IBYME   02675
INSTITUTO DE BIOLOGIA Y MEDICINA EXPERIMENTAL
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Effect of FSH and its glycosylation variants on proliferation, steroid and inhibin production in a human ovarian granulosa-like tumor cell line (KGN)
Autor/es:
LORETI N; IRUSTA G; ANDREONE L; TESONE M; CAMPO S
Lugar:
Wisconsin
Reunión:
Congreso; 43rd Annual Meeting of the Society for the Study of Reproduction (SSR); 2010
Institución organizadora:
SSR
Resumen:
KGN is a steroidogenic human ovarian
granulosa-like tumor cell line that expresses functional FSH receptor; in these
cells steroid production is stimulated by FSH. This glycoprotein is released
from the pituitary gland as a mixture of glycosylation variants that act on the
target cells inducing different biological responses in vitro and in vivo. The
aim of the present study was to determine the effect of recombinant human FSH (rhFSH) and its glycosylation variants on proliferation and estradiol (E2),
progesterone (P4), monomeric (Pro-αC) and dimeric
inhibin (inhibin A and B) production. Preparative
isoelectrofocusing was used to isolate rhFSH charge analogues in a pH range of
2.6-7.5. Two preparations were obtained by combining pH 3 to 4 (more acidic/sialylated, AC)
and pH 5 to 7 (less
acidic/sialylated, BA) fractions. Concanavalin-A chromatography was
used to isolate rhFSH glycosylation variants on the basis of glycan complexity.
Two preparations were obtained: unbound rhFSH isoforms (UB) bearing complex,
highly branched carbohydrate chains and firmly bound rhFSH isoforms (FB)
bearing hybrid type oligosaccharides. Cells were cultured on 24-well plate treated with or without rhFSH or
its glycosylation variants (20 ng/mL) for 24h or 72h; E2 and P4 (24h) were determined
by RIA and inhibins (72h) by specific ELISAs. [Methyl-3H]- thymidine
incorporation was used as a measure of proliferative activity (24h). Under
basal conditions KGN cells were able to produce E2 in the presence
of androstenedione (100nM), P4 in the absence of a steroidogenic
substrate (25-OH-Cholesterol), Pro-αC and inhibin A. Inhibin B was not
detected. There was a direct correlation between basal P4 production
and cell density. This condition did not affect either E2 or inhibin
production. rhFSH significantly stimulated the production of E2, P4,
Pro-αC, inhibin A and the incorporation of thymidine. Less acidic/sialylated
rhFSH was more biopotent to stimulate the production of E2 and
inhibins than its more sialylated counterpart. Progesterone production was not
significantly affected by the degree of sialylation of the hormone. UB isoforms
showed lower biopotency to stimulate steroids and inhibin production when
compared to FB isoforms. These glycosylation variants, bearing hybrid type oligosaccharides, exerted a potent stimulatory effect on E2, P4,Pro-αC and inhibin A production.
These results suggest that the degree of sialylation
as well as the complexity of carbohydrate chains present in rhFSH molecules may
be considered as additional factors that differentially regulate KGN cells
function. (Supported by CONICET, PIP 5479 and FONCYT, BID1728 OC/AR PICT 2004,
Nº 25365, Argentina)