Morning Of Pro-oestrus Research Articles

Cyclic changes in the responsiveness of regressing corpora lutea to the luteolytic effects of prolactin in rats.

In cyclic rats, apoptosis of luteal cells during structural luteolysis occurs cyclically at the transition from pro-oestrus to oestrus in response to the preovulatory prolactin surge. This finding indicates that cyclic changes in apoptosis during luteolysis are dependent on prolactin surge cyclicity. In this study, the effects of prolactin on structural luteolysis were studied under different experimental conditions in relation to the phase of the oestrous cycle. In rats treated with prolactin at metoestrus and dioestrus, apoptosis did not occur in regressing corpora lutea, whereas in rats treated with prolactin on the morning of pro-oestrus, a 12.3-fold and 3.4-fold increase were observed in the number of apoptotic cells in regressing corpora lutea of the current and previous oestrous cycles, respectively. However, when the preovulatory prolactin surge and hence the subsequent apoptotic burst were blocked, prolactin treatment at the dioestrus phase induced a 13-fold increase in the number of apoptotic cells and significant changes in the volume of the corpus luteum (38% decrease) and the number of steroidogenic cells per corpus luteum (70% decrease). The results of this study indicate that the responsiveness of the regressing corpus luteum to the pro-apoptotic effects of prolactin are dependent on the phase of the oestrous cycle and on the presence or absence of an apoptotic burst in response to the preovulatory prolactin surge on the evening of pro-oestrus. Steroidogenic cells surviving to the apoptotic burst during the transition from pro-oestrus to oestrus became refractory to the lytic effect of prolactin. Furthermore, these cells also responded to the luteotrophic effects of prolactin, reaching full morphological luteinization, as indicated by the rescue of regressing cyclic corpora lutea during pregnancy.

Cyclic changes in the responsiveness of regressing corpora lutea to the luteolytic effects of prolactin in rats

In cyclic rats, apoptosis of luteal cells during structural luteolysis occurs cyclically at the transition from pro-oestrus to oestrus in response to the preovulatory prolactin surge. This finding indicates that cyclic changes in apoptosis during luteolysis are dependent on prolactin surge cyclicity. In this study, the effects of prolactin on structural luteolysis were studied under different experimental conditions in relation to the phase of the oestrous cycle. In rats treated with prolactin at metoestrus and dioestrus, apoptosis did not occur in regressing corpora lutea, whereas in rats treated with prolactin on the morning of pro-oestrus, a 12.3-fold and 3.4-fold increase were observed in the number of apoptotic cells in regressing corpora lutea of the current and previous oestrous cycles, respectively. However, when the preovulatory prolactin surge and hence the subsequent apoptotic burst were blocked, prolactin treatment at the dioestrus phase induced a 13-fold increase in the number of apoptotic cells and significant changes in the volume of the corpus luteum (38% decrease) and the number of steroidogenic cells per corpus luteum (70% decrease). The results of this study indicate that the responsiveness of the regressing corpus luteum to the pro-apoptotic effects of prolactin are dependent on the phase of the oestrous cycle and on the presence or absence of an apoptotic burst in response to the preovulatory prolactin surge on the evening of pro-oestrus. Steroidogenic cells surviving to the apoptotic burst during the transition from pro-oestrus to oestrus became refractory to the lytic effect of prolactin. Furthermore, these cells also responded to the luteotrophic effects of prolactin, reaching full morphological luteinization, as indicated by the rescue of regressing cyclic corpora lutea during pregnancy.

Effect of exogenous melatonin on neuroendocrine-reproductive function of middle-aged female rats.

The possible role of melatonin in the regulation of the reproductive system of female rats during ageing was investigated in middle-aged female rats showing irregular duration of the oestrous cycle (n = 30). Blood samples were obtained by jugular venepuncture during the oestrous cycle in control rats. After this experiment was completed, the female rats were treated with melatonin for 2 months and blood samples were obtained at different stages of the oestrous cycle. Plasma LH, FSH and prolactin concentrations were significantly increased in the afternoon of the day of pro-oestrus after melatonin treatment compared with control rats. Moreover, FSH concentrations too were significantly increased on the morning of pro-oestrus and oestrus in melatonin treated rats compared with control rats. Similarly, oestradiol concentrations were significantly higher on the morning of pro-oestrus in melatonin treated rats compared with controls. Another group of rats showing irregular duration of the oestrous cycle was used to study the possible effect of melatonin treatment on the timing of pro-oestrous surges of LH and FSH. The results showed that LH and FSH peak values occurred at 5 h after melatonin treatment. Pituitary responsiveness to LHRH in a 90 min test was also studied in middle-aged rats showing irregular duration of the oestrous cycle that had been injected for 1 month with either melatonin or saline. Prolactin response was unaffected by exogenous melatonin, but a stimulatory effect of melatonin on LH and FSH pituitary responsiveness to LHRH was observed. The results indicate an improved function of the neuroendocrine-reproductive axis in middle-aged rats after melatonin treatment.

GnRH and oxytocin have nonidentical effects on the cellular LH response by gonadotrophs at pro-oestrus.

For full fertility in the female ovulation is necessary, which is dependent on the production of a surge of LH. An understanding of the processes which result in the high levels of LH requires delineation of the activities of individual component cells. In this study the responses of gonadotrophs to two signalling hypothalamic peptides, GnRH and oxytocin, were investigated. A cell immunoblot method was used to identify and distinguish between cells which secrete LH and those which contain LH but do not secrete the glycohormone. Rats were killed on the morning of pro-oestrus, the pituitary collected and the cells dispersed onto a protein-binding membrane for study. Cells were then incubated with GnRH and oxytocin, after which the membranes including the attached cells were stained by immunocytochemistry for LH. GnRH increased the total number of immunopositive cells which were present in a concentration-dependent manner. The most prominent change after 2 h incubation was in the number of secreting cells, whereas after 4 h there was also a marked increase in numbers of nonsecreting cells. Oxytocin also increased the total number of immunopositive cells in a concentration-responsive manner, however the profile of action of oxytocin was different from that observed for GnRH. Oxytocin had a relatively greater effect on numbers of immunopositive nonsecreting cells. Thus, the results reveal the potential for gonadotrophs to be flexibly and appropriately modulated by selected hypothalamic peptides. When cells were preincubated with oxytocin prior to GnRH there was not an additive increase in the numbers of immunopositive cells, suggesting that the two agonists act, in a nonidentical manner, on similar cells. The increase in the total number of immunopositive cells implies that there was a production of LH or post-translational processing, induced by exposure to GnRH or oxytocin. The results confirmed the heterogeneity of gonadotrophs and the existence of functionally distinguishable subpopulations, and revealed a difference between the effects of GnRH and oxytocin on expression and secretion of LH.

Temporal changes in inhibin subunit mRNAs during atresia of preovulatory follicles in the rat.

This study aimed to investigate the time course of disappearance of the mRNAs of the various subunits of inhibin in follicles which become atretic. An animal model was used in which atresia of preovulatory follicles could be studied in a chronological order. Injection of gonadotrophin-releasing hormone (GnRH) antagonist (20 microg) at the morning of pro-oestrus (P) blocked ovulation and the 10-12 preovulatory follicles became gradually atretic. A second injection was given the next day to prevent delayed ovulation. The rate of atresia could be delayed by simultaneous administration of a subovulatory dose of human chorionic gonadotrophin (hCG) (0.5 IU) and could be advanced by administration of a fivefold larger amount of GnRH antagonist. Functional activity of follicles becoming atretic was studied by measuring oestradiol production after incubation of individual follicles for 4 h. Follicles isolated 24 h after the first injection of GnRH antagonist (P+24) already secreted significantly less oestradiol in vitro than follicles isolated at pro-oestrus, although they were morphologically not different from pro-oestrous follicles. Follicles isolated at P+24 from hCG-treated rats secreted more oestradiol compared with follicles from rats not treated with hCG. In contrast, follicles isolated at P+24 from rats that were given a fivefold larger amount of GnRH antagonist secreted less oestradiol. Once this model was validated, temporal changes in inhibin subunit mRNAs in follicles undergoing atresia were measured by in situ hybridization and RNase protection assay. In situ hybridization showed abundant alpha- and betaA-subunit mRNA in the whole granulosa layer of preovulatory follicles at P and P+24, while betaB-subunit mRNA was restricted to the antral layer and cumulus. At P+48 the amount of alpha- and betaA-subunit mRNA had declined and was restricted to the cumulus, whereas betaB-subunit mRNA was absent. In the atretic follicles present at P+72 and P+96, mRNAs of all three inhibin subunits were absent. Administration of 0.5 IU hCG delayed the decline in the amount of alpha, betaA and betaB mRNA in preovulatory follicles at P+48. RNase protection assay of inhibin subunits in isolated follicles revealed no changes between P and P+24. However, at P+48, the mRNAs of alpha- and betaA-subunits were decreased. Expression of the mRNA of betaB-subunit declined gradually from P to P+48. The present study demonstrates that in follicles which are becoming atretic, mRNAs of alpha- and betaA-subunits decline simultaneously with the appearance of pycnotic cells in the granulosa layer, while betaB-subunit mRNA declines earlier, simultaneously with the decrease in the ability to secrete oestradiol in vitro.

Changes in inhibin-A (alpha-beta A dimer) and total alpha inhibin in the peripheral circulation and ovaries of rats after gonadotrophin-induced follicular development and during the normal oestrous cycle.

Recent modifications to a previously reported two-site IRMA have permitted the measurement of serum/plasma concentrations and ovarian contents of inhibin-A (alpha-beta A dimer) in pregnant mare serum gonadotrophin (PMSG)-treated immature female rats and adult rats throughout the 4-day oestrous cycle. For comparison, total alpha inhibin levels were also measured by alpha subunit-directed inhibin RIA and found to be at least tenfold higher (relative to the same 32 kDa bovine inhibin standard used to calibrate both assays). In immature female rats, serum levels of inhibin-A dimer and total alpha inhibin increased within 3 h of PMSG injection and rose in parallel over the next 48 h to values four- to fivefold higher than pretreatment levels. Ovariectomy led to a rapid and parallel fall in both inhibin-A dimer and total alpha inhibin; initial half-lives (+/- 95% confidence intervals) were 22 +/- 4 and 20 +/- 5 min respectively. In adult rats, marked fluctuations in plasma concentrations and ovarian contents of inhibin-A dimer and total alpha inhibin occurred during the 4-day oestrous cycle, most notably between the morning of pro-oestrus and the morning of oestrus. Plasma levels of inhibin-A dimer and total alpha inhibin peaked on the afternoon of pro-oestrus just before the preovulatory gonadotrophin surge. After ovulation, both inhibin-A dimer and total alpha inhibin fell abruptly (two- to threefold by 0200 h on oestrus; P < 0.001), while FSH showed a secondary rise which peaked at 0700 h on oestrus. Although IRMA- and RIA-derived inhibin values generally followed a similar pattern across the 4-day cycle (plasma: r = 0.52, P < 0.001; ovary: r = 0.41, P < 0.001), a transient rise in plasma and ovarian inhibin-A dimer was detected at 0700 h on oestrus (P < 0.01) which was unaccompanied by a rise in total alpha inhibin. This rise in plasma inhibin-A dimer was probably responsible for terminating the post-ovulatory FSH surge since FSH levels declined steadily over the next 15 h. Overall, plasma inhibin-A dimer and FSH concentrations across the whole cycle were negatively correlated (r = -0.22, P < 0.01) whereas no correlation existed between total alpha inhibin and FSH (r = -0.11, P = 0.12).

Methodology and characterization of an in vitro perfusion model for the mouse ovary

Perfusion of intact ovaries in vitro has proved to be a suitable model for the study of ovarian events, such as ovulation and corpus luteum function, with advantages over cell culture systems of preserved three-dimensional structure with intact intercellular communication. This methodology has been described for several larger experimental animals. Development of genetically manipulated mouse strains and the larger availability of recombinant proteins and monoclonal antibodies in this species prompted the development of a mouse ovary perfusion system. In the present study, we describe the methodology for perfusion of mouse ovaries in vitro and characterize the model with respect to steroidogenesis, ovulatory efficiency and morphology. Swiss mice (30-45 g) were synchronized with a single injection of a GnRH agonist and laparotomy was performed on the morning of pro-oestrus. The right ovary and its vasculature, after cannulation on the arterial side, were surgically isolated and transferred to a recirculating perfusion system, and were perfused for 20 h. The ultrastructure of the ovaries after 20 h of perfusion was examined by transmission electron microscopy, which showed well-preserved cellular organelles and nuclei and distinct tight junctions between endothelial cells. Unstimulated ovaries did not ovulate and secreted small quantities of progesterone. After addition of hCG (50 iu) or ovine LH (0.1 microgram ml-1), ovulations were observed in all perfused ovaries (2.6 +/- 0.5 and 2.0 +/- 0.3 ovulations per treated ovary, respectively). There was a distinct rise in progesterone output after hormone addition. When a phosphodiesterase inhibitor, isobutylmethylxanthine (0.2 mumol l-1), was added in combination with LH, the ovulation rate (7.4 +/- 1.2) was similar to that in vivo and progesterone secretion was further increased compared with other treatments. Since the mouse has become a very important experimental animal in medical research and we have the ability to genetically manipulate this species, this methodology for perfusing mouse ovaries in vitro may be a useful tool in future studies of ovarian physiology.

Hypothalamic release of atrial natriuretic factor and β-endorphin into rat hypophysial portal plasma: relationship to oestrous cycle and effects of hypophysectomy

The release of beta-endorphin and atrial natriuretic factor (ANF) into hypophysial portal plasma was investigated in male and female Wistar rats. The principal aim of the study was to investigate the possible role of beta-endorphin and ANF in the hypothalamic control of LH and prolactin secretion. In male rats, anaesthetized with urethane, the concentrations of beta-endorphin in portal blood collected immediately after hypophysectomy were within the same range as those in peripheral plasma. Furthermore, electrical stimulation of the median eminence did not increase the portal plasma concentrations of beta-endorphin. In female rats, anaesthetized with alphaxalone, the portal plasma concentrations in long-term (6-8 weeks) or acutely hypophysectomized rats were significantly greater than those in peripheral plasma. In acutely hypophysectomized female rats the concentrations and contents of beta-endorphin in portal plasma collected at 10.00-11.30 h of pro-oestrus were significantly (approximately sixfold) greater than at dioestrus or at 20.00-21.00 h of pro-oestrus, but these changes were not consistently seen in all experiments. In female rats in which the pituitary gland was not removed for portal blood collection, portal plasma contents of ANF remained unchanged throughout the day of pro-oestrus, suggesting that it is unlikely that ANF is involved in the spontaneous LH or prolactin surge. The effects of ovarian steroids on the secretion of hypothalamic ANF and beta-endorphin were determined by measuring the portal plasma concentration of ANF and beta-endorphin on the morning of presumptive pro-oestrus in rats ovariectomized 24 h previously and injected with either oil or oestradial benzoate (OB). Portal plasma contents of ANF were significantly lower in OB- compared with oil-treated rats, suggesting that oestradiol inhibits ANF release into rat hypophysial portal plasma. In contrast, there were no significant between-group differences in the content or concentration of beta-endorphin in portal plasma. Thus, the increased beta-endorphin in the portal plasma of some of the intact animals during the morning of pro-oestrus is not due to the preovulatory surge of oestradiol-17 beta. The output of beta-endorphin into portal blood in long-term hypophysectomized rats was lower than in dioestrous or pro-oestrous rats in which the pituitary gland was removed immediately before portal blood collection.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of neonatal androgenization on the chromatofocusing pattern of anterior pituitary FSH in the female rat

Anterior pituitary glands were removed from neonatally androgenized (100 micrograms testosterone propionate) female rats and normal controls at 5, 10, 18, 21, 30, 60 and 90 days of age, and the multiple forms of FSH present within them were separated by chromatofocusing (pH range 7.5-4.0). Additional pituitary glands from intact adult males (90 days old) were also studied for comparative purposes. All animal groups exhibited multiple forms of immunoactive FSH within a pH range of 7.5-4.0, as well as an additional FSH form obtained after the addition of 1.0 mol NaCl/l to the chromatofocusing column (salt peak). In animals 5-30 days old (controls and androgenized) the majority of FSH applied to the chromatofocusing columns was recovered within the salt peak (45-85% of total FSH immunoactivity recovered). However, as the animals aged, more FSH immunoactivity focused within less acidic regions (isoelectric point (pI) 5.9-5.0); pituitaries from animals 60 days old contained the greatest proportion of FSH focused within this pH range (controls, 39.2 +/- 0.6%; androgenized, 23.1 +/- 0.9% of total immunoactivity recovered; P less than 0.03 vs animals 30 days old for both experimental groups). This shift towards less acidic FSH was attenuated in androgenized animals compared with the controls (P less than 0.01). In control adult rats, the chromatofocusing distribution pattern of pituitary FSH varied according to the day of the oestrous cycle. Pituitary extracts from control rats decapitated during the morning of pro-oestrus, oestrus and day 1 of dioestrus exhibited the highest proportion of immunoactive FSH (23.2-28.8% of total) focused within a pH range of 5.9-5.0, whilst only 10.4-11.6% of FSH from androgenized rats and those on day 1 of dioestrus was recovered within this pH range (P less than 0.05). In control animals decapitated during the morning of pro-oestrus and oestrus, 10-26% of FSH focused within the most alkaline region (pI 7.5-6.0); the chromatofocusing pattern of pituitary FSH from the neonatally androgenized animals was characteristic, in that no more than one peak (1.5 +/- 0.5% of total) was detected in this alkaline region. In the adult male rats, the majority of pituitary FSH eluted from the chromatofocusing columns within a pH of 4.9-4.0 (52.4 +/- 1.2% of total FSH immunoactivity) and the salt peak (pH less than 4.0) (33.1 +/- 2.4 of total). All FSH isoforms obtained after chromatofocusing represented alpha and beta dimers as disclosed by size exclusion chromatography.(ABSTRACT TRUNCATED AT 400 WORDS)

Dynamics of ovarian inhibin secretion during the oestrous cycle of the rat

Plasma and ovarian concentrations of inhibin were determined at 3-h intervals throughout the 4-day oestrous cycle of rats by a radioimmunoassay (RIA) based on a bovine RIA. Plasma concentrations of LH, FSH, progesterone, testosterone, oestradiol-17 beta, and pituitary contents of FSH and LH were also determined during the cycle. Plasma levels of inhibin showed a marked increase on the morning of oestrus and the afternoon of metoestrus, and a further increase was noted on the morning of pro-oestrus. These increases in plasma levels of inhibin were probably due to the following three events in the ovary, follicular recruitment on the morning of oestrus, selection of follicles on the day of metoestrus, and final maturation of follicles for ovulation on the morning of pro-oestrus with an increase in oestradiol-17 beta secretion. A striking decrease in inhibin secretion occurred during the process of ovulation after the preovulatory gonadotrophin surge on the afternoon of pro-oestrus. Basal levels of plasma FSH gradually decreased from metoestrus to pro-oestrus as plasma levels of inhibin increased. There was a significant inverse relationship between plasma levels of FSH and inhibin throughout the oestrous cycle (r = -0.51). The present findings suggest that changes in the plasma levels of inhibin during the oestrous cycle provide a precise indicator for follicular recruitment, selection and ovulation, and that changes in concentrations of oestradiol-17 beta in the plasma are associated with follicular maturation.

Serotonin enhances oestradiol production by hamster preovulatory follicles in vitro: effects of experimentally induced atresia

Preovulatory follicles from adult hamsters on the morning of pro-oestrus were used in this study. Serotonin stimulated oestradiol production by preovulatory follicles during a 5-h incubation in 1 ml Krebs-Ringer bicarbonate glucose medium containing isobutylmethylxanthine (0.1 mmol/l; IBMX) and androstenedione (1 mumol/l). The enhanced oestradiol production by serotonin was dependent on the dose of IBMX and androstenedione. Mianserin, a serotonin type-1 and serotonin type-2 receptor antagonists, prevented the serotonin-enhanced oestradiol production in a dose-dependent manner. Ketanserin, a specific serotonin type-2 receptor antagonist, was ineffective in blocking the action of serotonin, indicating that the effect of serotonin was mediated by the serotonin type-1 receptor. In the presence of androstenedione (1 mumol/l), serotonin was unable to enhance oestradiol production in isolated granulosa cells. It was also unable to enhance oestradiol production in early atretic follicles; atresia was induced experimentally by an injection of phenobarbital in order to prevent ovulation. The data indicate that serotonin stimulates oestradiol production by hamster preovulatory follicles in vitro. The mechanism of action of serotonin involves an intact healthy follicle, a serotonin type-1 receptor and possibly cyclic AMP. The increased oestradiol secretion might be related to increased androgen production by the follicle and increased permeability (leakiness) of the follicle to androstenedione which serves as substrate for aromatization to oestradiol by the granulosa cell.

Effect of ovariectomy and treatment with ovarian steroids on vasopressin release and fluid balance in the rat

Plasma vasopressin concentrations have previously been shown to vary during the oestrous cycle of the rat, being highest on the morning of pro-oestrus and lowest on dioestrus day 1. To determine the effect of gonadal steroids on vasopressin secretion and fluid balance, mature rats were ovariectomized and given oestrogen, progesterone or vehicle alone s.c. for periods of up to 16 days. Plasma vasopressin concentrations fell after ovariectomy and this was reflected in an increase in 24-h urine volume. The normal increase in plasma vasopressin concentrations seen over day-light hours was also suppressed. The change in vasopressin concentrations observed on steroid treatment depended upon both the dose and the duration. High doses of oestrogen were associated with a fall in plasma vasopressin, probably as a result of fluid retention. Thus, of an initial group of rats given silicone elastomer implants containing 50, 500 or 1000 micrograms oestradiol in oil, plasma vasopressin concentrations were reduced after 7 days treatment with 1000 micrograms oestradiol implants in association with reduced plasma sodium concentrations. Daily s.c. injections of 100 micrograms oestradiol benzoate/100 g body weight produced an immediate small increase in plasma vasopressin concentrations, but by 14 days the plasma concentrations of 0.7 +/- 0.16 pmol/l (mean +/- S.E.M.) had fallen significantly and were less than those in the vehicle-treated group (1.2 +/- 0.26 pmol/l). However, after treatment for 14 days with implants containing only 50 micrograms oestradiol, plasma vasopressin concentrations were higher compared with the group receiving vehicle alone, despite the fact that the plasma osmolality was lower in the latter group, suggesting a long term resetting of the osmoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in serum concentrations of immunoreactive inhibin during the oestrous cycle of the rat.

Serum and ovarian concentrations of inhibin during the oestrous cycle of rats were determined by a radioimmunoassay (RIA) based on a porcine inhibin RIA. Serum concentrations of LH, FSH, oestradiol-17 beta, progesterone and testosterone were also determined during the cycle. Serum concentrations of inhibin were high (8.47 +/- 0.58 micrograms/l) during the morning of pro-oestrus and then dropped sharply to the lowest level (3.21 +/- 0.38 micrograms/l) at 24.00 h on the day of pro-oestrus after the preovulatory LH and FSH surge. Inhibin concentrations then recovered rapidly, with a peak (7.94 +/- 0.97 micrograms/l) at 15.00 h on the day of oestrus. Inhibin concentrations in serum dropped again to their second lowest value (4.00 +/- 0.12 micrograms/l) at 24.00 h on the day of oestrus. The largest FSH surge observed from midnight of pro-oestrus to the morning of oestrus (the so-called second FSH surge) was shown to be inversely related to the changes in serum concentrations of inhibin, suggesting a close relationship between the serum FSH and inhibin concentrations in cyclic rats.

Inhibition of first ovulation: administration of an LHRH antagonist to immature female rats.

Subcutaneous injections of an LHRH antagonist (ALHRH; Org.30093) were administered to immature female rats. Neither a single high dose (50 micrograms) nor repeated daily doses of 5-30 micrograms ALHRH/day, administered between 28 and 38 days of age, influenced the age and body weight at the time of vaginal opening or first ovulation. If repeated daily doses of 2 X 10 micrograms ALHRH were given from 32 to 42 or from 37 to 47 days of age, first ovulation was delayed by 3.0 and 6.3 days respectively. Administration of 10 micrograms ALHRH at 09.00 h and again at 17.00 h on the day of first pro-oestrus was found to be sufficient to block the expected first ovulation in 36 out of 38 rats. This effect could be repeated by administering the same doses of ALHRH at pro-oestrus and again on the next day: ovulation was blocked in eight out of eight rats. A single dose of 10 micrograms ALHRH, administered on the morning of pro-oestrus, blocked ovulation in five out of twelve rats. Both the preovulatory LH and FSH surge, as measured at 16.00 h on pro-oestrus, were found to be inhibited by ALHRH treatment. On the day after pro-oestrus no recruitment of new small antral follicles had occurred in rats with ovulatory blockade. Delayed ovulation took place 2-5 days after ALHRH injection at pro-oestrus; until 3 days after injection rats were able to ovulate their original preovulatory follicles, thereafter newly developed follicles ovulated and large ovarian cysts were found in the ovaries, next to fresh corpora lutea.(ABSTRACT TRUNCATED AT 250 WORDS)

Oestrous cycle-related LH responsiveness to naloxone: effect of high oestrogen levels on the activity of opioid receptors.

Endogenous opioid peptides have a tonic inhibitory control on LH secretion, participating in the functional changes of the hypothalamic-pituitary-ovarian axis. To evaluate the activity of the endogenous opioid systems during the oestrous cycle, we measured plasma LH levels after naloxone administration (5 mg/kg, s.c.) at 09.00 and 16.00 h on all days of the cycle (two further measurements were taken at 14.00 and 18.00 h on the day of pro-oestrus) and after one dose or one week's treatment with oestradiol benzoate (OB; 0.2 micrograms/rat). Concentrations of LH were measured in the same experimental models after injection of LH-releasing hormone (LHRH; 1 microgram/kg, i.p.) or saline. Naloxone induced a significant rise in LH levels on the day of oestrus, dioestrus day-1 and dioestrus day-2; this response was blunted on the morning of pro-oestrus and absent in the afternoon and after acute and chronic OB treatment. Conversely LHRH was most effective in increasing LH levels on the day of pro-oestrus and in OB-treated rats. These results indicate that opioid mechanisms, independently of the time of day and the pituitary responsiveness, exhibit a reduced activity when preovulatory changes occur, probably as a result of increased oestrogen levels.

Ovum transport in cyclic rats rendered hypo-oestrogenic by treatment with 4-hydroxy-4-androstene-3,17-dione

The effects of decreasing oestrogen secretion upon the rate of oocyte transport achieved by the administration of 4-hydroxy-4-androstene-3,17-dione were investigated in cyclic rats. Control animals examined during late pro-oestrus showed that the majority of oocytes had entered the uterus. In contrast, when inhibitor was administered from the afternoon of metoestrus or from late dioestrus through prooestrus, oviducal retention of oocytes was observed. When treatment was delayed until the morning of pro-oestrus, only uterine retention of oocytes was observed. The inhibitor decreased oestradiol concentrations in ovarian vein, while systemic testosterone values were increased. Treatment with exogenous oestradiol counteracted the effect of the inhibitor on ovum transport. The elevation of systemic testosterone concentrations by means of subdermal implants of testosterone failed to alter the normal pattern of ovum transport. These results demonstrate that normal oestrogen secretion during late metoestrus and dioestrus is required for the movement of oocytes from the oviduct to the uterus, whereas the preovulatory oestrogen surge is needed for the expulsion of ova from the uterus.

Evidence for a stimulatory beta-adrenergic component in the release of the ovulatory LH surge in pro-oestrous rats.

The effect on ovulation of intraventricular infusions of noradrenaline, adrenaline and various pharmacological agents acting on the adrenergic receptor subtypes were investigated in cyclic female rats on the day of pro-oestrus. The inhibitory effects on ovulation of the different infusions were monitored by administering the drugs before 11.00 h (several hours before the critical period for the ovulatory LH surge). In experiments designed to show how the drugs under investigation might stimulate ovulation, pentobarbitone sodium (35 mg/kg) was given at 14.30 h; this anaesthetic inhibits ovulation and its effects can be overcome by substances that advance the preovulatory LH surge. Noradrenaline (an alpha-agonist) stimulated ovulation when administered on the morning of pro-oestrus to rats injected with pentobarbitone early in the afternoon of the same day. Phenoxybenzamine and phentolamine (non-selective alpha-antagonists) and clonidine (a selective alpha 2-agonist) all inhibited ovulation when infused on the morning of pro-oestrus. Yohimbine (a moderately selective alpha 2-antagonist) neither stimulated nor inhibited ovulation. Both isoprenaline (a non-selective beta-agonist) and fenoterol (a selective beta 2-agonist) stimulated ovulation in pentobarbitone-treated rats when administered on the morning of pro-oestrus and fenoterol was also able to overcome the pentobarbitone block when infused later in the afternoon. Propranolol (a non-selective beta-antagonist) and metoprolol (a selective beta 1-antagonist) were stimulatory only when administered in the afternoon. Adrenaline (both an alpha- and beta-agonist), prenalterol (a selective beta 1-agonist), atenolol (a selective beta 1-antagonist) and ICI 118,551 (a selective beta 2-antagonist) neither stimulated nor inhibited ovulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of abdominal vagotomy on serum LH concentrations in female rats.

Vagotomy on the morning of pro-oestrus did not prevent the pro-oestrous LH surge and rats became oestrous on the following day. However, vagotomized rats then exhibited a period of acyclicity which lasted for 20.4 +/- 1.3 (s.e.m.) days. Food intake and body weight also declined after vagotomy. During the first week after vagotomy, afternoon LH surges generally did not occur, a pattern which was similar in animals pair fed with vagotomized rats. However, pair-fed rats showed oestrous cycles while vagotomized rats were acyclic. At 7 days after vagotomy, LH surges were induced by oestradiol benzoate and progesterone treatment of ovariectomized rats. Vagotomy suppressed the post-ovariectomy increase in serum LH at 7 and 21 days after surgery. These results, combined with those of other studies, suggest impairment of LH release in vagotomized rats.

Evidence that the dopaminergic incerto-hypothalamic tract has a stimulatory effect on ovulation and gonadotrophin release.

2 micrograms dopamine (DA) were injected into particular hypothalamic sites containing dopaminergic nerve terminals, on the morning of pro-oestrus in cyclic rats, in which ovulation was blocked by administration of 35 mg/kg pento-barbitone Na, in the afternoon of pro-oestrus. DA in a specific area of the zona incerta (ZI) (medial area at A 5.4 mm; de Groot atlas), and medial anterior hypothalamus (AH) overcame the pentobarbitone block and induced ovulation. Injections into the median eminence and preoptic area were ineffective, as were injections of 5-hydroxytryptamine and nor-adrenaline into the ZI. Injection of 2 micrograms haloperidol and lesions in the ZI inhibited ovulation in otherwise untreated cyclic rats. Lesions induced constant dioestrus for 14 +/- 0.7 days, but the animals were not pseudopregnant. 2 micrograms DA were also injected into the ZI of ovariectomised rats primed with 2 micrograms oestradiol benzoate 48 h before; this treatment stimulated a rise in plasma LH approximately 40 min later. These findings indicate that the dopaminergic incerto-hypothalamic tract which has nerve terminals in the ZI and AH has a stimulatory role in the control of gonadotrophin release.

Oestradiol synthesis by granulosa cells from immature rats treated with pregnant mare's serum gonadotrophin

Granulosa cells harvested from follicles in hypophysectomized or intact immature rats treated with 20 IU of pregnant mare's serum gonadotrophin (PMS) produced immunoreactive oestradiol (E2) when incubated in Krebs Ringer bicarbonate buffer containing an NADPH generating system; inclusion of steroid substrates in the medium increased the rate of synthesis. Further, tritiated E2 was synthesized when labelled progesterone was used as substrate. Granulosa cells removed from pre-ovulatory follicles on the morning of pro-oestrus in adult females also produced E2 in vitro. Although E2 synthesis was apparent by cells from immature hypophysectomized rats within 12 h of PMS treatment, it increased greatly with longer in vivo exposure to the gonadotrophin. Production was linear with the number of cells incubated and with time, at least through the first 30 min; the production rate decreased slightly with longer incubations. Exposure of the cells in vivo to hCG or ovine LH, before incubation, destroyed most of their ability to synthesize E2 even if progesterone or pregnenolone was added to the medium, but conversion of testosterone to E2 was reduced by only about 50%. Inhibitors of steroid synthesis, i.e. 4-OH-androstenedione, SU-10603, cyanoketone, or aminoglutethimide, greatly reduced the amount of E2 synthesized by the cells. The results indicate that granulosa cells exposed in vivo to gonadotrophins can synthesize E2 without the addition of androgenic substrate provided that cofactors are supplied. This finding has important implications for the current 'two cell' theory for oestrogen production by the ovary. A deficiency in steroidogenic enzymes within the granulosa cell appears to be an inadequate basis for the theory.(ABSTRACT TRUNCATED AT 250 WORDS)