Effects of bromocriptine administration during the follicular phase of the oestrous cycle on prolactin and gonadotrophin secretion and follicular dynamics in merino monovular ewes

Two experiments using Spanish Merino ewes were conducted to investigate whether the secretion of prolactin during the follicular phase of the sheep oestrous cycle was involved in the patterns of growth and regression of follicle populations. In both experiments, oestrus was synchronized with two cloprostenol injections which were administered 10 days apart. Concurrent with the second injection (time 0), ewes (n = 6 per group) received one of the following treatments every 12 h from time 0 to 72 h: group 1: vehicle injection (control); group 2: 0.6 mg bromocriptine (0.03 mg per kg per day); and group 3: 1.2 mg bromocriptine (0.06 mg per kg per day). In Expt 1, blood samples were collected every 3 h from 0 to 72 h, and also every 20 min from 38 to 54 h to measure prolactin, LH and FSH concentrations. In Expt 2, transrectal ultrasonography was carried out every 12 h from time 0 until oestrus, and blood samples were collected every 4 h to measure prolactin, LH and FSH concentrations. Ovulation rates were determined by laparoscopy on day 4 after oestrus. Bromocriptine markedly decreased prolactin secretion, but did not affect FSH concentrations, the mean time of the LH preovulatory surge or LH concentrations in the preovulatory surge. Both doses of bromocriptine caused a similar decrease in LH pulse frequency before the preovulatory surge. The highest bromocriptine dose led to a reduction (P < 0.01) in the number of 2-3 mm follicles detected in the ovaries at each time point. However, bromocriptine did not modify the total number or the number of newly detected 4-5 mm follicles at each time point, the number of follicles > 5 mm or the ovulation rate. In conclusion, the effects of bromocriptine on gonadotrophin and prolactin secretion and on the follicular dynamics during the follicular phase of the sheep oestrous cycle indicate that prolactin may influence the viability of gonadotrophin-responsive follicles shortly after luteolysis.     

Peripheral plasma concentrations of oestradiol, progesterone, cortisol, LH and prolactin during the oestrous cycle in the cow, with emphasis on the peri-oestrous period

Interrelationships of circulating hormone levels and their implications for follicular development were studied throughout the oestrous cycle with emphasis on the perioestrous period in heifers and cows. The oestradiol level showed a major peak (45 pmol/1) before and coinciding with oestrus, and a second peak (27 pmol/1) around day 5–6 (day 0: day of first standing oestrus); it was low during the luteal phase of the cycle when progesterone was higher than 14 nmol/1 from day −12 to day −2. Large antral follicles, which had developed during the luteal phase, did not secrete significant amounts of oestradiol, degenerated after luteolysis, and were replaced by a newly developing follicle which became preovulatory. Parallel with this development the oestradiol level increased from the onset of luteolysis to reach a plateau about 26 h before the onset of oestrus. The interval between the onset of luteolysis and the onset of oestrus was 58 h; luteolysis proceeded at a slower rate in heifers than in cows. At 4.6 h after the onset of oestrus the maximum of the LH surge was recorded; the LH surge appeared to be postponed in the period October–December in comparison to the period August–September. The maximum of the LH surge was higher in heifers (45 μg/l) than in cows (30 μg/l), but its duration was similar (8.0 h). The oestradiol level decreased significantly from 6 h after the maximum of the LH surge, and standing oestrus (duration 18 h) was terminated almost at the same time as the return to basal values of oestradiol. Cortisol and prolactin levels did not show a peak during the peri-oestrus period. Cortisol fluctuated irrespective of the stage of the oestrus cycle and prolactin was significantly higher during the luteal phase.

The results of this study indicate that development of the preovulatory follicle starts in the cow at the onset of luteolysis, about 2.5 days before the preovulatory LH surge, and that oestradiol secretion by this follicle is possibly inhibited by the LH surge.     

Changes in oestrogen biosynthesis in preovulatory rat follicles after blockage of ovulation with pentobarbitone sodium

Follicles isolated 1 and 2 days after pentobarbitone sodium injection at pro-oestrus were incubated with C-21 steroids or aromatizable C-19 steroids. Addition of testosterone or androstenedione (50 ng/ml) increased oestradiol production by ovulation-blocked follicles, while addition of progesterone or 17 alpha-hydroxyprogesterone was ineffective. LH-stimulated oestradiol production was lower in follicles isolated 1 and 2 days after pentobarbitone sodium injection, but progesterone production was elevated compared to pro-oestrous follicles. Total steroidogenesis, measured by pregnenolone production in the presence of inhibitors of pregnenolone conversion, did not differ on the 3 days. The activity of C17-20 lyase, measured in follicular homogenates, decreased between pro-oestrus and the next day. Aromatase and 17 alpha-hydroxylase activities also decreased, but the activity of these enzymes was always considerably higher than that of C17-20 lyase. It is concluded that the decrease in follicular oestradiol production after injection of pentobarbitone sodium was due primarily to a decrease in the activity of the enzyme system responsible for the conversion of 17 alpha-hydroxyprogesterone to androstenedione, thereby limiting the amount of substrate available for aromatization to oestrogen.     

Exogenous progesterone reduces follicular prostaglandin E and 6-keto prostaglandin F-1 alpha in the cyclic hamster

In cyclic hamsters, exogenous progesterone (100 micrograms) administered s.c. at 09:00 h on the day of dioestrus II reduced prostaglandin (PG) E and 6-keto PGF-1 alpha but not PGF concentrations in preovulatory follicles measured at 09:00 h of pro-oestrus. The injection of 10 micrograms ovine LH (NIADDK-oLH-25) concurrently with 100 micrograms progesterone on dioestrus II prevented the decline in follicular PGE and 6-keto PGF-1 alpha values. Administration of LH alone did not significantly alter follicular PG concentrations. Inhibition of follicular PGE accumulation by progesterone was due to a decline in granulosa PGE concentration and not thecal PGE. Progesterone administration also reduced follicular oestradiol concentrations. Administration of oestradiol-17-cyclopentanepropionate (ECP) (10 micrograms) with progesterone did not prevent the decline in follicular PGE and 6-keto PGF-1 alpha but did increase follicular PGF concentrations. However, ECP given alone on dioestrus II reduced follicular PGE and increased PGF concentrations in preovulatory follicles on pro-oestrus. It is concluded that exogenous progesterone administered on dioestrus II inhibits granulosa PGE and 6-keto PGF-1 alpha accumulation in preovulatory follicles, probably by reducing serum LH concentrations, and that the granulosa cells, which are LH-dependent, are a major source of follicular PGE.     

Effect of LH on progesterone and oestradiol production in vivo and in vitro by preovulatory rat follicles

Temporal changes in follicular oestradiol production induced in vitro and in vivo by LH were studied. In-vitro changes were measured by incubating preovulatory rat follicles for 12 h, changing the medium every 2 h. Follicles isolated at various intervals after an injection of 10 i.u. hCG were incubated for 2 h to measure changes in oestradiol production in vivo. In both studies there was an increase in oestradiol production lasting 4 h followed by a sharp decline. Progesterone production was also increased by LH in vitro or hCG in vivo, but remained high. A second exposure to LH did not raise oestradiol synthesis, but increased progesterone synthesis in vitro only. The decline in oestradiol production is most probably due to a decrease in C17-20 lyase activity, because addition of testosterone, but not of 17 alpha-hydroxyprogesterone, increased oestradiol production. Incubation of preovulatory follicles in the absence of LH or incubation of follicles derived from animals in which the spontaneous LH surge was blocked by an injection of pentobarbitone sodium also resulted in a decrease of oestradiol and an increase in progesterone production. This oestrogen-progesterone shift was also caused by a decrease in C17-20 lyase activity. The results demonstrate that the changes in steroid production in vivo and in vitro are similar and occur in the presence and absence of LH. It is concluded that the decrease in oestradiol production is dependent on the decrease in the activity of enzymes converting progesterone to aromatizable androgens.     

Production of oestradiol, camp and 125I-hcg binding by rat ovaries during the reproductive cycle.

Plasma concentration of oestradiol in female rats is dynamically altered in the reproductive cycle, depending on the biosynthesis and secretion of oestradiol secretion by the ovaries. The present study has followed oestradiol and cAMP concentration in the plasma and ovaries, and 125I-HCG binding during the pro-oestrus, oestrus and di-oestrus. Oestradiol concentration in plasma and its production by the ovaries were found to be highest in the pro-oestrus and lowest in the oestrus. These changes depend on the production and secretion of gonadotrophins. The mediator of gonadotropin action is cAMP whose ovarian concentration is lowest in the oestrus. LH stimulates the cAMP synthesis in the ovaries during the cyclic stages, without any significant difference being apparent between the various stages. 125I-HCG binding is highest in the di-oestrus stage, when concentration of endogenous gonadotrophins is low.     

Decline in circulating estradiol during the periovulatory period is correlated with decreases in estradiol and androgen, and in messenger RNA for p450 aromatase and p450 17alpha-hydroxylase, in bovine preovulatory follicles

The preovulatory surge of gonadotropins induces meiotic maturation of the oocyte, the follicular/luteal phase shift in hormone production, and ovulation. This complex and rapid series of developmental changes is difficult to study in large mammals, such as primates and ruminants, because variability in the length of individual reproductive cycles makes it virtually impossible to predict the time of the LH surge. We have validated an experimental model for inducing the LH surge and ovulation in cattle and used it to study the sequence of changes in hormone secretion and some of the mechanisms of these changes. Luteolysis and a follicular phase were induced by injection of prostaglandin F(2alpha); injection of a GnRH analogue 36 h later induced an LH surge and ovulation. The LH surge peaked 2 h after GnRH and ovulation followed 22-31 h after the surge, consistent with the periovulatory interval in natural cycles. The ensuing luteal phase was normal, both in length and in concentrations of circulating progesterone. In experiment I, the uteroovarian effluent was collected, via cannulation of the vena cava, at frequent intervals relative to GnRH injection. Circulating estradiol declined progressively after GnRH, reaching a nadir by 8-10 h before ovulation, whereas concentrations of androstenedione and testosterone remained constant. In experiment II, preovulatory follicles were obtained at 0, 3.5, 6, 12, 18, or 24 h after GNRH: Concentrations of androgens and estradiol were measured in follicular fluid and medium from cultures of follicle wall (theca + granulosa cells); steady-state levels of mRNA for 17alpha-hydroxylase (17alphaOH) and P450 aromatase were measured in follicular tissue. Shortly after the LH surge (3.5 h post-GnRH) there was an acute increase in the capacity of follicular tissue to secrete androstenedione, but not estradiol, in vitro. Thereafter, both androgens and estradiol declined, both in follicular fluid and in medium collected from cultures of follicle wall. Levels of mRNA for 17alphaOH and aromatase in follicle wall decreased significantly by 6 h after GnRH, suggesting that declining levels of these enzymes underlie the decreases in steroid production by follicular cells. These results show that in cattle the preovulatory decrease in follicular estradiol production is mediated by redundant mechanisms, because androgen production and the capacity of granulosa cells to convert androgens to estradiol decline coordinately, in concert with decreases in mRNA for 17alphaOH and P450 aromatase.     

Oxygen consumption by rat oocytes and cumulus cells during induced atresia

Oxygen consumption was measured in denuded oocytes and oocyte-cumulus complexes isolated from atretic rat follicles. Adult cyclic rats or immature PMSG-treated rats were used, and follicular atresia was induced by hypophysectomy on the morning of pro-oestrus or by repeated pentobarbitone injections beginning on the day of pro-oestrus. The later stages of atresia were accompanied by meiosis-like changes in the oocytes. Oxygen consumption by oocytes that had resumed meiosis (germinal vesicle breakdown, GVB) was higher than in oocytes with an intact germinal vesicle, a change similar to that observed in oocytes maturing in healthy follicles. This may indicate that the meiotic process in the atretic follicles is similar to that in normal ones. Oxygen consumption by the cumulus cells was not altered during pentobarbitone-induced atresia. Hypophysectomy led to a rapid and marked increase in cumulus oxygen consumption in cyclic rats but there was no change in PMSG-treated young animals. Since both pentobarbitone-treatment and hypophysectomy result in follicular atresia, but changes in cumulus respiration occurred only in hypophysectomized adult rats, it is concluded that an increase in cumulus respiration is not inherent to the atretic process.     

Effects of withholding food for 0-72 h on mating, pregnancy rate and pituitary function in female rats

Food was withheld from female rats for 0-72 h at various stages of the oestrous cycle. Withholding food for periods of 24 h ending at 12:00 h on the day of pro-oestrus reduced the mating rate from 61 to 25% (P less than 0.05) but not the pregnancy rate of those rats that mated. Fasting for 24 h ending at 18:00 h on the day of pro-oestrus reduced the pregnancy rate from 82 to 18% (P less than 0.05) without affecting the mating rate and a 48-h fast starting at 12:00 h on the day of pro-oestrus reduced the pregnancy rate from 82 to 25% (P less than 0.05). Withholding food for 23 h ending at 17:00 h on the day of pro-oestrus prevented the LH and prolactin surges normally present at 17:00 h on this day. The treatments had no apparent effect on the ability of the adenohypophysis to release LH in response to injections of GnRH. When ovariectomized female rats fasted for 0-72 h and given 2 injections of oestradiol dibenzoate to test the ability of the hypothalamus to respond to an increasing plasma oestradiol concentration by stimulating the release of LH, a fast for 24 h reduced and a fast for 72 h completely prevented LH release.     

Elevated peripheral concentrations of LH block ovulation and increase thecal and serum progesterone in the hamster

Insertion of osmotic minipumps containing 1 mg ovine LH on Day 1 (oestrus) elevated circulating serum concentrations of LH, progesterone and androstenedione when compared with values at pro-oestrus. Ovulation was blocked for at least 2 days at which time there were twice the normal numbers of preovulatory follicles. Follicular and thecal progesterone production in vitro was elevated when compared with that in pro-oestrous controls. Follicular and thecal androstenedione production in vitro was lower than in controls even though serum concentrations of androstenedione were elevated; the higher androstenedione values may be due to the increase in number of preovulatory follicles when compared with pro-oestrous controls. Follicles from LH-treated hamsters aromatized androstenedione to oestradiol and follicular production of oestradiol was similar to that in pro-oestrous follicles despite low follicular androstenedione production in the LH-treated group. Treatment with 20 i.u. hCG on Days 4 or 6 after insertion of an LH osmotic minipump on Day 1 induced ovulation of approximately 30 ova, indicating that the blockade of ovulation was not due to atresia of the preovulatory follicles. Serum progesterone concentrations on Days 2, 4 and 6 in LH-treated hamsters were greater than 17 nmol/l, suggesting that the blockade of ovulation might have been due to prevention of the LH surge by high serum progesterone concentrations.     

Development of progesterone dependency in the appearance of the nocturnal prolactin surge in immature rats

Basal concentrations of plasma prolactin in immature, Wistar-Imamichi strain rats at 25, 28 and 31 days of age were 5-12 ng/ml and no prolactin surges were observed in intact immature rats. Plasma progesterone values ranged from 5 to 9 ng/ml, while plasma oestradiol concentrations increased from 11 to 27 pg/ml between 25 and 31 days of age. When oestradiol was administered to ovariectomized 25- or 28-day-old rats by s.c. insertion of an implant, plasma prolactin concentrations at 05:00 and 12:00 h were similarly elevated 3 days after the operation. Oestradiol did not induce a nocturnal prolactin surge. The progesterone implants in ovariectomized rats at 28 days of age or on the first day of oestrus increased plasma prolactin values at 05:00 h. The magnitude of the progesterone-induced prolactin surge was greater when progesterone was given closer to the time of the first ovulation (about 34 days old). Pretreatment with oestradiol amplified the progesterone-induced prolactin surge. Mechanisms causing nocturnal prolactin surges are more sensitive to, and respond over a longer time period, to progesterone in pubertal rats than in adult animals. The results suggest that progesterone initiates the nocturnal surge of prolactin release and that oestradiol can amplify the effects of progesterone.     

Effects of ovarian hyperstimulation and isolated preovulatory follicles on LH responses to GnRH in rats

Adult rats were pretreated with a 3-day regimen of human menopausal gonadotrophin (hMG), PMSG, human FSH or hCG and experiments were carried out on the day of pro-oestrus. Treatment with hMG and hFSH induced a significant increase in the number of preovulatory follicles on the day of pro-oestrus and this was correlated with increased circulating concentrations of oestradiol. There was a parallel increase in the self-priming effect of GnRH, as observed from the biphasic LH response to a continuous GnRH challenge. PMSG treatment did not stimulate increased numbers of maturing follicles and was less effective in raising circulating oestrogen concentrations compared with hMG and hFSH. However, pituitary responsiveness was much higher after PMSG treatment and the biphasic response to continuous perfusion with GnRH was absent; LH release was high from the initiation of the stimulus. hCG alone failed to stimulate follicular maturation but enhanced pituitary LH responses. Hemi-pituitary glands perfused in the presence of isolated preovulatory follicles also showed augmented biphasic LH responses to GnRH compared with control hemi-pituitary glands. The apparent dissociation which can occur between follicular maturation, circulating oestrogen concentrations and pituitary responsiveness to GnRH supports the idea of non-steroidal ovarian factors modulating LH release.     

Gonadotropin surge-induced up-regulation of the plasminogen activators (tissue plasminogen activator and urokinase plasminogen activator) and the urokinase plasminogen activator receptor within bovine periovulatory follicular and luteal tissue

This study examined the effect of the preovulatory gonadotropin surge on the temporal and spatial regulation of tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), and uPA receptor (uPAR) mRNA expression and tPA, uPA, and plasmin activity in bovine preovulatory follicles and new corpora lutea collected at approximately 0, 6, 12, 18, 24, and 48 h after a GnRH-induced gonadotropin surge. Messenger RNAs for tPA, uPA, and uPAR were increased in a temporally specific fashion within 24 h of the gonadotropin surge. Localization of tPA mRNA was primarily to the granulosal layer, whereas both uPA and uPAR mRNAs were detected in both the granulosal and thecal layers and adjacent ovarian stroma. Activity for tPA was increased in follicular fluid and the preovulatory follicle apex and base within 12 h after the gonadotropin surge. The increase in tPA activity in the follicle base was transient, whereas the increased activity in the apex was maintained through the 24 h time point. Activity for uPA increased in the follicle apex and base within 12 h of the gonadotropin surge and remained elevated. Plasmin activity in follicular fluid also increased within 12 h after the preovulatory gonadotropin surge and was greatest at 24 h. Our results indicate that mRNA expression and enzyme activity for both tPA and uPA are increased in a temporally and spatially specific manner in bovine preovulatory follicles after exposure to a gonadotropin surge. Increased plasminogen activator and plasmin activity may be a contributing factor in the mechanisms of follicular rupture in cattle.     

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.     

Evidence to support a follicle-stimulating hormone threshold theory for follicle selection in ewes chronically treated with gonadotrophin-releasing hormone agonist

The mean and peak concentrations of follicle-stimulating hormone (FSH) during the luteal phase of a normal cycle were measured in 8 Welsh Mountain ewes. Gonadotrophin secretion and follicle growth were then suppressed by the chronic administration of the GnRH agonist buserelin for 5 weeks. During the 6th week of agonist treatment, each ewe was given a continuous infusion of FSH to produce a peripheral concentration of FSH equal to either the mean or peak of the gonadotrophin measured for that individual in the cycle preceding agonist treatment. Treatment had no effect on the total number of follicles, the number of follicles less than or equal to 2.5 mm in diameter or the in-vitro production of oestradiol by the small follicles when compared with control animals. None of the animals infused with the mean luteal-phase FSH equivalent developed large follicles greater than 2.5 mm diameter which could be classified as preovulatory follicles (oestradiol greater than 1000 pg/follicle/h). All of the animals infused with the peak luteal-phase FSH equivalent developed large follicles, some of which were preovulatory. The results suggest that an individual threshold concentration exists for FSH above which the later stages of preovulatory follicular development are stimulated.     

Direct effect of prolactin, induced by TRH injection, on ovarian oestradiol secretion in the ewe

The effect of sustained high plasma levels of prolactin, induced by repeated 2-h i.v. injections of thyrotrophin-releasing hormone (TRH; 20 micrograms), on ovarian oestradiol secretion and plasma levels of LH and FSH was investigated during the preovulatory period in the ewe. Plasma levels of progesterone declined at the same rate after prostaglandin-induced luteal regression in control and TRH-treated ewes. However, TRH treatment resulted in a significant increase in plasma levels of LH and FSH compared to controls from 12 h after luteal regression until 5 to 6 h before the start of the preovulatory surge of LH. In spite of this, and a similar increase in pulse frequency of LH in control and TRH-treated ewes, ovarian oestradiol secretion was significantly suppressed in TRH-treated ewes compared to that in control ewes. The preovulatory surge of LH and FSH, the second FSH peak and subsequent luteal function in terms of plasma levels of progesterone were not significantly different between control and TRH-treated ewes. These results show that TRH treatment, presumably by maintaining elevated plasma levels of prolactin, results in suppression of oestradiol secretion by a direct effect on the ovary in the ewe.     

Changes in oestrogen-synthesizing ability of preovulatory bovine follicles relative to the peak of LH

Preovulatory bovine follicles (n = 28) were collected at different times after the onset of standing oestrus until shortly before ovulation. In-vitro conversion of tritiated androstenedione in the presence of NADPH by homogenates of the follicular wall was compared in phases relative to the LH peak. During phase 0 (before the LH surge) conversion into oestradiol-17 beta was high and production of oestrone was about 8-fold lower. During phases 1 (0-6 h after the LH peak) and 2A (6-14 h after the LH peak) the production of oestradiol and oestrone remained constant; the percentage of remaining androstenedione increased. In phase 2B (14-20 h after the LH peak) conversion into oestradiol and oestrone had decreased to about one third correlating with a higher percentage of remaining androstenedione. In phase 3 (20 h after the LH peak until ovulation) conversion into oestradiol and oestrone remained constant. The ratio between the production of oestrone and oestradiol remained constant throughout the phases of preovulatory development (0.13), indicating a concurrent inhibition of aromatase and 17 beta-hydroxysteroid dehydrogenase activities. Conversion into 19-hydroxyandrostenedione showed a pattern similar to that of oestradiol, and testosterone was produced in minute quantities. The results indicate that in preovulatory bovine follicles eventual inhibition of aromatization takes place at about 14 h after the preovulatory LH peak.     

Changes in follicular steroidogenic enzymes following the preovulatory surge of gonadotropins and experimentally-induced atresia

Atresia that is induced experimentally and the preovulatory surge of gonadotropins stimulate similar changes in follicular steroidogenesis in the rat, i.e., both enhance production of progesterone and reduce production of androgen and 17 beta-estradiol. In this study, mature cycling rats were either stimulated with human chorionic gonadotropin (hCG) or atresia was induced by blocking the proestrous surge of gonadotropins through the use of pentobarbitone or hypophysectomy. Changes in activity of C17,20-lyase (lyase) and 20 alpha-hydroxysteroid dehydrogenase (20 alpha SDH) were estimated from homogenates of 10-15 Graafian follicles by evaluating conversion of precursors to products that were separated and quantified by high performance liquid chromatography (HPLC). Within 3 h of administration to proestrous rats, hCG reduced follicular lyase activity (pmole androstenedione produced per mg protein during 30 min incubation) from (mean +/- SEM) 221.3 +/- 24.2 to 120.2 +/- 30.4, and to 8.5 +/- 0.1 after 9 h. By contrast, 20 alpha SDH activity increased somewhat after hCG stimulation. Similar changes were observed after follicular atresia was induced, with hypophysectomy causing the most striking changes. Lyase was reduced to 60% within 6 h after the operation, and to 2% within 24 h. Activity of 20 alpha SDH was doubled within 6 h of hypophysectomy and remained high even 24 h later. Thus, in preovulatory rat follicles, luteinizing hormone (LH)/hCG reduces lyase activity and similar changes occur in such follicles undergoing atresia. There was no clear correlation between 20 alpha SDH and lyase activities; our results did not support the argument that 20 alpha SDH products regulate lyase following the ovulatory stimulus and atresia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Matrix metalloproteinase-2 and -9 secretion by the equine ovary during follicular growth and prior to ovulation

Profound hormonally controlled tissue remodelling occurs in the equine ovary for follicle growth and development, and also for the alteration in follicle shape directed towards the ovulation fossa, the site where ovulation occurs. The aim of this study was to examine the spatial and temporal regulation of matrix metalloproteinases (MMP)-2 and MMP-9, important enzymes in tissue remodelling, during follicle growth, and ovulation. Using gelatin substrate zymography, we measured these MMPs in follicular fluid of large anovulatory follicles collected during spring transition, early dominant follicles (> 23 mm), and at oestrus in follicles approximately 3 days prior to ovulation, and post-hCG treatment when ovulation was predicted in approximately 4 h. The most abundant activity detected in follicular fluid was MMP-2, although there were no changes in secretion or activation in association with ovulation. The activity of MMP-9 was detected in lower amounts, with no changes prior to ovulation, although it decreased significantly (P < 0.05) post-hCG treatment. At oestrus, when different regions of the ovary were maintained in explant culture for 24 h, there were no significant changes in either MMP-2 or MMP-9 secretion by stromal tissues collected at the ovarian fossa, adjacent to the preovulatory follicle but away from the fossa, and a further site remote from the preovulatory follicle. Over this same time period, follicular progesterone (P < 0.01) and oestradiol (P < 0.05) increased significantly, although oestradiol tended to decrease after hCG administration. These findings indicate that MMP-2 and MMP-9 are not key acute regulators for the changes in follicle shape immediately prior to ovulation.     

Follicular and hormonal dynamics during the first follicular wave in heifers

A few days after the first follicular wave emerges as 4-mm follicles, follicular deviation occurs wherein 1 follicle of the wave continues to grow (dominant follicle) while the others regress. The objectives of this study were to characterize follicle growth and associated changes in systemic concentrations of gonadotropins and estradiol at 8-h intervals encompassing the time of follicle deviation. Blood samples from heifers (n = 11) were collected and the ovaries scanned by ultrasound every 8 h from 48 h before to 112 h after the maximal value for the preovulatory LH surge. The follicular wave emerged at 5.8 +/- 5.5 h (mean +/- SEM) after the LH surge, and at this time the future dominant follicle (4.2 +/- 0.8 mm) was larger (P < 0.001) than the future largest subordinate follicle (3.6 +/- 0.1 mm). There was no difference in growth rates between the 2 follicles from emergence to the beginning of the deviation (0.5 mm/8 h for each follicle), indicating that, on average, the future dominant follicle maintained a size advantage over the future subordinate follicle. Deviation occurred when the 2 largest follicles were 8.3 +/- 0.2 and 7.8 +/- 0.2 mm in diameter, at 61.0 +/- 3.7 h after wave emergence. Diameter deviation was manifested between 2 adjacent examinations at 8-h intervals. Mean concentrations of FSH decreased, while mean concentrations of LH increased 24 and 32 h before deviation, respectively, and remained constant (no significant differences) for several 8-h intervals encompassing deviation. In addition to the increase and decrease in circulating estradiol concentrations associated with the preovulatory LH surge, an increase (P < 0.05) occurred between the beginning of deviation and 32 h after deviation. The results supported the hypotheses that deviation occurs rapidly (within 8 h), that elevated systemic LH concentrations are present during deviation, and that deviation is not preceded by an increase in systemic estradiol.