Prostaglandin metabolism in the ovine corpus luteum: catabolism of prostaglandin F(2alpha) (PGF(2alpha)) coincides with resistance of the corpus luteum to PGF(2alpha)

To examine possible mechanisms involved in resistance of the ovine corpus luteum to the luteolytic activity of prostaglandin (PG)F(2alpha), the enzymatic activity of 15-hydroxyprostaglandin dehydrogenase (PGDH) and the quantity of mRNA encoding PGDH and cyclooxygenase (COX-2) were determined in ovine corpora lutea on Days 4 and 13 of the estrous cycle and Day 13 of pregnancy. The corpus luteum is resistant to the action of PGF(2alpha) on Days 4 of the estrous cycle and 13 of pregnancy while on Day 13 of the estrous cycle the corpus luteum is sensitive to the actions PGF(2alpha). Enzymatic activity of PGDH, measured by rate of conversion of PGF(2alpha) to PGFM, was greater in corpora lutea on Day 4 of the estrous cycle (P < 0.05) and Day 13 of pregnancy (P < 0.05) than on Day 13 of the estrous cycle. Levels of mRNA encoding PGDH were also greater in corpora lutea on Day 4 of the estrous cycle (P < 0. 01) and Day 13 of pregnancy (P < 0.01) than on Day 13 of the estrous cycle. Thus, during the early estrous cycle and early pregnancy, the corpus luteum has a greater capacity to catabolize PGF, which may play a role in the resistance of the corpus luteum to the actions of this hormone. Levels of mRNA encoding COX-2 were undetectable in corpora lutea collected on Day 13 of the estrous cycle but were 11 +/- 4 and 44 +/- 28 amol/microgram poly(A)(+) RNA in corpora lutea collected on Day 4 of the estrous cycle and Day 13 of pregnancy, respectively. These data suggest that there is a greater capacity to synthesize PGF(2alpha), early in the estrous cycle and early in pregnancy than on Day 13 of the estrous cycle. In conclusion, enzymatic activity of PGDH may play an important role in the mechanism involved in luteal resistance to the luteolytic effects of PGF(2alpha).     

Luteinizing hormone receptors and progesterone content in porcine corpora lutea after prostaglandin F2 alpha

The effect of prostaglandin F2 alpha (PGF2 alpha) on luteinizing hormone (LH) receptors, weight and progesterone content of corpora lutea (CL), and serum progesterone concentrations was studied in gilts. Fifteen gilts were hysterectomized between Days 9 to 11 of the estrous cycle. Twelve gilts were injected i.m. with 10 mg of PGF2 alpha and 3 with saline on Day 20. Ovaries were surgically removed from each of 3 gilts at 4, 8, 12 and 24 h following PGF2 alpha treatment and from the 3 control gilts 12 h following saline injection. Jugular blood samples for progesterone analysis were collected from all gilts at 0, 2 and 4 h following treatment and at 8, 12 and 24 h for gilts from which ovaries were removed at 8, 12 and 24 h, respectively. Mean serum progesterone and CL progesterone concentrations decreased within 4 h after PGF2 alpha treatment (P less than 0.05) and remained low through 24 h after treatment. The number of unoccupied LH receptors decreased by 4 h (P less than 0.05) and this trend continued through 24 h. There were no differences in luteal weight or affinity of unoccupied LH receptors of luteal tissue at 4, 8 12 and 24 h after PGF2 alpha when compared to luteal tissue from controls. These data indicate that during PGF2 alpha-induced luteolysis in the pig, luteal progesterone, serum progesterone concentrations and the number of LH receptors decrease simultaneously.     

Prostaglandin F2 alpha, oxytocin and progesterone secretion by bovine luteal cells at several stages of luteal development: effects of oxytocin, luteinizing hormone, prostaglandin F2 alpha and estradiol-17 beta

Bovine luteal cells from Days 4, 8, 14 and 18 of the estrous cycle were incubated for 2 h (1 x 10(5) cells/ml) in serum-free media with one or a combination of treatments [control (no hormone), prostaglandin F2 alpha (PGF), oxytocin (OT), estradiol-17 beta (E) or luteinizing hormone (LH)]. Luteal cell conditioned media were then assayed by RIA for progesterone (P), PGF, and OT. Basal secretion of PGF on Days 4, 8, 14 and 18 was 173.8 +/- 66.2, 111.1 +/- 37.8, 57.7 +/- 15.4 and 124.3 +/- 29.9 pg/ml, respectively. Basal release of OT and P was greater on Day 4 (P less than 0.01) than on Day 8, 14 and 18 (OT: 17.5 +/- 2.6 versus 5.6 +/- 0.7, 6.0 +/- 1.4 and 3.1 +/- 0.4 pg/ml; P: 138.9 +/- 19.5 versus 23.2 +/- 7.5, 35.4 +/- 6.5 and 43.6 +/- 8.1 ng/ml, respectively). Oxytocin increased (P less than 0.01) PGF release by luteal cells compared with control cultures irrespective of day of estrous cycle. Estradiol-17 beta stimulated (P less than 0.05) PGF secretion on Days 8, 14 and 18, and LH increased (P less than 0.01) PGF production only on Day 14. Prostaglandin F2 alpha, E and LH had no effect on OT release by luteal cells from any day. Luteinizing hormone alone or in combination with PGF, OT or E increased (P less than 0.01) P secretion by cells from Days 8, 14 and 18. However on Day 8, a combination of PGF + OT and PGF + E decreased (P less than 0.05) LH-stimulated P secretion. These data demonstrate that OT stimulates PGF secretion by bovine luteal cells in vitro. In addition, LH and E also stimulate PGF release but effects may vary with stage of estrous cycle.     

Synthesis of prostaglandin F2 alpha, E2 and prostacyclin in isolated corpora lutea of adult pseudopregnant rats throughout the luteal life-span.

The ability of de novo biosynthesis of prostaglandins (PGs) in individual whole corpora lutea (CL) obtained from sterile-mated adult pseudopregnant rats on different days of the luteal phase and the post-luteolytic period was evaluated. Production of PGs, progesterone and 20 alpha-dihydroprogesterone were determined after in vitro incubation of CL extirpated from Day 2 to Day 19 after mating. A time-relationship with increased accumulation of PGs in the medium was demonstrated from 18 s to 5 h, with large increments during the first 30 min. Basal accumulation of PGs in the incubation medium was highest for 6-keto-PGF1 alpha (the stable metabolite of prostacyclin) greater than PGE2 greater than PGF2 alpha greater than thromboxane B2 (TXB2) and basal accumulation of PGF2 alpha and PGE2 measured in the medium was maximal on Day 10-11 of pseudopregnancy, concomitantly with a decline in secretion of progesterone. Addition of arachidonic acid (AA) dose-dependently increased synthesis of PGs, with absolute amounts of PGE2 greater than 6-keto-PGF1 alpha greater than PGF2 alpha greater than TXB2 and addition of 14 microM indomethacin markedly inhibited accumulation of all PGs measured. Luteinizing hormone (LH, 10 micrograms/ml) stimulated progesterone secretion on all days during pseudopregnancy, but not on the post-luteolytic Day 19. LH increased PGF2 alpha, PGE2 and 6-keto-PGF1 alpha secretion on Day 13 of pseudopregnancy by 76%, 91% and 28%, respectively, but not on the other days tested. Furthermore, stimulation of PG-synthesis by addition of AA abrogated the LH-induced progesterone accumulation markedly, but only on Day 13 of pseudopregnancy. Epinephrine (5 micrograms/ml) increased production of progesterone and also PGs, but only on Day 2 of pseudopregnancy, whereas oxytocin (100 mIU/ml) was found to be without effect on progesterone as well as PG secretion on all days tested. The results of the present study demonstrates the independent ability of the rat CL to synthesize PGG/PGH2-derived prostaglandins, including the putative luteolysin PGF2 alpha. Secondly, we demonstrate that LH and AA-induced increases in PGF2 alpha and PGE2 production during the luteolytic period, may be an autocrine or paracrine mechanism involved in luteolysis.     

Regulation of prostaglandin synthesis by progesterone in the bovine corpus luteum

The objective of the present study was to investigate the influence of progesterone on prostaglandin synthesis by the corpus luteum (CL). Corpora lutea were obtained from dairy cows on days 4, 6, 10, and 18 of the estrous cycle, dissociated, and placed in serum-free culture. The addition of luteinizing hormone (LH) resulted in a slight, but non-significant (p greater than 0.05), increase in levels of 6-keto-PGF1 alpha, and had no effect on PGF2 alpha. Progesterone treatment caused a significant, dose-dependent decrease in both PGF2 alpha and 6-keto-PGF1 alpha in 6-day and 10-day corpora lutea, but not in 4-day or 18-day corpora lutea. In the 6- and 10-day corpora lutea, progesterone treatment resulted in a greater inhibition of PGF2 alpha than 6-keto-PGF1 alpha production. Therefore, progesterone treatment brought about an increase in the 6-keto-PGF1 alpha to PGF2 alpha ratio in these cells (12.9 vs. 21.3). It is concluded from these studies that progesterone can modulate luteal prostacyclin and PGF2 alpha synthesis, suggesting an interaction of progesterone and prostaglandin production within the corpus luteum.     

Effects of TPA, A23187, and prostaglandin F2 alpha on progesterone synthesis by dispersed ovine luteal cells

The possible roles of protein kinase C, intracellular calcium, and oxygen environment in luteal progesterone (P4) production and their interaction with prostaglandin (PGF2 alpha) were investigated in dispersed ovine luteal cells. The following experiments were performed: 1) dose response to TPA and A23187, 2) interactions between the phorbol ester TPA and PGF2 alpha at 5% or 18% O2, 3) effect of TPA and PGF2 alpha on basal and luteinizing hormone (LH)-stimulated P4 secretion, 4) interaction of submaximal inhibitory concentrations of TPA with PGF2 alpha and the effect of indomethacin (IN) on the TPA response. Day 9 (Day 0 = first day of estrus) corpora lutea (CL) from ewes exhibiting estrous cycles of normal duration (15-17 days) were dispersed and 50,000-150,000 cells were cultured for 4 h in Dulbecco's Modified Eagle Medium. The proportion of luteal cells greater than 22 microns in diameter in these preparations averaged 17.8 +/- 2.1%. P4 in medium and cells was measured by radioimmunoassay. Both TPA and A23187 inhibited basal P4 accumulation in a dose-dependent manner. Maximum inhibition (500 nM) by TPA was greater than by A23187 at the same concentration (66.4 +/- 3.4 and 83.2 +/- 7.2% of controls, respectively; p less than 0.05), and the two were not additive in their effects. Reducing O2 did not affect P4 accumulation with or without TPA, PGF2 alpha, or both. Basal P4 accumulation was inhibited 30% by TPA and 10% by PGF2 alpha, but no additivity was seen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandin F2 alpha-stimulated release of ovarian oxytocin in the sheep in vivo: threshold and dose dependency

To determine the threshold of prostaglandin F2 alpha (PGF2 alpha)-stimulated oxytocin secretion from the ovine corpus luteum, low levels of PGF2 alpha (5-100 pg/min) were infused into the ovarian arterial blood supply of sheep with ovarian autotransplants. PGF2 alpha was infused for six sequential 10-min periods at hourly intervals, 6, 12, or 24 days after estrus (n = 3 for each day). Each cycle day was studied during a separate cycle. Oxytocin and progesterone in ovarian venous and carotid arterial plasma was measured by radioimmunoassay, and secretion rates were determined (venous-arterial concentration x plasma flow). In animals treated on Day 6, 5 pg/min PGF2 alpha caused a significant release of oxytocin (p less than 0.01), whereas in animals treated on Day 12, this threshold was 40 pg/min (p less than 0.05). In animals treated on Day 24, the threshold for oxytocin release was greater than 100 pg/min. PGF2 alpha did not significantly change ovarian blood flow or progesterone secretion rate on any day (p greater than 0.05). To determine residual luteal oxytocin after each threshold experiment, 5 mg PGF2 alpha was given i.m. to all animals. Significantly more oxytocin was released by Day 6 than by Day 12 and Day 24 corpora lutea, and by Day 12 than by Day 24 corpora lutea (1.2 micrograms, 0.7 microgram, and 0.3 microgram, respectively; p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Gonadotropin-releasing hormone 1 directly affects corpora lutea lifespan in Mediterranean buffalo (Bubalus bubalis) during diestrus: presence and in vitro effects on enzymatic and hormonal activities

The expression of gonadotropin-releasing hormone (GNRH) receptor (GNRHR) and the direct role of GNRH1 on corpora lutea function were studied in Mediterranean buffalo during diestrus. Immunohistochemistry evidenced at early, mid, and late luteal stages the presence of GNRHR only in large luteal cells and GNRH1 in both small and large luteal cells. Real-time PCR revealed GNRHR and GNRH1 mRNA at the three luteal stages, with lowest values in late corpora lutea. In vitro corpora lutea progesterone production was greater in mid stages and lesser in late luteal phases, whereas prostaglandin F2 alpha (PGF2alpha) increased from early to late stages, and PGE2 was greater in the earlier-luteal phase. Cyclooxygenase 1 (prostaglandin-endoperoxide synthase 1; PTGS1) activity did not change during diestrus, whereas PTGS2 increased from early to late stages, and PGE2-9-ketoreductase (PGE2-9-K) was greater in late corpora lutea. PTGS1 activity was greater than PTGS2 in early corpora lutea and lesser in late luteal phase. In corpora lutea cultured in vitro, the GNRH1 analog (buserelin) reduced progesterone secretion and increased PGF2alpha secretion as well as PTGS2 and PGE2-9-K activities at mid and late stages. PGE2 release and PTGS1 activity were increased by buserelin only in late corpora lutea. These results suggest that GNRH is expressed in all luteal cells of buffalo, whereas GNRHR is only expressed in large luteal phase. Additionally, GNRH directly down-regulates corpora lutea progesterone release, with the concomitant increases of PGF2alpha production and PTGS2 and PGE2-9-K enzymatic activities.     

Growth hormone, but not luteinizing hormone, acts with luteal peptides on prostaglandin F2alpha and progesterone secretion by bovine corpora lutea in vitro*

Prostaglandin F2alpha (PGF2alpha) is a major physiological luteolysin in the cow. However, injection of PGF2alpha before day 5 (day 0 = estrus) of the estrous cycle dose not induce luteolysis. On the other hand, the early corpus luteum (CL) actively produces PGF2alpha. This indicates that luteal PGF2alpha may play a key role in the refractoriness to PGF2alpha injected during the early luteal phase when angiogenesis is active in the CL. Thus, this study aimed to investigate the possible interaction between pituitary hormones and local factors (luteal peptides) on secretion of PGF2alpha and progesterone (P) by the early bovine CL, and to evaluate the effect of growth hormone (GH) as well as its interactions on production of PGF2alpha in the developing CL. A RT-PCR analysis revealed that mRNA for GH receptor in CL was fully expressed from early in the luteal phase throughout the estrous cycle, while luteinizing hormone (LH) receptor mRNA was expressed less by the early and regressing CL than those at mid or late luteal phases (P < 0.05). For the stimulation test, an in vitro microdialysis system (MDS) was used as a model. Each bovine early CL (days 3-4) was implanted with the MDS, and maintained in an organ culture chamber. The infusion of GH, insulin-like growth factor-1 (IGF-1) and oxytocin (OT) increased (P < 0.05) PGF2alpha and P release. In contrast, LH had no effect (P > 0.05) on PGF2alpha secretion and little effect on P release. Unexpectedly, there was no distinct interaction between pituitary hormones and luteal peptides on secretion of PGF2alpha and P. These results indicate that GH is a more powerful stimulator of PGF2alpha and P production in the early bovine CL than LH and suggest that GH and luteal peptides, IGF-1 and OT, contribute to maintenance of elevated PGF2alpha production in the developing bovine CL.     

Prostaglandin f(2alpha) induces distinct physiological responses in porcine corpora lutea after acquisition of luteolytic capacity

This study examines differences in intracellular responses to cloprostenol, a prostaglandin (PG)F(2alpha) analog, in porcine corpora lutea (CL) before (Day 9 of estrous cycle) and after (Day 17 of pseudopregnancy) acquisition of luteolytic capacity. Pigs on Day 9 or Day 17 were treated with saline or 500 microgram cloprostenol, and CL were collected 10 h (experiment I) or 0.5 h (experiment III) after treatment. Some CL were cut into small pieces and cultured to measure progesterone and PGF(2alpha) secretion. In experiment I, progesterone remained high and PGF(2alpha) low in luteal incubations from either Day 9 or Day 17 saline-treated pigs. Cloprostenol increased PGF(2alpha) production 465% and decreased progesterone production 87% only from Day 17 luteal tissue. Cloprostenol induced prostaglandin G/H synthase (PGHS)-2 mRNA (0.5 h) and protein (10 h) in both groups. In cell culture, cloprostenol or phorbol 12, 13-didecanoate (PDD) (protein kinase C activator), induced PGHS-2 mRNA in luteal cells from both groups. However, acute cloprostenol treatment (10 min) decreased progesterone production and increased PGF(2alpha) production only from Day 17 luteal cells. Thus, PGF(2alpha) production is induced by cloprostenol in porcine CL with luteolytic capacity (Day 17) but not in CL without luteolytic capacity (Day 9). However, this change in PGF(2alpha) production is not explained by a difference in induction of PGHS-2 mRNA or protein.     

Growth hormone, but not luteinizing hormone, acts with luteal peptides on prostaglandin F2alpha and progesterone secretion by bovine corpora lutea in vitro

Prostaglandin F2alpha (PGF2alpha) is a major physiological luteolysin in the cow. However, injection of PGF2alpha before day 5 (day 0 = estrus) of the estrous cycle dose not induce luteolysis. On the other hand, the early corpus luteum (CL) actively produces PGF2alpha. This indicates that luteal PGF2alpha may play a key role in the refractoriness to PGF2alpha injected during the early luteal phase when angiogenesis is active in the CL. Thus, this study aimed to investigate the possible interaction between pituitary hormones and local factors (luteal peptides) on secretion of PGF2alpha and progesterone (P) by the early bovine CL, and to evaluate the effect of growth hormone (GH) as well as its interactions on production of PGF2alpha in the developing CL. A RT-PCR analysis revealed that mRNA for GH receptor in CL was fully expressed from early in the luteal phase throughout the estrous cycle, while luteinizing hormone (LH) receptor mRNA was expressed less by the early and regressing CL than those at mid or late luteal phases (P < 0.05). For the stimulation test, an in vitro microdialysis system (MDS) was used as a model. Each bovine early CL (days 3-4) was implanted with the MDS, and maintained in an organ culture chamber. The infusion of GH, insulin-like growth factor-I (IGF-I) and oxytocin (OT) increased (P < 0.05) PGF2alpha and P release. In contrast, LH had no effect (P > 0.05) on PGF2alpha secretion and little effect on P release. Unexpectedly, there was no distinct interaction between pituitary hormones and luteal peptides on secretion of PGF2alpha and P. These results indicate that GH is a more powerful stimulator of PGF2alpha and P production in the early bovine CL than LH and suggest that GH and luteal peptides, IGF-1 and OT, contribute to maintenance of elevated PGF2alpha production in the developing bovine CL.     

Effects of prostaglandins on the bovine corpus luteum: granules, lipid inclusions and progesterone secretion

Corpora lutea collected at 15, 30 and 60 min after prostaglandin F2 alpha (PGF2 alpha) treatment were compared to control corpora lutea at 60 min after saline treatment. There were decreases (P less than 0.05) in the relative percentages of cytoplasm occupied by granules in large luteal cells (LLC) by 30 min and in small luteal cells (SLC) by 60 min. Differences were not observed among the groups for lipid inclusions. Luteal progesterone was decreased at all post-PGF2 alpha treatment times when compared to 60-min controls (P less than 0.05). PGF2 alpha was then compared with prostaglandin F1 alpha (PGF1 alpha), prostaglandin E1 (PGE1), and 17-phenyl-18,19,20-trinor-prostaglandin F2 alpha (17-phenyl-PGF2 alpha) in 60-min trials with plasma progesterone and luteinizing hormone (LH) determined every 5 min. LH was not affected by these treatments. Like PGF2 alpha, 17-phenyl-PGF2 alpha induced a greater loss of granules from LLC then SLC. 17-phenyl-PGF2 alpha also induced an increase in the lipid content of LLC. Treatments with PGF2 alpha and 17-phenyl-PGF2 alpha were associated with decreased concentrations of luteal progesterone but PGF1 alpha and PGE1 were without effect on this variable. In contrast to PGF1 alpha, PGE1 increased both luteal progesterone and the area occupied by cytoplasmic granules. The latter effect was greater in LLC than SLC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Destruction of bovine ovarian follicles: effects on the pulsatile release of luteinizing hormone and prostaglandin F2 alpha-induced luteal regression

Destruction of ovarian follicles during diestrus prolongs the lifespan of corpora lutea in cows, but the site(s) of action is unclear. Thus, ovarian follicles were destroyed in 10 beifers (X-IRRAD) on Day 9 postestrus, while 10 additional beifers (SHAM) served as a control group. To investigate changes in luteotropic support resulting from destruction of ovarian follicles, pulses of luteinizing hormone (LH) were characterized on Days 8, 13, and 15 postestrus. To study the interaction between products from ovarian follicles and prostaglandin F2 alpha (PGF2 alpha) in luteolysis, changes in serum concentrations of progesterone were monitored after an injection of saline or PGF2 alpha on Day 14 postestrus. Frequency and amplitude of pulses of LH increased by Day 13 in X-IRRAD beifers. An increase of similar magnitude in amplitude but not frequency of pulses of LH occurred between Day 13 and Day 15 postestrus in SHAM beifers. Exogenous PGF2 alpha was significantly less efficacious in causing luteolysis in X-IRRAD animals. We suggest that increased luteotropic support may be involved in but is not the only cause for lengthening the lifespan of corpora lutea following destruction of ovarian follicles. Additionally, we suggest that regression of bovine corpora lutea involves a synergistic action between products from ovarian follicles and PGF2 alpha.     

Second messenger systems and progesterone secretion in the small cells of the bovine corpus luteum: effects of gonadotropins and prostaglandin F2a

The present studies were conducted to determine the effects of gonadotropins (LH and hCG) and prostaglandin F2a (PGF2a) on the production of "second messengers" and progesterone synthesis in purified preparations of bovine small luteal cells. Corpora lutea were removed from heifers during the luteal phase of the normal estrous cycle. Small luteal cells were isolated by unit-gravity sedimentation and were 95-99% pure. LH provoked rapid and sustained increases in the levels of [3H]inositol mono-, bis-, and trisphosphates (IP, IP2, IP3, respectively), cAMP and progesterone in small luteal cells. LiCl (10 mM) enhanced inositol phosphate accumulation in response to LH but had no effect on LH-stimulated cAMP or progesterone accumulation. Time course studies revealed that LH-induced increases in IP3 and cAMP occurred simultaneously and preceded the increases in progesterone secretion. Similar dose-response relationships were observed for inositol phosphate and cAMP accumulation with maximal increases observed with 1-10 micrograms/ml of LH. Progesterone accumulation was maximal at 1-10 ng/ml of LH. LH (1 microgram/ml) and hCG (20 IU/ml) provoked similar increases in inositol phosphate, cAMP and progesterone accumulation in small luteal cells. 8-Bromo-cAMP (2.5 mM) and forskolin (1 microM) increased progesterone synthesis but did not increase inositol phosphate accumulation in 30 min incubations. PGF2a (1 microM) was more effective than LH (1 microgram/ml) at stimulating increases in inositol phosphate accumulation (4.4-fold vs 2.2-fold increase for PGF2a and LH, respectively). The combined effects of LH and PGF2a on accumulation of inositol phosphates were slightly greater than the effects of PGF2a alone. In 30 min incubations, PGF2a had no effect on cAMP accumulation and provoked small increases in progesterone secretion. Additionally, PGF2a treatment had no significant effect on LH-induced cAMP or progesterone accumulation in 30 min incubations of small luteal cells. These findings provide the first evidence that gonadotropins stimulate the cAMP and IP3-diacylglycerol transmembrane signalling systems in bovine small luteal cells. PGF2a stimulated phospholipase C activity in small cells but did not reduce LH-stimulated cAMP or progesterone accumulation. These results also demonstrate that induction of functional luteolysis in vitro requires more than the activation of the phospholipase C-IP3/calcium and -diacylglycerol/protein kinase C transmembrane signalling system.     

Effects of indomethacin, luteinizing hormone (LH), prostaglandin E2 (PGE2), trilostane, mifepristone, ethamoxytriphetol (MER-25) on secretion of prostaglandin E (PGE), prostaglandin F2alpha (PGF2alpha) and progesterone by ovine corpora lutea of pregnancy or the estrous cycle

Two experiments were conducted to determine the luteotropin of pregnancy in sheep and to examine autocrine and paracrine roles of progesterone and estradiol-17 beta on progesterone secretion by the ovine corpus luteum (CL). Secretion of progesterone per unit mass by day-8 or day-11 CL of the estrous cycle was similar to day-90 CL of pregnancy (P > or = 0.05). In experiment 1, secretion of progesterone in vitro by slices of CL from ewes on day-8 of the estrous cycle was increased (P < or = 0.05) by LH or PGE2. Secretion of progesterone in vitro by CL slices from day-90 pregnant ewes was not affected by LH (P > or = 0.05) while PGE2 increased (P < or = 0.05) secretion of progesterone. Day 8 ovine CL of the estrous cycle did not secrete (P > or = 0.05) detectable quantities of PGF2alpha or PGE while day-90 ovine CL of pregnancy secreted PGE (P < or = 0.05) but not PGF2alpha. Secretion of progesterone and PGE in vitro by day-90 CL of pregnancy was decreased (P < or = 0.05) by indomethacin. The addition of PGE2, but not LH, in combination with indomethacin overcame the decreases in progesterone by indomethacin (P < or = 0.05). In experiment 2, secretion of progesterone in vitro by day-11 CL of the estrous cycle was increased at 4-h (P < or = 0.05) in the absence of treatments. Both day-11 CL of the estrous cycle and day-90 CL of pregnancy secreted detectable quantities of PGE and PGF2alpha (P < or = 0.05). In experiment 1, PGF2alpha secretion by day-8 CL of the estrous cycle and day-90 ovine CL of pregnancy was undetectable, but was detectable in experiment 2 by day-90 CL. Day 90 ovine CL of pregnancy also secreted more PGE than day-11 CL of the estrous cycle (P < or = 0.05), whereas day-8 CL of the estrous cycle did not secrete detectable quantities of PGE (P > or = 0.05). Trilostane, mifepristone, or MER-25 did not affect secretion of progesterone, PGE, or PGF2alpha by day- 11 CL of the estrous cycle or day-90 CL of pregnancy (P > or = 0.05). It is concluded that PGE2, not LH, is the luteotropin at day-90 of pregnancy in sheep and that progesterone does not modify the response to luteotropins. Thus, we found no evidence for an autocrine or paracrine role for progesterone or estradiol-17 36 on luteal secretion of progesterone, PGE or PGF2alpha.     

Effects of indomethacin, luteinizing hormone (LH), prostaglandin E(2) (PGE(2)), trilostane, mifepristone, ethamoxytriphetol (MER-25) on secretion of prostaglandin E (PGE), prostaglandin F(2alpha) (PGF(2alpha)) and progesterone by ovine corpora lutea of pregnancy or the estrous cycle

Two experiments were conducted to determine the luteotropin of pregnancy in sheep and to examine autocrine and paracrine roles of progesterone and estradiol-17 beta on progesterone secretion by the ovine corpus luteum (CL). Secretion of progesterone per unit mass by day-8 or day-11 CL of the estrous cycle was similar to day-90 CL of pregnancy (P >/= 0.05). In experiment 1, secretion of progesterone in vitro by slices of CL from ewes on day-8 of the estrous cycle was increased (P /= 0.05) while PGE(2) increased (P /= 0.05) detectable quantities of PGF(2alpha) or PGE while day-90 ovine CL of pregnancy secreted PGE (P /= 0.05). Trilostane, mifepristone, or MER-25 did not affect secretion of progesterone, PGE, or PGF(2alpha) by day-11 CL of the estrous cycle or day-90 CL of pregnancy (P >/= 0.05). It is concluded that PGE(2), not LH, is the luteotropin at day-90 of pregnancy in sheep and that progesterone does not modify the response to luteotropins. Thus, we found no evidence for an autocrine or paracrine role for progesterone or estradiol-17 36 on luteal secretion of progesterone, PGE or PGF(2alpha).     

Effect of dose of prostaglandin F(2alpha) on steroidogenic components and oligonucleosomes in ovine luteal tissue

To determine whether prostaglandin (PG) F(2alpha) had a dose-dependent effect upon secretion of progesterone, oligonucleosome formation, or loss of luteal weight, ewes on Day 9 or 10 of the estrous cycle were administered 0, 3, 10, or 30 mg PGF(2alpha) per 60 kg BW (i.v.), and luteal tissue was collected 9 and 24 h after injection. All doses of PGF(2alpha) decreased (P < 0. 05) concentrations of progesterone in sera by 9 h; however, in ewes treated with 3 mg PGF(2alpha), concentrations of progesterone were similar to control values at 24 h and higher (P < 0.05) than those in the 10- or 30-mg groups. Concentrations of progesterone in sera over all dose levels were highly correlated to luteal concentrations of mRNA encoding steroidogenic acute regulatory protein (P < 0.001), cytochrome P450 side-chain cleavage (P < 0.02), and 3beta-hydroxysteroid dehydrogenase (P < 0.01). Corpora lutea collected at 24 h from ewes treated with the 10- and 30-mg doses of PGF(2alpha) weighed less (P < 0.05) than those from controls. Oligonucleosomes were not present in luteal tissues from control ewes. Surprisingly, all doses of PGF(2alpha)-induced oligonucleosomes in a majority of animals at 9 h and in a majority of ewes treated with 10 and 30 mg of PGF(2alpha) at 24 h. In conclusion, 3 mg of PGF(2alpha) per 60 kg BW transiently decreased serum concentrations of progesterone and induced oligonucleosome formation, but did not result in reduced luteal weight. The 10- and 30-mg doses of PGF(2alpha) decreased secretion of progesterone and induced oligonucleosome formation and luteolysis.     

Effects of in vivo and in vitro administration of prostaglandin F2 alpha on lipoprotein utilization in cultured bovine luteal cells

Two experiments were conducted to determine if a loss in the ability to utilize lipoprotein-cholesterol is one mechanism whereby prostaglandin F2 alpha (PGF2 alpha) decreases steroidogenesis in bovine luteal cells. In the first experiment, serum-free cultures of bovine luteal cells were treated with PGF2 alpha (100 ng/ml) for 5 days prior to addition of lipoproteins. Exposure to PGF2 alpha completely suppressed low-density lipoprotein (LDL)- and high-density lipoprotein (HDL)-stimulated progesterone production (p less than 0.01) compared to control (no PGF2 alpha) cultures. Luteal cells cultured in the presence of LDL + luteinizing hormone (LH, 10 ng/ml) + PGF2 alpha produced significantly less progesterone than luteal cells cultured with LDL + LH (p less than 0.05). Treatment with PGF2 alpha had no significant effect on HDL + LH-stimulated progesterone synthesis. In the second experiment, cows were injected with a luteolytic dose of PGF2 alpha (25 mg), and the corpora lutea were removed at 0 (no PG), 1, 4, or 12 h post-injection. Dissociated luteal cells were placed in culture for 7 days, either with or without LH (10 ng/ml), and lipoproteins were added on Days 5-7. LH stimulation of progesterone production was apparent in cultures obtained at 0 and 12 (p less than 0.05) but not 1 and 4 h post-PGF2 alpha. Addition of either LDL or HDL increased progesterone synthesis in all cultures, regardless of time following in vivo administration of PGF2 alpha. It is concluded that PGF2 alpha can inhibit bovine luteal cell utilization of either LDL or HDL in vitro. However, luteal cell utilization of lipoproteins in vitro is not adversely affected by in vivo exposure to PGF2 alpha, if collected within 12 h post-PGF2 alpha.     

Endothelin-1 mediates prostaglandin F(2alpha)-induced luteal regression in the ewe

A diversified series of experiments was conducted to determine the potential role of endothelin-1 (ET-1) in ovine luteal function. Endothelin-1 inhibited basal and LH-stimulated progesterone production by dispersed ovine luteal cells during a 2-h incubation. This inhibition was removed when cells were preincubated with cyclo-D-Asp-Pro-D-Val-Leu-D-Trp (BQ123), a highly specific endothelin ET(A) receptor antagonist. Administration of a luteolytic dose of prostaglandin F(2alpha) (PGF(2alpha)) rapidly stimulated gene expression for ET-1 in ovine corpora lutea (CL) collected at midcycle. Intraluteal administration of a single dose of BQ123 to ewes on Day 8 or 9 of the estrous cycle mitigated the luteolytic effect of PGF(2alpha). Intramuscular administration of 100 microg ET-1 to ewes at midcycle reduced plasma progesterone concentrations for the remainder of the estrous cycle. Following pretreatment with a subluteolytic dose of PGF(2alpha), i.m. administration of 100 microg ET-1 caused a rapid decline in plasma progesterone and shortened the length of the estrous cycle. These data complement and extend previously published reports in the bovine CL and are the strongest evidence presented to date in support of a role for ET-1 in PGF(2alpha)-mediated luteal function in domestic ruminants.