Aberrant oogenesis in the patterning mutant dicephalic of Drosophila melanogaster: time-lapse recordings and volumetry in vitro

Summary Drosophila females homozygous for the mutation dicephalic occasionally produce ovarian follicles with a nurse-cell cluster on each oocyte pole (dic follicles). Most dic follicles contain 15 nurse cells as in the normal follicle, but the total nurse-cell volume is larger in dic follicles; this is in keeping with the increase in DNA content recently described. However, the relative increase in oocyte volume during nurse-cell regression (from stage 10B onward) is not significantly larger in dic than in normal follicles. Time-lapse recordings in vitro show that, as a rule, both nurse cell clusters in a dic follicle export cytoplasm to the oocyte but nurse-cell regression remains incomplete at both poles and the persisting remnants of the nurse cells cause anomalies in chorion shape. The kinematics of cytoplasmic transfer are less aberrant at that oocyte pole which harbours the germinal vesicle. Possible links are discussed between these anomalies of oogenesis and the double-anterior embryonic patterns observed in the majority of developing dic eggs.     

Development of the rat oocyte in vitro: inhibition and induction of maturation in the presence or absence of the cumulus oophorus

The effect in vitro of oocyte maturation inhibitors and the ability of preparations of luteinizing hormone to relieve the arrest induced by these substances was studied in preparations of cumulus-free (naked) rat oocytes, and compared to previously obtained results from oocytes enclosed by their cumulus cells. The development of both the cumulus-oocyte complex and naked oocyte is arrested in vitro by cyclic AMP derivatives or cyclic nucleotide phosphodiesterase inhibitors. While gonadotropins can overcome the effect of these substances in the cumulus oocyte complex, they have no effect on naked oocytes. Cholera enterotoxin, an irreversible activator of adenylate cyclase, maintains developmental arrest in cultured cumulus oocyte complexes but not in naked oocytes. Preparations of luteinizing hormone can partially overcome the effect of cholera enterotoxin in the complexes. Furthermore, the acceleration of oocyte maturation in vitro observed in the presence of gonadotropins, which is seen in cumulus oocyte complexes, can be mimicked by stripping the oocyte of its associated cumulus cells. The results of these and other studies suggest that: (1) the cytoplasmic levels of cyclic AMP in the isolated oocyte are high enough to maintain meiotic arrest if a phosphodiesterase inhibitor is present; (2) the oocyte contains an active phosphodiesterase; (3) the oolemma may lack the adenylate cyclase system and; (4) gonadotropins seem to affect the oocyte indirectly, via the cumulus cells, possibly by interrupting communication between the two cell types.     

Translational repression of gurken mRNA in the Drosophila oocyte requires the hnRNP Squid in the nurse cells

Establishment of the Drosophila dorsal-ventral axis depends upon the correct localization of gurken mRNA and protein within the oocyte. gurken mRNA becomes localized to the presumptive dorsal anterior region of the oocyte, but is synthesized in the adjoining nurse cells. Normal gurken localization requires the heterogeneous nuclear ribonucleoprotein Squid, which binds to the gurken 3′ untranslated region. However, whether Squid functions in the nurse cells or the oocyte is unknown. To address this question, we generated genetic mosaics in which half of the nurse cells attached to a given oocyte are unable to produce Squid. In these mosaics, gurken mRNA is localized normally but ectopically translated during the dorsal anterior localization process, even though the oocyte contains abundant Squid produced by the wild type nurse cells. These data indicate that translational repression of gurken mRNA requires Squid function in the nurse cells. We propose that Squid interacts with gurken mRNA in the nurse cell nuclei and, together with other factors, maintains gurken in a translationally silent state during its transport to the dorsal anterior region of the oocyte. This translational repression is not required for gurken mRNA localization, indicating that the information repressing translation is separable from that regulating localization.     

INTERCELLULAR MIGRATION OF CENTRIOLES IN THE GERMARIUM OF DROSOPHILA MELANOGASTER : An Electron Microscopic Study

A cluster of centrioles has been found in the early Drosophila oocyte. Since the oocyte is connected to 15 nurse cells by a system of intercellular bridges or ring canals, the possibility that the cluster of centrioles arose in the germarium from an intercellular migration of centrioles from the nurse cells to the oocyte was analyzed in serial sections for the electron microscope. Initially, all of the 16 cells of the future egg chambers possess centrioles, which are located in a juxtanuclear position. At the time the 16 cell cluster becomes arranged in a lens-shaped layer laterally across the germarium, the centrioles lose their juxtanuclear position and move towards the oocyte. By the time the 16 cell cluster of cells is surrounded by follicle cells (Stage 1), between 14 and 17 centrioles are found in the oocyte. Later, these centrioles become located between the oocyte nucleus and the follicle cell border and become aggregated into a cluster less than 1.5 µ in its largest dimension. The fate of these centrioles in the oocyte is not known. The fine structure of the germarium and the early oocyte is also described.     

Opposing roles for Egalitarian and Staufen in transport,anchoring and localization of oskar mRNA in the Drosophila oocyte

Localization of oskar mRNA includes two distinct phases: transport from nurse cells to the oocyte, a process typically accompanied by cortical anchoring in the oocyte, followed by posterior localization within the oocyte. Signals within the oskar 3’ UTR directing transport are individually weak, a feature previously hypothesized to facilitate exchange between the different localization machineries. We show that alteration of the SL2a stem-loop structure containing the oskar transport and anchoring signal (TAS) removes an inhibitory effect such that in vitro binding by the RNA transport factor, Egalitarian, is elevated as is in vivo transport from the nurse cells into the oocyte. Cortical anchoring within the oocyte is also enhanced, interfering with posterior localization. We also show that mutation of Staufen recognized structures (SRSs), predicted binding sites for Staufen, disrupts posterior localization of oskar mRNA just as in staufen mutants. Two SRSs in SL2a, one overlapping the Egalitarian binding site, are inferred to mediate Staufen-dependent inhibition of TAS anchoring activity, thereby promoting posterior localization. The other three SRSs in the oskar 3’ UTR are also required for posterior localization, including two located distant from any known transport signal. Staufen, thus, plays multiple roles in localization of oskar mRNA.     

Observation des spermatozoïdes au fort grossissement (MSOME): intérêt et perspectives

Motile Sperm Organelle Morphology Examination (MSOME) constitutes a real improvement in ART management and outcome, as it allows detection of specific sperm anomalies on living cells, which cannot be detected by routine analysis. MSOME applied to the selection of sperm injected into the oocyte is called IMSI (Intracytoplasmic Morphologically Selected sperm Injection) and is associated with a considerable improvement of implantation, clinical pregnancy and delivery rates. A high-power microscope (X 2,000 to X 10,000) and video enhancement system are necessary and technical limitations are related to cryptozoospermia and/or severe teratozoospermia. Compared to routine sperm morphology assessment, MSOME allows the detection of subtle cephalic anomalies, such as vacuoles. These vacuoles seem to have a deleterious effect on fertilization and embryo developmentin vitro. These observations have led to a detailed classification of anomalies and this morphological diagnosis on living sperm demonstrates that most sperm selected for conventional ICSI at X 400 are actually abnormal on MSOME at X 6,000 or more. In addition to the very good results obtained in IMSI, this new approach opens up interesting prospects concerning the relationship between the phenotype of the injected sperm and its fertilization capacity and embryo development. In terms of diagnosis, MSOME could be used to select and study homogeneous groups of normal sperm or homogeneous groups of sperm exhibiting the same well defined anomaly. Such studies, associated with fine analysis of injected sperm and follow-up of each oocyte and each embryo, should provide more information about the relationship between sperm structure and function and should help to define the relevant indications for IMSI and the choice of spermatozoa to be injected.     

Mago Nashi, Tsunagi/Y14, and Ranshi form a complex that influences oocyte differentiation in Drosophila melanogaster

During Drosophila melanogaster oogenesis, a germline stem cell divides forming a cyst of 16 interconnected cells. One cell enters the oogenic pathway, and the remaining 15 differentiate as nurse cells. Although directed transport and localization of oocyte differentiation factors within the single cell are indispensible for selection, maintenance, and differentiation of the oocyte, the mechanisms regulating these events are poorly understood. Mago Nashi and Tsunagi/Y14, core components of the exon junction complex (a multiprotein complex assembled on spliced RNAs), are essential for restricting oocyte fate to a single cell and for localization of oskar mRNA. Here we provide evidence that Mago Nashi and Tsunagi/Y14 form an oogenic complex with Ranshi, a protein with a zinc finger-associated domain and zinc finger domains. Genetic analyses of ranshi reveal that (1) 16-cell cysts are formed, (2) two cells retain synaptonemal complexes, (3) all cells have endoreplicated DNA (as observed in nurse cells), and (4) oocyte-specific cytoplasmic markers accumulate and persist within a single cell but are not localized within the posterior pole of the presumptive oocyte. Our results indicate that Ranshi interacts with the exon junction complex to localize components essential for oocyte differentiation within the posterior pole of the presumptive oocyte.     

Cell-cell communication and axis specification in the Drosophila oocyte

Intercellular communication between the somatic and germline cells is vital to development of the Drosophila egg chamber. One critical outcome of this communication is the polarization of the oocyte along the anterior-posterior axis, a process induced by an unknown signal from the somatic follicle cells to the oocyte. The existence of this signal has been inferred from several reports demonstrating that the differentiation and patterning of the follicle cells by the spatially restricted activation of certain cell-signaling pathways is necessary for axis formation in the oocyte. These reports have also provided a framework for understanding how these signaling pathways are integrated to generate the follicle-cell pattern, but the precise role of the follicle cells in anterior-posterior axis formation remains enigmatic. Research has identified several genes that appear to be involved in the polarizing communication from the follicle cells to the oocyte. Interestingly the proteins encoded by most of these genes are associated with the extracellular matrix, suggesting a pivotal role for this complex biological component in the polarizing communication between the follicle cells and the oocyte. This review summarizes the findings in this area, and uses the experimental analyses of these genes to evaluate various models describing the possible nature of the polarizing signal, and the role of these genes in it.     

A stem–loop structure directs oskar mRNA to microtubule minus ends

mRNA transport coupled with translational control underlies the intracellular localization of many proteins in eukaryotic cells. This is exemplified in Drosophila, where oskar mRNA transport and translation at the posterior pole of the oocyte direct posterior patterning of the embryo. oskar localization is a multistep process. Within the oocyte, a spliced oskar localization element (SOLE) targets oskar mRNA for plus end-directed transport by kinesin-1 to the posterior pole. However, the signals mediating the initial minus end-directed, dynein-dependent transport of the mRNA from nurse cells into the oocyte have remained unknown. Here, we show that a 67-nt stem–loop in the oskar 3′ UTR promotes oskar mRNA delivery to the developing oocyte and that it shares functional features with the fs(1)K10 oocyte localization signal. Thus, two independent cis-acting signals, the oocyte entry signal (OES) and the SOLE, mediate sequential dynein- and kinesin-dependent phases of oskar mRNA transport during oogenesis. The OES also promotes apical localization of injected RNAs in blastoderm stage embryos, another dynein-mediated process. Similarly, when ectopically expressed in polarized cells of the follicular epithelium or salivary glands, reporter RNAs bearing the oskar OES are apically enriched, demonstrating that this element promotes mRNA localization independently of cell type. Our work sheds new light on how oskar mRNA is trafficked during oogenesis and the RNA features that mediate minus end-directed transport.     

A Dual Role for Actin and Microtubule Cytoskeleton in the Transport of Golgi Units from the Nurse Cells to the Oocyte Across Ring Canals

Axis specification during Drosophila embryonic development requires transfer of maternal components during oogenesis from nurse cells (NCs) into the oocyte through cytoplasmic bridges. We found that the asymmetrical distribution of Golgi, between nurse cells and the oocyte, is sustained by an active transport process. We have characterized actin basket structures that asymmetrically cap the NC side of Ring canals (RCs) connecting the oocyte. Our results suggest that these actin baskets structurally support transport mechanisms of RC transit. In addition, our tracking analysis indicates that Golgi are actively transported to the oocyte rather than diffusing. We observed that RC transit is microtubule-based and mediated at least by dynein. Finally, we show that actin networks may be involved in RC crossing through a myosin II step process, as well as in dispatching Golgi units inside the oocyte subcompartments.     

Regulation of oocyte number during oocyte differentiation in the lizard Podarcis sicula

This paper concerns the differentiation process of germ cells from oogonia to primary follicles in the lizard Podarcis sicula. The study was carried out at the morphological level and using a cytophotometric analysis for determining the number of differentiating germ cells undergoing degeneration. The progressive disorganization of the germ cell clusters during the early diplotene stage and the role played by the prefollicular cells during this process are described. Oocyte degeneration has been observed between the mid-zygotene and the early diplotene stages. When the primary follicle (oocyte plus follicular cells) is being formed, the degeneration process stops and the oocyte undergoes regular growth and ovulation.     

Mitochondrial development during Drosophila oogenesis: distribution,density and in situ RNA hybridizations

The changes in distribution and density of mitochondria and the level of mitochondrial RNA during Drosophila oogenesis were studied simultaneously in the 3 cell types ie follicle cells, nurse cells and oocyte, making up the egg chamber. Up to stage 6, mitochondrial density (mitochondrial and cellular areas ratio) was elevated and increased similarly in both follicle and nurse cells. Thereafter the mitochondrial density of follicle cells continued to increase and that of the nurse cells declined markedly while the nurse cell mitochondria assembled in dense groups and decreased in size. This can be related to a transfer of nurse cell cytoplasm, including mitochondria, to the oocyte. In the oocyte from stage 4 to stage 7 we observed a significant decrease of the mitochondrial density due to the absence of mitochondrial biogenesis. Then the cytoplasm transfer caused mitochondrial density to increase up to the level found in the nurse cells at the end of oogenesis. The mature oocyte contains enough mitochondria to supply 15 000 somatic cells. Our results strongly suggest that the variations in size, distribution and density of mitochondria relate to the particular energetic requirements of the different cell types during the first half of oogenesis. Later they relate to the developmental requirements of the nurse cells and the oocyte, in particular the storage of mitochondria in the oocyte. The level of mitochondrial RNA was studied through in situ hybridization. Throughout oogenesis the follicle and nurse cell RNA evolved similarly. Up to stage 9, there was no change in RNA densities in these cells, suggesting a correlation with the cell volume and/or the nuclear DNA content. Thereafter the cellular RNA concentration declined rapidly. In the oocyte the RNA concentration evolved differently especially from stage 10 to the end, the RNA density being stabilized. This can be related to the injection of nurse cell mitochondria, followed by their assignment to reserve status. Our results suggest that the mt RNA density is under extramitochondrial control mechanisms.     

Microtubule Actin Crosslinking Factor 1 Regulates the Balbiani Body and Animal-Vegetal Polarity of the Zebrafish Oocyte

Although of fundamental importance in developmental biology, the genetic basis for the symmetry breaking events that polarize the vertebrate oocyte and egg are largely unknown. In vertebrates, the first morphological asymmetry in the oocyte is the Balbiani body, a highly conserved, transient structure found in vertebrates and invertebrates including Drosophila, Xenopus, human, and mouse. We report the identification of the zebrafish magellan (mgn) mutant, which exhibits a novel enlarged Balbiani body phenotype and a disruption of oocyte polarity. To determine the molecular identity of the mgn gene, we positionally cloned the gene, employing a novel DNA capture method to target region-specific genomic DNA of 600 kb for massively parallel sequencing. Using this technique, we were able to enrich for the genomic region linked to our mutation within one week and then identify the mutation in mgn using massively parallel sequencing. This is one of the first successful uses of genomic DNA enrichment combined with massively parallel sequencing to determine the molecular identity of a gene associated with a mutant phenotype. We anticipate that the combination of these technologies will have wide applicability for the efficient identification of mutant genes in all organisms. We identified the mutation in mgn as a deletion in the coding sequence of the zebrafish microtubule actin crosslinking factor 1 (macf1) gene. macf1 is a member of the highly conserved spectraplakin family of cytoskeletal linker proteins, which play diverse roles in polarized cells such as neurons, muscle cells, and epithelial cells. In mgn mutants, the oocyte nucleus is mislocalized; and the Balbiani body, localized mRNAs, and organelles are absent from the periphery of the oocyte, consistent with a function for macf1 in nuclear anchoring and cortical localization. These data provide the first evidence for a role for spectraplakins in polarization of the vertebrate oocyte and egg.     

Essential role of ubiquitin C-terminal hydrolases UCHL1 and UCHL3 in mammalian oocyte maturation

Ubiquitin C-terminal hydrolases (UCHs) comprise a family of deubiquitinating enzymes that play a role in the removal of multi-ubiquitin chains from proteins that are posttranslationally modified by ubiquitination to be targeted for proteolysis by the 26S proteasome. The UCH-enzymes also generate free monomeric ubiquitin from precursor multi-ubiquitin chains and, in some instances, may rescue ubiquitinated proteins from degradation. This study examined the roles of two oocyte-expressed UCHs, UCHL1, and UCHL3 in murine and rhesus monkey oocyte maturation. The Uchl1 and Uchl3 mRNAs were highly expressed in GV and MII oocytes, and were associated with the oocyte cortex (UCHL1) and meiotic spindle (UCHL3). Microinjection of the UCH-family enzyme inhibitor, ubiquitin-aldehyde (UBAL) to GV oocytes prevented oocyte meiotic progression beyond metaphase I in a majority of treated oocytes and caused spindle and first polar body anomalies. Injection of antibodies against UCHL3 disrupted oocyte maturation and caused meiotic anomalies, including abnormally long meiotic spindles. A selective, cell permeant inhibitor of UCHL3, 4, 5, 6, 7-tetrachloroidan-1, 3-dione also caused meiotic defects and chromosome misalignment. Cortical granule localization in the oocyte cortex was disrupted by UBAL injected after oocyte maturation. We conclude that the activity of oocyte UCHs contributes to oocyte maturation by regulating the oocyte cortex and meiotic spindle.     

Biochemical research on oogenesis: Oocytes and liver cells of the teleost fish Tinca tinca contain different kinds of 5S RNA

Previtellogenic oocytes of Tinca tinca accumulate very large amounts of 5S RNA. We show here that 5S RNA stored in oocytes differs from liver 5S RNA in 3 out of 120 nucleotides. Liver and oocyte 5S RNAs, therefore, are produced by different genes. Both kinds of 5S genes are active in oocytes. However, only 5S RNA of the oocyte type accumulates in these cells. In Tinca tinca as in Xenopus laevis, oocyte-type and somatic-type 5S RNAs differ by three properties, ie., primary structure, conformation, and metabolic stability. Nucleotide substitutions occur in different positions in oocyte and somatic 5S RNAs of Tinca tinca and Xenopus laevis. We do not understand how different sets of nucleotide substitutions confer to 5S RNAs of both species similar properties in vivo, namely, increased metabolic stability.