The effect of ovarian steroidogenesis on ovulation and fertilizability in the in vitro perfused rabbit ovary

The effects of aminoglutethimide phosphate (AGP) on ovulation, ovum maturation, fertilizability, and steroid production were studied with the use of an isolated perfused rabbit ovary preparation. AGP (10(-3) or 10(-4) M) was added to the perfusate of one ovary. The contralateral control ovary was perfused in medium alone. Thirty minutes later human chorionic gonadotropin (hCG) (50 IU) was added to the perfusate of all ovaries. No difference was observed in time of ovulation or ovulatory efficiency between controls and AGP-treated ovaries. The degree of ovum maturity and degeneration was also comparable in the two groups. Progesterone and estradiol production were significantly reduced by AGP treatment. A second experiment examined fertilizability of ova ovulated in vitro after perfusion with 10(-3) M AGP. AGP significantly reduced the rate of normal fertilization as observed 12 h after insemination. The percentage of inseminated ova with evidence of degeneration was greater in ova from AGP-treated ovaries than in those from controls, however, this difference was not significant. The study indicates that AGP affects neither hCG-induced ovulation nor meiotic resumption; however, fertilizability of ova from ovaries treated with AGP is impaired. These data suggest that the intrafollicular steroid environment may participate in cytoplasmic maturation of ovulated ova.     

Effect of histamine and histamine blockers on the ovulatory process in the vitro perfused rabbit ovary

An increase in the content of histamine in the ovary following luteinizing hormone (LH) release and the inhibition of ovulation in the rabbit by antihistamines suggest that histamine may be involved in the ovulatory process. The effects of various doses of histamine and antihistamines on ovulation were investigated using the in vitro perfused rabbit ovary system. Histamine (100 ng/ml) added to the perfusate at hourly intervals induced ovulation, although at a rate below that observed following human chorionic gonadotropin (hCG) administration. Cimetidine (10 micrograms/ml), an H2 blocker, inhibited histamine-induced ovulation, while the H1 blocker, chlorpheniramine (66.7 micrograms/ml), failed to do so. Neither cimetidine nor chlorpheniramine was able to block ovulation following hCG (50 IU). In all experimental groups in which histamine was used to induce ovulation, both extruded ova and follicular oocytes remained in an immature stage and displayed little evidence of degeneration. In contrast, a high percentage of ova exposed to hCG were mature. Ovarian edema was increased in ovaries in which ovulation occurred, regardless of treatment. A linear correlation was noted between ovulatory efficiency and degree of ovarian edema. Histamine may be an intermediary in the mechanism of follicular rupture, but does not support ovum maturation. However, the inability of H1 and H2 antagonists to block hCG-induced ovulation raises questions regarding the role of histamine in the physiologic process of ovulation.     

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.     

Role of cGMP in the response of frog skin to noradrenaline

The effects of noradrenaline on cGMP levels of epithelial cells isolated from frog skin are reported. Noradrenaline does not change cGMP levels either in a medium containing Ca++ and Mg++, or without Ca++ or without Mg++.     

The involvement of oxytocin in ovulation and in the outputs of cyclo-oxygenase and 5-lipoxygenase products from isolated rat ovaries

The effects on ovulation of a specific anti-oxytocin rabbit serum (anti-OT) (50.0 microliters) given by intrabursal injection into the right ovaries of etherized adult female rats at proestrus, were explored by counting the number of ovulated ova present within the right oviducts. Left ovaries were not treated and served as control ovaries. Control rats were treated with male normal rabbit serum (NRS) (50.0 microliters) given by intrabursal injections into the right ovaries of animals at proestrus. Ovulation was induced by injection of human chorionic gonadotrophin (hCG). Anti-OT administered into the right ovarian bursae of proestrous rat ovaries evoked a significant 51% inhibition of ovulation in comparison with that observed in control non-injected left ovaries (p less than 0.01). Also, when the ovulation of right ovaries injected with anti-OT was compared with that of left ovaries injected with NRS, the number of ovulated ova in the right side was significantly smaller (30%) than on the contralateral side (p less than 0.02). However, in rats pre-treated with hCG the intrabursal injection of oxytocin (OT) (50.0 mU/ml) into right and left ovaries failed to alter the number of ovulated ova compared with that of rats receiving intrabursal injections of saline. The basal control and the OT-evoked synthesis and release of endogenous prostaglandin E2 (PGE2) and PGF2 alpha were explored in ovaries isolated from prepuberal rats injected with pregnant mare's serum gonadotrophin (PMSG), two days prior to sacrifice. OT augmented the basal release of PGF2 alpha but did not influence that of PGE2. Moreover, the conversion of exogenous 14C-arachidonic acid (14C-AA) into different prostanoids and into 5-HETE, in the presence and in the absence of added OT (50.0 mU/ml), was studied in rat ovaries isolated in proestrus. The challenge with OT augmented the basal synthesis and release of PGF2 alpha and of 5-HETE from 14C-AA, but failed to influence the formation of products generated via the cyclo-oxygenase pathway, namely 6-keto-PGF1 alpha, PGE2 and thromboxane B2 (TXB2). Therefore, the present results suggest that ovarian OT may play a role in the ovulatory process, via generation of PGF2 alpha to enhance contractions of ovarian smooth muscle and of 5-HETE to promote follicular collagenolysis.     

Ca++ dependence of the contraction of coronary artery segments in response to norepinephrine after beta-adrenergic blockade

The effect of external calcium concentration on the NE-induced contraction after beta-adrenergic blocking was studied in vitro. It resulted that the effect of NE was enhanced by increase, or reduced by decrease of calcium concentration. NE-induced contraction was not abolished when the bathing fluid was Ca++-free. The disappearance of the NE effect was only obtained in preparations treated with EDTA and perfused with Ca++-free Ringer-Locke solution. It is concluded that NE induced contraction after beta-adrenergic blocking is Ca++-dependent and on the tissue bound Ca++.     

Effects of delta 9-tetrahydrocannabinol on testosterone production in vitro: influence of Ca++, Mg++ or glucose

The major psychoactive component of marihuana, delta 9-tetrahydrocannabinol (THC), influences testicular function. In the present experiments, the addition of THC to incubations of whole decapsulated mouse testes altered testosterone (T) production differentially, depending on the specific gonadotropin used, the dose of THC and/or the amount of divalent cation present in the media. In the presence of luteinizing hormone (LH; 10 ng/ml), and a dose of 25 micrograms THC/ml, T production was significantly decreased, compared to that by testes incubated with LH and vehicle at all Ca++ levels, except at 0.127 or 1.0 mM Ca++. The production of T by these paired testes exposed to either THC or vehicle (ethanol; ETOH), increased as Ca++ concentration approached physiological levels. In contrast, in the presence of follicle-stimulating hormone (FSH; 1 microgram/ml), THC-induced suppression of T production was significant in the absence of Ca++ from the media, and at 12.7 mM Ca++. However, it appeared that the levels of Ca++ did not differentially affect T production in the presence of FSH, whether or not THC was also added. In the presence of human chorionic gonadotropin (hCG; 12.5 mIU/ml), a lower dose of THC (25 ng/ml), stimulated T production at 0.25 to 1 mM Ca++, but had no effect as Ca++ reached 2.5 mM. Without additional Ca++ in the media, this dose of THC significantly reduced T secretion. In contrast, in the presence of hCG, a higher THC dose (25 micrograms/ml), suppressed T accumulation at 0.127, and from 1.0 to 12.7, but had no effect at 0.25 mM, or in the absence of Ca++. In the presence of hCG, the high 25 micrograms/ml dose of THC stimulated T production, in the absence of additional Mg++, and at 0.01 mM Mg++, but THC had no effect at 0.1 mM Mg++, but inhibited T production at 1.1 mM Mg++. In the presence of hCG, 25 micrograms THC/ml produced a consistent suppression of T production across glucose concentrations examined. These findings suggest that the mechanisms by which THC effects testicular steroidogenesis may involve Ca++- and/or Mg++-dependent processes. Differential requirements for these divalent cations by the gonadotropins may explain the interactive effects of THC with LH, hCG or FSH.     

Calcium current activated by muscarinic receptors and thapsigargin in neuronal cells

The activation of muscarinic receptors in N1E-115 neuroblastoma cells elicits a voltage-independent calcium current. The current turns on slowly, reaches its maximum value approximately 45 s after applying the agonist, is sustained as long as agonist is present, and recovers by one half in approximately 10 s after washing the agonist away. The current density is 0.11 +/- 0.08 pA/pF (mean +/- SD; n = 12). It is absent in zero-Ca++ saline and reduced by Mn++ and Ba++. The I(V) curve characterizing the current has an extrapolated reversal potential > +40 mV. The calcium current is observed in cells heavily loaded with BAPTA indicating that the calcium entry pathway is not directly gated by calcium. In fura-2 experiments, we find that muscarinic activation causes an elevation of intracellular Ca++ that is due to both intracellular calcium release and calcium influx. The component of the signal that requires external Ca++ has the same time course as the receptor operated calcium current. Calcium influx measured in this way elevates (Ca++)i by 89 +/- 41 nM (n = 7). Thapsigargin, an inhibitor of Ca++/ATPase associated with the endoplasmic reticulum (ER), activates a calcium current with similar properties. The current density is 0.22 +/- 0.20 pA/pF (n = 6). Thapsigargin activated current is reduced by Mn++ and Ba++ and increased by elevated external Ca++. Calcium influx activated by thapsigargin elevates (Ca++)i by 82 +/- 35 nM. The Ca++ currents due to agonist and due to thapsigargin do not sum, indicating that these procedures activate the same process. Carbachol and thapsigargin both cause calcium release from internal stores and the calcium current bears strong similarity to calcium-release-activated calcium currents in nonexcitable cells (Hoth, M., and R. Penner. 1993. Journal of Physiology. 465:359-386; Zweifach, A., and R. S. Lewis, 1993. Proceedings of the National Academy of Sciences, USA. 90:6295- 6299).     

Ovulation in the isolated perfused rat ovary as documented by intravital microscopy

Surface cell changes at the apices of preovulatory follicles and ovulations were documented in isolated perfused ovaries from immature rats treated with pregnant mare serum gonadotropin (20 IU) and 48 h later with human chorionic gonadotropin (hCG) (10 IU). A video camera coupled to an inverted microscope and a video recorder captured the preovulatory and ovulatory events at a cellular level. At around 8 h post-hCG, the follicular apex changed from a smooth and optically homogeneous appearance into a rough surface with bleb formation and extrusions of single cells through minute perforations (early stigma formation). At approximately 10 h, a sticky material formed a basketlike structure with trapped cells (late stigma formation). At 12 to 15 h, ovulation took place at a constant speed and with no contractions of the follicular wall. This indicates that ovulation can occur with no visible circumfollicular muscular activity. Furthermore, the observations of a leakage of cells over an extended period of time indicates that the follicular wall is partly digested several hours before ovulation occurs.     

Effect of calcium on creatine kinase activity of cerebellum.

The effect of Ca++ ions on the histochemical activity of creatine kinase (CK) was investigated in striated muscle and cerebellum of the rat. The intensity and pattern of CK activity was unchanged in the striated muscle when Ca++ was present in the incubation medium instead of Mg++. In the cerebellum, however, Ca++ inhibited the enzymatic activity except in the Purkinje cells.     

Conformational change on calcium binding by the lipopeptide antibiotic amphomycin. A C.D. and monolayer study

The acidic linear lipopeptide amphomycin is a calcium dependent antibiotic which is thought to bind to carrier lipids such as dolichol monophosphate. The actual role of Ca++ is not definitely established and in this article we have examined the peptides interactions with a range of divalent cations. By CD we have shown that a conformational change is induced by Ca++, Sr++ and Ba++ but not by Mg++, Zn++, Cd++ or Gd+++. Monolayer studies show a decrease in molecular area and an increase in film stability when the subphase contains Ca++. The ensemble of results provides preliminary evidence for the formation of a beta hairpin structure on ion binding (Ka (Ca++) = 2.4 x 10(3)M-1) which could enhance amphomycin's bilayer solubility.     

Depolarizing response of rat parathyroid cells to divalent cations

Membrane potentials were recorded from rat parathyroid glands continuously perfused in vitro. At 1.5 mM external Ca++, the resting potential averages -73 +/- 5 mV (mean +/- SD, n = 66). On exposure to 2.5 mM Ca++, the cells depolarize reversibly to a potential of -34 +/- 8 mV (mean +/- SD). Depolarization to this value is complete in approximately 2-4 min, and repolarization on return to 1.5 mM Ca++ takes about the same time. The depolarizing action of high Ca++ is mimicked by all divalent cations tested, with the following order of effectiveness: Ca++ greater than Sr++ greater than Mg++ greater than Ba++ for alkali-earth metals, and Ca++ greater than Cd++ greater than Mn++ greater than Co++ greater than Zn++ for transition metals. Input resistance in 1.5 mM Ca++ was 24.35 +/- 14 M omega (mean +/- SD) and increased by an average factor of 2.43 +/- 0.8 after switching to 2.5 mM Ca++. The low value of input resistance suggests that cells are coupled by low-resistance junctions. The resting potential in low Ca++ is quite insensitive to removal of external Na+ or Cl-, but very sensitive to changes in external K+. Cells depolarize by 61 mV for a 10- fold increase in external K+. In high Ca++, membrane potential is less sensitive to an increase in external K+ and is unchanged by increasing K+ from 5 to 25 mM. Depolarization evoked by high Ca++ may be slowed, but is unchanged in amplitude by removal of external Na+ or Cl-. Organic (D600) and inorganic (Co++, Cd++, and Mn++) blockers of the Ca++ channels do not interfere with the electrical response to Ca++ changes. Our results show remarkable parallels to previous observations on the control of parathormone (PTH) release by Ca++. They suggest an association between membrane voltage and secretion that is very unusual: parathyroid cells secrete when fully polarized, and secrete less when depolarized. The extraordinary sensitivity of parathyroid cells to divalent cations leads us to hypothesize the existence in their membranes of a divalent cation receptor that controls membrane permeability (possibly to K+) and PTH secretion.     

The calcium dependence of pigment translocation in freshwater shrimp red ovarian chromatophores

The roles of calcium in cell signaling consequent to chromatophorotropin action and as an activator of mechanochemical transport proteins responsible for pigment granule translocation were investigated in the red ovarian chromatosomes of the freshwater shrimp Macrobrachium olfersii. Chromatosomes were perfused with known concentrations of free Ca++ (10(-3) to 10(-9) M) prepared in Mg(++)-EGTA-buffered physiological saline after selectively permeabilizing with 25 microM calcium ionophore A23187 or with 10(-8) M red pigment concentrating hormone (RPCH). The degree of pigment aggregation and the translocation velocity of the leading edges of the pigment mass were recorded in individual chromatosomes during aggregation induced by RPCH or A23187 and dispersion induced by low Ca++. Aggregation is Ca++ dependent, showing a dual extracellular and intracellular requirement. After perfusion with reduced Ca++ (10(-4) to 10(-9) M), RPCH triggers partial aggregation (approximately 65%), although the maximum translocation velocities (approximately 16.5 microns/min) and velocity profiles are unaffected. After aggregation induced at or below 10(-5) M Ca++, spontaneous pigment dispersion ensues, suggesting a Ca++ requirement for RPCH coupling to its receptor, or a concentration-dependent, Ca(++)-induced Ca(++)-release mechanism. The Ca(++)-channel blockers Mn++ (5 mM) and verapamil (50 microM) have no effect on RPCH-triggered aggregation. An intracellular Ca++ requirement for aggregation was demonstrated in chromatosomes in which the Ca++ gradient across the cell membrane was dissipated with A23187. At free [Ca++] above 10(-3) M, aggregation is complete; at 10(-4) M, aggregation is partial, followed by spontaneous dispersion; below 10(-5) M Ca++, pigments do not aggregate but disperse slightly. Aggregation velocities diminish from 11.6 +/- 1.2 microns/min at 5.5 mM Ca++ to 7.4 +/- 1.3 microns/min at 10(-4) M Ca++. Half-maximum aggregation occurs at 3.2 x 10(-5) M Ca++ and half-maximum translocation velocity at 4.8 x 10(-5) M Ca++. Pigment redispersion after 5.5 mM Ca(++)-A23187-induced aggregation is initiated by reducing extracellular Ca++: slight dispersion begins at 10(-7) M, complete dispersion being attained at 10(-9) M Ca++. Dispersion velocities increase from 0.6 +/- 0.2 to 3.1 +/- 0.5 microns/min. Half-maximum dispersion occurs at 7.6 x 10(-9) M Ca++ and half-maximum translocation velocity at 2.9 x 10(-9) M Ca++. These data reveal an extracellular and an intracellular Ca++ requirement for RPCH action, and demonstrate that the centripetal or centrifugal direction of pigment movement, the translocation velocity, and the degree of pigment aggregation or dispersion attained are calcium-dependent properties of the granule translocation apparatus.     

Gonadotropin-releasing hormone: effects on the in vitro perfused rabbit ovary

Gonadotropin-releasing hormone (GnRH) has been shown to inhibit ovulation in gonadotropin-primed hypophysectomized rats and steroid production in cultured rat granulosa cells. To determine if similar effects of GnRH can be observed in another species, the extracorporeal perfused rabbit ovary was utilized. Two groups of rabbit ovaries were exposed to GnRH in a pulsatile fashion at two dose levels (Group I, 2.56 X 10(-8) M; Group II, 2.56 X 10(-7) M). Contralateral ovaries were not perfused with GnRH. Human chorionic gonadotropin (hCG) was added to the perfusate of all ovaries 30 min after the onset of perfusion. Ovulation occurred in all ovaries exposed to hCG in the presence or absence of GnRH. Ovulatory efficiency was similar in both the experimental and control groups. No statistical difference could be determined in the time of ovulation, stage of maturity of oocytes, or percent of degeneration of ovulated or follicular oocytes. Progesterone production was not inhibited in the GnRH-treated ovaries. In contrast to observations in the rat, GnRH does not exhibit a direct inhibitory effect on ovulation or steroid production in the rabbit.     

Diamide inhibited (Ca++ + Mg++) and (Mg++) dependent ATPase in erythrocyte membranes: activity at different temperatures.

After inhibition of the monovalent cation dependent ATPase, a (Ca++ + Mg++) and a (Mg++) dependent ATPase activity can be detected. The inhibition due to diamide on the (Mg++) ATPase, assayed in the 12.5 degrees C - 30 degrees C temperature range, is almost complete. On the contrary the diamide induced inhibition of (Ca++ + Mg++) ATPase, in the same temperature range, is not complete and the residual activity increases with temperature. The reported data indicate that the ATPase activity induced by calcium is much less diamide-sensitive and -SH-dependent than that elicited by Mg++ alone.     

Effect of papaverine on the longitudinal and circular smooth muscle of the guinea-pig caecum.

Electrical and mechanical activity of smooth muscle of the guinea-pig caecum was recorded by means of the sucrose-gap technique. The responses of longitudinal and circular smooth muscle to acetycholine were differently affected by changes of extracellular calcium concentration (0.8, 2.5 and 7.5 mM). The contractions of both preparations were depressed at high Ca++ concentrations, whereas at low Ca++ concentrations only contractions of the circular smooth muscle were augmented. The stimulatory effect of acetylcholine was decreased by papaverine in both preparations at all three concentrations of Ca++. This inhibition was the greater the lower the concentration of extracellular Ca++ and this process was more pronounced in the circular muscle. The ability of papaverine to counteract the effect of lowered concentrations of extracellular Ca++ on membrane excitability may well explain its inhibitory effect upon intestinal smooth muscle.     

Immature rats show ovulatory defects similar to those in adult rats lacking prostaglandin and progesterone actions

Gonadotropin-primed immature rats (GPIR) constitute a widely used model for the study of ovulation. Although the equivalence between the ovulatory process in immature and adult rats is generally assumed, the morphological and functional characteristics of ovulation in immature rats have been scarcely considered. We describe herein the morphological aspects of the ovulatory process in GPIR and their response to classical ovulation inhibitors, such as the inhibitor of prostaglandin (PG) synthesis indomethacin (INDO) and a progesterone (P) receptor (PR) antagonist (RU486). Immature Wistar rats were primed with equine chorionic gonadotropin (eCG) at 21, 23 or 25 days of age, injected with human chorionic gonadotropin (hCG) 48 h later, and sacrificed 16 h after hCG treatment, to assess follicle rupture and ovulation. Surprisingly, GPIR showed age-related ovulatory defects close similar to those in adult rats lacking P and PG actions. Rats primed with eCG at 21 or 23 days of age showed abnormally ruptured corpora lutea in which the cumulus-oocyte complex (COC) was trapped or had been released to the ovarian interstitum, invading the ovarian stroma and blood and lymphatic vessels. Supplementation of immature rats with exogenous P and/or PG of the E series did not significantly inhibit abnormal follicle rupture. Otherwise, ovulatory defects were practically absent in rats primed with eCG at 25 days of age. GPIR treated with INDO showed the same ovulatory alterations than vehicle-treated ones, although affecting to a higher proportion of follicles. Blocking P actions with RU486 increased the number of COC trapped inside corpora lutea and decreased ovulation. The presence of ovulatory defects in GPIR, suggests that the capacity of the immature ovary to undergo the coordinate changes leading to effective ovulation is not fully established in Wistar rats primed with eCG before 25 days of age.     

Separate effects of mercurial compounds on the ionophoric and hydrolytic functions of the (Ca++ +Mg++)-ATPase of sarcoplasmic reticulum.

We have shown that a Ca++-ionophore activity is present in the (Ca++ +Mg++)-ATPase of rabbit skeletal muscle sarcoplasmic reticulum (A. E. Shamoo & D. H. MacLennan, 1974. Proc. Nat. Acad. Sci. USA 71:3522). Methylmercuric chloride inhibited the (Ca++ +Mg++)-ATPase and Ca++ transport, but had no effect on the activity of the Ca++ ionophore. Mercuric chloride inhibited ATPase, transport and ionophore activity. The ATPase and transport functions were more sensitive to methylmercuric chloride than to mercuric chloride. The two functions were inhibited concomitantly by methylmercuric chloride but slightly lower concentrations of mercuric chloride were required to inhibit Ca++ transport than were required to inhibit ATPase. Methylmercuric chloride and mercuric chloride probably inhibited ATPase and Ca++ transport by blocking essential -SH groups. However, it appears that there are no essential -SH groups in the Ca++ ionophore and that mercuric chloride inhibited the Ca++ ionophore activity by competition with Ca++ for the ionophoric site. Blockage of Ca++ transport by mercuric chloride probably occurs both at sites of essential -SH groups and at sites of ionophoric activity. These data suggest the separate identity of the sites of ATP hydrolysis and of Ca++ ionophoric activity.     

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.     

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.