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RESEARCH ARTICLE

Cumulus cell-conditioned medium supports embryonic stem cell differentiation to germ cell-like cells

Syed Mohmad Shah A , Neha Saini A , Syma Ashraf A , Manoj Kumar Singh A , Radhey Sham Manik A , Suresh Kumar Singla A , Prabhat Palta A and Manmohan Singh Chauhan A B
+ Author Affiliations
- Author Affiliations

A Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute, Karnal 132001, India.

B Corresponding author. Email: chauhanabtc@gmail.com

Reproduction, Fertility and Development 29(4) 679-693 https://doi.org/10.1071/RD15159
Submitted: 21 April 2015  Accepted: 17 October 2015   Published: 24 November 2015

Abstract

Cumulus cells provide cellular interactions and growth factors required for oogenesis. In vitro studies of oogenesis are limited primarily because of the paucity of their source, first trimester fetal gonads, and the small number of germ lineage precursor cells present within these tissues. In order to understand this obscure but vitally important process, the present study was designed to direct differentiation of embryonic stem (ES) cells into germ lineage cells. For this purpose, buffalo ES cells were differentiated, as embryoid bodies (EBs) and monolayer adherent cultures, in the presence of different concentrations of cumulus-conditioned medium (CCM; 10%, 20% and 40%) for different periods of culture (4, 8 and 14 days) to identify the optimum differentiation-inducing concentration and time. Quantitative polymerase chain reaction analysis revealed that 20%–40% CCM induced the highest expression of primordial germ cell-specific (deleted in Azoospermia- like (Dazl), dead (Asp-Glu-Ala-Asp) box polypeptide 4 (Vasa also known as DDX4) and promyelocytic leukemia zinc finger protein (Plzf)); meiotic (synaptonemal complex protein 3 (Sycp3), mutl homolog I (Mlh1), transition protein 1/2 (Tnp1/2) and protamine 2 (Prm2); spermatocyte-specific boule-like RNA binding protein (Boule) and tektin 1 (Tekt1)) and oocyte-specific growth differentiation factor 9 (Gdf9) and zona pellucida 2 /3 (Zp2/3)) genes over 8–14 days in culture. Immunocytochemical analysis revealed expression of primordial germ cell (c-KIT, DAZL and VASA), meiotic (SYCP3, MLH1 and PROTAMINE 1), spermatocyte (ACROSIN and HAPRIN) and oocyte (GDF9 and ZP4) markers in both EBs and monolayer differentiation cultures. Western blotting revealed germ lineage-specific protein expression in Day 14 EBs. The significantly lower (P < 0.05) concentration of 5-methyl-2-deoxycytidine in differentiated EBs compared to undifferentiated EBs suggests that methylation erasure may have occurred. Oocyte-like structures obtained in monolayer differentiation stained positive for ZONA PELLUCIDA protein 4 and progressed through various embryo-like developmental stages in extended cultures.

Additional keywords: buffalo, germ lineage differentiation, epigenetic reprogramming.


References

Aflatoonian, B., Ruban, L., Jones, M., Aflatoonian, R., Fazeli, A., and Moore, H. (2009). In vitro post-meiotic germ cell development from human embryonic stem cells. Hum. Reprod. 24, 3150–3159.
In vitro post-meiotic germ cell development from human embryonic stem cells.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhsVKls7rL&md5=53943bde016d3069da8d2bf5f7c91572CAS | 19770126PubMed |

Clark, A. T., Bodnar, M. S., Fox, M., Rodriquez, R. T., Abeyta, M., Firpo, M. T., and Pera, R. A. R. (2004). Spontaneous differentiation of germ cells from human embryonic stem cells in vitro. Hum. Mol. Genet. 13, 727–739.
Spontaneous differentiation of germ cells from human embryonic stem cells in vitro.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXitVyrs74%3D&md5=1e1cb856697847d9d76d9ca7120bcfb5CAS | 14962983PubMed |

De Felici, M., Dolci, S., and Pesce, M. (1993). Proliferation of mouse primordial germ cells in vitro: a key role for cAMP. Dev. Biol. 157, 277–280.
Proliferation of mouse primordial germ cells in vitro: a key role for cAMP.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK3sXkt1Gnt7k%3D&md5=5bc8df342ca1921c4f44cf870588f508CAS | 8387035PubMed |

Demeestere, I., Gervy, C., Centner, J., Devreker, F., Englert, Y., and Delbaere, A. (2004). Effect of insulin-like growth factor-I during preantral follicular culture on steroidogenesis, in-vitro oocyte maturation, and embryo development in mice. Biol. Reprod. 70, 1664–1669.
Effect of insulin-like growth factor-I during preantral follicular culture on steroidogenesis, in-vitro oocyte maturation, and embryo development in mice.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXktlOmtrs%3D&md5=9f2f92e28fd8994145cde220913b31b4CAS | 14960488PubMed |

Dong, J., Albertini, D., Nishimori, K., Kumar, T., Lu, N., and Matzuk, M. (1996). Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature 383, 531–535.
Growth differentiation factor-9 is required during early ovarian folliculogenesis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK28Xmt1GrsL0%3D&md5=5c10c4ac41fd02bc37d86bca11314897CAS | 8849725PubMed |

Durcova-Hills, G., Ainscough, J., and McLaren, A. (2001). Pluripotential stem cells derived from migrating primordial germ cells. Differentiation 68, 220–226.
Pluripotential stem cells derived from migrating primordial germ cells.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD38%2Fks1Krug%3D%3D&md5=176237c1816607f16453a0962fbd6e2aCAS | 11776474PubMed |

Eyestone, W. H., and First, N. L. (1989). Co-culture of early cattle embryos to the blastocyst stage with oviductal tissue or in conditioned medium. J. Reprod. Fertil. 85, 715–720.
Co-culture of early cattle embryos to the blastocyst stage with oviductal tissue or in conditioned medium.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaL1M7pvFGmtA%3D%3D&md5=fce12667ae11b9ebbf38bdaa21a68101CAS | 2704004PubMed |

Fabbri, R., Porcu, E., Marsella, T., Primavera, M., Cecconi, S., Nottola, S., Motla, P., Venturoli, S., and Flamigni, C. (2000). Human embryo development and pregnancies in homologous granulosa co-culture system. J. Assist. Reprod. Genet. 17, 1–12.
Human embryo development and pregnancies in homologous granulosa co-culture system.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD3c3hslCqsw%3D%3D&md5=5ede4381dc5947ce130d2d5189ee3ad5CAS | 10754777PubMed |

Findlay, J. K., Drummond, A. E., Dyson, M., Baillie, A. J., Robertson, D. M., and Ethier, J.-F. (2001). Production and actions of inhibin and activin during folliculogenesis in the rat. Mol. Cell. Endocrinol. 180, 139–144.
Production and actions of inhibin and activin during folliculogenesis in the rat.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXltVaqs7g%3D&md5=a352d4b9589cafc0249d4a140a3b5e3aCAS | 11451583PubMed |

Fitzpatrick, S. L., Sindoni, D. M., Shughrue, P. J., Lane, M. V., Merchenthaler, I. J., and Frail, D. E. (1998). Expression of growth differentiation factor-9 messenger ribonucleic acid in ovarian and nonovarian rodent and human tissues. Endocrinology 139, 2571–2578.
| 1:CAS:528:DyaK1cXivFSiu7w%3D&md5=442d90f1b9529a9ba25853e0081b1310CAS | 9564873PubMed |

Geijsen, N., Horoschak, M., Kim, K., Gribnau, J., Eggan, K., and Daley, G. (2004). Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 427, 148–154.
Derivation of embryonic germ cells and male gametes from embryonic stem cells.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXhtFOntA%3D%3D&md5=661bbd48894b0b0a22522f240a796fbcCAS | 14668819PubMed |

Hernandez-Ledezma, J., Villanueva, C., Sikes, J., and Reberts, R. (1995). Comparison of co-culture and conditioned medium on expansion and hatching in vitro derived bovine blastocysts. Theriogenology 43, 233.
Comparison of co-culture and conditioned medium on expansion and hatching in vitro derived bovine blastocysts.Crossref | GoogleScholarGoogle Scholar |

Hsiao, L.-L., Dangond, F., Yoshida, T., Hong, R., Jensen, R. V., Misra, J., Dillon, W., Lee, K. F., Clark, K. E., Haverty, P., et al. (2001). A compendium of gene expression in normal human tissues. Physiol. Genomics 7, 97–104.
A compendium of gene expression in normal human tissues.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XhtFagsLs%3D&md5=76b9c668c0cd7095567f95032eaa6cacCAS | 11773596PubMed |

Hübner, K., Fuhrman, G., Christensen, L. K., Kehler, J., Reinbold, R., De La Fuente, R., Wood, J., Strauss, J. F., Boiani, M., and Schöler, H. R. (2003). Derivation of oocytes from mouse embryonic stem cells. Science 300, 1251–1256.
Derivation of oocytes from mouse embryonic stem cells.Crossref | GoogleScholarGoogle Scholar | 12730498PubMed |

Ivanova, N. B., Dimos, J. T., Schaniel, C., Hackney, J. A., Moore, K. A., and Lemischka, I. R. (2002). A stem cell molecular signature. Science 298, 601–604.
A stem cell molecular signature.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XnvFSqur8%3D&md5=b53335653e494d00b574ad535f4522b3CAS | 12228721PubMed |

Jaatinen, R., Laitinen, M., Vuojolainen, K., Aaltonen, J., Heikinheimo, K., Lehtonen, E., and Ritvos, O. (1999). Localization of growth differentiation factor-9 (GDF-9) mRNA and protein in rat ovaries and cDNA cloning of rat GDF-9 and its novel homolog GDF-9B. Mol. Cell. Endocrinol. 156, 189–193.
Localization of growth differentiation factor-9 (GDF-9) mRNA and protein in rat ovaries and cDNA cloning of rat GDF-9 and its novel homolog GDF-9B.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXmsFaitbs%3D&md5=3beb031c6caf2b1913a11b1e6293f07bCAS | 10612437PubMed |

Kee, K., Vanessa, T., Martha, F., Nguyen, N., and Pera, R. (2009). Human DAZL, DAZ and BOULE genes modulate primordial germ-cell and haploid gamete formation. Nature 462, 222–225.
Human DAZL, DAZ and BOULE genes modulate primordial germ-cell and haploid gamete formation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhtlaksr3L&md5=42a21b146c9a7ef314eb902d3918d513CAS | 19865085PubMed |

Klinger, F. G., and De Felici, M. (2002). In vitro development of growing oocytes from fetal mouse oocytes: stage-specific regulation by stem cell factor and granulosa cells. Dev. Biol. 244, 85–95.
In vitro development of growing oocytes from fetal mouse oocytes: stage-specific regulation by stem cell factor and granulosa cells.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XitVaju78%3D&md5=aa197315edd4930f1022c864fcf047f7CAS | 11900461PubMed |

Knight, P. G., and Glister, C. (2001). Potential local regulatory functions of inhibins, activins and follistatin in the ovary. Reproduction 121, 503–512.
Potential local regulatory functions of inhibins, activins and follistatin in the ovary.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXivFKgs7Y%3D&md5=f2152565c96ca191accd13dbcad54bfeCAS | 11277869PubMed |

Kobayashi, K., Yamashita, S., and Hoshi, H. (1994). Influence of epidermal growth factor and transforming growth factor-α on in vitro maturation of cumulus cell-enclosed bovine oocytes in a defined medium. J. Reprod. Fertil. 100, 439–446.
Influence of epidermal growth factor and transforming growth factor-α on in vitro maturation of cumulus cell-enclosed bovine oocytes in a defined medium.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2cXlt1Gqur4%3D&md5=fff238b17955f22ff8e06a0391d8ec55CAS | 8021861PubMed |

Lacham-Kaplan, O., Chy, H., and Trounson, A. (2006). Testicular cell conditioned medium supports differentiation of embryonic stem cells into ovarian structures containing oocytes. Stem Cells 24, 266–273.
Testicular cell conditioned medium supports differentiation of embryonic stem cells into ovarian structures containing oocytes.Crossref | GoogleScholarGoogle Scholar | 16109761PubMed |

Lee, J. B., Song, J. M., Lee, J. E., Park, J. H., Kim, S. J., Kang, S. M., Kwon, J. N., Kim, M. K., Roh, S. I., and Yoon, H. S. (2004). Available human feeder cells for the maintenance of embryonic stem cells. Reproduction 128, 727–735.
Available human feeder cells for the maintenance of embryonic stem cells.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXhtFSktg%3D%3D&md5=43fece7fa1d4f362d6a5b41fb1a4e26bCAS | 15579590PubMed |

Liu, F., Ventura, F., Doody, J., and Massague, J. (1995). Human type II receptor for bone morphogenic proteins (BMPs): extension of the two-kinase receptor model to the BMPs. Mol. Cel. Biol. 15, 3479–3486.
| 1:CAS:528:DyaK2MXmsVCmu7w%3D&md5=6c97a526fa0fe7386b227e95ea99094aCAS |

Maatouk, D. M., Kellam, L. D., Mann, M. R. W., Lei, H., Li, E., Bartolomei, M. S., and Resnick, J. L. (2006). DNA methylation is a primary mechanism for silencing postmigratory primordial germ cell genes in both germ cell and somatic cell lineages. Development 133, 3411–3418.
DNA methylation is a primary mechanism for silencing postmigratory primordial germ cell genes in both germ cell and somatic cell lineages.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XhtV2hsLnE&md5=d80d528d87e82462ed9c42120eef6325CAS | 16887828PubMed |

Matsui, Y., Zsebo, K., and Hogan, B. (1992). Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 70, 841–847.
Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK38XmtVGrtrw%3D&md5=8fd1e54a94eaac6b47af7d3cc3b2fd49CAS | 1381289PubMed |

Matzuk, M. M., Burns, K. H., Viveiros, M. M., and Eppig, J. J. (2002). Intercellular communication in the mammalian ovary: oocytes carry the conversation. Science 296, 2178–2180.
Intercellular communication in the mammalian ovary: oocytes carry the conversation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XkvFGhsbw%3D&md5=66c61d3a07d8e332c110c1555ec9076eCAS | 12077402PubMed |

Mise, N., Fuchikami, T., Sugimoto, M., Kobayakawa, S., Ike, F., Ogawa, T., Tada, T., Kanaya, S., Noce, T., and Abe, K. (2008). Differences and similarities in the developmental status of embryo-derived stem cells and primordial germ cells revealed by global expression profiling. Genes Cells 13, 863–877.
Differences and similarities in the developmental status of embryo-derived stem cells and primordial germ cells revealed by global expression profiling.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXpsFKrurY%3D&md5=7df6cc135fe5d2dda5752b0987fc23efCAS | 18782224PubMed |

Niederreither, K., and Dolle, P. (2008). Retinoic acid in development: towards an integrated view. Nat. Rev. Genet. 9, 541–553.
Retinoic acid in development: towards an integrated view.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXnt1ehs7c%3D&md5=8e4ac51522520e51593bbefa33a8dac6CAS | 18542081PubMed |

Nilsson, E., and Skinner, M. (2001). Cellular interactions that control primordial follicle development and folliculogenesis. J. Soc. Gynecol. Invest. 8, S17–S20.
Cellular interactions that control primordial follicle development and folliculogenesis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXht1Oqsb8%3D&md5=30167fed8d79b84c0e66b010894c0792CAS |

Nilsson, E. E., and Skinner, M. K. (2002). Growth and differentiation factor-9 stimulates progression of early primary but not primordial rat ovarian follicle development. Biol. Reprod. 67, 1018–1024.
Growth and differentiation factor-9 stimulates progression of early primary but not primordial rat ovarian follicle development.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XmsV2jt78%3D&md5=7a24ae2318cdc1ab9120946ea8c882a5CAS | 12193416PubMed |

Nilsson, E. E., and Skinner, M. K. (2003). Bone morphogenetic protein-4 acts as an ovarian follicle survival factor and promotes primordial follicle development. Biol. Reprod. 69, 1265–1272.
Bone morphogenetic protein-4 acts as an ovarian follicle survival factor and promotes primordial follicle development.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXnsV2nsLw%3D&md5=bce8ced5544eca1c3e4d3ed7852a1ef4CAS | 12801979PubMed |

Nilsson, E. E., and Skinner, M. K. (2004). Kit ligand and basic fibroblast growth factor interactions in the induction of ovarian primordial to primary follicle transition. Mol. Cell. Endocrinol. 214, 19–25.
Kit ligand and basic fibroblast growth factor interactions in the induction of ovarian primordial to primary follicle transition.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXhtFWgsr8%3D&md5=8465a6cae4dc9732a20b7fec98947ee8CAS | 15062541PubMed |

Odorico, J. S., Kaufman, D. S., and Thomson, J. A. (2001). Multilineage differentiation from human embryonic stem cell lines. Stem Cells 19, 193–204.
Multilineage differentiation from human embryonic stem cell lines.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXkslGqtr8%3D&md5=4568327a3d6219fbe6d6746fd4a944eeCAS | 11359944PubMed |

Ohinata, Y., Payer, B., O’Carroll, D., Ancelin, K., Ono, Y., Sano, M., Barton, S., Obukhanych, T., Nussenzweig, M., and Tarakhovsky, A. (2005). Blimp1 is a critical determinant of the germ cell lineage in mice. Nature 436, 207–213.
Blimp1 is a critical determinant of the germ cell lineage in mice.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXmtVersrY%3D&md5=2b8027f4dfb66cd44ef19e6c317d9182CAS | 15937476PubMed |

Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Generation of germline-competent induced pluripotent stem cells. Nature 448, 313–317.
Generation of germline-competent induced pluripotent stem cells.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXnvVeqsL0%3D&md5=7a99bb9c3984f4c8a3cbe73af7e22fb7CAS | 17554338PubMed |

Pangas, S. A., Jorgez, C. J., and Matzuk, M. M. (2004). Growth differentiation factor 9 regulates expression of the bone morphogenetic protein antagonist gremlin. J. Biol. Chem. 279, 32 281–32 286.
Growth differentiation factor 9 regulates expression of the bone morphogenetic protein antagonist gremlin.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXmtVChs7o%3D&md5=e4abe0df9300d14737f99d977ddd46dcCAS |

Qing, T., Shi, Y., Qin, H., Ye, X., Wei, W., Liu, H., Ding, M., and Deng, H. (2007). Induction of oocyte-like cells from mouse embryonic stem cells by co-culture with ovarian granulosa cells. Differentiation 75, 902–911.
Induction of oocyte-like cells from mouse embryonic stem cells by co-culture with ovarian granulosa cells.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXmtFamsQ%3D%3D&md5=572b3a4d13e2b0670e08b9218b8beec5CAS | 17490416PubMed |

Ramalho-Santos, M., Yoon, S., Matsuzaki, Y., Mulligan, R., and Melton, D. (2002). ‘Stemness’: transcriptional profiling of embryonic and adult stem cells. Science 298, 597–600.
‘Stemness’: transcriptional profiling of embryonic and adult stem cells.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XnvFSqur4%3D&md5=4d1c3c0a14bcacfae0c86e2d4b30969bCAS | 12228720PubMed |

Resnick, J. L., Bixler, L. S., Cheng, L., and Donovan, P. J. (1992). Long-term proliferation of mouse primordial germ cells in culture. Nature 359, 550–551.
Long-term proliferation of mouse primordial germ cells in culture.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaK3s%2FisV2htA%3D%3D&md5=63663e42f1e2176cbede996f921452b1CAS | 1383830PubMed |

Ricci, G., Catizone, A., and Galdieri, M. (2004). Embryonic mouse testis development: role of platelet derived growth factor (PDGF-BB). J. Cell. Physiol. 200, 458–467.
Embryonic mouse testis development: role of platelet derived growth factor (PDGF-BB).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXmtlKisrg%3D&md5=f42cc10af7f003cc264f10536f0e8a34CAS | 15254974PubMed |

Saitou, M., Payer, B., O’Carroll, D., Ohinata, Y., and Surani, M. (2005). Blimp1 and the emergence of the germ line during development in the mouse. Cell Cycle 4, 1736–1740.
| 1:CAS:528:DC%2BD28XktVChu7w%3D&md5=7494dbb42edcb00c3d273b4096a607fdCAS | 16294024PubMed |

Schmittgen, T. D., and Livak, K. J. (2008). Analyzing real-time PCR data by the comparative C(T) method. Nat. Protoc. 3, 1101–1108.
Analyzing real-time PCR data by the comparative C(T) method.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXmvVemt7c%3D&md5=2631c2b2b1241a121f40c7c7f9c099ceCAS | 18546601PubMed |

Schotanus, K., Hage, W., Vanderstichele, H., and Hurk, R. (1997). Effects of conditioned media from murine granulosa cell lines on the growth of isolated bovine prenatal follicles. Theriogenology 48, 471–483.
Effects of conditioned media from murine granulosa cell lines on the growth of isolated bovine prenatal follicles.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD28zgtVyqtg%3D%3D&md5=41f5c03fa2404500a17dccefb5aa5fe2CAS | 16728143PubMed |

Shah, S. M., Saini, N., Ashraf, S., Zandi, M., Manik, R. S., Singla, S. K., Palta, P., and Chauhan, M. S. (2015a). Development, characterization and pluripotency analysis of buffalo (Bubalus bubalis) embryonic stem cell lines derived from in vitro fertilized, hand-guided cloned and parthenogenetic embryos. Cell. Reprogram. 17, 306–322.
Development, characterization and pluripotency analysis of buffalo (Bubalus bubalis) embryonic stem cell lines derived from in vitro fertilized, hand-guided cloned and parthenogenetic embryos.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2MXht1Ons7%2FL&md5=bec105b4b64c5f8012bcbb58809431a6CAS |

Shah, S. M., Saini, N., Ashraf, S., Zandi, M., Manoj, K. S., Manik, R. S., Singla, S. K., Palta, P., and Chauhan, M. S. (2015b). Comparative expression analysis of gametogenesis-associated genes in foetal and adult bubaline (Bubalus bubalis) ovaries and testes. Reprod. Domest. Anim. 50, 365–377.
Comparative expression analysis of gametogenesis-associated genes in foetal and adult bubaline (Bubalus bubalis) ovaries and testes.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2MXntFSgsr8%3D&md5=92df018a0220a30fe03de7700969b2b6CAS | 25703697PubMed |

Tanaka, S. S., Yamaguchi, Y. L., Tsoi, B., Lickert, H., and Tam, P. P. L. (2005). IFITM/Mil/fragilis family proteins IFITM1 and IFITM3 play distinct roles in mouse primordial germ cell homing and repulsion. Dev. Cell 9, 745–756.
IFITM/Mil/fragilis family proteins IFITM1 and IFITM3 play distinct roles in mouse primordial germ cell homing and repulsion.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XptV2i&md5=c216dab1ac779dc043ded7e250d0c1cdCAS | 16326387PubMed |

Tilgner, K., Atkinson, S., Golebiewska, A., Stojkovic, M., Lako, M., and Armstrong, L. (2008). Isolation of primordial germ cells from differentiating human embryonic stem cells. Stem Cells 26, 3075–3085.
Isolation of primordial germ cells from differentiating human embryonic stem cells.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhtFSkt7w%3D&md5=445e0acd9bfbcefb3eac2c1f28d8d7b0CAS | 18802037PubMed |

Toyooka, Y., Tsunekawa, N., Akasu, R., and Noce, T. (2003). Embryonic stem cells can form germ cells in vitro. Proc. Natl Acad. Sci. USA 100, 11 457–11 462.
Embryonic stem cells can form germ cells in vitro.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXotFKmsb4%3D&md5=f23a4d07b1369e9d4edec51b5ba583bdCAS |

Tres, L. L., Rosselot, C., and Kierszenbaum, A. L. (2004). Primordial germ cells: what does it take to be alive? Mol. Reprod. Dev. 68, 1–4.
Primordial germ cells: what does it take to be alive?Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXivFyku70%3D&md5=9a6af4e2998118f59ea861f0a5a67bceCAS | 15039942PubMed |

Wang, J., and Roy, S. (2004). Growth differentiation factor-9 and stem cell factor promote primordial follicle formation in the hamster: modulation by follicle-stimulating hormone. Biol. Reprod. 70, 577–585.
Growth differentiation factor-9 and stem cell factor promote primordial follicle formation in the hamster: modulation by follicle-stimulating hormone.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXhs1Chsrc%3D&md5=2ee97cf3bd24a8bdea3c48d90686f2f5CAS | 14585807PubMed |

Warrington, J. A., Nair, A., Mahadevappa, M., and Tsyganskaya, M. (2000). Comparison of human adult and fetal expression and identification of 535 housekeeping/maintenance genes. Physiol. Genomics 2, 143–147.
| 1:CAS:528:DC%2BD3cXltFKns70%3D&md5=8aaaa064fb242f3c4137a9ad28ac17e8CAS | 11015593PubMed |

West, J. A., and Daley, G. Q. (2004). In vitro gametogenesis from embryonic stem cells. Curr. Opin. Cell Biol. 16, 688–692.
In vitro gametogenesis from embryonic stem cells.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXpsVOrur8%3D&md5=a220bbd5af49b6cf5f15292a4d5c709eCAS | 15530782PubMed |

Xu, X., Pantakani, D. V., Luhrig, S., Tan, X., Khromov, T., Nolte, J., Dressel, R., Zechner, U., and Engel, W. (2011). Stage-specific germ-cell marker genes are expressed in all mouse pluripotent cell types and emerge early during induced pluripotency. PLoS One 6, e22413.
Stage-specific germ-cell marker genes are expressed in all mouse pluripotent cell types and emerge early during induced pluripotency.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXhtVKgtLnF&md5=7e89b47b2248569ba4372f8685346191CAS | 21799849PubMed |

Zolti, M., Ben-Rafel, Z., Meirom, R., Shemesh, M., Bider, D., Mashiach, S., and Apte, R. (1991). Cytokine involvement in oocytes and early embryos. Fertil. Steril. 56, 265–272.
| 1:STN:280:DyaK3MzgtFansA%3D%3D&md5=5fbbe38735c5a1e77366792670aac6a2CAS | 2070856PubMed |