An Improved in Vitro Bioassay Method for Measuring Luteinizing Hormone (Lh) Activity Using Mouse Leydig Cell Preparations

Damme, M.-P. Van; Robertson, David; Diczfalusy, E.

doi:10.1530/acta.0.0770655

Cited by 388 publications

(126 citation statements)

References 4 publications

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“…Plasma concentrations of LH were determined for both baseline and post-GnRH samples using an in vitro bioassay based on the production of testosterone by dispersed mouse Leydig cells (Van Damme et al 1974), as previously described and validated for Damaraland molerats Molteno and Bennett 2002). Plasma samples were assayed in duplicate at a dilution of 1:20 and compared with a rat LH standard (the rLH antigen preparation: rLH-1-7 from NIDDK Baltimore) over the range 0.031-400 miu/ml.…”

Section: Blood Sampling Gnrh Administration and Lh Bioassaymentioning

confidence: 99%

Physiological suppression eases in Damaraland mole-rat societies when ecological constraints on dispersal are relaxed

Young

Oosthuizen

Lutermann

et al. 2010

Hormones and Behavior

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In many vertebrate societies, subordinate females exhibit down-regulated reproductive physiologies relative to those of dominants, a condition commonly termed physiological suppression. Research into the causes of physiological suppression has focused principally on the role of the subordinate's social environment (typically the presence of the dominant female and/or an absence of unrelated males within the group), while few studies have considered the additional role that the physical environment may play. Here we present new evidence from wild Damaraland mole-rats, Fukomys damarensis, revealing that physiological suppression among subordinate females eases markedly during the annual rains (a time when ecological constraints on dispersal are relaxed), despite the continued presence of the dominant female and in groups that contain no new immigrant males. Subordinate females showed substantially higher pituitary sensitivities to GnRH challenge during the wet period than the dry, a contrast that cannot be attributed to between-female differences (as it holds for paired within-female comparisons), associated changes in body mass (as our analyses control for this), or concomitant reductions in physiological stress (as their urinary cortisol concentrations were actually higher in the wet period). We suggest that our findings reflect selection for the maintenance of reproductive readiness among subordinate females during high rainfall periods, given the increased likelihood of encountering dispersal and/or mating opportunities with extra-group males when ecological constraints on dispersal are relaxed.These findings reveal new complexity in the processes that regulate physiological suppression, suggesting a key role in some species for changes in the physical as well as social environment.

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Section: Blood Sampling Gnrh Administration and Lh Bioassaymentioning

confidence: 99%

Physiological suppression eases in Damaraland mole-rat societies when ecological constraints on dispersal are relaxed

Young

Oosthuizen

Lutermann

et al. 2010

Hormones and Behavior

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“…A Leydig cell-rich fraction from the testes of adult Wistar rats was prepared according to the procedure of Van Damme et al (1974). Granulosa cells were isolated from equine chorionic gonadotropin (eCG)-primed immature female rats according to the procedure described elsewhere (Selvaraj and Moudgal, 1994).…”

Section: Preparation Of Leydig Cells and Granulosa Cells For Determinmentioning

confidence: 99%

Immunobiology of a Synthetic Luteinizing Hormone Receptor Peptide 21–41

Moudgal

Surekha

Krishnamurthy

et al. 2001

Journal of Andrology

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ABSTRACT:Immunization of adult male rabbits with a synthetic luteinizing hormone-receptor peptide (LH-RP; representing amino-acids 21-41 of the extracellular domain of the rat LH receptor) resulted in production of high-titer antibodies capable of interacting with particulate and cell-based LH receptors. The antibody produced was able to inhibit binding of 125 I-labeled human chorionic gonadotropin (hCG) to a particulate sheep luteal LH receptor preparation by 40%-50%. Maximal inhibitory activity was correlated with high antibody titer. Immunocytometry revealed that the antibody could directly bind to cells having LH receptors, such as rat granulosa and Leydig cells. The antibodies recognized a 77-kilodalton membrane protein in Western blots of mouse testicular extracts. Interaction of endogenous Leydig cell LH receptor with the LH-RP antibody resulted in both hormone agonist and antagonistic activities. The hormone-mimicking activity (increase in serum testosterone over control) was confined only to the early phase of immunization when the antibody titer was low. Blockade of LH receptor during the later part of immunization resulted in a significant reduction in serum testosterone over controls and inhibition of spermatogenesis. DNA flow cytometry showed that a specific and significant inhibition of meiosis (transformation of primary spermatocytes to round and elongated spermatids P Ͻ .01) and spermiogenesis (transformation of round spermatids to elongated spermatids P Ͻ .0001) occurred following blockade of LH function.Key words: Testicular function, spermatogenesis, receptor antibodies. J Androl 2001;22:992-998S permatogenesis in mammals is mainly regulated by 2 glycoprotein hormones; namely, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Immunization of adult male bonnet monkeys (Macaca radiata) with ovine FSH (Aravindan et al, 1993) as well as a recombinant FSH receptor protein has been shown to result in impairment of spermatogenesis . Immunization of male rabbits (Jeyakumar et al, 1995) and monkeys with ovine LH also results in blockade of testosterone production and spermatogenesis. Immunization of rabbits with a holo LH receptor (LH-R) preparation isolated from sheep corpora lutea led to the production of receptor-specific antibodies, but not to testicular dysfunction (Jeyakumar and Moudgal, 1996). This was shown to be due to the presence of antibodies expressing both hormone-mimicking and antagonistic activities throughout the period of immunization. During the early period of immunization, when only hormone-mim- icking antibodies were present, there was a sustained (lasting over several weeks) and significant (fourfold to sevenfold) increase in serum testosterone levels. The return of serum testosterone to normal levels during later periods of immunization could be correlated to the appearance of receptor antagonistic (blocking) antibodies. The occurrence of both types of antibodies in the serum could be demonstrated by in vitro tests (Jeyakumar and Moudgal, 1996). The presence of ...

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“…In vitro bioassay A modification of a bioassay using mouse Leydig cells (Van Damme et al, 1974) Immunoassays UGP and the fractions obtained from HPLC chromatography, were measured for immunological activity using both radioimmunoassay (RIA) and immunoradiometric assay (IRMA) in the systems previously described (Kardana et al, 1989). A radioimmunoassay for peptide 3 was generated using the AK12 antibody system with 251I-peptide 3 instead of 251I-UGP.…”

Section: Purification Of Ugpmentioning

confidence: 99%

Characterisation of UGP and its relationship with beta-core fragment

Kardana

Bagshawe²,

Coles³

et al. 1993

Br J Cancer

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Summary Urinary gonadotrophin peptide (UGP) was originally identified by immunoassay in the urine of patients with various types of cancer and by immunohistochemistry in human cancers of various histological types. Extracts of normal adult male urine also contained UGP by immunoassay. Purified UGP from different starting material was subjected to high pressure liquid chromatography (HPLC) prior to defining amino acid sequences. Chromatographed UGP after HPLC showed three distinct fractions. The N-terminal sequence of peptide 2 was completely homologous with the beta-core fragment of human chorionic gonadotrophin (hCG) and this was found associated with two smaller peptides. The N-terminal sequence of peptide has not been described previously whilst the N-terminus of peptide 3 that was sequenced showed complete homology with the N-terminal sequence of eosinophil derived neurotoxin and non-secretory ribonuclease. The monoclonal antibodies 2C2 and 6D3 only bind beta core-fragment (peptide 2) whilst the polyclonal (rabbit) antibody AK12 could bind all three peptides. The radioimmunoassay system using AK12 could be inhibited by all three peptides and the immunoradiometric assay although based on a capture antibody (2C2) that only bound peptide 2, had the potential to measure all three peptides (when bound together as UGP) at the second step when '25l-AK12 was introduced as the detector. A specific radioimmunoassay for peptide 3 was generated using 1251-peptide 3 and the AK12 antibody. Beta core-fragment on iso-electric focusing was found to have a pl > 9.5, peptide 3 showed two bands at pl = 3.5 and 3.8 whilst insufficient purified peptide 1 was available to determine its iso-electric point. Bioassay studies on UGP showed that any biological activity could be attributed to trace contamination with hCG.Urinary gonadotrophin peptide (UGP) has been purified from the urine of patients with trophoblastic and nontrophoblastic disease (Kardana et al., 1988). It has also been detected in 93% (77/83) of tumours examined immunohistochemically (Kardana et al., 1988). The apparent molecular weight is 15,000. UGP cross reacts with antisera to hCG beta-core fragment, which contains at least one epitope in common with the beta-subunit of hCG, as seen by the ability to measure beta-core fragment using antisera raised to hCG beta-subunit Papapetrou & Nicopoulou, 1986;Wehmann & Nisula, 1980). Initial screening of urines using specific immunoassays for UGP (Kardana et al., 1989) has shown detectable levels of UGP in normal subjects and elevated levels in the urine of some patients with neoplasms (manuscript submitted for publication).The origin of beta-core fragment (BCF) is unclear and there are reports that it is a renal breakdown product of either hCG or its beta-subunit . It has been shown that the major form of betacore fragment in serum exists as a high molecular weight complex (Mr >60,000) which can be dissociated with 3M ammonium thiocyanate to release beta-core fragment (Mr= 15,000) (Kardana & .In this paper we report further ...

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An Improved in Vitro Bioassay Method for Measuring Luteinizing Hormone (Lh) Activity Using Mouse Leydig Cell Preparations

Cited by 388 publications

References 4 publications

Physiological suppression eases in Damaraland mole-rat societies when ecological constraints on dispersal are relaxed

Physiological suppression eases in Damaraland mole-rat societies when ecological constraints on dispersal are relaxed

Immunobiology of a Synthetic Luteinizing Hormone Receptor Peptide 21–41

Characterisation of UGP and its relationship with beta-core fragment

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