Increased sperm output of rabbits and bulls treated with prostaglandin F2α

Seven rabbits were ejaculated four times once weekly, and saline or 2.5 mg PGF_2α tromethamine salt was injected sc at 4 and 2 hours or at 8 and 4 hours before ejaculation. First ejacula taken at 2 hours after the second injection of PGF_2α contained 150% more (P.07) sperm than those after injections of saline. The comparable difference (60%) at 4 hours after PGF_2α was not significant. PGF_2α did not influence sperm output in second, third or fourth ejacula. After 28 daily sc injections of 5 mg PGF_2α in another experiment, the fertility of four treated rabbits was as high as that for four controls. Without sexual preparation in seven bulls, im injections of 40 or 80 mg PGF_2α 30 minutes before ejaculation resulted in 33% greater (P<.05) sperm output than that after injection of 0, 7 or 20 mg PGF_2α, but the highest sperm output after PGF_2α was 30% less (P<.05) than that after sexual preparation in the same bulls. We conclude that injections of PGF_2α result in increased sperm output in ejacula taken without sexual preparation within 2 hours in rabbits and in bulls.     

Increased blood LH and testosterone after administration of prostaglandin F2α in bulls

Two types of experiments were conducted to determine the relationship of changes in blood luteinizing hormone (LH) and testosterone in bulls given prostaglandin F_2α (PGF_2α). Episodic surges of LH and testosterone occurred in tandem, apparently at random intervals, on the average once during the 8-hr period after bulls were given saline. In contrast, after sc injection of 20 mg PGF_2α, blood serum testosterone increased synchronously to a peak within 90 minutes four-fold greater than pre-injection values, and the testosterone surges were prolonged about three-fold compared to those in controls. Each of the PGF_2α-induced surges of testosterone was preceded by a surge of blood serum LH which persisted for about 45 minutes and peaked at about 3 ng/ml. In a second experiment, PGF_2α was infused (iv, 0.2 mg/min) for 20 hr; blood plasma testosterone increased from 7.0 ± 0.6 to 16.0±1.5 ng/ml within 2.5 hr and remained near this peak for 10 hr. Then testosterone gradually declined to about 9 ng/ml at the conclusion of the 20-hr infusion. These changes in testosterone were paralleled by similar changes in blood plasma LH, although LH declined 3 hr earlier than testosterone. Random episodic peaks of blood plasma LH and testosterone typical of untreated bulls resumed within 8 hr after conclusion of PGF_2α infusion. In both experiments, the surge of testosterone after PGF_2α was preceded by increased blood LH. We conclude that increased LH after administration of PGF_2α probably caused the increased testosterone. However the mechanisms of these actions of PGF_2α remain to be determined.     

Prostaglandin E2 opunteracts the effects of PGF2α in indomethacin treated cycling gilts

Twenty crossbred gilts with at least 2 consecutive estrous cycles of 18 to 21 days in length were used to study the effects of prostaglandins E₂ and F₂α (PGE₂ and PGF₂α) on luteal function in indomethacin (INDO) treated cycling gilts. Intrauterine and jugular vein catheters were surgically palced before day 7 of the treatment estrous cycle and gilts were randomly assigned to 1 of 5 treatment groups (4/groups). With exception of the controls (Group I) all gilts received 3.3 mg/kg INDO every 8 h, Groups III, IV and V received 2.5 mg PGF₂; 2.5 mg PGF₂α + 400 μg PGE₂ every 4 hr, or 400μg PGE₂ every 4 h, respectively. All treatments were initiated on day 7 and continued until estrus or day 23. Jugular blood for progesterone analysis was collected twice daily from day 7 to 30. Estradiol-17β (E₂-17β) concentrations were dtermined in samples collected twice daily, from 2 d before until 2 d following the day of estrus onset. When compared to pretreatment values, estrous cycle length was unaffected (P>0.05) in Group I, prolonged (P<0.05) in Groups II, IV and V; and shortened (P<0.05) in Group III. The decline in plasma progesterone concentration that normally occurs around day 15 was unaffected (P>.05) in Group I; delayed (P<0.05) in Groups II, IV and V; and occurred early (P<0.05) in Group III. Mean E₂-17β remained high (31.2 ± 4.9 to 49.3 ± 3.1 pg/ml) in Groups III and IV, while the mean concentrations in Groups III and V varied considerably (17.0 ± 2.0 to 52.2 ± 3.5 pg/ml). The results of this study have shown that PGE₂ will counteract the effects of PGF₂α in INDO treated cycling gilts. The inclusion of PGF₂α appeared to either stimulate E₂-17β secretion or maintain it at a higher level than other treatments.     

Radioimmunological and biological measurement of prostaglandins in rabbit urine : Decrease of PGE2 excretion at high NaCl intake

The estimation of prostaglandin (PG) E₂ and of PGF_2α by radioimmunoassay is described in detail. PGE₂ was measured after conversion to either PGB₂ or PGF_2α and the results compared to bioassay. The methods were used to follow the excretion of PGE₂ and PGF_2α after salt loading in rabbits. A marked reduction of PGE₂ levels was observed at high NaCl intake, while PGF_2α excretion remained unchanged.     

Effects of prostaglandins E1 and F2α on serum luteinizing hormone concentration and on some sexual functions in male rabbits

PGE₁(50μg/animal) and PGF_2α (250 μg/animal) caused a transient in serum LH at 5 min after injection. PGE₁ (250 μg/animal) had a biphasic effect on serum LH. A small peak was obtained at 5 min, and a second, larger peak at 60 min after injection. It is suggested that the first peak is a result of the stress associated with injection of the PGs, whereas the second peak represents a physiological effect of PGE. Subcutaneous injection of PGE₁ (1 mg in arachis oil b.i.d.) for 10 days did not affect the concentration of LH in serum, the function of the accessory sexual glands or the sexual activity. PGF_2α, given at the same dose and in the same manner, increased the sexual activity but left all other variables unaffected. The pituitary responsiveness to LH-RH was unaltered by the treatment with PGE₁ and PGF_2α.     

Stereospecificity of enzymatic reduction of prostaglandin E2 to F2α

Antibodies directed toward PGF_2β were prepared in rabbits. The serologic specificity of the immune reaction was determined by inhibition of sodium borohydride-reduced (³H) PGE₂ anti-PGF_2β binding by several prostaglandins. The antibodies to PGF_2β recognize the β-hydroxyl configuration in the cyclopentane ring of PGF_2β. With the use of both anti-PGF_2α and anti-PGF_2β, the product of PGE₂ reduction by 9-ketoreductase purified from chicken heart was identified as PGF_2α. Guinea pig liver and kidney homogenates were examined for PGE 9-ketoreductase activity. Although enzyme activity was present, no evidence of PGF_2β production was found.     

Blood LH after PGF2α in diestrous and ovariectomized cattle

Two experiments were conducted to determine whether the increased serum LH which occurs within 12 hr after a luteolytic dose of PGF_2α is dependent upon changes in progesterone or estradiol secretion. In the first experiment, exogenous progesterone abolished the increase in serum LH caused by a subcutaneous injection of 25 mg PGF_2α in diestrous heifers, but not in ovariectomized heifers. In the second experiment, progesterone pessaries were removed at 6 hr after a subcutaneous injection of 25 mg PGF_2α. LH remained at pre-PGF_2α values while the pessaries were in place, but began to increase within 1 hr after they were removed. Blood estradiol also remained at pre-PGF_2α values until the pessaries were removed, and began to increase at 2 hr after pessary removal. We conclude that the increase in serum LH within 12 hr after PGF_2α treatment in diestrous cattle is dependent upon withdrawal of progesterone; it is not due to increased serum estradiol.     

Interactions of prostaglandins with subpopulations of ovine luteal cells. II. Inhibitory effects of PGF2α and protection by PGE2

Purified preparations of ovine large luteal cells were utilized in a series of experiments to test the effects of prostaglandins (PG) E₂ abd F₂α on cell morphology, viability and secretion of progesterone. Luteal cells were allowed to attach to culture dishes overnight before experiments. In the first series of experiments incubation of large steroidogenic cells with PGF₂α for 6 hr resulted in morphological changes including a retraction of the cell cytoplasm and apparent extrusion of cytoplasmic components which became more pronounced after 12 hr. In a second series of experiments, PGF₂α decreased and PGE₂ increased progesterone accumulation in media after 6 hr when media were not replaced during the incubation period, while progesterone accumulation was not different than that observed in control dishes when both prostaglandins were present. Hourly replacement of the media negated the inhibitory effects of PGF₂α but had no effect on the stimulated secretion of progesterone induced by PGE₂. Finally, in incubations without media replacement, PGF₂α induced a dose-dependent decrease in progesterone accumulation while PGE₂ elicited a biphasic response with progesterone secretion increasing from 0.1 ng/ml to maximal levels at 10 ng/ml followed by a dose-dependent decrease at 100 and 1000 ng/ml. These data are compatible with the hypotheses that: 1) luteolysis is initiated, at least in part, by an action of PGF₂α on large luteal cells; and 2) the embryonic signal from the pregnant uterus which rescues the ovine corpus luteum may be PGE₂.     

PGE2 attenuates PGF2α-induced increases in free intracellular calcium in ovine large luteal cells

When ovine large luteal cells are placed in culture and exposed to PGF_2α, there is a rapid and sustained increase in the concentration of free intracellular calcium which is believed to play a major role in the luteolytic and cytotoxic effects of PGF_2α. Since administration of exogenous PGE₂ can prevent spontaneous and PGF_2α-induced luteolysis in vivo, and the cytotoxic effects of PGF_2α on large luteal cells in vitro, the objective of this study was to determine if one mechanism by which PGE₂ acts is to attenuate increases in free intracellular calcium induced by PGF_2α. At concentrations of 10 nM or greater, PGF_2α caused a significant and sustained increase in free intracellular calcium in large luteal cells. Similarly, PGE₂ also induced increases in free intracellular calcium but required doses 20-fold greater than PGF_2α. When PGE₂ (1, 10 or 100 nM) was incubated with PGF_2α (100 nM) increases in free intracellular calcium induced by PGF_2α were attenuated (P<0.05) when measured 5 min, but not at 30 min, after initiation of treatment. The observed decrease in the concentration of free intracellular calcium at 5 min in response to PGF_2α was the result of fewer cells responding to PGF_2α. In addition, the concentrations of free intracellular calcium attained in the cells that did respond was reduced 25% compared to cells treated with PGF_2α alone. Thus, part of the luteal protective actions of PGE₂ appears to involve an inhibition of the early (5 min) increase in free intracellular calcium induced by PGF_2α.     

Prostaglandin E2: Midterm abortion in rats using Silastic-PVP tubes

The results of the present study establish that 1.5 mg PGE₂ (lyophilized sodium salt) incorporated in one cm long open-ended Silastic-polyvinylpyrrolidone (PVP) tube when inserted into 10 day pregnant rats induced abortion within 70–72 hours in all the treated rats. A combined treatment of PGE₂ and 17β-estradiol failed to increase the abortion inducing effect of a Silastic-PVP-PGE₂ tube. It is observed that PGE₂ is about 4 times less potent than PGF_2α in inducing midterm abortion in rats. It is suggested that either PGE₂ exerts luteolytic effect after being converted to PGF_2α, although how it occurs is not clear; or PGE₂ causes expulsion of the fetuses by its uterine stimulating property. 17β- estradiol increases the uterine synthesis of PGF_2α as described earlier but seems not affecting the production of PGE₂ by the uterus. The release rate of ³H-PGE₂ from Silastic-PVP tube and is also described.     

Characterization and ontogeny of PGE2 and PGF2α receptors on the retinal vasculature of the pig

The vasoconstrictor effects of PGE₂ and PGF_2α are less pronounced on retinal vessels of the newborn than of the adult pig. We tested the hypothesis that the decreased vasomotor response to these prostaglandins might be due to relatively fewer receptors and/or different receptor subtypes (in the case of PGE₂) on retinal vessels of the newborn animal. Binding studies using [³H]PGE₂ and [³H]PGF_2α revealed that PGE₂ (EP) and PGF_2α (FP) receptor densities in retinal microvessel membrane preparations from newborn animals were approximately 25% of those found in vessels from the adult. The K_d for PGF_2α did not differ; however, the K_d for PGE₂ was less in newborn than in adult vessels. Competition binding studies using AH 6809 (EP₁ antagonist), butaprost (EP₂ agonist), M&B 28,767 (EP₃ agonist), and AH 23848B (EP₄ antagonist) suggested that the retinal vessels of the newborn contained approximately equal number of EP₁ and EP₂ receptor subtypes whereas the main receptor subtype in the adult vessels was EP₁. In addition, PGE₂ and butaprost produced comparable increases in adenosine 3′,5′-cyclic monophosphate synthesis in newborn and adult vessels. PGE₂, 17-phenyl trinor PGE₂ (EP₁agonist) and PGF_2α caused a 2.5 to 3-fold greater increase in inositol1,4,5-triphosphate (IP₃) formation in adult than in newborn preparations. It is concluded that fewer PGF_2α receptors and an associated decrease in receptor-coupled IP₃ formation in the retinal vessels of the newborn could lead to weaker vasoconstrictor effects of PGF_2α on retinal vessels of the newborn than of adult pigs; fewer EP₁ receptors (associated with vasoconstriction) and a relatively greater proportion of EP₂ receptors (associated with vasodilation) might be responsible for the reduced retinal vasoconstrictor effects of PGE₂ in the newborn.     

Differential effects of detergents and dimethylsulfoxide on membrane prostaglandin E1 and F2α receptors

Pretreatment of membranes for 1 hr at 4° with up to 0.1% Triton X-100 (TX-100) and sodium desoxycholate (SDC), resulted in a greater loss of [³H] prostaglandin (PG)F₂α binding compared to E₁ binding. Lubrol WX (LWX) tended to cause a greater loss of [³H]PGF₂α than E₁ binding. However, the differential loss was not as marked as with TX-100 or SDC. Triton X-305 was relatively ineffective, but loss of [³H]PGE₁ binding was greater than for PGF₂α. Increasing concentrations of dimethylsulfoxide (DMSO) progressively inhibited PGF₂α binding without affecting PGE₁ binding. The detergent, but not DMSO, induced losses of membrane PG binding were due to solubilization of the receptors. Greater amounts of membrane protein and phospholipids were solubilized at detergent (TX-100 and SDC) concentrations that solubilized 100% of PGE₁ receptors compared to 100% solubilization of F₂α receptors. Neither the duration of preincubation nor the amount of membrane protein chosen were responsible for differential PGE₁ and F₂α receptor losses. These differential membrane PG receptor losses raise the possibility of differences in PGE₁ and F₂α receptors association with the membrane structure.     

Radioimmunoassays of prostaglandin F2α and prostaglandin F2α-main urinary metabolite with prostaglandin-I-tyrosine methylester amide

Radioimmunoassays for measuring prostaglandin F_2α (PGF_2α) and 5α, 7α-dihydroxy-11-keto tetranorprosta-1,16-dioic acid, PGF_2α-main urinary metabolite (PGF_2α-MUM), with ¹²⁵I-tyrosine methylester amide (TMA) of PGF_2α and PGF_2α-MUM were developed.Antibody to PGF_2α was produced in rabbits immunized with conjugates of PGF_2α coupled to bovine serum albumine. Antibody to PGF_2α-MUM was also produced in rabbits immunized with conjugates of PGF_2α-MUM coupled to bovine serum albumin.PGF_2α-¹²⁵I-TMA had an affinity to antiserum to PGF_2α. PGF_2α-MUM-¹²⁵I-TMA also responded to antiserum to PGF_2α-MUM.     

Radioimmunoassay of prostaglandins E1, E2, and F2α in unextracted plasma, serum and myocardium

A radioimmunoassay procedure for the determination of PGE₁, PGE₂, and PGF₂α is presented. The procedure involves the pre-precipitation of each prostaglandin specific antiserum with the precipitating antisera (ARGG), and the use of these antisera mixtures in assaying for PGE₁, PGE₂, and PGF₂α. Applicability of the methods to unextracted plasma, serum and myocardial homogenate has been demonstrated through tests of specificity, recovery, reproducability and parallelism. A mathematical correction for cross-reactivity between PGE₁ and PGE₂, and their opposing antisera is given. To demonstrate the utility of the methodology in differentiation of experimental variables, prostaglandin concentrations in unincubated serum, incubated serum, and the rate of prostaglandin production in serum of dogs are given.     

Influence of prostaglandin F2α on sperm production and seminal characteriticss of the stallion

Six mature stallions were used to test the effect of prostaglandin F_2α (PGF_2α) on sperm production and seminal characteristics. Semen was collected from each stallion twice weekly 1 hr following a 10 mg intramuscular injection of PGF_2α or a sham injection. A switchback design was used so that three stallions received PGF₂ and three served as controls during the first 9 weeks (period 1). Threatment regimens were reversed during the second 9 weeks (period 2). Treatment of stallions with PGF_2α resulted in an increase (/prmP<.05) in gel free seminla voume and a decrease in sperm cell concentration. Total spermatozoa, sperm cell motility, and percentage of primary and secondary sperm abnormalities or ejaculates were not significantly affected by treatment of stallions with PGF_2α before semen collection. All treated stallions exhibited a pronounced sweating response to the drug. During the experiment, two of the six stallions masturbated within 20 to 30 minutes after PGF_2α treatment without achieving an erection.     

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

The role of progesterone in regulation of uteroovarian venous concentrations of prostaglandins F₂ α (PGF₂α) and E₂ (PGE₂) during days 13 to 16 of the ovine estrous cycle or early pregancy was examined. At estrus, ewes were either mated to a fertile ram or unmated. On day 12 postesturus, ewes were laparotomized and a catheter was inserted into a uteroovarian vein. Six mated and 7 unmated ewes received no further treatment. Fifteen mated and 13 unmated ewes were ovariectomized on day 12 and of these, 7 mated and 5 unmated ewes were given 10 mg progesteron sc and an intravaginal pessary containing 30 mg of progesterone. Uteroovarian venous samples were collected every 15 min for 3 h on days 13 to 16 postestrus. Mating resulted in higher mean daily concentrations of PGE₂ in the uterovarian vein than in unmated ewes. Ovariectomy prevented the rise in PGE₂ with day in mated ewes but had no effect in unmated ewes. Progesterone treatment restored PGE₂ in ovariectomized, mated ewes with intact embros. Mating had no effect on mean daily concentrations of PGF₂α or the patterns of the natural logarithm (ln) of the invariance of PGF₂α. Ovariectomy resulted in higher mean concentrations and ln invariances of PGF₂α on day 13 and lower mean concentrations and ln invariances of PGF₂α on days 15 and 16. Replacement with progesterone prevented these changes in patters of mean concentrations and ln variances of PGF₂α following ovariectomy. It is concluded that progesterone regulates the release of PGF₂α from the uterus, maintaining high concentrations while also preventing the occurrence of the final peaks of PGF₂α which are seen with falling concentrations of progesterone. This occurs in both pregnant and non-pregnant ewes. Progesterone is also needed to maintain increasing concentrations of PGE₂ in mated ewes.     

The effect of adrenergic stimulation on urinary prostaglandin E2 and 6 keto PGF1α in man

To evaluate the details of the adrenergic stimulation of urinary prostaglandins in man, ten normal volunteers were given various agonists and antagonists. The effect of 4 hour IV infusions of norepinephrine (NE), NE + phentolamine (PHT), NE + phenoxybenzamine (PHB), NE + prazosin (PZ), isoproterenol (ISO), and PHT alone on urinary PGE₂ and PGI₂ (6 keto PGF_1α) were determined. PGE₂ and 6 keto PGF_1α were measured by radioimmunoassay from 4 hour urine samples. NE stimulated both PGE₂ (196±40 to 370±84 ng/4 hrs/g creatinine and 6 keto PGF_1α(184±30 to 326±36), both p<0.01. In contrast, ISO had no effect on either PGE₂ or 6 keto PGF_1α excretion. Alpha blockade with PHT. PHB, or PZ inhibited the NE induced systemic pressor effect. However, the effect of the alpha blockers on the NE induced stimulation of PGE₂ and 6 keto PGF_1α varied. PHT did not alter the NE stimulated PGE₂ or 6 keto PGF_1α release (370±84 vs. 381±80) PGE₂ and (326±50 vs. 315±40) 6 keto PGF_1α, both p>0.2). PHT alone stimulated only 6 keto PGF_1α. PHB and the specific α₁ antagonist PZ similarly eliminated the NE induced prostaglandin release. These results suggest that adrenergically mediated urinary prostaglandin release in man is via an alpha receptor with α₁ characteristics.     

The effects of prostaglandins PGE1, PGE2, PGF1a, and PGF2a on canine synovial perfusion

In these experiments we have examined the effects of PGE₁, PGE₂, PGF_1α and PGF_2α on synovial perfusion in the normal canine synovial microcirculation. The effects of the drugs on synovial perfusion were determined indirectly from the changes produced in the rate of clearance of ¹³³Xenon from the joint by their intra-articular injection. Prostaglandins PGE₁ and PGE₂ were found to be strongly vasodilator with PGE₁ being the more active. PGF_1α appeared to have little or no vasoactive properties in doses up to 1 ugm. (2.8 × 10^−5M) in our I preparation while PGF_2α was vasodilator at this high dosage only. Neither SC19920 nor diphloretin phosphate antagonised the effects of PGE₁ in these experiments.     

Biosynthesis of prostaglandins and thromboxane B2 by fetal lung homogenates

The conversion of arachidonic acid to prostaglandins (PG's) and thromboxane B₂ (TXB₂) was investigated in homogenates from fetal and adult bovine and rabbit lungs. Adult bovine lungs were very active in converting arachidonic acid (100 μg/g tissue) to both PGE₂ (10.7 μg/g tissue) and TXB₂ (6.2 μ/g tissue). Smaller amounts of PGF_2α (0.9 μ/g) and 6-oxoPGF_1α were formed. Homogenates from fetal calf lungs during the third trimester of pregnancy were quite active in converting arachidonic acid to PGE₂, but formed very little TXB₂, PGF_2α or 6-oxoPGF_1α. Homogenates from rabbit lungs converted arachidonic acid (100 μg/g) mainly to PGE₂, both before and after birth. The amount of PGE₂ formed increased during gestation to a maximum of about 6 μg/g tissue at 28 days of gestation. It then decreased to a minimum (1.5 μg/g) which was observed 8 days after birth, followed by an increase to about 4 μg/g in older rabbits.     

Prostaglandin E1 and F2α specific binding in bovine corpora lutea: Comparison with luteolytic effects

Preliminary characterization indicated the presence of separate prostaglandin (PG)E₁ and (PG)F_2α binding sites in membrane fractions prepared from bovine corpora lutea. These differ in the rate and temperature dependence of the specific binding. Equilibrium binding data indicate the apparent dissociation constants as 1.32 × 10⁻⁹M and 2.1 × 10⁻⁸M for PGE₁ and PGF_2α, respectively. Competition of several natural prostaglandins for the PGE₁ and PGF_2α bovine luteal specific binding sites indicates specificity for the 9-keto or 9α-hydroxyl moiety, respectively. Differences in relative ability to inhibit ³H-PG binding were found due to sensitivity to the absence or presence of the 5,6-cis-double bond as well.Bovine luteal function was affected following treatment of heifers with 25 mg PGF_2α as measured by reduced estrous cycle length, decreased corpus luteum size and significantly decreased plasma progesterone levels. In contrast, treatment with 25 mg PGE₁ resulted in cycle lengths comparable to those of non-treated herdmates with no apparent modification in corpus luteum size. However, plasma progesterone levels were increased significantly following PGE₁ treatment compared to pretreatment values. In so far as data obtained on PGF_2α relative binding affinity to the bovine CL can be compared to data obtained independently on PGF_2α induced luteolysis in the bovine, PGF_2α relative binding to the CL and luteolysis appeared to be associated. By similar reasoning, there was no apparent relationship between PGE₁ relative binding affinity in the luteal fractions and luteolysis in estrous cyclic cattle.