Prostaglandin dehydrogenase (PGDH) in granulosa cells of primate periovulatory follicles is regulated by the ovulatory gonadotropin surge via multiple G proteins

Abstract

The ovulatory gonadotropin surge increases granulosa cell prostaglandin synthesis as well as prostaglandin dehydrogenase (PGDH), the key enzyme responsible for prostaglandin metabolism. To investigate gonadotropin regulation of PGDH in the primate follicle, monkey granulosa cells were obtained across the 40-h periovulatory interval. PGDH activity was low before the ovulatory hCG stimulus, peaked 12-24 h after hCG, and was low again 36 h after hCG administration. Granulosa cells maintained in vitro with hCG showed a similar temporal pattern of PGDH. The LH/CG receptor can utilize multiple signaling pathways to regulate intracellular events. Gonadotropin-stimulated cAMP appears to act primarily via the Epacs to increase PGDH mRNA, protein, and activity. In contrast, PLC activation of PKC likely decreases PGDH mRNA, protein, and activity late in the periovulatory interval. Increased, then decreased PGDH activity may delay accumulation of prostaglandins in the follicle until late in the periovulatory interval, contributing to timely ovulation in primates.

Figure 1

PGDH activity as assessed by conversion of PGE2 to PGEM. Panel A. Granulosa cells (10,000 cells/reaction) were incubated in the presence of PGE2 (▲, 1.0 μg/ml; •, 0.1 μg/ml; ■, 0.01 μg/ml) for 10-120 minutes. Panel B. Granulosa cells (2,500-25,000 cells/reaction) were incubated in the presence of PGE2 (▲, 1.0 μg/ml; •, 0.1 μg/ml) for 60 minutes. Media PGEM was determined by EIA.

Figure 2

PGDH activity in monkey granulosa cells obtained throughout the periovulatory interval. Panel A. Granulosa cells from monkeys experiencing controlled ovarian stimulation were obtained before (0 hour) and 12, 24, and 36 hours after administration of an ovulatory dose of hCG. PGDH activity was assessed by conversion of PGE2 to PGEM (see Figure 1). Panels B-C. Ciglitazone inhibition of PGE2 accumulation in media of granulosa cell cultures. Granulosa cells obtained before (B) or 36 hours after (C) hCG administration were incubated for 24 hours with vehicle (0.1% DMSO; control) or with the PGDH inhibitor ciglitazone (0.3-3.0 μM); all cultures contained arachidonic acid (10 μM) as substrate for PGE2 synthesis. Media PGE2 levels were determined by EIA. For each animal, PGE2 levels in ciglitazone-treated cultures are expressed as a percentage of control PGE2 levels. Within each panel, treatment groups with no common superscripts are different, p<0.05. Data are expressed as mean ± SEM; n=3-4 animals/treatment group.

Figure 3

hCG regulates PGDH mRNA, protein, and activity in monkey granulosa cells in vitro. Granulosa cells obtained from large follicles before administration of hCG were treated without (control) or with hCG (100 ng/ml) for 12, 24, or 36 hours in vitro. A. RNA harvested from cultured cells was assessed for PGDH mRNA by real time RT-PCR and expressed relative to the β-actin mRNA level in each granulosa cell sample (n=4-5 animals/treatment group). B. PGDH activity was assessed by conversion of PGE2 to PGEM (n=4 animals/treatment group). For Panels A and B, within each time point, control and hCG are different as indicated by the asterisk (*), p<0.05. Within hCG-treated cells, groups with no common superscripts are different, p<0.05. Data are expressed as mean ± SEM. Panels C-J. Modest levels of PGDH protein were detected by immunostaining in granulosa cells cultured without hCG for 12 hours (C), 24 hours (D), and 36 hours (E). Granulosa cells cultured with hCG for 12 hours (G) and 24 hours (H) showed stronger immunodetection of PGDH; little PGDH was detected after 36 hours with hCG in vitro (J). Dark precipitate represents immunodetection of PGDH; cells were not counterstained. Reduced immunostaining was observed when the primary antibody was omitted (not shown) or preabsorbed with the peptide used to generate the primary antibody (F). PGDH protein detection is representative of granulosa cells from n=3 animals.

Figure 4

cAMP regulates PGDH mRNA, protein, and activity. Granulosa cells obtained from large follicles before administration of hCG were maintained in vitro without treatment (control) or with treatments as described below for 12, 24, or 36 hours. A. RNA harvested from untreated cells (control) and cells treated with the cAMP analog 8BR (0.5 mM) was assessed for PGDH mRNA by real time RT-PCR and expressed relative to the β-actin mRNA level in each sample (n=4-5 animals/treatment group). B. PGDH activity was assessed by conversion of PGE2 to PGEM (n=4 animals/treatment group). Within each time point, control and 8BR are different as indicated by the asterisk (*), p<0.05. Modest levels of PGDH protein were detected by immunostaining in granulosa cells cultured with 8BR for 12 hours (D); strong PGDH immunodetection was observed after treatment with 8BR for 24 hours (E) and 36 hours (F). PGDH immunostaining in control cells after 24 hours in vitro is also shown (C). PGDH protein detection is representative of granulosa cells from n=3 animals. G. Additional granulosa cells were treated for 12-36 hours in vitro with hCG (100 ng/ml), the PKA inhibitor H89 (0.01 mM), hCG+H89, or no treatment (control); PGDH mRNA was assessed as described above (n=4 animals/treatment group). H. Granulosa cell treated for 12-36 hours in vitro with the cAMP analogs 6BNZ (0.1 mM) or 8CPT (0.1 mM); PGDH mRNA was assessed as described above (n=5-8 animals/treatment group). For Panels G-H, within each time point, groups with no common superscripts are different, p<0.05. Data are expressed as mean ± SEM.

Figure 5

PLC and PKC regulate PGDH mRNA, protein, and activity. Granulosa cells obtained from large periovulatory follicles before administration of hCG were maintained in vitro without treatment (control) or with treatments as described below for 12, 24, or 36 hours. A. RNA harvested from untreated cells (control) and cells treated with the PKC activator PMA (50 nM) was assessed for PGDH mRNA by real time RT-PCR and expressed relative to the β-actin mRNA level in each sample (n=4 animals/treatment group). Within each time point, control and PMA are different as indicated by the asterisk (*), p<0.05. B. PGDH activity was assessed by conversion of PGE2 to PGEM (n=4/group). Within PMA-treated cells, groups with no common superscripts are different, p<0.05. Immunodetection of PGDH protein in granulosa cells was similar to or less than that observed in control cells after treatment with PMA for 12 hours (D), 24 hours (E), and 36 hours (F) in vitro. PGDH immunostaining in control cells after 24 hours in vitro is also shown (C). PGDH protein detection is representative of granulosa cells from n=3 animals. G. Additional granulosa cells were treated for 12-36 hours in vitro with hCG (100 ng/ml), the PLC inhibitor U-71322 (U7, 0.01 mM), hCG+U7, or no treatment (control); PGDH mRNA was assessed as described above (n=4-6 animals/treatment group). Within each time point, groups with no common superscripts are different, p<0.05. Data are expressed as mean ± SEM.