Effect of luteinizing hormone (LH), pregnancy-specific protein B (PSPB), or arachidonic acid (AA) on secretion of progesterone and prostaglandins (PG) E (PGE; PGE(1) and PGE(2)) and F(2alpha) (PGF(2alpha)) by ovine corpora lutea of the estrous cycle or pregnancy in vitro

LH regulates luteal progesterone secretion during the estrous cycle in ewes and cows. However, PGE, not LH, stimulated ovine luteal progesterone secretion in vitro at day 90 of pregnancy and at day 200 in cows. The hypophysis is not obligatory after day 50 nor the ovaries after day 55 to maintain pregnancy in ewes. LH has been reported to regulate ovine placental PGE secretion up to day 50 of pregnancy and by pregnancy-specific protein B (PSPB) after day 50 of pregnancy. The objective of this experiment was to determine if and when a switch from LH to PGE occurred as the luteotropin regulating luteal progesterone secretion during pregnancy in ewes. Ovine luteal tissue slices of the estrous cycle (days 8, 11, 13, and 15) or pregnancy (days 8, 11, 13, 15, 20, 30, 40, 50, 60, and 90) were incubated in vitro with vehicle, LH, AA (precursor to PGE(2) and PGF(2alpha) synthesis), or PSPB in M199 for 4 h and 8 h. Concentrations of progesterone in jugular venous plasma of bred ewes increased (P< or =0.05) after day 50 and continued to increase through day 90. Secretion of progesterone by luteal tissue of non-bred ewes on days 8, 11, 13 and 15 and by bred ewes on days 8, 11, 13, 15, 20, 30, 40, and 50 was increased (P< or =0.05) by LH, but not by luteal tissue from pregnant ewes after day 50 (P> or =0.05). LH-stimulated progesterone secretion by luteal tissue from day 15 bred ewes was greater (P< or =0.05) than day 15 luteal tissue from non-bred ewes. Concentrations of progesterone in media were increased (P< or =0.05) when luteal tissue from pregnant ewes on day 50, 60, or 90 were incubated with AA or PSPB. Concentrations of PGE in media of non-bred ewes on days 8, 11, 13, or 15 and bred ewes on days 8 and 11 did not differ (P> or =0.05). Concentrations of PGE were increased (P< or =0.05) in media by luteal slices from bred ewes on days 13, 15, 20, 30, 40, 50, 60, and 90 of vehicle, LH, AA or PSPB-treated ewes. In addition, PSPB increased (P< or =0.05) PGE in media by luteal slices from pregnant ewes only on days 40, 50, 60, and 90. Concentrations of PGF(2alpha) were increased in media (P<0.05) of vehicle, AA, LH, or PSPB-treated luteal tissue from non-bred ewes and bred ewes on day 15 and by luteal tissue from bred ewes on days 20 and 30 after which concentrations of PGF(2alpha) in media declined (P< or =0.05) and did not differ (P> or =0.05) from non-bred or bred ewes on days 8, 11, or 13. It is concluded that LH regulates luteal progesterone secretion during the estrous cycle of non-bred ewes and up to day 50 of pregnancy, while only PGE regulates luteal progresterone secretion by ovine corpora lutea from days 50 to 90 of pregnancy. In addition, PSPB appears to regulate luteal secretion of progesterone from days 50 to 90 of pregnancy through stimulation of PGE secretion by ovine luteal tissue.     

Intraluteal infusions of prostaglandins of the E, D, I, and A series prevent PGF2 alpha-induced, but not spontaneous, luteal regression in rhesus monkeys

A luteotropic role for prostaglandins (PGs) during the luteal phase of the menstrual cycle of rhesus monkeys was suggested by the observation that intraluteal infusion of a PG synthesis inhibitor caused premature luteolysis. This study was designed to identify PGs that promote luteal function in primates. First, the effects of various PGs on progesterone (P) production by macaque luteal cells were examined in vitro. Collagenase-dispersed luteal cells from midluteal phase of the menstrual cycle (Day 6-7 after the estimated surge of LH, n = 3) were incubated with 0-5,000 ng/ml PGE2, PGD, 6 beta PGI1 (a stable analogue of PGI2), PGA2, or PGF2 alpha alone or with hCG (100 ng/ml). PGE2, PGD2, and 6 beta PGI1 alone stimulated (p less than 0.05) P production to a similar extent (2- to 3-fold over basal) as hCG alone, whereas PGA2 and PGF2 alpha alone had no effect on P production. Stimulation (p less than 0.05) of P synthesis by PGE2, PGD2, and 6 beta PGI1 in combination with hCG was similar to that of hCG alone. Whereas PGA2 inhibited gonadotropin-induced P production (p less than 0.05), that in the presence of PGF2 alpha plus hCG tended (p = 0.05) to remain elevated. Second, the effects of various PGs on P production during chronic infusion into the CL were studied in vivo. Saline with or without 0.1% BSA (n = 12), PGE2 (300 ng/h; n = 4), PGD2 (300 ng/h; n = 4), 6 beta PGI1 (500 ng/h; n = 3), PGA2 (300 ng/h; n = 4), or PGF2 alpha (10 ng/h; n = 8) was infused via osmotic minipump beginning at midluteal phase (Days 5-8 after the estimated LH surge) until menses. In addition, the same dose of PGE, PGD, PGI, or PGA was infused in combination with PGF2 alpha (n = 3-4/group) for 7 days. P levels over 5 days preceding treatment were not different among groups. In 5 of 8 monkeys receiving PGF2 alpha alone, P declined to less than 0.5 ng/ml within 72 h after initiation of infusion and was lower (p less than 0.05) than controls. The length of the luteal phase in PGF2 alpha-infused monkeys was shortened (12.3 +/- 0.9 days; mean +/- SEM, n = 8; p less than 0.05) compared to controls (15.8 +/- 0.5). Intraluteal infusion of PGE, PGD, PGI, or PGA alone did not affect patterns of circulating P or luteal phase length.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Effect of prostaglandins on luteal function during early pregnancy in pigs.

Luteal cells were obtained by digestion of luteal tissue of cyclic (day 12) and early pregnant (days 12, 20 and 30) pigs. Suspensions of the dispersed luteal cells (5 x 10(4) cells ml-1) were incubated for 2 h in minimum essential medium (MEM) alone (control) and MEM with different concentrations of prostaglandin F2 alpha (PGF2 alpha) and PGE2 (0.01, 0.1, 1, 10, 100 and 1000 ng ml-1) and luteinizing hormone (LH) 100 and 1000 ng ml-1, or with combinations of LH + PGF2 alpha and LH + PGE2. Net progesterone production was measured in the incubation media by direct radioimmunoassay. The overall response pattern of the luteal cells to exogenous hormones on day 12 of the oestrous cycle and pregnancy differed (P < 0.05) from treatment on day 20 and 30 of pregnancy. In general progesterone production was higher (P < 0.05) and the response to PGF2 alpha and PGE2 treatment was most obvious on day 12 of the oestrous cycle and pregnancy. Overall, PGF2 alpha stimulated progesterone production in a dose-dependent manner (P < 0.05). The response to PGE2 was of a quadratic nature (P < 0.05) in which the lowest and the highest doses of PGE2 were associated with a greater production of progesterone than were the intermediate doses. Treatment of luteal cells with PGF2 alpha + LH or PGE2 + LH caused overall inhibition (P < 0.05) of progesterone production compared with treatment with each hormone alone. This interaction was not affected by the dose of LH used. These findings indicate that PGF2 alpha and PGE2 are involved in the autocrine control of corpus luteum function.     

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).     

In vitro prostaglandin production by bovine corpora lutea destined to be normal or short-lived

Basal and calcium ionophore (CaI)-influenced production of prostaglandins (PGs) by corpora lutea (CL) destined to be normal or short-lived were compared. Ovulation was induced in 24 lactating beef cows with human chorionic gonadotropin (hCG, 1000 IU) administered between 35 and 40 days postpartum. Ten cows received norgestomet implants for 9 days prior to induced ovulation (Normal CL) and 14 served as untreated controls (Subnormal CL). Five cows in each treatment were unilaterally ovariectomized on Day 6 (Day 0 = day of hCG administration) and CL were collected. Blood samples were collected daily through-out the experimental period from cows not ovariectomized. Plasma progesterone (P4) in ovary-intact animals indicated that short-lived CL were induced in 8/8 cows not pretreated with norgestomet, and normal luteal lifespan was observed in 4/5 implanted cows. Dispersed luteal cells were incubated for 8 h with 0, 0.05, 0.5, or 5 microM CaI (A23187). Incubation media were analyzed for P4, PGF2 alpha, 6-keto-PGF1 alpha (PGI), and PGE2. The weight, cell number, and basal or CaI-influenced production of P4 did not differ between Normal CL and Subnormal CL. Basal production of PGF2 alpha, PGI, and PGE2 was higher in Subnormal CL than in Normal CL (p less than 0.05). In response to 0.05 microM CaI, PGF2 alpha was stimulated in Subnormal CL (p less than 0.01), while PGI (p less than 0.05) and PGE2 (p less than 0.1) were increased in Normal CL. Production of PGs was reduced by 5 microM CaI in Subnormal CL (p less than 0.01), but not in Normal CL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of prostaglandins E2 and F2alpha (PGE2; PGF2alpha), trilostane,mifepristone, palmitic acid (PA), indomethacin (INDO), ethamoxytriphetol (MER-25), PGE2 + PA,or PGF2alpha + PA on PGE2, PGF2alpha,and progesterone secretion by bovine corpora lutea of mid-pregnancy in vitro

The effects of PGE2, PGF2alpha, trilostane, RU-486, PA, INDO, MER-25, PGE2, or PGF2alpha + PA on secretion of progesterone, PGE2, or PGF2alpha by bovine corpora lutea (CL) of mid-pregnancy in vitro for 4 and 8 hr was examined. Secretion of PGE2 and PGF2alpha increased with time in culture (P < or = 0.05). PGE2 and PGE2 + PA increased (P < or = 0.05) secretion of progesterone at 4 and 8 h, progesterone secretion was increased (P < or = 0.05) at 4 h; but not at 8 h (P > or = 0.05) by trilostane, mifepristone, PGF2alpha and PGF2alpha + PA, and was decreased at 8 h by PGF2alpha and PGF2alpha + PA. Indomethacin decreased (P < or = 0.05) secretion of PGE2, PGF2alpha, and progesterone at 4 and 8 h. Trilostane, PA, PGF2alpha, RU-486 and PGF2alpha + PA increased (P < or = 0.05) PGE2 at 4 h only. Palmitic acid decreased (P < or = 0.05) PGF2alpha at 4 h, while trilostane, RU-486, or MER-25 did not affect (P < or = 0.05) PGE2 of PGF2alpha secretion. It is concluded that PGE2 of luteal tissue origin is the luteotropin at mid-pregnancy in cows. Also, it is suggested that PA may alter progesterone secretion by affecting the inter conversion of PGE2 and PGF2alpha.     

Effects of PGE1 or PGE2 on luteal function in pseudopregnant rats

Effects of PGE1 or PGE2 on luteal function were studied in 163 pseudopregnant rats. PGE1 (10, 100, or 300 micrograms) given intrauterine every 6 hr did not shorten pseudopregnancy (P greater than 0.05), however, the same doses of PGE2 given intrauterine every 6 hr advanced luteolysis (P less than 0.05). PGE1 (100 or 300 micrograms) given every 4 hr intramuscular maintained levels of progesterone in peripheral blood above controls (P less than 0.05) while 100 or 300 micrograms of PGE2 hastened the decline in progesterone (P less than 0.05). The antiluteolytic effect of PGE1 was not via an inhibition of PGF secretion (P greater than 0.05) by the uterus or by induction of ovulation in treated animals. Moreover, PGE1 (100, 200, or 500 micrograms) given intramuscular every 4 hr from day 4 of pseudopregnancy until the next proestrus delayed luteal regression around 3 days (P less than 0.05). PGE2 at doses of 100, 200, or 500 micrograms every 4 hr given intramuscular consistently shortened pseudopregnancy (P less than 0.05). Lower doses were without effect (P greater than 0.05). Based on the above data it is concluded that PGE2 is consistently luteolytic whereas PGE1 is not luteolytic in pseudopregnant rats and that PGE1 may be an antiluteolysin.     

Activity of phospholipase C in ovine luteal tissue in response to PGF2 alpha, PGE2 and luteinizing hormone.

Four ewes were utilized to determine the effects of prostaglandin (PG) F2 alpha, PGE2 and luteinizing hormone (LH) on activity of phospholipase C (PLC) in ovine luteal tissue. Corpora lutea were collected on d 10 post-estrus and six slices from one corpus luteum from each ewe were pre-incubated with [3H]-inositol prior to incubation with one of 6 treatments. Treatments were 1) control, 2) PGF2 alpha (100 ng/ml), 3) PGE2 (10 ng/ml), 4) LH (10 ng/ml), 5) PGF2 alpha + PGE2 and 6) PGF2 alpha + LH. Phospholipase C was determined indirectly by measuring the accumulation of [3H]-inositol mono-, bis- and tris-phosphates (IP, IP2, IP3). Effects of PGF2 alpha (0 vs. PGF2 alpha) and luteotropic treatment (0 vs. PGE2 vs. LH) and their interactions were determined by analysis of variance. There was a significant main effect of PGF2 alpha (P less than 0.01) as concentrations of IP, IP2, IP3 and total [3H]-inositol phosphates were greater in tissue slices treated with PGF2 alpha, regardless of luteotropic treatment. Within groups receiving no PGF2 alpha (1,3,4), no effect of luteotropic treatment was observed. Within groups receiving PGF2 alpha (2,5,6), LH caused a significant (P less than .05) increase in the accumulation of total [3H]-inositol phosphates. Thus, PGF2 alpha can stimulate the activity of PLC in ovine luteal tissue and LH can potentiate this effect.     

A comparison of the effects of cyclooxygenase prostanoids on progesterone production by small and large bovine luteal cells

Highly purified preparations of small and large bovine luteal cells were utilized to examine the effects of prostaglandins F2 alpha (PGF2 alpha), E2 (PGE2) and I2 (PGI2) analog on progesterone production. Corpora lutea were obtained from Holstein heifers between days 10 and 12 of the estrous cycle. Purified small and large cells were obtained by unit gravity sedimentation and flow cytometry. Progesterone accumulation was determined in 1 x 10(5) small and 5 x 10(3) large cells after 2 and 4 h incubations respectively. Progesterone synthesis was increased (p less than 0.05) in the small cells by the increasing levels of PGF2 alpha, PGE2, carba-PGI2 and LH. PGF2 alpha, but not PGE2 or carba-PGI2 increased (p less than 0.05) LH-stimulated progesterone production. There was no interaction of various combinations of prostaglandins on progesterone production in the small cells. In the large cells, PGF2 alpha had no effect on basal progesterone production. However, it inhibited LH-stimulated progesterone synthesis. In contrast, PGE2 and carba-PGI2 stimulated (p less than 0.05) basal progesterone production in the large cells. In the presence of LH, high levels of carba-PGI2 inhibited (p less than 0.05) progesterone synthesis. The PGE2 and PGI2-stimulated progesterone production in the large luteal cells was also inhibited in the presence of PGF2 alpha. These data suggest all of the prostaglandins used exert a luteotropic action in the small cells. In the large cells only PGE2 and carba-PGI2 are luteotropic, while PGF2 alpha exerts a luteolytic action. The effects of the prostaglandins in the small and large luteal cells suggest that their receptors are present in both cell types.     

Effects of prostaglandin E1 or E2 (PGE1; PGE2) on luteal function and binding of luteinizing hormone in nonpregnant ewes

Two studies were conducted to determine the effects of PGE1 or PGE2 on luteal function and binding of luteinizing hormone (LH) to luteal cell membranes in nonpregnant ewes. In Study I, ewes (n=5 per group) received an injection of vehicle (VEH) or 333 micrograms of PGE1 or PGE2 into the tissue surrounding the ovarian vascular pedicle (intrapedicle) on day 7 postestrus. Systemic progesterone concentrations of PGE1-treated ewes were greater (P less than 0.01) than those of VEH-treated ewes at 24 and 48 hr after injection. For PGE2-treated ewes, progesterone concentrations were greater (P less than 0.01) than for VEH-treated ewes only at 24 hr. Neither PGE1 nor PGE2 affected luteal weights or LH binding capacity at 48 hr. Treatment with PGE1, however, increased (P less than 0.10) endogenously bound LH at this time. In Study II, ewes (n=5 per group) received an intrapedicle injection of VEH, or 10 mg of PGE1 or PGE2 on day 8 postestrus. Systemic progesterone concentrations in PGE1-treated ewes were less (P less than 0.01) than for VEH-treated ewes at 24 hr, but by 72 hr were not different from those of VEH-treated ewes. For PGE2-treated ewes, systemic progesterone declined steadily to reach low values by 72 hr. Prostaglandin E2 had no effect on luteal binding of LH at 72 hr, whereas PGE1 increased (P less than 0.05) LH binding capacity and endogenously bound LH. Although PGE2 had no apparent affect on luteal binding of LH in these studies, PGE1 may enhance the function of ovine corpora lutea by stimulating an increase in their binding of LH and capacity to bind LH when the CL receives a luteolytic signal.     

Localization of prostaglandin F2 alpha inhibition of lipoprotein use by bovine luteal cells.

Prostaglandin F2 alpha (PGF2 alpha) inhibits lipoprotein-stimulated progesterone production by bovine luteal cells in vitro and the objective of this study was to localize the site of action of PGF2 alpha. Cultured bovine luteal cells were treated with PGF2 alpha for seven days, and then with either lipoproteins or 25-hydroxycholesterol in the presence of aminoglutethimide (which inhibits cholesterol side-chain cleavage) for the final 48 h. The effects of PGF2 alpha on progesterone production, cellular cholesterol content, mitochondrial cholesterol content and cholesterol side-chain cleavage activity were determined. As expected, PGF2 alpha inhibited (P less than 0.05) lipoprotein-stimulated progesterone production. However, PGF2 alpha did not inhibit low-density lipoprotein-stimulated, or high density lipoprotein-stimulated, increases in cellular cholesterol (P less than 0.05) or inhibit lipoprotein-induced increases in mitochondrial cholesterol content (P less than 0.05). Additionally, cholesterol content of mitochondria increased (P less than 0.05) in the presence of PGF2 alpha alone. To determine if the PGF2 alpha-induced inhibition of steroidogenesis occurred at, or after, the side-chain cleavage reaction, we treated cells with the readily diffusable sterol, 25-hydroxycholesterol. Prostaglandin F2 alpha did not inhibit 25-hydroxycholesterol-stimulated progesterone production (P less than 0.05). Prostaglandin F2 alpha may therefore exert its luteolytic effect at a site after cholesterol transport to the mitochondria but before cholesterol side-chain cleavage.     

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)     

Effects of estrous synchronization on response to nitric oxide donors, nitric oxide synthase inhibitors, and endothelin-1 in vitro

Two experiments were conducted to determine the effects of nitric oxide (NO) donors, endothelin-(ET-1), and NO synthase (NOS) inhibitors on bovine luteal function in vitro. In experiment 1, estrus in Brahman cows was synchronized with Synchro-Mate-B (SMB) and day-13-14 corpora luteal slices were weighed, diced and incubated in vitro. Treatments (100 ng/ml) were: vehicle, N[see symbol in text]-nitro-L-arginine-L-methyl ester (L-NAME), N(G)-monomethyl-L-arginine acetate (L-NMMA), diethylenetriamine (DETA), DETA-NONOate, sodium nitroprusside (SNP), or ET-1. In experiment 2, estrus was synchronized with Lutalyse, a Controlled Intravaginal Progesterone Releasing Device (CIDR), or cows were not synchronized. Corpora lutea were collected, weighed, and luteal slices were weighed, diced and incubated in vitro with treatments. Treatments (100ng/ml) were: vehicle, L- NAME, L-NMMA, DETA, DETA-NONOate, sodium nitroprusside, S-nitroso-N-acetylpenicillamine (SNAP) or endothelin-1. Tissues were incubated in M- 199 for 1 h without treatments and for 4 and 8 h in both experiments with treatments in both experiments. Media were analyzed for progesterone, prostaglandins E2 and F2alpha (PGE2, PGF2alpha) by radioimmunoassay (RIA). Hormone data in experiments 1 and 2 were analyzed by 2 x 7 and 3 x 2 x 8 factorial design for analysis of variance (ANOVA), respectively. Luteal weights in experiment 2 were analyzed by a one-way ANOVA. Concentrations of progesterone in media were similar (P > or = 0.05) among treatments within experiments. Concentrations of PGE2 in media in experiment 1 were undetectable in 90 and 57% of the samples at 4 and 8 h, respectively. PGF2alpha increased (P < or = 0.05) with time, but did not differ (P > or = 0.05) among treatments. Secretion of PGF2alpha was not affected by treatments (P > or = 0.05). In experiment 2, luteal weights of the induced estrous cycle were decreased (P < or = 0.05) by Lutalyse. Concentrations of PGE2 and PGF2alpha increased (P < or = 0.05) with time in control of all three synchronization regimens. DETA-NONOate, SNAP, sodium nitroprusside (NO donors) and ET-1 increased (P < or = 0.05) PGE2 except in the CIDR synchronized group (P > or = 0.05). No treatment increased (P > or = 0.05) PGF2alpha in any synchronization regimen. It is concluded that either SMB containing norgestomet or a CIDR containing progesterone alters luteal secretion of PGE2, Lutalyse lowers luteal weights in the induced estrous cycle, and NO or ET-1 given alone are not luteolytic agents. It is suggested that NO and ET-1 could have indirect antiluteolytic/luteotropic effects via increasing PGE2 secretion by luteal tissue rather than being luteolytic.     

Anti-apoptotic roles of prostaglandin E2 and F2alpha in bovine luteal steroidogenic cells

Production of prostaglandins (PGs) and expression of their receptors have been demonstrated in bovine corpus luteum (CL). The aim of the present study was to determine whether PGE2 and PGF2alpha have roles in bovine luteal steroidogenic cell (LSC) apoptosis. Cultured bovine LSCs obtained at the midluteal stage (Days 8-12 of the cycle) were treated for 24 h with PGE2 (0.001-1 microM) and PGF2alpha (0.001-1 microM). Prostaglandin E2 (1 microM) and PGF2alpha (1 microM) significantly stimulated progesterone (P4) production and reduced the levels of cell death in the cells cultured with or without tumor necrosis factor alpha (TNF)/interferon gamma (IFNG), in the presence and absence of FAS ligand (P < 0.05). Furthermore, DNA fragmentation induced by TNF/IFNG was observed to be suppressed by PGE2 and PGF2alpha. Prostaglandin E2 and PGF2alpha also attenuated mRNA expression of caspase 3 and caspase 8, as well as caspase 3 activity (P < 0.05) in TNF/IFNG-treated cells. FAS mRNA and protein expression were decreased only by PGF2alpha (P < 0.05). A specific P4 receptor antagonist (onapristone) attenuated the apoptosis-inhibitory effects of PGE2 and PGF2alpha in the absence of TNF/IFNG (P < 0.05). A PG synthesis inhibitor (indomethacin) reduced cell viability in PGE2- and PGF2alpha-treated cells (P < 0.05). A specific inhibitor of cyclooxygenase (PTGS), PTGS2 (NS-398), also reduced cell viability, whereas an inhibitor of PTGS1 (FR122047) did not affect it. The overall results suggest that PGE2 and PGF2alpha locally play luteoprotective roles in bovine CL by suppressing apoptosis of LSCs.     

In-vitro and in-vivo responsiveness of the corpus luteum of the mare to gonadotrophin stimulation

Dispersed horse luteal cells were used to evaluate the ability of horse LH, hCG and PMSG to stimulate progesterone secretion in vitro. Morphological characterization of these cells before gonadotrophin stimulation indicated the presence of two populations of cells based on cell diameters. In luteal cells incubated as suspended cells, horse LH and hCG stimulated (P less than or equal to 0.05) progesterone production at all levels of treatment. Stimulation of progesterone secretion by hCG was greater (P less than or equal to 0.05) than by horse LH over the range of concentrations utilized. When mares (N = 7) received an intramuscular injection of 1000 i.u. hCG on Days 3, 4 and 5 after the end of oestrus, there was an increase (P less than or equal to 0.05), in peripheral progesterone concentrations beginning on Day 7 and continuing until Day 14 compared with controls (N = 7). Peripheral progesterone concentrations continued to be elevated in hCG-treated mares for Days 15-30 after oestrus in those mares that conceived. Although treatment with hCG increased progesterone concentrations, it had no influence on anterior pituitary release of LH as measured by frequency and amplitude of LH discharge. We conclude that the mare corpus luteum is responsive to gonadotrophins in vitro and that exogenous hCG can enhance serum progesterone concentrations throughout the oestrous cycle and early pregnancy.     

Effects of PGF2alpha and PGF2alpha, 1-15 lactone on the corpus luteum and on early pregnancy in the rhesus monkey.

The effects of prostaglandin (PG)F2alpha and PGF2alpha, 1-15 lactone were compared in luteal phase, non-pregnant and in early pregnant rhesus monkeys. Animals treated with either PG after pretreatment with human chorionic gonadotropin (hCG) had peripheral plasma progesterone concentrations that were not statistically different from those in animals treated with hCG and vehicle. However, menstrual cycle lengths in monkeys treated with PGF2alpha, 1-15 lactone were significantly (P less than 0.02) shorter than those in vehicle treated animals. In the absence of hCG pretreatment, plasma progesterone concentrations were significantly (P less than 0.008) lower by the second day after the initial treatment with either PGF2alpha or PGF2alpha, 1-15 lactone than in vehicle treated monkeys. Menstrual cycle lengths in monkeys treated with either PG were significantly (P less than 0.04) shorter than those in animals treated with vehicle. There were no changes in plasma progesterone concentrations in early pregnant monkeys treated with PGF2alpha, and pregnancy was not interrupted. In contrast, plasma progesterone declined and pregnancy was terminated in 5 of 6 early pregnant monkeys treated with PGF2alpha, 1-15 lactone. These data indicate that PGF2alpha, 1-15 lactone decreases menstrual cycle lengths in non-pregnant rhesus monkeys. More importantly, PGF2alpha, 1-15 lactone terminates early pregnancy in the monkey at a dose which is less than an ineffective dose of PGF2alpha.     

Effect of prostaglandin F2 alpha (PGF2 alpha) on sources of progesterone and pregnancy in intact, ovariectomized and hysterectomized 90-100 day pregnant ewes.

A single dose of 8 or 16 mg of PGF2 alpha per 58 kg body weight was injected intramuscular into intact, ovariectomized or hysterectomized 90-100 day pregnant sheep in three separate experiments. Both doses of PGF2 alpha decreased the weights of the corpora lutea (P less than or equal to 0.05) and the concentration of progesterone in ovarian venous plasma at 72 hr (P less than or equal to 0.05) compared to the 0 hr sample within treatment groups and to control ewes at 72 hr in intact and hysterectomized pregnant ewes. In hysterectomized pregnant ewes, progesterone in jugular plasma declined (P less than or equal to 0.05) from 0 to 72 hr but never fell below 4 mg/ml and this decrease in progesterone after 8 or 16 mg PGF2 alpha was greater than in control hysterectomized ewes (P less than or equal to 0.05). There was a significant decrease in progesterone over time in jugular or uterine venous plasma in the presence of absence of the ovaries in 90-100 day pregnant ewes (P less than or equal to 0.05) but the profiles of progesterone were not different between vehicle and PGF2 alpha-treated ewes (P greater than or equal to 0.05). Uterine venous progesterone never declined below 30 ng/ml in the presence or absence of the ovaries and there was a significant quadratic increase (P less than or equal to 0.05) in uterine venous progesterone toward the end of the 72 hr sampling period indicating an increase in steroidogenic activity of the placenta. PGF2 alpha did not affect the number of abortions in intact or ovariectomized pregnant ewes (P greater than 0.05). Thus, the corpus luteum of sheep at 90-100 days of pregnancy is functional and responsive to PGF2 alpha, placentomes are functional but do not appear to be responsive to the doses of PGF2 alpha tested and PGF2 alpha was not an abortifacient over the 72 hr treatment period.     

Progesterone and prostaglandin production by primate luteal cells collected at various stages of the luteal phase: modulation by calcium ionophore

Prostaglandins (PG) are produced by the corpus luteum (CL) of the rhesus monkey and may be involved in luteal regulation. Intracellular calcium has also been implicated as a mediator of luteolysis in domestic and laboratory species; however, its role in primate luteal function has not been investigated. The objectives of this study were to characterize temporal changes in basal and stimulated luteal PG production by CL of rhesus monkeys, and to examine the effects of calcium ionophore (CaI) on basal and gonadotropin-stimulated progesterone (P) production by the CL. CL were collected at various times after the estimated day of the luteinizing hormone (LH) surge: 5 days (early luteal phase, n = 4), 8-10 days (mid-luteal phase, n = 8), and 12-14 days (late luteal phase, n = 5). Dispersed luteal cells were incubated in the absence and presence of CaI, or with human chorionic gonadotropin (hCG) plus CaI at 37 degrees C for 8 h. PG and P concentrations in the medium were measured by radioimmunoassay. PGE2 and 6-keto-PGF1 alpha production decreased (p less than 0.05) from early luteal phase to mid-luteal phase and remained lower (p less than 0.05) during late luteal phase for all treatment groups. PGF2 alpha production decreased (p less than 0.05) from early to mid-luteal phase and rebounded in late luteal phase to the same level (p greater than 0.05) found in early luteal phase. CaI stimulated (p less than 0.05) basal PG production. The degree of stimulation was similar throughout the luteal phase (p greater than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism whereby nitric oxide (NO) infused chronically intrauterine in ewes is antiluteolytic rather than being luteolytic

Nitric oxide (NO) has been reported to be luteolytic in vitro and in vivo in cows. However, an NO donor reversed PGF2alpha-induced inhibition of rat luteal progesterone secretion in vitro and an NO donor or endothelin-1 stimulated bovine luteal tissue secretion of prostaglandins E (PGE; PGE1, PGE2) in vitro without affecting progesterone or PGF2alpha secretion. In addition, chronic infusion of an NO donor into the interstitial tissue of the ovarian vascular pedicle adjacent the luteal-containing ovary prevented the decline in circulating progesterone, while a nitric oxide synthase (NOS) inhibitor did not affect luteolysis. The objective of this experiment was to determine whether an NO donor or NOS inhibitor infused chronically intrauterine adjacent to the luteal-containing ovary during the ovine estrous cycle was luteolytic or antiluteolytic. Ewes were treated either with vehicle (N=5), diethylenetriamine (DETA-control for DETANONOate; N=5), (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETANONOate-long acting NO donor; N=6), l-arginine (N=5), l-nitro-arginine methyl ester (l-NAME-NOS inhibitor; N=6), or NG-monomethyl-l-arginine acetate (l-NMMA; NOS inhibitor; N=5) every 6h from 2400h (0h) on day 8 through 1800h on day 18 of the estrous cycle. Jugular venous blood and inferior vena cava plasma via a saphenous vein cathether 5cm anterior to the juncture of the ovarian vein and inferior vena cava were collected every 6h for analysis for progesterone and PGF2alpha and PGE, respectively, by RIA. Corpora lutea were collected at 1800h on day 18 and weighed. Weights of corpora lutea were heavier (P< or =0.05) in DETANONOate-treated ewes when compared to vehicle, DETA, l-arginine, l-NAME, or l-NMMA-treated ewes, l-arginine luteal weights were heavier than vehicle, DETA, l-arginine, l-NAME, or l-NMMA-treated ewes, and luteal weights of vehicle, DETA, l-NAME, or l-NMMA-treated ewes did not differ amongst each other (P> or =0.05). Profiles of progesterone in jugular venous blood on days 8-18 differed (P< or =0.05) in DETANONOate-treated ewes when compared to vehicle, DETA, l-arginine, l-NMMA or l-NAME-treated ewes, which did not differ (P> or =0.05) amongst each other. The PGE:PGF2alpha ratio profile in inferior vena cava plasma of DETANONOate-treated ewes was increased (P< or =0.05) when compared to all other treatment groups. In a second experiment, conversion of [3H PGE2] to [3H PGF2alpha] by day 15 ovine caruncular endometrium in vitro was determined in vehicle, DETA, or DETANONOate-treatment groups. Conversion of [3H PGE2] to [3H PGF2alpha] was decreased (P< or =0.05) only by DETANONOate. It is concluded that NO is not luteolytic during the ovine estrous cycle, but may instead be antiluteolytic and prevent luteolysis by altering the PGE:PGF2alpha ratio secreted by the uterus.