Differential effects of pulsatile versus continuous follicle-stimulating hormone delivery on the production of cyclic adenosine 5'-monophosphates and the accumulation of cytochrome P450 cholesterol side-chain cleavage enzyme mRNA in perifused porcine granulosa cells.
Although administration of FSH to porcine granulosa cells in static primary cultures increased accumulation of cAMP in the culture medium and cytochrome P450 cholesterol side chain cleavage enzyme (SCC) mRNA in the cells, the importance of the physiologically pulsatile pattern of FSH secretion in regulating the expression of specific genes required for ovarian steroidogenesis is unknown. To evaluate the impact of the mode of FSH delivery on ovarian target cell responses, we examined cAMP production and SCC mRNA accumulation in granulosa cells perifused with continuous or pulsatile FSH stimuli. Porcine granulosa cells were plated onto Cytodex 3 microcarrier beads, placed into perifusion columns, and perifused at 25 or 37 degrees C with 190 microliters/min Eagle's minimum essential medium (MEM) or MEM containing 100 ng/ml ovine (o) FSH delivered continuously, or 100 or 600 ng/ml oFSH delivered in 15-min pulses every 90 or 180 min. After perifusion, total RNA was isolated from granulosa cells for Northern analysis of specific SCC mRNA normalized to the constitutively expressed cyclophilin mRNA. Cyclic AMP in control columns remained basal throughout the perifusion. In contrast, cAMP increased 10- to 15-fold within 30-50 min of the onset of tonic FSH delivery. This increase in cAMP was maintained at 25 degrees C, but gradually decreased to a 2-fold increase in columns perifused at 37 degrees C. Release of cAMP increased 5- to 7-fold in response to 100 ng/ml oFSH pulses given at 90 or 180 min intervals, and 15- to 20-fold in response to 600 ng/ml oFSH pulses, and decreased to near control levels between FSH pulses.(ABSTRACT TRUNCATED AT 250 WORDS)
Benoit
A M
AM
Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, USA.
Veldhuis
J D
JD
D000818:Animals; D002478:Cells, Cultured; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000235:genetics*; D000242:Cyclic AMP / Q000096:biosynthesis*; D005260:Female; D005312:Fetal Blood; D005640:Follicle Stimulating Hormone / Q000008:administration & dosage*; D006107:Granulosa Cells / Q000187:drug effects / Q000378:metabolism*; D010477:Perfusion; D010507:Periodicity; D012333:RNA, Messenger / Q000096:biosynthesis*; D013552:Swine; D013696:Temperature
D012333:RNA, Messenger; D005640:Follicle Stimulating Hormone; D000242:Cyclic AMP; D002786:Cholesterol Side-Chain Cleavage Enzyme