Articles de revues : « Transzonal projections »

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Perecin, Felipe. « 412 Germ and somatic cell interactions during oocyte development and maturation ». Journal of Animal Science 98, Supplement_4 (3 novembre 2020) : 189. http://dx.doi.org/10.1093/jas/skaa278.349.

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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.

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Stoecklein, K. S., M. S. Ortega, L. Spate, C. N. Murphy et R. S. Prather. « 188 Improvement of bovine oocyte maturation invitro through cytokine supplementation ». Reproduction, Fertility and Development 32, n^o 2 (2020) : 222. http://dx.doi.org/10.1071/rdv32n2ab188.

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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.

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Feng, Xiaoyi, Chongyang Li, Hang Zhang, Peipei Zhang, Muhammad Shahzad, Weihua Du et Xueming Zhao. « Heat-Stress Impacts on Developing Bovine Oocytes : Unraveling Epigenetic Changes, Oxidative Stress, and Developmental Resilience ». International Journal of Molecular Sciences 25, n^o 9 (28 avril 2024) : 4808. http://dx.doi.org/10.3390/ijms25094808.

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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.

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Chen, Mingyue, Chengyong He, Kongyang Zhu, Zihan Chen, Zixiao Meng, Xiaoming Jiang, Jiali Cai, Chunyan Yang et Zhenghong Zuo. « Resveratrol ameliorates polycystic ovary syndrome via transzonal projections within oocyte-granulosa cell communication ». Theranostics 12, n^o 2 (2022) : 782–95. http://dx.doi.org/10.7150/thno.67167.

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Clarke, Hugh J. « History, origin, and function of transzonal projections : the bridges of communication between the oocyte and its environment ». Animal Reproduction 15, n^o 3 (2018) : 215–23. http://dx.doi.org/10.21451/1984-3143-ar2018-0061.

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Lee, Seunghoon, Yuuki Hiradate, Yumi Hoshino, Yeoung-gyu Ko, Kentaro Tanemura et Eimei Sato. « Localization and quantitative analysis of Cx43 in porcine oocytes during in vitro maturation ». Zygote 24, n^o 3 (21 juillet 2015) : 364–70. http://dx.doi.org/10.1017/s0967199415000271.

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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.

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Robert, Claude. « Nurturing the egg : the essential connection between cumulus cells and the oocyte ». Reproduction, Fertility and Development 34, n^o 2 (2022) : 149. http://dx.doi.org/10.1071/rd21282.

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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.

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Crozet, Flora, Gaëlle Letort, Rose Bulteau, Christelle Da Silva, Adrien Eichmuller, Anna Francesca Tortorelli, Joséphine Blévinal et al. « Filopodia-like protrusions of adjacent somatic cells shape the developmental potential of oocytes ». Life Science Alliance 6, n^o 6 (21 mars 2023) : e202301963. http://dx.doi.org/10.26508/lsa.202301963.

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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.

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Xu, Rui, Menghao Pan, Lu Yin, Yiqian Zhang, Yaju Tang, Sihai Lu, Yan Gao, Qiang Wei, Bin Han et Baohua Ma. « C-Type Natriuretic Peptide Pre-Treatment Improves Maturation Rate of Goat Oocytes by Maintaining Transzonal Projections, Spindle Morphology, and Mitochondrial Function ». Animals 13, n^o 24 (16 décembre 2023) : 3880. http://dx.doi.org/10.3390/ani13243880.

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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.

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Nagyová, Eva, Lucie Němcová et Antonella Camaioni. « Cumulus Extracellular Matrix Is an Important Part of Oocyte Microenvironment in Ovarian Follicles : Its Remodeling and Proteolytic Degradation ». International Journal of Molecular Sciences 23, n^o 1 (21 décembre 2021) : 54. http://dx.doi.org/10.3390/ijms23010054.

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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.

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Appeltant, Ruth, Tamás Somfai, Elisa C. S. Santos, Thanh Quang Dang-Nguyen, Takashi Nagai et Kazuhiro Kikuchi. « Effects of vitrification of cumulus-enclosed porcine oocytes at the germinal vesicle stage on cumulus expansion, nuclear progression and cytoplasmic maturation ». Reproduction, Fertility and Development 29, n^o 12 (2017) : 2419. http://dx.doi.org/10.1071/rd16386.

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Although offspring have been produced from porcine oocytes vitrified at the germinal vesicle (GV) stage, the rate of embryo development remains low. In the present study, nuclear morphology and progression, cumulus expansion, transzonal projections (TZPs), ATP and glutathione (GSH) levels were compared between vitrified cumulus–oocyte complexes (COCs) and control COCs (no cryoprotectant treatment and no cooling), as well as a toxicity control (no cooling). Vitrification was performed with 17.5% (v/v) ethylene glycol and 17.5% (v/v) propylene glycol. Vitrification at the GV stage caused premature meiotic progression, reflected by earlier GV breakdown and untimely attainment of the MII stage. However, cytoplasmic maturation, investigated by measurement of ATP and GSH levels, as well as cumulus expansion, proceeded normally despite detectable damage to TZPs in vitrified COCs. Moreover, treatment with cryoprotectants caused fragmentation of nucleolus precursor bodies and morphological changes in F-actin from which oocytes were able to recover during subsequent IVM culture. Reduced developmental competence may be explained by premature nuclear maturation leading to oocyte aging, although other mechanisms, such as initiation of apoptosis and reduction of cytoplasmic mRNA, can also be considered. Further research will be required to clarify the presence and effects of these phenomena during the vitrification of immature COCs.

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Wang, Yan, Hualin Huang, Minghua Zeng, Ru-Ping Quan, Jun-Ting Yang, Dan Guo, Ying Sun, Hongwen Deng et Hongmei Xiao. « Mutation of rat Zp2 causes ROS-mediated oocyte apoptosis ». Reproduction 160, n^o 3 (septembre 2020) : 353–65. http://dx.doi.org/10.1530/rep-20-0037.

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In this study, we investigated a gene-edited (Zp2MT/MT) rat model of infertility caused by the failure to express the zona pellucida glycoprotein 2 (ZP2) due to the significant reduction of mRNA amount. We examined the defects in the zona pellucida (ZP) caused by ZP2 nullification and the influence of these defects on aspects of oocyte development, including apoptosis and fertilization ability. To investigate the cause of the influence to the oocytes’ development, we evaluated the morphology of follicular transzonal projections (TZPs), known as ‘bridges’, which mediate the bidirectional signaling between the oocyte and surrounding granulosa cells and the level of reactive oxygen species (ROS) in ovulated eggs. Our results showed that two types of ZP defects were generated in the Zp2MT/MT rat,that is, ZP intact but thinned and ZP cracked (or even absent). The fertilization rate of the ovulated eggs reduced in both types, while increased oocyte apoptosis was observed only in the latter type. Moreover, the increased oocyte apoptosis rate correlated closely with the reduction in follicular TZPs and increased ROS levels in ovulated egg. In conclusion, nullification of rat ZP2 destroyed the integrity of the ZP, impaired the bidirectional signaling between the oocyte and surrounding granulosa cells. Therefore, the resulting infertility likely occurs via elevation of oxidative stress and oocytes apoptosis.

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Caballero, Julieta, Isabelle Gilbert, Eric Fournier, Dominic Gagné, Sara Scantland, Angus Macaulay et Claude Robert. « Exploring the function of long non-coding RNA in the development of bovine early embryos ». Reproduction, Fertility and Development 27, n^o 1 (2015) : 40. http://dx.doi.org/10.1071/rd14338.

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Now recognised as part of the cellular transcriptome, the function of long non-coding (lnc) RNA remains unclear. Previously, we found that some lncRNA molecules in bovine embryos are highly responsive to culture conditions. In view of a recent demonstration that lncRNA may play a role in regulating important functions, such as maintenance of pluripotency, modification of epigenetic marks and activation of transcription, we sought evidence of its involvement in embryogenesis. Among the numerous catalogued lncRNA molecules found in oocytes and early embryos of cattle, three candidates chosen for further characterisation were found unexpectedly in the cytoplasmic compartment rather than in the nucleus. Transcriptomic survey of subcellular fractions found these candidates also associated with polyribosomes and one of them spanning transzonal projections between cumulus cells and the oocyte. Knocking down this transcript in matured oocytes increased developmental rates, leading to larger blastocysts. Transcriptome and methylome analyses of these blastocysts showed concordant data for a subset of four genes, including at least one known to be important for blastocyst survival. Functional characterisation of the roles played by lncRNA in supporting early development remains elusive. Our results suggest that some lncRNAs play a role in translation control of target mRNA. This would be important for managing the maternal reserves within which is embedded the embryonic program, especially before embryonic genome activation.

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Wang, Yan, Chao Lv, Hua-Lin Huang, Ming-Hua Zeng, Da-Jing Yi, Hang-Jing Tan, Tian-Liu Peng, Wen-Xian Yu, Hong-Wen Deng et Hong-Mei Xiao. « Influence of mouse defective zona pellucida in folliculogenesis on apoptosis of granulosa cells and developmental competence of oocytes† ». Biology of Reproduction 101, n^o 2 (4 juin 2019) : 457–65. http://dx.doi.org/10.1093/biolre/ioz093.

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Abstract Zona pellucida (ZP), which enwraps the oocyte during folliculogenesis, initially forms in the primary follicle and plays an important role in female fertility. Here, we investigated a mouse strain (“mutant mice” for short) carrying two types of ZP defects in folliculogenesis, i.e., ZP thinned (but intact) and ZP cracked, caused by targeted mutation in the Zp1 gene. Using this mutant mouse strain and wild-type mouse as control, we studied the effects of the ZP defects on the development of oocytes and granulosa cells during folliculogenesis. For each ZP defect, we examined the morphology of transzonal projections and apoptosis of granulosa cells in the corresponding growing follicles, as well as the morphology of corresponding ovulated eggs and their abilities to develop into viable individuals. Our results suggested that ZP integrity rather than thickness or porosity is crucial for preventing the ectopia of granulosa cells, maintaining adequate routine bilateral signaling between oocyte and surrounding granulosa cells, and thus for ensuring the survival of granulosa cells and the establishment of the full developmental competence of oocytes. This is the first study to elucidate the effects of different degrees of ZP defects caused by the same gene mutation, on the apoptosis of granulosa cells and developmental competence of oocytes, and to explore the potential mechanisms underlying these effects.

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Stoecklein, Katy S., M. Sofia Ortega, Lee D. Spate, Clifton N. Murphy et Randall S. Prather. « Improved cryopreservation of in vitro produced bovine embryos using FGF2, LIF, and IGF1 ». PLOS ONE 16, n^o 2 (3 février 2021) : e0243727. http://dx.doi.org/10.1371/journal.pone.0243727.

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In vitro embryo production systems are limited by their inability to consistently produce embryos with the competency to develop to the blastocyst stage, survive cryopreservation, and establish a pregnancy. Previous work identified a combination of three cytokines [fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1)], called FLI, that we hypothesize improve preimplantation development of bovine embryos in vitro. To test this hypothesis, FLI was supplemented into oocyte maturation or embryo culture medium. Embryos were produced in vitro using abattoir-derived oocytes and fertilized with sperm from a single bull known to have high fertility. After an 18–20 h fertilization period, putative zygotes were cultured in synthetic oviductal fluid (SOF) for 8 days. The addition of FLI to the oocyte maturation medium increased (P< 0.05) the dissociation of transzonal projections at 12, 18, and 24 h of maturation, as well as, the proportion of oocytes that reached the metaphase II stage of meiosis. Additionally, lipid content was decreased (P< 0.05) in the blastocyst stage embryo. The addition of FLI during the culture period increased development to the blastocyst stage, cytoskeleton integrity, and survival following slow freezing, as well as, decreased post thaw cell apoptosis (P< 0.05). In conclusion, the supplementation of these cytokines in vitro has the potential to alleviate some of the challenges associated with the cryo-survival of in vitro produced bovine embryos through improving embryo development and embryo quality.

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Grosbois, J., M. Vermeersch, M. Devos, H. J. Clarke et I. Demeestere. « Ultrastructure and intercellular contact-mediated communication in cultured human early stage follicles exposed to mTORC1 inhibitor ». Molecular Human Reproduction 25, n^o 11 (7 octobre 2019) : 706–16. http://dx.doi.org/10.1093/molehr/gaz053.

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Abstract The reproductive lifespan of a woman is determined by the gradual recruitment of quiescent follicles into the growing pool. In humans, ovarian tissue removal from its in vivo environment induces spontaneous activation of resting follicles. Similarly, pharmacological activation of the PI3K/Akt pathway leads to accelerated follicle recruitment, but has been associated with follicular damage. Recent findings demonstrate that everolimus (EVE), an mTORC1 inhibitor, limits primordial follicle activation. However, its potential benefit regarding growing follicle integrity remains unexplored. Ovarian cortical fragments were exposed to ± EVE for 24 h and cultured for an additional 5 days. After 0, 1 and 6 days of culture, fragments were either processed for ultrastructural analysis or subjected to follicular isolation for gene expression and immunofluorescence assessments. Data from transmission electron microscopy showed that growing follicles displayed similar ultrastructural features irrespective of the conditions and maintained close contacts between germinal and stromal compartments. Establishment of intra-follicular communication was confirmed by detection of a gap junction component, Cx43, in both groups throughout culture, whereas transzonal projections, which physically link granulosa cells to oocyte, formed later in EVE-treated follicles. Importantly, levels of GJA1 mRNA, encoding for the Cx43 protein, significantly increased from Day 0 to Day 1 in the EVE group, but not in the control group. Given that EVE-treated follicles were smaller than controls, these findings suggest that EVE might facilitate the establishment of appropriate intercellular communications without impairing follicle ultrastructure. Therefore, mTORC1 inhibitors might represent an attractive tool to delay the culture-induced primordial follicle activation while maintaining follicles in a functionally integrated state.

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Zhang, Peipei, Baigao Yang, Xi Xu, Hang Zhang, Xiaoyi Feng, Haisheng Hao, Weihua Du et al. « Combination of CNP, MT and FLI during IVM Significantly Improved the Quality and Development Abilities of Bovine Oocytes and IVF-Derived Embryos ». Antioxidants 12, n^o 4 (7 avril 2023) : 897. http://dx.doi.org/10.3390/antiox12040897.

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Oocyte maturation is a critical step in the completion of female gametogenesis in the ovary; thus, for subsequent fertilization and embryogenesis. Vitrification of embryo also has been shown to be closely associated with oocyte maturation. To improve the quality and developmental potential of bovine oocytes derived from in vitro maturation (IVM), Pre-IVM with C-type natriuretic peptide (CNP), melatonin (MT) and in combination, IGF1, FGF2, LIF (FLI) were supplemented in the IVM medium. In this current study, we cultured bovine oocytes in Pre-IVM with CNP for 6 h before transferring them to the IVM medium supplemented with MT and FLI. The developmental potential of bovine oocytes was then investigated by measuring the reactive oxygen species (ROS), the intracellular glutathione (GSH) and ATP levels, the transzonal projections (TZP), the mitochondrial membrane potential (ΔΨm), cacline-AM, and the expression of related genes (cumulus cells (CCs), oocytes, blastocysts). The results revealed that oocytes treated with a combination of CNP, MT, and FLI had dramatically improved the percentage of oocytes developed to blastocyst, ATP content, GSH levels, TZP intensity, the ΔΨm, cacline-AM fluorescence intensity, and considerably reduced ROS levels of oocytes. Furthermore, the survival rate and the hatched rate after vitrification of the CNP+MT+FLI group were significantly higher than those other groups. Thus, we speculated that CNP+MT+FLI increases the IVM of bovine oocytes. In conclusion, our findings deepen our understanding and provide new perspectives on targeting the combination of CNP, MT and FLI to enhance the quality and developmental potential of bovine oocytes.

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Martínez-Moro, Á., I. Lamas-Toranzo et P. Bermejo-Álvarez. « 127 Metabolomics analysis of human cumulus cells from oocytes exhibiting different developmental competence ». Reproduction, Fertility and Development 33, n^o 2 (2021) : 172. http://dx.doi.org/10.1071/rdv33n2ab127.

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Cumulus cells play fundamental metabolic roles during folliculogenesis, being closely connected to the oocyte through transzonal projections. These oocyte-supporting cells are removed and discarded before intracytoplasmic sperm injection (ICSI), thereby constituting an interesting biological material on which to perform molecular analyses aimed to predict oocyte developmental potential. The objective of this study was to determine the possible differences in the amount of biochemical compounds in human cumulus cells from oocytes exhibiting different developmental competence: (1) oocytes not developing to blastocyst following ICSI (Bl−), (2) oocytes developing to blastocyst but failing to establish pregnancy following embryo transfer (P−), and (3) oocytes developing to blastocyst able to establish a pregnancy (P+). Cumulus cells were removed following conventional chemical and mechanical treatments before ICSI. Following cell dissociation, cumulus cells from individual oocytes were pelleted by centrifugation at 1500×g for 10min, snap frozen in liquid nitrogen, and kept at −80°C until analysis. Once the developmental potential was known, metabolomics analysis was performed in 12 samples per group, each composed of cumulus cells attached to an individual oocyte. Metabolomics analyses were performed by Metabolon Inc., which provides unbiased metabolite analyses based on ultra-high-performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS). Welch’s two-sample t-test was used to identify biochemicals that differed significantly between groups (P<0.05). The analysis identified 97 compounds of known identity in human cumulus cells, excluding xenobiotics. Of these, only 3 showed significant differences between groups: (1) cysteine, more abundant (1.5-fold increase) in P− compared with Bl−; (2) erythronate, more abundant (1.25-fold increase) in P+ compared with Bl−; and (3) malonate, more abundant (1.55-fold increase) in Bl− compared with P−. No differences were found in other comparisons for these compounds, although tendencies (0.05 < P <0.1) were noted for cysteine (1.68-fold increase in P+ vs. P−) and malonate (1.92-fold increase in P− vs. P+). These results suggest that metabolomics analysis of human cumulus cells provides a poor predictive value for the developmental potential of the enclosed oocyte.

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Martínez-Moro, Á., I. Lamas-Toranzo et P. Bermejo-Álvarez. « 127 Metabolomics analysis of human cumulus cells from oocytes exhibiting different developmental competence ». Reproduction, Fertility and Development 33, n^o 2 (2021) : 172. http://dx.doi.org/10.1071/rdv33n2ab127.

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Cumulus cells play fundamental metabolic roles during folliculogenesis, being closely connected to the oocyte through transzonal projections. These oocyte-supporting cells are removed and discarded before intracytoplasmic sperm injection (ICSI), thereby constituting an interesting biological material on which to perform molecular analyses aimed to predict oocyte developmental potential. The objective of this study was to determine the possible differences in the amount of biochemical compounds in human cumulus cells from oocytes exhibiting different developmental competence: (1) oocytes not developing to blastocyst following ICSI (Bl−), (2) oocytes developing to blastocyst but failing to establish pregnancy following embryo transfer (P−), and (3) oocytes developing to blastocyst able to establish a pregnancy (P+). Cumulus cells were removed following conventional chemical and mechanical treatments before ICSI. Following cell dissociation, cumulus cells from individual oocytes were pelleted by centrifugation at 1500×g for 10min, snap frozen in liquid nitrogen, and kept at −80°C until analysis. Once the developmental potential was known, metabolomics analysis was performed in 12 samples per group, each composed of cumulus cells attached to an individual oocyte. Metabolomics analyses were performed by Metabolon Inc., which provides unbiased metabolite analyses based on ultra-high-performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS). Welch’s two-sample t-test was used to identify biochemicals that differed significantly between groups (P<0.05). The analysis identified 97 compounds of known identity in human cumulus cells, excluding xenobiotics. Of these, only 3 showed significant differences between groups: (1) cysteine, more abundant (1.5-fold increase) in P− compared with Bl−; (2) erythronate, more abundant (1.25-fold increase) in P+ compared with Bl−; and (3) malonate, more abundant (1.55-fold increase) in Bl− compared with P−. No differences were found in other comparisons for these compounds, although tendencies (0.05 < P <0.1) were noted for cysteine (1.68-fold increase in P+ vs. P−) and malonate (1.92-fold increase in P− vs. P+). These results suggest that metabolomics analysis of human cumulus cells provides a poor predictive value for the developmental potential of the enclosed oocyte.

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Keim, J., Y. Liu et I. Polejaeva. « 177 Increasing in vitro embryonic development through improved oocyte maturation in cattle oocytes ». Reproduction, Fertility and Development 31, n^o 1 (2019) : 213. http://dx.doi.org/10.1071/rdv31n1ab177.

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In vitro maturation (IVM) is an important process in the in vitro production of embryos. It has been recently shown that 3 cytokines: fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1) have increased the efficiency of IVM, blastocyst production, and in vivo development in pig (Yuan et al. 2017 Proc. Natl. Acad. Sci. USA 114, E5796-E5804). In vitro maturation in medium supplemented with cytokines doubled the blastocyst rate and quadrupled the litter size when transferred. It was observed that the addition of cytokines to IVM medium had an effect on the regulation of pMAPK1/3, cumulus cell expansion, and transzonal projections in cumulus-oocyte complexes (COC). This study was designed to assess the effect of these 3 cytokines on IVM in bovine oocytes and their consecutive development to blastocyst. Intracellular glutathione level (GSH), frequently used as an indicator of metaphase II (MII) oocyte quality, was also evaluated. The COC were retrieved from abattoir-derived ovaries and matured for 21h in either our standard maturation medium [TCM-199 (Gibco/Life Technologies, Grand Island, NY, USA), containing 10% fetal bovine serum, 0.5µg mL−1 FSH, 5µg mL−1 LH, and 100U mL−1 penicillin/streptomycin] or maturation medium supplemented with 20ng mL−1 human LIF, 20ng mL−1 human IGF1, and 40ng mL−1 human FGF2. After IVM, COC were placed in fertilization medium and incubated with frozen-thawed sperm for 20h. Cumulus cells were removed from fertilized COC and cultured in SOF culture medium at 38.5°C in 5% CO2/humidified air. Cleavage and blastocyst rates were assessed at 48h and Day 8 post-IVF, respectively. To assess GSH level, MII oocytes were incubated in 20 µM CellTracker Blue CMF2HC (Thermo Fisher Scientific, Waltham, MA, USA) and observed under blue fluorescent light. All statistical analysis was performed using one-way ANOVA and data are presented as mean±s.e.m. The MII rate, assessed by the presence of the first polar body, was significantly higher in the maturation medium supplemented with cytokines compared with the control medium (167/202; 82.4±2.02% v. 136/198; 68.8±1.1%; P<0.05, 4 replicates). For IVF, no statistical difference was found in the cleavage rate between oocytes matured in the medium supplemented with cytokines compared with control medium (351/473; 74.3±4.86% v. 358/573; 63.9±4.03%; P>0.05, 5 replicates), respectively. However, a significant increase in blastocyst rate was observed in the cytokine-containing medium (64/351; 17.7±2.06%) compared with the control group (42/358; 11.0±1.96%; P<0.05, 5 replicates). Furthermore, our preliminary data indicate an increase in GSH in MII oocytes matured in the cytokine-containing medium. In conclusion, the addition of FGF2, LIF, and IGF1 to maturation media improves bovine IVM efficiency and quality of the MII oocytes, leading to a greater blastocyst development rate. Supported by RFBR (18-29-07089) and UAES (1343).

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Buratini, Jose, Thaisy Tino Dellaqua, Mariabeatrice Dal Canto, Antonio La Marca, Domenico Carone, Mario Mignini Renzini et Robert Webb. « The putative roles of FSH and AMH in the regulation of oocyte developmental competence : from fertility prognosis to mechanisms underlying age-related subfertility ». Human Reproduction Update 28, n^o 2 (30 décembre 2021) : 232–54. http://dx.doi.org/10.1093/humupd/dmab044.

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Abstract BACKGROUND Fertility loss during female ageing is associated with increasing basal FSH and decreasing anti-Müllerian hormone (AMH) concentrations, together with compromised oocyte quality, presumably due to increased oxidative stress (OS) and DNA damage, as well as reduced metabolic and meiotic competences. Basal FSH and AMH circulatory concentrations have been broadly utilized as IVF success predictors, regardless of fluctuations in prognostic accuracy; basal FSH and AMH perform better in pre-advanced maternal age (AMA: >35 years) and AMA patients, respectively. The relationships between FSH and AMH intrafollicular levels and IVF outcomes suggest, nevertheless, that both hormones regulate oocyte competence, supporting the hypothesis that changes in FSH/AMH levels cause, at least in part, oocyte quality degradation during ageing. To understand the reasons behind the fluctuations in FSH and AMH prognostic accuracies and to clarify their participation in mechanisms determining oocyte competence and age-related subfertility, a deeper knowledge of the regulation of FSH and AMH intrafollicular signalling during the female reproductive lifespan, and of their effects on the cumulus–oocyte complex, is required. OBJECTIVE AND RATIONALE An extensive body of information on the regulation of FSH and AMH intrafollicular availability and signalling, as well as on the control of folliculogenesis and oocyte metabolism, has been accumulated. However, these datasets have been explored within the relatively narrow boundaries of their specific subjects. Given the aforementioned gaps in knowledge and their clinical relevance, herein we integrate clinical and basic data, within a wide biological perspective, aiming to shed light on (i) the reasons for the variability in the accuracy of serum FSH and AMH as fertility markers, and on (ii) the potential roles of these hormones in mechanisms regulating oocyte quality, particularly those associated with ageing. SEARCH METHODS The PubMed database encompassing the period between 1960 and 2021 was searched. Principal search terms were FSH, FSH receptor, AMH, oocyte, maternal age, cumulus, transzonal projections (TZPs), actin, OS, redox, reactive oxygen species, mitochondria, DNA damage, DNA repair, aneuploidy, spindle, meiosis, gene expression, transcription, translation, oocyte secreted factors (OSFs), cAMP, cyclic guanosine monophosphate, natriuretic peptide C, growth differentiation factor 9, bone morphogenetic protein 15 and fibroblast growth factor. OUTCOMES Our analysis suggests that variations in the accuracy of fertility prognosis reflect a modest association between circulatory AMH levels and oocyte quality as well as increasing basal FSH inter-cycle variability with age. In addition, the basic and clinical data articulated herein support the hypothesis that increased intrafollicular FSH levels, as maternal age advances, may override the physiological protective influences of AMH and OSFs against excessive FSH signalling in cumulus cells. This would result in the disruption of oocyte homeostasis via reduced TZP-mediated transfer of cumulus-derived molecules essential for meiotic competence, gene expression, redox activity and DNA repair. WIDER IMPLICATIONS In-depth data analysis, encompassing a wide biological perspective has revealed potential causative mechanisms of age-related subfertility triggered by alterations in FSH/AMH signalling during the female reproductive life. Insights from new mechanistic models arising from this analysis should contribute to advancing our comprehension of oocyte biology in humans and serve as a valuable reference for novel AMA subfertility treatments aimed at improving oocyte quality through the modulation of AMH/FSH action.

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Yang, Sha, Yuze Yang, Haisheng Hao, Weihua Du, Yunwei Pang, Shanjiang Zhao, Huiying Zou, Huabin Zhu, Peipei Zhang et Xueming Zhao. « Supplementation of EGF, IGF-1, and Connexin 37 in IVM Medium Significantly Improved the Maturation of Bovine Oocytes and Vitrification of Their IVF Blastocysts ». Genes 13, n^o 5 (30 avril 2022) : 805. http://dx.doi.org/10.3390/genes13050805.

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The quality and developmental capacity of oocytes derived from in vitro maturation (IVM) remain unsatisfactory, which greatly impairs the efficiency and application of embryo technologies. The present experiment was designed to investigate the effect of the supplementation of EGF, IGF-1, and Cx37 in an IVM medium on the maturation quality and development ability of bovine oocytes. The cytoplasmic maturation events of oocytes and the quality of in vitro fertilization (IVF) blastocysts were examined to investigate the relative mechanisms. Our results showed that the nuclear maturation and blastocyst development after the IVF of oocytes treated with 25 μg/mL Cx37 or the combination of 50 ng/mL EGF and 100 ng/mL IGF-1 were significantly increased compared to those of the control group (p < 0.05). Furthermore, the blastocyst rate, and blastocyst total cell number and survival rate after vitrification of the EGF+IGF-1+Cx37 group, were significantly higher than those of the control group (p < 0.05), but lower than those of the FSH+LH+EGF+IGF-1+Cx37 group (p < 0.05). The transzonal projection (TZP) intensity, glutathione (GSH) level, and mitochondrial function of the EGF+IGF-1+Cx37 group were significantly higher than that of the control group, and lower than those of the FSH+LH+EGF+IGF-1+Cx37 group, in contrast to the results of the reactive oxygen species (ROS) levels. In conclusion, our results showed that the supplementation of 50 ng/mL EGF, 100 ng/mL IGF-1, and 25 μg/mL Cx37 in the IVM of bovine oocytes significantly improved their quality and developmental ability by increasing the TZP, mitochondrial function, and GSH level.

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Albertini, DF, CM Combelles, E. Benecchi et MJ Carabatsos. « Cellular basis for paracrine regulation of ovarian follicle development ». Reproduction, 1 mai 2001, 647–53. http://dx.doi.org/10.1530/rep.0.1210647.

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Paracrine factors secreted by oocytes and somatic cells regulate many important aspects of early ovarian follicle development in mammals. From activation of dormant primordial follicles to selection of secondary follicles, locally acting factors have been identified that appear to exert important effects on the growth and differentiation of oocytes and granulosa cells. This article summarizes evidence to support a model for bi-directional paracrine communication that is based on developmental regulation of the delivery and reception of paracrine factors at the oocyte-granulosa cell interface. Transzonal projections that originate from granulosa cells and terminate at the oocyte plasma membrane provide a polarized means to orient the secretory organelles of somatic cells. Characterization of transzonal projections in follicles from normal and genetically modified mice reveals dynamic changes in the density and stability of transzonal projections. On the basis of new data analysing the orientation and cytoskeletal content of transzonal projections in mammalian oocytes, a model is proposed for regulation of paracrine growth factor secretion by follicle-stimulating hormone. These findings have immediate implications for ovarian hyperstimulation protocols and follicle culture models as related to the production of mammalian embryos by assisted reproductive technologies.

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Nenonene, Elolo Karen, Mallorie Trottier-Lavoie, Mathilde Marchais, Alexandre Bastien, Isabelle Gilbert, Angus Macaulay, Edouard W. Khandjian et al. « Roles of the cumulus-oocyte transzonal network and the Fragile X protein family in oocyte competence ». Reproduction, novembre 2022. http://dx.doi.org/10.1530/rep-22-0165.

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The determinants of oocyte developmental competence have puzzled scientists for decades. It is known that follicular conditions can nurture the production of a high-quality oocyte, but the underlying mechanisms remain unknown. Somatic cumulus cells most proximal to the oocyte are known to have cellular extensions that reach across the zona pellucida and contact with the oocyte plasma membrane. Herein, it was found that transzonal projections (TZPs) network quality is associated with developmental competence. Knowing that ribonucleo-particles are abundant within TZPs, the distribution of RNA binding proteins were studied. The Fragile X-Related Proteins (FMRP, FXR1P, and FXR2P) and two partnering protein families, namely cytoplasmic FMRP interacting protein (CYFIP) and nuclear FMRP interacting protein (NUFIP), exhibited distinctive patterns consistent with roles in regulating mRNA packaging, transport and translation. Expression of GFP-FMRP fusion protein in cumulus cells showed active granule formation and their transport and transfer through filipodia connecting with neighboring cells. Near the projections’ ends was found the cytoskeletal anchoring protein Filamin A and active protein synthesis sites. This study highlights key proteins involved in delivering mRNA to the oocyte. Thus, cumulus cells appear to indeed support the development of high-quality oocytes via the transzonal network.

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Clarke, Hugh J. « Transzonal projections : Essential structures mediating intercellular communication in the mammalian ovarian follicle ». Molecular Reproduction and Development, 16 septembre 2022. http://dx.doi.org/10.1002/mrd.23645.

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FUSHII, Mihoko, Rie YAMADA, Jibak LEE et Takashi MIYANO. « Reestablishment of transzonal projections and growth of bovine oocytes in vitro ». Journal of Reproduction and Development, 2021. http://dx.doi.org/10.1262/jrd.2021-036.

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Baena, Valentina, et Mark Terasaki. « Three-dimensional organization of transzonal projections and other cytoplasmic extensions in the mouse ovarian follicle ». Scientific Reports 9, n^o 1 (4 février 2019). http://dx.doi.org/10.1038/s41598-018-37766-2.

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Granados-Aparici, Sofia, Alexander Volodarsky-Perel, Qin Yang, Sibat Anam, Togas Tulandi, William Buckett, Weon-Young Son et al. « MYO10 promotes transzonal projection (TZP)-dependent germ line-somatic contact during mammalian folliculogenesis ». Biology of Reproduction, 26 avril 2022. http://dx.doi.org/10.1093/biolre/ioac078.

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Abstract Granulosa cells of growing ovarian follicles elaborate filopodia-like structures termed transzonal projections (TZPs) that supply the enclosed oocyte with factors essential for its development. Little is known, however, of the mechanisms underlying the generation of TZPs. We show in mouse and human that filopodia, defined by an actin backbone, emerge from granulosa cells in early-stage primary follicles and that actin-rich TZPs become detectable as soon as a space corresponding to the zona pellucida appears. mRNA encoding Myosin10 (MYO10), a motor protein that accumulates at the base and tips of filopodia and has been implicated in their initiation and elongation, is present in granulosa cells and oocytes of growing follicles. MYO10 protein accumulates in foci located mainly between the oocyte and innermost layer of granulosa cells, where it co-localizes with actin. In both mouse and human, the number of MYO10 foci increases as oocytes grow, corresponding to the increase in the number of actin-TZPs. RNAi-mediated depletion of MYO10 in cultured mouse granulosa cell-oocyte complexes is associated with a 52% reduction in the number of MYO10 foci and a 28% reduction in the number of actin-TZPs. Moreover, incubation of cumulus-oocyte complexes in the presence of epidermal growth factor, which triggers a 93% reduction in the number of actin-TZPs, is associated with a 55% reduction in the number of MYO10 foci. These results suggest that granulosa cells possess an ability to elaborate filopodia, which when directed towards the oocyte become actin-TZPs, and that MYO10 increases the efficiency of formation or maintenance of actin-TZPs.

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Granados-Aparici, Sofia, Alexander Volodarsky-Perel, Qin Yang, Sibat Anam, Togas Tulandi, William Buckett, Weon-Young Son et al. « MYO10 promotes transzonal projection (TZP)-dependent germ line-somatic contact during mammalian folliculogenesis ». Biology of Reproduction, 26 avril 2022. http://dx.doi.org/10.1093/biolre/ioac078.

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Abstract Granulosa cells of growing ovarian follicles elaborate filopodia-like structures termed transzonal projections (TZPs) that supply the enclosed oocyte with factors essential for its development. Little is known, however, of the mechanisms underlying the generation of TZPs. We show in mouse and human that filopodia, defined by an actin backbone, emerge from granulosa cells in early-stage primary follicles and that actin-rich TZPs become detectable as soon as a space corresponding to the zona pellucida appears. mRNA encoding Myosin10 (MYO10), a motor protein that accumulates at the base and tips of filopodia and has been implicated in their initiation and elongation, is present in granulosa cells and oocytes of growing follicles. MYO10 protein accumulates in foci located mainly between the oocyte and innermost layer of granulosa cells, where it co-localizes with actin. In both mouse and human, the number of MYO10 foci increases as oocytes grow, corresponding to the increase in the number of actin-TZPs. RNAi-mediated depletion of MYO10 in cultured mouse granulosa cell-oocyte complexes is associated with a 52% reduction in the number of MYO10 foci and a 28% reduction in the number of actin-TZPs. Moreover, incubation of cumulus-oocyte complexes in the presence of epidermal growth factor, which triggers a 93% reduction in the number of actin-TZPs, is associated with a 55% reduction in the number of MYO10 foci. These results suggest that granulosa cells possess an ability to elaborate filopodia, which when directed towards the oocyte become actin-TZPs, and that MYO10 increases the efficiency of formation or maintenance of actin-TZPs.

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Herta, Anamaria-Cristina, Nazli Akin, Katy Billooye, Laura Saucedo-Cuevas, Francesca Lolicato, Ingrid Segers, Ellen Anckaert et Johan Smitz. « Reversing complete mechanical transzonal projections disruption during mouse in vitro follicle culture with unaltered oocyte competence† ». Biology of Reproduction, 11 mars 2021. http://dx.doi.org/10.1093/biolre/ioab045.

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Abstract In vitro oocyte growth is widely studied as an alternative fertility preservation approach. Several animal models are used to generate extensive information on this complex process regulated by the constant and dynamic interaction between the oocyte and its somatic compartment throughout follicle growth and maturation. A two-dimensional attachment mouse secondary follicle culture system was used to assess the oocyte’s capacity to overcome disconnection from its somatic companions at different developmental stages for final competence acquisition. To test this, complete mechanical denudation of oocytes from preantral (PA) and early antral (EA) follicles was performed. Established endpoints were the oocyte’s potential to reconnect with somatic cells and the impact of connectivity disruption on mature oocyte quality. This study proves that oocytes from PA and EA cultured mouse follicles can overcome complete denudation, restoring likely functional transzonal projections with no significant differences in meiotic and developmental competence compared with those from intact cultured follicles. These novel findings constitute good premises for developing successful strategies to rescue human oocyte competence in the context of in vitro culture approaches such as nonhuman chorionic gonadotropin triggered in vitro maturation.

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Bus, Anniek, Katarzyna Szymanska, Isabel Pintelon, Jo L. M. R. Leroy, Luc Leybaert et Peter E. J. Bols. « Preservation of connexin 43 and transzonal projections in isolated bovine pre-antral follicles before and following vitrification ». Journal of Assisted Reproduction and Genetics, 6 novembre 2020. http://dx.doi.org/10.1007/s10815-020-01993-2.

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Bus, Anniek, Katarzyna Szymanska, Isabel Pintelon, Jo L. M. R. Leroy, Luc Leybaert et Peter E. J. Bols. « Correction to : Preservation of connexin 43 and transzonal projections in isolated bovine pre-antral follicles before and following vitrification ». Journal of Assisted Reproduction and Genetics, 17 décembre 2020. http://dx.doi.org/10.1007/s10815-020-02040-w.

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del Collado, Maite, Juliano Coelho da Silveira, Juliano Rodrigues Sangalli, Gabriella Mamede Andrade, Letícia Rabello da Silva Sousa, Luciano Andrade Silva, Flavio Vieira Meirelles et Felipe Perecin. « Fatty Acid Binding Protein 3 And Transzonal Projections Are Involved In Lipid Accumulation During In Vitro Maturation Of Bovine Oocytes ». Scientific Reports 7, n^o 1 (1 juin 2017). http://dx.doi.org/10.1038/s41598-017-02467-9.

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Yin, Chao, Jie Liu, Zhanglin Chang, Bin He, Yang Yang et Ruqian Zhao. « Heat exposure impairs porcine oocyte quality with suppressed actin expression in cumulus cells and disrupted F-actin formation in transzonal projections ». Journal of Animal Science and Biotechnology 11, n^o 1 (6 juillet 2020). http://dx.doi.org/10.1186/s40104-020-00477-8.

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Abstract Background Transzonal projections (TZPs) constitute a structural basis for the communication between the oocyte and its surrounding cumulus cells (CCs), which play critical roles in promoting the oocyte maturation. Previously we found that heat stress (HS) causes loss of TZPs in porcine cumulus-oocyte complexes (COCs) with decreased density of filamentous actin (F-actin). However, the time-course responses of F-actin and its monomeric actins (β-actin and γ-actin) during the in vitro maturation of oocytes remain unclear. Results In this study, excised porcine ovaries were exposed to HS at 41.5 °C for 1 h before COCs were isolated and matured in vitro for 44 h. HS significantly reduced oocyte quality, characterized by impaired cumulus expansion, delayed meiotic resumption and lower survival rate and polar body extrusion rate, as well as decreased expression of mitochondrial DNA-encoded genes and elevated mitochondrial reactive oxygen species concentration. Expression of β-actin and γ-actin in CCs increased gradually with oocytes maturation, which was significantly reduced in HS group, especially at 24 h and/or 44 h of in vitro maturation. By contrast, the number of TZPs and the fluorescence intensity of F-actin in zona pellucida decreased gradually during oocytes maturation, which were significantly reduced by HS at 24 h of in vitro maturation. Moreover, colocalization analyses revealed both β-actin and γ-actin contribute to the F-actin formation in porcine TZPs, and the colocalization of F-actin with GJ protein connexin 45 was significantly reduced in heat-exposed COCs. Conclusions The results indicate that the suppression of actin expressions in CCs, which may lead to the F-actin unstabilization in TZPs, will subsequently contribute to the compromised quality of oocytes under HS.

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Jankovičová, Jana, Petra Sečová, Ľubica Horovská, Lucia Olexiková, Linda Dujíčková, Alexander V. Makarevich, Katarína Michalková et Jana Antalíková. « Distribution of tetraspanins in bovine ovarian tissue and fresh/vitrified oocytes ». Histochemistry and Cell Biology, 15 octobre 2022. http://dx.doi.org/10.1007/s00418-022-02155-4.

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AbstractTetraspanin proteins are mostly known as organizers of molecular complexes on cell membranes, widely expressed on the surface of most nucleated cells. Although tetraspanins participate in many physiological processes of mammals, including reproduction, their relevance to the processes of folliculogenesis and oogenesis has not yet been fully elucidated. We bring new information regarding the distribution of tetraspanins CD9, CD81, CD151, CD82, and CD63 at different stages of follicular development in cattle. The found distribution of tetraspanin CD9, CD63, and integrin alpha V in similar areas of ovarian tissue outlined their possible cooperation. We also describe yet-unknown distribution patterns of CD151, CD82, and CD63 on immature and mature bovine oocytes. The unique localization of tetraspanins CD63 and CD82 in the zona pellucida of bovine oocytes suggested their involvement in transzonal projections. Furthermore, we present an unchanged distribution pattern of the studied tetraspanins in vitrified mature bovine oocytes. The immunofluorescent analysis was supplemented by in silico data addressing tetraspanins expression in the ovarian cells and oocytes across several species. The obtained results suggest that in the study of the oocyte development and potentially the fertilization process of cattle, the role of tetraspanins and integrins should also be taken into account.

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Cheng, Kaixin, Xie’an Feng, Chen Yang, Chiyuan Ma, Shudong Niu, Longzhong Jia, Xuebing Yang et al. « Simulated microgravity reduces quality of ovarian follicles and oocytes by disrupting communications of follicle cells ». npj Microgravity 9, n^o 1 (23 janvier 2023). http://dx.doi.org/10.1038/s41526-023-00248-5.

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AbstractOvarian follicles are the fundamental structures that support oocyte development, and communications between oocytes and follicle somatic cells are crucial for oogenesis. However, it is unknown that whether exposure to microgravity influences cellular communications and ovarian follicle development, which might be harmful for female fertility. By 3D culturing of ovarian follicles under simulated microgravity (SMG) conditions in a rotating cell culture system, we found that SMG treatment did not affect the survival or general growth of follicles but decreased the quality of cultured follicles released oocytes. Ultrastructure detections by high-resolution imaging showed that the development of cellular communicating structures, including granulosa cell transzonal projections and oocyte microvilli, were markedly disrupted. These abnormalities caused chaotic polarity of granulosa cells (GCs) and a decrease in oocyte-secreted factors, such as Growth Differentiation Factor 9 (GDF9), which led to decreased quality of oocytes in these follicles. Therefore, the quality of oocytes was dramatically improved by the supplementations of GDF9 and NADPH-oxidase inhibitor apocynin. Together, our results suggest that exposure to simulated microgravity impairs the ultrastructure of ovarian follicles. Such impairment may affect female fertility in space environment.

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Converse, Aubrey, Zhenghui Liu, Jai C. Patel, Sushil Shakyawar, Chittibabu Guda, George R. Bousfield, T. Rajendra Kumar et Francesca E. Duncan. « Hypoglycosylated FSH enhances oocyte quality via increased cell-to-cell interaction during mouse follicle development ». Development, 23 octobre 2023. http://dx.doi.org/10.1242/dev.202170.

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Macroheterogeneity in follicle-stimulating hormone (FSH) β-subunit N-glycosylation results in distinct FSH glycoforms. Hypoglycosylated FSH21 is the abundant and more bioactive form in pituitaries of young women, whereas fully glycosylated FSH24 is less bioactive and increases with age. To investigate if the shift in FSH glycoform abundance contributes to the age-dependent decline in oocyte quality, the direct effects of FSH glycoforms on folliculogenesis and oocyte quality were determined using an encapsulated in vitro follicle growth system. Long-term culture (10-12 days) with FSH21 (10 ng/ml) enhanced follicle growth, estradiol secretion, and oocyte quality compared to FSH24 (10 ng/ml) treatment. FSH21 enhanced establishment of transzonal projections, gap junctions, and cell-to-cell communication within 24 hrs in culture. Transient inhibition of FSH21-mediated bidirectional communication abrogated the positive effects of FSH21 on follicle growth, estradiol secretion and oocyte quality. Our data indicate that FSH21 promotes folliculogenesis and oocyte quality in vitro through increasing cell-to-cell communication early in folliculogenesis and that the shift in in vivo abundance of low FSH21 to high FSH24 with reproductive aging may contribute to the age-dependent decline in oocyte quality.

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Ang, Li, Guo Xingping, Cao Haixia, Wang Zhulin et Wang Huaixiu. « Assessment of cGMP level in medium during in vitro growth period of murine preantral follicles with and without supplementation of C-type natriuretic peptide ». Zygote, 22 juin 2021, 1–5. http://dx.doi.org/10.1017/s0967199421000393.

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Summary To enhance the developmental competency of murine ovarian follicles cultured in vitro, C-type natriuretic peptide (CNP) was supplemented in the culture system. Although the mechanism is not fully elucidated, it was reported that the effect of CNP supplementation was mediated by increased cyclic guanosine monophosphate (cGMP). In the present study, cGMP levels in media for murine preantral follicle culture were compared both between a control group without CNP supplementation and an experimental group with CNP supplementation and between days in each group. In addition, follicle growth patterns and oocyte maturity were assessed and compared between the two groups. Results demonstrated that along with in vitro culture, cGMP levels increased (P < 0.05) both in the control group and the experimental group, whereas cGMP levels were not significantly different between the two groups on the same day of in vitro culture (P > 0.05). The oocyte’s maturity was superior in the experimental group compared with the control group (P < 0.05). As ovarian follicles grew three-dimensionally in the experimental group but were flattened in the control group, CNP might improve oocyte maturity through maintaining the three-dimensional architecture of the ovarian follicle because of increased transzonal projections (TZP) and functional gap junctions between oocyte and surrounding granulosa cells.

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Catandi, G. D., D. R. Bresnahan, S. O. Peters, K. J. Fresa, L. J. Maclellan, C. D. Broeckling et E. M. Carnevale. « Equine maternal aging affects the metabolomic profile of oocytes and follicular cells during different maturation time points ». Frontiers in Cell and Developmental Biology 11 (25 septembre 2023). http://dx.doi.org/10.3389/fcell.2023.1239154.

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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.

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Xie, Jun, Xiao Xu et Suying Liu. « Intercellular communication in the cumulus–oocyte complex during folliculogenesis : A review ». Frontiers in Cell and Developmental Biology 11 (19 janvier 2023). http://dx.doi.org/10.3389/fcell.2023.1087612.

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During folliculogenesis, the oocyte and surrounding cumulus cells form an ensemble called the cumulus-oocyte complex (COC). Due to their interdependence, research on the COC has been a hot issue in the past few decades. A growing body of literature has revealed that intercellular communication is critical in determining oocyte quality and ovulation. This review provides an update on the current knowledge of COC intercellular communication, morphology, and functions. Transzonal projections (TZPs) and gap junctions are the most described structures of the COC. They provide basic metabolic and nutrient support, and abundant molecules for signaling pathways and regulations. Oocyte-secreted factors (OSFs) such as growth differentiation factor 9 and bone morphogenetic protein 15 have been linked with follicular homeostasis, suggesting that the communications are bidirectional. Using advanced techniques, new evidence has highlighted the existence of other structures that participate in intercellular communication. Extracellular vesicles can carry transcripts and signaling molecules. Microvilli on the oocyte can induce the formation of TZPs and secrete OSFs. Cell membrane fusion between the oocyte and cumulus cells can lead to sharing of cytoplasm, in a way making the COC a true whole. These findings give us new insights into related reproductive diseases like polycystic ovary syndrome and primary ovarian insufficiency and how to improve the outcomes of assisted reproduction.

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Marchais, Mathilde, Isabelle Gilbert, Alexandre Bastien, Angus Macaulay et Claude Robert. « Mammalian cumulus-oocyte complex communication : a dialog through long and short distance messaging ». Journal of Assisted Reproduction and Genetics, 2 mai 2022. http://dx.doi.org/10.1007/s10815-022-02438-8.

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Abstract Communications are crucial to ovarian follicle development and to ovulation, and while both folliculogenesis and oogenesis are distinct processes, they share highly interdependent signaling pathways. Signals from distant organs such as the brain must be processed and compartments within the follicle have to be synchronized. The hypothalamic–pituitary–gonadal (HPG) axis relies on long-distance signalling analogous to wireless communication by which data is disseminated in the environment and cells equipped with the appropriate receptors receive and interpret the messages. In contrast, direct cell-to-cell transfer of molecules is a very targeted, short distance messaging system. Numerous signalling pathways have been identified and proven to be essential for the production of a developmentally competent egg. The development of the cumulus-oocyte complex relies largely on short distance communications or direct transfer type via extensions of corona radiata cells through the zona pellucida. The type of information transmitted through these transzonal projections is still largely uncharacterized. This review provides an overview of current understanding of the mechanisms by which the gamete receives and transmits information within the follicle. Moreover, it highlights the fact that in addition to the well-known systemic long-distance based communications from the HPG axis, these mechanisms acting more locally should also be considered as important targets for controlling/optimizing oocyte quality.

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Barrozo, Laryssa G., Bianca R. Silva, Laís R. F. M. Paulino, Efigênia C. Barbalho, Danisvânia R. Nascimento, Francisco C. Costa, Ana L. P. S. Batista, Everton P. F. Lopes, Ana P. R. Rodrigues et José R. V. Silva. « N-Acetyl cysteine reduces the levels of reactive oxygen species and improves in vitro maturation of oocytes from medium-sized bovine antral follicles ». Zygote, 23 septembre 2022, 1–9. http://dx.doi.org/10.1017/s0967199422000429.

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Summary This study aims to evaluate the effects of N-acetylcysteine (NAC) on bovine oocyte maturation, mitochondrial activity and transzonal projections (TZP), as well as on the levels of reactive oxygen species (ROS) and messenger RNA (mRNA) for catalase (CAT) superoxide dismutase (SOD), periredoxin-6 (Prdx6), glutathione peroxidase (GPx), growth and differentiation factor-9 (GDF9), histone H1Foo, cyclin B1 (CCNB1) and c-Mos. Bovine cumulus–oocyte complexes (COC) of medium-sized antral follicles (3.0–6.0 mm) were prematured in TCM-199 for 8 h at 38.5°C in 5% CO2. After prematuration in the presence of forskolin and C-type natriuretic peptide, COCs were matured in TCM-199 alone or with 0.1, 0.5 or 2.5 mM NAC. Then, oocytes were classified according to the stage of chromatin. Furthermore, mitochondrial activity and intracellular levels of ROS and TZP were also evaluated. The levels of mRNAs for CAT, SOD, Prdx6, GPx, GDF9, H1Foo, CCNB1 and c-Mos were evaluated using real-time polymerase chain reaction (RT-PCR). The results showed that NAC significantly increased the percentages of oocytes with resumption of meiosis when compared with those oocytes matured in control medium. Oocytes had homogeneous mitochondrial distribution, and those cultured with 0.1 and 0.5 mM NAC had lower levels of ROS when compared with the control. In addition, 0.5 mM NAC reduced TZP and the levels of mRNA for CCNB1. In contrast, NAC did not influence the expression of CAT, GPx, Prdx6, SOD, GDF9, H1Foo, and c-Mos. In conclusion, 0.5 mM NAC reduced the levels of ROS, TZP and mRNA for CCNB1, and improved in vitro resumption of meiosis in oocytes from medium-sized bovine antral follicles.

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Dubuc, Karine, Mathilde Marchais, Isabelle Gilbert, Alexandre Bastien, Karen E. Nenonene, Edward W. Khandjian, Robert S. Viger, Géraldine Delbes et Claude Robert. « Epitranscriptome marks detection and localization of RNA modifying proteins in mammalian ovarian follicles ». Journal of Ovarian Research 16, n^o 1 (10 mai 2023). http://dx.doi.org/10.1186/s13048-023-01172-8.

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Abstract Background Most of the resources that support the early development of the embryo are stored in the oocyte. Clearing of maternal resources and activation of the embryonic genome to produce its own mRNA transcripts marks the maternal-to-embryo transition. Dependence on stored mRNA can last from a few hours to several days, depending on animal species. The mechanisms regulating stabilization and recruitment of stored maternal transcripts have not yet been described in full detail but are known to involve reversible polyadenylation and modulation of 3’UTR-mediated elements. RNA epigenetic modifications, new players in this field, have an important role in RNA regulation and stabilization. Results The objectives of this study were first to determine if some of post-transcriptional methylation of stored mRNA is greater in oocytes than in somatic cells. We found that m6A, known to be the most prevalent and involved in various aspects of RNA metabolism and physiological functions, is particularly abundant in porcine oocyte mRNA compared to liver used as a somatic tissue reference. The second objective was to compare the epitranscriptome machinery, such as methyltransferases (“writers”), binding proteins (“readers”) and demethylases (“erasers”) catalyzing the different process, in follicles and oocytes of different mammalian species by immunofluorescence and confocal microscopy. The expression and localization patterns of these proteins differ between mice, pigs and cows ovaries and oocytes. m5C-associated proteins were generally less abundant. In contrast, m6A-associated proteins were expressed strongly during the early and late stages of folliculogenesis. Transzonal projections were found to contain more granules bearing the m5C mark in mice but both m5C and m6A methylation marks in association with mature oocytes of pigs and cows. Eraser proteins showed the greatest interspecies diversity in terms of distribution in the germinal tissues. Conclusions So far, few studies have looked at the oocyte and ovarian epitranscriptomic profile. Our findings indicate that a hitherto unrecognized species-specific layer of transcript regulation occurs at the RNA level and might be consequential during the oocyte transcriptional silencing period.

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Martínez-Moro, Álvaro, Ismael Lamas-Toranzo, Leopoldo González-Brusi, Alba Pérez-Gómez, Ester Padilla-Ruiz, Javier García-Blanco et Pablo Bermejo-Álvarez. « mtDNA content in cumulus cells does not predict development to blastocyst or implantation ». Human Reproduction Open, 6 juillet 2022. http://dx.doi.org/10.1093/hropen/hoac029.

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Abstract STUDY QUESTION Is relative mitochondrial DNA (mtDNA) content in cumulus cells related to embryo developmental competence in humans and/or the bovine model? SUMMARY ANSWER mtDNA content in cumulus cells provides poor predictive value of oocyte developmental potential, both in vitro and following embryo transfer. WHAT IS KNOWN ALREADY Cumulus cells are closely connected to the oocyte through transzonal projections, serving essential metabolic functions during folliculogenesis. These oocyte-supporting cells are removed and discarded prior to ICSI, thereby providing interesting biological material on which to perform molecular analyses designed to identify markers that predict oocyte developmental competence. Previous studies have positively associated oocyte mtDNA content with developmental potential in animal models and women. However, it remains debatable whether mtDNA content in cumulus cells could be used as a proxy to infer oocyte developmental potential STUDY DESIGN, SIZE, DURATION mtDNA content was analyzed in cumulus cells obtained from 109 human oocytes unable to develop to blastocyst, able to develop to blastocyst but failing to establish pregnancy, or able to develop to blastocyst and to establish pregnancy. mtDNA analysis was also performed on bovine cumulus samples collected from 120 oocytes unable to cleave, oocytes developing into cleaved embryos but arresting development prior to the blastocyst stage, or oocytes developing to blastocysts. PARTICIPANTS/MATERIALS, SETTING, METHODS Human cumulus cells samples were obtained from women undergoing IVF. Only unfrozen oocytes and embryos not submitted to preimplantation genetic testing were included in the analysis. Bovine samples were obtained from slaughtered cattle and individually matured, fertilized and cultured in vitro. Relative mtDNA was assessed by quantitative PCR analysis. MAIN RESULTS AND THE ROLE OF CHANCE mtDNA content in human and bovine cumulus cells did not differ according to the developmental potential of their enclosed oocyte. Moreover, mtDNA content in bovine oocytes did not correlate with that of their corresponding cumulus cells. LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION The lack of correlation found between mtDNA content in human cumulus cells and oocytes was also assessed in bovine samples. Although bovine folliculogenesis, monoovulatory ovulation and early embryo development exhibit considerable similarities with that of humans, they may not be fully comparable. WIDER IMPLICATIONS OF THE FINDINGS The use of molecular markers for oocyte developmental potential in cumulus cells could be used to enhance success rates following single embryo transfer. However, our data indicate that mtDNA in cumulus cells is not a good proxy for oocyte quality. STUDY FUNDING/COMPETING INTEREST(S) Research was supported by the Industrial Doctorate Project IND2017/BIO-7748 funded by Madrid Region Government. The authors declare no competing interests.

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Martíne. Moro, Á., I. Lamas-Toranzo, L. González-Brusi, A. Pérez-Gómez et P. Bermejo-Álvarez. « P–219 mtDNA content in bovine cumulus cells does not predict oocytés developmental competence ». Human Reproduction 36, Supplement_1 (1 juillet 2021). http://dx.doi.org/10.1093/humrep/deab130.218.

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Abstract Study question Does cumulus cell mtDNA content correlate with oocyte developmental potential in the bovine model? Summary answer The relative amount of mtDNA content did not vary significantly in oocytes showing different developmental outcomes following IVF What is known already Cumulus cells are closely connected to the oocyte through transzonal projections, serving essential metabolic functions during folliculogenesis. These oocyte-supporting cells are removed and discarded prior to ICSI, thereby constituting an interesting biological material on which to perform molecular analysis aimed to predict oocyte developmental competence. Previous studies have positively associated oocytés mtDNA content with developmental potential in both animal models and women. However, it remains debatable whether mtDNA content in cumulus cells could be used as a proxy to infer oocyte developmental potential. Study design, size, duration Bovine cumulus cells were allocated into three groups according to the developmental potential of the oocyte: 1) oocytes developing to blastocysts following IVF (Bl+Cl+), 2) oocytes cleaving following IVF but arresting their development prior to the blastocyst stage (Bl-Cl+), and 3) oocytes not cleaving following IVF (Bl-Cl-). Relative mtDNA content was analysed in 40 samples/group, each composed by the cumulus cells from one cumulus-oocyte complex (COC). Participants/materials, setting, methods Bovine cumulus-oocyte complexes were obtained from slaughtered cattle and individually matured in vitro (IVM). Following IVM, cumulus cells were removed by hyaluronidase treatment, pelleted, snap frozen in liquid nitrogen and stored at –80 ºC until analysis. Cumulus-free oocytes were fertilized and cultured in vitro individually and development was recorded for each oocyte. Relative mtDNA abundance was determined by qPCR, amplifying a mtDNA sequence (COX1) and a chromosomal sequence (PPIA). Statistical differences were tested by ANOVA. Main results and the role of chance Relative mtDNA abundance did not differ significantly (ANOVA p > 0.05) between the three groups exhibiting different developmental potential (1±0.06 vs. 1.19±0.05 vs. 1.11±0.05, for Bl+Cl+ vs. Bl-Cl+ vs. Bl-Cl-, mean±s.e.m.). Limitations, reasons for caution Experiments were conducted in the bovine model. Although bovine folliculogenesis, monoovulatory ovulation and early embryo development exhibit considerable similarities with that of humans, caution should be taken when extrapolating these data to humans. Wider implications of the findings: The use of molecular markers for oocyte developmental potential in cumulus cells could be used to enhance success rates following single-embryo transfer. Unfortunately, mtDNA in cumulus cells was not found to be a good proxy for oocyte quality. Trial registration number Not applicable

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Converse, Aubrey, T. Rajendra Kumar et Francesca E. Duncan. « OR19-04 Hypoglycosylated FSH Enhances Ovarian Follicle Development and Gamete Quality ». Journal of the Endocrine Society 7, Supplement_1 (octobre 2023). http://dx.doi.org/10.1210/jendso/bvad114.1650.

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Abstract Disclosure: A. Converse: None. T. Kumar: None. F.E. Duncan: None. Gamete quality depends on the proper growth and maturation of the ovarian follicle, which consists of the oocyte and its supporting somatic cells. FSH is essential for follicle growth as female Fshb and Fshr null mice are infertile due to arrested follicle development. Macroheterogeneity in N-glycosylation on the FSHß-subunit confers differential bioactivities with hypoglycosylated FSH21 exhibiting higher FSHR binding affinity and bioactivity compared to fully glycosylated FSH24. In females, there is a shift from the highly bioactive FSH21 to FSH24 with increasing age which occurs coordinately with the age-dependent decline in female fertility. In this study, we used an encapsulated in vitro follicle growth assay to test the hypothesis that FSH glycoforms have differential abilities to promote follicle development and gamete quality. Early-stage (preantral) ovarian follicles were isolated from CD-1 mice, cultured in media containing 10 ng/ml of either FSH21 or FSH24 for up to 12 days, and then ex vivo ovulation was induced. While both glycoforms maintained >75% follicle survival through day 12, follicles treated with FSH21 were significantly larger than FSH24-treated follicles between days 4 and 12 of culture. Over the 12 day culture, FSH21-treated follicles grew from 110.9 ± 9.2 µm to 251.3 ± 24.4 µm, while FSH24-treated follicles grew from 109.3 ± 7.1 µm to 229.7 ± 15.4 µm. This difference in growth correlated with a 13.4 and 4.5-fold increase in estradiol secretion by FSH21-treated follicles compared to FSH24 treatment on days 8 and 12, respectively. After ex vivo maturation, FSH21-treated follicles produced a higher proportion (76.9%) of meiotically competent gametes that reached the metaphase II (MII)-arrested state compared to FSH24-treated follicles (62.5%). Furthermore, normal meiotic spindle configuration was observed in 76% of MII eggs from the FSH21 cohort but only 50% of the FSH24 cohort. These results indicate that FSH21 is more bioactive than FSH24 in promoting follicle development and high-quality oocytes. Mechanistically, this is likely due in part to the differential effects of FSH21 and FSH24 on the formation of transzonal projections (TZPs), which are cytoplasmic filaments essential for granulosa-oocyte communication. By 24 hrs, FSH21-treated follicles had more TZPs than FSH24-treated follicles, as demonstrated by increased actin staining (1.6x) and Myo10 foci (5.1x) in the zona pellucida region. Thus, the early establishment of TZPs and increased somatic-oocyte communication in FSH21-treated follicles ultimately results in a more developmentally competent gamete. In addition, we anticipate that ongoing transcriptomic analysis of 24 hr FSH glycoform-treated follicles will highlight other processes involved in early follicle development that contribute to the observed differential effects on oocyte quality. Presentation Date: Saturday, June 17, 2023

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Kumar, K., L. Nesbeth, M. Venturas, D. Needleman, C. Racowsky et D. Wells. « P-175 Human cumulus cell telomere length and its association with assisted reproduction outcomes ». Human Reproduction 38, Supplement_1 (1 juin 2023). http://dx.doi.org/10.1093/humrep/dead093.535.

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Abstract Study question Is there any relationship between the relative telomere length (RTL) within cumulus cells (CCs) and the outcome of assisted reproductive treatment using the corresponding oocyte? Summary answer Lower RTLs in CCs were significantly associated with embryos chosen for transfer or cryopreservation. In contrast, embryos considered non-viable (discarded) tended to have higher RTLs. What is known already Cumulus cells fulfil vital roles in support of oocyte development, including the transduction of external signals and the provision of resources via transzonal projections. Given their essential role in the acquisition of oocyte developmental competence, the biology of CCs is of clinical relevance. Telomeres are specialised structures protecting the ends of chromosomes, composed of repetitive DNA sequences and associated proteins. Telomeres shorten with each mitotic division, as well as due to oxidative damage, eventually reaching a critical threshold at which point cellular senescence occurs. Currently, published data on CCs telomere length and relationship with oocyte potential are conflicting. Study design, size, duration The study involved 182 human CC samples collected from 52 IVF patients. Quantitative PCR (qPCR) was used to measure the relative telomere length in each of the CC samples. Telomere lengths were assessed for associations with various patient characteristics (e.g. age, body mass index, infertility diagnosis). Additionally, potential relationships with clinically relevant oocyte/embryo features were investigated (fertilisation; development/morphology), as well as the eventual fate of the associated embryo (transferred; cryopreserved for potential future use; discarded). Participants/materials, setting, methods Real-time quantitative PCR was carried out using PCR primers specific for the telomere repeat. A single-copy gene was also amplified from each CC sample. Quantification of this gene was used for normalisation of the telomere data, allowing control for variation in the number of cumulus cells in each sample. Associations between RTL in CCs and patient, embryonic, and clinical factors were assessed using various statistical methods, with P-values <0.05 considered significant. Main results and the role of chance No associations were identified between RTL and any patient characteristics, except for BMI. The amount of CC telomeric DNA tended to be greater for patients with higher BMI (P = 0.002). When considering links between RTL and oocyte or embryonic factors, a significant relationship was detected between the quantity of telomeric DNA in CCs and whether the corresponding embryo was considered non-viable (discarded) or whether it was transferred or cryopreserved (P = 0.019). This finding raises the possibility that measurement of RTL in CCs could provide a pre-conception, non-invasive assessment of oocyte quality. In the context of fertility preservation, RTL measurement could assist in evaluating a cohort of oocytes, indicating whether the cryopreserved eggs are likely to be sufficient or whether additional cycles to generate more would be advisable. If future studies confirm that CC RTL has a strong predictive value, the possibility of limiting fertilisation to oocytes considered to have high likelihood of viability could also be considered for routine IVF cycles. It is unclear why shorter CC telomeres might be associated with oocytes of superior potential, but one possibility is that the cells may have undergone a greater proliferation (more mitoses), resulting in a more extensive cumulus mass supporting the enclosed oocyte. Limitations, reasons for caution Before drawing definitive conclusions, confirmation of the findings within a larger, independent data set is necessary. Even if confirmed, determination of the true clinical value of telomere assessment will require further, appropriately designed studies. The current study was not powered to evaluate relationships between CC telomere lengths and IVF outcomes. Wider implications of the findings Currently, simplistic morphological evaluation is the only method for assessing oocyte competence prior to fertilisation. If CCs telomere measurement is confirmed to have predictive value, a preconception test of oocyte potential could be offered. This would be extremely valuable for patients cryopreserving oocytes for fertility preservation and for donor banks. Trial registration number NA

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Dadashzadeh, Arezoo, Saeid Moghassemi, Alexis Peaucelle, Carolina M. Lucci et Christiani A. Amorim. « Mind the mechanical strength : tailoring a 3D matrix to encapsulate isolated human preantral follicles ». Human Reproduction Open, 17 février 2023. http://dx.doi.org/10.1093/hropen/hoad004.

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Abstract STUDY QUESTION Would a hydrogel with similar mechanical properties to the human ovarian cortex support preantral follicle development? SUMMARY ANSWER Yes, our tailored PEGylated fibrin hydrogel was shown to significantly improve follicle growth in vitro. WHAT IS KNOWN ALREADY One of the main challenges in developing an engineered ovary is to provide a 3D matrix that supports the follicle architecture and the interaction between granulosa cells and the oocyte as they are essential for folliculogenesis. Thanks to its biocompatibility and bioactivity, fibrin has been employed to fabricate a 3D matrix to encapsulate ovarian follicles. However, follicles lose their physical support within a few days owing to rapid fibrin degradation. Therefore, different strategies, including physical and chemical modifications, have been developed to enhance the stability of fibrin. STUDY DESIGN, SIZE, DURATION By developing a matrix made of a synthetic (polyethylene glycol: PEG) and natural polymer (fibrin), we aimed to overcome fibrin degradation by the chemical reaction of PEGylation and tailor a PEGylated fibrin hydrogel formulation with mechanical strength similar to the ovarian cortex in women of reproductive age. To this end, response surface methodology was employed to obtain a tailored formulation of PEGylated fibrin. This hydrogel was then tested to encapsulate and support isolated human preantral follicles in vitro. PARTICIPANTS/MATERIALS, SETTING, METHODS A PEGylated fibrin formulation was tailored using mathematical modeling software to mimic the mechanical properties of human ovarian tissue at reproductive age. Human preantral follicles were isolated from 11 patients of reproductive age and encapsulated in the tailored hydrogels, which were cultured in vitro for 4 or 7 days. Follicle survival and diameter were assessed on days 1 and 7. Furthermore, the follicles were subjected to confocal microscopy to evaluate their growth (Ki67 staining) on day 7 and analyze cell-cell communication (connexin 43 and transzonal projection staining) on day 4. MAIN RESULTS AND THE ROLE OF CHANCE In this study, mathematical modeling was applied to achieve the biomechanically tailored PEGylated fibrin formulation by targeting the specific goal of 3178 ± 245 Pascal, Young’s modulus of ovarian cortical tissue in reproductive-age women. Our results demonstrated that the PEGylated fibrin hydrogel consisting of 39.06 mg/ml of PEGylated fibrinogen and 50.36 IU/ml of thrombin was the optimum condition with the desirability of 97.5%. This tailored hydrogel yielded a high follicle survival rate (83%) after 7 days of in vitro culture and supported its development up to the secondary stage. Follicle growth was confirmed by the presence of Ki67-positive granulosa cells on day 7. Additionally, connexin 43 and Phalloidin staining indicated the retention of connections between granulosa cells and the oocyte. LARGE SCALE DATA Not applicable. LIMITATIONS, REASONS FOR CAUTION In this study, our tailored hydrogel was only tested in vitro, which is not the same as the physiological environment. It is crucial to conduct a study assessing the follicles following their encapsulation in the tailored hydrogel and transplantation, which will be the next step of our investigation. WIDER IMPLICATIONS OF THE FINDINGS The findings from this study introduced a suitable biomaterial similar to the ovarian cortex in reproductive-age women in terms of biomechanical properties for encapsulating human preantral follicles. This biomaterial allowed the radial growth of follicles and preserved their viability. Furthermore, PEGylation improved the stability of fibrin and the physical support of follicles. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by a grant from the Fondation Louvain (awarded to C.A.A.). The authors declare no competing interests. WHAT DOES THIS MEAN FOR PATIENTS? Survival rates of leukemia patients have been steadily increasing but, owing to cancer treatment, some survivors will face infertility at a very young age. One of the strategies under development to restore fertility in these patients is the transplantable ‘engineered ovary’, which should mimic the normal ovary as closely as possible. To assemble the engineered ovary, it is necessary to encase isolated preantral follicles (follicles at an early stage of growth that contain one immature egg) and ovarian cells in a 3D matrix. Developing this matrix is one of the main challenges in creating an engineered ovary, as it should support the follicle architecture as well as the interaction between granulosa cells (which produce estrogen, progesterone) and the oocyte (egg), as they are essential for follicle development. This study demonstrates that a matrix made of a synthetic (polyethylene glycol) and natural polymer (fibrin), with mechanical strength similar to that of the outer layer of the ovary in women of reproductive age, can successfully be used to encapsulate human preantral follicles. Indeed, we have shown that our 3D matrix yielded a high follicle survival rate and supported follicle growth to the secondary stage.

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