Histamine induces ovulation in the isolated perfused rat ovary

Ovulatory effects of histamine and specific antagonists were studied in isolated perfused ovaries from immature rats treated with 10 i.u. PMSG to stimulate follicular growth and maturation. Histamine alone, like LH, induced ovulation in all ovaries tested, but the number of follicular ruptures was lower after histamine (7.0 and 2.2 ruptures, respectively, per ovary). The histamine-induced ovulations could be inhibited dose-dependently by the H1-receptor antagonist, pyrilamine, or the H2-antagonists, cimetidine and ranitidine. At the concentrations tested, these antagonists did not, when given separately, reduce the LH-induced ovulations significantly, but pyrilamine and cimetidine in combination lowered the ovulation frequency by 65%. The prostaglandin synthesis inhibitor, indomethacin, was not able to block the histamine-induced ovulations.     

Cyclic adenosine 3',5'-monophosphate-induced ovulation in the perfused rat ovary and its mediation by prostaglandins

The role and mechanism of action of cyclic adenosine 3',5'-monophosphate (cAMP) in the ovulatory process was investigated by using the in vitro-perfused rat ovary model. Ovaries of pregnant mare's serum gonadotropin (PMSG, 20 IU)-primed rats were perfused for 21 h beginning in the morning of induced proestrus. In vitro stimulation with luteinizing hormone (LH; 0.1 micrograms/ml) resulted in 2.4 +/- 0.7 ovulations per treated ovary. Ovulations could also be induced by the addition of forskolin (30 microM) or dibutyryl cAMP (dbcAMP, 1 mM) with isobutylmethylxanthine (IBMX, 0.2 mM), with 11.8 +/- 1.9 and 18.6 +/- 4.4 ovulations per treated ovary, respectively. Indomethacin (5 micrograms/ml) significantly decreased the number of ovulations in the forskolin and dbcAMP + IBMX groups. The addition of prostaglandin E2 (PGE2; 1 micrograms/ml three times during the perfusion) to the forskolin + indomethacin group reversed the inhibition of ovulation (21.6 +/- 5.4 ovulations per treated ovary). Ovarian PGE tissue levels were significantly higher 10 h after stimulation with either LH, forskolin, or dbcAMP + IBMX compared to the unstimulated control group. Ovulated oocytes in the LH and forskolin groups resumed meiosis but oocytes in the dbcAMP + IBMX groups remained immature. This study shows that an increase in ovarian cAMP, even if not induced by LH, is sufficient to cause ovulation of preovulatory rat follicles, supporting the involvement of cAMP in the normal ovulatory process of the PMSG-treated rat. Furthermore, prostaglandin involvement in cAMP-induced ovulations is demonstrated.     

Histamine modulates the production of interferon-gamma and interleukin-2 by mitogen-activated human mononuclear blood cells

Histamine inhibited the production of interferon-gamma and interleukin 2 (IL-2) induced in human peripheral blood mononuclear cells by Staphylococcal Enterotoxin A (SEA) but had no effect on the expression of IL-2 receptors. The effects on lymphokine production were dose dependent with maximal inhibition occurring at histamine concentrations of 10(-4) to 10(-6) M. The H2-agonist 4-methylhistamine but not the H1-agonist 2-methylhistamine modulated lymphokine production in a similar manner as histamine. Histamine at concentrations of 10(-3) to 10(-8) M had no inhibitory effect directly on the activity of admixed IL-2 containing medium. The inhibitory effects of histamine could be reversed by the H2-antagonist cimetidine but not by the H1-antagonist diphenhydramine. This indicates that the inhibitory effects of histamine on lymphokine production are mediated through H2-receptors on mononuclear cells.     

Histaminergic effects on the isolated rat ovarian artery during the estrous cycle

Histamine may play a role in many of the events occurring in the ovarian tissue and leading to ovulation. To elucidate the histaminergic influence on the ovarian vasculature, the mechanical response of the isolated rat ovarian artery to histamine and histamine agonists was investigated. Histamine relaxed the precontracted vessel segments in a concentration-dependent way, amounting to 82.7 +/- 4.3% of the papaverine-induced relaxation. This relaxant effect was counteracted by both the H1 antagonist, pyrilamine, and the H2 antagonist, cimetidine. That the effect of histamine was mediated by both histamine receptor subtypes was further confirmed by the relaxant effect produced in the presence of either of the H1-specific agonists, 2-pyridylethylamine and 2-methylhistamine on the one hand, and the H2-specific agonists, impromidine and 4-methylhistamine on the other. The H1 receptor-induced relaxation was mediated via an effect on the endothelium, whereas the H2 receptor-mediated relaxation was mostly a direct effect on the smooth musculature in the vessel wall. No major differences in the mechanical response of the rat ovarian artery were seen during the different stages of the estrous cycle, although at late proestrus, just before ovulation, the maximum relaxation induced by histamine was particularly high, in spite of a low sensitivity of the receptors for the amine.     

Modulatory effect of HCO3- on rat mast cell exocytosis: cross-talks between bicarbonate and calcium.

HCO-3 modulation of histamine release and its relationship with the Ca2+ signal were studied in serosal rat mast cells. Histamine release was induced by Ca2+ mobilizing stimuli, namely compound 48/80, thapsigargin, Ca2+ chelators, ionophore A23187, and PMA and ionophore A23187 in a HCO-3-buffered medium or a HCO-3-free medium. The presence of HCO-3 reduced histamine release by 48/80, Ca2+ chelators, A23187, and PMA/A23187, but increased histamine release induced by thapsigargin. Histamine release by PMA was significantly higher in a HCO-3-free medium than in a HCO-3-free medium, as it was the PMA potentiation of histamine release by A23187. [Ca2+]i changes induced by these drugs were measured in fura-2-loaded mast cells. In thapsigargin and EGTA or BAPTA preincubated mast cells [Ca2+]i increase was higher in a HCO-3-buffered medium than in a HCO-3-free medium in the presence of Ca2+. On the contrary, in compound 48/80 and PMA/A23187 activated mast cells the [Ca2+]i increase is the same both in the presence and in the absence of HCO-3. The effect of HCO-3 on histamine release in serosal rat mast cells depends on the stimulus, but it is not related to the presence of Cl-. In thapsigargin-stimulated mast cells the effect of HCO-3 on histamine release may be related to the Ca2+ signal, but in compound 48/80, EGTA, and PMA/A23187-activated mast cells there is no relationship between intracellular Ca2+ and the inhibitory effect of HCO-3 on histamine release. Additionally, the PKC pathway is implicated in the inhibitory effect of HCO-3 on histamine release, the higher the chelation of calcium rendering the higher the enhancement of the response after adding calcium in the absence of HCO-3.     

Can reduced consumption of gonadotrophins account for ovarian compensation in unilaterally ovariectomized, immature mice injected with gonadotrophins?

The effect of unilateral ovariectomy on ovulation rates in immature mice was studied. Ovulations were induced by injecting PMSG and hCG and their number was determined by counting tubal oocytes. A 2--3-fold increase in number of ovulations per ovary was observed after unilateral ovariectomy, and daily injections of progesterone abolished this ovulatory compensation. No significant increase in serum concentrations of immunoreactive FSH and LH was observed at 4, 8, 32 and 51 h after unilateral ovariectomy. Progesterone treatment lowered FSH levels at all times, while LH was unaffected. In intact mice, ovarian sensitivity to PMSG and hCG was not substantially affected by progesterone. Ovulatory compensation in immature gonadotrophin-injected mice appears to arise through a negative feedback mechanism and transiently increased secretion of pituitary gonadotrophin rather than through a greater utilization of a fixed amount of gonadotrophin.     

Stimulatory effects of bradykinin on the ovulatory process in the in vitro-perfused rat ovary

The role of bradykinin in the ovulatory process was investigated using an in vitro-perfused rat ovary model. Stimulation with LH (0.1 micrograms/ml) resulted in 2.6 +/- 0.5 (mean +/- SEM) ovulations per ovary, whereas no ovulations occurred in the nonstimulated control group. Bradykinin (5 microM) added to the perfusion system hourly for 10 h induced 2 of 5 ovaries to ovulate, with 2 and 3 ovulations, respectively. When bradykinin (5 microM) was given as a single dose at 5 or 10 h after LH, the ovulation rate was significantly increased to 11.0 +/- 2.8 and 8.6 +/- 2.0 ovulations per ovary, respectively. A competitive bradykinin antagonist, phenylalanine bradykinin, inhibited the bradykinin-induced increase in LH-stimulated ovulations. The addition of LH, but not of bradykinin, increased the levels of prostaglandin endoperoxide synthase in granulosa cells, but the levels of the enzyme in the residual ovarian tissue were negligible. In contrast, prostacyclin synthase was predominantly located in the residual ovarian tissue. This enzyme was not affected by LH or bradykinin. LH increased the tissue levels of prostaglandins, predominantly prostaglandin E2 (PGE2), at 7 h, whereas the stimulatory effect of bradykinin was smaller, with a preferential increase in prostacyclin (prostaglandin I2) levels. This study indicates a modulatory role of bradykinin, possibly involving prostacyclin late in the ovulatory process, in the rat.     

Cytotoxicity of Vibrio vulnificus cytolysin on rat peritoneal mast cells

Histamine has been thought to be a permeability enhancing factor in Vibrio vulnificus infection. The injection of living bacteria or purified V. vulnificus cytolysin (VVC) can cause lethality in mice by inducing hemoconcentration and increased vascular permeability. In the present study, we tried to identify whether histamine release causes the increased vascular permeability that is responsible for the lethal effect of VVC. Treatment of rat peritoneal mast cells with high concentrations of VVC caused the release of whole cellular histamine and lactate dehydrogenase (LDH). At concentrations less than 10 HU/ml, histamine and LDH were not released whereas preloaded 2-deoxy-D-glucose was rapidly effluxed with the concomitant decrease in cellular ATP. VVC-treated mast cells were refractory to the stimulation of histamine secretion by Compound 48/80 but remained fully responsive to Ca2+ plus GTP-gamma-S. These results indicate that histamine can be released from mast cells only when the concentration of VVC is high enough to cause the lysis of cells. At low concentrations, VVC does not induce the release of stored histamine from damaged cells. The intravenous injection of 80 HU purified VVC to rats, which can produce the calculated blood concentration of about 3 HU/ml, caused a marked increase in pulmonary vascular permeability, hemoconcentration and death. However, no increase in blood histamine level was detected. This level of VVC in rat blood was enough to cause severe hemoconcentration and lethality but might not be enough to cause cytolysis of the mast cells and resulting histamine release.     

Steroidogenic effects of lipoproteins and 25-hydroxycholesterol on luteal and ovarian cells: a comparison of two pseudopregnant rat models

Steroidogenesis was compared between luteal cells from immature pseudopregnant (PSP) rats induced by either 5 IU pregnant mare serum gonadotropin (PMSG) alone or 50 IU PMSG combined with 25 IU human chorionic gonadotropin (hCG). It was also determined whether differences in steroidogenesis existed when the entire ovary (ovarian cells) or just luteal cells from Day 4 PSP rats were exposed in vitro to lipoproteins or 25-hydroxycholesterol (25-OH chol). In the absence of luteinizing hormone (LH), basal steroid accumulation, especially progesterone (P4) was around fourfold greater in luteal cells from rats treated with PMSG alone than from rats receiving PMSG-hCG. However, serum P4 and LH were about fivefold greater in the latter group. It is therefore likely that net cellular cholesterol uptake per luteal cell is lower in the PMSG-hCG treated rats, but this is offset by a much greater mass and number of corpora lutea. Lipoproteins (HDL and LDL) and 25-OH chol stimulated in vitro luteal steroidogenesis from rats treated with PMSG alone or PMSG-hCG, and their responses were virtually identical. Therefore, luteal steroidogenesis in the rat always depends on exogenous cholesterol even though treatment in the preovulatory period with PMS or PMSG-hCG and serum LH and follicle-stimulating hormone (FSH) levels on Day 4 PSP are very different. When ovarian cells from PMSG-hCG treated rats were incubated with LH plus HDL or 25-OHP, the production of 20 alpha-DHP was considerably greater than luteal cell production which may be due to a contribution from nonluteal cells. Indeed, about 30% of the cells in the PMSG-hCG group represent nonluteal components as estimated by weight and deoxyribonucleic acid content.     

Alterations in mast cell degranulation and ovarian histamine in the proestrous hamster

Mast cells in the ovary of cyclic hamsters were observed exclusively in the hilum and in the vicinity of blood vessels that enter and exit the ovary. Ovaries were collected on proestrus from hamsters at 0900 h preluteinizing hormone (LH) surge, 1500 h (peak LH surge), and 2100 h (post-LH surge) and processed for routine histologic staining with toluidine blue. A significant increase in the percentage of extensively degranulating mast cells was observed coincident with the gonadotropin surge (0900 h: 5.39 +/- 0.97%; 1500 h: 20.39 +/- 2.76%). At the peak of the LH surge the ovarian histamine concentration was also significantly higher than those before and after the surge (1500 h: 5.13 +/- 0.94 ng/mg ovary; 0900 h and 2100 h: 2.84 +/- 0.35 and 3.02 +/- 0.48 ng/mg, respectively). The results indicate that a major source of ovarian histamine may be mast cells residing in the ovarian hilum and surrounding the ovarian blood vessels that enter and exit the ovary. In addition, the gonadotropin surge on the day of proestrus may be a trigger for release of mast cell histamine.     

Participation of mast cell 5-hydroxytryptamine in the vasoconstrictor effect of neurotensin in the rat perfused hindquarter

Neurotensin (NT) (1 X 10(-8) - 1.5 X 10(-6) g ml-1) caused a transient, dose-dependent increase in perfusion pressure in the rat perfused hindquarter. The vasoconstrictor effect of NT was associated with a short-lived, dose-dependent release of histamine and 5-hydroxytryptamine (5-HT) in the hindquarter effluent. Compound 48/80, a classical mast cell secretagogue, also elicited a vasoconstrictor effect in, and release of histamine from, the rat hindquarter. The vasoconstrictor effect and the release of histamine and 5-HT evoked by NT were much smaller in hindquarters derived from rats pretreated with compound 48/80 for 4 days to cause mast cell depletion than in hindquarters derived from control rats. The mast cell inhibitor cromoglycate (4 mg ml-1) inhibited by about 50% the histamine releasing effect and vasoconstriction produced by the lowest concentrations of NT utilized. The histamine releasing effect of compound 48/80 was more sensitive to blockade by cromoglycate than that of NT. The steroidal antiinflammatory and antiallergic drug dexamethasone did not affect the histamine and 5-HT releasing effect of NT. The vasoconstrictor effects of NT, compound 48/80 and 5-HT were markedly reduced by the 5-HT receptor antagonist methysergide (1 X 10(-7) g ml-1). Histamine (1 X 10(-6) - 10(-4) g ml-1) evoked a decrease in perfusion pressure in hindquarters pre-exposed to noradrenaline. The results suggest the participation of mast cell 5-HT in the vasoconstrictor effect of NT in the rat perfused hindquarter.     

Direct ovarian effects of a GnRH agonist in hypophysectomized immature rats: inhibition of theca-interstitial luteinizing hormone receptor mRNA expression and induction of interstitial cell differentiation

The effect of a gonadotropin-releasing hormone (GnRH) agonist on luteinizing hormone (LH) receptor mRNA expression was examined histologically in the ovaries of immature hypophysectomized (HPX) rats by in situ hybridization. In the ovaries of HPX rats treated with diethylstilbestrol (DES) and pregnant mare serum gonadotropin (PMSG), LH receptor mRNA was expressed in the granulosa cells of mature follicles as well as the theca-interstitial cells. In DES-primed ovaries of rats treated with both GnRH agonist plus PMSG, many follicles were luteinized without ovulation, and the signal of LH receptor mRNA disappeared completely in the theca-interstitial cells as well as the luteinized cells, but remained in the granulosa cells of unaffected mature follicles. The complete suppression of the theca-interstitial LH receptor expression by GnRH agonist was also observed in HPX rats that received no other treatment. On the other hand, the coadministration of a GnRH antagonist with PMSG resulted in the hyperstimulation of follicular growth, accompanied by very strong expression of LH receptor mRNA in the granulosa cells as well as the thecainterstitial cells. In addition, morphological changes in the ovarian interstitial cells were also induced by the administration of GnRH agonist in HPX rats: loose connective tissue decreased and the interstitial cell mass markedly increased. The increase of the interstitial cells became more prominent when rats were treated with GnRH agonist and testosterone simultaneously. These results suggest that GnRH may be an important factor for modulating the interstitial cell function and differentiation in the rat ovary.     

Neurotensin stimulates histamine release from the isolated, spontaneously beating heart of rats

Neurotensin (NT) evoked a transient, dose-dependent histamine release (ED50 170 ng ml-1) from the rat perfused heart. Histamine release by NT occurred within seconds and lasted less than 2 min. The histamine releasing effect of NT was followed by a dose-dependent increase of the perfusion pressure and a slight tachycardia. The histamine releasing effect of NT was completely abolished in hearts derived from rats pretreated for 3 days with high doses of compound 48/80. The coronary vasoconstrictor effect of NT was increased in hearts derived from compound 48/80-pretreated rats. The mast cell inhibitor cromoglycate markedly inhibited NT-induced histamine release without affecting the coronary vasoconstrictor effect of NT. The histamine releasing effect of NT was inhibited, while its coronary vasoconstrictor effect was markedly potentiated, in hearts derived from rats pretreated with the antiallergic and antiinflammatory steroid dexamethasone. The increase of perfusion pressure evoked by NT was not modified by antihistamine drugs. Infusions of exogenous histamine (10(-6)-10(-5) g ml-1) caused a dose-dependent coronary vasodilation in the rat perfused heart. The results suggest that NT stimulates histamine release from cardiac mast cells. These results together with those obtained in previous studies suggest that mast cell mediators (particularly histamine and serotonin) are unlikely to be responsible for the coronary vasoconstrictor effect of NT in the rat perfused heart.     

Effects of compound 48/80 and exogenous histamine on wound healing in mice

Compound 48/80 has previously been shown to improve wound healing in rats, presumably through stimulation of histidine decarboxylase activity and mobilization of histamine from mast cells. In the present study, C57Bl/6 mice were wounded by dorsal skin incision followed by treatment with compound 48/80, exogenous histamine, or the combination of 48/80 plus histamine. Skin-breaking strength was significantly increased over saline-injected controls by the combined treatment with 48/80 and histamine. Neither 48/80 or histamine alone had any influence on wound healing. Histamine content of skin at the wound site was significantly reduced by 48/80 treatment, but was unaffected by 48/80 plus histamine or histamine given alone. In contrast, stomach and leg muscle histamine levels were significantly increased beyond those of unwounded, wounded saline- or 48/80-injected mice. These results were also confirmed in CD mice, and are in contrast to findings in rats in which treatment with 48/80 alone significantly improved wound healing of similarly injured animals.     

H1 and H2 histamine actions on lung vessels; their relevance to hypoxic vasoconstriction.

Pulmonary vasomotor actions of histamine and the possible relationship of histamine to hypoxic pulmonary vasconstriction were studied in anaesthetized cats with one lobe of lung perfused at constant flow and in isolated perfused rat and ferret lungs. In the cat histamine caused dilatation, biphasic responses and constriction with increasing doses. Histamine induced dilatation was better demonstrated during hypoxic vasoconstriction and was reduced by an H2 histamine antagonist; constriction with histamine was abolished by an H1 antagonist. Histamine also caused both vasodilatation and vasoconstriction in ferret lungs. A mast cell stabilizing agent had no effect on hypoxic pulmonary vasoconstriction in cats or rats. This response was unaffected in cats but greatly reduced in rats and ferrets by cyproheptadine, a combined histamine and 5-hydroxy-tryptamine inhibitor. It was unaffected in cats but abolished in ferrets an H1 histamine inhibitor. It was again unaffected in cats but greatly reduced in rats and ferrets by an H2 histamine inhibitor. These species differences may reflect differences in mechanism but more probably reflect non-specific effects of the inhibitors in certain circumstances. However, when drugs nearly abolished hypoxic vasoconstriction, ATP still caused vasoconstriction.     

A prostacyclin analogue, iloprost, augments the ovulatory response of the LH-stimulated in vitro perfused rat ovary.

Ovaries from immature rats, primed with pregnant mare's serum gonadotropin (PMSG; 20 IU, on day 28), were perfused in vitro in a recirculating system for 21 h from the morning of day 30 of age. Stimulation with luteinizing hormone (LH; 0.1 micrograms/ml) in vitro at 0 h of perfusion resulted in 2.4 +/- 0.75 (mean +/- SEM) ovulations per treated ovary, whereas no ovulations occurred in the unstimulated group. When the addition of LH was supplemented hourly for 10 h with a stable prostacyclin analogue, Iloprost, at concentrations of 0.01 microM or 0.1 microM, the ovulation rate increased significantly (p less than 0.05) to 6.6 +/- 1.3 and 10.2 +/- 2.4 ovulations per treated ovary, respectively. Iloprost (0.1 microM) did not cause any follicular ruptures when added by itself at every hour up to 10 h. The addition of Iloprost did not affect the release of cyclic adenosine 3',5'-monophosphate (cAMP), progesterone or estradiol from unstimulated or LH-stimulated ovaries. All ovulated oocytes had resumed meiosis as judged from the absence of a germinal vesicle. These data indicate a positive modulatory role of prostacyclin in the LH-induced ovulatory process for the rat.     

Subcellular localization of brain mast cell histamine in developing rat

The intracerebroventricular administration of compound 48/80 or polymixin B to rats 0 to 60 days old, produced a decrease both in the histamine which sediments in the crude nuclear fraction, as well as in the number of mast cells in the brain. In contrast, the histamine-releasers did not affect histamine levels in subcellular fractions where neuronal histamine is found. Once released, histamine disappeared rapidly (t 1/2 = 3.8 min). In untreated animals and in those treated with histamine releasers, the number of mast cells/g in the whole brains of developing rats and in the cerebral regions of adult rats showed a close correlation with the histamine levels in the crude nuclear fraction. The content of histamine per mast cell in adult rat brain was estimated to be about 13 pg/cell. Histologic examination of the subcellular fractions revealed the presence of intact mast cells in the crude nuclear fraction obtained from untreated animals, and of degranulated mast cells in the same fraction obtained from animals treated with histamine releasers. The mast cell contribution to adult rat brain histamine levels was about 22%. Our results strongly support that most of the histamine which sediments in the crude nuclear fraction of the rat brain is located in mast cells. Determination of histamine in the crude nuclear fraction and in the supernatant of this fraction is proposed as an easy way for identifying the cellular pool altered by any treatment affecting brain histamine levels.     

Effects of inhibitors of protein synthesis on the ovulatory process of the perfused rat ovary

The effects of two different protein synthesis inhibitors (cycloheximide and puromycin) on the ovulatory process were examined in vitro using a perfused rat ovary model. Ovaries of PMSG (20 i.u.)-primed rats were perfused for 21 h. Release of cyclic adenosine 3',5'-monophosphate (cAMP) and steroids (progesterone, testosterone, and oestradiol) was measured and the number of ovulations was estimated by counting released oocytes. Unstimulated control ovaries did not ovulate whereas addition of LH (0.1 microgram/ml) plus 3-isobutyl-1-methylxanthine (IBMX; 0.2 mM) resulted in 16.7 +/- 3.5 ovulations per treated ovary. Cycloheximide (5 micrograms/ml) totally inhibited the ovulatory effect of LH + IBMX when present from the beginning of the perfusions and also when added 8 h after LH + IBMX. No inhibition was seen when cycloheximide was added 10 h after LH + IBMX (1-1.5 h before the first ovulation; 15.2 +/- 4.4 ovulations per treated ovary). Puromycin (200 micrograms/ml) completely blocked ovulation when present from the beginning of the perfusions and the inhibition was congruent to 60% (6.5 +/- 2.2 ovulations per treated ovary) when the compound was added 8 h after LH + IBMX. Both inhibitors increased LH + IBMX-stimulated cAMP release substantially, but decreased the release of progesterone, testosterone and oestradiol. These results indicate that de-novo protein synthesis is important late in the ovulatory process for follicular rupture to occur.     

Inhibition of ovulation in the perfused rat ovary by the synthetic collagenase inhibitor SC 44463.

A new, powerful, synthetic inhibitor of mammalian tissue collagenases and related metalloproteinases is inhibitory to ovulation in perfused rat ovaries. Ovaries of immature rats, primed with 20 IU of eCG, were dissected and perfused with 0.1 micrograms/ml LH and 0.2 mM 3-isobutyl-1-methylxanthine (IBMX) for 20 h. Addition of SC 44463 (N4-hydroxy-N1-[1S [(4-methoxphenyl)methyl]-2-(methylamino)-2-oxoethyl]- 2R-(2-methylpropyl)butane-diamide) at a concentration of 25 nM inhibited ovulation by 55% (9.6 +/- 1.7 ovulations per ovary, mean +/- SEM, compared to a control value of 21.7 +/- 1.7); and 250 nM inhibited ovulation by 75% (5.3 +/- 1.1 ovulations per ovary). We previously showed that the related compound SC 40827 inhibited ovulation by 70% when used at a concentration of 25 microM (Br?nnstr?m et al., Endocrinology 1988; 122:1715-1721). We now show that SC 44463 is 100, 500, and 75 times more powerful than SC 40827 in blocking ovulation, inhibiting action of ovarian interstitial collagenase, and inhibiting action of the small metalloproteinase of the rat uterus, respectively. SC 44463 also inhibits ovarian type IV collagen-digesting activity 50% at a concentration of 18 nM. Ovulation occurs after 9-12 h of perfusion with LH. Compound SC 44463 (25 nM) showed its full inhibitory capacity when added to the medium as late as 7 h after LH, but there was no significant inhibition when it was added at 9 h. This suggests that the major collagenolytic events occur beyond 7 h after stimulation by LH.