Effect of indomethacin on collagenolytic enzyme activities in rabbit ovary

To investigate the possible participation of prostaglandins in the activation of collagenolytic enzymes of the follicular wall at ovulation, we measured the activities of neutral and acid collagenase in the rabbit ovaries at various stages of follicular development by using synthetic substrate DNP-Pro-Gln-Ile-Ala-Gly-Gln-D-Arg OH (DNP peptide) with its optimal pH 7.6 and alpha-N-benzoyl-DL-arginine-2-naphthylamide HCl (BANA) with pH 6.0, and the effect of ovulation-blocking doses of indomethacin (4 mg/kg) on DNP peptidase and BANA hydrolase activities were investigated. DNP peptidase and BANA hydrolase activities were increased toward ovulation with the highest level 7 to 9hrs after the hCG injection and then decreased significantly at 10hr. At 11hr, around the time of ovulation, the activity stayed at its low level, then rose by 13hr. Following the concomitant administration of IM with hCG, ovulation was blocked and the preovulatory increases in DNP peptidase and BANA hydrolase activities were not observed and their activities stayed at the low level until 20hr. It is suggested that collagenolytic activity for the ovulatory process started to intensiby 6hrs and ended 3hrs prior to ovulation and PGs are necessary for the enzymatic activation of DNP peptidase and BANA hydrolase.     

BANA hydrolase and DNP peptidase activities in the ovulatory process of PMS-hCG treated immature rat

To investigate the roles of collagenolytic enzymes in the ovulatory process of PMS-hCG treated immature female rats (22 days old), we measured the activities of two of them in the ovary by the using synthetic substrates alpha-N-benzoyl-DL-arginine-2-naphthylamide HCl (BANA) (a collagenolytic cathepsin) and dinitrophenol-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg OH (DNP peptide) (a neutral collagenase). BANA hydrolase activities significantly began to increase after the hCG injection, reaching a peak 8-9 hours later, and then decreased sharply 10 hours later. There was also a significant increase in DNP peptidase activities 7-10 hours after the hCG injection and a significant decrease 12 hours after the injection. The present study has shown that BANA hydrolase and DNP peptidase appear during the ovulatory process of PMS-hCG-treated immature female rats, and that their significant preovulatory increases are contributory to collagenolysis in follicle rupture.     

Effect of RU486 on collagenolytic enzyme activities in immature rat ovary

To investigate the role of progesterone (P) in the ovulatory process, RU486 (RU) (10mg/kg), an antiprogesterone, was administered to PMS-hCG-treated immature female rats (22 days old). As for the collagenolytic enzymes (CE), the activities of BANA hydrolase and DNP peptidase were measured by using synthetic substrates alpha-N-benzoyl-DL-arginine-2-naphthylamide HCl and DNP Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH, respectively. Serial assays were performed at 0, 2, 4, 6, 7, 8, 9, 10 and 12 hours after the hCG injection. In the control, the CE activities were significantly increased after the hCG injection at 8-9 hours in BANA hydrolase and 7-10 hours in DNP peptidase, respectively. By administering RU concomitant with hCG, the preovulatory increase in the CE activities was totally suppressed. Following the P(10mg/kg) injection at 4 hours after hCG and RU injections, the CE activities showed the greatest recovery, approaching the control range. In the ovulatory process of the rat ovary, P plays an important role through its regulatory effects on the collagenolytic enzymes, and this P effect seems to be most prominent around 4 hours after a LH surge.     

Prevention of pregnancy in rabbits using vaginal application of prostaglandin f2alpha

Seventy-two rabbits were artificially inseminated at the time of ovulation induced by intravenous human chorionic gonadotropin. Twenty-four hours later vaginal suppositories containing graded amounts of prostaglandin F2alpha (PGF2alpha) (5 to 25 mg) were inserted into test rabbits in each group. A control group received plain vaginal suppositories. Ten days later the rabbits were killed and examined for numbers of corpora lutea and implantation sites. The results indicate that 5 to 15 mg of PGF2alpha had no effect or a minimal effect on the number of implantation sites, that 20 mg of PGF2alpha reduced the number of implantation sites, and that 25 mg of PGF2alpha was the most effective dose. Fifteen of twenty treated rabbits were not pregnant, despite the presence of ovarian corpora lutea. Four remaining rabbits were pregnant but had markedly reduced implantation sites (four, two, three, and two fetuses, respectively. PGF2alpha is known to increase tubal motility markedly. These data are consistent with such a mechanism's inhibiting pregnancy when used postcoitally in rabbits.     

Correlation between progesterone and plasminogen activator in rat ovaries during the ovulatory process

The role of progesterone in the action of hCG on follicular rupture is unclear at present. In this present study, we investigated the correlation between the preovulatory increase in progesterone and the periovulatory increase in plasminogen activator (PA) activities was completely suppressed with simultaneous injection with hCG of a minimum effective dose of cycloheximide to block ovulation completely. Suppression of hCG-induced acute rise of preovulatory progesterone after simultaneous injection with hCG of a minimum effective dose of cyanoketone to block ovulation completely, was accompanied by an only partial but significant suppression of hCG-induced increase in PA activities. Furthermore this suppression of PA activities after concurrent injection of cyanoketone with hCG was reversed by a supplementary injection of a dose of progesterone which was enough to restore ovulatory responses completely against the inhibitory action of cyanoketone. These facts suggest that PA in rat ovaries is synthesized de novo and progesterone may be responsible for the increase of PA in rat ovaries during the periovulatory process.     

Collagen synthetic activity in rabbit ovary during ovulation and its blockage by indomethacin

In order to investigate the possibility of the participation of prostaglandins (PGs) in the collagen synthetic activity during the ovulatory process, we measured prolyl hydroxylase (PH) activity and the hydroxyproline (Hyp) content of ovarian follicles in rabbits after the administration of hCG and indomethacin (IM). The minimum dose of IM (4mg/kg) effective in blocking ovulation was given simultaneously with hCG to mature female Japanese white rabbits. PH activity in the ovary reached its peak level at 13hr after the hCG injection, immediately after ovulation. This increase was not observed in other tissues such as skin, aorta or ear obtained from ovulating rabbits. In the IM-treated rabbits in which ovulation was blocked, PH activity was rather suppressed and did not show any prominent peaks, although IM did not directly suppress PH activity in the in vitro assay system. The amount of Hyp did not show any significant changes during the ovulatory process or during ovulation blockage by IM. It is suggested that PGs play an important role in controlling collagen synthetic activity of the ovary through the ovulatory process.     

Collagenolytic enzyme activity in human ovary: an ovulatory enzyme system

Collagenolytic enzyme activities presumed to play an important role in ovulation were investigated in the human follicular apex, base, and granulosa cell layer throughout the ovarian cycle. Those analyzed were human ovarian collagenase, 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH peptidase, N-carbobenzoxy-Gly-Pro-Gly-Gly-Pro-Ala peptidase, and alpha-N-benzoyl-DL-arginine beta-naphthylamide hydrolase. Collagenase activity was also measured in human granulosa cell cultures. The activities of all four enzymes showed a marked preovulatory decrease in the apex. The activities in the apex were slightly higher than those in the base throughout the ovarian cycle. However, the activities in the granulosa cell layer and collagenase activity in the granulosa cell cultures increased toward preovulation and decreased after ovulation. These findings suggest that collagenase is synthesized in the granulosa cells maximally at preovulation and is consumed in the follicular apex at the same time, resulting in collagen degradation and disruption of the follicular wall in collaboration with other collagenolytic enzymes investigated here.     

Role of endosalpinx in the oviductal environment

The oviducal fluid and serum samples from rabbits were obtained daily during estrus and pseudopregnancy and were analyzed for electrolytes, steroid hormones and prostaglandins (PGs). The oviducal fluid volume reached its maximum (1.72 +/- 0.39 ml/day) 48 hours after hCG exposure and then declined gradually. Although the ratios of Na+/K+ and Ca++/Mg++ in the oviducal fluid did not change substantially throughout the observation period, the levels of K+ in the oviducal fluid were consistently 2.5 times greater than those in the serum. The concentrations of progesterone in the oviducal fluid were significantly smaller than those found in the serum. The levels of estradiol in the oviducal fluid were consistently elevated above the serum levels. The concentrations of PGF2 alpha in the oviducal fluid increased rapidly following ovulation and reached levels that were 10-14 times greater than those found in the estrus, whereas PGF2 alpha levels in the serum did not change significantly during the observation period. A significant difference between PGF2 alpha/PGE2 in the oviducal fluids (3.66 +/- 0.72) and serum (0.25 +/- 0.11) was observed at 24 hours after hCG. A surface morphologic study revealed a decrease in the number of ciliated cells, and an increase and expansion of secretory cells after hCG administration. The qualitative and qualitative differences between the oviducal fluid and serum suggest the involvement of secretory cells in the microenvironment for fertilization and preimplantation embryonic development.     

Mechanism of luteolysis: effect of estradiol and prostaglandin F2 alpha on corpus luteum luteinizing hormone/human chorionic gonadotropin receptors and cyclic nucleotides in the rhesus monkey

Recent studies from our laboratory suggest that estrogen-induced luteolysis in the primate may be mediated through synthesis of prostaglandin F2 alpha (PGF2 alpha). To elucidate further the mechanism of luteolysis, normally cycling rhesus monkeys received intra-corpus luteum (CL) injections of estradiol (100 microgram), PGF2 alpha (500 microgram), or the appropriate vehicles on the seventh day after the preovulatory estradiol surge. Peripheral vein blood was drawn, at 30-minute intervals for 6 hours for progesterone and luteinizing hormone (LH) assays. Five hours after intra-CL injection, the CL was excised and the LH/human chorionic gonadotropin (hCG) receptor-binding activity and cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) were measured. PGF2 alpha significantly (p < 0.05) lowered progesterone within 1 hour, while the time required for estradiol to lower progesterone significantly was 3.5 hours; there was no significant change in LH, Estradiol and PGF2 alpha significantly (p < 0.05) decreased the LH receptor-binding capacity at 5 hours, without any change in the binding affinity. Also PGF2 alpha significantly (p < 0.05) increased cGMP in the CL, while cAMP remained unchanged; estradiol treatment resulted in a significant (p < 0.05) increase in cAMP with no change in cGMP. This study suggests that estradiol and PGF alpha cause a decrease in progesterone secretion by a loss of the LH/hCG receptor and the PGF2 alpha may act further through the cGMP system.     

The relationship between prostaglandins and histamine in the ovulatory process as determined with the in vitro perfused rabbit ovary

The process of follicle rupture has been described as an inflammatory reaction in which prostaglandins (PGs) and/or histamine may be involved. With an in vitro perfused rabbit ovary preparation, experiments were carried out for determination of whether a relationship exists among PGs, histamine, and ovulation. PGF2 alpha alone was capable of inducing ovulation when added to the perfusion fluid at 1, 10, and 100 ng/ ml. Effectiveness in achieving ovulation varied directly with the dosage; however, the ovulatory efficiency of PGF2 alpha-treated ovaries was lower than that of ovaries exposed to human chorionic gonadotropin (hCG, 100 IU). PGF2 alpha-induced ovulation could not be blocked by the H2 receptor antagonist, cimetidine. The PG synthesis inhibitor, indomethacin, did not prevent histamine-induced ovulation. Ovulation induced by hCG was partially blocked by the administration of indomethacin; however, the concomitant administration of cimetidine was not associated with further reduction in ovulation. In all but one experimental group, the majority of ovulated ova did not progress beyond the intact germinal vesicle stage unless the ovaries had been exposed to hCG. On the basis of these experiments, PGs and histamine do not appear to be interdependent in their effects on the ovulatory process in vitro.     

Prostaglandin F2 alpha, progesterone and estradiol concentrations in human follicular fluid and their relation to success of in vitro fertilization

Prostaglandin F2 alpha (PGF2 alpha), progesterone (P4) and estradiol-17 beta (E2) in follicular fluid were measured by radioimmunoassay in patients undergoing in vitro fertilization and embryo transfer. Follicular growth was induced using clomiphene citrate-hMG-hCG (15 patients) and FSH-hMG-hCG (4 patients). There was no significant difference in follicular fluid PGF2 alpha and P4 concentrations relative to oocyte maturity as assessed morphologically. The highest PGF2 alpha concentration was found in fluid from FSH-hMG-hCG cycles where fertilization occurred. The value is significantly higher (p less than 0.002) than in fluid from clomiphene-hMG-hCG cycles whether fertilization took place or not. There was no significant difference in P4 and E2 levels in relation to the type of ovarian induction or success in fertilization. Positive correlation between P4 and E2 in follicular fluid was found (r = 0.404). The positive correlation between total dose of hMG given to the patients and PGF2 alpha concentration in their preovulatory follicular fluid (r = 0.434) suggests that PGF2 alpha is secreted locally as the result of hMG and hCG stimulation. It is proposed that PGF2 alpha could be a biochemical marker for assessing the success of in vitro fertilization.     

The effect of prostaglandin F2 alpha infusion on corpus luteum function

The effect of prostaglandin (PG) F2 alpha on corpus luteum function in normal women with regular menstrual cycles was investigated. 21 women were recruited as volunteers and PGF2 alpha was infused for 8 hours (25 mgm). Infusions were given between Day 14 and 27 of the luteal phase of the cycle, with Day 14 arbitrarily considered the time of ovulation. 13 of 21 women showed a shortened luteal phase of at least 1 day or less. 1 of 2 women, given PGF2 alpha before ovulation, failed to ovulate, and 2 other patients had anovulatory cycles in the immediate postinfusion cycle. Steroid parameters (serum progesterone, urinary pregnanediol, and serum estradiol) failed to show a consistent change. Measurements of serum progesterone and estradiol during infusion showed an initial depression and subsequent elevation of steroid levels measured. It is concluded that an 8-hour infusion of PGF2 alpha in women fails to disrupt corpus luteum function.     

Intrauterine PGF2 alpha infusion for termination of pregnancies with second-trimester rupture of membranes.

Intrauterine prostaglandin (PG) F2 alpha infusion and intravenous (IV) oxytocin infusion were compared to evaluate the effectiveness of the two methods for termination of pregnancies with second-trimester rupture of membranes. Twenty-two women with this complication were randomly allocated to receive either 20 mg PGF2 alpha, diluted in 500 mL of NaCl 0.9% and administered through a Foley catheter inserted through the cervix, or IV oxytocin infusion in increasing doses. All subjects in the PGF2 alpha group aborted after the first administration. Repeat infusion was necessary in three oxytocin-treated subjects. The mean (+/- SD) induction-abortion interval was significantly shorter in those receiving PGF2 alpha (6.7 +/- 1.2 hours) than in those receiving oxytocin (8.8 +/- 2.7 hours). Minor side effects, such as nausea and vomiting, were observed in three women during PGF2 alpha infusion and were treated symptomatically and by temporary interruption of the infusion. Uterine hypertonus, observed in one subject in each group, was treated by temporary cessation of the infusion. We conclude that intrauterine PGF2 alpha infusion seems more effective than IV oxytocin for termination of pregnancies with second-trimester rupture of membranes.     

In vitro steroidogenesis by human corpora lutea of pregnancy. Effects of human chorionic gonadotropin and prostaglandin F2alpha.

Human corpora lutea of pregnancy obtained at 5 to 11 weeks of gestation were incubated in vitro in the presence of various concentrations of human chorionic gonadotropin (hCG) and prostaglandin F2alpha (PGF2alpha). Progesterone (P) and 17beta-estradiol (E2) released into the medium during incubation were measured by radioimmunoassay. Pieces of the corpora lutea were also examined ultrastructurally before and after incubation. Release of P and E2 into the medium was significantly increased by the addition of hCG and PGF2alpha in some cases. However, the response to hCG and PGF2alpha appeared to vary according to the age of the corpus luteum. Ultrastructurally, the lutein cells were well maintained with respect to the appearance of the smooth endoplasmic reticulum and mitochondria after 120-min incubations. On the basis of these observations, the following conclusions have been reached: 1) This incubation system seems to be suitable for investigating the direct effects of chemicals on lutein cells in vitro. 2) Both hCG and PFG2alpha directly stimulate steroidogenesis in the human corpus luteum of pregnancy, demonstrating their luteotropic actions in vitro. 3) Observed variations in P and E2 secretion might have been due to the different activities of aromatizing enzymes at different stages of gestation.     

Intravenous administration of prostaglandin F2alpha or prostaglandin E2 in labor pain induction in fetal death

Successful treatment of intrauterine fetal death by intravenous administration of (PGF2alpha) prostaglandin F2alpha in 20 patients (gestational age 22-40 weeks) and of PGE2 in 6 patients (gestational age 24-36 weeks) is reported. Infusion of 5-20 mcg PGF2alpha/minute and of 0.5-2.0 mcg PGE2/minute resulted in regular uterine contractions within the first 2 hours. The average dosage of PGF2alpha was 7.06 mg in cephalic presentations (infusion time=7 hours, 2 minutes) and 23.4 mg in anomalous presentations (infusion time=16 hours, 54 minutes). The average dose of PGE2 was 1.17 mg (infusion time=9 hours, 23 minutes). Coagulation investigations were performed before, during, and after PG infusion. These investigations suggest that the coagulation potential of patients with intratuerine fetal death by intravenous PG administration is not influenced to any extent. Few undesirable side effects were seen using this PG. We consider that the intravenous PG application for the induction of labor in intrauterine fetal death is a useful method because of the effectiveness and few undersirable side effects. (author's)     

Do prostaglandins lead to ovulation in the rabbit by stimulating proteolytic enzyme activity?

OBJECTIVE: To determine if prostaglandins (PGs) have a direct effect on the ovarian proteolytic enzyme system by examining ultrastructure of the follicle wall and the microvasculature in the presence and absence of indomethacin and by using the isolated perfused rabbit ovary. DESIGN: Nine hours after administration of human chorionic gonadotropin (hCG) or hCG plus indomethacin, follicles were removed and processed for scanning and transmission electron microscopy. Isolated perfused rabbit ovaries were induced to ovulate with PGF2 alpha (100 ng/mL) in the presence and absence of tranexamic acid (0.1, 1.0, or 10 mM), a plasminogen activator inhibitor. RESULTS: The addition of indomethacin to hCG inhibited ovulation and production of PGs without affecting the follicular microvasculature. However, the changes in follicle wall architecture were less pronounced after treatment with indomethacin. Ovulatory efficiency in response to PGF2 alpha (the percent of follicles greater than 1.5 mm that ovulate) was significantly reduced (P less than 0.01) by 10 mM tranexamic acid. CONCLUSIONS: These results suggest that PGs induce follicular rupture by activation of proteolytic enzymes located in the follicle wall.     

Prostaglandin E and F concentration in the fimbria of the rabbit fallopian tube increases at the time of ovulation

To see if the fimbrial concentrations of Prostaglandin E (PGE) and Prostaglandin F (PGF) change in the process of ovulation, these prostaglandins were measured in the fimbriae of one group of 18 doe rabbits using the specific radioimmunoassay; 13 rabbits were bred so as to induce ovulation and 5 rabbits were not bred. The PGE and PGF in the fimbriae of the 13 bred rabbits were 105.4 +/- 18.5 pg/mg of protein and 186 +/- 26.9 pg/mg protein, respectively. In the 5 control unbred animals, the fimbrial PGE and PGF concentrations were 39.6 +/- 5.9 pg/mg protein and 111 +/- 14.7 pg/mg protein, respectively (p less than 0.05). In an additional group of 8 rabbits (2 before ovulation and 6 after ovulation), it was found that the rise in the PGE and PGF concentration association with ovulation was maximal in fimbriae harvested immediately before ovulation (8 hours-8 hours 30 postcoitally) (p less than 0.001). These findings are consistent with the hypothesis that fimbrial PGE and PGF play a role in the process of ovum retrieval in the rabbit.     

A histochemical study of the respiratory system in rabbit ovarian follicles during ovulation

Histochemical studies on the activities of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), succinate dehydrogenase (SDH) and cytochrome oxidase (CY-O) in rabbit ovarian follicles at various times after the administration of hCG were performed to investigate the relationship between the activities of steroid biosynthesis and the respiratory system. The activities of 3 beta-HSD and SDH were studied by using the tetrazolium salt and CY-O activities by the method of Seligman with prefixation. On the granulosa cell layer prior to the administration of hCG, no activities of 3 beta-HSD and CY-O were detectable, while slight activities of SDH were observed. These three enzymes showed intense activities on the granulosa cell layer 3 hours after the administration of hCG, which were maintained till the time of ovulation. The results indicated that increased activities of steroid biosynthesis in the granulosa cell layer after LH-surge were accompanied by the accelerated TCA cycle and respiratory chain. Energy from accelerated glycolysis may be utilized for steroid biosynthesis in preovulatory granulosa cells. Increased O2 consumption due to accelerated aerobic glycolysis is in accordance with hyperactivity of perifollicular capillaries, which finally leads to rapid follicular expansion and ovulation.     

Production of 6-keto-PG F1 alpha in hyperstimulated cycles: in vivo and in vitro studies.

Prostacyclin (PG12), a potent vasodilator, is believed to be involved in increasing permeability within the wall of the preovulatory follicle. In ovarian hyperstimulation syndrome (OHSS), increased vascular permeability has been shown to lead to massive fluid accumulation in ovarian cysts and the peritoneal cavity. The objective of the in vitro and in vivo studies reported herein was to examine the production of 6-keto PGF1 alpha (a breakdown metabolite of PG12) during the luteal phase of women who develop OHSS, as well as the capacity of human granulosa luteal cells (GLC) obtained from stimulated in vitro fertilization (IVF) cycles to synthesize PGF1 alpha in vitro in long-term cultures. The in vivo results showed that during the luteal phase of women with OHSS, there is no increase in 6-keto PGF1 alpha production compared with the levels obtained from the luteal phase of normal ovulatory women. The GLC (representing early corpus luteum function) secreted significant amounts of 6-keto PGF1 alpha, but only in the first 48 hours of culture. Human chorionic gonadotropin (hCG) had no additional augmentative effect upon the production of 6-keto PGF1 alpha throughout the culture period. It is concluded that 6-keto PGF1 alpha is not produced in significant amounts during the luteal phase, and therefore PG12 probably does not play a major role in the etiology of OHSS.     

Subcutaneously administered Repronex in oligoovulatory female patients undergoing ovulation induction is as effective and well tolerated as intramuscular human menopausal gonadotropin treatment.

OBJECTIVE: To determine the efficacy and safety of Repronex SC as compared with Repronex IM and Pergonal IM in patients undergoing ovulation induction. DESIGN: Randomized, open-label, multicenter, parallel group study. SETTING: Ten academic and private fertility clinics with expertise in ovualtion induction. PATIENT(S): Premenopausal anovulatory and oligoovulatory females (n = 115) undergoing ovulation induction. INTERVENTION(S): Down-regulation with leuprolide acetate followed by up to 12 days of treatment with gonadotropins and hCG administration and luteal phase progesterone support. MAIN OUTCOME MEASURE(S): Percentage of patients ovulating; percentage of cycles with follicular development meeting criteria for hCG administration; number of follicles recruited per cycle meeting hCG criteria; peak serum E(2) levels; rates of chemical, clinical and ongoing pregnancies; adverse events; injection-site pain scores. RESULT(S): There was no statistically significant difference in the percentage of women who ovulated among the treatment groups. However, Repronex SC was significantly more effective than Pergonal IM in producing follicular development in patients who met hCG criteria. There were no significant differences in clinical, ongoing, or continuing pregnancy rates or in multiple pregnancies among the treatment groups. No differences were found in the safety assessments, proportions or seriousness of adverse events or treatment discontinuations. Also, there were no differences between the three treatment groups in patient-recorded scores of injection-site pain or injection-site reactions. CONCLUSION(S): Repronex SC is as efficacious and well tolerated as Repronex IM or Pergonal IM in ovulation induction. Self-administration of Repronex SC provides a convenient treatment alternative to daily IM injections.