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

A. M. Benoit, Johannes D Veldhuis

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

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°C with 190 μl/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°C, but gradually decreased to a 2-fold increase in columns perfused at 37°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. The magnitude of the cAMP response to successive pulses was maintained at 25°C, but diminished at 37°C such that the final pulse of FSH resulted in a 2-fold increase in cAMP. Tonic oFSH delivery and 90-min pulses of 100 ng/ml oFSH both increased SCC mRNA (1.9- and 2.1-fold, respectively; p < 0.05). Pulses of 100 ng/ml oFSH given every 180 min, or 600 ng/ml given every 90 or 180 min did not increase SCC mRNA (p > 0.1). These data indicate that SCC mRNA can be increased by both continuous delivery and frequent (90 min) pulses of a low dosage of FSH, but not by relatively infrequent (180 min) pulses of FSH, or high dosages of FSH at either pulse frequency. Thus, up-regulation of SCC mRNA in granulosa cells appears to be dependent on both the amplitude (concentration) and frequency of FSH exposure. Furthermore, treatments that increased the accumulation of SCC mRNA similarly evoked widely differing temporal patterns of cAMP production, suggesting a dissociation between the time course and/or amount of cAMP generated, and the ability of FSH to increase SCC mRNA accumulation. We conclude that both continuous FSH delivery and an appropriate temporal pattern and concentration of pulsatile FSH delivery to granulosa cells can induce expression of SCC mRNA despite widely differing cAMP signals.

Original languageEnglish (US)
Pages (from-to)46-54
Number of pages9
JournalBiology of Reproduction
Volume53
Issue number1
DOIs
StatePublished - 1995
Externally publishedYes

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Cholesterol Side-Chain Cleavage Enzyme
Granulosa Cells
Follicle Stimulating Hormone
Cyclic AMP
Cytochrome P-450 Enzyme System
Swine
Messenger RNA
Enzymes
Cyclophilins
Eagles
Culture Media
Sheep
Up-Regulation

ASJC Scopus subject areas

  • Cell Biology
  • Developmental Biology
  • Embryology

Cite this

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title = "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",
abstract = "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°C with 190 μl/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°C, but gradually decreased to a 2-fold increase in columns perfused at 37°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. The magnitude of the cAMP response to successive pulses was maintained at 25°C, but diminished at 37°C such that the final pulse of FSH resulted in a 2-fold increase in cAMP. Tonic oFSH delivery and 90-min pulses of 100 ng/ml oFSH both increased SCC mRNA (1.9- and 2.1-fold, respectively; p < 0.05). Pulses of 100 ng/ml oFSH given every 180 min, or 600 ng/ml given every 90 or 180 min did not increase SCC mRNA (p > 0.1). These data indicate that SCC mRNA can be increased by both continuous delivery and frequent (90 min) pulses of a low dosage of FSH, but not by relatively infrequent (180 min) pulses of FSH, or high dosages of FSH at either pulse frequency. Thus, up-regulation of SCC mRNA in granulosa cells appears to be dependent on both the amplitude (concentration) and frequency of FSH exposure. Furthermore, treatments that increased the accumulation of SCC mRNA similarly evoked widely differing temporal patterns of cAMP production, suggesting a dissociation between the time course and/or amount of cAMP generated, and the ability of FSH to increase SCC mRNA accumulation. We conclude that both continuous FSH delivery and an appropriate temporal pattern and concentration of pulsatile FSH delivery to granulosa cells can induce expression of SCC mRNA despite widely differing cAMP signals.",
author = "Benoit, {A. M.} and Veldhuis, {Johannes D}",
year = "1995",
doi = "10.1095/biolreprod53.1.46",
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volume = "53",
pages = "46--54",
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TY - JOUR

T1 - 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

AU - Benoit, A. M.

AU - Veldhuis, Johannes D

PY - 1995

Y1 - 1995

N2 - 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°C with 190 μl/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°C, but gradually decreased to a 2-fold increase in columns perfused at 37°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. The magnitude of the cAMP response to successive pulses was maintained at 25°C, but diminished at 37°C such that the final pulse of FSH resulted in a 2-fold increase in cAMP. Tonic oFSH delivery and 90-min pulses of 100 ng/ml oFSH both increased SCC mRNA (1.9- and 2.1-fold, respectively; p < 0.05). Pulses of 100 ng/ml oFSH given every 180 min, or 600 ng/ml given every 90 or 180 min did not increase SCC mRNA (p > 0.1). These data indicate that SCC mRNA can be increased by both continuous delivery and frequent (90 min) pulses of a low dosage of FSH, but not by relatively infrequent (180 min) pulses of FSH, or high dosages of FSH at either pulse frequency. Thus, up-regulation of SCC mRNA in granulosa cells appears to be dependent on both the amplitude (concentration) and frequency of FSH exposure. Furthermore, treatments that increased the accumulation of SCC mRNA similarly evoked widely differing temporal patterns of cAMP production, suggesting a dissociation between the time course and/or amount of cAMP generated, and the ability of FSH to increase SCC mRNA accumulation. We conclude that both continuous FSH delivery and an appropriate temporal pattern and concentration of pulsatile FSH delivery to granulosa cells can induce expression of SCC mRNA despite widely differing cAMP signals.

AB - 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°C with 190 μl/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°C, but gradually decreased to a 2-fold increase in columns perfused at 37°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. The magnitude of the cAMP response to successive pulses was maintained at 25°C, but diminished at 37°C such that the final pulse of FSH resulted in a 2-fold increase in cAMP. Tonic oFSH delivery and 90-min pulses of 100 ng/ml oFSH both increased SCC mRNA (1.9- and 2.1-fold, respectively; p < 0.05). Pulses of 100 ng/ml oFSH given every 180 min, or 600 ng/ml given every 90 or 180 min did not increase SCC mRNA (p > 0.1). These data indicate that SCC mRNA can be increased by both continuous delivery and frequent (90 min) pulses of a low dosage of FSH, but not by relatively infrequent (180 min) pulses of FSH, or high dosages of FSH at either pulse frequency. Thus, up-regulation of SCC mRNA in granulosa cells appears to be dependent on both the amplitude (concentration) and frequency of FSH exposure. Furthermore, treatments that increased the accumulation of SCC mRNA similarly evoked widely differing temporal patterns of cAMP production, suggesting a dissociation between the time course and/or amount of cAMP generated, and the ability of FSH to increase SCC mRNA accumulation. We conclude that both continuous FSH delivery and an appropriate temporal pattern and concentration of pulsatile FSH delivery to granulosa cells can induce expression of SCC mRNA despite widely differing cAMP signals.

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