Synchronized breeding in cattle using PGF2α and LHRH/FSHRH stimulatory analogs

Two studies were conducted to synchronize breeding in cattle using PGF₂α and LHRH/FSHRH analogs. In the first study, 60 mature lactating Angus cows were assigned at random to 4 treatment groups: saline and saline (SS); 30 mg PGF₂α tham salt + saline (PS); saline + 2 mg D-ala⁶-des-GlyNH₂¹⁰ LHRH/FSHRH ethylamide (D-ala⁶) (SA); 30 mg PGF₂α tham salt + 2 mg D-ala⁶ (PA). The first letter of the two-letter code for each group always indicates a dual injection at an 11-day interval. PGF₂α or saline was administered intramuscular (im) twice at an 11 day interval. D-ala⁶ or saline was administered 48 hr after PGF₂α treatment. In the SA group, the D-ala⁶ was administered at first signs of estrus, and cows were then artificially inseminated (AI) 24 hr later. All cows in the PS group were inseminated 72 hr after the second PGF₂α injection. In the SS group, cows were inseminated 24 hr after first signs of estrus. An additional 6 mature lactating Angus cows were added equally to the PS and PA groups to evaluate changes in serum LH. The percent calf crops was: SS = 40% (frsol|6/15); PS = 47% ($\text{7}\text{15}$); SA = 47% ($\text{7}\text{15}$); PA = 53% ($\text{8}\text{15}$). In the second study, 51 mature lactating Angus cows and 39 Holstein heifers were assigned at random to 3 treatment groups: saline + saline (SS); 33.4 mg PGF₂α tham salt + saline (PS); 33.4 mg PGF₂α tham salt + 1 mg D-leu⁶-des GlyNH₂¹⁰ LHRH/FSHRH ethylamide (D-leu⁶) (PL). PGF₂α tham salt or saline was administered im twice at an 11 day interval. D-leu⁶ or saline was administered 68 hr following the second PGF₂α treatment. Cows pretreated with PGF₂α were inseminated 80 hr after the second PGF₂α injection. In the SS group, cows were administered saline at the time of natural estrus and were artificially inseminated 12 hr later. Calving percent to the first AI was SS = 70% ($\text{21}\text{30}$); PS = 53% ($\text{16}\text{30}$) and PL = 40% ($\text{12}\text{30}$). An additional 10 mature lactating Angus cows were used to evaluate changes in serum LH. Five of the cows were assigned to the PS treatment and five to the PL treatment. Sequential blood samples were collected to monitor serum LH levels. Using the Chi-square test, there were no significant differences between calving percentages of the control and PGF₂α treated cows in either study. These results indicate that the PGF₂α treatments were successful in timed artificial insemination of cows without detection of estrus. The LHRH/FSHRH analogs did not improve the conception even though they appear to induce a pituitary release of LH simultaneously in all cows within 1 hr of treatment.     

Effect of mifepristone and antiestrogens on uterine PGF2α and PGE2 concentrations in ovariectomized and pregnant rats

Four antiestrogens (anordiol, tamoxifen, RU 39411, ICI 182780) and the antiprogestin, mifepristone (RU 486), were administered to the following three animal models: (1) ovariectomized rats, (2) mated rats treated post-coitally; and (3) pregnant rats treated post-implantation. The antiestrogens were administered alone or in combination with mifepristone at doses effective in preventing and/or terminating pregnancy in rats. The objective of the study was to determine whether these drugs influenced uterine concentrations of prostaglandins (PGF_2α and PGE₂).Antiestrogens administered alone to ovariectomized rats did not effect uterine PGE₂ or PGF_2α concentrations; whereas the combination of anordiol/mifepristone increased uterine PGF_2α concentration, resulting in an increase in the PGF_2α/PGE₂ ratio.Mated rats were treated post-coitally for three consecutive days with anordiol, tamoxifen, estradiol and mifepristone alone and with the combination of anordiol/mifepristone and tamoxifen/mifepristone. An increase in uterine PGF_2α concentrations and in the PGF_2α/PGE₂ ratio occurred only in anordiol/mifepristone treated group. A decrease in uterine PGE₂ concentrations occurred in animals treated with anordiol, tamoxifen and estradiol, resulting in an increase in the PGF_2α/PGE₂ ratio.Anordiol (5.0 mg/kg/day) and mifepristone (4.0 mg/kg/day) alone and the combination of anordiol/mifepristone (2.5/1.0 mg/kg/day) administered to pregnant rats on days 7, 8 and 9 of pregnancy induced an increase in PGF_2α levels without affecting uterine PGE₂ concentration. The changes in uterine PGF_2α concentrations induced by anordiol and the combination of anordiol/mifepristone resulted in an increase in the PGF_2α/PGE₂ ratio.The antiestrogens tested except for ICI 182780 possessed agonist activity when assayed by measuring their capacity to increase the uterine weights in ovariectomized rats. Also, ICI 182789 was the only antiestrogen that did not influence uterine PG concentrations. It can be concluded that ICI 182780 is the only “pure” antiestrogen among those tested.The present results show that antiestrogens and the combination of mifepristone plus anordiol at doses preventing implantation and terminating pregnancy increase uterine PGF_2α and/or decrease PGE₂ concentrations, resulting in an alteration of PGF_2α/PGE₂ ratio. These findings suggest that there exists a critical balance of PGF_2α to PGE₂ concentrations in the uterus required for the normal passage of fertilized ova through the oviduct, initiating implantation of the blastocysts, development of embryos, and maintenance of pregnancy.     

The roles of PGF2α and PGE2 in regression of the corpus luteum after intrauterine infusion of Arcanobacterium pyogenes in cows

To study the effect of bacteria in the uterus on the fate of the corpus luteum (CL), Arcanobacterium pyogenes was inoculated into the uteri of cows on Day 3 (Day 0 = day of spontaneous ovulation). Plasma concentrations of 13,14-dihydro-15-keto-PGF_2α (PGFM), 13,14-dihydro-15-keto-PGE₂ (PGEM) and progesterone (P₄) were determined. In five cows, the developing CL regressed and first-wave dominant follicles, which normally become atretic, ovulated (Group OV) after bacterial inoculation. In another five cows (Group NOV) and five control cows, the developing CL did not regress and first-wave dominant follicles did not ovulate. In Group OV, PGFM concentrations increased by 126.2 pg/mL (from 36.8 ± 7.8 pg/mL on Day 3 to 163 ± 37.2 pg/mL on Day 6), with an increase ratio of 5.8-fold. Conversely, in Group NOV, PGFM had a greater increase of 198.4 pg/mL (from 128.2 ± 27.8 pg/mL on Day 3 to 326.6 ± 115.1 pg/mL on Day 5), but the increase ratio was only 2.3-fold. Although PGEM tended to increase in both groups, raw increases and increase ratios were small. Bacterial inoculation into the uterus stimulated the release of prostaglandins and affected the fate of the CL; in that regard, the CL was affected more by PGF_2α than by PGE₂, and the increase ratio of PGF_2α was more important than the raw increase.     

Effect of PGD2, PGE2, PGF2α and PGI2 on blood pressure,heart rate and plasma catecholamine responses to spinal cord stimulation in the rat

The following experiments were designed in order to examine the inter-relationships of various prostaglandins (PG's) and the adrenergic nervous system, in conjunction with blood pressure and heart rate responses, $\text{in vivo}$. Stimulation of the entire spinal cord (50v, 0.3–3 Hz, 1.0 msec) of the pithed rat increased blood pressure, heart rate and plasma epinephrine (EPI) and norepinephrine (NE) concentration (radioenzymatic-thin layer chromatographic assay). Infusion of PGE₂(10–30 μg/kg. min, i.v.) suppressed blood pressure and heart rate responses to spinal cord stimulation while plasma EPI (but not NE) was augmented over levels found in control animals. PGI₂ (0.03–3.0 μg/kg. min, i.v.) suppressed the blood pressure response to spinal cord stimulation without any effect on heart rate or the plasma catecholamine levels. PGE₂ and PGF_2α(10–30 μg/kg. min, i.v.) did not change the blood pressure, heart rate or plasma EPI and NE responses to the spinal cord stimulation although PGF_2α disclosed an overall vasopressor effect during the pre-stimulation period. At the pre-stimulation period it was also observed that PGE₂, PGF_2α and PGI₂, had a positive chronotropic effect on the heart rate, the cardiac accelerating effect of PGE₂ was not abolished by propanolol. These $\text{in vivo}$ studies suggest that in the rat, PGE₂ and PGI₂ modulate sympathetic responses, primarily by interaction with the post-synaptic elements — PGE₂ on both blood vessels and the heart and PGI₂ by acting principally on blood vessels.     

Effect of PGF2α and neostigmine on induction of parturition,farrowing interval and litter survival in swine

An investigation of the effect of PGF₂α induced parturition alone or in combination with neostigmine (NEO) on subsequent productivity was conducted in a total confinement swine production unit. Forty-five crossbred gilts and 106 sows were randomly assigned to one of six treatments: (1) $\text{0 mg PGF}{}_2\text{α}\text{0 mg NEO}$, (2) $\text{10 mg PGF}{}_2\text{α}\text{0 mg NEO}$, (3) $\text{0 mg PGF}{}_2\text{α}\text{5 mg NEO}$, (4) $\text{10 mg PGF}{}_2\text{α}\text{5 mg NEO}$, (5) $\text{0 mg PGF}{}_2\text{α}\text{10 mg NEO}$, (6) $\text{10 mg PGF}{}_2\text{α}\text{10 mg NEO}$. PGF₂α injections (IM) were given 2 days prior to the mean expected farrowing day for this farm (day 112). NEO injections (IM) were given after the fourth pig born in gilt litters and after the fifth pig born in sow litters. Females injected with 0 or 10 mg PGF₂α farrowed 71.7 ± 3.7 and 31.4 ± 3.4 hours after treatment, respectively (P<0.01). No other parameters measured were significantly affected by injection of PGF₂α. These data show that PGF₂α effectively induced parturition and did not adversely affect subsequent litter productivity. NEO was found to be ineffective in reducing total farrowing time (P>0.05).     

Luteal function in the hysterectomized bitch following treatment with prostaglandin F2α (PGF2α)

Estrus and ovulation were induced in ten mature, mixed-breed, anestrous bitches (10 to 20 kg) using exogenous gonadotropins. Bitches were bred once, on the second day of estrus. Between 11 and 13 days following estrus, bitches were bilaterally hysterectomized and randomly divided into two treatment groups of five bitches each. Four days following surgery, Group A (treated) was given a single subcutaneous injection of PGF₂α (Prostin F₂ alpha^®) at a dose of 1 mg/kg body weight and Group B (controls) similarly given an equal volume of .9% saline. Blood samples were collected daily by cephalic venipuncture prior to surgery and for 75 days thereafter. Plasma progesterone was monitored by a radioimmunoassay method. Although bitches were teased daily following PGF₂α or saline treatments, estrual behavior was not exhibited. In both the PGF₂α and saline treatment groups, plasma progesterone levels showed a transient decline by 12 hours following injection, although a more dramatic decrease was observed at this time in the prostaglandin-treated bitches. Subsequently, progesterone concentrations tended to increase in both groups at 6 days following treatment, however, not to pre-treatment levels. Within 20 to 32 days following treatment in both groups, plasma progesterone levels declined to <1 ng/ml and remained depressed at least 60 days post-injection. In this study, complete luteal regression was not induced following PGF₂α treatment. Luteal function in both groups, as indicated by plasma progesterone concentrations, was shortened in the absence of the uterus.     

Effect of exogenous estradiol and progesterone on the uterine tissue levels of prostaglandin F2α (PGF2α) in ovariectomized mice

Mice ovariectomized for 14 days were treated for 6 days with estradiol and/or progesterone. Both the steroids were effective in increasing the levels of PGF_2α in the uterine tissue, but the treatment with progesterone for 3 days followed by 3 days of estrogen resulted in a highly significant production of PGF_2α. It is concluded that for the production of PGF_2α both estrogen and progesterone are necessary and that the pretreatment with progesterone followed by estrogen results in the maximum production of PGF_2α.     

Localization and function of cytosolic phospholipase A2α at the Golgi

Cytosolic phospholipase A₂α (cPLA₂α, Group IVA phospholipase A₂) is a central mediator of arachidonate release from cellular phospholipids for the biosynthesis of eicosanoids. cPLA₂α translocates to intracellular membranes including the Golgi in response to a rise in intracellular calcium level. The enzyme’s calcium-dependent phospholipid-binding C2 domain provides the targeting specificity for cPLA₂α translocation to the Golgi. However, other features of cPLA₂α regulation are incompletely understood such as the role of phosphorylation of serine residues in the catalytic domain and the function of basic residues in the cPLA₂α C2 and catalytic domains that are proposed to interact with anionic phospholipids in the membrane to which cPLA₂α is targeted. Increasing evidence strongly suggests that cPLA₂α plays a role in regulating Golgi structure, tubule formation and intra-Golgi transport. For example, recent data suggests that cPLA₂α regulates the transport of tight junction and adherens junction proteins through the Golgi to cell–cell contacts in confluent endothelial cells. However, there are now examples where data based on knockdown using siRNA or pharmacological inhibition of enzymatic activity of cPLA₂α affects fundamental cellular processes yet these phenotypes are not observed in cells from cPLA₂α deficient mice. These results suggest that in some cases there may be compensation for the lack of cPLA₂α. Thus, there is continued need for studies employing highly specific cPLA₂α antagonists in addition to genetic deletion of cPLA₂α in mice.     

Decreased blood flow through rat testis after intratesticular injection of PGF2α

The blood flow through the testes was calculated for 26 rats after measurements of the clearance rate of intratesticularly injected ¹³³Xenon dissolved in saline.The testicular blood flow in the rats was measured immediately after injection of 1 or 10 μg PGF_2α into each testis, as well as one and four weeks later. The range of group mean flows in the contral groups was 27.5–37.0 ml/100 g/min. The injection of 1 or 10 μg PGF_2α immediately decreased (p < 0.025 and p < 0.0005) the flow to 12.0 and 4.3 ml/100 g/min, respectively. One and four weeks later, no difference was found between the control and experimental groups. At autopsy during the fourth week, no difference was found between the control and PGF_2α - treated groups with regard to the weight of the adrenals, testes, prostate gland and seminal vesicles, or in the histological appearance of the testes.Unilateral injection of 10 μg PGF_2α decreased the flow through the injected testis and tended to decrease the flow through the non-injected testis.     

Comparison of the actions of the 15-methyl analogs of prostaglandins E2 and F2α on hormone levels after their administration for therapeutic abortion

Plasma levels of progesterone, total estrogens and HCG were measured after the administration of 15 (S) 15-methyl prostaglandin E₂ methyl ester (15-methyl PGE₂) or 15 (S) 15-methyl prostaglandin F_2α free acid (15-methyl PGF_2α) for therapeutic abortion during the first trimester of pregnancy. 15-methyl PGE₂ given intramuscular (im) in a dose of 50μg resulted in the termination in pregnancy in four out of five patients; these subjects exhibited falls in hormone concentrations. However, an im injection of 500μg 15-methyl PGF_2α did not affect the hormone levels nor did it produce abortion in any of the five subjects studied. The results confirm that 15-methyl PGE₂ is a potent abortifacient and this action may be related to an effect that this compound has on hormone production by the corpus luteum or the feto-placental unit; 15-methyl PGF_2α does not share the same action in the dose range investigated.     

HT-29 human colon cancer cell proliferation is regulated by cytosolic phospholipase A2α dependent PGE2via both PKA and PKB pathways

Cytosolic phospholipase A₂α (cPLA₂α) up-regulation has been reported in human colorectal cancer cells, thus we aimed to elucidate its role in the proliferation of the human colorectal cancer cell line, HT-29. EGF caused a rapid activation of cPLA₂α which coincided with a significant increase in cell proliferation. The inhibition of cPLA₂α activity by pyrrophenone or by antisense oligonucleotide against cPLA₂α (AS) or inhibition of prostaglandin E₂ (PGE₂) production by indomethacin resulted with inhibition of cell proliferation, that was restored by addition of PGE₂. The secreted PGE₂ activated both protein kinase A (PKA) and PKB/Akt pathways via the EP2 and EP4 receptors. Either, the PKA inhibitor (H-89) or the PKB/Akt inhibitor (Ly294002) caused a partial inhibition of cell proliferation which was restored by PGE₂. But, inhibited proliferation in the presence of both inhibitors could not be restored by addition of PGE₂. AS or H-89, but not Ly294002, inhibited CREB activation, suggesting that CREB activation is mediated by PKA. AS or Ly294002, but not H-89, decreased PKB/Akt activation as well as the nuclear localization of β-catenin and cyclin D1 and increased the plasma membrane localization of β-catenin with E-cadherin, suggesting that these processes are regulated by the PKB pathway. Similarly, Caco-2 cells exhibited cPLA₂α dependent proliferation via activation of both PKA and PKB/Akt pathways. In conclusion, our findings suggest that the regulation of HT-29 proliferation is mediated by cPLA₂α-dependent PGE₂ production. PGE₂via EP induces CREB phosphorylation by the PKA pathway and regulates β-catenin and cyclin D1 cellular localization by PKB/Akt pathway.     

Extra-amniotic 15(S)-15 methyl PGF2α to induce abortion: A study of three administration schedules

Abortion was induced in 60 patients between 8 and 18 weeks gestation using 15(S)-15 methyl PGF₂α in one of three extra-amniotic administration schedules: 1.0 mg in viscous medium (Tylose), 1 mg in viscous medium (Hyskon) or 0.5 mg in non-viscous medium repeated at 12 hours. Eighty per cent of patients aborted within 24 hours in each group. The overall mean induction-abortion interval (± S.E.) was 17.6 ± 2.0: there was no significant difference between the three groups. Twenty patients treated with 1.0 mg in viscous medium had the catheter removed immediately following the prostaglandin injection and the success rate was not significantly altered.Gastro-intestinal side effects (vomiting in 50%, diarrhoea in 32.5%) were more frequent in the patients treated with the larger dose though the difference was not statistically significant. No significant haematological or biochemical changes were detected during the 24 hours following the start of treatment in 24 patients investigated. Thirty seven of the 60 patients (61.5%) aborted completely and did not require surgical evacuation, and none lost more than 500 ml of blood, nor required transfusion.It is concluded that abortion can be induced with a single extra-amniotic injection of 1 mg of 15(S)-15 methyl PGF₂α in viscous medium in a large percentage of patients but that the incidence of side effects is high.     

Alkylsulfanyl analogs as potent α2δ ligands

We identified novel (3R, 5S)-3-aminomethyl-5-methanesulfanyl hexanoic acid (5a: DS75091588) and (3R, 5S)-3-aminomethyl-5-ethanesulfanyl hexanoic acid (6a: DS18430756) as sulfur-containing γ-amino acid derivatives that were useful for the treatment of neuropathic pain. These two compounds exhibited a potent analgesic effect in animal models of both type I diabetes and type II diabetes, and good pharmacokinetics.     

Side chain SAR of bicyclic β-lactamase inhibitors (BLIs). 2. N-Alkylated and open chain analogs of MK-8712

The bridged monobactam β-lactamase inhibitor MK-8712 (1) effectively inhibits class C β-lactamases. Side chain N-alkylated and ring-opened analogs of 1 were prepared and evaluated for combination with imipenem to overcome class C β-lactamase mediated resistance. Although some analogs were more potent inhibitors of AmpC, none exhibited better synergy with imipenem than 1.     

The effects of prostaglandins E2 and F2α on synaptosomal accumulation and release of 3H-norepinephrine

Prostaglandins (PG) of both the E and F series may serve as modulators of norepinephrine (NE) release from peripheral sympathetic neurons. We have studied the effects of PGE₂ and PGF_2α on the accumulation and release of ³H-NE in the CNS using synaptosomes isolated from rat hypothalami.The release of ³H-NE from synaptosomes superfused with Krebs-Ringer bicarbonate buffer was multiphasic with an initial fast release phase followed by a slower release. Raising KC1 concentration of the superfusion medium to 56mM during the slow release phase is known to stimulate ³H-NE release. PGE₂ (1 × 10^?6M) attenuated ³H-NE release during the fast phase and reduced the amount of ³H-NE released due to KC1 stimulation. At lower concentrations of PGE₂ there was no change in the release profile. PGF_2α was without effect on ³H-NE release at all concentrations tested.The accumulation of ³H-NE was significantly diminished by PGE₂ at a concentration of 1 × 10^?6M, while a lower concentration (1 × 10^?7M) was ineffective. PGF_2α had no effect on ³H-NE accumulation at all concentrations investigated.     

Biophysical studies on molecular mechanism of abortificient action of prostaglandins—IV. Conformation energy calculations on PGA1, PGB1 and PGE1

This paper reports the conformation energy (CE) calculations on three forms of prostaglandins (PGs) PGA₁, PGB₁ and PGE₁ on the basis of the empirical potential energy functions, for the simultaneous rotations around C₇–C₈ (θ), C₁₂–C₁₃ (β) and C₁₄–C₁₅ (β) bonds [Fig. 1(a)]. The isoenergy contours plotted for θβ rotations for the different β values show the existence of two low energy regions for thg equal to about 90° and 240° in all the three cases. The absolute minimum was obtained for thg = 240° and almost coincided with the crystallographic conformation for PGE₁ and PGA₁. In the case of PGB₁ series of low energy conformations were obtained with the thg values equal to about 90° and 270°, but none of them coincided with the observed crystallographic conformation. The paper discusses the comparison of the different low energy conformations in these three molecules, their biological relevance and the cause of disagreement in the case of PGB₁ with the crystallographic data.     

Comparison of the effects of prostaglandins D2, F2α and E1 on spontaneous contractions of rabbit oviduct

The effects of PGD₂, PGF_2α and PGE₁ were studied on the circular muscle of post-ovulatory rabbit oviducts $\text{in vitro}$. PGE₁ inhibited spontaneous contractile activity. Lower concentrations of PGD₂ and PGF_2α were stimulatory and higher concentrations were inhibitory. Since PGD₂ may be produced in the oviduct, any hypothesis concerning the role of prostaglandins in the control of oviductal motility and ovum transport should include PGD₂ as well as PGFs and PGEs.     

Effect of gonadotropin-releasing hormone and prostaglandin F2α on reproduction in postpartum dairy cows

Thirty dairy cows serving as the treated group (Group A) were injected intramuscularly with 100 mcg gonadotropin-releasing hormone (GnRH) at 10 to 16 days postpartum followed by 25 mg prostaglandin F₂α (PGF₂α) 14 days later. Twenty-nine herdmate dairy cows (Group B) serving as controls were treated in a similar manner using saline injections rather than GnRH or PGF₂α treatments. Only cows without obvious uterine infection were assigned to the experimental groups, and any uterine pathology that developed during the treatment interval was treated accordingly following the experimental period. Internal genitalia were evaluated via rectal palpation prior to each injection. Blood samples were collected for progesterone analysis before each injection and at 30 hours following the PGF₂α or the second saline injection. Experimental animals were artificially inseminated at the first detected postpartum estrus starting 35 to 40 days following calving. Results indicated evidence of enhanced cyclicity when Group A cows were compared with those in Group B. However, there were no significant differences between the two groups for interval to first observed estrus, interval to first serive, first serive pregnancy rate, services per pregnancy and days open. Furthermore, no difference in the incidence of follicular or luteal cysts, incidence of repeat breeders or number of reproductive culls was observed. From observations in this study, the GnRH and PGF₂α treatment scheudule might not be economically beneficial in lactating dairy cows as long as reproductive tract abnormalities are promptly diagnosed and subsequently treated by the attending practitioner.     

High performance liquid chromatography studies on the interactions of cis-Pt(NH3)2−(H2O)22+ with guanine and methylated g

Time dependence studies, using high performance liquid chromatography (HPLC), on the reaction between cis-diamminediaquoplatinum and guanine, N1-methylguanine, N7-methylguanine, N9-methylguanine, and N1 ,N7-dimethylguanine are reported. Each reaction gave rise to eight or more compounds; the major components have been prepared and characterization by ¹H and ¹⁹⁵Pt nuclear magnetic resonance has been attempted. Species of the form ((NH₃)₂Pt(NO₃)-(G-H)-(NO₃)-Pt(NH₃)₂)⁺, (NH₃)₂,Pt(G-H)(NO₃) monomer and (NH₃)₂Pt(G-H)(NO₃) dimer, where G-H indicates the guanine monoanion, are postulated.