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Follicular Fluid-Derived Extracellular Vesicles Influence on In Vitro Maturation of Equine Oocyte: Impact on Cumulus Cell Viability, Expansion and Transcriptome.

ABSTRACT: Cumulus cell (CC) expansion is pivotal for oocyte maturation, during which CCs release factors that initiate paracrine signaling within the follicular fluid (FF). The FF is abundant in extracellular vesicles (EVs) that facilitate intercellular communication. Although bovine and murine EVs can control cumulus expansion, these effects have not been observed in equines. This study aimed to assess the impact of FF-derived EVs (ffEVs) on equine CC expansion, viability, and transcriptome. Cumulus-oocyte complexes (COCs) that underwent in vitro maturation (IVM) in the presence (200 µg protein/mL) or absence (control) of ffEVs were assessed for cumulus expansion and viability. CCs were isolated after 12 h of IVM, followed by RNA extraction, cDNA library generation, and subsequent transcriptome analysis using next-generation sequencing. Confocal microscopy images illustrated the internalization of labeled ffEVs by CCs. Supplementation with ffEVs significantly enhanced cumulus expansion in both compacted (Cp, p < 0.0001) and expanded (Ex, p < 0.05) COCs, while viability increased in Cp groups (p < 0.01), but decreased in Ex groups (p < 0.05), compared to the controls. Although transcriptome analysis revealed a subtle effect on CC RNA profiles, differentially expressed genes encompassed processes (e.g., MAPK and Wnt signaling) potentially crucial for cumulus properties and, consequently, oocyte maturation.

SUBMITTER: Gabrys J

PROVIDER: S-EPMC10970002 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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Follicular Fluid-Derived Extracellular Vesicles Influence on In Vitro Maturation of Equine Oocyte: Impact on Cumulus Cell Viability, Expansion and Transcriptome.

Gabryś Julia J Gurgul Artur A Szmatoła Tomasz T Kij-Mitka Barbara B Andronowska Aneta A Karnas Elżbieta E Kucharski Mirosław M Wojciechowska-Puchałka Joanna J Kochan Joanna J Bugno-Poniewierska Monika M

International journal of molecular sciences 20240313 6

Cumulus cell (CC) expansion is pivotal for oocyte maturation, during which CCs release factors that initiate paracrine signaling within the follicular fluid (FF). The FF is abundant in extracellular vesicles (EVs) that facilitate intercellular communication. Although bovine and murine EVs can control cumulus expansion, these effects have not been observed in equines. This study aimed to assess the impact of FF-derived EVs (ffEVs) on equine CC expansion, viability, and transcriptome. Cumulus-oocy ...[more]

PMID: 38542236

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Project description:In this study, we tested the overall hypothesis that CC expansion and early embryo development would be improved by including follicular fluid (FF) from small or large follicles in the oocyte maturation medium. In the first experiment, FF aspirated from bovine abattoir ovaries was added to the maturation medium at 0, 25, 50, 75 or 100%. Images of individual COCs were captured at 0, 6, 12 and 19 hours (h) of the maturation period and analyzed to calculate change in the total area over time. Cumulus cell expansion was greatest in COCs matured in 75% and 50% FF, and these differences were detectable at 12 (75% FF only) and 19 h (50% and 75% FF) of maturation. The improvement in CC expansion was greatest when FF from small follicles was used. Treatments for the subsequent experiments were selected based upon the results of the first experiment. Oocyte nuclear maturation rates were observed after supplementing the maturation medium with 0 or 75% FF and maturing for 19 h. The rate of nuclear maturation as determined by the presence or absence of the first polar body was similar between control (0% FF) and treated (75% FF) groups. In the final experiment, COCs were matured in 0%, 50% or 75% FF in preparation for IVF. Duration of the maturation period (12, 19 or 22 h) and size of the follicles from which FF was collected (small or large) also varied. In general, FF supplementation at 50% did not affect the zygotes' developmental potential (neither increased nor decreased). Supplementation of maturation medium with 75% FF from small follicles consistently reduced measures of embryo development while 75% FF from large follicles yielded mixed results. It is concluded that FF supplementation improves CC expansion, but the greater CC expansion does not benefit subsequent embryo development. Notably, however, the 50% FF treatment did not reduce blastocyst rates, indicating that FF can be included in maturation media at concentrations of 50% or less with no detriment to IVF outcomes.

Project description:BackgroundIn vitro embryo production is a highly demanded reproductive technology in horses, which requires the recovery (in vivo or post-mortem) and in vitro maturation (IVM) of oocytes. Oocytes subjected to IVM exhibit poor developmental competence compared to their in vivo counterparts, being this related to a suboptimal composition of commercial maturation media. The objective of this work was to study the effect of different concentrations of secretome obtained from equine preovulatory follicular fluid (FF) on cumulus-oocyte complexes (COCs) during IVM. COCs retrieved in vivo by ovum pick up (OPU) or post-mortem from a slaughterhouse (SLA) were subjected to IVM in the presence or absence of secretome (Control: 0 µg/ml, S20: 20 µg/ml or S40: 40 µg/ml). After IVM, the metabolome of the medium used for oocyte maturation prior (Pre-IVM) and after IVM (Post-IVM), COCs mRNA expression, and oocyte meiotic competence were analysed.ResultsIVM leads to lactic acid production and an acetic acid consumption in COCs obtained from OPU and SLA. However, glucose consumption after IVM was higher in COCs from OPU when S40 was added (Control Pre-IVM vs. S40 Post-IVM: 117.24 ± 7.72 vs. 82.69 ± 4.24; Mean µM ± SEM; p < 0.05), while this was not observed in COCs from SLA. Likewise, secretome enhanced uptake of threonine (Control Pre-IVM vs. S20 Post-IVM vs. S40 Post-IVM: 4.93 ± 0.33 vs. 3.04 ± 0.25 vs. 2.84 ± 0.27; Mean µM ± SEM; p < 0.05) in COCs recovered by OPU. Regarding the relative mRNA expression of candidate genes related to metabolism, Lactate dehydrogenase A (LDHA) expression was significantly downregulated when secretome was added during IVM at 20-40 µg/ml in OPU-derived COCs (Control vs. S20 vs. S40: 1.77 ± 0.14 vs. 1 ± 0.25 vs. 1.23 ± 0.14; fold change ± SEM; p < 0.05), but not in SLA COCs.ConclusionsThe addition of secretome during in vitro maturation (IVM) affects the gene expression of LDHA, glucose metabolism, and amino acid turnover in equine cumulus-oocyte complexes (COCs), with diverging outcomes observed between COCs retrieved using ovum pick up (OPU) and slaughterhouse-derived COCs (SLA).

Project description:BackgroundOvarian follicular fluid (FF) is a dynamic environment that changes with the seasons, affecting follicle development, ovulation, and oocyte quality. Cells in the follicles release tiny particles called extracellular vesicles (EVs) containing vital regulatory molecules, such as microRNAs (miRNAs). These miRNAs are pivotal in facilitating communication within the follicles through diverse signaling and information transfer forms. EV-coupled miRNA signaling is implicated to be associated with ovarian function, follicle and oocyte growth and response to various environmental insults. Herein, we investigated how seasonal variations directly influence the ovulatory and anovulatory states of ovarian follicles and how are they associated with follicular fluid EV-coupled miRNA dynamics in horses.ResultsUltrasonographic monitoring and follicular fluid aspiration of preovulatory follicles in horses during the anovulatory (spring: non-breeding) and ovulatory (spring, summer, and fall: breeding) seasons and subsequent EV isolation and miRNA profiling identified significant variation in EV-miRNA cargo content. We identified 97 miRNAs with differential expression among the groups and specific clusters of miRNAs involved in the spring transition (miR-149, -200b, -206, -221, -328, and -615) and peak breeding period (including miR-143, -192, -451, -302b, -100, and let-7c). Bioinformatic analyses showed enrichments in various biological functions, e.g., transcription factor activity, transcription and transcription regulation, nucleic acid binding, sequence-specific DNA binding, p53 signaling, and post-translational modifications. Cluster analyses revealed distinct sets of significantly up- and down-regulated miRNAs associated with spring anovulatory (Cluster 1) and summer ovulation-the peak breeding season (Clusters 4 and 6).ConclusionsThe findings from the current study shed light on the dynamics of FF-EV-coupled miRNAs in relation to equine ovulatory and anovulatory seasons, and their roles in understanding the mechanisms involved in seasonal shifts and ovulation during the breeding season warrant further investigation.

Project description:BackgroundImmature cumulus-oocyte complexes are retrieved to obtain mature oocytes by in vitro maturation (IVM), a laboratory tool in reproductive medicine to obtain mature oocytes. Unfortunately, the efficiency of IVM is not satisfactory. To circumvent this problem, we therefore intended to commence with the composition of ovarian follicular fluid (FF), an important microenvironment influencing oocyte growth. It is well known that FF has a critical role in oocyte development and maturation. However, the components in human FF remain largely unknown, particularly with regard to small molecular peptides.ResultsIn current study, the follicular fluid derived from human mature and immature follicles were harvested. The peptide profiles of FF were further investigated by using combined ultrafiltration and LC-MS/MS. The differential peptides were preliminary determined by performing differentially expressed analysis. Human and mouse oocyte culture were used to verify the influence of differential peptides on oocyte development. Constructing plasmids, cell transfecting, Co-IP, PLA etc. were used to reveal the detail molecular mechanism. The results from differentially expressed peptide as well as cultured human and mouse oocytes analyses showed that highly conserved C3a-peptide, a cleavage product of complement C3a, definitely affected oocytes development. Intriguingly, C3a-peptide possessed a novel function that promoted F-actin aggregation and spindle migration, raised the percentage of oocytes at the MII stage, without increasing the chromosome aneuploidy ratio, especially in poor-quality oocytes. These effects of C3a-peptide were attenuated by C3aR morpholino inhibition, suggesting that C3a-peptide affected oocytes development by collaborating with its classical receptor, C3aR. Specially, we found that C3aR co-localized to the spindle with β-tubulin to recruit F-actin toward the spindle and subcortical region of the oocytes through specific binding to MYO10, a key regulator for actin organization, spindle morphogenesis and positioning in oocytes.ConclusionsOur results provide a new perspective for improving IVM culture systems by applying FF components and also provide molecular insights into the physiological function of C3a-peptide, its interaction with C3aR, and their roles in enabling meiotic division of oocytes.

Dataset Information

Follicular Fluid-Derived Extracellular Vesicles Influence on In Vitro Maturation of Equine Oocyte: Impact on Cumulus Cell Viability, Expansion and Transcriptome.

Publications

Follicular Fluid-Derived Extracellular Vesicles Influence on In Vitro Maturation of Equine Oocyte: Impact on Cumulus Cell Viability, Expansion and Transcriptome.

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