Mouse oocytes develop in cysts with the help of nurse cells.

Mouse germline cysts, on average, develop into six oocytes supported by 24 nurse cells that transfer cytoplasm and organelles to generate a Balbiani body. We showed that between E14.5 and P5, cysts periodically activate some nurse cells to begin cytoplasmic transfer, which causes them to shrink and turnover within 2 days. Nurse cells die by a programmed cell death (PCD) pathway involving acidification, similar to Drosophila nurse cells, and only infrequently by apoptosis. Prior to initiating transfer, nurse cells co-cluster by scRNA-seq with their pro-oocyte sisters, but during their final 2 days, they cluster separately. The genes promoting oocyte development and nurse cell PCD are upregulated, whereas the genes that repress transfer, such as Tex14, and oocyte factors, such as Nobox and Lhx8, are under-expressed. The transferred nurse cell centrosomes build a cytocentrum that establishes a large microtubule aster in the primordial oocyte that organizes the Balbiani body, defining the earliest oocyte polarity.

Sexually dimorphic dynamics of the microtubule network in medaka (Oryzias latipes) germ cells.

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.

Comparative analysis of vertebrates reveals that mouse primordial oocytes do not contain a Balbiani body.

Oocytes spend the majority of their lifetime in a primordial state. The cellular and molecular biology of primordial oocytes is largely unexplored; yet, it is necessary to study them to understand the mechanisms through which oocytes maintain cellular fitness for decades, and why they eventually fail with age. Here, we develop enabling methods for live-imaging-based comparative characterization of Xenopus, mouse and human primordial oocytes. We show that primordial oocytes in all three vertebrate species contain active mitochondria, Golgi and lysosomes. We further demonstrate that human and Xenopus oocytes have a Balbiani body characterized by a dense accumulation of mitochondria in their cytoplasm. However, despite previous reports, we did not find a Balbiani body in mouse oocytes. Instead, we demonstrate that what was previously used as a marker for the Balbiani body in mouse primordial oocytes is in fact a ring-shaped Golgi that is not functionally associated with oocyte dormancy. This study provides the first insights into the organization of the cytoplasm in mammalian primordial oocytes, and clarifies the relative advantages and limitations of choosing different model organisms for studying oocyte dormancy.

Germ plasm dynamics during oogenesis and early embryonic development in Xenopus and zebrafish.

Specification of the germline and its segregation from the soma mark one of the most crucial events in the lifetime of an organism. In different organisms, this specification can occur through either inheritance or inductive mechanisms. In species such as Xenopus and zebrafish, the specification of primordial germ cells relies on the inheritance of maternal germline determinants that are synthesized and sequestered in the germ plasm during oogenesis. In this review, we discuss the formation of the germ plasm, how germline determinants are recruited into the germ plasm during oogenesis, and the dynamics of the germ plasm during oogenesis and early embryonic development.

A proteomics approach identifies novel resident zebrafish Balbiani body proteins Cirbpa and Cirbpb.

The Balbiani body (Bb) is the first marker of polarity in vertebrate oocytes. The Bb is a conserved structure found in diverse animals including insects, fish, amphibians, and mammals. During early zebrafish oogenesis, the Bb assembles as a transient aggregate of mRNA, proteins, and membrane-bound organelles at the presumptive vegetal side of the oocyte. As the early oocyte develops, the Bb appears to grow slowly, until at the end of stage I of oogenesis it disassembles and deposits its cargo of localized mRNAs and proteins. In fish and frogs, this cargo includes the germ plasm as well as gene products required to specify dorsal tissues of the future embryo. We demonstrate that the Bb is a stable, solid structure that forms a size exclusion barrier similar to other biological hydrogels. Despite its central role in oocyte polarity, little is known about the mechanism behind the Bb's action. Analysis of the few known protein components of the Bb is insufficient to explain how the Bb assembles, translocates, and disassembles. We isolated Bbs from zebrafish oocytes and performed mass spectrometry to define the Bb proteome. We successfully identified 77 proteins associated with the Bb sample, including known Bb proteins and novel RNA-binding proteins. In particular, we identified Cirbpa and Cirbpb, which have both an RNA-binding domain and a predicted self-aggregation domain. In stage I oocytes, Cirbpa and Cirbpb localize to the Bb rather than the nucleus (as in somatic cells), indicating that they may have a specialized function in the germ line. Both the RNA-binding domain and the self-aggregation domain are sufficient to localize to the Bb, suggesting that Cirbpa and Cirbpb interact with more than just their mRNA targets within the Bb. We propose that Cirbp proteins crosslink mRNA cargo and proteinaceous components of the Bb as it grows. Beyond Cirbpa and Cirbpb, our proteomics dataset presents many candidates for further study, making it a valuable resource for building a comprehensive mechanism for Bb function at a protein level.

Balbiani body formation and cytoplasmic zonation during early oocyte development in two Clupeiform fishes.

The Balbiani body (Bb) was examined in primary growth phase oocytes for the first time in two clupeoid fish species, the Mediterranean sardine, Sardina pilchardus, and the European anchovy, Engraulis encrasicolus, which belong to different families, Clupeidae and Engraulidae, respectively. Cytoplasmic morphological changes of early secondary growth oocytes were also investigated using confocal laser scanning microscopy, light and transmission electron microscopy. The ultrastructural observations showed that the two species develop a distinct spherical Bb. However, differences in the cytoplasm, mainly in the perinuclear area, were observed. Briefly, in sardine the Bb coexists with a thick perinuclear ring containing mitochondria, nuage, endoplasmic reticulum and small vesicles, while in anchovy this perinuclear ring is thinner, consisting of complexes of nuage and mitochondria. After the disassembly of the Bb, a prominent cytoplasmic zonation develops in the secondary growth oocytes of sardine and anchovy, although with different organelle distribution between the two species. Sardine oocytes exhibit a thick zone of endoplasmic reticulum around the nucleus, whereas in those of anchovy, a thick mitochondria-rich ring surrounding the nucleus was observed. The cytoplasmic characteristics, such as the perinuclear ring in primary oocytes in sardine and the mitochondria-rich ring of early secondary oocytes in anchovy, are also discernible in histological sections by standard procedures and could thus be used as indicators of maturity or imminent spawning period in routine light microscopy observations, providing a valuable tool for applied fisheries biology.

In situ localization of diacylglycerol lipase α and ß producing an endocannabinoid 2-arachidonoylglycerol and of cannabinoid receptor 1 in the primary oocytes of postnatal mice.

In order to understand the mechanism of the endocannabinoid (eCB) signal, which has so far been shown to work in oocyte genesis and maturation, it is critical to clarify detailed localization of the eCB synthesizing enzyme molecules as well as receptors for eCBs in oocytes in the ovary in situ. For this purpose, diacylglycerol lipase (DGL) α and ß are involved in the synthesis of an eCB 2-arachidonoylglycerol (2-AG). DGLα/ß and the cannabinoid receptor 1 (CB1) for 2-AG were shown to be localized to the primary oocytes of postnatal mice using immuno-light and electron microscopy. It was found that two types of localization existed: first, immunoreactivities for DGLα and ß were weakly detected throughout the ooplasm in light microscopy for which the intracellular membranes of vesicles forming tiny scattered aggregates were responsible. Secondly, DGLß-immunoreactivity was distinctly confined to the nuage of Balbiani bodies and small nuage-derivative structures; both amorphous materials and membranes of vesicles were responsible for their localization. On the other hand, the weak immunoreactivity for CB1 was localized in a pattern similar to the first one for DGLs, but not found in a pattern for the Balbiani nuage. Two routes of functional exertion of 2-AG synthesized by DGLs were suggested from the two types of localization: one was that the eCB synthesized at all the sites of DGLs is released from the oocytes and exerts paracrine or autocrine effects on adjacent intra-ovarian cells as well as the oocytes themselves. The other was that the eCB synthesized within the nuage was involved in the modulation of the posttranscriptional processing of oocytes. Owing to the failure in the detection of CB1 in the Balbiani nuage, however, the validity of the latter possibility remains to be elucidated.

Coordination of cellular differentiation, polarity, mitosis and meiosis - New findings from early vertebrate oogenesis.

A mechanistic dissection of early oocyte differentiation in vertebrates is key to advancing our knowledge of germline development, reproductive biology, the regulation of meiosis, and all of their associated disorders. Recent advances in the field include breakthroughs in the identification of germline stem cells in Medaka, in the cellular architecture of the germline cyst in mice, in a mechanistic dissection of chromosomal pairing and bouquet formation in meiosis in mice, in tracing oocyte symmetry breaking to the chromosomal bouquet of meiosis in zebrafish, and in the biology of the Balbiani body, a universal oocyte granule. Many of the major events in early oogenesis are universally conserved, and some are co-opted for species-specific needs. The chromosomal events of meiosis are of tremendous consequence to gamete formation and have been extensively studied. New light is now being shed on other aspects of early oocyte differentiation, which were traditionally considered outside the scope of meiosis, and their coordination with meiotic events. The emerging theme is of meiosis as a common groundwork for coordinating multifaceted processes of oocyte differentiation. In an accompanying manuscript we describe methods that allowed for investigations in the zebrafish ovary to contribute to these breakthroughs. Here, we review these advances mostly from the zebrafish and mouse. We discuss oogenesis concepts across established model organisms, and construct an inclusive paradigm for early oocyte differentiation in vertebrates.

Methods for the analysis of early oogenesis in Zebrafish.

Oocyte differentiation is a highly dynamic and intricate developmental process whose mechanistic understanding advances female reproduction, fertility, and ovarian cancer biology. Despite the many attributes of the zebrafish model, it has yet to be fully exploited for the investigation of early oocyte differentiation and ovarian development. This is partly because the properties of the adult zebrafish ovary make it technically challenging to access early stage oocytes. As a result, characterization of these stages has been lacking and tools for their analysis have been insufficient. To overcome these technical hurdles, we took advantage of the juvenile zebrafish ovary, where early stage oocytes can readily be found in high numbers and progress in a predictable manner. We characterized the earliest stages of oocyte differentiation and ovarian development and defined accurate staging criteria. We further developed protocols for quantitative microscopy, live time-lapse imaging, ovarian culture, and isolation of stage-specific oocytes for biochemical analysis. These methods have recently provided us with an unprecedented view of early oogenesis, allowing us to study formation of the Balbiani body, a universal oocyte granule that is associated with oocyte survival in mice and required for oocyte and egg polarity in fish and frogs. Despite its tremendous developmental significance, the Bb has been little investigated and how it forms was unknown in any species for over two centuries. We were able to trace Balbiani body formation and oocyte symmetry breaking to the onset of meiosis. Through this investigation we revealed novel cytoskeletal structures in oocytes and the contribution of specialized cellular organization to differentiation. Overall, the juvenile zebrafish ovary arises as an exciting model for studies of cell and developmental biology. We review these and other recent advances in vertebrate oogenesis in an accompanying manuscript in this issue of Developmental Biology. Here, we describe the protocols for ovarian investigation that we developed in the zebrafish, including all experimental steps that will easily allow others to reproduce such analysis. This juvenile ovary toolbox also contributes to establishing the zebrafish as a model for post-larval developmental stages.

Oocyte polarity requires a Bucky ball-dependent feedback amplification loop.

In vertebrates, the first asymmetries are established along the animal-vegetal axis during oogenesis, but the underlying molecular mechanisms are poorly understood. Bucky ball (Buc) was identified in zebrafish as a novel vertebrate-specific regulator of oocyte polarity, acting through unknown molecular interactions. Here we show that endogenous Buc protein localizes to the Balbiani body, a conserved, asymmetric structure in oocytes that requires Buc for its formation. Asymmetric distribution of Buc in oocytes precedes Balbiani body formation, defining Buc as the earliest marker of oocyte polarity in zebrafish. Through a transgenic strategy, we determined that excess Buc disrupts polarity and results in supernumerary Balbiani bodies in a 3'UTR-dependent manner, and we identified roles for the buc introns in regulating Buc activity. Analyses of mosaic ovaries indicate that oocyte pattern determines the number of animal pole-specific micropylar cells that are associated with an egg via a close-range signal or direct cell contact. We demonstrate interactions between Buc protein and buc mRNA with two conserved RNA-binding proteins (RNAbps) that are localized to the Balbiani body: RNA binding protein with multiple splice isoforms 2 (Rbpms2) and Deleted in azoospermia-like (Dazl). Buc protein and buc mRNA interact with Rbpms2; buc and dazl mRNAs interact with Dazl protein. Cumulatively, these studies indicate that oocyte polarization depends on tight regulation of buc: Buc establishes oocyte polarity through interactions with RNAbps, initiating a feedback amplification mechanism in which Buc protein recruits RNAbps that in turn recruit buc and other RNAs to the Balbiani body.

Localization in Oogenesis of Maternal Regulators of Embryonic Development.

Cell polarity generates intracellular asymmetries and functional regionalization in tissues and morphogenetic processes. Cell polarity in development often relies on mechanisms of RNA localization to specific subcellular domains to define the identity of future developing tissues. The totipotent egg of most animals illustrates in a grand way the importance of cell polarity and RNA localization in regulating multiple crucial developmental events. The polarization of the egg arises during its development in oogenesis. RNAs localize asymmetrically in the early oocyte defining its animal-vegetal (AV) axis, which upon further elaboration in mid- and late-oogenesis stages produces a mature egg with specific localized factors along its AV axis. These localized factors will define the future anterior-posterior (AP) and dorsal-ventral (DV) axes of the embryo. Furthermore, AV polarity confines germ cell determinants to the vegetal pole, from where they redistribute to the cleavage furrows of the 2- and 4-cell stage embryo, ultimately specifying the primordial germ cells (PGCs). The sperm entry region during fertilization is also defined by the AV axis. In frogs and fish, sperm enters through the animal pole, similar to the mouse where it enters predominantly in the animal half. Thus, AV polarity establishment and RNA localization are involved in all the major events of early embryonic development. In this chapter, we will review the RNA localization mechanisms in vertebrate oocytes that are key to embryonic patterning, referring to some of the groundbreaking studies in frog oocytes and incorporating the current genetic evidence from the zebrafish.

Selection of mitochondria in female germline cells: is Balbiani body implicated in this process?

Early oocytes of nearly all animal species contain a transient organelle assemblage termed the Balbiani body. Structure and composition of this assemblage may vary even between closely related species. Despite this variability, the Balbiani body always comprises of numerous tightly clustered mitochondria and accumulations of nuage material. It has been suggested that the Balbiani body is an evolutionarily ancestral structure, which plays a role in various processes such as the localization of organelles and macromolecules to the germ plasm, lipidogenesis, as well as the selection/elimination of dysfunctional mitochondria from female germline cells. We suggest that the selection/elimination of mitochondria is a primary and evolutionarily ancestral function of Balbiani body, and that the other functions are secondary, evolutionarily derived additions. We propose a simple model explaining the role of the Balbiani body in the selection of mitochondria, i.e., in the mitochondrial DNA (mtDNA) bottleneck phenomenon.

Localization of germ plasm-related structures during sea urchin oogenesis.

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.

Multiplicity of Buc copies in Atlantic salmon contrasts with loss of the germ cell determinant in primates, rodents and axolotl.

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.

Exclusion of dysfunctional mitochondria from Balbiani body during early oogenesis of Thermobia.

Oocytes of many invertebrate and vertebrate species contain a characteristic organelle complex known as the Balbiani body (Bb). Until now, three principal functions have been ascribed to this complex: delivery of germ cell determinants and localized RNAs to the vegetal cortex/posterior pole of the oocyte, transport of the mitochondria towards the germ plasm, and participation in the formation of lipid droplets. Here, we present the results of a computer-aided 3D reconstruction of the Bb in the growing oocytes of an insect, Thermobia domestica. Our analyses have shown that, in Thermobia, the central part of each fully developed Bb comprises a single intricate mitochondrial network. This "core" network is surrounded by several isolated bean-shaped mitochondrial units that display lowered membrane potential and clear signs of degeneration. In light of the above results and recent theoretical models of mitochondrial quality control, the role of the Bb is discussed. We suggest that, in addition to the aforementioned functions, the Bb is implicated in the selective elimination of dysfunctional mitochondria during oogenesis.

Asymmetry in previtellogenic and early vitellogenic oocytes, ultrastructure of follicular cells and egg envelope in the pigmented sterlet, Acipenser ruthenus L. 1758 (Chondrostei, Acipenseriformes).

Ovarian follicles of sterlets (Acipenser ruthenus) are composed of a single oocyte surrounded by follicular cells (FCs), basal lamina, and thecal cells. Previtellogenic oocytes are polarized. Homogeneous ooplasm (contains ribosomes) and granular ooplasm (comprises nuage aggregations of nuclear origin, rough endoplasmic reticulum (RER), Golgi complexes, ribosomes, and mitochondria) are distinguished. Granular ooplasm is initially located near the nucleus, contacts the plasma membrane of the oocyte (oolemma) and forms a thin layer underneath its entire perimeter. Next, a ring that surrounds the nucleus is formed and sends strands directed toward the oolemma. The lipid body composed of lipid droplets forms adjacent to this ring. Later, the granular ooplasm and strands enlarge toward the oolemma, lipid body disperses, and homogeneous ooplasm is no longer present. A thin cortical ooplasm is formed underneath the oolemma and does not contain any organelles. The oocyte nucleus moves to the center. The nucleoplasm contains lampbrush chromosomes, nuclear bodies, and multiple nucleoli. Early vitellogenic oocytes are polarized, too. Three regions in the ooplasm are distinguished: the perinuclear (contains lipid droplets near the nuclear envelope), the endoplasm (contains yolk platelets and lipid droplets), and the periplasm (contains yolk spheres, pigment granules, and microtubules). In all these regions the RER, Golgi complexes, nuage, and mitochondria are present. Micropinocytotic vesicles, Golgi vesicles and precursors of the internal layer of the egg envelope are in the cortical ooplasm. Some FCs delaminate from the follicular epithelium, degenerate and vesicles are released into the perioocytic space. They may contain precursors of egg envelope and may be involved in "cell-cell" communication. The egg envelope (zona radiata, zona pellucida) is made up of three layers: the vitelline envelope (inner layer), the middle layer, and the outer layer. In its deposition, both the oocyte and FCs are engaged.

Microscopic study of the primary growth ovarian follicles of the pike-perch Sander lucioperca (Linnaeus 1758) (Actinopterygii, Perciformes).

The ovaries of Sander lucioperca (Actinopterygii, Perciformes) are made up of the germinal epithelium and ovarian follicles, in which primary oocytes grow. Each follicle is composed of an oocyte surrounded by flattened follicular cells, the basal lamina, and thecal cells. The early stages of oocyte development (primary growth = previtellogenesis) are not fully explained in this species. The results of research with the use of stereoscopic, light, fluorescence, and transmission electron microscopes on ovarian follicles containing developing primary oocytes of S. lucioperca are presented. The polarization and ultrastructure of oocytes are described and discussed. The deposition of egg envelopes during the primary growth and the ultrastructure of the eggshell in maturing follicles of S. lucioperca are also presented. Nuclei in primary oocytes comprise lampbrush chromosomes, nuclear bodies, and nucleoli. Numerous additional nucleoli arise in the nucleoplasm during primary growth and locate close to the nuclear envelope. The Balbiani body in the cytoplasm of oocytes (ooplasm) is composed of nuage aggregations of nuclear origin and mitochondria, endoplasmic reticulum (ER), and Golgi apparatus. The presence of the Balbiani body was reported in oocytes of numerous species of Actinopterygii; however, its ultrastructure was investigated in a limited number of species. In primary oocytes of S. lucioperca, the Balbiani body is initially located in the perinuclear ooplasm on one side of the nucleus. Next, it surrounds the nucleus, expands toward the plasma membrane of oocytes (oolemma), and becomes fragmented. Within the Balbiani body, the granular nuage condenses in the form of threads, locates near the oolemma, at the vegetal oocyte pole, and then dissolves. Mitochondria and cisternae of the rough endoplasmic reticulum (RER) are present between the threads. During primary growth micropylar cells differentiate in the follicular epithelium. They contain cisternae and vesicles of the RER and Golgi apparatus as well as numerous dense vesicles suggesting high synthetic and secretory activity. During the final step of primary growth several follicular cells delaminate from the follicular epithelium, migrate toward the oocyte and submerge in the most external egg envelope. In the ooplasm, three regions are distinguished: perinuclear, endoplasm, and periplasm. Cortical alveoli arise in the perinuclear ooplasm and in the endoplasm as a result of the fusion of RER vesicles with Golgi ones. They are evenly distributed. Lamellar bodies in the periplasm store the plasma membrane and release it into a space between the follicular cells and the oocyte. The developing eggshell in this space is made up of two egg envelopes (the internal one and the external) that are pierced by canals formed around the microvilli of oocytes and the processes of follicular cells. In the deposition of egg envelopes the oocyte itself and follicular cells are engaged. In maturing ovarian follicles the eggshell is solid and the internal egg envelope is covered with protuberances.

Conservation of oocyte development in germline cysts from Drosophila to mouse.

Recent studies show that pre-follicular mouse oogenesis takes place in germline cysts, highly conserved groups of oogonial cells connected by intercellular bridges that develop as nurse cells as well as an oocyte. Long studied in Drosophila and insect gametogenesis, female germline cysts acquire cytoskeletal polarity and traffic centrosomes and organelles between nurse cells and the oocyte to form the Balbiani body, a conserved marker of polarity. Mouse oocyte development and nurse cell dumping are supported by dynamic, cell-specific programs of germline gene expression. High levels of perinatal germ cell death in this species primarily result from programmed nurse cell turnover after transfer rather than defective oocyte production. The striking evolutionary conservation of early oogenesis mechanisms between distant animal groups strongly suggests that gametogenesis and early embryonic development in vertebrates and invertebrates share even more in common than currently believed.

Microinjection Method for Analyzing Zebrafish Early Stage Oocytes.

Maternal factors which accumulate and establish oocyte polarity during the early stages of oogenesis play key roles in embryonic development, as well as germ cell formation. However, vertebrate oogenesis, especially early stages of oogenesis, is not well understood due to the difficulty of accessing these oocytes and the lack of analytical methods. Here, we report on a microinjection method for analyzing zebrafish early-stage oocytes and some artifacts to be aware of when performing oocyte injections or analyzing oocytes. Using this method, we successfully injected mRNAs encoding fluorescent-tagged proteins into early-stage oocytes and observed subcellular localization in the live oocytes. This method is expected to advance the functional analysis of genes involved in oogenesis.

The need for high-quality oocyte mitochondria at extreme ploidy dictates mammalian germline development.

Selection against deleterious mitochondrial mutations is facilitated by germline processes, lowering the risk of genetic diseases. How selection works is disputed: experimental data are conflicting and previous modeling work has not clarified the issues; here, we develop computational and evolutionary models that compare the outcome of selection at the level of individuals, cells and mitochondria. Using realistic de novo mutation rates and germline development parameters from mouse and humans, the evolutionary model predicts the observed prevalence of mitochondrial mutations and diseases in human populations. We show the importance of organelle-level selection, seen in the selective pooling of mitochondria into the Balbiani body, in achieving high-quality mitochondria at extreme ploidy in mature oocytes. Alternative mechanisms debated in the literature, bottlenecks and follicular atresia, are unlikely to account for the clinical data, because neither process effectively eliminates mitochondrial mutations under realistic conditions. Our findings explain the major features of female germline architecture, notably the longstanding paradox of over-proliferation of primordial germ cells followed by massive loss. The near-universality of these processes across animal taxa makes sense in light of the need to maintain mitochondrial quality at extreme ploidy in mature oocytes, in the absence of sex and recombination.