Role of prostaglandin E2 in basal and noradrenaline-induced progesterone secretion by the bovine corpus luteum

The role of prostaglandin E2 (PGE2) in basal and noradrenaline (NA)-stimulated utilization of high density lipoprotein (HDL) as a source of cholesterol for progesterone synthesis was examined. In Experiment 1, a cannula was inserted into the aorta abdominalis through the coccygeal artery (cranial to the origin of the ovarian artery) in mature heifers, to facilitate infusion of NA (4 mg/30 min; n = 3) on day 10 of the estrous cycle. Three other heifers were similarly cannulated to serve as control. Before, during, and after NA or saline infusion, blood samples from the vena cava were collected every 5-15 min for analysis of PGE2, progesterone, and cholesterol. Each NA infusion stimulated (P < 0.01) secretion of both hormones in heifers. Short-duration increases (P < 0.05) in progesterone were observed due to the infusion of NA while cholesterol was not altered significantly. In addition, increases in PGE2 concentrations (P < 0.05) compared to controls were seen after NA infusion. Therefore, we used an in vitro model to verify the effect of PGE2 on HDL utilization by luteal cells from day 5 to 10 of the estrous cycle. In the preliminary experiment, 10(-6) M of PGE2 out of four different doses examined was selected for further studies, since it evoked the highest release of progesterone. In the next experiment, it was found that HDL increases progesterone secretion by luteal cells and both PGE2 and LH increased (P < 0.05) the response to HDL while NA did not. In the last in vitro experiment, progesterone stimulated PGE2 secretion by luteal cells. In conclusion, PGE2 may be directly involved in the utilization of cholesterol from HDL for progesterone synthesis. Furthermore, PGE2 may influence NA-stimulated progesterone secretion by the corpus luteum (CL). It is concluded that there is a positive feedback loop between progesterone and luteal PGE2 during days 5-10 of the estrous cycle.     

Role of arachidonic acid and its metabolites in the regulation of progesterone and oxytocin release from the bovine corpus luteum.

We have examined the effects of arachidonic acid (AA) and some of its metabolites on progesterone (P4) and oxytocin (OT) release by corpora lutea obtained from Holstein heifers at day 8 of the estrous cycle (Day 0 = estrus). The luteal cells were dispersed with collagenase and small and large cells were separated by unit gravity sedimentation and flow cytometry. After an 18-hr preincubation period, the cells were incubated in the presence of various treatments for 1 hr, followed by a 23-hr incubation period with no treatment. OT was secreted by the large, but not by the small, luteal cells into the incubation medium. AA elicited a significant (P less than 0.05) release of OT from the large cells and P4 from both the large and small cells within 1 hr of incubation, having a specific effect at a concentration of 10 microM. Larger doses (25 and 100 microM) of AA adversely affected the cell viability. Phospholipases A2 (0.5 unit/ml) and C (0.05 unit/ml) and calcium ionophore A23187 (0.1 microM) stimulated OT release from the large cells to the same extent as AA (10 microM). Inhibition of the AA cyclooxygenase metabolic pathway by indomethacin did not affect AA-induced release of OT and P4, although exogenous prostaglandins F2 alpha and I2 (5-25 ng/ml) stimulated the release of OT. Lipoxygenase products of AA (hydroxyeicosatetraenoic acid and leukotrienes; 25 ng/ml) also stimulated OT release. Inhibition of the lipoxygenase metabolic pathway by nordihydroguaiaretic acid abolished AA-induced release of both OT and P4. These results suggest that intracellular accumulation of free AA may modulate secretory functions in the bovine corpora lutea, including OT and P4 release.     

Oxytocin stimulates progesterone release from microdialyzed bovine corpus luteum in vitro

A microdialysis system (MDS) was implanted in corpora lutea (CL) from cows (Days 5-7, 8-12, and 15-18 of the estrous cycle); the CL were maintained in organ culture chambers. With this system, active substances can be applied, and a collection of steroids released from luteal cells surrounding the microcapillary (cut-off point = 100 kDa) is possible, while luteal cells maintain cell-to-cell contact. Spontaneous pulses of progesterone release were observed in 90% of control (perfused with Ringer's solution only) at 60-80 min intervals. The infusion of bovine LH (bLH) for 20 min (0.1-10 micrograms/ml) stimulated dose-dependent release of progesterone. Both results indicate that the CL maintains the activity of progesterone release and the ability to respond to LH stimulation in this system. Oxytocin (1-100 microM) also stimulated progesterone release in a dose-dependent manner. Preexposure with oxytocin antagonist blocked the stimulatory effect of oxytocin (p less than 0.01) but not of LH (p less than 0.05), confirming the specificity of the effect. When CL were prestimulated with a low dose of oxytocin (1 microM, 20 min) twice before bLH application, the release of progesterone by bLH (1 micrograms/ml, 20 min) was more pronounced (p less than 0.05). A long-term infusion (3 h) with oxytocin and/or bLH stimulated the release of progesterone for the whole period of time. Oxytocin was most stimulative during the early luteal phase (Days 5-7) and decreased continuously from Days 8-12 to Days 15-18.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of prostaglandin synthesis by progesterone in the bovine corpus luteum

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

Changes in ovarian oxytocin secretion as an indicator of corpus luteum response to prostaglandin F(2alpha) treatment in cattle

Exogenous prostaglandin F(2alpha) (PGF(2alpha)) rapidly increases ovarian oxytocin (OT) release and decreases progesterone (P4) secretion in cattle. Hence, the measurement of OT secretion (the area under the curve and the height of the peak) after different doses of Oestrophan - PGF(2alpha) analogue (aPGF(2alpha)) on Days 12 and 18 of the estrous cycle (estrus = day 0), could be a suitable indicator of corpus luteum (CL) sensitivity to PGF(2alpha) treatment. Mature heifers (n = 36) were used in this study. Blood samples were collected from the jugular vein for the estimation of OT, P4 and 13, 14-dihydro-15-keto-prostaglandin F(2alpha) (PGFM). In Experiment 1, different doses of aPGF(2alpha) (400, 300, 200 and 100 microg) given on Day 12 of the estrous cycle (n = 8) shortened (P < 0.05) the cycle duration (15.2 +/- 0.6 d) compared with that of the control (21.7 +/- 0.4 d). Successive heifers were also treated on Day 12 with 200 (n = 2), 100 (n = 2), 75 (n = 2) or 50 microg aPGF(2alpha) (n = 2). Only the 50 microg aPGF(2alpha) dose did not cause CL regression, although it increased OT concentrations to levels comparable to those observed during spontaneous luteolysis (50 to 70 pg/ml). In Experiment 2, on Day 18 of the cycle heifers (n = 8) were treated with 50, 40, 30 and 20 microg aPGF(2alpha). There was a dose-dependent effect of aPGF(2alpha) on OT secretion on Day 18 of the estrous cycle (r = 0.77; P < 0.05). In Experiment 3, an injection of 500 microg aPGF(2alpha) on Day 12 (n = 4) and 50 microg aPGF(2alpha) on Day 18 (n = 4) caused a similar (P > 0.05) increase in the OT concentration (288.5 +/- 23.0 and 261.5 +/- 34.7 pg/ml, respectively). Thus the effect of the same dose of aPGF(2alpha) (50 microg) on OT secretion was different on Days 12 and 18 of the cycle. To evoke similar OT secretion on Days 12 and 18 the dose of aPGF(2alpha) on Day 18 could be reduced 10-fold, confirming that CL sensitivity to PGF(2alpha) appears to increase in the late luteal phase.     

Treatment with prostaglandin F2alpha increases expression of prostaglandin synthase-2 in the rat corpus luteum

Recent studies indicate that the corpus luteum (CL) may be a source of prostaglandin F2alpha (PGF2alpha) for regression. We investigated expression of mRNA and protein for prostaglandin G/H synthase (PGHS) in the CL of immature superovulated rats following administration of PGF2alpha. We observed an increase in mRNA for PGHS-2, the induced isoform, at 1 h and protein at 8 and 24 h after treatment. One hour after PGF2alpha, there was also a progressive decrease in plasma progesterone concentration. There were no changes, however, in expression of PGHS-1, the constitutive isoform, over the 24 h sampling period. These results indicate that PGHS-2 increases following PGF2alpha treatment and that expression of this enzyme in the rat CL may contribute to the luteolytic mechanism.     

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.     

Inhibition of prostaglandin F2alpha synthesis and oxytocin receptor by progesterone antagonists in bovine endometrial cells in vitro

Oxytocin receptor (OTR) expression is suppressed by progesterone (P4) during the luteal phase of the estrous cycle and then it increases at the time of luteolysis, but its regulation is still not completely understood. In vitro studies to determine the mechanism of action are hindered because OTR spontaneously upregulates in vitro and it is impossible to alter expression with P4 or estradiol. During recent studies examining the effect of P4 and an antagonist (mifepristone) on PG secretion, we found that mifepristone attenuated OT-stimulated PG secretion from endometrial epithelial cells. The objective of the present study was to determine, whether this effect of mifepristone was due to changes in prostaglandin synthesis and/or OTR. A time-course showed that mifepristone (5 microM) had no significant effect after 24 h but by 72 h it decreased PGF(2alpha) secretion (P<0.01) and abolished the response of the cells to OT (P<0.01). The presence or absence of P4 did not affect the response to mifepristone. To determine the site of action of mifepristone, cells were cultured for 72 h with or without mifepristone and then COX-1 and COX-2 were measured by Western blotting and OTR was measured by saturation analysis. The results showed that mifepristone did not affect basal or PMA-stimulated expression of either COX-1 or COX-2 but did, however, decrease OTR number (P<0.05). These data demonstrate that OTR and the response to OT can be downregulated in endometrial epithelial cells in vitro via a mechanism involving the P4 receptor.     

Lack of negative cooperativity among binding sites for prostaglandin F2alpha in bovine corpus luteum cell membranes.

The Scatchard analysis of equilibrium prostaglandin (PG) F₂α binding revealed that the binding was heterogeneous. The Hill plot of the same data had a slope of 0.68. This suggested that the heterogeneous nature of [³H] PGF₂α binding was either due to the presence of negative cooperativity or to the presence of two groups of independent binding sites. The kinetic experiments revealed that the presence of excess unlabeled PGF₂α in a diluting medium had no effect on dissociation rates at 25 fold dilution and it even retarded dissociation at higher dilutions. Furthermore, the observations that the low affinity PGF₂α binding sites can exist in the absence of high affinity binding sites and high affinity binding sites can be selectively abolished by treatment with N-ethylmaleimide suggest that negative cooperativity was not responsible for heterogeneous [³H] PGF₂α binding.     

Effect of exogenous progesterone on prostaglandin F2 alpha release and the interestrous interval in the bovine

The present study was developed to determine if administration of progesterone, early in the estrous cycle of the cow, stimulated an advanced pulsatile release of PGF2 alpha from the uterine endometrium resulting in a decreased interestrous interval. Twenty-three cyclic beef cows were randomly assigned to receive either sesame oil or progesterone (100 mg) on Day 1, 2, 3 and 4 of the estrous cycle. Peripheral plasma concentrations of progesterone and the metabolite of prostaglandin F2 alpha, 15-keto-13,14-dihydro-prostaglandin F2 alpha (PGFM) were measured by radioimmunoassay. Administration of exogenous progesterone increased peripheral plasma concentration of progesterone in treated (3.67 ng/ml) compared to control (1.28 ng/ml) cows from Day 2 through 5 of the estrous cycle. Progesterone administration shortened the interestrous interval (16.7 d) compared to controls (21.6 d). The shortened interestrous intervals in treated cows resulted from an earlier decline in peripheral plasma progesterone. Decline of peripheral plasma progesterone concentrations is coincident with an increased pulsatile release of PGFM in both progesterone treated and control cows. Results indicate that administration of exogenous progesterone stimulates an earlier maturation of endometrial development, causing an advanced release of PGF2 alpha which shortens the interestrous interval of the cow.     

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

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

Control of bovine uterine prostaglandin F2 alpha release in vitro

Prostaglandin F2 alpha (PGF2 alpha) release from the uterus causes luteolysis in ruminants, and oxytocin is thought to be a regulator of this release. In the present study, we have examined the mechanisms involved in oxytocin stimulation of PGF2 alpha secretion by bovine endometrium in vitro. Endometrial tissue explants, obtained from heifers at Day 19 or 20 (n = 3) and Day 0 (estrus, n = 5) of the estrous cycle, were incubated for 2 h and 6 h, and PGF2 alpha concentration in the medium was determined by radioimmunoassay (RIA). Basal PGF2 alpha release increased for up to 6 h and was significantly stimulated after 2 h of incubation with 100 microU and 1000 microU of oxytocin at Day 0 but not at Day 19 or 20. Secretion of PGF2 alpha was not affected by cholera toxin (10 ng/ml) or the cyclic nucleotide analogs dibutyryl cyclic adenosine 3',5'-monophosphate and dibutyryl cyclic guanosine 3',5'-monophosphate at a concentration of 1 mM. A protein kinase A inhibitor (500 microM) had no effect on the oxytocin-induced release of PGF2 alpha. Both the phorbol ester, 12-myristate-13-acetate (100 mM), and the non-phorbol stimulator of protein kinase C, 1-octanoyl-2-acetylglycerol (500 microM), significantly stimulated PGF2 alpha secretion to the same extent as oxytocin. Neither basal nor stimulated PGF2 alpha release was affected by the calcium ionophore A23187 (0.1-5.0 microM). However, PGF2 alpha secretion was sensitive to cycloheximide (1 microgram/ml) suggesting that protein synthesis may be involved. In conclusion, these data suggest that the stimulation of PGF2 alpha by oxytocin is via the protein kinase C effector pathway.     

Expression and activation of protein kinase C isozymes by prostaglandin F(2alpha) in the early- and mid-luteal phase bovine corpus luteum

Western blotting was used to identify the array of protein kinase C (PKC) isozymes expressed in the early (Day 4) and midcycle (Day 10) bovine corpus luteum (CL). PCKalpha, betaI, betaII, epsilon, and micro isozymes were detected in total protein samples prepared from both Day-4 and Day-10 corpora lutea. In contrast, specific antibodies for PKCgamma, eta, lambda, and theta isozymes failed to detect protein bands in the luteal samples. PKCbetaII and epsilon isozymes were expressed differentially at these two developmental stages of the bovine CL. In the Day-4 luteal samples, PKCepsilon was barely detectable; in contrast, in the Day-10 samples, the actin-corrected ratio for PKCepsilon was 1.16 +/- 0.13. This ratio was higher than the detected ratio for PKCbetaI and micro at this developmental phase of the CL (P < 0.01), but it was comparable with the ratio detected for the PCKalpha and betaII. The amount of PKCbetaII was, although not as dramatic, also greater in the Day-10 CL (actin-corrected ratio was 0.85 +/- 0.2) than in the Day-4 CL (0.35 +/- 0.09 [P < 0.01]). The actin-corrected ratios for all other PKC isozymes, alpha (Day 4 = 0.93 +/- 0.16, Day 10 = 0.97 +/- 0.09), betaI (Day 4 = 0.54 +/- 0.073, Day 10 = 0.48 +/- 0.74), and micro (Day 4 = 0.21 +/- 0.042, Day 10 = 0.21 +/- 0.38) were not different at these 2 days of the cycle. An experiment was designed to test whether activation of specific isozymes differed between CL that do or do not regress in response to PGF(2alpha). Bovine CL from Day 4 and Day 10 of the estrous cycle were collected and 1 mm CL fragments were treated in vitro for 0, 2.5, 5, 10 or 20 min with PGF(2alpha) (0.1, 1.0, and 10 nM) or minimal essential medium-Hepes vehicle. Translocation of PKC from cytoplasm to membrane fraction was used as indication of PKC activation by PGF(2alpha). Evidence for PKC activation was observed in both Day-4 and Day-10 luteal samples treated with 10 nM PGF(2alpha). Therefore, if PKC, an intracellular mediator associated with the luteal PGF(2alpha) receptor, contributes to the lesser sensitivity of the Day-4 CL, it is likely due to the differential expression of the epsilon and betaII isozymes of PKC at this stage and not due to an inability of the PGF(2alpha) receptor to activate the isozymes expressed in the early CL.     

Patterns of gene expression in the bovine corpus luteum following repeated intrauterine infusions of low doses of prostaglandin F2alpha

Natural luteolysis involves multiple pulses of prostaglandin F2alpha (PGF) released by the nonpregnant uterus. This study investigated expression of 18 genes from five distinct pathways, following multiple low-dose pulses of PGF. Cows on Day 9 of the estrous cycle received four intrauterine infusions of 0.25 ml of phosphate-buffered saline (PBS) or PGF (0.5 mg of PGF in 0.25 ml of PBS) at 6-h intervals. A luteal biopsy sample was collected 30 min after each PBS or PGF infusion. There were four treatment groups: Control (n = 5; 4 PBS infusions), 4XPGF (4 PGF infusions; n = 5), 2XPGF-non-regressed (2 PGF infusions; n = 5; PGF-PBS-PGF-PBS; no regression after treatments), and 2XPGF-regressed (PGF-PBS-PGF-PBS; regression after treatments; n = 5). As expected, the first PGF pulse increased mRNA for the immediate early genes JUN, FOS, NR4A1, and EGR1 but unexpectedly also increased mRNA for steroidogenic (STAR) and angiogenic (VEGFA) pathways. The second PGF pulse induced immediate early genes and genes related to immune system activation (IL1B, FAS, FASLG, IL8). However, mRNA for VEGFA and STAR were decreased by the second PGF infusion. After the third and fourth PGF pulses, a distinctly luteolytic pattern of gene expression was evident, with inhibition of steroidogenic and angiogenic pathways, whereas, there was induction of pathways for immune system activation and production of PGF. The pattern of PGF-induced gene expression was similar in corpus luteum not destined for luteolysis (2X-non-regressed) after the first PGF pulse but was very distinct after the second PGF pulse. Thus, although the initial PGF pulse induced mRNA for many pathways, the second and later pulses of PGF appear to have set the distinct pattern of gene expression that result in luteolysis.     

Response of the 1-5 day-aged ovine corpus luteum to prostaglandin F2alpha

The hypothesis that, in the ewe, prostaglandin (PG) F2alpha administration on day 3 after ovulation is followed by luteolysis and ovulation was tested using 24 animals. The ewes were treated with a dose of a PGF2alpha analogue (delprostenate, 160 microg) on days 1 (n=8), 3 (n=8) or 5 (n=8) after ovulation, was established by transrectal ultrasonography. Daily scanning and blood sampling were performed to determine ovarian changes and progesterone serum concentrations by radioinmunoassay. The treatment induced a sharp decrease of progesterone concentrations followed by oestrus and ovulation in all ewes treated on days 3 and 5 and in one ewe treated on day 1 (8/8, 8/8, 1/8; P<0.05). Seven ewes treated on day 1 did not respond to PGF2alpha treatment and had an inter-ovulatory cycle of normal length (17.4 +/- 0.5 days). However, the profile of progesterone concentrations during the cycle of these ewes was delayed 1 day (P<0.05) compared with a control cycle. The overall interval between PGF2alpha and oestrus for the 17 responding ewes was 42.4 +/- 2.3 h. In 15 of these ewes the ovulatory follicle was originated from the first follicular wave and the ovulation occurred at 60.8 +/- 1.8 h after PGF2alpha treatment. The other two responding ewes ovulated an ovulatory follicle originated from the second follicular wave between 72 and 96 h after treatment. These results support the hypothesis and suggest that refractoriness to PGF2alpha of the recently formed corpus luteum (CL) may be restricted to the first 1-2 days post-ovulation.     

Changes in corpus luteum content of prostaglandin F2 alpha and E in the adult pseudopregnant rat

Conflicting reports exist regarding the source of luteolytic PGF2 alpha in the rat ovary. To assess the quantities of different PGs, measurements of PGF2 alpha, PGE and PGB were performed by radioimmunoassay in the adult pseudopregnant rat ovary throughout the luteal lifespan. Ovaries of 84 rats were separated by dissection into two compartments, corpora lutea of pseudopregnancy and remainder of ovary. Tissue samples were homogenized and prostaglandins extracted and determined by radioimmunoassay. During the mid-luteal and late-luteal phases, levels of PGs were significantly higher in the corpora lutea of pseudopregnancy than in the remainder of ovary. An increase of PGF2 alpha-content in the corpus luteum was registered with peak-levels of 53.9 +/- 8.5 (mean +/- SEM, N = 18) ng/g tissue wet weight at day 13 of pseudopregnancy. PGE-levels reached peak-values at day 11 of pseudopregnancy (271.6 +/- 28.4 ng/g w w, mean +/- SEM, N = 12). PGB-levels were below detection limits in all compartments for all ages studied. The present study demonstrates increased availability of PGF2 alpha in the corpus luteum during the luteolytic period, and points toward either increased luteal synthesis or luteal binding of PGF2 alpha during the luteolytic period.     

Trypanosome-induced increase in prostaglandin F(2alpha) and its relationship with corpus luteum function in the goat

Plasma progesterone and 13,14-dihydro-15-keto Prostaglandin F(2alpha) (PGFM) were measured in normal (uninfected) and Trypanosoma congolense -infected adult goats for a period of 121 d, from May to August, during the breeding season in Kenya. Chronic trypanosomiasis rapidly increased the baseline plasma PGFM levels and the occurrence of irregular PGFM peaks in several infected goats. Progesterone luteal levels declined rapidly from the second and subsequent cycles post patency. Estrous cycles also became irregular but predominately shorter (8 to 19 d) before cessation from the second to fourth cycle following infection. The PGFM levels were still high during the acyclic period in all goats when progesterone levels were very low (1.4 to 2.4 nmol/l). The reciprocal increase in peripheral PGFM and decline in progesterone in these goats would suggest, in part, a trypanosome-induced PGF(2alpha) mediated luteolysis, and the possible involvement of prostaglandins in trypanosome-induced infertility in female goats.     

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.     

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

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