Kisspeptin and Its Receptor Are Expressed in Canine Trophoblast Cells

In humans, placental kisspeptin contributes to the regulation of implantation. During a previous study, we detected the kisspeptin and -receptor gene in the canine placenta. The aim of this study was to assess the protein expression in uterine and placental cells. Tissues were collected from bitches after ovariohysterectomy and assigned to 4 groups: preimplantation, day 10-12, n=9; post-implantation, day 18-25, n=13; mid-gestation, day 30-40, n=7; early metestrus, day 10-12, non-pregnant, n=3. Immunohistochemistry was performed by using the streptavidin-biotin peroxidase complex technique for detection of kisspeptin (kiss), kisspeptin receptor (kissR), pan-Cytokeratin and Vimentin. Serial sections were prepared and incubated with each of the primary antibodies (kisspeptin10, Rabbit PAb, Merck Millipore; Kiss1R/GPR54, Rabbit PAb, Novus Biologicals). Sections were then incubated with biotin-conjugated second step antibody (Zymed Laboratories, Inc.; San Fransisco, CA, USA) and thereafter with streptavidin-peroxidase complex (Zymed Laboratories). Labelling was ‘visualized’ with aminoethyl carbazole (AEC substrate kit; Invitrogen, Camarillo, Canada). Sections were counterstained with Gill's haematoxylin. Negative controls were incubated with normal serum instead of primary antibody. The immunoreactivity was evaluated semi-quantitatively at x200 magnification over an area of 0.050 mm2 and the percentage of positively stained cells assessed. The labelling intensity was quantified using a four-point scale: 0=no labelling; 1=weak; 2=moderate; 3=intense labelling. An immunoreactivity score (IRS) was obtained by multiplying the labelling intensity score (0-3) with the percentage of immunolabelled cell types investigated. The IRS ranged fron 0 to 300. The statistical significance was analysed by Wilcoxon test using PASW-Statistic 18 (SPSS, IBM). Homogeneity of groups were determined by the Shapiro-Wilk test. The confidence interval was determined as P<0.05. All values are expressed as mean ± SE. The kiss and kissR signals varied among groups and individuals. Labeling for kiss in the vascular endothelial cells and tunica media of larger vessels were present in the preimplantation period only (IRS:33). KissR staining of vessels was recorded in all gestational periods including non-pregnant uteri with highest IRS score in the postimplantation period (IRS:53). In general, luminal (surface) epithelial cells were negative for both kiss and kissR with the exception of kiss in the controls (IRS:100). Staining for kiss was more intense in the superficial uterine glands of the non-pregnant than the pregnant uterus; expression dimished following implantation. KissR staining was negative in the superfical uterine glands in all groups. Deep uterine glands stained negative or weak for both kiss and kissR. IRS for kiss was more pronounced in the non-pregnant uterus (IRS:26) compared to all gestational periods investigated. In myocytes, kiss expression was significantly higher in midgestation than in the preimplantation period (in the deep glandular layer P<0.05; sponge zone P<0.01). For kissR, the highest IRS scores were seen in mid-gestation (IRS:70) and post-implantation (IRS:56). In the placental labyrinth, fetal trophoblast cells, predominantly syncytiotrophoblasts, stained weak to moderately for kiss in the post-implantation (IRS:22) and mid-gestation (IRS:29) periods. A similar localization and staining intensity was observed in the post-implantation period for kissR (IRS:24).