Transplantation of Spermatogonial Stem Cells Suspension into Rete Testis of Azoospermia Mouse Model

Arefeh Jafarian, Niknam Lakpour, Mohammad Reza Sadeghi, Sheida Salehkhou, Mohammad Mehdi Akhondi

Abstract


595

Purpose: The loss of spermatogonia following chemo-or radiotherapy leading to temporary or permanent infertility of the patient is a well known and unwanted side effect of many oncological therapies.
Materials and Methods: In this study, germ cells were isolated from 4 days old mouse testis cells. Busulfan treatment was used to the eliminate proliferating cells in the testis of recipient mice. The donor cells suspended in DMEM, were introduced into the rete testis of recipient mice via microinjection method. To distinguish the progeny of the transplanted donor stem cells from endogenous germ cells, BrdU-labeled cells were used. In addition, real time PCR was performed to determine expression levels of ngn3 and LIN28 (spermatogonia stem cells markers)
before and after transplantation. Western blot analysis was further performed to detect an increase in - ngn3 expression after transplantation.
Results: Transplantations of stem cells into rete testis of the recipients was done. Our results clearly showed a significant increase in spermatozoa number in epididymal luman Spermatogonial stem cells (SSCs) did not show alkaline phosphatase activities while ngn3 and LIN28 were clearly expressed. Ngn3 and LIN28 expression were reduced after busulfan treatment compared to untreatmented mice. However, the expression of ngn3 and LIN28 increased after transplantation . BrdU-labeled testis cells were successfully transplanted into rete testis of recipient mice. These cells remained in rete testis of all recipient mice up to two months after transplantation.
Conclusion: The present study clearly confirme that a regeneration after cytotoxic treatment was based on morphological criteria. We demonstrated the increase in stem cell numbers during regeneration and after transplantation. Transplantation of spermatogonial stem cells suspension by the injection of cells via the rete testis of recipient azoospermia model considerably enhances the efficiency of this procedure.

Full Text:

PDF

190

References


Ogawa T, Dobrinski I, Avarbock MR, Brinster RL. Transplantation of male germ line stem cells restores fertility in infertile mice. Nature medicine. 2000;6:29-34.

Brinster CJ, Ryu B-Y, Avarbock MR, Karagenc L, Brinster RL, Orwig KE. Restoration of fertility by germ cell transplantation requires effective recipient preparation. Biology of Reproduction. 2003;69:412-20.

Revel A, Revel-Vilk S. Pediatric fertility preservation: is it time to offer testicular tissue cryopreservation? Molecular and cellular endocrinology. 2008;282:143-9.

Wu X, Goodyear SM, Abramowitz LK, et al. Fertile offspring derived from mouse spermatogonial stem cells cryopreserved for more than 14 years. Human reproduction. 2012des077.

Huckins C. The spermatogonial stem cell population in adult rats. I. Their morphology, proliferation and maturation. The Anatomical Record. 1971;169:533-57.

Nagano M, Avarbock MR, Brinster RL. Pattern and kinetics of mouse donor spermatogonial stem cell colonization in recipient testes. Biology of Reproduction. 1999;60:1429-36.

Brinster RL, Zimmermann JW. Spermatogenesis following male germ-cell transplantation. Proceedings of the National Academy of Sciences. 1994;91:11298-302.

Brinster RL, Avarbock MR. Germline transmission of donor haplotype following spermatogonial transplantation. Proceedings of the National Academy of Sciences. 1994;91:11303-7.

Lodi D, Iannitti T, Palmieri B. Stem cells in clinical practice: applications and warnings. J Exp Clin Cancer Res. 2011;30:1-20.

Yoshida S, Takakura A, Ohbo K, et al. Neurogenin3 delineates the earliest stages of spermatogenesis in the mouse testis. Dev Biol. 2004;269:447-58.

Zheng K, Wu X, Kaestner KH, Wang PJ. The pluripotency factor LIN28 marks undifferentiated spermatogonia in mouse. BMC Dev Biol. 2009;9:38.

Yang DH, Moss EG. Temporally regulated expression of Lin-28 in diverse tissues of the developing mouse. Gene Expr Patterns. 2003;3:719-26.

Schlatt S, Ehmcke J, Jahnukainen K. Testicular stem cells for fertility preservation: preclinical studies on male germ cell transplantation and testicular grafting. Pediatric blood & cancer. 2009;53:274-80.

Dobrinski I. Germ cell transplantation. Paper presented at: Seminars in reproductive medicine, 2005.

Jafarian A, Sadeghi M, Pejhan N, Salehkhou S, Lakpour N, Akhondi M. Regeneration of spermatogenesis in a mouse model of azoospermia by follicle‐stimulating hormone and oestradiol. Andrologia. 2014;46:1098-106.

Brinster RL. Germline stem cell transplantation and transgenesis. Science. 2002;296:2174-6.

Akhondi MM, Najar RA, Jeddi-Tehrani M, et al. The effect of human chorionic gonadotropin treatment on recipient mouse germ cell proliferation following spermatogonial stem cell transplantation of neonatal donor mice. Avicenna journal of medical biotechnology. 2010;2:23.

Schlatt S, Rosiepen G, Weinbauer G, Rolf C, Brook P, Nieschlag E. Germ cell transfer into rat, bovine, monkey and human testes. Human reproduction. 1999;14:144-50.

Anderson WA. Cytochemistry of sea urchin gametes: III. Acid and alkaline phosphatase activity of spermatozoa and fertilization. Journal of ultrastructure research. 1968;25:1-14.

Heidari B, Gifani M, Shirazi A, et al. Enrichment of undifferentiated type A spermatogonia from goat testis using discontinuous percoll density gradient and differential plating. Avicenna journal of medical biotechnology. 2014;6:94.

Guerin J. [Testicular tissue cryoconservation for prepubertal boy: indications and feasibility]. Gynecologie, obstetrique & fertilite. 2005;33:804-8.

Bellve AR, Cavicchia J, Millette CF, O'brien DA, Bhatnagar Y, Dym M. Spermatogenic cells of the prepuberal mouse: isolation and morphological characterization. The Journal of cell biology. 1977;74:68-85.

Oatley JM, Kaucher AV, Avarbock MR, Brinster RL. Regulation of mouse spermatogonial stem cell differentiation by STAT3 signaling. Biol Reprod. 2010;83:427-33.

Zhang M, Zhou H, Zheng C, et al. The roles of testicular c-kit positive cells in de novo morphogenesis of testis. Sci Rep. 2014;4:5936.

Kramer MF, de Rooij DG. The effect of three alkylating agents on the seminiferous epithelium of rodents. II. Cytotoxic effect. Virchows Arch B Cell Pathol. 1970;4:276-82.

Bellve AR. Purification, culture, and fractionation of spermatogenic cells. Methods Enzymol. 1993;225:84-113.

de Rooij DG, Russell LD. All you wanted to know about spermatogonia but were afraid to ask. J Androl. 2000;21:776-98.

Meistrich ML. Effects of chemotherapy and radiotherapy on spermatogenesis. Eur Urol. 1993;23:136-41; discussion 42.

Hemsworth BN, Jackson H. Effect of Busulphan on the developing gonad of the male rat. J Reprod Fertil. 1963;5:187-94.

Ishii K, Kanatsu-Shinohara M, Shinohara T. Cell-cycle-dependent colonization of mouse spermatogonial stem cells after transplantation into seminiferous tubules. J Reprod Dev. 2014;60:37-46.

Cheung HH, Rennert OM. Generation of fertile sperm in a culture dish: clinical implications. Asian J Androl. 2011;13:618-9. SSCs transplantation in azoospermic mice-Jafarian et al.




DOI: http://dx.doi.org/10.22037/uj.v0i0.3777


Creative Commons License 
This work is licensed under a Creative Commons Attribution 3.0 License