Immunocytochemistry of luteinizing hormone releasing hormone (LHRH) in the sheep hypothalamus during various reproductive stages

Summary Using the labeled and unlabeled immunoperoxidase methods, the distribution and concentration (1) of immunoreactive LHRH-material in the hypothalamus, and (2) of gonadotropic hormones in the adenohypophysis of the ewe were determined during various reproductive stages, including two phases of the estrous cycle, anestrus, lactation, and the state after ovariectomy. The concentration of LHRH-immunoreactive material varied in particular regions of the median eminence (ME) and was closely dependent on the physiological state. The immunoreactive material was most abundant in hypothalami of lactating animals, exceeding gradually the corresponding deposits in ovariectomized animals, in ewes during the 16th day of the estrous cycle (before ovulation), and in anestrous ewes. A severe depletion of LHRH from the rostral and central parts of the ME was observed 24h after ovulation. This depletion was accompanied by a degranulation of LH-and prolactin-producing cells in the adenohypophysis. It is postulated that LHRH is stored in the ME of the sheep in all examined endocrine stages. Only circumscribed regions of the ME take part in the cyclic release of LHRH during the ovulatory phase; this suggests a functional differentiation of the nerve terminals in this neurohemal area.     

Norepinephrine-stimulated in vitro release of luteinizing hormone-releasing hormone (LHRH) from median eminence tissue is facilitated by inhibition of LHRH-degrading activity in hens

We and others have previously reported the existence of hypothalamic and anterior pituitary (AP) enzymes that degrade luteinizing hormone (LH)-releasing hormone (LHRH). We have further characterized these LHRH-degrading activities (LHRH-DA) and in addition assessed the role of LHRH-DA in LHRH release from median eminence (ME) tissue in vitro. Major LHRH-DA components were separated and their molecular weights were estimated by gel filtration chromatography. The role of LHRH-DA in LHRH release was determined by release studies from isolated ME, in the presence and absence of N-tosyl L-phenylalanine chloromethyl ketone (TPCK) and/or norepinephrine (NEpi). Degradation and in vitro release studies were performed by using LHRH analogs with amino acid substitutions at their 5-6 bond. Biological activity of these analogs was assessed by measuring in vitro LH release from dispersed anterior pituitary cells. LHRH-DA was determined by high-performance liquid chromatography; LH and LHRH were measured by radioimmunoassay. Separation of LHRH-DA by gel filtration chromatography yielded two major enzymatic activities: a Tyr5-Gly6 cleaving endopeptidase and a post-proline cleaving enzyme. Although LHRH-DA from AP and ME produced identical degradation fragments, the former had 3-fold greater specific activity than the latter. LHRH moieties with a Tyr5-Gly6 bond substitution were more resistant to enzymatic degradation and had greater biological activity than LHRH moieties with a Tyr5-Gly6 bond. TPCK decreased LHRH-DA and increased NEpi-stimulated in vitro release of LHRH from isolated ME.(ABSTRACT TRUNCATED AT 250 WORDS)     

In-vitro pulsatile luteinizing hormone-releasing hormone output is dependent on hypothalamic region and the stage of the estrous cycle

In the following experiments, the role of the preoptic-suprachiasmatic area (POA-SCN) in the control of luteinizing hormone-releasing (LHRH) release was examined by in vitro superfusion of either mediobasal hypothalamus (MBH) or MBH-POA-SCN fragments obtained from cycling rats killed on various days of the estrous cycle. The lowest level of LHRH output occurred during estrus, highest levels during diestrus, and intermediate levels on proestrus in the MBH-POA-SCN preparation. As expected, significant decreases in LHRH output from the MBH alone occurred during both days of diestrus and on proestrus, as compared to output from the MBH-POA-SCN tissue, since this structure contains most of the LHRH perikarya. However, similar LHRH secretion patterns were detected in estrus from both preparations. The average period of the LHRH pulses for the estrous cycle in the MBH-POA-SCN was 30.9 +/- 1.2 min compared to 97.7 +/- 25.1 min in the MBH, with significant differences occurring on diestrus 2. The increase and extreme variability of the period of LHRH pulses in the MBH region, compared to the MBH-POA-SCN region, suggests that it is the latter region that contains the neural circuits that control the LHRH pulse generator. The LHRH pulse amplitudes from both hypothalamic regions were similar during all phases of the estrous cycle, except diestrus 2, when the LHRH pulse amplitude from the MBH region was significantly lower than the LHRH pulse amplitude from MBH-POA-SCN. The percentage of LHRH released in the MBH did not vary with the estrous cycle, however, in the MBH-POA-SCN significant changes were observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diurnal variations of medial basal and anterior hypothalamic thyroliberin [TRH] and serum thyrotropin [TSH] concentrations in male rats.

The diurnal variation of TRH concentrations in different parts of hypothalamus was studied in 80 male rats, which were killed in groups of 5 at 3 h intervals. The hypothalamus was dissected into three parts: I) medial basal hypothalamus (MBH), II) anterior hypothalamus, and III) dorsal hypothalamus. Anterior pituitary and serum TSH concentrations were also measured. TRH concentrations were higher in MBH than in the other parts of the hypothalamus: at night 300–450 pg/mg of wet weight of tissue. When the lights were turned on, MBH-TRH levels began to decrease, reaching a nadir of 210 pg/mg at 12 noon. After 15 h, MBH-TRH levels began to increase again. The changes in TRH levels in anterior hypothalamus were usually opposite to those in MBH (r = ?0.6185). Serum TSH levels were about 800 ng/ml during the day and were decreased to about one half of these levels when the lights were turned off. Serum TSH levels were positively correlated with anterior hypothalamic TRH levels (r = 0.6457) and inversely correlated with MBH-TRH levels (r = ?0.7747). Anterior pituitary TSH levels showed small but statistically insignificant variations. In conclusion, there were statistically interrelated diurnal rhythms in anterior hypothalamic and MBH-TRH levels and serum TSH concentrations.     

Ontogeny of neuropeptidergic systems: luteinizing hormone releasing hormone (LHRH); somatostatin (SRIF) and neurophysin (NF) in the hypothalamus of the domestic pig by immunocytochemistry

The ontogenic development of some hypothalamic neuropeptides: luteinizing hormone releasing hormone (LHRH); somatostatin (SRIF) and neurophysin (NF) and their localization in the hypothalamus of fetuses in different stages of the fetal life were studied by immunoperoxidase method. It was found that differentiation of the neurons which produce the examined hormones begins in the midstage of pregnancy. LHRH is stored in the nerve terminals of the median eminence (ME) and organum vasculosum of the lamina terminalis (OVLT) since 72 day of gestation and its amount gradually increases with the development of the embryo. In this stage a few immunoreactive (ir) LHRH perikarya appear but they are most numerous in the last days of pregnancy (110 day). They are localized in the most anterior periventricular parts of the hypothalamus, area preoptica, diagonal band of Broca and very rare in the medial-basal hypothalamus. Somatostatin is produced in the separate neuronal system and appears in the last days of fetal life. Neurophysin is present in both magnocellular nuclei in 72 day-old fetuses, but at the end of gestation it is seen also in some preoptico-septal region.     

Functional and morphological changes in the hypothalamo-pituitary-gonadal axis of aged female rats.

Age-related functional and morphological alterations in the hypothalamo-pituitary-gonadal axis were investigated in old recurrently pseudopregnant (RPP) female rats, and these alterations were compared with those in young diestrous rats. LHRH in the median eminence (ME) and mediobasal hypothalamus (MBH) as well as plasma FSH, LH, and progesterone were measured by RIA. LHRH in the lateral ME (LME) and pituitary FSH and LH were evaluated by morphometry and densitometrical immunocytochemistry. Furthermore, by light microscopy, we classified and counted the number of ovarian follicles and corpora lutea. LHRH concentrations in the ME and MBH were similar in old and young rats, whereas in old rats, plasma FSH was markedly increased, LH was moderately increased, and plasma progesterone was unchanged. The number and the total area and immunoreactivity of LHRH-labeled axon cross sections in the LME were reduced in old rats. The number of nucleated FSH-labeled cells and total FSH area and immunoreactivity were almost twice in old compared with young animals. The measurements of LH-labeled cells were not different between the two groups. In old rats, the numbers of ovarian follicles and corpora lutea were reduced and that of atretic follicles increased. In conclusion, age-related morphological impairments of LHRH axons associated with an increased number of FSH gonadotropes and higher plasma FSH in our old RPP rats suggest hypothalamic and pituitary disturbances, which may largely contribute to the complex hormonal disarrangement responsible for the decline of reproductive functions in old female rats.     

Projections to the median eminence and the arcuate nucleus with special reference to monoamine systems: Effects of lesions

The external layer of the median eminence (ELME) and the arcuate nucleus of male rats were studied with the Falck-Hillarp technique and electron microscopy of aldehyde-OsO4 or KMnO4 fixed material after various types of hypothalamic deafferentation experiments with the Haláz knife. Special reference was paid to the monoamine systems and the results can be summarized as follows. 1. The main monoaminergic input to the ELME comes from the arcuate nucleus-periventricular area via a dorsal approach. A horizontal transection through the arcuate nucleus decreases the percentage of monoamine boutons i.e. boutons with small granular vesicles, from 31.6% in the controls to 4.4% in the lesion group, whereas only a small effect is seen after anterior (or complete) deafferentations. 2. A major input to the ELME enters the basal hypothalamus at the anterior-lateral aspects (see Réthelyi and Halsáz, 1970). The fibers cut after anterior deafferentations in all probability mainly come from cell bodies localized in the anterior hypothalamus or even further rostrally but some may represent Na axons ascending from the lower brain stem. 3. The degeneration course of nerve endings in the ELME both after anterior deafferentations as well as after lesions in the arcuate nucleus is rapid (within 2-3 days) and morphologically characterized by an initial aggregation of large dence cored vesicles seemingly to electron dense bodies within the boutons and probably also to a closer spacing of the small electron lucent synaptic vesicles (see Raisman, 1972). This type of degeneration seems to take place both in monoamine and non-monoamine neurons. 4. Degenerating boutons are found in the arcuate nucleus after anterior and complete deafferentations. Thus, the anterior hypothalamus may exert an "indirect" control of the pituitary gland via synapses on arcuate neurons although quantitavely the direct influence through the projection to the ELME is of more importance. 5. After anterior deafferentations enlarged axons containing large amounts of large dense cored vesicles and other organelles are found caudally of the cut indicating the existence of rostral projections from the medial hypothalamus.     

Changes in substance P content at the hypothalamic-pituitary axis during the Wallerian degeneration of peripheral sympathetic neurons after superior cervical ganglionectomy in male rats: effect of hyperprolactinemia

The effects of Wallerian degeneration of the peripheral sympathetic neurons projecting to the hypothalamus on the mechanism of interaction between prolactin and substance P (SP) were examined. The effects of superior cervical ganglionectomy (SCGx) on SP content in various hypothalamic regions and in the hypophysis were evaluated in control and hyperprolactinemic rats. Male rats that received pituitary transplants at the age of 5 days and age-matched sham-operated controls were used. Pituitary grafting significantly increased circulating values of prolactin, as did SCGx. In hyperprolactinemic rats, SCGx partially decreased plasma prolactin levels. Neonatal hyperprolactinemia decreased SP content in the anterior (AH) and posterior (PH) hypothalamus and in the median eminence (ME), but increased it in the mediobasal hypothalamus (MBH). Acute SCGx significantly increased SP in the MBH, PH, and ME. SCGx in hyperprolactinemic animals further increased SP content in MBH. In the ME and Ah, SCGx in pituitary grafted rats decreased SP content as compared with the controls. In the pituitary gland (PG), SCGx only decreased SP content in hyperprolactinemic, but not in control rats. An interaction between peripheral nor-adrenergic neurons and prolactin to regulate SP within the hypothalamus was positive in the MBH, AH, ME, and PG, but not in the PH. These data indicate the existence of interactive mechanisms between prolactin and the peripheral sympathetic neurons to regulate SP content at the hypothalamic-pituitary axis. Interrelationships between prolactin and SP were also observed.     

Development of the hypothalamic luteinizing hormone-releasing hormone-containing neuron system in the rat: in vivo and in transplantation studies

The development of the hypothalamic LHRH-containing neuron system was immunohistochemically investigated in vivo and in tissue transplantation using rat embryos aged from 12.5 to 17.5 days of gestation. The sera used were generated against rat gonadotropic hormone-releasing hormone-associated peptide (28-56) (rGAP) and LHRH. Immunoreaction for rGAP was first found in cells migrated from and in the vomeronasal organ on Days 13.5 and 14.5 of gestation. Immunoreactive cells seem to ascend along the terminal nerves, reaching the medial surface of the forebrain vesicles. Subsequently the cells occurred in the septum and further into their final position in the septopreoptic-diagonal band area on Days 16.5-17.5 of gestation; during this traverse the cells become secretory neurons after changes in morphology and in behavior. Intraventricular transplantation revealed that nasal epithelia of Day 12.5 embryos raised only a few cells immunoreactive both for LHRH and rGAP, but a great number of immunoreactive cells and fibers in the presence of the medial basal hypothalamus (MBH). The fibers formed a median eminence-like structure together with dense capillary plexus that had grown in the cografted MBH. The same phenomenon was apparently observed in the grafts obtained from older embryos of gestation, but not in the combined grafts of the anterior septum and the nasal epithelium or the MBH. We conclude that hypothalamic LHRH neurons originate from the nasal placode and acquire secretory behavior in the presence of the MBH.     

Median eminence lesions reveal separate hypothalamic control of pulsatile follicle-stimulating hormone and luteinizing hormone release

The effects of hypothalamic lesions designed to destroy either the anterior median eminence (ME) or the posterior and mid-ME on pulsatile release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were determined in castrated male rats. In sham-operated animals, mean plasma FSH concentrations rose to peak at 10 min after the onset of sampling, whereas LH declined to a nadir during this time. In the final sample at 120 min, the mean FSH concentrations peaked as LH decreased to its minimal value. In rats with anterior ME lesions, there was suppression of LH pulses with continuing FSH pulses in 12 of 21 rats. On the other hand, in animals with posterior to mid-ME lesions, 3 out of 21 rats had elimination of FSH pulses, whereas LH pulses were maintained. Fifteen of 42 operated rats had complete ME lesions, and pulses of both hormones were abolished. The remaining 12 rats had partial ME lesions that produced a partial block of the release of both hormones. The results support the concept of separate hypothalamic control of FSH and LH release with the axons of the putative FSH-releasing factor (FSHRF) neuronal system terminating primarily in the mid- to caudal ME, whereas those of the LHRH neuronal system terminate in the anterior and mid-median eminence. We hypothesize that pulses of FSH alone are mediated by release of the FSHRF into the hypophyseal portal vessels, whereas those of LH alone are mediated by LHRH. Pulses of both gonadotropins simultaneously may be mediated by pulses of both releasing hormones simultaneously. Alternatively, relatively large pulses of LHRH alone may account for simultaneous pulses of both gonadotropins since LHRH has intrinsic FSH-releasing activity.     

Development of the hypothalamic luteinizing hormone-releasing hormone-containing neuron system in the rat: In vivo and in transplantation studies

The development of the hypothalamic LHRH-containing neuron system was immunohistochemically investigated in vivo and in tissue transplantation using rat embryos aged from 12.5 to 17.5 days of gestation. The sera used were generated against rat gonadotropic hormone-releasing hormone-associated peptide (28–56) (rGAP) and LHRH. Immunoreaction for rGAP was first found in cells migrated from and in the vomeronasal organ on Days 13.5 and 14.5 of gestation. Immunoreactive cells seem to ascend along the terminal nerves, reaching the medial surface of the forebrain vesicles. Subsequently the cells occurred in the septum and further into their final position in the septopreoptic-diagonal band area on Days 16.5–17.5 of gestation; during this traverse the cells become secretory neurons after changes in morphology and in behavior. Intraventricular transplantation revealed that nasal epithelia of Day 12.5 embryos raised only a few cells immunoreactive both for LHRH and rGAP, but a great number of immunoreactive cells and fibers in the presence of the medial basal hypothalamus (MBH). The fibers formed a median eminence-like structure together with dense capillary plexus that had grown in the cografted MBH. The same phenomenon was apparently observed in the grafts obtained from older embryos of gestation, but not in the combined grafts of the anterior septum and the nasal epithelium or the MBH. We conclude that hypothalamic LHRH neurons originate from the nasal placode and acquire secretory behavior in the presence of the MBH.     

Effects of ethanol on rat hypothalamic luteinizing hormone releasing hormone. A study utilizing radioimmunoassay

To further understand the mechanism of action by which ethanol (ETOH) decreases plasma luteinizing hormone (LH) levels, the effects of multiple i.p. injections of EOH (1.0--1.5 g/kg) or saline on hypothalamic luteinizing hormone releasing hormone (LHRH) and plasma LH concentrations were evaluated in intact and castrate male rats. After injections, animals were decapitated, brains rapidly removed, and blocks containing the hypothalamus [with median eminence (ME)] were isolated. Hypothalami were subjected to acetic acid extraction and LHRH content quantitated via radioimmunoassay (RIA). Hypothalamic LHRH was found to be inversely correlated with plasma LH. In response to castration, both saline and ETOH-treated rats showed a decrease in hypothalamic LHRH content with a concomitant increase in plasma LH; however, the ETOH-treated animals retained significantly greater concentrations of LHRH and showed significantly lower plasma LH levels when compared to saline-treated controls. Likewise, ETOH-treated intact animals showed significant increases in LHRH content, with LH levels remaining significantly lower than the saline-treated intact controls. Thus, these data from both intact and castrate rats provide evidence to support the hypothesis that alcohol-induced decreases in LH levels are due to a diminished release rate of hypothalamic LHRH.     

Growth hormone and prolactin secretion after hypothalamic deafferentation in pigs

Control of growth hormone (GH) and prolactin (PRL) secretion was investigated in ovariectomized, prepuberal Yorkshire gilts by comparing the effects of anterior (AHD), complete (CHD), and posterior (PHD) hypothalamic deafferentation with sham-operated controls (SOC). Blood samples were collected sequentially via an indwelling jugular catheter at 20-min intervals during surgery and recovery from anesthesia (Day 0) and Days 1 and 2 after cranial surgery. Mean serum concentrations of GH after AHD, CHD, and PHD were reduced (P less than 0.01) when compared with SOC gilts. Furthermore, episodic GH release evident in SOC animals was obliterated after hypothalamic deafferentation. PRL concentrations in peripheral serum of hypothalamic deafferentated gilts remained similar (P greater than 0.05) to those of SOC animals. These results indicate that anterior and posterior hypothalamic neural pathways play a minor role in the control of PRL secretion in the pig in as much as PRL levels remained unchanged after hypothalamic deafferentation. These findings may be interpreted to suggest that the hypothalamus by itself seems able to maintain tonic inhibition of PRL release. In contrast, the maintenance of episodic GH secretion depends upon its neural connections traversing the anterior and posterior aspects of the hypothalamus in the pig.     

Thermal stress reduces serum luteinizing hormone and bioassayable hypothalamic content of luteinizing hormone-releasing hormone in hens

Studies were conducted to evaluate the effects of acute (24 h) thermal stress on anterior pituitary function in hens. Circulating levels of luteinizing hormone (LH) were measured and the ability of the pituitary to respond to luteinizing hormone-releasing hormone (LHRH) challenge was determined. Moreover, bioassayable hypothalamic LHRH content was assessed by using dispersed anterior pituitary cells. In two separate experiments, circulating levels of LH were reduced in hens exposed to acute thermal stress (35 degrees C). Injection of LHRH did not result in significant differences in release of LH between normothermic and hyperthermic hens. However, the hypothalamic content of bioassayable hypothalamic releasing activity from hyperthermic hens were significantly reduced compared with normothermic hens. Taken together, these data suggest that the reproductive decline in the acutely heat-stressed hen is mediated by reduced LH releasing ability of the hypothalamus.     

Hypothalamic sites of catecholamine inhibition of luteinizing hormone in the anestrous ewe.

Norepinephrine (NE) and dopamine (DA) actively inhibit the release of LH in anestrous ewes. This can be detected as an increase in LH pulse frequency following i.v. injection of NE and DA antagonists. The objective of this study was to determine the sites of these inhibitory actions in the ovine hypothalamus by using local administrations of the NE antagonist, phenoxybenzamine (PBZ), or the DA antagonist, pimozide (PIM), into specific hypothalamic areas. Each neurotransmitter antagonist was administered via a chronically implanted steel guide tube into either the preoptic area (POA), retrochiasmatic area (RCh), or the median eminence region (ME). Blood samples were taken every 15 min for 2 h before and 4 h during implantation of these drugs and were analyzed for LH and prolactin by RIA. Control (no treatment) samples were obtained similarly from the same animals on another day. Placement of PBZ into the POA significantly increased LH pulse frequency and mean LH concentrations over control values whereas PIM did not. In contrast, PIM significantly increased LH pulse frequency and mean LH concentrations when placed in the ME or in the RCh, but PBZ did not. No effects of PIM on prolactin concentrations were detected. These results suggest that an NE neural system operates in the POA and that a DA system acts in the medial basal hypothalamus (RCh or ME) to suppress GnRH pulse frequency in the ovary-intact anestrous ewe.     

Release of LHRH in vitro and anterior pituitary responsiveness to LHRH in vivo during sexual maturation in pullets (Gallus domesticus)

An in-vitro superfusion technique was used to study basal and depolarization-induced (32 mmol K+/l) release of LHRH from the mediobasal hypothalamus (MBH) of pullets at 8-25 weeks of age. Plasma LH concentrations and the incremental change (delta LH) after an i.v. injection of 1 or 15 micrograms synthetic ovine LHRH/kg body weight were also determined. Between 8 and 25 weeks of age, significant (P less than 0.01) increases in basal and depolarization-induced release of LHRH (93 and 330%, respectively) were accompanied by a significant (P less than 0.01) rise in the residual LHRH content of MBH tissue (152%), observations which suggest that the ability of the hypothalamus to synthesize and secrete LHRH increases as sexual maturation proceeds. However, plasma LH, which reached a maximum concentration of 2.05 +/- 0.43 micrograms/l at 15 weeks, fell significantly (P less than 0.05) to 1.14 +/- 0.05 micrograms/l at 25 weeks. Since delta LH in response to exogenous LHRH showed a marked and progressive decline between 12 and 20 weeks of age, the low plasma concentration of LH typical of the mature hen is probably attributable to a direct negative-feedback action of ovarian steroids on the anterior pituitary gland rather than to an impaired secretion of LHRH from the median eminence. It is suggested that a dramatic increase in the responsiveness of LHRH nerve terminals in the MBH to depolarization by 32 mmol K+/l between 20 and 25 weeks of age (mean age at onset of lay 21.9 weeks; range 19-25 weeks) may reflect the development of hypothalamic responsiveness to the positive feedback action of progesterone.     

Hypothalamic deafferentation and luteinizing hormone-releasing hormone effects on secretion of luteinizing hormone in prepubertal pigs

The control of luteinizing hormone (LH) secretion was investigated in ovariectomized, prepubertal Yorkshire pigs by comparing the effects of anterior (AHD), complete (CHD), and posterior (PHD) hypothalamic deafferentation to sham-operated controls (SOC). Gilts (n = 16) were assigned randomly to treatments, fitted with an indwelling jugular catheter, and ovariectomized 2 days before deafferentation or sham-operation (Day 0). Blood for radioimmunoassay (RIA) of LH was collected sequentially at 20-min intervals for a period of 2 h before and 24, 48, 72, and 96 h after hypothalamic deafferentation or SOC. Episodic LH release after AHD or CHD was abolished (p less than 0.01), but not after PHD or SOC. Concentrations of serum LH in AHD and CHD dropped (p less than 0.01) at 24 and 48 h after surgery. Levels of LH before and after surgery in PHD and SOC were similar (p greater than 0.05). Infusion of 25 micrograms LH-releasing hormone (LHRH) i.v. at 72 and 96 h after hypothalamic deafferentation and SOC increased (p less than 0.01) serum LH to peak levels within 15 min. after infusion; LH returned to basal levels 60-80 min later. By 96 h after surgery, LH response to LH-releasing hormone (LHRH) was less in AHD and CHD as compared with the response at 72 h postinjection. Concentrations of LH in PHD and SOC were similar (p greater than 0.05) at 72 and 96 h, respectively. The results from this study clearly indicate that neural stimuli originating or traversing the neural areas rostral to the median eminence are required for secretion of LH in the pig.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ascorbic acid enhances the release of luteinizing hormone-releasing hormone from the mediobasal hypothalamus in vitro

Ascorbic acid is frequently used in in vitro studies of neurotransmitter-evoked release of luteinizing hormone-releasing hormone (LHRH) from hypothalamic fragments. Although it is assumed that ascorbate merely prevents the oxidative degradation of catecholamines, we have discovered that ascorbic acid itself produces significant increases in the release of LHRH. Our studies showed that ascorbic acid, at concentrations below 1 mM, produced a dose-dependent release of LHRH from incubated rat mediobasal hypothalamus (MBH). The magnitude of the ascorbate-induced release was in the range of 100-200% above controls; significant amounts of LHRH were released only if the MBH were incubated with ascorbate for time periods longer than 30 minutes. We also found that ascorbate-induced increases in LHRH were equivalent to those produced by another LHRH secretagogue, naloxone, and that the combined effects of the two substances were additive in nature. Although the mechanisms underlying this effect are not fully understood, nonspecific chemical reduction is probably not a factor since sodium metabisulfite did not induce the release of LHRH. It seems probable that ascorbate may enhance the activity of endogenous norepinephrine in the MBH and, thereby, lead to increased release of LHRH.     

Simultaneous measurement of luteinizing hormone-releasing hormone and luteinizing hormone during estradiol-induced luteinizing hormone surges in the ovariectomized ewe

Sequential bleeding and push-pull perfusion of the hypothalamus were used to characterize luteinizing hormone (LH) and LH-releasing hormone (LHRH) release in ovariectomized (OVX) ewes after injection of corn oil or estradiol benzoate (EB). Push-pull cannulae were surgically implanted into the stalk median eminences of 24 OVX ewes. Seven to 14 days later each of 20 animals was given an i.m. injection of 50 micrograms EB. Blood samples and push-pull perfusate were collected at 10-min intervals for 6-12 h beginning 12-15 h after EB injection. Four OVX ewes were given i.m. injections of corn oil 7 days after implantation of push-pull cannulae. Blood samples and push-pull perfusate were collected at 10-min intervals for 4 h between 18 and 22 h after injection of corn oil. Luteinizing hormone remained below 2 ng/ml throughout most of the sampling periods in 9 of 20 EB-treated ewes. In 5 of these 9 LHRH also was undetectable, whereas in 4 LHRH was detectable (1.84 +/- 0.29 pg/10 min), but did not increase with time. Preovulatory-like surges of LH occurred in 11 EB-treated ewes, but LHRH was undetectable in 5. In 4 of 6 ewes showing LH surges and detectable LHRH, sampling occurred during the onset of the LH surge.(ABSTRACT TRUNCATED AT 250 WORDS)     

Frontal deafferentation of the mediobasal hypothalamus in the neonatal rat and its effects on the preoptico-infundibular LHRH-tract

Summary By use of the peroxidase-antiperoxidase-complex (PAP) immunohistological method, the preoptico-infundibular LHRH-tract was studied in adult female rats in which frontal hypothalamic deafferentation was performed at the third or tenth postnatal day. In the former group, this LHRH-tract appeared to be similar to that of the intact controls; the animals showed regular vaginal cycles and ova were present in their oviducts. In the latter group, however, marked reduction in the number of the LHRH-nerve fibers was observed behind the sites of the deafferentation in the mediobasal hypothalamus (MBH), whereas LHRH-immunoreactive perikarya and nerve fibers containing the immunoreactive material were seen rostral to the plane of severance. In these animals reduction of LHRH-fibers in the MBH was accompanied by an anovulatory syndrome characterized by constant vaginal cornification and polyfollicular ovaries. Comparing the glial scar formation induced by the cut, significant differences were detected between the two experimental groups. In the animals deafferented on the 3rd day of life, reduction of nerve cells was seen along the cut, but LHRH-fibers crossing the thin glial scar were detectable in large numbers. On the other hand, in the animals deafferented on the 10th postnatal day, extensive glial scar tissue appeared to interrupt the LHRH-fibers rostral to the cut.