Skip to main content

Live-cell imaging and ultrastructural analysis reveal remarkable features of cultured porcine gonocytes

Cell and Tissue Research volume 381, pages 361–377 (2020)Cite this article

Abstract

Gonocytes in the neonatal testis have male germline stem cell potential. The objective of the present study was to examine the behavior and ultrastructure of gonocytes in culture. Neonatal porcine testis cells were cultured for 4 weeks and underwent live-cell imaging to explore real-time interactions among cultured cells. This included imaging every 1 h from day 0 to day 3, every 2 h from day 4 to day 7, and every 1 h for 24 h at days 14, 21, and 28. Samples also underwent scanning electron microscopy, transmission electron microscopy, morphometric evaluations, immunofluorescence, and RT-PCR. Live-cell imaging revealed an active amoeboid-like movement of gonocytes, assisted by the formation of extensive cytoplasmic projections, which, using scanning electron microscopy, were categorized into spike-like filopodia, leaf-like lamellipodia, membrane ruffles, and cytoplasmic blebs. In the first week of culture, gonocytes formed loose attachments on top of a somatic cell monolayer and, in week 2, formed grape-like clusters, which, over time, grew in cell number. Starting at week 3 of culture, some of the gonocyte clusters transformed into large multinucleated embryoid body–like colonies (EBLCs) that expressed both gonocyte- and pluripotent-specific markers. The number and diameter of individual gonocytes, the number and density of organelles within gonocytes, as well as the number and diameter of the EBLCs increased over time (P < 0.05). In conclusion, cultured porcine gonocytes displayed extensive migratory behavior facilitated by their various cytoplasmic projections, propagated, and transformed into EBLCs that increased in size and complexity over time.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

References

  • Altman E, Yango P, Moustafa R, Smith JF, Klatsky PC, Tran ND (2014) Characterization of human spermatogonial stem cell markers in fetal, pediatric, and adult testicular tissues. Reproduction 148(4):417–427

    CAS  PubMed  PubMed Central  Google Scholar 

  • Aoshima K, Baba A, Makino Y, Okada Y, Sun Y (2013) Establishment of alternative culture method for spermatogonial stem cells using knockout serum replacement. PLoS One 8(10):e77715

    CAS  PubMed  PubMed Central  Google Scholar 

  • Aponte PM, Soda T, van de Kant HJG, De Rooij DG (2006) Basic features of bovine spermatogonial culture and effects of glial cell line-derived neurotrophic factor. Theriogenology 65(9):1828–1847

    CAS  PubMed  Google Scholar 

  • Arregui L, Rathi R, Megee SO, Honaramooz A, Gomendio M, Roldan ERS, Dobrinski I (2008) Xenografting of sheep testis tissue and isolated cells as a model for preservation of genetic material from endangered ungulates. Reproduction 136(1):85–93

    CAS  PubMed  Google Scholar 

  • Awang-Junaidi AH, Honaramooz A (2018) Optimization of culture conditions for short-term maintenance, proliferation, and colony formation of porcine gonocytes. J Anim Sci Biotechnol 9(1):8

    PubMed  PubMed Central  Google Scholar 

  • Baillie AH (1964) The histochemistry and ultrastructure of the gonocyte. J Anat 98(4):641–645

    CAS  PubMed  PubMed Central  Google Scholar 

  • Borjigin U, Zhou X, Han X, Li R, Herrid M, Bou S (2011) Enrichment and short term culture of the ovine gonocyte. J Anim Vet Adv 10(22):2936–2942

    Google Scholar 

  • Bowman PD, Meek RL, Daniel CW (1975) Aging of human fibroblasts in vitro. Correlations between DNA synthetic ability and cell size. Exp Cell Res 93(1):184–190

    CAS  PubMed  Google Scholar 

  • Bratt-Leal AM, Carpenedo RL, McDevitt TC (2009) Engineering the embryoid body microenvironment to direct embryonic stem cell differentiation. Biotechnol Prog 25(1):43–51

    CAS  PubMed  PubMed Central  Google Scholar 

  • Buzzard JJ, Wreford NG, Morrison JR (2002) Marked extension of proliferation of rat Sertoli cells in culture using recombinant human FSH. Reproduction (Cambridge, England) 124(5):633–641

    CAS  Google Scholar 

  • Castrillon DH, Quade BJ, Wang TY, Quigley C, Crum CP (2000) The human VASA gene is specifically expressed in the germ cell lineage. Proc Natl Acad U S A 97(17):9585–9590

    CAS  Google Scholar 

  • Chuma S, Kanatsu-Shinohara M, Inoue K, Ogonuki N, Miki H, Toyokuni S, Hosokawa M, Nakatsuji N, Ogura A, Shinohara T (2005) Spermatogenesis from epiblast and primordial germ cells following transplantation into postnatal mouse testis. Development 132(1):117–122

    CAS  PubMed  Google Scholar 

  • Cristofalo VJ, Kritchevsky D (1969) Cell size and nucleic acid content in the diploid human cell line WI-38 during aging. Med Exp Int J Exp Med 19(6):313–320

    CAS  PubMed  Google Scholar 

  • Dang SM, Gerecht-Nir S, Chen J, Itskovitz-Eldor J, Zandstra PW (2004) Controlled, scalable embryonic stem cell differentiation culture. Stem Cells 22(3):275–282

    PubMed  Google Scholar 

  • Dann CT, Alvarado AL, Molyneux LA, Denard BS, Garbers DL, Porteus MH (2008) Spermatogonial stem cell self-renewal requires OCT4, a factor downregulated during retinoic acid-induced differentiation. Stem Cells 26(11):2928–2937

    CAS  PubMed  Google Scholar 

  • Dasgupta A, Hughey R, Lancin P, Larue L, Moghe PV (2005) E-cadherin synergistically induces hepatospecific phenotype and maturation of embryonic stem cells in conjunction with hepatotrophic factors. Biotechnol Bioeng 92(3):257–266

    CAS  PubMed  Google Scholar 

  • De Miguel MP, De Boer-Brouwer M, Paniagua R, van den Hurk R, De Rooij DG, Van Dissel-Emiliani FM (1996) Leukemia inhibitory factor and ciliary neurotropic factor promote the survival of Sertoli cells and gonocytes in coculture system. Endocrinology 137(5):1885–1893

    PubMed  Google Scholar 

  • De Rooij DG (1998) Stem cells in the testis. Int J Exp Pathol 79(2):67–80

    PubMed  Google Scholar 

  • De Rooij DG, Russell LD (2000) All you wanted to know about spermatogonia but were afraid to ask. J Androl 21(6):776–798

    PubMed  Google Scholar 

  • Fok EYL, Zandstra PW (2005) Shear-controlled single-step mouse embryonic stem cell expansion and embryoid body-based differentiation. Stem Cells 23(9):1333–1342

    CAS  PubMed  Google Scholar 

  • Fujihara M, Kim S-M, Minami N, Yamada M, Imai H (2011) Characterization and in vitro culture of male germ cells from developing bovine testis. J Reprod Dev 57(3):355–364

    CAS  PubMed  Google Scholar 

  • Goel S, Imai H (2011) Pluripotent stem cells from testis. In: Kallos MS (ed) Embryonic stem cells—differentiation and pluripotent alternatives. IntechOpen, London, pp 473–492

    Google Scholar 

  • Goel S, Sugimoto M, Minami N, Yamada M, Kume S, Imai H (2007) Identification, isolation, and in vitro culture of porcine gonocytes. Biol Reprod 77(1):127–137

    CAS  PubMed  Google Scholar 

  • Goel S, Fujihara M, Minami N, Yamada M, Imai H (2008) Expression of NANOG, but not POU5F1, points to the stem cell potential of primitive germ cells in neonatal pig testis. Reproduction 135:785–795

    CAS  PubMed  Google Scholar 

  • Goel S, Fujihara M, Tsuchiya K, Takagi Y, Minami N, Yamada M, Imai H (2009) Multipotential ability of primitive germ cells from neonatal pig testis cultured in vitro. Reprod Fertil Dev 21(5):696–708

    CAS  PubMed  Google Scholar 

  • Gondos B, Hobel CJ (1971) Ultrastructure of germ cell development in the human fetal testis. Z Zellforch Microsk Anat Histochem 119(1):1–20

    CAS  Google Scholar 

  • Hadley MA, Byers SW, Suárez-Quian CA, Kleinman HK, Dym M (1985) Extracellular matrix regulates Sertoli cell differentiation, testicular cord formation, and germ cell development in vitro. J Cell Biol 101(4):1511–1522

    CAS  PubMed  Google Scholar 

  • Hamra FK, Chapman KM, Nguyen DM, Williams-Stephens AA, Hammer RE, Garbers DL (2005) Self renewal, expansion, and transfection of rat spermatogonial stem cells in culture. Proc Natl Acad Sc U S A 102(48):17430–17435

    CAS  Google Scholar 

  • Han SY, Gupta MK, Uhm SJ, Lee HT (2009) Isolation and in vitro culture of pig spermatogonial stem cell. Asian-Australas J Anim Sci 22(2):187–193

    CAS  Google Scholar 

  • Hasthorpe S (2003) Clonogenic culture of normal spermatogonia: in vitro regulation of postnatal germ cell proliferation. Biol Reprod 68(4):1354–1360

    CAS  PubMed  Google Scholar 

  • Hasthorpe S, Barbic S, Farmer PJ, Hutson JM (1999) Neonatal mouse gonocyte proliferation assayed by an in vitro clonogenic method. J Reprod Fertil 116(2):335–344

    CAS  PubMed  Google Scholar 

  • Hasthorpe S, Barbic S, Farmer PJ, Hutson JM (2000) Growth factor and somatic cell regulation of mouse gonocyte-derived colony formation in vitro. J Reprod Fertil 119(1):85–91

    CAS  PubMed  Google Scholar 

  • Hermann BP, Orwig KE (2011) Translating spermatogonial stem cell transplantation to the clinic. In: Herman B, Orwig K (eds) Male germline stem cells, developmental and regenerative potential. Humana, Totowa, pp 227–253

    Google Scholar 

  • Herrid M, Olejnik J, Jackson M, Suchowerska N, Stockwell S, Davey R, Hutton K, Hope S, Hill JR (2009) Irradiation enhances the efficiency of testicular germ cell transplantation in sheep. Biol Reprod 81(5):898–905

    CAS  PubMed  Google Scholar 

  • Honaramooz A (2012) Cryopreservation of testicular tissue. In: Katkov I (ed) Current frontiers in cryobiology. IntechOpen, London, pp 209–228

    Google Scholar 

  • Honaramooz A (2014) Potential and challenges of testis tissue xenografting from diverse ruminant species. In: Juengel JL, Miyamoto A, Price C, Reynolds LP, Smith MP, Webb R (eds) Reproduction in domestic ruminants VII. Leicestershire, Context Products Ltd, pp 257–275

    Google Scholar 

  • Honaramooz A, Behboodi E, Megee SO, Overton SA, Galantino-Homer H, Echelard Y, Dobrinski I (2003) Fertility and germline transmission of donor haplotype following germ cell transplantation in immunocompetent goats. Biol Reprod 69(4):1260–1264

    CAS  PubMed  Google Scholar 

  • Honaramooz A, Megee SO, Rathi R, Dobrinski I (2007) Building a testis: formation of functional testis tissue after transplantation of isolated porcine (Sus scrofa) testis cells. Biol Reprod 76(1):43–47

    CAS  PubMed  Google Scholar 

  • Izadyar F, Den Ouden K, Stout TAE, Stout J, Coret J, Lankveld DPK, Spoormakers TJP, Colenbrander B, Oldenbroek JK, Van der Ploeg KD, Woelders H, Kal HB, De Rooij DG (2003) Autologous and homologous transplantation of bovine spermatogonial stem cells. Reproduction 126(6):765–774

    CAS  PubMed  Google Scholar 

  • Jeong D, McLean DJ, Griswold MD (2003) Long-term culture and transplantation of murine testicular germ cells. J Androl 24(5):661–669

    PubMed  Google Scholar 

  • Kanatsu-Shinohara M, Ogonuki N, Inoue K, Miki H, Ogura A, Toyokuni S, Shinohara T (2003) Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol Reprod 69(2):612–616

    CAS  PubMed  Google Scholar 

  • Kanatsu-Shinohara M, Inoue K, Ogonuki N, Morimoto H, Ogura A, Shinohara T (2011) Serum- and feeder-free culture of mouse germline stem cells. Biol Reprod 84(1):97–105

    CAS  PubMed  Google Scholar 

  • Kanatsu-Shinohara M, Ogonuki N, Matoba S, Morimoto H, Ogura A, Shinohara T (2014) Improved serum- and feeder-free culture of mouse germline stem cells. Biol Reprod 9188:1–11

    Google Scholar 

  • Kim KJ, Cho CM, Kim BG, Lee YA, Kim BJ, Kim YH, Kim CG, Schmidt JA, Ryu BY (2014) Lentiviral modification of enriched populations of bovine male gonocytes. J Anim Sci 92(1):106–118

    CAS  PubMed  Google Scholar 

  • Korn ED, Carlier MF, Pantaloni D (1987) Actin polymerization and ATP hydrolysis. Science 238(4827):638–644

    CAS  PubMed  Google Scholar 

  • Kubota H, Avarbock MR, Brinster RL (2004) Culture conditions and single growth factors affect fate determination of mouse spermatogonial stem cells. Biol Reprod 71(3):722–731

    CAS  PubMed  Google Scholar 

  • Kubota H, Wu X, Goodyear SM, Avarbock MR, Brinster RL (2011) Glial cell line-derived neurotrophic factor and endothelial cells promote self-renewal of rabbit germ cells with spermatogonial stem cell properties. FASEB J 25(8):2604–2614

    CAS  PubMed  PubMed Central  Google Scholar 

  • Kuijk EW, Colenbrander B, Roelen BAJ (2009) The effects of growth factors on in vitro-cultured porcine testicular cells. Reproduction 138(4):721–731

    CAS  PubMed  Google Scholar 

  • Kurosawa H (2007) Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells. J Biosci Bioeng 103(5):389–398

    CAS  PubMed  Google Scholar 

  • Larsen H-PE, Thorup J, Skovgaard LT, Cortes D, Byskov AG (2002) Long-term cultures of testicular biopsies from boys with cryptorchidism: effect of FSH and LH on the number of germ cells. Hum Reprod 17(2):383–389

    CAS  PubMed  Google Scholar 

  • Lauffenburger DA, Horwitz AF (1996) Cell migration: a physically integrated molecular process. Cell 84(3):359–369

    CAS  PubMed  Google Scholar 

  • Lee WY, Park HJ, Lee R, Lee KH, Kim YH, Ryu BY, Kim NH, Kim JH, Kim JH, Moon SH, Park JK, Chung HJ, Kim DH, Song H (2013) Establishment and in vitro culture of porcine spermatogonial germ cells in low temperature culture conditions. Stem Cell Res 11(3):1234–1249

    CAS  PubMed  Google Scholar 

  • Llames S, García-Pérez E, Meana Á, Larcher F, del Río M (2015) Feeder layer cell actions and applications. Tissue Eng Part B Rev 21(4):345–353

    CAS  PubMed  PubMed Central  Google Scholar 

  • Luo J, Megee S, Rathi R, Dobrinski I (2006) Protein gene product 9.5 is a spermatogonia-specific marker in the pig testis: application to enrichment and culture of porcine spermatogonia. Mol Reprod Dev 73(12):1531–1540

    CAS  PubMed  Google Scholar 

  • McGuinness MP, Orth JM (1992) Reinitiation of gonocyte mitosis and movement of gonocytes to the basement membrane in testes of newborn rats in vivo and in vitro. Anat Rec 233(4):527–537

    CAS  PubMed  Google Scholar 

  • Miething A (1993) Multinucleated spermatocytes in the aging human testis: formation, morphology, and degenerative fate. Andrologia 25(6):317–323

    CAS  PubMed  Google Scholar 

  • Miething A (1995) Multinuclearity of germ cells in the senescent human testis originates from a process of cell-cell fusion. J Submicroc Cytol Pathol 27(1):105–113

    CAS  Google Scholar 

  • Nagano R, Tabata S, Nakanishi Y, Ohsako S, Kurohmaru M, Hayashi Y (2000) Reproliferation and relocation of mouse male germ cells (gonocytes) during prespermatogenesis. Anat Rec 258(2):210–220

    CAS  PubMed  Google Scholar 

  • Nagano M, Ryu BY, Brinster CJ, Avarbock MR, Brinster RL (2003) Maintenance of mouse male germ line stem cells in vitro. Biol Reprod 68:2207–2214

    CAS  PubMed  Google Scholar 

  • Niu Z, Wu S, Wu C, Li N, Zhu H, Liu W, Hua J (2016) Multipotent male germline stem cells (mGSCs) from neonate porcine testis. Braz Arch Biol Technol 59:e16150449

    Google Scholar 

  • Oatley JM, Brinster RL (2008) Regulation of spermatogonial stem cell self-renewal in mammals. Annu Rev Cell Dev Biol 24:263–286

    CAS  PubMed  PubMed Central  Google Scholar 

  • Ohbo K, Yoshida S, Ohmura M, Ohneda O, Ogawa T, Tsuchiya H, Kuwana T, Kehler J, Abe K, Schöler HR, Suda T (2003) Identification and characterization of stem cells in prepubertal spermatogenesis in mice. Dev Biol 258(1):209–225

    CAS  PubMed  Google Scholar 

  • Ohmura M, Yoshida S, Ide Y, Nagamatsu G, Suda T, Ohbo K (2004) Spatial analysis of germ stem cell development in Oct-4/EGFP transgenic mice. Arch Histol Cytol 67(4):285–296

    CAS  PubMed  Google Scholar 

  • Orth JM, Boehm R (1990) Functional coupling of neonatal rat Sertoli cells and gonocytes in coculture. Endocrinology 127(6):2812–2820

    CAS  PubMed  Google Scholar 

  • Orth JM, Jester WF (1995) NCAM mediates adhesion between gonocytes and Sertoli cells in cocultures from testes of neonatal rats. J Androl 16(5):389–399

    CAS  PubMed  Google Scholar 

  • Orwig KE, Ryu B-Y, Avarbock MR, Brinster RL (2002) Male germ-line stem cell potential is predicted by morphology of cells in neonatal rat testes. Proc Natl Acad Sci U S A 99(18):11706–11711

    CAS  PubMed  PubMed Central  Google Scholar 

  • Park JH, Kim SJ, Oh EJ, Moon SY, Il RS, Kim CG, Yoon HS (2003) Establishment and maintenance of human embryonic stem cells on STO, a permanently growing cell line. Biol Reprod 69(6):2007–2014

    CAS  PubMed  Google Scholar 

  • Perrett RM, Turnpenny L, Eckert JJ, O’Shea M, Sonne SB, Cameron IT, Wilson DI, Meyts ER-D, Hanley NA (2008) The early human germ cell lineage does not express SOX2 during in vivo development or upon in vitro culture. Biol Reprod 78(5):852–858

    CAS  PubMed  Google Scholar 

  • Pesce M, Farrace MG, Piacentini M, Dolci S, De Felici M, DeChiara TM, Yancopoulous GD (1993) Stem cell factor and leukemia inhibitory factor promote primordial germ cell survival by suppressing programmed cell death (apoptosis). Development (Cambridge, England) 118(4):1089–1094

    CAS  Google Scholar 

  • Rajpert-De Meyts E, Hanstein R, Jørgensen N, Graem N, Vogt PH, Skakkebaek NE (2004) Developmental expression of POU5F1 (OCT-3/4) in normal and dysgenetic human gonads. Hum Reprod 19(6):1338–1344

    CAS  PubMed  Google Scholar 

  • Reijo RA, Dorfman DM, Slee R, Renshaw AA, Loughlin KR, Cooke H, Page DC (2000) DAZ family proteins exist throughout male germ cell development and transit from nucleus to cytoplasm at meiosis in humans and mice. Biol Reprod 63(5):1490–1496

    CAS  PubMed  Google Scholar 

  • Ryu B-Y, Kubota H, Avarbock MR, Brinster RL (2005) Conservation of spermatogonial stem cell self-renewal signaling between mouse and rat. Proc Proc Natl Acad Sci U S A 102(40):14302–14307

    CAS  PubMed  Google Scholar 

  • Sahare M, Kim S-M, Otomo A, Komatsu K, Minami N, Yamada M, Imai H (2016) Factors supporting long-term culture of bovine male germ cells. Reprod Fertil Dev 28(12):2039–2050

    CAS  PubMed  Google Scholar 

  • Schneider EL, Fowlkes BJ (1976) Measurement of DNA content and cell volume in senescent human fibroblasts utilizing flow multiparameter single cell analysis. Exp Cell Res 98(2):298–302

    CAS  PubMed  Google Scholar 

  • Schneider EL, Mitsui Y (1976) The relationship between in vitro cellular aging and in vivo human age. Proc Natl Acad Sci U S A 73:3584–3588

    CAS  PubMed  PubMed Central  Google Scholar 

  • Shinohara T, Avarbock MR, Brinster RL (1999) Beta1- and alpha6-integrin are surface markers on mouse spermatogonial stem cells. Proc Natl Acad Sci U S A 96(10):5504–5509

    CAS  PubMed  PubMed Central  Google Scholar 

  • Shinohara T, Orwig KE, Avarbock MR, Brinster RL (2000) Spermatogonial stem cell enrichment by multiparameter selection of mouse testis cells. Proc Natl Acad Sci U S A 97(15):8346–8351

    CAS  PubMed  PubMed Central  Google Scholar 

  • Tiptanavattana N, Radtanakatikanon A, Hyttel P, Holm H, Buranapraditkun S, Setthawong P, Techakumphu M, Tharasanit T (2015) Determination phase at transition of gonocytes to spermatogonial stem cells improves establishment efficiency of spermatogonial stem cells in domestic cats. J Reprod Dev 61(6):581–588

    CAS  PubMed  PubMed Central  Google Scholar 

  • Tu J, Fan L, Tao K, Zhu W, Li J, Lu G (2007) Stem cell factor affects fate determination of human gonocytes in vitro. Reproduction 134(6):757–765

    CAS  PubMed  Google Scholar 

  • van den Ham R, van Pelt AMM, de Miguel MP, Kooten PJS, Walther N, van Dissel-Emiliani FM (1997) Immunomagnetic isolation of fetal rat gonocytes. Am J Reprod Immunol 38(1):39–45

    PubMed  Google Scholar 

  • van Dissel-Emiliani F, Boer-Brouwer M, Spek E (1993) Survival and proliferation of rat gonocytes in vitro. Cell Tissue Res 273:141–147

    PubMed  Google Scholar 

  • van Dissel-Emiliani FM, De Boer-Brouwer M, De Rooij DG (1996) Effect of fibroblast growth factor-2 on Sertoli cells and gonocytes in coculture during the perinatal period. Endocrinology 137(2):647–654

    PubMed  Google Scholar 

  • Wang X, Chen T, Zhang Y, Li B, Xu Q, Song C (2015) Isolation and culture of pig spermatogonial stem cells and their in vitro differentiation into neuron-like cells and adipocytes. Int J Mol Sci 16(11):26333–26346

    CAS  PubMed  PubMed Central  Google Scholar 

  • Wu X, Schmidt JA, Avarbock MR, Tobias JW, Carlson CA, Kolon TF, Ginsberg JP, Brinster RL (2009) Prepubertal human spermatogonia and mouse gonocytes share conserved gene expression of germline stem cell regulatory molecules. Proc Natl Acad Sci U S A 106(51):21672–21677

    CAS  PubMed  PubMed Central  Google Scholar 

  • Yang Y, Honaramooz A (2011) Efficient purification of neonatal porcine gonocytes with Nycodenz and differential plating. Reprod Fertil Dev 23(3):496–505

    CAS  PubMed  Google Scholar 

  • Yang Y, Yarahmadi M, Honaramooz A (2010) Development of novel strategies for the isolation of piglet testis cells with a high proportion of gonocytes. Reprod Fertil Dev 22(7):1057–1065

    CAS  PubMed  Google Scholar 

  • Yang J, Dungrawala H, Hua H, Manukyan A, Abraham L, Lane W, Mead H, Wright J, Schneider BL (2011) Cell size and growth rate are major determinants of replicative lifespan. Cell Cycle 10(1):144–155

    CAS  PubMed  PubMed Central  Google Scholar 

  • Yu X, Sidhu JS, Hong S, Faustman EM (2005) Essential role of extracellular matrix (ECM) overlay in establishing the functional integrity of primary neonatal rat sertoli cell/gonocyte co-cultures: an improved in vitro model for assessment of male reproductive toxicity. Toxicol Sci 84(2):378–393

    CAS  PubMed  Google Scholar 

  • Zadrag-Tecza R, Kwolek-Mirek M, Bartosz G, Bilinski T (2009) Cell volume as a factor limiting the replicative lifespan of the yeast Saccharomyces cerevisiae. Biogerontology 10(4):481–488

    CAS  PubMed  Google Scholar 

  • Zheng Y, He Y, An J, Qin J, Wang Y, Zhang Y, Tian X, Zeng W (2014) THY1 is a surface marker of porcine gonocytes. Reprod Fertil Dev 26(4):533–539

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Brian Andries and Tatjana Ometlic at the Prairie Swine Centre for their assistance in the collection of neonatal porcine testes. We also thank Drs. Muhammad Anzar and Patrick Krone for their insightful discussions.

Funding

This study was supported by grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada to A. Honaramooz. The University of Saskatchewan College of Graduate and Postdoctoral Studies and the University of Saskatchewan Western College of Veterinary Medicine provided scholarships to A.H. Awang-Junaidi and M.A. Fayaz. The Malaysian Ministry of Higher Education also provided financial support to A.H. Awang-Junaidi.

Author information

Author notes

  1. Awang Hazmi Awang-Junaidi

    Present address: Department of Veterinary Preclinical Sciences, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia

Authors and Affiliations

  1. Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, S7N 5B4, SK, Canada

    Awang Hazmi Awang-Junaidi, Mohammad Amin Fayaz, Daniel J. MacPhee & Ali Honaramooz

  2. WCVM Imaging Centre, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, S7N 5B4, SK, Canada

    Eiko Kawamura & LaRhonda Sobchishin

Authors
  1. Awang Hazmi Awang-Junaidi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mohammad Amin Fayaz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Eiko Kawamura
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. LaRhonda Sobchishin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Daniel J. MacPhee
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ali Honaramooz
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.H.A.J. contributed to conceiving and designing of the study, performed the experiments, and wrote the first draft of the manuscript. M.A.F. contributed to the experimental analyses and revising the manuscript. E.K. and L.S. contributed to the live-cell imaging and ultrastructural studies. D.J.M. contributed to designing of the study and revising the manuscript. A.H. supervised the work and contributed to conceiving and designing of the study, as well as writing and revising the manuscript.

Corresponding author

Correspondence to Ali Honaramooz.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

All experimental procedures involving animals were approved by the University of Saskatchewan’s Institutional Animal Care and Use Committee.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Portions of this study were presented as an abstract at the 51st Annual Meeting of Society for the Study of Reproduction, 2018.

Electronic supplementary material

Supplementary Video 1

Organization of neonatal porcine testis cells in prolonged culture at 0–24 h (00:04 s), 25–48 h (00:09 s), 17–120 h (00:15 s), D7 (00:20 s), D14 d (00:27 s), D21 (00.33 s) and D28 (00:39 s) (time lapse 15 fps). Images were taken every 1 h for 24 h from day 0 to day 3, every 2 h for 24 h from day 4 to day 7, and finally every 1 h for 24 h at days 14, 24, and 28. (WMV 42141 kb)

Supplementary Video 2

Cytoplasmic projections and amoeboid-like movement of gonocytes at 1-week (a) in the media (00:04 s) and (b) on top of somatic cell monolayer (00:12 s). The images were taken every 2.5 s for 30 s (time lapse 1 fps) and 5 min for gonocytes (time lapse 4 fps) floating in the media and on the somatic monolayer, respectively. (WMV 15346 kb)

Supplementary Video 3

The stages in the formation of embryoid body–like colony (EBLC) formation. The images were taken every 2.5 s for 5 min (time lapse 4 fps). (a) Gonocytes first settled on the somatic cell monolayer either singles or paired (00:04 s). (b) Continuous migration of gonocytes towards each other results in the formation of morula-like (grape-like) colonies (00:11 s). (c) The morula-like colonies increased in size over time (00:18 s). (d) The EBCL formed after ~14 days of culture (00:24 s). Cytoplasmic fusions of adjacent gonocytes seemed to transform the morula-like colonies into an embryoid body–like colony (00:34 s). (e) Continued fusion of migrating gonocytes increased the mass of the EBLC (00:44 s). (g) Note the presence of extensive cytoplasmic projections possessed by EBLC which seemed to assist with its movement, and further attachment of migrating gonocytes (00:56 s). (h) Higher magnification of active movement of cytoplasmic projections of EBLC (01:06). (WMV 122001 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Awang-Junaidi, A.H., Fayaz, M.A., Kawamura, E. et al. Live-cell imaging and ultrastructural analysis reveal remarkable features of cultured porcine gonocytes. Cell Tissue Res 381, 361–377 (2020). https://doi.org/10.1007/s00441-020-03218-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00441-020-03218-5

Keywords

  • Gonocytes
  • Ultrastructure
  • Cell culture
  • Germline stem cells
  • Live-cell imaging