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.     

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.     

Regulation of prostaglandin endoperoxide synthase by cyclic adenosine 3',5'-monophosphate in the in vitro-perfused rat ovary

The preovulatory regulation of two enzymes in the prostaglandin biosynthetic pathway, prostaglandin endoperoxide synthase (PGS) and prostacyclin synthase (ISN), was examined in granulosa cells and residual tissue of rat ovaries perfused in vitro. Ovaries from rats primed with pregnant mare's serum gonadotropin (20 IU) were perfused for up to 20 h starting the morning of induced proestrus. The amounts of PGS and ISN present were analyzed with immunoblotting techniques. Soluble extracts from granulosa cells and residual ovarian tissues were obtained at different times (0 h, 3 h, 7 h, 12 h) after treatment in vitro with luteinizing hormone (LH, 0.1 microgram/ml) and 3-isobutyl-1-methylxanthine (IBMX; 0.2 mM) and at 7 h in untreated control ovaries or after treatment with forskolin (30 microM) or LH (0.1 microgram/ml). The levels in the perfusion medium of cyclic adenosine 3',5'-monophosphate (cAMP), progesterone, testosterone, and estradiol were measured and the number of ovulations were examined. The levels of PGS after treatment with LH + IBMX increased up to 7 h and remained high at 12 h, a time that is close to the time of ovulation. The increase was more pronounced in the granulosa cells than in the residual tissue. Treatment with forskolin induced synthesis of PGS in granulosa cells, and the levels at 7 h were similar to those after stimulation with LH + IBMX. The levels of PGS were lower in granulosa cells of the group stimulated with LH alone than in granulosa cells from ovaries stimulated with LH + IBMX or forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Time-dependent ovulation inhibition of a selective progesterone-receptor antagonist (Org 31710) and effects on ovulatory mediators in the in vitro perfused rat ovary

Progesterone (P) is one of several local mediators in the ovulatory cascade in the rat. The precise mechanisms of action for P in ovulation and in what phase of the ovulatory process P is critical, however, need to be clarified. The present study used a selective P-receptor antagonist, Org 31710, in the in vitro perfused rat ovary model to examine the local role of P and possible effects on prostaglandin (PG) and plasminogen-activator (PA) release in ovulation. Ovaries from eCG (15 IU)-primed rats were perfused for 20 h with LH (0.2 microg/ml) and 3-isobutyl-1-methylxanthine (IBMX, 200 microM) to induce ovulation (median = 10.0, 25%-75% range = 8.5-13). Org 31710 was added at either 0, 3.5, 7, or 9 h after LH+IBMX, resulting in significant suppression of ovulation after addition at 0 and 3.5 h (1.0, 1-5.5; and 5.0, 2.5-7.75 ovulations, respectively) but no suppressive effect when added at later time points. Progesterone and estradiol levels in the perfusion media were increased after LH+IBMX but were not affected by the presence of Org 31710. Ovarian tissue levels of PGE(2), PGF(2 alpha), and PA activity were measured in ovaries that had been perfused for 10 h, a time that was 2 to 5 h before anticipated ovulation. The presence of Org 31710 significantly decreased the levels of PGE(2), PGF(2 alpha), and PA activity. These results suggest that P is essential in ovulation during the initial stages of the ovulatory process. The effect of P to facilitate ovulation seems to relate to stimulation of the PG- and PA-mediator systems.     

Progesterone is a mediator in the ovulatory process of the in vitro-perfused rat ovary

The role of steroids in the ovulatory process of the rat was explored in an in vitro perfusion system. Immature rat ovaries were primed with pregnant mare's serum gonadotropin (20 IU) and perfused in a recirculating perfusion system for up to 20 h. Unstimulated ovaries did not ovulate whereas the addition of luteinizing hormone (LH; 0.1 micrograms/ml) plus 3-isobutyl-1-methylxanthine (IBMX; 0.2 mM) resulted in 13.6 +/- 1.0 ovulations per treated ovary. Addition of an inhibitor of 3 beta-hydroxysteroid dehydrogenase (Compound A; 10 micrograms/ml) significantly (p less than 0.01) decreased the number of ovulations after LH plus IBMX stimulation (1.6 +/- 0.8 ovulations per treated ovary). This inhibition was reversed by the addition of progesterone, with 6.6 +/- 2.1 ovulations at approximately 100 ng/ml progesterone in the perfusion medium and 15.2 +/- 3.4 ovulations at approximately 3000 ng/ml progesterone. The addition of testosterone (10 micrograms/ml) did not reverse the inhibition of ovulations by Compound A. High levels of progesterone in the perfusion medium (greater than 3000 ng/ml) did not significantly (p greater than 0.05) increase the number of ovulations after stimulation with LH plus IBMX (20.2 +/- 4.8 ovulations), and progesterone (greater than 3000 ng/ml) was not by itself able to induce ovulations. Addition of LH plus IBMX resulted in a marked increase in the levels of progesterone, testosterone, and estradiol in the perfusion medium. The production of these steroids was almost completely inhibited by the addition of Compound A, and the levels of testosterone and estradiol were restored by the addition of high concentrations of progesterone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of prostaglandin biosynthesis by luteinizing hormone and bradykinin in rat preovulatory follicles in vitro.

Luteinizing hormone (LH) stimulates prostaglandin biosynthesis and steroidogenesis in preovulatory (PO) follicles prior to ovulation. Since the ovulatory process shares many similarities with an inflammatory reaction, mediators of the inflammatory response, such as bradykinin (BK) have been suggested to modulate the effects of LH. In the present study the effect of BK (5 microM) on: 1) prostaglandin biosynthesis (PGE2, PGF2 alpha and 6-keto-PGF1 alpha), 2) the levels of two enzymes in the cyclo-oxygenase pathway, prostaglandin endoperoxide synthase (PGS) and prostacyclin synthase (PCS), and 3) cyclic adenosine 3'5'-monophosphate (cAMP) and progesterone response of PO follicles incubated in vitro were examined. LH (0.1 microgram/ml) stimulated the accumulation of cAMP and progesterone in the medium, while BK had no effect on these parameters. BK exerted a slight stimulatory effect on PGE2, and PGF2 alpha, (p less than or equal to 0.01) but not on 6-keto-PGF1 alpha synthesis, but no changes in PGS or PCS levels could be detected. The effect of LH on prostaglandin biosynthesis was much more pronounced, with an increase of PGE2, PGF2 alpha and 6-keto-PGF1 alpha. LH also induced PGS. The combination of LH and BK did not alter these responses compared to that of LH alone. This study demonstrates that BK stimulates prostaglandin biosynthesis in PO follicles. In contrast to LH, this effect of BK does not seem to involve the adenylate cyclase system, since BK did not stimulate cAMP production. BK did not affect the levels of PGS or PCS, and the stimulatory effect of BK is suggested to involve an increase in the availability of substrate for the cyclo-oxygenase pathway.     

Ovulations in rat ovaries perfused in vitro with follicle-stimulating hormone

Using the model of the isolated perfused rat ovary, we have found that highly purified ovine follicle-stimulating hormone (FSH) preparations cause ovulation and that this effect is not due to luteinizing hormone (LH) contamination. Ovine FSH-13 at a concentration of 1.5 mU/ml induced ovulations in all perfused ovaries (8.8 +/- 2.3 ovulations/ovary), as did a more purified preparation, ovine FSH-211B, at concentrations of 0.5 mU/ml (15.0 +/- 6.4 ovulations/ovary) and 5 mU/ml (11.3 +/- 2.6 ovulations/ovary). This ovulation-inducing effect of FSH is accompanied by a marked stimulation of estradiol levels in the perfusion medium without stimulation of progesterone levels. Furthermore, a purified rat FSH preparation (15 mU/ml) also induced ovulation in all ovaries (13.8 +/- 2.2 ovulations/ovary) as well as a stimulation of both estradiol and progesterone in the medium. These data clearly confirm the direct ovulatory effect of FSH on the ovary.     

Inhibition of ovulation by tyrosine kinase inhibitors in the in vitro perfused rat ovary

Protein tyrosine kinase activity, leading to tyrosine phosphorylation of the intracellular domains of receptors or non-receptor proteins, is an important feature of downstream signalling after receptor binding of a variety factors, such as growth factors and cytokines. Since several members of these classes of paracrine-autocrine mediator may be involved in the intraovarian events of ovulation, the present study was designed to evaluate the effect of protein tyrosine kinase inhibition on the in vitro perfused rat ovary. Immature rats were primed with 20 iu pregnant mares' serum gonadotrophin 48 h before surgical isolation of the right ovary with connecting vasculature. The ovary was placed in a perfusion system for either 10 h, to examine ovarian concentrations of the established ovulatory mediators plasminogen activator, prostaglandins E2 and F2 alpha, or for 20 h, enabling a complete ovulatory process to occur in vitro. Ovulation was induced by ovine LH (0.2 microgram ml-1) in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.2 mmol l-1) and the effects of two different protein tyrosine kinase inhibitors, genistein and tyrphostin A25, were studied. Unstimulated control ovaries did not ovulate and showed low secretion of progesterone and oestradiol. Addition of LH + 3-isobutyl-1-methylxanthine resulted in a marked stimulation of steroid release, and ovulations occurred in all ovaries (9.0 +/- 0.9; mean +/- SEM). The protein tyrosine kinase inhibitors, genistein and tyrphostin A25, significantly inhibited ovulation at the higher concentrations tested (3.0 +/- 0.3 at 100 mumol genistein l-1; 5.8 +/- 1.0 at 500 mumol tyrphostin A25 l-1) but no effect was seen at lower concentrations. The presence of genistein and tyrphostin A25 at any concentration used did not significantly decrease the LH + 3-isobutyl-1-methylxanthine-induced progesterone or oestradiol concentrations. The intraovarian concentrations of plasminogen activator activity, and prostaglandin E2 and F2 alpha were not altered by the presence genistein (100 mumol l-1). In conclusion, the results of the present study indicate that protein tyrosine kinase signalling pathways are integral parts of the mammalian ovulatory process but do not involve actions on the synthesis of steroids, plasminogen activator or prostaglandins.     

Leukocyte supplementation increases the luteinizing hormone-induced ovulation rate in the in vitro-perfused rat ovary

Ovaries from eCG-primed (20 IU s.c. on Day 28) rats were perfused from the morning of Day 30 of age in a recirculating system initially containing a buffered blood cell-free medium (M199 + 4% BSA) for periods of up to 21 h. The addition of ovine LH (0.1 micrograms/ml) at 0 h of perfusion resulted in ovulations in all 6 ovaries perfused (3.2 +/- 0.7 ovulations per treated ovary; mean +/- SEM), whereas none of the 6 control ovaries ovulated. Rat leukocytes (50 x 10(6)), added at 7 h of perfusion significantly increased the number of LH-induced ovulations (7.8 +/- 0.5; p less than 0.05). All ovulated oocytes showed resumption of meiosis as judged from the presence of germinal vesicle breakdown. Ovaries perfused with leukocytes but without LH did not ovulate. Histological examination of ovaries 14 h after leukocyte administration showed a considerable number of perifollicular extravasated white blood cells. These findings indicate that leukocytes participate in the normal ovulatory process as part of an inflammation-like reaction.     

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.     

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 luteinzing hormone (LH; 0.1 μg/ml) in vitro at 0 h of perfusion resulted in 2.4 ± 0.75 (mean ± SEM) ovulatioons per treated ovary, whereas no ovulations occured 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 μM or 0.1 μM, the ovulation rate increase significantly (p<0.05) to 6.6 ± 1.3 and 10.2 ± 2.4 ovulations per treated ovary, respectively. Iloprost (0.1 μM) 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 or LH-stimulated ovaries. All ovulated oocytes had resumed meiosis as judged from the absence of a germinal vesicle. These data indicate a positve modulatory role of prostacyclin in the LH-induced ovulatory process for the rat.     

Inhibition of follicle-stimulating hormone-induced ovulation by indomethacin in the perfused rat ovary

In isolated, perfused ovaries of rats treated with pregnant mare's serum gonadotropin (PMSG), purified preparations of ovine follicle-stimulating hormone (FSH) (oFSH-211B) and rat FSH (rFSH-I-6), 100 ng/ml, were found to induce ovulations (4.8 +/- 0.9, n = 4, and 6.4 +/- 2.0, n = 5, ovulations per ovary, respectively). Indomethacin (5 micrograms/ml) added to the perfusate inhibited this ovulatory effect and exogenous prostaglandin F2 alpha (PGF2 alpha) (1 microgram/ml), or prostaglandin E2 (PGE2) (0.5 microgram/ml), reversed the blockade. Ovine FSH and rFSH had only a weak stimulatory effect on estradiol release, and only rFSH caused a significant increase in progesterone accumulation. Indomethacin reduced the stimulatory effect of rFSH on progesterone release, and this effect was reversed by PGE2 but not by PGF2 alpha. In a 6-h incubation experiment with preovulatory rat follicles, we tested the biological activity of gonadotropins used to induce oocyte maturation. The concentration of FSH used in the perfusion experiments induced oocyte maturation in more than 88% of the oocytes studied. The data confirm earlier findings that FSH can induce ovulations and show that prostaglandins are involved in this process. The data also indicate that prostaglandins might be involved in the FSH-induced increase of progesterone levels.     

Induction of multiple ovulations during the ovulatory season in mares

The efficacy of an equine pituitary extract for induction of multiple ovulations during the ovulatory season was studied in 112 horse mares in four experiments. Combined for all experiments, 70% of the mares (78 112 ) had multiple ovulations for an average of 3.0 ovulations per mare. The interval between first and last ovulation was decreased (P<0.01) when human chorionic gonadotropin (hCG) was included in the treatment regimen (0.0 +/- 0.0 versus 1.6 +/- 0.4 days). Ovulation rate was lower (P<0.01) when extract treatment was initiated at day 19 (1.3 +/- 0.2) than when initiated at day 15 post-ovulation (2.9 +/- 0.5). Initiation of extract treatment at day 11 was as effective as initiation at day 15, with or without an injection of prostaglandin F(2alpha) on the first day of extract treatment. Increasing the dose of extract from 750 to 1500 or 2250 units per day did not significantly increase the ovulatory response.     

Episodic LH secretion patterns in the mare during the oestrous cycle.

Jugular blood samples were obtained from 8 mares at 5- and/or 20-min intervals for 2 to 5 days during various phases of the oestrous cycle for plasma LH determination. An episodic release pattern was observed in 1 of 3 mares sampled during the ovulatory period. One mare had one secretory burst and the other mare had several periods of fluctuating plasma LH concentration. During dioestrus, episodic secretions were observed in 2 mares sampled 11 to 13 days before and, in 1 mare, 9 days after ovulation. During the 2 to 5-day period before ovulation, episodic secretion was not observed (3 mares) but plasma LH concentrations fluctuated as much as 6 ng/ml during a period of 3--4 h. Daily plasma samples were obtained form 10 mares (1--8 oestrous cycles/mare) during which 22 single, 18 double and 2 luteal-phase ovulations occurred. Dioestrous ovulations were accompanied by small increases in plasma LH (1--4 ng/ml), but many similar increases in LH were not accompanied by ovulation. No significant differences in secretory patterns were observed between single and multiple ovulations. In one mare, 4 ovulations occurred in the presence of a prolonged luteal phase; 3 were accompanied by increasing LH concentrations and the other occurred when LH was at a low concentration.     

Examination of the relative role of FSH and LH in the mechanism of ovulatory follicle selection in sheep

The GnRH-antagonist suppression-ovarian autotransplant model (n = 18) was used to examine the relative roles of temporal changes in FSH and LH stimulation on follicle development and selection. Follicle development was stimulated by infusion with oFSH for 3 days and treatments applied for 60 h after progestagen sponge withdrawal and before delivery of an ovulatory stimulus. In Expt 1, there was continuous infusion of FSH with or without small amplitude high frequency LH pulses, or withdrawal of FSH with or without pulsatile LH. In Expt 2, there was acute or gradual withdrawal of FSH at sponge withdrawal with pulsatile LH. The patterns of follicle development and basal and pulsatile ovarian hormone secretion were determined. The maintenance of FSH throughout the artificial follicular phase resulted in multiple follicle development and ovulation (3.3 +/- 0.3). Pulsatile LH stimulated steroid secretion (P < 0.001) but had little effect on ovulation rates (3.8 +/- 0.8) when FSH was maintained. However, withdrawal of FSH in the absence of LH resulted in atresia of the ovulatory follicles and anovulation whereas, when FSH was withdrawn in the presence of LH, preovulatory follicle development was maintained in some animals (3/6 and 5/9 in Expts 1 and 2, respectively) and these ewes had lower (P < 0.05) ovulation rates (1-2 ovulations per ewe). When FSH was withdrawn gradually in the presence of pulsatile LH, 9/9 animals ovulated with ovulation rates in the normal range. These results indicate that ovulatory follicles can transfer their gonadotrophic dependence from FSH to LH. It is hypothesized that the ability of a follicle to respond to this switch in gonadotrophic support is central to the mechanism of follicle selection.     

Control of prostacyclin synthesis in pregnancy-induced hypertension

Uterine prostacyclin synthase (PGI synthase) and prostaglandin endoperoxide synthase (PGH synthase) concentrations, measured by specific immunoradiometric assays, did not differ between patients with severe pregnancy-induced hypertension (syn. pre-eclampsia; n = 5) and normotensive gravidae (n = 6) with comparable gestational ages (34 - 38 weeks). Myometrial microsomes from pre-eclamptic women contained ten times more PGI synthase than PGH synthase and the ratio of PGI synthase to PGH synthase (mean +/- SD; 10.1 +/- 3.9) was not different from that in normotensive pregnancies. None of the pre-eclamptic patients had myometrial enzyme levels that were more than one standard deviation below the mean established previously for pregnancies ranging from 32 to 42 weeks of gestation. These findings indicate that the commonly observed association between deficient PGI2 production and pregnancy-induced hypertension cannot be explained in terms of a generalized lack of immunoassayable prostacyclin or prostaglandin endoperoxide synthases.     

Resveratrol prevents apoptosis in K562 cells by inhibiting lipoxygenase and cyclooxygenase activity.

The natural polyphenolic compound resveratrol (trans-3,4', 5-trihydroxystilbene) is shown to prevent apoptosis (programmed cell death) induced in human erythroleukemia K562 cells by hydrogen peroxide and other unrelated stimuli. Resveratrol reversed the elevation of leukotriene B4 (from 6.40 +/- 0.65 to 2.92 +/- 0.30 pmol.mg protein-1) and prostaglandin E2 (from 11.46 +/- 1.15 to 8.02 +/- 0.80 nmol.mg protein-1), induced by H2O2 challenge in K562 cells. The reduction of leukotriene B4 and prostaglandin E2 correlated with the inhibition of the 5-lipoxygenase activity, and the cyclooxygenase and peroxidase activity of prostaglandin H synthase, respectively. Resveratrol also blocked lipoperoxidation induced by hydrogen peroxide in K562 cell membranes. Resveratrol was found to act as a competitive inhibitor of purified 5-lipoxygenase and 15-lipoxygenase and prostaglandin H synthase, with inhibition constants of 4.5 +/- 0.5 microM (5-lipoxygenase), 40 +/- 5.0 microM (15-lipoxygenase), 35 +/- 4.0 microM (cyclooxygenase activity of prostaglandin H synthase) and 30 +/- 3.0 microM (peroxidase activity of prostaglandin H synthase). Altogether, the results reported here suggest that the anti-apoptotic activity of resveratrol depends on the direct inhibition of the main arachidonate-metabolizing enzymes.     

Effects of unilateral ovariectomy on follicular development and ovulation in cattle

In a study of 4 cyclic dry cows (Trial I) and 6 cyclic puberal heifers (Trial II), unilateral ovariectomy increased the number of ovulatory follicles, did not alter the hormone profile, cycle length or the number of follicular waves. Ovarian follicular development in all 4 cows was monitored daily using transrectal ultrasonography until the day of ovulation, during which period daily blood samples were also taken from the tail vein for determination of plasma FSH, LH and P4 concentrations. Unilateral ovariectomy was performed on the day after ovulation and ovarian activity was again monitored daily (ultrasonography and blood sampling for FSH, LH and P4) for 2 consecutive cycles (8 cycles in all). Estrus in all 6 heifers was synchronized using 2 injections of PGF2 alpha given 12 d apart. Similarly, ovarian activity in the 6 puberal heifers was monitored daily using ultrasonography and blood sampling for 1 complete control cycle. Following estrus and ovulation the left ovary was removed in all the animals, and thereafter 1 complete cycle was followed. Mean cycle length, FSH, LH and P4 concentrations before and after unilateral ovariectomy were compared using paired sample t-test. The results show that unilateral ovariectomy neither altered the cycle length nor the number of follicular waves in the cows, but it increased the number of ovulatory follicles (2 follicles developed and ovulated in 6 of the 8 cycles). The mean diameter of the largest follicle was 16.1 +/- 0.9 mm and the second largest 12.5 +/- 0.9 mm. No significant (P > 0.05) differences were observed in FSH (0.72 +/- 0.09 vs 0.71 +/- 0.07), LH (0.42 +/- 0.1 vs 0.37 +/- 0.07) and P4 (2.8 +/- 0.6 vs 2.6 +/- 0.4) levels before and after unilateral ovariectomy. Of the 6 heifers, 5 had 2 waves and 1 heifer had 3 waves of follicular growth during the control cycle, and this pattern did not change after the procedure. Mean cycle length (20.7 +/- 0.9 vs 21 +/- 0.9) did not differ before and after unilateral ovariectomy, and 4 of the 6 heifers ovulated twin follicles following ovariectomy. The mean diameter of the largest follicle was 14.5 +/- 0.7 mm and second largest measured 12.1 +/- 0.8 mm. No significant (P > 0.05) differences were observed in FSH (0.16 +/- 0.09 vs 0.21 +/- 0.07), LH (0.11 +/- 0.1 vs 0.15 +/- 0.07) and P4 levels (3.6 +/- 0.26 vs 3.8 +/- 0.29) before and after unilateral ovariectomy. Based on these results, we conclude that unilateral ovariectomy is an ideal method for obtaining twin ovulations in cows and heifers.     

Prostaglandin F2a, an ovulatory intermediate in the in vitro perfused rabbit ovary model

PGF_2a has been proposed as a mediator of mammalian ovulation. To elucidate further the role of PGF_2a in the process of ovulation, PGF and PGF_2a metabolite were measured by radioimmunoassay in the perfusate of an perfused rabbit ovary preparation.Perfusion medium samples were collected over a 10 to 12 hour period from ovaries perfused with tissue culture M199 (total volume 150 ml, sample volume 3 ml) to which varying amounts of hCG had been added. [The PGF_2a antisera a 40% cross reaction with PGF_1a, hence total PGF was measured with this antisera.] Both PGF and PGF_2a metabolite showed a linear increase with time and numbers of ovulations.PGF media accumulation was 575 pg/ovary/ovulation/hr and PGF_2a metabolite accumulation was 367 pg/ovary/ ovulation/hr. Medium prostaglandin content could be correlated with numbers of ovulations, ovulatory efficiency (number of ovulations/total follicles) but total follicles. These data best fit a model of independent ovulatory units producing PFG_2a without recruitment or interaction between them. We infer the PGF and PGF_2a metabolites in this system can be used as a direct index of the ovulation process.