Interplacental uterine expression of genes involved in prostaglandin synthesis during canine pregnancy and at induced prepartum luteolysis/abortion

Abstract

Background: In the non-pregnant dog, ovarian cyclicity is independent of a uterine luteolysin. This is in contrast to pregnant animals where a prepartum increase of luteolytic PGF2α occurs, apparently originating in the pregnant uterus. Recently, the placenta as a source of prepartum prostaglandins (PGs) was investigated, indicating fetal trophoblast cells as the likely main source. However, the possible contribution of uterine interplacental tissues to the production of these hormones has not yet been thoroughly examined in the dog.

Methods: Several key factors involved in the production and/or actions of PGs were studied: cyclooxygenase 2 (COX2, PTGS2), PGF2α-synthase (PGFS/AKR1C3), PGE2-synthase (PGES), and the respective receptors FP (PTGFR), EP2 (PTGER2) and EP4 (PGTER4), 15-hydroxyprostaglandin dehydrogenase (HPGD), PG-transporter (PGT, SLCO2A1) and progesterone receptor. Their expression and localization patterns were assessed by Real Time PCR and immunohistology in the interplacental uterine sites from pregnant dogs during the pre-implantation period (days 8-12), post-implantation (days 18-25), mid-gestation (days 35-40) and during antigestagen-induced luteolysis/abortion.

Results: Whereas only low COX2 expression was observed in uterine samples at all the selected time points, expression of PGFS/AKR1C3 strongly increased post-implantation. A gradual increase in PGES-mRNA expression was noted towards mid-gestation. FP-mRNA expression decreased significantly with the progression of pregnancy until mid-gestation. This was associated with clearly detectable expression of HPGD, which did not change significantly over time. The expression of FP and EP2-mRNA decreased significantly over time while EP4-mRNA expression remained unaffected. The antigestagen-treatment led to a significant increase in expression of COX2, PGES, EP2 and PGT (SLCO2A1) mRNA. COX2 was localized predominantly in the myometrium. The expression of PGFS/AKR1C3, which was unchanged, was localized mostly to the surface luminal epithelium. The expression of EP4, PGT and HPGH did not change during treatment, they were co-localized with PGES and EP2 in all uterine compartments.

Conclusions: The data clearly demonstrate the basic capability of the canine pregnant uterus to produce and respond to PGs and suggests their functions both as local regulatory factors involved in the establishment and maintenance of pregnancy, as well as potential contributors to the process of parturition, supporting the myometrial contractility associated with fetal expulsion.

Figure 1

Expression of cyclooxygenase 2 (COX2, PTGS2), prostaglandin F2α synthase (PGFS) and of prostaglandin E2 synthase (PGES). Expression of COX2 (PTGS2), PGFS/AKR1C3 and PGES was determined by Real Time (TaqMan) PCR (mean ± SD) in the interplacental sites of canine uterus from the pre-implantation period until mid-gestation (A,C,E) and during Aglepristone®-induced luteolysis/abortion (B,D,F, compared with the mid-gestation group as a non-treated control). Bars with different asterisks differ either at P < 0.01 in (C) and (E), or at P < 0.05 in (B) and (F).

Figure 2

Expression of PGF2α receptor (FP, PTGFR) and of PGE2 receptors, EP2 (PTGER2) and EP4 (PTGER4). Expression of FP (PTGFR) and of EP2 and EP4 (PTGER2 and PTGER4, respectively) was determined by Real Time (TaqMan) PCR (mean ± SD) in the interplacental sites of canine uterus from the pre-implantation period until mid-gestation (A,C,E) and during Aglepristone®-induced luteolysis/abortion (B,D,F, compared with the mid-gestation group as non-treated control). Bars with different asterisks differ either at P < 0.01 in (A) or at P < 0.05 in (C,D).

Figure 3

Expression of prostaglandin transporter (PGT, SLCO2A1), 15-prostaglandin dehydrogenase (HPGD) and progesterone receptor (PGR). Expression of PGT (SLCO2A1), HPGD and PGR was determined by Real Time (TaqMan) PCR (mean ± SD) in the interplacental sites of canine uterus from the pre-implantation period until mid-gestation (A,C,E) and during Aglepristone®-induced luteolysis/abortion (B,D,F, compared with the mid-gestation group as non-treated control). Bars with different asterisks differ either at P < 0.001 in (F) or at P < 0.05 (A,B,E).

Figure 4

Immunohistochemical localization of cyclooxygenase 2 (COX2, PTGS2) and prostaglandin F2α synthase (PGFS/AKR1C3). Immunohistochemical (IHC) localization of COX2 (PTGS2) at interplacental uterine sites during mid-gestation (A), as well as in the utero-placental compartment (B,C), and interplacental uterine sites (D,E) during Aglepristone® (Agl.)-induced abortion. IHC localization of PGFS/AKR1C3 in the uterus pre-implantation (F), post-implantation (G) and in the utero-placental compartment (H,I) and interplacental uterine sites (J,K) during Aglepristone® (Agl.)-induced abortion. Whereas no or only very weak COX2 IHC signals are observed in uterine endometrium (open arrows = surface luminal epithelium, solid arrowhead = uterine gland), clearly visible staining is localized in myometrium (A). In the utero-placental compartment during Agl.-induced parturition, placental COX2 is localized to the fetal trophoblast cells (solid arrows in B); there are only very weak signals in the uterine glands (solid arrowhead in C), but strong ones are present in myometrium (open arrowhead in C). In the interplacental sites, following Agl. treatment, strong COX2 staining is localized in myometrium (open arrowhead in E), and only very weak staining is present in surface (luminal) uterine epithelium (open arrows in D) and uterine glands (solid arrowheads in D and E). No or only very weak uterine signals are observed for PGFS/AKR1C3 pre-implantation (F). Following implantation, PGFS/AKR1C3 protein is localized in surface (luminal) uterine epithelium and endometrial superficial glands (open and solid arrows in G). In the utero-placental compartment during Agl.-induced parturition, placental PGFS/AKR1C3 protein is localized to the fetal trophoblast cells (solid arrows in H); strong signals are localized also in the superficial endometrial glands, the so-called glandular chambers (open arrows in I). Within the interplacental uterine sites, in Agl.-treated animals, PGFS/AKR1C3 staining is localized predominantly in surface (luminal) uterine epithelium (open arrows in J); the open and solid arrowheads in (K) indicate myometrium and uterine glands, respectively, with barely detectable signals.

Figure 5

Immunohistochemical localization of prostaglandin E2 synthase (PGES), PGE2 receptors EP2 and EP4 (PTGER2 and PTGER4), prostaglandin transporter (PGT, SLCO2A1) and of 15-prostaglandin dehydrogenase (HPGD). Immunohistochemical (IHC) localization of: (A-C) PGES, and (D-F) EP2 (PTGER2) in interplacental uterine sites during mid-gestation and during Aglepristone® (Agl.)-induced abortion. (G) Representative picture of EP4 (PTGER4) expression in interplacental site during Aglepristone® (Agl.)-induced abortion in mid-gestation dog. (H,I) Expression of PGT (SLCO2A1) in interplacental uterine site during mid-gestation and during Aglepristone® (Agl.)-induced abortion. (J-L) Uterine expression of HPGD in interplacental uterine sites during mid-gestation and after Aglepristone® (Agl.) treatment. Open arrows = surface (luminal) uterine epithelium, solid arrowheads = uterine glands, open arrowheads in (C), (F), (K), (L) = myometrium.