Expression pattern of testis-specific expressed gene 2 in cryptorchidism model and its role in apoptosis of spermatogenic cells

Abstract

In our previous study, we identified a novel testis-specific expressed gene 2 (TSEG-2) from mouse testis. To further investigate its functions, 35 male Balb/c mice (8 weeks old) were divided into cryptorchidism group (n=20), sham group (n=10), and control group (n=5). In cryptorchidism group, the right testes were anchored to the inner lateral abdominal wall. In situ hybridization (ISH) was applied to measure the localization of TSEG-2 in mouse testis. Real-time quantitative PCR was performed to detect the expression of TSEG-2 gene. Meanwhile, under the mediation of polyethylenimine (PEI), the recombinant vector pEGFP-TSEG-2 (n=5) or empty vector (mock, n=5) was transfected into the testis of male mice. The transfection efficiencies were measured under a fluorescence microscope. The apoptosis of spermatogenic cells was detected by terminal deoxynuleotidyl-mediated nick end labeling (TUNEL). The results showed that TSEG-2 was expressed in convoluted seminiferous tubules, more precisely, in spermatogonia and spermatocytes. As compared with sham and control groups, the TSEG-2 transcription was significantly enhanced (P<0.05) and was correlated with apoptosis of spermatogenic cells in cryptorchid testes (P<0.05). PEI was efficient in mediating transfection of TSEG-2 into seminiferous tubules of testis. One week post-transfection, intratesticular injection of TSEG-2 resulted in increased apoptosis of spermatogenic cells in vivo (P<0.05). These results indicate that TSEG-2 may participate in the apoptosis of spermatogenic cells and the pathogenesis of cryptorchidism.

Keywords

animal model / testis-specific expressed gene 2 / polyethylenimine / apoptosis

References

[1] ShaJ.H., ZhouZ.M., LiJ.M., et al.. Expression of a novel bHLH-Zip gene in human testis. Asian J Androl, 2003, 5(2): 83-87

[2] KashiwabaraS., NoguchiJ., ZhuangT., et al.. Regulation of spermatogenesis by testis-specific, cytoplasmic poly(A) polymerase TPAP. Science, 2002, 298(5600): 1999-2002

[3] YuZ., GuoR., GeY., et al.. Gene expression profiles in different stages of mouse spermatogenic cells during spermatogenesis. Biol Reprod, 2003, 69(1): 37-47

[4] WangZ.Y., TongQ.S., ZengF.Q., et al.. Cloning and expression of a novel mouse testis gene TSEG-2. Zhonghua Nan Ke Xue (Chinese), 2009, 15(2): 99-105

[5] BraissantO., WahliW.. A simplified in situ hybridization protocol using non-radioactively labeled probes to detect abundant and rare mrnas on tissue sections. Biochemica, 1998, 1: 10-16

[6] FarooquiS.M., Al-BagdadiF., HouslayM.D., et al.. Surgically induced cryptorchidism-related degenerative changes in spermatogonia are associated with loss of cyclic adenosine monophosphate-dependent phosphodiesterases type 4 in abdominal testes of rats. Biol Reprod, 2001, 64(6): 1583-1589

[7] Mendis-HandagamaS.M., KerrJ.B., De KretserD.M.. Experimental cryptorchidism in the adult mouse. III. Qualitative and quantitative electron microscopic morphology of Leydig cells. J Androl, 1991, 12(5): 335-343

[8] LiL., MinL., MinX., et al.. Gene functional research using polyethylenimine-mediated in vivo gene transfection into mouse spermatogenic cells. Asian J Androl, 2006, 8(1): 53-59

[9] OgawaT., ArechagaJ.M., AvarbockM.R., et al.. Transplantation of testis germinal cells into mouse seminiferous tubules. Int J Dev Biol, 1997, 41(1): 111-122

[10] VirtanenH.E., BjerknesR., CortesD., et al.. Cryptorchidism: classification, prevalence and long-term consequences. Acta Paediatr, 2007, 96(5): 611-616

[11] RockettJ.C., MappF.L., GargesJ.B., et al.. Effects of hyperthermia on spermatogenesis, apoptosis, gene expression, and fertility in adult male mice. Biol Reprod, 2001, 65(1): 229-239

[12] HunterE.S.3rd, DixD.J.. Heat shock proteins Hsp70-1 and Hsp70-3 are necessary and sufficient to prevent arsenite-induced dysmorphology in mouse embryos. Mol Reprod Dev, 2001, 59(3): 285-293

[13] PeluffoH., ArisA., AcarinL., et al.. Gene delivery to the central nervous system based on a novel integrin-targeting multifunctional protein. Hum Gene Ther, 2003, 14(13): 1215-1223

[14] PackD.W., HoffmanA.S., PunS., et al.. Design and development of polymers for gene delivery. Nat Rev Drug Discov, 2005, 4(7): 581-593

[15] BoussifO., LezoualchF., ZantaM.A., et al.. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine. PNAS, 1995, 92(16): 7297-7301

[16] GodbeyW.T., BarryM.A., SaggauP., et al.. Poly(-ethyleni-mine)-mediated transfection: a new paradigm for gene delivery. J Biomed Mater Res, 2000, 51(3): 321-328

[17] BmnnerS., SauserT., CarottaS. e. a1.. Cell cycle dependence of gene transfer by lipoplex, polyplex and recombinant adenovirus. Gene Ther, 2000, 7(5): 401-407