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Drosophila oogenesis is an excellent system for the study of developmental cell biology. Active areas of research include stem cell maintenance, gamete development, pattern formation, cytoskeletal regulation, intercellular communication, intercellular transport, cell polarity, cell migration, cell death, morphogenesis, cell cycle control, and many more. The large size and relatively simple organization of egg chambers make them ideally suited for microscopy of both living and fixed whole mount tissue. A wide range of tools is available for oogenesis research. Newly available shRNA transgenic lines provide an alternative to classic loss-of-function F2 screens and clonal screens. Gene expression can be specifically controlled in either germline or somatic cells using the Gal4/UAS system. Protein trap lines provide fluorescent tags of proteins expressed at endogenous levels for live imaging and screening backgrounds. This review provides information on many available reagents and key methods for research in oogenesis.
Butterflies are popular model organisms to study physiological mechanisms underlying variability in oogenesis and egg provisioning in response to environmental conditions. Nothing is known, however, about; the developmental mechanisms governing butterfly oogenesis, how polarity in the oocyte is established, or which particular maternal effect genes regulate early embryogenesis. To gain insights into these developmental mechanisms and to identify the conserved and divergent aspects of butterfly oogenesis, we analysed a de novo ovarian transcriptome of the Speckled Wood butterfly Pararge aegeria (L.), and compared the results with known model organisms such as Drosophila melanogaster and Bombyx mori.
Drosophila oogenesis provides a developmental system with which to study nuclear actin. During Stages 5-9, nuclear actin levels are high in the oocyte and exhibit variation within the nurse cells. Cofilin and Profilin, which regulate the nuclear import and export of actin, also localize to the nuclei. Expression of GFP-tagged Actin results in nuclear actin rod formation. These findings indicate that nuclear actin must be tightly regulated during oogenesis. One factor mediating this regulation is Fascin. Overexpression of Fascin enhances nuclear GFP-Actin rod formation, and Fascin colocalizes with the rods. Loss of Fascin reduces, whereas overexpression of Fascin increases, the frequency of nurse cells with high levels of nuclear actin, but neither alters the overall nuclear level of actin within the ovary. These data suggest that Fascin regulates the ability of specific cells to accumulate nuclear actin. Evidence indicates that Fascin positively regulates nuclear actin through Cofilin. Loss of Fascin results in decreased nuclear Cofilin. In addition, Fascin and Cofilin genetically interact, as double heterozygotes exhibit a reduction in the number of nurse cells with high nuclear actin levels. These findings are likely applicable beyond Drosophila follicle development, as the localization and functions of Fascin and the mechanisms regulating nuclear actin are widely conserved.
Normal mammalian development requires the action of DNA methyltransferases (DNMTs) for the establishment and maintenance of DNA methylation within repeat elements and imprinted genes. Here we report the expression dynamics of Dnmt3a and Dnmt3b, as well as a regulator of DNA methylation, Dnmt3L, in isolated female germ cells.
The molecular events that direct nuclear pore complex (NPC) assembly toward nuclear envelopes have been conceptualized in two pathways that occur during mitosis or interphase, respectively. In gametes and embryonic cells, NPCs also occur within stacked cytoplasmic membrane sheets, termed annulate lamellae (AL), which serve as NPC storage for early development. The mechanism of NPC biogenesis at cytoplasmic membranes remains unknown. Here, we show that during Drosophila oogenesis, Nucleoporins condense into different precursor granules that interact and progress into NPCs. Nup358 is a key player that condenses into NPC assembly platforms while its mRNA localizes to their surface in a translation-dependent manner. In concert, Microtubule-dependent transport, the small GTPase Ran and nuclear transport receptors regulate NPC biogenesis in oocytes. We delineate a non-canonical NPC assembly mechanism that relies on Nucleoporin condensates and occurs away from the nucleus under conditions of cell cycle arrest.
Argonaute2 protein (Ago2) is a key component of RNA-induced gene silencing complex, which is crucial for microRNA-mediated repression of target genes. The function of Ago2 in the mouse oocyte and early embryonic development is less well characterized but it is likely to have an important role in regulating maternally inherited mRNA. We have examined the role of Ago2 by conditional deletion of the gene in developing oocytes.
Ten years ago, we reported that in adult human females the ovarian surface epithelium (OSE) is a source of germ cells. Recently, we also demonstrated that new primary follicles are formed by assembly of oocytes with nests of primitive granulosa cells in the ovarian cortex. The components of the new primary follicles, primitive granulosa and germ cells, differentiated sequentially from the OSE, which arises from cytokeratin positive mesenchymal progenitor cells residing in the ovarian tunica albuginea. In the present study, we investigated the possibility that the oocytes and granulosa cells may differentiate in cultures derived from adult human ovaries. Cells were scrapped from the surface of ovaries and cultured for 5 to 6 days, in the presence or absence of estrogenic stimuli [phenol red (PhR)]. The OSE cells cultured in the medium without PhR differentiated into small (15 micron) cells of granulosa phenotype, and epithelial, neural, and mesenchymal type cells. In contrast, OSE cells cultured in the presence of PhR differentiated directly into large (180 micron) cells of the oocyte phenotype. Such cells exhibited germinal vesicle breakdown, expulsion of the polar body, and surface expression of zona pellucida proteins, i.e. characteristics of secondary oocytes. These in vitro studies confirm our in vivo observations that in adult human ovaries, the OSE is a bipotent source of oocytes and granulosa cells. Development of numerous mature oocytes from adult ovarian stem cells in vitro offers new strategies for the egg preservation, IVF utilization, and treatment of female infertility. In addition, other clinical applications aiming to utilize stem cells, and basic stem cell research as well, may employ totipotent embryonic stem cells developing from fertilized oocytes.
Although DNA methylation is an important gene regulatory mechanism in mammals, its function in arthropods remains poorly understood. Studies in eusocial insects have argued for its role in caste development by regulating gene expression and splicing. However, such findings are not always consistent across studies, and have therefore remained controversial. Here we use CRISPR/Cas9 to mutate the maintenance DNA methyltransferase DNMT1 in the clonal raider ant, Ooceraea biroi. Mutants have greatly reduced DNA methylation, but no obvious developmental phenotypes, demonstrating that, unlike mammals, ants can undergo normal development without DNMT1 or DNA methylation. Additionally, we find no evidence of DNA methylation regulating caste development. However, mutants are sterile, whereas in wild-type ants, DNMT1 is localized to the ovaries and maternally provisioned into nascent oocytes. This supports the idea that DNMT1 plays a crucial but unknown role in the insect germline.
Poly (A)-specific ribonuclease (PARN) is the most important 3'-5'exonuclease involved in the process of deadenylation, the removal of poly (A) tails of mRNAs. Although PARN is primarily known for its role in mRNA stability, recent studies suggest several other functions of PARN including a role in telomere biology, non-coding RNA maturation, trimming of miRNAs, ribosome biogenesis and TP53 function. Moreover, PARN expression is de-regulated in many cancers, including solid tumours and hematopoietic malignancies. To better understand the in vivo role of PARN, we used a zebrafish model to study the physiological consequences of Parn loss-of-function. Exon 19 of the gene, which partially codes for the RNA binding domain of the protein, was targeted for CRISPR-Cas9-directed genome editing. Contrary to the expectations, no developmental defects were observed in the zebrafish with a parn nonsense mutation. Intriguingly, the parn null mutants were viable and fertile, but turned out to only develop into males. Histological analysis of the gonads in the mutants and their wild type siblings revealed a defective maturation of gonadal cells in the parn null mutants. The results of this study highlight yet another emerging function of Parn, i.e., its role in oogenesis.
The Notch gene in Drosophila encodes a transmembrane protein with homology to EGF that, in a variety of tissues, appears to mediate cell interactions necessary for cell fate choices. Here we demonstrate that oogenesis and spermatogenesis depend on Notch. We examine the phenotypes of the temperature-sensitive Notch allele, Nts1, and, using a monoclonal antibody, determine the cellular and subcellular distribution of Notch protein during oogenesis. We show that Nts1 is associated with a missense mutation in the extracellular, EGF homologous region of Notch and that at non-permissive temperatures oogenesis is blocked and the subcellular distribution of the protein is altered. In wild-type ovaries, Notch protein is found on the apical surface of somatically derived follicle cells, while in the germline-derived cells the protein is not polarized. These findings are discussed in view of the hypothesis that Notch acts as a multifunctional receptor to mediate developmentally important cell interactions.
During oogenesis, the egg prepares for fertilization and early embryogenesis. As a consequence, vesicle transport is very active during vitellogenesis, and oocytes are an outstanding system to study regulators of membrane trafficking. Here, we combine zebrafish genetics and the oocyte model to identify the molecular lesion underlying the zebrafish souffle (suf) mutation. We demonstrate that suf encodes the homolog of the Hereditary Spastic Paraplegia (HSP) gene SPASTIZIN (SPG15). We show that in zebrafish oocytes suf mutants accumulate Rab11b-positive vesicles, but trafficking of recycling endosomes is not affected. Instead, we detect Suf/Spastizin on cortical granules, which undergo regulated secretion. We demonstrate genetically that Suf is essential for granule maturation into secretion competent dense-core vesicles describing a novel role for Suf in vesicle maturation. Interestingly, in suf mutants immature, secretory precursors accumulate, because they fail to pinch-off Clathrin-coated buds. Moreover, pharmacological inhibition of the abscission regulator Dynamin leads to an accumulation of immature secretory granules and mimics the suf phenotype. Our results identify a novel regulator of secretory vesicle formation in the zebrafish oocyte. In addition, we describe an uncharacterized cellular mechanism for Suf/Spastizin activity during secretion, which raises the possibility of novel therapeutic avenues for HSP research.
Recent studies have investigated whether the Wnt family of extracellular ligands can signal at long range, spreading from their source and acting as morphogens, or whether they signal only in a juxtacrine manner to neighboring cells. The original evidence for long-range Wnt signaling arose from studies of Wg, a Drosophila Wnt protein, which patterns the wing disc over several cell diameters from a central source of Wg ligand. However, the requirement of long-range Wg for patterning was called into question when it was reported that replacing the secreted protein Wg with a membrane-tethered version, NRT-Wg, results in flies with normally patterned wings. We and others previously reported that Wg spreads in the ovary about 50 μm or 5 cell diameters, from the cap cells to the follicle stem cells (FSCs) and that Wg stimulates FSC proliferation. We used the NRT-wg flies to analyze the consequence of tethering Wg to the cap cells. NRT-wg homozygous flies are sickly, but we found that hemizygous NRT-wg/null flies, carrying only one copy of tethered Wingless, were significantly healthier. Despite their overall improved health, these hemizygous flies displayed dramatic reductions in fertility and in FSC proliferation. Further, FSC proliferation was nearly undetectable when the wg locus was converted to NRT-wg only in adults, and the resulting germarium phenotype was consistent with a previously reported wg loss-of-function phenotype. We conclude that Wg protein spreads from its source cells in the germarium to promote FSC proliferation.
Efficient replication of the genome and the establishment of endogenous chromatin states are processes that are essential to eukaryotic life. It is well documented that Mcm10 is intimately linked to both of these important biological processes; therefore, it is not surprising that Mcm10 is commonly misregulated in many human cancers. Most of the research regarding the biological roles of Mcm10 has been performed in single-cell or cell-free in-vitro systems. Though these systems are informative, they are unable to provide information on the cell-specific function of Mcm10 in the context of the tissue and organ systems that comprise multicellular eukaryotes. We therefore sought to identify the potential biological functions of Mcm10 in the context of a complex multicellular organism by continuing our analysis in Drosophila using three novel hypomorphic alleles. Observation of embryonic nuclear morphology and quantification of embryo hatch rates reveal that maternal loading of Mcm10 is required for embryonic nuclear stability, and suggest a role for Mcm10 post zygotic transition. Contrary to the essential nature of Mcm10 depicted in the literature, it does not appear to be required for adult viability in Drosophila if embryonic requirements are met. Although not required for adult somatic viability, analysis of fecundity and ovarian morphology in mutant females suggest that Mcm10 plays a role in maintenance of the female germline. Taken together, our results demonstrate critical roles for Mcm10 during early embryogenesis, and mark the first data linking Mcm10 to female specific reproduction in multicellular eukaryotes.
Tudor family proteins exist in all eukaryotic organisms and play a role in many cellular processes by recognizing and binding to proteins with methylated arginine or lysine residues. TDRD5, a member of Tudor domain-containing proteins (TDRDs), has been implicated in the P-element-induced wimpy testis-interacting RNA (piRNA) pathway and germ cell development in some model species, but little is known about its function in other species. Therefore, we identified and characterized LmTDRD5, the TDRD5 ortholog in Locusta migratoria, a hemimetabolous pest. The LmTdrd5 gene has 19 exons that encode a protein possessing a single copy of the Tudor domain and three LOTUS domains at its N-terminus. qRT-PCR analysis revealed a high LmTdrd5 expression level in genital glands. Using RNA interference, LmTdrd5 knockdown in males led to a lag in meiosis phase transition, decreased spermatid elongation and sperm production, and downregulated the expression of the two germ cell-specific transcription factors, LmCREM and LmACT, as well as the sperm tail marker gene LmQrich2.LmTdrd5 knockdown in females reduced the expression levels of vitellogenin (Vg) and Vg receptor (VgR) and impaired ovarian development and oocyte maturation, thus decreasing the hatchability rate. These results demonstrate that LmTdrd5 is essential for germ cell development and fertility in locusts, indicating a conserved function for TDRD5.
Female gamete production relies on coordinated molecular and cellular processes that occur in the ovary throughout oogenesis. In fish, as in other vertebrates, these processes have been extensively studied both in terms of endocrine/paracrine regulation and protein expression and activity. The role of small non-coding RNAs in the regulation of animal reproduction remains however largely unknown and poorly investigated, despite a growing interest for the importance of miRNAs in a wide variety of biological processes. Here, we analyzed the role of miR-202, a miRNA predominantly expressed in male and female gonads in several vertebrate species. We studied its expression in the medaka ovary and generated a mutant line (using CRISPR/Cas9 genome editing) to determine its importance for reproductive success with special interest for egg production. Our results show that miR-202-5p is the most abundant mature form of the miRNA and that it is expressed in granulosa cells and in the unfertilized egg. The knock out (KO) of mir-202 gene resulted in a strong phenotype both in terms of number and quality of eggs produced. Mutant females exhibited either no egg production or produced a dramatically reduced number of eggs that could not be fertilized, ultimately leading to no reproductive success. We quantified the size distribution of the oocytes in the ovary of KO females and performed a large-scale transcriptomic analysis approach to identified dysregulated molecular pathways. Together, cellular and molecular analyses indicate that the lack of miR-202 impairs the early steps of oogenesis/folliculogenesis and decreases the number of large (i.e. vitellogenic) follicles, ultimately leading to dramatically reduced female fecundity. This study sheds new light on the regulatory mechanisms that control the early steps of follicular development, including possible targets of miR-202-5p, and provides the first in vivo functional evidence that a gonad-predominant microRNA may have a major role in female reproduction.
Understanding the control of stem cell (SC) differentiation is important to comprehend developmental processes as well as to develop clinical applications. Lin28 is a conserved molecule that is involved in SC maintenance and differentiation by regulating let-7 miRNA maturation. However, little is known about the in vivo function of Lin28. Here, we report critical roles for lin-28 during oogenesis. We found that let-7 maturation was increased in lin-28 null mutant fly ovaries. We showed that lin-28 null mutant female flies displayed reduced fecundity, due to defects in egg chamber formation. More specifically, we demonstrated that in mutant ovaries, the egg chambers fuse during early oogenesis resulting in abnormal late egg chambers. We also showed that this phenotype is the combined result of impaired germline SC differentiation and follicle SC differentiation. We suggest a model in which these multiple oogenesis defects result from a misregulation of the ecdysone signaling network, through the fine-tuning of Abrupt and Fasciclin2 expression. Our results give a better understanding of the evolutionarily conserved role of lin-28 on GSC maintenance and differentiation.
Public concerns on phthalates distributions in the environment have been increasing since they can cause liver cancer, structural abnormalities and reduce sperm counts in male reproductive system. However, few data are actually available on the effects of Di-(2-ethylhexyl)-phthalate (DEHP) in female reproductive system. The aim of this study was to assess the impacts of DEHP on zebrafish oogenesis and embryo production. Female Danio rerio were exposed to environmentally relevant doses of DEHP and a significant decrease in ovulation and embryo production was observed. The effects of DEHP on several key regulators of oocyte maturation and ovulation including bone morphogenetic protein-15 (BMP15), luteinizing hormone receptor (LHR), membrane progesterone receptors (mPRs) and cyclooxygenase (COX)-2 (ptgs2) were determined by real time PCR. The expressions of BMP15 and mPR proteins were further determined by Western analyses to strengthen molecular findings. Moreover, plasma vitellogenin (vtg) titers were assayed by an ELISA procedure to determine the estrogenic effects of DEHP and its effects on oocyte growth. A significant reduction of fecundity in fish exposed to DEHP was observed. The reduced reproductive capacity was associated with an increase in ovarian BMP15 levels. This rise, in turn, was concomitant with a significant reduction in LHR and mPRbeta levels. Finally, ptgs2 expression, the final trigger of ovulation, was also decreased by DEHP. By an in vitro maturation assay, the inhibitory effect of DEHP on germinal vesicle breakdown was further confirmed. In conclusion, DEHP affecting signals involved in oocyte growth (vtg), maturation (BMP15, LHR, mPRs,) and ovulation (ptgs2), deeply impairs ovarian functions with serious consequences on embryo production. Since there is a significant genetic similarity between D.rerio and humans, the harmful effects observed at oocyte level may be relevant for further molecular studies on humans.
During oocyte differentiation in mouse fetal ovaries, sister germ cells are connected by intercellular bridges, forming germline cysts. Within the cyst, primary oocytes form via gaining cytoplasm and organelles from sister germ cells through germ cell connectivity. To uncover the role of intercellular bridges in oocyte differentiation, we analyzed mutant female mice lacking testis-expressed 14 (TEX14), a protein involved in intercellular bridge formation and stabilization. In Tex14 homozygous mutant fetal ovaries, germ cells divide to form a reduced number of cysts in which germ cells remained connected via syncytia or fragmented cell membranes, rather than normal intercellular bridges. Compared with wild-type cysts, homozygous mutant cysts fragmented at a higher frequency and produced a greatly reduced number of primary oocytes with precocious cytoplasmic enrichment and enlarged volume. By contrast, Tex14 heterozygous mutant germline cysts were less fragmented and generate primary oocytes at a reduced size. Moreover, enlarged primary oocytes in homozygous mutants were used more efficiently to sustain folliculogenesis than undersized heterozygous mutant primary oocytes. Our observations directly link the nature of fetal germline cysts to oocyte differentiation and development.
Oogenesis in the domestic silkworm (Bombyx mori) is a complex process involving previtellogenesis, vitellogenesis and choriogenesis. During this process, follicles show drastic morphological and physiological changes. However, the genome-wide regulatory profiles of gene expression during oogenesis remain to be determined.
The determination of cell fate and the establishment of polarity axes during Drosophila oogenesis depend upon pathways that localize mRNAs within the egg chamber and control their on-site translation. One factor that plays a central role in regulating on-site translation of mRNAs is Orb. Orb is a founding member of the conserved CPEB family of RNA-binding proteins. These proteins bind to target sequences in 3' UTRs and regulate mRNA translation by modulating poly(A) tail length. In addition to controlling the translation of axis-determining mRNAs like grk, fs(1)K10, and osk, Orb protein autoregulates its own synthesis by binding to orb mRNA and activating its translation. We have previously shown that Rasputin (Rin), the Drosophila homologue of Ras-GAP SH3 Binding Protein (G3BP), associates with Orb in a messenger ribonucleoprotein (mRNP) complex. Rin is an evolutionarily conserved RNA-binding protein believed to function as a link between Ras signaling and RNA metabolism. Here we show that Orb and Rin form a complex in the female germline. Characterization of a new rin allele shows that rin is essential for oogenesis. Co-localization studies suggest that Orb and Rin form a complex in the oocyte at different stages of oogenesis. This is supported by genetic and biochemical analyses showing that rin functions as a positive regulator in the orb autoregulatory pathway by increasing Orb protein expression. Tandem Mass Spectrometry analysis shows that several canonical stress granule proteins are associated with the Orb-Rin complex suggesting that a conserved mRNP complex regulates localized translation during oogenesis in Drosophila.
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