Perfused Rabbit Ovary Preparation Research Articles

Interactions between insulin-like growth factor-I (IGF-I) and the renin-angiotensin system in follicular growth and ovulation.

The interactions between insulin-like growth factor-I (IGF-I) and the renin-angiotensin system (RAS) in follicular growth and ovulation were studied with the use of an isolated perfused rabbit ovary preparation. Ovulation failed to occur in either control ovaries or the experimental ovaries perfused with IGF-I in a concentration of 1, 10, or 100 ng/ml in the absence of gonadotropin. Exposure to IGF-I stimulated the secretion rate of angiotensin II-like immunoreactivity (Ang II-IR) in perfused rabbit ovaries in a dose-dependent manner. The percent increase in follicle diameter in ovaries perfused with IGF-I for 12 h was significantly correlated with the secretion rate of Ang II-IR at 12 h after exposure to IGF-I. The addition of IGFBP-3 to the perfusate did not induce ovulation in the absence of gonadotropin, but exposure to IGFBP-3 inhibited hCG-induced ovulation in a dose-dependent manner. In addition, IGFBP-3 significantly reduced the ovarian secretion rate of Ang II-IR and prostaglandins stimulated by hCG administration. Intrafollicular plasminogen activator (PA) activity significantly increased within 4 h after exposure to 100 ng/ml of IGF-I, compared with that in control ovaries perfused with medium alone. The concomitant addition of IGFBP-3 to the perfusate significantly reduced the IGF-I-stimulated PA activity in the preovulatory follicles at 4, 6, and 8 h after exposure to IGF-I. However, IGFBP-3 alone affected neither the ovarian secretion rate of Ang II-IR nor intrafollicular PA activity. Exposure to streptokinase, an exogenous PA, in vitro stimulated both follicular growth and the intrafollicular Ang II-IR content. In conclusion, IGF-I enhances both ovarian Ang II production and follicular development by stimulating intrafollicular PA activity.

Effects of insulin-like growth factor-I on follicle growth, oocyte maturation, and ovarian steroidogenesis and plasminogen activator activity in the rabbit.

We examined the effects of insulin-like growth factor (IGF)-I on follicular growth, oocyte maturation, and ovarian steroidogenesis and plasminogen activator (PA) activity in vitro, using a perfused rabbit ovary preparation in order to determine whether the follicle-stimulating effects of growth hormone (GH) are mediated by IGF-I. The addition of IGF-I to the perfusate stimulated follicular growth and the resumption of meiosis in follicular oocytes in a dose-dependent manner. There was no significant difference in the production of progesterone by perfused rabbit ovaries between IGF-I-treated and control ovaries, whereas IGF-I increased the production of estradiol (E2) by perfused rabbit ovaries in a dose-dependent manner. The concomitant addition of a monoclonal antibody recognizing the type I IGF receptor, alpha IR-3, to the perfusate significantly blocked IGF-I-stimulated follicular growth, oocyte maturation, and E2 production. Intrafollicular PA activity increased significantly 4 h after exposure to 10 or 100 ng/ml of IGF-I and reached maximal levels at 6 h. The percentage increase in follicle diameter at 6 h after exposure to IGF-I was significantly correlated with the intrafollicular PA activity. Treatment with GH resulted in a 2.7-fold increase in intrafollicular levels of IGF-I mRNA. The binding of [125I]-IGF-I to rabbit ovarian membrane preparations was inhibited by unlabeled IGF-I and IGF-II in a concentration-dependent manner. The relative affinity of the IGF-I receptor for IGF-I, IGF-II, and insulin was typical of type I binding (IGF-I > IGF-II > insulin). Affinity cross-linking of ovarian membranes with [125I]-IGF-I revealed a radiolabeled band corresponding to a molecular weight of 135,000, the alpha subunit of the type I IGF receptor. This band was totally displaced by IGF-I and alpha IR-3. It was concluded that IGF-I stimulated follicular development, E2 production, and oocyte maturation by interacting with its specific receptor located in rabbit ovarian membranes.

Involvement of leukotriene B4 in ovulation in the rabbit.

The present study was undertaken to assess the effects of lipoxygenase products on ovulation, oocyte maturation, and steroid production in the perfused rabbit ovary preparation. Ovulatory efficiency was significantly reduced when rabbit ovaries were perfused with human CG (hCG) plus nordihydroguaiaretic acid (NDGA) at 10(-5) or 10(-6) M, as compared to contralateral hCG-treated controls. The addition of NDGA to the perfusate inhibited hCG-induced ovulation in a dose-related manner. The percentage of ovulated ova and follicular oocytes achieving germinal vesicle breakdown did not differ significantly between NDGA-treated ovaries and contralateral controls. Leukotriene B4 (LTB4) production by the perfused rabbit ovaries reached its maximum 6 h after exposure to hCG and then declined. The addition of NDGA at 10(-5) M significantly inhibited hCG-stimulated LTB4 production by rabbit ovaries throughout the entire perfusion periods. The ovulatory efficiency in ovaries treated with hCG alone or with hCG plus NDGA correlated significantly with LTB4 production by perfused rabbit ovaries 6 h after exposure to hCG (alpha = 0.8893, P less than 0.01). Furthermore, the addition of LTB4 at 100 ng/ml to the perfusate reversed the inhibitory effects of NDGA on hCG-induced ovulation. However, exposure to NDGA affected neither progesterone nor estradiol production elicited by hCG administration. These results suggest that NDGA may block hCG-induced ovulation in vitro, probably via the inhibition of LTB4 production by rabbit ovaries.

Gonadotropin-releasing hormone agonists induce meiotic maturation and degeneration of oocytes in the in vitro perfused rabbit ovary

The present study was undertaken to assess the effects of gonadotropin-releasing hormone agonists (GnRH-a, buserelin and leuprolide acetate [LA]) on ovulation, oocyte maturation and degeneration, and steroid and prostaglandin production in the perfused rabbit ovary preparation. Ovulation did not occur in any of ovaries treated with buserelin or LA (10(2) to 10(4) ng/mL) in the absence of gonadotropin. Gonadotropin-releasing hormone agonists were associated with the resumption of meiosis in follicular oocytes in a dose-related manner. Furthermore, the addition of GnRH-a to the perfusate significantly increased the percentage of follicular oocytes that showed evidence of degeneration compared with contralateral untreated or human chorionic gonadotropin-treated controls. Prostaglandin E2 and prostaglandin F2 alpha production by the perfused rabbit ovaries were stimulated significantly by GnRH-a treatment. Exposure to GnRH-a failed to increase either progesterone or estradiol production by the perfused rabbit ovaries. These data demonstrate that GnRH-a act directly in the rabbit ovary to trigger meiotic maturation in oocytes within the follicles, concomitantly increasing oocyte degeneration.

Effect of inhibition of oxygen free radical on ovulation and progesterone production by the in-vitro perfused rabbit ovary

The potential role of oxygen free radicals in hCG-induced ovulation was investigated using the free radical scavenging enzymes superoxide dismutase (SOD) and/or catalase with the in-vitro perfused rabbit ovary preparation. SOD (25 micrograms/ml) and SOD + catalase (25 micrograms/ml) significantly reduced the % of large follicles that ovulated during perfusion (P less than 0.005). Neither maturity nor degeneration of ovulated ova and follicular oocytes was affected by SOD and/or catalase. Progesterone concentration in the perfusate was significantly increased in the SOD + catalase treatment group (P less than 0.01). These results indicate a significant role for oxygen free radicals in the process of ovulation.

Progesterone secretion by corpora lutea of the isolated perfused rabbit ovary during pseudopregnancy.

An ovarian in vitro perfusion method was adapted to examine rabbit corpus luteum (CL) function during pseudopregnancy. Ovaries were perfused in vitro with tissue culture Medium 199 with or without 3% bovine serum albumin (BSA). Samples were obtained from both arterial and venous cannulae, allowing rates of progesterone secretion to be determined. Two perfusion methods were compared: a closed system in which perfusion medium continuously recirculated through the tissue, and an open system in which the ovarian vein cannula was left outside the perfusion chamber. The addition of 3% BSA was found to prevent edema and distortion of the interstitial space, and to result in increased progesterone secretion. With the closed perfusion system, the progesterone secretion rate measured over the 6-h perfusion was significantly higher on Day 11 than on Day 1 of pseudopregnancy and had declined significantly on Day 18. There was no difference in secretion rates whether the perfusion system was open or closed, and the results obtained with both systems closely approximated in vivo progesterone secretion rates. Measurements of CL tissue progesterone content before and after in vitro perfusion indicated that the changes in progesterone secretion seen during pseudopregnancy resulted from differences in the synthesis and secretion of progesterone and not from leakage of progesterone already present in the CL prior to perfusion. Taken together, these results indicate that the modified in vitro perfused rabbit ovary preparation described herein is an appropriate model to examine progesterone secretion by ovaries bearing CL.

The effects of bradykinin on ovulation and prostaglandin production by the perfused rabbit ovary.

Involvement of the vasoactive peptide bradykinin (BK) in ovulation, oocyte maturation, and prostaglandin (PG) production was assessed using an in vitro perfused rabbit ovary preparation. In the first experiment, BK at a concentration of 0.033, 0.33, or 3.3 micrograms/ml was added to the perfusate of one ovary at hourly intervals for the first 10 h of perfusion. The contralateral control ovary was treated with medium alone parallel to the experimental ovary. Ovaries were perfused for a total of 12 h. BK induced ovulation in the absence of gonadotropin in a dose-related fashion, but did not induce maturation of ovulated ova or follicular oocytes. BK significantly stimulated PG production at all concentrations tested, but the effect was not dose related. Prostacyclin, as reflected by the concentration of 6-keto-PGF1 alpha in the perfusate, was the major PG produced. Smaller quantities of PGE2 and PGF2 alpha were present in the perfusate. After a single injection of BK (3.3 micrograms/ml), 6-keto-PGF1 alpha and PGF2 alpha production increased within 15 min and reached a maximum at 60-90 min. PGE2 did not change significantly over this time period. The addition of 1 microgram/ml indomethacin to the perfusate completely inhibited BK-stimulated PG production. However, indomethacin did not significantly affect the ovulatory efficiency of BK-treated ovaries. Neither BK nor indomethacin induced any degenerative changes in ovulated ova or follicular oocytes. The addition of a BK antagonist at 1 microgram/ml every 30 min to the perfusate resulted in an effective blockade of hCG-induced ovulation. These results suggest that BK is involved in the process of follicle rupture, but BK may induce ovulation by a mechanism(s) other than through PG stimulation.

Are Ovarian Steroids Required for Ovum Maturation and Fertilization? Effects of Cyanoketone on the in Vitro Perfused Rabbit Ovary

YOSHIMURA, Y.; HOSOI, Y.; BONGIOVANNI, A. M.; SANTULLI, R.; ATLAS, S. J.; WALLACH, E. E. Author Information

Are ovarian steroids required for ovum maturation and fertilization? Effects of cyanoketone on the in vitro perfused rabbit ovary.

An isolated perfused rabbit ovary preparation was used to determine the effects of cyanoketone, a potent inhibitor of 3 beta-hydroxysteroid dehydrogenase, on ovulation, ovum maturation and fertilizability, and steroid production. In the first experiment, cyanoketone (10(-4) M) was added to the perfusate of one ovary. The contralateral control ovary was perfused with medium alone. Thirty minutes after the onset of perfusion, hCG (50 IU) was added to the perfusate of both ovaries. The ovulatory efficiency of ovaries treated with cyanoketone plus hCG (82.3 +/- 4.6%) was similar to that of ovaries treated with hCG alone (84.8 +/- 4.4%). No difference was observed in the degree of ovum maturity or degeneration between control and cyanoketone-treated ovaries. Progesterone and estradiol production were significantly reduced by cyanoketone treatment; concentrations in the perfusate of ovaries treated with cyanoketone were 9.7% and 8.0% of the control values, respectively, 2 h after exposure to hCG. The concentration of 17-hydroxypregnenolone was not affected by cyanoketone treatment. Exposure to cyanoketone resulted in a significant (P less than 0.005) reduction in the fertilizability of ova ovulated and fertilized in vitro. In the second experiment, the percentage of ova that showed evidence of normal fertilization was significantly (P less than 0.025) increased in ovaries perfused with cyanoketone plus estradiol (64.5%) compared to that in ovaries perfused with cyanoketone alone (32.4%). In the third experiment, the addition of progesterone to the perfusate did not affect fertilizability of ovulated ova in ovaries perfused with cyanoketone plus estradiol. These results suggest that the presence of estradiol in the ovarian steroid environment may be essential for fertilizability of ova, but not for the processes of ovulation or meiotic maturation.

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.

Examination of the role of calcium in ovulation in the in vitro perfused rabbit ovary with use of ethyleneglycol-bis(β-aminoethyl ether)-n,n′-tetraacetic acid and verapamil

The in vitro perfused rabbit ovary preparation was used to examine the role of calcium in the ovulatory process. Two groups of rabbits were studied. In the first group, verapamil hydrochloride (10(-4) mol/L), a calcium channel blocker, was used together with human chorionic gonadotropin (50 IU) in the perfusate. Verapamil had no apparent effect on human chorionic gonadotropin-induced ovulation. Verapamil treatment, however, significantly reduced the percentage of ovulated ova that were mature (68.8%) in comparison to ovulated ova from human chorionic gonadotropin-treated control ovaries (95.0%). In a second experimental group, ethyleneglycol-bis(beta-aminoethyl ether)-n,n'-tetraacetic acid (2.0 mmol/L), a calcium ion chelator, was included in the perfusate with gonadotropin. The ethyleneglycol-bis(beta-aminoethyl ether)-n,n'-tetraacetic acid significantly reduced ovulatory efficiency (16.7% +/- 9.43%) in comparison to that of controls exposed to human chorionic gonadotropin alone (79.5% +/- 11.1%). In addition, ovulation occurred at an earlier time in ovaries perfused with ethyleneglycol-bis(beta-aminoethyl ether)-n,n'-tetraacetic acid; however, only four ovulations occurred in these ovaries. These four ovulated ova were immature, probably reflecting the early time of ovulation. Furthermore, both verapamil and ethyleneglycol-bis(beta-aminoethyl ether)-n,n'-tetraacetic acid blocked ovarian smooth muscle contractions during ovarian perfusion. These data provide additional support for the concept that calcium dynamics influence the processes of ovulation and ovum maturation. Furthermore ovarian smooth muscle contractions do not appear to be essential for ovulation in this model.

The relationship between prostaglandins and histamine in the ovulatory process as determined with the in vitro perfused rabbit ovary

The process of follicle rupture has been described as an inflammatory reaction in which prostaglandins (PGs) and/or histamine may be involved. With an in vitro perfused rabbit ovary preparation, experiments were carried out for determination of whether a relationship exists among PGs, histamine, and ovulation. PGF2 alpha alone was capable of inducing ovulation when added to the perfusion fluid at 1, 10, and 100 ng/ ml. Effectiveness in achieving ovulation varied directly with the dosage; however, the ovulatory efficiency of PGF2 alpha-treated ovaries was lower than that of ovaries exposed to human chorionic gonadotropin (hCG, 100 IU). PGF2 alpha-induced ovulation could not be blocked by the H2 receptor antagonist, cimetidine. The PG synthesis inhibitor, indomethacin, did not prevent histamine-induced ovulation. Ovulation induced by hCG was partially blocked by the administration of indomethacin; however, the concomitant administration of cimetidine was not associated with further reduction in ovulation. In all but one experimental group, the majority of ovulated ova did not progress beyond the intact germinal vesicle stage unless the ovaries had been exposed to hCG. On the basis of these experiments, PGs and histamine do not appear to be interdependent in their effects on the ovulatory process in vitro.

Prostaglandin F 2a, 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 in vitro 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 not 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.

The effect of adrenergic agents on the ovulatory process in the in vitro perfused rabbit ovary

The presence of adrenergic nerves and smooth muscle in the mammalian ovary has prompted an investigation of the role of this neuromuscular complex in the ovulatory process. The in vitro perfused rabbit ovary preparation was used to study the effects of adrenergic agents on ovulation. Ovaries were exposed to norepinephrine (NE) at four dose levels (6 × 10−6M, 10−7M, 10−8M, and 10−9M). Ovulation occurred in a dose-dependent fashion with 10−6M NE resulting in the greatest ovulatory efficiency (64.7%). Addition of NE to ovaries treated with the minimal effective dose of human chorionic gonadotropin (hCG) (5 IU) led to an increase (59.9%) in ovulatory efficiency when compared to that of the ovaries treated with hCG alone (25.9%) but was not associated with an increase when compared to that of the ovaries treated with NE alone at the same dose level. Phenoxybenzamine (PBZ), an irreversible α-receptor antagonist, or phentolamine (POA), a competitive α-adrenergic inhibitor, was added to perfused ovaries to determine if either agent could block hCG- or NE-induced ovulation. Neither PBZ (10−6M) nor POA (10−5M) had any effect on NE (10−8M)-induced ovulation. The addition to the perfusate of either PBZ or POA significantly reduced ovulatory efficiency associated with hCG stimulation (p < 0.02) but did not completely block ovulation. Determination of the stage of maturity of ovulated ova and follicular oocytes demonstrated that more than 85% of ova exposed to NE remained in the germinal vesicle stage. Only those ova exposed to hCG consistently achieved metaphase I or II. A high percentage of degenerated ova was observed in both ovulated ova and follicular oocytes exposed to POA. A positive correlation was observed between ovarian edema and ovulatory efficiency (p < 0.005). These experiments indicate that adrenergic receptors can contribute to follicle rupture. However, the failure of PBZ and POA to inhibit NE- or hCG-induced ovulation indicates that adrenergic receptors are not essential mediators of the ovulatory process.

The effect of prostaglandin synthesis inhibition by indomethacin on ovulation and ovum maturation in the in vitro perfused rabbit ovary

Prostaglandins (PGs) have been implicated in the mechanism of ovulation in several species through the use of PG synthesis inhibitors such as indomethacin. Studies of ovulation in the in vitro perfused rabbit ovary have aided in the delineation of the process of ovulation. This study was designed to determine the effects of indomethacin on follicle rupture and ovum development in the in vitro perfused rabbit ovary preparation. Indomethacin treatment (0.5 μg/ml) significantly reduced the occurrence of ovulation in gonadotropin-treated ovaries. The percentage of in vitro ovulated ova and ova recovered from unruptured follicles during ovarian perfusion which achieved Metaphase II did not significantly differ between indomethacin-treated and control ovaries (hCG alone). However, increased degeneration of Metaphase II ova was associated with indomethacin treatment. In in vitro culture experiments this degeneration could be prevented by supplemental PGF2α. These results indicate that inhibition of PG synthesis by indomethacin prevents follicle rupture but does not affect ovum maturation, thus providing further evidence that these two processes are distinct phenomena in vitro.

Inhibitory effect of prolactin on ovulation in the in vitro perfused rabbit ovary.

Prolactin is best known for its effects on the breast, promoting mammary growth and lactation. In some species, including rat, mouse, hamster, sheep and rabbit, prolactin is necessary for the maintenance of the corpus luteum. Further, a relationship has been recognised between hyperpromus and or adenohypophysis. McNatty et al. have postulated a direct ovarian effect of prolactin, reporting that low prolactin levels were essential for progesterone production by preovulatory human granulosa cells cultured in vitro. However, high levels of prolactin decreased progesterone production. The present investigation sought to determine the effect of prolactin on ovulation using the in vitro perfused rabbit ovary preparation. We report that its effects are inhibitory.

The Effects of Progesterone and Human Chorionic Gonadotropin on Ovulation in the in Vitro Perfused Rabbit Ovary

The in vitro perfused rabbit ovary preparation was used to study the effect of progesterone on follicle rupture and to determine the minimum level of human chorionic gonadotropin (hCG) required to induce ovulation in vitro. Both ovaries from an intact female rabbit were removed and simultaneously placed in separate perfusion systems. In four groups of five rabbits each, hCG at a dose of 25, 10, 5, or 1 IU was added to the perfusate of both ovaries. An additional group received no hCG. One ovary from each rabbit was treated with progesterone (1 mg/150 ml of perfusate) together with hCG. The contralateral ovary served as a control, receiving only hCG. The number of ovaries ovulating in each group varied directly with the amount of hCG added to the perfusate (four of five with 25 IU, three of five with 10 IU, two of five with 5 IU, one of five with 1 IU, and none of five without hCG). In each group the number of ovulations per ovary did not increase significantly by the addition of progesterone to the perfusate. These data suggest that a dose-response relationship can be established between hCG and ovulation in the in vitro model and that amounts of hCG as small as 1 IU can produce ovulation in an in vitro perfused rabbit ovary preparation. Although exogenous progesterone did not appear to influence ovulation in this model, these data do not exclude the possibility that progesterone exerts a direct facilitatory effect on ovulation.

Investigation of mammalian ovulation with an in vitro perfused rabbit ovary preparation

An in vitro perfused rabbit ovary preparation has been developed to study local factors responsible for the ovulatory process. This preparation enables: (1) isolation of the ovary from systemic influences, (2) direct serial observations of follicle development and ovulation, (3) addition to the perfusing medium of substances that may influence ovulation acting at the level of the ovary, and (4) correlation of the times of individual follicle rupture with ovarian contractile patterns. With this model the temporal requirements for human chorionic gonadotropin in the process of ovulation have been studied. The direct effects on ovulation of prostaglandin F3 alpha (PGF 2 alpha), the antihistamine chlorpheniramine, and calcium deprivation were also investigated. Chlorpheniramine and EDTA each led to inhibition of ovulation and depressed ovarian contractility. The use of the isolated in vitro ovary preparation permits characterization of the local requirements for ovulation.