A role for lipoxygenase metabolites of arachidonic acid in porcine ovulation

Prostaglandins, products of arachidonic acid via the cyclooxygenase pathway, are essential to the porcine ovulatory process in that inhibition of their synthesis results in ovulation failure. Studies in the rat have shown that ovulation is also preceded by a rise in three ovarian hydroxyeicosatetraenoic acids, products of the lipoxygenase pathway, and inhibition of this pathway also inhibits ovulation. Experiments were designed, using a pregnant mare serum gonadotropin/human chorionic gonadotropin (hCG)-treated prepuberal gilt model, to measure pre-ovulatory changes in follicular fluid concentrations of 15-hydroxyeicosatetraenoic acid (15-HETE), and to compare the effects of indomethacin and nordihydroguaiaretic acid (NDGA) on ovulation in the pig and on 15-HETE and prostaglandin F₂α synthesis both in vivo and in vitro. Follicular fluid concentrations of 15-HETE were elevated significantly just prior to the expected time of ovulation (40 h after hCG). When indomethacin (10 mg) was injected into the ovarian stalk at 24 h after hCG, follicular fluid concentrations of both 15-HETE and prostaglandin F₂α were lower (P<0.01) than controls at 40 h and ovulation rate was suppressed (P<0.01). When NDGA (5 mg) was administered in the same manner, ovulation rate was suppressed (P<0.01), but the levels of 15-HETE and prostaglandin F₂α were not altered. Synthesis of 15-HETE by cultured granulosa and theca interna cells was reduced by the presence of NDGA (1 mg/ml), whereas indomethacin (100 ng/ml) lowered 15-HETE production in theca interna cells only. These results clearly demonstrate that indomethacin can block the lipoxygenase as well as the cyclooxygenase pathways, depending on the dose used, and suggest that lipoxygenase metabolites of arachidonic acid are involved in the ovulatory process in the pig.     

Effect of various agents on ovarian plasminogen activator activity during ovulation in pregnant mare's serum gonadotropin-primed immature rats

The plasminogen activator/plasmin synthetic substrate S-2251 was used to measure the effect of indomethacin, cycloheximide, colchicine, dexamethasone, tranexamic acid, and aprotinin on the elevation of ovarian plasminogen activator (PA) that normally occurs during ovulation in the rat. Young Wistar rats were weaned on the morning of Day 21, given 4.0 IU of pregnant mare's serum gonadotropin (PMSG) s.c. at 0800 h on Day 22, and given 10.0 IU of human chorionic gonadotropin (hCG) on Day 24. These animals normally began ovulating between 0000 and 0200 h on Day 25. The induced ovulation rate was 11.5 +/- 2.2 ova/rat, based on the number of ova in the oviducts of control animals at 0900 h on Day 25. In the controls, PA activity in extracts of homogenized ovaries increased 3-fold from 0.125 +/- 0.010 OD units just before the administration of hCG to 0.371 +/- 0.021 at 12 h after hCG, i.e., near the time of ovulation. Indomethacin, in doses of 0.1-1.0 mg/rat, inhibited ovulation but did not inhibit the normal increase in PA activity, whereas indomethacin at the high dose of 10.0 mg/rat inhibited both ovulation and PA activity. Cycloheximide, at a dose of 0.1 mg/rat, was given at 12 h before hCG, immediately after hCG, and at 9 h after hCG. This agent inhibited ovulation most effectively when given at 12 h before hCG, yet it inhibited PA activity most effectively when given immediately after or at 9 h after hCG. Colchicine, at a dose of 0.1 mg/rat, inhibited ovulation, but not PA activity, when it was given 1 h before hCG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gonadotropin uptake in genetic and irradiation models of ovarian tumorigenesis

Genetic and irradiation models of ovarian tumorigenesis were investigated for evidence that elevated gonadotropins have a role in tumorigenesis. Wx/Wv mice lack oocytes at birth, develop complex mesothelial adenomas by 6 mo, and additional ovarian tumor types later. Uptake of iodinated human chorionic gonadotropin (125I-hCG) was measured in mice aged 1 to 30 mo, and uptake iodinated human follicle-stimulating hormone (125I-hFSH) was measured in mice aged 1 to 12 mo. Gonadotropin uptake by Wx/Wv ovaries in vivo declined quickly and was undetectable by 6 mo. Irradiated ovaries rapidly lost oocytes and follicular structures, formed mesothelial adenomas by 5 mo, and later formed additional types of ovarian tumors. In the irradiation model, 125I-hCG uptake also declined quickly and was undetectable by 3 mo of age. Neither the surface nor the tubular epithelium of the mesothelial adenoma were consistently labeled by 125I-hCG in autoradiography studies with either model. Although these data do not exclude an acute role for gonadotropins in initiation of preneoplastic events, they do indicate that ovarian cells do not require chronic gonadotropin stimulation during subsequent tumorigenesis. These findings are discussed in relation to additional models of ovarian tumorigenesis.     

Vascular endothelial growth factor production in growing pig antral follicles

Angiogenesis is the process that drives blood vessel development in growing tissues in response to the local production of angiogenic factors. With the present research the authors have studied vascular endothelial growth factor (VEGF) production in ovarian follicles as a potential mechanism of ovarian activity regulation. Prepubertal gilts were treated with 1250 IU equine chorionic gonadotropin (eCG) followed 60 h later by 750 IU of human chorionic gonadotropin (hCG) in order to induce follicle growth and ovulation. Ovaries were collected at different times of the treatment and single follicles were isolated and classified according to their diameter as small (<4 mm), medium (4-5 mm), or large (>5 mm). VEGF levels were measured in follicular fluid by enzyme immunoassay, and VEGF mRNA content was evaluated in isolated theca and granulosa compartments. Equine chorionic gonadotropin stimulated a prompt follicular growth and induced a parallel evident rise in VEGF levels in follicular fluid of medium and large follicles. Analysis of VEGF mRNA levels confirmed the stimulatory effect of eCG, showing that it is confined to granulosa cells, whereas theca cells maintained their VEGF steady state mRNA. Administration of hCG 60 h after eCG caused a dramatic drop in follicular fluid VEGF that reached undetectable levels in 36 h. A parallel reduction in VEGF mRNA expression was recorded in granulosa cells. The stimulating effect of eCG was also confirmed by in vitro experiments, provided that follicles in toto were used, whereas isolated follicle cells did not respond to this hormonal stimulation. Consistent with the observation in vivo, granulosa cells in culture reacted to hCG with a clear block of VEGF production. These results demonstrate that while follicles of untreated animals produce stable and low levels of the angiogenic factor, VEGF markedly rose in medium and large follicles after eCG administration. The increasing levels, essentially attributable to granulosa cells, are likely to be involved in blood vessel development in the wall of growing follicles, and may play a local key role in gonadotropin-induced follicle development. When ovulation approaches, under the effect of hCG, the production of VEGF is switched off, probably creating the safest conditions for the rupture of the follicle wall while theca cells maintained unaltered angiogenic activity, which is probably required for corpus luteum development.     

Localization of relaxin in the pig follicle during preovulatory development

The avidin-biotin immunoperoxidase method and antisera to purified porcine relaxin were used to localize relaxin in sections of follicles from pregnant mare's serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG)-primed pigs during preovulatory development. Prepubertal pigs were treated i.m. with PMSG (750 IU) and 72 h later with hCG (500 IU) to induce follicular development and ovulation. Follicles were collected from untreated gilts or from gilts 24, 48, 60, 72, 84, 96, or 108 h after PMSG treatment. Light immunostaining in the theca interna was observed early in follicular development, at 48 and 60 h post-PMSG. At 72 h post-PMSG, relaxin immunostaining in the theca interna of the preovulatory follicle was more intense. After hCG treatment, the intense thecal immunostaining persisted and was apparent 84 and 96 h after PMSG. At about 6 h prior to expected ovulation (108 h post-PMSG), there was thinning of the follicle wall and a reduction in relaxin immunostaining in the theca interna. Immunoactive relaxin was not detected in follicles from untreated gilts, follicles 24 h post-PMSG, small healthy or atretic follicles, or in granulosa cells, theca externa or ovarian stroma, at any of the time points studied. These studies support the hypothesis that the theca interna is the primary source of follicular relaxin and provide further evidence for a paracrine role for relaxin in the ovulatory process.     

Morphological analyses of interleukin-8 effects on rat ovarian follicles at ovulation and luteinization in vivo

The aim of the present study was to elucidate functions of the interleukin (IL)-8 at ovulation and luteinization in vivo. To compare the morphological differences between human chorionic gonadotropin (hCG) and IL-8 stimulation, scanning electron microscopy was employed to study rat ovarian vascular corrosion casts. Follicular growth and increased capillary vessel densities around the follicles were seen in vascular corrosion casts after IL-8 injection, similar to the result of hCG administration. This result indicated that exogenous IL-8 could play a role in the neovascularization during follicular development as an angiogenetic factor. Many fenestrations were observed in the vascular endothelium by hCG administration. In contrast, no fenestrations were observed with IL-8 injection, indicating that IL-8 may not be sufficient to increase the vascular permeability directly. Although germinal vesicle breakdown (GVBD) occurred at rates of 82% after the hCG injection, only 20% GVBD was observed after the IL-8 injection. The present study indicated that IL-8 might have important effects on rat follicles at ovulation and luteinization via vascularization in a similar manner to hCG. However, IL-8 was not effective on vascular permeability and oocyte maturation, which were different from hCG. Thus, we can conclude that IL-8 can participate in follicular development in part and may play important roles in ovulation and luteinization as one of some mediators induced by endogenous luteinizing hormone.     

Progesterone and estrogens in the pregnant and nonpregnant dolphin, Tursiops truncatus, and the effects of induced ovulation

Baseline serum levels of progesterone and total immunoreactive estrogens were determined for intact and ovariectomized captive female Atlantic bottlenose dolphins (Tursiops truncatus), as well as newly captured wild adult females. Stimulation of ovarian follicular growth and ovulation was attempted by intramuscular injection of pregnant mare's serum gonadotropin (PMSG). High doses of PMSG were required to increase serum estrogen levels. When PMSG was followed by an injection of human chorionic gonadotropin (hCG), ovulation was presumed to have occurred as indicated by subsequent high levels of serum progesterone. From these observations, it appears that 1) females with progesterone levels greater than 3000 pg/ml over an extended period are pregnant, 2) Tursiops truncatus is capable of spontaneous ovulation in captivity without gonadotropin therapy, 3) captive female dolphins, although relatively resistant to PMSG, can be induced to ovulate using a combination of high intramuscular-injected doses of PMSG followed by hCG, and 4) spontaneous ovulation is likely to follow an induced ovulation.     

Pre-ovulatory changes in cyclic AMP and prostaglandin concentrations in follicular fluid of gilts

The concentrations of cyclic adenosine 3′,5′-monophosphate (cyclic AMP) and prostaglandins E and F (PGE and PGF) were determined in follicular fluid collected from follicles of prepubertal gilts at various times after treatment with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) to induce ovulation. The concentrations of cyclic AMP, PGE and PGF in the follicular fluid after PMSG treatment but prior to hCG injection were about 1 pmol/ml, 1 ng/ml and 0.2 ng/ml, respectively. After hCG administration, the follicular fluid levels of cyclic AMP increased markedly, reaching a peak (400-fold increase) about 4 h after injection and then declined gradually to pre-hCG levels. A second rise (2.5- to 5-fold increase) occurred about 30 h after hCG with the levels being sustained up to the expected time of ovulation. In contrast, the levels of PGE and PGF remained relatively constant until 28–30 h after hCG treatment. Thereafter, the concentrations of both prostaglandins began to rise with the increases becoming more pronounced and reaching maximal values as the expected time of ovulation approached. These data provide further evidence for a physiological role of follicular prostaglandins in the process of ovulation but do not support an obligatory role for prostaglandins in the acute gonadotropin stimulation of cyclic AMP formation.     

Pre-ovulatory changes in cyclic AMP and prostaglandin concentrations in follicular fluid of gilts.

The concentrations of cyclic adenosine 3', 5'-monophosphate (cyclic AMP) and prostaglandins E and F (PGE and PGF) were determined in follicular fluid collected from follicles of prepubertal gilts at various times after treatment with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) to induced ovulation. The concentrations of cyclic AMP, PGE and PGF in the follicular fluid after PMSG treatment but prior to hCG injection were about 1 pmol/ml, 1 ng/ml and 0.2 ng/ml, respectively. After hCG administration, the follicular fluid levels of cyclic AMP increased markedly, reaching a peak (400-fold increase) about 4 h after injection and then declined gradually to pre-hCG levels. A second rise (2.5- to 5-fold increase) occurred about 30 h after hCG with the levels being sustained up to the expected time of ovulation. In contrast, the levels of PGE and PGF remained relatively constant until 28-30 h after hCG treatment. Thereafter, the concentrations of both prostaglandins began to rise with the increases becoming more pronounced and reaching maximal values as the expected time of ovulation approached. These data provide further evidence for a physiological role of follicular prostaglandins in the process of ovulation but do not support an obligatory role for prostaglandins in the acute gonadotropin stimulation of cyclic AMP formation.     

Induction of ovulation in gilts with cloprostenol

Prepuberal gilts were treated with 750 IU pregnant mare serum gonadotropin (PMSG) followed 72 h later by 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. In this model, ovulation occurred at 42 +/- 2 h post hCG treatment. When 500 mug of cloprostenol was injected at 34 and of 36 h after hCG injection, 78% of the preovulatory follicles ovulated by 38 h compared with 0% in the control gilts. In addition, plasma progesterone concentrations were significantly higher in the cloprostenol-treated group than in the control group (P<0.01) at 38 h, indicating luteinization along with premature ovulation. These results suggest that prostaglandin F(2)alpha (PGF(2)alpha) or an analog can be used to advance, synchronize or induce ovulation in gilts.     

Clinical use of human chorionic gonadotropin in dairy cows: An update

Human chorionic gonadotropin (hCG) has a potent luteinizing hormone (LH)-like effect in cattle that extends the life span of the corpus luteum (CL) and increases progesterone synthesis, induces ovulation throughout the estrous cycle, promotes the formation of accessory corpora lutea when applied in the early luteal phase, and modifies follicular wave dynamics increasing the frequency of three-wave dominant follicular cycles. As hCG acts on ovarian cells independently of the pituitary gland and its effect is longer lasting than that produced by endogenous LH release, use of hCG rather than gonadotropin-releasing hormone (GnRH) could be targeted at populations of subfertile cows. This review describes the clinical use of hCG to improve the reproductive performance of dairy cows. In addition, we describe recent developments in the therapeutic use of hCG and studies addressing the benefits of including hCG in estrus and ovulation synchronization protocols. Our review ends with a critical discussion of how earlier findings related to ovarian responses to hCG treatment can be interpreted in the light of recent advances in the clinical applications of hCG.     

Human chorionic gonadotropin administration is associated with high pregnancy rates during ovarian stimulation and timed intercourse or intrauterine insemination

Background  

There are different factors that influence treatment outcome after ovarian stimulation and timed-intercourse or intrauterine insemination (IUI). After patient age, it has been suggested that timing of insemination in relation to ovulation is probably the most important variable affecting the success of treatment. The objective of this study is to study the value of human chorionic gonadotropin (hCG) administration and occurrence of luteinizing hormone (LH) surge in timing insemination on the treatment outcome after follicular monitoring with timed-intercourse or intrauterine insemination, with or without ovarian stimulation.     

Increase in ovarian blood volume during ovulation in the gonadotropin-primed immature rat

This study quantifies ovarian blood volume in Wistar rats by measuring the optical density (414 nm) of hemoglobin in ovarian extracts and comparing this measurement to the optical density of known amounts of whole blood. Immature rats were primed with pregnant mare's serum gonadotropin (PMSG), 10 IU s.c., at 23 days of age. On Day 25, the ovulatory process was initiated by human chorionic gonadotropin (hCG), 10 IU s.c., and ova began to appear in the oviducts 10 h later. At 2-h intervals, the ovaries were extirpated and homogenized in 1.0 ml of 0.05 M tris (hydroxymethyl)aminomethane buffer (pH 7.4) for 30 s. Homogenates were centrifuged for 20 min and the supernatant fluids were analyzed with a Gilford RESPONSE UV/VIS spectrophotometer. The hemoglobin in these ovarian extracts had the same peak absorbance of 414 nm characteristic of oxyhemoglobin in whole blood taken by cardiac puncture of the rats. There was a linear relationship between the absorbance and the volume of whole blood in the samples. The volume of blood per ovary from groups of 8 rats was 0.60 +/- 0.07 microL at 0 h after hCG. The volume increased to 1.37 +/- 0.26 microL at 4 h after hCG and reached a peak of 4.55 +/- 0.72 microL at 10 h. Indomethacin treatment (0.3-10.0 mg/rat, s.c.) partially inhibited this 7-fold increase in ovarian blood volume. In conclusion, the increase in ovarian blood volume during ovulation may reflect the vasodilation and hyperemia that are characteristic of inflamed tissues.     

Localized increases in ovarian vascular permeability and leucocyte accumulation after induced ovulation in rabbits.

Colloidal carbon was injected i.v. in mature virgin rabbits at different times after induction of ovulation by human chorionic gonadotrophin (hCG, 100 iu) or mating. Before induction of ovulation, slight carbon leakage was observed in the inner vascular ring of the theca interna of antral follicles, but blood vessels in the other ovarian compartments were unstained. Between 4 and 10.5 h after hCG-treatment or mating, vascular leakage was most marked in the blood vessels of the interstitial gland and in the theca interna of antral follicles. Just before ovulation, carbon particles were observed between granulosa cells and some carbon was seeping into the follicular fluid of preruptured follicles. Vascular leakage was also observed over the follicle dome before rupture as well as at the dorsomedial junction between the mesovarium and the ovary. The blood vessels stained with carbon were 7-70 microns diameter, representing capillaries and postcapillary venules. About 6 h after hCG injection, an increased number of polymorphonuclear leucocytes migrated from the vessels of these ovarian compartments into the surrounding interstitial tissue. The number of leucocytes seen in the follicular wall and ovarian medulla increased markedly towards ovulation. During early corpus luteum formation, the number of leucocytes decreased markedly. The localized vascular changes seen after mating and hCG stimulation were similar to an inflammatory reaction and could form the basis for the formation of peritoneal exudate after ovulation in rabbits and periovulatory ascitic accumulation seen in the peritoneal cavity of women during the menstrual cycle.     

Ovarian follicular fluid eicosanoid concentrations during the pre-ovulatory period in humans

Prostaglandins are involved in ovulation and in every mammal studied so far, ovulation has been inhibited by prostaglandin inhibition. Information regarding the role of leukotrienes and thromboxanes in ovulation is more limited. In order to study the production of eicosanoids in human pre-ovulatory follicular fluid, follicular aspiration was timed by means of serial ultrasound scans and human chorionic gonadotrophin (hCG) to be immediately pre-ovulatory. 11 women were studied and the eicosanoids measured by radioimmunoassay (RIA). The follicular fluid was found to contain leukotrienes (LT) B4, LTC4 (D4, E4), prostaglandin (PG) E2, PGF2 alpha 6 keto PGF1 alpha k and thromboxane (TX) B2. This is the first published report of leukotrienes in human follicular fluid in spontaneous cycles, and is one of the few reports showing prostaglandins and thromboxanes. The significance of demonstrating leukotrienes in human follicular fluid is discussed as is the correlation between individual eicosanoids in the human ovary.     

Inhibition of human chorionic gonadotropin-induced ovulation and steroidogenesis by short-term hyperprolactinemia in female rabbits

To clarify the possible direct effects of hyperprolactinemia on the ovulatory process, we experimentally established hyperprolactinemia in female rabbits with 4 daily injections of sulpiride (SLP) at different doses and induced ovulation with human chorionic gonadotropin (hCG). Plasma levels of prolactin (PRL) were increased significantly before hCG injection in each SLP-treated group compared with the corresponding values for the controls. The ovulation rates at 14 h after hCG were significantly reduced in the 16 and 24 mg/kg/day SLP-treated groups. An inverse correlation (r = -0.74, P less than 0.001) was found between the ovulation rate and the increasing in plasma PRL measured just prior to hCG injection. The increase in peripheral as well as ovarian venous progesterone and 20 alpha-hydroxypregn-4-en-3-one(20 alpha-OHP) at 4 and 14 h after hCG injection in inhibited ovulation groups was much less than in the control group. However, the estradiol, androstenedione and testosterone concentrations were comparable with the control values. These results indicate that hypersecretion of PRL induced by SLP has a direct effect on ovary by inhibiting follicular rupture induced by hCG and this inhibitory effect was partly due to the suppression of progesterone secretion during the course of ovulation. This may be one of the causes leading to hypogonadism during hyperprolactinemia.     

Granulosa cell proliferation is inhibited by PGE2 in the primate ovulatory follicle

ABSTRACT

Prostaglandin E2 (PGE2) is a key paracrine mediator of ovulation. Few specific PGE2-regulated gene products have been identified, so we hypothesized that PGE2 may regulate the expression and/or activity of a network of proteins to promote ovulation. To test this concept, Ingenuity Pathway Analysis (IPA) was used to predict PGE2-regulated functionalities in the primate ovulatory follicle. Cynomolgus macaques underwent ovarian stimulation. Follicular granulosa cells were obtained before (0 h) or 36 h after an ovulatory dose of human chorionic gonadotropin (hCG), with ovulation anticipated 37–40 h after hCG. Granulosa cells were obtained from additional monkeys 36 h after treatment with hCG and the PTGS2 inhibitor celecoxib, which significantly reduced hCG-stimulated follicular prostaglandin synthesis. Granulosa cell RNA expression was determined by microarray and analyzed using IPA. No granulosa cell mRNAs were identified as being significantly up-regulated or down-regulated by hCG?+?celecoxib compared with hCG only. However, IPA predicted that prostaglandin depletion significantly regulated several functional pathways. Cell cycle/cell proliferation was selected for further study because decreased granulosa cell proliferation is known to be necessary for ovulation and formation of a fully-functional corpus luteum. Prospective in vivo and in vitro experiments confirmed the prediction that hCG-stimulated cessation of granulosa cell proliferation is mediated via PGE2. Our studies indicate that PGE2 provides critical regulation of granulosa cell proliferation through mechanisms that do not involve significant regulation of mRNA levels of key cell cycle regulators. Pathway analysis correctly predicted that PGE2 serves as a paracrine mediator of this important transition in ovarian structure and function.     

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.     

Preovulatory changes in the perifollicular capillary network in the rat: role of eicosanoids.

The aim of this study was to evaluate morphometrically the influence of ovulation-inhibiting doses of indomethacin, an inhibitor of the cyclooxygenase pathway, and esculetin and caffeic acid, inhibitors of the lipoxygenase pathway, on the dilatation of the perifollicular capillary network in the theca interna. The development of the perifollicular capillary network as a function of follicular size and the changes in the vascular lumen were examined by light microscopy on a series of semithin cross sections of rat ovaries. The number of capillaries in the theca interna increased linearly with increasing follicle diameter. Thus, the relative number of capillaries in the theca interna supplying the avascular stratum granulosum remained constant. This indicates that follicular function is not regulated through changes in the number of capillaries in the theca interna. After hCG injection, an increase in the capillary area could be observed in follicles having a diameter of more than 600 microns. Indomethacin administration increased the capillary area of the ovulatory follicles as compared to the untreated side only at 6 h after treatment. By contrast, treatment with inhibitors of lipoxygenase resulted in a significant decrease in the capillary area of large follicles at all times examined (3, 6, and 9 h after hCG injection). Nevertheless, since both types of eicosanoid inhibitors suppressed follicle rupture, in spite of their opposing actions on the capillary area, it seems unlikely that their action on ovulation is primarily due to their effect on this parameter.     

GnRH agonist Lupron (leuprolide acetate) pre-treatments prevent ovulation in response to gonadotropin stimulation in the clouded leopard (Neofelis nebulosa)

In many species, controlling the ovary prior to induction of ovulation improves the success of ovarian response and artificial insemination (AI). We assessed the impact of suppression of estrus with the GnRH agonist, Lupron, on ovarian sensitivity to equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) in the clouded leopard. Seven female clouded leopards were given two injections of Lupron (3.75 mg IM) 23 d apart, followed 44 d later by eCG and hCG. Daily fecal samples were collected from 60 d before Lupron to 60 d after hCG. Fecal metabolites of estrogen (E) and progesterone (P) were measured by radioimmunoassay. Lupron decreased (P < 0.05) the number of E peaks during Lupron treatment compared to pre-Lupron. All females had baseline E and six of seven (86%) had nadir P on day of eCG. Exogenous gonadotropins induced E elevations in all females. However, mean E in the gonadotropin-provoked estrus was decreased (P < 0.05) compared to pre-Lupron estrous periods. Only one of seven (14%) females ovulated after eCG/hCG. In conclusion, estrous cycle control with Lupron resulted in predictable ovarian suppression prior to gonadotropin stimulation but altered ovarian sensitivity by an as yet unknown mechanism so that ovulation was inhibited, even when using a proven exogenous gonadotropin protocol.