Luteal and placental function in the bitch: spatio-temporal changes in prolactin receptor (PRLr) expression at dioestrus, pregnancy and normal and induced parturition

Abstract

Background: Endocrine mechanisms governing canine reproductive function remain still obscure. Progesterone (P4) of luteal origin is required for maintenance of pregnancy. Corpora lutea (CL) are gonadotrop-independent during the first third of dioestrus; afterwards prolactin (PRL) is the primary luteotropic factor. Interestingly, the increasing PRL levels are accompanied by decreasing P4 concentrations, thus luteal regression/luteolysis occurs in spite of an increased availability of gonadotropic support. PRL acts through its receptor (PRLr), the expression of which has not yet been thoroughly investigated at the molecular and cellular level in the dog.

Methods: The expression of PRLr was assessed in CL of non-pregnant dogs during the course of dioestrus (days 5, 15, 25, 35, 45, 65 post ovulation; p.o.) as well as in CL, the utero/placental compartments (Ut/Pl) and interplacental free polar zones (interplacental sites) from pregnant dogs during the pre-implantation, post-implantation and mid-gestation period of pregnancy and during the normal and antigestagen-induced luteolysis. Expression of PRLr was tested by Real Time PCR, immunohistochemistry and in situ hybridization.

Results: In non-pregnant CL the PRLr expression was significantly upregulated at day 15 p.o. and decreased significantly afterwards, towards the end of dioestrus. CL of pregnancy showed elevated PRLr expression until mid gestation while prepartal downregulation was observed. Interestingly, placental but not interplacental expression of PRLr was strongly time-related; a significant upregulation was observed towards mid-gestation. Within the CL PRLr was localized to the luteal cells; in the Ut/Pl it was localized to the fetal trophoblast and epithelial cells of glandular chambers. Moreover, in mid-pregnant animals treated with an antigestagen, both the luteal and placental, but not the uterine PRLr were significantly downregulated.

Conclusions: The data presented suggest that the luteal provision of P4 in both pregnant and non-pregnant dogs may be regulated at the PRLr level. Furthermore, a role of PRL not only in maintaining the canine CL function but also in regulating the placental function is strongly suggested. A possible functional interrelationship between luteal P4 and placental and luteal PRLr expression also with respect to the prepartal luteolysis is implied.

Figure 1

Expression of canine PRLr (1) and GAPDH (2) in anterior pituitary (positive control) and corpus luteum by conventional RT-PCR. The amplified PRLr fragments were used for the cloning procedure.

Figure 2

Time-dependent expression of canine prolactin receptor (PRLr) as determined by Real Time (TaqMan) PCR (mean ± SD). (A) Cyclic CL, day 5-65 after ovulation. (B) CL of pregnancy. (C) Utero/placental compartment throughout pregnancy. Bars with different letters differ either at p < 0.05 (A,B) or at p < 0.01 (C).

Figure 3

Immunohistochemical localization of PRLr in canine CL during pregnancy from the pre-implantation period until prepartal luteolysis and on days 15 and 65 post-ovulation in non-pregnant animals; (open arrowheads) lutein cells.

Figure 4

Expression of PRLr as determined by Real Time (TaqMan) PCR (mean ± SD) in the interplacental polar zones (interplacental sites of uterus) from the pre-implantation period until mid-gestation (A) and during Aglepristone^®- induced luteolysis (B; compared with the mid-gestation group as non-treated control).

Figure 5

Immunohistochemical (IHC) localization of PRLr in the uterus pre-implantation and in the utero/placental compartment. (A, B) IHC localization of PRLr in canine endometrium at the pre-implantation stage of pregnancy. (C, D) IHC localization of PRLr within the utero/placental compartment (mid-gestation). (E, F) Localization of PRLr-mRNA in utero/placental compartment by in situ hybridization (ISH). (A, B) Pre-implantation PRLr is localized to the endometrial surface epithelial cells (solid arrowheads) and glandular epithelial cells of the superficial (open arrowheads) and deep (solid arrows) uterine glands. (C, D) Within the utero/placental compartment signals are localized to the endometrial glands including the superficial glands (solid arrowheads) (the so-called glandular chambers) and to the fetal trophoblast cells (open arrows). (E, F) The same localization pattern was observed at the mRNA-level by ISH. Thus, strong signals are localized to the trophoblast cells (open arrows) including those strongly invading maternal blood vessels at the base of placental labyrinth (open arrows).

Figure 6

The effects of antiprogestin treatment on luteal and placental expression of PRLr as determined by Real Time (TaqMan) PCR. The fold changes in luteal (A) and utero/placental (B) PRLr mRNA expression compared with the mid-gestation group (non-treated control) are shown (mean ± SD). Bars with different asterisks in A differ at p < 0.01. In B "a, b" vs. "c" differ at p < 0.01 and "b" vs. "c" differ at p < 0.05.