Recent studies in several non-primate species have suggested that prostaglandin F2 alpha (PGF2 alpha) inhibits luteal cell progesterone production by activating the calcium and phospholipid-dependent protein kinase, protein kinase C (PKC). This study investigated the presence of PKC in human ovarian cells and assessed the ability of PGF2 alpha and its structural analogue, cloprostenol, to generate inositol polyphosphates and activate PKC. PKC was detected in cultured human granulosa-lutein cells and human luteal cells (from mid-late luteal phase). The major proportion of PKC detected was cytosol-associated in both cell types. Cloprostenol increased the generation of inositol polyphosphates in cultured human granulosa-lutein cells in a dose- and time-dependent manner. In addition both cloprostenol and PGF2 alpha activated PKC (as assessed by redistribution of enzyme activity from a principally cytosol-associated form to a membrane-associated form) in both granulosa-lutein and luteal cells. Short-term exposure of both cell types to phorbol myristate acetate (4 beta-PMA) activated PKC, whilst prolonged exposure of human granulosa-lutein cells to 4 beta-PMA led to a>> 85% loss of total PKC activity. The inactive phorbol ester, 4 alpha-PMA, had no effect on PKC activity when exposed to cells for up to 20 h. These results demonstrate the presence of PKC in human ovarian cells and the ability of PGF2 alpha to induce translocation/activation of this kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulators of G protein signaling (RGS) proteins bind the G protein Galpha subunit in its active GTP-bound state and accelerate its GTPase activity, thus halting Galpha activity. Induction of RGS2 expression has been previously shown in the rat ovary in response to ovulatory stimulation; however, the significance of RGS2 in the ovary has not been established. This study reports the potential role of RGS2 in the signaling of two G protein-coupled receptors, the LH and PGF2alpha (FP) receptors, in the human and the mouse granulosa cell lines, KGN and NT-1. The RGS2 mRNA concentration was rapidly and transiently elevated by human chorionic gonadotropin (hCG) or PGF2alpha analogue cloprostenol and this was followed by a decline to basal level at 24 h. Expression of the downstream critical target gene of the LH and FP receptor signaling pathways, namely, cyclooxygenase 2 (COX2), was induced by hCG but was inhibited by cloprostenol. Overexpression of RGS2 attenuated hCG-induced COX2 transcription. However, this augmented cloprostenol-mediated suppression of COX2 transcription. Confocal microscopy and immunoblot analysis were adopted to monitor the intracellular localization of RGS2 in COS-7 cells carrying the FP receptor and expressing RGS2-GFP or FLAG-RGS2. RGS2 was initially located predominantly in the nucleus and activation of the FP receptor resulted in RGS2 translocation from nucleus to the cell membrane. Thus, RGS2 expression was upregulated by LH receptor and FP receptor activation and modulation of partner receptor signaling by RGS2 may require RGS2 translocation from the nucleus to the plasma membrane.

This study aimed to characterize the endometrial transcriptome and functional pathways overrepresented in the endometrium of cows treated to ovulate larger (≥13 mm) versus smaller (≤12 mm) follicles. Nelore cows were presynchronized prior to receiving cloprostenol (large follicle [LF] group) or not (small follicle [SF] group), along with a progesterone (P4) device on Day (D) -10. Devices were withdrawn and cloprostenol administered 42-60 h (LF) or 30-36 h (SF) before GnRH agonist treatment (D0). Tissues were collected on D4 (experiment [Exp.] 1; n = 24) or D7 (Exp. 2; n = 60). Endometrial transcriptome was obtained by RNA-Seq, whereas proliferation and apoptosis were assessed by immunohistochemistry. Overall, LF cows developed larger follicles and corpora lutea, and produced greater amounts of estradiol (D-1, Exp. 1, SF: 0.7 ± 0.2; LF: 2.4 ± 0.2 pg/ml; D-1, Exp. 2, SF: 0.5 ± 0.1; LF: 2.3 ± 0.6 pg/ml) and P4 (D4, Exp. 1, SF: 0.8 ± 0.1; LF: 1.4 ± 0.2 ng/ml; D7, Exp. 2, SF: 2.5 ± 0.4; LF: 3.7 ± 0.4 ng/ml). Functional enrichment indicated that biosynthetic and metabolic processes were enriched in LF endometrium, whereas SF endometrium transcriptome was biased toward cell proliferation. Data also suggested reorganization of the extracellular matrix toward a proliferation-permissive phenotype in SF endometrium. LF endometrium showed an earlier onset of proliferative activity, whereas SF endometrium expressed a delayed increase in glandular epithelium proliferation. In conclusion, the periovulatory endocrine milieu regulates bovine endometrial transcriptome and seems to determine the transition from a proliferation-permissive to a biosynthetic and metabolically active endometrial phenotype, which may be associated with the preparation of an optimally receptive uterine environment.

In order to characterize prostanoid receptors present in the non-pregnant porcine uterus, the effects of naturally occurring prostaglandins (D2, E2, F2alpha, I2) and synthetic prostanoid receptor agonists on contractility of the longitudinal and circular muscles were examined in vitro. The potent contractile actions of prostaglandin F2alpha and cloprostenol indicate the presence of excitatory FP receptors in the porcine uterus. The longitudinal muscle was more sensitive to FP receptor agonists than was the circular muscle. Prostaglandin D2 produced an excitatory response in the longitudinal muscle but completely inhibited the spontaneous contraction of the circular muscle. BW-245C (5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl)hydantoin, 1 nM-10 microM, a DP receptor agonist) inhibited the spontaneous contractions of both muscles, but the inhibition was conspicuously stronger in the circular muscle. Prostaglandin I2 caused excitatory and inhibitory responses in the longitudinal and circular muscles, respectively, at relatively high concentrations (10-100 microM). Cicaprost, an IP receptor agonist caused inhibition of the contraction in the circular muscle but contracted the longitudinal muscle. Iloprost, an EP(1)/IP receptor agonist, caused excitatory responses in both muscles at relative high concentrations. Prostaglandin E2 caused excitatory responses at 1-100 nM and inhibitory responses at 100 nM-10 microM in both muscle layers. ONO-DI-004 ((17S)-2,5-ethano-6-oxo-17,20-dimethyl prostaglandin E1, an EP1 receptor agonist) and ONO-AE-248 ((16S)-9-deoxy-9beta-chloro-15-deoxy-16-hyfroxy-17,17-trimethylene-19,20-didehydro prostaglandin F2, an EP3 receptor agonist) contracted the longitudinal muscle but had little effect on the circular muscle. ONO-AE1-259 (11,15-O-dimethyl prostaglandin E2, an EP2 receptor agonist) inhibited the spontaneous contractions of both muscle layers to almost the same degree, but ONO-AE1-329 (16-(3-methoxymethyl)phenyl-omega-tetranor-3,7-dithia prostaglandin E1, an EP4 receptor agonist) did not inhibit the myometrial contraction. The present results indicate that contractile (FP, EP1, EP3) and relaxatory (DP, IP, EP2) prostanoid receptors are present in the non-pregnant porcine uterus. There are marked muscle layer-related differences in the degree of responsiveness of prostanoid receptor agonists, and these differences suggest that there is a heterogeneous distribution of prostanoid receptors in the longitudinal and circular muscles (FP, EP1 and EP3, longitudinal muscle>circular muscle; DP, circular muscle>longitudinal muscle).

It is well documented that prostaglandin F2 alpha (PGF2 alpha) inhibits progesterone production in luteal cells, but its mode of action is uncertain. It has recently been suggested that PGF2 alpha acts by activating the calcium and phospholipid-dependent protein kinase, protein kinase C (PKC). This hypothesis has been tested by comparing the site and mode of action of PGF2 alpha, a PGF2 alpha analogue (cloprostenol) and the PKC activator phorbol myristate acetate (4 beta PMA) in human granulosa-lutein cells. PGF2 alpha and cloprostenol exerted similar concentration-dependent inhibitory actions on gonadotrophin-stimulated cyclic AMP (cAMP) accumulation and progesterone production by human granulosa-lutein cells. The similarity in the actions of PGF2 alpha and cloprostenol in human granulosa-lutein cells suggests that they can be used interchangeably to study the role of PGF2 alpha in the regulation of steroidogenesis in the human ovary. Gonadotrophin-stimulated cAMP accumulation and progesterone production was also concentration-dependently inhibited by 4 beta PMA. In addition, cloprostenol and 4 beta PMA also inhibited dibutyryl cAMP-stimulated progesterone production, suggesting that these compounds inhibit LH action at sites before and after the generation of cAMP. The pre-cAMP site of action can be localised to the stimulatory G-protein (Gs) as both compounds inhibited cholera toxin-stimulated cAMP accumulation without affecting forskolin-stimulated cAMP accumulation. The post cAMP site of action can be localised to actions on cholesterol side chain cleavage enzyme, as both cloprostenol and 4 beta PMA inhibited 22R hydroxycholesterol-supported progesterone production without affecting pregnenolone-supported progesterone production. The finding that cloprostenol and 4 beta PMA interact with the steroidogenic cascade in a similar manner is indicative of a shared common mediator of their actions in human granulosa-lutein cells, i.e. PKC. The inhibitory actions of PGF2 alpha and 4 beta PMA on hLH-stimulated progesterone production were abolished in the presence of the PKC inhibitor, staurosporine. In addition, in PKC-depleted cells (achieved by exposure to 4 beta PMA for 20 h) the inhibitory actions of PGF2 alpha and 4 beta PMA were abolished. These results support the hypothesis that the inhibitory actions of PGF2 alpha are mediated by PKC in human granulosa-lutein cells.

In this study, we investigated follicular dynamics and ovarian steroid secretion during the follicular and early luteal phases of the estrous cycle in sheep. Six Finn-Merino ewes with ovarian autotransplanted ovaries were monitored for 10 days during the follicular phase and subsequent early luteal phase after luteal regression was induced with cloprostenol (a potent analogue of prostaglandin F2alpha). Over this period, follicular diameter was measured by serial ultrasound scans, and the concentration of gonadotropins and steroids in ovarian venous blood was measured at intervals of 6-12 h. All animals had an LH surge (Day 0) 59 +/- 4.7 h after injection of cloprostenol. The ovulatory follicles were derived mainly from large antral follicles present at the time of injection of cloprostenol (5.1 +/- 0.4 mm; mean +/- SEM, n = 6), although in some animals recruitment of additional small follicles was observed after luteolysis. The concentration of FSH decreased during the follicular phase and peaked synchronously with the LH surge, while estradiol and androstenedione concentrations in ovarian venous plasma increased progressively from luteal regression to a maximum at the LH surge. The rise in concentration of FSH on Day 1 was followed by the growth of a new cohort of follicles. Follicular size and ovarian steroid secretion increased in a linear fashion from Day 1 to Day 3, with ovarian steroid secretion reaching a maximum when the first wave of luteal phase follicles achieved a diameter of 5 mm or more. On Day 4, steroid secretion began to decline without significant changes in follicular diameter, and a second wave of follicles emerged. We conclude that 1) the preovulatory follicles are usually derived from the large follicle population present at the time of luteal regression, but the sheep has the ability to promote smaller follicles if required; and 2) the second peak of FSH stimulates the development of large estrogenic follicles during the early luteal phase, but the period of functional dominance is shorter than the period of morphological dominance.

Poor detection of estrus, still a major problem in the dairy industry, has prompted the development of electronic estrous detection technologies. One of the features of estrous behavior is a marked increase in walking activity. The objectives of the present study were to evaluate the effects of various management factors on walking activity increase at estrus, and the relationship between this trait and fertility. Data from 5883 artificial inseminations (AI) conducted in two high-producing dairy herds were analyzed. Detection of estrus was performed using a pedometer system. Of the total AI investigated, 2072 (35.2%) resulted in pregnancy. The following data were recorded for each animal at AI: herd, lactation number, milk production (average for the 3 days prior to AI), lactation stage (early, mid, and late lactation), previous estrous synchronization (cloprostenol or progesterone releasing intravaginal device [PRID] for animals showing estrus within 7 days of treatment), season (warm versus cool period), insemination number following parturition, inseminating bull, inseminator, and pedometer measurements. Variables were screened for associations with walking activity by analysis of variance (ANOVA) through generalized linear model procedures (PROC GLM). Increased parity and milk production, and insemination during the warm period were associated with lower pedometer measurements. No significant effects of the herd, estrous synchronization, and lactation stage were observed. The link between walking activity and fertility was determined by applying logistic regression models. We detected no significant effects of herd, milk production, estrous synchronization, lactation stage, and inseminator on pregnancy rate. A higher lactation and insemination number, and insemination during the warm period were negatively correlated with the pregnancy rate. The likelihood of pregnancy was greater when semen from one of the bulls was used and when physical activity at estrus was increased. Our findings indicate that cow and management factors contribute intensely to walking activity at estrus, and also reveal a close link between increased walking activity and fertility.

The pharmacologic characteristics of a number of FP-class prostaglandin (PG) analogs were determined by using the cat iris sphincter smooth-muscle-contraction assay. Cumulative concentration-response curves were generated for each compound. The relative agonist potencies (EC(50)) of the compounds were: cloprostenol (0.0012 +/- 0.0004 nM)>>) travoprost acid (0.46 +/- 0.13 nM) = bimatoprost acid (0.99 +/- 0.19 nM)>> (+/-)-fluprostenol (15.8 +/- 2.6 nM) = PGF(2alpha) (18.6 +/- 1.8 nM)>> latanoprost acid (29.9 +/- 1.6 nM)>> bimatoprost (140 +/- 45 nM)>> S-1033 (588 +/- 39 nM)>> unoprostone (UF-021; 1280 +/- 50 nM; n = 4-14). The maximum response induced by travoprost acid (122% +/- 2.3% maximum response relative to PGF(2alpha)) was significantly greater than that induced by all the other PG compounds (P < 0.001 - P < 0.02). Interestingly, the FP-receptor antagonist, AL-8810, behaved as a moderate efficacy partial agonist (EC(50) = 2140 +/- 190 nM; 63 +/- 4.3% maximum response relative to PGF(2alpha)), indicating that the cat iris contains an extremely well-coupled FP-receptor population, and/or the tissue contains an extremely high density of the FP-receptor and/or spare receptors. The cat iris contraction data were well correlated with other FP-receptor-mediated signal-transduction processes, including FP-receptor binding in bovine corpus luteum (r = 0.86), FP-receptor binding in human iris (r = 0.61), phosphoinositide (PI) hydrolysis in human ciliary muscle and trabecular meshwork cells (r = 0.77 - 0.86), PI turnover in rat and mouse cells (r = 0.73 - 0.76) and via cloned human FP-receptor (r = 0.9), and rat uterus contraction (r = 0.84). These data confirm the presence of functional FP-receptors in the cat iris sphincter, which are exquisitely well coupled and which respond to a variety of FP-class PG analogs with differing potencies.

A detailed pharmacological characterization of the prostaglandin (PG) receptor coupled to phosphoinositide (PI) turnover and intracellular calcium mobilization in Swiss 3T3 mouse fibroblast cells was undertaken. The pharmacological profile of this functional receptor was compared with the pharmacological profile of specific [3H]PGF2 alpha binding to bovine corpus luteum membranes, which are known to contain a bona fide FP receptor. PGs that were potent stimulators and full agonists in the PI turnover assay in the 3T3 cells were the following (for all, n = 3-45): 16-phenoxy-PGF2 alpha (EC50 = 0.61 +/- 0.1 nM), cloprostenol (EC50 = 0.73 +/- 0.04 nM), 17-phenyl-PGF2 alpha (EC50 = 2.71 +/- 0.35 nM), fluprostenol (EC50 = 3.67 +/- 0.61 nM), PhXA85 (EC50 = 27.3 +/- 5.63 nM) and PGF2 alpha (EC50 = 28.5 +/- 5.26 nM). However, PGD2 (EC50 = 155 +/- 29.9 nM; Emax = 49% of cloprostenol), PGE2 (EC50 = 2570 +/- 566 nM; Emax = 59%) and U46619 (EC50 = 1060 +/- 310 nM; Emax = 63%) were less potent and were partial agonists, and iloprost and BW245C were inactive. Although the PGs tested exhibited lower affinities in the 3[H]PGF2 alpha binding assay than their functional potencies in the PI turnover assay, the rank orders of potencies and affinities were well correlated (r = 0.94; n = 15 compounds). However, the PI turnover assay was more sensitive than the calcium mobilization assay for rank ordering PG agonists. In conclusion, the Swiss 3T3 cells express an FP receptor coupled to PI turnover and intracellular Ca+2 mobilization signal transduction pathways. The pharmacological profile of this receptor was similar to that of the FP receptor found in the bovine corpus luteum, a tissue previously used to clone the first pharmacologically defined FP receptor.

BACKGROUND

There are over 5000 approved prescription and over-the-counter medications, as well as vaccines, with labeled indications for veterinary patients. Of these, there are several products that have significant human health hazards upon accidental or intentional exposure or ingestion in humans: carfentanil, clenbuterol (Ventipulmin), ketamine, tilmicosin (Micotil), testosterone/estradiol (Component E-H and Synovex H), dinoprost (Lutalyse/Prostamate), and cloprostenol (Estromate/EstroPlan). The hazards range from mild to life-threatening in terms of severity, and include bronchospasm, central nervous system stimulation, induction of miscarriage, and sudden death.

OBJECTIVE

To report medication descriptions, human toxicity information, and medical management for the emergent care of patients who may have had exposure to veterinary medications when they present to an emergency department (ED).

CONCLUSIONS

The intended use of this article is to inform and support ED personnel, drug information centers, and poison control centers on veterinary medication hazards.

CONCLUSIONS

There is a need for increased awareness of the potential hazards of veterinary medications within human medicine circles. Timely reporting of veterinary medication hazards and their medical management may help to prepare the human medical community to deal with such exposures or abuses when time is of the essence.

In Experiments 1 and 2, ultrasound-guided transvaginal follicle aspiration was used as a method of follicle ablation to induce and synchronize subsequent follicular wave emergence and enhance ovulation synchrony following PGF2alpha administration. Heifers were at unknown stages of the estrous cycle at the start of both experiments in which all follicles>or=5 mm in diameter were ablated; luteolysis was induced 4 d later with cloprostenol (500 ug/dose, im). In Experiment 1, heifers were randomly assigned to either an ablation (n=17) or a procedural control (no follicle ablation, n=17) group. Ablation-induced wave emergence was indicated by a significant increase in the total number of follicles>or=5 mm within 2 d of ablation (mean, 1.5 d), which was preceded by a significant surge in circulating FSH. Although the mean (+/-SEM) interval from PGF2alpha administration to ovulation did not differ between follicle-ablated heifers (5.1+/-0.5 d range, 3 to 9 d) and control heifers (5.1+/-1.0 d; range, 1 to 5 d), the variability of the interval was different (P<0.05). Inequality of variance between the 2 groups was attributed to a greater (P<0.08) degree of ovulation synchrony in the ablation group than in the control group; 13/16 (81%) versus 9/17 (53%), respectively, ovulated within 5 d of cloprostenol administration. Relative asynchrony of ovulations in control heifers was associated with the status of the follicular wave at the time of PGF2alpha administration and, in part, to incomplete luteolysis following a single dose of PGF2alpha. Experiment 2 was designed to examine the efficacy of 2 doses of cloprostenol 12 h apart (n=7) versus a single dose (n=8) to induce complete luteolysis subsequent to follicle ablation-induced wave emergence. Two doses of cloprostenol potentiated ovulation synchrony; more (P<0.05) 2-dose heifers (7/7, 100%) than single-dose heifers (4/8, 50%) ovulated within 5 d after PGF2alpha administration. In summary, ultrasound-guided transvaginal follicle ablation, done at random during the estrous cycle, induced and synchronized subsequent follicular wave emergence, and resulted in a high degree of ovulation synchrony among heifers after PGF2alpha induced luteolysis, especially when 2 doses of PGF2alpha were administered 12 h apart.

A total of 177 Nelore heifers were examined by ultrasonography to determine the presence or absence of a corpus luteum (CL) and received a 3mg norgestomet ear implant plus 2mg of estradiol benzoate i.m. On Day 8, implants were removed and 150 microg of d-cloprostenol i.m. was administered. At the time of norgestomet implant removal, heifers with or without CL at the time of initiating treatment were assigned equally and by replicate to be treated with 0IU (n=87) or 400IU (n=90) eCG i.m. All heifers received 1mg of EB i.m. on Day 9 and were submitted to fixed-time artificial insemination (FTAI) 30-34h later. The addition of eCG increased the diameter of the largest follicle (LF) at FTAI (10.6+/-0.2mm vs. 9.5+/-0.2mm; P=0.003; mean+/-SEM), the final growth rate of the LF (1.14+/-0.1mm/day vs. 0.64+/-0.1mm/day; P=0.0009), ovulation rate [94.4% (85/90) vs. 73.6% (64/87); P=0.0006], the diameter of the CL at Day 15 (15.5+/-0.3mm vs. 13.8+/-0.3mm; P=0.0002), serum concentrations of progesterone 5 days after FTAI (6.6+/-1.0 ng/ml vs. 3.6+/-0.7ng/ml; P=0.0009), and pregnancy per AI [P/AI; 50.0% (45/90) vs. 36.8% (32/87); P=0.04]. The absence of a CL at the beginning of the treatment negatively influenced the P/AI [30.2% (16/53) vs. 49.2% (61/124); P=0.01]. Therefore, the presence of a CL (and/or onset of puberty) must be considered in setting up FTAI programs in heifers. In addition, eCG may be an important tool for the enhancement of follicular growth, ovulation, size and function of the subsequent CL, and pregnancy rates in progestin-based FTAI protocols in Bos indicus heifers.

Luteolytic capacity is defined as the ability of corpora lutea (CL) to undergo luteolysis after prostaglandin (PG) F2alpha treatment. The mechanisms causing acquisition of luteolytic capacity are not yet identified but CL without luteolytic capacity have PGF2alpha receptors and respond to PGF2alpha with some changes in gene expression. Inhibition of progesterone biosynthesis is a key feature of luteolysis and therefore we postulated that genes involved in progesterone biosynthesis would be regulated by PGF2alpha differently in CL with or without luteolytic capacity. Gilts on day 9 after estrus (lack luteolytic capacity) or day 17 of pseudopregnancy (with luteolytic capacity) were treated with saline or a PGF2alpha analog (cloprostenol) and CL were collected 0.5 (Experiment I) or 10 h (Experiment II) later. In Experiment III, large luteal cells from CL on day 9 or 17 were cultured for 1, 12 and 24h with or without PGF2alpha. PGF2alpha decreased LDL receptor mRNA (27%), steroidogenic acute regulatory protein (StAR) mRNA (41%), StAR protein (75%), LH receptor mRNA (55%), and LH receptor protein (45%) at 10 h after treatment in day 17 but not day 9 CL. PGF2alpha increased DAX-1 mRNA at 0.5 h (43%) and 10 h (46%) after PGF2alpha in day 17 but not day 9 CL but decreased 3betaHSD mRNA ( approximately 20% at 10 h) in both days 9 and 17 CL. In vitro, PGF2alpha decreased StAR mRNA at 12 h only in day 17 luteal cells; however, continuous treatment with PGF2alpha for 24 h decreased StAR mRNA in both days 9 and 17 luteal cells. Thus, luteolytic capacity involves a critical change in responsiveness of DAX-1, StAR, and LH receptor to PGF2alpha that results in inhibition of luteal progesterone biosynthesis.

Spontaneous contractile activity of strips of human myometrium obtained from non-pregnant donors at the time of hysterectomy was inhibited by the selective prostanoid DP receptor agonists BW 245C (5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl)hydantoin) and ZK110841 ((5Z,13E)-(9R,11R,15S)-9 beta-chlor-15-cyclohexyl-11,15-dihydroxy-16,17,18,19, 20-pentanor-5,13-prostadienoic acid) with pEC50 values of 8.4 and 7.3 respectively but prostaglandin D2 produced a biphasic effect. In the presence of the TP receptor antagonist L670596 ((-)-6,8-difluoro-9-p-methylsulfonyl benzyl-1,2,3,4-tetrahydrocarbazol-1-yl-acetic acid), contractile activity induced by the FP receptor agonist, cloprostenol ([1R-[1 alpha(Z),2 beta(1E,3R),3 alpha,5 alpha]]-7-[2-[4-(3- chlorophenoxy)-3-hydroxy-7-butenyl]-3,5-dihydroxycyclopentyl]-5-he ptenoic acid), was inhibited by BW 245C (pEC50 = 7.5), ZK110841 (pEC50 = 6.7) and prostaglandin D2 (pEC50 = 6.3). Under these conditions both prostaglandin J2 and 9 alpha,11 beta-prostaglandin F2 were inhibitory partial agonists. All compounds were antagonized by the selective DP receptor antagonist BW A868C (3-benzyl-5-(6-carboxyhexyl)-1-(2-cyclohexyl-2-hydroxyethylamino)h ydantoin), but the pKB values were both concentration-dependent (pKB versus BW 245C at 10 nM = 9.1, at 50 nM = 8.3) and agonist-dependent (pKB at 10 nM versus BW 245C = 9.1, versus ZK110841 = 7.4). Both agonist and antagonist potencies support the existence of DP receptors in human myometrium. The concentration and agonist dependence of the action of BW A868C suggests that putative DP receptor agonists relax human myometrium by more than one mechanism. These observations may be explained by the existence of subtypes of DP receptor in human myometrium.

Ninety-two cows that calved between April and August 1984 in a commercial dairy herd were studied. The cows were randomly assigned to receive either cloprostenol (500 mug) or saline placebo on Day 26 postpartum. In addition, each cow was examined per rectum, had three endometrial biopsies taken and milk progesterone level determined. Double-blind techniques were used in administering treatments and in assessing response to treatment. The results of histological and bacteriological assessment of endometrial biopsies and clinical findings at Day 40 were compared between the cows that received prostaglandin at Day 26 and those that did not. Each Day 40 variable which was significantly associated with prostaglandin treatment at Day 26 was regressed against the treatment variable and progesterone level at Day 26 as well as a prostaglandin progesterone interaction term. Prostaglandin-treated cows had less vaginal discharge, smaller diameter uterine horns, less inflammation and fibrosis in the endometrium, and were less likely to have Actinomyces pyogenes isolated from a biopsy at Day 40 than untreated cows. These effects were independent of progesterone level at the time of treatment.

OBJECTIVE

Surprisingly high contractile activity was reported for 11-deoxy-16,16-dimethyl prostaglandin E₂ (DX-DM PGE₂) on pig cerebral artery when used as a selective EP₃ receptor agonist. This study investigated the selectivity profile of DX-DM PGE₂, focusing on the interaction between its EP₃ and TP (thromboxane A₂-like) agonist activities.

METHODS

Contraction of guinea-pig trachea (EP₁ system) and aorta (EP₃ and TP systems) was measured in conventional organ baths.

RESULTS

Strong contraction of guinea-pig aorta to sulprostone and 17-phenyl PGE₂ (EP₃ agonists) was only seen under priming with a second contractile agent such as phenylephrine, histamine or U-46619 (TP agonist). In contrast, DX-DM PGE₂ induced strong contraction, which on the basis of treatment with (DG)-3ap (EP₃ antagonist) and/or BMS-180291 (TP antagonist) was attributed to self-synergism arising from co-activation of EP₃ and TP receptors. EP₃/TP self-synergism also accounted for contraction induced by PGF(2α) and its analogues (+)-cloprostenol and latanoprost-FA. DX-DM PGE₂ also showed significant EP₁ agonism on guinea-pig trachea as defined by the EP₁ antagonists SC-51322, (ONO)-5-methyl-1 and AH-6809, although AH-6809 exhibited poor specificity at concentrations ≥3 µM.

CONCLUSIONS

EP₃/TP self-synergism, as seen with PGE/PGF analogues in this study, may confound EP₃ agonist potency comparisons and the characterization of prostanoid receptor systems. The competitive profile of a TP antagonist may be distorted by variation in the silent/overt contraction profile of the EP₃ system in different studies. The relevance of self-synergism to in vivo actions of natural prostanoid receptor agonists is discussed.

Three hundred and five Holstein Friesian cows were given either 250 micrograms gonadotrophin releasing hormone (GnRH) or saline on day 15 postpartum followed by 500 micrograms cloprostenol or saline on day 24 postpartum. Four treatment groups were formed using random allocation: Group I -- placebo (Day 15)/placebo (Day 24), Group II -- GnRH (Day 15)/placebo (Day 24), Group III -- placebo (Day 15)/cloprostenol (Day 24), Group IV -- GnRH (Day 15)/cloprostenol (Day 24). Double blind techniques were used during the follow-up period. Rectal palpation, to assess uterine involution and ovarian activity was performed just prior to each treatment and again at 28 days postpartum. In addition blood samples were collected at 15, 24 and 28 days postpartum for measurement of plasma progesterone. There were no significant differences among treatment groups with respect to services per conception, number of heats detected before first service and culling for infertility. Cows treated only with GnRH had an increased calving to first estrus and calving to first breeding interval, and tended to have an increased calving to conception interval. Treatment with cloprostenol significantly decreased calving to conception and calving to first observed estrus intervals. Treatment with GnRH on day 15 postpartum resulted in a significant increase in the subsequent incidence of pyometra and prebreeding anestrus. On the other hand, cloprostenol treatment on day 24 postpartum resulted in a decreased incidence of pyometra, regardless of GnRH treatment and a decreased incidence of prebreeding anestrus in GnRH treated cows compared to cows receiving only GnRH at day 15 postpartum.(ABSTRACT TRUNCATED AT 250 WORDS)

The objective was to determine the accuracy of a pregnancy test for predicting nonpregnant cattle based on the evaluation of corpus luteum (CL) blood flow at 20 d (CLBF-d20) after timed artificial insemination (TAI). Crossbred Holstein-Gir dairy heifers (n=209) and lactating cows (n=317) were synchronized for TAI using the following protocol: intravaginal implant (1.0 g of progesterone) and 2mg of estradiol benzoate i.m. on d -10, implant removal and 0.526 mg of sodium cloprostenol i.m. on d -2, 1mg of estradiol benzoate i.m. on d -1, and TAI on d 0. On d 20, animals underwent grayscale ultrasonography (US) to locate the CL and color flow Doppler to evaluate CLBF-d20 using a portable ultrasound equipped with a 7.5-MHz rectal transducer. Based only on a visual, subjective CLBF evaluation, the animals were classified as pregnant or not pregnant. On d 30 to 35, blinded from results of the previous diagnosis, the same operator performed a final pregnancy diagnosis using US to visualize the fetal heartbeat (gold standard; US-d30). A second evaluator also analyzed the CLBF-d20 in the same animals by watching 7-s recorded videos. Blood samples were collected from a subset of 171 females to determine, by RIA, plasma progesterone (P4) concentrations, which indicate CL function. The final pregnancy outcome (US-d30) was retrospectively compared with the CLBF-d20 diagnoses and then classified either as correct or incorrect. The number of true positive, true negative, false positive, and false negative decisions were inserted into a 2 × 2 decision matrix. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the CLBF-d20 test were calculated using specific equations. Binomial variables (pregnancy rate and proportions) were analyzed using Fisher's exact test for the effect of parity and to compare between evaluators and tests (CLBF-d20 vs. plasma P₄). The kappa values were calculated to quantify the agreement between CLBF-d20 and the gold standard (US-d30) and between evaluators. The performance parameters of CLBF-d20 test were as follows: sensitivity=99.0%, specificity=53.7%, positive predictive value=65.1%, negative predictive value=98.5%, and accuracy=74.8%. False negatives represented only 0.4% of the exams. No differences existed in these parameters between evaluators (no. 1 vs. no. 2) and tests (CLBF-d20 vs. plasma P4). Moreover, a high level of agreement was observed between evaluators (0.91). In conclusion, visual evaluation of CLBF-d20 represents a quick, reliable, and consistent diagnostic test that enables the early detection of nonpregnant cattle.

Recent observations suggest that the endothelial cell-derived vasoconstrictive peptide endothelin-1 (ET-1) interacts with prostaglandin F2alpha (PGF2alpha) and that luteal ET-1 participates in the rapid cascade of functional luteolysis in vivo. Thus, the present study aimed to determine in detail the real-time changes in ET-1, oxytocin (OT), and progesterone (P4) concentrations within the regressing corpus luteum (CL), along with the changes in ovarian venous plasma (OVP) ipsilateral to the CL as well as in jugular venous plasma (JVP) in the cow. In the first study, peripheral plasma from daily sampling during the estrous cycle (n = 6) showed clear changes in ET-1 concentration with the stage of the cycle (p < 0.05). ET-1 remained at basal concentrations (23.2+/-1.3 pg/ml) on Days 2-12, increased (p < 0.05) on Days 13-19 (33.5+/-2.6 pg/ml), and reached the highest (p < 0.001) concentrations (45.6+/-4.4 pg/ml) on Days 20-22 after estrus. These data indicate that plasma ET-1 concentration increases around luteolysis and estrus. In the second study, a microdialysis system (MDS) was surgically implanted into the CL of 11 cows in the midluteal phase. In 4 of the 11 cows, the catheter was also fitted to the ovarian vein ipsilateral to the CL at surgery. A PGF2alpha analogue (cloprostenol; 500 microg) was then injected (designated as 0 h) i.m. to induce luteolysis. In the cows fitted with an MDS, the PGF2alpha injection clearly induced a rapid decrease in intraluteal P4 release within 4 h (p < 0.05), and the levels decreased to 20% of the baseline after 24 h. Intraluteal release of ET-1 increased (p < 0.05) to 160% within 4 h after PGF2alpha injection, when an enormous OT release (to 950%) occurred, which reached a plateau of 250% after 20 h that persisted until 72 h. ET-1 release into the ovarian vein began to increase at 2 h after PGF2alpha injection, when the acute OT release almost dropped to the baseline. The ET-1 concentration was temporarily (between 0 and 24 h after PGF2alpha) 2-3 times higher in OVP than in JVP (p < 0.05), and increased again to higher levels than in JVP from 32 to 64 h (p < 0.05). ET-1 concentrations in JVP gradually increased from 10 pg/ml to 30 pg/ml during PGF2alpha-induced luteolysis (p < 0.05). In conclusion, PGF2alpha injection rapidly increased ET-1 release within the regressing CL as well as into the ovarian venous blood in the cow. The overall results strongly support the hypothesis that luteal ET-1 is a local luteolytic mediator/promotor in the regressing bovine CL.

The objective of this study was to determine whether nitric oxide (NO) is produced locally in the bovine corpus luteum (CL) and whether NO mediates prostaglandin F2alpha (PGF2alpha)-induced regression of the bovine CL in vivo. The local production of NO was determined in early I, early II, mid, late, and regressed stages of CL by determining NADPH-d activity and the presence of inducible and endothelial NO synthase immunolabeling. To determine whether inhibition of NO production counteracts the PGF2alpha-induced regression of the CL, saline (10 ml/h; n = 10) or a nonselective NOS inhibitor (Nomega-nitro-l-arginine methyl ester dihydrochloride [L-NAME]; 400 mg/h; n = 9) was infused for 2 h on Day 15 of the estrous cycle into the aorta abdominalis of Holstein/Polish Black and White heifers. After 30 min of infusion, saline or cloprostenol, an analogue of PGF2alpha (aPGF2alpha; 100 microg) was injected into the aorta abdominalis of animals infused with saline or L-NAME. NADPH-diaphorase activity was present in bovine CL, with the highest activity at mid and late luteal stages (P < 0.05). Inducible and endothelial NO synthases were observed with the strongest immunolabeling in the late CL (P < 0.05). Injection of aPGF2alpha increased nitrite/nitrate concentrations (P < 0.01) and inhibited P4 secretion (P < 0.05) in heifers that were infused with saline. Infusion of L-NAME stimulated P4 secretion (P < 0.05) and concomitantly inhibited plasma concentrations of nitrite/nitrate (P < 0.05). Concentrations of P4 in heifers infused with L-NAME and injected with aPGF2alpha were higher (P < 0.05) than in animals injected only with aPGF2alpha. The PGF2alpha analogue shortened the cycle length compared with that of saline (17.5 +/- 0.22 days vs. 21.5 +/- 0.65 days P < 0.05). L-NAME blocked the luteolytic action of the aPGF2alpha (22.6 +/- 1.07 days vs. 17.5 +/- 0.22 days, P < 0.05). These results suggest that NO is produced in the bovine CL. NO inhibits luteal steroidogenesis and it may be one of the components of an autocrine/paracrine luteolytic cascade induced by PGF2alpha.

One hundred and seventy Holstein Friesian cows were randomly assigned to receive either 500 ug cloprostenol or saline placebo on Day 26 postpartum followed by 500 ug cloprostenol or saline on Day 40 postpartum. Four treatment groups were formed: Group 1-saline (Day 26)/saline (Day 40); Group 2-cloprostenol/(Day 26) saline (Day 40); Group 3-saline (Day 26)/cloprostenol (Day 40); Group 4-cloprostenol (Day 26)/cloprostenol (Day 40). Double blind techniques were used in administering treatments and in assessing the response to treatment. Palpation of the reproductive tract per tectum and uterine biopsies were performed on 92 cows prior to each treatment at Day 26 and Day 40 postpartum. Progesterone concentrations were determined on milk samples collected prior to treatment. There were no significant differences among treatment groups with respect to services per conception, number of heats detected before first service and culling for infertility. Cloprostenol treatment at Day 26 appeared to delay the first estrus, but it reduced the number of days to conception after the first service. Cows receiving cloprostenol at Days 26 and/or 40 had a decreased calving-to-conception interval compared to controls (P=0.01). Sequential therapy with two doses of cloprostenol resulted in slightly better reproductive performance than either treatment on Day 26 or 40 alone. Treatment with cloprostenol resulted in a decrease in the subsequent incidence of pyometra (P<0.05). It is concluded that in the herd studied, cloprostenol therapy at Day 26 and/or 40 postpartum was beneficial to reproductive performance. Although it was anticipated that cloprostenol would be more effective in cows with elevated progesterone levels, the opposite was observed at the Day 26 cloprostenol treatment. Uterine biopsy at Days 26 and/or 40 had a detrimental effect on subsequent reproductive performance.

Differences in the function and composition of individual ovarian follicles were noted in Booroola Merino ewes which had previously been segregated on at least one ovulation rate record of greater than 5 (FF ewes, N = 15), 3-4 (F+ ewes, N = 18) or less than 3 (++ ewes, N = 18). Follicles in FF and F+ ewes produced oestradiol and reached maturity at a smaller diameter than in ++ ewes. In FF (N = 3), F+ (N = 3) and ++ (N = 3) ewes, the respective mean +/- s.e.m. diameters for the presumptive preovulatory follicles were 3.4 +/- 0.3, 4.1 +/- 0.2 and 6.8 +/- 0.3 mm and in each of these follicles the respective mean +/- s.e.m. numbers of granulosa cells (X 10(6)) were 1.8 +/- 0.3, 2.2 +/- 0.3 and 6.6 +/- 0.3. During a cloprostenol-induced follicular phase, the oestradiol secretion rates from FF ewes with 4.8 +/- 0.4 'oestrogenic' follicles, F+ ewes with 3.2 +/- 0.2 'oestrogenic' follicles and ++ ewes with 1.5 +/- 0.02 'oestrogenic' follicles were not significantly different from one another. Moreover, the mean total numbers of granulosa cells from the 'oestrogenic' follicles from each genotype were identical, namely 5.4 X 10(6) cells. Irrespective of genotype the mean weight of each corpus luteum was inversely correlated to the ovulation rate (R = 0.91, P less than 0.001). Collectively, these findings support the notion that the maturation of greater than or equal to 5 follicles in FF ewes and 3-4 follicles in F+ ewes may each be necessary to provide a follicular-cell mass capable of producing the same quantity of oestradiol as that from 1-2 preovulatory follicles in ++ ewes.

Angiogenic factors, like vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF), and their receptors, are strongly regulated during the development of bovine corpus luteum (CL). The aim of this study was to investigate real-time changes of these factors in luteal tissue of cows (n = 4-5 per group) in the mid-luteal phase (day 8-12) after intramuscular injection of the PGF2alpha-analog Cloprostenol. Before (control) and 2, 4, 12, 48, and 64 hr after prostaglandin (PG) injection, CL were collected by transvaginal ovariectomy. RT-PCR for VEGF, VEGF-receptor type 1 (VEGF-R1), VEGF-R2, acidic FGF (FGF-1), basic FGF (FGF-2), and FGF-receptor (FGF-R) was performed. Additionally, the protein concentration for VEGF was determined. The mRNA expression of VEGF and its two receptors (VEGF-R1 and -R2) was significantly downregulated during structural luteolysis (after 12 hr). VEGF protein concentration already significantly declined 2 hr after PGF2alpha. Surprisingly FGF-1 and FGF-2 were significantly and maximally upregulated during functional luteolysis (until 12 hr). Furthermore, FGF-R mRNA was significantly upregulated at 2 hr after PGF2alpha, when compared with the control group. During structural luteolysis, the expression of FGFs and their receptors was not significantly different from control, except FGF-2 mRNA, which was downregulated at 64 hr. We conclude that the cessation of VEGF-support for the CL plays a role during structural luteolysis, whereas FGFs seem to have a major impact on functional luteolysis. The possible role of these growth factors could be a transient counter-regulation of luteolysis, but also an involvement in preventing inflammatory reactions during luteal regression.

AL-5848 (5Z,13E)-(9 S,11R,15S)-9,11,15-trihydroxy-5,13-prostadienoic acid) is the carboxylic acid of travoprost (AL-6221), a single (+)-isomer of (+/-)-fluprostenol, an FP-class prostaglandin agonist which lowers intraocular pressure. We have prepared a radioligand from this selective prostaglandin and demonstrated its utility for studying the pharmacology and autoradiographic location of the FP-receptor. Specific [3H]AL-5848 binding (84% of total) was linearly related to bovine corpus luteum tissue concentration and reached equilibrium within 275 min at 23 degrees C. Scatchard analysis of saturation isotherms indicated interaction of [3H]AL-5848 with a single class of high-affinity (dissociation constant, Kd, = 33.8+/-2.9 nM, n = 4) and saturable (Bmax = 37.3+/-3.0 pmol (g wet weight tissue)(-1)) FP receptor-binding sites in bovine corpus luteum. Specific [3H]AL-5848 binding was potently inhibited by the FP-receptor ligands 16-phenoxyPGF2alpha (inhibition constant Ki = 17.3 nM); cloprostenol (Ki = 56.8 nM); 17-phenyl PGF2alpha (Ki = 87.0 nM); AL-5848 (Ki = 52.1 nM); PGF2alpha (Ki = 195 nM); PHXA85 (Ki = 223 nM); (n = 3-11) but very weakly by PGD2, ZK118182, BW245C, PGE2, PGI2 and U-46619. The pharmacology of specific [3H]AL-5848 binding correlated well with the pharmacology of [3H]PGF2alpha binding in the bovine corpus luteum preparation (r = 0.98, n = 14, P<0.0001) and also with functional responses in Swiss 3T3 and rat vascular smooth muscle cells (A7r5) (r = 0.96) expressing FP receptors. Autoradiographic studies revealed high levels of specific FP-receptor binding with [3H]AL-5848 on granulosa cells in the bovine corpus luteum sections, and on longitudinal ciliary muscle, the ciliary process, the iris sphincter and the retina in eye sections from man. These studies show [3H]AL-5848 to be a high-affinity agonist radioligand capable of selectively labelling the FP prostaglandin receptor.