Effects Of Ovarian Steroids Research Articles

The effects of estrogen and progesterone on corticotropin-releasing hormone and arginine vasopressin messenger ribonucleic acid levels in the paraventricular nucleus and supraoptic nucleus of the rhesus monkey.

Ovarian steroids increase hypothalamic-pituitary-adrenal (HPA) axis activity and sensitize the hypothalamic-pituitary-ovarian (HPO) axis to stress-induced inhibition. The present study investigated the effect of ovarian steroids on CRH and arginine vasopressin (AVP) messenger RNA (mRNA) levels in the rhesus monkey hypothalamus, as both neuropeptides have been shown to stimulate the HPA axis and inhibit the HPO axis in this species. This was accomplished by measuring CRH and AVP mRNA in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) by in situ hybridization histochemistry. Menstrual cycles were simulated in ovariectomized (OVX) rhesus monkeys by sequential addition and removal of SILASTIC brand (Dow Corning Corp.) tubing containing either 17beta-estradiol (E2) or progesterone (P4). On the morning of day 11 of the simulated follicular phase (E2 alone) or day 21 of the luteal phase (E2 + P4), animals were anesthetized, and the brains were perfused with paraformaldehyde via the carotid artery. Coronal sections (30 microm) were cut, and mRNA for CRH and AVP in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) were semiquantified by in situ hybridization. CRH mRNA in the PVN of E2-replaced OVX animals (n = 7) was 2-fold greater than that in untreated OVX controls (n = 4), whereas CRH mRNA after E2 + P4 (n = 4) was no different from that in controls (optical density + SEM, 0.38 +/- 0.06, 0.13 +/- 0.08, and 0.14 +/- 0.09 for OVX + E2, OVX + E2 + P4, and OVX, respectively; P = 0.02). CRH in the SON was undetectable. In contrast to CRH, AVP mRNA in the PVN and the SON was similar in the three treatment groups. We conclude that E2 and E2 + P4 replacement to OVX monkeys exert different effects on CRH and AVP gene expression, as estrogen stimulation of CRH mRNA in the PVN was abrogated by progesterone, whereas no effect of ovarian steroids on AVP mRNA in either the PVN or SON was observed. We postulate that ovarian steroid regulation of CRH synthesis and release may in part explain the central nervous system mechanisms by which ovarian steroids affect the HPA and HPO axes during basal and stress conditions.

The Effects of Estrogen and Progesterone on Corticotropin-Releasing Hormone and Arginine Vasopressin Messenger Ribonucleic Acid Levels in the Paraventricular Nucleus and Supraoptic Nucleus of the Rhesus Monkey

Ovarian steroids increase hypothalamic-pituitary-adrenal (HPA) axis activity and sensitize the hypothalamic-pituitary-ovarian (HPO) axis to stress-induced inhibition. The present study investigated the effect of ovarian steroids on CRH and arginine vasopressin (AVP) messenger RNA (mRNA) levels in the rhesus monkey hypothalamus, as both neuropeptides have been shown to stimulate the HPA axis and inhibit the HPO axis in this species. This was accomplished by measuring CRH and AVP mRNA in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) by in situ hybridization histochemistry. Menstrual cycles were simulated in ovariectomized (OVX) rhesus monkeys by sequential addition and removal of SILASTIC brand (Dow Corning Corp.) tubing containing either 17β-estradiol (E2) or progesterone (P4). On the morning of day 11 of the simulated follicular phase (E2 alone) or day 21 of the luteal phase (E2 + P4), animals were anesthetized, and the brains were perfused with paraformaldehyde via the carotid artery. Coronal sections (30 μm) were cut, and mRNA for CRH and AVP in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) were semiquantified by in situ hybridization. CRH mRNA in the PVN of E2-replaced OVX animals (n = 7) was 2-fold greater than that in untreated OVX controls (n = 4), whereas CRH mRNA after E2 + P4 (n = 4) was no different from that in controls (optical density ± sem, 0.38 ± 0.06, 0.13 ± 0.08, and 0.14 ± 0.09 for OVX + E2, OVX + E2 + P4, and OVX, respectively; P = 0.02). CRH in the SON was undetectable. In contrast to CRH, AVP mRNA in the PVN and the SON was similar in the three treatment groups. We conclude that E2 and E2 + P4 replacement to OVX monkeys exert different effects on CRH and AVP gene expression, as estrogen stimulation of CRH mRNA in the PVN was abrogated by progesterone, whereas no effect of ovarian steroids on AVP mRNA in either the PVN or SON was observed. We postulate that ovarian steroid regulation of CRH synthesis and release may in part explain the central nervous system mechanisms by which ovarian steroids affect the HPA and HPO axes during basal and stress conditions.

The Effect of Ovarian Steroids on Epithelial Ciliary Beat Frequency in the Human Fallopian Tube

Using a method that detects variations in light intensity we have studied the effect of ovarian steroids on human Fallopian tube epithelial ciliary beat frequency in vitro. We have found that baseline ciliary beat frequency averages between 5-6 Hz. Cilia from ampullary segments of the Fallopian tube beat significantly faster (5.4 Hz+/-0.2) than those from fimbrial segments (4.8 Hz+/-0.2). There was no significant difference in baseline ciliary beat frequency at any other anatomical site in the Fallopian tube. Incubation with progesterone (10 micromol/l) suppresses human Fallopian tube epithelial ciliary beat frequency by 40-50%. This inhibition was observed at similar magnitudes in all Fallopian tubes studied irrespective of anatomical site. Progesterone-induced reductions in ciliary beat frequency were concentration dependent and prevented by the progesterone receptor antagonist mifepristone (RU486). Oestradiol alone (10 micromol/l) had no effect on ciliary beat frequency at any anatomical site in the Fallopian tube but did prevent the reduction in ciliary beat frequency seen with progesterone when tissues were incubated with these two steroids together.

Effect of ovarian steroids and oxytocin on the production of prostaglandin E2, prostaglandin F2alpha and endothelin-1 from cow oviductal epithelial cell monolayers in vitro.

Cyclic physio-anatomical variation in the oviducts is mediated by the local countercurrent transfer of ovarian products. Thus, in this study cow oviductal epithelial cells (COEC) culture were utilized to investigate the effects of ovarian products such as progesterone (P4), estradiol 17beta (E2) and oxytocin (OT) on local oviductal prostaglandin E2 (PGE2), F2alpha (PGF2alpha) and endothelin-1 (ET-1) production. COEC were collected from non-pregnant Holstein cows (n = 8) during the follicular phase and cultured in M199 under standard culture conditions until monolayer formation. Cells in first passage were incubated for 24 or 48 h with P4 (500 ng/ml), E2 (1 ng/ml), OT (10(-9) M) or combination of E2 + P4. Administration of E2 significantly increased the production of PGE2, PGF2alpha and ET-1. However, simultaneous administration of P4 blocked the effect of E2. OT did not show any effect on oviductal productions of either PGs or ET-1. The results of this study show that E2 stimulates PG and ET-1 production by COEC in vitro. Thus, it can be suggested that locally transferred E2 from the ovarian follicles may be important for oviductal contraction and gamete/zygote transport during the peri-ovulatory period.

The differential effects of oestrogens and progestins on vascular tone.

The purpose of this paper is to present reported findings of the effects of ovarian steroids on vascular tone. The medical literature was reviewed for relevant contributions. Oestrogen replacement therapy in postmenopausal women is associated with a reduction in mortality from coronary artery disease. Many different cellular actions have been described which help explain the cardioprotective effects of oestrogens, and among these are effects on vascular tone. Oestrogens induce vasodilation through mechanisms involving the arterial endothelium and through endothelial-independent actions. Progestins have varying effects on arterial tone, including induction of vascular smooth muscle relaxation as well as induction of smooth muscle constriction. The effects of oestrogens and progestins on vascular tone are clinically meaningful. Pathophysiological arterial conditions, including angina pectoris and migraine headaches, have been associated with oestradiol deficiency and improvement has been associated with oestradiol replacement. Women with coronary artery disease show improved arterial vasodilator responses after oestradiol treatment which can be reduced by the addition of progestin treatment. Androgens are also vasoactive. Study of the effects of ovarian hormones on vascular tone has become an important area for basic and clinical research.

Hormonal Regulation of Glutamate Receptor Gene Expression in the Anteroventral Periventricular Nucleus of the Hypothalamus

Glutamate plays an important role in mediating the positive feedback effects of ovarian steroids on gonadotropin secretion, and the preoptic region of the hypothalamus is a likely site of action of glutamate. The anteroventral periventricular nucleus (AVPV) of the preoptic region is an essential part of neural pathways mediating hormonal feedback on gonadotropin secretion, and it appears to provide direct inputs to gonadotropin releasing hormone (GnRH)-containing neurons. Immunohistochemistry and in situ hybridization were used in this study to define the distribution and hormonal regulation of glutamate receptor subtypes in the AVPV of juvenile female rats. Neurons that express the NMDAR1 receptor subtype are abundant in the AVPV, as are cells that express AMPA receptor subtypes (GluR1, GluR2, and GluR3 but not GluR4), and the AVPV appears to contain a dense plexus of NMDAR1-immunoreactive presynaptic terminals. However, AVPV neurons do not seem to express detectable levels of kainate receptor (GluR5, GluR6, and GluR7) or metabotropic receptor (mGluR1-6) subtypes. Treatment of ovariectomized juvenile rats with estradiol induced expression of GluR1 mRNA but did not alter levels of GluR2 or GluR3 mRNA. Treatment of estrogen-primed ovariectomized juvenile rats with progesterone caused an initial increase in GluR1 mRNA expression, followed by a small decrease 24 hr after treatment. In contrast, estrogen appears to suppress levels of NMDAR1 mRNA in the AVPV, which remained unchanged after progesterone treatment. Thus, one mechanism whereby ovarian steroids may provide positive feedback to GnRH neurons is by altering the sensitivity of AVPV neurons to glutamatergic activation.

Participation of the opioid system in the regulation of prolactin secretion in androgenized rats: effect of ovarian steroids

We examined the role of the opioid system on the regulation of prolactin secretion in neonatally androgenized rats and evaluated the participation of ovarian steroids in this regulation. Androgenized rats exhibited an increase of prolactin secretion with higher serum circulating levels in the afternoon (1800) than in the morning (1000). The administration of the opioid antagonist naloxone (2 mg/kg, 30 min before decapitation) reduced serum prolactin levels in both groups. To identify the opioid receptor subtypes involved in this regulation, opioid agonists were administered i.c.v. 15 min before the decapitation (1000). The μ-opioid receptor agonist DAMGO ([ d-Ala 2, NMe-Phe 4, Gly 5-ol]-enkephalin) caused a significant increase in serum prolactin concentration. The selective κ-opioid receptor agonist U-50, 488H ( trans-(±)-3,4-dichloro- N-[2(1-pyrrolidinyl)-cyclohexyl]-benzene acetamide methane sulfonate salt) induced a small but significant increase in serum prolactin levels but no effect was observed after administration of the δ-opioid agonist DPDPE ([ d-Pen 2, d-Pen 5]-enkephalin). The role of oestradiol and the opioid system in the continuous secretion of prolactin was also study. Chronic gonadectomy (3–4 weeks) reduced serum prolactin concentrations measured at 1000 but the administration of naloxone had no effect. Three days of oestrogen treatment (2 μg/rat in oil) restored serum prolactin levels compared with ovariectomized animals and this effect was abolished by naloxone treatment. Interestingly, acute ovariectomy or administration of tamoxifen to intact androgenized rats did not prevent the continuous secretion of prolactin observed in these animals and naloxone treatment reduced serum prolactin levels in both groups of rats. We also examine the participation of adrenal progesterone and the endogenous opioid peptides on the regulation of prolactin levels in androgenized rats. After adrenalectomy, no changes in serum prolactin levels (1000) were observed compared with the control animal and naloxone treatment significantly reduced circulating prolactin levels. Progesterone treatment to intact androgenized rats significantly increased prolactin levels and the administration of naloxone blocked the stimulatory effect of the steroid. These results suggest that the opioid system play a role in the regulation of prolactin secretion in androgenized rats modulated by the persistence of oestrogen action. Moreover, the presence or absence of progesterone did not modify the regulation of prolactin secretion by the opioids. The μ- and κ-opioid receptor subtypes are the ones involved in the modulation of pituitary prolactin secretion.

The Expression of Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) and Its Regulation by Ovarian Steroids in Rat Uterine Stromal Cells

The effects of ovarian steroids on the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in rat uterus were examined. Intense expression of GM-CSF mRNA was dispersedly located in the endometrial-myometrial junction and stroma of the uterus. GM-CSF was immuno-histochemically localized in stromal cells and luminal epithelium. Ovariectomy significantly reduced the appearance of GM-CSF-mRNA-positive cells and the levels of expression for GM-CSF mRNA in the whole uterus, whereas treatment with 17β-estradiol (E2) or a combination of E2 and progesterone (P4) for 5 days on ovariectomized animals recruited GM-CSF-mRNA-positive cells and stimulated its expression. The combined treatment with E2 and P4 also stimulates the expression for GM-CSF mRNA and the production of immunoreactive GM-CSF in the stromal tissues. These data suggest that the expression of GM-CSF in uterine stromal cells is partially regulated by ovarian steroids.

Ovarian steroid effects on serotonin 1A, 2A and 2C receptor mRNA in macaque hypothalamus

This study mapped the location of serotonin (5HT) 1A, 2A and 2C receptor mRNA expression in the female macaque hypothalamus and determined whether the expression was regulated by estrogen plus or minus progesterone treatment using in situ hybridization (ISH) and densitometric analysis of autoradiographic films. The experimental groups of pigtail macaques ( Macaca nemestrina) were spayed controls ( n=4), estrogen treated (28 days, n=4) and estrogen+progesterone-treated animals (14 days estrogen+14 days estrogen and progesterone, n=4). Monkey specific 5HT1A (432 bp), 2A (411 bp) and 2C (294 bp) receptor probes were generated with PCR. Moderate 5HT1A receptor hybridization signal was detected in the preoptic area and the ventromedial nuclei. Less intense 5HT1A receptor signal was detected in a contiguous area from the dorsomedial nuclei through the posterior hypothalamus and in the supramammillary area. There was no change in 5HT1A receptor hybridization signal in any area with ovarian steroid treatment. Dense 5HT2A receptor hybridization signal was morphologically confined to the paraventricular, supraoptic, and mammillary nuclei and the external capsule of the thalamus. Light 5HT2A mRNA signal was inconsistently observed in the ventromedial nuclei. There was no change in the 5HT2A receptor hybridization signal in any area with ovarian steroid treatment. The 5HT2C receptor mRNA was widely distributed in the macaque hypothalamus. The preoptic area and anterior hypothalamus were largely positive for 5HT2C mRNA with a more concentrated signal in a narrow periventricular area. Dense 5HT2C receptor signal was detected lateral to the ventromedial nuclei (capsule), in the tuberomammillary nuclei, arcuate nucleus, dorsomedial nuclei, infundibular area and choroid plexus. Moderate 5HT2C receptor signal was detected in the ventromedial nuclei, lateral hypothalamus and dorsal to posterior hypothalamus. There was a significant decrease in total 5HT2C mRNA hybridization signal with ovarian steroid treatment in the ventromedial nuclei, dorsal and posterior hypothalamus. In summary, macaque 5HT1A, 2A and 2C receptor mRNAs are located in distinct hypothalamic loci which play a role in a number of autonomic functions and behavior. Ovarian steroids decreased the expression of 5HT2C receptor mRNA in the ventromedial nuclei, dorsal and posterior hypothalamus. The expression of 5HT1A and 5HT2A receptor mRNA was not altered by treatment with ovarian steroids.

The effect of ovarian steroids on epithelial ciliary beat frequency in the human Fallopian tube.

Using a method that detects variations in light intensity we have studied the effect of ovarian steroids on human Fallopian tube epithelial ciliary beat frequency in vitro. We have found that baseline ciliary beat frequency averages between 5-6 Hz. Cilia from ampullary segments of the Fallopian tube beat significantly faster (5.4 Hz+/-0.2) than those from fimbrial segments (4.8 Hz+/-0.2). There was no significant difference in baseline ciliary beat frequency at any other anatomical site in the Fallopian tube. Incubation with progesterone (10 micromol/l) suppresses human Fallopian tube epithelial ciliary beat frequency by 40-50%. This inhibition was observed at similar magnitudes in all Fallopian tubes studied irrespective of anatomical site. Progesterone-induced reductions in ciliary beat frequency were concentration dependent and prevented by the progesterone receptor antagonist mifepristone (RU486). Oestradiol alone (10 micromol/l) had no effect on ciliary beat frequency at any anatomical site in the Fallopian tube but did prevent the reduction in ciliary beat frequency seen with progesterone when tissues were incubated with these two steroids together.

Ovarian steroids differentially regulate the expression of PRL-R in neuroendocrine dopaminergic neuron populations: a double label confocal microscopic study

The aims of this study were (1) to identify the possible hypothalamic targets for a short prolactin (PRL) feedback in the adult female rat by identifying DAergic neuron populations expressing PRL receptor (PRL-R); (2) to describe the effect of ovarian steroids on the expression of PRL-R and (3) to compare the distribution of both the extracellular (EC) and ligand binding (LB) domains of the PRL-R on the hypothalamic dopaminergic neurons by applying double label immunocytochemistry for the different domains of PRL-R and for tyrosine hydroxylase (TH). Five- to six-month-old female rats were ovariectomized (OVX) and implanted with either 17β-estradiol (E 2), progesterone (P 4) or received an E 2 and a P 4 implant (E 2+P 4) at the same time. In the periventricular nucleus and in the dorsomedial portion of the middle arcuate nucleus, a dramatic increase in PRL-R EC immunoreactivity was observed in E 2 implanted rats. This increase was attenuated in E 2+P 4 rats, but P 4 treatment alone had no effect. Changes in PRL-R EC expression were paralleled by changes in serum PRL levels. Interestingly, PRL-R EC expression in the rostral arcuate nucleus decreased in P 4 implanted rats, however, P 4 did not attenuate the E 2-induced increase in PRL-R EC density. PRL-R EC immunostaining was observed on the membrane, in the cytoplasm and in the nucleus. PRL-R LB immunoreactivity was also detectable in the TH positive neurons, but no nuclear staining was observed with this antibody. However, we found a strong PRL-R LB immunostaining in the ependymal lining of the 3rd ventricle and in the processes of tanycytes projecting to the median eminence. These data indicate that (1) all neuroendocrine DAergic cells can be targets for PRL, (2) expression of PRL-R is differentially affected by ovarian steroids in the different TH cell populations, (3) PRL-R LB domain may be involved in trafficking PRL in the median eminence.

The effect of ovarian steroids on sickle cell deformability.

Sickle cell disease (SCD) is an incurable debilitating disease affecting the Afro-Caribbean population. The combined oral contraceptive pill (COCP), an effective and popular method of contraception, is often denied to women with SCD for fear that the disease process may have a synergistic effect on the coagulation changes associated with contraceptive steroids. In this study red cell deformability was assessed in 10 women with SCD and 10 comparable women with normal AA haemoglobin. Neither group was on exogenous hormones. The red cells were taken in the follicular phase of the menstrual cycle when women have low endogenous levels of oestradiol and progesterone. The effect of the steroids contained in the COCP on red blood cells was simulated by incubation with therapeutic concentrations of oestradiol and progesterone. Red cell deformability is a measure of the ease with which erythrocytes flow through small capillaries and was assessed using the parameters red cell transit time (RCTT) and clogging rate (CR). Therapeutic concentrations of oestradiol and progesterone did not appear to influence red cell deformability in women with SCD or normal AA haemoglobin.

Effects of ovarian steroid hormones and thyroxine on calcitonin secretion in pregnant rats.

In the present study, the roles of ovarian steroid hormones and thyroxine (T4) in regulating the secretion of calcitonin (CT) in pregnant rats were examined. The levels of plasma progesterone, pre- and post-CaCl2 plasma CT, and recovery time of plasma CT and calcium after calcium challenge were greatest in midterm pregnant rats. The levels of basal plasma progesterone, CT, calcium, and recovery time of plasma CT after calcium challenge were less in late pregnant rats, but basal plasma estradiol was highest in late pregnancy. The concentrations of plasma T4 were gradually decreased in rats during pregnancy. Regardless of the presence of estradiol, administration of progesterone in ovariectomized (Ovx) rats resulted in an increase of plasma T4 as well as the basal and calcium-induced secretion of CT. Administration of estradiol alone did not alter the CaCl2-induced levels but decreased the post-CaCl2 levels of plasma calcium in Ovx rats. The basal levels of plasma CT were decreased in Ovx rats treated with T4. These results suggest that the hypercalcitoninemia in midterm pregnant rats is due to an increased secretion of progesterone. Hypocalcitoninemia in late pregnant rats, however, is due in part to lower plasma calcium.

Effects of ovarian steroids on uterine growth, morphology, and cell proliferation in ovariectomized, steroid-treated ewes.

Uterine growth, morphology, and endometrial cell proliferation were evaluated in intact estrous (EST; Day 0 of the estrous cycle) or luteal (LUT; Day 10 of the estrous cycle) ewes or in ovariectomized ewes receiving no hormone (OVX); estradiol-17beta (OVX+E); progesterone for 8 (OVX+P8), 30 (OVX+P30), or 60 (OVX+P60) days; estradiol followed by progesterone (OVX+E+P); or progesterone followed by estradiol (OVX +P+E). Uterine weights were greater for EST than for LUT ewes, were reduced 2- to 4-fold in OVX ewes, and were increased in estradiol-treated (OVX+E, OVX+E+P, and OVX+P+E) ewes. The effects of estradiol treatment on uterine growth in OVX ewes were primarily on cellular hypertrophy rather than hyperplasia. Endometrial labeling index (LI) was greater for EST than for LUT ewes and was low in OVX ewes. Compared with OVX ewes, OVX+E and OVX+P+E ewes had increased LI for all endometrial tissues, whereas OVX+P8 and OVX+E+P ewes had increased LI only for luminal epithelium. These data will help us determine the mechanisms by which ovarian steroids regulate uterine growth in ewes.

Effects of ovarian steroid hormones on dopamine-controlled prolactin secretory responses in vitro.

Dopamine (DA), a well-established inhibitor of prolactin (PRL) secretion, has also been shown to stimulate PRL secretion from the anterior lobe of the pituitary gland of the rat. It has been reported that low doses of DA stimulate PRL whereas high doses inhibit PRL secretion. Our laboratory has previously reported that these PRL secretory responses are dependent upon the stages of the estrous cycle of the rat. The objective of the present study was to determine the steroid requirements for the differing PRL secretory responses to low and high doses of DA in perifusion. Animals were ovariectomized (OVX) and immediately given Silastic implants containing estradiol (E2) which has previously been shown in our laboratory to produce blood levels of 70-100 pg/ml, progesterone which has previously been shown in our laboratory to produce blood levels of 30-40 ng/ml, or the combination. OVX rats served as controls. Ten days later, the anterior lobes of the pituitary glands were harvested and enzymatically dissociated. Cells were mixed with Sephadex G-10 and placed in six 0.5-ml perifusion chambers (1 x 10(6) cells/chamber). Cells were perifused for 24 h with Dulbecco's modified Eagle's medium containing 0.2% bovine serum albumin and 0.1 mM ascorbic acid. The PRL secretory pattern was characterized in response to the following treatment sequence: (1) 30 min media alone (maximally uninhibited); (2) 24 min 100 pM DA; (3) 30 min media alone (DA withdrawal); (4) 24 min 1 microM DA, and (5) 30 min media alone (DA withdrawal). PRL secretion in the presence of 100 pM DA was unchanged in cells obtained from OVX animals, but exposure to 1 microM DA inhibited PRL release in these cells. Subsequent withdrawal stimulated PRL secretion relative to that of the initial exposure to media alone. The responses of cells from OVX rats implanted with progesterone alone was indistinguishable from those of the OVX controls. In cells obtained from animals implanted with E2 alone, 100 pM DA and its subsequent withdrawal neither stimulated nor inhibited PRL secretion. In contrast, 1 microM DA exposure initially stimulated and then inhibited PRL secretion in cells from E2-treated animals. Here, subsequent withdrawal of DA enhanced PRL secretion. In cells obtained from E2+progesterone-treated animals, 100 pM DA exposure robustly enhanced PRL secretory responses. Withdrawal of this dose of DA had no further effect on PRL secretion. However, exposure of E2+progesterone-treated cells to 1 microM DA robustly stimulated and subsequently only slightly inhibited PRL secretion. The results of these studies suggest that inhibition of PRL secretion by DA is independent of ovarian steroids while E2 and progesterone in combination favors stimulation of PRL secretion in response to DA. Taken together, these data suggest that PRL secretory responses in this system are determined by the ovarian steroid milieu.

Nocturnal changes in pineal melatonin synthesis during puberty: relation to estrogen and progesterone levels in female rats.

Our objective was to evaluate the changes in melatonin synthesis during the peripubertal period in the female rat and to determine the effects of ovarian steroid hormones on melatonin synthesis. Pineal levels of tryptophan, 5-hydroxytryptamine (5-HT), melatonin and norepinephrine were determined in female Sprague Dawley rats (between 2 and 12 weeks of age) in the mid-dark during the daily light/dark cycle. Melatonin levels increased with age, parallel to pineal growth, until 6 weeks of age, when the vaginal opening was found in 66.7% of rats, and significantly decreased until 8 weeks of age, when the vaginal opening was found in all rats. Norepinephrine began to increase earlier and reached a mature level at 4 weeks of age. Treatments with bilateral ovariectomy at 4, 6, and 8 weeks of age resulted in significant increases in melatonin and 5-HT levels, and significant decrease in tryptophan level at 2 weeks after ovariectomy. Treatments with ovariectomy at 6 weeks of age produced a consistent increase in 5-HT level and a consistent decrease in tryptophan level until 6 weeks after ovariectomy. However, melatonin levels increased until 2 weeks after ovariectomy, then decreased and reached a control level at 6 weeks after ovariectomy. Subcutaneous implantation of estradiol-17 beta capsule and daily subcutaneous injection of estradiol benzoate (E2B) (1.0 microgram, 20 micrograms) for two weeks in the rats ovariectomized at 4, 6, and 8 weeks of age resulted in significant decreases in melatonin and 5-HT levels and a significant increase in tryptophan level at 2 weeks after ovariectomy. A smaller dose of E2B (0.1 microgram) produced the same effects in the rats ovariectomized at 4, but not at 6 and 8 weeks of age. Administration of progesterone (200 micrograms/day) for 2 weeks did not produce any significant changes in melatonin, 5-HT, and tryptophan levels. Norepinephrine levels were not changed by any of the above treatments. These results suggest that estrogen, but not progesterone, can modulate nocturnal pineal melatonin synthesis in peripubertal female rats, and that the decline in the melatonin synthetic activity during the pubertal period might be related to the increasing levels of endogenous estrogen, which is secreted from the maturing ovary. The sites of action of the inhibitory effect of estrogen on the pineal melatonin synthesis may be multiple.

The milk-ejection reflex in the peri-partum rat: effects of oestradiol and progesterone on basal milk-ejection frequency and the facilitatory response to central oxytocin.

Experiments were undertaken to examine the effects of ovarian steroids on the functional characteristics of the milk-ejection reflex during late pregnancy. Basal milk-ejection frequency and response to i.c.v. oxytocin (OT) were compared in different experimental groups, using intramammary pressure recordings obtained in suckling tests under urethane anaesthesia. Ovariectomy (OVX) on day 20 of pregnancy significantly (P < 0.05) increased milk-ejection frequency on day 22, compared with sham-OVXed animals. I.c.v. injection of 2.2 ng OT during suckling had no consistent effect on milk ejection in either of these groups. Pretreatment with oestradiol (5 micrograms per day, s.c.) or progesterone (5 mg per day, s.c.) both resulted in a fall in milk-ejection frequency compared to oil-treated OVXed controls. However, whereas oestradiol-treated OVXed rats showed a facilitatory response to i.c.v. OT, with a significant (P < 0.05) increase in milk-ejection frequency in the 20 min period after injection, progesterone-treated OVXed rats showed only a delayed decrease in milk-ejection frequency (significant at P < 0.05 between 20-40 min after injection). Oil-treated OVXed rats showed no significant response to i.c.v. OT at any stage. Electrophysiological recordings from supraoptic OT neurones confirmed that bursting activity was increased by i.c.v. injection of OT in oestradiol-treated, but not progesterone-treated rats. Further experiments with hysterectomized ovariectomized rats indicated that the difference in response to i.c.v. OT in oestradiol- vs progesterone-treated rats was not related to changes in the timing of birth induced steroid treatments. These findings demonstrate the ability of ovarian steroids to alter the characteristics of the milk-ejection reflex in the peri-partum rat. In particular, the rise in oestradiol and fall in progestesterone near term, may contribute to programming of the facilitary response to central OT in preparation for lactation.

Effects of nitric oxide on stimulated release of norepinephrine from female rat hypothalamic slices

Norepinephrine (NE) is an important neurotransmitter involved in ovarian steroid hormone regulation of female reproductive function in rats. Nitric oxide (NO) has also been suggested to be an essential mediator of gonadotropin-releasing hormone release and of lordosis behavior of female rats. These studies used a superfusion system to investigate the hypothesis that NO regulates [ 3H]NE release in the preoptic area (POA) and hypothalamus (HYP), brain regions that mediate ovarian steroid effects on reproductive function. The NO synthase inhibitors N-nitro- l-arginine and N G-nitro- l-arginine methyl ester did not modify either basal or N-methyl- l-aspartate (NMDA)-stimulated NE release in either brain region of ovariectomized, hormone-treated or control animals. The NO precursor l-arginine ( l-Arg) reduced NMDA-stimulated NE release in POA but had no effect on KCl- or electrically-stimulated release. l-Arg did not influence basal or evoked release of [ 3H]NE from HYP slices. Sodium nitroprusside (SNP), a NO-generating compound, blocked the release of NE in response to NMDA stimulation but not in response to KCl or electrical stimulation. Thus, SNP is probably reducing NE release by acting as an NMDA antagonist rather than via NO production. There was a tendency for administration of both estrogen and progesterone to ovariectomized females to facilitate NMDA-stimulated NE release, particularly in the POA. Our data suggest that NO does not mediate basal or NMDA-stimulated NE release in rat POA and HYP. Therefore, NO regulation of lordosis behavior and gonadotropin release in female rats is probably not exerted at the level of NE release.

Neural systems mediating the negative feedback actions of estradiol and progesterone in the ewe.

The ewe shows a marked seasonal variation in the effects of ovarian steroids on pulsatile GnRH secretion. In the breeding season, progesterone inhibits GnRH pulse frequency, while estradiol suppresses pulse amplitude. In anestrus, both steroids inhibit pulse frequency. The effects of progesterone in both seasons are mediated by endogenous opioid peptides (EOP) that act in the preoptic area (POA) and medial basal hypothalamus (MBH). However, knife cut studies indicate that actions in the MBH are most important. Moreover, blockade of EOP receptors activates (e.g., induces Fos) GnRH perikarya in the MBH, but not those in the POA. Thus interactions between EOP and GnRH neurons within the MBH may be critical for progesterone negative feedback. The neural systems mediating estradiol suppression of GnRH pulse amplitude in the breeding season are largely unknown, although alpha-adrenergic neurons may be involved. The seasonal variation in inhibition of GnRH pulse frequency by estradiol is postulated to be mediated by a group of dopaminergic (DA) neurons that have three important properties: (1) they inhibit GnRH pulse frequency; (2) their activity is stimulated by estradiol; and (3) they are functional in anestrus, but not the breeding season. Recent work examining the effects of lesions of DA neurons and the ability of estradiol to induce Fos in DA cells strongly suggests that DA neurons in the retrochiasmatic area (A15) and POA (A14) have all three characteristics. We thus propose that these DA neurons are responsible for the seasonal variation in the ability of estradiol to inhibit GnRH pulse frequency.

Glutamic acid decarboxylase in rat olfactory bulb: effect of ovarian steroids or male pheromones

The effect of ovarian steroids and pheromones on the activity of glutamic acid decarboxylase, the enzyme that synthesizes γ-aminobutyric acid (GABA), was studied in the rat olfactory bulbs. The enzyme activity was measured in the main and accessory olfactory bulbs at 11:00 h and 17:00 h in ovariectomized rats, and in rats treated with ovarian steroids or exposed to male pheromones. The enzyme activity in both bulbs showed a diurnal fluctuation that was not affected in the accessory bulbs by the exposure to pheromones while the rhythm disappeared in the main bulbs. Estrogen and estrogen-progesterone treatments decreased the enzyme activity in both bulbs either in the morning or in the afternoon. The exposure of ovariectomized estrogen-primed rats to male pheromones reversed the effect of estrogen on the enzyme activity in the morning but not in the afternoon. Ovarian hormones plus pheromones prevented the steroid effect only in the morning. These results support the view that in olfactory bulbs, the GABAergic system can be modulated by endocrine and pheromonal factors.