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© PhD Students Symposium 2005. All rights reserved.
Mario Sousa, MD. PhD
Instituto Ciencias Biomedicas Abel Salazar, Porto, Portugal
Sertoli cells (SC), diploid germ cells (DGC) and round spermatids
(Sa1) were isolated by micromanipulation from testicular biopsies of
men with conserved spermatogenesis, normal karyotypes and absence of
Yq11.2 microdeletions. Cell purity was confirmed by FISH, transmission
electron microscopy and gene expression. Cells were cultured for 2wk
in medium, FSH and/or testosterone. Cell kinetics were evaluated daily
under inverted Hoffman optics. Proliferation was quantified by live
BrdU incorporation and anti-BrdU staining. Apoptosis was quantified
with a profluorescent caspase-3 substrate and pathways examined by
gene expression. FSH+T elicited 7% of meiotic index and
differentiation into elongating (49%) and late (17%) spermatids. FSH
acted over DGC and Sa1, whereas T acted in all steps of
spermatogenesis. Developmental arrest mainly occurred at flagellum
extrusion and late spermatid development. Although meiotic divisions
were observed, new Sa1 were mainly derived from late pachytene and
secondary spermatocytes. TEM demonstrated partial reestablishment of
cell-junctions between SC and DGC. FISH demonstrated normal meiotic
chromosomal synapsis and segregation in vitro. DNA replication (4%)
of DGC occurred only in the 1wk. Although inhibited by FSH+T, cultures
were limited by apoptosis, which caused degeneration of DGC (90%) and
developmental arrest of round (46%) and elongating (32%) spermatids.
FCT (POCTI/SAU-MMO/60709/04, 60555/04, 59997/04, UMIB).