Colonization Enhancing in Recipient Testes after Spermatogonial Transplantation by Leuprolide Is Through Improving Donor Cells Both in Homing and Proliferating Processes.

Abstract

Administration of leuprolide, a GnRH agonist, could promote the formation of donor cell colonies in seminiferous tubules after spermatogonial transplantation. Both homing and proliferating processes might involved in the colony enhancement after transplantation. To our knowledge there was no report to address this issue yet. Present study carried out several experiments to illustrate at which processes (homing or proliferation) leuprolide enhancing donor cell colonies in seminiferous tubules after the transplantation. In experiment I, donor spermatogonial cells from GFP transgenetic mice were transplanted through efferent duct with micromanipulator into recipient testes (depleting endogenous germ by busulfan, 44mg/BW, i.p.) with or without administration of leuprolide (7.6mg/kg BW, s.c.). Recipient testes were examined under fluorescence and routine microscopy at 1wk, 1mo and 2mo, respectively. Donor derived cells showed significantly more density in leuprolide group (15-37%) at all time points examined (p<0.05), indicating that leuprolide could at least enhance homing process, thereafter, increase efficiency of spermatogonial transplantation at consequent points. Enhancement of homing by leuprolide may act through increase of beta1-intergrin since mRNA level was higher (36%) at same time (p<0.05). However, due to stimulation of homing occurred, whether or not leuprolide play a role in consequent proliferating process could not be excluded. Therefore, Experiment II were designed to answer the question. To override the homing process, with similar leuprolide protocol restoration of proliferation of endogenous spermatogonial germ cells remained in testes (mild treated by busulfan 22mg/BW, i.p.) were checked by routine and immunohistological processes at different time. By checking seminiferous tubules diameter, thickness of seminiferous epitheliums, and appearance of spermide in testes, diameters of seminiferous tubules in leuprolide groups at 1 and 2 mo were larger (1.2 and 2.2 folds, respectively) compared with sham groups (p<0.05). All other indexes checked in leuprolide group were significant improved compared with sham groups at 2 mo (p<0.01). This implicates the role of leuprolide in proliferation of germ cells after mild germ cell depriving. Therefore, leuprolide (with impression of testosterone) promoted colony enhancement of donor cells after the transplantation and endogenous spermatogenesis restoration after busulfan treatment are through improvement in both homing and proliferating processes.