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Small-extracellular vesicles and their microRNA cargo from porcine follicular fluids: the potential association with oocyte quality

Small-extracellular vesicles and their microRNA cargo from porcine follicular fluids: the... Background: Ovarian follicular fluids (FFs) contain several kinds of regulatory factors that maintain a suitable micro - environment for oocyte development. Extracellular vesicles (EVs) are among the factors that play essential roles in regulating follicle and oocyte development through their cargo molecules that include microRNAs (miRNAs). This study aimed to investigate small-EV (s-EV ) miRNAs in porcine FFs and their potential association with oocyte quality. Methods: Individual aspirated oocytes were stained with lissamine green B stain (LB), a vital stain for oocyte qual- ity, and each oocyte was classified as high-quality (unstained; HQ) or low-quality (stained; LQ). FFs corresponding to oocytes were pooled together into HQ and LQ groups. Small-EVs were isolated from FFs, characterized, and their miRNA cargo was identified using the Illumina NovaSeq sequencing platform. Additionally, s-EVs from the HQ and LQ groups were utilized to investigate their effect on oocyte development after co-incubation during in vitro maturation. Results: A total of 19 miRNAs (including miR-125b, miR-193a-5p, and miR-320) were significantly upregulated, while 23 (including miR-9, miR-206, and miR-6516) were downregulated in the HQ compared to the LQ group. Apoptosis, p53 signaling, and cAMP signaling were among the top pathways targeted by the elevated miRNAs in the HQ group while oocyte meiosis, gap junction, and TGF-beta signaling were among the top pathways targeted by the elevated miRNAs in the LQ group. The supplementation of small-EVs during maturation does not affect the oocyte develop - mental rates. However, LQ s-EVs increase the proportion of oocytes with homogeneous mitochondrial distribution and decrease the proportion of heterogeneous distribution. Conclusion: Our findings indicated that FF-EVs contain different miRNA cargos associated with oocyte quality and could affect the mitochondrial distribution patterns during oocyte maturation. Keywords: Extracellular vesicles, Follicular fluids, MiRNA, Oocyte quality, Porcine oocyte quality, could influence this ability and subse - Background quently determine developmental competence. Various Oocyte developmental competence is commonly defined methods including morphological, biochemical, and as the ability of the oocyte to mature, fertilize, and molecular techniques, are being used to assess oocyte develop to the blastocyst stage. Several factors, including quality [1], with the aim of enhancing the efficiency of assisted reproductive technologies (ARTs). At the molec- *Correspondence: Murin@iapg.cas.cz ular level, several studies have been done to determine molecular markers from follicular cells surrounding the Institute of Animal Physiology and Genetics, Czech Academy of Sciences, 27721 Liběchov, Czech Republic oocyte or follicular fluids (FFs) as a non-invasive method Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Gad et al. Journal of Animal Science and Biotechnology (2022) 13:82 Page 2 of 16 that predicts oocyte quality [2]. Ovarian FFs contain sev- Merck (Kenilworth, NJ, USA), respectively, unless stated eral kinds of regulatory factors that maintain a suitable otherwise. All media were prepared fresh and sterilized microenvironment for oocyte development and follicular using 0.22 μm syringe filters. intercellular communication [3]. This kind of communi - cation within the follicle is essential and could determine Collection of follicular fluids, COCs staining, oocyte quality and consequently developmental compe- and classification tence [4]. One of the recently discovered mechanisms Porcine ovaries of prepubertal gilts (Landrace × Large that facilitate and modulate intercellular crosstalk is via White, 6–8 months of age, 90–120 kg) were collected the extracellular vesicles (EVs). EVs, including exosomes from a local slaughterhouse and transported to the and microvesicles, are cell-derived lipid bilayer mem- lab in a thermos flask within 2 h. Ovaries were washed branous particles that contain different biomolecules, three times with saline solution. Follicular fluids and including proteins, lipids, RNAs, and microRNAs (miR- COCs were aspirated from individual healthy follicles NAs). These particles are secreted by almost all cell types (3–6 mm in diameter, measured as previously recom- and can be found in all body fluids [5 ]. The ability of EVs mended [15]) using a 25-gauge needle attached to a 1-mL to modulate intercellular crosstalk is through the capac- syringe (Braun, Germany). Each COC and its corre- ity to transfer their biomolecular cargos between differ - sponding FF were allocated in separate wells in a 96-well ent cells after being secreted in body fluids [5 ]. After this plate. COCs were washed once in PXM-HEPES (HEPES discovery, several studies revealed the fundamental roles buffered porcine X medium [16]) and then stained for of EVs in the different reproduction processes in domes - 15 min at room temperature with 0.5% lissamine green B tic animals (reviewed by Llobat [6]) and the subsequent stain (LB), a vital synthetic stain for determining oocyte improvement of ART outcomes [7]. quality and competence [17, 18]. Each COC was classi- MiRNAs, a small non-coding RNA class, are post- fied separately according to the oocyte stain into high- transcriptional regulators of gene expression by bind- quality (unstained; HQ) and low-quality (stained; LQ) as ing specific mRNA target sequences for degradation or presented in Fig.  1. FFs corresponding to oocytes were translational repression [8]. Extracellular miRNAs were pooled together into HQ and LQ groups and used for the discovered in various body fluids in a stable form, which isolation of small-extracellular vesicles (s-EVs). supports their potential utilization as non-invasive bio- markers for several physiopathological conditions [9]. Isolation of small‑extracellular vesicles from follicular fluid The availability and stability of extracellular miRNAs To isolate s-EVs (< 200 nm), pooled FFs (~ 1 mL/repli- within body fluids are supported by either the incor - cate) from each group were centrifuged at 700  ×  g for poration of miRNAs within the EVs or by binding with 10 min to pellet cells, at 2000  ×  g for 10 min to remove specific protein complexes [10]. In 2007, miRNAs were cell debris, and at 12,000 ×  g for 30 min to remove large identified for the first time within cell-secreted EVs that particles and protein aggregates. All centrifugation steps can be delivered and regulate several functions in the were performed at 4 °C. The remaining supernatants target cells [11]. In ovarian FFs, it has been reported were filtrated through a 0.2-μm syringe filter to eliminate that the majority of miRNAs are found within EVs [12] larger vesicles. Small-EVs were isolated from 0.5 mL fil - and are suggested to play a role in follicle development tered FFs (3 replicates/group) using an Exo-spin kit (Cell and other ovarian functions [13]. Consequently, altera- Guidance Systems, UK) and eluted in PBS according to tions in the FF miRNAs could reflect the status of the the manufacturer’s protocol. For electron microscope oocyte quality and its developmental competence [14]. imaging and western blot analysis, samples were kept at Therefore, the objectives of this study were to identify 4 °C until analysed. The remaining samples were stored small-EV (s-EV) miRNA differences in porcine FFs in at − 80 °C for further analysis. All relevant data regarding association with oocyte quality and to investigate the s-EV isolation and characterization were submitted to effect of s-EVs on oocyte developmental competence. the EV-TRACK knowledgebase [19] with the EV-TRACK The identified miRNAs could be used as non-invasive ID EV210251. biomarkers for oocyte selection. Moreover, our results provided more insights into the potential role of FF-EVs during oocyte maturation. Nanoparticle tracking analysis (NTA) To determine vesicle size and concentration, samples Materials and methods were diluted in PBS (1:200) and NTA was conducted Chemicals and supplements in a ZetaView instrument (Particle Metrix, Germany) All plastic materials and chemicals were purchased from in scatter mode (488 nm laser). Measurements were Thermo Fisher Scientific (Waltham, MA, USA) and performed in two cycles by scanning 11 cell positions G ad et al. Journal of Animal Science and Biotechnology (2022) 13:82 Page 3 of 16 Fig. 1 Classification of porcine COCs after lissamine green B (LB) staining. Left: Unstained COC (high-quality; HQ). Right: Stained COC (low-quality; LQ). Scale bar, 60 μm each, and the videos were analysed using the software in 2% BSA) and anti-ATP5A (Abcam, Ab14748, diluted ZetaView (version 8.05.12). 1:2000 in 5% milk) raised against somatic cell-specific markers. The secondary antibodies (Amersham ECL anti-mouse or anti-rabbit IgG, GE Healthcare, Little Transmission electron microscopy (TEM) Chalfont, UK) were diluted 1:5000 in 2% BSA in TBS- For morphological evaluation, isolated s-EV samples Tween. The membranes were incubated with the sec - were placed into formvar/carbon-coated 400 mesh cop- ondary antibody for 1 h at room temperature and then per grids for 20 min at room temperature. Then, the grids washed intensively in TBS-Tween. The immune reac - were incubated with 2% formaldehyde in PBS for 20 min, tions were detected by enhanced chemiluminescence followed by 6 washes for 2 min each with ultrapure water. (Pierce, Rockford, IL, USA) according to the manufac- The grids were stained with uranyl acetate (2%) for turer’s instructions and captured on CL-XPosure film 12 min, washed in water, left to dry, and then examined (ThermoFisher Scientific). in a Jeol JEM-1400 FLASH transmission electron micro- scope (Tokyo, Japan) equipped with Matataki 2k×2k CMOS camera at 80 kV. Total RNA extraction, library preparation, and sequencing Total RNA, including miRNA was isolated from EV sam- Western blot analysis ples using a miRNeasy Micro Kit (Qiagen, Hilden, Ger- Samples of s-EV, filtered follicular fluid, or follicu - many) that combines the phenol/guanidine-based lysis lar cells containing 40 μg of total protein were mixed of samples and silica membrane-based purification of with Laemmli buffer containing 2% sodium dodecyl total RNA, according to the manufacturer’s instructions. sulphate (SDS) and 5% 2-mercaptoethanol. Samples The RNA concentration and size distribution were ana - were boiled at 100 °C for 3 min and stored frozen at lyzed using an Agilent RNA 6000 Pico kit in an Agilent − 20 °C. Subsequently, proteins were separated in 10% 2100 Bioanalyzer (Agilent Technologies, Santa Clara, or 12% acrylamide/SDS gels and transferred to Immo- CA, USA). Small-RNA libraries were prepared for next- bilon-P membranes (Millipore, Bedford, MA, USA). generation sequencing (NGS) using a QIAseq miRNA Membranes were blocked in 5% low-fat dry milk in Library Kit (Qiagen) according to the manufacturer’s Tris-buffered saline (TBS) with 0.5% Tween 20 for 2 h instructions. Library quantity and quality assessments at room temperature and then incubated with a pri- were performed using a Qubit DNA HS Assay Kit in a mary antibody at 4 °C overnight. The primary anti - Qubit 4 Fluorometer (Thermo Fisher Scientific) and Agi - bodies were anti-CD63 (Abcam, Ab 118307, diluted lent DNA High Sensitivity kit in an Agilent 2100 Bioana- 1:1000 in 2% BSA in TBS-Tween), anti-Alix (Abcam, lyzer (Agilent Technologies), respectively. The libraries Ab 88388, diluted 1:500 in 5% milk), and anti-TSG101 were pooled in equimolar ratios and then sequenced in a (Santa Cruz Biotechnology, Sc-7964, diluted in 1:200 in NovaSeq6000 sequencing instrument (Illumina, Inc., San 5% milk) raised against exosome-specific markers, and Diego, CA, USA) as single-end reads. anti-Cytochrome C (Abcam, Ab 90529, diluted 1:1000 Gad et al. Journal of Animal Science and Biotechnology (2022) 13:82 Page 4 of 16 Sequencing data analysis samples, according to the analysis with the software FASTQ files were generated for each sample using the NormFinder. software bcl2fastq (Illumina Inc., San Diego, CA), and their quality was checked using the FastQC tool ver- Co‑incubation of s‑EVs with COCs during maturation sion 0.11.9. Data were analyzed using the software CLC COCs were collected as mentioned above using a Genomics Workbench, version 21 (www. qiage nbioi 20-gauge needle attached to a 10-mL syringe and then nform atics. com). Raw sequencing reads were trimmed morphologically evaluated under a stereomicroscope based on quality score (Q-score > 30), ambiguous nucle- (Zeiss Stemi 508, magnification × 50). Only those with at otides (maximum two nucleotides allowed), read length least three layers of cumulus cells and an evenly granu- (≥15 nucleotides) and removing adapter sequences. lated ooplasm were used. COCs were washed twice in Reads were mapped to the porcine (Sus scrofa) refer- the maturation medium (Medium 199) supplemented ence genome (Sscrofa11.1) and annotated against por- with 0.005% gentamicin (Roth 0233), 0.0022% sodium cine precursor and mature miRNAs listed in the mirBase pyruvate, 0.01% L-glutamine, 0.1% BSA, 10 ng/mL database (release 22) using the CLC Genomics Work- EGF, 40 ng/mL FGF2, 20 ng/mL IGF1, 2000 IU/mL LIF, bench RNA-Seq Analysis and Quantify miRNA tools, 0.57 mmol/L L-Cysteine, 10 IU/mL PMSG and 10 IU/mL respectively, applying the default software parameters. HCG. COCs were cultivated in 4-well dishes (30–50 per Raw expression data were normalized using the trimmed well) for 44 h at 38.5 °C under a 5% C O atmosphere in mean of M-values normalization method (TMM nor- 500 μL maturation medium supplemented with or with- malization) [20] and presented as TMM-adjusted Counts out (control) s-EV particles (~ 200 million particles/mL) Per Million (CPM). The CLC Genomics Workbench Dif - isolated from HQ or LQ FF groups. COCs cultivated in ferential Expression tool was used for the expression the same maturation media supplemented with a volume analysis comparison of the two groups. MiRNAs with of PBS similar to the s-EVs were used as the negative fold change (FC) ≥ 2, P-adjusted value (FDR [21]) < 0.05, control group (NC). and CPM > 5 in the enriched group were considered dif- ferentially expressed (DE). The raw FASTQ files and pro - Parthenogenetic activation and embryo cultivation cessed CSV files have been deposited in the NCBI’s Gene After maturation, cumulus cells were removed from Expression Omnibus (GEO) with the accession number COCs by pipetting and washed twice in PXM-HEPES. GSE181182. Oocytes were activated using 10 μmol/L ionomycin in PXM-HEPES for 5 min, washed twice in porcine zygote Target gene prediction and ontological classification medium 3 (PZM 3) [27] supplemented with 2 mmol/L Genes targeted by DE-miRNA were identified using the 6-dimethylaminopurine, and cultivated for 5 h at 38.5 °C miRWalk database [22]. Within the miRWalk, validated under a 5% CO atmosphere. A group of 30–50 putative target genes from miRTarBase (version 7.0) and com- parthenotes was washed twice in PZM 3 and cultivated monly target genes predicted by TargetScan (version for 7 d in 4-well dishes in 500 μL of PZM 3 medium at 7.1) and miRDB (release 5.0) were selected for ontologi- 38.5 °C under a 5% CO atmosphere. The cleavage rate cal classification and pathway analysis using the DAVID was assessed after 40 h, and the ability of the embryos to bioinformatics web tool (https:// david. abcc. ncifc rf. gov/). reach the blastocyst stage as well as the number of nuclei Pathways were determined from the KEGG database in blastocysts were analyzed after 168 h of cultivation. [23], and interaction networks of the targeted genes and Blastocysts were fixed using 4% paraformaldehyde and the identified pathways were constructed with Cytoscape mounted on glass slides using DAPI mounting medium. [24] and its plug-in ClueGO [25]. Each blastocyst was scanned using a confocal microscope (Leica SP5, Germany), and the number of nuclei was DE‑miRNA validation using droplet digital PCR (ddPCR) counted using the software ImageJ. To validate the miRNA-seq data, we performed a PCR analysis for a selected group of 11 DE-miRNAs. The Oocyte mitochondrial activity and distribution patterns absolute copy numbers of the selected DE-miRNAs were Matured COCs were denuded as described above. quantified in the EVs samples using specific TaqMan Oocytes were washed three times in PBS, stained with miRNA Assays (Applied Biosystems, Foster City, CA, 300 nmol/L Mitotracker Orange kit (Thermo Fisher USA) in a ddPCR system (Bio-Rad Inc., Hemel Hemp- Scientific, USA) for 30 min in the dark at 38.5 °C. Then, stead, UK) according to the manufacturer’s instructions oocytes were washed three times in PBS at 38.5 °C, and as previously described [26]. The copy numbers fixed for 15 min in 4% paraformaldehyde, and mounted of the selected DE-miRNAs were normalized to miR- on glass slides using DAPI mounting medium. Oocytes 26b-5p, the most stably expressed miRNA across all were scanned in a confocal microscope (Leica SP5, G ad et al. Journal of Animal Science and Biotechnology (2022) 13:82 Page 5 of 16 Germany) and mitochondrial activity was analyzed using Student’s t-test. The statistical significance level using the software ImageJ. The distribution patterns of was defined at P < 0.05. mitochondria were characterized by observing (at 630- fold magnification) labeled mitochondria with an active Results oxidative state. We classified the distribution and aggre - Characterization of s‑EVs from porcine follicular fluids gation patterns mainly as homogeneous, heterogeneous, Different morphological and molecular analyses were and clustered distribution according to the previously done to determine the characteristics and the purity reported classification [28, 29]. of s-EV isolated samples. Vesicle size and concentra- tion were determined in each sample using NTA. The Statistical analysis concentration of s-EVs from the HQ and LQ FF groups All experiments were done in at least three replicates. was 8.98E+ 09 ± 3.43E+ 08 and 8.97E+ 09 ± 1.25E+ 09 Data normality was checked using the Shapiro–Wilk particles/mL, the median size was 135.7 ± 5.3 and test. Maturation and developmental rates and blas- 132.6 ± 3.3 nm, respectively, and the mode was 135 nm tocyst nuclei numbers were analyzed using one-way in both groups, with no significant differences between ANOVA (SigmaPlot 12, US) followed by Tukey’s test to them (Fig. 2A). Imaging with TEM revealed the presence detect differences between the means and expressed as of s-EVs with visible lipid bilayer membranes (Fig.  2B). mean ± SD. Categorical variables were identified using Western blot analysis identified the presence of EV-spe - the chi-square test. PCR data were statistically analyzed cific markers (ALIX, TSG101, and CD63) in both s-EV Fig. 2 Morphological and molecular characterization of s-EVs. A The concentration and size distribution of s-EVs isolated from follicular fluids (FFs) corresponding to high- (HQ) or low-quality (LQ) oocytes analysed by NTA. B Transmission electron microscopy ( TEM) representative photos of isolated s-EVs showing the lipid bilayer membrane and the cup-shaped EV particles (black arrows). Scale bar of 500 and 200 nm for the upper left and right photos, respectively, and of 200 and 100 nm for the lower left and right photos, respectively). C Immunoblotting analysis of EV-specific protein markers (ALIX, TSG101, and CD63) and cellular specific protein markers (CytC and ATP5A) in the HQ and LQ EV groups, as well as in filtered FF and follicular cell lysate (FC) as positive and negative controls, respectively. Scale numbers are in kDa. D RNA size distribution from s-EV samples of HQ (sample 1–3) and LQ (sample 4–6) groups analysed with a bioanalyzer (Agilent Technologies) Gad et al. Journal of Animal Science and Biotechnology (2022) 13:82 Page 6 of 16 groups and the filtered FFs. Additionally, cellular specific Validated and predicted target gene analysis revealed protein markers (CytC and ATP5A) were only detected a total of 860 and 1308 genes are targeted by up- and in the cell lysates, but not in the isolated s-EVs, indicating downregulated miRNAs, respectively, with 193 genes the absence of other cellular membrane contamination in targeted by both. In addition, 702 genes were among the the EV samples (Fig.  2C). Lastly, the size distribution of validated and predicted genes and targeted by the top the total RNA isolated from s-EV samples exhibited clear five most abundant miRNAs in both groups. Apoptosis, peaks for small-RNA and the absence of ribosomal-RNA p53 signaling, and cAMP signaling were among the top peaks, confirming the samples to be free of cells and cel - pathways targeted by the elevated miRNAs in the HQ lular components (Fig. 2D). group (Fig. 5A, Additional file  1: Table S3). On the other hand, oocyte meiosis, gap junction, TGF-beta signal- MiRNA expression profiles of s‑EVs isolated from follicular ing, and estrogen signaling were among the top path- fluids ways targeted by the elevated miRNAs in the LQ group Small-RNA libraries were prepared from six different EV (Fig. 5B, Additional file  1: Table S3). Signaling pathways samples (3 replicates per group) to identify the expressed including PI3K-Akt, MAPK, AMPK, and FoxO were miRNAs in each. RNA-seq analysis gave an average num- the top commonly targeted pathways by the elevated ber of 27 million raw reads per library with an average of miRNAs in the HQ and LQ s-EV groups, as well as by 16 million reads being retained after trimming and qual- the top five most abundant miRNAs in both groups ity control. An average of 89% of reads were mapped to (Fig.  5C and D, Additional file  1: Table  S3). However, the porcine genome, and the average proportion of anno- genes involved in these common pathways were differ - tated miRNAs was 15% (Additional file  1: Table S1). Vari- entially targeted by each miRNA group, with few genes ous types of small non-coding RNA (sncRNA), including commonly targeted by two or three miRNA groups, as misc_RNA, snRNA, and snoRNA were also identified shown for instance for the PI3K-Akt and MAPK signal- in all samples. However, the majority of sncRNA reads ing pathways (Additional file 3 : Fig. S2). were assigned to miRNAs (Additional  file  2: Fig. S1). Heatmap clustering and principal component analysis (PCA) were performed on the miRNA CPM values. The Validation of DE‑miRNA results showed a clear clustering of the three biological A group of 11 DE-miRNAs was selected to validate the replicates of each group, with a clear separation between miRNA-seq data using ddPCR. All selected miRNAs the two groups. The first two components of the PCA exhibited the same expression pattern as in the miRNA- explained around 68.5% of the existing variances (Fig. 3). seq data (P < 0.05) except for two miRNAs (ssc-miR-125b A miRNA with an average value of CPM > 1 was con- and ssc-miR-1306-5p), which did not differ significantly sidered to be expressed. Accordingly, a total of 303 and between the HQ and LQ s-EV groups (Fig. 6). 301 miRNAs were expressed in the HQ and LQ groups, respectively, with 295 miRNAs being expressed in both Oocyte maturation, developmental competence, groups (Fig.  4A). A complete list of all expressed miR- and mitochondrial activity after s‑EVs co‑incubation NAs is presented in Additional file  1: Table  S2, and To investigate the effect of s-EVs and their cargos on por - the top 20 most abundant miRNAs in each group are cine oocyte maturation and embryonic development, presented in Table  1. Interestingly, miR-27b-3p, miR- COCs were supplemented with s-EVs of the HQ or LQ 140-3p, miR-29a-3p, miR-202-5p, and miR-16 were the groups. We evaluated the nuclear maturation, cleav- top 5 expressed miRNAs in both groups, in which they age, blastocyst rates, and blastocyst cell count in com- accounted for 42% and 49.2% of the miRNAs sequence parison to the non-supplemented control group (C) or reads in the HQ and LQ groups, respectively (Table 1). PBS-supplemented group (NC). There were no signifi - cant differences in nuclear maturation or developmen - Differentially expressed miRNAs and ontological tal rates, as well as in the blastocyst cell counts among classification the experimental groups (Table  3). To examine whether Differential expression analysis of miRNAs revealed that s-EVs might modulate changes in mitochondrial distribu- 19 miRNAs (including miR-193a-5p, miR-339-3p, ssc- tion or activity in oocytes after maturation, metaphase miR-132, ssc-miR-125b and ssc-miR-320) and 23 miR- II (MII) oocytes were stained with MitoTracker Orange NAs (including miR-9-1, miR-9, miR-206, miR-133b, and after co-incubation with s-EVs. Based on the mitochon- miR-133a-3p) were significantly up- and downregulated drial distribution patterns, oocytes were categorized into (FC ≥ 2, FDR < 0.05, and CPM > 5 in the enriched group) three main categories, homogeneous, heterogeneous, or in the HQ compared to the LQ s-EVs group, respectively cluster distribution (Fig.  7A). The results showed that a (Fig. 4B, Table 2). significantly higher proportion of oocytes co-incubated G ad et al. Journal of Animal Science and Biotechnology (2022) 13:82 Page 7 of 16 Fig. 3 Samples clustering. A Principal component analysis (PCA). B Heatmap and hierarchical clustering. HQ1-HQ3: high-quality s-EVs replicates, LQ1-LQ3: low-quality s-EVs replicates Gad et al. Journal of Animal Science and Biotechnology (2022) 13:82 Page 8 of 16 Fig. 4 Differential expression analysis. A Venn diagram for commonly and exclusively expressed miRNAs in HQ and LQ s-EVs groups. B Volcano plot of expressed miRNAs. Up- and downregulated miRNAs in the HQ compared to the LQ s-EVs groups are labeled with red and green points, respectively G ad et al. Journal of Animal Science and Biotechnology (2022) 13:82 Page 9 of 16 Table 1 List of top 20 most abundant miRNAs in small-extracellular vesicles obtained from follicular fluids of high (HQ) or low (LQ) quality corresponding oocytes a a miRNA HQ group % miRNA LQ group % CPM CPM ssc-miR-27b-3p 102,691.3 10.9 ssc-miR-27b-3p 155,625.9 14.8 ssc-miR-140-3p 80,017.22 8.6 ssc-miR-16 115,577.2 11.4 ssc-miR-29a-3p 78,734.08 8.3 ssc-miR-29a-3p 99,770.91 9.2 ssc-miR-202-5p 71,447.84 7.5 ssc-miR-140-3p 82,856.62 7.4 ssc-miR-16 62,302.65 6.7 ssc-miR-202-5p 70,526.08 6.4 ssc-let-7c 39,828.22 4.3 ssc-miR-29c 51,664.11 4.7 ssc-miR-423-5p 32,738.73 3.6 ssc-miR-152 44,794.39 4.3 ssc-miR-128 31,129.2 3.4 ssc-let-7c 30,586.53 2.5 ssc-miR-152 28,718.56 3.1 ssc-miR-146a-5p 26,660.02 2.5 ssc-miR-29c 28,297.13 3.0 ssc-let-7i-5p 26,275.77 2.5 ssc-let-7a 26,486.27 2.9 ssc-miR-676-3p 22,826.69 2.2 ssc-miR-676-3p 24,661.24 2.7 ssc-miR-30e-5p 22,798.43 2.2 ssc-miR-146a-5p 23,950.27 2.6 ssc-miR-128 22,443.82 2.2 ssc-miR-24-3p 21,119.86 2.3 ssc-let-7a 16,416.26 1.4 ssc-miR-10b 19,325.57 2.1 ssc-miR-24-3p 16,005.13 1.5 ssc-miR-191 17,564.45 1.9 ssc-miR-30a-5p 15,408.66 1.4 ssc-let-7f-5p 17,071.41 1.8 ssc-miR-423-5p 14,893.75 1.3 ssc-let-7i-5p 16,857.99 1.8 ssc-miR-10b 13,573.69 1.1 ssc-miR-30e-5p 12,606.02 1.3 ssc-let-7f-5p 12,654.58 1.1 ssc-miR-30a-5p 11,671.15 1.2 ssc-miR-19b 11,413.06 1.2 CPM average Counts Per Million mapped reads Percentage of the miRNAs sequence reads with the LQ s-EV group was in the homogeneous cat- homogeneous and decreasing the proportion of hetero- egory and a lower proportion was in the heterogene- geneous distribution patterns. ous category compared to the other groups (Chi-square Oocyte quality is one of the key factors that determine test, N = 130, P < 0.001; Fig.  7B). The overall mitochon - the success of IVM, IVF, and the developmental poten- drial activity measured as the intensity signal of the stain tial of the produced embryo [30]. Several methods are exhibited no significant differences between the experi - used to evaluate oocyte quality based on morphology, mental groups (Fig. 7C). biomarkers expression, and machine learning assistance using oocyte images [31]. Staining GV oocytes with vital stains is another method that can predict oocyte qual- Discussion ity and developmental competence. For instance, bril- In this study, we identified the miRNA cargo of the s-EVs liant cresyl blue (BCB) staining has been widely used to isolated from porcine FFs corresponding to different differentiate between growing and fully grown oocytes oocyte qualities. Furthermore, we investigated the effect based on the activity of the G6PDH enzyme [32], since of s-EV supplementation during maturation on oocyte oocytes stained with BCB are more competent than the developmental competence. We mainly found that s-EV unstained ones. Another interesting synthetic non-toxic miRNA expression profiles in FFs differed between high- stain that has been used to detect cellular membrane and low-quality corresponding oocytes. These identi - damages is LB stain [33]. It has been used previously for fied miRNAs could be used as non-invasive biomarkers the non-invasive morphological assessment of porcine to predict oocyte developmental competence and could oocyte quality since it enables the detection of oocytes in provide more insights into the potential role of miR- the pre-apoptotic stage, expressing high levels of TP53, NAs in follicular cell-cell communications and develop- but still with low levels of pro-apoptotic genes [18]. ment. Moreover, the supplementation of s-EVs from the Moreover, in another study from our group, Bartkova FFs of the low-quality group to oocytes during matura- et  al. [17] reported LB staining as a non-invasive oocyte tion modulates changes in mitochondrial distribution selection method that can detect cellular membrane patterns in MII oocytes by increasing the proportion of Gad et al. Journal of Animal Science and Biotechnology (2022) 13:82 Page 10 of 16 Table 2 Differentially expressed (DE) miRNAs in small-extracellular vesicles obtained from follicular fluids of high (HQ) compared to low (LQ) quality corresponding oocytes Upregulated miRNAs FC FDR Downregulated miRNAs FC FDR ssc-miR-193a-5p 4.48 4.83E-05 ssc-miR-9-1 −51.16 4.90E-22 ssc-miR-339-3p 3.34 1.38E-03 ssc-miR-9 −12.67 2.60E-17 ssc-miR-671-3p 3.26 1.48E-04 ssc-miR-338 −8.39 2.15E-04 ssc-miR-1306-5p 3.22 4.63E-09 ssc-miR-6516 −7.95 3.34E-05 ssc-miR-885-5p 2.75 4.28E-05 ssc-miR-206 −5.67 3.51E-07 ssc-miR-7142-3p 2.67 1.07E-04 ssc-miR-133b −4.34 6.23E-06 ssc-miR-125b 2.60 3.40E-06 ssc-miR-133a-3p −3.14 3.04E-05 ssc-miR-132 2.60 7.14E-05 ssc-miR-101 −3.10 9.57E-12 ssc-miR-6529 2.54 1.62E-07 ssc-miR-219b-3p −3.07 3.67E-02 ssc-miR-296-5p 2.28 1.82E-03 ssc-miR-143-5p −2.90 2.04E-03 ssc-miR-125a 2.27 8.51E-04 ssc-miR-142-5p −2.74 1.94E-07 ssc-miR-423-3p 2.22 6.35E-06 ssc-miR-451 −2.48 2.34E-02 ssc-miR-423-5p 2.21 1.92E-04 ssc-miR-10,388 −2.43 1.44E-02 ssc-miR-7144-5p 2.19 3.78E-02 ssc-miR-199b-5p −2.33 3.51E-07 ssc-let-7d-3p 2.17 6.33E-06 ssc-miR-20a-5p −2.29 7.58E-04 ssc-miR-551a 2.09 5.36E-03 ssc-miR-19a −2.19 7.14E-05 ssc-miR-149 2.09 7.98E-03 ssc-miR-301 −2.18 1.34E-04 ssc-miR-320 2.07 1.85E-06 ssc-miR-126-5p −2.18 5.10E-07 ssc-miR-425-5p 2.02 1.29E-04 ssc-miR-218b −2.16 1.29E-04 ssc-miR-424-5p −2.14 4.15E-03 ssc-miR-190b −2.13 5.58E-04 ssc-miR-545-5p −2.08 2.04E-02 ssc-miR-95 −2.04 9.49E-03 FC Fold Change, FDR False Discovery Rate damage in porcine COCs. Although oocyte staining cellular-specific markers. In addition, the RNA electro - with such stains is considered a non-invasive method, pherogram clearly showed the absence of the ribosomal it is still not the optimal approach for oocyte selection, RNA peaks after total RNA isolation. These verified the since further treatment and incubation steps need to be isolation procedure and confirmed the degree of purity of incorporated to evaluate the oocytes. Therefore, search - the s-EV preparations from other intracellular compart- ing for specific biomarkers in follicular cells or FFs which ments or cellular contamination [34]. are associated with oocyte quality could be a much Since the first reported evidence on the EV-medi- more appropriate strategy for oocyte quality assessment ated transfer of miRNAs between cells [11], EV-miR- and selection. To avoid any variations in the follicular NAs have been considered to be novel non-invasive stages or the physiological conditions of the donors, we molecular markers for the prediction and diagnosis collected the FFs from individual healthy ovarian fol- of various pathophysiological conditions [10]. From licles of similar size from prepubertal gilts within simi- the FFs, several EV-miRNAs have been reported to lar weight and age ranges. Then, we used LB staining to be associated with follicular and oocyte development differentiate between high- and low-quality COCs, and in different mammalian species including porcines we subsequently collected the corresponding FFs for [35], humans [36], bovines [37, 38], and equines [39]. each category for s-EVs isolation and miRNA identifica - However, the mechanism of EV-miRNAs in FFs that tion. We characterized the s-EVs isolated from porcine influences the oocyte developmental competence FFs using three different methods. The size distribu - remains unclear. In this study, miRNA sequencing tion, shape, and the analysis of specific protein markers analysis identified a total of 42 significantly DE miR- revealed the successful isolation of s-EVs in agreement NAs between the HQ and LQ s-EV groups. Among with the minimal information for studies of extracellu- the DE miRNAs, the mir-9 family-related miRNAs lar vesicles (MISEV2018) [34]. Importantly, s-EV sam- (miR-9-1 and miR-9) were the most highly elevated ples were negative for CytC and ATP5A proteins as miRNAs in the LQ compared to the HQ group, with G ad et al. Journal of Animal Science and Biotechnology (2022) 13:82 Page 11 of 16 Fig. 5 Pathway analysis of differentially expressed miRNA target genes. Bubble plots for the pathways targeted by the elevated miRNAs in the high (A) and low quality (B) s-EVs groups and by the top five most abundant miRNAs in both groups (C) . The color and size of each bubble represent the P-value and the number of miRNA target genes in each pathway, respectively. Exclusive and common pathways targeted by elevated miRNAs in the high- (HQ) and low-quality (LQ) s-EVs groups and by the top five most abundant miRNAs in both groups (D) more than 50-fold miR-9-1 in the LQ group. The large follicles. These findings may indicate a possible miR-9 family was previously known to play roles as correlation between the cellular and/or extracellular repressor mediators of proliferation promoting tran- expression of miR-9 with the oocyte quality. In addi- scription factors [40] and as tumor suppressors [41]. tion, miR-101 was among the upregulated miRNAs In humans, miR-9 exhibited a higher expression in the in the LQ compared to the HQ group and was one FFs (104 folds) [42] and granulosa cells [43] of women of the highly abundant DE-miRNAs. Recently, it has with polycystic ovary syndrome (PCOS) compared to been reported that miR-101-3p inhibits goat granu- normal women. It has been previously reported that losa cells in  vitro proliferation by regulating CDK4, PCOS is highly correlated with poor oocyte qual- CCND1, CCNE1, and PCNA expressions and promotes ity and subsequently low developmental competence the apoptotic rate by regulating Bcl-2, Bax, p53, and due to consecutive disturbances in the paracrine and/ caspase3 expression [46]. The same study reported or endocrine follicular microenvironment [44, 45]. that, in mouse ovaries, miR-101-3p exhibited unu- Moreover, in our previous study [26] we reported an sual ovarian development functions, as reflected by increase in the expression of miR-9-1 in low-compe- decreased numbers of various follicles as well as small tence porcine oocytes derived from small compared to and stunted ovarian fragments. Another interesting Gad et al. Journal of Animal Science and Biotechnology (2022) 13:82 Page 12 of 16 Fig. 6 Droplet digital PCR (ddPCR) validation of the selected DE-miRNAs in comparison to the small RNAseq (sRNAseq) analysis. *Statistical significance between the high- (HQ) and low- quality (LQ) s-EVs groups (P < 0.05) group of miRNAs including miR-206, miR-133b, and oocytes in this study. On the other hand, a group of miR-133a-3p showed a significant up-regulation in the 19 miRNAs including miR-132 and miR-320 (one of LQ compared to the HQ group. The same three miR- the highly abundant DE-miRNAs) was significantly NAs exhibited a higher expression pattern in the EVs up-regulated in the FF-EVs of the corresponding HQ isolated from the blood plasma of low- compared to oocytes. Similar to our findings, miR-132 and miR-320 high-response heifers to ovarian stimulation [47]. Both were detected as extracellular miRNAs with a higher miR-206 and miR-133a are highly correlated with E2 expression level in human ovarian FFs of oocytes that deficiency by targeting and reducing the expression yielded top-quality embryos [55]. Moreover, both of E2 receptor-α, which mediates the biological activ- miRNAs were highly expressed in the ovarian FFs of ity of E2 in ovarian follicular cells and subsequently healthy compared to PCOS patients [56]. In another affects oocyte quality [48–51]. In mice, theca-spe- study, the expression of miR-320 in FFs was posi- cific E2 receptor-α knockout leads to a reduction in tively correlated with human embryonic quality and the oocyte quality and decreased ovulation capacity development. The same study showed that miR-320 [52]. Recently, it has been demonstrated that miR- knockdown in mouse oocytes strongly decreased their 206 decreased the viability and induced apoptosis developmental competence [57]. The expression level levels in ovarian granulosa cells by targeting CCND2 of miR-132 was higher in the FFs of equine preovula- mRNA [53]. Similarly, miR-133a was reported to be a tory compared to dominant follicles, with an indica- cell proliferation inhibitor and apoptosis promotor in tion of the physiological involvement of miR-132 in the intestinal epithelial cells [54]. These could explain steroidogenesis, follicle selection, and ovulation [58]. the higher expression of this group of miRNAs in the Other interesting miRNAs which were highly abun- FF-EVs of the LQ compared to the corresponding HQ dant in the FF-EVs and exhibited a higher expression Table 3 Maturation and developmental rates of porcine oocytes co-incubated with s-EVs isolated from HQ or LQ FF groups Group MII rate Cleavage rate Blastocyst rate Blastocyst cell % ± SD (n) % ± SD (n) % ± SD (n) number ± SD (n) C 90.07 ± 9.95% (47) 85.34 ± 7.21% (124) 32.40 ± 2.37% (124) 39 ± 5 (27) NC 85.35 ± 10.95% (55) 86.79 ± 6.17% (125) 29.33 ± 7.93% (125) 39 ± 10 (17) HQ 75.74 ± 14.29% (53) 76.68 ± 3.83% (129) 25.38 ± 5.13% (129) 42 ± 6 (20) LQ 87.74 ± 5.75% (55) 75.84 ± 7.98% (154) 25.70 ± 7.71% (154) 45 ± 4 (18) C Control, NC negative control, HQ high quality, LQ low quality G ad et al. Journal of Animal Science and Biotechnology (2022) 13:82 Page 13 of 16 Fig. 7 Mitochondrial activity and distribution patterns in porcine MII oocytes. A Oocytes were classified into three main distribution patterns: Homogeneous (left), Heterogeneous (middle), or Cluster (right). Scale bar, 60 μm. B The proportion of each distribution pattern in porcine MII oocytes after co-incubation with s-EVs of high- or low-quality groups. Bars with different letters indicate significant differences (P < 0.05). C The relative intensity signal of mitochondrial activity in the different oocyte groups. C: control; NC: negative control; HQ: high-quality; LQ: low-quality level in the HQ compared to the LQ group are miR- cells was associated with an increase in endogenous 125a and miR-125b. Both are members of the mir-10 miRNA levels and altered gene expression in in  vitro family and were detected in human FF microvesicles cultured follicular cells [12]. However, the results of [56]. A recent study demonstrated that miR-125a can studies on the effect of EVs on developmental rates con - be transcribed by all follicular components and play tradict each other. For instance, de Ávila et al. reported a role in intercellular communication within the folli- that the supplementation of bovine oocytes with FF- cle [59]. In mice, miR-125a and miR-125b were highly isolated EVs does not affect the maturation rate [37]. expressed in GV compared to MII oocytes, and it was Moreover, in pigs, it has been suggested that exosomes also found that they play an important role in regulat- from FFs are not effective in inducing cumulus cell ing maternal genes and zygotic genome activation [60]. expansion [61]. In contrast, bovine follicular EVs To investigate the effect of s-EVs on porcine oocyte induced mouse and bovine cumulus cell expansion [38] maturation and embryonic development, we performed and improve oocyte competence and survival of heat a functional experiment by coincubating COCs with stress [62]. Several factors including the source of EVs, s-EVs of the HQ or LQ groups. However, we didn’t isolation method, concentration, and incubation time observe any significant differences in maturation or could influence the extent of the EV impact on oocyte embryonic developmental rates after coincubation or embryonic developmental competence, which could compared to the control groups. In different mamma - explain these contrary results. For instance, the supple- lian species, several studies have demonstrated that, mentation of EVs from early antral follicles increased during in  vitro culture, EVs could be uptaken by gran- bovine blastocyst rates compared to the control, how- ulosa or cumulus cells and could be found within the ever, EVs derived from pre-ovulatory follicles exhib- zona pellucida and transzonal projections of cumulus ited no significant difference [63]. In another study, the cells [37–39]. Moreover, the uptake of EVs by follicular supplementation of maturation media with follicular Gad et al. Journal of Animal Science and Biotechnology (2022) 13:82 Page 14 of 16 component analysis; PCOS: Polycystic ovary syndrome; PZM 3: Porcine zygote EVs derived by density gradient ultracentrifugation medium 3; PXM-HEPES: HEPES buffered porcine X medium; SDS: Sodium improved the blastocyst rate compared to EVs derived dodecyl sulphate; sRNAseq: Small RNAseq; s-EV: Small-EV; sncRNA: Small non- by size-exclusion chromatography [64]. In the same coding RNA; TBS: Tris buffered saline; TEM: Transmission electron microscopy; TMM: Trimmed mean of M-values normalization. study, EVs derived by both isolation methods improved embryo quality measured as blastocyst cell number and Supplementary Information apoptotic cell ratio. More studies considering these fac- The online version contains supplementary material available at https:// doi. tors are needed to optimize the EV co-culture condi- org/ 10. 1186/ s40104- 022- 00723-1. tions and to determine the mechanisms that regulate oocyte and embryonic development under in vitro con- Additional file 1: Table S1. Summary of sequence reads mapped to the ditions. Our results showed that a higher proportion of porcine reference genome and annotated against porcine miRNAs listed in the mirBase database. Table S2. A complete list of all expressed miRNAs MII oocytes that were co-cultured with the LQ s-EVs in the high (HQ) and low (LQ) quality s-EVs groups indicated as TMM- exhibited a homogeneous mitochondrial distribution adjusted Counts Per Million (CPM). Table S3. KEGG pathway analysis for pattern, and a lower proportion of them was in the het- genes targeted by the differentially expressed miRNAs in HQ vs. LQ group and top 5 miRNAs in both groups. erogeneous pattern compared to the other groups. It is Additional file 2: Fig. S1. Mapped reads proportions of small non-coding well known that homogeneous and heterogeneous dis- RNA types in high- (HQ) and low-quality (LQ) s-EVs. tribution patterns of mitochondria are more commonly Additional file 3: Fig. S2. Interaction networking of genes involved in observed in GV and MII oocytes, respectively [28]. As PI3K-Akt (A) and MAPK signaling (B) pathways and targeted by elevated oocyte maturation progresses, the mitochondrial dis- miRNAs in high- (HQ) and low-quality (LQ) s-EVs groups and by top five tribution changes from homogeneous to a heteroge- most abundant miRNAs in both groups. neous pattern as a sign of cytoplasmic maturation [28, 65]. Several studies reported that released EVs could Acknowledgements We acknowledge the Electron Microscopy Core Facility, IMG CAS, Prague, regulate the function and composition of mitochondria Czech Republic, supported by MEYS CR (LM2018129 Czech-BioImaging) and in receptor cells via their metabolite, miRNA, and pro- ERDF (projects: Z.02.1.01/0.0/0.0/18_046/0016045, CZ.02.1.01/0.0/0.0/16_01 tein cargos (reviewed in [66]). Not only that, but whole 3/0001775) for their support with obtaining the microscopic data presented in this paper. mitochondria or their component parts could be trans- ferred between cells via EVs [67]. This could explain the Authors’ contributions negative effect of LQ-EVs on the cytoplasmic matura - Conceptualization: AG; Experimental work: AG, MM, AB, VK, RP; Data analysis: AG, MM, AB, VK; Interpretation of data: AG, MM, AB, RP; Funding acquisition: tion of MII oocytes by affecting the mitochondrial dis - AG, JL, RP; Literature review: AG, KM; Writing the original draft: AG, MM. All the tribution pattern. However, the precise mechanism by authors reviewed and approved the final version of the manuscript. which the EVs could regulate oocyte mitochondrial dis- Funding tribution is still unclear. This work was supported by the Institute of Animal Physiology and Genetics “IAPG-Matoušek Award 2020” Grant number: DRMA-2020-0002 and the Minis- Conclusion try of Education, Youth and Sports of the Czech Republic, Operational Program Research, Development and Education, the project “EXCELLENCE in molecular Our results indicated that s-EVs purified from porcine aspects of the early development of vertebrates” Grant number: CZ.02.1.01/0.0 ovarian FFs contain different miRNA cargos that are /0.0/15_003/0000460. associated with the quality of the corresponding oocytes. Availability of data and materials These miRNAs could be used as non-invasive biomarkers The datasets supporting the conclusions of this article are included within for oocyte selection. Moreover, the supplementation of the article and as additional files. The raw FASTQ files and processed CSV files maturation media with s-EVs of the LQ FF group modu- have been deposited in the NCBI’s Gene Expression Omnibus (GEO) with an accession number GSE181182. All relevant data regarding s-EV isolation and lates the cytoplasmic maturation of the matured oocytes characterization were submitted to the EV-TRACK knowledgebase with an by affecting the mitochondrial distribution patterns. EV-TRACK ID EV210251. Further functional studies on the mechanisms by which FF-EVs and their molecular cargos could regulate and Declarations maintain oocyte developmental competence will enhance Consent for publication ART outcomes. Not applicable. Competing interests Abbreviations The authors declare that they have no competing interests. ARTs: Assisted reproductive technologies; BCB: Brilliant cresyl blue; C: Control group; COCs: Cumulus-oocyte complexes; CPM: Counts per million; ddPCR: Author details Droplet digital PCR; DE: Differentially expressed; EVs: Extracellular vesicles; FC: Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Fold change; FFs: Follicular fluids; GEO: Gene expression omnibus; HQ: High 27721 Liběchov, Czech Republic. Department of Animal Production, Faculty quality; LB: Lissamine green B stain; LQ: Low quality; MII: Metaphase II; miRNA: of Agriculture, Cairo University, Giza 12613, Egypt. Department of Botany MicroRNA; NC: Negative control group (PBS-supplemented group); NGS: Next and Genetics, Faculty of Natural Sciences, Constantine the Philosopher Univer- generation sequencing; NTA: Nanoparticle tracking analysis; PCA: Principle sity in Nitra, 94901 Nitra, Slovakia. G ad et al. 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Small-extracellular vesicles and their microRNA cargo from porcine follicular fluids: the potential association with oocyte quality

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Abstract

Background: Ovarian follicular fluids (FFs) contain several kinds of regulatory factors that maintain a suitable micro - environment for oocyte development. Extracellular vesicles (EVs) are among the factors that play essential roles in regulating follicle and oocyte development through their cargo molecules that include microRNAs (miRNAs). This study aimed to investigate small-EV (s-EV ) miRNAs in porcine FFs and their potential association with oocyte quality. Methods: Individual aspirated oocytes were stained with lissamine green B stain (LB), a vital stain for oocyte qual- ity, and each oocyte was classified as high-quality (unstained; HQ) or low-quality (stained; LQ). FFs corresponding to oocytes were pooled together into HQ and LQ groups. Small-EVs were isolated from FFs, characterized, and their miRNA cargo was identified using the Illumina NovaSeq sequencing platform. Additionally, s-EVs from the HQ and LQ groups were utilized to investigate their effect on oocyte development after co-incubation during in vitro maturation. Results: A total of 19 miRNAs (including miR-125b, miR-193a-5p, and miR-320) were significantly upregulated, while 23 (including miR-9, miR-206, and miR-6516) were downregulated in the HQ compared to the LQ group. Apoptosis, p53 signaling, and cAMP signaling were among the top pathways targeted by the elevated miRNAs in the HQ group while oocyte meiosis, gap junction, and TGF-beta signaling were among the top pathways targeted by the elevated miRNAs in the LQ group. The supplementation of small-EVs during maturation does not affect the oocyte develop - mental rates. However, LQ s-EVs increase the proportion of oocytes with homogeneous mitochondrial distribution and decrease the proportion of heterogeneous distribution. Conclusion: Our findings indicated that FF-EVs contain different miRNA cargos associated with oocyte quality and could affect the mitochondrial distribution patterns during oocyte maturation. Keywords: Extracellular vesicles, Follicular fluids, MiRNA, Oocyte quality, Porcine oocyte quality, could influence this ability and subse - Background quently determine developmental competence. Various Oocyte developmental competence is commonly defined methods including morphological, biochemical, and as the ability of the oocyte to mature, fertilize, and molecular techniques, are being used to assess oocyte develop to the blastocyst stage. Several factors, including quality [1], with the aim of enhancing the efficiency of assisted reproductive technologies (ARTs). At the molec- *Correspondence: Murin@iapg.cas.cz ular level, several studies have been done to determine molecular markers from follicular cells surrounding the Institute of Animal Physiology and Genetics, Czech Academy of Sciences, 27721 Liběchov, Czech Republic oocyte or follicular fluids (FFs) as a non-invasive method Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Gad et al. Journal of Animal Science and Biotechnology (2022) 13:82 Page 2 of 16 that predicts oocyte quality [2]. Ovarian FFs contain sev- Merck (Kenilworth, NJ, USA), respectively, unless stated eral kinds of regulatory factors that maintain a suitable otherwise. All media were prepared fresh and sterilized microenvironment for oocyte development and follicular using 0.22 μm syringe filters. intercellular communication [3]. This kind of communi - cation within the follicle is essential and could determine Collection of follicular fluids, COCs staining, oocyte quality and consequently developmental compe- and classification tence [4]. One of the recently discovered mechanisms Porcine ovaries of prepubertal gilts (Landrace × Large that facilitate and modulate intercellular crosstalk is via White, 6–8 months of age, 90–120 kg) were collected the extracellular vesicles (EVs). EVs, including exosomes from a local slaughterhouse and transported to the and microvesicles, are cell-derived lipid bilayer mem- lab in a thermos flask within 2 h. Ovaries were washed branous particles that contain different biomolecules, three times with saline solution. Follicular fluids and including proteins, lipids, RNAs, and microRNAs (miR- COCs were aspirated from individual healthy follicles NAs). These particles are secreted by almost all cell types (3–6 mm in diameter, measured as previously recom- and can be found in all body fluids [5 ]. The ability of EVs mended [15]) using a 25-gauge needle attached to a 1-mL to modulate intercellular crosstalk is through the capac- syringe (Braun, Germany). Each COC and its corre- ity to transfer their biomolecular cargos between differ - sponding FF were allocated in separate wells in a 96-well ent cells after being secreted in body fluids [5 ]. After this plate. COCs were washed once in PXM-HEPES (HEPES discovery, several studies revealed the fundamental roles buffered porcine X medium [16]) and then stained for of EVs in the different reproduction processes in domes - 15 min at room temperature with 0.5% lissamine green B tic animals (reviewed by Llobat [6]) and the subsequent stain (LB), a vital synthetic stain for determining oocyte improvement of ART outcomes [7]. quality and competence [17, 18]. Each COC was classi- MiRNAs, a small non-coding RNA class, are post- fied separately according to the oocyte stain into high- transcriptional regulators of gene expression by bind- quality (unstained; HQ) and low-quality (stained; LQ) as ing specific mRNA target sequences for degradation or presented in Fig.  1. FFs corresponding to oocytes were translational repression [8]. Extracellular miRNAs were pooled together into HQ and LQ groups and used for the discovered in various body fluids in a stable form, which isolation of small-extracellular vesicles (s-EVs). supports their potential utilization as non-invasive bio- markers for several physiopathological conditions [9]. Isolation of small‑extracellular vesicles from follicular fluid The availability and stability of extracellular miRNAs To isolate s-EVs (< 200 nm), pooled FFs (~ 1 mL/repli- within body fluids are supported by either the incor - cate) from each group were centrifuged at 700  ×  g for poration of miRNAs within the EVs or by binding with 10 min to pellet cells, at 2000  ×  g for 10 min to remove specific protein complexes [10]. In 2007, miRNAs were cell debris, and at 12,000 ×  g for 30 min to remove large identified for the first time within cell-secreted EVs that particles and protein aggregates. All centrifugation steps can be delivered and regulate several functions in the were performed at 4 °C. The remaining supernatants target cells [11]. In ovarian FFs, it has been reported were filtrated through a 0.2-μm syringe filter to eliminate that the majority of miRNAs are found within EVs [12] larger vesicles. Small-EVs were isolated from 0.5 mL fil - and are suggested to play a role in follicle development tered FFs (3 replicates/group) using an Exo-spin kit (Cell and other ovarian functions [13]. Consequently, altera- Guidance Systems, UK) and eluted in PBS according to tions in the FF miRNAs could reflect the status of the the manufacturer’s protocol. For electron microscope oocyte quality and its developmental competence [14]. imaging and western blot analysis, samples were kept at Therefore, the objectives of this study were to identify 4 °C until analysed. The remaining samples were stored small-EV (s-EV) miRNA differences in porcine FFs in at − 80 °C for further analysis. All relevant data regarding association with oocyte quality and to investigate the s-EV isolation and characterization were submitted to effect of s-EVs on oocyte developmental competence. the EV-TRACK knowledgebase [19] with the EV-TRACK The identified miRNAs could be used as non-invasive ID EV210251. biomarkers for oocyte selection. Moreover, our results provided more insights into the potential role of FF-EVs during oocyte maturation. Nanoparticle tracking analysis (NTA) To determine vesicle size and concentration, samples Materials and methods were diluted in PBS (1:200) and NTA was conducted Chemicals and supplements in a ZetaView instrument (Particle Metrix, Germany) All plastic materials and chemicals were purchased from in scatter mode (488 nm laser). Measurements were Thermo Fisher Scientific (Waltham, MA, USA) and performed in two cycles by scanning 11 cell positions G ad et al. Journal of Animal Science and Biotechnology (2022) 13:82 Page 3 of 16 Fig. 1 Classification of porcine COCs after lissamine green B (LB) staining. Left: Unstained COC (high-quality; HQ). Right: Stained COC (low-quality; LQ). Scale bar, 60 μm each, and the videos were analysed using the software in 2% BSA) and anti-ATP5A (Abcam, Ab14748, diluted ZetaView (version 8.05.12). 1:2000 in 5% milk) raised against somatic cell-specific markers. The secondary antibodies (Amersham ECL anti-mouse or anti-rabbit IgG, GE Healthcare, Little Transmission electron microscopy (TEM) Chalfont, UK) were diluted 1:5000 in 2% BSA in TBS- For morphological evaluation, isolated s-EV samples Tween. The membranes were incubated with the sec - were placed into formvar/carbon-coated 400 mesh cop- ondary antibody for 1 h at room temperature and then per grids for 20 min at room temperature. Then, the grids washed intensively in TBS-Tween. The immune reac - were incubated with 2% formaldehyde in PBS for 20 min, tions were detected by enhanced chemiluminescence followed by 6 washes for 2 min each with ultrapure water. (Pierce, Rockford, IL, USA) according to the manufac- The grids were stained with uranyl acetate (2%) for turer’s instructions and captured on CL-XPosure film 12 min, washed in water, left to dry, and then examined (ThermoFisher Scientific). in a Jeol JEM-1400 FLASH transmission electron micro- scope (Tokyo, Japan) equipped with Matataki 2k×2k CMOS camera at 80 kV. Total RNA extraction, library preparation, and sequencing Total RNA, including miRNA was isolated from EV sam- Western blot analysis ples using a miRNeasy Micro Kit (Qiagen, Hilden, Ger- Samples of s-EV, filtered follicular fluid, or follicu - many) that combines the phenol/guanidine-based lysis lar cells containing 40 μg of total protein were mixed of samples and silica membrane-based purification of with Laemmli buffer containing 2% sodium dodecyl total RNA, according to the manufacturer’s instructions. sulphate (SDS) and 5% 2-mercaptoethanol. Samples The RNA concentration and size distribution were ana - were boiled at 100 °C for 3 min and stored frozen at lyzed using an Agilent RNA 6000 Pico kit in an Agilent − 20 °C. Subsequently, proteins were separated in 10% 2100 Bioanalyzer (Agilent Technologies, Santa Clara, or 12% acrylamide/SDS gels and transferred to Immo- CA, USA). Small-RNA libraries were prepared for next- bilon-P membranes (Millipore, Bedford, MA, USA). generation sequencing (NGS) using a QIAseq miRNA Membranes were blocked in 5% low-fat dry milk in Library Kit (Qiagen) according to the manufacturer’s Tris-buffered saline (TBS) with 0.5% Tween 20 for 2 h instructions. Library quantity and quality assessments at room temperature and then incubated with a pri- were performed using a Qubit DNA HS Assay Kit in a mary antibody at 4 °C overnight. The primary anti - Qubit 4 Fluorometer (Thermo Fisher Scientific) and Agi - bodies were anti-CD63 (Abcam, Ab 118307, diluted lent DNA High Sensitivity kit in an Agilent 2100 Bioana- 1:1000 in 2% BSA in TBS-Tween), anti-Alix (Abcam, lyzer (Agilent Technologies), respectively. The libraries Ab 88388, diluted 1:500 in 5% milk), and anti-TSG101 were pooled in equimolar ratios and then sequenced in a (Santa Cruz Biotechnology, Sc-7964, diluted in 1:200 in NovaSeq6000 sequencing instrument (Illumina, Inc., San 5% milk) raised against exosome-specific markers, and Diego, CA, USA) as single-end reads. anti-Cytochrome C (Abcam, Ab 90529, diluted 1:1000 Gad et al. Journal of Animal Science and Biotechnology (2022) 13:82 Page 4 of 16 Sequencing data analysis samples, according to the analysis with the software FASTQ files were generated for each sample using the NormFinder. software bcl2fastq (Illumina Inc., San Diego, CA), and their quality was checked using the FastQC tool ver- Co‑incubation of s‑EVs with COCs during maturation sion 0.11.9. Data were analyzed using the software CLC COCs were collected as mentioned above using a Genomics Workbench, version 21 (www. qiage nbioi 20-gauge needle attached to a 10-mL syringe and then nform atics. com). Raw sequencing reads were trimmed morphologically evaluated under a stereomicroscope based on quality score (Q-score > 30), ambiguous nucle- (Zeiss Stemi 508, magnification × 50). Only those with at otides (maximum two nucleotides allowed), read length least three layers of cumulus cells and an evenly granu- (≥15 nucleotides) and removing adapter sequences. lated ooplasm were used. COCs were washed twice in Reads were mapped to the porcine (Sus scrofa) refer- the maturation medium (Medium 199) supplemented ence genome (Sscrofa11.1) and annotated against por- with 0.005% gentamicin (Roth 0233), 0.0022% sodium cine precursor and mature miRNAs listed in the mirBase pyruvate, 0.01% L-glutamine, 0.1% BSA, 10 ng/mL database (release 22) using the CLC Genomics Work- EGF, 40 ng/mL FGF2, 20 ng/mL IGF1, 2000 IU/mL LIF, bench RNA-Seq Analysis and Quantify miRNA tools, 0.57 mmol/L L-Cysteine, 10 IU/mL PMSG and 10 IU/mL respectively, applying the default software parameters. HCG. COCs were cultivated in 4-well dishes (30–50 per Raw expression data were normalized using the trimmed well) for 44 h at 38.5 °C under a 5% C O atmosphere in mean of M-values normalization method (TMM nor- 500 μL maturation medium supplemented with or with- malization) [20] and presented as TMM-adjusted Counts out (control) s-EV particles (~ 200 million particles/mL) Per Million (CPM). The CLC Genomics Workbench Dif - isolated from HQ or LQ FF groups. COCs cultivated in ferential Expression tool was used for the expression the same maturation media supplemented with a volume analysis comparison of the two groups. MiRNAs with of PBS similar to the s-EVs were used as the negative fold change (FC) ≥ 2, P-adjusted value (FDR [21]) < 0.05, control group (NC). and CPM > 5 in the enriched group were considered dif- ferentially expressed (DE). The raw FASTQ files and pro - Parthenogenetic activation and embryo cultivation cessed CSV files have been deposited in the NCBI’s Gene After maturation, cumulus cells were removed from Expression Omnibus (GEO) with the accession number COCs by pipetting and washed twice in PXM-HEPES. GSE181182. Oocytes were activated using 10 μmol/L ionomycin in PXM-HEPES for 5 min, washed twice in porcine zygote Target gene prediction and ontological classification medium 3 (PZM 3) [27] supplemented with 2 mmol/L Genes targeted by DE-miRNA were identified using the 6-dimethylaminopurine, and cultivated for 5 h at 38.5 °C miRWalk database [22]. Within the miRWalk, validated under a 5% CO atmosphere. A group of 30–50 putative target genes from miRTarBase (version 7.0) and com- parthenotes was washed twice in PZM 3 and cultivated monly target genes predicted by TargetScan (version for 7 d in 4-well dishes in 500 μL of PZM 3 medium at 7.1) and miRDB (release 5.0) were selected for ontologi- 38.5 °C under a 5% CO atmosphere. The cleavage rate cal classification and pathway analysis using the DAVID was assessed after 40 h, and the ability of the embryos to bioinformatics web tool (https:// david. abcc. ncifc rf. gov/). reach the blastocyst stage as well as the number of nuclei Pathways were determined from the KEGG database in blastocysts were analyzed after 168 h of cultivation. [23], and interaction networks of the targeted genes and Blastocysts were fixed using 4% paraformaldehyde and the identified pathways were constructed with Cytoscape mounted on glass slides using DAPI mounting medium. [24] and its plug-in ClueGO [25]. Each blastocyst was scanned using a confocal microscope (Leica SP5, Germany), and the number of nuclei was DE‑miRNA validation using droplet digital PCR (ddPCR) counted using the software ImageJ. To validate the miRNA-seq data, we performed a PCR analysis for a selected group of 11 DE-miRNAs. The Oocyte mitochondrial activity and distribution patterns absolute copy numbers of the selected DE-miRNAs were Matured COCs were denuded as described above. quantified in the EVs samples using specific TaqMan Oocytes were washed three times in PBS, stained with miRNA Assays (Applied Biosystems, Foster City, CA, 300 nmol/L Mitotracker Orange kit (Thermo Fisher USA) in a ddPCR system (Bio-Rad Inc., Hemel Hemp- Scientific, USA) for 30 min in the dark at 38.5 °C. Then, stead, UK) according to the manufacturer’s instructions oocytes were washed three times in PBS at 38.5 °C, and as previously described [26]. The copy numbers fixed for 15 min in 4% paraformaldehyde, and mounted of the selected DE-miRNAs were normalized to miR- on glass slides using DAPI mounting medium. Oocytes 26b-5p, the most stably expressed miRNA across all were scanned in a confocal microscope (Leica SP5, G ad et al. Journal of Animal Science and Biotechnology (2022) 13:82 Page 5 of 16 Germany) and mitochondrial activity was analyzed using Student’s t-test. The statistical significance level using the software ImageJ. The distribution patterns of was defined at P < 0.05. mitochondria were characterized by observing (at 630- fold magnification) labeled mitochondria with an active Results oxidative state. We classified the distribution and aggre - Characterization of s‑EVs from porcine follicular fluids gation patterns mainly as homogeneous, heterogeneous, Different morphological and molecular analyses were and clustered distribution according to the previously done to determine the characteristics and the purity reported classification [28, 29]. of s-EV isolated samples. Vesicle size and concentra- tion were determined in each sample using NTA. The Statistical analysis concentration of s-EVs from the HQ and LQ FF groups All experiments were done in at least three replicates. was 8.98E+ 09 ± 3.43E+ 08 and 8.97E+ 09 ± 1.25E+ 09 Data normality was checked using the Shapiro–Wilk particles/mL, the median size was 135.7 ± 5.3 and test. Maturation and developmental rates and blas- 132.6 ± 3.3 nm, respectively, and the mode was 135 nm tocyst nuclei numbers were analyzed using one-way in both groups, with no significant differences between ANOVA (SigmaPlot 12, US) followed by Tukey’s test to them (Fig. 2A). Imaging with TEM revealed the presence detect differences between the means and expressed as of s-EVs with visible lipid bilayer membranes (Fig.  2B). mean ± SD. Categorical variables were identified using Western blot analysis identified the presence of EV-spe - the chi-square test. PCR data were statistically analyzed cific markers (ALIX, TSG101, and CD63) in both s-EV Fig. 2 Morphological and molecular characterization of s-EVs. A The concentration and size distribution of s-EVs isolated from follicular fluids (FFs) corresponding to high- (HQ) or low-quality (LQ) oocytes analysed by NTA. B Transmission electron microscopy ( TEM) representative photos of isolated s-EVs showing the lipid bilayer membrane and the cup-shaped EV particles (black arrows). Scale bar of 500 and 200 nm for the upper left and right photos, respectively, and of 200 and 100 nm for the lower left and right photos, respectively). C Immunoblotting analysis of EV-specific protein markers (ALIX, TSG101, and CD63) and cellular specific protein markers (CytC and ATP5A) in the HQ and LQ EV groups, as well as in filtered FF and follicular cell lysate (FC) as positive and negative controls, respectively. Scale numbers are in kDa. D RNA size distribution from s-EV samples of HQ (sample 1–3) and LQ (sample 4–6) groups analysed with a bioanalyzer (Agilent Technologies) Gad et al. Journal of Animal Science and Biotechnology (2022) 13:82 Page 6 of 16 groups and the filtered FFs. Additionally, cellular specific Validated and predicted target gene analysis revealed protein markers (CytC and ATP5A) were only detected a total of 860 and 1308 genes are targeted by up- and in the cell lysates, but not in the isolated s-EVs, indicating downregulated miRNAs, respectively, with 193 genes the absence of other cellular membrane contamination in targeted by both. In addition, 702 genes were among the the EV samples (Fig.  2C). Lastly, the size distribution of validated and predicted genes and targeted by the top the total RNA isolated from s-EV samples exhibited clear five most abundant miRNAs in both groups. Apoptosis, peaks for small-RNA and the absence of ribosomal-RNA p53 signaling, and cAMP signaling were among the top peaks, confirming the samples to be free of cells and cel - pathways targeted by the elevated miRNAs in the HQ lular components (Fig. 2D). group (Fig. 5A, Additional file  1: Table S3). On the other hand, oocyte meiosis, gap junction, TGF-beta signal- MiRNA expression profiles of s‑EVs isolated from follicular ing, and estrogen signaling were among the top path- fluids ways targeted by the elevated miRNAs in the LQ group Small-RNA libraries were prepared from six different EV (Fig. 5B, Additional file  1: Table S3). Signaling pathways samples (3 replicates per group) to identify the expressed including PI3K-Akt, MAPK, AMPK, and FoxO were miRNAs in each. RNA-seq analysis gave an average num- the top commonly targeted pathways by the elevated ber of 27 million raw reads per library with an average of miRNAs in the HQ and LQ s-EV groups, as well as by 16 million reads being retained after trimming and qual- the top five most abundant miRNAs in both groups ity control. An average of 89% of reads were mapped to (Fig.  5C and D, Additional file  1: Table  S3). However, the porcine genome, and the average proportion of anno- genes involved in these common pathways were differ - tated miRNAs was 15% (Additional file  1: Table S1). Vari- entially targeted by each miRNA group, with few genes ous types of small non-coding RNA (sncRNA), including commonly targeted by two or three miRNA groups, as misc_RNA, snRNA, and snoRNA were also identified shown for instance for the PI3K-Akt and MAPK signal- in all samples. However, the majority of sncRNA reads ing pathways (Additional file 3 : Fig. S2). were assigned to miRNAs (Additional  file  2: Fig. S1). Heatmap clustering and principal component analysis (PCA) were performed on the miRNA CPM values. The Validation of DE‑miRNA results showed a clear clustering of the three biological A group of 11 DE-miRNAs was selected to validate the replicates of each group, with a clear separation between miRNA-seq data using ddPCR. All selected miRNAs the two groups. The first two components of the PCA exhibited the same expression pattern as in the miRNA- explained around 68.5% of the existing variances (Fig. 3). seq data (P < 0.05) except for two miRNAs (ssc-miR-125b A miRNA with an average value of CPM > 1 was con- and ssc-miR-1306-5p), which did not differ significantly sidered to be expressed. Accordingly, a total of 303 and between the HQ and LQ s-EV groups (Fig. 6). 301 miRNAs were expressed in the HQ and LQ groups, respectively, with 295 miRNAs being expressed in both Oocyte maturation, developmental competence, groups (Fig.  4A). A complete list of all expressed miR- and mitochondrial activity after s‑EVs co‑incubation NAs is presented in Additional file  1: Table  S2, and To investigate the effect of s-EVs and their cargos on por - the top 20 most abundant miRNAs in each group are cine oocyte maturation and embryonic development, presented in Table  1. Interestingly, miR-27b-3p, miR- COCs were supplemented with s-EVs of the HQ or LQ 140-3p, miR-29a-3p, miR-202-5p, and miR-16 were the groups. We evaluated the nuclear maturation, cleav- top 5 expressed miRNAs in both groups, in which they age, blastocyst rates, and blastocyst cell count in com- accounted for 42% and 49.2% of the miRNAs sequence parison to the non-supplemented control group (C) or reads in the HQ and LQ groups, respectively (Table 1). PBS-supplemented group (NC). There were no signifi - cant differences in nuclear maturation or developmen - Differentially expressed miRNAs and ontological tal rates, as well as in the blastocyst cell counts among classification the experimental groups (Table  3). To examine whether Differential expression analysis of miRNAs revealed that s-EVs might modulate changes in mitochondrial distribu- 19 miRNAs (including miR-193a-5p, miR-339-3p, ssc- tion or activity in oocytes after maturation, metaphase miR-132, ssc-miR-125b and ssc-miR-320) and 23 miR- II (MII) oocytes were stained with MitoTracker Orange NAs (including miR-9-1, miR-9, miR-206, miR-133b, and after co-incubation with s-EVs. Based on the mitochon- miR-133a-3p) were significantly up- and downregulated drial distribution patterns, oocytes were categorized into (FC ≥ 2, FDR < 0.05, and CPM > 5 in the enriched group) three main categories, homogeneous, heterogeneous, or in the HQ compared to the LQ s-EVs group, respectively cluster distribution (Fig.  7A). The results showed that a (Fig. 4B, Table 2). significantly higher proportion of oocytes co-incubated G ad et al. Journal of Animal Science and Biotechnology (2022) 13:82 Page 7 of 16 Fig. 3 Samples clustering. A Principal component analysis (PCA). B Heatmap and hierarchical clustering. HQ1-HQ3: high-quality s-EVs replicates, LQ1-LQ3: low-quality s-EVs replicates Gad et al. Journal of Animal Science and Biotechnology (2022) 13:82 Page 8 of 16 Fig. 4 Differential expression analysis. A Venn diagram for commonly and exclusively expressed miRNAs in HQ and LQ s-EVs groups. B Volcano plot of expressed miRNAs. Up- and downregulated miRNAs in the HQ compared to the LQ s-EVs groups are labeled with red and green points, respectively G ad et al. Journal of Animal Science and Biotechnology (2022) 13:82 Page 9 of 16 Table 1 List of top 20 most abundant miRNAs in small-extracellular vesicles obtained from follicular fluids of high (HQ) or low (LQ) quality corresponding oocytes a a miRNA HQ group % miRNA LQ group % CPM CPM ssc-miR-27b-3p 102,691.3 10.9 ssc-miR-27b-3p 155,625.9 14.8 ssc-miR-140-3p 80,017.22 8.6 ssc-miR-16 115,577.2 11.4 ssc-miR-29a-3p 78,734.08 8.3 ssc-miR-29a-3p 99,770.91 9.2 ssc-miR-202-5p 71,447.84 7.5 ssc-miR-140-3p 82,856.62 7.4 ssc-miR-16 62,302.65 6.7 ssc-miR-202-5p 70,526.08 6.4 ssc-let-7c 39,828.22 4.3 ssc-miR-29c 51,664.11 4.7 ssc-miR-423-5p 32,738.73 3.6 ssc-miR-152 44,794.39 4.3 ssc-miR-128 31,129.2 3.4 ssc-let-7c 30,586.53 2.5 ssc-miR-152 28,718.56 3.1 ssc-miR-146a-5p 26,660.02 2.5 ssc-miR-29c 28,297.13 3.0 ssc-let-7i-5p 26,275.77 2.5 ssc-let-7a 26,486.27 2.9 ssc-miR-676-3p 22,826.69 2.2 ssc-miR-676-3p 24,661.24 2.7 ssc-miR-30e-5p 22,798.43 2.2 ssc-miR-146a-5p 23,950.27 2.6 ssc-miR-128 22,443.82 2.2 ssc-miR-24-3p 21,119.86 2.3 ssc-let-7a 16,416.26 1.4 ssc-miR-10b 19,325.57 2.1 ssc-miR-24-3p 16,005.13 1.5 ssc-miR-191 17,564.45 1.9 ssc-miR-30a-5p 15,408.66 1.4 ssc-let-7f-5p 17,071.41 1.8 ssc-miR-423-5p 14,893.75 1.3 ssc-let-7i-5p 16,857.99 1.8 ssc-miR-10b 13,573.69 1.1 ssc-miR-30e-5p 12,606.02 1.3 ssc-let-7f-5p 12,654.58 1.1 ssc-miR-30a-5p 11,671.15 1.2 ssc-miR-19b 11,413.06 1.2 CPM average Counts Per Million mapped reads Percentage of the miRNAs sequence reads with the LQ s-EV group was in the homogeneous cat- homogeneous and decreasing the proportion of hetero- egory and a lower proportion was in the heterogene- geneous distribution patterns. ous category compared to the other groups (Chi-square Oocyte quality is one of the key factors that determine test, N = 130, P < 0.001; Fig.  7B). The overall mitochon - the success of IVM, IVF, and the developmental poten- drial activity measured as the intensity signal of the stain tial of the produced embryo [30]. Several methods are exhibited no significant differences between the experi - used to evaluate oocyte quality based on morphology, mental groups (Fig. 7C). biomarkers expression, and machine learning assistance using oocyte images [31]. Staining GV oocytes with vital stains is another method that can predict oocyte qual- Discussion ity and developmental competence. For instance, bril- In this study, we identified the miRNA cargo of the s-EVs liant cresyl blue (BCB) staining has been widely used to isolated from porcine FFs corresponding to different differentiate between growing and fully grown oocytes oocyte qualities. Furthermore, we investigated the effect based on the activity of the G6PDH enzyme [32], since of s-EV supplementation during maturation on oocyte oocytes stained with BCB are more competent than the developmental competence. We mainly found that s-EV unstained ones. Another interesting synthetic non-toxic miRNA expression profiles in FFs differed between high- stain that has been used to detect cellular membrane and low-quality corresponding oocytes. These identi - damages is LB stain [33]. It has been used previously for fied miRNAs could be used as non-invasive biomarkers the non-invasive morphological assessment of porcine to predict oocyte developmental competence and could oocyte quality since it enables the detection of oocytes in provide more insights into the potential role of miR- the pre-apoptotic stage, expressing high levels of TP53, NAs in follicular cell-cell communications and develop- but still with low levels of pro-apoptotic genes [18]. ment. Moreover, the supplementation of s-EVs from the Moreover, in another study from our group, Bartkova FFs of the low-quality group to oocytes during matura- et  al. [17] reported LB staining as a non-invasive oocyte tion modulates changes in mitochondrial distribution selection method that can detect cellular membrane patterns in MII oocytes by increasing the proportion of Gad et al. Journal of Animal Science and Biotechnology (2022) 13:82 Page 10 of 16 Table 2 Differentially expressed (DE) miRNAs in small-extracellular vesicles obtained from follicular fluids of high (HQ) compared to low (LQ) quality corresponding oocytes Upregulated miRNAs FC FDR Downregulated miRNAs FC FDR ssc-miR-193a-5p 4.48 4.83E-05 ssc-miR-9-1 −51.16 4.90E-22 ssc-miR-339-3p 3.34 1.38E-03 ssc-miR-9 −12.67 2.60E-17 ssc-miR-671-3p 3.26 1.48E-04 ssc-miR-338 −8.39 2.15E-04 ssc-miR-1306-5p 3.22 4.63E-09 ssc-miR-6516 −7.95 3.34E-05 ssc-miR-885-5p 2.75 4.28E-05 ssc-miR-206 −5.67 3.51E-07 ssc-miR-7142-3p 2.67 1.07E-04 ssc-miR-133b −4.34 6.23E-06 ssc-miR-125b 2.60 3.40E-06 ssc-miR-133a-3p −3.14 3.04E-05 ssc-miR-132 2.60 7.14E-05 ssc-miR-101 −3.10 9.57E-12 ssc-miR-6529 2.54 1.62E-07 ssc-miR-219b-3p −3.07 3.67E-02 ssc-miR-296-5p 2.28 1.82E-03 ssc-miR-143-5p −2.90 2.04E-03 ssc-miR-125a 2.27 8.51E-04 ssc-miR-142-5p −2.74 1.94E-07 ssc-miR-423-3p 2.22 6.35E-06 ssc-miR-451 −2.48 2.34E-02 ssc-miR-423-5p 2.21 1.92E-04 ssc-miR-10,388 −2.43 1.44E-02 ssc-miR-7144-5p 2.19 3.78E-02 ssc-miR-199b-5p −2.33 3.51E-07 ssc-let-7d-3p 2.17 6.33E-06 ssc-miR-20a-5p −2.29 7.58E-04 ssc-miR-551a 2.09 5.36E-03 ssc-miR-19a −2.19 7.14E-05 ssc-miR-149 2.09 7.98E-03 ssc-miR-301 −2.18 1.34E-04 ssc-miR-320 2.07 1.85E-06 ssc-miR-126-5p −2.18 5.10E-07 ssc-miR-425-5p 2.02 1.29E-04 ssc-miR-218b −2.16 1.29E-04 ssc-miR-424-5p −2.14 4.15E-03 ssc-miR-190b −2.13 5.58E-04 ssc-miR-545-5p −2.08 2.04E-02 ssc-miR-95 −2.04 9.49E-03 FC Fold Change, FDR False Discovery Rate damage in porcine COCs. Although oocyte staining cellular-specific markers. In addition, the RNA electro - with such stains is considered a non-invasive method, pherogram clearly showed the absence of the ribosomal it is still not the optimal approach for oocyte selection, RNA peaks after total RNA isolation. These verified the since further treatment and incubation steps need to be isolation procedure and confirmed the degree of purity of incorporated to evaluate the oocytes. Therefore, search - the s-EV preparations from other intracellular compart- ing for specific biomarkers in follicular cells or FFs which ments or cellular contamination [34]. are associated with oocyte quality could be a much Since the first reported evidence on the EV-medi- more appropriate strategy for oocyte quality assessment ated transfer of miRNAs between cells [11], EV-miR- and selection. To avoid any variations in the follicular NAs have been considered to be novel non-invasive stages or the physiological conditions of the donors, we molecular markers for the prediction and diagnosis collected the FFs from individual healthy ovarian fol- of various pathophysiological conditions [10]. From licles of similar size from prepubertal gilts within simi- the FFs, several EV-miRNAs have been reported to lar weight and age ranges. Then, we used LB staining to be associated with follicular and oocyte development differentiate between high- and low-quality COCs, and in different mammalian species including porcines we subsequently collected the corresponding FFs for [35], humans [36], bovines [37, 38], and equines [39]. each category for s-EVs isolation and miRNA identifica - However, the mechanism of EV-miRNAs in FFs that tion. We characterized the s-EVs isolated from porcine influences the oocyte developmental competence FFs using three different methods. The size distribu - remains unclear. In this study, miRNA sequencing tion, shape, and the analysis of specific protein markers analysis identified a total of 42 significantly DE miR- revealed the successful isolation of s-EVs in agreement NAs between the HQ and LQ s-EV groups. Among with the minimal information for studies of extracellu- the DE miRNAs, the mir-9 family-related miRNAs lar vesicles (MISEV2018) [34]. Importantly, s-EV sam- (miR-9-1 and miR-9) were the most highly elevated ples were negative for CytC and ATP5A proteins as miRNAs in the LQ compared to the HQ group, with G ad et al. Journal of Animal Science and Biotechnology (2022) 13:82 Page 11 of 16 Fig. 5 Pathway analysis of differentially expressed miRNA target genes. Bubble plots for the pathways targeted by the elevated miRNAs in the high (A) and low quality (B) s-EVs groups and by the top five most abundant miRNAs in both groups (C) . The color and size of each bubble represent the P-value and the number of miRNA target genes in each pathway, respectively. Exclusive and common pathways targeted by elevated miRNAs in the high- (HQ) and low-quality (LQ) s-EVs groups and by the top five most abundant miRNAs in both groups (D) more than 50-fold miR-9-1 in the LQ group. The large follicles. These findings may indicate a possible miR-9 family was previously known to play roles as correlation between the cellular and/or extracellular repressor mediators of proliferation promoting tran- expression of miR-9 with the oocyte quality. In addi- scription factors [40] and as tumor suppressors [41]. tion, miR-101 was among the upregulated miRNAs In humans, miR-9 exhibited a higher expression in the in the LQ compared to the HQ group and was one FFs (104 folds) [42] and granulosa cells [43] of women of the highly abundant DE-miRNAs. Recently, it has with polycystic ovary syndrome (PCOS) compared to been reported that miR-101-3p inhibits goat granu- normal women. It has been previously reported that losa cells in  vitro proliferation by regulating CDK4, PCOS is highly correlated with poor oocyte qual- CCND1, CCNE1, and PCNA expressions and promotes ity and subsequently low developmental competence the apoptotic rate by regulating Bcl-2, Bax, p53, and due to consecutive disturbances in the paracrine and/ caspase3 expression [46]. The same study reported or endocrine follicular microenvironment [44, 45]. that, in mouse ovaries, miR-101-3p exhibited unu- Moreover, in our previous study [26] we reported an sual ovarian development functions, as reflected by increase in the expression of miR-9-1 in low-compe- decreased numbers of various follicles as well as small tence porcine oocytes derived from small compared to and stunted ovarian fragments. Another interesting Gad et al. Journal of Animal Science and Biotechnology (2022) 13:82 Page 12 of 16 Fig. 6 Droplet digital PCR (ddPCR) validation of the selected DE-miRNAs in comparison to the small RNAseq (sRNAseq) analysis. *Statistical significance between the high- (HQ) and low- quality (LQ) s-EVs groups (P < 0.05) group of miRNAs including miR-206, miR-133b, and oocytes in this study. On the other hand, a group of miR-133a-3p showed a significant up-regulation in the 19 miRNAs including miR-132 and miR-320 (one of LQ compared to the HQ group. The same three miR- the highly abundant DE-miRNAs) was significantly NAs exhibited a higher expression pattern in the EVs up-regulated in the FF-EVs of the corresponding HQ isolated from the blood plasma of low- compared to oocytes. Similar to our findings, miR-132 and miR-320 high-response heifers to ovarian stimulation [47]. Both were detected as extracellular miRNAs with a higher miR-206 and miR-133a are highly correlated with E2 expression level in human ovarian FFs of oocytes that deficiency by targeting and reducing the expression yielded top-quality embryos [55]. Moreover, both of E2 receptor-α, which mediates the biological activ- miRNAs were highly expressed in the ovarian FFs of ity of E2 in ovarian follicular cells and subsequently healthy compared to PCOS patients [56]. In another affects oocyte quality [48–51]. In mice, theca-spe- study, the expression of miR-320 in FFs was posi- cific E2 receptor-α knockout leads to a reduction in tively correlated with human embryonic quality and the oocyte quality and decreased ovulation capacity development. The same study showed that miR-320 [52]. Recently, it has been demonstrated that miR- knockdown in mouse oocytes strongly decreased their 206 decreased the viability and induced apoptosis developmental competence [57]. The expression level levels in ovarian granulosa cells by targeting CCND2 of miR-132 was higher in the FFs of equine preovula- mRNA [53]. Similarly, miR-133a was reported to be a tory compared to dominant follicles, with an indica- cell proliferation inhibitor and apoptosis promotor in tion of the physiological involvement of miR-132 in the intestinal epithelial cells [54]. These could explain steroidogenesis, follicle selection, and ovulation [58]. the higher expression of this group of miRNAs in the Other interesting miRNAs which were highly abun- FF-EVs of the LQ compared to the corresponding HQ dant in the FF-EVs and exhibited a higher expression Table 3 Maturation and developmental rates of porcine oocytes co-incubated with s-EVs isolated from HQ or LQ FF groups Group MII rate Cleavage rate Blastocyst rate Blastocyst cell % ± SD (n) % ± SD (n) % ± SD (n) number ± SD (n) C 90.07 ± 9.95% (47) 85.34 ± 7.21% (124) 32.40 ± 2.37% (124) 39 ± 5 (27) NC 85.35 ± 10.95% (55) 86.79 ± 6.17% (125) 29.33 ± 7.93% (125) 39 ± 10 (17) HQ 75.74 ± 14.29% (53) 76.68 ± 3.83% (129) 25.38 ± 5.13% (129) 42 ± 6 (20) LQ 87.74 ± 5.75% (55) 75.84 ± 7.98% (154) 25.70 ± 7.71% (154) 45 ± 4 (18) C Control, NC negative control, HQ high quality, LQ low quality G ad et al. Journal of Animal Science and Biotechnology (2022) 13:82 Page 13 of 16 Fig. 7 Mitochondrial activity and distribution patterns in porcine MII oocytes. A Oocytes were classified into three main distribution patterns: Homogeneous (left), Heterogeneous (middle), or Cluster (right). Scale bar, 60 μm. B The proportion of each distribution pattern in porcine MII oocytes after co-incubation with s-EVs of high- or low-quality groups. Bars with different letters indicate significant differences (P < 0.05). C The relative intensity signal of mitochondrial activity in the different oocyte groups. C: control; NC: negative control; HQ: high-quality; LQ: low-quality level in the HQ compared to the LQ group are miR- cells was associated with an increase in endogenous 125a and miR-125b. Both are members of the mir-10 miRNA levels and altered gene expression in in  vitro family and were detected in human FF microvesicles cultured follicular cells [12]. However, the results of [56]. A recent study demonstrated that miR-125a can studies on the effect of EVs on developmental rates con - be transcribed by all follicular components and play tradict each other. For instance, de Ávila et al. reported a role in intercellular communication within the folli- that the supplementation of bovine oocytes with FF- cle [59]. In mice, miR-125a and miR-125b were highly isolated EVs does not affect the maturation rate [37]. expressed in GV compared to MII oocytes, and it was Moreover, in pigs, it has been suggested that exosomes also found that they play an important role in regulat- from FFs are not effective in inducing cumulus cell ing maternal genes and zygotic genome activation [60]. expansion [61]. In contrast, bovine follicular EVs To investigate the effect of s-EVs on porcine oocyte induced mouse and bovine cumulus cell expansion [38] maturation and embryonic development, we performed and improve oocyte competence and survival of heat a functional experiment by coincubating COCs with stress [62]. Several factors including the source of EVs, s-EVs of the HQ or LQ groups. However, we didn’t isolation method, concentration, and incubation time observe any significant differences in maturation or could influence the extent of the EV impact on oocyte embryonic developmental rates after coincubation or embryonic developmental competence, which could compared to the control groups. In different mamma - explain these contrary results. For instance, the supple- lian species, several studies have demonstrated that, mentation of EVs from early antral follicles increased during in  vitro culture, EVs could be uptaken by gran- bovine blastocyst rates compared to the control, how- ulosa or cumulus cells and could be found within the ever, EVs derived from pre-ovulatory follicles exhib- zona pellucida and transzonal projections of cumulus ited no significant difference [63]. In another study, the cells [37–39]. Moreover, the uptake of EVs by follicular supplementation of maturation media with follicular Gad et al. Journal of Animal Science and Biotechnology (2022) 13:82 Page 14 of 16 component analysis; PCOS: Polycystic ovary syndrome; PZM 3: Porcine zygote EVs derived by density gradient ultracentrifugation medium 3; PXM-HEPES: HEPES buffered porcine X medium; SDS: Sodium improved the blastocyst rate compared to EVs derived dodecyl sulphate; sRNAseq: Small RNAseq; s-EV: Small-EV; sncRNA: Small non- by size-exclusion chromatography [64]. In the same coding RNA; TBS: Tris buffered saline; TEM: Transmission electron microscopy; TMM: Trimmed mean of M-values normalization. study, EVs derived by both isolation methods improved embryo quality measured as blastocyst cell number and Supplementary Information apoptotic cell ratio. More studies considering these fac- The online version contains supplementary material available at https:// doi. tors are needed to optimize the EV co-culture condi- org/ 10. 1186/ s40104- 022- 00723-1. tions and to determine the mechanisms that regulate oocyte and embryonic development under in vitro con- Additional file 1: Table S1. Summary of sequence reads mapped to the ditions. Our results showed that a higher proportion of porcine reference genome and annotated against porcine miRNAs listed in the mirBase database. Table S2. A complete list of all expressed miRNAs MII oocytes that were co-cultured with the LQ s-EVs in the high (HQ) and low (LQ) quality s-EVs groups indicated as TMM- exhibited a homogeneous mitochondrial distribution adjusted Counts Per Million (CPM). Table S3. KEGG pathway analysis for pattern, and a lower proportion of them was in the het- genes targeted by the differentially expressed miRNAs in HQ vs. LQ group and top 5 miRNAs in both groups. erogeneous pattern compared to the other groups. It is Additional file 2: Fig. S1. Mapped reads proportions of small non-coding well known that homogeneous and heterogeneous dis- RNA types in high- (HQ) and low-quality (LQ) s-EVs. tribution patterns of mitochondria are more commonly Additional file 3: Fig. S2. Interaction networking of genes involved in observed in GV and MII oocytes, respectively [28]. As PI3K-Akt (A) and MAPK signaling (B) pathways and targeted by elevated oocyte maturation progresses, the mitochondrial dis- miRNAs in high- (HQ) and low-quality (LQ) s-EVs groups and by top five tribution changes from homogeneous to a heteroge- most abundant miRNAs in both groups. neous pattern as a sign of cytoplasmic maturation [28, 65]. Several studies reported that released EVs could Acknowledgements We acknowledge the Electron Microscopy Core Facility, IMG CAS, Prague, regulate the function and composition of mitochondria Czech Republic, supported by MEYS CR (LM2018129 Czech-BioImaging) and in receptor cells via their metabolite, miRNA, and pro- ERDF (projects: Z.02.1.01/0.0/0.0/18_046/0016045, CZ.02.1.01/0.0/0.0/16_01 tein cargos (reviewed in [66]). Not only that, but whole 3/0001775) for their support with obtaining the microscopic data presented in this paper. mitochondria or their component parts could be trans- ferred between cells via EVs [67]. This could explain the Authors’ contributions negative effect of LQ-EVs on the cytoplasmic matura - Conceptualization: AG; Experimental work: AG, MM, AB, VK, RP; Data analysis: AG, MM, AB, VK; Interpretation of data: AG, MM, AB, RP; Funding acquisition: tion of MII oocytes by affecting the mitochondrial dis - AG, JL, RP; Literature review: AG, KM; Writing the original draft: AG, MM. All the tribution pattern. However, the precise mechanism by authors reviewed and approved the final version of the manuscript. which the EVs could regulate oocyte mitochondrial dis- Funding tribution is still unclear. This work was supported by the Institute of Animal Physiology and Genetics “IAPG-Matoušek Award 2020” Grant number: DRMA-2020-0002 and the Minis- Conclusion try of Education, Youth and Sports of the Czech Republic, Operational Program Research, Development and Education, the project “EXCELLENCE in molecular Our results indicated that s-EVs purified from porcine aspects of the early development of vertebrates” Grant number: CZ.02.1.01/0.0 ovarian FFs contain different miRNA cargos that are /0.0/15_003/0000460. associated with the quality of the corresponding oocytes. Availability of data and materials These miRNAs could be used as non-invasive biomarkers The datasets supporting the conclusions of this article are included within for oocyte selection. Moreover, the supplementation of the article and as additional files. The raw FASTQ files and processed CSV files maturation media with s-EVs of the LQ FF group modu- have been deposited in the NCBI’s Gene Expression Omnibus (GEO) with an accession number GSE181182. All relevant data regarding s-EV isolation and lates the cytoplasmic maturation of the matured oocytes characterization were submitted to the EV-TRACK knowledgebase with an by affecting the mitochondrial distribution patterns. EV-TRACK ID EV210251. Further functional studies on the mechanisms by which FF-EVs and their molecular cargos could regulate and Declarations maintain oocyte developmental competence will enhance Consent for publication ART outcomes. Not applicable. Competing interests Abbreviations The authors declare that they have no competing interests. ARTs: Assisted reproductive technologies; BCB: Brilliant cresyl blue; C: Control group; COCs: Cumulus-oocyte complexes; CPM: Counts per million; ddPCR: Author details Droplet digital PCR; DE: Differentially expressed; EVs: Extracellular vesicles; FC: Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Fold change; FFs: Follicular fluids; GEO: Gene expression omnibus; HQ: High 27721 Liběchov, Czech Republic. Department of Animal Production, Faculty quality; LB: Lissamine green B stain; LQ: Low quality; MII: Metaphase II; miRNA: of Agriculture, Cairo University, Giza 12613, Egypt. Department of Botany MicroRNA; NC: Negative control group (PBS-supplemented group); NGS: Next and Genetics, Faculty of Natural Sciences, Constantine the Philosopher Univer- generation sequencing; NTA: Nanoparticle tracking analysis; PCA: Principle sity in Nitra, 94901 Nitra, Slovakia. G ad et al. 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Journal

Journal of Animal Science and Biotechnology – Springer Journals

Published: Jun 20, 2022

Keywords: Extracellular vesicles; Follicular fluids; MiRNA; Oocyte quality; Porcine

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