Characterization and In Vitro Culture of Male Germ Cells from Developing Bovine Testis

Abstract

Abstract. The transition from male primitive germ cells (gonocytes) to type A spermatogonia in the neonatal testis is the initial process and a crucial process in spermatogenesis. However, in large domestic animals, the physiological and biochemical characteristics of germ cells during the developmental processes remain largely unknown. In this study, we characterized bovine germ cells in the developing testis from the neonatal stage to the adult stage. The binding of the lectin Dolichos biflorus agglutinin (DBA) and the expression of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) were restricted to gonocytes in the neonatal testis and spermatogonia in the adult testis. Gonocytes also expressed a germ cell marker (VASA) and stem cell markers (NANOG and OCT3/4), while the expressions of these markers in the adult testis were restricted to differentiated spermatic cells and were rarely expressed in spermatogonia. We subsequently utilized these markers to characterize gonocytes and spermatogonia after culture in vitro. Spermatogonia that were collected from the adult testis formed colonies in vitro only for one week. On the other hand, gonocytes from the neonatal testis could proliferate and form colonies after every passage for 1.5 months in culture. These colonies retained undifferentiated states of gonocytes as confirmed by the expression of both germ cell and stem cell markers. Moreover, a transplantation assay using immunodeficient mice testes showed that long-term cultured cells derived from gonocytes were able to colonize in the recipient testis. These results indicated that bovine gonocytes could maintain germ cell and stem cell potential in vitro. Key words: Gonocytes, Spermatogonia, Spermatogenesis, Stem cells, Testis (J. Reprod. Dev. 57: [355][356][357][358][359][360][361][362][363][364] 2011) erm cells originate from primordial germ cells (PGCs), which are primary cells on the germline lineage in embryos. After migration to the genital ridge, male germ cells become gonocytes [1]. At a certain period after birth, gonocytes migrate to the basement membrane of the testis and differentiate to type A spermatogonia including spermatogonial stem cells (SSCs). SSCs have the potential to self-renew and generate differentiated germ cells, resulting in the production of large numbers of spermatozoa throughout most or all of adult life. Thus, gonocytes have key roles in producing SSCs and initiating spermatogenesis. In mice, the transition of gonocytes to SSCs begins 3 days after birth [2]. Although some report have shown the postnatal testis development in large domestic species including cattle (Bos indicus [3], Bos taurus [4,5]), little is known about gonocytes during their development in cattle.Specific germ cell markers have been identified in the mouse testis. One such marker is VASA, a DEAD (asparagineglutamine-alanine-asparagine) box protein 4 (DDX4) that is required for male germ cell development in mice [6,7]. Additionally, stem cell characteristics of mouse SSCs were determined by a transplantation a...

Effects of extracellular matrices and lectin Dolichos biflorus agglutinin on cell adhesion and self-renewal of bovine gonocytes cultured in vitro

“…3) suggests that proper stimulation of somatic cells, including Leydig cells, Sertoli cells and endothelial cells (which are necessary for the survival and proliferation of germ cells; Aponte et al 2008), supports the colony formation of gonocytes on the DBA-precoated plates. In fact, the colony formation of bovine gonocytes can be supported on GN-coated plates for the first week of culture (Fujihara et al 2011). However, adhesion affinity and colony formation of gonocytes were lower on GN-compared with DBA-precoated plates (Fig.…”

“…Colonies of bovine gonocytes have stem cell potential, as identified by the expression of stem cell-specific genes (NANOG and POU5F1; Fujihara et al 2011). The colony formation of testicular cells in culture depends on the presence of germ cell populations (Aponte et al 2008), and these cell populations were strongly associated with the expression of NANOG and POU5F1 (Fig.…”

“…To collect testicular cells, the testes were treated with threestep enzymatic digestions and isolated cells were subjected to discontinuous density gradient Percoll centrifugation, as described previously (Fujihara et al 2011) with some modifications. Briefly, to obtain a testicular cell suspension, the decapsulated testicular tissue was minced into small pieces and treated with a first enzymatic solution that was supplemented with 2 mg mL À1 collagenase (Type IV; Sigma-Aldrich) and 1 mg mL À1 DNase I (Sigma-Aldrich) in DMEM/F12 for 30 min at 378C.…”

“…The presence of Sertoli cells in cultured testicular cells was confirmed by using anti-vimentin (clon v9; 1 : 100; Sigma-Aldrich). The expression of pluripotencyspecific markers on gonocytes in bovine testis and cultured testicular cells was examined using anti-NANOG (1 : 200; Chemicon International, Temecula, CA, USA) and anti-POU5F1 (1 : 50; C-10; Santa Cruz Biotechnology, Santa Cruz, CA, USA), as described previously (Goel et al 2008;Fujihara et al 2011). Briefly, testis sections were fixed with Bouin's fixative or 4% PFA, washed several times with 0.2% (v/v) Tween 20 in Tris-buffered saline (TBST), incubated in 5% (w/v) BSA in Tris-buffered saline (TBS) for 90 min to block non-specific binding, incubated with DBA-FITC and primary antibodies overnight at 48C, washed with TBST three times, incubated with the corresponding secondary antibody (Alexa 546-conjugated anti-rabbit IgG antibody (1 : 500; Molecular Probes, Eugene, OR, USA) or Alexa 546-conjugated antimouse IgG antibody (1 : 500; Molecular Probes)) for 1 h at 378C, rinsed three times with TBST, stained with Hoechst 33342 (Sigma-Aldrich) for 10 min, mounted with 50% glycerol in phosphate-buffered saline (PBS) and observed under an immune fluorescence microscope (BX 50; Olympus, Tokyo, Japan).…”

Pluripotent Stem Cells from Testis

“…Certain rodent markers for germ cells and SSCs such as VASA, DAZL, PLZF, and GFR1are also of monkey (Hermann et al, 2007) and in other species. VASA expression marks spermatogonia in sheep (Borjigin et al, 2010), buffalo (Goel et al, 2010b) and bull (Fujihara et al, 2011) testis. It has been demonstrated that GFR1 is a marker for mouse SSCs and probably their progeny (Meng et al, 2000;Buageaw et al, 2005;Hofmann et al, 2005;Naughton et al, 2006;He et al, 2007), and that KIT is a characteristic for the more differentiated spermatogonia, including type A 1-4 spermatogonia (Yoshinaga et al, 1991).…”