Progesterone protects oocytes from premature degeneration within the follicle

The present study was designed to determine the effects of gestrinone (R2323) in the process of follicle rupture and oocyte maturation and degeneration in an in vitro perfused rabbit ovary model. In the first experiment, R2323 at 10(2), 10(3), or 10(4) ng/ml was added to the perfusate of one ovary. The contralateral control ovary was perfused simultaneously with medium alone. Thirty minutes after the onset of perfusion, 50IU of human chorionic gonadotropin (hCG) was added to the perfusate of both ovaries. All ovaries exposed to R2323 plus hCG or hCG alone ovulated. The addition of R2323 to the perfusate did not affect the ovulatory efficiency of ovaries treated with hCG. No significant difference in the percentage of ovulated ova or follicular oocytes demonstrating germinal vesicle breakdown was seen with R2323 treatment. R2323 increased the degeneration rate of ovulated ova in a dose-dependent fashion. In the second experiment, in which experimental ovaries were perfused with R2323 (10(4) ng/ml) plus progesterone (10(3) ng/ml) and the control ovaries with R2323 (10(4) ng/ml) alone ovulation occurred in response to hCG. However, the addition of progesterone to the perfusate reduced the degeneration-inducing effect of R2323 on both ovulated ova and follicular oocytes. In conclusion, R2323 appears to act as an antiprogesterone, thereby promoting the degeneration of oocytes. The increased production of progesterone in the preovulatory follicle following the gonadotropin surge protects oocytes from premature degeneration within the follicles.     

Direct ovarian effect of clomiphene citrate in the rabbit

The effects of clomiphene citrate (CC) on ovulation and ovum maturation were studied using the isolated perfused rabbit ovary. CC (10(-5) M) added to the perfusate with human chorionic gonadotropin (50 IU) did not affect ovulatory efficiency, ovulation time, oocyte maturation, or degeneration of ovulated ova and follicular oocytes. During perfusion without human chorionic gonadotropin, the percentage of follicular oocytes with germinal vesicle breakdown was significantly increased in response to CC (10(-5) M or 10(-7) M); a greater percentage of follicular oocytes was degenerated. Estradiol (100 ng/ml) added to the perfusate reversed the effect of CC on degeneration of follicular oocytes. Of follicular oocytes from ovaries perfused with CC, 79.3% were degenerated; in contrast, 25% were degenerated in ovaries treated with CC plus estradiol. These data suggest that CC has a direct ovarian effect and that ovum degeneration associated with CC may be related to an antiestrogenic action.     

The effects of proteolytic enzymes on in vitro ovulation in the rabbit

The involvement of proteolytic enzymes in follicle rupture was assessed by use of the in vitro perfused rabbit ovary. Streptokinase (10 and 100 units/ml) induced ovulation in the absence of gonadotropin. Ovulation failed to occur in contralateral control ovaries. The time of ovulation in streptokinase- and human chorionic gonadotropin--treated ovaries was similar, but significantly more ova from streptokinase-treated ovaries were immature (p less than 0.001). Other ovaries were pretreated with trans-4-(aminomethyl)-cyclohexane-carboxylic acid, an inhibitor of the conversion of plasminogen to plasmin, and then perfused with human chorionic gonadotropin (50 IU). Ovulatory efficiency was significantly reduced by trans-4-(aminomethyl)-cyclohexane-carboxylic acid at 10 or 1 mmol/L (p less than 0.001), but ovum maturity was unaffected. Aprotinin (100 or 10 micrograms/ml), a potent inhibitor of plasmin, significantly inhibited human chorionic gonadotropin-induced ovulation (p less than 0.001) but did not affect oocyte maturation. Scanning electron microscopy of detergent-treated streptokinase-perfused ovaries revealed loosening and decomposition of collagen in the tunica albuginea. These results suggest proteolytic enzyme involvement in follicle rupture.     

Observation of the dynamics of follicular development in the ovary

The number of ovulated oocytes is different among mammals but does not vary much within the same species. In order to sustain periodic ovulation, follicular development must be coordinated at the tissue level. Elucidating the regulatory mechanisms of follicular development is difficult because the ovary has a complicated structure and it takes a long time for primordial follicles to develop into Graafian follicles. Therefore, it is not possible to observe follicular development by conventional experiments. The authors previously developed a new ovarian tissue culture method that enabled the observation of follicular development from the early follicle stage. These findings indicated that follicular interactions are important in regulating follicular development and ovulation. This review describes the current methods of observing follicular development in the ovary and the regulatory mechanisms of follicular development.     

卵泡期经阴道小卵泡穿刺抽吸术治疗多囊卵巢综合征排卵障碍

目的 探讨对克罗米芬治疗无反应的多囊卵巢综合征患者在卵泡期经阴道小卵泡穿刺抽吸术后,使用促性腺激素诱发排卵时卵泡的发育及其结局。方法 选择17例对克罗米芬治疗无反应,或对促性腺激素治疗发生卵巢过度刺激或无反应,但输卵管通畅、男方精液正常的多囊卵巢综合征不孕患者,在月经(人工周期)第5天给予促性腺激素治疗,给药5d后,在B超指引下经阴道行小卵泡穿刺抽吸术,双侧卵巢仅留1—2个较大卵泡,术后继续给予促性腺激素,观察卵泡发育、排卵和妊娠情况及血中性激素水平变化。结果 17例中除2例(11．8％)对该治疗方法无反应外,15例出现优势卵泡发育和排卵,其中单卵泡发育9例(52．9％),双卵泡发育4例(23.5％),3卵泡发育2例(11．8％),发育的优势卵泡全部排卵。总共有7例妊娠,全部为单胎妊娠,单周期治疗妊娠率41．2％(7／17)。结论 卵泡期经阴道小卵泡穿刺抽吸术能使对克罗米芬治疗无反应的多囊卵巢综合征不孕患者,使用促性腺激素治疗获得良好的单卵泡发育和单胎妊娠率。     

Direct inhibitory ovarian effects of prolactin in the process of ovulation

The present study was designed to determine the effects of prolactin (PRL) on the process of follicle rupture and oocyte maturation using an in vitro perfused rabbit ovary model. In the first experiment ovaries were perfused for 12 hours with or without PRL at 10(2) or 10(3) ng/ml. Ovulation did not occur in any ovaries in the absence of gonadotropin. The majority of follicular oocytes did not progress beyond the germinal vesicle stage following treatment with PRL. The percentage of follicular oocytes which showed evidence of degeneration was also comparable in both groups. The concentrations of progesterone in the perfusate did not differ significantly between PRL-treated and control ovaries. In the second experiment, ovaries were perfused with or without PRL at 10, 10(2), or 10(3) ng/ml. Thirty minutes later 50 IU of human chorionic gonadotropin (hCG) was added to the perfusate of all ovaries. The addition of PRL to the perfusate inhibited hCG-induced ovulation in a dose-related fashion. The degree of ovum maturity and degeneration was comparable in the two groups. PRL did not affect hCG-stimulated progesterone production by the perfused rabbit ovaries. The present study demonstrates that PRL acts directly on the ovary to influence the process of ovulation, resulting in the inhibition of hCG-induced follicle rupture. These data suggest that PRL inhibits ovulation by mechanism(s) independent of ovarian progesterone synthesis.     

Estradiol reverses the limiting effects of clomiphene citrate on early embryonic development in the in vitro perfused rabbit ovary

This study examined whether the addition of estradiol (E2) to the perfused rabbit ovary would reverse the deleterious effects of clomiphene citrate (CC) on early embryonic development. Ovaries were perfused with CC (10(-5) M) or CC + E2 (1 to 1000 ng/ml). Human chorionic gonadotropin (hCG, 50 IU) was added to the perfusate of each ovary. In vitro ovulated ova in cumulus were retrieved and inseminated in vitro. E2 significantly increased the percentage of ovulated ova achieving (1) the 2-cell stage at 36 hours, (2) the morula stage by 84 hours, and (3) the blastocyst stage at 132 hours. The percentage of inseminated ova showing evidence of degeneration was reduced in ovaries treated with E2. These data suggest that CC may exert an antiestrogenic effect on the intrafollicular oocyte, which interferes with postfertilization development.     

Changes in intrafollicular pressure in the rat ovary by nitric oxide and by alteration of systemic blood pressure

BACKGROUND: ovulation is associated with degradation of the follicular apex vasodilatation and increased permeability of ovarian vessels. These changes may maintain or increase intrafollicular pressure (IFP) at ovulation to cause rupture of the follicular wall. OBJECTIVE: to investigate the possible regulation of IFP during the ovulatory process. STUDY DESIGN: immature Sprague-Dawley rats were primed with pregnant mare serum gonadotrophin (PMSG; 10IU) and given hCG (10IU) 48h later. The ovary was exposed 48-60h after PMSG, micropipette inserted into the Graafian follicle and the IFP measured at three time periods: preovulatory (PO) 48h after PMSG; midovulatory (MO) 4-7h after hCG; late ovulatory (LO) 9-12h after hCG. The offset of the nitric oxide synthase (NOS) inhibitor L-arginine methyl ester (L-NAME), the alpha(1)-adrenoceptor agonist phenylephrine and the beta-adrenoceptor agonist isoprenaline were tested. RESULTS: phenylephrine given i.v. increased the systemic blood pressure, and significantly decreased the IFP in the LO phase (78% of pre-treatment value). Local administration of phenylephrine or isoprenaline (1ml of 1.5-15 microM) by superfusion over the ovary did not change the IFP. Local administration of L-NAME (1ml of 2 microM) significantly lowered (P<0.05) the IFP in the MO and LO phases, but was without effect in the PO phase. CONCLUSION: this study reveals that IFP regulation may be related to changes of the systemic blood pressure and that NO may be one local ovarian mediator in IFP regulation.     

Involvement of plasminogen activator activity in the process of ovulation

The present study was undertaken to determine the changes in follicular plasminogen activator (PA) activity during ovulation. In the first experiment, hCG (100 IU) administration in vivo enhanced PA activity in mature follicles within 1 hour. PA activity reached its maximum at 4 hours and then declined. A second peak occurred 8 hours after hCG administration. In the second experiment, the effects of an ovulatory dose (50 IU) of hCG on ovulatory efficiency and PA activity were assessed in mature and immature follicles with an in vitro perfused rabbit ovary preparation. Ovulatory efficiency in immature follicles was significantly less (p less than 0.01) (16.7 +/- 4.5%) than that in mature follicles (86.7 +/- 11.0%). During the perfusion, the growth of mature follicles was observed but not that of immature follicles. PA activity in mature follicles derived from unstimulated ovaries was 1.43 +/- 0.17 IU/g tissue. PA activity in mature follicles (28.4 +/- 4.2 IU/g tissue) 4 hours after in vitro exposure to hCG was significantly greater (p less than 0.01) than that found in immature follicles. In conclusion, exposure to gonadotropin stimulates PA activity in preovulatory follicles. Failure to increase PA activity in immature follicles impairs follicular development, resulting in reduced ovulatory efficiency. The increase in PA activity observed in ovulatory follicles implicates the plasmin-generating system in follicle rupture.     

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.     

Ablation of folliculogenesis in women by a single dose of gonadotropin-releasing hormone agonist: significance of time in cycle

Effects of single subcutaneous doses (1, 5, 20, and 100 micrograms) of nafarelin, a potent gonadotropin-releasing hormone agonist, on the physiologic events of the human menstrual cycle were studied in 28 normal women. Nafarelin entered the circulation rapidly after injection. Peak concentrations were observed within 1 hour, and the plasma half-life was 4 to 5 hours. Maximal concentrations of luteinizing hormone and follicle-stimulating hormone were reached 3 to 4 hours after nafarelin administration. The magnitude of the gonadotropin responses depended both on the phase of the menstrual cycle (smallest responses during the early follicular phase) and the dose of nafarelin. Nafarelin administration during the early follicular phase delayed ovulation by 4.6 +/- 1.7 (standard deviation) days and prolonged the duration of the menstrual cycle from a pretreatment length of 29.2 +/- 2.1 days to 33.4 +/- 4.0 days (P less than 0.001). When nafarelin was administered shortly before or after ovulation, cycle length was not altered consistently. Administration 5 to 10 days after ovulation resulted in a truncated luteal phase. These observations suggest that the hormonal events triggered by nafarelin during the early follicular phase temporarily arrest the process of selection of the dominant follicle. Repeated intermittent administration of nafarelin or other gonadotropin-releasing hormone agonists in the early follicular phase may prevent follicular maturation and ovulation and may be a practical approach to contraceptive development.     

The transperitoneal migration of ova in the rabbit

The purpose of this study was to assess the incidence and direction of the transperitoneal migration of ova in the rabbit. Sixteen New Zealand white rabbits underwent unilateral microsurgical enclosure of either the left (n = 9) or right (n = 7) ovary within a peritoneal bursa. Each animal was bred later to induce ovulation. The fallopian tubes were removed and flushed retrogadely to retrieve ova. The number of transmigrated ova retrieved from the oviduct ipsilateral to the ovary within the bursa was compared with the number of ovulated follicles present on the contralateral ovary. Three ova migrated to the left fallopian tube from a total of 49 ovulation sites on the right ovary. No ova transmigrated to the right oviduct from 33 ovulation sites on the left ovary. Chi-square analysis shows that there is no statistical difference between the tendency to right or left transmigration. This finding substantiates the numerous studies of reproductive physiology in the rabbit that have assumed previously the net transmigration of ova to be zero.     

Effect of the exposure of intrafollicular oocytes to clomiphene citrate on pregnancy outcome in the rabbit

This study was designed to determine if exposure of rabbit intrafollicular oocytes to clomiphene citrate (CC) affects pregnancy outcome after in vitro ovulation, in vitro fertilization (IVF), and embryo transfer (ET). Ovaries were perfused in the presence or absence of CC (10(-5) M) and estradiol (E2, 100 ng/ml). Human chorionic gonadotropin (hCG, 50 IU) was added to the perfusate of all ovaries. In vitro ovulated ova were retrieved, inseminated, and transferred to host rabbits. Neither CC nor CC + E2 significantly affected hCG-induced ovulation or fertilization. CC significantly reduced (P less than 0.025) percentage of ovulated ova resulting in offspring. Addition of E2 significantly (P less than 0.05) reversed the reduction in offspring associated with CC alone. These results may be due to the antiestrogenic effects of CC on the intrafollicular oocyte, which compromises postfertilization development.     

Effect of clomiphene citrate on in vitro ovulated ova

The effect of clomiphene citrate (CC) on developmental capacity of ovulated ova was studied with isolated in vitro perfused rabbit ovaries. Fifty-two ovulated ova were recovered from ovaries perfused with human chorionic gonadotropin (hCG) in a medium containing CC and 45 ova from ovaries perfused with hCG in a CC-free medium. Ova were cultured and inseminated with capacitated sperm and observed serially for evidence of fertilization and stage of development. CC did not affect the fertilization of ovulated ova. However, the percentage of ova which had reached the morula stage by 60 hours was significantly reduced in the CC-treated (15.4%) group, compared with the control group of ovaries (48.9%). A significant percentage of inseminated ova from CC-treated ovaries (65.4%) showed evidence of degeneration, as compared with control ovaries (37.8%). Thus, a partial loss of developmental capacity may explain the discrepancy observed between the rate of successful ovulation induction and the establishment of pregnancy associated with CC administration in humans.     

Cyclic changes of the functional ovarian compartments: echographic assessment

Using bidimensional echography, 25 normal ovulatory cycles were evaluated. The following parameters were assessed: 1) ovarian volume, 2) volume of the dominant follicle, 3) corpus luteum volume, 4) residual follicular volume, and 5) stromal volume. Ovarian volume and dominant follicle volumes reached their maximum on day -1: 11.68 +/- 2.87 ml and 3.60 +/- 1.20 ml respectively. The maximum stromal volume was seen during the follicular phase: 7.98 +/- 2.29 ml. In the dominant ovary the maximum corpus luteum volume was observed on day +1 and the maximum stromal volume on day +7 (8.64 +/- 2.53 ml). In the contralateral ovary, the stromal volume did not show significant changes. The residual follicular volume in both ovaries diminished gradually from the early follicular phase except for a slight postovulatory rise. In this study, normal values during the ovulatory cycle were obtained as indicated above. The use of bidimensional echography in the diagnosis of functional disorders of the ovary is stressed.     

Placental prostacyclin production in normal and toxemic pregnancies

Prostacyclin is a potent vasodilator and inhibitor of platelet aggregation. Because toxemia is characterized by increased vasoconstriction frequently associated with increased platelet aggregation and reduced uteroplacental blood flow, a deficiency in prostacyclin production during pregnancy could contribute to the development of toxemia. Placentally produced prostacyclin could have both local effects on the uteroplacental vasculature and systemic effects because prostacyclin, unlike the other prostaglandins, is not extensively metabolized by the lungs. Fresh human term placentas were obtained immediately after delivery from 12 normal and 12 toxemic (blood pressure greater than or equal to 140/90 mm Hg, urinary protein greater than 0.3 gm/24 hours) pregnancies. Tissues (300 mg) were incubated in a sterile manner in 5 ml of Dulbecco's Modified Eagle's Medium for 48 hours at 37 degrees C with 95% oxygen and 5% carbon dioxide in a metabolic shaker. Samples were collected at 8, 20, 32, and 48 hours and analyzed for prostacyclin by radioimmunoassay of its stable metabolite, 6-keto-prostaglandin F1 alpha. Prostacyclin production was significantly decreased in toxemic placental tissue compared with normal placental tissue (2.72 +/- 0.49 versus 7.22 +/- 0.44 pg/mg/hr, mean +/- SE, p less than 0.01). In both normal and toxemic placentas, prostacyclin production was inhibited by indomethacin (5 or 50 mumol/L) and not affected (p greater than 0.10) by arachidonic acid (5 or 100 mumol/L). Lowering the oxygen concentration from 95% to 20% significantly (p less than 0.01) decreased prostacyclin production in normal but not toxemic placentas. Prostacyclin production rates in the amnion and chorion were not affected (p greater than 0.10) by toxemia. The amniotic and chorionic prostacyclin production rates were not different from each other (p greater than 0.10) and were only one seventh of the normal placental production rate. These data indicate that placental prostacyclin production is decreased in toxemia; therefore, this vasoactive prostaglandin may be involved in the causation and the associated hypertension and coagulation abnormalities of this disorder.     

Measurement of noradrenaline, dopamine and serotonin contents in follicular fluid of human graafian follicles after superovulation treatment.

We measured the noradrenaline (NA), serotonin (5-HT) and dopamine (DA) contents of 47 normally maturated and 16 cystically degenerated follicular fluid samples obtained from patients involved in the in vitro fertilization and gamete transfer program. The patients were given human menopausal gonadotropin (HMG), as a superovulation treatment, and 7,500 IU human chorionic gonadotropin (HCG) to induce ovulation 34-36 h prior to the follicular puncture done by laparoscope. The NA content of the normally developed follicles was 11.4 + 8.4 micrograms/100 ml on average. For cystically degenerated follicles, the following data were obtained: 1.1 + 0.7 micrograms/100 ml (p less than 0.001). 5-HT and DA contents in the preovulatory follicles are 14.3 +/- 8.9 and 19.3 +/- 8.2 micrograms/100 ml, respectively; at the same time, 5-HT and DA contents in the cystically degenerated follicles were 12.2 +/- 6.2 and 12.7 +/- 6.8 micrograms/100 ml, respectively. They suggest that the higher amount of NA in the follicular fluid might play an important role in the mechanism of ovulation, the regulation of postovulatory tubal motility and the release of progesterone from granulosa cells.     

In vitro studies of ovulation in the perfused rabbit ovary.

A system has been developed for the perfusion of the rabbit ovary in vitro. At laparotomy, the ovarian artery is cannulated and perfused with M 199 tissue culture medium containing insulin and heparin, then removed with its vascular pedicle intact. Perfusion at 37 degrees C is maintained by using a capillary oxygenator and Buchler roller pump. The functional integrity of the perfused ovary is confirmed by serial determinations of the perfusate pH, glucose and lactate concentrations, and by ovarian histology. This in vitro model was used to study the mechanism of ovulation. One group of isolated rabbits received human chorionic gonadotropin (50 IU, intravenously) and, 8 hours later, one ovary was removed and perfused; the contralateral ovary remained in situ, serving as an in vivo control. Serial observations for follicle development and rupture were made over the subsequent 7-hour interval. The occurrence of ovulation in vitro was documented by time-lapse photography. In each animal, comparisons made between the in vitro and in vivo ovary indicated that the rate and time of follicle maturation and ovulation were comparable. Ovulation occurred between 10 and 15 hours after administration of human chorionic gonadotropin in both preparations.     

Comparison of in vitro fertilization results in women with one or two ovaries

A study compared the in vitro fertilization results in women with one or two ovaries. Eighteen percent (23/125) of in vitro fertilization/embryo transfer cycles were in patients with one ovary. One-ovary patients averaged 4.2 follicles (greater than or equal to 10 mm) as compared to 7.9 in two-ovary patients, and significantly fewer ova were recovered from one-ovary patients (4.0 vs. 5.3). The total follicular volume was significantly less in one-ovary patients as compared to two-ovary patients, and the serum estradiol per follicle was less in one-ovary patients. However, the volume of the dominant follicle, the maximum serum estradiol levels, the number of ampules of Pergonal given and the ovum fertilization rate were not significantly different in one- and two-ovary patients. The mean number of embryos transferred was 3.9 +/- 1.9 in one-ovary patients and 4.5 +/- 1.8 in two-ovary patients (NS). Two pregnancies occurred in one-ovary patients (8.3% per laparoscopy) and 13 in two-ovary patients (12.9% per laparoscopy). The two one-ovary patients who achieved pregnancy behaved more like two-ovary patients in terms of peak serum estradiol levels and number of ova recovered.     

Anti-macrophage inhibitory factor antibody inhibits PMSG-hCG-induced follicular growth and ovulation in mice

Purpose : To investigate the effect of an anti-MIF antibody on PMSG-hCG-induced murine follicular growth and ovulation and to determine whether MIF plays an essential role in this process. Methods : Mice were primed with an intraperitoneal injection of pregnant mare serum gonadotropin (PMSG) and were treated with an anti-rat MIF antibody and human chorionic gonadotropin (hCG) to induce ovulation. After that, the ovulated ova were counted. The ovaries were studied using standard histological procedures. Results : Ovaries treated with the anti-MIF antibody showed reduced numbers of growing follicles surrounded by granulosa cells and theca cells with a little proliferation compared with the control. The average numbers of ova collected from mice treated with the anti-MIF antibody were reduced compared with those collected from control mice. Conclusions : Anti-MIF antibody inhibits the follicular growth and ovulation in mice, and MIF may play an important role in the inflammatory reactions during follicle growth and ovulation.