The present studies were undertaken to determine the involvement of protein kinase C (PKC) in the regulation of rat granulosa cell steroidogenesis in vitro. The effects of PKC activators (1-oleoyl-2-acetylglycerol (OAG); 1,2-dioctanoylglycerol (DiC$\sb8$) and phorbol 12-myristate 13-acetate (TPA)) and inhibitors (DL-Sphingosine (ESP) and 1-(5-Isoquinolinylsulfonyl)-3-methylpiperazine free base (H$\sb7)\rbrack$ on basal and FSH-, (Bu)$\sb2$cAMP-, forskolin- and calcium ionophore A23187-stimulated pregnenolone (P$\sb5$), progesterone (P) and 20$\alpha$-hydroxy-pregn-4-en-3-one (20$\alpha$-OH-P) secretion by granulosa cells were studied. OAG, when continually present in the culture medium (MEM), significantly stimulated P$\sb5$, P and 20$\alpha$-OH-P secretion during 6 to 24 h culture periods. It also markedly increased the conversion of exogenous P$\sb5$ to P and 20$\alpha$-OH-P and exogenous P to 20$\alpha$-OH-P during 24 h cultures. Pretreatment of granulosa cells with TPA for 1 h or treatment for up to 6 h resulted in a significant increase in P$\sb5$, P and 20$\alpha$-OH-P secretion. Except for 20$\alpha$-OH-P production, which was stimulated by the phorbol ester during all culture periods studied, secretion of P$\sb5$ and P (in the presence or absence of exogenous hormones and the inhibitors of steroidogenic enzymes) were substantially inhibited by TPA during a 24 h incubation. However, when granulosa cells were incubated with both OAG (20 $\mu$g/ml) and TPA (40 ng/ml), progestin secretion was increased irrespective of the duration of incubation. PKC inhibitors dose-dependently suppressed the stimulatory effect of OAG (20 $\mu$g/ml) and TPA (40 ng/ml) with complete inhibition noted at 100 $\mu$M of H$\sb7$ and 10 $\mu$M of ESP. Diacylglycerols and TPA exerted divergent effects on FSH-, (Bu)$\sb2$cAMP- and forskolin-stimulated progestin secretion. FSH-stimulated accumulation of P$\sb5$ throughout the culture periods (1-24 h) was markedly increased by OAG (20 $\mu$g/ml) but inhibited by TPA (40 ng/ml). OAG (5-80 $\mu$g/ml) and DiC$\sb8$ (20 $\mu$g/ml) significantly enhanced FSH-induced progestin secretion during 6 h and 24 h culture periods and increased steroid synthesis in 24 h cultures in the presence of (Bu)$\sb2$cAMP or forskolin. In contrast, TPA significantly inhibited FSH- and (Bu)$\sb2$cAMP-stimulated progestin secretion during both 6 h and 24 h of incubation. Pretreatment of granulosa cells with TPA (40 ng/ml) for 20 h to down-regulate PKC, decreased progestin secretion during subsequent incubation with FSH (150 ng/ml) and prevented any stimulation by OAG (20 $\mu$g/ml). The effects of OAG (20 $\mu$g/ml) and TPA (40 ng/ml) on FSH-induced steroid secretion appeared to be additive when both PKC activators were present together and differed significantly from those when OAG and TPA were present with FSH separately. Diolein (a nonpermeable diacylglycerol), 4$\alpha$-phorbol 12,13-didecanoate and phorbol 13-monoacetate (two phorbol esters with no tumor promoting activity) did not influence basal or FSH-stimulated steroid secretion by granulosa cells. (Abstract shortened by UMI.)
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/6745 |
| Date | January 1993 |
| Creators | Komorowski, Joanna Irena. |
| Contributors | Tsang, B., |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Detected Language | English |
| Type | Thesis |
| Format | 276 p. |
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