The retino-hypothalamic tract is involved in prolactin regulation in fetal sheep.

To investigate the role of the retino-hypothalamic tract on fetal prolactin regulation, we examined the effect of ocular enucleation on fetal plasma prolactin. Eleven fetuses of Suffolk ewes were chronically catheterized during fall, and six of them were subjected to bilateral ocular enucleation. All ewes were kept at 12h:12h light:dark cycle (lights on at 0800 and off at 2000). The experiments were performed 5-9 days after surgery (GA control fetuses 125 +/- 1.5, optical enucleation 121.3 +/- 1.5 days). Blood samples were taken from fetuses hourly around the clock, and plasma prolactin and cortisol were measured by radioimmunoassay (RIA). Luteinizing hormone (LH) and Growth hormone (GH) were measured in pooled plasma samples from control and enucleated fetuses by RIA. Average plasma prolactin was 5-fold lower in enucleated than in control fetuses (9.6 +/- 0.5 and 54.2 +/- 3.3 ng/ml, SEM; P < 0.005). Both control and enucleated fetuses presented circadian rhythm of prolactin with acrophase between 1400 and 1830 h. An enucleated fetus was tested for response of prolactin to TRH. Prolactin increased as described in the literature. There was no change in plasma concentration of cortisol, LH or GH after ocular enucleation. Our data indicate that the optical pathway participates in prolactin regulation in the fetal sheep.     

Episodic gonadotropin secretion in the mature fowl: serial blood sampling from unrestrained male broiler breeders (Gallus domesticus)

Forty-week-old male broiler breeders were used in two experiments. Males were reared as recommended by the breeder, housed in individual cages, and cannulated to facilitate blood sampling. In experiment 1, blood samples were collected at 10- min intervals for 4 h commencing the day of cannulation (Day 0) and for 12 h on each of Days 1 and 2. In experiment 2, blood samples were collected at 10-min intervals for 8 h on Day 1. After centrifugation, plasma was stored at -20 degrees C until LH, FSH (experiment 1 and 2), testosterone, and corticosterone (experiment 1) concentrations were determined by RIA. Different statistical methods used to identify hormone secretion profiles revealed a characteristic pulsatile pattern of LH and FSH in plasma. However, LH pulses were more frequent and had greater amplitude than FSH pulses. Less than 32% of the FSH pulses were associated with LH episodes. Conversely, the association between LH and testosterone pulses averaged 83% in birds with testis weight greater than 10 g. Concentrations of corticosterone tended to increase after cannulation and remained elevated for only 3-4 h. Our data indicate that LH, FSH, and testosterone secretion is pulsatile in male broiler breeders. Additionally, LH pulses are associated with testosterone episodes but not with FSH pulses. The pulsatile pattern of FSH secretion, which is unique from those of LH, in adult males suggests that FSH secretion is independently regulated in the adult male fowl.     

虎纹蛙促性腺激素含量随年龄及季节的变化(英文)

促性腺激素（ＧｔＨ;或ＬＨ和ＦＳＨ）在脊椎动物生殖调节中起中Ｃ作用;脑垂体和血浆ＧｔＨ水平在一定程度上反映着动物体的生殖生理状态。在蛙类,对脑垂体和血浆的ＬＨ及ＦＳＨ含量进行过较全面研究的,仅在牛蛙和日本蟾蜍有过报道,结果显示它们的ＬＨ和ＦＳＨ的含量存在有种类差异性。虎纹蛙属中国二级保护动物,也是唯一受保护的蛙类,对其生殖生物学的基础内容进行研究具有重要的意义。 为此,本文利用放射免疫测定法,测定了虎纹蛙幼蛙和不同性腺发育阶段（季节）成蛙脑垂体与血浆的ＬＨ及ＦＳＨ含量,以弄清这些激素的含量变化与年龄、季节（性腺发育阶段）变化的关系,以期为虎纹蛙的基础生殖生物学及蛙类的生殖内分泌学充实新的内容,为虎纹蛙的人工繁殖和保护提供理论依据。结果是：幼蛙血浆ＬＨ水平显著高于各期成蛙（Ｆｉｇ．Ａ）,血浆ＦＳＨ水平显著低于成熟前期成蛙,而和其它各性腺发育阶段成蛙相当（Ｆｉｇ．Ｂ）。而脑垂体ＬＨ或ＦＳＨ的含量显著低于各期成蛙（Ｆｉｇ．Ｃ＆Ｄ）。这说明,幼蛙脑垂体已具有一定的合成和释放ＬＨ及ＦＳＨ的能力。 成蛙脑垂体和血浆ＬＨ及ＦＳＨ水平随性腺发育阶段（季节）的不同而有一定的变化：雌蛙血浆ＬＨ水平在成熟期最高,性腺再发育期最低;     

Development of an enzyme‐linked immunosorbent assay (ELISA) for Luteinizing Hormone (LH) in the elephant (Loxodonta africana and Elephas maximus)

A simple, rapid enzyme‐linked immunosorbent assay (ELISA) for the measurement of LH in plasma and serum of elephants (Loxodonta africana and Elephas maximus) has been developed, validated, and used for comparative studies. Purified elephant LH (eleLH) diluted in elephant plasma was used as standards (0.78–50 ng/ml). A monoclonal antibody against the β‐subunit of bovine LH (518B₇) was used as the capture antibody. The second antibody (a polyclonal rabbit anti‐human LH antibody), conjugated to horseradish peroxidase, cleaved a substrate (tetramethyl benzidine), resulting in a color change. The total assay time was approximately 2½ hr, with incubations at room temperature. Sensitivity was found to be 1.56 ng/ml. Cross‐reactivities to elephant FSH and TSH were low: 0.9% and 0.15%, respectively. The accuracy of the assay was demonstrated by comparing the ELISA with a validated eleLH radioimmunoassay (RIA), progesterone data, and ultrasound observations. Blood samples from 18 Asian and African elephant cows were analyzed with the ELISA and RIA, and an additional 11 cows were used to describe endocrine parameters for LH and progesterone using only RIA. No difference was found in LH peak concentrations between the ELISA and RIA. The time from the progesterone decline to the first LH peak, and the time between the two peaks were similar between species. Asian cows had higher LH peaks than African cows. Ultrasound confirmed the time of ovulation occurring with the second LH peak. Three cows were inseminated and confirmed to be pregnant using this ELISA as a timing device. Instrumentation is not always required, as LH peaks approximating 3 ng/ml can be visually observed. In conclusion, this ELISA can be used as a field test to determine time of ovulation for artificial insemination (AI) or natural breeding of both species of elephants, and thus is an important tool for the preservation of captive populations worldwide. Zoo Biol 23:65–78, 2004. © 2004 Wiley‐Liss, Inc.     

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.     

Validation of radioimmunoassays for LH and FSH in the sooty mangabey (Cercocebus atys): Characterization of LH and the response to GnRH

Heterologous radioimmunoassays (RIA) for macaque LH and FSH were validated for the measurement of these hormones in the sooty mangabey and mangabey pituitary LH was characterized relative to rhesus monkey LH. Dilutions of a pituitary mangabey extract and a partially purified preparation of mangabey LH ran parallel to a rhesus monkey standard (LER 1909-2) in the ovine-ovine (o-o) LH assay but showed some deviation from parallelism in the rhesus monkey FSH assay. The LH potency of the mangabey extract and standard were six and 190 times more potent, respectively, than LER 1909-2 in the LH RIA. Mangabey LH was estimated to have a molecular weight of 40,000–42,000 daltons vs 35,000–38,000 daltons for rhesus LH on Sephadex G-100 chromatography. Plasma levels of radioimmunoreactive LH, FSH, and testosterone were assayed before and after a bolus administration of 25, 50, or 100 μg synthetic go-nadotropin releasing hormone (GnRH) to adult male mangabeys. A significant increase in serum levels of LH was seen within 30 min with levels more than fourfold higher than the basal level of LH after administration of 100 μg GnRH. However, no consistent increases in plasma FSH values were detected. The integrated mean LH response above preinjection levels following 25, 50, or 100 μg GnRH was dose related. Serum levels of testosterone were also elevated after administration of GnRH, but peak concentrations of testosterone lagged behind peak levels of LH by approximately 30 min. These studies indicate that the heterologous RIAs may be used for measuring gonadotropins in the mangabey and that the male mangabey is apparently more sensitive to GnRH than the rhesus monkey.     

Radioimmunoassay of luteinizing hormone in the baboon (Papio hamadryas)

A heterologous radioimmunoassay (RIA) for luteinizing hormone (LH) consisting of a cynomolgus LH tracer and an antiserum raised against human chorionic gonadotropin (cynLH:anti-hCG) fulfilled the recognized criteria of reliability when applied to baboon (Papio hamadryas) plasma and pituitary extracts obtained in different endocrine conditions. This RIA is 5.5 times more sensitive than the ovine (oLH:anti-oLH) system, yields estimates of baboon LH (bLH) fairly close to those obtained by in vitro bioassay, and recognizes all bioactive molecular species of bLH present in male and female pituitary extracts. However, the system yields slightly but significantly lower estimates of bLH than the in vitro bioassay.     

Radioimmunoassay with heterologous antibody (hetero-antibody RIA): utilization of highly cross-reactive antibody present in polyclonal antiserum.

To develop a homologous radioimmunoassay (RIA) for a hormone of a small or rare animal often meets difficulty in collecting a large amount of purified antigen required for antibody production. On the other hand, to employ a heterologous RIA to estimate the hormone often gives poor sensitivity. To overcome this difficulty, a "hetero-antibody" RIA was studied. In a hetero-antibody RIA system, a purified preparation of a hormone is used for radioiodination and standardization and a heterologous antibody to the hormone is used for the first antibody. Canine motilin and rat LH were selected as examples, and anti-porcine motilin and anti-hCG, anti-hCG beta or anti-ovine LH beta was used as the heterologous antibody. The sensitivities of the hetero-antibody RIAs were much higher than those of heterologous RIAs in any case, showing that these hetero-antibody RIA systems were suitable for practical use. To clarify the principle of hetero-antibody RIA, antiserum to porcine motilin was fractionated on an affinity column where canine motilin was immobilized. The fraction bound had greater constants of affinity with both porcine and canine motilins than the rest of the antibody fractions. This fraction also reacted with a synthetic peptide corresponding to the C-terminal sequence common to porcine and canine motilins in a competitive binding test with labeled canine motilin. These results suggest that an antibody population having high affinity and cross-reactivity is present in polyclonal antiserum and indicate that the population can be used in hetero-antibody RIA at an appropriate concentration.     

Pituitary and plasma levels of growth hormone (GH), follicle stimulating hormone (FSH) and luteinizing hormone (LH) in hereditary dwarf rats (rdw/rdw)]

Koto et al found a new hereditary dwarf mutation from breeding colony of Wistar-Imamichi rat and named 'rdw'. To characterize endocrinological functions in rdw rats, pituitary and plasma levels of pituitary hormones including growth hormone (GH), follicle stimulating hormone (FSH) and luteinizing hormone (LH) were compared between rdw and normal rats. The hormone levels were estimated with radioimmunoassay (RIA). It was found that pituitary and plasma levels of GH of rdw were drastically decreased and those of FSH and LH were inclined to decrease but not remarkable as compared with normal. Rats of rdw were, therefore, considered to be useful as a model animal for endocrinological defects.     

Absence of effect of estrogen on dopamine, norepinephrine and beta-endorphin-like immunoreactivity in human plasma

The plasma concentrations of immunoreactive norepinephrine (NE), dopamine (DA), beta-endorphin (beta-E), luteinizing hormone (LH) and follicle stimulating hormone (FSH) were determined by RIA and HPLC every 6 h until 72 h after iv administration of conjugated estrogens during the midfollicular phase. The LH level showed a biphasic pattern after the injection of conjugated estrogens, i.e. significant suppression (-50%) for 6-42 h after the injection, followed by a rebound increase with a peak (+85%) at 72 h. The plasma levels of immunoreactive beta-E, NE and DA did not change significantly for 72 h after the injection.     

Active immunization of intact mares against gonadotropin-releasing hormone: differential effects on secretion of luteinizing hormone and follicle-stimulating hormone

Five lighthorse mares were actively immunized against gonadotropin releasing hormone (GnRH) to determine the relative importance of this hypothalamic hormone in the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Five mares immunized against the conjugation protein served as controls. Mares were initially immunized in November and received secondary immunizations 4 wk later, and then at 6-wk intervals until ovariectomy in June. All mares immunized against GnRH exhibited an increase (p less than 0.01) in the binding of tritiated GnRH by plasma, an indication that antibodies against this hormone had been elicited. Concentrations of LH, FSH and progesterone in weekly blood samples were lower (p less than 0.05) in GnRH-immunized mares than in controls after approximately 4 mo of immunization. However, the LH concentrations were affected to a greater degree than were FSH concentrations. All five control mares exhibited normal cycles of estrus and diestrus in spring, whereas no GnRH-immunized mare exhibited cyclic displays of estrus up to ovariectomy. All mares were injected intravenously with a GnRH analog (which cross-reacted less than 0.1% with the anti-GnRH antibodies) in May, after all control mares had displayed normal estrous cycles, to characterize the response of LH and FSH in these mares; two days later, the mares were injected with GnRH. The LH response to the analog, which was assessed by net area under the curve, was lower (p less than 0.01) by approximately 99% in mares immunized against GnRH than in control mares. In contrast, the FSH response to the analog was similar for both groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduced pulsatile luteinizing hormone and testosterone secretion with aging in the male rat

To identify possible age-dependent changes in the feedback relationship between the brain-pituitary and testes, we examined the minute-to-minute patterns of plasma luteinizing hormone (LH) and testosterone (T) in intact, young male rats and compared these profiles to those of old animals. Young (3 mo; n = 11) and old (22 mo; n = 12) Sprague-Dawley rats were fitted with indwelling venous catheters and between 24 and 48 h later, were bled without anesthesia, by remote sampling, at 10-min intervals for 8 h. Blood samples of 400 microliter were withdrawn, and an equivalent volume of a blood replacement mixture was infused after each sample. Plasma LH and T levels in each sample were measured by radioimmunoassay (RIA). Plasma T levels in old animals failed to show the transient oscillations observed in young animals. Mean plasma T levels were 50% lower in old compared to young animals (P less than 0.001). Plasma patterns of LH in old animals, like their younger counterparts, showed statistically significant episodic increases, whose apparent pulse frequency was inappropriately low for their circulating T level (although not statistically different from the young group). Pulse amplitude in the old animals was 66% lower in the old compared to the young group (P less than 0.015). We conclude that age-associated alterations in brain mechanisms governing LH secretion underline these endocrine changes.     

Simultaneous radioimmunoassay for luteinizing hormone and prolactin

A combined radioimmunoassay (RIA) for the measurement of the anterior pituitary proteins luteinizing hormone (LH) and prolactin (PRL) is described and compared with individual RIAs for these hormones. The standard curves and the sample values for LH and PRL were identical when determined in a combined or in an individual RIA. This technique may prove useful to a number of laboratories where it is desirable to determine levels of more than one hormone in limited sample volumes.     

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.     

Characterization of equine luteinizing hormone by chromatofocusing

Three equine luteinizing hormone (LH) preparations (eLH-A, -B, and -C) recently have been isolated in our laboratory and were shown to differ in average basicity (eLH-A greater than -B greater than -C). The present study further characterizes these preparations by chromatofocusing. Each of these preparations are comprised of a family of isohormones, with 5 major immunoreactive peaks in the pH range of 7 to 4 (approx. pIs = 6.6, 6.1, 5.7, 5.2, and 4.8), with varying amounts of material eluting to either side of the pH gradient. Although similar isoforms are seen in all three LH preparations, the relative proportions of different isoforms vary in a manner reflecting the average charge properties of eLH-A, -B, and -C. While eLH-A contains predominantly basic forms, eLH-C consists largely of acidic material, and eLH-B is composed mostly of isohormones with pIs intermediate to eLH-A and -C. Chromatofocusing of a crude extract from a single horse pituitary gland revealed isohormone peaks corresponding to those found in the highly purified LH preparations. Peak fractions of the various isoforms were used to generate a variety of activity ratios (LH bioactivity:LH radioimmunoassay (RIA), LH radioreceptorassay (RRA):LH RIA, LH bioactivity:LH RRA, follicle-stimulating hormone (FSH) RRA:LH RIA, and FSH RRA:LH RRA activity ratios). The LH bioactivity:LH receptor binding potency ratio showed a linear increase with increasing isohormone acidity (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Partial purification and characterization of rhinoceros gonadotropins, growth hormone, and prolactin: comparison with the horse and sheep

The rhinoceros is an endangered species related to the horse family. Little is known of its reproductive endocrinology. The objectives of this study were to partially purify rhinoceros pituitary hormones, determine which assays could be used for their assessment, and to ascertain whether rhinoceros LH possesses the intrinsic FSH activity of equine LH. A single pituitary each from a White (1.3 g) and a Black (1.2 g) Rhinoceros was homogenized and extracted (pH 9.5), then subjected to pH and salt fractionation, and ion-exchange chromatography (DEAE and Sephadex SP-C50) to yield partially purified fractions of LH, FSH, growth hormone (GH), and prolactin (PRL). LH was readily measured by a rat Leydig cell assay (0.1-1% x equine LH) and an RIA using a monoclonal antibody to bovine LH (6-11% x equine LH). FSH activity detected in the LH by either an FSH RIA or a calf testis radioreceptor assay (RRA) was extremely low. No FSH activity could be detected in the White Rhinoceros pituitary "FSH" fraction, but was readily detected in the Black Rhinoceros fraction (RIA: 0.2% x equine FSH: RRA: 0.8% x equine FSH). The presence of GH and PRL was determined by SDS-PAGE and Western blots. Results showed a single immunoreactive GH band and multiple immunoreactive PRL bands. Adsorption with Concanavalin A-Sepharose indicated that some of the PRL bands are glycosylated.     

Radioimmunoassay of serum follicle-stimulating hormone and luteinizing hormone in the bottlenosed dolphin

Commercially available radioimmunoassay (RIA) kits for human follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were adapted for quantitation of these hormones in serum from bottlenosed dolphins (Tursiops truncatus). Serum samples from over 160 wild and 70 captive animals were assayed in order to determine basal concentrations of FSH and LH in these animals, as well as to detect possible differences between various groups. Mean FSH and LH levels for all animals were 0.22 +/- 0.08 and 0.37 +/- 0.18 ng/ml, respectively. Although wild animals had higher FSH and LH levels than captive ones, the differences were not statistically significant (P less than 0.07). However, both FSH and LH were significantly (P less than 0.01 and P less than 0.05, respectively) elevated in females when compared to males. Adults and peripubescent animals had significantly (P less than 0.01) higher LH levels than did juveniles. Among wild animals, serum concentrations of FSH and LH reflected seasonal differences. Samples obtained in early summer (Gulf of Mexico population) contained significantly (P less than 0.01) higher concentrations of FSH and LH than samples obtained in the fall (Indian River, Florida population). Both FSH and LH were significantly elevated in samples from confirmed pregnant animals as compared to the overall mean and to a sample from a confirmed nonpregnant female. Our observations indicate that these RIAs can reliably detect serum FSH and LH from bottlenosed dolphins and represent the first quantitation of these hormones in cetaceans.     

Short-term effects of vasoligation upon plasma follicle-stimulating hormone, luteinizing hormone, and prolactin in the adult rat: further evidence for a direct neural connection between the testes and the central nervous system

The purpose of these experiments was to determine whether bilateral vasoligation of adult male rats had any short-term effects upon plasma levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin. Adult male rats (250-300 g) were either bilaterally vasoligated or sham vasoligated, and blood samples were obtained by cardiac puncture preoperatively and at 24 h and 7 days following surgery. Plasma levels of both FSH and LH were significantly (P less than 0.01) decreased at 24 h following vasoligation compared to preoperative levels and those of sham-operated controls. However, the response was differential since, at 7 days following vasoligation, plasma FSH was still significantly decreased while LH was returning to control levels. Conversely, plasma prolactin levels were significantly (P less than 0.01) increased at 24 h compared to preoperative values and those in sham-operated controls, and at 7 days prolactin had returned to preoperative control levels. Sham vasoligation did not significantly change plasma levels of FSH, LH, or prolactin at any of the time intervals investigated. These results provide further evidence that suggests that there may be a direct connection between the testis and central nervous system that may be involved in the short-term regulation of gonadotropin and prolactin secretion.     

Proteome and radioimmunoassay analyses of pituitary hormones and proteins in response to feed restriction of dairy cows

The hypothalamic-pituitary system controls homeostasis during feed energy reduction. In order to examine which pituitary proteins and hormone variants are potentially associated with metabolic adaptation, pituitary glands from ad libitum and energy restrictively fed dairy cows were characterized using RIA and 2-DE followed by MALDI-TOF-MS. We found 64 different spots of regulatory hormones: growth hormone (44), preprolactin (16), luteinizing hormone (LH) (1), thyrotropin (1), proopiomelanocortin (1) and its cleavage product lipotropin (1), but none of these did significantly differ between feeding groups. Quantification of total pituitary LH and prolactin concentrations by RIA confirmed the results obtained by proteome analysis. Also, feed energy restriction provoked increasing non-esterified fatty acid, decreasing prolactin, but unaltered glucose, LH and growth hormone plasma concentrations. Energy restriction decreased the expression of glial fibrillary acidic protein, triosephosphate isomerase, purine-rich element-binding protein A and elongation factor Tu, whereas it increased expression of proline synthetase co-transcribed homolog, peroxiredoxin III, β-tubulin and annexin A5 which is involved in the hormone secretion process. Our results indicate that in response to feed energy restriction the pituitary reservoir of all posttranslationally modified hormone forms remains constant. Changing plasma hormone concentrations are likely attributed to a regulated releasing process from the gland into the blood.