BACKGROUND: Ovarian follicular fluids (FFs) contain several kinds of regulatory factors that maintain a suitable microenvironment 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 quality, 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 developmental 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.

Department of Animal Production Faculty of Agriculture Cairo University Giza 12613 Egypt

Department of Botany and Genetics Faculty of Natural Sciences Constantine the Philosopher University in Nitra 94901 Nitra Slovakia

Institute of Animal Physiology and Genetics Czech Academy of Sciences 27721 Liběchov Czech Republic

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Bukowska D, Kempisty B, Piotrowska H, Walczak R, Sniadek P, Dziuban J, et al. The invasive and new non-invasive methods of mammalian oocyte and embryo quality assessment: a review. Vet Med (Praha) 2012;57:169–176. doi: 10.17221/5913-VETMED. DOI

Aguila L, Treulen F, Therrien J, Felmer R, Valdivia M, Smith LC. Oocyte selection for in vitro embryo production in bovine species: noninvasive approaches for new challenges of oocyte competence. Animals. 2020;10:2196. doi: 10.3390/ani10122196. PubMed DOI PMC

Revelli A, Piane LD, Casano S, Molinari E, Massobrio M, Rinaudo P. Follicular fluid content and oocyte quality: from single biochemical markers to metabolomics. Reprod Biol Endocrinol. 2009;7:40. doi: 10.1186/1477-7827-7-40. PubMed DOI PMC

Matzuk MM, Burns KH, Viveiros MM, Eppig JJ. Intercellular communication in the mammalian ovary: oocytes carry the conversation. Science. 2002;296:2178–2180. doi: 10.1126/science.1071965. PubMed DOI

Yáñez-Mó M, Siljander PR-M, Andreu Z, Zavec AB, Borràs FE, Buzas EI, et al. Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles. 2015;4:27066. doi: 10.3402/jev.v4.27066. PubMed DOI PMC

Llobat L. Extracellular vesicles and domestic animal reproduction. Res Vet Sci. 2021;136:166–173. doi: 10.1016/j.rvsc.2021.02.016. PubMed DOI

Gervasi MG, Soler AJ, González-Fernández L, Alves MG, Oliveira PF, Martín-Hidalgo D. Extracellular vesicles, the road toward the improvement of ART outcomes. Animals. 2020;10:2171. doi: 10.3390/ani10112171. PubMed DOI PMC

Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116:281–297. doi: 10.1016/s0092-8674(04)00045-5. PubMed DOI

Weber JA, Baxter DH, Zhang S, Huang DY, Huang KH, Lee MJ, et al. The microRNA spectrum in 12 body fluids. Clin Chem. 2010;56:1733–1741. doi: 10.1373/clinchem.2010.147405. PubMed DOI PMC

Mori MA, Ludwig RG, Garcia-Martin R, Brandão BB, Kahn CR. Extracellular miRNAs: from biomarkers to mediators of physiology and disease. Cell Metab. 2019;30:656–673. doi: 10.1016/J.CMET.2019.07.011. PubMed DOI PMC

Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 2007;9:654–659. doi: 10.1038/ncb1596. PubMed DOI

Sohel MMH, Hoelker M, Noferesti SS, Salilew-Wondim D, Tholen E, Looft C, et al. Exosomal and non-exosomal transport of extra-cellular microRNAs in follicular fluid: implications for bovine oocyte developmental competence. PLoS One. 2013;8:e78505. doi: 10.1371/journal.pone.0078505. PubMed DOI PMC

Martinez RM, Liang L, Racowsky C, Dioni L, Mansur A, Adir M, et al. Extracellular microRNAs profile in human follicular fluid and IVF outcomes. Sci Rep. 2018;8:17036. doi: 10.1038/s41598-018-35379-3. PubMed DOI PMC

Scalici E, Traver S, Mullet T, Molinari N, Ferrières A, Brunet C, et al. Circulating microRNAs in follicular fluid, powerful tools to explore in vitro fertilization process. Sci Rep. 2016;6:24976. doi: 10.1038/srep24976. PubMed DOI PMC

Ireland JJ, Murphee RL, Coulson PB. Accuracy of predicting stages of bovine estrous cycle by gross appearance of the corpus luteum. J Dairy Sci. 1980;63:155–160. doi: 10.3168/jds.S0022-0302(80)82901-8. PubMed DOI

Yoshioka K, Suzuki C, Onishi A. Defined system for in vitro production of porcine embryos using a single basic medium. J Reprod Dev. 2008;54:208–213. doi: 10.1262/jrd.20001. PubMed DOI

Bartkova A, Morovic M, Strejcek F, Murin M, Benc M, Percinic FP, et al. Characterization of porcine oocytes stained with Lissamine green B and their developmental potential in vitro. Anim Reprod. 2020;17:e20200533. doi: 10.1590/1984-3143-ar2020-0533. PubMed DOI PMC

Dutta R, Li S, Fischer K, Kind A, Flisikowska T, Flisikowski K, et al. Non-invasive assessment of porcine oocyte quality by supravital staining of cumulus-oocyte complexes with lissamine green B. Zygote. 2016;24:418–427. doi: 10.1017/S0967199415000349. PubMed DOI

Van Deun J, Mestdagh P, Agostinis P, Akay Ö, Anand S, Anckaert J, et al. EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research. Nat Methods. 2017;14:228–232. doi: 10.1038/nmeth.4185. PubMed DOI

Robinson MD, Oshlack A. A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol. 2010;11:R25. doi: 10.1186/gb-2010-11-3-r25. PubMed DOI PMC

Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Ser B. 1995;57:289–300. doi: 10.1111/j.2517-6161.1995.tb02031.x. DOI

Sticht C, De La Torre C, Parveen A, Gretz N. miRWalk: an online resource for prediction of microRNA binding sites. PLoS One. 2018;13:e0206239. doi: 10.1371/journal.pone.0206239. PubMed DOI PMC

Ogata H, Goto S, Sato K, Fujibuchi W, Bono H, Kanehisa M. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 1999;27:29–34. doi: 10.1093/nar/27.1.29. PubMed DOI PMC

Shannon P. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13:2498–2504. doi: 10.1101/gr.1239303. PubMed DOI PMC

Bindea G, Mlecnik B, Hackl H, Charoentong P, Tosolini M, Kirilovsky A, et al. ClueGO: a Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks. Bioinformatics. 2009;25:1091–1093. doi: 10.1093/bioinformatics/btp101. PubMed DOI PMC

Gad A, Nemcova L, Murin M, Kanka J, Laurincik J, Benc M, et al. microRNA expression profile in porcine oocytes with different developmental competence derived from large or small follicles. Mol Reprod Dev. 2019;86:426–439. doi: 10.1002/mrd.23121. PubMed DOI

Yoshioka K, Suzuki C, Tanaka A, Anas IM-K, Iwamura S. Birth of piglets derived from porcine zygotes cultured in a chemically defined medium. Biol Reprod. 2002;66:112–119. doi: 10.1095/biolreprod66.1.112. PubMed DOI

Torner H, Brüssow K-P, Alm H, Ratky J, Pöhland R, Tuchscherer A, et al. Mitochondrial aggregation patterns and activity in porcine oocytes and apoptosis in surrounding cumulus cells depends on the stage of pre-ovulatory maturation. Theriogenology. 2004;61:1675–1689. doi: 10.1016/j.theriogenology.2003.09.013. PubMed DOI

Egerszegi I, Alm H, Rátky J, Heleil B, Brüssow K-P, Torner H. Meiotic progression, mitochondrial features and fertilisation characteristics of porcine oocytes with different G6PDH activities. Reprod Fertil Dev. 2010;22:830–838. doi: 10.1071/RD09140. PubMed DOI

Sirard M-A, Richard F, Blondin P, Robert C. Contribution of the oocyte to embryo quality. Theriogenology. 2006;65:126–136. doi: 10.1016/j.theriogenology.2005.09.020. PubMed DOI

Fischer NM, Nguyen HV, Singh B, Baker VL, Segars JH. Prognostic value of oocyte quality in assisted reproductive technology outcomes: a systematic review. F S Rev. 2021;2:120–139. doi: 10.1016/j.xfnr.2021.03.001. DOI

Wu Y-G, Liu Y, Zhou P, Lan G-C, Han D, Miao D-Q, et al. Selection of oocytes for in vitro maturation by brilliant cresyl blue staining: a study using the mouse model. Cell Res. 2007;17:722–731. doi: 10.1038/cr.2007.66. PubMed DOI

Hamrah P, Alipour F, Jiang S, Sohn J-H, Foulks GN. Optimizing evaluation of Lissamine green parameters for ocular surface staining. Eye (Lond) 2011;25:1429–1434. doi: 10.1038/eye.2011.184. PubMed DOI PMC

Théry C, Witwer KW, Aikawa E, Alcaraz MJ, Anderson JD, Andriantsitohaina R, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 2018;7:1535750. doi: 10.1080/20013078.2018.1535750. PubMed DOI PMC

Inoue Y, Munakata Y, Shinozawa A, Kawahara-Miki R, Shirasuna K, Iwata H. Prediction of major microRNAs in follicular fluid regulating porcine oocyte development. J Assist Reprod Genet. 2020;37:2569–2579. doi: 10.1007/s10815-020-01909-0. PubMed DOI PMC

Machtinger R, Rodosthenous R, Mansour A, Adir M, Racowsky C, Hauser R, et al. Mirnas isolated from extracellular vesicles in follicular fluid and oocyte development potential. Fertil Steril. 2015;104:e54. doi: 10.1016/j.fertnstert.2015.07.162. DOI

de Ávila ACFCM, Bridi A, Andrade GM, del Collado M, Sangalli JR, Nociti RP, et al. Estrous cycle impacts microRNA content in extracellular vesicles that modulate bovine cumulus cell transcripts during in vitro maturation. Biol Reprod. 2020;102:362–375. doi: 10.1093/biolre/ioz177. PubMed DOI

Hung W-T, Hong X, Christenson LK, McGinnis LK. Extracellular vesicles from bovine follicular fluid support cumulus expansion. Biol Reprod. 2015;93:117–118. doi: 10.1095/biolreprod.115.132977. PubMed DOI PMC

da Silveira JC, Veeramachaneni DNR, Winger QA, Carnevale EM, Bouma GJ. Cell-secreted vesicles in equine ovarian follicular fluid contain miRNAs and proteins: a possible new form of cell communication within the ovarian follicle. Biol Reprod. 2012;86:71. doi: 10.1095/biolreprod.111.093252. PubMed DOI

Ristori E, Nicoli S. Comparative functions of miRNAs in embryonic neurogenesis and neuronal network formation. Essentials Noncoding RNA Neurosci. 2017:265–82. 10.1016/B978-0-12-804402-5.00015-7.

Zhang J, Cheng J, Zeng Z, Wang Y, Li X, Xie Q, et al. Comprehensive profiling of novel microRNA-9 targets and a tumor suppressor role of microRNA-9 via targeting IGF2BP1 in hepatocellular carcinoma. Oncotarget. 2015;6:42040–42052. doi: 10.18632/oncotarget.5969. PubMed DOI PMC

Roth LW, McCallie B, Alvero R, Schoolcraft WB, Minjarez D, Katz-Jaffe MG. Altered microRNA and gene expression in the follicular fluid of women with polycystic ovary syndrome. J Assist Reprod Genet. 2014;31:355–362. doi: 10.1007/s10815-013-0161-4. PubMed DOI PMC

Luo H, Han Y, Liu J, Zhang Y. Identification of microRNAs in granulosa cells from patients with different levels of ovarian reserve function and the potential regulatory function of miR-23a in granulosa cell apoptosis. Gene. 2019;686:250–260. doi: 10.1016/j.gene.2018.11.025. PubMed DOI

Patel SS, Carr BR. Oocyte quality in adult polycystic ovary syndrome. Semin Reprod Med. 2008;26:196–203. doi: 10.1055/s-2008-1042958. PubMed DOI

Decanter C. Oocyte quality in PCOS. In: Palomba S, editors. Infertility women with polycystic ovary syndrome. Cham: Springer; 2018. p. 31–9. 10.1007/978-3-319-45534-1_4.

An X, Ma H, Liu Y, Li F, Song Y, Li G, et al. Effects of miR-101-3p on goat granulosa cells in vitro and ovarian development in vivo via STC1. J Anim Sci Biotechnol. 2020;11:1–20. doi: 10.1186/S40104-020-00506-6/TABLES/2. PubMed DOI PMC

Gad A, Sánchez JM, Browne JA, Nemcova L, Laurincik J, Prochazka R, et al. Plasma extracellular vesicle miRNAs as potential biomarkers of superstimulatory response in cattle. Sci Rep. 2020;10:19130. doi: 10.1038/s41598-020-76152-9. PubMed DOI PMC

Adams BD, Furneaux H, White BA. The micro-ribonucleic acid (miRNA) miR-206 targets the human estrogen receptor-α (ERα) and represses ERα messenger RNA and protein expression in breast cancer cell lines. Mol Endocrinol. 2007;21:1132–1147. doi: 10.1210/me.2007-0022. PubMed DOI

Zhang Y. MiR-133 is involved in estrogen deficiency-induced osteoporosis through modulating osteogenic differentiation of mesenchymal stem cells. Med Sci Monit. 2015;21:1527–1534. doi: 10.12659/MSM.894323. PubMed DOI PMC

Hewitt S, Korach K. Oestrogen receptor knockout mice: roles for oestrogen receptors alpha and beta in reproductive tissues. Reproduction. 2003;125:143–149. doi: 10.1530/rep.0.1250143. PubMed DOI

Wang Y, Schatten H, Cui X-S, Sun S-C. Editorial: quality control of mammalian oocyte meiotic maturation: causes, molecular mechanisms and solutions. Front Cell Dev Biol. 2021;9:2163. doi: 10.3389/fcell.2021.736331. PubMed DOI PMC

Lee S, Kang D-W, Hudgins-Spivey S, Krust A, Lee E-Y, Koo Y, et al. Theca-specific estrogen receptor-alpha knockout mice lose fertility prematurely. Endocrinology. 2009;150:3855–3862. doi: 10.1210/en.2008-1774. PubMed DOI PMC

Zhou J, Jin X, Sheng Z, Zhang Z. miR-206 serves an important role in polycystic ovary syndrome through modulating ovarian granulosa cell proliferation and apoptosis. Exp Ther Med. 2021;21:179. doi: 10.3892/etm.2021.9610. PubMed DOI PMC

Tian X, Li L, Fu G, Wang J, He Q, Zhang C, et al. miR-133a-3p regulates the proliferation and apoptosis of intestinal epithelial cells by modulating the expression of TAGLN2. Exp Ther Med. 2021;22:824. doi: 10.3892/etm.2021.10256. PubMed DOI PMC

Machtinger R, Rodosthenous RS, Adir M, Mansour A, Racowsky C, Baccarelli AA, et al. Extracellular microRNAs in follicular fluid and their potential association with oocyte fertilization and embryo quality: an exploratory study. J Assist Reprod Genet. 2017;34:525–533. doi: 10.1007/s10815-017-0876-8. PubMed DOI PMC

Sang Q, Yao Z, Wang H, Feng R, Wang H, Zhao X, et al. Identification of microRNAs in human follicular fluid: characterization of microRNAs that govern steroidogenesis in vitro and are associated with polycystic ovary syndrome in vivo. J Clin Endocrinol Metab. 2013;98:3068–3079. doi: 10.1210/jc.2013-1715. PubMed DOI

Feng R, Sang Q, Zhu Y, Fu W, Liu M, Xu Y, et al. MiRNA-320 in the human follicular fluid is associated with embryo quality in vivo and affects mouse embryonic development in vitro. Sci Rep. 2015;5:8689. doi: 10.1038/srep08689. PubMed DOI PMC

Schauer SN, Sontakke SD, Watson ED, Esteves CL, Donadeu FX. Involvement of miRNAs in equine follicle development. Reproduction. 2013;146:273–282. doi: 10.1530/REP-13-0107. PubMed DOI

Grossman H, Har-Paz E, Gindi N, Miller I, Shalgi R. Pre-ovulatory intercellular regulation of miR-125a-3p within mouse ovarian follicles. Reproduction. 2020;159:215–225. doi: 10.1530/REP-19-0419. PubMed DOI

Kim K-H, Seo Y-M, Kim E-Y, Lee S-Y, Kwon J, Ko J-J, et al. The miR-125 family is an important regulator of the expression and maintenance of maternal effect genes during preimplantational embryo development. Open Biol. 2016;6:160181. doi: 10.1098/rsob.160181. PubMed DOI PMC

Matsuno Y, Onuma A, Fujioka YA, Yasuhara K, Fujii W, Naito K, et al. Effects of exosome-like vesicles on cumulus expansion in pigs in vitro. J Reprod Dev. 2017;63:51–58. doi: 10.1262/jrd.2016-124. PubMed DOI PMC

Rodrigues TA, Tuna KM, Alli AA, Tribulo P, Hansen PJ, Koh J, et al. Follicular fluid exosomes act on the bovine oocyte to improve oocyte competence to support development and survival to heat shock. Reprod Fertil Dev. 2019;31:888–897. doi: 10.1071/RD18450. PubMed DOI

da Silveira JC, Andrade GM, del Collado M, Sampaio RV, Sangalli JR, Silva LA, et al. Supplementation with small-extracellular vesicles from ovarian follicular fluid during in vitro production modulates bovine embryo development. PLoS One. 2017;12:e0179451. doi: 10.1371/journal.pone.0179451. PubMed DOI PMC

Asaadi A, Dolatabad NA, Atashi H, Raes A, Van Damme P, Hoelker M, et al. Extracellular vesicles from follicular and ampullary fluid isolated by density gradient ultracentrifugation improve bovine embryo development and quality. Int J Mol Sci. 2021;22:578. doi: 10.3390/ijms22020578. PubMed DOI PMC

Wang L, Wang D, Zou X, Xu C. Mitochondrial functions on oocytes and preimplantation embryos. J Zhejiang Univ Sci B. 2009;10:483–492. doi: 10.1631/jzus.B0820379. PubMed DOI PMC

Zhang Y, Tan J, Miao Y, Zhang Q. The effect of extracellular vesicles on the regulation of mitochondria under hypoxia. Cell Death Dis. 2021;12:358. doi: 10.1038/s41419-021-03640-9. PubMed DOI PMC

Valenti D, Vacca RA, Moro L, Atlante A. Mitochondria can cross cell boundaries: an overview of the biological relevance, pathophysiological implications and therapeutic perspectives of intercellular mitochondrial transfer. Int J Mol Sci. 2021;22:8312. doi: 10.3390/ijms22158312. PubMed DOI PMC