gene expression and thus preventing Primordial Germ Cell (PGC) loss and sterility. Hermes/Rbpms protein and RNA co-localize within germinal granules, diagnostic electron dense particles found within the germ plasm. Previous work indicates that accumulates within the germ plasm through a diffusion/entrapment mechanism. Here we show that Hermes/Rbpms interacts with through sequence specific RNA localization signals found in the -3'UTR. Importantly, Hermes/Rbpms specifically binds , but not RNA in the nucleus of stage I oocytes. binding data show that Hermes/Rbpms requires additional factors that are present in stage I oocytes in order to bind . One such factor may be hnRNP I, identified in a yeast-2-hybrid screen as directly interacting with Hermes/Rbpms. We suggest that Hermes/Rbpms functions as part of a RNP complex in the nucleus that facilitates selection of germline RNAs for germ plasm localization. We propose that Hermes/Rbpms is required for RNA to form within the germinal granules and in this way, participates in the germline specific translational repression and sequestration of .
PubMed: 26998427
DOI: 10.3390/jdb4010002