Single-cell profiling reveals transcriptome dynamics during bovine oocyte growth.

BACKGROUND: Mammalian follicle development is characterized by extensive changes in morphology, endocrine responsiveness, and function, providing the optimum environment for oocyte growth, development, and resumption of meiosis. In cattle, the first signs of transcription activation in the oocyte are observed in the secondary follicle, later than during mouse and human oogenesis. While many studies have generated extensive datasets characterizing gene expression in bovine oocytes, they are mostly limited to the analysis of fully grown and matured oocytes. The aim of the present study was to apply single-cell RNA sequencing to interrogate the transcriptome of the growing bovine oocyte from the secondary follicle stage through to the mid-antral follicle stage. RESULTS: Single-cell RNA-seq libraries were generated from oocytes of known diameters (< 60 to > 120 µm), and datasets were binned into non-overlapping size groups for downstream analysis. Combining the results of weighted gene co-expression network and Trendy analyses, and differently expressed genes (DEGs) between size groups, we identified a decrease in oxidative phosphorylation and an increase in maternal -genes and transcription regulators across the bovine oocyte growth phase. In addition, around 5,000 genes did not change in expression, revealing a cohort of stable genes. An interesting switch in gene expression profile was noted in oocytes greater than 100 µm in diameter, when the expression of genes related to cytoplasmic activities was replaced by genes related to nuclear activities (e.g., chromosome segregation). The highest number of DEGs were detected in the comparison of oocytes 100-109 versus 110-119 µm in diameter, revealing a profound change in the molecular profile of oocytes at the end of their growth phase. CONCLUSIONS: The current study provides a unique dataset of the key genes and pathways characteristic of each stage of oocyte development, contributing an important resource for a greater understanding of bovine oogenesis.

Bellymount-Pulsed Tracking: A Novel Approach for Real-Time In vivo Imaging of Drosophila Oogenesis.

Quantitative live imaging is a valuable tool that offers insights into cellular dynamics. However, many fundamental biological processes are incompatible with current live imaging modalities. Drosophila oogenesis is a well-studied system that has provided molecular insights into a range of cellular and developmental processes. The length of the oogenesis coupled with the requirement for inputs from multiple tissues has made long-term culture challenging. Here, we have developed Bellymount-Pulsed Tracking (Bellymount-PT), which allows continuous, non-invasive live imaging of Drosophila oogenesis inside the female abdomen for up to 16 hours. Bellymount-PT improves upon the existing Bellymount technique by adding pulsed anesthesia with periods of feeding that support the long-term survival of females during imaging. Using Bellymount-PT we measure key events of oogenesis including egg chamber growth, yolk uptake, and transfer of specific proteins to the oocyte during nurse cell dumping with high spatiotemporal precision within the abdomen of a live female.

Developmental changes in nuclear lamina components during germ cell differentiation.

The nuclear lamina (NL) changes composition for regulation of nuclear events. We investigated changes that occur in Drosophila oogenesis, revealing switches in NL composition during germ cell differentiation. Germline stem cells (GSCs) express only LamB and predominantly emerin, whereas differentiating nurse cells predominantly express LamC and emerin2. A change in LamC-specific localization also occurs, wherein phosphorylated LamC redistributes to the nuclear interior only in the oocyte, prior to transcriptional reactivation of the meiotic genome. These changes support existing concepts that LamC promotes differentiation, a premise that was tested. Remarkably ectopic LamC production in GSCs did not promote premature differentiation. Increased LamC levels in differentiating germ cells altered internal nuclear structure, increased RNA production, and reduced female fertility due to defects in eggshell formation. These studies suggest differences between Drosophila lamins are regulatory, not functional, and reveal an unexpected robustness to level changes of a major scaffolding component of the NL.

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.

Methylosome protein 50 is necessary for oogenesis in medaka.

Methylosome protein 50 (Mep50) functions as a partner to protein arginine methyltransferase 5. MEP50 serves as a coactivator for both the androgen receptor and estrogen receptor in humans. Mep50 plays a crucial role in the development of germ cells in Drosophila. The precise role of Mep50 in oogenesis remains unclear in vertebrates. The objective of this study was to investigate the role of Mep50 in oogenesis in medaka fish. Disruption of Mep50 resulted in impaired oogenesis and the formation of multiple oocyte follicles in medaka. RNA-seq analysis revealed significant differential gene expression in the mutant ovary, with 4542 genes up-regulated and 1264 genes down-regulated. The regulated genes were found to be enriched in cellular matrices and ECM-receptor interaction, the Notch signaling pathway, the PI3K-Akt signaling pathway, the MAPK signaling pathway, the Hippo signaling pathway, and the Jak-Stat pathway, among others. In addition, the genes related to the hypothalamus-pituitary-gonad axis, steroid metabolism, and IGF system were impacted. Furthermore, the mutation of mep50 caused significant alterations in alternative splicing of pre-mRNA in ovarian cells. Quantitative RT-PCR results validated the findings from RNA-seq analysis in the specific genes, including akt2, map3k5, yap1, fshr, cyp17a, igf1, ythdc2, cdk6, and col1, among others. The findings of this study demonstrate that Mep50 plays a crucial role in oogenesis, participating in a diverse range of biological processes such as steroid metabolism, cell matrix regulation, and signal pathways. This may be achieved through the regulation of gene expression via mRNA splicing in medaka ovarian cells.

A Cytological F1 RNAi Screen for Defects in Drosophila melanogaster Female Meiosis.

Genetic screens for recessive alleles induce mutations, make the mutated chromosomes homozygous, and then assay those homozygotes for the phenotype of interest. When screening for genes required for female meiosis, the phenotype of interest has typically been nondisjunction from chromosome segregation errors. As this requires that mutant females be viable and fertile, any mutants that are lethal or sterile when homozygous cannot be recovered by this approach. To overcome these limitations, we have screened the VALIUM22 collection of RNAi constructs that target germline-expressing genes in a vector optimized for germline expression by driving RNAi with GAL4 under control of a germline-specific promoter (nanos or mat-alpha4). This allowed us to test genes that would be lethal if knocked down in all cells, and by examining unfertilized metaphase-arrested mature oocytes, we could identify defects in sterile females. After screening >1450 lines of the collection for two different defects (chromosome congression and the hypoxic sequestration of Mps1-GFP to ooplasmic filaments), we obtained multiple hits for both phenotypes, identified novel meiotic phenotypes for genes that had been previously characterized in other processes, and identified the first phenotypes to be associated with several previously uncharacterized genes.

Integrated stress response signaling acts as a metabolic sensor in fat tissues to regulate oocyte maturation and ovulation.

Reproduction is an energy-intensive process requiring systemic coordination. However, the inter-organ signaling mechanisms that relay nutrient status to modulate reproductive output are poorly understood. Here, we use Drosophila melanogaster as a model to establish the integrated stress response (ISR) transcription factor, Atf4, as a fat tissue metabolic sensor that instructs oogenesis. We demonstrate that Atf4 regulates lipase activity to mediate yolk lipoprotein synthesis in the fat body. Depletion of Atf4 in the fat body also blunts oogenesis recovery after amino acid deprivation and re-feeding, suggestive of a nutrient-sensing role for Atf4. We also discovered that Atf4 promotes secretion of a fat-body-derived neuropeptide, CNMamide, which modulates neural circuits that promote egg-laying behavior (ovulation). Thus, we posit that ISR signaling in fat tissue acts as a "metabolic sensor" that instructs female reproduction-directly by impacting yolk lipoprotein production and follicle maturation and systemically by regulating ovulation.

Collective cell migration relies on PPP1R15-mediated regulation of the endoplasmic reticulum stress response.

Collective cell migration is integral to many developmental and disease processes. Previously, we discovered that protein phosphatase 1 (Pp1) promotes border cell collective migration in the Drosophila ovary. We now report that the Pp1 phosphatase regulatory subunit dPPP1R15 is a critical regulator of border cell migration. dPPP1R15 is an ortholog of mammalian PPP1R15 proteins that attenuate the endoplasmic reticulum (ER) stress response. We show that, in collectively migrating border cells, dPPP1R15 phosphatase restrains an active physiological protein kinase R-like ER kinase- (PERK)-eIF2α-activating transcription factor 4 (ATF4) stress pathway. RNAi knockdown of dPPP1R15 blocks border cell delamination from the epithelium and subsequent migration, increases eIF2α phosphorylation, reduces translation, and drives expression of the stress response transcription factor ATF4. We observe similar defects upon overexpression of ATF4 or the eIF2α kinase PERK. Furthermore, we show that normal border cells express markers of the PERK-dependent ER stress response and require PERK and ATF4 for efficient migration. In many other cell types, unresolved ER stress induces initiation of apoptosis. In contrast, border cells with chronic RNAi knockdown of dPPP1R15 survive. Together, our results demonstrate that the PERK-eIF2α-ATF4 pathway, regulated by dPPP1R15 activity, counteracts the physiological ER stress that occurs during collective border cell migration. We propose that in vivo collective cell migration is intrinsically "stressful," requiring tight homeostatic control of the ER stress response for collective cell cohesion, dynamics, and movement.

The molecular mechanisms underpinning maternal mRNA dormancy.

A large number of mRNAs of maternal origin are produced during oogenesis and deposited in the oocyte. Since transcription stops at the onset of meiosis during oogenesis and does not resume until later in embryogenesis, maternal mRNAs are the only templates for protein synthesis during this period. To ensure that a protein is made in the right place at the right time, the translation of maternal mRNAs must be activated at a specific stage of development. Here we summarize our current understanding of the sophisticated mechanisms that contribute to the temporal repression of maternal mRNAs, termed maternal mRNA dormancy. We discuss mechanisms at the level of the RNA itself, such as the regulation of polyadenine tail length and RNA modifications, as well as at the level of RNA-binding proteins, which often block the assembly of translation initiation complexes at the 5' end of an mRNA or recruit mRNAs to specific subcellular compartments. We also review microRNAs and other mechanisms that contribute to repressing translation, such as ribosome dormancy. Importantly, the mechanisms responsible for mRNA dormancy during the oocyte-to-embryo transition are also relevant to cellular quiescence in other biological contexts.

Dynamic Cytoophidia during Late-Stage Drosophila Oogenesis.

CTP synthase (CTPS) catalyzes the final step of de novo synthesis of CTP. CTPS was first discovered to form filamentous structures termed cytoophidia in Drosophila ovarian cells. Subsequent studies have shown that cytoophidia are widely present in cells of three life domains. In the Drosophila ovary model, our previous studies mainly focused on the early and middle stages, with less involvement in the later stages. In this work, we focus on the later stages of female germline cells in Drosophila. We use live-cell imaging to capture the continuous dynamics of cytoophidia in Stages 10-12. We notice the heterogeneity of cytoophidia in the two types of germline cells (nurse cells and oocytes), manifested in significant differences in morphology, distribution, and dynamics. Surprisingly, we also find that neighboring nurse cells in the same egg chamber exhibit multiple dynamic patterns of cytoophidia over time. Although the described dynamics may be influenced by the in vitro incubation conditions, our observation provides an initial understanding of the dynamics of cytoophidia during late-stage Drosophila oogenesis.

Ras/MAPK signaling mediates adipose tissue control of ovarian germline survival and ovulation in Drosophila melanogaster.

From insects to humans, oogenesis is tightly linked to nutritional input, yet little is known about how whole organism physiology matches dietary changes with oocyte development. Considering that diet-induced adipose tissue dysfunction is associated with an increased risk for fertility problems, and other obesity-associated pathophysiologies, it is critical to decipher the cellular and molecular mechanisms linking adipose nutrient sensing to remote control of the ovary and other tissues. Our previous studies in Drosophila melanogaster have shown that amino acid sensing, via the amino acid response pathway and mTOR-mediated signaling function within adipocytes to control germline stem cell maintenance and ovulation, respectively. Additionally, we demonstrated that insulin/insulin-like growth factor signaling within adipocytes employs distinct effector axes, PI3K/Akt1-dependent and -independent, downstream of insulin receptor activity to mediate fat-to-ovary communication. Here, we report that the Ras/MAPK signaling axis functions in adipocytes to regulate early germline cyst survival and ovulation of mature oocytes but is not important for germline stem cell maintenance or the progression through vitellogenesis. Thus, these studies uncover the complexity of signaling pathway activity that mediates inter-organ communication.

Premature endocycling of Drosophila follicle cells causes pleiotropic defects in oogenesis.

Endocycling cells grow and repeatedly duplicate their genome without dividing. Cells switch from mitotic cycles to endocycles in response to developmental signals during the growth of specific tissues in a wide range of organisms. The purpose of switching to endocycles, however, remains unclear in many tissues. Additionally, cells can switch to endocycles in response to conditional signals, which can have beneficial or pathological effects on tissues. However, the impact of these unscheduled endocycles on development is underexplored. Here, we use Drosophila ovarian somatic follicle cells as a model to examine the impact of unscheduled endocycles on tissue growth and function. Follicle cells normally switch to endocycles at mid-oogenesis. Inducing follicle cells to prematurely switch to endocycles resulted in the lethality of the resulting embryos. Analysis of ovaries with premature follicle cell endocycles revealed aberrant follicular epithelial structure and pleiotropic defects in oocyte growth, developmental gene amplification, and the migration of a special set of follicle cells known as border cells. Overall, these findings reveal how unscheduled endocycles can disrupt tissue growth and function to cause aberrant development.

Regulation and Functions of Autophagy During Animal Development.

Autophagy is used to degrade cytoplasmic materials, and is critical to maintain cell and organismal health in diverse animals. Here we discuss the regulation, utilization and impact of autophagy on development, including roles in oogenesis, spermatogenesis and embryogenesis in animals. We also describe how autophagy influences postembryonic development in the context of neuronal and cardiac development, wound healing, and tissue regeneration. We describe recent studies of selective autophagy during development, including mitochondria-selective autophagy and endoplasmic reticulum (ER)-selective autophagy. Studies of developing model systems have also been used to discover novel regulators of autophagy, and we explain how studies of autophagy in these physiologically relevant systems are advancing our understanding of this important catabolic process.

Ana1/CEP295 is an essential player in the centrosome maintenance program regulated by Polo kinase and the PCM.

Centrioles are part of centrosomes and cilia, which are microtubule organising centres (MTOC) with diverse functions. Despite their stability, centrioles can disappear during differentiation, such as in oocytes, but little is known about the regulation of their structural integrity. Our previous research revealed that the pericentriolar material (PCM) that surrounds centrioles and its recruiter, Polo kinase, are downregulated in oogenesis and sufficient for maintaining both centrosome structural integrity and MTOC activity. We now show that the expression of specific components of the centriole cartwheel and wall, including ANA1/CEP295, is essential for maintaining centrosome integrity. We find that Polo kinase requires ANA1 to promote centriole stability in cultured cells and eggs. In addition, ANA1 expression prevents the loss of centrioles observed upon PCM-downregulation. However, the centrioles maintained by overexpressing and tethering ANA1 are inactive, unlike the MTOCs observed upon tethering Polo kinase. These findings demonstrate that several centriole components are needed to maintain centrosome structure. Our study also highlights that centrioles are more dynamic than previously believed, with their structural stability relying on the continuous expression of multiple components.

Disruption of Zar1 leads to arrested oogenesis by regulating polyadenylation via Cpeb1 in tilapia (Oreochromis niloticus).

Oogenesis is a complex process regulated by precise coordination of multiple factors, including maternal genes. Zygote arrest 1 (zar1) has been identified as an ovary-specific maternal gene that is vital for oocyte-to-embryo transition and oogenesis in mouse and zebrafish. However, its function in other species remains to be elucidated. In the present study, zar1 was identified with conserved C-terminal zinc finger domains in Nile tilapia. zar1 was highly expressed in the ovary and specifically expressed in phase I and II oocytes. Disruption of zar1 led to the failed transition from oogonia to phase I oocytes, with somatic cell apoptosis. Down-regulation and failed polyadenylation of figla, gdf9, bmp15 and wee2 mRNAs were observed in the ovaries of zar1-/- fish. Cpeb1, a gene essential for polyadenylation that interacts with Zar1, was down-regulated in zar1-/- fish. Moreover, decreased levels of serum estrogen and increased levels of androgen were observed in zar1-/- fish. Taken together, zar1 seems to be essential for tilapia oogenesis by regulating polyadenylation and estrogen synthesis. Our study shows that Zar1 has different molecular functions during gonadal development by the similar signaling pathway in different species.

Auxin exposure disrupts feeding behavior and fatty acid metabolism in adult Drosophila.

The ease of genetic manipulation in Drosophila melanogaster using the Gal4/UAS system has been beneficial in addressing key biological questions. Current modifications of this methodology to temporally induce transgene expression require temperature changes or exposure to exogenous compounds, both of which have been shown to have detrimental effects on physiological processes. The recently described auxin-inducible gene expression system (AGES) utilizes the plant hormone auxin to induce transgene expression and is proposed to be the least toxic compound for genetic manipulation, with no obvious effects on Drosophila development and survival in one wild-type strain. Here, we show that auxin delays larval development in another widely used fly strain, and that short- and long-term auxin exposure in adult Drosophila induces observable changes in physiology and feeding behavior. We further reveal a dosage response to adult survival upon auxin exposure, and that the recommended auxin concentration for AGES alters feeding activity. Furthermore, auxin-fed male and female flies exhibit a significant decrease in triglyceride levels and display altered transcription of fatty acid metabolism genes. Although fatty acid metabolism is disrupted, auxin does not significantly impact adult female fecundity or progeny survival, suggesting AGES may be an ideal methodology for studying limited biological processes. These results emphasize that experiments using temporal binary systems must be carefully designed and controlled to avoid confounding effects and misinterpretation of results.

Comparative Transcriptome Analysis Reveals the Role of Ribosome Reduction in Impeding Oogenesis in Female Triploid Crassostrea Gigas.

The fecundity of triploid female Crassostrea gigas exhibited significant variation and was lower compared to diploid individuals. Previous studies categorized mature stage triploid female C. gigas into two groups: female α, characterized by a high number of oocytes, and female ß, displaying few or no oocytes. To investigate the molecular mechanisms underlying irregular oogenesis and fecundity differences in triploid C. gigas, we performed a comparative analysis of gonad transcriptomes at different stages of gonadal development, including female α, female ß, and diploids. During early oogenesis, functional enrichment analysis between female diploids and putative female ß triploids revealed differently expressed genes (DEGs) in the ribosome and ribosome biogenesis pathways. Expression levels of DEGs in these pathways were significantly decreased in the putative female ß triploid, suggesting a potential role of reduced ribosome levels in obstructing triploid oogenesis. Moreover, to identify regulatory pathways in gonad development, female oysters at the early and mature stages were compared. The DNA repair and recombination proteins pathways were enriched in female diploids and female α triploids but absent in female ß triploids. Overall, we propose that decreased ribosome biogenesis in female triploids hinders the differentiation of germ stem cells, leading to the formation of a large number of abnormal germ cells and ultimately resulting in reduced fecundity. The variation in fertility among triploids appeared to be related to the degree of DNA damage repair during female gonad development. This study offers valuable insights into the oogenesis process in female triploid C. gigas.

Maternal High-Fat Diet Affects the Contents of Eggs and Causes Abnormal Development in the Medaka Fish.

Maternal nutritional status can affect development and metabolic phenotypes of progeny in animals. The effects of maternal diet are thought to be mediated mainly by changes inside oocytes such as organelles, maternal RNAs, and metabolites. However, to what extent each factor contributes to offspring phenotypes remains uncertain, especially in viviparous mammalian systems, where factors other than oocytes, such as placenta and milk, need to be considered. Here, using the medaka fish as an oviparous vertebrate model, we examined whether maternal high-fat diet (mHFD) feeding affects offspring development and what kind of changes occur in the contents of mature eggs. We found that mHFD caused the high frequency of embryonic deformities of offspring, accompanied by downregulation of transcription- and translation-related genes and zygotic transcripts at the blastula stage. Transcriptomic and metabolomic analyses of mature eggs suggested decreased catabolism of amino acids and glycogen, moderate upregulation of endoplasmic reticulum stress-related genes, and elevated lipid levels in mHFD eggs. Furthermore, high-fat diet females showed a higher incidence of oocyte atresia and downregulation of egg protein genes in the liver. These data suggest that attenuated amino acid catabolism triggered by decreased yolk protein load/processing, as well as elevated lipid levels inside eggs, are the prime candidates that account for the higher incidence of embryonic deformities in mHFD offspring. Our study presents a comprehensive data on the changes inside eggs in a mHFD model of nonmammalian vertebrates and provides insights into the mechanisms of parental nutritional effects on offspring.

"A comparison of thermal stress response between Drosophila melanogaster and Drosophila pseudoobscura reveals differences between species and sexes".

The environment is changing faster than anticipated due to climate change, making species more vulnerable to its impacts. The level of vulnerability of species is influenced by factors such as the degree and duration of exposure, as well as the physiological sensitivity of organisms to changes in their environments, which has been shown to vary among species, populations, and individuals. Here, we compared physiological changes in fecundity, critical thermalmaximum (CTmax), respiratory quotient (RQ), and DNA damage in ovaries in response to temperature stress in two species of fruit fly, Drosophila melanogaster (25 vs. 29.5 °C) and Drosophila pseudoobscura (20.5 vs. 25 °C). The fecundity of D. melanogaster was more affected by high temperatures when exposed during egg through adult development, while D. pseudoobscura was most significantly affected when exposed to high temperatures exclusively during egg through pupal development. Additionally, D. melanogaster males exhibited a decrease of CTmax under high temperatures, while females showed an increase of CTmax when exposed to high temperatures during egg through adult development. while D. pseudoobscura females and males showed an increased CTmax only when reared at high temperatures during egg through pupae development. Moreover, both species showed an acceleration in oogenesis and an increase in apoptosis due to heat stress. These changes can likely be attributed to key differences in the geographic range, thermal range, development time, and other different factors between these two systems. Through this comparison of variation in physiology and developmental response to thermal stress, we found important differences between species and sexes that suggest future work needs to account for these factors separately in understanding the effects of constant increased temperatures.

Parasitoid cues modulate Drosophila germline development and stem cell proliferation.

Environmental factors influence an organism's reproductive ability by regulating germline development and physiology. While the reproductive adaptations in response to extrinsic stress cues offer fitness and survival advantages to individuals, the mechanistic understanding of these modifications remains unclear. Here, we find that parasitoid wasps' stress signaling regulates Drosophila melanogaster oogenesis. We show that fruit flies dwelling in the wasp-infested area elevate their fecundity, and the observed reproductive response is specific to Pachycrepoideus sp., a pupal parasitoid wasp. Pachycrepoideus-specific olfactory and visual cues recruit the signaling pathways that promote germline stem cell proliferation and accelerate follicle development, increasing egg production in Drosophila females. Downregulation of signaling engaged in oocyte development by shifting flies to a non-wasp-infested environment increases apoptosis of the developing follicles. Thus, this study establishes host germline responsiveness to parasitoid-specific signals and supports a predator strategy to increase hosts for infection.