Dose-response study with canine kisspeptin-10 in vivo and in vitro
M. van Elderen
Dose-response study with canine kisspeptin-10
in vivo and in vitro
M. van Elderen*
*Solis-ID: 3515230, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
Supervisor: Drs. C.H.J. Albers-Wolthers
Examiner: Dr. A.C. Schaefers-Okkens, Dipl. ECAR
__________________________________________________________________________________
ABSTRACT
Kisspeptin stimulates gonadotropic-releasing hormone (GnRH) and is a key player in puberty onset and
maintenance of normal reproductive function in several mammalian species. However, doses below 1
μg/kg BW have not been studied in dogs. The aims of this study were to find the lowest dose of canine
kisspeptin-10 that significantly increases the plasma LH concentration above the 95 percentile of the
measured basal plasma LH concentrations in dogs and to investigate the corresponding effects of human
kisspeptin-10 (hKP10) and canine kisspeptin-10 (cKP10) in vitro. cKP10 was administered intravenously
at weekly intervals to adult Beagle bitches during anestrus in doses of 0.1, 0.5, 1 and 10 μg/kg BW. Blood
samples were collected at 40 and 0 minutes before and at 10, 20, 30, 40, 60, 90, and 120 minutes after
KP10 administration for measurement of plasma LH concentration. The doses 0.5, 1 and 10 μg/kg BW
resulted in an increase in plasma LH concentration above the 95 percentile of the measured basal plasma
LH concentrations. Chem-1 cells transfected with GPR54 cDNA were exposed to 10-6,10-8 or 10-10 M hKP10
or cKP10 and the maximum amplitude of intracellular Ca2+ concentrations, [Ca2+]i, was calculated for each
cell. The maximum amplitudes of [Ca2+]i differed between hKP10 and cKP10 for only 10-8 M. In conclusion,
0.5 μg/kg BW cKP10 is the lowest dose that significantly increases the plasma LH concentration above the
95 percentile of the measured basal plasma LH concentrations in the dog and hKP10 and cKP10 give a
similar [Ca2+]i response after binding to the human kisspeptin receptor GPR54 in vitro.
_________________________________________________________________________________________________________________
INTRODUCTION
Each day, thousands of dogs are brought to animal shelters all over the world, where the majority is
euthanized due to overpopulation (Chan et al. 2009, Popa, Clifton & Steiner 2008). In recent years, this
population has not declined, but just increases (Harris 2012, Panda, Thakur & Katoch 2008). One possible
solution to this problem is to interfere with the reproduction of stray dogs by means of a cheap, fast and
animal-friendly method. Chemical castration offers us the means of achieving this. However, a nonsurgical method to neuter male and female dogs for more than twelve months does still not exist (Miller,
Zawistowski 2013). Fortunately, the discovery of kisspeptin and its antagonist gives us opportunities to
find a new non-surgical castration method.
Kisspeptins (KP), a family of neuropeptides encoded by the gene KISS1, were discovered in 1996 and
have recently emerged as essential regulators of gonadotropin-releasing hormone (GnRH) neurons across
mammalian species (Irwig et al. 2004, Oakley, Clifton & Steiner 2009). The KISS1 gene encodes for a
hydrophobic 145 amino acid secretory polypeptide. This 145 amino acid protein (KP145), depending on
the species (d'Anglemont de Tassigny, Colledge 2010), can be cleaved into a smaller 54 amino acid protein
(52 in rodents) and from this larger peptide the smaller peptides KP14, KP13 and KP10 are processed
(Irwig et al. 2004, Gottsch, Clifton & Steiner 2009, Ohtaki et al. 2001, Lee et al. 1996). It is unclear how
these shorter peptides are processed from the larger peptide (Ohtaki et al. 2001). KP10 has full intrinsic
biological activity and is the minimal sequence required for full receptor binding and activation (Roseweir
et al. 2009, Millar et al. 2010). The G-protein coupled receptor of kisspeptin (GPR54), encoded by the
KISS1R gene, has been located on GnRH neurons (Popa, Clifton & Steiner 2008). Kisspeptin stimulates
GnRH secretion by binding on this receptor which in turn stimulates the pituitary to secrete luteinizing
hormone (LH) and follicle-stimulating hormone (FSH) which is associated with the initiation of
reproductive function (Thompson et al. 2004). Kisspeptins are also involved in the regulation of the
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Dose-response study with canine kisspeptin-10 in vivo and in vitro
M. van Elderen
hypothalamic-pituitary-gonadal (HPG) axis by positive and negative feedback mechanisms (Oakley, Clifton
& Steiner 2009). Expression of KISS1 and KISS1R genes increases during pubertal development and
inactivating or loss-of-function mutations in respectively KISS1 or KISS1R, demonstrated in humans and
mice, cause hypogonadotropic hypogonadism (HH) and impairment of pubertal development
(d'Anglemont de Tassigny, Colledge 2010, de Roux et al. 2003, Silveira et al. 2010). Furthermore,
administration of a potent kisspeptin antagonist, p234, prevented the post-castration rise of LH in rats,
mice and sheep and the preovulatory LH surge in rats (Roseweir et al. 2009, Pineda et al. 2010). Therefore
kisspeptin seems to be a key player in puberty onset and maintenance of normal reproductive function in
several mammalian species. However, the role of kisspeptin and its receptor in the dog are hardly studied.
Recently, Albers-Wolthers et al. (2013) showed that the KISS1 and the KISS1R genes are present in the
canine genome and that both human kisspeptin-10 (hKP10) as canine kisspeptin-10 (cKP10)
administration (1, 5, 10, 30, 50 and 100 μg/kg BW) in the dog results in a significant increase of the
plasma LH concentration at 10 minutes after administration. This study strongly suggests that kisspeptin
regulation and GPR54 signaling plays an important role in reproductive function in the dog, as it does in
many other species. However, there is no literature about the antagonist p234 in dogs. P234 was
developed by Roseweir et al. (2009) via amino acid substitution of kisspeptin-10 analogs and has the
ability to block the effects of KP in vivo and in vitro. P234 has the same C-terminal sequence (RF-NH2) as
KP10, which is essential for receptor binding. Pineda et al. (2010) used the addition of a penetratin tag to
p234 to increase its penetration of the blood-brain-barrier. This tag did not affect the binding affinity of
p234 to GPR54 and can be used when p234 is administered peripherally (Pineda et al. 2010). P234
suppresses pulsatile LH secretion in female rats and GnRH release in female monkeys and reduces FSH
responses after administration of 100 pmol KP10 in male rats (Pineda et al. 2010, Li et al. 2009, Guerriero
et al. 2012). Therefore, p234 seems to have high binding affinity, specificity and efficacy. In practice, p234
might be developed into a contraceptive. Before this antagonist can be used for clinical application, further
research is necessary. P234 is probably a competitive antagonist. To determine the effect of p234 on
hormone levels after kisspeptin administration in the dog, p234 must be administered in higher doses
than the doses of administered kisspeptin, because the antagonist has to be able to compete with
kisspeptin. Therefore, before this antagonist is tested in dogs, it would be convenient to determine the
dose response relationship between cKP10 and plasma LH concentration in the dog to find the lowest
effective dose of cKP10 that still gives a significant rise in plasma LH levels.
The first aim of this study was to find the lowest dose of cKP10 that significantly increases the plasma
LH concentration above the 95 percentile of the measured basal plasma LH concentrations. In order to
investigate the corresponding effects of hKP10 and cKP10 in vitro, we also studied the intracellular Ca2+
concentration response after acute exposure to different doses of hKP10 and cKP10 in Chem-1 cells
transfected with GPR54 cDNA.
MATERIALS AND METHODS
Animals
Six healthy Beagle bitches (1-7 years of age) were used in this study. The dogs were born and raised at the
Department of Clinical Sciences of Companion Animals and were accustomed to the laboratory
environment and procedures, such as blood collection. They were housed in pairs in indoor–outdoor runs
and fed a standard commercial dog food once daily. Water was available ad libitum.
The bitches were in anestrus and did not show signs of proestrus (swelling of the vulva and
serosanguineous vaginal discharge) during the experimental period (de Gier et al. 2006). Anestrus was
defined as the period starting 100 days after ovulation and ending when signs of proestrus were shown
(Kooistra et al. 1999). During anestrus plasma progesterone levels are below 1 ng/ml (Okkens et al. 1985,
Hazewinkel 1996). Plasma progesterone concentration was measured thrice weekly from the start of
proestrus until the day on which it reached values above 4 ng/ml and ovulation was assumed to occur,
and on the first day of the experimental period.
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Dose-response study with canine kisspeptin-10 in vivo and in vitro
M. van Elderen
In vivo experimental design and collection of blood samples
A single injection of cKP10 (0.1, 0.5, 1 and 10 μg/kg BW) was given into the vena cephalica to six Beagle
bitches during anestrus at weekly intervals. Blood samples were collected from the jugular vein in
heparinized tubes at 40 and 0 minutes before cKP10 administration and at 10, 20, 30, 40, 60, 90 and 120
minutes after cKP10 administration. Individual plasma samples were obtained after centrifugation and
stored at -20°C until assayed. Before and after each collected blood sample respiratory rate, pulse rate and
perfusion indicators (mucous membrane color and capillary refill time) were measured.
Hormone assays
Plasma progesterone concentrations were measured using a 125I-radioimmunoassay (RIA) validated for
the dog (Risvanli et al. 2010, Poling, Kauffman 2012, Garcia-Galiano, Pinilla & Tena-Sempere 2012). The
intra-assay and interassay coefficients of variation (CVs) were 6% and 10.8%, respectively. The lower
limit of quantitation was 0.15 nmol/L.
Plasma LH concentrations were measured with a heterologous RIA as described previously (Nett et al.,
1975). The intra-assay and interassay CVs for values above 0.5 µg/L were 2.3% and 10.5%, respectively,
and the limit of quantitation was 0.3 μg/L.
Peptide
cKP10 (YNWNVFGLRY-NH2) was synthesized by American Peptide Company (Sunnyvale, USA) and
dissolved in sterile 0.09% NaCl and stored at -20°C.
Data analysis
For each dog, basal plasma LH concentrations were calculated as the mean of the values in the samples
collected at 40 and 0 minutes before cKP10 administration. The 95 percentile of all plasma LH
concentrations at T=-40 and T=0 was determined. Nonparametric test were used, because the plasma LH
data were not normally distributed. A Wilcoxon signed rank test and a Friedman test were used to
determine differences in plasma LH concentrations before and after cKP10 administration. A Friedman
test was used to determine differences between the different doses of cKP10. P < 0.05 was considered
statistically significant. Statistical analysis was performed using IBM SPSS for Windows, version 20.
Ethics of experimentation
The experimental protocol was approved by the Ethics Committee of the Faculty of Veterinary Medicine,
Utrecht University, The Netherlands.
In vitro experiments to compare [Ca2+]i response to hKP10 and cKP10 after binding to human
GPR54
Chemicals and peptides. Human KP10 was produced by Eurogentec (Maastricht, The Netherlands) and
cKP10 was produced by American Peptide Company (Sunnyvale, USA). Both peptides were dissolved in
saline and stored at -20°C. A saline solution, called external medium, was prepared with deionized water
(Milli-Q®; resistivity >10 MΩ∙cm) obtained from Millipore (Bedford, MA) and contained (in mM) 125
NaCl, 5.5 KCl, 2 CaCl2, 0.8 MgCl2, 10 HEPES, 24 glucose, and 36.5 sucrose at pH 7.3 (adjusted with NaOH).
Ethylenediaminetetraacetic acid (EDTA; 17mM) and stock solutions of 2 mM ionomycin in
dimethylsulfoxide (DMSO) were kept at -20°C and thawed before the experiment. NaCl, KCl and HEPES
were obtained from Merck (Whitehouse Station, NJ, USA); MgCl 2, CaCl2, glucose, sucrose and NaOH were
obtained from BDH Laboratory Supplies (Poole, UK). Fura-2 AM was obtained from Molecular Probes
(Invitrogen, Breda, The Netherlands). All other chemicals were obtained from Sigma-Aldrich (St. Louis
MO, USA), unless otherwise noted. Stock solutions were prepared just prior to the experiments.
Cell culture. Chem-1 cells, an adherent cell line, transfected with full-length human GPR54 cDNA were
obtained from Millipore (Billerica, MA, USA) and were frozen in liquid nitrogen at 2∙106 cells/ml in DMEM
with 20% fetal bovine serum, 100 U/ml penicillin and streptomycin, and 10% DMSO upon receipt. Cell
line tests were negative for mycoplasma. After reception, the Chem-1 cells were thawed in a 37°C water
bath. After the ice had thawed, the exterior of the vial was sterilized with 70% ethanol and the cells were
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Dose-response study with canine kisspeptin-10 in vivo and in vitro
M. van Elderen
transferred to a T75 flask containing 20 mL growth medium (Opti-MEM® supplemented with 4% fetal
calf serum and 1% glutamine 200 mM) obtained from Life Technologies (Bleiswijk, The Netherlands) and
placed in a humidified 37°C incubator with 5% CO2 for two days to recover. For Ca2+ imaging experiments,
the cells were detached with 0.05% Trypsin-EDTA (Life Technologies) and subcultured in 35 mm
diameter uncoated glass-bottom dishes (MatTek, Ashland, MA) in 2 mL growth medium at a density of 5 ∙
105 cells/dish and placed in a 37°C incubator with 5% CO2. Trypsinization was done by first washing the
cells with 10 mL Dulbecco’s Phosphate Buffered Saline (DPBS, obtained from Life Technologies) and then
incubating the cells with 0.05% trypsin/0.2 g/L EDTA (1 mL/T75) for 5-10 minutes at RT. After
incubation, the cells were dislodged by rapping the side of the flask and the cells were observed under the
microscope. When the cells were rounded and loosen they were transferred to a culture tube and growth
medium with serum was added to neutralize the trypsin. The cells were centrifuged at 1500-2000 RPM for
15 minutes at ambient temperature and the supernatant was removed carefully and replaced with fresh
medium. The cells were counted in a hemocytometer and transferred to glass-bottom dishes as described
above. Ca2+ imaging experiments were performed 1 day after subculture.
Ca2+ imaging. To measure changes in the intracellular Ca2+ concentration ([Ca2+]i) a Ca2+-sensitive
fluorescent ratio dye Fura-2 AM was used. Briefly, cells were incubated with 5 μM Fura-2 AM in external
medium for 20 minutes at room temperature (RT), followed by 15 minutes de-esterification in external
medium at RT. After de-esterification the cells were placed on the stage of an Axiovert 35M inverted
microscope (Zeiss, Göttingen, Germany) equipped with a TILL Photonics Polychrome IV (TILL Photonics
GmBH, Gräfelfing, Germany). Fluorescence, evoked by 340 and 380 nm excitation wavelengths (F340 and
F380), was collected at 510 nm with an Image SensiCam digital camera (TILL Photonics GmBH). This
digital camera and polychromator were controlled by imaging software (TILLvisION, version 4.01), which
was also used for data collection and processing. The F340/F380 ratio (R), which is a qualitative measure
for [Ca2+]i, was collected every 6 seconds. Changes in R were further analyzed using custom-made MSExcel macros and included background correction. The recording of each experiment lasted 45 minutes.
After 5 minutes baseline recording with external medium, cells were exposed to hKP10 (10-6,10-8 or 10-10
M) or cKP10 (10-6,10-8 or 10-10 M) for 18 seconds. At the end of the recording maximum and minimum
ratios (Rmax and Rmin) were determined by addition of 5μM ionomycin and 17 mM EDTA respectively as a
control for the experimental conditions.
Data analysis and Statistics All data are presented as mean [Ca2+]i ± standard error of the mean (SEM)
from the number of cells (n) indicated, obtained from 1-3 independent experiments (N) per dose. For each
cell, basal intracellular calcium concentrations were calculated as the mean over the first 5 minutes.
Furthermore, the maximum amplitude of [Ca2+]i after KP10 exposure was calculated. To determine
whether all basal[Ca2+]i were equal, a one-way ANOVA was used. A Paired Samples T-test was used to
determine differences between basal [Ca2+]i and the maximum amplitude of [Ca2+]i after KP10 exposure
for both hKP10 and cKP10. For each dose, an Independent Samples T-test was used to determine
differences in the maximum amplitude of [Ca2+]i after hKP10 and cKP10 exposure. Furthermore, for both
hKP10 and cKP10, a one-way ANOVA was performed to determine differences in the maximum amplitude
of [Ca2+]i between doses. Significant main effects were followed up by Bonferroni corrected pairwise
comparisons. P < 0.05 was considered statistically significant. Statistical analysis was performed using
IBM SPSS for Windows, version 20.
RESULTS
In vivo dose-response study with cKP10
The 95 percentile of all plasma LH concentrations at T=-40 and T=0 was 3.88. A Wilcoxon signed rank test
indicated that administration of all different doses of cKP10 resulted in a significant increase of the plasma
LH concentration at T=10 (Table 1A and Figure 1). A Friedman test indicated that there was no significant
difference in plasma LH concentrations between all the moments before and after 0.1 μg/kg BW cKP10
administration. Plasma LH concentrations at 10 minutes after 0.1 μg/kg BW cKP10 administration were
significantly lower compared to the plasma LH concentrations at 10 minutes after 0.5, 1, and 10 μg/kg BW
cKP10 administration (P=0.03). The plasma LH concentrations at T=10 did not differ significantly
between 0.5, 1, and 10 μg/kg BW cKP10 (Table 1B). For 0.5, 1 and 10 μg/kg BW cKP10 plasma LH
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Dose-response study with canine kisspeptin-10 in vivo and in vitro
M. van Elderen
concentrations at T=10 were significantly higher than plasma LH concentrations at 20, 30, 40, 60, 90 and
120 minutes after cKP10 administration (P=0.02). For 0.1 μg/kg BW cKP10 the plasma LH concentration
at T=10 was significantly higher than the plasma LH concentration at T=30 (P=0.03). After 0.1 μg/kg BW
cKP10 administration basal LH levels were reached again at T=20, after 1 μg/kg at T=90 and after 0.5
μg/kg and 10 μg/kg BW cKP10 administration at T=120. The median plasma LH concentrations at T=10
after administration of 0.5, 1, and 10 μg/kg BW cKP10 were higher than the 95 percentile of the measured
basal plasma LH concentrations (Figure 2). The median plasma LH concentrations at T=10 after
administration of 0.5, 1 and 10 μg/kg BW cKP10 (14.3 μg/L) were 6.5 times larger than the median basal
plasma LH concentration (2.2 μg/L). For 0.1 μg/kg BW cKP10 this median plasma LH concentration (3.0
μg/L) was 1.3 times larger than the median basal plasma LH concentration (2.3 μg/L) and was lower than
the 95 percentile of the measured basal plasma LH concentrations. Respiratory rate, pulse rate and
perfusion indicators remained stable in all dogs throughout the experimental period (data not shown).
In vitro Ca2+ imaging experiments with hKP10 and cKP10
One of the results is presented in Figure 3 as a typical example of the [Ca2+]i. For each dose, the results of
one experiment are presented in Figure 4. A one-way ANOVA indicated that there were significant
differences between the basal [Ca2+]i (F (5,146) = 8.74, P < 0.001). For all doses, the maximum amplitude
of [Ca2+]i was significantly higher compared to the basal [Ca2+]i after hKP10 and cKP10 exposure (P <
0.001)(Table 2A). The maximum amplitude of [Ca2+]i after hKP10 exposure was significantly different
from cKP10 exposure only for the 10-8 M dose (P < 0.001, 95% CI [-1737 -749])(Table 2B). Significant
differences between doses were only found after cKP10 exposure (Table 2C); the 10-10 M dose differed
significantly from both the 10-6 M and 10-8 M dose (P < 0.001, 95% CI [-1337 -451] and [-1981 -808]
respectively).
DISCUSSION
In vivo dose-response study
The purpose of this study was to investigate the minimal dose of intravenously administered cKP10 in
adult female dogs in anestrus that results in a significant LH response above the 95 percentile of the
measured basal plasma LH concentrations. One dog (0.1 μg/kg BW cKP10) had a basal plasma LH
concentration that exceeded this 95 percentile, probably due to pulsatile LH secretion, and was excluded
from the statistical analysis. The results showed that a statistically significant increase in plasma LH
concentrations was reached at 10 minutes after administration of all doses of cKP10. These findings
explain that all doses are effective, while the lowest dose is 10 times lower than the lowest doses used in
the study of Albers-Wolthers et al. (2013). However, a Friedman test indicated that there was no
significant difference in plasma LH concentrations between all blood samples collected before and after
0.1 μg/kg BW cKP10 administration. Furthermore, there was a significant difference in plasma LH
concentrations at 10 minutes after cKP10 administration between 0.1 μg/kg BW cKP10 and the three
highest doses of cKP10 and the median plasma LH concentration at T=10 after 0.1 μg/kg BW cKP10
administration was 1.3 times larger than the median basal plasma LH concentration. For the other doses
this was 6.5 times larger. 0.1 μg/kg BW cKP10 administration did not result in an increase above the 95
percentile of the measured basal plasma LH concentrations. According to these data we suggest that 0.5
μg/kg BW cKP10 (i.v. administration) is more effective than 0.1 μg/kg BW cKP10 and is the lowest dose
that gives an increase in plasma LH concentrations above the 95 percentile of the measured basal plasma
LH concentrations. In men the peripherally administered lowest effective dose was 0.1 μg/kg BW hKP10,
in ovariectomized cows 0.13 μg/kg BW hKP10 and in ewes 0.16 μg/kg BW hKP10 (George et al. 2011,
Whitlock et al. 2008, Caraty et al. 2007). In male rats, goats, prepubertal heifers, calves, prepubertal gilts
and monkeys the lowest peripherally administered doses were respectively 0.39, 1, 4.8, 5, 13.4-16.6 and
25.6-34.5 μg/kg hKP10 and were all effective ( Tovar et al. 2006, Hashizume et al. 2010, Ezzat Ahmed et al.
2009, Shahab et al. 2005, Kadokawa et al. 2008, Lents et al. 2008). However, only George et al. (2011),
Whitlock et al. (2008) and Caraty et al. (2007) did a dose-response study with lower non-effective doses.
Therefore, in the other mammals lower doses may also be effective.
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Dose-response study with canine kisspeptin-10 in vivo and in vitro
M. van Elderen
There was no significant difference in plasma LH concentrations at T=10 after cKP10 administration
between the three highest doses of cKP10, which suggests that a maximum LH response had been reached
after administration of 0.5 μg/kg BW. cKP10. Furthermore, Albers-Wolthers et al. (2013) showed that
there was no significant difference in plasma LH concentrations at T=10 after cKP10 administration
between all the doses (1-100 μg/kg). This does not support a dose-dependent increase in plasma LH
concentrations in response to cKP10 administration in the dog. However, analysis of Figure 1 and 2
suggest that there is a dose-dependent increase in plasma LH concentrations in response to cKP10
administration. Furthermore, Albers-Wolthers et al. (2013) showed that the median increase in plasma LH
concentration after administration of higher doses of cKP10 (1, 5, 10, 30, 50 and 100 μg/kg) was slightly
more than 10-fold, while the median increase after 0.5, 1 and 10 μg/kg BW cKP10 was 6.5-fold. Plasma LH
concentrations due to pulsatile LH secretion in Beagle bitches in anestrus can be 10- to 20-fold higher
than basal plasma LH concentrations (Kooistra et al. 1999, Beijerink et al. 2007). George et al. (2011)
showed a dose-dependent rise in serum LH concentration in men. However, before this can be concluded
in dogs, more research is necessary and doses between 0.1 and 0.5 μg/kg BW cKP10 must be
administered to dogs in anestrus to determine the rise in plasma LH concentrations.
In vitro Ca2+ imaging experiments
The purpose of this study was to compare the [Ca2+]i response to hKP10 and cKP10 after binding to the
human kisspeptin receptor GPR54 in vitro. The results showed that there were significant differences
between the basal [Ca2+]i for both hKP10 and cKP10. This can be due to differences in the experimental
setup between experiments. For example differences in the homogeneity of the cells, in room
temperature, in the flow of external medium over the cells and in time of incubation of the cells with Fura2 AM. Based on these results, the significant differences between maximum [Ca 2+]i amplitudes are less
reliable and the interpretation is more difficult. To get more reliable results, the experimental setup must
be the same between different experiments and for each dose more experiments must be performed.
When there are no significant differences between maximum amplitudes of [Ca2+]i for different doses,
there can be significant differences in [Ca2+]i after the amplitude. It is possible that a high dose of KP10 is
toxic or harmful to a cell. The cell will react on KP10 exposure, but will not return to basal [Ca2+]i or the
cell will oscillate. In a figure as Figure 3 this toxic effect can be seen as a messy image where the lines lie
further upwards after the maximum amplitude and are not perfectly horizontal, but show random peaks.
In Figure 4 this toxic effect can be seen after exposure of 10-6 and 10-8 hKP10 and cKP10 to the cells.
Conclusions
In this study we have conclusively demonstrated that 0.5 μg/kg BW cKP10 is the lowest dose that
significantly increases the plasma LH concentration above the 95 percentile of the measured basal plasma
LH concentrations in the dog and that hKP10 and cKP10 give a similar [Ca2+]i response after binding to the
human kisspeptin receptor GPR54 in vitro.
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Dose-response study with canine kisspeptin-10 in vivo and in vitro
M. van Elderen
Fig. 1. Median plasma LH concentrations in response to intravenous administration of 0.1, 0.5, 1 and 10
μg/kg BW cKP10 (T=0) to Beagle bitches (n=5 for 0.1 μg/kg; n=6 for 0.5, 1 and 10 μg/kg).
Fig. 2. Boxplots of LH concentrations 10 minutes after 0.1, 0.5, 1 and 10 μg/kg BW cKP10 administration
to Beagle bitches (n=5 for 0.1 μg/kg; n=6 for 0.5, 1 and 10 μg/kg). The horizontal line corresponds with
the 95 percentile of all plasma LH concentrations at T=-40 and T=0 (3.88).
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Dose-response study with canine kisspeptin-10 in vivo and in vitro
M. van Elderen
Fig. 3. [Ca2+]i before and after 10-10 hKP10 exposure. The mean [Ca2+]i over the first 5 minutes was taken as
the basal value and the maximum amplitude of [Ca2+]i after KP10 exposure was calculated for each dose.
Fig. 4. F340/F380 ratio (R) before and after KP10 exposure.
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Dose-response study with canine kisspeptin-10 in vivo and in vitro
M. van Elderen
Table 1. Exact Significances (1-tailed). A. Wilcoxon signed rank test. B. Friedman test.
Table 2. A. Paired Samples T-test: differences between basal [Ca2+]i and the maximum amplitude of [Ca2+]i.
B. Independent Samples T-test: differences between hKP10 and cKP10 exposure. C. One-way ANOVA:
differences between doses.
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Dose-response study with canine kisspeptin-10 in vivo and in vitro
M. van Elderen
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