Peripheral administration of κ-opioid receptor antagonist stimulates gonadotropin-releasing hormone pulse generator activity in ovariectomized, estrogen-treated female goats.

Pulsatile gonadotropin-releasing hormone (GnRH) secretion is indispensable for reproduction in mammals. Kisspeptin neurons in the hypothalamic arcuate nucleus (ARC), referred to as KNDy neurons because of the coexpression of neurokinin B and dynorphin A, are considered as components of the GnRH pulse generator that produces rhythmic GnRH secretion. The present study aimed to investigate if peripheral administration of PF-4455242, a κ-opioid receptor (KOR, a dynorphin A receptor) antagonist, facilitates pulsatile luteinizing hormone (LH) secretion and GnRH pulse generator activity in estrogen-treated ovariectomized Shiba goats to determine the possibility of using KOR antagonists to artificially control ovarian activities. PF-4455242 was intravenously infused for 4 h (1 or 10 µmol/kg body weight/4 h) or as a single subcutaneous injection (1 or 10 µmol/kg body weight). In a separate experiment, the same KOR antagonist (10 µmol/kg body weight/4 h) was intravenously infused during the recording of multiple unit activity (MUA) in the ARC that reflects the activity of the GnRH pulse generator to test the effects of KOR antagonist administration on GnRH pulse generator activity. Intravenous infusion and single subcutaneous injection of the KOR antagonist significantly increased the frequency of LH pulses compared with controls. Intravenous infusion of KOR antagonist also significantly increased the frequency of episodic bursts in the MUA. The present study demonstrates that peripherally administered KOR antagonist stimulates pulsatile LH secretion by acting on the GnRH pulse generator, and peripheral administration of PF-4455242 can be used to facilitate pulsatile LH secretion, which in turn facilitates ovarian activities in farm animals.

Inhibition of metastin (kisspeptin-54)-GPR54 signaling in the arcuate nucleus-median eminence region during lactation in rats.

Follicular development and ovulation are suppressed during lactation in various mammalian species, mainly due to the suppression of pulsatile GnRH/LH secretion. Metastin (kisspeptin-54), a KiSS-1 gene product, is an endogenous ligand for GPR54, a G-protein-coupled receptor, and suggested to play a critical role in regulating the gonadal axis. The present study therefore aims to determine whether metastin (kisspeptin-54)-GPR54 signaling in discrete brain areas is inhibited by the suckling stimulus that causes suppression of LH secretion in lactating rats. Quantitative RT-PCR revealed that the KiSS-1 mRNA level was significantly lower in the arcuate nucleus (ARC)-median eminence region in lactating ovariectomized (OVX) and estrogen-treated OVX rats than in nonlactating controls. KiSS-1 mRNA in the anteroventral periventricular nucleus was kept at a low level in both lactating and nonlactating rats despite estrogen treatment. GPR54 mRNA levels were significantly lower in lactating than nonlactating rats in the anteroventral periventricular nucleus, but the levels in lactating mothers of the preoptic area and ARC-median eminence were comparable with nonlactating controls. Although KiSS-1 mRNA-expressing cells or metastin (kisspeptin-54) immunoreactivities were densely located in the ARC of nonlactating controls, few were found in the ARC of lactating OVX animals. Various doses of metastin (kisspeptin-54) (0.02, 0.2, and 2 nmol) injected into the third ventricle caused a significant increase in LH secretion in both lactating and nonlactating OVX rats, suggesting that lactating rats are responsive to metastin (kisspeptin-54) stimulus. Thus, the present study demonstrated that KiSS-1 mRNA/metastin (kisspeptin-54) expression is inhibited in the ARC by the suckling stimulus, suggesting that the inhibition is most probably involved in suppressing LH secretion in lactating rats.

Enhancement of the luteinising hormone surge by male olfactory signals is associated with anteroventral periventricular Kiss1 cell activation in female rats.

Olfactory stimuli play an important role in regulating reproductive functions in mammals. The present study investigated the effect of olfactory signals derived from male rats on kisspeptin neuronal activity and luteinising hormone (LH) secretion in female rats. Wistar-Imamichi strain female rats were ovariectomised (OVX) and implanted with preovulatory levels of 17ß-oestradiol (E2 ). OVX+E2 rats were killed 1 hour after exposure to either: clean bedding, female-soiled bedding or male-soiled bedding. Dual staining for Kiss1 mRNA in situ hybridisation and c-Fos immunohistochemistry revealed that the numbers of Kiss1-expressing cells and c-Fos-immunopositive Kiss1-expressing cells in the anteroventral periventricular nucleus (AVPV) were significantly higher in OVX+E2 rats exposed to male-soiled bedding than those of the other groups. No significant difference was found with respect to the number of c-Fos-immunopositive Kiss1-expressing cells in the arcuate nucleus and c-Fos-immunopositive Gnrh1-expressing cells between the groups. The number of c-Fos-immunopositive cells was also significantly higher in the limbic system consisting of several nuclei, such as the bed nucleus of the stria terminalis, the cortical amygdala and the medial amygdala, in OVX+E2 rats exposed to male-soiled bedding than the other groups. OVX+E2 rats exposed to male-soiled bedding showed apparent LH surges, and the peak of the LH surge and area under the curve of LH concentrations in the OVX+E2 group were significantly higher than those of the other two groups. These results suggest that olfactory signals derived from male rats activate AVPV kisspeptin neurones, likely via the limbic system, resulting in enhancement of the peak of the LH surge in female rats. Taken together, the results of the present study suggests that AVPV kisspeptin neurones are a target of olfactory signals to modulate LH release in female rats.

Evidence of involvement of neurone-glia/neurone-neurone communications via gap junctions in synchronised activity of KNDy neurones.

Pulsatile secretion of gonadotrophin-releasing hormone (GnRH)/luteinising hormone is indispensable for the onset of puberty and reproductive activities at adulthood in mammalian species. A cohort of neurones expressing three neuropeptides, namely kisspeptin, encoded by the Kiss1 gene, neurokinin B (NKB) and dynorphin A, localised in the hypothalamic arcuate nucleus (ARC), so-called KNDy neurones, comprises a putative intrinsic source of the GnRH pulse generator. Synchronous activity among KNDy neurones is considered to be required for pulsatile GnRH secretion. It has been reported that gap junctions play a key role in synchronising electrical activity in the central nervous system. Thus, we hypothesised that gap junctions are involved in the synchronised activities of KNDy neurones, which is induced by NKB-NK3R signalling. We determined the role of NKB-NK3R signalling in Ca2+ oscillation (an indicator of neuronal activities) of KNDy neurones and its synchronisation mechanism among KNDy neurones. Senktide, a selective agonist for NK3R, increased the frequency of Ca2+ oscillations in cultured Kiss1-GFP cells collected from the mediobasal hypothalamus of the foetal Kiss1-green fluorescent protein (GFP) mice. The senktide-induced Ca2+ oscillations were synchronised in the Kiss1-GFP and neighbouring glial cells. Confocal microscopy analysis of these cells, which have shown synchronised Ca2+ oscillations, revealed close contacts between Kiss1-GFP cells, as well as between Kiss1-GFP cells and glial cells. Dye coupling experiments suggest cell-to-cell communication through gap junctions between Kiss1-GFP cells and neighbouring glial cells. Connexin-26 and -37 mRNA were found in isolated ARC Kiss1 cells taken from adult female Kiss1-GFP transgenic mice. Furthermore, 18ß-glycyrrhetinic acids and mefloquine, which are gap junction inhibitors, attenuated senktide-induced Ca2+ oscillations in Kiss1-GFP cells. Taken together, these results suggest that NKB-NK3R signalling enhances synchronised activities among neighbouring KNDy neurones, and that both neurone-neurone and neurone-glia communications via gap junctions possibly contribute to synchronised activities among KNDy neurones.

Anti-diabetic potentials of Momordica charantia and Andrographis paniculata and their effects on estrous cyclicity of alloxan-induced diabetic rats.

Momordica charantia and Andrographis paniculata are the commonly used herbs by the diabetic patients in Pampanga, Philippines. While the anti-diabetic potential of Momordica charantia is well established in streptozocin- or alloxan-induced diabetic animals, the anti-diabetic potential of Andrographis paniculata in alloxan-induced diabetic rat is not known. Neither the effects of these herbs on estrous cyclicity of alloxan-induced diabetic rats are elucidated. Thus, in these experiments, Momordica charantia fruit juice or Andrographis paniculata decoction was orally administered to alloxan-induced diabetic rats. Rats that were treated with Momordica charantia and Andrographis paniculata had higher body weight (BW) compared with diabetic positive control (P < 0.01) from day 22 to day 27 (D27) but exhibited lower BW than the non-diabetic control (P < 0.05). These rats had lower feed (P < 0.05) and liquid intakes (P < 0.01) compared with diabetic positive control from day 17 to D27, but similar with the non-diabetic control. The blood glucose levels in these groups were significantly reduced from day 12 to D27 compared with diabetic positive control (P < 0.01), however, comparable with non-diabetic control. The diabetic positive control had extended mean estrous cycles (8 days) compared to Momordica charantia and Andrographis paniculata-treated diabetic rats (5 days; P < 0.05). Our results suggest that the anti-diabetic potentials of Momordica charantia and Andrographis paniculata could restore impaired estrous cycle in alloxan-induced diabetic rats.

Neonatal Kisspeptin is Steroid-Independently Required for Defeminisation and Peripubertal Kisspeptin-Induced Testosterone is Required for Masculinisation of the Brain: A Behavioural Study Using Kiss1 Knockout Rats.

Rodents show apparent sex differences in their sexual behaviours. The present study used Kiss1 knockout (KO) rats to evaluate the role of kisspeptin in the defeminisation/masculinisation of the brain mechanism that controls sexual behaviours. Castrated adult Kiss1 KO males treated with testosterone showed no male sexual behaviours but demonstrated the oestrogen-induced lordosis behaviours found in wild-type females. The sizes of some of the sexual dimorphic nuclei of Kiss1 KO male rats are similar to those of females. Plasma testosterone levels at embryonic day 18 and postnatal day 0 (PND0) in Kiss1 KO males were high, similar to wild-type males, indicating that perinatal testosterone is secreted in a kisspeptin-independent manner. Long-term exposure to testosterone from peripubertal to adult periods restored mounts and intromissions in KO males, suggesting that kisspeptin-dependent peripubertal testosterone secretion is required to masculinise the brain mechanism. This long-term testosterone treatment failed to abolish lordosis behaviours in KO males, whereas kisspeptin replacement at PND0 reduced lordosis quotients in Kiss1 KO males but not in KO females. These results suggest that kisspeptin itself is required to defeminise behaviour in the perinatal period, in cooperation with testosterone. Oestradiol benzoate treatment at PND0 suppressed lordosis quotients in Kiss1 KO rats, indicating that the mechanisms downstream of oestradiol work properly in the absence of kisspeptin. There was no significant difference in aromatase gene expression in the whole hypothalamus between Kiss1 KO and wild-type male rats at PND0. Taken together, the present study demonstrates that both perinatal kisspeptin and kisspeptin-independent testosterone are required for defeminisation of the brain, whereas kisspeptin-dependent testosterone during peripuberty to adulthood is needed for masculinisation of the brain in male rats.

The luteinising hormone surge-generating system is functional in male goats as in females: involvement of kisspeptin neurones in the medial preoptic area.

A luteinising hormone (LH) surge is fundamental to the induction of ovulation in mammalian females. The administration of a preovulatory level of oestrogen evokes an LH surge in ovariectomised females, whereas the response to oestrogen in castrated males differs among species; namely, the LH surge-generating system is sexually differentiated in some species (e.g. rodents and sheep) but not in others (e.g. primates). In the present study, we aimed to determine whether there is a functional LH surge-generating system in male goats, and whether hypothalamic kisspeptin neurones in male goats are involved in the regulation of surge-like LH secretion. By i.v. infusion of oestradiol (E2; 6 µg/h) for 16 h, a surge-like LH increase occurred in both castrated male and ovariectomised female goats, although the mean peak LH concentration was lower and the mean peak of the LH surge was later in males compared to females. Dual staining with KISS1 in situ hybridisation and c-Fos immunohistochemistry revealed that E2 treatment significantly increased c-Fos expression in the medial preoptic area (mPOA) KISS1 cells in castrated males, as well as ovariectomised females. By contrast, dual-labelled cells were scarcely detected in the arcuate nucleus (ARC) after E2 treatment in both sexes. These data suggest that kisspeptin neurones in the mPOA, but not those in the ARC, are involved in the induction of surge-like LH secretion in both male and female goats. In summary, our data show that the mechanism that initiates the LH surge in response to oestrogen, the mPOA kisspeptin neurones, is functional in male goats. Thus, sexual differentiation of the LH surge-generating system would not be applicable to goats.

Lack of pulse and surge modes and glutamatergic stimulation of luteinising hormone release in Kiss1 knockout rats.

Kisspeptin, encoded by the Kiss1 gene, has attracted attention as a key candidate neuropeptide in controlling puberty and reproduction via regulation of gonadotrophin-releasing hormone (GnRH) secretion in mammals. Pioneer studies with Kiss1 or its cognate receptor Gpr54 knockout (KO) mice showed the indispensable role of kisspeptin-GPR54 signalling in the control of animal reproduction, although detailed analyses of gonadotrophin secretion, especially pulsatile and surge-mode of luteinising hormone (LH) secretion, were limited. Thus, in the present study, we have generated Kiss1 KO rats aiming to evaluate a key role of kisspeptin in governing reproduction via pulse and surge modes of GnRH/LH secretion. Kiss1 KO male and female rats showed a complete suppression of pulsatile LH secretion, which is responsible for folliculogenesis and spermatogenesis, and an absence of puberty and atrophic gonads. Kiss1 KO female rats showed no spontaneous LH/follicle-stimulating hormone surge and an oestrogen-induced LH surge, suggesting that the GnRH surge generation system, which is responsible for ovulation, does not function without kisspeptin. Furthermore, challenge of major stimulatory neurotransmitters, such as monosodium glutamate, NMDA and norepinephrine, failed to stimulate LH secretion in Kiss1 KO rats, albeit they stimulated LH release in wild-type controls. Taken together, the results of the present study confirm that kisspeptin plays an indispensable role in generating two modes (pulse and surge) of GnRH/gonadotrophin secretion to regulate puberty onset and normal reproductive performance. In addition, the present study suggests that kisspeptin neurones play a critical role as a hub integrating major stimulatory neural inputs to GnRH neurones, using newly established Kiss1 KO rats, which serve as a useful model for detailed analysis of hormonal profiles.

The LHRH pulse generator: a mediobasal hypothalamic location.

The location and mechanism of LHRH pulse generator are discussed based on our series of experiments. Suckling stimulus is a novel stimulus that inhibits LH pulses without any cooperation from ovarian steroids, unlike other stimuli such as stress, photoperiod etc. It is directly involved in suppressing the activity of the LHRH pulse generator. The information from teats suckled by pups or babies is conveyed dorsally to the mediobasal hypothalamus (MBH), where the LHRH pulse generator may be located. Experiments using various types of deafferentation and fetal brain tissue transplantation confirmed that the LHRH pulse generator is located in the MBH and suggested that LHRH pulse generator consists of nonLHRH neurons. Endogenous excitatory amino acid is one of the possible neurotransmitters that regulate LHRH release at the nerve terminal in ME.

Estrogen feedback needed at the paraventricular nucleus or A2 to suppress pulsatile luteinizing hormone release in fasting female rats.

The feedback sites of estrogen within the hypothalamus and lower brain stem involved in the suppression of pulsatile LH secretion during 48-h fasting were examined in ovariectomized rats with local estradiol (E2) implants. The animals were ovariectomized and immediately implanted stereotaxically with stainless steel cannula containing crystal E2 diluted 10 times with crystal cholesterol into the medial preoptic area, paraventricular nucleus (PVN), arcuate nucleus (ARC), locus ceruleus, or A1 or A2 region of the brain. Five days later, animals were deprived of food for 48 h, and blood samples were collected every 6 min for 3 h. Animals were immediately refed for 45 h and bled again as described above. Changes in the mean LH concentrations over the 3-h sampling period and the frequency and amplitude of LH pulses were determined by calculating the differences in these parameters between the first and second blood samplings in each animal. Fasting significantly lowered mean LH concentrations in animals implanted with E2 in the A2. The more potent suppression of pulsatile LH release during fasting was found in rats with E2 implants in the PVN: the mean LH concentrations and LH pulse frequency were significantly reduced by fasting in this group. In the animals with E2 implants in the medial preoptic area, ARC, locus ceruleus, or A1, 48-h fasting did not induce any significant changes in LH pulse parameters compared to those in cholesterol-implanted controls. A decrease in LH pulse amplitude was apparent in refed rats as well as fasted animals only when E2 was implanted in the ARC. These results suggest that the feedback action of estrogen at the PVN and/or A2 is required for fasting-induced suppression of pulsatile LH release, as opposed to the so-called negative feedback action of estrogen, which tonically suppresses LH release in nonfasting rats.

Involvement of the catecholaminergic input to the paraventricular nucleus and of corticotropin-releasing hormone in the fasting-induced suppression of luteinizing hormone release in female rats.

The roles of the adrenergic projection to the paraventricular nucleus (PVN) and of central CRH in the suppression of pulsatile LH secretion during 48-h fasting were examined in ovariectomized estradiol (E2)-treated rats. The animals were ovariectomized and immediately implanted with Silastic tubing containing E2. One week after ovariectomy and E2 implantation, the animals were implanted stereotaxically with a guide cannula for microinjection into the PVN or intracerebroventricular (icv) injection. One week later, some of the animals were deprived of food for 48 h. The unfasted controls were provided with food ad libitum. At this point, blood samples were collected every 6 min for 3 h. Animals received an injection of 50 micrograms alpha-methyl-p-tyrosine (AMPT), a catecholamine synthesis inhibitor, into the PVN 3 h before the sampling started or an icv injection of 26 nmol alpha-helical CRF-(9-41), a CRH antagonist, after the first hour of blood sampling; control animals were given the vehicle at the equivalent time. The fasted animals injected with AMPT showed a significantly higher mean LH concentration and LH pulse frequency over the 3-h sampling period compared with the vehicle-injected controls. Treatment with AMPT had no significant effect on LH secretion in unfasted animals. The icv injection of alpha-helical CRF-(9-41) reinstated the suppressed LH release in fasted rats, but had no significant effect in unfasted animals. These results suggest that the adrenergic projection to the PVN and central CRH are involved in the suppression of pulsatile LH release during food deprivation. The possibility that fasting activates an ascending adrenergic projection that stimulates CRH release and thus suppresses pulsatile LH secretion is discussed.

Corticotropin-releasing hormone mediates suppression of pulsatile luteinizing hormone secretion induced by activation of alpha-adrenergic receptors in the paraventricular nucleus in female rats.

The present study examined which subtype of adrenergic receptor in the paraventricular nucleus (PVN) has a role in regulating pulsatile LH secretion and whether CRH mediates the effect of norepinephrine (NE) injection into the PVN on pulsatile LH secretion in ovariectomized (OVX) and ovariectomized estradiol (E2)-treated (OVX + E2) rats. All animals were OVX, and some were sc implanted with Silastic capsules containing E2 dissolved in peanut oil. One week after ovariectomy and E2 implantation, guide cannulae for injection of agent and alpha-helical CRF-(9-41) (alpha-hel CRF), an antagonist of CRH, were stereotaxically implanted into the PVN and third ventricle, respectively. Animals were bled for 3 h at 6-min intervals through an atrial cannula immediately after PVN injection of NE or various adrenergic receptor agonists (phenylephrine, an alpha 1-adrenergic receptor agonist; clonidine, an alpha 2-agonist; and isoproterenol, a beta-agonist). Some of the animals were injected with alpha-hel CRF 5 min before NE injection. NE and both alpha-receptor agonists inhibited pulsatile LH secretion throughout the 3-h sampling period in OVX + E2 rats and significantly reduced the mean plasma LH levels and the LH pulse frequency. On the other hand, LH secretion was inhibited transiently for the first 1-2 h by an injection of NE or an alpha 2-receptor agonist into the PVN in OVX rats, resulting in a significant decrease only in mean plasma LH levels in the NE-injected group. Injection of a beta-agonist did not affect pulsatile LH secretion in either OVX or OVX + E2 animals. Intracerebroventricular injection of alpha-hel CRF before PVN injection of NE completely blocked the NE-induced suppression of pulsatile LH secretion in OVX+E2 rats. These results suggest that 1) the noradrenergic system projecting to the PVN suppresses pulsatile LH secretion via the activation of alpha-adrenergic receptors; 2) this inhibition is mediated by CRH release; and 3) estrogen enhances this suppression.

Paraventricular norepinephrine release mediates glucoprivic suppression of pulsatile luteinizing hormone secretion.

Restriction of glucose availability by 2-deoxyglucose (2DG) suppresses pulsatile LH release. The aim of the present study was to determine whether norepinephrine (NE) release in the paraventricular nucleus (PVN) is involved in the glucoprivic suppression of LH secretion in ovariectomized rats. Twelve days after ovariectomy, animals were stereotaxically implanted with a guide cannula for microdialysis in the PVN. Two days later, the PVN was perfused continuously with Ringer's solution or Ringer's solution containing a catecholamine synthesis inhibitor, alpha-methyl-p-tyrosine (100 microM), through a microdialysis probe inserted in the guide cannula 2 h before the beginning of sampling, which lasted 3 h. Blood samples were collected every 6 min through an atrial cannula, and dialysates were collected every 20 min. One hour after the beginning of sampling, 2DG (400 mg/kg BW) was administered iv through the atrial cannula. Paraventricular NE levels significantly increased immediately after 2DG injection (P < 0.05), and both mean LH concentrations and the frequency of LH pulses decreased. By contrast, when alpha-methyl-p-tyrosine was administered into the PVN, 2DG did not produce an increase in paraventricular NE, and no depression of LH secretion occurred. These results suggest that the PVN mediates the glucoprivic suppression of LH pulses via the release of NE.

Central, but not peripheral, glucose-sensing mechanisms mediate glucoprivic suppression of pulsatile luteinizing hormone secretion in the sheep.

Changes in glucose availability are proposed to modulate pulsatile GnRH secretion, and at least two anatomical sites, the liver and hindbrain, may serve as glucose sensors. The present study determined the relative importance of these putative glucose-sensing areas in regulating pulsatile LH secretion in the sheep. Our approach was to administer the antimetabolic glucose analog, 2-deoxy-D-glucose (2DG) into either the hepatic portal vein or the fourth ventricle in gonadectomized females in which LH pulse frequency was high. In the first study, a catheter was placed in the ileocolic vein to determine the effects of local injection of 2DG into the hepatic portal system on the release of LH. After monitoring the pattern of LH secretion for 4 h, 2DG (250 mg/kg) was infused (500 microl/min) into the liver for 2 h. For comparison, animals were also given the same dose of 2DG into a jugular vein for 2 h. Administration of 2DG into either the hepatic portal or jugular vein reduced LH pulse frequency to the same extent. Infusion of the lower dose (50 mg/kg) locally into the hepatic portal vein did not affect plasma LH profiles. Collectively, these results are interpreted to indicate that the liver does not contain special glucose-sensing mechanisms for the glucoprivic suppression of LH pulses. In the second study, 2DG (5 mg/kg) was infused (50 l/min) for 30 min into the fourth ventricle or lateral ventricle. During the subsequent 4-h sampling period, pulsatile LH secretion was significantly suppressed, but there was no significant difference in LH pulse frequency between sites of infusion. Peripheral 2DG concentrations were not detectable after either fourth or lateral ventricle infusions, indicating that the 2DG had acted centrally to suppress LH pulses. Plasma cortisol concentrations increased more in animals infused with 2DG into the fourth ventricle than in those infused into the lateral ventricle, suggesting that 2DG infused into lateral ventricle is transported caudally into the fourth ventricle and acts within the area surrounding the fourth ventricle. Overall, these findings suggest that an important glucose-sensing mechanism is located circumventricularly in the fourth ventricle. Moreover, the liver does not appear to play an important role in detecting glucoprivic action of 2DG to suppress pulsatile LH secretion.

Differential expression of epidermal growth factor receptor (EGF-R) gene and regulation of EGF-R bioactivity by progesterone and estrogen in the adult mouse uterus.

The present study examined several aspects of epidermal growth factor receptor (EGF-R) in the mouse uterus during the peri-implantation period and after ovarian hormone treatment of adult ovariectomized mice. The cell-specific distribution, regulation of expression, and binding kinetics were assessed by immunohistochemistry, Northern blot analysis, and ligand binding assays, respectively. Affinity cross-linking studies ascertained the size of the EGF-R, and its bioactivity was examined by determining EGF-dependent subcellular protein tyrosine kinase activity and receptor autophosphorylation. In the intact uterus and separated cell types, EGF-R was detected in the stroma, deciduum, and myometrium, but not in the luminal or glandular epithelium. Uterine EGF-R mRNA transcript profiles showed some differences between pregnant and ovariectomized mice regardless of steroid hormone treatments. Two major [6.5- and 2.7-kilobase (kb)] and two less abundant (9.6- and 5.0-kb) transcripts were detected in pregnant uterine poly(A)+ RNA. Three additional transcripts (< 2.0 kb) were detected in decidual poly(A)+ RNA, and a larger transcript (8.0 kb) was detected in uterine poly(A)+ RNA isolated from ovariectomized mice. Scatchard analysis of EGF binding also revealed apparent differences in binding kinetics between pregnant and ovariectomized mice, although EGF was cross-linked to a 170-kilodalton protein under these conditions. Two classes (Kd, approximately 0.2 and approximately 2.0 nM) of binding sites were noted in pregnant mice, whereas a single class (Kd, approximately 1.0 nM) was found in ovariectomized mice. 17 beta-Estradiol (E2) caused a rapid transient upregulation of uterine EGF-R mRNA levels and increased the number of EGF-binding sites in ovariectomized mice, as did an injection of progesterone (P4). However, the bioactivity of EGF-R could not be detected in uteri of ovariectomized mice treated with oil or P4. E2 treatment was found to be essential for EGF-R bioactivity. Taken together, the results suggest that in the adult mouse uterus, EGF-R status is influenced by factors other than P4 and E2, the epithelium is not the direct target for the actions of EGF-related growth factors as thought previously, the mitogenic effects of these growth factors on epithelial cells in vivo are perhaps mediated by other uterine cell-types expressing EGF-R, and, lastly, these growth factors are not likely to be functional in the uterus in the absence of estrogen. The present observations are supportive of the concept of paracrine or juxtacrine interactions between EGF-related growth factor ligands of luminal epithelial origin and blastocyst EGF-R in the process of implantation.

Evidence for terminal regulation of GnRH release by excitatory amino acids in the median eminence in female rats: a dual immunoelectron microscopic study.

The present study was designed to determine whether excitatory amino acids directly act on the gonadotropin-releasing hormone (GnRH) nerve terminals to release the peptide. The median eminence taken from ovariectomized rats was dual immunostained with GnRH and ionotropic glutamate receptor subtypes (NR1, GIuR1, GluR2/3, GluR6/7 and KA2), and their colocalization was examined under electron microscopy. The connection of fibers immunopositive for GnRH and glutamate was also examined. Of the glutamate receptor subtypes, NR1- and KA2-immunoreactivities were colocalized with GnRH-immunoreactivity in nerve terminals of the median eminence. In addition, some glutamate-immunopositive nerve terminals were shown to abut the many GnRH-immunopositive nerve terminals. No synaptic contacts were observed on these immunopositive nerve terminals. These results suggest that GnRH release is regulated at the GnRH nerve terminals by excitatory amino acids in a non-synaptic manner in the median eminence.

Reduction of glucose availability suppresses pulsatile luteinizing hormone release in female and male rats.

Glucose availability controls reproductive activity through modulation of LH secretion. The aim of the present study was to determine whether the glucoprivic suppression is potentiated by gonadal steroids and if glucoprivic suppression of pulsatile LH release is sexually differentiated. Pulsatile LH secretion was examined in rats after peripheral (jugular) administration of the competitive inhibitor of glycolysis, 2-deoxyglucose (2DG). Fourteen days after gonadectomy, blood samples were collected every 6 min for 3 h. One hour after the onset of sampling, 2DG was administered peripherally (200, 400, or 800 mg/kg BW, iv), and food intake was determined after 2DG injection in gonadectomized males and females in the presence or absence of sex steroids (testosterone or estradiol). To test the ability of the pituitary to produce LH under glucoprivic conditions, LHRH was injected every 30 min for 2.5 h in ovariectomized (OVX) rats 30 min after treatment with 400 mg/kg 2DG. At all peripheral doses of 2DG in females and at the middle and high doses of 2DG in males, mean plasma LH and LH pulse frequency decreased (P < 0.05) in the presence of steroids. However, in the absence of sex steroids, the lowest dose in females and the middle dose in males were not effective. Pituitary function appeared normal, because increases in mean plasma LH in response to the exogenous LHRH occurred in OVX rats treated with the middle dose of 2DG. Food intake significantly (P < 0.05) increased after 2DG injection in all groups except estrogen-treated OVX females at the low and high doses of 2DG. These findings suggest that glucoprivic suppression of LH pulses is potentiated by gonadal steroids in both sexes. Moreover, the hypothalamo-hypophyseal axis of the female rat seems to be more sensitive to the decreased glucose availability induced by 2DG than that of the male.

Suppression of luteinizing hormone pulses by restriction of glucose availability is mediated by sensors in the brain stem.

The availability of metabolic fuels such as glucose is known to influence reproductive function. Peripheral administration of 2-deoxyglucose (2DG), a competitive inhibitor of glycolysis, inhibits pulsatile LH secretion in the rat and growth-retarded lamb. We hypothesized that such glucoprivic suppression of LH secretion is mediated by the lower brain stem, because studies of both ingestive and reproductive behavior implicate lower brain stem structures, such as the area postrema, as a site that is sensitive to glucose availability. In the present study, the effect of a 2DG infusion, targeted to the fourth ventricle, on pulsatile LH secretion was examined in male rats. The males were castrated or castrated and immediately implanted with testosterone. Blood samples were collected through an indwelling atrial cannula every 6 min for 4 h for LH determination. After the first hour of blood sampling, 2DG (4 or 40 mg/kg) was infused into the fourth ventricle at a flow rate of 0.2 microliter/min through a cannula that had been stereotaxically implanted 1 week before sampling. The high dose of 2DG (40 mg/kg), but not the low dose (4 mg/kg), suppressed pulsatile LH secretion and increased food intake in both castrated and testosterone-treated castrated rats. LH secretion and food intake were not affected by the infusion of xylose (40 mg/kg) as an isoosmotic control. The site specificity of the 2DG treatment was confirmed by histological examination after an isovolumetric infusion of dye (0.2 microliter/min). These results suggest that glucose availability could influence LH secretion as well as feeding through a central sensor in the lower brain stem and are consistent with the idea that the area postrema might be an important glucosensor involved in the modulation of LH secretion.

Localization of glucokinase-like immunoreactivity in the rat lower brain stem: for possible location of brain glucose-sensing mechanisms.

Pancreatic glucokinase (GK) is considered an important element of the glucose-sensing unit in pancreatic beta-cells. It is possible that the brain uses similar glucose-sensing units, and we employed GK immunohistochemistry and confocal microscopy to examine the anatomical distribution of GK-like immunoreactivities in the rat brain. We found strong GK-like immunoreactivities in the ependymocytes, endothelial cells, and many serotonergic neurons. In the ependymocytes, the GK-like immunoreactivity was located in the cytoplasmic area, but not in the nucleus. The GK-positive ependymocytes were found to have glucose transporter-2 (GLUT2)-like immunoreactivities on the cilia. In addition, the ependymocytes had GLUT1-like immunoreactivity on the cilia and GLUT4-like immunoreactivity densely in the cytoplasmic area and slightly in the plasma membrane. In serotonergic neurons, GK-like immunoreactivity was found in the cytoplasm and their processes. The present results raise the possibility that these GK-like immunopositive cells comprise a part of a vast glucose-sensing mechanism in the brain.

Effect of the suckling stimulus on daily LH surges induced by chronic oestrogen treatment in ovariectomized lactating rats.

Effects of the suckling stimulus on the daily LH surge induced by chronic oestrogen treatment were examined in ovariectomized lactating rats. Wistar-Imamichi strain rats were kept under 14 h light:10 h darkness (lights on at 05.00 h). Litter size was adjusted to eight on day 1 (day 0 = day of parturition) and ovariectomy performed on day 2. Lactating rats deprived of their litters on day 0 served as nonlactating controls. Silicone elastomer tubing filled with oestradiol was implanted on day 6 or 15. Blood samples were collected through an indwelling cannula at 10.00 and 17.00 h on each day after implantation to detect daily LH surges. Daily LH surges occurred in the late afternoon in both lactating and non-lactating rats implanted with oestradiol on day 6 or 15. The amplitude of daily LH surges in lactating rats implanted on day 6 declined much more rapidly than in non-lactating rats implanted on day 6, but no significant difference was found in the profile of the LH surge between lactating and non-lactating rats implanted on day 15. Pituitary LH contents just before the daily LH surge (12.00-12.30 h) 4 days after implantation in lactating rats implanted with oestradiol on day 6 were significantly less than those in nonlactating rats implanted with oestradiol on day 6 or 15 and in lactating rats implanted on day 15.(ABSTRACT TRUNCATED AT 250 WORDS)