Effects of the antiandrogen hydroxyflutamide on progesterone secretion by preovulatory rat follicles in vivo and in vitro.

Serum and ovarian progesterone levels and in vitro production of progesterone by preovulatory follicles were measured on proestrus in pregnant mare's serum gonadotropin (PMSG) primed immature rats in which the luteinizing hormone (LH) surge and ovulation were blocked by administration of the antiandrogen hydroxyflutamide. Serum progesterone levels observed at 12:00 on proestrus were significantly elevated, twofold above those observed in vehicle-treated controls, by in vivo administration of 5 mg hydroxyflutamide 4 h earlier. In control rats, proestrous progesterone did not increase until 16:00, in parallel with rising LH levels of the LH surge. No LH surge occurred in the hydroxyflutamide-treated rats, ovulation was blocked, and serum progesterone declined throughout the afternoon of proestrus, from the elevated levels present at 12:00. Administration of human chorionic gonadotropin (hCG) at 11:00 advanced the elevation of serum progesterone by 2 h in vehicle-treated controls and prevented the decline in progesterone levels in hydroxyflutamide-treated rats. The patterns of change in ovarian tissue concentrations with time and treatment were essentially similar to those observed for serum progesterone. In in vitro experiments, progesterone secretion during 24 h culture of preovulatory follicles obtained on PMSG-induced proestrus was significantly increased, sixfold, by addition to the culture media of 370 microM but not of 37 microM hydroxyflutamide. Testosterone (50 nM) and hCG (20 mIU/mL) caused 26- and 14-fold increases, respectively, in progesterone secretion by cultured follicles. Hydroxyflutamide significantly reduced the stimulatory effect of testosterone but not of hCG on progesterone secretion in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ability of progesterone to reverse anti-androgen (hydroxyflutamide)-induced interference with the preovulatory LH surge and ovulation in PMSG-primed immature rats

In Exp. 1, PMSG was injected to 26-day-old prepubertal rats to induce ovulations. On Day 2 (2 days later, the equivalent of the day of pro-oestrus) they received at 08:00 h 5 mg hydroxyflutamide or vehicle and at 12:00 h 2 mg progesterone or testosterone or vehicle. Animals were killed at 18:00 h on Day 2 or at 09:00 h on Day 3. Progesterone but not testosterone restored the preovulatory LH surge and ovulation in hydroxyflutamide-treated rats. In Exp. 2, 2 mg progesterone or testosterone were injected between 10:30 and 11:00 h on Day 2, to advance the pro-oestrous LH surge and ovulation in PMSG-primed prepubertal rats. Injection of hydroxyflutamide abolished the ability of progesterone to advance the LH surge or ovulation. Testosterone did not induce the advancement of LH surge or ovulation. In Exp. 3, ovariectomized prepubertal rats implanted with oestradiol-17 beta showed significantly (P less than 0.01) elevated serum LH concentrations at 18:00 h over those observed at 10:00 h. Progesterone injection to these animals further elevated the serum LH concentrations at 18:00 h, in a dose-dependent manner, with maximal values resulting from 1 mg progesterone. Hydroxyflutamide treatment significantly (P less than 0.003) reduced the serum LH values in rats receiving 0-1 mg progesterone but 2 mg progesterone were able to overcome this inhibition. It is concluded that progesterone but not testosterone can reverse the effects of hydroxyflutamide on the preovulatory LH surge and ovulation. It appears that hydroxyflutamide may interfere with progesterone action in induction of the LH surge, suggesting a hitherto undescribed anti-progestagenic action of hydroxyflutamide.     

A secondary surge of prolactin on the estrus afternoon

It has been described that throughout the estrous cycle of the rat, plasma prolactin (PRL) is basal except on proestrus afternoon when a preovulatory surge occurs. However, there have been controversies about PRL levels on the estrus day. Thus, the aim of this study was to evaluate the existence of a secondary surge of PRL on estrus afternoon and correlate it with plasma estradiol levels. The jugular vein of cycling rats was cannulated at 14:00 h on proestrus and a blood sample was withdrawn at 17:00 h for plasma LH measurement and determination of the preovulatory LH surge occurrence. In order to exclude the regular cycling rats that do not present the gonadotropins preovulatory surge and do not ovulate, only rats showing the LH surge on proestrus were considered in this study. Blood samples were collected hourly during estrus from midnight to 9:00 h (group 1) and from 10:00 to 18:00 h (group 2). In group 1, PRL showed a descending profile from midnight to 9:00 h, whereas the estradiol concentrations were constant. In group 2, a secondary surge of PRL was observed in 20 of 25 (80%) rats and plasma estradiol remained constant, but was higher in animals with the PRL surge. Thus the present data suggest the occurrence of a secondary surge of PRL in the afternoon of estrus that seems to be related to plasma estradiol levels of estrus day, which might exert only a permissive role in this surge generation.     

Does corpus luteum function autonomously during estrous cycle in the rat? A possible involvement of LH and prolactin

This study examined the of LH and prolactin in the control of corpus luteum function during 4-day cycles in the rat. Bromocriptine (BRC) treatment was performed on proestrus or/and estrus morning that means before or after the preovulatory release of LH. This caused complete blood prolactin depression from the time of injection until diestrus 1 afternoon. This decrease in blood prolactin concentration was associated with a rise in the tonic level of LH secretion in those females which received BRC as soon as on proestrus. We first observed that injection on the morning of proestrus of doses of BRC capable of blunting prolactin secretion on proestrus afternoon did not significantly impair the preovulatory release of LH and did not prevent ovulation occurring during the following night. The life span of the corpora lutea edified from ovarian follicles rupturing before or under BRC administration did not exceed that of those formed under physiological circumstances since 4-day cycles culminating in ovulation constantly took place in all the treated animals whatever the time of BRC injection. To determine the pattern of luteal activity in the absence of prolactin secretion, we measured blood progesterone concentration from estrus until late diestrus in female rats injected with BRC on proestrus and/or estrus at 1100 h. The initiation of the function of corpus luteum on estrus and the achievement of its full activity on diestrus 1 did not appear to be affected by BRC. By contrast the level of blood progesterone declined more rapidly on the morning of diestrus 2 in BRC-treated than in control females. The capacity for autonomous progesterone secretion by corpus luteum of the cycle was discussed in the light of previous and present observations.     

Role of progesterone in the modulation of the preovulatory surge of gonadotropins and ovulation in the pregnant mare's serum gonadotropin-primed immature rat and the adult rat

The role of progesterone in the regulation of the preovulatory surge in gonadotropins and ovulation was examined in this study by use of a potent antagonist of progesterone, RU 486 (17 beta-hydroxy-11 beta-[4-dimethyl-aminophenyl]-17 alpha- [prop-1-ynyl]estra-4,9-diene-3-one). The immature rat primed with pregnant mare's serum gonadotropin (PMSG) and the cycling adult animal were the models used to verify the role of progesterone. When RU 486 (200 micrograms/rat) was given as a single dose on the morning of proestrus, there was a significant reduction in the preovulatory surge levels of gonadotropins and ovulation in both animal models. Serum progesterone levels in both models at the time of death on the evening of proestrus were unaltered upon treatment with RU 486. RU 486 did not have any effect on gonadotropin levels in immature rats 7 days after castration. These results show that the actin of RU 486 on the preovulatory gonadotropin surge is due to an antagonism of the action of progesterone on the hypothalamic-pituitary axis. Thus, a role for progesterone in modulating the preovulatory surge of gonadotropins and, consequently, ovulation is strongly suggested.     

Patterns of circulating gonadotropins and ovarian steroids during the first periovulatory period in the developing sheep.

This report provides evidence that an increment in serum gonadotropin levels occurs at puberty in the sheep and that this reflects the critical hormonal event culminating in first ovulation in this species. Blood samples were collected from 6 female lambs at 4-h intervals for a period of approximately 2 mo around the expected time of puberty (32 wk of age) until behavioral estrus was observed and ovulation was verified by assay of serum progesterone. Patterns of circulating LH, FSH, progesterone, and estradiol concentrations were characterized during the peripubertal period for each lamb. A rise in serum levels of both LH and FSH began approximately 7-10 days before the first preovulatory surge of gonadotropins. Although the increase in gonadotropin levels occurred gradually over several days, serum estradiol levels rose only during the final 40-60 h prior to the preovulatory surge of gonadotropin. Serum progesterone profiles revealed, however, that normal (14-16-day) luteal phases were induced in only 2 of 6 females as a result of the first surge. In four lambs, a short luteal phase of 2.5 days' duration occurred, which was followed by another estradiol rise and a preovulatory surge that then resulted in a full luteal phase of 14 days' duration. These data demonstrate clearly that the precipitating event at puberty in the female sheep is an increase in circulating gonadotropin levels and that the estradiol secreted from the newly stimulated follicle provides the signal for the first preovulatory surge.     

Human chorionic gonadotropin and luteinizing hormone-releasing hormone reverse the blockade of ovulation in pregnant mare's serum gonadotropin-primed immature rats by the anti-androgenic drug, hydroxyflutamide

The present study was designed to examine mechanism(s) of the anti-ovulatory action of the anti-androgen, hydroxyflutamide (OH-F). Prepubertal rats were treated with 4 IU pregnant mare's serum gonadotropin (PMSG) (day -2) to induce first estrus and ovulation. They received OH-F in sesame oil or oil alone at 08:00 and 20:00 h on day 0 (the day of proestrus) and ovulations were assessed on the morning of day 1. Eighty-three percent of control animals ovulated with a mean of 7.7 +/- 1.1 corpora lutea per rat. Hydroxyflutamide blocked ovulation in all but 2 of the 12 rats receiving this drug alone. All of OH-F treated rats that received 5 and 25 IU human chorionic gonadotropin (hCG) ovulated with means +/- SEM of 9.1 +/- 0.1 and 7.3 +/- 1.4 corpora lutea per rat, respectively. The dose of 0.2 IU hCG was essentially ineffective, while the effect of 1.0 IU hCG was intermediate. At the dose of 20 ng and above (100 and 500 ng) luteining hormone-releasing hormone (LHRH) completely overcame the ovulation blockade in the OH-F treated animals, while a 4-ng dose was ineffective. At 18:00 h on the day of proestrus, serum LH levels in control animals were 17.56 +/- 2.60 ng/mL, which were 920% above basal levels (1.90 +/- 0.13) indicating a spontaneous LH surge. This surge was suppressed in OH-F treated rats. Injection of LHRH, at the dose of 20 ng and above, reinstated the LH release in OH-F treated animals. Thus, the anti-androgen, OH-F, inhibits ovulation in PMSG-treated immature rats through its interference with the preovulatory LH surge; the inhibition can be reversed by hCG or LHRH. Hydroxyflutamide does not appear to interfere at the level of the pituitary, but may have direct action at the hypothalamic and (or) extrahypothalamic sites involved in the generation of positive feedback signals that control LH release.     

Follicle population, cumulus mucification, and oocyte chromatin configuration during the periovulatory period in the female dog

This study was designed to describe the follicular population present on the canine ovary (Canis familiaris) during the preovulatory period and essentially the changes in oocyte size, mucification, and chromatin configuration occurring from before the luteinizing hormone (LH) surge up to postovulation. In a first experiment, ovaries of beagle bitches were collected before (n = 21) or after LH surge but before ovulation (post-LH surge/preovulation stage, n = 24) as determined using hormone (LH, estradiol, progesterone) assays and ultrasonography. All large (>2 mm) follicles were measured and punctured. The numbers of oocytes collected per follicle and the degree of cumulus mucification were recorded. In a second experiment, ovaries were similarly collected before (n = 13) and after the LH surge but before ovulation (n = 11) as well as after ovulation as determined by ultrasonography (n = 9). Chromatin configuration of the oocytes was observed by DNA staining and confocal microscopy. In Experiment 1, before the LH peak, an average of 13.5 ± 0.7 follicles per bitch (total 284 follicles) were detected, and the maximal follicle diameter reached 6.5 mm. Large follicles were observed already in this period of the cycle and as early as when progesterone was still below 0.5 ng/mL. After the LH peak but before ovulation, 11.0 ± 0.7 follicles were present (total 264 follicles). Fully mucified cumulus cells were observed only in follicles larger than 4 mm. Multi-oocytic follicles represented 7% (before LH peak) and 4% (after LH peak) of the follicular population. In Experiment 2, all the oocytes were at the germinal vesicle (GV) stage, but three chromatin configurations could be distinguished: diffuse, partly grouped, and fully grouped chromatin. The proportion of oocytes with fully grouped chromatin increased with the follicular diameter and the time in estrus, the maximum being observed after the LH peak. These results suggest that (1) before LH peak, follicles are already of large diameter, similar to the ones at ovulation; (2) the ability for cumulus mucification is acquired during the late steps of follicular growth; (3) three GV patterns may be observed during the periovulatory period.     

Preovulatory secretion of progesterone, luteinizing hormone, and prolactin in 4-day and 5-day cycling rats

Timing of ovulation and changes in plasma progesterone, luteinizing hormone (LH), and prolactin (PRL) during periovulatory stages were determined in Holtzman rats exhibiting regular 4- or 5-day cycles under a daily artificial illumination from 0500 to 1900 h. The 5-day cycling rats ovulated between 0130 and 0930 h on estrus, whereas some of the 4-day cycling animals ovulated as early as about 0130 h and others as late as 1130 h on estrus. Onset time of preovulatory LH and progesterone surges was about 1500 h on proestrus in both the 4- and the 5-day cycling rats. Peak levels of plasma LH and progesterone were measured at 1700 to 1900 h on proestrus, while the first rises and peak values of plasma PRL were evident a few hours earlier than those of plasma LH in the rats with two cycle lengths. Plasma LH levels at 1900 h on proestrus as well as plasma progesterone levels at 1600 and 2300 h on proestrus and at 0130 and 0330 h on estrus were significantly lower in the 5-day cycling rats than in the 4-day cycling animals (p less than 0.05). In contrast, PRL levels from 1500 through 2300 h on proestrus remained consistently higher in 5-day cycling rats than in 4-day cycling rats, and significant differences in PRL levels between these rats were apparent at 1500, 1600, and 2100 h (p less than 0.05-0.01). Thus, these results demonstrate that the 5-day cycling rats exhibit the attenuated magnitude of LH surge accompanied by the augmented preovulatory PRL release, and that plasma progesterone levels reflect the magnitude of LH surge. A tentative working hypothesis concerning the etiology of the 5-day cycle has been proposed.     

Daily variations in the sensitivity of proestrous LH surge in the inhibitory effect of intraventricular injection of 5-HT or GABA in rats.

Intraventricular injection of 5-hydroxytryptamine (5-HT) into female rats at 11:00 h on the day of proestrus inhibited the preovulatory surge of luteinizing hormone (LH) and ovulation. A similar response was observed after the activation of the serotonergic system by stimulation of the median raphe nucleus. A diurnal rhythm of these responses was observed. In rats acclimated to a 14-h:10-h light:dark cycle the potency of 5-HT to inhibit the LH surge and ovulation was 2.06 and 2.3 times greater, respectively, when injected at 11:00 h than at 13:00 h. Also stimulation of the median raphe nucleus at 11:00 h was significantly more effective in inhibiting these parameters than stimulation at 13:00 h. Similarly, the ability of gamma-amino-butyric acid (GABA) to inhibit the preovulatory LH surge and ovulation was greater in rats injected in the morning than in the afternoon. The results of this study indicate that during proestrus the sensitivity of 5-HT and GABA to induce inhibition of preovulatory LH release and ovulation shows daily variations with maximal effect before the critical period.     

Interaction between ovarian and adrenal steroids in the regulation of gonadotropin secretion

Recent work from our laboratory suggests that a complex interaction exists between ovarian and adrenal steroids in the regulation of preovulatory gonadotropin secretion. Ovarian estradiol serves to set the neutral trigger for the preovulatory gonadotropin surge, while progesterone from both the adrenal and the ovary serves to (1) initiate, (2) synchronize, (3) potentiate and (4) limit the preovulatory LH surge to a single day. Administration of RU486 or the progesterone synthesis inhibitor, trilostane, on proestrous morning attenuated the preovulatory LH surge. Adrenal progesterone appears to play a role in potentiating the LH surge since RU486 still effectively decreased the LH surge even in animals ovariectomized at 0800 h on proestrus. The administration of ACTH to estrogen-primed ovariectomized (ovx) immature rats caused a LH and FSH surge 6 h later, demonstrating that upon proper stimulation, the adrenal can induce gonadotropin surges. The effect was specific for ACTH, required estrogen priming, and was blocked by adrenalectomy or RU486, but not by ovariectomy. Certain corticosteroids, most notably deoxycorticosterone and triamcinolone acetonide, were found to possess "progestin-like" activity in the induction of LH and FSH surges in estrogen-primed ovx rats. In contrast, corticosterone and dexamethasone caused a preferential release of FSH, but not LH. Progesterone-induced surges of LH and FSH appear to require an intact N-methyl-D-aspartate (NMDA) neurotransmission line, since administration of the NMDA receptor antagonist, MK801, blocked the ability of progesterone to induce LH and FSH surges. Similarly, NMDA neurotransmission appears to be a critical component in the expression of the preovulatory gonadotropin surge since administration of MK801 during the critical period significantly diminished the LH and PRL surge in the cycling adult rat. FSH levels were lowered by MK801 treatment, but the effect was not statistically significant. The progesterone-induced gonadotropin surge appears to also involve mediation through NPY and catecholamine systems. Immediately preceding the onset of the LH and FSH surge in progesterone-treated estrogen-primed ovx. rats, there was a significant elevation of MBH and POA GnRH and NPY levels, which was followed by a significant fall at the onset of the LH surge. The effect of progesterone on inducing LH and FSH surges also appears to involve alpha 1 and alpha 2 adrenergic neuron activation since prazosin and yohimbine (alpha 1 and 2 blockers, respectively) but not propranolol (a beta-blocker) abolished the ability of progesterone to induce LH and FSH surges. Progesterone also caused a dose-dependent decrease in occupied nuclear estradiol receptors in the pituitary.(ABSTRACT TRUNCATED AT 400 WORDS)     

Timing of ovulation in relation to onset of estrus and LH peak in yak (Poephagus grunniens L.)

The objective of the study was to determine the timing of ovulation in relation to onset of estrus and the preovulatory LH peak in yaks. For this purpose, a sensitive LH enzymeimmunoassay previously established in buffaloes was successfully validated for measuring the hormone in yak plasma. Plasma LH and progesterone were estimated from blood samples collected from eight non-lactating cycling yaks at 2 h intervals after estrus onset until 6 h after ovulation (ovulation was confirmed by palpation of ovaries per rectum). The mean+/-S.E.M. preovulatory plasma LH peak was 10.11+/-0.35 ng/ml with the values ranging from 8.75 to 11.51 ng/ml in individual yaks. The mean+/-S.E.M. duration of the LH surge was 7.25+/-0.55 h with a range of 6-10 h. Onset of LH surge (mean+/-S.E.M.) occurred 3.0+/-0.65 h after the onset of estrus. Mean plasma progesterone stayed low (<0.25 ng/ml) during the entire duration of sampling. Ovulation occurred 30.5+/-0.82 h (range, 28-34 h) after the onset of estrus and 20.25+/-1.03 h after the end of LH surge. The occurrence of the LH peaks within a narrow time frame of 4-8h post estrus onset in yaks could have contributed to the animals ovulating within a narrow time interval.     

Changes in plasma progesterone, estradiol, follicle-stimulating hormone and luteinizing hormone during diestrus and ovulation in rats with 5-day estrous cycles: effect of antibody against progesterone

Progesterone secretion remained significantly higher during diestrus in the 5-day cyclic rat than in the 4-day cyclic animal. Injection of a sufficient amount of antiprogesterone serum (APS) at 2300 h on metestrus in a 5-day cycle advances ovulation and completion of the cycle by 1 day in the majority of animals (75 and 80%, respectively). Progesterone (250 micrograms) administered with APS eliminated the effect of the antiserum. Within 2 h after administration of APS, levels of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) elevated significantly, while a significant elevation of plasma estradiol above the control value followed as late as 36 h after the treatment. None of the 5-day cyclic rats treated with APS showed ovulatory increases of FSH and LH at 1700 h on the second day of diestrus, although 3 of the 4 animals receiving the same treatment ovulated by 1100 h on the following day. The onset of ovulatory release of gonadotropins might have been delayed for several hours in these animals. These results indicate that recurrence of the 5-day cycle is due to an elevated progesterone secretion on the morning of diestrus, and suggest that a prolongation of luteal progesterone secretion in an estrous cycle suppresses gonadotropin secretion. Rather than directly blocking the estrogen triggering of ovulatory LH surge, the prolonged secretion of luteal progesterone may delay the estrogen secretion itself, which decreases the threshold of the neural and/or hypophyseal structures for ovulatory LH release.     

Differential expression of estradiol receptors alpha and beta by gonadotropes during the estrous cycle.

This study focused on expression of estradiol receptors (ER) during the estrous cycle. Labeling for ERalpha or beta antigens and luteinizing hormone (LH) or follicle-stimulating hormone (FSH) beta-subunits was done on freshly dispersed pituitary cells. The lowest expression of ERalpha and beta was seen in estrus (23% and 12%, respectively). Expression increased to 42-54% of pituitary cells by diestrus. In males, cells with ERalpha or beta were 37% or 20% of the population, respectively. ERalpha or beta and gonadotropin antigens were in 6-9% of pituitary cells from male rats. Early in the cycle (estrus and metestrus), less than 5% of pituitary cells expressed ERalpha or beta with gonadotropins. These values doubled to reach a peak of 10% during proestrus (just before ovulation). These data show that a rise in expression of both ERalpha and ERbeta is a part of preovulatory differentiation of pituitary gonadotropes.(J Histochem Cytochem 49:665-666, 2001)     

Importance of intense male sexual behavior for inducing the preovulatory LH surge and ovulation in seasonally anovulatory female goats

The present study was carried out to determine whether the presence of photostimulated sedated male goats could stimulate the LH preovulatory surge and ovulation in seasonal anestrous goats. Sexually experienced male goats were treated with artificial long days (16 hours light per day) from 1 November to 15 January to stimulate their sexual activity in March and April, corresponding to the natural sexual rest. A female group of goats (n = 20) was exposed to non-sedated males who displayed an intense sexual behavior and provided strong odor (non-sedated group). Another female group of goats (n = 20) was exposed to the photo-stimulated male goats, but these males were sedated with Xylazine 2% to prevent the expression of sexual behavior (sedated group). The sedated males also provided a strong odor. Females of both groups had full physical and visual contact with non-sedated or sedated males. In both groups, the males remained with females during 4 days. The LH preovulatory surge of 10 female goats per group was measured by determination of LH plasma concentrations in samples taken every 3 hours. In addition, in all goats, (n = 20 by group), ovulation was determined by measuring plasma concentrations of progesterone. The proportion of female goats showing a preovulatory LH surge was higher in goats exposed to non-sedated (10/10) than in those exposed to sedated bucks (0/10; P < 0.0001). Similarly, most of does in contact with non-sedated males ovulated (19/20), but none of those in contact with sedated males did so (0/20; P < 0.0001). We conclude that the expression of an intense sexual behavior by male goats is necessary to induce LH preovulatory surge and ovulation in seasonally anovulatory goats.     

Pituitary and ovarian hormone secretion and ovulation in gilts injected with gonadotropins during and after oral administration of progesterone agonist (Altrenogest)

We determined changes in plasma hormone concentrations in gilts after treatment with a progesterone agonist, Altrenogest (AT), and determined the effect of exogenous gonadotropins on ovulation and plasma hormone concentrations during AT treatment. Twenty-nine cyclic gilts were fed 20 mg of AT/(day X gilt) once daily for 15 days starting on Days 10 to 14 of their estrous cycle. The 16th day after starting AT was designated Day 1. In Experiment 1, the preovulatory luteinizing hormone (LH) surge occurred 5.6 days after cessation of AT feeding. Plasma follicle-stimulating hormone (FSH) increased simultaneously with the LH surge and then increased further to a maximum 2 to 3 days later. In Experiment 2, each of 23 gilts was assigned to one of the following treatment groups: 1) no additional AT or injections, n = 4; 2) no additional AT, 1200 IU of pregnant mare's serum gonadotropin (PMSG) on Day 1, n = 4); 3) AT continued through Day 10 and PMSG on Day 1, n = 5, 4) AT continued through Day 10, PMSG on Day 1, and 500 IU of human chorionic gonadotropin (hCG) on Day 5, n = 5; or 5) AT continued through Day 10 and no injections, n = 5. Gilts were bled once daily on Days 1-3 and 9-11, bled twice daily on Days 4-8, and killed on Day 11 to recover ovaries. Termination of AT feeding or injection of PMSG increased plasma estrogen and decreased plasma FSH between Day 1 and Day 4; plasma estrogen profiles did not differ significantly among groups after injection of PMSG (Groups 2-4). Feeding AT blocked estrus, the LH surge, and ovulation after injection of PMSG (Group 3); hCG on Day 5 following PMSG on Day 1 caused ovulation (Group 4). Although AT did not block the action of PMSG and hCG at the ovary, AT did block the mechanisms by which estrogen triggers the preovulatory LH surge and estrus.     

Changes in hypothalamic LH-RH content and blood levels of LH-RH, gonadotropin and estradiol during the preovulatory stage of rat estrous cycle.

In order to investigate the sequence of events concerning gonadotropin surge, serum LH, FSH and estradiol concentrations were measured during the rat estrous cycle as well as hypothalamic and blood levels of LH-RH in the preovulatory stage. Normally cyclic female Wistar rats kept on 12 hr light (from 22.00 hr to 10.00 hr) and 12 hr dark were killed at different times of day during each stage of the cycle. The hypothalamus was quickly dissected out, divided into 3 portions (the anterior, middle and posterior) and extracted in 90% methanol. Blood LH-RH was extracted by affinity chromatography prior to radioimmunoassay. The content of LH-RH in the anterior and middle hypothalamus started to decrease between 1.00 hr-3.00 hr, reached its nadir at 6.00 hr on proestrus and recovered to its previous values on estrus. Almost simultaneously blood LH-RH concentration showed an increase of 18.3 pg/ml-8.8 pg/ml between 1.00 hr-3.00 hr, and then fell to less than 1.0 pg/ml at 6.00 hr. On the other hand, serum estradiol level began to elevate on diestrus II followed by its peak at 6.00 hr on proestrus, while the peaks of serum LH and FSH were observed at 8.00 hr and 10.00 hr, respectively. These studies indicate that the elevation of serum estradiol was followed by the release of LH-RH from the hypothalamus and the LH-RH may be responsible for the preovulatory discharge of gonadotropin.     

2-Hydroxylation of estrogens in the brain participates in the initiation of the preovulatory LH surge in the rat

Estradiol-2-hydroxylase, the enzyme responsible for the conversion of estrogens to catechol estrogens was measured in the brain of female rats at specific stages of the estrus cycle. Radiometric measurements of the enzyme activity in microsomal, mitochondrial, and synaptosomal fractions of the brain revealed a sharp increased in activity at proestrus just prior to the preovulatory LH surge. The enzyme activity declined to lower levels at diestrus and metestrus. No comparable fluctuations were noted in the liver enzyme. These changes in brain enzyme activity in conjunction with demonstrated positive feedback of exogenous catechol estrogens on pituitary LH release, suggest that a rise in endogenous catechol estrogen formation in the brain may be responsible for the physiological induction of the preovulatory LH surge.     

Control of follicular development and luteal function in the mare: effects of a GnRH antagonist

Control of the equine estrous cycle was studied by suppressing gonadotropin secretion by administration of a GnRH antagonist to cyclic pony mares. Four mares received vehicle (control cycle) or a GnRH antagonist, Antarelix (100 microg/kg) on Day 8 of diestrus, and blood samples were collected at 15-min intervals from 0 to 16 h, 24 to 36 h, and daily until the next ovulation. Ovarian activity was monitored by transrectal ultrasonography, and measurement of plasma concentrations of progesterone and estradiol. Antagonist treatment eliminated large diestrous pulses of LH. Progesterone concentrations had fallen significantly in all mares by the day after treatment and, in three of the four mares, remained low until luteolysis. However timing of luteolysis (ie., progesterone concentrations <1 ng/mL) was not affected by antagonist treatment. The preovulatory surges of estradiol and LH were significantly delayed in the treatment cycle, as was the appearance of a preovulatory follicle >30 mm. Cycle length was significantly longer during the treatment than the control cycle. These results show that treatment of diestrous mares with a GnRH antagonist attenuated progesterone secretion, indicating a role for LH in control of CL function in the mare, and delayed ovulation presumably because of lack of gonadotropic support.     

LH surge in Nelore cows (Bos indicus), after induced estrus or after ovarian superestimulation

Considering that there is limited information about the preovulatory LH surge in Zebu cattle (Bos indicus), the purpose of the present work was to assess the LH surge in Nelore cows during the estrous cycle and after ovarian superestimulation of ovarian follicular development with FSH. This information is particularly important to improve superovulatory protocols associated with fixed-time artificial insemination. Nelore cows (n=12) had their estrus synchronized with an intravaginal device containing progesterone (CIDR-B) associated with estradiol benzoate administration (EB, 2.5 mg, i.m., Day 0). Eight days later all animals were treated with PGF2alpha (Day 8) in the morning (8:00 h) and at night, when CIDR devices were removed (20:00 h). Starting 38h after the first PGF2alpha injection, blood sampling and ovarian ultrasonography took place every 4h, during 37 consecutive hours. Frequent handling may have resulted in a stress-induced suppression of LH secretion resulting in only 3 of 12 cows having ovulations at 46.7+/-4.9 and 72.3+/-3.8 h, respectively, after removal of CIDR-B. Thirty days later, the same animals received the described hormonal treatment associated with FSH (Folltropin), total dose=200 mg) administered twice a day, during 4 consecutive days, starting on Day 5. Thirty-six hours after the first injection of PGF2alpha, to minimize stress, only seven blood samples were collected at 4h interval each, and ultrasonography was performed every 12 h until ovulation. In 11 of 12 cows (92%) the LH surge and ovulation were observed 34.6+/-1.6 and 59.5+/-1.9 h, respectively, after removal of progesterone source. The maximum values for LH in those animals were 19.0+/-2.6 ng/ml (mean+/-S.E.M.). It is concluded that, in Nelore cows submitted to a ovarian superstimulation protocol, the LH surge occurs approximately 35 h after removal of intravaginal device containing progesterone, and approximately 12h before the LH surge observed after an induced estrus without ovarian superstimulation.