Decrease in ovarian platelet-activating factor during ovulation in the gonadotropin-primed immature rat

Platelet-activating factor (PAF) is a biologically active phospholipid that is released locally during acute inflammatory reactions and tissue injury. Since there is evidence that the biochemical events of mammalian ovulation resemble an inflammatory reaction, the objective of this study was to determine whether ovarian levels of PAF change during ovulation. At 2-h intervals during the ovulatory process in gonadotropin-primed 25-day-old Wistar rats, the ovaries were extirpated, homogenized, and extracted for lipids. The extracts were subjected to thin-layer chromatography (TLC), and the portion of the silica gel that comigrated with PAF was re-extracted and assayed for PAF activity. The PAF was measured (in fmole equivalents of synthetic PAF) by a bioassay based on the capacity of aliquots of the extracts to release [3H]-serotonin from platelets isolated from whole blood of rabbits and prelabeled with [3H]-serotonin. The ovarian level of PAF decreased (p less than 0.01) by 36% from 6.67 +/- 0.77 to 4.27 +/- 0.45 fmoles/mg ovary by 2 h after treatment with human chorionic gonadotropin (hCG), and it declined another 14% by 4 h after hCG. The ovarian PAF remained at this reduced level for up to 24 h after hCG. The administration of indomethacin (5 mg/rat, s.c.) or epostane (5 mg/rat, s.c.) at 1 h after hCG prevented ovulation, but neither drug affected the decline in ovarian PAF. Preliminary tests showed that the lipid extracts from the ovaries also contained PAF inhibitor(s) that comigrated with PAF on the TLC plates. Similar to PAF, the lipid-soluble inhibitor(s) decreased (p less than 0.05) in the ovaries within 4 h after hCG treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increase in ovarian kallikrein activity during ovulation in the gonadotrophin-primed immature rat

The ovulatory process was initiated in 25-day-old rats by injecting them with hCG (10 i.u., s.c.) 2 days after the animals had been primed with PMSG (10 i.u., s.c.). At 2-h intervals after hCG, the ovaries were extracted and assayed for glandular kallikrein activity by using a chromogenic substrate (H-D-Val-Leu-Arg-p-nitroanilide) which exhibits optical density (at 405 nm) upon hydrolysis. In 0-h control ovaries the activity was 12.5 x 10(-3) kallikrein units (KU)/mg protein and it increased to a peak of 56.6 x 10(-3) KU/mg at 12 h after hCG, when the follicles first began to rupture. The kallikrein activity was distinguishable from ovarian plasminogen activator activity on the basis of pH optima and response to trypsin inhibitor (SBTI). The activity was inhibited by a s.c. dose of indomethacin of 0.3 mg/rat, or higher, and this dosage inhibited ovulation. The results suggest that kallikrein activity contributes to the degradation of Graafian follicles during ovulation in mammals.     

Epostane and indomethacin actions on ovarian kallikrein and plasminogen activator activities during ovulation in the gonadotropin-primed immature rat.

Kallikrein and plasminogen activator (PA) are serine proteases that have been implicated in the ovulatory process. Epostane and indomethacin are anti-ovulatory agents that inhibit steroid and eicosanoid synthesis, respectively. This study examines the effects of these two anti-ovulatory agents on ovarian kallikrein and PA activities during ovulation. The proteases were assayed by their actions on chromogenic peptide substrates S-2266 and S-2251, respectively. The ovulatory process was induced in 25-day-old Wistar rats by giving them hCG (10 IU, s.c.) 2 days after the animals had been primed with eCG (10 IU, s.c.). Control animals ovulated approximately 60-70 ova/rat, with the first ova appearing in the oviducts at 10-12 h after hCG administration, and this was the same time ovarian kallikrein and PA activities reached a peak. When doses of epostane ranging from 0.1-5.0 mg/rat or doses of indomethacin ranging from 0.03 to 3.16 mg/rat were administered s.c. at 3 h after hCG, the two drugs inhibited ovulation and ovarian kallikrein and PA activities in a dose-dependent manner. However, the anti-ovulatory action of the two drugs was more closely correlated with suppression of kallikrein activity than with PA activity. Treatment of the animals with exogenous progesterone reversed the inhibitory action of epostane, but not of indomethacin. The results suggest that the increase in ovarian progesterone at the time of ovulation may influence ovarian kallikrein and PA activities.     

Effect of indomethacin, cycloheximide, aminoglutethimide on ovarian steroid and prostanoid levels during ovulation in the gonadotropin-primed immature rat

It has become popular to use the gonadotropin-primed immature rat to study ovulation. The ovarian content of progesterone, estradiol, PGE₂, PGF_2α, and 6-keto-PGF_1α during the ovulatory process was determined in this model. Also, the effect of three anti-ovulatory agents on the ovarian levels of the above substances was determined. At 23 days of age, Wistar rats were primed with pregnant mares serum gonadotropin (PMSG) sc, and two days later the ovulatory process was initiated with human chorionic gonadotropin (hCG) sc. The ovarian follicles began rupturing 12 h later. Ovaries were assayed for the two steroids and prostanoids at 2-h intervals befored and several 4-h intervals after ovulation. The ovarian estradiol level increased slightly between 0 and 2 h after hCG, while the progesterone level increased sharply between 2 and 4 h after hCg--at a time when the estradiol declined markedly. All three prostanoids increased concomitantly with progesterone. When the PG synthesis was blocked by indomethacin treatment at 1 h before hCG, ovarian progesterone levels still incrased. In contrast, when steroidogenic activity was inhibited by aminoglutethimide, the ovarian prostanoid levels also decreased. Cycloheximide had little effect on the steroids and prostanoids. It is concluded that ovarian prostanoid synthesis might be influenced by ovarian steroid output.     

Effect of indomethacin, cycloheximide, and aminoglutethimide on ovarian steroid and prostanoid levels during ovulation in the gonadotropin-primed immature rat

It has become popular to use the gonadotropin-primed immature rat to study ovulation. The ovarian content of progesterone, estradiol, PGE2, PGF2 alpha, and 6-keto-PGF1 alpha during the ovulatory process was determined in this model. Also, the effect of three anti-ovulatory agents on the ovarian levels of the above substances was determined. At 23 days of age, Wistar rats were primed with pregnant mares serum gonadotropin (PMSG) sc, and two days later the ovulatory process was initiated with human chorionic gonadotropin (hCG) sc. The ovarian follicles began rupturing 12 h later. Ovaries were assayed for the two steroids and prostanoids at 2-h intervals before and several 4-h intervals after ovulation. The ovarian estradiol level increased slightly between 0 and 2 h after hCG, while the progesterone level increased sharply between 2 and 4 h after hCG--at a time when the estradiol declined markedly. All three prostanoids increased concomitantly with progesterone. When the PG synthesis was blocked by indomethacin treatment at 1 h before hCG, ovarian progesterone levels still increased. In contrast, when steroidogenic activity was inhibited by aminoglutethimide, the ovarian prostanoid levels also decreased. Cycloheximide had little effect on the steroids and prostanoids. It is concluded that ovarian prostanoid synthesis might be influenced by ovarian steroid output.     

Effect of various agents on ovarian plasminogen activator activity during ovulation in pregnant mare's serum gonadotropin-primed immature rats

The plasminogen activator/plasmin synthetic substrate S-2251 was used to measure the effect of indomethacin, cycloheximide, colchicine, dexamethasone, tranexamic acid, and aprotinin on the elevation of ovarian plasminogen activator (PA) that normally occurs during ovulation in the rat. Young Wistar rats were weaned on the morning of Day 21, given 4.0 IU of pregnant mare's serum gonadotropin (PMSG) s.c. at 0800 h on Day 22, and given 10.0 IU of human chorionic gonadotropin (hCG) on Day 24. These animals normally began ovulating between 0000 and 0200 h on Day 25. The induced ovulation rate was 11.5 +/- 2.2 ova/rat, based on the number of ova in the oviducts of control animals at 0900 h on Day 25. In the controls, PA activity in extracts of homogenized ovaries increased 3-fold from 0.125 +/- 0.010 OD units just before the administration of hCG to 0.371 +/- 0.021 at 12 h after hCG, i.e., near the time of ovulation. Indomethacin, in doses of 0.1-1.0 mg/rat, inhibited ovulation but did not inhibit the normal increase in PA activity, whereas indomethacin at the high dose of 10.0 mg/rat inhibited both ovulation and PA activity. Cycloheximide, at a dose of 0.1 mg/rat, was given at 12 h before hCG, immediately after hCG, and at 9 h after hCG. This agent inhibited ovulation most effectively when given at 12 h before hCG, yet it inhibited PA activity most effectively when given immediately after or at 9 h after hCG. Colchicine, at a dose of 0.1 mg/rat, inhibited ovulation, but not PA activity, when it was given 1 h before hCG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of ovulation in the gonadotropin-primed immature rat by exogenous prostaglandin E2.

In the past two decades there have been innumerable reports that prostaglandins (PGs) are essential for mammalian ovulation. However, we have recently found that a relatively low dose of 0.03 mg indomethacin (INDO) sc to PMSG/hCG-primed immature Wistar rats can significantly reduce ovarian PG levels without inhibiting the control ovulation rate of 60+ ova/rat (1-3). In view of this information, the present study was an effort to duplicate the earlier reports that PGs can reverse the "inhibitory" effect of INDO on ovulation. In control animals, which received PMSG and hCG only, the ovulation rate was 63.8 +/- 4.5 ova/rat. This rate was reduced to 4.1 +/- 1.1 ova/rat when the animals were injected with 1.0 mg INDO at 3 h after hCG. In no instance was this inhibition reversed when the animals were treated with 1.0 mg of PGE2 or PGF2 alpha, or a combination of both prostanoids in either a single dose at 3 h after hCG, or in 4x doses at 2-h intervals beginning at 3 h after hCG. Furthermore, in animals that did not receive INDO, the ovulation rate in PGE2-treated animals was reduced to 20.0 +/- 6.7 ova/rat, and in animals treated with PGE2 and PGF2 alpha (combined) it was reduced to 19.4 +/- 6.5 ova/rat. In summary, not only did the PGs fail to reverse the anti-ovulatory effect of INDO, PGE2 actually suppressed the ovulation rate.     

Inhibition of ovulation in the gonadotropin-primed immature rat by exogenous prostaglandin E2

In the past two decades there have been innumerable reports that prostaglandins (PGs) are essential for mammalian ovulation. However, we have recently found that a relatively low dose of 0.03 mg indomethacin (INDO) sc to PMSG/hCG-primed immature Wistar rats can significantly reduce ovarian PG levels without inhibiting the control ovulation rate of 60+ ova/rat (1–3). In view of this information, the present study was an effort to duplicate the earlier reports that PGs can reverse the “inhibitory” effect of INDO on ovulation. In control animals, which received PMSG and hCG only, the ovulation rate was 63.8 ± 4.5 ova/rat. This rate was reduced to 4.1 ± 1.1 ova/rat when the animals were injected with 1.0 mg INDO at 3 h after hCG. In no instance was this inhibition reversed when the animals were treated with 1.0 mg of PGE₂ or PGF_2α, or a combination of both prostanoids in either a single dose at 3 h after hCG, or in 4× doses at 2-h intervals beginning at 3 h after hCG. Furthermore, in animals that did not receive INDO, the ovulation rate in PGE₂-treated animals was reduced to 20.0 ± 6.7 ova/rat, and in animals treated with PGE₂ and PGF_2α (combined) it was reduced to 19.4 ± 6.5 ova/rat. In summary, not only did the PGs fail to reverse the anti-ovulatory effect of INDO, PGE₂ actually suppressed the ovulation rate.     

The relationship between ovarian progesterone and proteolytic enzyme activity during ovulation in the gonadotropin-treated immature rat.

To clarify the mechanisms by which progesterone acts as a mediator in the ovulatory process, ovulation rate and proteolytic enzyme activities were investigated in immature 22-day-old rats treated with PMSG/hCG, RU486 (10 mg/kg), synthetic antiprogesterone, and RU486 (10 mg/kg) + progesterone (10 mg/kg). The number of ova was significantly decreased when RU486 (10 mg/kg) was given from 2 h before to 4 h after the hCG injection. In addition, its inhibitory action on ovulation was reversed by exogenous progesterone (10 mg/kg) at 2 or 4 h after the hCG injection. Serum progesterone and estradiol concentrations in the rats treated with RU486 did not show any significant differences compared to controls. The proteolytic enzyme activities were measured by using the synthetic substrates alpha-N-benzoyl-DL-Arg-beta naphthylamide (BANA) and dinitrophenyl peptide (DNP). Activities were significantly increased after hCG injection in the control group during 8-9 h for BANA hydrolase and 7-10 h for DNP peptidase. The preovulatory increase of these activities was totally suppressed by RU486 with hCG. After administration of progesterone (10 mg/kg) following hCG and RU486 injection, the elevation of proteolytic, enzyme activities in the preovulatory phase was effectively reversed, and levels became similar to those in the control group. These results suggest that progesterone plays an indispensable role in the first 4 h of the ovulatory process by regulating proteolytic enzyme activities.     

Role of progesterone in the modulation of the preovulatory surge of gonadotropins and ovulation in the pregnant mare's serum gonadotropin-primed immature rat and the adult rat

The role of progesterone in the regulation of the preovulatory surge in gonadotropins and ovulation was examined in this study by use of a potent antagonist of progesterone, RU 486 (17 beta-hydroxy-11 beta-[4-dimethyl-aminophenyl]-17 alpha- [prop-1-ynyl]estra-4,9-diene-3-one). The immature rat primed with pregnant mare's serum gonadotropin (PMSG) and the cycling adult animal were the models used to verify the role of progesterone. When RU 486 (200 micrograms/rat) was given as a single dose on the morning of proestrus, there was a significant reduction in the preovulatory surge levels of gonadotropins and ovulation in both animal models. Serum progesterone levels in both models at the time of death on the evening of proestrus were unaltered upon treatment with RU 486. RU 486 did not have any effect on gonadotropin levels in immature rats 7 days after castration. These results show that the actin of RU 486 on the preovulatory gonadotropin surge is due to an antagonism of the action of progesterone on the hypothalamic-pituitary axis. Thus, a role for progesterone in modulating the preovulatory surge of gonadotropins and, consequently, ovulation is strongly suggested.     

Interference with the preovulatory luteinizing hormone surge and blockade of ovulation in immature pregnant mare's serum gonadotropin-primed rats with the anti-androgenic drug, hydroxyflutamide

The involvement of androgens in the control of ovulation has been assessed by administration of the androgen antagonist, hydroxyflutamide, to prepubertal rats treated with pregnant mare's serum gonadotropin (PMSG) to induce first estrus and ovulation. Without human chorionic gonadotropin (hCG) injection, only 46% of rats that received six 5-mg, s.c. injections of hydroxyflutamide at 12-h intervals, beginning an hour before s.c. injection of 4 IU PMSG on Day-2 (Day 0 = the day of proestrus), had ovulated a mean of 1.3 +/- 0.4 oocytes per rat when killed on the morning of Day 1, whereas 92% of sesame oil-treated controls had ovulated a mean of 6.9 +/- 0.6 oocytes. After i.p. injection of hCG at 1600 h on Day 0, 92% of hydroxyflutamide-treated rats ovulated a mean of 8.3 +/- 1.2 oocytes compared to 100% of controls, which ovulated 7.3 +/- 0.4 oocytes per rat: these groups were not significantly different from each other, nor from control rats that received no hCG. Thus, exogenous hCG completely overcame the inhibitory effect of hydroxyflutamide on ovulation. Rats treated with PMSG and hydroxyflutamide without hCG were killed either on the morning of Day 0 to determine serum and ovarian steroid levels or on the afternoon of Day 0 to determine serum LH levels. Serum levels of estradiol-17 beta and testosterone in hydroxyflutamide-treated rats were significantly higher (178% and 75%, respectively; p less than 0.01) than levels observed in controls on the morning of Day 0. Ovarian concentrations of the steroids were also elevated in hydroxyflutamide-treated rats (p less than 0.01 for testosterone only).(ABSTRACT TRUNCATED AT 250 WORDS)     

Human chorionic gonadotropin and luteinizing hormone-releasing hormone reverse the blockade of ovulation in pregnant mare's serum gonadotropin-primed immature rats by the anti-androgenic drug, hydroxyflutamide

The present study was designed to examine mechanism(s) of the anti-ovulatory action of the anti-androgen, hydroxyflutamide (OH-F). Prepubertal rats were treated with 4 IU pregnant mare's serum gonadotropin (PMSG) (day -2) to induce first estrus and ovulation. They received OH-F in sesame oil or oil alone at 08:00 and 20:00 h on day 0 (the day of proestrus) and ovulations were assessed on the morning of day 1. Eighty-three percent of control animals ovulated with a mean of 7.7 +/- 1.1 corpora lutea per rat. Hydroxyflutamide blocked ovulation in all but 2 of the 12 rats receiving this drug alone. All of OH-F treated rats that received 5 and 25 IU human chorionic gonadotropin (hCG) ovulated with means +/- SEM of 9.1 +/- 0.1 and 7.3 +/- 1.4 corpora lutea per rat, respectively. The dose of 0.2 IU hCG was essentially ineffective, while the effect of 1.0 IU hCG was intermediate. At the dose of 20 ng and above (100 and 500 ng) luteining hormone-releasing hormone (LHRH) completely overcame the ovulation blockade in the OH-F treated animals, while a 4-ng dose was ineffective. At 18:00 h on the day of proestrus, serum LH levels in control animals were 17.56 +/- 2.60 ng/mL, which were 920% above basal levels (1.90 +/- 0.13) indicating a spontaneous LH surge. This surge was suppressed in OH-F treated rats. Injection of LHRH, at the dose of 20 ng and above, reinstated the LH release in OH-F treated animals. Thus, the anti-androgen, OH-F, inhibits ovulation in PMSG-treated immature rats through its interference with the preovulatory LH surge; the inhibition can be reversed by hCG or LHRH. Hydroxyflutamide does not appear to interfere at the level of the pituitary, but may have direct action at the hypothalamic and (or) extrahypothalamic sites involved in the generation of positive feedback signals that control LH release.     

Involvement of cytosolic phospholipase A2 in the ovulatory process in gonadotropin-primed immature rats

The preovulatory LH surge induces a remarkable increase in ovarian prostaglandins (PGs) which help to mediate the ovulatory process. We investigated whether cytosolic phospholipase A2 (cPLA2) has a role in this PG production in PMSG/hCG-primed immature rats. The immunoreactive signal for cPLA2 was localized in both thecal and granulosa layers of mature follicles and became evident in response to gonadotropins. The PLA2 activity in the whole ovarian cytosol rose slightly after PMSG stimulation, persisted relatively constant until 24 h after hCG injection and thereafter increased gradually. Intra-ovarian bursal injection of arachidonyl trifluoromethyl ketone, a specific inhibitor for cPLA2 ( 1.0-3.0 mg/ovary), significantly reduced ovarian PGE2 content and the ovulation rate. These results suggest that cPLA2 exists in periovulatory follicles and functions in PG production related to the ovulation process.     

Effect of insulin-like growth factor-binding protein 7 on steroidogenesis in granulosa cells derived from equine chorionic gonadotropin-primed immature rat ovaries

Insulin-like growth factor (IGF)-binding protein (IGFBP) 7 is a secreted protein that regulates cellular proliferation, adhesion, and angiogenesis, and has low affinity for IGF compared with that of IGFBP1-IGFBP6. We sought to determine whether IGFBP7 is present in follicular fluid and to elucidate whether IGFBP7 participates in the steroidogenesis of rat mature follicles. Follicular fluid and granulosa cells (GCs) were collected from immature rats 2 days after their treatment with equine chorionic gonadotropin (eCG). IGFBP7 protein was detected in the follicular fluid and the conditioned medium of cultured ovarian GCs by immunoblot analysis. When subconfluent GCs were cultured and treated with FSH and activin, coincubation with FSH and activin markedly increased GC expression of Cyp19a1 (aromatase) mRNA and 17beta-estradiol (E(2)) secretion. The addition of recombinant murine IGFBP7 to these cultures decreased in the activin-enhanced, FSH-stimulated Cyp19a1 mRNA levels in the cells and suppressed the 17beta-E(2) levels in the culture medium. Treatment of GCs with Igfbp7-specific small interfering RNA (siRNA), which knocked down Igfbp7 expression, increased the FSH-stimulated levels of Cyp19a1 but not Cyp11a1 expression. Basal and FSH-stimulated 17beta-E(2) secretion into the culture medium was also enhanced by Igfbp7 siRNA. These results suggest that IGFBP7 suppresses estrogen production in GCs. These observations support the notion that this protein, which is secreted into the follicular fluid, may serve as an intraovarian factor that negatively regulates GC differentiation.