Follicular Somatic Cells Research Articles

Rejuvenation of aged oocyte through exposure to young follicular microenvironment.

Reproductive aging is a major cause of fertility decline, attributed to decreased oocyte quantity and developmental potential. A possible cause is aging of the surrounding follicular somatic cells that support oocyte growth and development by providing nutrients and regulatory factors. Here, by creating chimeric follicles, whereby an oocyte from one follicle was transplanted into and cultured within another follicle whose native oocyte was removed, we show that young oocytes cultured in aged follicles exhibited impeded meiotic maturation and developmental potential, whereas aged oocytes cultured within young follicles were significantly improved in rates of maturation, blastocyst formation and live birth after in vitro fertilization and embryo implantation. This rejuvenation of aged oocytes was associated with enhanced interaction with somatic cells, transcriptomic and metabolomic remodeling, improved mitochondrial function and higher fidelity of meiotic chromosome segregation. These findings provide the basis for a future follicular somatic cell-based therapy to treat female infertility.

Follicular oocyte as a potential target for severe acute respiratory syndrome coronavirus 2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported in December 2019 and rapidly became a pandemic as coronavirus disease 2019 (COVID-19). Apart from other organs, presence of specific receptor angiotensin-converting enzyme (ACE2) and corresponding proteases such as transmembrane serine protease 2, basigin and cysteine protease cathepsin L make follicular somatic cells as well as oocyte as potential targets for SARS-CoV-2 infection. The SARS-CoV-2 causes inflammation and hypoxia that generate reactive oxygen species (ROS) in critically ill patients. In addition, a large number of casualties and insecurity of life due to repeated waves of SARS-CoV-2 infection generate psychological stress and cortisol resulting in the further generation of ROS. The excess levels of ROS under physiological range cause meiotic instability, while high levels result in oxidative stress that trigger various death pathways and affect number as well as quality of follicular oocytes. Although, emerging evidence suggests that the SARS-CoV-2 utilises cellular machinery of ovarian follicular cells, generates ROS and impairs quality of follicular oocytes, the underlying mechanism of viral entry into host cell and its negative impact on the follicular oocyte remains poorly understood. Therefore, this review summarises emerging evidence on the presence of cellular machinery for SARS-CoV-2 in ovarian follicles and the potential negative impact of viral infection on the follicular oocytes that affect ovarian functions in critically ill and stressed women.

Dynamics of extracellular vesicle uptake by mural granulosa cells in mice.

Extracellular vesicles (EVs) play an important role in the development and function of mammalian ovarian follicles. However, the mechanisms by which they are taken up by the follicular granulosa cells remain unclear. In addition, while oocytes play a pivotal role in follicular development, the possible interactions between oocyte-derived paracrine factors (ODPFs) and EV signals are unknown. Therefore, this study aimed to elucidate the mechanism of EV uptake and the effects of ODPFs on EV uptake by follicular somatic mural granulosa cells in mice. Fluorescence-labeled transferrin (TRF) and cholera toxin B (CTB), substrates for clathrin- and caveolae-mediated endocytosis, respectively, were taken up by mural granulosa cells in vitro. Their uptake was inhibited by Pitstop 2 and genistein, inhibitors of clathrin and caveolae pathways, respectively. Mural granulosa cells took up EVs, and this uptake was suppressed by Pitstop 2 and genistein. Moreover, ODPFs promoted the uptake of EVs and CTB, but not TRF, by mural granulosa cells. These results suggest that mural granulosa cells take up EVs through both clathrin- and caveolae-mediated endocytosis and that oocytes may promote caveolae-mediated endocytosis to facilitate the uptake of EVs.

Maturation and culture affect the metabolomic profile of oocytes and follicular cells in young and old mares.

Introduction: Oocytes and follicular somatic cells within the ovarian follicle are altered during maturation and after exposure to culture in vitro. In the present study, we used a nontargeted metabolomics approach to assess changes in oocytes, cumulus cells, and granulosa cells from dominant, follicular-phase follicles in young and old mares. Methods: Samples were collected at three stages associated with oocyte maturation: (1) GV, germinal vesicle stage, prior to the induction of follicle/oocyte maturation in vivo; (2) MI, metaphase I, maturing, collected 24 h after induction of maturation in vivo; and (3) MIIC, metaphase II, mature with collection 24 h after induction of maturation in vivo plus 18 h of culture in vitro. Samples were analyzed using gas and liquid chromatography coupled to mass spectrometry only when all three stages of a specific cell type were obtained from the same mare. Results and Discussion: Significant differences in metabolite abundance were most often associated with MIIC, with some of the differences appearing to be linked to the final stage of maturation and others to exposure to culture medium. While differences occurred for many metabolite groups, some of the most notable were detected for energy and lipid metabolism and amino acid abundance. The study demonstrated that metabolomics has potential to aid in optimizing culture methods and evaluating cell culture additives to support differences in COCs associated with maternal factors.

Is FSH combined with equine chorionic gonadotropin able to modify lipid metabolism in bovine superstimulated antral follicles?

Lipid metabolism is essential for ensuring oocyte maturation and embryo development. β-Oxidized fatty acids (FA) are a potent source of energy for cells, particularly for bovine somatic follicular cells. Superstimulatory protocols using follicle stimulating hormone (FSH) or FSH combined with equine chorionic gonadotropin (eCG) are capable of stimulating the follicular microenvironment and drive the expression of biomarker genes associated with lipid metabolism in the cumulus-oocyte complex (COC) for better embryo development. In this study, we assesed the effects of FSH and FSH/eCG protocols on the expression of genes related to lipid metabolism in bovine granulosa cells (GCs). Further, we measured triglyceride levels in follicular fluid (FF) obtained from both superstimulatd and non-superstimulated cows (synchronized cows). In summary, superstimulation with gonadotropins maintained the TG levels in bovine FF and ensured GCs mRNA abundance of ACSL1, ACSL3, ACSL6, SCD, ELOVL5, ELOVL6, FASN, FADS2, and SREBP1. We, however, found the abundance of CPTIB mRNA to be lower in GCs obtained from cows subjected to FSH/eCG protocols than synchronized cows. In conclusion, the findings of this study showed that ovarian superstimulation around the preovulatory phase has a mild impact on the lipid metabolism in GCs.

Equine maternal aging affects the metabolomic profile of oocytes and follicular cells during different maturation time points.

Introduction: Oocyte quality and fertility decline with advanced maternal age. During maturation within the ovarian follicle, the oocyte relies on the associated somatic cells, specifically cumulus and granulosa cells, to acquire essential components for developmental capacity. Methods: A nontargeted metabolomics approach was used to investigate the effects of mare age on different cell types within the dominant, follicular-phase follicle at three time points during maturation. Metabolomic analyses from single oocytes and associated cumulus and granulosa cells allowed correlations of metabolite abundance among cell types. Results and Discussion: Overall, many of the age-related changes in metabolite abundance point to Impaired mitochondrial metabolic function and oxidative stress in oocytes and follicular cells. Supporting findings include a higher abundance of glutamic acid and triglycerides and lower abundance of ceramides in oocytes and somatic follicular cells from old than young mares. Lower abundance of alanine in all follicular cell types from old mares, suggests limited anaerobic energy metabolism. The results also indicate impaired transfer of carbohydrate and free fatty acid substrates from cumulus cells to the oocytes of old mares, potentially related to disruption of transzonal projections between the cell types. The identification of age-associated alterations in the abundance of specific metabolites and their correlations among cells contribute to our understanding of follicular dysfunction with maternal aging.

Immunolocalization of activin and inhibin at different stages of follicular development in the lizard Sceloporus torquatus

The activins and inhibins are glycoproteins with a role in the follicular development of vertebrates, that are found in follicular fluid and somatic follicular cells, with a different pattern among taxa. The principal function of activin (Act) is to modulate the follicle-stimulating hormone (FSH) synthesis and secretion, whereas inhibin (Inh) downregulates it. Both factors are modulators of intraovarian follicular recruitment, oocyte maturation, cell proliferation, and steroidogenic activity. Our aim was to characterize the immunolocalization of Act and Inh in the ovarian follicles during the reproductive cycle of the lizard Sceloporus torquatus. Act was detected in the granulosa cells and oocyte cortex in the different stages of follicular development. On the other hand, we identified Inh in the oocyte cortex and the cytoplasm of pyriform and small cells of previtellogenic follicles. Also, we found immunoreactivity in the oocyte cortex, theca, and small cells of vitellogenic and preovulatory follicles. Our data provide evidence that Act and Inh have changes related to the stage of follicular development. This dynamic appears to be conserved among vertebrates and is fundamental to ensure an adequate follicular development in this specie.

Translocation of Oocytic HES1 into Surrounding Cumulus Cells in Bovine: Mechanism of Cellular Interaction during IVM?

HES1 (hairy and enhancer of split-1, effector of the NOTCH pathway) plays a role in oocyte maturation and has been detected so far mainly in somatic follicular cells. In this study, we aimed to investigate whether HES1 is present in both compartments of bovine cumulus oocyte complexes (COCs) and whether in vitro maturation itself has an effect on its distribution. We investigated the abundance of HES1 mRNA and protein in bovine COCs characterized by Brilliant-Cresyl-Blue (BCB) stainability by RT-PCR and immunofluorescence before and after in vitro maturation (IVM). To study the interaction of the compartments and the possible translocation of HES1, we injected GFP-HES1 mRNA into oocytes before maturation and analyzed fluorescence recovery after photobleaching (FRAP). The results showed that HES1 mRNA was detectable in oocytes but not in cumulus cells. The number of transcripts increased with maturation, especially in BCB-positive oocytes. In contrast, the protein was mainly visible in cumulus cells both before and after maturation. After GFP-HES1-mRNA injection into oocytes, a signal could be detected not only in the oocytes but also in cumulus cells. Our result shows a nearly exclusive distribution of HES1 mRNA and protein in oocytes and cumulus cells, respectively, that might be explained by the transfer of the protein from the oocyte into cumulus cells.

From in vivo to in vitro: exploring the key molecular and cellular aspects of human female gametogenesis.

Human oogenesis is a highly complex and not yet fully understood process due to ethical and technological barriers that limit studies in the field. In this context, replicating female gametogenesis in vitro would not only provide a solution for some infertility problems, but also be an excellent study model to better understand the biological mechanisms that determine the formation of the female germline. In this review, we explore the main cellular and molecular aspects involved in human oogenesis and folliculogenesis in vivo, from the specification of primordial germ cells (PGCs) to the formation of the mature oocyte. We also sought to describe the important bidirectional relationship between the germ cell and the follicular somatic cells. Finally, we address the main advances and different methodologies used in the search for obtaining cells of the female germline in vitro.

Metabolite abundance in bovine preovulatory follicular fluid is influenced by follicle developmental progression post estrous onset in cattle.

Introduction: Preovulatory follicle response to the luteinizing hormone (LH) surge leads to metabolic, molecular, and functional changes in the oocyte and somatic follicular cells from the onset of estrus to ovulation. Follicular fluid contains metabolites, miRNAs, proteins, and hormones that are byproducts of follicular metabolism and support cellular processes of oocyte, cumulus, and granulosa constituents. Numerous studies have highlighted the importance of follicular fluid composition to support fertility, but critical gaps exist toward understanding dynamic modifications in the follicular fluid metabolome from estrous onset to ovulation. The hypothesis was that abundance of follicular fluid metabolites is dependent on follicle progression post LH surge and variability in follicular fluid metabolome profiles indicate key processes required for preparation of the follicle and oocyte for optimal fertility. The objective was to generate preovulatory follicular fluid metabolome profiles and discern differences in the metabolome of preovulatory follicular fluid samples collected at onset of estrus, 11 h post estrous onset, and 18 h post estrous onset. Methods: Estrus was synchronized in non-lactating Jersey cows (n=40) and follicular fluid was collected immediately after the first observed standing mount (hr 0) or at approximately h 11 or 18 after the first standing mount. Ultra-High-Performance Liquid Chromatography-High Resolution Mass Spectrometry was performed on preovulatory follicular fluid samples (n = 9 collected at hr 0, 9 at h 11, and 10 at h 18) and a multiple linear model was performed to determine if time post estrous onset impacted metabolite abundance. Results: Metabolites influenced by time post estrous onset were tested for enrichment in KEGG pathways. Ninety metabolites were identified in follicular fluid samples. Twenty metabolites differed in abundance among timepoints post estrous onset (p ≤ 0.05). Pathways corresponding to amino acid and energy metabolism were enriched with metabolites impacted by time post estrous onset (FDR ≤ 0.10). Discussion: Results from the current study indicate early response to the LH surge to increase bioavailability of amino acids and metabolites used by the cumulus and granulosa cells for energy production and shuttled into the oocyte to support meiotic maturation. Such metabolites may later be used by the ovulatory follicle for protein production.

Bitter Taste Receptors and Endocrine Disruptors: Cellular and Molecular Insights from an In Vitro Model of Human Granulosa Cells.

Endocrine disrupting chemicals (EDCs) are compounds that interfere with the synthesis, transport and binding action of hormones responsible for reproduction and homeostasis. Some EDCs compounds are activators of Taste bitter Receptors, a subclass of taste receptors expressed in many extraoral locations, including sperm and follicular somatic cells. This makes TAS2Rs attractive molecules to study and investigate to shed light on the effect of EDCs on female reproduction and fertility. This study aims to assess the effect of selected EDCs [namely Biochanin A (BCA), caffeine, Daidzein, Genistein and Isoflavone] on hGL5, an immortalized cell line exhibiting characteristics coherent with primary follicular granulosa cells. After demonstrating that this model expresses all the TAS2Rs (TAS2R3, TAS2R4, TAS2R14, TAS2R19, TAS2R43) specifically expressed by the primary human granulosa cells, we demonstrated that BCA and caffeine significantly affect mitochondrial footprint and intracellular lipid content, indicating their contribution in steroidogenesis. Our results showed that bitter taste receptors may be involved in steroidogenesis, thus suggesting an appealing mechanism by which these compounds affect the female reproductive system.

P-591 Cellular heterogeneity in the preovulatory follicle correlates with response to stimulation with recombinant FSH and affects somatic cell gene expression levels

Abstract Study question What is the difference in the composition and gene expression of pre-ovulatory follicular somatic cells between normo- and hyporesponder patients to rFSH stimulation? Summary answer Gene expression changes in the somatic cells of the preovulatory follicles of hyporesponder patients is partly attributable to distinct cell populations. What is known already Some women undergoing IVF respond suboptimally to the preceding ovarian stimulation without an indication of poor ovarian insufficiency nor advanced age. The gene expression levels in the somatic cells of their pre-ovulatory follicles have not been determined. However, the somatic cells play key roles in the transfer of gonadotrophin signals and in steroidogenesis. In the preovulatory follicle, being >20 mm in size, the somatic cells are exposed to various molecular gradients that drive their differentiation. Due to advancements in sequencing technologies the importance of somatic cell sub-populations in the etiologies of ovarian infertility can now be investigated by bioinformatic methods. Study design, size, duration Consecutive IVF patients <41 years of age were recruited at Nova Vita Clinic, Estonia, undergoing the antagonist stimulation protocol with rFSH administration. Ovarian puncture was performed for all patients after 36 hours of hCG stimulaton. rFSH dose >200 IU per one retrieved oocyte was considered as an indication for hyporesponse. Ten normo- and 9 hyporesponder patients were enrolled. Participants/materials, setting, methods Cells from the follicular fluid devoid of the cumulus oocyte complex were collected by centrifugation. RNA-seq was performed on the pooled cells of all participants. The study groups were compared by differential gene expression analysis by DESeq2. Single-cell RNA-seq was performed for >6000 individual cells of 3 normoresponder patients and cellular sub-populations were determined with Seurat package. CIBERTSORTx software was used to deconvolute the proportion of cell types from bulk RNA-seq data of all participants. Main results and the role of chance Alterations in the gene expression of ovarian somatic cells have been previously attributed to female age. We demonstrate that 407 genes are differentially expressed (FDR<0.05) between normo- and hyporesponder IVF patients after age adjustment. These genes were enriched into pathways of extracellular matrix reorganization (31 genes, FDR<0.005), post-translational protein phosphorylation (16 genes, FDR<0.006) and regulation of insulin-like growth factor transport and uptake by insulin-like growth factor binding proteins (16 genes, FDR<0.019). In addition, the sequencing of > 24 000 individual cells from 3 normoresponder patients revealed the somatic cell types of the preovulatory follicle in high resolution indicating the presence of immune cell populations (N = 4), epithelial cells, theca, cumulus cells and mural granulosa cell sub-populations (N = 7). Bioinformatic cell type deconvolution demonstrated significant differences between proportions of distinct cell sub-populations between normo- and hyporesponder patients and that several differentially expressed genes between the study groups can be attributed to a specific mural granulosa sub-population. Limitations, reasons for caution The number of study participants is small due to the high cost of the study methods. Wider implications of the findings The study demonstrates that hyporesponse to stimulation is associated with age-unrelated disturbances in gene expression of preovulatory somatic cells and that different somatic cell populations may have important underlying functions in the ovarian etiologies of infertility. Trial registration number not applicable

Dynamic transcriptome analysis of ovarian follicles in artificial maturing Japanese eel (Anguilla japonica)

Inducing maturation of the ovaries to enable the production of good-quality eggs is critical for the successful artificial breeding of Anguilla japonica. During the spawning season, however, the ovaries of A. japonica have been found to develop into asynchronous clutches, impeding the success of artificial breeding on a commercial scale. The dynamic molecular regulation of follicular development in the same individual was assessed by transcriptome analysis of the five stages of follicles, the pre-vitellogenic, early vitellogenic, midvitellogenic, late vitellogenic, and migratory nucleus stages in artificial maturing A. japonica. Comparisons across these developmental stages identified a total of 19,298 differentially expressed transcripts (DETs). Short time-series expression miner analysis across these DETs revealed four significant expression profiles. Gene Ontology function and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses found that some of the significantly enriched biological processes and metabolic pathways included those related to steroid hormone biosynthesis (cyp11a1, cyp17a1, cyp17a2, hsd17b1, and hsd17b12), cargo receptor activity (vtgr and vldlr), meiosis and ovulation (pgrs and mPRγ), hydration (cts and aqp1), and egg coat formation (zp). These genes and pathways were associated with serum 17β-estradiol concentrations and morphological changes. The levels of hsd17b12 and mPRγ mRNAs were much higher during the migratory nucleus stage, suggesting their respective involvement in the biosynthesis and functional pathway of the maturation-inducing steroid 17α,20β-dihydroxy-4-pregnen-3-one. The gene subtypes aqp1b and ctsd may regulate water influx into oocytes and yolk protein proteolysis, respectively. To our knowledge, the present study is the first to describe combined transcriptome profiling of asynchronously developing follicles in the same individual. The findings suggest that steroid hormone synthesis and nutrient absorption in follicular somatic cells play important roles during follicular development and maturation, despite the same external physiological surroundings.

Bitter Taste Receptors Expression in Human Granulosa and Cumulus Cells: New Perspectives in Female Fertility.

Bitter taste receptors (TAS2RS) expression is not restricted to the oral cavity and the presence of these receptors in the male reproductive system and sperm provides insights into their possible role in human reproduction. To elucidate the potential role of TAS2Rs in the female reproductive system, we investigated the expression and localization of bitter taste receptors and the components of signal transduction cascade involved in the pathway of taste receptors in somatic follicular cells obtained from women undergoing assisted reproductive techniques. We found that TAS2R genes are expressed in both cumulus (CCs) and granulosa (GCs) cells, with TAS2R14 being the most highly expressed bitter receptor subtype. Interestingly, a slight increase in the expression of TAS2R14 and TAS2R43 was shown in both GCs and CCs in young women (p < 0.05), while a negative correlation may be established between the number of oocytes collected at the pickup and the expression of TAS2R43. Regarding α-gustducin and α-transducin, two Gα subunits expressed in the taste buds on the tongue, we provide evidence for their expression in CCs and GCs, with α-gustducin showing two additional isoforms in GCs. Finally, we shed light on the possible downstream transduction pathway initiated by taste receptor activation in the female reproductive system. Our study, showing for the first time the expression of taste receptors in the somatic ovarian follicle cells, significantly extends the current knowledge of the biological role of TAS2Rs for human female fertility.

MiR-23b-3p inhibits chicken granulosa cell proliferation and steroid hormone synthesis via targeting GDF9

MicroRNAs (miRNAs) are ∼22 nt RNAs that direct post-transcriptional repression of mRNA targets in diverse eukaryotic lineages. Granulosa cells (GCs) are the earliest differentiated follicular somatic cells. From the initiation of primordial follicles, their differentiation and growth are closely related to the development of follicles. The research on follicular development mostly focused on the granular layer, as well as the hormone synthesis induced by granulosa cell differentiation before and after follicular selection. In this study, we evaluated the effects of miR-23b-3p on chicken granulosa cells, including granulosa cell proliferation and steroid hormone synthesis. Elevated expression of miR-23b-3p significantly inhibited granulosa cell proliferation and steroid hormone synthesis, but did not affect apoptosis. Furthermore, it was observed that the forecast growth differentiation factor 9 (GDF9) is a target gene of miR-23b-3p and miR-23b-3p can down-regulate expression of GDF9. Overall, this study demonstrated that miR-23b-3p can regulate the proliferation and steroid hormone synthesis of chicken granulosa cells by inhibiting the expression of GDF9.

Metabolic exchanges between the oocyte and its environment: focus on lipids.

Finely regulated fatty acid (FA) metabolism within ovarian follicles is crucial to follicular development and influences the quality of the enclosed oocyte, which relies on the surrounding intra-follicular environment for its growth and maturation. A growing number of studies have examined the association between the lipid composition of follicular compartments and oocyte quality. In this review, we focus on lipids, their possible exchanges between compartments within the ovarian follicle and their involvement in different pathways during oocyte final growth and maturation. Lipidomics provides a detailed snapshot of the global lipid profiles and identified lipids, clearly discriminating the cells or fluid from follicles at distinct physiological stages. Follicular fluid appears as a main mediator of lipid exchanges between follicular somatic cells and the oocyte, through vesicle-mediated and non-vesicular transport of esterified and free FA. A variety of expression data allowed the identification of common and cell-type-specific actors of lipid metabolism in theca cells, granulosa cells, cumulus cells and oocytes, including key regulators of FA uptake, FA transport, lipid transformation, lipoprotein synthesis and protein palmitoylation. They act in harmony to accompany follicular development, and maintain intra-follicular homeostasis to allow the oocyte to accumulate energy and membrane lipids for subsequent meiotic divisions and first embryo cleavages.

Single-cell RNA sequencing analysis of mouse follicular somatic cells†.

Within the development of ovarian follicle, in addition to cell proliferation and differentiation, sophisticated cell-cell cross talks are established among follicular somatic cells such as granulosa cells (GCs) and theca cells. To systematically reveal the cell differentiation and signal transductions in follicular somatic cells, we collected the mouse follicular somatic cells from secondary to ovulatory stage, and analyzed the single cell transcriptomes. Having data filtered and screened, we found 6883 high variable genes in 4888 single cells. Then follicular somatic cells were clustered into 26 cell clusters, including 18 GC clusters, 4 theca endocrine cell (TEC) clusters, and 4 other somatic cell clusters, which include immune cells and Acta2 positive theca externa cells. From our data, we found there was metabolic reprogramming happened during GC differentiation. We also found both Cyp19a1 and Cyp11a1 could be expressed in TECs. We analyzed the expression patterns of genes associated with cell-cell interactions such as steroid hormone receptor genes, insulin signaling genes, and cytokine/transformation growth factor beta associated genes in all cell clusters. Lastly, we clustered the highly variable genes into 300 gene clusters, which could be used to search new genes involved in follicle development. These transcriptomes of follicular somatic cells provide us potential clues to reveal how mammals regulating follicle development and could help us find targets to improve oocyte quality for women with low fertility.

MALDI-TOF Mass Spectrometry Revealed Significant Lipid Variations in Follicular Fluid and Somatic Follicular Cells but Not in Enclosed Oocytes between the Large Dominant and Small Subordinate Follicles in Bovine Ovary.

Lipid metabolism in ovarian follicular cells supports the preparation of an enclosed oocyte to ovulation. We aimed to compare lipid composition of a dominant large follicle (LF) and subordinated small follicles (SFs) within the same ovaries. Mass spectrometry imaging displayed the differences in the distribution of several lipid features between the different follicles. Comparison of lipid fingerprints between LF and SF by Matrix Assisted Laser Desorption/Ionisation Time-Of-Flight (MALDI-TOF) mass spectrometry revealed that in the oocytes, only 8 out of 468 detected lipids (1.7%) significantly changed their abundance (p < 0.05, fold change > 2). In contrast, follicular fluid (FF), granulosa, theca and cumulus cells demonstrated 55.5%, 14.9%, 5.3% and 9.8% of significantly varied features between LF and SF, respectively. In total, 25.2% of differential lipids were identified and indicated potential changes in membrane and signaling lipids. Tremendous changes in FF lipid composition were likely due to the stage specific secretions from somatic follicular cells that was in line with the differences observed from FF extracellular vesicles and gene expression of candidate genes in granulosa and theca cells between LF and SF. In addition, lipid storage in granulosa and theca cells varied in relation to follicular size and atresia. Differences in follicular cells lipid profiles between LF and SF may probably reflect follicle atresia degree and/or accumulation of appropriate lipids for post-ovulation processes as formation of corpus luteum. In contrast, the enclosed oocyte seems to be protected during final follicular growth, likely due in part to significant lipid transformations in surrounding cumulus cells. Therefore, the enclosed oocyte could likely keep lipid building blocks and energy resources to support further maturation and early embryo development.

Asymmetry in the cytoplasm of oocytes of largescale yellowfish Labeobarbus marequensis Smith 1841 (Teleostei: Cypriniformes: Cyprinidae).

The ovaries of the largescale yellowfish, Labeobarbus marequensis (Teleostei: Cypriniformes: Cyprinidae), are made up of the germinal epithelium, nests of late chromatin nucleolus stage oocytes, and ovarian follicles. Each follicle is composed of a single oocyte, which is surrounded by somatic follicular cells and a basal lamina covered by thecal cells. We describe polarization and ultrastructure of oocytes during the primary growth stage. The oocyte nucleus contains lampbrush chromosomes, nuclear bodies and fibrillar material in which multiple nucleoli arise. Nuage aggregations composed of material of a nuclear origin are present in the perinuclear cytoplasm. The Balbiani body (Bb) contains aggregations of nuage, rough endoplasmic reticulum, individual mitochondria and complexes of mitochondria with nuage (cement). Some mitochondria in the Bb come into close contact with endoplasmic reticulum cisternae and vesicles that contain granular material. At the start of primary growth, the Bb is present in the cytoplasm close to the nucleus. Next, it expands towards the oocyte plasma membrane. In these oocytes, a spherical structure, the so-called yolk nucleus, arises in the Bb. It consists of granular nuage in which mitochondria and vesicles containing granular material are immersed. Later, the Bb becomes fragmented and a fully grown yolk nucleus is present in the vegetal region. It contains numerous threads composed of granular nuage, mitochondria, lysosome-like organelles and autophagosomes. We discuss the formation of autophagosomes in the cytoplasm of primary growth oocytes. During the final step of primary growth, the cortical alveoli arise in the cytoplasm and are distributed evenly. The eggshell is deposited on the external surface of the oocyte plasma membrane and is made up of two egg envelopes that are pierced by numerous pore canals. The external egg envelope is covered in protuberances. During primary growth no lipid droplets are synthesized or stored in the oocytes.

Mitofusin1 is required for oocyte growth and communication with follicular somatic cells.

Mitochondrial function, largely regulated by the dynamics of this organelle, is inextricably linked to the oocyte health. In comparison with most somatic cells, mitochondria in oocytes are smaller and rounder in appearance, suggesting limited fusion. The functional implications of this distinct morphology, and how changes in the mitochondrial shape translate to mitochondrial function in oogenesis is little understood. We, therefore, asked whether the pro-fusion proteins mitofusins 1 (MFN1) and 2 (MFN2) are required for the oocyte development. Here we show that oocyte-specific deletion of Mfn1, but not Mfn2, prevents the oocyte growth and ovulation due to a block in folliculogenesis. We pinpoint the loss of oocyte growth and ovulation to impaired PI3K-Akt signaling and disrupted oocyte-somatic cell communication. In support, the double loss of Mfn1 and Mfn2 partially rescues the impaired PI3K-Akt signaling and defects in oocyte development secondary to the single loss of Mfn1. Together, this work demonstrates that the mitochondrial function influences the cellular signaling during the oocyte development, and highlights the importance of distinct, nonredundant roles of MFN1 and MFN2 in oogenesis.