Luteinizing hormone-releasing hormone and thyrotropin-releasing hormone in human and bovine milk

Two hypothalamic peptide hormones, luteinizing hormone-releasing hormone (LHRH) and thyrotropin-releasing hormone (TRH), have been isolated from human milk and bovine colostrum. Acidified methanolic extracts, prepared from human milk, bovine colostrum and rat hypothalami, as well as synthetic LHRH and TRH markers were subjected to high-pressure liquid chromatography (HPLC). The eluates were tested for the presence of LHRH and TRH by specific radioimmunoassays. It was found that milk extracts contain significant amounts of LHRH (3.9 - 11.8 ng/ml) and TRH (0.16 - 0.34 ng/ml), which comigrate with the corresponding marker hormones and with those of hypothalamic origin. The HPLC-purified LHRH from both human and bovine milk was bioactive in a dose-response manner similar to synthetic LHRH.     

Alteration in the normal pattern of serum testosterone and 5α-androstane-3α,17β-diol in the immature male rat following chronic treatment with luteinizing hormone releasing hormone or luteinizing hormone

A major component of sexual maturation in the male rat is a progressive decline in serum concentrations of 5α-androstane-3α,17β-diol (3α-diol) and a concomitant increase in testicular testosterone biosynthesis and secretion. Chronic administration of synthetic luteinizing hormone releasing hormone (LHRH) or luteinizing hormone (LH)/human chorionic gonadotropin (hCG) to immature male rats has been shown to result in a delay in sexual maturation as evidenced by decreased sex accessory gland weights and altered testicular testosterone production. We have examined the postulate that such treatments may either reverse or retard the normal developmental pattern of serum testosterone and 3α-diol concentrations. Chronic $\text{in vivo}$ treatment of 28 day old immature male rats for 2 weeks with daily injections of either 0.5 μg of LHRH, 1.0 μg of LHRH, or 30 μg of LH was found to result in significant reductions in weights of the seminal vesicles and ventral prostate glands and diminutions in serum testosterone concentrations. Serum content of 3α-diol was either unchanged or slightly elevated in the LHRH treated animals and increased significantly in the LH treated animals. These data suggest that either a reversal of or retardation in the normal developmental pattern of serum testosterone and 3α-diol content has been achieved in the immature male rat by chronic LHRH or LH treatment.     

The human hypothalamic LHRH precursor is the same size as that in rat and mouse hypothalamus

The synthesis of the decapeptide luteinizing hormone releasing hormone (LHRH) in human, rat and mouse brain has been investigated by studying the in vitro translation products of Poly A+ mRNA extracts from the hypothalamus. The translation products of all three species contained a single 28000 MW polypeptide which immunoprecipitated with a specific anti-LHRH serum. This polypeptide was not present in the translation products of Poly A+ mRNA extracts from the hypothalamus of the hypogonadal mouse, a mutant strain totally deficient in LHRH. These results show that in the human, rat and normal mouse, LHRH is synthesized as a component of a precursor peptide with a molecular weight of 28000.     

Differential actions of GnRH and rat hypothalamic extracts in release of hypophysial FSH and LH in vitro.

A study was made of the separate patterns of luteinizing hormone (LH) and follicle stimulating hormone (FSH) release from isolated rat pituitary tissue evoked by synthetic gonadotropin releasing hormone (GnRH) or female hypothalamic extracts (HE), respectively, in a continuous perifusion system. Under defined conditions, gonadotropin release from hemipituitaries was relatively stable and reproducible. Absolute levels of LH and FSH release evoked by HE in terms of their GnRH content were always greater than those following exposure to synthetic GnRH at varying doses. Synthetic GnRH released more FSH than LH. In contrast, the HE released slightly higher levels of LH than FSH. The data suggest that the female rat hypothalamus contains substances other than GnRH, capable of releasing both LH and FSH. It is possible that such unidentified components can modify the hypophysial action of GnRH, resulting in particular circumstances in a differential release of LH and FSH.     

Luteinizing hormone secretion from the quail pituitary in vitro

An enzymatically dispersed pituitary preparation from Japanese quail (Coturnix coturnix) was used to study the dynamics of gonadotropin release. After an 18-h incubation, the cells were challenged with different luteinizing hormone-releasing hormones (LHRH) for 90 min. Using pituitary cells from mature males, mammalian and chicken LHRH I (Gln8-LHRH) had approximately equal luteinizing hormone (LH)-releasing activity whereas chicken LHRH II (His5, Trp7, Tyr8-LHRH) was 8-9 times more potent. The LHRH agonist (Trp6, Pro9-NEt-LHRH) had 15 times greater potency than chicken LHRH I. Pre-incubation with an LHRH antagonist (D-Phe2, D-Trp6-LHRH) significantly suppressed LH release. Acid extracts of median eminence released LH from pituitary cells, extracts from short-day and long-day males had equal activity, while tissue extracts from castrated males had significantly greater LH-releasing activity. Pituitary cells from sexually immature males released LH in response to chicken LHRH I in a similar profile to cells from mature males. These data indicate that the quail LHRH receptor in the male recognizes several different molecular species of LHRH and the response to LHRH is comparable between short- and long-day males. Pituitary cells from ovulating females were variably sensitive to LHRH peptides, possibly due to changes in pituitary sensitivity during the ovulatory cycle. Pituitary cells from immature females did not release LH in response to chicken LHRH I. However, pituitary cells from immature females photostimulated for 1 wk displayed a response to chicken LHRH I and II similar to that of pituitary cells from males.(ABSTRACT TRUNCATED AT 250 WORDS)     

Organization of LHRH cells: differential apposition of neurotensin, substance P and catecholamine axons

A wealth of evidence suggests that catecholamines (particularly norepinephrine) influence gonadotropin secretion via a direct interaction with the LHRH neurons. Neuropeptides such as neurotensin (NT) and substance P (SP) are likewise implicated in the control of LHRH secretion, based on pharmacological and preliminary anatomical studies. Since sub-populations of LHRH neurons project to areas of the brain other than the median eminence, a detailed analysis of the topography of axonal interactions of catecholamines (CA), substance P and neurotensin with LHRH cells was conducted in adult male mice using dual immunocytochemical techniques. An analysis of the patterns of apparent contact of NT or SP axons on LHRH cells as determined by close apposition of immunoreactive axons to LHRH cells when viewed under a light microscope at high magnification revealed that the density of NT or SP axons was not a reliable index of the degree of contact; in many locations, NT and SP had similar densities yet a greater portion of the LHRH cells appeared contacted by SP than NT. NT axons were in close contact with up to one-third of the LHRH cells. Analysis of the location of these "contacted" cells did not reveal a discrete subnucleus controlled by NT. Rather, the NT-contacted cells were scattered throughout the LHRH cell field. Interactions of LHRH cells with SP axons were likewise uniform throughout most of the LHRH cell field, with the exception of the most anterior portion of the field. In the anterior septum, few SP axons appeared to contact LHRH cells. Elsewhere, most of the LHRH cells were in contact with SP axons. For the CAs, the fiber density in the regions of the LHRH cells was uniformly moderate, yet the pattern of cells contacted showed variation across the LHRH cell field, with most of the "contacted" cells located near the OVLT and medial preoptic area. These data suggest that LHRH cells may be differentially regulated by NT, SP and the CAs.     

Inhibition of release of a novel pituitary polypeptide, 7B2, follicle-stimulating hormone, and luteinizing hormone from rat anterior pituitary cells in vitro by human beta-inhibin

We have recently purified a novel pituitary polypeptide designated 7B2. By raising polyclonal antibodies to a synthetic 7B2 fragment in rabbits, we have developed a sensitive and specific radioimmunoassay for this novel polypeptide, and it has been used for the study of the release of immunoreactive 7B2 from rat anterior pituitary cells in vitro. In addition, immunocytochemical study shows that 7B2 is present in the gonadotropin cells of rat anterior pituitary. The aim of the present studies is to investigate the effect of human beta-inhibin, testosterone, and combined testosterone plus human beta-inhibin on the induced release of immunoreactive 7B2, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) in rat anterior pituitary cell culture in vitro. Our results show that both human beta-inhibin and testosterone effectively suppress the stimulatory effect of luteinizing hormone-releasing hormone (LHRH) on immunoreactive 7B2, FSH, and LH release. The present data indicate that the regulation of secretion of 7B2 and pituitary gonadotropins may be under a similar type of feedback mechanism.     

Immunocytochemical distribution of LHRH neurons and processes in the rat: Hypothalamic and extrahypothalamic locations

Summary The distribution of luteinizing hormone-releasing hormone (LHRH)-immunoreactive perikarya and processes was examined, in the untreated rat, with the unlabeled antibody enzyme method of immunocytochemistry on thick 50 m vibratome sections. LHRH neurons were primarily observed in the preoptico-anterior hypothalamic and septal areas. Projections from these cell bodies to the median eminence form three distinct pathways, one laterally along the course of the optic tracts, one medially through the periventricular stratum of the third ventricle, and one through the tractus infundibularis. In addition, some of these cell bodies project to the organum vasculosum of the lamina terminalis (OVLT) and the subfornical organ (SFO). LHRH immunoreactive neurons were also noted in the anterior olfactory regions; they project along the medial olfactory tract to the olfactory bulb.     

Effect of luteinizing hormone releasing hormone pulse characteristics on comparative luteinizing hormone and follicle stimulating hormone secretion from superfused rat anterior pituitary cell cultures

We have shown that 4 ng luteinizing hormone releasing hormone (LHRH) pulses induced significantly greater luteinizing hormone (LH) release from proestrous rat superfused anterior pituitary cells with no cycle related differences in follicle stimulating hormone (FSH). Current studies gave 8 ng LHRH in various pulse regimens to study amplitude, duration and frequency effects on LH and FSH secretion from estrous 0800, proestrous 1500 and proestrous 1900 cells. Regimen 1 gave 8 ng LHRH as a single bolus once/h; regimen 2 divided the 8 ng into 3 equal 'minipulses' given at 4 min intervals to extend duration; regimen 3 gave the 3 'minipulses' at 10 min intervals, thereby further extending duration: regimen 4 was the same as regimen 2, except that the 3 'minipulses' were given at a pulse frequency of 2 h rather than 1 h. In experiment 1, all four regimens were employed at proestrus 1900. FSH was significantly elevated by all 8 ng regimens as compared to 4 ng pulses; further, 8 ng divided into 3 equal 'minipulses' separated by 4 min at 1 and 3 h frequencies (regimens 2 and 4) resulted in FSH secretion that was significantly greater than with either a single 8 ng bolus (regimen 1) or when the 'minipulses' were separated by 10 min (regimen 3). In experiment 2, at proestrus 1500, FSH response to the second pulse of regimen 4 was significantly greater than in regimen 2; LH release was significantly suppressed at pulse 2 compared to regimen 2 accentuating divergent FSH secretion. At estrus 0800, FSH response to the second pulse of regimen 4 was significantly stimulated FSH at proestrus 1900, 1500 and estrus 0800, FSH divergence was most marked at proestrus 1500. These data indicate a potential role for hypothalamic LHRH secretory pattern in inducing divergent gonadotropin secretion in the rat.     

Changes in the isoelectric focusing profile of pituitary follicle-stimulating hormone in the developing male rat

Pituitary glands, hypothalami, and trunk blood were obtained from male rats at 5, 15, 18, 21, and 29 days of age, on the day of balanopreputial separation (Days 42-45), and during adulthood. The forms of follicle-stimulating hormone (FSH) present within each pituitary were separated by polyacrylamide gel isoelectric focusing. Serum and pituitary gonadotropins, hypothalamic luteinizing hormone-releasing hormone (LHRH), and the profile of FSH forms across the isoelectric focusing gel were determined by radioimmunoassay. No change in the relative proportions of FSH forms were observed between 5 and 21 days of age. Likewise, only slight changes in serum and pituitary gonadotropin levels and hypothalamic LHRH content were observed at these times. After 21 days of age, dramatic increases in serum and pituitary gonadotropin levels were observed. Similarly, a shift in FSH forms within the pituitary to more basic and bioactive forms was observed at this time. These results demonstrate that, during the transition through puberty in the male rat, not only the absolute amount, but also the isoelectric focusing profile, of FSH change.     

Identification of luteinizing hormone releasing hormone-like immunoactivity in ovine cotyledons

Luteinizing hormone releasing hormone (LHRH)-like immunoactivity has been identified in cotyledonary extracts prepared from pregnant ewes. This activity displayed similar physico-chemical properties as synthetic LHRH, as determined by reverse-phase HPLC and size-exclusion HPLC. Under reverse-phase conditions, cotyledonary LHRH-like immunoactivity displayed a retention time (10.5 +/- 0.1 min) which was not significantly different from that of synthetic LHRH. When subjected to size-exclusion HPLC, cotyledonary LHRH-like immunoactivity eluted in fractions which corresponded to a molecular weight range of 1100-1200 Da, which was not significantly different from the elution profile observed for synthetic LHRH. The cotyledonary tissue content of LHRH-like immunoactivity averaged 94 +/- 24 pg/mg (n = 6). The results of this study demonstrate the presence of LHRH-like immunoactivity in ovine cotyledons. Although placental synthesis of LHRH-like immunoactive material has been demonstrated in other species, it remains to be established whether this activity, demonstrated in ovine placenta, is the consequence of de novo placental synthesis or represents uptake from the maternal (and/or fetal) circulation.     

Development of hypothalamic neurons in intraventricular grafts: expression of specific transmitter phenotypes

The anlages of the medial-basal hypothalamus (MBH), septopreoptic area (POA), Rathke's pouch, and the parietal cortex (CC) of rats (at 12.5, 14.5 and 16.5 days of gestation) were transplanted singly or in combination into the third ventricle of adult female rats, and the development of neurons in the grafts was investigated immunohistochemically with the use of antisera to tyrosine hydroxylase (TH), somatostatin (SRIH), ACTH, methionine enkephalin-Arg6-Gly7-Leu8 (Enk-8), rat corticotropin-releasing factor (rCRF), rat hypothalamic growth hormone-releasing factor (rhGRF), and luteinizing hormone-releasing hormone (LHRH). TH and all the peptides examined except LHRH were detected in distinct neurons in MBH grafts and in cografts of MBH plus Rathke's pouch from 12.5-day-old embryos. SRIH, rCRF, Enk-8, and TH were found in POA grafts from embryos of the same age. Although immunoreactive LHRH was first detected in neurons in POA grafts from 16.5-day-old embryos, it appeared in cografts of POA and MBH from 12.5-day-old embryos. The immunoreactive fibers developed in the grafts expressed the same characteristic behaviors as in intact brain; the fibers containing hormonal substances formed complexes with the vasculature like in the organum vasculosum laminae terminalis (OVLT) or in the median eminence, while the fibers containing neurotropic signals formed fiber networks surrounding other nerve cell bodies as if they synaptically associate. In CC grafts, the neurons contained TH, SRIH, rCRF, or Enk-8, and their axonal processes formed fiber networks. These findings suggest that all the hypothalamic neurons examined are committed by 12.5 days of gestation to develop maintaining transmitter phenotype and target recognition capacity.     

Variations in the number of pituitary LHRH receptors correlated with altered responsiveness to LHRH

Isolated pituitary cells from metestrous, ovariectomized (OVX), and ovariectomized-estradiol treated (OVX-EB) rats were employed to study the gonadotropin response to luteinizing hormone-releasing hormone (LHRH) challenge and to quantitate LHRH receptors, using a labeled LHRH analog. Ovariectomy (3–4 weeks post castration) resulted in a reduction of LHRH receptor concentration from 34.4 ± 2.1 in metestrous females to 14.3 ± 0.9 fmoles/10⁶ cells. Concomitantly, the luteinizing hormone (LH) response to a near-maximal dose of LHRH (5 ng/ml) decreased from a 3-fold stimulation in intact females to 1.13-fold stimulation in cells from OVX rats. Replacement therapy with EB (50 ug/rat for 2 days) to OVX rats restored LH response and LHRH binding sites (a 2.5-fold stimulation in LH secretion and 32.0 ± 2.1 fmoles/10⁶ cells, respectively). The LH response to LHRH stimulation was not altered after one day of EB treatment although the number of LHRH binding sites was increased. The changes in the number of LHRH binding sites were not accompanied by any alterations in the affinity of the LHRH analog ($\text{K}{}_{\mathrm{d}}\text{? 0.5 × 10}{}^{\mathrm{?9}}\text{M}$). It is concluded that variations in LHRH receptor number reflect the degree of pituitary sensitivity to LHRH and it may suggest that LHRH and estradiol modulation of gonadotropin release is mediated by these receptors.     

Processing of the luteinizing hormone-releasing hormone precursor in the preoptic area and hypothalamus of the rat

The LHRH precursor is known to contain the decapeptide and a 56 amino acid peptide termed gonadotropin-releasing hormone-associated peptide (GAP). The purpose of our study was to characterize the proLHRH and its processed products from the cell body and fiber region and from the nerve terminal region of LHRH neurons. The median eminence (ME) and a tissue block containing the preoptic area and hypothalamus (POH) were dissected separately. Tissues were homogenized and peptides were separated according to mol wt. Three different LHRH antisera bound to one immunoreactive (IR) substance which eluted at approximately 1200 mol wt. Subsequently, this material coeluted with synthetic LHRH on a reversed-phase column as a single peak. There was approximately 1.6-fold more LHRH-like IR in the ME than in the POH. The four different GAP antisera recognized multiple mol wt forms of GAP-like IR at approximately 16,000 to 14,000, 8,200, 6,500, 3,500, and 2,800 mol wt. There were more of the high mol wt materials and less of the 6500 and lower mol wt materials in the POH than in the ME. The most abundant species in both regions was the 6500 mol wt form. This IR substance coeluted with synthetic rat GAP1-56 on a reversed-phase column as a single peak. These experiments demonstrate 1) that multiple IR forms of the LHRH prohormone exist in the POH of the rat and 2) that nerve terminals of the LHRH neurons contain LHRH, GAP1-56, and some lower mol wt GAP-like substances. These results provide the first information concerning the processing scheme for the LHRH prohormone in the rat brain.     

Studies on the physiological role and mechanism of action of neuropeptide Y in the regulation of luteinizing hormone secretion in the rat

It has been recently shown that intraventricular or systemic injection of neuropeptide Y (NPY) can produce a decrease in plasma luteinizing hormone (LH) levels in castrated rats of both sexes. In order to evaluate the physiological role of NPY in the regulation of LH secretion in the female rat, we proceeded to immunoneutralization experiments using specific antibodies to NPY. Injection of 0.5 ml antiserum to NPY produce a 20-fold increase of LH plasma levels, whereas injection of preimmune serum did not modify the plasma concentrations of LH. To investigate the possibility that catecholamines or serotonin might be involved in the effect of NPY in LH secretion, castrated rats were treated with alpha-methylparatyrosine (alpha-MPT), an inhibitor of catecholamine biosynthesis, or received an i.c.v. injection of the neurotoxin 5-7-dihydroxytryptamine (5,7-DHT) prior to the intraventricular injections of NPY. The pretreatment with alpha-MPT could not prevent the decrease of plasma LH induced by NPY injection whereas the pretreatment with 5,7-DHT reversed the effect of NPY injection. The anatomical connection between LH-releasing hormone (LHRH) and NPY neuronal systems were also investigated using double immunostaining technique. It appeared that NPY endings are in apposition to LHRH cell bodies in the preoptic area in proximity to the organum vasculosum of the lamina terminalis (OVLT).(ABSTRACT TRUNCATED AT 250 WORDS)     

Peptidase activity in the hypothalamus of the rat: utilization of leucine-p-nitroanilide to monitor the activity degrading luteinizing hormone releasing hormone

Previous studies by other investigators have shown that luteinizing hormone releasing hormone (LHRH) and amino acid derivatives of p-nitroanilide are probably degraded by a common enzymatic activity; however, most of these studies are inferential in that they are largely based upon kinetic inhibition data derived from relatively crude tissue preparations. The purpose of this work was to determine whether the synthetic substrate leucine-p- nitroanilide (Leu-p-NA) and LHRH were degraded by the same peptidase activity. Supernatants (10,000 X g) from homogenates of rat hypothalami were eluted from Sephadex G-200, and the resultant fractions were assayed for degrading activity toward LHRH and Leu-p-NA. Radioimmunoassay (RIA) indicated that loss of immunologically active LHRH occurred in the same fractions in which maximal Leu-p-NA degrading activity eluted. Kinetically, exogenous LHRH inhibited degradation of Leu-p-NA in a concentration-dependent manner. When fractions evidencing Leu-p-NA degrading activity were incubated with 125I-LHRH, polyacrylamide gel electrophoresis (PAGE) indicated a time-dependent loss of LHRH with the concomitant production of a radioactive peptide fragment. High-performance liquid chromatography (HPLC) analysis of unlabeled LHRH incubations revealed, within the Leu-p-NA degrading fractions, the formation of two peptide fragments. These studies have further substantiated the likelihood that LHRH and Leu-p-NA are degraded by a common enzyme activity as indicated not only by kinetic inhibition data, but also by cofractionation of activity toward both substrates and by two analytical methods capable of detecting LHRH fragmentation (PAGE and HPLC).     

Alterations in hypothalamic content of luteinizing hormone-releasing hormone associated with pineal-mediated testicular regression in the golden hamster

The adult male golden hamster will undergo testicular regression when exposed to a short photoperiod, blinding, or late afternoon injections of melatonin. The present study was conducted to compare the effects of all three treatments on serum gonadotropin levels and testicular weights, and to evaluate the effects of these treatments on hypothalamic content of both immunoreactive and bioactive luteinizing hormone-releasing hormone (LHRH) levels. Hamsters were blinded (BL), exposed to a short photoperiod (SP), or received daily injections of melatonin (MEL) for 15 wk. Each treatment (BL, SP, MEL) induced a temporally similar decline in serum luteinizing hormone (LH), serum follicle-stimulating hormone (FSH), and testicular weight. Spontaneous recrudescence occurred earliest in the MEL group, with serum gonadotropins and testicular weight returning to normal by 15 wk. The SP group exhibited recovery of serum gonadotropins but not testicular weight by 15 wk. The BL group demonstrated partial recovery of serum FSH levels by 15 wk, with no recovery in either serum LH or testicular weight. Each treatment group demonstrated increased hypothalamic content of immunoreactive LHRH which was temporally correlated with the decreases of serum gonadotropins. Additionally, the MEL and SP groups demonstrated decreased immunoreactive LHRH levels during spontaneous recrudescence. Extracts of hypothalami from all treatment groups were bioactive on control hamster pituitary cells. These results indicate that there are temporal differences among the three common treatments and that these differences are manifested in serum gonadotropins, testicular weight and hypothalamic LHRH. Hypothalamic LHRH levels determined by radioimmunoassay and bioassay show periods of increase and decrease which coincide with periods of altered serum gonadotropin levels in all groups.     

Increased LH and FSH release from the anterior pituitary of ovariectomized rat, in vivo, by copper-, nickel-, and zinc-LHRH complexes.

The effect of Cu2+, Ni2+, Zn2+ and their complexes with LHRH on the release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) was estimated in in vivo experiments with the use of the method proposed by Ramirez and McCann. Ovariectomized, estradiol, and progesterone pretreated rats were injected intravenously either with LHRH alone, a metal ion alone, a mixture of metal and hormone, or a metal-LHRH complex. A metal alone or a mixture of it with LHRH did not affect gonadotropin release at all or no more than LHRH alone. However, the complex of Cu2+ with LHRH brought about a high release of LH and even higher release of FSH. This indicates that copper complex is more effective than metal-free LHRH. The nickel complex showed a similar although lesser effect. The zinc complex had similar potency to free LHRH though higher FSH-releasing ability was noticed. We conclude that copper-, nickel-, and zinc-LHRH complexes were more potent than the peptide hormone itself and promoted the FSH release in the ovariectomized, estradiol, and progesterone pretreated rats.     

Gonadal steroids and neuropeptide Y-opioid-LHRH axis: interactions and diversities

We report that the two classes of regulatory neuropeptides, neuropeptide Y (NPY) and endogenous opioid peptides (EOP), modulate luteinizing hormone (LH) release in diverse fashion in gonad-intact rats. Each neuropeptide acts at two loci, the hypothalamus and pituitary, to excite (NPY) or inhibit (EOP) LH release. At the hypothalamic level, NPY stimulates luteinizing hormone releasing hormone (LHRH) release, a response mediated by alpha 2-adrenoreceptors and amplified in the presence of adrenergic agonists. At the pituitary level, NPY acts in concert with LHRH to amplify the LH response. In contrast, EOP inhibit LHRH release by decreasing the supply of excitatory adrenergic signals in the vicinity of LHRH neurons in the preoptic-tuberal pathway, and at the pituitary level, they decrease LH release in response to LHRH. Further, the gonadal steroidal milieu facilitates NPY neurosecretion and postsynaptic expression of NPY in concert with adrenergic system; a similar clear-cut facilitatory effect of gonadal steroids on EOP secretion is not yet obvious. Our additional studies imply that the EOP system has the potential to increase sensitivity towards gonadal steroids and that to induce the preovulatory LH surge the neural clock may decrease the inhibitory EOP tone prior to the critical period on proestrus. This antecedent neural event allows the excitatory adrenergic and NPY signals to evoke LHRH secretion at a higher frequency approximating that seen in ovariectomized rats. Further studies are under way to delineate the steroid-induced subcellular events that integrate the action of these regulatory peptides in the control of the episodic LHRH secretion pattern which sustains basal and cyclic gonadotropin release in the rat.     

Regulation of the pulsatile releases of luteinizing and follicle-stimulating hormones in ovariectomized hamsters

We have combined for modifications of common radioimmunoassay (RIA) techniques to increase the sensitivity of the gonadotropin assays by an order of magnitude compared with those generated according to the instructions provided by the National Pituitary Agency. The four modifications are: a) enzymatic radioiodination, b) purification of radiolabeled hormones by Sephadex and concanavalin A chromatography, c) reduced first antibody concentration, and d) a prolonged incubation time. These methods increase the sensitivities of the RIAs and allow for the quantitation of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels in small volumes of plasma. We have used these methods to measure the changes in pulse frequency and amplitude of LH and FSH in ovariectomized hamsters after a variety of neuroendocrine manipulations. Alterations in catecholaminergic neurotransmission affect the frequency and amplitude of LH but not FSH release, and suggest that the hypothalamic mechanisms responsible for LH releasing hormone (LHRH)-mediated LH release are distinct from those that regulate FSH secretion. Further, alterations in LHRH-pituitary interactions (elicited by injections of LHRH antisera or a potent LHRH agonist), suggest the existence of separate control mechanisms responsible for LH and FSH release at the level of the adenohypophysis. Combined, these studies provide further evidence for complex and separate neuroendocrine regulatory control over the secretion of each gonadotropin.