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.     

Biological and immunological characterization of purified follicle-stimulating hormone preparations from monkey pituitaries

The suitability of a purified rhesus (rhFSH) and cynomolgus (cynFSH) follicle-stimulating hormone (FSH) preparation to replace the expired LER 1909-2 reference standard in the immunoassay of monkey FSH was assessed using an in vitro bioassay and two heterologous radioimmunoassay (RIA) procedures in combination with fractionation by high-resolution isoelectrofocusing. The results presented indicate that rhFSH is unsuitable for the replacement of LER 1909-2 as a standard, though cynFSH might be suitable after the removal of its LH contamination.     

Characterization of various molecular species of follicle-stimulating hormone in baboon pituitary preparations

The properties of baboon (Papio hamadryas) follicle-stimulating hormone (bFSH) have been studied after isoelectrofocusing (IEF) of individual pituitary extracts from five female and three male baboons and of a partially purified bFSH preparation (CM-1). The in vitro bioactivities of the female and male pituitary extracts and of CM-1 were 6.0 (range 4.1-9.6), 10.8 (6.9-18.2), and 34.1 (30.3-38.3) mg equivalent of LER 1909-2 reference standard per mg protein, respectively. The corresponding ratios of bioactivity to immunoreactivity (B/I) were 1.72 (range 1.31-2.06), 1.82 (1.53-2.25), and 1.54 (1.51-1.58), respectively. There was a significant increase (p less than 0.05) in all B/I ratios after IEF, due to a diminished immunoreactivity without any loss in bioactivity. Several molecular forms of bFSH were observed with pI values ranging from 4.5 to 7.5, with maximum activity between pH 4.5 to 6.0. The IEF profiles of female and male pituitaries and of the purified bFSH preparation were similar. However, the B/I ratios of the different molecular species of bFSH increased with increasing pI values in every case. It is concluded that there is a significant heterogeneity of bFSH in crude as well as in purified pituitary extracts, but neither the sex of the animals nor the process of purification influenced the quantity and distribution of various molecular species.     

Influence of gonadectomy on isoelectrofocusing profiles of pituitary gonadotropins in rhesus monkeys

Isoelectrofocusing (IEF) profiles of bioactive and immunoreactive follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were studied in saline extracts of pituitary glands from rhesus monkeys. No sex differences were found in the IEF profiles of FSH and LH in intact or gonadectomized animals. Gonadectomy in both sexes resulted in a marked increase in the formation of relatively more acidic molecular species of FSH and LH in parallel with the production of an unusual strongly alkaline FSH species.     

Changes in circulating levels of immunoreactive follicle stimulating hormone, luteinizing hormone, and testosterone during sexual development in the rhesus monkey, Macaca mulatta

Basal serum levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) and the responsiveness of these hormones to a challenge dose of luteinizing hormone releasing hormone (LHRH), were determined in juvenile, pubertal, and adult rhesus monkeys. The monkey gonadotrophins were analyzed using RIA reagents supplied by the World Health Organization (WHO) Special Programme of Human Reproduction. The FSH levels which were near the assay sensitivity in immature monkeys (2.4 +/- 0.8 ng/ml) showed a discernible increase in pubertal animals (6.4 +/- 1.8 ng/ml). Compared to other two age groups, the serum FSH concentration was markedly higher (16.1 +/- 1.8 ng/ml) in adults. Serum LH levels were below the detectable limits of the assay in juvenile monkeys but rose to 16.2 +/- 3.1 ng/ml in pubertal animals. When compared to pubertal animals, a two-fold increase in LH levels paralleled changes in serum LH during the three developmental stages. Response of serum gonadotrophins and T levels to a challenge dose of LHRH (2.5 micrograms; i.v.) was variable in the different age groups. The present data suggest: an asynchronous rise of FSH and LH during the pubertal period and a temporal correlation between the testicular size and FSH concentrations; the challenge dose of LHRH, which induces a significant rise in serum LH and T levels, fails to elicit an FSH response in all the three age groups; and the pubertal as compared to adult monkeys release significantly larger quantities of LH in response to exogenous LHRH.     

Expression of luteinizing hormone and follicle-stimulating hormone receptor in the dog prostate

A possible role for gonadotrophins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the prostate physiology has been suggested in humans and rats. This study aimed at investigating the presence of receptors for LH and FSH (LHR and FSHR) in the canine prostate. Prostates were collected at post mortem from 6 clinically healthy, sexually intact beagles free from any prostatic disorder. Tissue was sampled from dorsal, middle and ventral regions of each prostate. Immunohistochemical localization was performed on wax-embedded sections using polyclonal antibodies for LHR or FSHR. The pattern and intensity of staining in the parenchyma (glandular epithelium) and stroma were determined using a semiquantitative histologic assessment. Receptors for LH and FSH were consistently present in both the glandular epithelium and the stroma in all tissue samples examined. Expression for both receptors was higher in the glandular epithelium than the stroma of all prostatic regions (P < 0.001). In the glandular epithelium, LHR (P < 0.01) and FSHR (P < 0.05) expression was lower in the lateral than the other regions, and there was no difference between dorsal and ventral regions. However, variations in the expression for LHR and FSHR among prostatic regions were not found in the stroma. These findings have demonstrated that LHR and FSHR are expressed in the dog prostate, and the variation observed in their levels of expression among its regions and tissue layers suggests a potential role of gonadotrophins LH and FSH in the regulation of the prostate physiology, particularly the glandular epithelium.     

Urinary androstenedione and luteinizing hormone concentrations during musth in a mature African elephant

Musth has not been well documented in captive African elephants. A 37-year-old African bull elephant in the Kansas City Zoological Park was observed during periods of behavioral musth and non-musth. Androstenedione and luteinizing hormone (LH) concentrations in urine were measured by radioimmunoassay. Urinary androstenedione and LH levels were significantly higher in musth urine than in non-musth samples. A positive correlation (P > 0.001) existed between urinary LH and androstenedione concentrations. These results indicate that musth can occur in a zoo-maintained African elephant and that urinary androgen levels are elevated during musth, possibly as the result of LH stimulation of testicular steroidogenesis.     

Insulin-like growth factor-I feedback regulation of growth hormone and luteinizing hormone secretion in the pig: evidence for a pituitary site of action

Three experiments (EXP) were conducted to determine the role of insulin-like growth factor-I (IGF-I) in the control of growth hormone (GH) and LH secretion. In EXP I, prepuberal gilts, 65 ± 6 kg body weight and 140 days of age received intracerebroventricular (ICV) injections of saline (n = 4), 25 μg (n = 4) or 75 μg (n = 4) IGF-I and jugular blood samples were collected. In EXP II, anterior pituitary cells in culture collected from 150-day-old prepuberal gilts (n = 6) were challenged with 0.1, 10 or 1000 nM [Ala15]-h growth hormone-releasing hormone-(1-29)NH2 (GHRH), or 0.01, 0.1, 1, 10, 30 nM IGF-I individually or in combinations with 1000 nM GHRH. Secreted GH was measured at 4 and 24 h after treatment. In EXP III, anterior pituitary cells in culture collected from 150-day-old barrows (n = 5) were challenged with 10, 100 or 1000 nM gonadotropin-releasing hormone (GnRH) or 0.01, 0.1, 1, 10, 30 nM IGF-I individually or in combinations with 100 nM GnRH. Secreted LH was measured at 4 h after treatment. In EXP I, serum GH and LH concentrations were unaffected by ICV IGF-I treatment. In EXP II, relative to control all doses of GHRH increased (P < 0.01) GH secretion. Only 1, 10, 30 nM IGF-I enhanced (P < 0.02) basal GH secretion at 4 h, whereas by 24 h all doses except for 30 nM IGF-I suppressed (P < 0.02) basal GH secretion compared to control wells. All doses of IGF-I in combination with 1000 nM GHRH increased (P < 0.04) the GH response to GHRH compared to GHRH alone at 4 h, whereas by 24 h all doses of IGF-I suppressed (P < 0.04) the GH response to GHRH. In EXP III, all doses of IGF-I increased (P < 0.01) basal LH levels while the LH response to GnRH was unaffected by IGF-I (P > 0.1). In conclusion, under these experimental conditions the results suggest that the pituitary is the putative site for IGF-I modulation of GH and LH secretion. Further examination of the role of IGF-I on GH and LH secretion is needed to understand the inhibitory and stimulatory action of IGF-I on GH and LH secretion.     

Ontention of antisera against a hypothalamic decapeptide (luteinizing hormone/follicle stimulating hormone releasing hormone) which stimulates the release of pituitary gonadotropins and development of its radioimmunoassay

Using the classical approach, a decapeptide was synthesized with the structure of porcine luteinizing hormone/follicle stimulating hormone releasing hormone reported by Matsuo, H., Baba, Y., Nair, R. M. G., Arimura, A. and Schally, A. V. (1971) Biochem. Biophys. Res. Commun. 43, 1393–1399. As already reported, this peptide was capable of inducing in vitro the release of luteinizing hormone and follicle stimulating hormone from rat pituitary glands. A specific antiserum against luteinizing hormone/follicle stimulating hormone releasing hormone has been generated in the guinea pig and this allowed the development of a radioimmunoassay for this peptide. The antisera, at a final dilution of to depending on the antiserum used, were able to bind 35% of the ¹³¹I-labelled antigen. The sensitivity of this assay method was 50 pg of luteinizing hormone/follicle stimulating hormone releasing hormone. The following substances did not cross-react: oxytocin, lysine-vasopressin, synthetic thyroid stimulating hormone releasing hormone, ovine luteinizing hormone, follicle stimulating hormone and prolactin. Des-Trp³ luteinizing hormone/follicle stimulating hormone releasing hormone, pyroglutamyl-histidyl-tryptophan and seryl-tyrosyl-glycyl-leucyl-arginyl-prolyl-glycinamide, exhibited flatter curves than luteinizing hormone/follicle stimulating hormone releasing hormone with a cross-reactivity of about . Using this method, luteinizing hormone/follicle stimulating hormone releasing hormone was assayed in extracts of the sheep stalk-median eminence and of the hypothalamus and in jugular vein blood from a normal ram and from normal male rats, from cyclic ewe and from hypophysectomized ram and rats. It was concluded that luteinizing hormone/follicle stimulating hormone releasing hormone is present in hypothalamic extracts and in plasma of sheep and rat.     

大鼠垂体细胞液泡内存在黄体生成激素（LH）

研究者普遍认为糖蛋白激素存在于促性腺激素（gonadotrophin,GTH)细胞的颗粒内,目前在生殖内分泌领域内对糖蛋白激素形成与释放的研究也主要集中在细胞内颗粒的变化上,我们近年的研究发现,大鼠垂体GTH细胞内黄体生成激素（luteinizing hormone,LH)的分泌与细胞内液泡的形态变化有密切的关系。铁形态也随液泡的形态变化而变化,因而推测“LH的储存与释放可能与液泡有极大的关系”,为进一步揭示垂体细胞的液泡内是否存在LH和探讨哺乳动物垂体细胞的液泡是否具有储存与释放LH的功能,本研究对大鼠垂体细胞的液泡进行了分离和纯化。用SDS－PAGE,Western immunobloting及Con A/HRP等方法分别对纯化的垂体,大脑皮层及肝脏组织的液泡进行了蛋白质,LH及糖蛋白的分析。结果显示：（1）垂体,皮层及肝脏细胞的液泡内均含有丰富的,分子量大小不等的蛋白质成分,不同组织的细胞液泡内蛋白质成分有许多是相似的;（2）垂体组织及其液泡内均含有LH,而且在相同浓度的蛋白量中,两者LH的水平并无明显差异;（3）垂体,皮层和肝脏组织液泡内均有分子量不同的糖蛋白,但只有垂体细胞的液泡内才有与LH位置相同的糖蛋白染色谱带。上述结果表明：虽然哺乳动物不同组织的细胞液泡内含有许多相似的蛋白质成分,但LH是特异性地存在于垂体细胞液泡内。在这些LH分子中,至少有一部分是已经装配了糖基的完整LH分子。因此,垂体细胞的液泡有可能具有储存与释放LH的功能。     

The structure and function of glycoprotein hormone receptors: ganglioside interactions with luteinizing hormone.

A minor glycopeptide was newly isolated from the exhaustive pronase digest of crystalline ovalbumin by Dowex-50w column chromatography, and its structure was determined as Manα1→3Manα1→6 (Manα1→3) Manβ1→4GlcNAcβ1→4GlcNAc→Asn. This glycopeptide (GP-VI) has the smallest carbohydrate unit among the ovalbumin glycopeptides so far reported, and is also the smallest glycopeptide of all which are susceptible to endo-β-N-acetylglucosaminidases C_II and H. This finding, together with the already reported data of the action of both enzymes to glycopeptides of known structures, elucidates that the structural requirement of C_II enzyme for its substrate is R→2Manα1→3 (R→6) Manα1→6 (R→2Manα1→3) (R→4) Manβ1→4GlcNAcβ1→4GlcNAc→Asn, in which R represents either hydrogen or sugars, and that of H enzyme is R→2Manα1→3 (R→6) Manα1→6 (R→4) Manβ1→4GlcNAcβ1→4GlcNAc→Asn.     

A correlative radioimmunoassay and immunohistochemical study of LHRH-induced LH depletion in the anterior pituitary of male and female neonatal rats in vitro

Summary After an exposure of 24 h to synthetic LHRH (100 ng/ml) in vitro, the anterior pituitaries of 4-day-old rats show a notable loss of immunoreactive material in most LH cells in males, but not in females. When radioimmunoassayed without incubation, the pituitary LH content of 4-day-old female rats is 2.8 times higher than that of males of the same age. LHRH treatment stimulates a higher rate of LH discharge in females than in males, but if LH release is expressed as a percentage of the initial pituitary LH content, there is no apparent difference. In both sexes, more than 70% of the initially stored LH is discharged into the medium after 24 h of LHRH stimulation. In males, this discharge produces a pronounced depletion, but in females, the pituitary still contains 78.2% of the initial LH content despite the large amount of hormone released.From these results, it is concluded that in newborn rats the LH synthetic rate in females is higher than that in males. This high synthetic activity, together with the large store of LH, may explain why prolonged LHRH treatment fails to cause LH depletion in females. At 4 days of age LHRH had no stimulatory effect on pituitary synthesis of LH in either sex.     

Acceleration of onset of oocyte maturation in vitro by luteinizing hormone

The purpose of this study was to analyze the effect of luteinizing hormone (LH) on the earliest stage of oocyte maturation - the stage of breakdown of the dictyate nucleus. Oocytes were isolated from the preovulatory follicles of adult, cyclic rats. They were incubated in culture medium with or without 10 μg/ml LH. The cultures were observed continuously for up to 3 hours. Analysis of the rate of disappearance of the germinal vesicle nucleolus revealed that LH accelerated the breakdown process. The median times of disappearance were 91.3 minutes without LH and 62.3 minutes with LH. This is in accord with earlier reports on enhancement of fertilizability of oocytes matured in vitro with LH. Thus, although oocytes mature spontaneously in culture, the maturation remains LH sensitive.     

Expression of follicle stimulating hormone and luteinizing hormone receptors during follicular growth in the domestic cat ovary

In order to better understand the pituitary regulation of follicular growth in the domestic cat, follicle stimulating hormone (FSH) and luteinizing hormone (LH) receptors (R) were localized and quantified in relation to follicle diameter and atresia using in situ ligand binding on ovarian sections. Expression of FSHR was homogeneous and restricted to follicle granulosa cells from the early antral stage onwards, whereas expression of LHR was heterogeneous on theca cells of all follicles from the early antral stage onward, and homogeneous on granulosa cells of healthy follicles larger than 800 microm in diameter and in corpora lutea. LHR were also widely expressed as heterogeneous aggregates in the ovarian interstitial tissue. Atretic follicles exhibited significantly reduced levels of both FSHR and LHR on granulosa cells, compared with healthy follicles whatever the follicular diameter, whereas levels of LHR on theca cells were lower only for atretic follicles larger than 1,600 microm in diameter. In healthy follicles, levels of FSHR and LHR in all follicular compartments increased significantly with diameter. Although generally comparable to that observed in other mammals, the expression pattern of gonadotropin receptors in the cat ovary is characterized by an early acquisition of LHR on granulosa cells of growing follicles and islets of LH binding sites in the ovarian interstitial tissue.     

Attenuation of the estradiol-induced surge release of luteinizing hormone by antihistamine in ovariectomized ewes

Ovariectomized ewes received intramuscular (i.m.) injections of an H₁-histamine receptor antagonist, diphenhydramine, or saline during the anestrous and breeding seasons to determine if histamine may regulate the estradiol-induced surge release of LH in ewes. In addition, concentrations of histamine and GnRH in hypothalamic regions and histamine and LH in the pituitary gland were determined during the estradiol-induced surge of LH. Pretreatment mean, basal, and estradiol-induced secretion of LH did not differ (P > 0.05) among seasons. However, the quantity of LH (ng) measured during the estradiol-induced surge of LH was less (P < 0.05) in ewes treated with diphenhydramine (411 ± 104) than saline (747 ± 133). Treatment with diphenhydramine did not (P > 0.05) influence steady-state concentrations of histamine in hypothalamic or pituitary gland tissues, hypothalamic concentrations of GnRH, or anterior pituitary concentrations of LH during the estradiol-induced surge of LH. It is concluded that histamine may modulate the estradiol-induced surge release of LH in ewes by affecting the secretion of GnRH.     

Plasma luteinizing hormone concentrations in cows given repeated treatments or three different doses of gonadotropin releasing hormone

We hypothesized that: (i) repeated GnRH treatments would increase the magnitude and duration of the LH surge and would increase progesterone (P4) concentrations after ovulation; and (ii) the release of pituitary LH would be greater in response to larger doses of GnRH. In Experiment 1, ovary-intact cows were given an intravaginal P4 (1.9 g) insert (CIDR) for 10 d and 500 μg cloprostenol (PGF) at CIDR removal to synchronize estrus. On Days 7 or 8 after estrus, cows received two PGF treatments (12 h apart) and 100 μg GnRH at 36 (Control), 36 and 38 (GnRH38), or 36 and 40 h (GnRH40) after the first PGF. Mean plasma LH concentration (ng/mL) was greater (P < 0.05) in GnRH38 (8.8 ± 1.1) than in Control (5.1 ± 1.3), with that in GnRH40 (5.8 ± 1.3) being intermediate. Although the duration (h) of the LH surge was longer in GnRH40 (8.0 ± 0.4) than in either GnRH38 (P < 0.05; 7.0 ± 0.3) or Control (P < 0.09; 7.1 ± 0.4), mean postovulatory P4 (ng/mL) was greater (P < 0.01) in Control (4.2 ± 0.7) than in GnRH38 (2.9 ± 0.6) or GnRH40 (3.0 ± 0.7) cows. In Experiment 2, ovariectomized cows were given a CIDR for 10 d and 2 mg of estradiol cypionate im at CIDR insertion. Thirty-six hours after CIDR removal, cows received, 50, 100, or 250 μg of GnRH. Cows given 250 μg GnRH released more LH (9.4 ± 1.4 ng/mL) than those given 50 or 100 μg (6.1 ± 1.3 and 5.4 ± 1.4 ng/mL, respectively), and had an LH surge of longer duration than those given 50 μg (6.8 ± 0.4 vs. 5.1 ± 0.3 h). In summary, ovary-intact cows in the GnRH38 group had greater mean and peak LH concentrations, but subsequent plasma P4 concentrations were lower than in Control cows. Ovariectomized cows given 250 μg GnRH had a greater pituitary release of LH.     

The extracellular domain of luteinizing hormone receptor dictates its efficiency of maturation

The processing of luteinizing hormone receptor (LHR) shows marked differences in different species. While the human LHR is predominantly expressed as the mature, 90 kDa species, rat LHR exists mostly in the 70 kDa precursor form. Since the extracellular domain of the LHR is unusually large in comparison with other G protein-coupled receptors, the present studies examined the role of extracellular domain in its processing. FLAG-tagged chimeric LH receptors were constructed by substituting the extracellular domain of the human receptor in rat LHR (hrr) and the extracellular domain of the rat receptor in human LHR (rhh). The intracellular processing, ligand binding and recycling of the chimeric receptors were compared with that of the wild type receptors in 293T cells. The results showed that the human and rat LHR were expressed predominantly as 90 and 70 kDa species, respectively, as expected. The introduction of the rat extracellular domain into the human LHR (rhh) decreased the abundance of the mature form with an increase in the precursor form. Conversely, substitution of the extracellular domain of the rat LHR by the extracellular domain of the human LHR (hrr) led to an increase in the mature form with a corresponding decrease in the precursor form. Changes were also observed in the ligand binding and recycling of the wild type and chimeric receptors. These results suggest that the extracellular domain of the LHR is one of the determinants that confer its ability for proper maturation and cell surface expression.