(W429) THE IN VITRO EFFECT OF EGF, FSH AND TESTOSTERONE ON SPERMATOGONIAL CELLS COLONIZATION.
Anjamrooz, SH1, Movahedin, M1, Tiraihi, T1, Mowla, SJ1, 1 Tarbiat Modares University, Tehran, Iran
ABSTRACT- Spermatogenesis is regulated by many growth factors and hormones. Failure of any of these processes leads to male infertility. Cytokines like epidermal growth factor (EGF) is the active ligand for EGF receptor reported in germ cell that stimulates mitosis and meiosis in spermatogenesis. Leydig cells are the principal source of EGF in the testis. Follicle-stimulating hormone (FSH) and testosterone (T) are the main hormones regulating spermatogenesis. The role of FSH and T in spermatogenesis has been a matter of controversy. Initiation of spermatogenesis is hormonally regulated by T. The FSH alone increased the absolute numbers of spermatogonia. In this research the effects of EGF, FSH and T on spermatogonial cell colonization in vitro were investigated. Sertoli and spermatogonial cells were isolated from neonatal mice testes by 2 steps: enzymatic digestion and DSA lectin. To confirm Sertoli and spermatogonial cells, we studied their morphology, alkaline phosphatase activity and immunohistochemistry techniques. Co-cultured Sertoli and spermatogonial cells were treated with EGF (4, 12 and 20 ng/ml), FSH (5, 10 and 20 IU/L) and T (0.1, 0.2 and 0.4 micromol) during 2 weeks culture. Colony assay was done by light microscopy. Results indicated that these factors influenced colony numbers, diameters, and formation rate. In EGF- and FSH-treated groups, colonies appeared with delay compared with control group but in T-treated group, colonies appeared earlier than in the control group. In T-treated group, the number of colonies and their diameters did not show significant differences compared with control group; in FSH-treated group, the number of colonies also did not show any changes during culture, but numbers of colonies in EGF-treated group were lower than control group. Colony diameters in both EGF- and FSH-treated groups were lower compared to control group until the 11th day of co-culture; after this time, their diameters increased significantly. In conclusion these factors influence spermatogonial proliferation in vitro. There are some differences between their mechanisms regarding in vivo. As in the designed cultural system the lack of hypothalamo-pituitary axis, Leydig cells, their interactions and etc caused observed different actions of the used factors.
KEY WORDS: Colonization, spermatogonia, FSH, EGF