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.


Introduction
The kisspeptins are a family of peptides essential for the onset of puberty and the regulation of reproductive function. The absence of a functional kisspeptin receptor (KiSS1r) or Kiss-1 gene results in low gonadotrophin levels and failure to undergo pubertal development in both mice and humans [1][2][3][4]. Administration of exogenous kisspeptin potently stimulates gonadotropin secretion in rodents and man [4][5][6][7]. Kisspeptin is the endogenous ligand of the G-protein coupled receptor Kiss1r [8][9][10]. The stimulatory effects of kisspeptin on the reproductive axis appear to be mediated by a direct activation of KiSS1r-expressing gonadotrophin releasing hormone (GnRH) neurons [11][12][13].
In humans, KISS1 encodes a 145 amino acid precursor peptide which is cleaved to form a 54-amino-acid peptide, known as kisspeptin-54 or metastin [10], and shorter fragments 14, 13 and 10 amino acids long [9]. All kisspeptin share a common 10 amino acid C-terminal section, which is required for kisspeptin receptor activation. Kisspeptin-54, -14, -13 and -10 all bind to Kiss1 with equal affinity and efficacy in vitro [9]. The largest proteolytic product in rodents is a 52 amino-acid peptide. Although this peptide shares only relatively low overall homology (52%) with human kisspeptin-54, the C-terminal 10 amino-acid signalling sequences is highly conserved between mouse and human KiSS1 proteins, varying by only one amino acid [Tyr 10 (Y) in rodents to Phe 10 (F) in humans] [14].
Expression of Kiss1 mRNA has been observed in tissues including the placenta, pancreas, small intestine, and brain in humans [8][9][10]. Within the human CNS, kisspeptin immunoreactive cell bodies are predominantly located in the infundibular nucleus of the hypothalamus [15]. In rodents, kisspeptin perikarya are located in two major populations in the hypothalamus, the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC) [5,16].
Kisspeptin mRNA expression has been quantified using in situ hybridisation (ISH) and quantitative polymerase chain reaction (qPCR). This provides valuable information on gene transcription, but it is widely recognised that changes in mRNA are not always mirrored by protein levels [17,18]. In humans, circulating kisspeptin levels have been quantified by radioimmunoassay, and immunocytochemical (ICC) methods have been used to localise kisspeptin expression in humans and rodents [7,19]. However, to date, kisspeptin peptide levels in the rodent have not been measured. Western blotting can be used to measure protein levels, but it can be challenging to detect small changes in kisspeptin-IR levels using this technique. In the current study we have developed a sensitive and specific radioimmunoassay (RIA) which enables the quantification of kisspeptin levels within rodent tissues.

Materials
Synthetic kisspeptin-10 was obtained from the Advanced Biotechnology Centre, Imperial College, London, UK. Kisspeptin-52 was purchased from Phoenix Pharmaceuticals (Burlingame, CA, USA). The kisspeptin polyclonal antibody was purchased from Millipore (Watford, UK).

Rat Tissue
Female Wistar rats (220-250 g) obtained from Charles River (Margate, UK) were housed in single cages (specific pathogen free, Imperial College London, UK) and maintained in a controlled environment (temperature 21-23uC, 12-h light-dark cycle, lights on at 07:00) with ad libitum access to food (RM1 diet; SDS UK, Witham, Essex, UK) and water. All animal procedures were approved by the British Home Office Animals (Scientific Procedures) Act 1986. Estrus cyclicty was monitored daily by vaginal lavage, slides were examined under a light microscope to determine the dominant cell type. On the morning of the diestrus phase of the cycle, animals were killed by decapitation, and either whole brain or hypothalamus dissected and snap frozen then stored at 280uC. Pregnant female rats were obtained from Charles River. At pregnancy day 20 rats were killed and the placenta removed, snap frozen and stored at 220uC. An additional cohort of female Wistar rats were bilaterally ovarectomized (OVX) and implanted with an estradiol (E 2 ) filled (150 ug E 2 /ml) silicone capsule. This model has previously been shown to mediate changes in kisspeptin expression [20]. Two weeks later animals were killed by decapitation and whole brain dissected, snap frozen and stored at 280uC.

Chromatography
Synthetic kisspeptin-52 (2 nmol in 150 ul water) was fractionated using RP-HPLC Phenomenex Jupiter 4 mm Proteo 90 Å column and eluted with a 30-35% gradient of ACN plus 0?05% (v/v) water/0?1% (v/v) 50 min at a flow rate of 1 ml/min per fraction. Fractions were collected every min. An additional set of hypothalamic tissue samples (n = 4) was extracted using the protocol described above, centrifuged at 15 000 g for 3 min, and the supernatants filtered through 0?2 mm hydrophilic membranes (Sartorius, Göttingen, Germany). Samples of 150 ul from each extract were then loaded separately onto the HPLC column and eluted under the same conditions described above. Fractions from all runs were freeze-dried, reconstituted in assay buffer and the kisspeptin content determined by RIA.

Quantification of AVPV, ARC and Placenta Kisspeptin
Several groups have confirmed kisspeptin expression in the placenta and the hypothalamic AVPV and ARC [25,26]. Brain sections (300 mm) were cut on a cryostat, and bilateral punches  (1 mm diameter) of the AVPV were taken from Bregma +0.2 to 2 0.4 mm, single midline punch (1 mm diameter) that included both ARC was taken from bregma 21.7 to 23.9 according to the rat brain atlas [27], following the micropunch method of Palkovits [28]. Hypothalamic punches, cerebellum and placental tissue (350 mg) were homogenised and extracted in acid ethanol buffer as described above, and kisspeptin content measured by radioimmunoassay. A separate set of tissues were also extracted by boiling for 15 min in 0?5 M acetic acid [24].

Statistical Analysis
All data are presented as mean 6 standard error of the mean (SEM). Levels of kisspeptin expression were analysed using an unpaired t-test (GraphPad Prism version 5 for Windows; GraphPad Software, San Diego, CA).

Chromatography
HPLC of hypothalamic extracts resulted in a distinct kisspeptin-IR peak at the same point at which synthetic kisspeptin-52 peptide eluted ( Figure 2). The recovery of kisspeptin-IR for each column run of the tissue extracts was .80%.

Discussion
In the present study, we have characterised the development of a novel kisspeptin RIA and demonstrated its utility in quantifying kisspeptin-IR within the AVPV and ARC of female rats. We have demonstrated that this assay can detect changes in kisspeptin expression in response to E 2 feedback. In accord with studies using other methods, we observed suppression of kisspeptin-IR in the ARC and increased kisspeptin-IR in the AVPV in response to E 2 in ovariectomised female rats [29]. Numerous studies have demonstrated a wide distribution of kisspeptin expression throughout the rodent brain [30,31]. Initial studies examining the distribution of kisspeptin-IR rodents were hampered by the limited specificity of available antibodies [30]. However, the development of reliable antibodies which recognise rodent, sheep [19] and human [7] kisspeptin has enabled the distribution of kisspeptin-IR to be fully mapped. Furthermore, we observe a single IR peak following HPLC of hypothalamic extracts, at the point we would expect kisspeptin-52 to elute, thus supporting previous findings regarding antibody specificity [19].
In addition to mapping kisspeptin distribution, ICC methodologies have been utilised to quantify changes in expression under different experimental and physiological conditions [31,32]. Kisspeptin-IR has been successfully quantified in the AVPV, and also in the ARC, though the ARC has presented difficulties due to the dense plexus of kisspeptin fibres present [31,33,34]. The limitations of ICC preclude the quantification of the absolute number of kisspeptin molecules or its concentration in a sample. Further, though semi-quantitative methodologies provide information about relative quantities of kisspeptin-IR within each study, it is difficult to compare the levels observed between different studies and models. Kisspeptin expression can be quantified using ISH [5] or qPCR [35]. However, it is becoming increasingly evident that examination of mRNA levels may be an unreliable proxy for protein concentrations [36,37], with only ,40% protein levels being explained by mRNA abundances in mammalian cell lines [17,18]. Differences in the distribution of kisspeptin mRNA and immunoreactivity have previously been reported. For example, kisspeptin-IR cell bodies and fibres were described in the dorsomedial nucleus of mice [31], though ISH failed to identify kisspeptin mRNA within this region [26]. A subsequent study suggested that the IR detected within the dorsomedial nucleus may be the result of cross-reactivity of the antibody used with neuropeptide FF [38], though it is interesting to note that a recent study detected kisspeptin-immunoreactivity in some DMN cells in the mouse using a different antibody (Franceschini et al 2013). In the current investigation, the higher level of kisspeptin-IR in the ARC than the AVPV is in accord with the relative levels predicted by assessment of mRNA levels.
Kisspeptin-IR was also present in the placenta, albeit at relatively low levels. Previous studies have suggested that the expression of kisspeptin mRNA in the placenta varies during pregnancy, and it may be that higher levels would have been detected at another time point [25]. Our data also suggest that the majority of hypothalamic kisspeptin-IR is in the form of kisspeptin-52, though it is possible that our extraction methods favour this particular form, and that other kisspeptin forms are present at lower levels. Further studies are thus necessary to conclusively demonstrate the relative quantities of the different kisspeptins in the hypothalamus.
In summary, we have developed a novel radioimmunoassay for the measurement of kisspeptin-IR within rat tissue. When combined with the Palkovits punch micro-dissection methodology, this assay enables quantification of kisspeptin-IR within specific brain nuclei.