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Estrous cycle impacts microRNA content in extracellular vesicles that modulate bovine cumulus cell transcripts during in vitro maturation

ABSTRACT: Purpose: Extracellular vesicles (EVs) are nanoparticles that can be secreted by different cells, including cells found within the ovarian follicle. Currently, EVs are considered an important form of intercellular communication, since they carry biological contents. The goal of this study was to survey the effects of small EVs from follicles at different estrous cycle stage in bovine cumulus cells. Methods: We used an established model to obtain follicular fluid (FF) at early and late estrous cycle stage according to corpus luteum appearance, corresponding to low and high progesterone (P4) levels, respectively. We collected FF from 3-6 mm follicles and isolated small EVs, which were used as a supplement during in vitro maturation (IVM). Cumulus cells were collected from cumulus-oocyte complexes (COCs) pools and the RNAs were obtained and subjected to RNA sequencing. Results: The results showed that small EVs from different estrous cycle stage are capable to affect transcripts in cumulus cells and modulate different pathways and biological processes related to oocyte maturation, ovulation and immune response. Conclusion: This study demonstrated that small EVs from low and high P4 group impact the RNA profile in cumulus cells after 9 hours of in vitro maturation.

ORGANISM(S): Bos taurus

PROVIDER: GSE133587 | GEO | 2019/12/02

REPOSITORIES: GEO

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Project description:Purpose: Preovualtory follicle diameter influences embryo quality in beef cattle that rely on a gonadotropin releasing hormone (GnRH) injection to induce the preovualtory gonadotropin surge and subsequent ovualtion. The goals of this study are to compare transcriptome profiles of pools of four oocytes or associated cumulus cells collected from small (≤11.7mm) and large follicles (≥12.7 mm) exposed to a GnRH-induced gonadotropin surge and follicles (11.7-14.0 mm) exposed to an endogenous gonadotropin surge (spontaneous follicles). Methods: Oocyte and cumulus cell mRNA profiles of pools of four oocytes or associated cumulus cells collected from small (≤11.7mm) and large follicles (≥12.7 mm) exposed to a GnRH-induced gonadotropin surge or spontaneous follicles (11.7-14.0 mm) which were exposed to an endogenous gonadotropin surge were generated by deep sequencing (n=6 small follicle oocyte, 6 small follicle cumulus, 6 large follicle oocyte, 6 large follicle cumulus, 4 spontaneous follcile oocyte, and 5 spontaneous follicle cumulus pools), using Illumina HiSeq 2000 . The sequence reads that passed quality filters were aligned to the Bos taurus reference genome UMD3.1 using Hisat2mapper. FeatureCounts was used to quantify transcript abundance in each library using Bos taurus gene annotation from Ensembl. Differences in gene transcript expression were then analyzed amoung oocyte or cumulus cell follcile classifications using the packages edgeR and DESeq2 in R software. Results: We mapped an average of 30 million sequence reads per sample to the Bos taurus reference genome UMD3.1 and identified 69, 94, and 83 differentially expressed gene transcripts (DEG) among oocyte libraries from small versus large, small versus spontaneous, and large versus spontaneous follicle classifications, respectively. We also identified 128, 98, and 80 DEG among cumulus cell libraries from small versus large, small versus spontaneous, and large versus spontaneous follicles, respectively. Conclusions: Our study represents detailed analysis of RNA-sequencing data generated from in vivo produced cumulus and oocyte samples collected from preovualtory follicles of varied physiological status after exposure to an endogenous or exogenously stimulated gonadotropin surge. The results of our study highlight key differences in the transcriptome of samples from small, large, or spontaneous follicles and provide insight into the reduced embryo quality observed when small follicles undergo a GnRH induced gonadotropin surge.

Project description:The mammalian follicular development is a well-orchestrated and complex molecular process characterized by sequences of follicular waves that yield the ovulatory follicle. It has been reported that bi-directional communication between the growing oocyte and the surrounding somatic cells is a hallmark of mammalian follicular development, partially mediated by extracellular vesicles (EVs) present in the follicular fluid (FF). Bioactive molecules such as the miRNAs encapsulated in the EVs mediate this communication within the follicular environment. Here, we aimed to decipher the dynamics of the miRNAs in the EVs of equine FF aspirated in vivo during different stages of follicular development. Hence, all follicles ≥6 mm were ablated on Days 10–11 of the estrous cycle (ovulation = Day 0) and the new induced wave was ultrasound-monitored daily until the largest growing follicle for at least three consecutive days was randomly allocated to one of the five groups (n = 8 follicles/group) namely: predeviation (preDEV; 18–20 mm), deviation (DEV; 22–25 mm), postdeviation (postDEV; 26–29 mm), preovulatory (preOV; 30–35 mm), and impending ovulation (IMP; ~40 mm). The FF was collected from each target group by ultrasound-guided transvaginal follicle aspiration. After removal of cells, cellular debris, and larger particles, EVs were isolated from FF groups (4 biological replicates/group; 0.5 mL FF/replicate) using high-speed ultracentrifugation (120,000xg for 70 min). Isolated EVs were characterized using western blot for EV marker proteins, NanoSight Tracking Analysis, and electron microscopy. MiRNA enriched total RNA was isolated from EVs using an Exosomal RNA Isolation kit and small-RNA library preparation and RNAseq (NextSeq500; Illumina) was performed by Novogene Co, Ltd. Approximately 180 known miRNAs were detected in each group with 148 mutually found in all groups. Among the expressed miRNAs, miR-148a, miR-21, miR-192, and miR-99a were the top highly abundant miRNAs in all groups. Cluster analysis exhibited 15 different expression patterns during follicular development. Among these patterns, a group of 23 miRNAs (including miR-106b, miR-199a-5p, and miR-125a-5p) exhibited a stable expression at the preDev until the postDev stage, a significant increase at the preOV stage, and then a significant decrease at the IMP stage. This miRNA cluster has been found to target genes involved in TGF-signaling and endocytosis pathways. Another interesting pattern with 22 miRNAs (including miR-146b-5p, miR-140, and miR-143) exhibited a sharp reduction in their expression from the preDEV until the preOV stage and was predicted to target genes associated with fatty acid biosynthesis, cell cycle, and oocyte meiosis pathways. In contrast, a group of 16 miRNAs (including miR-483, miR-128, and miR-192) gradually increased in expression from preDEV to the postDEV and then downregulated at the preOV stage. This miRNA cluster targets several signaling pathways, including ErbB, thyroid, and estrogen signaling. In conclusion, this study provides greater insights into the stage-specific expression dynamics of the FF EV-miRNAs during equine follicular development, which may play a role in folliculogenesis in mares.

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Estrous cycle impacts microRNA content in extracellular vesicles that modulate bovine cumulus cell transcripts during in vitro maturation

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