Quantification of Rat Kisspeptin Using a Novel Radioimmunoassay
File(s)
Author(s)
Type
Journal Article
Abstract
Kisspeptin is a hypothalamic peptide hormone that plays a pivotal role in pubertal onset and reproductive function. Previous studies have examined hypothalamic kisspeptin mRNA expression, either through in situ hybridisation or real-time RT-PCR, as a means quantifying kisspeptin gene expression. However, mRNA expression levels are not always reflected in levels of the translated protein. Kisspeptin-immunoreactivity (IR) has been extensively examined using immunohistochemistry, enabling detection and localisation of kisspeptin perikaya in the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV). However, quantification of kisspeptin-IR remains challenging. We developed a specific rodent radioimmunoassay assay (RIA) capable of detecting and quantifying kisspeptin-IR in rodent tissues. The RIA uses kisspeptin-10 as a standard and radioactive tracer, combined with a commercially available antibody raised to the kisspeptin-10 fragment. Adult female wistar rat brain samples were sectioned at 300 µm and the ARC and AVPV punch micro-dissected. Brain punches were homogenised in extraction buffer and assayed with rodent kisspeptin-RIA. In accord with the pattern of kisspeptin mRNA expression, kisspeptin-IR was detected in both the ARC (47.1±6.2 fmol/punch, mean±SEM n = 15) and AVPV (7.6±1.3 fmol/punch, mean±SEM n = 15). Kisspeptin-IR was also detectable in rat placenta (1.26±0.15 fmol/mg). Reverse phase high pressure liquid chromatography analysis showed that hypothalamic kisspeptin-IR had the same elution profile as a synthetic rodent kisspeptin standard. A specific rodent kisspeptin-RIA will allow accurate quantification of kisspeptin peptide levels within specific tissues in rodent experimental models.
Date Issued
2014-05-20
Date Acceptance
2014-04-21
Citation
PLoS ONE, 2014, 9 (5)
ISSN
1932-6203
Publisher
Public Library of Science
Journal / Book Title
PLoS ONE
Volume
9
Issue
5
Copyright Statement
© 2014 Kinsey-Jones et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
License URL
Subjects
Science & Technology
Multidisciplinary Sciences
Science & Technology - Other Topics
MULTIDISCIPLINARY SCIENCES
GONADOTROPIN-RELEASING-HORMONE
PROTEIN-COUPLED RECEPTOR
MESSENGER-RNA EXPRESSION
PITUITARY-GONADAL AXIS
ARCUATE NUCLEUS
SEXUAL-DIMORPHISM
GENE-EXPRESSION
FEMALE MOUSE
NEURONS
KISS-1
Publication Status
Published
Article Number
e97611