Dataset Information

Study of the Metabolomics of Equine Preovulatory Follicular Fluid: A Way to Improve Current In Vitro Maturation Media.

ABSTRACT: Production of equine embryos in vitro is currently a commercial technique and a reliable way of obtaining offspring. In order to produce those embryos, immature oocytes are retrieved from postmortem ovaries or live mares by ovum pick-up (OPU), matured in vitro (IVM), fertilized by intracytoplasmic sperm injection (ICSI), and cultured until day 8-10 of development. However, at best, roughly 10% of the oocytes matured in vitro and followed by ICSI end up in successful pregnancy and foaling, and this could be due to suboptimal IVM conditions. Hence, in the present work, we aimed to elucidate the major metabolites present in equine preovulatory follicular fluid (FF) obtained from postmortem mares using proton nuclear magnetic resonance spectroscopy (1H-NMR). The results were contrasted against the composition of the most commonly used media for equine oocyte IVM: tissue culture medium 199 (TCM-199) and Dulbecco's modified eagle medium/nutrient mixture F-12 Ham (DMEM/F-12). Twenty-two metabolites were identified in equine FF; among these, nine of them are not included in the composition of DMEM/F-12 or TCM-199 media, including (mean ± SEM): acetylcarnitine (0.37 ± 0.2 mM), carnitine (0.09 ± 0.01 mM), citrate (0.4 ± 0.04 mM), creatine (0.36 ± 0.14 mM), creatine phosphate (0.36 ± 0.05 mM), fumarate (0.05 ± 0.007 mM), glucose-1-phosphate (6.9 ± 0.4 mM), histamine (0.25 ± 0.01 mM), or lactate (27.3 ± 2.2 mM). Besides, the mean concentration of core metabolites such as glucose varied (4.3 mM in FF vs. 5.55 mM in TCM-199 vs. 17.5 mM in DMEM/F-12). Hence, our data suggest that the currently used media for equine oocyte IVM can be further improved.

SUBMITTER: Fernandez-Hernandez P

PROVIDER: S-EPMC7278476 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Study of the Metabolomics of Equine Preovulatory Follicular Fluid: A Way to Improve Current In Vitro Maturation Media.

Fernández-Hernández Pablo P Sánchez-Calabuig María Jesús MJ García-Marín Luis Jesús LJ Bragado María J MJ Gutiérrez-Adán Alfonso A Millet Óscar Ó Bruzzone Chiara C González-Fernández Lauro L Macías-García Beatriz B

Animals : an open access journal from MDPI 20200519 5

Production of equine embryos in vitro is currently a commercial technique and a reliable way of obtaining offspring. In order to produce those embryos, immature oocytes are retrieved from postmortem ovaries or live mares by ovum pick-up (OPU), matured in vitro (IVM), fertilized by intracytoplasmic sperm injection (ICSI), and cultured until day 8-10 of development. However, at best, roughly 10% of the oocytes matured in vitro and followed by ICSI end up in successful pregnancy and foaling, and th ...[more]

PMID: 32438699

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Project description:BACKGROUND: Follicular fluid contains substances involved in follicle activity, cell differentiation and oocyte maturation. Studies of its components may contribute to better understanding of the mechanisms underlying follicular development and oocyte quality. The canine species is characterized by several ovarian activity features that are not extensively described such as preovulatory luteinization, oocyte ovulated at the GV stage (prophase 1) and poly-oocytic follicles. In this study, we examined the hypothesis that the preovulatory LH surge is associated with changes in steroid and protein content of canine follicular fluid prior to ovulation. METHODS: Follicular fluid samples were collected from canine ovaries during the preovulatory phase, before (pre-LH; n = 16 bitches) and after (post-LH; n = 16) the LH surge. Blood was simultaneously collected. Steroids were assayed by radioimmunoassay and proteomic analyses were carried out by 2D-PAGE and mass spectrometry. RESULTS: The concentrations of 17beta-estradiol and progesterone at the pre-LH stage were 737.2 +/- 43.5 ng/ml and 2630.1 +/- 287.2 ng/ml in follicular fluid vs. 53 +/- 4.1 pg/ml and 3.9 +/- 0.3 ng/ml in plasma, respectively. At that stage, significant positive correlations between follicular size and intra-follicular steroid concentrations were recorded. After the LH peak, the intrafollicular concentration of 17beta-estradiol decreased significantly (48.3 +/- 4.4 ng/ml; p < 0.001), whereas that of progesterone increased (11690.2 +/- 693.6 ng/ml; p < 0.001). Plasmatic concentration of 17beta-estradiol was not modified (49 +/- 9.6 pg/ml) after the LH peak, but that of progesterone significantly increased (9.8 +/- 0.63 ng/ml).Proteomic analysis of canine follicular fluid identified 38 protein spots, corresponding to 21 proteins, some of which are known to play roles in the ovarian physiology. The comparison of 2D-PAGE patterns of follicular fluids from the pre- and post-LH stages demonstrated 3 differentially stained single spot or groups of spots. One of them was identified as complement factor B. A comparison of follicular fluid and plasma protein patterns demonstrated a group of 4 spots that were more concentrated in plasma than in follicular fluid, and a single spot specific to follicular fluid. These proteins were identified as gelsolin and clusterin, respectively. CONCLUSION: Our results provide the first demonstration of size-related changes in the steroid concentrations in canine follicular fluid associated with the LH surge. 2D protein mapping allowed identification of several proteins that may play a role in follicle physiology and ovarian activity at the preovulatory stage. This may help in the future to explain and to better understand the species specificities that are described in dogs.

Project description:Ovarian follicular fluid (FF) comprises a dynamic milieu whose composition alters under seasonal variation, influencing follicle development and oocyte developmental competence. Various cell subtypes (e.g. granulosa, theca, and cumulus) produce cell-secreted nanoparticles, coined extracellular vesicles (EVs), which contain bioactive regulatory molecules such as microRNAs (miRNAs) known to mediate intrafollicular communication through various forms of signaling and information transfer. Markedly, the hallmark feature of EVs transferring cellular knowledge propagates their appealing nature as direct indicators of cellular status, albeit homeostasis, dysfunction, or disease. Photoperiod alterations influencing the regulatory content of FF-EVs as downstream modifiers remain largely unknown, particularly in seasonal breeding animals. Here, we aimed to unpack the impacts of seasonal variation on the miRNA expression profiles of FF-EVs in the mare. Equine pre-ovulatory follicles were monitored and aspirated using ultrasound-guided transvaginal techniques to obtain follicular fluids during non-breeding (spring anovulatory, SAN) and breeding seasons (spring ovulatory, SOV; summer, SUM; and fall ovulatory, FOV). 97 miRNAs were found to be differentially expressed between groups. Pairwise comparisons revealed specific clusters, including six miRNAs involved in the spring transition (miR-149-200b-206-221-328-615) and many others dysregulated during that of the summer (miR-143-192451- 302b-100, and let 7c), the peak time point for breeding. Bioinformatic analyses led to unveiling significant enrichments in various biological functions, such as transcription factor activity, transcription and transcription regulation, nucleic acid binding, sequence-specific DNA binding, p53 signaling, and post-translational modifications. Ultimately, the miRNA content of seasonally divergent FF-EVs poses as potentially relevant indicators of intrafollicular mechanisms directing seasonal transitions and their functional regulatory roles in the pre-ovulatory stage, governing subsequent ovulation.

Project description:Cattle induced to ovulate a small, physiologically immature preovulatory follicle had reduced oocyte developmental competence that resulted in decreased embryo cleavage and day 7 embryo quality compared with animals induced to ovulate a more advanced follicle. RNA-sequencing was performed on oocytes and their corresponding cumulus cells approximately 23 h after gonadotropin-releasing hormone (GnRH) administration to induce the preovulatory gonadotropin surge suggested reduced capacity for glucose metabolism and oxidative phosphorylation in the cumulus cells and oocytes from follicles ≤11.7 mm, respectively. We hypothesized that induced ovulation of a small, physiologically immature preovulatory follicle results in a suboptimal follicular microenvironment and reduced oocyte metabolic capacity. We performed a study with the objective to determine the impact of preovulatory follicle diameter and serum estradiol concentration at GnRH administration on oocyte metabolic competence and follicular fluid metabolome profiles. We synchronized the development of a preovulatory follicle and collected the follicle contents via transvaginal aspiration approximately 19 h after GnRH administration in lactating beef cows (n = 319). We determined ATP levels and mitochondrial DNA (mtDNA) copy number in 110 oocytes and performed ultra-high-performance liquid chromatography-high resolution mass spectrometry metabolomic studies on 45 follicular fluid samples. Intraoocyte ATP and the amount of ATP produced per mtDNA copy number were associated with serum estradiol concentration at GnRH and time from GnRH administration to follicle aspiration (P < 0.05). mtDNA copy number was not related to follicle diameter at GnRH, serum estradiol concentration at GnRH, or any potential covariates (P > 0.10). We detected 90 metabolites in the aspirated follicular fluid. We identified 22 metabolites associated with serum estradiol concentration at GnRH and 63 metabolites associated with follicular fluid progesterone concentration at the time of follicle aspiration (FDR < 0.10). Pathway enrichment analysis of significant metabolites suggested altered proteinogenesis, citric acid cycle, and pyrimidine metabolism in follicles of reduced estrogenic capacity pre-gonadotropin surge or reduced progesterone production by the time of follicle aspiration.

Project description:Background and aimOur objective was to use metabolomics in a toxicological-relevant target tissue to gain insight into the biological processes that may underlie the negative association between air pollution exposure and oocyte quality.MethodsOur study included 125 women undergoing in vitro fertilization at an academic fertility center in Massachusetts, US (2005-2015). A follicular fluid sample was collected during oocyte retrieval and untargeted metabolic profiling was conducted using liquid chromatography with ultra-high-resolution mass spectrometry and two chromatography columns (C18 and HILIC). Daily exposure to nitrogen dioxide (NO2), ozone, fine particulate matter, and black carbon was estimated at the women's residence using spatiotemporal models and averaged over the period of ovarian stimulation (2-weeks). Multivariable linear regression models were used to evaluate the associations between the air pollutants, number of mature oocytes, and metabolic feature intensities. A meet-in-the-middle approach was used to identify overlapping features and metabolic pathways.ResultsOf the air pollutants, NO2 exposure had the largest number of overlapping metabolites (C18: 105; HILIC: 91) and biological pathways (C18: 3; HILIC: 6) with number of mature oocytes. Key pathways of overlap included vitamin D3 metabolism (both columns), bile acid biosynthesis (both columns), C21-steroid hormone metabolism (HILIC), androgen and estrogen metabolism (HILIC), vitamin A metabolism (HILIC), carnitine shuttle (HILIC), and prostaglandin formation (C18). Three overlapping metabolites were confirmed with level-1 or level-2 evidence. For example, hypoxanthine, a metabolite that protects against oxidant-induced cell injury, was positively associated with NO2 exposure and negatively associated with number of mature oocytes. Minimal overlap was observed between the other pollutants and the number of mature oocytes.ConclusionsHigher exposure to NO2 during ovarian stimulation was associated with many metabolites and biologic pathways involved in endogenous vitamin metabolism, hormone synthesis, and oxidative stress that may mediate the observed associations with lower oocyte quality.

Project description:Oocyte competence for early embryo development relies on intercellular communication between the maturing oocyte and preovulatory follicle. Preovulatory follicle maturity, as indicated by serum estradiol concentration or follicle diameter, has previously been linked to pregnancy, follicular fluid metabolites, cumulus-oocyte metabolism, and oocyte competency for embryo development. Such relationships indicate metabolic and developmental programming of the oocyte based on the preovulatory follicle’s physiological status, but downstream impacts on the molecular signature of blastocysts have not been examined. We hypothesized that supplementing maturing oocytes with follicular fluid originating from preovulatory follicles of greater or lesser maturity would impact the transcriptome of resulting blastocysts and indicate metabolic programming of the embryo that originated from the oocyte’s maturation environment. The objective was to investigate the effect of follicle maturity on metabolic preparation of the oocyte by examining the functional genome of blastocysts originating from oocytes matured in the presence of follicular fluid from preovulatory follicles of greater or lesser maturity. In vitro maturing oocytes were supplemented with follicular fluid collected from preovulatory follicles of greater or lesser maturity. Following identical embryo culture procedures, RNA-sequencing was performed on pools of 2 blastocysts (Greater, n = 12; Lesser, n = 15; all with stage code = 7 and quality code = 1). A total of 12,310 gene transcripts were identified in blastocysts after filtering to remove lowly abundant transcripts. There were 113 transcripts that differed in abundance between blastocysts originating from oocytes matured in greater versus lesser maturity follicular fluid (eFDR < 0.01). Although no pathways were significantly enriched with differentially abundant transcripts, transcriptome profiles suggested improved Wnt/β-catenin signaling, metabolism, and protection from oxidative stress in blastocysts derived from oocytes matured in greater maturity follicular fluid, while unregulated cell growth presented in blastocysts resulting from the lesser follicle maturity treatment.

Dataset Information

Study of the Metabolomics of Equine Preovulatory Follicular Fluid: A Way to Improve Current In Vitro Maturation Media.

Publications

Study of the Metabolomics of Equine Preovulatory Follicular Fluid: A Way to Improve Current In Vitro Maturation Media.

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