Perfused Rabbit Ovary Research Articles

Angiotensin-(1-7) induces ovulation and steroidogenesis in perfused rabbit ovaries

A local renin-angiotensin system has been described in several organs, including the ovary; however, data indicating a role for angiotensin II in the induction of ovulation are controversial. We have previously shown the presence of a novel peptide, angiotensin-(1-7) [Ang-(1-7)], in the rat ovary and its effect on steroidogenesis. The objective of the present study was to determine whether Ang-(1-7) plays a role in ovulation. We first determined the presence and distribution of Ang-(1-7) and the receptor Mas in rabbit ovaries by immunohistochemistry. Angiotensin-(1-7) and Mas immunoreactivity were observed in interstitial cells and oocytes of immature ovaries. Immunoreactivity for Ang-(1-7) and Mas was also observed in theca and granulosa cells of preovulatory follicles in ovaries of gonadotrophin-stimulated rabbits. To verify the effect of Ang-(1-7) in ovulation and steroidogenesis, we used isolated ovaries from immature rabbits pretreated with equine chorionic gonadotrophin (50 i.u., 48 h before the experiment) and then perfused in vitro. The ovulatory efficiency was determined by the number of oocytes compared with the number of preovulatory follicles present in the ovary. Angiotensin-(1-7) stimulated oestradiol production and enhanced ovulatory efficiency, which was blocked by the specific Ang-(1-7) antagonist, A-779. Ovulation induced by human chorionic gonadotrophin was also antagonized by A-779. These results show, for the first time, the involvement of a novel regulatory peptide system, Ang-(1-7) and Mas, in the ovulatory process. More importantly, because A-779 antagonized hCG-induced ovulation, it may be inferred that Ang-(1-7) plays an important role in ovulation, possibly as a mediator of gonadotrophin action.

31P NMR studies of phosphate metabolism in the ovulatory process induced by gonadotropins in perfused rabbit ovary

The concentrations of phosphate metabolites were measured in perfused rabbit ovaries before, during and after ischemia, and during the ovulatory process induced by the administration of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG) using 31P NMR spectroscopy. A decrease in adenosine triphosphate (ATP) and an increase in inorganic phosphate (Pi) during ischemia were observed. After reperfusion of the ovary, both ATP and Pi levels returned to their initial control levels. These results are in agreement with the results observed in other organ tissues. On the other hand, ATP levels rapidly decreased dose dependently with infusion of HCG during 10 h of perfusion. A decrease in ovarian ATP concentration after infusion of HCG was also confirmed by enzymatic analysis of the extracts from ovarian tissues. On the other hand, significant changes in Pi levels were not observed throughout the perfusion period. Furthermore, significant changes in phosphomonoester (PME) levels, adenosine diphosphate (ADP) levels and intracellular pH were not observed before and during the infusion of HCG. The concentration of cyclic adenosine 3′,5′-monophosphate (cAMP) in the perfusate after the perfusion was also measured during the ovulatory period. Within 30 min cAMP significantly increased dose dependently and reached peak levels within 60 min. The concentration of cAMP then gradually decreased. Ovulatory efficacy during the infusion of HCG was also observed. These results suggest that the decreased ATP in the successive processes leading to ovulation probably reflects protein phosphorylation through the activation of the cAMP-dependent protein kinases prior to induction of various chemical reactions related to the initiation of ovulation. Copyright © 1999 John Wiley & Sons, Ltd.

31P NMR studies of phosphate metabolism in the ovulatory process induced by gonadotropins in perfused rabbit ovary.

The concentrations of phosphate metabolites were measured in perfused rabbit ovaries before, during and after ischemia, and during the ovulatory process induced by the administration of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG) using 31P NMR spectroscopy. A decrease in adenosine triphosphate (ATP) and an increase in inorganic phosphate (Pi) during ischemia were observed. After reperfusion of the ovary, both ATP and Pi levels returned to their initial control levels. These results are in agreement with the results observed in other organ tissues. On the other hand, ATP levels rapidly decreased dose dependently with infusion of HCG during 10 h of perfusion. A decrease in ovarian ATP concentration after infusion of HCG was also confirmed by enzymatic analysis of the extracts from ovarian tissues. On the other hand, significant changes in Pi levels were not observed throughout the perfusion period. Furthermore, significant changes in phosphomonoester (PME) levels, adenosine diphosphate (ADP) levels and intracellular pH were not observed before and during the infusion of HCG. The concentration of cyclic adenosine 3',5'-monophosphate (cAMP) in the perfusate after the perfusion was also measured during the ovulatory period. Within 30 min cAMP significantly increased dose dependently and reached peak levels within 60 min. The concentration of cAMP then gradually decreased. Ovulatory efficacy during the infusion of HCG was also observed. These results suggest that the decreased ATP in the successive processes leading to ovulation probably reflects protein phosphorylation through the activation of the cAMP-dependent protein kinases prior to induction of various chemical reactions related to the initiation of ovulation.

Effects of nitric oxide on ovulation and ovarian steroidogenesis and prostaglandin production in the rabbit.

Evidence supports the involvement of nitric oxide (NO) in ovarian physiology. The present study was undertaken to investigate the role of the NO/NO synthase (NOS) systems in ovulation, oocyte maturation, ovarian steroidogenesis, and PG production using in vitro perfused rabbit ovaries. The addition of the NOS inhibitors, aminoguanidine hemisulfate salt (AG) and N-omega-nitro-L-arginine methyl ester (L-NAME), to the perfusate inhibited the ovulation induced by hCG in a dose-dependent manner, whereas D-NAME had no significant effect. Neither AG nor L-NAME affected the hCG-induced meiotic maturation of the ovulated ova. The exogenous administration of the NO generator, sodium nitroprusside (NP), induced follicle rupture in the absence of gonadotropin, but did not induce oocyte maturation. Inhibition of endogenous NOS by AG and L-NAME resulted in a significant elevation in the production of estradiol (E2), but not of progesterone, stimulated by hCG. The concomitant administration of NP significantly reduced the AG-stimulated production of E2 by ovaries perfused in the presence of hCG, which suggests that NO down-regulates ovarian E2 synthesis. Ovarian production of PGE2 and PGF2alpha in response to hCG was significantly blocked by L-NAME, and exogenous administration of NP stimulated the production of PGs in the absence of gonadotropin. Significant correlations were observed between the ovulatory efficiencies and the production of PGs by rabbit ovaries perfused with or without L-NAME. In conclusion, the ovarian NO/NOS system is involved in follicle rupture during the ovulatory process. NO may induce follicle rupture in rabbit ovaries at least in part by the stimulation of PG production.

Dietary carrot results in diminished ovarian progesterone secretion, whereas a metabolite, retinoic acid, stimulates progesterone secretion in the in vitro perfused rabbit ovary

Objective: To determine the effects of dietary carrots and retinoic, acid on P secretion in the in vitro perfused rabbit ovary. Design: Controlled experiment. Setting: Laboratory. Subjects: Sexually mature New Zealand white rabbits. Intervention(s): Experiments were done in vitro using an isolated rabbit ovarian perfusion system after acute feeding of carrots or with in vitro exposure to retinoic acid. Main Outcome Measure(s): Progesterone and E 2 were measured in aliquots taken from arterial and venous cannulas. Result(s): Progesterone secretion during the in vitro perfusion of six ovaries is significantly diminished after the acute feeding of carrots. Human chorionic gonadotropin-induced P secretion also is significantly reduced. Progesterone secretion during in vitro perfusion on day 1 of pseudopregnancy was significantly increased in the perfusate samples from ovaries taken from rabbits not acutely fed carrots but that were exposed to retinoic acid. Progesterone secretion was increased but only marginally significant on day 11 of pseudopregnancy in perfusate samples from ovaries exposed to retinoic acid. Conclusion(s): Rabbit ovarian P secretion may be modified by carrots and carotene metabolites. This effect on steroid secretion may contribute to the relationship between hypercarotenemia and alterations in menstrual function.

The role of insulin-like growth factor-I (IGF-I) and estradiol in rabbit corpus luteum progesterone production.

To determine whether insulin-like growth factor-I (IGF-I) plays a role in rabbit corpus luteum (CL) physiology the authors examined: IGF-I expression, the effect of IGF-I on progesterone (P) production in vitro, and the interaction of IGF-I with estradiol, the primary luteotropin in the rabbit. Northern blot analysis revealed that IGF-I mRNA is present in the rabbit CL throughout pseudopregnanacy. An intact ovarian in vitro perfusion model and dispersed luteal cell culture were used to determine effects of IGF-I on P production and interactions with estradiol. IGF-I significantly stimulated P production compared to control medium by the isolated, intact perfused rabbit ovary and by dispersed, cultured luteal cells. Estradiol alone did not stimulate P production in vitro. Estradiol did augment IGF-I stimulated P production in the intact perfused ovary and in luteal cell culture. These findings support a role for IGF-I in rabbit CL P production.

Involvement of angiotensin II in the process of gonadotropin-induced ovulation in rabbits.

In the present study we investigated the role of angiotensin II (Ang II) receptor subtypes in gonadotropin-induced ovulation, oocyte maturation, and ovarian steroidogenesis and prostaglandin (PG) production in in vitro-perfused rabbit ovaries. The addition to the perfusate of PD123319, a nonpeptide Ang II antagonist with a high affinity for AT2 receptors, inhibited hCG-induced ovulation in a dose-dependent manner, whereas CV-11974, a nonpeptide AT1 receptor antagonist, had no effect. The majority of ovulated ova and follicular oocytes resumed meiotic maturation in response to hCG; and PD123319, but not CV-11974, significantly inhibited hCG-induced oocyte maturation. The addition of both Ang II receptor antagonists to the perfusate had no significant effect on the concentration of progesterone in the perfusate of hCG-treated ovaries, whereas PD123319 inhibited the hCG-stimulated production of estradiol. The production of PGE2 and PGF2 alpha was significantly increased at 6 h in hCG-treated ovaries compared with ovaries before hCG administration. PD123319 inhibited the hCG-stimulated production of PGs by perfused rabbit ovaries in a dose-dependent manner, indicating that hCG-induced PG synthesis is mediated, at least in part, via the activation of AT2 receptors. Ovulatory efficiency in ovaries perfused with or without PD123319 in the presence of hCG was significantly correlated with PG production by perfused rabbit ovaries 12 h after exposure to hCG (r = 0.6553 for PGE2, p < 0.001; r = 0.4758 for PGF2 alpha, p < 0.05). In conclusion, Ang II exerts complex and coordinated control on at least two distinct aspects in the normal ovulatory process, ovulation and oocyte maturation. Ang II produced locally by gonadotropin exposure may be a part of a novel intraovarian paracrine or autocrine control mechanism that operates via the AT2 receptor in the ovary.

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.

Angiotensin II induces ovulation and oocyte maturation in rabbit ovaries via the AT2 receptor subtype.

The present study was undertaken to investigate the role of angiotensin II (Ang II) in ovulation and ovarian steroidogenesis and prostaglandin (PG) production via the Ang II receptors in rabbit ovaries. In in vitro perfused rabbit ovaries, PD123319, a selective nonpeptide antagonist for AT2 receptors, reduced the Ang II-induced ovulation in a dose-dependent manner, whereas CV-11974, a selective nonpeptide antagonist for AT1 receptor, did not affect the Ang II-induced ovulation. Ang II also significantly stimulated the meiotic maturation of ovulated ova and follicular oocytes in the absence of gonadotropin. The addition of PD123319 at 10 (-6) M to the perfusate significantly inhibited the Ang II-induced oocyte maturation. Ang II did not stimulate the production of progesterone by perfused rabbit ovaries but significantly stimulated the production of estradiol (E2) and PGs. When PD123319 at 10(-6) M was added to the perfusate 30 min before the onset of Ang II administration, the Ang II-stimulated production of E2 and PGs was significantly blocked. Saralasin, a peptide analog of Ang II, inhibited the specific binding of [125I] iodo-[Sar1, Ile8] Ang II to rabbit ovarian membranes in a concentration-dependent manner, yielding an inhibitory constant (IC50) value of 1.58 x 10(-9) M. PD123319 and CV-11974 also inhibited the binding of [125I]iodo-[Sar1, Ile8] Ang II; however, PD123319 and CV-11974 were 15 and 40 times less potent than saralasin, respectively. Autoradiographic study revealed that an intense localization of Ang II receptors in the rabbit ovaries was present in the granulosa cell layers and the stroma of the preovulatory follicles. AT2 receptors were predominantly located in granulosa cells, whereas AT1 receptors were more concentrated in the stroma and thecal cell layers. In summary, Ang II induced ovulation and oocyte maturation and stimulated the production of E2 and PG by perfused rabbit ovary in vitro via the AT2 receptor. Thus, locally produced Ang II may be part of a novel intraovarian paracrine or autocrine control mechanism during the ovulatory process.

Role of calcium/calmodulin-dependent protein kinase II in gonadotrophin-induced ovulation in in vitro perfused rabbit ovaries

The objectives of these experiments were to determine (i) the role of calcium/calmodulin-dependent protein kinase II-mediated signal transduction in hCG-induced ovulation and (ii) whether there is an association between arachidonic acid metabolites, nitric oxide and calcium/calmodulin-dependent protein kinase II in the overall scheme of ovulation induction. Ovarian arteries were cannulated in situ, and the ovaries were excised and perfused in vitro. Ovulatory efficiency ([number of ovulated follicles/number of mature follicles > 1.5 mm] x 100) was calculated for each experiment. Calcium/calmodulin-dependent protein kinase II substrate induced ovulation in the absence of gonadotrophin (calcium/calmodulin-dependent protein kinase II substrate: 66.3%; control: 0%). In the next experiment, perfusion medium of the experimental ovary was supplemented with KN 62, a potent inhibitor of calcium/calmodulin-dependent protein kinase II, while the contralateral ovary served as control. Ovulations were induced in both ovaries with hCG (50 iu (150 ml)-1) and perfusion was continued for 8 h. In the third experiment, ovaries were perfused with prostaglandin F2 alpha (PGF2 alpha) with and without KN 62, while the contralateral ovary was perfused with medium alone. KN 62 reduced the ovulatory efficiency of hCG-treated ovaries in vitro during perfusion (hCG + 10(-7) mol KN 62 l-1: 32.9%; hCG: 80.9%). Furthermore, it significantly reduced the ovulatory efficiency of PGF2 alpha-treated ovaries (PGF2 alpha + KN 62 = 21.5%; PGF2 alpha = 59.9%).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Varying Modes of Perfusion on Ovum Maturation and Ovulation in the Perfused Rabbit Ovary.

Using a perfused rabbit ovary, we studied the effect of luteinizing hormone (LH) secretion on the ovulation time and the maturity of ova with 2 modes of perfusion. One mode consisted of a constant level of human chorionic gonadotropin (hCG) in the medium (the constant mode of perfusion), and the other mode mimicked the profile of LH surge in the rabbit (the surge mode of perfusion). The mean numbers of ova ovulated in the 2 modes were almost equal. The mean ovulation time in the constant mode was shorter than that in the surge mode. In the constant mode, the percentage of ova in germinal vesicle stage was larger, and in metaphase II was smaller, than those in the surge mode, while the difference in metaphase I was insignificant. The results indicate that the maturities of the ova in the surge mode were more advanced than those in the constant mode. A reason for the occurrence of the difference might be the early occurrence of ovulation in the constant mode due to excessive action on the follicular wall by various proteolytic enzymes, which are activated by the higher concentration of hCG, when compared with that of the surge mode, in the latter half of the perfusion.

Growth hormone stimulates follicular development by stimulating ovarian production of insulin-like growth factor-I

The present study was undertaken to investigate the effects of GH on follicular growth, oocyte maturation, ovulation, and production of insulin-like growth factor-I (IGF-I) in the in vitro perfused rabbit ovaries. Ovulation did not occur in any ovaries perfused with GH at a concentration of 1, 10, 100, or 200 ng/ml, but the addition of GH to the perfusate increased the follicle diameter in a dose-dependent manner. The production of IGF-I by ovaries perfused with medium alone was very low throughout the perfusion period. The addition of 100 ng/ml GH to the perfusate significantly increased ovarian production of IGF-I at 4, 6, 8, and 12 h compared with the contralateral control ovaries. Changes in the tissue concentrations of IGF-I in ovaries perfused with 100 ng/ml GH paralleled those triggered by exposure to 50 IU human CG (hCG). When the effect of GH on the tissue concentration of IGF-I was determined at 4 h, GH stimulated the tissue concentration of IGF-I in perfused rabbit ovaries in a dose-dependent manner. The percent increase in follicle diameter in ovaries treated with GH was significantly correlated with the intraovarian IGF-I content. The mean number of ovulations per ovary and the ovulatory efficiency were significantly reduced in ovaries perfused with 5 IU hCG, compared with those in ovaries perfused with 50 IU hCG. The addition of 100 ng/ml GH to the perfusate significantly increased the ovulatory efficiency and follicle diameter in the 5 IU hCG-treated ovaries. Exposure to GH significantly stimulated the resumption of meiosis in the follicular oocytes compared with that in ovaries perfused with medium alone. Furthermore, GH significantly stimulated the resumption of meiosis in ovulated ova and follicular oocytes in ovaries treated with 5 IU hCG. Thus, exposure to GH-stimulated follicular growth, oocyte maturation, and production of IGF-I in the in vitro perfused rabbit ovaries, which indicates that the ovary is in fact a site of GH reception and action. Additionally, GH enhanced the effects of gonadotropins, acting synergistically to promote the ovulatory process. These observations suggest that GH may amplify gonadotropin actions in the process of follicular development and ovulation, at least in part, by stimulating ovarian IGF-I production.

Growth hormone stimulates follicular development by stimulating ovarian production of insulin-like growth factor-I.

The present study was undertaken to investigate the effects of GH on follicular growth, oocyte maturation, ovulation, and production of insulin-like growth factor-I (IGF-I) in the in vitro perfused rabbit ovaries. Ovulation did not occur in any ovaries perfused with GH at a concentration of 1, 10, 100, or 200 ng/ml, but the addition of GH to the perfusate increased the follicle diameter in a dose-dependent manner. The production of IGF-I by ovaries perfused with medium alone was very low throughout the perfusion period. The addition of 100 ng/ml GH to the perfusate significantly increased ovarian production of IGF-I at 4, 6, 8, and 12 h compared with the contralateral control ovaries. Changes in the tissue concentrations of IGF-I in ovaries perfused with 100 ng/ml GH paralleled those triggered by exposure to 50 IU human CG (hCG). When the effect of GH on the tissue concentration of IGF-I was determined at 4 h, GH stimulated the tissue concentration of IGF-I in perfused rabbit ovaries in a dose-dependent manner. The percent increase in follicle diameter in ovaries treated with GH was significantly correlated with the intraovarian IGF-I content. The mean number of ovulations per ovary and the ovulatory efficiency were significantly reduced in ovaries perfused with 5 IU hCG, compared with those in ovaries perfused with 50 IU hCG. The addition of 100 ng/ml GH to the perfusate significantly increased the ovulatory efficiency and follicle diameter in the 5 IU hCG-treated ovaries. Exposure to GH significantly stimulated the resumption of meiosis in the follicular oocytes compared with that in ovaries perfused with medium alone. Furthermore, GH significantly stimulated the resumption of meiosis in ovulated ova and follicular oocytes in ovaries treated with 5 IU hCG. Thus, exposure to GH-stimulated follicular growth, oocyte maturation, and production of IGF-I in the in vitro perfused rabbit ovaries, which indicates that the ovary is in fact a site of GH reception and action. Additionally, GH enhanced the effects of gonadotropins, acting synergistically to promote the ovulatory process. These observations suggest that GH may amplify gonadotropin actions in the process of follicular development and ovulation, at least in part, by stimulating ovarian IGF-I production.

Prolactin Inhibits Ovulation by Reducing Ovarian Plasmin Generation

The present study was undertaken to investigate the effects of prolactin (PRL) on gonadotropin-induced ovulation and the biosynthesis of prostaglandin (PG), leukotriene (LT), and plasmin in in vitro perfused rabbit ovaries. The addition of PRL to the perfusate inhibited hCG-induced ovulation in vitro in a dose-dependent manner. Although exposure to hCG significantly increased PGF2 alpha, PGE2, and LTB4 production by perfused rabbit ovaries, PRL did not affect the secretion rates of PGs and LTB4 stimulated by hCG administration. The ovarian plasmin generation was determined by measuring the amount of plasmin bound to its major inhibitor, alpha 2-plasmin inhibitor (alpha 2PI-Plm). Exposure to hCG enhanced biphasically the ovarian secretion rate of alpha 2 PI-Plm, while PRL at a dose of 10(3) ng/ml significantly inhibited the hCG-stimulated generation of alpha 2 PI-Plm in ovaries throughout the entire perfusion period. A significant correlation was observed between ovulatory efficiency and ovarian plasmin generation in the PRL-treated ovaries. Additionally, PRL inhibited intrafollicular concentrations of alpha 2 PI-Plm in hCG-treated ovaries in a dose-dependent manner. These observations substantiate an essential role for a plasma-generating system in the cascade of events leading to ovulation. In conclusion, PRL may act directly on the ovary and block ovulation, at least in part, via the inhibition of ovarian plasmin generation.

Effect of interleukin-1 beta on ovulation in the in vitro perfused rabbit ovary.

Interleukin-1 (IL-1), a prominent 17-kilodalton member of a group of immune mediators referred to as cytokines, is secreted by a variety of immuno- and nonimmunocompetent cells. As IL-1 is an established mediator of inflammation, and ovulation may constitute an inflammatory-like reaction, consideration may be given to the possibility that IL-1 may play an intermediary role in the ovulatory process. Such a hypothesis is supported by the recent demonstration of the gonadotropin-dependent preovulatory induction of IL-1 transcripts at the level of the murine and human ovary. To date, however, the direct effect of IL-1 beta on the ovulatory process has not been examined. The objective of this study was to investigate the potential role of IL-1 beta in ovulation, oocyte maturation (nuclear and cytoplasmic), and subsequent fertilizability of in vitro ovulated oocytes. Rabbit ovaries perfused in vitro were used for these experiments. Ovarian arteries were cannulated in situ, and the ovaries were excised and perfused in vitro with or without IL-1 beta (18 ng/ml). The ovulatory efficiency of 18 ng/ml IL-1 beta-treated ovaries was 73.1%, similar to that of hCG (71.2%). Recovered oocytes were examined for their maturation and were inseminated in vitro to investigate fertilization, cleavage, and embryonic development. The fertilization rates of the 18 ng/ml IL-1 beta-treated and hCG-treated groups were 65.8% and 95.8% (P < 0.01), respectively. Cleavage rates of the IL-1 beta-treated and hCG-treated groups were 50% and 83.3% (P < 0.01), respectively. Most of the cleaved embryos from the IL-1 beta-treated group arrested at the four-cell stage, and only 2.6% of the fertilized embryos developed into the morula stage, whereas 54.2% of the hCG-treated group developed to the morula stage (P < 0.01). A cytotoxic effect of IL-1 beta is unlikely in this model. A more likely explanation is the induction of other factors by IL-1 beta, which may inhibit cytoplasmic maturation. Taken together, our findings demonstrate that in the absence of an ovulatory gonadotropic trigger, IL-1 beta can induce ovulation and oocyte maturation, facilitate fertilization, and influence subsequent embryonic development. Although fertilization and embryonic development occurred after IL-1 beta treatment, these rates were lower than those after hCG treatment. These observations give credence to the possibility that IL-1 may play an intermediary role in the ovulatory process.

Effect of interleukin-1 beta on ovulation in the in vitro perfused rabbit ovary

Effect of interleukin-1 beta on ovulation in the in vitro perfused rabbit ovary

Direct ovarian effect of growth hormone in the rabbit.

This study was undertaken to assess whether growth hormone (GH) can stimulate follicle growth and ovarian steroidogenesis via putative GH receptors. In vitro perfused rabbit ovary. Ovulation occurred in neither the control ovaries nor experimental ovaries treated with 100 ng/ml of GH, whereas all ovaries exposed to 50 IU of human chorionic gonadotropin (hCG) ovulated. The addition of GH to the perfusate significantly stimulated the follicle growth in the absence of gonadotropin. The percent change in follicle diameter in GH-treated ovaries did not differ significantly from that in hCG-treated ovaries. Exposure to GH significantly stimulated the meiotic maturation in the follicular oocytes, as compared with the contralateral control ovaries. Although the concentration of progesterone in the perfusate did not differ significantly between GH-treated and control ovaries, GH stimulated estradiol production by the perfused rabbit ovaries. Rabbit ovary membranes exhibited high affinity binding sites of hGH (Kd = 6.1 x 10(-9) M). GH acts on the rabbit ovary to stimulate the follicle growth, oocyte maturation, and ovarian estradiol production by interacting with the specific receptors located in ovarian plasma membranes.

Gonadotropin stimulates ovarian renin-angiotensin system in the rabbit.

The present study was undertaken to assess the role of ovarian renin-angiotensin system (RAS) in the preovulatory cascade induced by gonadotropin exposure. In the in vitro perfused rabbit ovaries, exposure to human chorionic gonadotropin (hCG) enhanced the secretion rate of angiotensin II (Ang II) within 1 h. The secretion rate reached maximal levels at 6 h and then declined thereafter. The intrafollicular Ang II content and renin-like activity were also significantly increased at 2 and 4 h after exposure to hCG, compared with control ovaries perfused with medium alone. The level of intrafollicular Ang II after hCG exposure significantly exceeded the concentration of Ang II in an equivalent volume of plasma. The addition of 1 microM captopril to the perfusate significantly inhibited the secretion rate of Ang II stimulated by hCG; however, captopril affected neither the ovulatory efficiency nor prostaglandin production in ovaries treated with hCG. Captopril significantly inhibited the resumption of meiosis in the ovulated ova and follicular oocytes stimulated by hCG. The administration of 100 micrograms Ang II at 2-h intervals to the perfusate reversed the inhibitory effects of captopril on hCG-induced oocyte maturation. In conclusion, these data indicate that gonadotropin stimulates renin-like activity and Ang II production in the rabbit ovary. Ovarian renin-angiotensin system may play an important role in the process of oocyte maturation after exposure to gonadotropin.

Locally produced angiotensin II induces ovulation by stimulating prostaglandin production in in vitro perfused rabbit ovaries.

The present study was undertaken to investigate the role of exogenous and endogenous angiotensin II (Ang II) in ovarian steroidogenesis and production of prostaglandin (PG) in in vitro perfused rabbit ovaries. The addition of 100 or 10 micrograms Ang II at 2-h intervals to the perfusate did not stimulate progesterone production, but significantly stimulated estradiol (E2) production by perfused rabbit ovaries. When the specific antagonist of Ang II, saralasin at 2 x 10(-6) M, was added to the perfusate 30 min before the onset of Ang II administration, Ang II-stimulated production of E2 was significantly blocked. Ang II also significantly stimulated both PGE2 and PGF2 alpha production, while the addition of saralasin to the perfusate significantly inhibited the Ang II-stimulated production of PG. The levels of PGs in ovaries perfused with saralasin plus 100 micrograms Ang II did not differ significantly from those in control ovaries perfused with medium alone. Exposure to human CG (hCG) significantly stimulated production of progesterone and E2 by perfused rabbit ovaries, while the concomitant administration of 2 x 10(-6) M saralasin significantly reduced only E2 production. Addition of saralasin to the perfusate inhibited hCG-stimulated PG production in a dose-dependent manner. The ovulatory efficiency in ovaries treated with hCG alone or hCG plus saralasin was significantly correlated with PG production by perfused rabbit ovaries at 12 h after exposure to hCG. The production of PG stimulated by Ang II was completely reduced by indomethacin treatment during the entire perfusion period. Indomethacin completely blocked Ang II-induced ovulation, but not Ang II-stimulated oocyte maturation. Concurrent administration of staurosporine, a protein kinase C inhibitor, at 10(-6) M significantly inhibited Ang II-stimulated meiotic maturation of ovulated ova and follicular oocytes. In conclusion, these results indicate that Ang II has a direct role in ovarian production of E2 and PG. An intrinsic renin-angiotensin system in the rabbit ovary may act as an intermediary of gonadotropin-stimulated PG production. Locally produced Ang II may induce ovulation in the rabbit ovary, at least in part, by stimulating PG production.