EFFECT OF IN-VIVO GONADOTROPIN TREATMENT ON THE ABILITY OF PROGESTERONE, ESTROGEN, AND CYCLIC ADENOSINE 5'-MONOPHOSPHATE TO INHIBIT INSULIN-DEPENDENT GRANULOSA-CELL MITOSIS IN-VITRO
Am. Luciano et Jj. Peluso, EFFECT OF IN-VIVO GONADOTROPIN TREATMENT ON THE ABILITY OF PROGESTERONE, ESTROGEN, AND CYCLIC ADENOSINE 5'-MONOPHOSPHATE TO INHIBIT INSULIN-DEPENDENT GRANULOSA-CELL MITOSIS IN-VITRO, Biology of reproduction, 53(3), 1995, pp. 664-669
The ability of progesterone (P-4), estradiol-17 beta (E(2)), and 8-bro
mo (br)-cAMP to inhibit smalt granulosa cells (GCs) from undergoing in
sulin-dependent mitosis was examined. Small GCs were isolated from imm
ature and eCG-primed rats and separated by Percoll fractionation. Smal
l GCs were cultured for 24 h with various combinations of insulin, ste
roids, steroid receptor antagonists, and 8-br-cAMP. Before and after c
ulture, the number of GCs was counted. Small GC proliferation was expr
essed as a percentage increase over the initial value. P-4 inhibited i
nsulin-dependent mitosis of small GCs isolated from both immature and
eCG-primed rats. The effects of P-4 were dose-dependent, steroid-speci
fic, and reversed by the progesterone antagonist RU486, E(2) inhibited
insulin-dependent mitosis of small GCs isolated from immature but not
eCG-primed rats. The action of E(2) was dose-dependent and inhibited
by the estrogen antagonist tamoxifen, Additional studies were conducte
d in which small GCs from immature rats were cultured with insulin in
the presence of both P-4 and E(2) and their respective antagonist. Bot
h antagonists were required for insulin to induce GC mitosis in the pr
esence of P-4 and E(2). Further, the ability of P-4 to suppress insuli
n-dependent mitosis was reduced if it was not present during the first
6 h of culture. In contrast, E(2) could be added up to 12 h after ins
ulin exposure and still completely prevent GC mitosis, 8-br-cAMP also
prevented insulin-dependent GC proliferation. The actions of 8-br-cAMP
could not be reversed by aminoglutethimide or RU486, This indicates t
hat 8-br-cAMP does not block mitosis by increasing steroid synthesis.
Taken together, these data demonstrate that insulin-dependent mitosis
of small GCs isolated from immature rats is negatively regulated by P-
4, E(2), and 8-br-cAMP. Each of these regulators appears to mediate th
eir antimitotic action through different cellular pathways, Further, i
n vivo treatment with eCG induces changes within small GCs that result
in the loss of E(2)'s anti mitogenic action.