Animal Reproduction (AR)
Animal Reproduction (AR)

Nanoparticles from culture media are internalized by in vitro-produced bovine embryos and its depletion affect expression of pluripotency genes

Bárbara Melo-Báez; Edwin A. Mellisho; Joel Cabezas; Alejandra E. Velásquez; Daniel Veraguas; Diego Andrés Caamaño Escobar; Fidel O. Castro; Lleretny Rodríguez-Álvarez

Downloads: 0
Views: 46


Abstract: Extracellular vesicles are nanoparticles secreted by cell and have been proposed as suitable markers to identify competent embryos produced in vitro. Characterizing EVs secreted by individual embryos is challenging because culture medium itself contributes to the pool of nanoparticles that are co-isolated. To avoid this, culture medium must be depleted of nanoparticles that are present in natural protein source. The aim of this study was to evaluate if the culture medium subjected to nanoparticle depletion can support the proper in vitro development of bovine embryos. Zygotes were cultured in groups on depleted or control medium for 8 days. Nanoparticles from the medium were characterized by their morphology, size and expression of EVs surface markers. Isolated nanoparticles were labelled and added to depleted medium containing embryos at different developmental stages and evaluated after 24 hours at 2, 8-16 cells, morula and blastocyst stages. There were no statistical differences on blastocyst rate at day 7 and 8, total cell count neither blastocyst diameter between groups. However, morphological quality was better in blastocysts cultured in non-depleted medium and the expression of SOX2 was significantly lower whereas NANOG expression was significantly higher. Few nanoparticles from medium had a typical morphology of EVs but were positive to specific surface markers. Punctuated green fluorescence near the nuclei of embryonic cells was observed in embryos from all developmental stages. In summary, nanoparticles from culture medium are internalized by in vitro cultured bovine embryos and their depletion affects the capacity of medium to support the proper embryo development.


EVs, nanoparticles, culture medium, bovine embryos, pluripotency genes


Al-Dossary AA, Strehler EE, Martin-DeLeon PA. Expression and secretion of plasma membrane Ca2+-ATPase 4a (PMCA4a) during murine estrus: association with oviductal exosomes and uptake in sperm. PLoS One. 2013;8(11):e80181. PMid:24244642.

Almiñana C, Bauersachs S. Extracellular vesicles in the oviduct: progress, challenges and implications for the reproductive success. Bioengineering. 2019;6(2):32. PMid:31013857.

Almiñana C, Bauersachs S. Extracellular vesicles: multi-signal messengers in the gametes/embryo-oviduct cross-talk. Theriogenology. 2020;150:59-69. PMid:32088033.

Almiñana C, Corbin E, Tsikis G, Alcântara-Neto AS, Labas V, Reynaud K, Galio L, Uzbekov R, Garanina AS, Druart X, Mermillod P. Oviduct extracellular vesicles protein content and their role during oviduct–embryo cross-talk. Reproduction. 2017;154(3):253-68. PMid:28630101.

Almiñana C, Heath PR, Wilkinson S, Sanchez-Osorio J, Cuello C, Parrilla I, Gil MA, Vazquez JL, Vazquez JM, Roca J, Martinez EA, Fazeli A. Early developing pig embryos mediate their own environment in the maternal tract. PLoS One. 2012;7(3):e33625. PMid:22470458.

Almiñana C, Tsikis G, Labas V, Uzbekov R, da Silveira JC, Bauersachs S, Mermillod P. Deciphering the oviductal extracellular vesicles content across the estrous cycle: implications for the gametes-oviduct interactions and the environment of the potential embryo. BMC Genomics. 2018;19(1):622. PMid:30134841.

Aswad H, Jalabert A, Rome S. Depleting extracellular vesicles from fetal bovine serum alters proliferation and differentiation of skeletal muscle cells in vitro. BMC Biotechnol. 2016;16(1):32. PMid:27038912.

Atay S, Gercel-Taylor C, Suttles J, Mor G, Taylor DD. Trophoblast-derived exosomes mediate monocyte recruitment and differentiation. Am J Reprod Immunol. 2011;65(1):65-77. PMid:20560914.

Ávila ACFCM, Silveira JC. Role of extracellular vesicles during oocyte maturation and early embryo development. Reprod Fertil Dev. 2019;32(2):56-64. PMid:32188558.

Berg DK, van Leeuwen J, Beaumont S, Berg M, Pfeffer PL. Embryo loss in cattle between Days 7 and 16 of pregnancy. Theriogenology. 2010;73(2):250-60. PMid:19880168.

Besenfelder U, Havlicek V, Brem G. Role of the oviduct in early embryo development. Reprod Domest Anim. 2012;47(Suppl 4):156-63. PMid:22827365.

Beyhan Z, Ross PJ, Iager AE, Kocabas AM, Cunniff K, Rosa GJ, Cibelli JB. Transcriptional reprogramming of somatic cell nuclei during preimplantation development of cloned bovine embryos. Dev Biol. 2007;305(2):637-49. PMid:17359962.

Bo G, Mapletoft R. Evaluation and classification of bovine embryos. Anim Reprod. 2013;10:344-8.

Brunner D, Frank J, Appl H, Schöffl H, Pfaller W, Gstraunthaler G. Serum-free cell culture: the serum-free media interactive online database. ALTEX. 2010;27(1):53-62. PMid:20390239.

Burns G, Brooks K, Wildung M, Navakanitworakul R, Christenson LK, Spencer TE. Extracellular vesicles in luminal fluid of the ovine uterus. PLoS One. 2014;9(3):e90913. PMid:24614226.

Cagnone G, Sirard M-A. The embryonic stress response to in vitro culture: insight from genomic analysis. Reproduction. 2016;152(6):R247-61. PMid:27601713.

Cahoy JD, Emery B, Kaushal A, Foo LC, Zamanian JL, Christopherson KS, Xing Y, Lubischer JL, Krieg PA, Krupenko SA, Thompson WJ, Barres BA. A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J Neurosci. 2008;28(1):264-78. PMid:18171944.

Colombo M, Raposo G, Théry C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol. 2014;30(1):255-89. PMid:25288114.

Diskin MG, Morris DG. Embryonic and early foetal losses in cattle and other ruminants. Reprod Domest Anim. 2008;43(Suppl 2):260-7. PMid:18638133.

Eitan E, Zhang S, Witwer KW, Mattson MP. Extracellular vesicle–depleted fetal bovine and human sera have reduced capacity to support cell growth. J Extracell Vesicles. 2015;4(1):26373. PMid:25819213.

Fazeli A. Maternal communication with gametes and embryos. Theriogenology. 2008;70(8):1182-7. PMid:18657312.

Giacomini E, Vago R, Sanchez AM, Podini P, Zarovni N, Murdica V, Rizzo R, Bortolotti D, Candiani M, Viganò P. Secretome of in vitro cultured human embryos contains extracellular vesicles that are uptaken by the maternal side. Sci Rep. 2017;7(1):5210. PMid:28701751.

Guerreiro EM, Vestad B, Steffensen LA, Aass HCD, Saeed M, Øvstebø R, Costea DE, Galtung HK, Søland TM. Efficient extracellular vesicle isolation by combining cell media modifications, ultrafiltration, and size-exclusion chromatography. PLoS One. 2018;13(9):e0204276. PMid:30260987.

Hackett AJ, Durnford R, Mapletoft RJ, Marcus GJ. Location and status of embryos in the genital tract of superovulated cows 4 to 6 days after insemination. Theriogenology. 1993;40(6):1147-53.

Hammond ER, McGillivray BC, Wicker SM, Peek JC, Shelling AN, Stone P, Chamley LW, Cree LM. Characterizing nuclear and mitochondrial DNA in spent embryo culture media: genetic contamination identified. Fertil Steril. 2017;107(1):220-8.e5. PMid:27865449.

Hansen PJ, Block J, Loureiro B, Bonilla L, Hendricks KEM. Effects of gamete source and culture conditions on the competence of in vitro-produced embryos for post-transfer survival in cattle. Reprod Fertil Dev. 2010;22(1):59-66. PMid:20003846.

Kim J, Lee J, Lee TB, Jun JH. Embryotrophic effects of extracellular vesicles derived from outgrowth embryos in pre- and peri-implantation embryonic development in mice. Mol Reprod Dev. 2019;86(2):187-96. PMid:30512223.

Kornilov R, Puhka M, Mannerström B, Hiidenmaa H, Peltoniemi H, Siljander P, Seppänen-Kaijansinkko R, Kaur S. Efficient ultrafiltration-based protocol to deplete extracellular vesicles from fetal bovine serum. J Extracell Vesicles. 2018;7(1):1422674. PMid:29410778.

Lane RE, Korbie D, Hill MM, Trau M. Extracellular vesicles as circulating cancer biomarkers: opportunities and challenges. Clin Transl Med. 2018;7(1):e14. PMid:29855735.

Lee K-F, Yao Y-Q, Kwok K-L, Xu J-S, Yeung WSB. Early Developing Embryos Affect the Gene Expression Patterns in the Mouse Oviduct. Biochem Biophys Res Commun. 2002;292(2):564-70. PMid:11906198.

Lee S-S, Won J-H, Lim GJ, Han J, Lee JY, Cho K-O, Bae YK. A novel population of extracellular vesicles smaller than exosomes promotes cell proliferation. Cell Commun Signal. 2019;17(1):95. PMid:31416445.

Lehrich B, Liang Y, Khosravi P, Federoff H, Fiandaca M. Fetal bovine serum-derived extracellular vesicles persist within vesicle-depleted culture media. Int J Mol Sci. 2018;19(11):3538. PMid:30423996.

Li J, Lee Y, Johansson HJ, Mäger I, Vader P, Nordin JZ, Wiklander OP, Lehtiö J, Wood MJ, Andaloussi SE. Serum-free culture alters the quantity and protein composition of neuroblastoma-derived extracellular vesicles. J Extracell Vesicles. 2015;4(1):26883. PMid:26022510.

Liao Z, Muth DC, Eitan E, Travers M, Learman LN, Lehrmann E, Witwer KW. Serum extracellular vesicle depletion processes affect release and infectivity of HIV-1 in culture. Sci Rep. 2017;7(1):2558. PMid:28566772.

Lopera-Vásquez R, Hamdi M, Fernandez-Fuertes B, Maillo V, Beltrán-Breña P, Calle A, Redruello A, López-Martín S, Gutierrez-Adán A, Yañez-Mó M, Ramirez MÁ, Rizos D. Extracellular vesicles from BOEC in in vitro embryo development and quality. PLoS One. 2016;11(2):e0148083. PMid:26845570.

Lopera-Vasquez R, Hamdi M, Maillo V, Gutierrez-Adan A, Bermejo-Alvarez P, Ramírez MÁ, Yáñez-Mó M, Rizos D. Effect of bovine oviductal extracellular vesicles on embryo development and quality in vitro. Reproduction. 2017;153(4):461-70. PMid:28104825.

Maillo V, Gaora PÓ, Forde N, Besenfelder U, Havlicek V, Burns GW, Spencer TE, Gutierrez-Adan A, Lonergan P, Rizos D. Oviduct-embryo interactions in cattle: two-way traffic or a one-way street? Biol Reprod. 2015;92(6):144. PMid:25926440.

Mathivanan S, Ji H, Simpson RJ. Exosomes: extracellular organelles important in intercellular communication. J Proteomics. 2010;73(10):1907-20. PMid:20601276.

Mellisho EA, Briones MA, Velásquez AE, Cabezas J, Castro FO, Rodríguez-Álvarez L. Extracellular vesicles secreted during blastulation show viability of bovine embryos. Reproduction. 2019;158(6):477-92. PMid:31600718.

Mellisho EA, Velásquez AE, Nuñez MJ, Cabezas JG, Cueto JA, Fader C, Castro FO, Rodríguez-Álvarez L. Identification and characteristics of extracellular vesicles from bovine blastocysts produced in vitro. PLoS One. 2017;12(5):e0178306. PMid:28542562.

Ng YH, Rome S, Jalabert A, Forterre A, Singh H, Hincks CL, Salamonsen LA. Endometrial exosomes/microvesicles in the uterine microenvironment: a new paradigm for embryo-endometrial cross talk at implantation. PLoS One. 2013;8(3):e58502. PMid:23516492.

Pallinger E, Bognar Z, Bogdan A, Csabai T, Abraham H, Szekeres-Bartho J. PIBF+ extracellular vesicles from mouse embryos affect IL-10 production by CD8+ cells. Sci Rep. 2018;8(1):4662. PMid:29549351.

Pan H, Schultz RM. SOX2 modulates reprogramming of gene expression in two-cell mouse embryos. Biol Reprod. 2011;85(2):409-16. PMid:21543769.

Pap E, Pállinger É, Falus A, Kiss AA, Kittel Á, Kovács P, Buzás EI. T lymphocytes are targets for platelet- and trophoblast-derived microvesicles during pregnancy. Placenta. 2008;29(9):826-32. PMid:18684502.

Pavani K, Hendrix A, Van Den Broeck W, Couck L, Szymanska K, Lin X, De Koster J, Van Soom A, Leemans B. Isolation and characterization of functionally active extracellular vesicles from culture medium conditioned by bovine embryos in vitro. Int J Mol Sci. 2018;20(1):38. PMid:30577682.

Perkel KJ, Tscherner A, Merrill C, Lamarre J, Madan P. The ART of selecting the best embryo: A review of early embryonic mortality and bovine embryo viability assessment methods. Mol Reprod Dev. 2015;82(11):822-38. PMid:26184077.

Qu P, Qing S, Liu R, Qin H, Wang W, Qiao F, Ge H, Liu J, Zhang Y, Cui W, Wang Y. Effects of embryo-derived exosomes on the development of bovine cloned embryos. PLoS One. 2017;12(3):e0174535. PMid:28350875.

Rizos D, Gutiérrez-Adán A, Pérez-Garnelo S, de la Fuente J, Boland MP, Lonergan P. Bovine Embryo Culture in the Presence or Absence of Serum: Implications for Blastocyst Development, Cryotolerance, and Messenger RNA Expression1. Biol Reprod. 2003;68(1):236-43. PMid:12493719.

Rodríguez-Alvarez L, Cox J, Tovar H, Einspanier R, Castro FO. Changes in the expression of pluripotency-associated genes during preimplantation and peri-implantation stages in bovine cloned and in vitro produced embryos. Zygote. 2010;18(3):269-79. PMid:20429963.

Rodríguez-Alvarez L, Manriquez J, Velasquez A, Castro FO. Constitutive expression of the embryonic stem cell marker OCT4 in bovine somatic donor cells influences blastocysts rate and quality after nucleus transfer. In Vitro Cell Dev Biol Anim. 2013;49(9):657-67. PMid:23846396.

Saadeldin IM, Kim SJ, Choi Y, Lee BC. Improvement of cloned embryos development by co-culturing with parthenotes: a possible role of exosomes/microvesicles for embryos paracrine communication. Cell Reprogram. 2014;16(3):223-34. PMid:24773308.

Shelke GV, Lässer C, Gho YS, Lötvall J. Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum. J Extracell Vesicles. 2014;3(1):24783. PMid:25317276.

Simmet K, Zakhartchenko V, Philippou-Massier J, Blum H, Klymiuk N, Wolf E. OCT4/POU5F1 is required for NANOG expression in bovine blastocysts. Proc Natl Acad Sci USA. 2018;115(11):2770-5. PMid:29483258.

Smits K, De Coninck DIM, Van Nieuwerburgh F, Govaere J, Van Poucke M, Peelman L, Deforce D, Van Soom A. The equine embryo influences immune-related gene expression in the oviduct. Biol Reprod. 2016;94(2):36. PMid:26740593.

Stolk M, Seifert M. Protein contaminations impact quantification and functional analysis of extracellular vesicle preparations from mesenchymal stromal cells. J Stem Cells Regen Med. 2015;11(2):44-7. PMid:27330254.

Szekeres-Bartho J, Šućurović S, Mulac-Jeričević B. The role of extracellular vesicles and PIBF in embryo-maternal immune-interactions. Front Immunol. 2018;9:2890. PMid:30619262.

Tannetta D, Dragovic R, Alyahyaei Z, Southcombe J. Extracellular vesicles and reproduction–promotion of successful pregnancy. Cell Mol Immunol. 2014;11(6):548-63. PMid:24954226.

Thatcher WW, Guzeloglu A, Mattos R, Binelli M, Hansen TR, Pru JK. Uterine-conceptus interactions and reproductive failure in cattle. Theriogenology. 2001;56(9):1435-50. PMid:11768809.

Théry C, Amigorena S, Raposo G, Clayton A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. 2006;30(1):3-22. PMid:18228490.

Théry C, Ostrowski M, Segura E. Membrane vesicles as conveyors of immune responses. Nat Rev Immunol. 2009;9(8):581-93. PMid:19498381.

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(6):654-9. PMid:17486113.

Vanroose G, Nauwynck H, Soom AV, Ysebaert MT, Charlier G, Van Oostveldt P, Kruif A. Structural aspects of the zona pellucida of in vitro: produced bovine embryos: a scanning electron and confocal laser scanning microscopic study. Biol Reprod. 2000;62(2):463-9. PMid:10642588.

Velasquez AE, Castro FO, Veraguas D, Cox JF, Lara E, Briones M, Rodriguez-Alvarez L. Splitting of IVP bovine blastocyst affects morphology and gene expression of resulting demi-embryos during in vitro culture and in vivo elongation. Zygote. 2016;24(1):18-30. PMid:25496989.

Velásquez AE, Veraguas D, Cabezas J, Manríquez J, Castro FO, Rodríguez-Alvarez LL. The expression level of SOX2 at the blastocyst stage regulates the developmental capacity of bovine embryos up to day-13 of in vitro culture. Zygote. 2019;27(6):398-404. PMid:31576792.

Wang P, Li X, Cao L, Huang S, Li H, Zhang Y, Yang T, Jiang J, Shi D. MicroRNA-148a overexpression improves the early development of porcine somatic cell nuclear transfer embryos. PLoS One. 2017;12(6):e0180535. PMid:28665977.

Witwer KW, Buzás EI, Bemis LT, Bora A, Lässer C, Lötvall J, Nolte-’t Hoen EN, Piper MG, Sivaraman S, Skog J, Théry C, Wauben MH, Hochberg F. Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles. 2013;2(1):20360. PMid:24009894.

Wrenzycki C, Herrmann D, Keskintepe L, Martins A Jr, Sirisathien S, Brackett B, Niemann H. Effects of culture system and protein supplementation on mRNA expression in pre-implantation bovine embryos. Hum Reprod. 2001;16(5):893-901. PMid:11331635.

Zhang H, Freitas D, Kim HS, Fabijanic K, Li Z, Chen H, Mark MT, Molina H, Martin AB, Bojmar L, Fang J, Rampersaud S, Hoshino A, Matei I, Kenific CM, Nakajima M, Mutvei AP, Sansone P, Buehring W, Wang H, Jimenez JP, Cohen-Gould L, Paknejad N, Brendel M, Manova-Todorova K, Magalhães A, Ferreira JA, Osório H, Silva AM, Massey A, Cubillos-Ruiz JR, Galletti G, Giannakakou P, Cuervo AM, Blenis J, Schwartz R, Brady MS, Peinado H, Bromberg J, Matsui H, Reis CA, Lyden D. Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation. Nat Cell Biol. 2018;20(3):332-43. PMid:29459780.

Zhang Q, Higginbotham JN, Jeppesen DK, Yang Y-P, Li W, McKinley ET, Graves-Deal R, Ping J, Britain CM, Dorsett KA, Hartman CL, Ford DA, Allen RM, Vickers KC, Liu Q, Franklin JL, Bellis SL, Coffey RJ. Transfer of functional cargo in exomeres. Cell Rep. 2019;27(3):940-54.e6. PMid:30956133.

Zhang Y, Chen K, Sloan SA, Bennett ML, Scholze AR, O’Keeffe S, Phatnani HP, Guarnieri P, Caneda C, Ruderisch N, Deng S, Liddelow SA, Zhang C, Daneman R, Maniatis T, Barres BA, Wu JQ. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J Neurosci. 2014;34(36):11929-47. PMid:25186741.

Zou G, Liu T, Guo L, Huang Y, Feng Y, Huang Q, Duan T. miR-145 modulates lncRNA-ROR and Sox2 expression to maintain human amniotic epithelial stem cell pluripotency and β islet-like cell differentiation efficiency. Gene. 2016;591(1):48-57. PMid:27346547.

Submitted date:

Accepted date:

6095335da9539531fb7bb512 animreprod Articles
Links & Downloads

Anim Reprod

Share this page
Page Sections