SPERMATOGENESIS IN VITRO INDUCTION OF PROLIFERATION, MEIOSIS AND DIFFERENTIATION Mário Sousa Lab Cell Biology Institute of Biomedical Sciences (ICBAS) University of Porto
[email protected] Spermatogonia A SPERMATOGENESIS IN VITRO Preleptotene Pachytene spermatocytes spermatocytes 26 days Spermatogonia B 16 days Elongated spermatids 2-3 days Secondary spermatocytes 7-11 days 5-8 days 2-3 days 2-3 days Elongating Round spermatids spermatids 16 days 16 days OBJECTIVES culture medium for long term cultures and cell differentiation cell and molecular processes at each germ cell stage germ cell lines homologous transplantation in vitro gene therapy 15 anejaculation cases M1 AB C D E Normal karyotypes Absence of Y microdeletions 600bp Conserved spermatogenesis SY254 (c) SY134 (b) SY142 (b) Mechanical dissociation SY152 (c) Erythrocyte lysis Enzymatic digestion Cell isolation by micromanipulation M2 Cell culture: SY14 (SRY) - Yp 5 CM SY84 (a) 5 CM + rFSH (25 U/L) SY157 (c) 5 rFSH + T (2 µmol/L) SY142 (b) Plated cells: 250 S + 100 SGA + 1000 ST1 + 100 ST2 Multiplex-PCR AZF a,b,c Yq11.2 Each testicle biopsy was collected in sperm preparation medium (SPM; Medicult, Copenhagen, Denmark) and squeezed with surgical blades. The resultant fluid was diluted with SPM and washed by centrifuging at 1,000 rpm (500-600 g), 2 times 5 minutes. The pellet was resuspended for 5 min in 2 ml of erythrocyte-lysing buffer (Verheyen et al., 1995), prepared with 155 mM NH4Cl, 10 mM KHCO3, and 2 mM EDTA in water, pH 7.2 with KOH (all from Sigma, Barcelone, Spain, cell culture tested), and filtered by 0.2 µm.