Gastrin-releasing peptide (GRP) in the ovine uterus: regulation by interferon tau and progesterone

Abstract

Gastrin-releasing peptide (GRP) is abundantly expressed by endometrial glands of the ovine uterus and processed into different bioactive peptides, including GRP1-27, GRP18-27, and a C-terminus, that affect cell proliferation and migration. However, little information is available concerning the hormonal regulation of endometrial GRP and expression of GRP receptors in the ovine endometrium and conceptus. These studies determined the effects of pregnancy, progesterone (P4), interferon tau (IFNT), placental lactogen (CSH1), and growth hormone (GH) on expression of GRP in the endometrium and GRP receptors (GRPR, NMBR, BRS3) in the endometrium, conceptus, and placenta. In pregnant ewes, GRP mRNA and protein were first detected predominantly in endometrial glands after Day 10 and were abundant from Days 18 through 120 of gestation. Treatment with IFNT and progesterone but not CSH1 or GH stimulated GRP expression in the endometrial glands. Western blot analyses identified proGRP in uterine luminal fluid and allantoic fluid from Day 80 unilateral pregnant ewes but not in uterine luminal fluid of either cyclic or early pregnant ewes. GRPR mRNA was very low in the Day 18 conceptus and undetectable in the endometrium and placenta; NMBR and BRS3 mRNAs were undetectable in ovine uteroplacental tissues. Collectively, the present studies validate GRP as a novel IFNT-stimulated gene in the glands of the ovine uterus, revealed that IFNT induction of GRP is dependent on P4, and found that exposure of the ovine uterus to P4 for 20 days induces GRP expression in endometrial glands.

FIG. 1.

In situ hybridization analysis of GRP mRNA in uteri of cyclic and pregnant ewes. Cross sections of the uterine wall from cyclic (C) and pregnant (P) ewes were hybridized with radiolabeled antisense or sense ovine GRP cRNAs. Note that GRP mRNA is most abundant in the endometrial glands during pregnancy. LE, Luminal epithelium; sGE, superficial glandular epithelium; GE, glandular epithelium; M, myometrium; S, stroma; Tr, trophectoderm. Bar = 25 μm.

FIG. 2.

In situ hybridization analysis of GRP mRNA in uteri of late pregnant ewes. Cross sections of the uterine wall and placentomes of pregnant (P) ewes were hybridized with radiolabeled antisense or sense ovine GRP cRNAs. Note that GRP mRNA is detected only in the endometrial glands. Bar = 10 μm.

FIG. 3.

Immunohistochemical localization of GRP protein in uteri from cyclic and pregnant ewes. A) Immunoreactive GRP protein was localized using a rabbit anti-porcine GRP polyclonal antibody. Note the presence of GRP protein in secretory vesicles (*). In the IgG control, normal rabbit IgG was substituted for rabbit polyclonal antibody to porcine GRP. Sections were not counterstained. B) Immunoreactive GRP protein was observed predominantly near the apical surface of endometrial glandular epithelium and on endometrial LE of uteri from Day 20 pregnant ewes. Sections were not counterstained. LE, Luminal epithelium. Bars = 25 μm (A); 2.5 μm (B).

FIG. 4.

Immunohistochemical localization of GRP protein in uterine wall and placentomes from late pregnant ewes. In the IgG control, normal rabbit IgG was substituted for rabbit polyclonal antibody to porcine GRP. Note the abundant levels of GRP protein in the endometrial glandular epithelium. Sections were not counterstained. Bar = 10 μm.

FIG. 5.

Effects of progesterone during early pregnancy on GRP mRNA and protein in the ovine uterus. A) Experimental design. See Materials and Methods for complete description. B) Steady-state levels of GRP mRNA in endometria determined by slot-blot hybridization analysis. Endometrial GRP mRNA levels were not different (P > 0.10) between CO- and P4-treated ewes on Day 9 but was about 23-fold higher (P < 0.01) in P4- than CO-treated ewes on Day 12. The asterisk denotes an effect of treatment (**P < 0.01). C) In situ hybridization and immunohistochemical analyses of GRP mRNA and protein expression. Cross sections of the uterine wall from treated ewes were hybridized with radiolabeled antisense or sense ovine GRP cRNA probes. Note the abundance of GRP mRNA and protein in endometrial glandular epithelium of P4+IFN-treated ewes. Sections for immunohistochemical localization were not counterstained. Hystx, Hysterectomy; CO, corn oil; P4, progesterone; INFN, interferon tau; RU, relative units; SE, standard error of the mean (SEM). Bar = 25 μm.

FIG. 6.

Effects of progesterone and IFNT on GRP mRNA and protein in the ovine uterus. A) Experimental design. See Materials and Methods for complete description. B) Steady-state levels of GRP mRNA in endometria were determined by slot-blot hybridization analysis. Treatment of ewes with ZK 136,317, PGR antagonist, did not affect (P > 0.10, P4+CX vs. P4+ZK+CX) endometrial GRP mRNA abundance. For ewes receiving P4 alone, intrauterine IFNT increased steady-state levels of GRP mRNA 3-fold in endometria (P < 0.05, P4+CX vs. P4+IFN). The asterisk (*) denotes an effect of treatment (P < 0.05). C) In situ hybridization and immunohistochemistry analyses of GRP mRNA expression. Cross sections of the uterine wall from treated ewes were hybridized with radiolabeled antisense or sense ovine GRP cRNA probes. Immunoreactive GRP protein in the uterus. Sections were not counterstained. CX, Control serum proteins; Hystx, hysterectomy; Ovx/Cath, ovariectomy and uterine catheterization; IFNT, recombinant ovine interferon tau; ZK, ZK137,316 anti-progestin. Bar = 25 μm.

FIG. 7.

Effects of long-term treatment with progesterone and IFNT on endometrial GRP mRNA. A) Experimental design (see Materials and Methods for complete description of experimental design). B) In situ hybridization analysis of GRP mRNA and immunohistochemistry of immunoreactive GRP protein in the uterus. C) Quantification of GRP mRNA in uterine endometrial glands. Intrauterine administration of IFNT increased GRP mRNA in uterine endometrial glands of P4-treated ewes about threefold (*P < 0.05) but not in P4+ZK-treated ewes (P > 0.10, P4+ZK+CX vs. P4+ZK+IFN). Treatment of ewes with the progesterone receptor antagonist decreased GRP mRNA abundance by fourfold in the endometrial glands (P < 0.01, P4+CX vs. P4+ZK+CX). Data are expressed as LSM (least squares means) and SEM for optical intensity. **P < 0.01. Bar = 10 μm.

FIG. 8.

Effects of intrauterine infusion of placental lactogen and growth hormone (GH) on endometrial GRP mRNA. A) Experimental design (see Materials and Methods for complete description of experimental design). B) In situ hybridization analysis of GRP mRNA and immunohistochemistry of immunoreactive GRP protein in the uterus. Bar = 10 μm. C) Quantification of GRP mRNA in the endometrial glands of uteri. Intrauterine administration of neither CSH1 nor GH affected (P > 0.01) GRP mRNA abundance in endometrial glands of P4 and IFNT treated ewes. Data are expressed as LSM and SEM for optical intensity. CSH1, Placental lactogen.

FIG. 9.

Analysis of immunoreactive GRP protein in allantoic fluid, amniotic fluid, and uterine luminal fluid from Day 80 unilaterally pregnant ewes. Proteins were separated by 15% SDS-PAGE under reducing conditions. Western blot analysis detected a single immunoreactive protein of ∼13 kDa in uterine luminal fluid and allantoic fluid from Day 80 unilaterally pregnant ewes but not in amniotic fluid. Positions of prestained molecular weight standards (×10⁻³) are indicated.

FIG. 10.

RT-PCR analysis of GRPR mRNA in ovine uteroplacental tissues. Representative analysis of Day 18 conceptus (lane 1), Day 18 pregnant endometrium (lane 2), Day 45 cotyledonary placenta (lane 3), and Day 45 intercotyledonary placenta (lane 4). Note the presence of GRPR mRNA only in the Day 18 conceptus, whereas actin beta (ACTB) mRNA was observed in all samples. M, 100-bp marker.