Quantification of rat kisspeptin using a novel radioimmunoassay.
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.
Section of Investigative Medicine, Department of Medicine, Imperial College London, London, United Kingdom.
James S
JS
Kinsey-Jones
Section of Investigative Medicine, Department of Medicine, Imperial College London, London, United Kingdom.
Kylie E
KE
Beale
Section of Investigative Medicine, Department of Medicine, Imperial College London, London, United Kingdom.
Joy
J
Cuenco
Division of Women's Health, School of Medicine, King's College London, London, United Kingdom.
Xiao Feng
XF
Li
Section of Investigative Medicine, Department of Medicine, Imperial College London, London, United Kingdom.
Stephen R
SR
Bloom
Division of Women's Health, School of Medicine, King's College London, London, United Kingdom.
Kevin T
KT
O'Byrne
Section of Investigative Medicine, Department of Medicine, Imperial College London, London, United Kingdom.
Kevin G
KG
Murphy
D000818:Animals; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology; D007031:Hypothalamus / Q000378:metabolism*; D059648:Kisspeptins / Q000276:immunology / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011863:Radioimmunoassay / Q000379:methods; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity
C491406:Kiss1 protein, rat; D059648:Kisspeptins; D009419:Nerve Tissue Proteins