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.     

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.     

Effects of endocannabinoid 1 and 2 (CB1; CB2) receptor agonists on luteal weight, circulating progesterone, luteal mRNA for luteinizing hormone (LH) receptors, and luteal unoccupied and occupied receptors for LH in vivo in ewes

Thirty to forty percent of ruminant pregnancies are lost during the first third of gestation due to inadequate progesterone secretion. During the estrous cycle, luteinizing hormone (LH) regulates progesterone secretion by small luteal cells (SLC). Loss of luteal progesterone secretion during the estrous cycle is increased via uterine secretion of prostaglandin F(2α) (PGF(2α)) starting on days 12-13 post-estrus in ewes with up to 4-6 pulses per day. Prostaglandin F(2α) is synthesized from arachidonic acid, which is released from phospholipids by phospholipase A2. Endocannabinoids are also derived from phospholipids and are associated with infertility. Endocannabinoid-induced infertility has been postulated to occur primarily via negative effects on implantation. Cannabinoid (CB) type 1 (CB1) or type 2 (CB2) receptor agonists and an inhibitor of the enzyme fatty acid amide hydrolase, which catabolizes endocannabinoids, decreased luteal progesterone, prostaglandin E (PGE), and prostaglandin F(2α) (PGF(2α)) secretion by the bovine corpus luteum in vitro by 30 percent. The objective of the experiment described herein was to determine whether CB1 or CB2 receptor agonists given in vivo affect circulating progesterone, luteal weights, luteal mRNA for LH receptors, and luteal occupied and unoccupied LH receptors during the estrous cycle of ewes. Treatments were: Vehicle, Methanandamide (CB1 agonist; METH), or 1-(4-chlorobenzoyl)-5-methoxy-1H-indole-3-acetic acid morpholineamide (CB2 agonist; IMMA). Ewes received randomized treatments on day 10 post-estrus. A single treatment (500 μg; N=5/treatment group) in a volume of 1 ml was given into the interstitial tissue of the ovarian vascular pedicle adjacent to the luteal-containing ovary. Jugular venous blood was collected at 0 h and every 6-48 h for the analysis of progesterone by radioimmunoassay (RIA). Corpora lutea were collected at 48 h, weighed, bisected, and frozen in liquid nitrogen until analysis of unoccupied and occupied LH receptors and mRNA for LH receptors. Profiles of jugular venous progesterone, luteal weights, luteal mRNA for LH receptors, and luteal occupied and unoccupied LH receptors were decreased (P≤0.05) by CB1 or CB2 receptor agonists when compared to Vehicle controls. Progesterone in 80 percent of CB1 or CB2 receptor agonist-treated ewes was decreased (P≤0.05) below 1 ng/ml by 48 h post-treatment. It is concluded that the stimulation of either CB1 or CB2 receptors in vivo affected negatively luteal progesterone secretion by decreasing luteal mRNA for LH receptors and also decreasing occupied and unoccupied receptors for LH on luteal membranes. The corpus luteum may be an important site for endocannabinoids to decrease fertility as well as negatively affect implantation, since progesterone is required for implantation.     

Effect of the aromatase inhibitor CGS-16949A on pregnancy and secretion of progesterone, estradiol-17beta, prostaglandins E and F2alpha (PGE; PGF2alpha) and pregnancy specific protein B (PSPB) in 90-day ovariectomized pregnant ewes

The aromatase inhibitor CGS-16949A was used to determine whether CGS-16949A altered secretion of progesterone, estradiol-17beta, PGE (PGE1 + PGE2), PGF2alpha and PSPB. Ninety day pregnant ewes were ovariectomized and received vehicle, PGF2alpha, CGS-16949A or PGF2alpha+CGS-16949A. None of the ewes treated with PGF2alpha, CGS-16949A or PGF2alpha+CGS-16949A aborted (P > or = 0.05) during the 108-h experimental period. Treatment with CGS-16949A lowered (P < or = 0.05) progesterone in jugular venous plasma but concentrations of progesterone were not affected (P > or = 0.05) by treatment with PGF2alpha. Concentrations of estradiol-17beta and PSPB in jugular venous plasma and PGE in inferior vena cava plasma were decreased (P < or = 0.05) by treatment with CGS-16949A. Concentrations of PGF2alpha in inferior vena cava plasma were not affected (P > or = 0.05) by treatment with CGS-16949A. Decreases in estradiol-17beta occurred before decreases in PSPB, which was then followed by decreases in PGE (P < or = 0.05). It is concluded that these data support the hypothesis that estradiol-17beta regulates placental secretion of PSPB; PSPB regulates placental secretion of PGE; and PGE regulates placental secretion of progesterone during mid-pregnancy in ewes.     

What regulates placental steroidogenesis in 90-day pregnant ewes?

By day-90, the placenta secretes half of the circulating progesterone and 85% of the circulating estradiol-17beta [Weems YS, Vincent D, Tanaka Y, et al. Effects of prostaglandin F(2alpha) on sources of progesterone and pregnancy in intact, ovariectomized, and hysterectomized 90-100 day pregnant ewes. Prostaglandins 1992;43:203-22; Weems YS, Vincent DL, Nusser K, et al. Effects of prostaglandin F(2alpha) (PGF(2alpha)) on secretion of estradiol-17beta and cortisol in 90-100 day hysterectomized, intact, or ovariectomized pregnant ewes. Prostaglandins 1994;48:139-57]. Ovariectomy (OVX) or prostaglandin (PG) F(2alpha) (PGF(2alpha)) does not abort intact or OVX 90-day pregnant ewes and PGF(2alpha) regresses the corpus luteum, but does not affect placental progesterone secretion in vivo [Weems YS, Vincent D, Tanaka Y, et al. Effects of prostaglandin F(2alpha) on sources of progesterone and pregnancy in intact, ovariectomized, and hysterectomized 90-100 day pregnant ewes. Prostaglandins 1992;43:203-22]. Luteal progesterone secretion in vitro at day-90 of pregnancy in ewes is regulated by PGE(1)and/or PGE(2), not by ovine luteinizing hormone (LH; 3). Concentrations of PGE in uterine or ovarian venous plasma averaged 6 ng/ml at 90-100 days of pregnancy in ewes [Weems YS, Vincent DL, Tanaka Y, Nusser K, Ledgerwood KS, Weems CW. Effect of prostaglandin F(2alpha) on uterine or ovarian secretion of prostaglandins E and F(2alpha) (PGE; PGF(2alpha)) in vivo in 90-100 day hysterectomized, intact or ovariectomized pregnant ewes. Prostaglandins. 1993;46:277-96]. Ovine placental PGE secretion is regulated by LH up to day-50 and by pregnancy specific protein B (PSPB) after day-50 of pregnancy [Weems YS, Kim L, Humphreys V, Tsuda V, Weems CW. Effect of luteinizing hormone (LH), pregnancy specific protein B (PSPB), or arachidonic acid (AA) on ovine endometrium of the estrous cycle or placental secretion of prostaglandins E(2) (PGE(2)) and F(2alpha) (PGF(2alpha)), and progesterone in vitro. Prostaglandins Other Lipid Mediators 2003;71:55-73]. Indomethacin (INDO), a prostaglandin synthesis inhibitor [Lands WEM. The biosynthesis and metabolism of prostaglandins. Annu Rev Physiol 1979;41:633-46], lowers jugular venous progesterone [Bridges PJ, Weems YS, Kim L, et al. Effect of prostaglandin F(2alpha) (PGF(2alpha)), indomethacin, tamoxifen or estradiol-17beta on pregnancy, progesterone and pregnancy specific protein B (PSPB) secretion in 88-90 day pregnant ewes. Prostaglandins Other Lipid Mediators 1999;58:113-24] and inferior vena cava PGE of pregnant ewes with ovaries by half at day-90 [Bridges PJ, Weems YS, Kim L, LeaMaster BR, Vincent DL, Weems CW. Effect of prostaglandin F(2alpha) (PGF(2alpha)), indomethacin, tamoxifen or estradiol-17beta on prostaglandin E (PGE), PGF(2alpha) and estradiol-17beta secretion in 88-90 day pregnant sheep. Prostaglandins Other Lipid Mediators 1999;58:167-78]. In addition, treatment of 90 day ovine diced placental slices with androstenedione in vitro increased placental estradiol-17beta, but treatment with PGF(2alpha)in vitro did not decrease placental progesterone secretion, which indicates that ovine placenta progesterone secretion is resistant to the luteolytic action of PGF(2alpha) [Weems YS, Bridges PJ, LeaMaster BR, Sasser RG, Vincent DL, Weems CW. Secretion of progesterone, estradiol-17beta, prostaglandins (PG) E (PGE), F(2alpha) (PGF(2alpha)), and pregnancy specific protein B (PSPB) by day 90 intact or ovariectomized pregnant ewes. Prostaglandins Other Lipid Mediators 1999;58:139-48]. This also explains why ovine uterine secretion of decreased around day-50 [Weems YS, Kim L, Humphreys V, Tsuda V, Weems CW. Effect of luteinizing hormone (LH), pregnancy specific protein B (PSPB), or arachidonic acid (AA) on ovine endometrium of the estrous cycle or placental secretion of prostaglandins E(2) (PGE(2)) and F(2alpha) (PGF(2alpha)), and progesterone in vitro. Prostaglandins Other Lipid Mediators 2003;71:55-73], when placental estradiol-17beta secretion is increasing [Weems C, Weems Y, Vincent D. Maternal recognition of pregnancy and maintenance of gestation in sheep. In: Reproduction and animal breeding: advances and strategies. Enne G, Greppi G, Lauria A, editors, Elsevier Pub., Amsterdam 1995. p. 277-93]. Treatment of 90 day pregnant ewes with estradiol-17beta+ PGF(2alpha), but not either treatment alone, caused a linear increase in both estradiol-17beta and PGF(2alpha) and ewes were aborting [Bridges PJ, Weems YS, Kim L, Sasser RG, LeaMaster BR, Vincent DL, Weems CW. Effect of prostaglandin F(2alpha) (PGF(2alpha)), indomethacin, tamoxifen or estradiol-17beta on pregnancy, progesterone and pregnancy specific protein B (PSPB) secretion in 88-90 day pregnant ewes. Prostaglandins Other Lipid Mediators 1999;58:113-24; Bridges PJ, Weems YS, Kim L, LeaMaster BR, Vincent DL, Weems CW. Effect of prostaglandin F(2alpha) (PGF(2alpha)), indomethacin, tamoxifen or estradiol-17beta on prostaglandin E (PGE), PGF(2alpha) and estradiol-17beta secretion in 88-90 day pregnant sheep. Prostaglandins Other Lipid Mediators 1999;58:167-78]. Pregnant ewes OVX on day 83 of pregnancy and placental slices cultured in vitro secretes 2-3-fold more estradiol-17beta, PSPB, PGE, and progesterone than placental slices from 90 day intact pregnant ewes, but placental PGF(2alpha) secretion by placental slices from intact or OVX ewes did not change [Denamur R, Kann G, Short R V. How does the corpus luteum of the sheep know that there is an embryo in the uterus? In: Pierrepont G, editor. Endocrinology of pregnancy and parturition, vol. 2. Cardiff, Wales, UK: Alpha Omega Pub Co.; 1973. p. 4-38]. The objective of these experiments was to determine what regulates ovine placental progesterone and estradiol-17beta secretion at day-90 of pregnancy, since the hypophysis [Casida LE, Warwick J. The necessity of the corpus luteum for maintenance of pregnancy in the ewe. J Anim Sci 1945;4:34-9] or ovaries [Weems CW, Weems YS, Randel RD. Prostaglandins and reproduction in female farm animals. Vet J 2006;171:206-28] are not necessary after day-55 to maintain pregnancy. In Experiment 1, diced placental slices from day-90 intact or OVX pregnant ewes that were ovariectomized or laparotomized and ovaries were not removed on day 83 were collected on day-90 and incubated in vitro in M-199 with Vehicle, ovine luteinizing hormone (oLH), ovine follicle stimulating hormone (oFSH), ovine placental lactogen (oPL), PGE(l), PGE(2), PGD(2), PGI(2), insulin-like growth factor (IGF) 1 or 2 (IGF(l); IGF(2)), leukotriene C(4) (LTC(4)), platelet activating factor (PAF) 16 or 18 (PAF-16; PAF-18) at doses of 0, 1, 10, or 100ng/ml for 4h. In Experiment 2, placental slices from day-90 intact and OVX (intact or OVX laporotomized 7 days earlier) pregnant ewes were incubated in vitro with vehicle, INDO, Meclofenamate (MECLO), PGE(l), PGE(2), INDO+PGE(1), MECLO+PGE(l), INDO+PGE(2), or MECLO+PGE(2) for 4h. Media were analyzed for progesterone, estradiol-17beta, PGE, or PGF(2alpha) by RIA. Hormone data in media were analyzed in Experiment 1 by a 2x3x13 and in Experiment 2 by a 2x9 Factorial Design for ANOVA. In Experiment 1, placental progesterone, PGE, or estradiol-17beta secretion were increased (P< or =0.05) two-fold by OVX. Progesterone was not increased (P> or =0.05) by any treatment other than OVX and only FSH increased (P< or =0.05) estradiol-17beta secretion by placental slices in both OVX and intact ewes 90-day pregnant ewes. In Experiment 2, INDO or MECLO decreased (P< or =0.05) placental progesterone secretion by 88% but did not decrease (P> or =0.05) placental estradiol-17beta secretion from intact or OVX ewes. PGE(l) or PGE(2) increased (P< or =0.05) progesterone secretion only in ewes treated with INDO or MECLO. It is concluded that FSH probably regulates day-90 ovine placental estradiol-17beta secretion, while PGE(l) or PGE(2) regulates day-90 placental progesterone secretion.     

Effect of mifepristone on pregnancy,pregnancy-specific protein B (PSPB), progesterone,estradiol-17beta,prostaglandin F2alpha (PGF2alpha) and prostaglandin E (PGE) in ovariectomized 90-day pregnant ewes

The objective of this experiment was to determine the effect of mifepristone, a progesterone receptor antagonist, on pregnancy and secretion of steroids, pregnancy-specific protein B (PSPB) and prostaglandins at mid-pregnancy in ewes. Ninety-day pregnant ewes were ovariectomized (OVX) and treatments were initiated 72 h post-OVX. Ewes received (1) vehicle, (2) prostaglandin F2alpha (PGF2alpha, 8 mg/58 kg/bw, i.m.) 84 h post-OVX, (3) mifepristone (50 mg intrajugular at 72, 84, 96, and 108 h post-OVX), (4) mifepristone (50mg) + PGF2alpha, (5) mifepristone (100 mg intrajugular at 72, 84, 96, and 108 h), and (6) mifepristone (100 mg) + PGF2alpha. Ewes treated with vehicle or PGF2alpha alone did not abort (P > or = 0.05). But, 60, 80, 60, and 100% of ewes treated with mifepristone (50 mg), mifepristone (50 mg) + PGF2alpha, mifepristone (100 mg), and mifepristone (100 mg) + PGF2alpha, respectively, aborted (P < or = 0.05). Profiles of progesterone, estradiol-17beta, prostaglandin E (PGE), or PSPB did not differ (P > or = 0.05) among treatment groups. Profiles of PGF2alpha of treatment groups receiving mifepristone with or without PGF2alpha differed (P < 0.05) from vehicle or PGF2alpha alone-treated ewes. It is concluded that progesterone actions are necessary to suppress uterine/placental secretion of PGF2alpha and that maintenance of critical progesterone: estradiol-17beta and PGE:PGF2alpha ratios are necessary for maintenance of pregnancy.     

Differential effects of progesterone treatment on the oxytocin-induced prostaglandin F2 alpha response and the levels of endometrial oxytocin receptors in ovariectomized ewes.

The oxytocin-induced uterine prostaglandin (PG) F2 alpha response and the levels of endometrial oxytocin receptors were measured in ovariectomized ewes after they had been given steroid pretreatment (SP) with progesterone and estrogen to induce estrus (day of expected estrus = Day 0) and had subsequently been treated with progesterone over Days 1-12 and/or PGF2 alpha over Days 10-12 postestrus. The uterine PGF2 alpha response was measured after an i.v. injection of 10 IU oxytocin on Days 13 and 14, using the PGF2 alpha metabolite, 13,14-dihydro-15-keto-PGF2 alpha (PGFM), as an indicator for PGF2 alpha release. The levels of oxytocin receptors in the endometrium were measured on Day 14. During the treatment with progesterone, the peripheral progesterone concentrations were elevated and remained above 1.8 ng/ml until the morning of Day 14. The PGFM responses to oxytocin in untreated controls and SP controls were low on both Days 13 and 14 whereas the levels of endometrial oxytocin receptors in the same ewes were high. Treatment with progesterone either alone or in combination with PGF2 alpha significantly (p less than 0.04) increased the PGFM response on Day 14 and reduced the levels of endometrial oxytocin receptors; treatment with PGF2 alpha alone had no effect. It is concluded that progesterone promotes the PGFM response to oxytocin while simultaneously suppressing the levels of endometrial oxytocin receptors. PGF2 alpha treatment had no effect on either the uterine secretory response to oxytocin or the levels of oxytocin receptors in the endometrium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of progesterone administered to dairy heifers on sensitivity of corpora lutea to PGF(2)alpha and on plasma LH concentration

To determine whether progesterone facilitates PGF(2)alpha-induced luteolysis prior to day 5 of the estrous cycle, 48 Holstein-Friestian heifers were assigned at random to four treatments: 1) 4 ml corn oil/day + 5 ml Tris-HCl buffer (control); 2) 25 mg prostaglandin F(2)alpha (PGF(2)alpha); 3) 100 mg progesterone/day (progesterone); 4) 100 mg progesterone/day + 25 mg PGF(2)alpha (combined treatment). Progesterone was injected subcutaneously daily from estrus (day 0) through day 3. The PGF(2)alpha was injected intramuscularly on day 3. Estrous cycle lengths were decreased by progesterone: 20.2 +/- 0.56, 19.2 +/- 0.31 (control and PGF(2)alpha); 13.2 +/- 1.40, and 11.7 +/- 1.27 (progesterone and combined). The combination of progesterone and PGF(2)alpha did not shorten the cycle any more than did progesterone alone (interaction, P>0.05). PGF(2)alpha treatment reduced progesterone concentrations on day 6 (P<0.05) and both progesterone and PGF(2)alpha reduced plasma progesterone on day 8 (P<0.01 and P<0.05, respectively). LH was measured in blood samples collected at 10- min intervals for 4 hr on day 4 from three heifers selected at random from each of the four treatment groups. Mean LH concentration for control heifers ranged from 0.35 to 0.63 ng/ml (overall mean, 0.49 ng/ml) and for progesterone-treated heifers ranged from 0.12 to 0.30 ng/ml (overall mean, 0.23 ng/ml). LH concentrations were greater in control heifers (P<0.01). The mean LH pulse rate for control heifers was 2.7 pulses/heifers/4 hr, while that for the progesterone-treated heifers was 1.7 pulses/heifer/4 hr. The mean pulse amplitude for control and progesterone treatments was 0.47 ng/ml and 0.36 ng/ml, respectively. Neither pulse amplitude nor frequency were different between treatment groups.     

Role of progesterone in regulating uteroovarian venous concentrations of PGF2 alpha and PGE2 during the estrous cycle and early pregnancy in ewes

The role of progesterone in regulation of uteroovarian venous concentrations of prostaglandins F2 alpha(PGF2 alpha) and E2 (PGE2) during days 13 to 16 of the ovine estrous cycle or early pregnancy was examined. At estrus, ewes were either mated to a fertile ram or unmated. On day 12 postestrus, ewes were laparotomized and a catheter was inserted into a uteroovarian vein. Six mated and 7 unmated ewes received no further treatment. Fifteen mated and 13 unmated ewes were ovariectomized on day 12 and of these, 7 mated and 5 unmated ewes were given 10 mg progesterone sc and an intravaginal pessary containing 30 mg of progesterone. Uteroovarian venous samples were collected every 15 min for 3 h on days 13 to 16 postestrus. Mating resulted in higher mean daily concentrations of PGE2 in the uteroovarian vein than in unmated ewes. Ovariectomy prevented the rise in PGE2 with day in mated ewes but had no effect in unmated ewes. Progesterone treatment restored PGE2 in ovariectomized, mated ewes with intact embryos. Mating had no effect on mean daily concentrations of PGE2 alpha or the patterns of the natural logarithm (1n) of the variance of PGF2 alpha. Ovariectomy resulted in higher mean concentrations and 1n variances of PGF2 alpha on day 13 and lower mean concentrations and 1n variances of PGF2 alpha on days 15 and 16. Replacement with progesterone prevented these changes in patterns of mean concentrations and 1n variances of PGF2 alpha following ovariectomy. It is concluded that progesterone regulates the release of PGF2 alpha from the uterus, maintaining high concentrations while also preventing the occurrence of the final peaks of PGF2 alpha which are seen with falling concentrations of progesterone. This occurs in both pregnant and non-pregnant ewes. Progesterone is also needed to maintain increasing concentrations of PGE2 in mated ewes.     

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.     

Increases in the oxytocin-induced prostaglandin F2 alpha response and reduction in the concentrations of endometrial oxytocin receptors in ewes in response to progesterone.

Ovariectomized ewes were given progesterone and oestrogen priming as steroid pretreatment and subsequently treated with progesterone, prostaglandin F2 alpha (PGF2 alpha), or both. In Expt 1, plasma concentrations of the metabolite 13,14-dihydro-15-keto-PGF2 alpha (PGFM) were measured after an i.v. injection of oxytocin. There was little PGFM response in the untreated control ewes or in the pretreated ewes. Treatment with PGF2 alpha alone had no effect (P greater than 0.05), whereas treatment with progesterone either alone or with PGF2 alpha significantly (P less than 0.05) increased the uterine PGFM response to oxytocin. In Expt 2, chronically ovariectomized ewes had high concentrations of endometrial oxytocin receptors. Treatment with PGF2 alpha alone did not alter the concentrations of the receptors. Treatment with progesterone either alone or with PGF2 alpha significantly (P less than 0.05) reduced the concentrations of the receptors. It is concluded that progesterone promotes the PGFM response to oxytocin, but simultaneously suppresses the concentrations of endometrial oxytocin receptors.     

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.     

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)     

Effect of exogenous melatonin on in vivo and in vitro prostaglandin secretion in Rasa Aragonesa ewes

The effect of exogenous melatonin on prostaglandin secretion was measured on Rasa Aragonesa ewes. Fourteen ewes received an 18 mg melatonin implant (M+) on 10 April and were compared with 13 control animals (without implants M-). Twenty days later, intravaginal pessaries were inserted in all animals to induce a synchronized oestrus (day 0). On day 14, ewes were injected, i.v., with 0.5 IU oxytocin. Plasma 15-ketodihydro-PGF(2alpha) (PGFM) concentrations were measured to assess uterine secretory responsiveness to oxytocin. After euthanasia, pieces of endometrium were collected to determine progesterone content and PGE(2) and PGF(2alpha) secretion in vitro, in the presence or absence of either 20 microg/ml recombinant ovine interferon-tau (roIFNt) or 1 nmol/l oxytocin in the medium. Endometrial progesterone content was similar in the two treatments (M+: 50.25+/-17.34 ng/mg tissue, M-: 43.08+/-11.21 ng/mg tissue). M+ ewes that responded to oxytocin had significantly higher plasma PGFM concentrations between 10 and 80 min after oxytocin administration, a higher mean PGFM peak (P<0.001), higher plasma PGFM levels after the challenge (P<0.05) and higher plasma progesterone concentrations (P<0.01) than control ewes. In the in vitro experiment, M+ and M- control samples secreted similar amounts of PGE(2). The presence of roIFNtau and oxytocin only stimulated PGE(2) production (P<0.05) in M- tissues. Control M+ tissues secreted higher amounts of PGF(2alpha) (P=0.07) and PGF(2alpha) secretion was significantly (P<0.01) stimulated by roIFNtau. Oxytocin produced this effect only in M- samples (P<0.01). In conclusion, although previous studies have demonstrated a positive effect of melatonin on lamb production, PGF(2alpha) secretion is higher in vitro and the PGE(2):PGF(2alpha) ratio is unfavourable in response to IFNtau, which could affect embryo survival. Whether or not these mechanisms are similar in pregnant ewes remains to be elucidated.     

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.     

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.     

Prostaglandin E1 or E2 (PGE1, PGE2) prevents premature luteolysis induced by progesterone given early in the estrous cycle in ewes

The objective of this study was to determine whether prostaglandin E₁ (PGE₁) or prostaglandin E₂ (PGE₂) prevents premature luteolysis in ewes when progesterone is given during the first 6 days of the estrous cycle. Progesterone (3 mg in oil, im) given twice daily from Days 1 to 6 (estrus = Day 0) in ewes decreased (P < 0.05) luteal weights on Day 10 postestrus. Plasma progesterone concentrations differed (P < 0.05) among the treatment groups; toward the end of the experimental period, concentrations in jugular venous blood decreased (P < 0.05) compared with the other treatment groups. Plasma progesterone concentrations in ewes receiving PGE₁ or PGE₁ + progesterone were greater (P < 0.05) than in vehicle controls or in ewes receiving PGE₂ or PGE₂ or PGE₂ + progesterone. Chronic intrauterine treatment with PGE₁ or PGE₂ prevented (P < 0.05) decreases in plasma progesterone concentrations, luteal weights, and the proportion of luteal unoccupied and occupied LH receptors on Day 10 postestrus in ewes given exogenous progesterone, but did not affect (P > 0.05) concentrations of PGF_2α in inferior vena cava blood. Progesterone given on Days 1 to 6 in ewes advanced (P < 0.05) increases in PGF_2α in inferior vena cava blood. We concluded that PGE₁ or PGE₂ prevented progesterone-induced premature luteolysis by suppressing loss of luteal LH receptors (both unoccupied and occupied).     

In vivo intra-luteal implants of prostaglandin (PG) E(1) or E(2) (PGE(1), PGE(2)) prevent luteolysis in cows. I. Luteal weight, circulating progesterone, mRNA for luteal luteinizing hormone (LH) receptor, and occupied and unoccupied luteal receptors for LH

Previously, it was reported that chronic intra-uterine infusion of PGE(1) or PGE(2) every four hours inhibited luteolysis in ewes. However, estradiol-17β or PGE(2) given intra-uterine every 8h did not inhibit luteolysis in heifers, but infusion of estradiol+PGE(2) inhibited luteolysis in heifers. The objective of this experiment was to determine whether and how intra-luteal implants containing PGE(1) or PGE(2) prevent luteolysis in Angus or Brahman cows. On day-13 post-estrus, Angus cows received no intra-luteal implant and corpora lutea were retrieved or Angus and Brahman cows received intra-luteal silastic implants containing Vehicle, PGE(1), or PGE(2) and corpora lutea were retrieved on day-19. Coccygeal blood was collected daily for analysis for progesterone. Breed did not influence the effect of PGE(1) or PGE(2) on luteal mRNA for LH receptors or unoccupied or occupied luteal LH receptors did not differ (P>0.05) so the data were pooled. Luteal weights of Vehicle-treated Angus or Brahman cows from days-13-19 were lower (P<0.05) than those treated with intra-luteal implants containing PGE(1) or PGE(2). Day-13 Angus luteal weights were heavier (P<0.05) than Vehicle-treated Angus cows on day-19 and luteal weights of day-13 corpora lutea were similar (P>0.05) to Angus cows on day-19 treated with intra-luteal implants containing PGE(1) or PGE(2). Profiles of circulating progesterone in Angus or Brahman cows treated with intra-luteal implants containing PGE(1) or PGE(2) differed (P<0.05) from controls, but profiles of progesterone did not differ (P>0.05) between breeds or between cows treated with intra-luteal implants containing PGE(1) or PGE(2). Intra-luteal implants containing PGE(1) or PGE(2) prevented (P<0.05) loss of luteal mRNA for LH receptors and unoccupied or occupied receptors for LH compared to controls. It is concluded that PGE(1) or PGE(2) alone delays luteolysis regardless of breed. We also conclude that either PGE(1) or PGE(2) prevented luteolysis in cows by up-regulating expression of mRNA for LH receptors and by preventing loss of unoccupied and occupied LH receptors in luteal tissue.     

Effect of prostaglandin F2alpha (PGF2alpha), indomethacin, tamoxifen or estradiol-17beta on prostaglandin E (PGE), PGF2alpha and estradiol-17beta secretion in 88 to 90-day pregnant sheep

Treatment with PGF2alpha plus estradiol-17beta aborts 90-day pregnant ewes, whereas PGF2alpha or estradiol-17beta alone does not abort ewes. The objective of this experiment was to evaluate whether tamoxifen, an estrogen receptor antagonist, estradiol-17beta, prostaglandin F2alpha (PGF2alpha), indomethacin, or some of their interactions affected ovine uterine/placental secretion of PGF2alpha, estradiol-17beta or prostaglandins E (PGE), because a single treatment with PGF2alpha and estradiol-17beta given every 6 h aborts 90-day pregnant ewes. Concentrations of PGF2alpha in uterine venous blood were increased (P < or = 0.05) by estradiol-17beta, PGF2alpha + estradiol-17beta, and PGF2alpha + tamoxifen, and decreased (P < or = 0.05) by indomethacin or PGF2alpha + indomethacin at 72 h when compared to the 0 h samples. Concentrations of PGE in uterine venous blood were decreased (P < or = 0.05) by indomethacin and PGF2alpha + indomethacin and increased (P < or = 0.05) by PGF2alpha + estradiol-17beta at 72 h when compared to the 0 h samples. Concentrations of PGF2alpha in inferior vena cava blood at 6 h were increased (P < or = 0.05) by PGF2alpha either alone or in combination with indomethacin, tamoxifen, or estradiol-17beta, which is due to the PGF2alpha injected. Concentrations of PGF2alpha in inferior vena cava blood in PGF2alpha + estradiol-17beta-treated 88- to 90-day pregnant ewes increased (P < or = 0.05) linearly over the 72-h sampling period and averaged 4.0 + 0.4 ng/ml. Concentrations of PGF2alpha in inferior vena cava blood of control, PGF2alpha, tamoxifen, PGF2alpha + indomethacin, PGF2alpha + tamoxifen, and estradiol-17beta-treated ewes did not differ (P > or = 0.05) and averaged 0.4 + 0.04 ng/ml. Profiles of PGE in inferior vena cava blood of 88- to 90-day pregnant ewes treated with vehicle, PGF2alpha, estradiol-17beta, tamoxifen, tamoxifen + PGF2alpha, or estradiol-17beta + PGF2alpha did not differ (P > or = 0.05). Concentrations of PGE in inferior vena cava blood of 88- to 90-day pregnant ewes treated with indomethacin or PGF2alpha + indomethacin were lower (P < or = 0.05) than in control ewes. Concentrations of estradiol-17beta in jugular venous plasma of PGF2alpha + estradiol-17beta-treated 88- to 90-day pregnant ewes increased linearly and differed (P < or = 0.05) from controls. Profiles of estradiol-17beta in jugular venous plasma of PGF2alpha, indomethacin, tamoxifen, and PGF2alpha + tamoxifen and PGF2alpha + indomethacin, estradiol-17beta, and controls did not differ (P > or = 0.05). It is concluded that treatment with a single injection of PGF2alpha and estradiol-17beta given every 6 h causes a linear increase in PGF2alpha and estradiol-17beta.     

Effect of luteinizing hormone (LH), pregnancy specific protein B (PSPB), or arachidonic acid (AA) on ovine endometrium of the estrous cycle or placental secretion of prostaglandins E2 (PGE2) and F2alpha (PGF2alpha) and progesterone in vitro

The objective of this experiment was to determine the effect of AA, LH, or PSPB on secretion of PGE2, PGF2alpha, or progesterone by ovine caruncular endometrium of the estrous cycle or placental tissue of pregnancy in vitro. Ovine caruncular endometrium of the estrous cycle (days 8, 11, 13, and 15) or caruncular/placental tissue on days 8, 11, 13, 15, 20, 30, 40, 50, 60, and 90 postbreeding were incubated in vitro with vehicle, AA, LH, or PSPB in M-199 for 4 and 8 h. Secretion of PGF2alpha by caruncular endometrium of non-bred ewes on days 13 and 15 and by caruncular/placental tissue of bred ewes on days 13, 15, 20, 30, and 40 was increased (P < or = 0.05) when incubated with vehicle and declined (P < or = 0.05) after day-40 in bred ewes. Secretion of PGF2alpha by day-15 caruncular endometrium of non-bred ewes and bred ewes was increased (P < or = 0.05) by AA on days 13 and 15 and by LH on day-15. Secretion of PGF2alpha by caruncular/placental tissue from bred ewes was (P < or = 0.05) by AA on days 13, 15, 20, 30, and 40 and by LH on days 15, 20, 30, and 40, after which the response decreased (P < or = 0.05). Secretion of PGF2alpha by caruncular endometrium of non-bred ewes during the estrous cycle or by caruncular/placental tissue of bred ewes during the first trimester was not affected by PSPB (P > or = 0.05). Secretion of PGE2 by caruncular endometrium of non-bred ewes did not change (P > or = 0.05) and was increased (P < or = 0.05) by caruncular/placental tissue on days 13-90 from bred ewes when incubated with vehicle. Secretion of PGE2 by endometrium from non-bred ewes was not affected (P > or = 0.05) by AA, LH, or PSPB, but was increased (P < or = 0.05) by AA or LH on days 13-50 and by PSPB on days 60 and 90 when incubated with caruncular/placental tissue from bred ewes. Secretion of progesterone by placental tissue of bred ewes increased (P < or = 0.05) on day-50 and continued to increase through day-90. In summary, uterine/placental tissue secretion of PGF2alpha is not reduced until the end of the first trimester of pregnancy in ewes. In addition, LH appears to play a role in luteolysis of non-bred ewes by stimulating caruncular endometrial secretion of PGF2alpha and on day-5 postbreeding to prevent luteolysis during early pregnancy by stimulating caruncular/placental secretion of PGE2 throughout the first trimester of pregnancy in sheep. Secretion of PGE2 by caruncular/placental tissue after day-50 of pregnancy appears to be regulated by PSPB, not LH.