Characterization of Immortalized Dairy Goat Male Germline Stem Cells (mGSCs)

Abstract

Male germline stem cells (mGSCs), in charge for the fertility in male testis, are the only kind of adult stem cells that transmit genetic information to next generation, with promising prospects in germplasm resources preservation and optimization, and production of transgenic animals. Mouse male germline stem cell lines have been established and are valuable for studying the mechanisms of spermatogenesis. However, there is a lack of stable mGSC cell lines in livestock, which restricts the progress of transgenic research and related biotechnology. Here, we firstly established an immortalized dairy goat mGSC cell line to study the biological properties and the signaling pathways associated with mGSCs self-renewal and differentiation. The ectopic factors SV40 large T antigen and Bmi1 genes were transduced into dairy goat mGSCs, and the results showed that the proliferation of these cells that were named mGSCs-I-SB was improved significantly. They maintained the typical characteristics including the expression of mGSC markers, and the potential to differentiate into all three germ layers, sperm-like cells in vitro. Additionally, mGSCs-I-SB survived and differentiated into three germ layer cell types when they were transplanted into chicken embryos. Importantly, the cells also survived in mouse spermatogenesis deficiency model testis which seemed to be the golden standard to examine mGSCs. Conclusively, our results demonstrate that mGSCs-I-SB present the characteristics of mGSCs and may promote the future study on goat mGSCs.

Melatonin promotes goat spermatogonia stem cells (SSCs) proliferation by stimulating glial cell line-derived neurotrophic factor (GDNF) production in Sertoli cells

“…In our study, melatonin had none effect of the proliferation of an immortalized CD49f positive dairy goat male germline stem cells (mGSCs-I-SB) [38]. However, we found that melatonin can promote the primary SSC colony formation and promote the proliferation markers expression, this indicated that melatonin may affect goat SSCs indirectly.…”

“…The un-adherent cells were transferred into 12-well plates. SSCs were cultured in Dulbecco's modified Eagle's medium (DMEM/F12, GIBCO, Carlsbad, CA) supplemented with 1% fetal bovine serum (FBS, GIBCO), 10% Knockout serum replacement (KSR, Invitrogen, Carlsbad, CA) medium, 4 mM L-glutamine (Invitrogen), 1% non-essential amino acids (Invitrogen), 100 IU/ml penicillin, 100 IU/ml streptomycin (Invitrogen), 10 ng/ml GDNF, 10 ng/ml epidermal growth factor (EGF), 10 ng/ml basic fibroblast growth factor (bFGF), and 20 ng/ml glial cell line derived neurotrophic factor family receptor alpha 1 (GFRa1) [25]. The cells were cultured at 37 °C in a humidified atmosphere with 5 % CO2 for 3 passages.…”

The Modification of Tet1 in Male Germline Stem Cells and Interact with PCNA, HDAC1 to promote their Self-renewal and Proliferation

“…We optimized in vitro culture systems for dairy goat mGSCs and have successfully established an immortalized dairy goat mGSC cell line42, enabling the long-term study of dairy goat mGSCs in vitro 424344. In order to maintain pluripotency and reprogramming efficiency of dairy goat mGSCs, we investigate the mGSC biology through Tet1 overexpression, verified the self-renewal and proliferation ability in vivo and in vitro to explore the role of Tet1 in mGSC characters and development potentiality.…”

Lin28a promotes self-renewal and proliferation of dairy goat spermatogonial stem cells (SSCs) through regulation of mTOR and PI3K/AKT

“…Hence, we hypothesize that the highly conserved RNA-binding protein is also involved in maintaining the identity of adult SSCs and promotes the self-renewal and proliferation of dairy goat SSCs. To test the hypothesis, we overexpressed Lin28a in mGSCs-I-SB cells41. The expression of the pluripotency factors SOX2 and OCT4 is up-regulated by overexpression of Lin28a.…”

“…These cells were primary cells isolated from dairy goat testes that were transduced with SV40 large T antigen and Bmi1 to establish immortalized male goat germline stem cells (GmGSC-I-SB)41.…”