Oocyte developmental competence is regulated by various mechanisms and molecules including microRNAs (miRNAs). However, the functions of many of these miRNAs in oocyte and embryo development are still unclear. In this study, we managed to manipulate the expression level of miR-152 during oocyte maturation to figure out its potential role in determining the developmental competence of porcine oocytes. The inhibition (Inh) of miR-152 during oocyte maturation does not affect the MII and cleavage rates, however it significantly enhances the blastocyst rate compared to the overexpression (OvExp) and control groups. Pathway analysis identified several signaling pathways (including PI3K/AKT, TGFβ, Hippo, FoxO, and Wnt signaling) that are enriched in the predicted target genes of miR-152. Gene expression analysis revealed that IGF1 was significantly up-regulated in the Inh group and downregulated in the OvExp group of oocytes. Moreover, IGF1R was significantly upregulated in the Inh oocyte group compared to the control one and IGFBP6 was downregulated in the Inh oocyte group compared to the other groups. Blastocysts developed from the OvExp oocytes exhibited an increase in miR-152 expression, dysregulation in some quality-related genes, and the lowest rate of blastocyst formation. In conclusion, our results demonstrate a negative correlation between miR-152 expression level and blastocyst rate in pigs. This correlation could be through targeting IGF system components during oocyte development.

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

Faculty of Natural Sciences Constantine the Philosopher University in Nitra 94901 Nitra Slovakia

Laboratory of Developmental Biology Institute of Animal Physiology and Genetics of the Czech Academy of Sciences 27721 Libechov Czech Republic

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Christou-Kent M., Dhellemmes M., Lambert E., Ray P.F., Arnoult C. Diversity of RNA-Binding Proteins Modulating Post-Transcriptional Regulation of Protein Expression in the Maturing Mammalian Oocyte. Cells. 2020;9:662. doi: 10.3390/cells9030662. PubMed DOI PMC

Weil T.T. Post-transcriptional regulation of early embryogenesis. F1000Prime Rep. 2015;7 doi: 10.12703/P7-31. PubMed DOI PMC

Bartel D.P. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell. 2004;116:281–297. doi: 10.1016/S0092-8674(04)00045-5. PubMed DOI

Reza A.M.M.T., Choi Y.-J., Han S.G., Song H., Park C., Hong K., Kim J.-H. Roles of microRNAs in mammalian reproduction: From the commitment of germ cells to peri-implantation embryos. Biol. Rev. 2019;94:415–438. doi: 10.1111/brv.12459. PubMed DOI PMC

Sinha P.B., Tesfaye D., Rings F., Hossien M., Hoelker M., Held E., Neuhoff C., Tholen E., Schellander K., Salilew-Wondim D. MicroRNA-130b is involved in bovine granulosa and cumulus cells function, oocyte maturation and blastocyst formation. J. Ovarian Res. 2017;10:37. doi: 10.1186/s13048-017-0336-1. PubMed DOI PMC

Chen H., Liu C., Jiang H., Gao Y., Xu M., Wang J., Liu S., Fu Y., Sun X., Xu J., et al. Regulatory Role of miRNA-375 in Expression of BMP15/GDF9 Receptors and its Effect on Proliferation and Apoptosis of Bovine Cumulus Cells. Cell. Physiol. Biochem. 2017;41:439–450. doi: 10.1159/000456597. PubMed DOI

Gittens J.E.I., Barr K.J., Vanderhyden B.C., Kidder G.M. Interplay between paracrine signaling and gap junctional communication in ovarian follicles. J. Cell Sci. 2005;118:113–122. doi: 10.1242/jcs.01587. PubMed DOI

Abd El Naby W.S., Hagos T.H., Hossain M.M., Salilew-Wondim D., Gad A.Y., Rings F., Cinar M.U., Tholen E., Looft C., Schellander K., et al. Expression analysis of regulatory microRNAs in bovine cumulus oocyte complex and preimplantation embryos. Zygote. 2013;21:31–51. doi: 10.1017/S0967199411000566. PubMed DOI

Miles J.R., McDaneld T.G., Wiedmann R.T., Cushman R.A., Echternkamp S.E., Vallet J.L., Smith T.P.L. MicroRNA expression profile in bovine cumulus-oocyte complexes: Possible role of let-7 and miR-106a in the development of bovine oocytes. Anim. Reprod. Sci. 2012;130:16–26. doi: 10.1016/j.anireprosci.2011.12.021. PubMed DOI

Tang F., Kaneda M., O’Carroll D., Hajkova P., Barton S.C., Sun Y.A., Lee C., Tarakhovsky A., Lao K., Surani M.A. Maternal microRNAs are essential for mouse zygotic development. Genes Dev. 2007;21:644–648. doi: 10.1101/gad.418707. PubMed DOI PMC

Chen L., Hu X., Dai Y., Li Q., Wang X., Li Q., Xue K., Li Y., Liang J., Wang Y., et al. MicroRNA-27a activity is not suppressed in porcine oocytes. Front. Biosci. 2012;4:2679–2685. doi: 10.2741/e574. PubMed DOI

Pan B., Toms D., Shen W., Li J. MicroRNA-378 regulates oocyte maturation via the suppression of aromatase in porcine cumulus cells. Am. J. Physiol. Endocrinol. Metab. 2015;308:E525–E534. doi: 10.1152/ajpendo.00480.2014. PubMed DOI PMC

Zhang J., Wang Y., Liu X., Jiang S., Zhao C., Shen R., Guo X., Ling X., Liu C. Expression and Potential Role of microRNA-29b in Mouse Early Embryo Development. Cell. Physiol. Biochem. 2015;35:1178–1187. doi: 10.1159/000373942. PubMed DOI

Liu X., Li J., Qin F., Dai S. miR-152 as a tumor suppressor microRNA: Target recognition and regulation in cancer. Oncol. Lett. 2016;11:3911–3916. doi: 10.3892/ol.2016.4509. PubMed DOI PMC

Ge S., Wang D., Kong Q., Gao W., Sun J. Function of miR-152 as a tumor suppressor in human breast cancer by targeting PIK3CA. Oncol. Res. 2017;25:1363–1371. doi: 10.3727/096504017X14878536973557. PubMed DOI PMC

Gad A., Nemcova L., Murin M., Kanka J., Laurincik J., Benc M., Pendovski L., Prochazka R. 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., 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

Ireland J.J., Murphee R.L., Coulson P.B. 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

Surani M.A., Barton S.C. Development of gynogenetic eggs in the mouse: Implications for parthenogenetic embryos. Science. 1983;222:1034–1036. doi: 10.1126/science.6648518. PubMed DOI

Yoshioka K., Suzuki C., Tanaka A., Anas I.M.-K., Iwamura S. Birth of Piglets Derived from Porcine Zygotes Cultured in a Chemically Defined Medium1. Biol. Reprod. 2002;66:112–119. doi: 10.1095/biolreprod66.1.112. PubMed DOI

Mahdipour M., van Tol H.T.A., Stout T.A.E., Roelen B.A.J. Validating reference microRNAs for normalizing qRT-PCR data in bovine oocytes and preimplantation embryos. BMC Dev. Biol. 2015;15:25. doi: 10.1186/s12861-015-0075-8. PubMed DOI PMC

Xiao F., Zuo Z., Cai G., Kang S., Gao X., Li T. miRecords: An integrated resource for microRNA-target interactions. Nucleic Acids Res. 2009;37:D105–D110. doi: 10.1093/nar/gkn851. PubMed DOI PMC

Huang D.W., Sherman B.T., Lempicki R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 2009;4:44–57. doi: 10.1038/nprot.2008.211. PubMed DOI

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., Fridman W.-H., Pagès F., Trajanoski Z., Galon J. 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

Pasquariello R., Manzoni E.F.M., Fiandanese N., Viglino A., Pocar P., Brevini T.A.L., Williams J.L., Gandolfi F. Implications of miRNA expression pattern in bovine oocytes and follicular fluids for developmental competence. Theriogenology. 2020;145:77–85. doi: 10.1016/j.theriogenology.2020.01.027. PubMed DOI

Song C., Yao J., Cao C., Liang X., Huang J., Han Z., Zhang Y., Qin G., Tao C., Li C., et al. PPARγ is regulated by miR-27b-3p negatively and plays an important role in porcine oocyte maturation. Biochem. Biophys. Res. Commun. 2016;479:224–230. doi: 10.1016/j.bbrc.2016.09.046. PubMed DOI

Tesfaye D., Worku D., Rings F., Phatsara C., Tholen E., Schellander K., Hoelker M. Identification and expression profiling of microRNAs during bovine oocyte maturation using heterologous approach. Mol. Reprod. Dev. 2009;76:665–677. doi: 10.1002/mrd.21005. PubMed DOI

Xu Y.-W., Wang B., Ding C.-H., Li T., Gu F., Zhou C. Differentially expressed micoRNAs in human oocytes. J. Assist. Reprod. Genet. 2011;28:559–566. doi: 10.1007/s10815-011-9590-0. PubMed DOI PMC

Mondou E., Dufort I., Gohin M., Fournier E., Sirard M.-A. Analysis of microRNAs and their precursors in bovine early embryonic development. MHR Basic Sci. Reprod. Med. 2012;18:425–434. doi: 10.1093/molehr/gas015. PubMed DOI

Li X., Wang H., Sheng Y., Wang Z. MicroRNA-224 delays oocyte maturation through targeting Ptx3 in cumulus cells. Mech. Dev. 2017;143:20–25. doi: 10.1016/j.mod.2016.12.004. PubMed DOI

Dehghan Z., Mohammadi-Yeganeh S., Salehi M. MiRNA-155 regulates cumulus cells function, oocyte maturation, and blastocyst formation. Biol. Reprod. 2020;103:548–559. doi: 10.1093/biolre/ioaa098. PubMed DOI

Kim Y.J., Ku S.-Y., Kim Y.Y., Liu H.C., Chi S.W., Kim S.H., Choi Y.M., Kim J.G., Moon S.Y. MicroRNAs transfected into granulosa cells may regulate oocyte meiotic competence during in vitro maturation of mouse follicles. Hum. Reprod. 2013;28:3050–3061. doi: 10.1093/humrep/det338. PubMed DOI

Ji W., Yang L., Yuan J., Yang L., Zhang M., Qi D., Duan X., Xuan A., Zhang W., Lu J., et al. MicroRNA-152 targets DNA methyltransferase 1 in NiS-transformed cells via a feedback mechanism. Carcinogenesis. 2013;34:446–453. doi: 10.1093/carcin/bgs343. PubMed DOI

Wang Z.-N. Altered expression of miR-152 and miR-148a in ovarian cancer is related to cell proliferation. Oncol. Rep. 2011;27:447–454. doi: 10.3892/or.2011.1482. PubMed DOI

Xu Q., Jiang Y., Yin Y., Li Q., He J., Jing Y., Qi Y.-T., Xu Q., Li W., Lu B., et al. A regulatory circuit of miR-148a/152 and DNMT1 in modulating cell transformation and tumor angiogenesis through IGF-IR and IRS1. J. Mol. Cell Biol. 2013;5:3–13. doi: 10.1093/jmcb/mjs049. PubMed DOI PMC

Gilchrist G.C., Tscherner A., Nalpathamkalam T., Merico D., LaMarre J. MicroRNA Expression during Bovine Oocyte Maturation and Fertilization. Int. J. Mol. Sci. 2016;17:396. doi: 10.3390/ijms17030396. PubMed DOI PMC

Hennebold J.D. Characterization of the ovarian transcriptome through the use of differential analysis of gene expression methodologies. Hum. Reprod. Update. 2004;10:227–239. doi: 10.1093/humupd/dmh017. PubMed DOI

Giudice L.C. Insulin-like growth factors and ovarian follicular development. Endocr. Rev. 1992;13:641–669. doi: 10.1210/edrv-13-4-641. PubMed DOI

Chen P., Pan Y., Cui Y., Wen Z., Liu P., He H., Li Q., Peng X., Zhao T., Yu S. Insulin-like growth factor I enhances the developmental competence of yak embryos by modulating aquaporin 3. Reprod. Domest. Anim. 2017;52:825–835. doi: 10.1111/rda.12985. PubMed DOI

Kowalik A., Liu H.-C., He Z.-Y., Mele C., Barmat L., Rosenwaks Z. Expression of the insulin-like growth factor-1 gene and its receptor in preimplantation mouse embryos; is it a marker of embryo viability? MHR Basic Sci. Reprod. Med. 1999;5:861–865. doi: 10.1093/molehr/5.9.861. PubMed DOI

Toori M.A., Mosavi E., Nikseresht M., Barmak M.J., Mahmoudi R. Influence of insulin-like growth factor-i on maturation and fertilization rate of immature oocyte and embryo development in NMRI mouse with TCM199 and α-MEM medium. J. Clin. Diagnostic Res. 2014;8:AC05–AC08. doi: 10.7860/JCDR/2014/9129.5242. PubMed DOI PMC

Yang R., Xiong X., Zi X. Effect of cysteine, insulin-like growth factor-1 and epidermis growth factor during in vitro oocyte maturation and in vitro culture of yak-cattle crossbred embryos. J. Appl. Anim. Res. 2019;47:463–466. doi: 10.1080/09712119.2019.1663353. DOI

Shabankareh H.K., Zandi M. Developmental potential of sheep oocytes cultured in different maturation media: Effects of epidermal growth factor, insulin-like growth factor I, and cysteamine. Fertil. Steril. 2010;94:335–340. doi: 10.1016/j.fertnstert.2009.01.160. PubMed DOI

Yu Y., Yan J., Li M., Yan L., Zhao Y., Lian Y., Li R., Liu P., Qiao J. Effects of combined epidermal growth factor, brain-derived neurotrophic factor and insulin-like growth factor-1 on human oocyte maturation and early fertilized and cloned embryo development. Hum. Reprod. 2012;27:2146–2159. doi: 10.1093/humrep/des099. PubMed DOI

Oberlender G., Murgas L.D.S., Zangeronimo M.G., da Silva A.C., Menezes T.d.A., Pontelo T.P., Vieira L.A. Role of insulin-like growth factor-I and follicular fluid from ovarian follicles with different diameters on porcine oocyte maturation and fertilization in vitro. Theriogenology. 2013;80:319–327. doi: 10.1016/j.theriogenology.2013.04.018. PubMed DOI

Xiao G., Xia C., Yang J., Liu J., Du H., Kang X., Lin Y., Guan R., Yan P., Tang S. MiR-133b Regulates the Expression of the Actin Protein TAGLN2 during Oocyte Growth and Maturation: A Potential Target for Infertility Therapy. PLoS ONE. 2014;9:e100751. doi: 10.1371/journal.pone.0100751. PubMed DOI PMC

Wen Y.-Y., Liu W.-T., Sun H.-R., Ge X., Shi Z.-M., Wang M., Li W., Zhang J.-Y., Liu L.-Z., Jiang B.-H. IGF-1-mediated PKM2/β-catenin/miR-152 regulatory circuit in breast cancer. Sci. Rep. 2017;7:15897. doi: 10.1038/s41598-017-15607-y. PubMed DOI PMC

Jones J.I., Clemmons D.R. Insulin-like growth factors and their binding proteins: Biological actions. Endocr. Rev. 1995;16:3–34. doi: 10.1210/edrv-16-1-3. PubMed DOI

Nuttinck F., Charpigny G., Mermillod P., Loosfelt H., Meduri G., Freret S., Grimard B., Heyman Y. Expression of components of the insulin-like growth factor system and gonadotropin receptors in bovine cumulus–oocyte complexes during oocyte maturation. Domest. Anim. Endocrinol. 2004;27:179–195. doi: 10.1016/j.domaniend.2004.03.003. PubMed DOI

Gad A., Nemcova L., Murin M., Kinterova V., Kanka J., Laurincik J., Benc M., Pendovski L., Prochazka R. Global transcriptome analysis of porcine oocytes in correlation with follicle size. Mol. Reprod. Dev. 2020;87:102–114. doi: 10.1002/mrd.23294. PubMed DOI

Menchero S., Rayon T., Andreu M.J., Manzanares M. Signaling pathways in mammalian preimplantation development: Linking cellular phenotypes to lineage decisions. Dev. Dyn. 2017;246:245–261. doi: 10.1002/dvdy.24471. PubMed DOI

Wang Q.T., Piotrowska K., Ciemerych M.A., Milenkovic L., Scott M.P., Davis R.W., Zernicka-Goetz M. A Genome-Wide Study of Gene Activity Reveals Developmental Signaling Pathways in the Preimplantation Mouse Embryo. Dev. Cell. 2004;6:133–144. doi: 10.1016/S1534-5807(03)00404-0. PubMed DOI

Aparicio I.M., Garcia-Herreros M., Fair T., Lonergan P. Identification and regulation of glycogen synthase kinase-3 during bovine embryo development. Reproduction. 2010;140:83–92. doi: 10.1530/REP-10-0040. PubMed DOI

Ashry M., Rajput S.K., Folger J.K., Knott J.G., Hemeida N.A., Kandil O.M., Ragab R.S., Smith G.W. Functional role of AKT signaling in bovine early embryonic development: Potential link to embryotrophic actions of follistatin. Reprod. Biol. Endocrinol. 2018;16:22. doi: 10.1186/s12958-017-0318-6. PubMed DOI PMC

Nie L., Zhao Y., Zhao D., Long Y., Lei Y., Liu M., Wang Y., Zhang X., Zhang J., Yuan D., et al. Progesterone-induced miR-152 interferes with embryonic implantation by downregulating GLUT3 in endometrial epithelium. Am. J. Physiol. Metab. 2019;316:E557–E567. doi: 10.1152/ajpendo.00245.2018. PubMed DOI

Ali T., Mushtaq I., Maryam S., Farhan A., Saba K., Jan M.I., Sultan A., Anees M., Duygu B., Hamera S., et al. Interplay of N acetyl cysteine and melatonin in regulating oxidative stress-induced cardiac hypertrophic factors and microRNAs. Arch. Biochem. Biophys. 2019;661:56–65. doi: 10.1016/j.abb.2018.11.007. PubMed DOI

Guerin P. Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings. Hum. Reprod. Update. 2001;7:175–189. doi: 10.1093/humupd/7.2.175. PubMed DOI

Cao Y.H., Li D.G., Xu B., Wang M.Q., Zhen N., Man L.X., Zhang Y.Y., Chi M. A microRNA-152 that targets the phosphatase and tensin homolog to inhibit low oxygen induced-apoptosis in human brain microvascular endothelial cells. Genet. Mol. Res. 2016;15 doi: 10.4238/gmr.15027371. PubMed DOI

Physiologically relevant miRNAs in mammalian oocytes are rare and highly abundant