Auswahl der wissenschaftlichen Literatur zum Thema „Transzonal projections“

Abstract Ovarian follicle development and oocyte competence acquisition is dependent upon continuous interactions between the somatic cells and the oocyte. Interactions between these cell types include bidirectional paracrine signaling and the exchange of small molecules, such and amino acids and cyclic nucleotides, through gap junctions located at the end of transzonal projections (TZPs). In the last decade, additional mechanisms of cell-to-cell interactions within the ovarian follicle were described. These mechanisms include the movement of small extracellular vesicles (EVs) within the follicular fluid and the delivery of its cargo to target cells; and the exchange of large molecules transiting from the cumulus cells to the oocytes via transzonal projections. Here, I will describe the investigations about these novel communication systems in the bovine ovarian follicle. The topics will include the content of EVs transiting in the bovine follicular fluid and its role regulating signaling pathways associated with oocyte competence, and the movement of large molecules from cumulus cell to the oocyte such as messenger RNAs and fatty acids. Finally, dysregulations of such communications mechanisms under in vitro culture conditions will also be reviewed. Emphasis will be given on the lipid metabolism in the cumulus-oocyte complex and lipid accumulation mediated by transzonal projections and fatty acid binding proteins in oocytes undergoing in vitro maturation.

Oocyte competence is one of the key factors determining the proportion of embryos that develop to the blastocyst stage. There is vast evidence that IVM oocytes exhibit less developmental potential than their invivo counterparts. Here, we tested whether supplementation of three cytokines [FGF2 (40 ngmL−1), LIF (20ngmL−1), and IGF1 (20 ngmL−1), termed FLI] improved oocyte maturation, and as a consequence, preimplantation development of bovine embryos invitro. In the first experiment, cumulus-oocyte complexes (COCs) were collected from abattoir-derived ovaries and placed in maturation medium,±FLI, for 18 to 22h. At the end of maturation, COCs were fertilized with sperm from a single Holstein bull known to have high fertility. After an 18- to 20-h fertilization period, putative zygotes were cultured in synthetic oviductal fluid for 8 days. The number of embryos that underwent at least one cellular division (cleavage) and the number of embryos that developed to the blastocyst stage was recorded on Days 3 and 8 after insemination, respectively. The COCs supplemented with FLI (n=554) and controls (n=534) were evaluated across 5 replicates. There was no difference in the cleavage rate (P>0.05) between the two treatments. Development to the blastocyst stage was higher (P=0.05) for FLI-treated COCs (34.9%±1.96) than for the control group (23.9%±1.96). In a second experiment, COCs (n=204) supplemented±FLI were collected and fixed at 6, 12, 18, and 24h after placement in maturation medium. The number of transzonal projections in the COCs was determined by localization of actin filaments by using confocal microscopy. Data were analysed by ANOVA using the GLM procedure of SAS software (version 9.4; SAS Institute Inc.). The model included treatment, time, and the interaction of treatment×time as fixed effects. There was no difference (P>0.05) in the number of transzonal projections at 6h (166.3±8.6 vs. 143.9±8.8) and 12h (107.8±8.4 vs. 128.3±7.7) between FLI-treated and control COCs. However, FLI-treated COCs had fewer (P<0.05) transzonal projections at 18h (67.9±7.8 vs. 100.1±7.7) and 24h (56.4±7.2 vs. 80.6±7.4) compared with the controls. There was a significant treatment×time interaction (P=0.006). In a third experiment, we tested whether the timing of transzonal projection disassociation affected lipid accumulation in the embryo. Blastocysts (n=59) on Day 8 produced from COCs matured±FLI were collected and lipid content was determined by using Nile Red staining. There was no difference (P>0.05) in lipid content between treatments. Thus, supplementation of maturation medium with FLI accelerates the disassociation of transzonal projections in COCs and improves subsequent embryonic development to the blastocyst stage while having no detectable effect on lipid content. Further research is necessary to understand how these cytokines modulate IVM of bovine oocytes. This project was supported by Food for the 21st Century and the Clifton Murphy scholarship fund.

Extreme temperature during summer may lead to heat stress in cattle and compromise their productivity. It also poses detrimental impacts on the developmental capacity of bovine budding oocytes, which halt their fertility. To mitigate the adverse effects of heat stress, it is necessary to investigate the mechanisms through which it affects the developmental capacity of oocytes. The primary goal of this study was to investigate the impact of heat stress on the epigenetic modifications in bovine oocytes and embryos, as well as on oocyte developmental capacity, reactive oxygen species, mitochondrial membrane potential, apoptosis, transzonal projections, and gene expression levels. Our results showed that heat stress significantly reduced the expression levels of the epigenetic modifications from histone H1, histone H2A, histone H2B, histone H4, DNA methylation, and DNA hydroxymethylation at all stages of the oocyte and embryo. Similarly, heat stress significantly reduced cleavage rate, blastocyst rate, oocyte mitochondrial-membrane potential level, adenosine-triphosphate (ATP) level, mitochondrial DNA copy number, and transzonal projection level. It was also found that heat stress affected mitochondrial distribution in oocytes and significantly increased reactive oxygen species, apoptosis levels and mitochondrial autophagy levels. Our findings suggest that heat stress significantly impacts the expression levels of genes related to oocyte developmental ability, the cytoskeleton, mitochondrial function, and epigenetic modification, lowering their competence during the summer season.

SummaryMany studies of the main gap junction protein, Cx43, have been conducted in porcine oocyte research, but they have been limited to investigations of cumulus–oocyte complexes (COCs). In this study, we verified Cx43 not in COCs, but in porcine oocytes during maturation, and conducted a quantitative time course analysis. The location and dynamics of Cx43 were examined by immunocytochemistry and western blotting, respectively. COCs were cultured in NCSU23 medium and processed for immunocytochemistry and western blotting at 0, 14, 28, and 42 h after denuding. A Cx43 signal was detected on oolemmas, transzonal projections and the surface of zona pellucidae. Western blotting showed that Cx43 band density increased from 0 to 14 h, and gradually decreased thereafter. Our results clarified that Cx43 is localized in the ooplasmic membrane through zona pellucidae and its level changes over time during culture in porcine oocytes.

The determinants of oocyte quality remain uncertain. Under suitable conditions, which have yet to be defined, the gamete grows and acquires the competence to resume meiosis, be fertilised and undergo embryonic development at least beyond genome activation, after which the blastomere is autonomous enough to adapt to the specificity of its environment. This review describes the central role played by the oocyte in reproductive success and how communication between cumulus cells and the oocyte are essential to proper oogenesis and the quality of the resulting gamete. While most attempts to improve oocyte quality have been directed at gonadotrophin-based systemic endocrine signalling, it is proposed that parallel control of fertility may act locally within ovarian follicles through intimate cooperation between somatic cells and the oocyte via the network of transzonal projections. This intercellular communication may prove to be more sensitive to environmental conditions than systemic endocrine signalling, which is essential for many non-reproductive tissues.

The oocyte must grow and mature before fertilization, thanks to a close dialogue with the somatic cells that surround it. Part of this communication is through filopodia-like protrusions, called transzonal projections (TZPs), sent by the somatic cells to the oocyte membrane. To investigate the contribution of TZPs to oocyte quality, we impaired their structure by generating a full knockout mouse of the TZP structural component myosin-X (MYO10). Using spinning disk and super-resolution microscopy combined with a machine-learning approach to phenotype oocyte morphology, we show that the lack ofMyo10decreases TZP density during oocyte growth. Reduction in TZPs does not prevent oocyte growth but impairs oocyte-matrix integrity. Importantly, we reveal by transcriptomic analysis that gene expression is altered in TZP-deprived oocytes and that oocyte maturation and subsequent early embryonic development are partially affected, effectively reducing mouse fertility. We propose that TZPs play a role in the structural integrity of the germline–somatic complex, which is essential for regulating gene expression in the oocyte and thus its developmental potential.

C-type natriuretic peptide (CNP) is a peptide molecule naturally found in follicles and can be used to extend meiotic resumption and enhance the potential for oocytes to develop. However, the mechanism by which CNP improves goat oocyte quality remains unclear. In this study, cumulus–oocyte complexes (COCs) from goats were pre-treated with CNP prior to IVM, and the results showed that pre-treatment with CNP enhanced goat oocyte maturation. First, we discovered that CNP maintained communication between cumulus cells and oocytes by regulating the transzonal projections (TZPs). We then found that CNP treatment reduced abnormal spindle formation and increased the expression of genes associated with spindle assembly and the spindle assembly checkpoint. Moreover, further analysis showed that oocytes exhibited better antioxidant ability in the CNP treatment group, which mainly manifested in higher glutathione (GSH) and lower reactive oxygen species (ROS) concentrations. Enhanced mitochondrial activity was signified via the augmented expression of mitochondrial oxidative metabolism and fusion and fission-related genes, thus diminishing the apoptosis of the oocytes. Overall, these results provide novel insights into the potential mechanism by which CNP treatment before IVM can improve oocyte quality.

The extracellular matrix (ECM) is an essential structure with biological activities. It has been shown that the ECM influences gene expression via cytoskeletal components and the gene expression is dependent upon cell interactions with molecules and hormones. The development of ovarian follicles is a hormone dependent process. The surge in the luteinizing hormone triggers ovulatory changes in oocyte microenvironment. In this review, we discuss how proteolytic cleavage affects formation of cumulus ECM following hormonal stimulation; in particular, how the specific proteasome inhibitor MG132 affects gonadotropin-induced cytoskeletal structure, the organization of cumulus ECM, steroidogenesis, and nuclear maturation. We found that after the inhibition of proteolytic cleavage, gonadotropin-stimulated oocyte–cumulus complexes (OCCs) were without any signs of cumulus expansion; they remained compact with preserved cytoskeletal F-actin-rich transzonal projections through the oocyte investments. Concomitantly, a significant decrease was detected in progesterone secretion and in the expression of gonadotropin-stimulated cumulus expansion–related transcripts, such as HAS2 and TNFAIP6. In agreement, the covalent binding between hyaluronan and the heavy chains of serum-derived the inter-alpha-trypsin inhibitor, essential for the organization of cumulus ECM, was missing.

Chez les femelles mammifères, les cellules somatiques qui entourent l’ovocyte, les cellules de la granulosa, coordonnent le développement ovocytaire post-partum, la croissance ovocytaire et la maturation méiotique, en dialoguant avec l’ovocyte. Ce dialogue est principalement établi par contact intercellulaire grâce à des protrusions des cellules de la granulosa, les projections transzonales (TZPs). Les TZPs ressemblent à des filopodes, résident dans la matrice extracellulaire entourant l’ovocyte, la zone pellucide, et établissent des jonctions gap et d’adhérence avec la membrane de l’ovocyte. Durant ma thèse, j’ai étudié le rôle des TZPs dans le développement ovocytaire. En générant un knockout total de la Myosine-X chez la souris, une protéine contenue dans les TZPs et participant à la formation des filopodes, nous avons obtenu des ovocytes avec une densité de TZPs réduite. Cela n’empêche pas l’ovocyte d’atteindre une taille canonique. Cependant, cela altère l’intégrité de la zone pellucide, l’adhésion ovocyte-matrice extracellulaire, et le transcriptome de l’ovocyte en fin de croissance, les transcrits dérégulés étant majoritairement sur-exprimés. Les ovocytes dépourvus de TZPs sont enclins à arrêter leur développement en métaphase de première division méiotique, bien que présentant un fuseau de division correctement assemblé et des chromosomes bien alignés. Nous proposons que les cellules somatiques modulent la synthèse ou la stabilité d’un sous-ensemble de transcrits ovocytaires par le biais de leurs protrusions cellulaires. Cette modulation améliore la capacité de l’ovocyte à terminer la maturation méiotique, et par extension, les chances de produire un embryon
In female mammals, somatic cells surrounding the oocyte, termed granulosa cells, coordinate the critical stages of post-partum oocyte development, i.e. oocyte growth and meiotic maturation, by dialoguing with the oocyte. This dialogue is primarily mediated by cell-cell contact carried out by granulosa cell protrusions termed transzonal projections (TZPs). TZPs are analogous to filopodia in their snake-like shape, but also in their structural composition. TZPs are located in the zona pellucida, the extracellular matrix surrounding the oocyte, and their extremities establish cellular junctions with the oocyte membrane, i.e. gap and adherens junctions. In my thesis, I investigated the role of TZP-mediated interaction between granulosa cells and the oocyte in oocyte development. By generating a total knockout of Myosin-X, a molecular motor contained in TZPs and participating in filopodia formation, we obtained mouse mutant oocytes with a reduced TZP density at the end of oocyte growth. This reduction does not impede the oocyte from reaching a canonical size. However, it impairs zona pellucida integrity, oocyte-matrix adhesion, and the oocyte transcriptome at the end of oocyte growth, with a subset of transcripts mostly up-deregulated. Importantly, TZP-deprived oocytes tend to cease their development at metaphase of the first meiotic division, despite a well-assembled division spindle and properly aligned chromosomes. We propose that somatic cells modulate the synthesis or stability of a subset of oocyte transcripts through their cellular protrusions. This modulation enhances the oocyte capacity to end meiotic maturation, and by extension, the chances of producing an embryo

La croissance ovocytaire comprend l’établissement de projections transzona les formant un réseau de communication entre le gamète et les cellules somatiques entourant ce dernier. Une accumulation des ARNm dans le cytoplasme se produit lors des premiers stades de croissance de l’ovocyte afin de soutenir la maturation durant le silence transcriptionnel qui perdure jusqu’à l’activation du génome embryonnaire. Selon l’espèce, cette période peut durer des jours voir des semaines nécessitant une grande stabilité des ARNm maternels. Il est connu que la communication entre l’ovocyte et les cellules somatiques ainsi que l’utilisation des réserves d’ARNm sont essentielles pour l’acquisition des compétences de l’ovocyte afin de soutenir la fécondation et le développement embryonnaire précoce. Des modifications chimiques connues comme ayant un rôle stabilisateur, tel que la méthylation, ont également été détectées sur les ARNm. L’hypothèse est que des modifications chimiques de l’ARNm, soit l’ajout de groupements méthyles, sont impliquées dans la stabilisation des transcrits au sein de l’ovocyte durant le silence transcriptionnel. Le réseau de communication pourrait jouer un rôle en faisant le transfert de protéines permettant la méthylation de l’ARNm. Les objectifs sont de détecter les différentes modifications chimiques ayant lieu dans le transcriptome de l’ovocyte ainsi que localiser et caractériser les protéines ayant pour rôle de catalyser, détecter et inverser les modifications chimiques identifiées sur l’ARN. Différents types de modifications post-transcriptionnelles ont été détectées au sein de l’ovocyte et les résultats montrent que m6A et m5C sont les modifications les plus abondantes. La caractérisation des protéines impliquées dans ces modifications dans l’ovaire et l’ovocyte de la souris, du porc et du bovin montre une activité de méthylation différente selon l’espèce durant la folliculogenèse et l’ovogenèse. De plus amples recherches devront être réalisées afin de bonifier la compréhension des mécanismes impliqués dans la production d’un ovocyte de qualité. Les travaux présentés contribuent à l’avancement de ces connaissances.
Oogenesis, which occurs within the ovarian follicle, is a process closely link tofolliculogenesis. The oocyte growth mainly involves the establishment of a communication network between the gamete and surrounding somatic cells. During the early stages ofoocyte growth, the gamete accumulates mRNAs in its cytoplasm to support maturation and first cell division during transcriptional silencing until the embryonic genome activation.This period can last from days to weeks depending on the species requiring long stability from maternal mRNAs. It is known that communication between oocyte and somatic cellsand use of mRNA reserves are essential for oocyte competence acquisition to sustain fertilization and early embryogenesis. Chemical modifications known to have stabilizing role, such as methylation, have also been detected on the mRNAs. The main hypothesis is the chemical modifications of mRNA, by the addition of methyl groups, that are involved in stabilizing and managing transcripts within oocyte during transcriptional silencing. The communication network could have a role in enabling proteins transfer from cumulus cells allowing mRNA methylation. Objectives are to detectvarious chemical modifications taking place in oocyte transcriptome and to locate and characterize proteins having role of managing the chemical modifications identified on RNA. Many post-transcriptional modifications have been detected within the oocyte and results have shown that m6A and m5C are modifications with higher abundant expression. Proteins characterization involved in these post-transcriptional modifications in ovary and oocyteof mouse, swine and bovine showed methylation activity during folliculogenesis and oogenesis. More research is needed to improve understanding of mechanisms involved inoocyte competences acquisition, but the present study has contributed to the advancement of this knowledge.