Table 1.
List of primers used for real time (TaqMan) PCR.
Fig 1.
Expression of (A) RLN, (B) RXFP1 and (C) RXFP2 in canine CL at selected time points of pregnancy.
Data are presented as geometric means Xg ± SD. (★) = P<0.01, (★★) = P<0.001.
Fig 2.
Antigestagen (aglepristone) effects on luteal expression of the RLN system (A) RLN, (B) RXFP1 and (C) RXFP2), as determined by real time (TaqMan) PCR.
Data are presented as geometric means Xg ± SD. Bars with different letters differ at P<0.01 compared to the mid-gestation group as a non-treated control.
Fig 3.
Representative microphotographs of immunohistochemical (IHC) localization of RLN in the canine CL at selected time points of pregnancy.
Pre-implantation (A), post-implantation (B), mid-gestation (C) and at prepartum luteolysis (D). RLN is localized to the lutein cells (open arrows in (A–D)). There is no background staining in the isotype control (insert to (D)).
Fig 4.
Representative microphotographs of immunohistochemical (IHC) localization of RXFP1 in the canine CL at selected time points of pregnancy.
Pre-implantation (A), post-implantation (B), mid-gestation (C) and at prepartum luteolysis (D). RXFP1 is localized to the luteal cells (open arrows in (A-D)), endothelial cells (solid arrows in A and C) and macrophages (open arrowheads in (A-D)). There is no background staining in the isotype control (insert to (D)).
Fig 5.
Representative microphotographs of immunohistochemical (IHC) localization of RXFP2 in the canine CL at selected time points of pregnancy.
Pre-implantation (A), post-implantation (B), mid-gestation (C) and at prepartum luteolysis (D); consecutive sections were stained against MHCII (E) and RXFP2 (F). While only weak RXFP2 signals were found in luteal cells from pre-implantation until mid-gestation, during prepartum luteolysis clearly detectable staining is present in these cells (open arrows in A-D). The detection of MHCII was performed in order to more easily differentiate cell types within canine CL. Accordingly, RXFP2 was found in interstitial cells, mostly those localized close to blood vessels expressing MHCII that were identified as macrophages (black arrows in E and F, open arrowheads in A-D). There is no background staining in the isotype control (insert to (D)).
Fig 6.
Isolation of hypophysis and adehohypophyseal expression of RLN-system.
Representative photographs of opened skull of a dog before (A, dorsal view and B, dorsocaudal view), and after (C, dorsocaudal view) removal of the brain including the hypophysis (D). The hypophysis was localized in the saddle-shaped depression of the sphenoid bone, so-called Turkish saddle (Sella turcica). Proximally, directly above the pituitary gland, the optic chiasm can be localized. Parts of the optic chiasm are attached to the isolated hypophysis (D). E) Expression of RLN, RXFP1, RXFP2 and GAPDH in canine adenohypophysis by conventional, qualitative PCR. The mRNA of all RLN system members was detectable, with RLN and RXFP2 signals appearing weaker than those for RXFP1.
Fig 7.
Hematoxylin-eosin (HE) staining (A, D) and immunolocalization of PRL (B, E) and RLN system (C, F, G) in canine adenohypophysis (two series of consecutive sections are presented: A-C and D-G).
HE staining was performed in order to distinguish basophilic (solid arrows in A and D) and acidophilic hypophyseal cells (open arrows in A and D). The latter include PRL-secreting cells, so-called lactotrophs, identified additionally by IHC with anti-PRL antibody (open arrows in B and E). RLN signals were observed in lactotrophs (open arrows in C) and in other hypophyseal cells, which remained negative for PRL (solid arrows in C). RXFP1 and RXFP2 stained extensively in hypophyseal cells with signals appearing more prominent for RXFP1 than RXFP2 (open arrows in F and G, respectively). There is no background staining in the isotype controls (H, I, J, K).
Fig 8.
A schematic model depicting proposed aspects of autocrine, paracrine and endocrine functions of RLN in canine reproductive tissues, emphasizing its role as a possible luteotropic factor.
(1) Local, luteal RLN acts in an auto/paracrine manner on steroidogenic, endothelial and immune cells. (2) Circulating RLN of placental origin acting as an endocrine factor. (3) Adenohypophysis, as a target of placental RLN during pregnancy. Additionally, locally produced hypophyseal RLN may exert auto/paracrine effects on pituitary cells (4); possible interactions of RLN with production and secretion of other hypophyseal hormones, including PRL are suggested. (5) PRL originating from the hypophysis is the main luteotropic hormone for mature canine CL [25]. Increased PRL levels observed following emergence of RLN (6) in circulation imply an indirect luteotropic function of RLN.