Hostname: page-component-586b7cd67f-dlnhk Total loading time: 0 Render date: 2024-11-26T14:10:02.547Z Has data issue: false hasContentIssue false
  • English
  • Français

Expression of markers for germ cells and oocytes in cow dermal fibroblast treated with 5-azacytidine and cultured in differentiation medium containing BMP2, BMP4 or follicular fluid

Published online by Cambridge University Press:  03 July 2017

José Jackson do Nascimento Costa ,
Glaucinete Borges de Souza ,
Joyla Maria Pires Bernardo ,
Regislane Pinto Ribeiro ,
José Renato de Souza Passos ,
Francisco Taiã Gomes Bezerra ,
Márcia Viviane Alves Saraiva  and
José Roberto Viana Silva
José Jackson do Nascimento Costa
Affiliation:
Biotechnology Nucleus of Sobral – NUBIS, Federal University of Ceara, CEP 62042–280, Sobral, CE, Brazil.
Glaucinete Borges de Souza
Affiliation:
Biotechnology Nucleus of Sobral – NUBIS, Federal University of Ceara, CEP 62042–280, Sobral, CE, Brazil.
Joyla Maria Pires Bernardo
Affiliation:
Biotechnology Nucleus of Sobral – NUBIS, Federal University of Ceara, CEP 62042–280, Sobral, CE, Brazil.
Regislane Pinto Ribeiro
Affiliation:
Biotechnology Nucleus of Sobral – NUBIS, Federal University of Ceara, CEP 62042–280, Sobral, CE, Brazil.
José Renato de Souza Passos
Affiliation:
Biotechnology Nucleus of Sobral – NUBIS, Federal University of Ceara, CEP 62042–280, Sobral, CE, Brazil.
Francisco Taiã Gomes Bezerra
Affiliation:
Biotechnology Nucleus of Sobral – NUBIS, Federal University of Ceara, CEP 62042–280, Sobral, CE, Brazil.
Márcia Viviane Alves Saraiva
Affiliation:
Biotechnology Nucleus of Sobral – NUBIS, Federal University of Ceara, CEP 62042–280, Sobral, CE, Brazil.
José Roberto Viana Silva*
Affiliation:
Biotechnology Nucleus of Sobral - NUBIS, Federal University of Ceara, Av. Comandante Maurocélio Rocha Ponte 100, CEP: 62.041–040, Sobral, CE, Brazil.
*
All correspondence to: J.R.V. Silva. Biotechnology Nucleus of Sobral – NUBIS, Federal University of Ceara, Av. Comandante Maurocélio Rocha Ponte 100, CEP: 62.041–040, Sobral, CE, Brazil. Tel:/Fax: +55 85 3611 8000. E-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

Summary

This study aims to investigate the effect 5-azacytidine (5-Aza) during induction of pluripotency in bovine fibroblasts and to evaluate the effects of BMP2, BMP4 or follicular fluid in the differentiation of reprogrammed fibroblasts in primordial germ cells and oocytes. It also analysis the mRNA levels for OCT4, NANOG, REX, SOX2, VASA, DAZL, cKIT, SCP3, ZPA and GDF9 after culturing 5-Aza treated fibroblasts in the different tested medium. Dermal fibroblasts were cultured and exposed to 0.5, 1.0 or 2.0 μM of 5-Aza for 18 h, 36 h or 72 h. Then, the cells were cultured in DMEM/F12 supplemented with 10 ng/ml BMP2, 10 ng/ml BMP4 or 5% follicular fluid. After culture, morphological characteristics, viability and gene expression were evaluated by qPCR. Treatment of skin fibroblasts with 2.0 μM 5-Aza for 72 h significantly increased expression of mRNAs for SOX2, OCT4, NANOG and REX. The culture in medium supplemented with BMP2, BMP4 or follicular fluid for 7 or 14 days induced formation of oocyte-like cells, as well as the expression of markers for germ cells and oocyte. In conclusion, treatment of bovine skin-derived fibroblasts with 2.0 μM 5-Aza for 72 h induces the expression of pluripotency factors. Culturing these cells in differentiation medium supplemented with BMP2, BMP4 or follicular fluid induces morphological changes and promotes expression of markers for germ cells, meiosis and oocyte.

Keywords

5-AzacytidineBMPFibroblastsFollicular fluidGene expression
Type
Research Article
Information
Zygote , Volume 25 , Issue 3 , June 2017 , pp. 341 - 357
Copyright
Copyright © Cambridge University Press 2017 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Aflatoonian, B., Ruban, L., Jones, M., Aflatoonian, R., Fazeli, A. & Moore, H.D. (2009). In vitro post-meiotic germ cell development from human embryonic stem cells. Hum. Reprod. 24, 3150–9.CrossRefGoogle ScholarPubMed
Angelos, M.G. & Kaufman, D.S. (2015). Pluripotent stem cell applications for regenerative medicine. Curr. Opin. Organ Transpl. 20, 663–70.CrossRefGoogle ScholarPubMed
Bertoldo, M.J., Nadal-Desbarats, L., Gérard, N., Dubois, A., Holyoake, P.K. & Grupen, C.G. (2013). Differences in the metabolomic signatures of porcine follicular fluid collected from environments associated with good and poor oocyte quality. Reproduction 146, 221–31.CrossRefGoogle ScholarPubMed
Bucay, N., Yebra, M., Cirulli, V., Afrikanova, I., Kaido, T. & Hayek, A. (2009). A novel approach for the derivation of putative primordial germ cells and Sertoli cells from human embryonic stem cells. Stem Cells 27, 68–77.CrossRefGoogle ScholarPubMed
Cai, L., Rothbart, S.B., Lu, R., Xu, B., Chen, W.Y. & Tripathy, A. (2013). An H3K36 methylation-engaging Tudor motif of polycomb-like proteins mediates PRC2 complex targeting. Mol. Cell 49, 571–82.CrossRefGoogle ScholarPubMed
Castrillon, D.H., Miao, L., Kollipara, R., Horner, J.W. & DePinho, R.A. (2003). Suppression of ovarian follicle activation in mice by the transcription factor Foxo3a. Science 301, 215–8.CrossRefGoogle ScholarPubMed
Chen, H.F., Kuo, H.C., Chien, C.L., Shun, C.T., Yao, Y.L. & Ip, P.L. (2007). Derivation, characterization and differentiation of human embryonic stem cells: comparing serum-containing versus serum-free media and evidence of germ cell differentiation. Hum. Reprod. 22, 567–77.CrossRefGoogle ScholarPubMed
Cheng, X., Chen, S., Yu, X., Zheng, P. & Wang, H. (2012). BMP15 gene is activated during human amniotic fluid stem cell differentiation into oocyte-like cells. DNA Cell Biol. 31, 1198–204.CrossRefGoogle ScholarPubMed
Clark, A.T., Bodnar, M.S., Fox, M., Rodriquez, R.T., Abeyta, M.J. & Firpo, M.T. (2004). Spontaneous differentiation of germ cells from human embryonic stem cells in vitro . Hum. Mol. Gen. 13, 727–39.CrossRefGoogle ScholarPubMed
Constantinides, P.G., Jones, P.A. & Gevers, W. (1977). Functional striated muscle cells from non-myoblast precursors following 5-azacytidine treatment. Nature 267, 364–6.CrossRefGoogle ScholarPubMed
Diecke, S., Jung, S.M., Lee, J. & Ju, J.H. (2014). Recent technological updates and clinical applications of induced pluripotent stem cells. Korean J. Intern. Med. 29, 547–57.CrossRefGoogle ScholarPubMed
Dyce, P.W., Zhu, H., Craig, J. & Li, J. (2004). Stem cells with multilineage potential derived from porcine skin. Biochem. Biophy. Res. Commun. 316, 651–8.CrossRefGoogle ScholarPubMed
Dyce, P.W., Liu, J., Tayade, C., Kidder, G.M., Betts, D.H. & Li, J. (2011). In vitro and in vivo germ line potential of stem cells derived from newborn mouse skin. PLoS One, 6, e20339.CrossRefGoogle ScholarPubMed
Dyce, P.W., Wen, L. & Li, J. (2006). In vitro germline potential of stem cells derived from fetal porcine skin. Nat. Cell. Biol. 8, 384–90.CrossRefGoogle ScholarPubMed
Eguizabal, C., Montserrat, N., Vassena, R., Barragan, M., Garreta, E. & Garcia-Quevedo, L. (2011). Complete meiosis from human induced pluripotent stem cells. Stem Cells 29, 1186–95.CrossRefGoogle ScholarPubMed
Enjoji, M., Nakashima, M., Honda, M., Sakai, H. & Nawata, H. (1997). Hepatocytic phenotypes induced in sarcomatous cholangiocarcinoma cells treated with 5-azacytidine. Hepatology 26, 288–94.CrossRefGoogle ScholarPubMed
Federation, A.J., Bradner, J.E. & Meissner, A. (2014). The use of small molecules in somatic-cell reprogramming. Trends Cell. Biol. 24, 179–87.CrossRefGoogle ScholarPubMed
Günesdogan, U., Magnúsdóttir, E. & Surani, M.A. (2014). Primordial germ cell specification: a context-dependent cellular differentiation event. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 369, 1657.Google ScholarPubMed
Hayashi, K., Kobayashi, T., Umino, T., Goitsuka, R., Matsui, Y. & Kitamura, D. (2002). SMAD1 signaling is critical for initial commitment of germ cell lineage from mouse epiblast. Mech. Dev. 118, 99–109.CrossRefGoogle ScholarPubMed
Hirschi, K.K., Li, S. & Roy, K. (2014). Induced pluripotent stem cells for regenerative medicine. Annu. Rev. Biomed. Eng. 16, 277–94.CrossRefGoogle ScholarPubMed
Hou, P., Li, Y., Zhang, X., Liu, C., Guan, J. & Li, H. (2013). Pluripotent stem cells induced from mouse somatic cells by small-molecule compound. Science 341, 651–4.CrossRefGoogle Scholar
Hu, X., Lu, H., Cao, S., Deng, Y.L., Li, Q.J. & Wan, Q. (2015). Stem cells derived from human first-trimester umbilical cord have the potential to differentiate into oocyte-like cells in vitro . Int. J. Mol. Med. 35, 1219–29.CrossRefGoogle ScholarPubMed
Ishii, T. (2014). Human iPS cell-derived germ cells: current status and clinical potential. J. Clin. Med. Res. 3, 1064– 83.Google ScholarPubMed
Jones, P.A. (1985). Effects of 5-azacytidine and its 2′-deoxyderivative on cell differentiation and DNA methylation. Pharmacol. Ther. 28, 17–27.CrossRefGoogle ScholarPubMed
Kee, K., Gonsalves, J., Clark, A. & Reijo Pera, R.A. (2006). Bone morphogenetic proteins induce germ cell differentiation from human embryonic stem cells. Stem Cells Dev. 15, 831–7.CrossRefGoogle ScholarPubMed
Kee, K., Angeles, V., Flores, M. & Nguyen, H. Human Reproduction Reijo Pera, R.A. (2009). Human DAZL, DAZ and BOULE genes modulate primordial germ cell and haploid gamete formation. Nature 462, 222–5.CrossRefGoogle ScholarPubMed
Keefer, C.L., Panta, D., Blomberg, L. & Talbot, N.C. (2007). Challenges and prospects for the establishment of embryonic stem cell lines of domesticated ungulates. Anim. Reprod. Sci. 98, 147–68.CrossRefGoogle ScholarPubMed
Kishigami, S. & Mishina, Y. (2005). BMP signaling and early embryonic patterning. Cytokine Growth Factor Rev. 16, 265–78.CrossRefGoogle ScholarPubMed
Kopper, O., Giladi, O., Golan-Lev, T. & Benvenisty, N. (2010). Characterization of gastrulation-stage progenitor cells and their inhibitory crosstalk in human embryoid bodies. Stem Cells 28, 75–83.CrossRefGoogle ScholarPubMed
Lefièvre, L., Conner, S.J., Salpekar, A., Olufowobi, O., Ashton, P. & Pavlovic, B. (2004). Four zona pellucida glycoproteins are expressed in the human. Hum. Reprod. 19, 1580–6.CrossRefGoogle ScholarPubMed
Lin, C.Y., Chen, C.Y., Yu, C.H., Yu, I.S., Lin, S.R. & Wu, J.T. (2016). Human X-linked intellectual disability factor CUL4B is required for post-meiotic sperm development and male. Fertil. Sci. Rep. 6, 20227.CrossRefGoogle ScholarPubMed
Lin, T. & Wu, S. (2015). Reprogramming with small molecules instead of exogenous transcription factors. Stem Cells Int. 2015, 794632.CrossRefGoogle ScholarPubMed
Linher, K., Dyce, P. & Li, J. (2009). Primordial germ cell-like cells differentiated in vitro from skin-derived stem cells. PLoS One 4, e8263.CrossRefGoogle ScholarPubMed
Livak, J.K., & Schmittgen, T.D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method. Methods 25, 402–8.CrossRefGoogle Scholar
Marthaler, A.G., Tubsuwan, A., Schmid, B., Poulsen, U.B., Hyttel, P. & Nielsen, J.E. (2016). Generation of spinocerebellar ataxia type 2 patient-derived iPSC line H271. Stem Cell Res. 16, 159–61.CrossRefGoogle ScholarPubMed
Masui, S., Nakatake, Y., Toyooka, Y., Shimosato, D., Yagi, R. & Takahashi, K. (2007). Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells. Nat. Cell Biol. 9, 625–35.CrossRefGoogle ScholarPubMed
Mohana Kumar, B., Jin, H.F., Kim, J.G., Song, H.J., Hong, Y. & Balasubramanian, S. (2006). DNA methylation levels in porcine fetal fibroblasts induced by an inhibitor of methylation, 5-azacytidine. Cell Tissue Res. 325, 445–54.CrossRefGoogle ScholarPubMed
Niikura, Y., Niikura, T. & Tilly, J.L. (2009). Aged mouse ovaries possess rare premeiotic germ cells that can generate oocytes following transplantation into a young host environment. Aging 1, 971–8.CrossRefGoogle Scholar
Ohinata, Y., Ohta, H., Shigeta, M., Yamanaka, K. & Wakayama, T. (2009). A signaling principle for the specification of the germ cell lineage in mice. Cell 137, 571–84.CrossRefGoogle ScholarPubMed
Pacchiarotti, J., Maki, C., Ramos, T., Marh, J., Howerton, K. & Wong, J. (2010). Differentiation potential of germ line stem cells derived from the postnatal mouse ovary. Differentiation 79, 159–70.CrossRefGoogle ScholarPubMed
Panula, S., Medrano, J.V., Kee, K., Bergström, R., Nguyen, H.N., Byers, B., Wilson, K.D., Wu, J.C., Simon, C., Hovatta, O. & Reijo Pera, R.A. (2011). Human germ cell differentiation from fetal- and adult-derived induced pluripotent stem cells. Hum. Mol. Gen. 20, 752–62.CrossRefGoogle ScholarPubMed
Park, T., Galic, Z., Conway, A., Lindgren, A., Van Handel, B. & Magnusson, M. (2009). Derivation of primordial germ cells from human embryonic and induced pluripotent stem cells is significantly improved by coculture with human fetal gonadal cells. Stem Cells 27, 783–95.CrossRefGoogle ScholarPubMed
Park, E.S., Woods, D.C. & Tilly, J.L. (2013). Bone morphogenetic protein 4 promote mammalian oogonial stem cell differentiation via Smad1/5/8 signaling. Fertil. Steril. 100, 1468–75.CrossRefGoogle ScholarPubMed
Pennarossa, G., Maffei, S., Campagnol, M., Tarantini, L., Gandolfi, F. & Brevini, T.A.L. (2013). Brief demethylation step allows the conversion of adult human skin fibroblasts into insulin-secreting cells. Proc. Natl. Acad. Sci. USA 110, 8948–53.CrossRefGoogle ScholarPubMed
Pennarossa, G., Maffei, S., Campagnol, M., Rahman, M.M., Brevini, T.A.L. & Gandolfi, F. (2014). Reprogramming of pig dermal fibroblast into insulin secreting cells by a brief exposure to 5-aza-cytidine. Stem Cell Rev. 10, 31–43.CrossRefGoogle ScholarPubMed
Pera, M.F., Andrade, J., Houssami, S., Reubinoff, B., Trounson, A. & Stanley, E.G. (2004). Regulation of human embryonic stem cell differentiation by BMP2 and its antagonist noggin. J. Cell Sci. 117, 1269–80.CrossRefGoogle ScholarPubMed
Picanço-Castro, V., Russo-Carbolante, E., Reis, L.C., Fraga, A.M., de Magalhães, D.A. & Orellana, M.D. (2011). Pluripotent reprogramming of fibroblasts by lentiviral mediated insertion of SOX2, C-MYC, and TCL-1A. Stem Cells Dev. 20, 169–80.CrossRefGoogle ScholarPubMed
Ratajczak, M.Z., Zuba-Surma, E., Kucia, M., Poniewierska, A., Suszynska, M. & Ratajczak, J. (2012). Pluripotent and multipotent stem cells in adult tissues. Adv. Med. Sci. 57, 1–17.CrossRefGoogle ScholarPubMed
Remenyi, A., Lins, K., Nissen, L.J., Reinbold, R., Schöler, H.R. & Wilmanns, M. (2003). Crystal structure of a POU/HMG/DNA ternary complex suggests differential assembly of Oct4 and Sox2 on two enhancers. Gene Dev. 17, 2048–59.CrossRefGoogle ScholarPubMed
Rodgers, R.J. & Irving-Rodgers, H.F. (2010). Formation of the ovarian follicular antrum and follicular fluid. Biol. Reprod. 82, 1021–9.CrossRefGoogle ScholarPubMed
Rossi, R.O.D, Cunha, E.V., Portela, A.M.L, Passos, J.R.S., Costa, J.J.N., Silva, A.W.B., Saraiva, M.V.A, Peixoto, C.A., Donato, M.A., van den Hurk, R. & Silva, J.R.V. (2016). Influence of BMP-2 on early follicular development and mRNA expression of oocyte specific genes in bovine preantral follicles cultured in vitro . Histol. Histopathol. 31, 339–48.Google ScholarPubMed
Salvador, L.M., Silva, C.P., Kostetskii, I., Radice, G.L. & Strauss, J.F. (2008). The promoter of the oocyte-specific gene, Gdf9, is active in population of cultured mouse embryonic stem cells with an oocyte-like phenotype. Methods 45, 172–81.CrossRefGoogle ScholarPubMed
Schenke-Layland, K. & Brucker, S.Y. (2015). Prospects for regenerative medicine approaches in women's health. J. Anat. 227, 781–5.CrossRefGoogle ScholarPubMed
Shah, S.M., Saini, N., Ashraf, S., Singh, M.K., Manik, R.S. & Singla, S.K. (2015). Bone morphogenetic protein 4 (BMP4). induces buffalo (Bubalus bubalis) embryonic stem cell differentiation into germ cells. Biochimie 119, 113–24.CrossRefGoogle ScholarPubMed
Shi, W., Wang, H., Pan, G., Geng, Y., Guo, Y. & Pei, D. (2006). Regulation of the pluripotency marker Rex-1 by Nanog and Sox2. J. Biol. Chem. 281, 23319–25.CrossRefGoogle ScholarPubMed
Singh, V.K., Kalsan, M., Kumar, N., Saini, A. & Chandra, R. (2015). Induced pluripotent stem cells: applications in regenerative medicine, disease modeling, and drug discovery. Front Cell Dev. Biol. 3, 2.CrossRefGoogle ScholarPubMed
Singhal, D.K., Singhal, R., Malik, H.N., Singh, S., Kumar, S. & Kaushik, J.K. (2015). Generation of germ cell-like cells and oocyte-like cells from goat induced pluripotent stem cells. J. Stem Cell Res. Ther. 5, 279.Google Scholar
Son, M.Y., Choi, H., Han, Y.M. & Cho, Y.S. (2013). Unveiling the critical role of REX1 in the regulation of human stem cell pluripotency. Stem Cells 31, 2374–87.CrossRefGoogle ScholarPubMed
Song, S.H., Kumar, B.M., Kang, E.J., Lee, Y.M., Kim, T.H. & Ock, S.A. (2011). Characterization of porcine multipotent stem/stromal cells derived from skin, adipose, and ovarian tissues and their differentiation in vitro into putative oocyte-like cells. Stem Cells Dev. 20, 1359–70.CrossRefGoogle ScholarPubMed
Sterneckert, J., Hoing, S. & Schöler, H.R. (2012). Concise review: Oct4 and more: the reprogramming expressway. Stem Cells 30, 15–21.CrossRefGoogle ScholarPubMed
Takahashi, K. & Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–76.CrossRefGoogle ScholarPubMed
Taylor, S.M. & Jones, P.A. (1979). Multiple new phenotypes induced in 10T1/2 and 3T3 cells treated with 5-azacytidine. Cell 17, 771–9.CrossRefGoogle ScholarPubMed
Taylor, S.M. & Jones, P.A. (1982). Changes in phenotypic expression in embryonic and adult cells treated with 5-azacytidine. J. Cell Physiol. 111, 187–94.CrossRefGoogle ScholarPubMed
Virant-Klun, I., Skutella, T., Kubista, M., Vogler, A., Sinkovec, J. & Meden-Vrtovec, H. (2013). Expression of pluripotency and oocyte-related genes in single putative stem cells from human adult ovarian surface epithelium cultured in vitro in the presence of follicular fluid. Biomed. Res. Int. 2013, 861460.CrossRefGoogle ScholarPubMed
Volarevic, V., Bojic, S., Nurkovic, J., Volarevic, A., Ljujic, B. & Arsenijevic, N. (2014). Stem cells as new agents for the treatment of infertility: current and future perspectives and challenges. Biomed Res. Int. 2014, 8.CrossRefGoogle ScholarPubMed
West, F.D., Roche-Rios, M.I., Abraham, S., Rao, R.R., Natrajan, M.S. & Bacanamwo, M. (2010). KIT ligand and bone morphogenetic protein signaling enhances human embryonic stem cell to germ-like cell differentiation. Hum. Reprod. 25, 168–78.CrossRefGoogle ScholarPubMed
Ying, Y. & Zhao, G.Q. (2001). Cooperation of endoderm-derived BMP2 and extraembryonic ectoderm-derived BMP4 in primordial germ cell generation in the mouse. Dev. Biol. 232, 484–92.CrossRefGoogle ScholarPubMed
Yu, H., Vu, T.H., Cho, K.S., Guo, C. & Chen, D.F. (2014). Mobilizing endogenous stem cells for retinal repair. Transl. Res. 163, 387–98.CrossRefGoogle ScholarPubMed
Zhang, Y., Li, W., Laurent, T. & Ding, S. (2012). Small molecules, big roles – the chemical manipulation of stem cell fate and somatic cell reprogramming. J. Cell Sci. 125, 5609–50.CrossRefGoogle ScholarPubMed
Zomer, H.D., Vidane, A.S., Gonçalves, N.N. & Ambrósio, C.E. (2015). Mesenchymal and induced pluripotent stem cells: general insights and clinical perspectives. Stem Cell Cloning 8, 125–34.Google ScholarPubMed