Maintenance of Mouse Male Germ Line Stem Cells In Vitro1

Nagano, Makoto; Ryu, Buom‐Yong; Brinster, Clayton J.; Avarbock, Mary R.; Brinster, Ralph L.

doi:10.1095/biolreprod.102.014050

Cited by 272 publications

(238 citation statements)

References 54 publications

Supporting

Mentioning

224

Contrasting

Unclassified

Order By: Relevance

“…GDNF is a member of the transforming growth factor-b superfamily and is the essential growth factor for SSCs selfrenewal. The addition of GDNF resulted in a significant improvement in stem cell maintenance (Nagano et al 2003). Regulation of spermatogonial proliferation by GDNF depends on Ras and PI3 kinase pathways since their activation has been shown in neurons and kidney cells.…”

Section: Discussionmentioning

confidence: 99%

Effects of GDNF and LIF on mouse spermatogonial stem cells proliferation in vitro

et al. 2013

View full text Add to dashboard Cite

Spermatogonial stem cells (SSCs) are the only type of cells that transmit genes to the subsequent generations. The proliferation, cultivation and identification of SSCs in vitro are critical to understanding of male infertility, genetic resources and conservation of endangered species. To investigate the effects of glial cell-derived neurotrophic factor (GDNF) and leukemia inhibitory factor (LIF) on the proliferation of mouse SSCs in vitro, supplement of GDNF and/or LIF were designed to culture SSCs. The testes of 6-8 d mouse were harvested and digested by two-step enzyme digestion method. The SSCs and Sertoli cells were separated by differential plating. Then the SSCs were identified by alkaline phosphatase staining, RT-PCR and indirect immunofluorescence cell analysis. The cellular proliferation capacity was measured by methyl thiazolyl tetrazolium assay. The results showed that addition of 20 and 40 ng/ml of GDNF could strongly promote growth of mouse SSCs (p \ 0.05). There was no significant difference between LIF treatment groups and the control group in promoting proliferation of the mouse SSCs (p[0.05). However, the combination of 20 ng/ml GDNF and 1,000 U/ml LIF could significantly enhance the invitro proliferation of mouse SSCs (p \ 0.05), and the OD 490 value was 0.696 at day 5 of culture when the density of SSCs was 5-10 9 10 4 cells/ml.

show abstract

Section: Discussionmentioning

confidence: 99%

Effects of GDNF and LIF on mouse spermatogonial stem cells proliferation in vitro

et al. 2013

View full text Add to dashboard Cite

show abstract

“…In vitro, activin and BMP4 can reduce the spermatogonial stem cell potential (Nagano et al, 2003) and BMP4 has been linked to spermatogonial differentiation (Pellegrini et al, 2003). The level of SMAD6 protein present in spermatogonia exposed to activin and BMP4 may govern which cells respond to these triggers; cells with higher levels of SMAD6 may not respond to these differentiation signals and, hence, may remain as stem cells.…”

Section: Discussionmentioning

confidence: 99%

SMAD expression in the testis: An insight into BMP regulation of spermatogenesis

Itman

Loveland

2007

Developmental Dynamics

View full text Add to dashboard Cite

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-␤ superfamily, extensively influence events that establish male fertility, affecting germ cells and somatic cells throughout fetal and postnatal life. BMP signals are relayed by SMAD proteins, transcription factors that translocate to the nucleus upon ligand stimulation. We show that BMP signaling in the testis may be regulated by selective expression of BMP-responsive and inhibitory SMADs, with expression differing between the first wave and adult spermatogenesis. Smad1, Smad5, Smad8, Smad4, Smad6, and Smad7 expression is ubiquitous during testis development but becomes cell-specific in the adult. Furthermore, regulated SMAD6 protein expression at the onset of spermatogenesis suggests differential responsiveness of spermatogonial subpopulations to ligands. In vitro, immature Sertoli cells and spermatogonia transduce BMP2 and BMP4 signals by means of SMAD1, SMAD5, and SMAD8. Based on these findings, we extrapolate these data to interpret BMP mutant testis phenotypes in terms of SMAD availability for signal transduction. Developmental Dynamics 237:97-111, 2008.

show abstract

“…Longand short-term culture of SSCs have been attempted in several laboratories (12)(13)(14), and in our previous studies, GDNF appeared to have a beneficial effect on maintenance during a 7-day culture period (6,14). In a recent report, SSCs derived from DBA͞2 background mouse gonocytes, precursors of SSCs in neonate testis, proliferated in culture using a complex, undefined medium containing leukemia inhibitory factor (LIF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), GDNF, and 1% FBS (13).…”

mentioning

confidence: 99%

Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells

Kubota

Avarbock²,

Brinster³

2004

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

831

943

View full text Add to dashboard Cite

Spermatogonial stem cells (SSCs) self-renew and produce large numbers of committed progenitors that are destined to differentiate into spermatozoa throughout life. However, the growth factors essential for self-renewal of SSCs remain unclear. In this study, a serum-free culture system and a transplantation assay for SSCs were used to identify exogenous soluble factors that promote proliferation of SSCs. Mouse pup testis cells were enriched for SSCs by selection with an anti-Thy-1 antibody and cultured on STO (SIM mouse embryo-derived thioguanine and ouabain resistant) feeders in a serum-free defined medium. In the presence of glial cell line-derived neurotrophic factor (GDNF), SSCs from DBA͞2J strain mice formed densely packed clumps of cells and continuously proliferated. However, other strains of mice required the addition of soluble GDNF-family receptor ␣-1 and basic fibroblast growth factor to support replication. The functional transplantation assay proved that the clump-forming cells are indeed SSCs. Thus, GDNFinduced cell signaling plays a central role in SSC self-renewal. The number of SSCs in culture doubled every 5.6 days, and the clumpforming cells strongly expressed Oct-4. Under these conditions, SSCs proliferated over 6 months, reconstituted long-term spermatogenesis after transplantation into recipient testes, and restored fertility to infertile recipients. The identification of exogenous factors that allow continuous proliferation of SSCs in vitro establishes the foundation to study the basic biology of SSCs and makes possible germ-line modification by sophisticated technologies. Moreover, the ability to recover, culture indefinitely, and transplant SSCs will make the germ-line of individual males available for periods extending beyond a normal lifetime.

show abstract

Maintenance of Mouse Male Germ Line Stem Cells In Vitro1

Cited by 272 publications

References 54 publications

Effects of GDNF and LIF on mouse spermatogonial stem cells proliferation in vitro

Effects of GDNF and LIF on mouse spermatogonial stem cells proliferation in vitro

SMAD expression in the testis: An insight into BMP regulation of spermatogenesis

Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells

Contact Info

Product

Resources

About