synthesis of progesterone and prostaglandin F by luteal tissue and prostaglandin F by endometrial tissue from the pig

The relationship between progesterone (P₄) synthesis by luteal tissue and prostaglandin F (PGF) synthesis by endometrium and luteal tissue from two stages of the cycle, Days 7 to 8 and 15 to 16, was determined. Luteal and endometrial tissues were collected from pigs in three experimental groups at two stages of the cycle: (A) 6 pigs on Days 7 to 8 with spontaneous, 5 to 6 day old corpora lutea (CL); (B) 5 pigs on Days 15 to 16 with spontaneous, 13 to 14 day old CL; and (C) 6 pigs on Days 15 to 16 with spontaneous, 13 to 14 day old CL and 5 to 6 day old CL induced by pregnant mares serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG) injections. Pigs with spontaneous, 13 to 14 day old CL of the cycle and PMSG-HCG induced accessory, 5 to 6 day old CL were used so that P₄ and PGF synthesis in tissue from old and new CL could be compared in the same pig on Day 15 to 16 of the cycle. Tissues (100 mg minces) were incubated in 5 ml of Krebs Ringer solution in an atmosphere of 95% 0₂:5% CO₂ for 2 hours at 0° C, 37° C, or 37° C with 1.3 x 10⁻⁴M indomethacin (IND). An aliquot of the incubation medium and an aliquot of the supernatant after homogenization of the tissue in the remaining medium of each flask was quantified for P₄ and PGF by radioimmunoassay. P₄ and PGF release into the medium and total accumulation of P₄ and PGF in the flasks indicated that synthesis had occured at 37° C. Compared to tissue from 13 to 14 day old CL, tissue from 5 to 6 day old CL synthesized more P₄ per flask (53.9 25.0 ng/mg tissue, P<.001) and released more P₄ into the medium (20.8 8.8 ng/mg, P<.001). P₄ synthesis by luteal tissue from 5 to 6 day old and 13 to 14 day old CL from pigs in group C was similar to P₄ synthesis by luteal tissue from pigs in group A and group B, respectively. Luteul PGF synthesis was not affected significantly by either the age of the CL or by PMSG-HCG treatment. For endometrial samples, the synthesis of PGF was not significantly different among pigs in groups A, B and C. If uterine PGF is involved in luteal regression in the pig, the sensitivity of the CL to PGF may be more important than an increase in PGF secretion during the late luteal phase of the estrous cycle.     

Prostacyclin, prostaglandin F2 alpha and progesterone production by bovine luteal cells during the estrous cycle

Corpora lutea (CL) were collected from Holstein heifers on Days 5, 10, 15 and 18 (5/day) of the estrous cycle. Dispersed luteal cell preparations were made and 10(6) viable luteal cells were incubated with bovine luteinizing hormone (LH) and different amounts of arachidonic acid in the presence and absence of the prostaglandin (PG) synthetase inhibitor indomethacin. The concentrations of progesterone, PGF2 alpha and 6-keto-PGF1 alpha, the stable inactive metabolite of prostacyclin (PGI2), were measured. Day 5 CL had the greatest initial content of 6-keto-PGF1 alpha (1.01 +/- 0.16 ng/10(6) cells), and synthesized more 6-keto-PGF1 alpha (2.55 +/- 0.43) than CL collected on Days 10 (0.57 +/- 0.11), 15 (0.08 +/- 0.05) and 18 (0.19 +/- 0.03) during a 2-h incubation period. Arachidonic acid stimulated the production of 6-keto-PGF1 alpha by Days 10, 15 and 18 luteal tissue. PGF2 alpha was produced at a greater rate on Day 5 (0.69 +/- 0.17 ng/10(6) cells) than on Days 10 (0.06 +/- 0.01), 15 (0.04 +/- 0.02) and 18 (0.08 +/- 0.01). Arachidonic acid stimulated and indomethacin inhibited the production of PGF2 alpha, in most cases. The initial content of 6-keto-PGF1 alpha was higher than that of PGF2 alpha on all days of the cycle and more 6-keto-PGF1 alpha was synthesized in response to arachidonic acid addition. The ratio of 6-keto-PGF1 alpha content to PGF2 alpha content was 4.39, 2.30, 1.25 and 1.13 on Days 5, 10, 15 and 18, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in steady-state concentrations of messenger ribonucleic acids in luteal tissue during prostaglandin F2alpha induced luteolysis in mares

Transvaginal ultrasound-guided luteal biopsy was used to evaluate the effects of prostaglandin (PG)F2alpha on steady-state concentrations of mRNA for specific genes that may be involved in regression of the corpus luteum (CL). Eight days after ovulation (Hour 0), mares (n=8/group) were randomized into three groups: control (no treatment or biopsy), saline+biopsy (saline treatment at Hour 0 and luteal biopsy at Hour 12), or PGF2alpha+biopsy (5mg PGF2alpha at Hour 0 and luteal biopsy at Hour 12). The effects of biopsy on CL were compared between the controls (no biopsy) and saline+biopsy group. At Hour 24 (12h after biopsy) there was a decrease in circulating progesterone in saline group to 56% of pre-biopsy values, indicating an effect of biopsy on luteal function. Mean plasma progesterone concentrations were lower (P<0.001) at Hour 12 in the PG group compared to the other two groups. The relative concentrations of mRNA for different genes in luteal tissue at Hour 12 was quantified by real time PCR. Compared to saline-treated mares, treatment with PGF2alpha increased mRNA for cyclooxygenase-2 (Cox-2, 310%, P<0.006), but decreased mRNA for LH receptor to 44% (P<0.05), steroidogenic acute regulatory protein to 22% (P<0.001), and aromatase to 43% (P<0.1) of controls. There was no difference in mRNA levels for PGF2alpha receptor between PG and saline-treated groups. Results indicated that luteal biopsy alters subsequent luteal function. However, the biopsy approach was effective for collecting CL tissue for demonstrating dynamic changes in steady-state levels of mRNAs during PGF2alpha-induced luteolysis. Increased Cox-2 mRNA concentrations suggested that exogenous PGF2alpha induced the synthesis of intraluteal PGF2alpha. Thus, the findings are consistent with the concept that an intraluteal autocrine loop augments the luteolytic effect of uterine PGF2alpha in mares.     

Lipid metabolism and in vitro production of progesterone and prostaglandin F during induced regression of porcine corpora lutea

The effects of Cloprostenol administration on porcine luteal lipid and arachidonic acid accumulation were examined in relation to luteal $\text{in vitro}$ progesterone and prostaglandin F synthesis in 18 mature gilts at day 12 of the estrous cycle. Basal and net $\text{in vitro}$ release of progesterone from luteal tissue was depressed at 8 hr after treatment whereas net $\text{in vitro}$ release of prostaglandin F was elevated at 8 hr. Inclusion of copper dithiothreitol or reduced glutathione in the incubation media resulted in minor alterations of $\text{in vitro}$ release of progesterone and prostaglandin F and no changes in composition of luteal lipids or fatty acids. Luteal contents of triglyceride had increased by 8 hr after treatment whereas contents of free and esterified cholesterols had increased by 32 hr after Cloprostenol administration. Luteal contents of phospholipid and free fatty acids were not affected by Cloprostenol administration. At 32 hr after treatment, percentages and content of arachidonic acid had increased in luteal cholesterol esters and triglycerides. Although arachidonic acid percentages increased in luteal free fatty acids and phospholipids, calculated arachidonic acid contents did not change following Cloprostenol administration. Induced luteal regression was associated with decreased $\text{in vitro}$ progesterone release, increased $\text{in vitro}$ prostaglandin F release, and accelerated lipid and arachidonic acid accumulation within the corpus luteum. The effects of altered lipid metabolism on release of prostaglandin F could not be defined. However, availability of arachidonic acid did not appear to be rate-limiting in relation to luteal $\text{in vitro}$ prostaglandin F synthesis.     

Sensitivity of bovine corpora lutea to prostaglandin F2alpha is dependent on progesterone, oxytocin, and prostaglandins.

Prostaglandin (PG) F2alpha that is released from the uterus is essential for spontaneous luteolysis in cattle. Although PGF2alpha and its analogues are extensively used to synchronize the estrous cycle by inducing luteolysis, corpora lutea (CL) at the early stage of the estrous cycle are resistant to the luteolytic effect of PGF2alpha. We examined the sensitivity of bovine CL to PGF2alpha treatment in vitro and determined whether the changes in the response of CL to PGF2alpha are dependent on progesterone (P4), oxytocin (OT), and PGs produced locally. Bovine luteal cells from early (Days 4-5 of the estrous cycle) and mid-cycle CL (Days 8-12 of the estrous cycle) were preexposed for 12 h to a P4 antagonist (onapristone: OP; 10(-4) M), an OT antagonist (atosiban: AT; 10(-6) M), or indomethacin (INDO; 10(-4) M) before stimulation with PGF2alpha. Although OP reduced P4 secretion (p < 0.001) only in early CL, it reduced OT secretion in the cells of both phases examined (p < 0.001). OP also reduced PGF2alpha and PGE2 secretion (p < 0.01) from early CL. However, it stimulated PGF2alpha secretion in mid-cycle luteal cells (p < 0.001). AT reduced P4 secretion in early and mid-cycle CL (p < 0.05). Moreover, PGF2alpha secretion was inhibited (p < 0.05) by AT in early CL. The OT secretion and the intracellular level of free Ca2+ ([Ca2+]i) were measured as indicators of CL sensitivity to PGF2alpha. PGF2alpha had no influence on OT secretion, although [Ca2+]i increased (p < 0.05) in the early CL. However, the effect of PGF2alpha was augmented (p < 0.01) in cells after pretreatment with OP, AT, and INDO in comparison with the controls. In mid-cycle luteal cells, PGF2alpha induced 2-fold increases in OT secretion and [Ca2+]i. However, in contrast to results in early CL, these increases were magnified only by preexposure of the cells to AT (p < 0.05). These results indicate that luteal P4, OT, and PGs are components of an autocrine/paracrine positive feedback cascade in bovine early to mid-cycle CL and may be responsible for the resistance of the early bovine CL to the exogenous PGF2alpha action.     

Prostaglandin F concentrations in utero-ovarian vein plasma of prepuberal and mature gilts

Prostaglandin F (PGF) and progestins in utero-ovarian vein (UOV) plasma during the late luteal phase of the estrous cycle in unbred mature gilts and following induced ovulation in unbred prepuberal gilts were determined. Prepuberal gilts (120 to 130 days of age) were induced to ovulate with Pregnant Mare Serum Gonadotropin and Human Chorionic Gonadotropin (HCG). The day following HCG was designated as Day 0. Mature gilts which had displayed two or more estrous cycles of 18 to 22 days (onset of estrus = Day 0) were used. Polyvinyl catheters were inserted into the UOV of all gilts and blood was collected at 15 min intervals from 0800 to 1045 hr on Days 10 through 20 or Days 12 through 18. Plasma PGF concentrations in the mature gilts were elevated on Days 13, 14, 15, 16 and 17, whereas, plasma PGF concentrations in the prepuberal gilts were elevated only on Days 15, 16 and 17 resulting in a reproductive age (mature vs prepuberal) by day interaction (P<.01). In addition, the PGF concentrations on Days 13 through 17 were consistently greater in the mature gilts than in the prepuberal gilts as was the overall mean PGF concentration (1.95 vs .83 ng/ml). The average peak PGF concentration throughout the sampling period (4.6 vs 2.5 ng/ml; P<.01) and the average peak PGF concentration prior to luteal regression (3.8 vs 1.1 ng/ml; P<.05) were also greater in the mature than in the prepuberal gilts. Based on these results, we suggest that luteal regression in the bred prepuberal gilt following induced ovulation may not be due to an excessive production of the uterine luteolysin, but rather that the induced corpora lutea (CL) of the prepuberal gilt may be more sensitive to the uterine luteolysin than the spontaneously formed CL of the mature gilt.     

Localization of prostaglandin F in the ovine uterus during early pregnancy

As part of a study on the anti-luteolytic action of the sheep conceptus, the distribution of prostaglandin F (PGF) within the uteri of 19 pregnant and 14 non-pregnant ewes was studied using three antisera to PGF_2α and fluorescent antibody tracing. In the uteri of seven non-pregnant ewes up to Day 11 after estrus (Day 0) and in uteri of $\text{18}\text{19}$ pregnant ewes up to Day 50, PGF was localized mainly in the lamina propria, with very little on epithelial cells lining the uterine lumen. After Day 11, in the uteri of seven non-pregnant ewes, PGF was localized on the surface of luminal epithelial cells and throughout their cytoplasm, and to a lesser extent in the lamina propria. The distribution of PGF on Days 14 and 17 in the gravid and non-gravid horns of the uteri of 13 ewes made unilaterally pregnant (conceptus confined to one uterine horn) was similar to that described above for normal pregnant and non-pregnant ewes after Day 11 respectively.These results are interpreted to indicate a change in the distribution of PGF in sheep uteri due to the presence of a conceptus.     

Involvement of ovarian steroids in basal and oxytocin-stimulated prostaglandin (PG) F2 alpha secretion by the bovine endometrium in vitro

It is assumed that exposure of endometrium to spontaneously secreted luteal hormones stimulates PGF2 alpha secretion and modifies oxytocin (OT) influence on the bovine uterus. At first, the time-dependent effect of endogenous luteal products on endometrial PGF2 alpha secretion was examined. Endometrial strips (100 mg) from slaughtered heifers (Days 11 to 17 of the cycle) were incubated alone or with luteal cells (1 x 10(5) cells/mL). The highest PGF2 alpha secretion by the endometrium under influence of hormones secreted from luteal cells was observed after 12 h of incubation compared with the control (P < 0.001). Then, endometrium (Days 11 to 17) was incubated with luteal cells and concomitantly with antagonists of P4 and OT. The P4 antagonist prevented the stimulatory effect of endogenous luteal hormones on PGF2 alpha secretion (P < 0.05), but the OT antagonist did not. Further, direct effects of exogenous P4, OT and estradiol (E2) on endometrial PGF2 alpha secretion (Days 11 to 17) were examined. Both OT and P4 increased PGF2 alpha secretion (P < 0.05); E2 alone had no effect on PGF2 alpha secretion, but it amplified the P4 effect (P < 0.05). Finally, we studied the effect of endogenous luteal products on OT-stimulated PGF2 alpha secretion from endometrium. When endometrium (Days 11 to 17) was incubated without luteal cells, OT stimulated PGF2 alpha secretion (P < 0.001), whereas incubation of endometrium with luteal cells abolished the stimulatory effect of OT on PGF2 alpha secretion (P < 0.001). These treatments did not affect PGF2 alpha secretion from the endometrium collected on Days 1 to 4. In conclusion, P4 stimulates PGF2 alpha secretion by the endometrium and E2 amplifies this effect. As long as the endometrium is under the influence of P4, ovarian OT does not affect PGF2 alpha secretion.     

Change in morphology of corpora lutea, central luteal cavities and steroid secretion patterns of postpartum suckled beef cows after melengestrol acetate with or without prostaglandin F2alpha

Change in morphology of the corpus luteum (CL) and patterns of progesterone and estradiol secretion after treatment with melengestrol acetate (MGA) were monitored in postpartum beef cows. Twenty Angus cows were randomly assigned to MGA or MGA + prostaglandin F(2alpha) (PGF) treatments. All cows were fed 0.5 mg of MGA per cow per day for 14 d. The MGA-treated cows (n = 10) were allowed to return to estrus spontaneously at the second estrus after withdrawal of MGA from the feed. The MGA + PGF-treated cows (n = 10) received an injection containing 25 mg of PGF(2alpha) 17 d after the last feeding of MGA. Cycle 1 was defined as the first luteal phase after MGA feeding and Cycle 2 represented the subsequent cycle or luteal phase after PGF. Blood sampling and transrectal ultrasonography of the ovaries was done daily through the completion of 2 estrous cycles upon removal of MGA from the feed. Blood samples were analyzed for plasma progesterone and estradiol concentrations. Area of CL and fluid-filled cavities within each CL were determined by ultrasonography. Concentrations of progesterone and area of CL were similar between cycles and treatments. Estradiol concentrations were higher (P < 0.05) in Cycle 2 than in Cycle 1. Fluid-filled cavities were larger (P < 0.001) in Cycle 1 than in Cycle 2 for both mid-luteal (Days 5 to 9) and late-luteal (Days 10 to 14) phases. Multiple CL (2 or more during 1 cycle) were observed in 5 cows. Progesterone concentrations and total area of luteal tissue did not change with respect to treatment or cycle, but CL morphology was altered in the first cycle after MGA treatment. Of the 19 cows that ovulated after withdrawal of MGA, 3 experienced a short luteal phase. These data characterize changes that occur among cows that are fed melengestrol acetate during the postpartum period and enhance observations from prior studies regarding MGA use.     

Differential suppression of endometrial prostaglandin F2alpha by the equine conceptus

Prostaglandin F2alpha secretion by the uterine endometrium between Days 13 and 14 postovulation causes luteal regression in mares. A mechanism involving interruption or suppression of this secretion causes pregnancy to be maintained. The present study was designed to determine the age of the conceptus when maximal suppression of PGF2alpha secretion occurs. Mares were examined daily during estrus with ultrasonography (day 0 = day of ovulation). Conceptus tissues were recovered nonsurgically on Days 9 (n = 7), 12 (n = 5), 13 (n = 5), and 16 (n = 7) and uterine biopsies on Day 14. Both uterine and conceptus tissues were washed in phosphate-buffered saline (PBS) with 100 units penicillin G/ml + 100 microg streptomycin/ml, pH 7.4. Endometrial tissue (approximately 200 mg) plus conceptus tissues were incubated in 15 ml of tissue culture medium 199 (M199) + 10% fetal calf serum and 10 units penicillin G/ml and 10 microg streptomycin/ml at 37 degrees C under 5% CO(2): 5% O(2) : 90% N(2). Samples were taken at 4, 8, and 24 h. Two plates that contained only endometrial tissue and two additional plates with 25 mg flunixin meglumine added along with endometrial tissue were also included in the incubations. Concentrations of PGF2alpha were measured in all samples using radioimmunoassay. There was a trend toward suppression of PGF2alpha secretion by conceptus tissues, regardless of age. However, Day 12 concepti significantly suppressed PGF2alpha secretion compared with that of endometrial tissue incubated alone (P = 0.03).     

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.     

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.     

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.     

Influence of nitric oxide and noradrenaline on prostaglandin F(2)(alpha)-induced oxytocin secretion and intracellular calcium mobilization in cultured bovine luteal cells

Although prostaglandin (PG) F(2alpha) released from the uterus has been shown to cause regression of the bovine corpus luteum (CL), the neuroendocrine, paracrine, and autocrine mechanisms regulating luteolysis and PGF(2alpha) action in the CL are not fully understood. A number of substances produced locally in the CL may be involved in maintaining the equilibrium between luteal development and its regression. The present study was carried out to determine whether noradrenaline (NA) and nitric oxide (NO) regulate the sensitivity of the bovine CL to PGF(2alpha) in vitro and modulate a positive feedback cascade between PGF(2alpha) and luteal oxytocin (OT) in cows. Bovine luteal cells (Days 8-12 of the estrous cycle) cultured in glass tubes were pre-exposed to NA (10(-5) M) or an NO donor (S-nitroso-N:-acetylpenicillamine [S-NAP]; 10(-4) M) before stimulation with PGF(2alpha) (10(-6) M). Noradrenaline significantly stimulated the release of progesterone (P(4)), OT, PGF(2alpha), and PGE(2) (P: < 0.01); however, S-NAP inhibited P(4) and OT secretion (P: < 0.05). Oxytocin secretion and the intracellular level of free Ca(2+) ([Ca(2+)](i)) were measured as indicators of CL sensitivity to PGF(2alpha). Prostaglandin F(2alpha) increased both the amount of OT secretion and [Ca(2+)](i) by approximately two times the amount before (both P: < 0.05). The S-NAP amplified the effect of PGF(2alpha) on [Ca(2+)](i) and OT secretion (both P: < 0.001), whereas NA diminished the stimulatory effects of PGF(2alpha) on [Ca(2+)](i) (P: < 0.05). Moreover, PGF(2alpha) did not exert any additionally effects on OT secretion in NA-pretreated cells. The overall results suggest that adrenergic and nitrergic agents play opposite roles in the regulation of bovine CL function. While NA stimulates P(4) and OT secretion, NO may inhibit it in bovine CL. Both NA and NO are likely to stimulate the synthesis of luteal PGs and to modulate the action of PGF(2alpha). Noradrenaline may be the factor that is responsible for the limited action of PGF(2alpha) on CL and may be involved in the protection of the CL against premature luteolysis. In contrast, NO augments PGF(2alpha) action on CL and it may be involved in the course of luteolysis.     

A study of prostaglandin F2α as the luteolysin in swine: V comparison of prostaglandin F, progestins, estrone and estradiol in uterine flushings from pregnant and nonpregnant gilts

Uterine flushings were collected from 38 gilts representing Days 6,8,10,12,14,15,16 and 18 of the estrous cycle and pregnancy. The same group of gilts were represented within each of the respective days of the estrous cycle and pregnancy, i.e., three to six gilts per day per status. Uterine flushings (about 40ml) were assayed for prostaglandin F (PGF), estrone (E₁), estradiol (E₂), progestins (P) and protein. Nonpregnant gilts had higher (P<.01) concentrations of P in uterine flushings than pregnant gilts, but pregnant gilts had higher (P<.01) E₁ and E₂ concentrations. Significant day by status interactions were detected for E₁ (P<.05), but not for E₂ concentrations in uterine flushings. Total recoverable PGF and PGF concentrations in uterine flushings were greater (P<.01) in pregnant than nonpregnant gilts and significant (P<.01) day by status interactions were detected. In nonpregnant gilts, PGF increased between Days 12 and 16, i.e., during the period of corpora lutea (CL) regression. In pregnant gilts, PGF in uterine flushings increased markedly between Days 10 and 18. Total recoverable PGF on Day 18 of the estrous cycle was only 464.5 ± 37.6 ng as compared to 22,688.1 ± 1772.4 ng on Day 18 of pregnancy. Total recoverable protein was also higher (P<.01) in pregnant gilts. These data indicate that PGF synthesis and secretion by the uterine endometrium and/or conceptuses is not inhibited during pregnancy and suggest that PGF is sequestered within the uterine lumen of pregnant gilts, as is the total protein component of endometrial secretions referred to as histotroph.     

Effects of cloprostenol administration on neutral lipid and prostaglandin F metabolism by porcine luteal tissue

The effects of Cloprostenol administration on porcine luteal lipid metabolism, progesterone production, and prostaglandin F production were examined in 32 pigs at day 12 of the estrous cycle. Pigs were killed between 0 and 18 hours after treatment. Recovered luteal tissue was incubated at 0 C and at 37 C in the absence and presence of dibutyryl cyclic AMP and indomethacin. Net release of progesterone from luteal tissue was depressed within 1 hour after Cloprostenol treatment whereas net release of prostaglandin F was accelerated 4 hours after Cloprostenol treatment. Inclusion of dibutyryl cyclic AMP in the incubation media did not alter progesterone production by did enhance prostaglandin F production at 0 and 1 hour after Cloprostenol treatment. Inclusion of indomethacin in the incubation media completely inhibited the Cloprostenol-induced acceleration of luteal PGF production. Cloprostenol treatment increased luteal triglycerides and decreased luteal free cholesterol and cholesterol esters within 1 hr after treatment. Arachidonic acid percentages in free fatty acids and triglycerides were also increased within 1 hr after treatment. When 37 C and 0 C incubations were compared, luteal accumulation of free fatty acids was maximum at 1 hr after Cloprostenol treatment. accumulation of triglycerides in luteal tissie was comparatively uniform at all times examined during the first 18 hr after Cloprostenol treatment. Comparison of 37 C and 0 C incubations further revealed that luteal triglycerides were active in accumulation of arachidonic acid. Inclusion of dibutyryl cyclic AMP and/or indomethacin in the incubation media did not alter luteal lipid contents or fatty acid compositions. Blood plasma progesterone was depressed at 4 hours after Cloprostenol whereas 13,14-dihydro-15-keto-prostaglandin F2a was elevated at 18 hours after treatment. Blood plasma free fatty acids increased 330 percetn at 4 hours and free fatty acid compositions also changed at this time. In both luteal tissue and blood plasma, changes in steroid and fatty acid metabolism occurred prior to changes in prostaglandin metabolism, suggesting that Cloprostenol induced functional luteal regression prior to altering prostaglandin metabolism.     

Reduction by human chorionic gonadotropin of the luteolytic effect of prostaglandin F2 alpha in ewes

The ability of human chorionic gonadotropin (HCG) to reduce the luteolytic effect of prostaglandin (PGF2 alpha) was demonstrated in cycling ewes. As expected, treatment with 10 mg of PGF2 alpha alone on Day 10 of the estrous cycle exerted a potent negative effect on the function and structure of corpus luteum (CL) as indicated by reduced plasma progesterone, CL progesterone, and CL weight. However, the identical PGF2 alpha treatment failed to significantly reduce either luteal function or luteal weight when administered to ewes that were also treated with HCG on Days 9 and 10 of the estrous cycle. Treatment with HCG alone had a positive effect on CL as indicated by increased plasma progesterone, CL progesterone, and CL weight. Treatment with HCG did not render the CL totally insensitive to the negative effects of PGF2 alpha because plasma progesterone was reduced when the dose of PGF2 alpha was doubled. Whether CL regressed or continued to function after treatment with both HCG and PGF2 alpha appeared to depend upon a balance between the positive and negative effects of the two hormones.     

Prostaglandin F2α, oxytocin and progesterone secretion by bovine luteal cells at several stages of luteal development: Effects of oxytocin, luteinizing hormone, prostaglandin F2α and estradiol-17β

Bovine luteal cells from Days 4, 8, 14 and 18 of the estrous cycle were incubated for 2 h (1 × 10⁵ cells/ml) in serum-free media with one or a combination of treatments [control (no hormone), prostaglandin F₂α (PGF), oxytocin (OT), estradiol-17β (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<0.01) than on Day 8, 14 and 18 (rmOT: 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<0.01) PGF release by luteal cells compared with control cultures irrespective of day of estrous cycle. Estradiol-17β stimulated (P<0.05) PGF secretion on Days 8, 14 and LH increased (P<0.01) PGF production only on Day 14. Prostaglandin F₂α, 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<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<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.     

Insulin-like growth factor (IGF)-I, IGF-I receptor, and IGF binding protein-3 messenger ribonucleic acids and protein in corpora lutea from prostaglandin F(2alpha)-treated gilts

Insulin-like growth factor-I (IGF-I) is produced within the porcine corpus luteum (CL) and is thought to play an autocrine/paracrine role in CL development/function during the early luteal phase. This study examines the hypotheses that the luteolytic actions of prostaglandin F(2alpha) (PGF(2alpha)) during the early luteal phase may involve either a decrease in IGF-I or IGF receptor (IGF-IR), or an increase in IGF binding protein (IGFBP)-3, expression, any of which could interfere with the luteotropic actions of IGF-I in this tissue. Cycling gilts were treated twice daily with PGF(2alpha) (or saline) on Days 5-9 of the cycle to induce premature luteolysis. CL were collected on Days 6-9, and RNA, protein, or progesterone was extracted. By slot blot analysis, steady-state levels of IGF-I and IGFBP-3 mRNA were not different in PGF(2alpha)-treated vs. control animals; however, IGF-IR mRNA was increased in treated animals on Day 9. No changes in IGF-I content (ng/CL measured by RIA) were observed with respect to treatment. According to ligand blot analysis, the levels of IGFBP-3 increased on Day 6 and decreased on Days 8-9, while IGFBP-2 was higher on Days 6-7 and decreased on Day 9 in treated animals. IGF-IR levels, determined from Western blots, were higher on Day 7 (P < 0.05) and lower on Day 9 in PGF(2alpha)-treated animals vs. control animals (P < 0.05). In conclusion, PGF(2alpha)-induced premature luteolysis was associated with an increase in steady-state levels of IGF-IR mRNA, but it did not appear to be linked to changes in mRNA levels for IGF-I or IGFBP-3. However, since IGFBP-2 and -3 protein levels increased early in the treatment period (Days 6-7), it is possible that they may mediate the luteolytic actions of PGF(2alpha) by sequestering IGF-I and preventing its interaction with the IGF-IR.     

Canine prostaglandin F2alpha receptor (FP) and prostaglandin F2alpha synthase (PGFS): molecular cloning and expression in the corpus luteum

In the dog luteolysis is not affected by hysterectomy. This observation led to the hypothesis that paracrine/autocrine rather than endocrine mechanisms of PGF2alpha are responsible for luteal regression in the dioestric bitch. The present experiments tested for the capacity of canine CL to produce and respond to PGF2alpha by qualitatively and quantitatively determining the expressions of PGFS, the enzyme converting PGH2 into PGF2alpha, and the PGF2alpha-receptor (FP) in CL of non-pregnant dogs during dioestrus. Canine PGFS and FP were isolated and cloned; both genes show a high homology (82-94%) when compared to those of other species. Relatively weak FP mRNA expression was detected on day 5 of dioestrus. It had increased by day 25 and remained constant thereafter. In situ hybridization (ISH) localized FP solely to the cytoplasm of the luteal cells, suggesting that these cells are the only luteal targets of PGF2alpha in this species. Only negative results were obtained for the expression of PGFS in canine CL by routine qualitative RT-PCR. When Real Time (TaqMan) PCR was applied, repetitively more negative than positive results were obtained at all timepoints. Any positive measurements observed at any point were neither repeatable nor related to the stage of dioestrus. This led us to conclude that expression of PGFS is either absent or present at very low level only. These data suggest that luteal regression in non-pregnant bitches is not modulated by PGF2alpha. However, the FP seems to be constitutionally expressed, explaining the receptivity of canine CL to exogenous PGF2alpha.