Differential effects of indomethacin on the sheep ovary: Prostaglandin biosynthesis, intracellular calcium, apoptosis, and ovulation

Cells of the apical wall of the dominant follicle and contiguous ovarian surface epithelium become apoptotic with the approach of ovulation in the sheep. It was hypothesized that indomethacin, an established inhibitor of prostaglandin biosynthesis and ovulation, would protect apical ovarian cells from programmed death. The anovulatory potencies of two systemic doses of indomethacin (200 and 800 mg) were tested in gonadotropin-stimulated ewes. A complete blockade of ovulation occurred at the higher dose of indomethacin. Ovulation was not inhibited by 200 mg indomethacin. Both doses of drug suppressed follicular prostaglandin production below pregonadotropin levels. Immunofluorescence detection of digoxigenin end-labeled (fragmented) DNA was used as a marker of apoptosis among ovarian surface epithelial and granulosa cells recovered from the apical hemisphere of preovulatory ovine follicles. Cellular DNA fragmentation was averted in animals given 800 mg indomethacin, whereas apoptosis ensued after 200 mg. A sustained increase in cytosolic calcium is generally a prerequisite to apoptotic DNA fragmentation and cell death. Indeed, intracellular calcium, detected by fluorescence of fura-2, was elevated in ovarian cells of animals destined to ovulate (controls, 200 mg indomethacin) in comparison to (safeguarded) cells of anovulatory ewes (800 mg indomethacin). These observations provide circumstantial evidence that apical ovarian cell degeneration by calcium-mediated apoptosis is a determinant of follicular instability and rupture, but that these events are unrelated to the gonadotropin-induced rise in prostanoid production characteristic of preovulatory follicles.     

Dose-dependent effects of indomethacin on ovulation in the sheep: relationship to follicular prostaglandin production, steroidogenesis, collagenolysis, and leukocyte chemotaxis.

A few recent investigations have indicated that it is possible for mammalian ovulation to progress to completion in the absence of a preovulatory rise in ovarian prostanoid production and that the antiovulatory mode of action of antiinflammatory agents (e.g., indomethacin) could be independent of their ability to inhibit the cyclooxygenase pathway of arachidonate metabolism. Mature ewes were treated during the preovulatory period with a systemic dosage of indomethacin that either consistently did (500 mg) or did not (100 mg) prevent follicular rupture. With both dosages, the rise in follicular production of prostaglandin F2 alpha following the surge in secretion of LH was negated. Indomethacin did not affect periovulatory patterns of change in follicular tissue concentrations of estradiol-17 beta, testosterone, or progesterone. The 500-mg dose of indomethacin inhibited collagen breakdown within the follicular wall as deduced from measurement of tissue levels of hydroxyproline. In vitro secretion of a follicular leukotactic agent and accumulation of extravascular white blood cells within the theca interna of periovulatory follicles were also suppressed by the ovulation-inhibiting dose of indomethacin. It appears that the blockage of ovulation induced by indomethacin in the sheep is largely unrelated to its capacity to suppress follicular prostaglandin biosynthesis; rather, it is more directly associated with effects on follicular collagenolysis and leukocyte chemoattraction.     

A role for lipoxygenase metabolites of arachidonic acid in porcine ovulation

Prostaglandins, products of arachidonic acid via the cyclooxygenase pathway, are essential to the porcine ovulatory process in that inhibition of their synthesis results in ovulation failure. Studies in the rat have shown that ovulation is also preceded by a rise in three ovarian hydroxyeicosatetraenoic acids, products of the lipoxygenase pathway, and inhibition of this pathway also inhibits ovulation. Experiments were designed, using a pregnant mare serum gonadotropin/human chorionic gonadotropin (hCG)-treated prepuberal gilt model, to measure pre-ovulatory changes in follicular fluid concentrations of 15-hydroxyeicosatetraenoic acid (15-HETE), and to compare the effects of indomethacin and nordihydroguaiaretic acid (NDGA) on ovulation in the pig and on 15-HETE and prostaglandin F₂α synthesis both in vivo and in vitro. Follicular fluid concentrations of 15-HETE were elevated significantly just prior to the expected time of ovulation (40 h after hCG). When indomethacin (10 mg) was injected into the ovarian stalk at 24 h after hCG, follicular fluid concentrations of both 15-HETE and prostaglandin F₂α were lower (P<0.01) than controls at 40 h and ovulation rate was suppressed (P<0.01). When NDGA (5 mg) was administered in the same manner, ovulation rate was suppressed (P<0.01), but the levels of 15-HETE and prostaglandin F₂α were not altered. Synthesis of 15-HETE by cultured granulosa and theca interna cells was reduced by the presence of NDGA (1 mg/ml), whereas indomethacin (100 ng/ml) lowered 15-HETE production in theca interna cells only. These results clearly demonstrate that indomethacin can block the lipoxygenase as well as the cyclooxygenase pathways, depending on the dose used, and suggest that lipoxygenase metabolites of arachidonic acid are involved in the ovulatory process in the pig.     

Effect of treatment of estrous ewes with indomethacin on the distribution of ovarian blood to the periovulatory follicle

The level of prostaglandin (PG) F2 alpha increases within the wall of the ovine follicle pending ovulation. Coincidently, the quantity of ovarian blood distributed to the follicular wall progressively declines. A potential cause(PGF2 alpha)-and-effect (impaired follicular blood supply) relationship was considered. At an early stage of estrus, ewes were injected systemically either with vehicle or indomethacin (an inhibitor of biosynthesis of prostaglandins). Abdominal laparotomies were carried out and the ovaries examined near the expected time of ovulation. The ovary containing the largest follicle or an ovulation point was perfused with radioactive microspheres via the ovarian artery. The periovulatory follicle was isolated from the ovary and the content of radioactivity monitored with respect to that of the whole ovary. Follicular tissue was analyzed for PGF2 alpha. Treatment with the drug was associated with: 1) failure of follicular rupture; 2) follicular hyperemia and edema; and 3) suppressed synthesis of PGF2 alpha. A reduction of the supply of ovarian blood reaching the preovulatory follicle, and a mediatory task of follicular prostaglandins in this process, could be a critical determinant of ovulation.     

The effect of indomethacin of HMG-HCG induced ovulation in the phesus monkey.

The investigation was designed to study the influence of indomethacin on gonadotropin induced ovulation in the rhesus monkey. Six mature female monkeys were treated with HMG-HCG for at least 2 control ovulatory cycles at dosage levels adjusted to induced ovulation while avoiding superovulation. Ovulation was confirmed by observation of the ovaries for fresh ovulation points at laparotomy. Following establishment of an appropriate dosage schedule, treatment was begun with indomethacin (5 mg/kg/day) starting 5 days prior to HCG and continuing to the time of laparotomy. In a second treatment cycle, indomethacin was administered at a dose of 5 mg/kg b.i.d. together with the established dose of HMG-HCG. Ovarian inspection was carried out as in the control cycles. Venous blood was obtained on treatment days 4, 7, 10 and 11 for determination of serum estrone, estradiol and progesterone. Indomethacin administration resulted in ovulation inhibition at a dose of 10 mg/kg/day when ovulation inducing doses of gonadotropins were administered. Peripheral blood steroid levels suggest that follicle maturation and estrogen production was unimparied by indomethacin. These findings indicate that the ovarian synthesis of prostaglandin may be essential in the process of ovulation.     

The effect of indomethacin on HMG-HCG induced ovulation in the rhesus monkey

The investigation was designed to study the influence of indomethacin on gonadotropin induced ovulation in the rhesus monkey. Six mature female monkeys were treated with HMG-HCG for at least 2 control ovulatory cycles at dosage levels adjusted to induce ovulation while avoiding superovulation. Ovulation was confirmed by observation of the ovaries for fresh ovulation points at laparotomy. Following establishment of an appropriate dosage schedule, treatment was begun with indomethacin (5 mg/kg/day) starting 5 days prior to HCG and continuing to the time of laparotomy. In a second treatment cycle, indomethacin was administered at a dose of 5 mg/kg b.i.d. together with the established dose of HMG-HCG. Ovarian inspection was carried out as in the control cycles. Venous blood was obtained on treatment days 4, 7, 10 and 11 for determination of serum estrone, estradiol and progesterone. Indomethacin administration resulted in ovulation inhibition at a dose of 10 mg/kg/day when ovulation inducing doses of gonadotropins were administered. Peripheral blood steroid levels suggest that follicle maturation and estrogen production are unimpaired by indomethacin. These findings indicate that the ovarian synthesis of prostaglandin may be essential in the process of ovulation.     

Rat ovarian prostaglandin levels and ovulation as indicators of the strength of non-steroidal anti-inflammatory drugs

Immature Wistar rats were treated with pregnant mare's serum gonadotropin and human chorionic gonadotropin to induce ovulation. The non-steroidal anti-inflammatory drugs indomethacin, diclofenac, flurbiprofen, and phenylbutazone inhibited both the ovulation rate and the normal increase in ovarian prostaglandin E during ovulation. Tolmetin, ibuprofen, and aspirin did not have any significant effect. There was a significant correlation between the ovulation rate and the level of ovarian prostaglandin E following treatment with these drugs. When indomethacin was given in graded doses, there was also a correlation between ovulation rate and the dose-dependent inhibition of ovarian prostaglandin E.     

Prostaglandin E(1) inhibits abnormal follicle rupture and restores ovulation in indomethacin-treated rats

In the presence of indomethacin, an inhibitor of prostaglandin (PG) synthesis, the gonadotropin surge induces abnormal follicle rupture at the basolateral follicle sides, thus preventing effective ovulation in rats. This study was undertaken to analyze whether exogenous prostaglandin administration can overcome the antiovulatory action of indomethacin. Cycling rats were treated with vehicle (olive oil) or indomethacin (1 mg/rat) on the morning of proestrus. Rats treated with indomethacin were injected with different doses (50, 250, or 500 micro g/rat) of PGE(1), PGE(2), PGF(2alpha), or vehicle (saline) at 1900 h in proestrus. The ovulatory response was analyzed on the morning of estrus by evaluating follicle rupture and the location of the oocytes in serially sectioned ovaries. The number of oocytes in the oviducts was also counted in rats treated with the highest prostaglandin doses. In indomethacin-treated rats, most newly formed corpora lutea showed abnormal follicle rupture at the basolateral sides. In addition, invasion of the ovarian stroma and blood and lymphatic vessels by granulosa cells and follicular fluid was observed. Prostaglandins of the E series, and especially PGE(1), inhibited abnormal follicle rupture and restored ovulation, although the number of oocytes in the oviducts were significantly decreased. PGF(2alpha) was only partially effective in inhibiting abnormal follicle rupture and restoring ovulation. These data suggest that prostaglandins of the E series, and particularly PGE(1), play a crucial role in ovulation by determining the targeting of follicle rupture at the apex, thus allowing release of oocytes to the periovarian space.     

Effects of indomethacin and prostaglandin F2α on ovulation and ovarian contractility in the rabbit

The present investigation was carried out to determine whether inhibition of ovulation in the rabbit by administration of indomethacin can be correlated with any change in ovarian contractility at ovulation time and can be reversed by administration of prostaglandins. Indomethacin was adminstered intra-muscularly using three different schedules in a dose of 5 mg/kg. A reduced number of ruptured follicles following HCG was noted in all groups treated with indomethacin. Infusion of PGF_2α into the aorta (1 μg/kg/min.) could reverse this effect. Less pronounced ovarian contractility was observed after indomethacin treatment, but infusion of PGF_2α immediately enhanced contractility in ovaries from indomethacin treated rabbits. The inhibition of ovulation in the rabbit associated with indomethacin adminstration may be related to suppression of ovarian contractions. These data also suggest that prostaglandins may play a significant role in the mechanism of ovulation through an influence on ovarian contractility.     

The effect of indomethacin on ovarian prostaglandin release in hens

Indomethacin, an inhibitor of prostaglandin (PG) synthetase, will block uterine muscle electromyographic activity (EMG activity) and oviposition at a midsequence oviposition and ovulation in domestic hens, but does not block the increase in EMG activity associated with the first ovulation of a sequence. To assess the potential relationship between prostaglandin release from the ovarian follicles and EMG activity in egg-laying hens, we determined the concentrations of PGF2 alpha, 13,14-dihydro-15-keto-PGF2 alpha (PGFM), and PGE2 in brachial, ovarian follicular and uterine venous plasma and tissues in relation to uterine muscle EMG activity at the first ovulation and at a midsequence oviposition. The concentrations were measured after an i.m. injection (25 mg/hen) of indomethacin. In control hens sampled hourly, beginning 4 h before the peak of EMG activity at the first ovulation of a sequence, there was a sharp increase (p less than 0.05) in concentrations of PGF2 alpha and PGFM in brachial vein plasma coincident with the increase (p less than 0.05) in uterine EMG activity. Hens pretreated with indomethacin also had increased plasma PGF2 alpha and PGFM levels (p less than 0.05) in brachial vein plasma and increased uterine EMG activity (p less than 0.05) at this time. Indomethacin treatment lowered but did not eliminate mean levels of PGF2 alpha in the venous effluent from the largest preovulatory follicle at the first ovulation (36.0 +/- 9.9 ng/ml vs. 14.4 +/- 1.8 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Significance of follicular cyclooxygenase and lipoxygenase pathways of metabolism of arachidonate in sheep

Concurrent changes in concentrations of a product of the cyclooxygenase (prostaglandin [PG] F2 alpha) and lipoxygenase (leukotriene [LT] B4) routes of metabolism of arachidonic acid were measured by radioimmunoassay within the wall of periovulatory ovine follicles. Increased concentrations of PGF2 alpha were detected within follicles before, during and following the time of ovulation. A significant rise in LTB4 was not observed until after follicular rupture had occurred. Inhibition of synthesis of PGF2 alpha by indomethacin was associated with a complete blockade of ovulation. Nordihydroguaiaretic acid, an inhibitor of 5-lipoxygenase, had no effect on ovulation. Periovulatory administration of either drug did not alter sera profiles of progesterone during subsequent luteal phases. These results reconfirm the importance of the cyclooxygenase system in the mechanism of ovulation. It does not appear that follicular LTB4 is a key component in the processes of ovulation or luteinization in sheep.     

Modulatory role of eicosanoids in vascular changes during the preovulatory period in the rat

Previous studies have demonstrated the involvement of eicosanoids (prostaglandins and hydroxyperoxides, including leukotrienes) in ovulation in several mammalian species. In this study, the role played by eicosanoids in the vascular changes that occur in the immediate preovulatory period after human chorionic gonadotropin (hCG) stimulation was examined in the rat. Changes in the ovarian uptake of two iodinated proteins were examined 30 minutes after i.v. injection of 125I-bovine serum albumin (BSA, Mr = 68,000) and 125I-alpha 2-macroglobulin (alpha 2M, Mr = 750,000). Uptake was measured during 30 min, 0, 3, 6, and 9 h after induction of ovulation by an i.p. injection of human chorionic gonadotropin (hCG, 10 IU). hCG enhanced the uptake of both iodinated proteins, with peak uptake values at 6 and 9 h. Intra-bursal injections of an ovulation inhibiting dose (0.5 mg/bursa) of indomethacin-a cycooxygenase inhibitor-and nordihydroguaiaretic acid (NDGA), esculetin, or caffeic acid--inhibitors of lipoxygenase--concomitantly with hCG attenuated the action of the hormone on 125I-BSA uptake. Indomethacin and esculetin were without effect on the uptake of alpha 2M. Ovarian and follicular blood flow was measured using 113Sn-microspheres. hCG increased ovarian and follicular blood flow with the most pronounced effect at the early time of 1.5 h. Indomethacin and NDGA did not attenuate this action of hCG. Accordingly, ovarian vascular resistance was reduced by hCG at 1.5, 6, and 9 h post-hCG, respectively, and indomethacin and NDGA had no significant effects. We suggest that one way in which eicosanoids are involved in follicular rupture is by their modulation of vascular permeability as revealed by uptake of the protein marker albumin.     

Expression of mRNAs encoding tumor necrosis factor-alpha and its receptor-I in buffalo ovary

The tumor necrosis factor-alpha (TNF-alpha) plays an important role in ovarian follicular development and ovulation process and acts through its receptor (TNFRI). The present investigation describes the expression of mRNAs encoding TNF-alpha and TNFRI in relation to glyceraldehyde-3-phosphate dehydrogenase (G3PDH) and beta-actin as control genes, using RT-PCR, in granulosa cells, intact follicles and luteal tissues from buffalo ovary. There was significant higher expression of mRNAs encoding TNF-alpha in granulosa cells from medium follicles and TNFRI expression increased with increase in size of follicles. Post-ovulatory structures (corpus luteum and corpus albicans) exhibited significantly higher expression of TNFRI mRNAs as compared to that obtained in intact follicles suggesting its immediate and critical role just after ovulation, for mediating TNF-alpha action on these tissues. Though the expression of TNF-alpha mRNA was stimulated by treatment of granulosa cells with FSH during culture, the expression of TNFRI mRNA did not change. The FSH alongwith IGF-I did not exert any effect. These results suggested an important role of TNF-alpha and its receptor in buffalo ovarian functions.     

Effects of pH on in vitro ovulation of goldfish (Carassius auratus) oocytes

The in vitro effects of pH on goldfish (Carassius auratus) ovulation were investigated. Final oocyte maturation and follicular detachment were induced in vivo in gravid goldfish by HCG injections and elevated holding temperatures. Females were biopsied to determine the time of germinal vesicle breakdown and when oocytes should be removed for in vitro incubations. Prior to ovulation, the ovaries were removed, dissected and mature intrafollicular oocytes were incubated in media of varying pH (7.3-8.9). There was a significant increase in ovulation as the pH of the incubation increased and this ovulation could be blocked by indomethacin. Indomethacin did not inhibit the actual mechanism of oocyte expulsion since exogenous PGF2 alpha induced ovulation in indomethacin blocked incubates. Increased pH did not increase the ovulatory response observed with exogenous PGF2 alpha. The combined results suggest that an increase in incubation pH stimulates prostaglandin synthesis that, in turn, stimulates ovulation.     

Effects of aromatizable and nonaromatizable androgen treatments on luteinizing hormone receptors and ovulation induction in immature rats

The effects of androgen pretreatment on follicle-stimulating hormone (FSH)-stimulated luteinizing hormone (LH) receptor induction in ovarian granulosa cells was examined. Immature female rats were treated with various doses (0.1-5 mg/rat) of testosterone (T), 5 alpha-dihydrotestosterone (DHT), 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol), or 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol). Subsequent follicular development was stimulated by treatment with ovine FSH. LH receptor induction in granulosa cells and ovulatory responses to 10 IU human chorionic gonadotropin (hCG) were examined. Since LH receptor induction requires the synergistic action of both FSH and estradiol, the effects of the androgen pretreatment on FSH-stimulated estradiol production were also examined. Dihydrotestosterone treatment at doses greater than 1 mg inhibited LH receptor induction by approximately 70%, which resulted in absent ovulatory responses. Treatment with 1 mg or more of T or 3 alpha-diol had no effect on LH receptor induction, yet the hCG-stimulated ovulation rate was reduced to 40% of that seen in vehicle-treated controls. 3 beta-Diol, at a dose of 1 mg/rat, did not affect LH receptor induction but did reduce hCG-stimulated ovulation responses. No significant effects of androgen treatment on ovarian or uterine weight or FSH-stimulated estradiol production were observed. These results suggest that androgens can act at multiple sites to inhibit ovarian follicular development and function. In addition these studies demonstrate that, although LH receptor induction is necessary, it may not be a sufficient condition to ensure ovulation of ovarian follicles.     

Effects of 2(p-biphenyl) propionic acid on the life span of the corpus luteum of pseudopregnant and cyclic rats

A potent prostaglandin synthesis inhibitor (cyclooxygenase inhibitor), 2(p-biphenyl) propionic acid (BPPA), was administered subcutaneously at the dose of 1 mg twice daily to pseudopregnant and cyclic rats. It was found to prolong the duration of pseudopregnancy by about 10 days and to have little or no effect on estrous cycle length. It did not block ovulation and had little effect on ovulation rate in cyclic rats. BPPA was given to pseudopregnant rats in two trials (one in October-December and the other in March-May) to determine its effect on ovarian weight and plasma progesterone concentration on Days 14, 15 and 16 (Day 0=day of induction of pseudopregnancy). BPPA significantly (P less than 0.001) increased plasma progesterone concentration and reduced ovarian weight. The present data support the hypothesis that prostaglandins cause the normal functional demise of the corpora lutea of pseudopregnancy in the intact rat, and that depressing their synthesis will prolong the functional life span of the corpora lutea.     

The influence of prostaglandin E2 and indomethacin on progesterone production and ovulation in the rabbit ovary perfused in vitro

The effects of prostaglandin E2 (PGE2) on the ovulation process were studied in a recirculating perfusion model using ovaries from virgin rabbits. Ovulation frequency, time of ovulation, and progesterone release from the ovaries were examined after the addition of PGE2, either alone or with luteinizing hormone (LH) in the presence or absence of indomethacin. The stimulatory effect of LH on ovulation was totally blocked if the ovaries were exposed to indomethacin at the same time. Ovaries treated with PGE2 alone did not ovulate, and PGE2 was unable to restore indomethacin-blocked ovulation. Conversely, the frequency of ovulation was reduced in ovaries treated with PGE2 and LH compared with controls receiving only LH. There was no measurable difference in the pattern of progesterone release between ovaries simultaneously treated with LH and indomethacin and LH-treated controls. Ovaries exposed to PGE2 alone showed only a slight increase of progesterone release in the medium, while those treated with PGE2 in combination with LH in the perfusate showed a smaller progesterone release than those treated with LH alone. The results confirm the blocking effect on ovulation by indomethacin. PGE2 could not reverse this effect, but instead reduced the number of LH-induced follicular ruptures. Indomethacin had no effect on progesterone levels, while PGE2 (which alone showed a slight stimulating effect on the steroid concentration) together with LH counteracted the effect of LH on progesterone release.     

Cycloheximide inhibition of ovulation, prostaglandin biosynthesis and steroidogenesis in rabbit ovarian follicles

Cycloheximide (5 mg/kg, i.v.) significantly inhibited ovulation in the rabbit when it was administered as early as 20 h before the ovulation process was initiated by hCG, and as late as 1 h after hCG. The ovulation rate was significantly reduced, but follicular biosynthesis of prostaglandins E and F was only partly inhibited. The biosynthesis of progesterone and oestradiol in follicles during the early stages of the ovulation process was also inhibited. Cycloheximide may therefore inhibit ovulation by a mechanism which is different from the action of indomethacin, and this mechanism may involve the suppression of ovarian steroidogenesis.     

Effect of a steroidal (prednisolone) and nonsteroidal (indomethacin) antiinflammatory agent on ovulation and follicular accumulation of prostaglandin F2 alpha in sheep

The antiinflammatory steroid, prednisolone, was administered to sheep during the preovulatory period. The drug did not produce a blockade of follicular rupture. However, prednisolone negated a rise in production of prostaglandin (PG) F2 alpha characteristic of preovulatory follicles. Indomethacin, a nonsteroidal antiinflammatory agent, was 100% effective in preventing ovulation. Levels of PGF2 alpha within follicular tissue were very low following treatment with indomethacin. These findings indicate that ovulation can occur in the absence of a preovulatory elevation in follicular accumulation of PGF2 alpha. Potency of antiinflammatory drugs as inhibitors of ovulation appears to hinge upon their ability to cause a marked suppression in follicular synthesis of prostaglandins.