Over-expression of testis-specific expressed gene 1 attenuates the proliferation and induces apoptosis of GC-1spg cells

Abstract

The effects of over-expression of testis-specific expressed gene 1 (TSEG-1) on the viability and apoptosis of cultured spermatogonial GC-1spg cells were investigated, and the immortal spermatogonial cell line GC-1spg (CRL-2053™) was obtained as the cell model in order to explore the function of TSEG-1. We transfected the eukaryotic vector of TSEG-1, named as pEGFP-TSEG-1 into cultured spermatogonial GC-1spg cells. Over-expression of TSEG-1 inhibited the proliferation of GC-1spg cells, and arrested cell cycle slightly at G₀/G₁ phase. Transfection of TSEG-1 attenuated the transcript levels of Ki-67, PCNA and cyclin D1. In addition, over-expression of TSEG-1 induced early and late apoptosis, and reduced the mitochondrial membrane potential of GC-1spg cells. Moreover, transfection of TSEG-1 significantly enhanced the ratio of Bax/Bcl-2 and transcript levels of caspase 9, and decreased the expression of Fas and caspase 8 in GC-1spg cells. These results indicated over-expression of TSEG-1 suppresses the proliferation and induces the apoptosis of GC-1spg cells, which establishes a basis for further study on the function of TSEG-1.

Keywords

testis-specific expressed gene 1 / apoptosis / gene expression

References

[1] GuCH, TongQS, ZengFQ, et al.. Cloning and sequence analysis of TSEG-1, a novel gene specifically expressed in mouse testis. Yichuan (Chinese), 2008, 30(3): 352-358

[2] GuC, TongQ, ZhengL, et al.. TSEG-1, a novel member of histone H2A variants, participates in spermatogenesis via promoting apoptosis of spermatogenic cells. Genomics, 2010, 95(5): 278-289

[3] GovinJ, EscoffierE, RousseauxS, et al.. Pericentric heterochromatin reprogramming by new histone variants during mouse spermiogenesis. J Cell Biol, 2007, 176(3): 283-294

[4] WuF, CaronC, De RobertisC, et al.. Testis-specific histone variants H2AL1/2 rapidly disappear from paternal heterochromatin after fertilization. J Reprod Dev, 2008, 54(6): 413-417

[5] MarchJC, RaoG, BentleyWE. Biotechnological applications of green fluorescent protein. Appl Microbiol Biotechnol, 2003, 62(4): 303-315

[6] TsienRY. The green fluorescent protein. Annu Rev Biochem, 1998, 67: 509-544

[7] MisteliT, SpectorDL. Applications of the green fluorescent protein in cell biology and biotechnology. Nat Biotechnol, 1997, 15(10): 961-964

[8] WangS, HazelriggT. Implications for bcd mRNA localization from spatial distribution of exu protein in Drosophila oogenesis. Nature, 1994, 369(6479): 400-403

[9] RosoriusO, HegerP, StelzG, et al.. Direct observation of nucleocytoplasmic transport by microinjection of GFP-tagged proteins in living cells. Biotechniques, 1999, 27(2): 350-355

[10] HofmannMC, NarisawaS, HessRA, et al.. Immortalization of germ cells and somatic testicular cells using the SV40 large T antigen. Exp Cell Res, 1992, 201(2): 417-435

[11] Castro-ConchaLA, EscobedoRM, Miranda-Ham MdeL. Measurement of cell viability in in vitro cultures. Methods Mol Biol, 2006, 318: 71-76

[12] LecoeurH. Nuclear apoptosis detection by flow cytometry: influence of endogenous endonucleases. Exp Cell Res, 2002, 277(1): 1-14

[13] LivakKJ, SchmittgenTD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods, 2001, 25(4): 402-408

[14] NolanT, HandsRE, BustinSA. Quantification of mRNA using real-time RT-PCR. Nat Protoc, 2006, 1(3): 1559-1582

[15] AlaviSH, TaghaviMM, MoallemSA. Evaluation of effects of methamphetamine repeated dosing on proliferation and apoptosis of rat germ cells. Syst Biol Reprod Med, 2008, 54(2): 85-91

[16] TakagiS, ItohN, KimuraM, et al.. Spermatogonial proliferation and apoptosis in hypospermatogenesis associated with nonobstructive azoospermia. Fertil Steril, 2001, 76(5): 901-907

[17] StegerK, AleitheI, BehreH, et al.. The proliferation of spermatogonia in normal and pathological human seminiferous epithelium: an immunohistochemical study using monoclonal antibodies against Ki-67 protein and proliferating cell nuclear antigen. Mol Hum Reprod, 1998, 4(3): 227-233

[18] SirianniR, ChimentoA, RuggieroC, et al.. The novel estrogen receptor, G protein-coupled receptor 30, mediates the proliferative effects induced by 17beta-estradiol on mouse spermatogonial GC-1 cell line. Endocrinology, 2008, 149(10): 5043-5051

[19] BeumerTL, Roepers-GajadienHL, GademanIS, et al.. Involvement of the D-type cyclins in germ cell proliferation and differentiation in the mouse. Biol Reprod, 2000, 63(6): 1893-1898

[20] WrightA, ReileyWW, ChangM, et al.. Regulation of early wave of germ cell apoptosis and spermatogenesis by deubiquitinating enzyme CYLD. Dev Cell, 2007, 13(5): 705-716

[21] RodriguezI, OdyC, ArakiK, et al.. An early and massive wave of germinal cell apoptosis is required for the development of functional spermatogenesis. EMBO J, 1997, 16(9): 2262-2270

[22] BoekelheideK. Mechanisms of toxic damage to spermatogenesis. J Natl Cancer Inst Monographs, 2005, 34: 6-8

[23] SheehanM. Is gene therapy for the treatment of male infertility ethical?. Nat Clin Pract Urol, 2008, 5(11): 596-597

[24] ChalfieM, TuY, EuskirchenG, et al.. Green fluorescent protein as a marker for gene expression. Science, 1994, 263(5148): 802-805

[25] StauberRH, HorieK, CarneyP, et al.. Development and applications of enhanced green fluorescent protein mutants. Biotechniques, 1998, 24(3): 462-466

[26] YazawaT, YamamotoT, NakayamaY, et al.. Conversion from mitosis to meiosis: morphology and expression of proliferating cell nuclear antigen (PCNA) and Dmc1 during newt spermatogenesis. Dev Growth Differ, 2000, 42(6): 603-611

[27] KojiT, HishikawaY. Germ cell apoptosis and its molecular trigger in mouse testes. Arch Histol Cytol, 2003, 66(1): 1-16

[28] KnudsonCM, TungKS, TourtellotteWG, et al.. Bax-deficient mice with lymphoid hyperplasia and male germ cell death. Science, 1995, 270(5233): 96-99

[29] LinWW, LambDJ, WheelerTM, et al.. In situ end-labeling of human testicular tissue demonstrates increased apoptosis in conditions of abnormal spermatogenesis. Fertil Steril, 1997, 68(6): 1065-1069

[30] KimuraM, ItohN, TakagiS, et al.. Balance of apoptosis and proliferation of germ cells related to spermatogenesis in aged men. J Androl, 2003, 24(2): 185-191

[31] LeeJ, RichburgJH, YounkinSC, et al.. The Fas system is a key regulator of germ cell apoptosis in the testis. Endocrinology, 1997, 138(5): 2081-2088

[32] GiampietriC, PetrungaroS, ColucciaP, et al.. Germ cell apoptosis control during spermatogenesis. Contraception, 2005, 72(4): 298-302

[33] LysiakJJ, ZhengS, WoodsonR, et al.. Caspase-9-dependent pathway to murine germ cell apoptosis: mediation by oxidative stress, BAX, and caspase 2. Cell Tissue Res, 2007, 328(2): 411-419

[34] OldereidNB, AngelisPD, WigerR, et al.. Expression of Bcl-2 family proteins and spontaneous apoptosis in normal human testis. Mol Hum Reprod, 2001, 7(5): 403-408

[35] MorenoRD, LizamaC, UrzuaN, et al.. Caspase activation throughout the first wave of spermatogenesis in the rat. Cell Tissue Res, 2006, 325(3): 533-540

[36] RussellLD, Chiarini-GarciaH, KorsmeyerSJ, et al.. Bax-dependent spermatogonia apoptosis is required for testicular development and spermatogenesis. Biol Reprod, 2002, 66(4): 950-958

[37] RuwanpuraSM, McLachlanRI, StantonPG, et al.. Pathways involved in testicular germ cell apoptosis in immature rats after FSH suppression. J Endocrinol, 2008, 197(1): 35-43

[38] HuJH, JiangJ, MaYH, et al.. Enhancement of germ cell apoptosis induced by ethanol in transgenic mice overexpressing Fas ligand. Cell Res, 2003, 13(5): 361-367

[39] KajiharaT, OkagakiR, IshiharaO. LPS-induced transient testicular dysfunction accompanied by apoptosis of testicular germ cells in mice. Med Mol Morphol, 2006, 39(4): 203-208

[40] SaradhaB, VaithinathanS, MathurPP. Lindane induces testicular apoptosis in adult Wistar rats through the involvement of Fas-FasL and mitochondria-dependent pathways. Toxicology, 2009, 255(3): 131-139

[41] YinY, StahlBC, DeWolfWC, et al.. P53 and Fas are sequential mechanisms of testicular germ cell apoptosis. J Androl, 2002, 23(1): 64-70