Corpus luteum function in the ewe: Effect of PGF2α and prostaglandin synthetase inhibitors

A series of experiments were conducted to evaluate the effects of mode and frequency of administration and estrous cycle stage on the response of the cycling ewe to PGF_2α. The effects of dexamethasone, arachadonic acid and prostaglandin synthetase inhibitors on estrous cycle length and plasma progesterone levels were also determined.Intramuscular administration of 5 or 10 mg of PGF_2α, on days 8 and 9 after estrus (5 ewes/group), significantly (p<.01) shortened the mean length of the estrous cycle and the interval from the end of treatment to estrus. Mean plasma progesterone levels, 24 hours after initial injection, were significantly (p<.01) lowered. When administered on day 8 only, these doses were considerably less effective in shortening estrous cycle length or lowering plasma progesterone levels. Intravaginal administration of PGF_2α, by polyurethane tampon, was also largely ineffective.Treatment of ewes with 10 mg of PGF_2α i.m., on days 3 and 4 of the estrous cycle, resulted in a return to estrus in 2 days in 25% of the treated animals. Plasma progesterone levels of PGF_2α-treated ewes were significantly lower than controls on the second, third and fourth days after the start of dosing. It would appear that PGF_2α exerts a retarding effect on developing CL functionality.The prostaglandin synthetase inhibitors, aspirin, flufenamic acid and 1-p-chlorobenzylidene-2-methyl-5-methoxy-3-indenylacetic acid, were administered orally or parenterally for 16 days beginning on day 8 of the estrous cycle. These compounds failed to prolong estrous cycle length. Parenteral administration of dexamethasone did not result in PGF_2α release in the cycling ewe, at least not in quantities sufficient to induce luteolysis. The prostaglandin precursor, arachadonic acid, also was not luteolytic when given parenterally to cycling ewes.     

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

The ability of human chorionic gonadotropin (HCG) to reduce the luteolytic effect of prostaglandin (PGF_2^α) was demonstrated in cycling ewes. As expected, treatment with 10 mg of PGF_2^α alone on Day 10 of the estrous cycle exerted a potent negative effect on the function and structure of corpus luteum (CL) as indicated by reduced plasma progesterone, CL progesterone, and CL weight. However, the identical PGF_2^α treatment failed to significantly reduce either luteal function or luteal weight when administered to ewes that were also treated with HCG on Days 9 and 10 of the estrous cycle. Treatment with HCG alone had a positive effect on CL as indicated by increased plasma progesterone, CL progesterone, and CL weight. Treatment with HCG did not render the CL totally insensitive to the negative effects of PGF_2^α because plasma progesterone was reduced when the dose of PGF_2^α was doubled. Whether CL regressed or continued to function after treatment with both HCG and PGF_2^α appeared to depend upon a balance between the positive and negative effects of the two hormones.     

Luteal function in sheep injected with prostaglandin F2α directly into the corpus luteum

Groups of ewes received either saline or prostaglandin F_2α (PGF_2α) as an injection directly into the corpus luteum. Changes in circulating progesterone levels were measured as well as subsequent histological examination of the corpora lutea. Saline, or PGF_2α given at the two lower doses (60 and 120 μg respectively), failed to suppress progesterone levels permanently, or to induce degenerative changes in the corpora lutea. Treatment with a higher dose of PGF_2α (240 μg) was followed by a marked elevation in progesterone levels. These results are discussed in relation to reported effects of PGF_2α arriving at the ovary via the arterial circulation.     

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.     

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 in vitro on PGF_2α relative binding affinity to the bovine CL can be compared to data obtained independently in vitro 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.     

Comparative luteolytic activity of estradiol cyclopentylpropionate and prostaglandin F2α in diestrous cows

The effect of estradiol cyclopentylpropionate (EC) on corpus luteum (CL) function in diestrous cows was evaluated. Two doses of EC (4 and 20 mg) were given by intramuscular (IM) injection and one dose of EC (4 mg) was given by intrauterine (IU) infusion. Control cows were treated with physiologic sterile saline (PSS) IU or corn oil IU (negative controls) or prostaglandin F_2α (PGF_2α, 30 mg IM, positive control). A total of 24 cows, four per treatment, were treated on days 8 to 12 of the estrous cycle (day 0 equals day of estrus). Luteal function was monitored by serum progesterone through 96 hours after treatment. A decrease in serum progesterone from pretreatment diestrous concentrations to less than 1.0 ng/ml was considered indicative of luteolysis.Intrauterine injection of PSS and corn oil had no effect on luteal function. Neither IM nor IU administration of EC caused consistent or rapid luteolytic effects. Prostaglandin F_2α consistently induced rapid luteal regression. These results indicate that EC should not be considered luteolytic in the same sense as is PGF_2α.     

Corpus luteum regression induced by ultra-low pulses of prostaglandin F2α

In view of the pulsatile nature of PGF_2α secretion from the ovine uterus at the time of luteolysis, experiments were designed to examine the effect of pulsed infusions of PGF_2α on luteal function and to re-examine the minimal effective levels of PGF_2α required to induce luteolysis. To mimic physiological conditions, hour-long infusions of PGF_2α in increasing concentrations were given either 4 times in 19 h or 5 times in 25 h into the arterial supply of the autotransplanted ovary in conscious sheep on day 12 of an induced cycle. Blood flow and progesterone secretion rate from the ovary were used to monitor directly the luteolytic effect of administered PGF_2α. The concentration of LH in peripheral plasma was measured throughout each infusion experiment and the presence of a preovulatory peak of LH was used as an indicator of the permanence of luteal regression. Four pulses of PGF_2α in 19 h caused complete corpus luteum regression in only 1 of 4 animals whereas the addition of a fifth pulse (5 pulses in 25 h) caused permanent regression in 4 out of 4 animals. Infusion of 5 hour-long pulses of saline or PGF_2α at a rate of <0.04 μg/h did not induce permanent suppression of progesterone secretion. The average total effective dose of PGF_2α required to induced luteal regression when given as 5 pulses was 1/40th of the amount currently regarded as the minimal effective one when given by constant infusion into the ovarian artery. In another series of experiments the luteolytic effect of a single hour-long pulse of 0.1 μg/h PGF_2α given daily for either 3 or 4 days was investigated. A significant fall (ANOVA, F_0.01) in progesterone secretion rate, which reached a nadir at $\text{5.3 ± 2.2}\text{h (}\text{x}\text{±}\text{S.D.}\text{, n=15)}$, was followed by a recovery of progesterone secretion rate. Permanent luteal regression did not occur with this protracted regimen, suggesting that a relatively short pulse frequency of PGF_2α over a minimal period of 24 h is a necessary condition for physiological regression of the corpus luteum in sheep.     

An intra-corpus luteum site for the luteolytic action of prostaglandin F2α in the rhesus monkey

It has not been possible to demonstrate prostaglandin F₂α (PGF₂α) participation in primate luteolysis under conditions of systemic administration or of acute intraluteal injection. These study designs were hampered by the short biological half-life in the first instance and brevity of administration in the latter. In this study, luteolysis has resulted from chronic, intraluteal delivery of PGF₂ α. Using the Alzet osmotic pump-cannula system, normally cycling rhesus monkeys were continuously infused, until menses occurred, with PGF₂ α (10 ng/1/hr) directly into the corpus luteum (CL, n=6), into the stroma of the ovary bot bearing the corpus luteum (NCL, n=3), or subcutaneously (SC, n=5). An additional 5 monkeys received vehicle (V) into the corpus luteum. All experiments commenced 5–7 days after the preovulatory estradiol surge. Luteal function was assessed by the daily measurements of plasma progesterone, estradiol, and LH. Intraluteal PGF₂α caused premature functional luteolysis in all monkeys, as reflected by a highly significant decline in circulating progesterone and estradiol and the early onset of menstruation, when compared to the other groups. V, NCL, and SC infusions had no effect on either circulating steroid levels or luteal phase lengths. None of the experimental groups showed any change in plasma LH concentrations. These are the first data to indicate that PGF₂α can induce functional luteolysis in the primate, and the site of action appears to be the corpus luteum.     

Assessment of possible luteolytic effect of intra-ovarian injection of prostaglandin F2α in the human

A group of five patients awaiting laparoscopic tubal diathermy were followed by daily assay of luteinising hormone (LH) and progesterone. Between five and eight days after the LH peak, prostaglandin F_2α (PGF_2α) was injected into either the corpus luteum or the ovarian stroma. Doses of 100 μg into the corpus luteum, 1000 μg into the adjacent stroma and 500 μg into an indeterminate ovarian structure had no effect on peripheral plasma progesterone levels or uterine bleeding.An injection of 500 μg or 1000 μg given unequivocally into the corpus luteum produced a rapid and profound fall in plasma progesterone levels, the nadir coinciding with the onset of uterine bleeding which commenced 24 hours after the injection and persisted for more than seven days. Injection of 100 μg in the same volume of saline had no such effect. Despite continued bleeding plasma progesterone levels returned to normal luteal levels for three days and then fell again.     

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.     

Effect of prostaglandin F2α on the hCG-stimulated progesterone production by human corpora lutea

The effect of prostaglandin PGF_2α on the hCG stimulated and basal progesterone production by human corpora lutea was examined . hCG (40 i.u./ml) stimulated progesterone formation in corpora lutea of early (days 16–19 of a normal 28 day cycle), mid (days 20–22) and late (days 23–27) luteal phases. This stimulation was inhibited by PGF_2α (10 μg/ml) in corpora lutea of mid and late luteal phases. PGF_2α alone did not show a consistent effect on basal progesterone production. The inhibition of hCG stimulated progesterone production by PGF_2α at times corresponding to luteolysis indicates a role for that prostaglandin in the process of luteolysis in the human corpus luteum.     

Jugular levels of 13,14-dihydro-15-keto-prostaglandin F and progesterone around luteolysis and early pregnancy in the ewe

Six non-pregnant ewes at day 12 of the estrous cycle each had a day-12 embryo transferred into the uterine horn ipsilateral to the corpus luteum, and 4 non-pregnant ewes at day 13 each had a day-13 embryo similarly transferred. Four control ewes, 2 at day 12 and 2 at day 13 received sheep serum into the uterine horn ipsilateral to the corpus luteum. Jugular blood samples were taken at 2-hourly intervals for 3 days post-surgery, then twice-daily for a further 4 days, and the plasma radioimmunoassayed for progesterone and 13,14-dihydro-15-keto-prostaglandin F. All control ewes exhibited estrus within the expected time range and pulsatile peaks of 13,14-dihydro-15-keto-prostaglandin F occurred coincident with declining progesterone levels. With one exception, the recipient ewes had prolonged cycles and those ewes found pregnant at necropsy, 30 days after transfer, showed no progesterone decline and no pulsatile peaks of prostaglandin during days 12 to 16 after estrus. These observations suggest that the presence of the embryo at a critical stage after mating suppresses the release of uterine prostaglandin F_2α.     

Luteolytic action of 15-keto prostaglandin F2α in the rhesus monkey

The naturally-occurring metabolite of prostaglandin F_2α, 15-keto prostaglandin F_2α (15-keto PGF_2α), elicited rapid and sustained declines in serum progesterone concentrations when administered to rhesus monkeys beginning on day 22 of normal menstrual cycles. Evidence for luteolysis of a more convincing nature was obtained in studies where a single dose of 15-keto PGF_2α was given on day 20 of ovulatory menstrual cycles in which intramuscular injections of hCG were also given on days 18–20; serum progesterone concentrations fell precipitously in monkeys within 24 hours following intramuscular administration of 15-keto PGF_2α. However, corpus luteum function was impaired in only 4 of 11 early pregnant monkeys when 15-keto PGF_2α was administered on days 30 and 31 from the last menses, a time when the ovary is essential for the maintenance of pregnancy. Gestation failed in 2 additional monkeys 32 and 60 days after treatment with 15-keto PGF_2α, but progressed in an apparently normal manner in the remaining 5 animals. Two pregnant monkeys treated with 15-keto PGF_2α on day 42 from the last menstrual period, a time when the ovary is no longer required for gestation, continued their pregnancies uneventfully. Corpus luteum function was not impaired in 9 control monkeys which received injections of vehicle or hCG at appropriate times during the menstrual cycle or pregnancy.     

11-Ketotetranor PGF metabolites,a suitable indicator for measuring prostaglandin release during the normal oestrous cycle and early pregnancy in the goat

Peripheral plasma levels of 15-ketodihydro-PGF_2α, 11-ketotetranor PGF metabolites and progesterone were measured during normal oestrous cycle and early pregnancy in six goats. The does were synchronized before the start of the study by means of 10 mg of PGF_2α. Blood samples were collected twice daily until day 12 of the oestrous cycle and subsequently every 3 h until the onset of oestrus, at which time the does were mated. The blood sampling protocol was repeated until day 28 of pregnancy. High pulsatile peaks of 15-ketodihydro-PGF_2α and 11-ketotetranor PGF metabolites were observed during the last days of the oestrous cycle, indicating PGF_2α releases. This coincided with a fall in progesterone levels. During early pregnancy no such peaks of prostaglandin metabolites were recorded and high levels of progesterone were maintained. In the goat, analysis of the 11-ketotetranor PGF metabolites seems to be a better indicator of PGF_2α release than the analysis of 15-ketodihydro-PGF_2α. The former metabolites are more long-lived in the circulation and are thus easier to detect.     

Effect of intrauterine iodine infusion on luteal function and blood PGF2a concentration in cycling goats

Studies were conducted to determine the effect of iodine infusion on the luteal function of goats, as evident by blood progesterone concentration, and on plasma PGF_2a levels. Ten cycling mixed breed goats were synchronized for estrus by PGF_2a (5 mg) and given a single intrauterine iodine infusion on day 5 and on day 15 of the estrous cycle.Iodine infusion on day 5 (group II) resulted in shorter estrous length (8.2 days) and a 7-fold increase in plasma PGF_2a concentration as compared to control animals (group I) given distilled water infusion. Similar infusion on day 15 (group III), on the other hand, failed to alter the estrcus cycle length but induced a moderate increase in PGF_2a concentration which lasted only for a brief period. The progesterone levels declined concomitantly as PGF_2a levels rose after iodine infusion in group II animals but failed to decline until after 24 hours in group III animals.The studies indicate that the endometrium reacts to the chemical stimuli and releases PGF_2a which, in turn, alters the luteal function.     

Sparing effects of intrauterine treatment with prostaglandin E2 on luteal function in cycling gilts

Twelve crossbred gilts, 8 to 9 months of age, were used to study the effects of prostaglandin E₂ (PGE₂) on luteal function during the estrous cycle. Intrataurine and jugular vein catheters were surgically placed before day 7 of the treatment estrous cycle and gilts were randomly assigned to 1 or 3 treatment groups. Groups I and II received constant intrauterine infusion of vehicle (6.0 ml/24 hr) or PGE₂ (2400 μg/day; 6.0 ml/24 hr) respectively; while group III was given intrauterine infusions of 400 μg PGE₂ every 4 hr. All infusions were initiated on day 7 and continued until estrus or through day 23. Jugular blood samples were collected twice daily from days 7 to 30 for progesterone analysis. Intrauterine infusion of PGE₂ at the dose and frequencies given in this study delayed the decline in jugular plasma progesterone and resulted in prolongation of the estrous cycle length. The results of this study have shown that PGE₂ at the dosage and frequency of administration used was capable of extending corpus luteum function.     

Hormonal asynchrony and embryonic development

Luteinizing hormone (LH), progesterone and estradiol profiles in peripheral blood serum were compared among parous and nonparous females with normal, abnormal or no embryonic development. Hormonal profiles between parous and nonparous females of the same embryonic status did not differ and the data were combined. Estrous cycle length was longer (P<.05) in parous (22.3±.4 days) than nonparous females (21.0±.4 days). Females with normal developing embryos had a higher serum progesterone concentration at Days 3 and 6 and a lower ratio of estradiol to progesterone than did females with abnormal embryonic development. Females with a normal embryo had higher (P<.05) preovulatory LH peaks than females with abnormal development or no recovery of an oocyte or embryo (34.3±4.7, 11.8±6.8 and 13.3±2.5 ng/ml, respectively). The interval from onset of estrus to LH peak was 8.9±2.1, 13.7±3.7 and 13.5±6.2 hr for females with normal, abnormal or no recovery of an embryo. The lower LH peak, the longer interval from onset of estrus to LH peak, and lower progesterone concentration in peripheral blood serum in females with abnormal embryos or no recovery indicated that these females had a hormonal asynchrony. The hormonal asynchrony may produce an undesirable uterine environment for male and female gametes or embryos which resulted in fertilization failure or embryonic death. In the second experiment, more transferable embryos were obtained when superovulated females received prostaglandin F_2α (PGF_2α) intravenously rather than intramuscularly. Administering PGF₂α intravenously rather than intramuscularly may have caused the demise of the corpus luteum sooner and thereby produced a more normal uterine environment which allowed more embryos to develop normally.     

Changes in plasma estriol and progesterone during labor induced with prostaglandin F2α or oxytocin

In a double blind study, 12 women received oxytocin for the induction of labor at term and 20 subjects received prostaglandin F_2α (PGF_2α). During the infusions, plasma progesterone and estriol levels were measured, and compared with pre-infusion levels of these steroids. From the analysis of the data, it is concluded that neither the infusion of oxytocin or PGF_2α per se alters the plasma levels of either progesterone or estriol in term pregnant subjects.     

Effect of oestradiol 17 β on prostaglandin biosynthesis by the uterus of ovariectomized rat and guinea pig

$\text{In vitro}$ prostaglandin biosynthesis by uteri of ovariectomized rats and guinea pigs treated or untreated with oestradiol 17 β, administered subsutaneously, was measured by R.I.A. of PGF_2α and PGE₂. Incubations with [1-¹⁴C] arachidonic acid were also performed and labelled metabolites were analyzed by TLC. The main metabolite in rats was 6 keto PGF_1α and in decreasing order of magniture, PGF_2α and PGE₂. In guinea pig PGF_2ga was the main product. Ovariectomy in rats completely changed the pattern of synthesized prostanoids: PGI₂ production was doubled when compared to cycling rats and PGE₂ increased 10 fold. PGF_2α walues were similar to the mean value measured during the cycle. OE₂ treatment almost completely inhibited PGI₂ synthesis and reduced PGE₂ by half. Total PG synthesis in OE₂ treated animals was decreased by 5 fold when compared to spayed rats. Endogenous PGF_sα synthesis was slightly stimulated. In the guinea pig OE₂ treatment of ovariectomized animals increased the total synthesis from 50 per cent. PGF_2α was always the main metabolite. In conclusion OE₂ regulation of uterine PG synthesis is depending on the animal species and cannot be explained by a unique effect on the cyclooxyhenase, but rather by an interplay on the various enzymes of the arachidonic acid cascade.     

Luteolysis in the rhesus monkey: Ovarian venous estrogen, progesterone, and prostaglandin F2α-metabolite

The role of prostaglandin F₂α (PGF₂α) in luteolysis in the non-human primate is poorly understood. We have recently reported that chronic PGF₂α infusion to the corpus luteum via Alzet pump, induced premature, functional luteolysis in the rhesus monkey. In the present study we sought to determine the ovarian events leading to spontaneous luteolysis in the monkey. Rhesus monkeys underwent laparotomy during the early luteal (4–5 days after the preovulatory estradiol surge, PES), mid-luteal (7–9 days PES), and late luteal (10–14 days PES) phases or at the first day of menses (M). Concentrations of progesterone, estradiol, estrone, and 13, 14-dihydro-15-keto-PGF₂α (PGFM) were measured in the ovarian venous effluents ipsilateral and contralateral to the ovary bearing the corpus luteum. Steroid levels in the ovarian vein on the corpus luteum side were significantly higher than the non-corpus luteum side throughout the cycle. PGFM levels were similar on both sides until the late luteal phase, when the effluent of the ovary bearing the corpus luteum contained significantly more PGFM (206±3) vs. 123±9 pg/ml, mean±sem); this disparity increased further at the time of menses (241±38 vs. 111±22 pg/ml). These data are the first to show an asymmetric secretion of PGFM in the ovarian venous effluent in the primate and suggest that PGF₂α of ovarian and possibly of corpus luteum origin may be directly involved in luteal demise.