"Rapid actions of the neurosteroid allopregnanolone on ovarian and hypothalamic steroidogenesis: Central and peripheral modulation".

The present study aimed to determine whether an i.c.v. administration of allopregnanolone (ALLO) rapidly modifies the hypothalamic and ovarian 3ß-hydroxysteroid dehydrogenase (3ß-HSD) enzymatic activity and gene expression in in vivo and ex vivo systems in pro-oestrus (PE) and dioestrus I (DI) rats. Animals were injected with vehicle, ALLO, bicuculline or bicuculline plus ALLO and were then killed. In the in vivo experiment, the hypothalamus, ovaries and serum were extracted and analysed. In the ex vivo experiment, the superior mesenteric ganglion - ovarian nerve plexus - ovary system was extracted and incubated during 120 minutes at 37 ºC. The serum and ovarian compartment fluids were used to determine progesterone by radioimmunoanalysis. In the in vivo experiments, ALLO caused a decrease in hypothalamic and ovarian 3ß-HSD enzymatic activity during PE. During DI, ALLO increased hypothalamic and ovarian 3ß-HSD activity and gene expression. The ovarian 3ß-HSD activity increased in both stages in the ex vivo system; gene expression increased only during DI. ALLO induced an increase in serum progesterone only in D1 and in the ovarian incubation liquids in both stages. All findings were reversed by an injection of bicuculline before ALLO. Ovarian steroidogenic changes could be attributed to signals coming from ganglion neurones, which are affected by the acute central neurosteroid stimulation. The i.c.v. administration of ALLO via the GABAergic system altered 3ß-HSD activity and gene expression, modulating the neuroendocrine axis. The present study reveals the action that ALLO exerts on the GABAA receptor in both the central and peripheral nervous system and its relationship with hormonal variations. ALLO is involved in the "fine tuning" of neurosecretory functions as a potent modulator of reproductive processes in female rats.

Evidence for a Putative Circadian Kiss-Clock in the Hypothalamic AVPV in Female Mice.

The kisspeptin (Kp) neurons in the anteroventral periventricular nucleus (AVPV) are essential for the preovulatory LH surge, which is gated by circulating estradiol (E2) and the time of day. We investigated whether AVPV Kp neurons in intact female mice may be the site in which both E2 and daily signals are integrated and whether these neurons may host a circadian oscillator involved in the timed LH surge. In the afternoon of proestrous day, Kp immunoreactivity displayed a marked and transient decrease 2 hours before the LH surge. In contrast, Kp content was stable throughout the day of diestrus, when LH levels are constantly low. AVPV Kp neurons expressed the clock protein period 1 (PER1) with a daily rhythm that is phase delayed compared with the PER1 rhythm measured in the main clock of the suprachiasmatic nuclei (SCN). PER1 rhythm in the AVPV, but not in the SCN, exhibited a significant phase delay of 2.8 hours in diestrus as compared with proestrus. Isolated Kp-expressing AVPV explants from PER2::LUCIFERASE mice displayed sustained circadian oscillations of bioluminescence with a circadian period (23.2 h) significantly shorter than that of SCN explants (24.5 h). Furthermore, in AVPV explants incubated with E2 (10 nM to 1 µM), the circadian period was lengthened by 1 hour, whereas the SCN clock remained unaltered. In conclusion, these findings indicate that AVPV Kp neurons display an E2-dependent daily rhythm, which may possibly be driven by an intrinsic circadian clock acting in combination with the SCN timing signal.

Middle-aged female rats retain sensitivity to the anorexigenic effect of exogenous estradiol.

It is well established that estradiol (E2) decreases food intake and body weight in young female rats. However, it is not clear if female rats retain responsiveness to the anorexigenic effect of E2 during middle age. Because middle-aged females exhibit reduced responsiveness to E2, manifesting as a delayed and attenuated luteinizing hormone surge, it is plausible that middle-aged rats are less responsive to the anorexigenic effect of E2. To test this we monitored food intake in ovariohysterectomized young and middle-aged rats following E2 treatment. E2 decreased food intake and body weight to a similar degree in both young and middle-aged rats. Next, we investigated whether genes that mediate the estrogenic inhibition of food intake are similarly responsive to E2 by measuring gene expression of the anorexigenic genes corticotropin-releasing hormone (CRH), proopiomelanocortin (POMC), the long form of the leptin receptor (Lepr) and serotonin 2C receptors (5HT2CR) and the orexigenic genes agouti-related peptide (AgRP), neuropeptide Y (NPY), prepromelanin-concentrating hormone (pMCH) and orexin in the hypothalamus of young and middle-aged OVX rats treated with E2. As expected, E2 increased expression of all anorexigenic genes while decreasing expression of all orexigenic genes in young rats. Although CRH, 5HT2CR, Lepr, AgRP, NPY and orexin were also sensitive to E2 treatment in middle-aged rats, POMC and pMCH expression were not influenced by E2 in middle-aged rats. These data demonstrate that young and middle-aged rats are similarly sensitive to the anorexigenic effect of E2 and that most, but not all feeding-related genes retain sensitivity to E2.

Histologic and cytologic detection of endocrine and reproductive tract effects of exemestane in female rats treated for up to twenty-eight days.

The objective of this study was to determine the shortest period of time necessary to detect histologic evidence of estrous cycle disruption in Sprague-Dawley rats treated for up to 28 days with the aromatase inhibitor exemestane at 1,000 mg/kg. Rats were evaluated on day 5, 8, 15, or 29. Vaginal mucification, uterine and cervical epithelial atrophy, uterine luminal epithelial vacuolation, decreased uterine granulocytes, and hypertrophy/hyperplasia of mammary ducts and alveoli were noted by day 5 and persisted throughout the study. From day 8 to day 29, absence of recent basophilic corpora lutea, increased atresia of antral follicles, interstitial cell hyperplasia, and increased luteinized follicles were present in the ovaries of treated rats. Vaginal smears detected persistent diestrus, confirming estrous cycle disruption between days 5 and 8. Ovary and uterine weights were largely unaffected. Serum hormone levels were not useful due to the study design employed. Other effects of exemestane included decreased adrenal weights and decreased cell size in both the adrenal zona fasciculata and the pituitary pars distalis. While early histologic changes were evident on day 5, only after 8 days of treatment were findings considered sufficient to clearly identify exemestane-induced estrous cycle disruption using microscopy alone.

Antifertility and fetotoxic activities of Acanthus montanus aqueous extract in Wistar rats.

Acanthus montanus T. Anderson (Acanthaceae) possesses several medicinal properties; it is used in Cameroon as a folk medicine to treat pain, inflammation and threatened abortion. The aim of this study was to determine the effect of A. montanus aqueous extract on the estrous cycle pre- and postimplantation in rats and its mechanism of action. The estrous cycles of Wistar rats were monitored before, during and after oral administration of distilled water (control) or aqueous extract (62.5, 125, 250, 500, 1000 mg/kg/day). Furthermore, pregnant rats received the above doses of aqueous extract on days 1-6 (preimplantation) or 6-15 (postimplantation) of gestation and were sacrificed on day 8 or 20 of pregnancy, respectively. Moreover, aqueous extract (500 and 1000 mg/kg/day) was given to ovariectomized rats in the presence or absence of exogenously administered estrogen and/or progesterone and uterine weight and deciduoma count were evaluated. The extract, irrespective of dose, reversibly prolonged the metestrous and occasionally the diestrous stages of the estrous cycle. The extract did not alter the uterine wet weight or deciduoma count, suggesting a lack of estrogenic and progestational effects. At 1000 mg/kg/day, the extract caused appreciable preimplantation losses of 36.8 +/- 6.5% (P < 0.05), while none of the doses caused postimplantation losses. The extract also caused delayed fetal growth.

In vitro PGF2alpha production by endometrium and corpus luteum explants from pregnant and nonpregnant diestrus bitches and placental explants from pregnant bitches.

To better understand the process of slow luteal regression of the nonpregnant cycle in dogs and the acute luteolysis that occurs prepartum, the present study investigated in vitro PGF2alpha production by the endometrium, corpus luteum and placental explants obtained at known times of the cycle from pregnant bitches (days 63, 64 and immediately postpartum; day 0 = estimated day of the ovulatory LH surge) and from nonpregnant diestrus bitches (approximately days 65, 75 and 85). Both basal PGF2alpha production and its production in the presence of the protein kinase C (PKC) stimulator 12,13-phorbol dibutyrate (PDBu) were determined. For PDBu-supplemented incubations, mean PGF2alpha production (pg/mL/mg/6 h) by endometrium explants of the nonpregnant bitches in late diestrus was highest on day 65 (205 +/- 87) and reduced to low levels (38 +/- 17 and 11 +/- 11) on days 75 and 85, respectively. The production by corpus luteum explants from these bitches was significantly less on day 65 (46 +/- 14) than that of the day 65 endometrium explants, and was slightly increased on day 85 (103 +/- 52). The corresponding mean PGF2alpha production by the endometrium explants of pregnant bitches was on average much greater (i.e., two to three-fold) compared to nonpregnant bitches (P < 0.01) and involved high concentrations at day 64 (1523 +/- 467) and postpartum, compared to somewhat lower levels on day 63 (830+/-65); luteal PGF production (165 +/- 4) was also higher than in nonpregnant bitches around day 65. For pregnant bitches, PGF production per gram of tissue in the endometrium explants was greater than for the CL or placenta explants (180 +/- 37). Therefore, the endometrium of the pregnant bitch has an increased capability to produce PGF2alpha immediately prepartum, which on a tissue weight basis, exceeds that of either corpora lutea or the placenta. However, assuming a larger mass of placental tissue in vivo, we inferred that the placenta may contribute substantially to peripheral PGF concentrations.

Pentobarbital stimulates the activity of the GnRH pulse generator interacting with opioid neurons in rats in proestrus.

In order to investigate the possibility that i.p. injection of pentobarbital sodium (PB, 32 mg/kg bw) potentiates the GnRH pulse generator activity, effects of i.v. infusions of an opiate receptor antagonist naloxone (NAL, 2 mg/h) on the pulsatile LH secretion were compared in saline (SAL)- and PB-injected rats in proestrus and diestrus 1. In SAL-injected rats in proestrus, NAL infusions significantly increased both the frequency and amplitude of LH pulses, and also the overall mean LH concentration. In PB-injected rats in proestrus, all the parameters of the pulsatile LH secretion were similar to those in SAL-injected rats in proestrus. The NAL infusion in PB-injected rats caused an increase in the frequency, but it was similar to that in SAL-injected rats. But, increases in the amplitude and the overall mean LH observed during NAL infusions in PB-injected rats were greater than in SAL-injected rats. In SAL-injected rats in diestrus 1, NAL infusions increased all the parameters, as in rats in proestrus. In PB-injected rats in diestrus 1, LH secretion was severely suppressed. NAL infusions recovered the pulsatile LH secretion, but the frequency and the overall mean LH of the secretion were smaller than those obtained during NAL infusions in SAL-injected rats. In addition, characteristic increases in the MUA (volleys), which occur in association with the initiation of an LH pulse and thus are considered to represent an increased activity of the GnRH pulse generator, appeared more frequently during NAL infusions in PB-injected rats in proestrus than in SAL-injected rats. These results suggest that the GnRH pulse generator in rats in proestrus, but not in rats in diestrus 1, is refractory to PB and further is potentiated by PB in the response to NAL. Together with the fact that this dosage of PB blocks the surge of LH secretion in rats in proestrus, the concept of the existence of separate neuronal mechanisms responsible for the surge and pulsatile secretion of LH are supported.

Differences in hypothalamic serotonin between estrous phases and gender: an in vivo microdialysis study.

The aim of the present study was to assess whether there are gender differences in (1) levels of extracellular serotonin (5-HT) in the forebrain, and (2) the effect on 5-HT of a reuptake inhibitor, paroxetine, or a releasing drug, fenfluramine. In vivo microdialysis was used to measure 5-HT in the hypothalamus of male and regularly cycling female rats. Hypothalamic 5-HT was significantly lower in estrous females (0.83 +/- 0.05 pg/sample, n=33) than in male rats (1.04 +/- 0.06 pg, n=38). Levels in diestrous females (0.98 +/- 0.09 pg, n=38) were not significantly different from males. Paroxetine (1 mg/kg) increased hypothalamic 5-HT in males, and diestrous and estrous females to approximately 2 pg/sample. However, the increase in hypothalamic 5-HT produced by a maximally effective dose of paroxetine (10 mg/kg) was significantly greater in male rats and during diestrous than during estrous. d,l-Fenfluramine (10 mg/kg) evoked an increase in extracellular 5-HT to approximately 15 pg/sample in all groups. A higher dose of d,l-fenfluramine (20 mg/kg) produced a significantly greater increase in hypothalamic 5-HT in males than in females during estrous or diestrous. These results are consistent with other evidence that during estrous, when rats are responding to peak levels of estrogen and progesterone, 5-HT release is decreased.

Disruption of estrous cyclicity following administration of a luteinizing hormone-releasing hormone antagonist to the preoptic area of the rat.

Experiments were designed to test the hypothesis that LHRH receptors in the preoptic area (POA) are of physiological importance for maintaining estrous cyclicity in the rat. Bilateral cannulae were implanted just dorsal to the POA. Estrous cycles were monitored daily by vaginal smears. Antide, a long-acting LHRH antagonist, was infused bilaterally (2.5 micrograms/side) in the POA or the hypothalamus on the mornings of diestrus I and II. As controls, at separate times, rats also received similar infusions of either vehicle (1:1 water:propylene glycol) or a bombesin antagonist (#B0650; Sigma, St. Louis, MO). Collection of daily vaginal smears continued, and the number of days from the first infusion to the next day of estrus that preceded a normal cycle was recorded. After infusion of Antide into the POA, rats demonstrated varying durations of interrupted cycles ranging from 11 days to more than 100 days. These periods of disruption were characterized by either long periods of diestrus, long periods of estrus, or an extended period of diestrus followed by an extended period of estrus. After infusion of Antide into the dorsomedial, ventromedial, or anterior hypothalamic areas, rats had either a 4- or 5-day estrous cycle and continued to cycle normally. Likewise, infusions into the septum had no effect. Infusion of vehicle or bombesin antagonist into any of the hypothalamic or POA sites tested also resulted in no interruptions in the cyclic activity of the rats. Therefore, it appears that functional LHRH receptors in the POA are necessary to drive the normal estrous cycle.

Wen-jing-tang, a traditional Chinese herbal medicine increases luteinizing hormone release in vitro.

For examination of the effect on luteinizing hormone (LH) release of Wen-Jing-Tang, a traditional Chinese herbal medicine, the pituitary from normal female rats in diestrus was perifused alone or in sequence with the mediobasal hypothalamus (MBH) in a sequential double-chamber perifusion system. Wen-Jing-Tang at 5 or 500 micrograms/ml induced significant LH release (60-95% increase) from the pituitary in series with the MBH, but had no effect on LH release from the pituitary perifused alone. These data suggest that Wen-Jing-Tang induces LH release from the pituitary through hypothalamic LH-RH.

Dopamine blockade reverses the inhibition of prolactin which results from intrahypothalamic pituitary grafts.

When mated with fertile bucks, rats with anterior pituitary (AP) tissue grafted into the hypothalamus did not exhibit prolongation of the diestrous cycle. Treatment of these rats with alpha-methyl-p-tyrosine, reserpine, or haloperidol for 1 or 2 days after mating increased the interestrous interval by a few days in all rats and to more than 8 days (leading to pseudopregnancy or pregnancy) in 20% of the cases. The same treatment in unmated normals resulted in 80% becoming pseudopregnant. To get more than 70% of rats with hypothalamic AP grafts pregnant or pseudopregnant required dopamine-blocking drugs for 3 or 4 consecutive days. Pregnancy was prolonged in 50% and lactation was impaired in 78% of the grafted rats which littered. Both impairments, like the original failure of the luteotrophic response, are attributed to the effects of PRL autofeedback from the hypothalamic AP grafts. These experiments provide further evidence that the mechanism whereby PRL in the hypothalamus inhibits PRL secretion involves elevation of dopamine.

LHRH-induced LH and FSH responses in the aged female rat.

LHRH-induced LH and FSH concentrations were measured in 8 to 12-month-old rats which had just begun to exhibit constant vaginal estrus (CE) or prolonged vaginal diestrus (PD) and in young rats on the day of proestrus (PE). All animals were anesthetized with sodium pentobarbital (31 mg/kg). They received (1) i.v. infusion of LHRH (Beckman 100 ng/ml at a rate of 0.5 ml/hr) for 3 hrs or (2) 2 i.v. injections of LHRH (50 ng/100 g body wt) spaced 120 min apart. LHRH stimulation resulted in a comparable gonadotropin response in young rats and old CE rats; however, old PD rats exhibited a markedly attenuated LH and FSH response to LHRH infusion. These data suggest that pituitary responsiveness to LHRH does not change in 8 to 12-month-old CE rats and therefore does not play a major role in the transition to acyclicity. In contrast, pituitary responsiveness is decreased in old PD rats early during the aging process and therefore may be an important factor in the transition to their acyclic vaginal smear pattern.

The effects of oestrogen on hypothalamic tissue.

Oestrogens exert potent influences on hypothalamic differentiation and on sexual function throughout life. During the period in which the hypothalamus differentiates, the availability of oestrogens (or aromatizable androgens) to the hypothalamus results in a male pattern of gonadotropin secretion and in male sexual behaviour in the adult. Lack of oestrogens during this period results in a cyclic schedule of gonadotropin secretion and in female sexual behaviour. Oestrogens or their metabolites determine future sex-specific activity by facilitating synapse formation in developing target circuits. The onset of puberty may also in part reflect oestrogen-stimulated synapse formation in the hypothalamus. In contrast, oestrogen given to an adult female rat causes specific multifocal lesions in the lateral arcuate nucleus. Such animals exhibit persistent oestrus and small multicystic ovaries. Since the same anovulatory syndrome can be produced by mechanical disruption of neuronal pathways from more anterior regions to the arcuate nucleus, it would appear that oestrogen could effect a 'disconnection' of the circuit responsible for cyclic drive of gonadotropin secretion. Furthermore, old female rats spontaneously exhibit the same type of reproductive derangement as well as the arcuate lesion. Thus direct effects of oestrogens on neuronal circuitry within the hypothalamus may be responsible for (1) sexual differentiation, (2) the onset of puberty and sexual activity, and (3) cessation of reproductive function in the female rat.

[Role of estrogen-sensitive neurons in the arcuate region of the hypothalamus in the mechanism of luteinizing hormone release]. / Rol' éstrogenchuvstvitel'nykh neironov arkuatnoi oblasti gipotalamusa v mekhanizme osvobozhdeniia liuteiniziruiushchego gormona.

Experiments were conducted on rats; estradiol brought to the arcuate region of the hypothalamus by means of microionophoresis led to the increase of the region of the hypothalamus by means of microionophoresis led to the increase of the blood luteinizing hormone (LH) level during the following stages of the estral cycle-diestrus 1, diestrus 2, and the first half day of the proestrus; as to the second half of the proestrus day--estradiol decreased its level. Changes in the LH level in the hypophysis under the influence of the microionophoretic introduction of estradiol into the arcuate region occurred during the second half of the day of diestrus 2 (reduction), and during the estrus (elevation). In the majority of cases a rise of the blood level was combined with the neuron activation in the arcuate region under the influence of estradiol.

[Effect of anticoagulants on the change of blood parameters in diestric gilts]. / Einfluss von Gerinnungshemmern auf die Veränderung von Blutparametern bei diöstrischen Jungsauen.

The above problem was studied by furnishing four groups of six gilts each with ear catheters for the following intravenous catheter treatment: 2,500 IU heparin per die over ten days, supported by two daily oral applications of 1 g Falithrom, or 2,500 IU heparin plus two daily applications of 1 g Falithrom, all intravenously. The last group remained untreated for control. One day of the dioestrus was chosen for catheter bleeding of all animals at 6 a.m., 8 a.m., noon, and 4 p.m. and subsequent determination from the plasma of free fatty acids, copper, inorganic phosphorus, total protein, albumin, globulin, chloride, urea, cholesterol, alkaline phosphatase, blood sugar, and beta-lipoproteides. Significant differences regarding these parameters between the various groups were not even established, if alpha = 0.25. The anticoagulants used in the study may be used without any reservation for catheter rinsing and clearing and will not cause any significant change in the levels of the blood parameters.

Inhibition of pituitary prolactin secretion by human placental lactogen in rats.

Intact female rats given twice daily injections of 1 mg human placental lactogen (HPL) showed continued dioestrous vaginal smears and their ovarian corpora lutea were found to be hypertrophied and functiona. The serum prolactin level was significantly lower in these rats than in the controls at dioestrus as well as at pro-oestrus. Twice-daily injections of 0.5 or 2 mg HPL to ovariectomized rats decreased serum and pituitary levels of prolactin and increased hypothalamic activity of prolactin inhibiting hormone, although the effect was less at the lower dose. Human placental lactogen had no direct effect on pituitary prolactin secretion in vitro. These findings have demonstrated that HPL, like prolactin itself, inhibits prolactin secretion by actin