Day Of Pro-oestrus Research Articles

Preovulatory secretion of steroids by cultured cumulus oophorus complexes of the rat: effects of FSH and LH.

The main objective of the present study was to establish whether a shift in steroid production, previously observed for preovulatory follicles, also takes place in preovulatory cumulus oophorus complexes (COCs). Female Wistar rats, displaying a regular 4-day oestrous cycle, were killed in succession every 2 or 3 h on the day of prooestrus and oestrus until ovulation (11.00-24.00 h). From excised ovaries preovulatory follicles were isolated. After puncturing cumuli oophori were aspirated and subsequently cultured for 24 h either in hormone-free or FSH- or LH- or FSH plus LH-supplemented medium. Cultured COCs released only a small amount of androgens, the main steroid produced being oestradiol. Its secretion decreased before ovulation (24.00 h). Relatively high progesterone release occured only in cultures set up at 22.00 and 24.00 h, thus during cumulus expansion. FSH and LH present in the medium effected above all oestradiol release, stimulating it before the presumptive endogenous gonadotrophin surge and inhibiting it thereafter. The activity of delta5-3beta-hydroxysteroid dehydrogenase (3betaHSD) investigated in cryostat sections appeared in COCs at 22.00h and was still present at 24.00 h. However, the activity was much weaker than in the granulosa cells lining the basal lamina. This in vivo study confirms in vitro results on more intense progesterone synthesis during cumulus expansion. The results indicate that in preovulatory COCs a shift in steroid production occurs after which the main steroid synthesized is progesterone, while oestradiol secretion decreases. However, this switch in COCs takes place later than the previously established shift in whole follicles.

Glial-neuronal-endothelial interactions are involved in the control of GnRH secretion.

In recent years compelling evidence has been provided that cell-cell interactions involving non-neuronal cells, such as glial and endothelial cells, are important in regulating the secretion of GnRH, the neuropeptide that controls both sexual development and adult reproductive function. Modification of the anatomical relationship that exist between GnRH nerve endings and glial cell processes in the external zone of the median eminence modulates the access of GnRH nerve terminals to the portal vasculature during the oestrous cycle. The establishment of direct neuro-haemal junctions between GnRH neuroendocrine terminals and the portal vasculature on the day of pro-oestrus may be critical for the transfer of GnRH upon its release into the fenestrated capillaries of the median eminence. Notwithstanding the importance of these plastic rearrangements, glial and endothelial cells also regulate GnRH neuronal function via specific cell-cell signalling molecules. While endothelial cells of the median eminence use nitric oxide to effect this regulatory control, astrocytes employ several growth factors, and in particular those of the EGF family and their erbB receptors to facilitate GnRH release during sexual development. Loss of function of each of these erbB receptors involved in the astroglial control of GnRH secretion leads to delayed sexual development. It is clear that regulation of GnRH secretion by cell-cell communication mechanisms other than transsynaptic inputs is an important component of the central neuroendocrine process controlling mammalian reproduction.

Changes in distribution of androgen receptor during maturation of rat ovarian follicles.

Sequential changes in androgen receptor (AR) distribution were investigated in rat ovarian follicles during their physiological development. Mature female Wistar rats, exhibiting a regular 4-day oestrous cycle, were killed in succession on the day of oestrus, metoestrus, dioestrus, and pro-oestrus. Excised ovaries were submitted to immunohistochemical procedure in which polyclonal androgen receptor antibody, avidin-biotin-peroxidase complex, and DAB were used. Strong AR immunostaining was located predominantly in the nuclei of the granulosa layer of preantral and very early antral follicles, present in the ovaries at all stages of the oestrous cycle. At early oestrus a decline in AR was noted in the mural granulosa cells of presumably recruited early antral follicles. The decline involved the area of appearing pseudostratification. During metoestrus and dioestrus AR decline proceeded towards the antrum, but the antral regions connected with COC by strings of granulosa cells or lying in close proximity to COC were always strongly AR-positive. It was only on the day of pro-oestrus that AR was confined to COCs and a few antral cells bordering the antrum. These findings indicate that during the oestrous cycle AR decline starts in the mural granulosa cells of oestrous antral follicles beginning to differentiate and is completed at pro-oestrus, but even before ovulation it does not extend to COC. The persistence of AR immunostaining in the latter region suggests that androgens can play here a paracrine role especially before ovulation. Atretic follicles showed a different pattern of AR distribution, dependent on their stage of development and the advancement of this process.

Apoptosis of postovulatory cumulus granulosa cells of the rat.

The process of apoptosis in the postovulatory cumulus granulosa cells was investigated in pregnant rats. Mature female Wistar rats, exhibiting a regular 4-day oestrous cycle, were placed with males on the day of pro-oestrus. The following day, on which spermatozoa were found in vaginal smears, was designated day 1 of pregnancy. The animals were killed just before ovulation (24.00 hours), on days 1 (5.00, 11.00, and 18.00 hours), and 2 ( 11.00 hours) of pregnancy. Excised ovaries and oviducts were submitted to a routine histological procedure and paraplast sections were subjected to detection of apoptotic cells using the TUNEL method. The cumulus granulosa cells of preovulatory follicles (24.00 hours) were negative for apoptotic staining. However, 5 h later a positive staining was observed in the oviduct ampulla and included the cumulus granulosa cells lying in the peripheral parts of postovulatory cumuli oophori, and the oviductal epithelial cells of this region. On the evening of day 1 almost all cumulus granulosa cells showed strong immunostaining while on day 2 at 11.00 hours only immunonegative clusters of remnants of cumulus granulosa cells were present in the distended ampulla region, while naked, two or more cell embryos were present in the further parts of oviduct. These results indicate that in the rat apoptosis of cumulus granulosa cells starts shortly after ovulation in the peripheral region. Epithelial ampullary cells surrounding ovulated cumuli show a massive apoptosis.

Oestrogens regulate pituitary alpha2,3-sialyltransferase messenger ribonucleic acid levels in the female rat.

Follicle-stimulating hormone (FSH) is synthesized by the anterior pituitary gland in multiple molecular forms. Increased acidic/sialylated FSH charge isoforms are associated with conditions characterized by a low oestrogen output. In the present study, we analysed the dynamics of the changes in mRNA levels of the enzyme Galbeta1,3[4]GlcNAc alpha2,3-sialyltransferase (2,3-STase) (one of the enzymes that incorporate sialic acid residues into the FSH molecule) in intact and ovariectomized rats. The anterior pituitaries of 4-day regularly cyclic adult female Wistar rats were obtained at 1000 h on the days of pro-oestrus (P), oestrus (O), dioestrus 1 (D1) and dioestrus 2 (D2), at 0200 h, 1400 h, 1800 h and 2200 h on D1, at 1800 h on day of O and at 1000 h after 7, 14, 21, 28 and 45 days of oophorectomy performed on the morning of P. Total RNA was isolated from each gland and the 2,3-STase levels were measured by Northern blot hybridization analysis employing a 346-base pair cDNA probe encoding for a non-conserved amino acid sequence of the catalytic domain of the enzyme. Maximal levels of the enzyme mRNA were detected at 1000 h on D1; thereafter, they progressively decreased by 60% during the ensuing 24 h, reaching the lowest concentration values (26% of the maximally observed level on D1) at 1000 h on day of P and remaining unchanged during the morning of O. Administration of the potent oestradiol receptor antagonist ICI 182,780 at 1000 h on D1 completely reverted the time-dependent decrease in 2,3-STase mRNA levels observed during the afternoon of D1, whereas oestradiol benzoate administered at 1000 h on day of O significantly reduced the enzyme mRNA levels (to 21% of the levels detected in vehicle-treated controls). In ovariectomized rats, the alpha2,3-STase mRNA progressively increased from day 21 to day 45 post castration. Administration of oestradiol benzoate on day 28 after oophorectomy significantly reduced the 2,3-STase mRNA levels (to 36% of the levels detected in vehicle-injected controls); ICI 182,780 partially counteracted this oestradiol-mediated effect. The dynamics of these changes in 2,3-STase mRNA levels partially correlated with changes in the relative abundance of the FSH charge isoforms separated by preparative chromatofocusing of anterior pituitary extracts, particularly in glands obtained during the morning of P and O. These data demonstrate for the first time that pituitary 2,3-STase is a hormonally-regulated enzyme and that the changes in transcription and/or stability of its mRNA may be involved, in part, in the post-translational processing of the FSH molecule during certain physiological conditions.

Stimulation of matrix-metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 gene expression in rats by the preovulatory prolactin peak.

Since structural luteolysis involves deterioration of tissue, the gene expression of matrix-metalloproteinase-1 (MMP-1) and the respective tissue inhibitor of this metalloprotease (TIMP-1) were measured at various times on the day of pro-oestrus and in animals in which the preovulatory prolactin surge was blocked for the duration of 3 cycles by bromocriptine. An additional group of prolactin-blocked rats received a prolactin replacement injection on the afternoon of pro-oestrus. In spontaneously pro-oestrous rats, MMP-1 and TIMP-1 gene expression increased significantly (P<0.01) prior to the occurrence of the preovulatory LH surge but simultaneously with the onset of the preovulatory prolactin surge. When prolactin release was blocked by bromocriptine for 3 cycles, no such changes were observed during the afternoon of pro-oestrus. However, an intraperitoneal injection of bovine prolactin at the time when the preovulatory prolactin surge occurs normally, increased MMP-1 and TIMP-1 gene expression (P<0.01). These results indicate that MMP-1 and TIMP-1 gene expression are stimulated by the preovulatory prolactin surge. Previous work has shown that the preovulatory LH surge activates the enzymatic cascade which leads to increased collagenase activity.

Granulocyte-macrophage colony-stimulating factor enhances interleukin-1β stimulated histamine release in the preovulatory rat ovary

The existence of immune cells including macrophages and mast cells in rat ovary implies that various cytokines from these cells may play a role in ovarian functions. The aim of the present study was to investigate whether granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-1β are capable of stimulating histamine release and steroidogenesis in rat ovary, and to determine the sites of histamine production in the ovary. Histamine release from preovulatory ovarian tissues was stimulated in a dose-dependent manner at 3–30 ng/ml of GM-CSF in the presence of interleukin-1β (10 ng/ml). However, treatment with GM-CSF and interleukin-1β did not cause any significant change in the levels of ovarian steroids. Intense staining of histidine decarboxylase in the ovary was immunohistochemically detected in large granular cells on the morning of the pro-oestrus day. These results indicate that GM-CSF may be involved in the regulation of ovarian histamine secretion in mast cells partially by enhancing interleukin-1β-induced histamine release in the process of ovulation.

Gonadotrophin-releasing hormone and oxytocin secretion from the hypothalamus in vitro during pro-oestrus: the effects of time of day and melatonin

An accurately timed surge of luteinizing hormone (LH), during the second half of the day of pro-oestrus in rats, is a crucial part of the endocrine signal that leads to expulsion of an ovum from an ovarian follicle. LH release is partly controlled by a number of peptides, including gonadotrophin-releasing hormone (GnRH) and oxytocin, which travel from the hypothalamus to the pituitary. The profile of secretion of these peptides is poorly understood. Therefore, the amounts of GnRH and oxytocin that were secreted from hypothalamic explants were determined at several time points during the day of pro-oestrus. Basal secretion of oxytocin from hypothalami taken later in pro-oestrus was greater than from hypothalami taken earlier in the day ( p < 0.02). On the other hand, basal secretion of GnRH decreased during the day of pro-oestrus ( p < 0.03). The different trends of GnRH and oxytocin secretion reveal that their secretion is regulated by distinct mechanisms. GnRH secretion was higher at midpro-oestrus than late in the day ( p < 0.05) consistent with a peak of GnRH having been observed by others in portal blood in the second half of the day of pro-oestrus. Responsiveness of oxytocin to stimulation by K + of the hypothalami declined from the early light hours to the evening dark hours ( p < 0.02). Thus, oxytocin modulation might be achieved partly by modification of intracellular processes. Melatonin, secreted during hours of darkness, is frequently involved in modulating time-dependent events in mammals, but its contribution to peptide regulation during the ovulatory cycle is unclear. Melatonin was observed to inhibit basal oxytocin secretion from hypothalami collected during light hours ( p < 0.05). The investigation has, therefore, revealed the potential for melatonin to modulate peptide secretion from the hypothalamus during the day of pro-oestrus. We also observed that secretion from the hypothalamus of the two LH-regulating peptides, GnRH and oxytocin, are differently regulated during the day of pro-oestrus.

Effects of DPDPE (a specific delta-opioid receptor agonist) and naloxone on hypothalamic monoamine concentrations during the pre-ovulatory LH surge in the rat.

We have investigated the inter-relationship between the opioid and aminergic systems in the control of secretion of the pro-oestrous LH surge and the involvement of delta-opioid receptor subtypes in this process. Conscious female rats bearing a cannula in the femoral artery were injected i.p. with a selective delta-opioid receptor agonist (DPDPE) either alone or with the opioid antagonist (naloxone) at 1300 h on the day of pro-oestrus. Blood samples were collected hourly between 1500 h and 1900 h, and plasma LH levels were measured by RIA. At the end of this period (1900 h), the animals were autopsied and the concentrations of the amines (noradrenaline (NA), dopamine (DA), 5-hydroxytryptamine (5HT)) and their metabolites (dihydroxyphenolglycol (DHPG) and 5-hydroxyindoleacetic acid (5HIAA), metabolites of NA and 5HT respectively) were determined by HPLC with electrochemical detection in the medial preoptic area, suprachiasmatic nucleus, median eminence and arcuate nucleus. DPDPE abolished the LH surge and concomitantly decreased hypothalamic NA and DHPG concentrations in all the areas examined. The levels of DA, 5HT and 5HIAA were also reduced in all hypothalamic regions studied, except DA and 5HIAA in the suprachiasmatic nucleus. Naloxone reversed these inhibitory effects of the delta-agonist. We conclude that activation of delta-opioid receptors may exert an inhibitory effect on LH release. The effect is probably an indirect one mediated by the monoaminergic systems, as they are suppressed by DPDPE in nearly all the hypothalamic regions studied.

Immunoneutralization of follicle stimulating hormone does not affect gonadotrophin surge-inhibiting factor/attenuating factor bioactivity during the rat ovarian cycle.

The physiological role of follicle stimulating hormone (FSH) in the regulation of the release of the putative ovarian factor gonadotrophin surge-inhibiting or -attenuating factor (GnSIF/AF) was investigated. Blood FSH concentrations were immunoneutralized in female rats during different days of the ovarian cycle. FSH antiserum was injected in different protocols to neutralize the FSH surge(s) and/or basal FSH concentrations. All animals were killed on the morning of the day of pro-oestrus of the control rats. Hemi-pituitaries were incubated and gonadotrophin-releasing hormone (GnRH) stimulated and basal luteinizing hormone (LH) and FSH releases and pituitary contents were measured. The biological effectiveness of the FSH antiserum was established by monitoring the histology of vaginal smears and inhibin concentrations in trunk-blood. The predominance of small leukocytes in the smears (low oestradiol bioactivity) and the decreased (bioactive) inhibin secretion imply that anti-FSH neutralizes FSH bioactivity. Simultaneously, the results showed that all anti-FSH injections did not affect the biphasic LH response to GnRH in vitro, which shows an unaltered presence of GnSIF/AF bioactivity in the blood circulation. The results suggest that under physiological conditions GnSIF/AF bioactivity, which keeps the LH responsiveness of the pituitary gland suppressed to the action of GnRH, is not supported by inhibin and not controlled by FSH.

Differential effects of ovarian local anaesthesia during pro-oestrus on ovulation by the right or left ovary in normal and hemi-ovariectomized adult rats.

The effects on spontaneous ovulation in normal and hemi-ovariectomized cyclic rats of an injection of saline or a local anaesthetic (2-diethylamine-2',6'-acetoxilidide hydrochloride with adrenaline, Xylocaine; administered at 13:00 h) into the bursa ovarica, were studied to investigate the participation of ovarian innervation in the regulation of the response of ovarian follicles to endogenous gonadotrophins on the day of pro-oestrus. The injection of saline or Xylocaine into both ovaries did not affect ovulation by the right ovary. Bilateral injection of Xylocaine did not affect ovulation by the left ovary. However, when saline was injected into the right ovary and Xylocaine into the left ovary, the number of ova shed by the left ovary decreased significantly (0.9 +/- 0.7 versus 4.4 +/- 0.2, P < 0.01; Mann-Whitney U test). In hemi-ovariectomized animals, when the right ovary was anaesthetized, ovulation was similar to that in saline-treated animals, and when the right ovary was anaesthetized, ovulation was blocked (one of six animals ovulated). The injection of hCG at 14:00 h to hemi-ovariectomized rats did not restore ovulation, whereas all the rats ovulated when anaesthesia occurred at 18:00 h. The present results suggest that on the afternoon of the day of pro-oestrus, before the LH peak, a neural signal modulates the response of the left ovary to gonadotrophins.

Intracellular antagonistic interaction between GnRH and gonadotrophin surge-inhibiting/attenuating factor bioactivity downstream of second messengers involved in the self-priming process.

The antagonistic control of LH concentrations by GnRH and the putative ovarian factor gonadotrophin surge-inhibiting or -attenuating factor (GnSIF/AF) was studied by perifusion of female rat pituitary glands in vitro. LH release and GnRH self-priming were measured in response to five (0.32-10 nmol l-1) or three (10 nmol l-1) 10 min pulses of GnRH. In the latter case, pulses were preceded by five 10 min pulses of either 1 mmol 8-bromo-cAMP l-1 plus 10 mmol theophylline l-1 or 1 mumol phorbol 12-myristate 13-acetate l-1. The stimulations were carried out in the presence or absence of steroid-free bovine follicular fluid, which possesses GnSIF/AF bioactivity, to study the effect of GnSIF/AF on the self-priming process during successive stimulations by GnRH. First, the effect of follicular fluid was studied on GnRH-induced LH release from pituitary glands collected during the ovarian cycle. Only when a clear self-priming effect was evident, as on day 2 of dioestrus and the day of pro-oestrus, follicular fluid antagonized the self-priming effect of GnRH. Second, when glands on day 2 of dioestrus were used, self-priming was displayed by various combinations of GnRH and follicular fluid. The pulses with the lowest concentrations of GnRH together with a high concentration of follicular fluid, however, led to stable low amplitude LH pulses. Finally, priming of the pituitary LH response to GnRH with 8-bromo-cAMP plus theophylline or phorbol 12-myristate 13-acetate was inhibited by follicular fluid. These results confirm the control of LH release by GnRH and GnSIF/AF bioactivity. The effect of GnSIF/AF in follicular fluid is most pronounced on the days before the LH surge, showing evidence of its important role in maintaining low LH concentrations during this period. GnSIF/AF neutralizes downstream actions of second messengers involved in GnRH self-priming.

Proliferative activity of preovulatory follicles and newly formed corpora lutea in cycling rats from late prooestrus to early oestrus

Ovaries from adult cycling rats were studied from 1600 h on the day of prooestrus to 0700 h on the day of oestrus in order to relate the cyclic hormonal changes to the proliferative activity of preovulatory and postovulatory (i.e. newly-formed corpora lutea) follicles. Proliferative activity was studied by the immunohistochemical demonstration of DNA-incorporated 5-bromodeoxyuridine (BrdU). The proliferative activity of granulosa cells (GC) in large preovulatory follicles showed a centripetal pattern and decreased during prooestrus, reaching a minimum at 2100 h. However, a proliferative wave was found in the GC of preovulatory follicles at 0200 h on the day of oestrus and in those of newly-formed corpora lutea at 0700 h on the day of oestrus. These results suggest that the granulosa cells of preovulatory follicles show maturational changes that followed a different pattern, depending on their location within the follicle, and that the proliferative wave found from 0200 to 0700 h on oestrus is important for the establishment of the number of steroidogenic cells in the cyclic corpus luteum.

Proliferative activity of preovulatory follicles and newly formed corpora lutea in cycling rats from late prooestrus to early oestrus

Ovaries from adult cycling rats were studied from 1600 h on the day of prooestrus to 0700 h on the day of oestrus in order to relate the cyclic hormonal changes to the proliferative activity of preovulatory and postovulatory (i.e. newly-formed corpora lutea) follicles. Proliferative activity was studied by the immunohistochemical demonstration of DNA-incorporated 5-bromodeoxyuridine (BrdU). The proliferative activity of granulosa cells (GC) in large preovulatory follicles showed a centripetal pattern and decreased during prooestrus, reaching a minimum at 2100 h. However, a proliferative wave was found in the GC of preovulatory follicles at 0200 h on the day of oestrus and in those of newly-formed corpora lutea at 0700 h on the day of oestrus. These results suggest that the granulosa cells of preovulatory follicles show maturational changes that followed a different pattern, depending on their location within the follicle, and that the proliferative wave found from 0200 to 0700 h on oestrus is important for the establishment of the number of steroidogenic cells in the cyclic corpus luteum.

The steroid antagonist RU486 given at pro-oestrus induces hypersecretion of follicle-stimulating hormone from oestrus afternoon to early metoestrus in the rat.

Administration of the steroid antagonist RU486 to cyclic rats at pro-oestrus blunts the preovulatory surge of LH and suppresses the first and second surges of FSH. In addition, administration of oestradiol to RU486-treated rats reactivates the LH surge the following day. The present study explored the effects of RU486 (4 mg/0.2 ml oil), administered at 0800 h on the day of pro-oestrus, on serum FSH and LH concentrations through oestrus and early metoestrus. RU486 induced a hypersecretion of FSH, which started at 1400 h on the day of oestrus and was maintained until 0800 h on the day of metoestrus. Because the timing and magnitude of this secretion of FSH were similar to those of the periovulatory secretion of FSH during pro-oestrus and early oestrus in intact cyclic rats, we investigated the effects of: 1) LHRH antagonist (LHRHa) injected at either 0900 h or 2000 h on the day of oestrus, 2) oestradiol benzoate injected at 1600 h on the day of pro-oestrus and at 0900 h on the day of oestrus, 3) bovine follicular fluid (bFF) given either at 1100 h or at 2000 h on the day of oestrus, or 4) adrenalectomy (ADX) at 1100 h on the day of oestrus, on serum FSH and LH concentrations at 1800 h on the day of oestrus and at 0200 h on the day of metoestrus in rats injected with RU486 at pro-oestrus. The results showed that 1) both components (late oestrus and early metoestrus) of FSH hypersecretion in RU486-injected rats in pro-oestrus were inhibited by oestradiol benzoate and bFF, 2) the metoestrous component was not affected by LHRHa, whereas the oestrous component was partially reduced, and 3) ADX partially reduced serum FSH concentrations only on the day of metoestrus, possibly because, as the serum concentrations of corticosterone reflected, the antiglucocorticoid activity of 4 mg RU486 lasted only 24 h. The results support the hypothesis that blockade of progesterone actions at pro-oestrus results in the maintenance of the daily neural signal that activates the release of gonadotrophins. Whereas the expression of LH secretion requires high levels of oestradiol, FSH secretion is expressed against a background of low oestradiol levels. The results of this study also indicate that the release of FSH during oestrus and metoestrus in rats injected with RU486 at pro-oestrus is a consequence of the lack of ovarian negative feedback inhibition on the pituitary.

Inhibitory effect of a new steroidal saponin, OSW-1, on ovarian functions in rats.

1. This study was undertaken to determine the effects of OSW-1 (3 beta, 16 beta, 17 alpha-trihydroxycholest-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-beta-D-xylopyranosyl)- (1-->3)-(2-O-acetyl-alpha-L-arabinopyranoside)) on the pituitary-ovarian system and the functions of aortic smooth muscle. 2. A single s.c. injection of OSW-1 (9 micrograms kg-1) on the morning of pro-oestrus inhibited the occurrence of the expected next pro-oestrus, whereas administration of OSW-1 at a dose of 4.5 micrograms kg-1 did not affect the oestrous cycle. OSW-1 treatment on the day of dioestrus-1 did not affect the oestrous cycle. 3. At doses of 4.5 and 9 micrograms kg-1 OSW-1, the serum oestradiol (E2) levels at the expected next pro-oestrus were significantly lower than in control (pro-oestrus). The serum luteinizing hormone (LH) levels 4 days after 9 micrograms kg-1 OSW-1 treatment were also markedly lower than those of control. OSW-1 (4.5 micrograms kg-1) did not affect the levels of inhibin, progesterone and gonadotrophins on the same day. 4. OSW-1 did not inhibit the preovulatory LH surge which occurs on the afternoon of pro-oestrus day. 5. The expression of mRNA coding for the cholesterol side chain cleavage cytochrome P-450 (p450scc), an ovarian steroidal limiting enzyme, was suppressed at 24 and 96 h after OSW-1 treatment. 6. Administration of OSW-1 (9 micrograms kg-1) tended to reduce the relaxation of isolated thoracic aorta ring preparations induced by acetylcholine, while there was no difference in the relaxation induced by sodium nitroprusside. 7. Our results show that OSW-1 inhibits ovarian E2 secretion and that the decrease in E2 secretion may contribute to its effects on the oestrous cycle and the sensitivity of the thoracic aorta to relaxation. The decrease in the levels of ovarian steroids induced by OSW-1 may be due to its direct inhibitory action on the gene expression of the steroidal enzyme and on the proliferation of granulosa cells in the ovary.

Induction of superovulation in cyclic rats by administration of decreasing doses of recombinant follicle stimulating hormone (Org32489).

The objective of this study was to set up a superovulation protocol in adult cyclic rats by using recombinant human follicle stimulating hormone (rhFSH; Org32489). Good results were obtained by treatment with decreasing doses of rhFSH (2.5 to 0.5 IU) during the dioestrus period. The number of corpora lutea (CL) found in rats treated with this protocol was 43.5 +/- 3.4; this is more than three times the number in saline-treated control rats (13.0 +/- 0.4). Fertilization of oocytes after superovulation was as good as after normal ovulation in terms of number of 2-cell stage embryos found 2 days after mating. The absolute number of implantations was twice the number observed in saline-treated control rats (23.3 +/- 1.8 versus 10.6 +/- 0.5); therefore the number of implantations per CL was lower in superovulated rats. The serum concentrations of luteinizing hormone (LH), endogenous FSH and oestradiol-17beta were decreased during rhFSH treatment, while the inhibin serum concentration was increased. The progesterone serum concentration was increased on the days of pro-oestrus and oestrus after treatment. No difference was observed in the testosterone serum concentration. Pretreatment with 10 IU rhFSH at oestrus before giving the decreasing doses of rhFSH during dioestrus reduced the ovulatory response. Finally, treatment with a constant low dose of rhFSH instead of a decreasing dose of rhFSH did not result in spontaneous ovulation. However, ovulation induction by means of a human chorionic gonadotrophin bolus resulted in superovulation in six out of eight rats. It is concluded that superovulation in cyclic rats can be achieved using rhFSH treatment. However, it was found that the type of rhFSH regimen was very important to achieve appropriate stimulation. The optimal protocol was treatment with decreasing doses of rhFSH during dioestrus. The oocytes retrieved could be fertilized as well as oocytes of saline-treated control rats. The results also indicate that treatment with higher doses of rhFSH might induce a desensitization for FSH and LH.

Effect of restraint stress on the preovulatory luteinizing hormone profile and ovulation in the rat.

Plasma profiles of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured during restraint stress on the day of pro-oestrus; these profiles were considered in relation to ovulation rate on the next day. Rats bearing a permanent jugular vein cannula were subjected to restraint, which was started 0, 1 or 2 h before the presumed onset of the LH surge and ended just before the beginning of the dark period. Exposure to restraint resulted in a suppression of the secretion of both gonadotrophins on the day of pro-oestrus. Suppression of the LH surge was virtually complete (plasma LH < or = 0.2 ng/ml) in 15 out of 32 stressed rats, and the ovaries of these rats contained graafian follicles with oocytes in germinal vesicle stage. In these rats, the LH surge did not occur 24 h later. In the remaining 17 rats, restraint resulted in a considerable suppression of the LH surge. Of these rats, five had an ovulation rate of 100% and four ovulated partially. In unruptured follicles of the latter, the oocyte had not resumed meiosis and the follicle wall was not luteinized. In the remaining eight rats with a reduced LH surge, ovulations had not occurred and graafian follicles were unaffected. The results of this study indicate that during pro-oestrus restraint stress suppresses and does not delay the release of preovulatory gonadotrophins. Partial suppression of LH by restraint does not result in induction of meiotic resumption without subsequent ovulation or in luteinized unruptured follicles.

Effect of hypothyroidism on the pituitary-gonadal axis in the adult female rat.

The pituitary-ovarian axis was studied after withdrawal of thyroid hormone in 131I-radiothyroidectomized adult female rats. Oestrous cycles became prolonged and irregular within 2 weeks after the supply of thyroid hormone was stopped. If an LH surge occurred in hypothyroid rats on the day of vaginal pro-oestrus it was significantly greater in rats which had been made hypothyroid for 4-5 weeks than in controls; in hypothyroid rats with an LH surge on pro-oestrus, plasma progesterone showed a rise similar to that in controls at pro-oestrus; the ovulation rate was decreased in hypothyroid rats. About half of the rats from which blood was sampled daily in the afternoon between 7 and 18 days after tri-iodothyronine (T3) withdrawal had 1 day of pro-oestrus; on this day the LH surge was higher than in controls. On days 2 and 1 before and days 1 and 2 after this pro-oestrus, plasma progesterone was similar to that of controls on days 2 and 1 before and days 1 and 2 after pro-oestrus respectively. However, progesterone was higher in the period before and after these days. The other hypothyroid rats showed no pro-oestrus and no LH surge during this period, while their plasma progesterone levels were high on all days. On the morning of day 10 after T3 withdrawal and 5 days after the preceding pro-oestrus, most hypothyroid rats had high progesterone and low oestradiol plasma levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of RU486 on ovarian progesterone production at pro-oestrus and during pregnancy: a possible dual regulation of the biosynthesis of progesterone

Changes in progesterone production were analysed after intrabursal ovarian administration of the antiprogesterone RU486, mifepristone, in rats at pro-oestrus and during pregnancy. RU486 was administered at 09:00-10:00 h and serum progesterone was measured 8 h after treatment, except for those on days 3 and 12 of pregnancy when the steroid was measured 4, 8 and 24 h later. RU486 stimulates progesterone production on the day of pro-oestrus and on days 3-5 and 15-20 of pregnancy. Conversely, treatment with the antiprogestagen inhibits progesterone production on days 7-14 of gestation. The inhibition (day 12) or stimulation (day 19) of progesterone production induced by RU486 could be correlated with the simultaneous inhibition or stimulation of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity observed in corpora lutea. A similar effect on 3 beta-HSD activity in corpora lutea was obtained by intrabursal ovarian administration of a specific progesterone antibody, indicating that the effect of RU486 is exerted through its antiprogesterone action. On the day of pro-oestrus and during early pregnancy the intrabursal ovarian injection of RU486 did not modify serum prolactin and LH concentrations, demonstrating that the antiprogesterone did not have a central action. The stimulatory action of RU486 on progesterone production on days 3 and 5 of pregnancy shifted to an inhibitory effect on progesterone production on day 7. Oestradiol treatment on day 6 of pregnancy reversed the effect of RU486 on progesterone production on day 7, inducing a response similar to that obtained on days 3 and 5 of gestation.(ABSTRACT TRUNCATED AT 250 WORDS)