dinoprost and 22-hydroxycholesterol

The present study investigated the induction and possible role of activating transcription factor 3 (ATF3) in the corpus luteum. Postpubertal cattle were treated at midcycle with prostaglandin F2α(PGF) for 0-4 hours. Luteal tissue was processed for immunohistochemistry, in situ hybridization, and isolation of protein and RNA. Ovaries were also collected from midluteal phase and first-trimester pregnant cows. Luteal cells were prepared and sorted by centrifugal elutriation to obtain purified small (SLCs) and large luteal cells (LLCs). Real-time PCR and in situ hybridization showed that ATF3 mRNA increased within 1 hour of PGF treatment in vivo. Western blot and immunohistochemistry demonstrated that ATF3 protein was expressed in the nuclei of LLC within 1 hour and was maintained for at least 4 hours. PGF treatment in vitro increased ATF3 expression only in LLC, whereas TNF induced ATF3 in both SLCs and LLCs. PGF stimulated concentration- and time-dependent increases in ATF3 and phosphorylation of MAPKs in LLCs. Combinations of MAPK inhibitors suppressed ATF3 expression in LLCs. Adenoviral-mediated expression of ATF3 inhibited LH-stimulated cAMP response element reporter luciferase activity and progesterone production in LLCs and SLCs but did not alter cell viability or change the expression or activity of key regulators of progesterone synthesis. In conclusion, the action of PGF in LLCs is associated with the rapid activation of stress-activated protein kinases and the induction of ATF3, which may contribute to the reduction in steroid synthesis during luteal regression. ATF3 appears to affect gonadotropin-stimulated progesterone secretion at a step or steps downstream of PKA signaling and before cholesterol conversion to progesterone.

Topics: Activating Transcription Factor 3; Adenoviridae; Animals; Cattle; Cell Survival; Corpus Luteum; Cyclic AMP-Dependent Protein Kinases; Dinoprost; Female; Gene Expression Regulation; Hydroxycholesterols; Luteinizing Hormone; Luteolysis; MAP Kinase Signaling System; Phosphorylation; Progesterone; Protein Kinase Inhibitors; Response Elements; RNA, Messenger; Signal Transduction; Time Factors; Transcription, Genetic

It is well documented that prostaglandin F2 alpha (PGF2 alpha) inhibits progesterone production in luteal cells, but its mode of action is uncertain. It has recently been suggested that PGF2 alpha acts by activating the calcium and phospholipid-dependent protein kinase, protein kinase C (PKC). This hypothesis has been tested by comparing the site and mode of action of PGF2 alpha, a PGF2 alpha analogue (cloprostenol) and the PKC activator phorbol myristate acetate (4 beta PMA) in human granulosa-lutein cells. PGF2 alpha and cloprostenol exerted similar concentration-dependent inhibitory actions on gonadotrophin-stimulated cyclic AMP (cAMP) accumulation and progesterone production by human granulosa-lutein cells. The similarity in the actions of PGF2 alpha and cloprostenol in human granulosa-lutein cells suggests that they can be used interchangeably to study the role of PGF2 alpha in the regulation of steroidogenesis in the human ovary. Gonadotrophin-stimulated cAMP accumulation and progesterone production was also concentration-dependently inhibited by 4 beta PMA. In addition, cloprostenol and 4 beta PMA also inhibited dibutyryl cAMP-stimulated progesterone production, suggesting that these compounds inhibit LH action at sites before and after the generation of cAMP. The pre-cAMP site of action can be localised to the stimulatory G-protein (Gs) as both compounds inhibited cholera toxin-stimulated cAMP accumulation without affecting forskolin-stimulated cAMP accumulation. The post cAMP site of action can be localised to actions on cholesterol side chain cleavage enzyme, as both cloprostenol and 4 beta PMA inhibited 22R hydroxycholesterol-supported progesterone production without affecting pregnenolone-supported progesterone production. The finding that cloprostenol and 4 beta PMA interact with the steroidogenic cascade in a similar manner is indicative of a shared common mediator of their actions in human granulosa-lutein cells, i.e. PKC. The inhibitory actions of PGF2 alpha and 4 beta PMA on hLH-stimulated progesterone production were abolished in the presence of the PKC inhibitor, staurosporine. In addition, in PKC-depleted cells (achieved by exposure to 4 beta PMA for 20 h) the inhibitory actions of PGF2 alpha and 4 beta PMA were abolished. These results support the hypothesis that the inhibitory actions of PGF2 alpha are mediated by PKC in human granulosa-lutein cells.

Topics: Bucladesine; Calcium; Cells, Cultured; Cholera Toxin; Cloprostenol; Colforsin; Cyclic AMP; Dinoprost; Female; Granulosa Cells; Humans; Hydroxycholesterols; Luteinizing Hormone; Pregnenolone; Progesterone; Protein Kinase C; Tetradecanoylphorbol Acetate

In an attempt to establish a defined model system for studies aimed at elucidating the mechanism of PGF2 alpha action, we examined the effects of medium supplementation with soluble hydroxycholesterol analogues, alone and in combination with ovine luteinizing hormone (oLH) in the presence and absence of PGF2 alpha, on progesterone secretion by mixed ovine luteal cells in vitro. In short-term cultures (2-6 h), supplementary 22R-hydroxycholesterol (22R-OHC; 0.16-20 micrograms ml-1) increased (P < 0.05) progesterone production in a dose-dependent manner, whereas similar concentrations of 22S-hydroxycholesterol (22S-OHC) and 25-hydroxycholesterol (25-OHC) had little effect. In incubations of < or = 24 h duration, 22R-OHC (1 micrograms ml-1) dramatically increased progesterone secretion, whereas oLH (100 ng ml-1) in the presence or absence of PGF2 alpha (250 ng ml-1) had no consistent effects, alone or in combination with 22R-OHC. In contrast, 22R-OHC (1 micrograms ml-1) alone had no effect in long-term incubations (72-192 h), nor did treatment with oLH (100 ng ml-1) in the presence or absence of PGF2 alpha (250 ng ml-1) in the absence of 22R-OHC. Together, however, 22R-OHC and oLH stimulated (P < 0.05) progesterone secretion, a synergistic effect consistently inhibited (P < 0.05) by PGF2 alpha. Equimolar (2.5 mumol l-1) concentrations of 22R-OHC and homologous serum low- or high-density lipoprotein cholesterol exhibited comparable capacities to maintain progesterone secretion in long-term cultures (24-168 h), with and without gonadotrophin (oLH or human chorionic gonadotrophin, 100 ng ml-1) stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cells, Cultured; Corpus Luteum; Culture Media; Dinoprost; Dose-Response Relationship, Drug; Drug Synergism; Female; Hydroxycholesterols; Lipoproteins; Luteinizing Hormone; Luteolysis; Models, Biological; Progesterone; Sheep; Stimulation, Chemical; Time Factors