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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.
Gastrin-releasing peptide (GRP), a mammalian homologue of bombesin from the amphibian Bombina bombina, was discovered and named after its first known activity of inducing gastrin secretion by porcine gastric tissue [1, 2]. GRP is widely expressed in mammalian organs, including hypothalamus, anterior pituitary, gastrointestinal tract, lung, and pancreas (see  for review) as well as pregnant uteri of sheep, cattle, and humans [3–17]. The inferred amino acid sequence of a GRP cDNA clone from the ovine endometrium suggests a preproprotein that, if processed similarly to human and rat preproGRP , would yield a signal peptide, GRP1-27 with an amidated C-terminus, and a C-terminal extension peptide. Transamidation and endoproteolytic cleavage of GRP1-27 releases the amidated decapeptide GRP18-27, and GRP18-27 is identical to the frog peptide bombesin or neuromedin-C . To date, three known receptors for the N-terminal-derived GRP1-27 and GRP18-27 peptides have been identified in mammals that include GRPR, neuromedin B receptor (NMBR), and BRS3 . GRPR has high affinity for GRP18-27, whereas NMBR has a 2500-fold higher affinity for NMB than GRP18-27 . A naturally occurring high affinity ligand has yet to be identified for BRS3, but BRS3 binds GRP18-27 with low affinity [21, 22].
GRP mRNA has been reported in cyclic and pregnant ovine uteri, and GRP-derived peptides are present in the uterine lumen and allantoic fluid [4, 6, 11–13]. There are no reports on expression of GRP receptors in the ovine uterus, although they are present in the kidneys of fetal and adult sheep . In the human uterus, GRP receptors were found to be expressed in the myometrium, subsets of secretory endometrial glands, and subsets of endometrial blood vessels [10, 24]. Thus, previous studies established that GRP is expressed abundantly by the endometrial glands of ovine uterus and implicated GRP as a candidate regulator of fetal kidney growth and development. In addition to providing histotroph for fetal growth and development, endometrial glands of the uterus synthesize or transport and secrete substances essential for peri-implantation blastocyst survival and growth in sheep, other domestic animals, and humans [25–27]. In the uterine gland knockout (UGKO) ewe, blastocysts fail to survive past Day 14 and do not elongate on Day 12 to form a filamentous conceptus . In addition to eliciting the release of gastrin, GRP can act as a potent mitogen for cancer cells of diverse origin both in vitro and in animal models of carcinogenesis (see ). Other actions of GRP include effects on cell morphogenesis, migration, and adhesion as well as angiogenesis [2, 28]. All those biological actions of GRP are consistent with a role for this protein in peri-implantation conceptus (embryo/fetus and associated extraembryonic membranes) growth in ruminants [29, 30].
Using a custom ovine endometrial cDNA array, we identified GRP as a candidate gene up-regulated in the endometrium of Day 14 pregnant as compared to Day 14 cyclic ewes and stimulated by interferon tau (IFNT) in ewes treated with progesterone (P4) but not regulated by P4 alone . Of particular note, Whitley and coworkers  found that administration of P4 for 10 days to ewes did not affect endometrial GRP mRNA levels and that estrogen and/or estrogen with P4 substantially decreased GRP mRNA levels in endometria of long-term ovariectomized ewes. However, endometrial functions during the peri-implantation period of pregnancy in sheep are regulated largely by P4 from the corpus luteum and cytokines/hormones from the conceptus, such as IFNT, chorionic somatomammotropin one or placental lactogen (CSH1), and growth hormone one (GH) [32, 33]. IFNT is the signal for maternal recognition of pregnancy in ruminants and is produced between Days 10 and 21 through 25 of pregnancy in sheep by the mononuclear trophectoderm cells of the conceptus [33, 34]. The antiluteolytic actions of IFNT are required for maintenance of a functional corpus luteum and continued secretion of P4, the essential hormone of pregnancy . IFNT also induces or stimulates expression of a number of genes, termed IFNT-stimulated genes (ISGs), in a cell-specific fashion within the endometrium, with emerging biological roles in uterine receptivity to conceptus implantation, as well as conceptus growth and development . Physiological studies on hormonal regulation of GRP expression in the ovine uterus have not been reported.
Collectively, available results support the working hypothesis that GRP is expressed by the endometrial glands and stimulated by IFNT, proGRP is processed into different forms that are secreted by the glands into the uterine lumen and absorbed by the conceptus trophectoderm, and proGRP-derived peptides modulate conceptus growth and development. Specific objectives of these studies were to determine 1) effects of pregnancy, P4, and placental hormones (IFNT, CSH1, and GH) on GRP expression in the ovine uterus and 2) if receptors for GRP1-27 and GRP18-27 (GRPR, NMBR, and BRS3) are expressed by the endometrium, conceptus, and placenta. Results of these studies found that IFNT and P4 but not CSH1 and GH stimulated GRP expression in the endometrial glands of the ovine uterus and, surprisingly, that GRPR, NMBR and BRS3 mRNAs are low or undetectable in the conceptus, placenta, or endometrium during pregnancy.
Crossbred Suffolk ewes (Ovis aries) were observed daily for estrus in the presence of vasectomized rams and used in the experiments after they exhibited at least two estrous cycles of normal duration (16–18 days). All experimental and surgical procedures were in compliance with the Guide for the Care and Use of Agriculture Animals in Teaching and Research and approved by the Institutional Animal Care and Use Committee of Texas A&M University.
At estrus (Day 0), ewes were mated to either an intact or vasectomized ram as described previously and then hysterectomized (n = 5 ewes/day) on either Day 10, 12, 14, or 16 of the estrous cycle or Day 10, 12, 14, 16, 18 or 20 of pregnancy. At hysterectomy, several sections (~0.5 cm) from the midportion of each uterine horn ipsilateral to the corpus luteum (CL) were fixed in fresh 4% paraformaldehyde in PBS (pH 7.2). After 24 h, fixed tissues were changed to 70% ethanol for 24 h and then dehydrated and embedded in Paraplast-Plus (Oxford Labware). Several sections (1–1.5 cm) from the middle of each uterine horn were embedded in Tissue-Tek OCT compound (Miles), frozen in liquid nitrogen vapor, and stored at −80°C. The remaining endometrium was physically dissected from myometrium, frozen in liquid nitrogen, and stored at −80°C for subsequent RNA extraction. In monovulatory pregnant ewes, uterine tissue samples were marked as either contralateral or ipsilateral to the ovary bearing the CL. No tissues from the contralateral uterine horn were used for this study. The uterine lumen was flushed with saline on Days 10 through 16 of pregnancy and the estrous cycle, and, in pregnant ewes, the flushing was examined for the presence of a morphologically normal conceptus. It was not possible to obtain uterine flushes on either Day 18 or Day 20 of pregnancy because the conceptus had firmly adhered to the endometrial luminal epithelium (LE) and basal lamina. Uterine flushings were clarified by centrifugation (3000 × g for 30 min at 4°C) and frozen at −80°C for Western blot analysis.
At estrus (Day 0), ewes were mated to an intact ram and then hysterectomized (n = 5 ewes/day) on either Day 40, 60, 80, 100, or 120 of pregnancy (gestation period is 147 days). At hysterectomy, the uterus was trimmed free of cervix and oviduct and opened along the mesometrial border. Several sections (~0.5 cm) of both intercarunucular and placentomal uterine wall regions from the midportion of each uterine horn were fixed in fresh 4% paraformaldehyde in PBS (pH 7.2). Placentomes were then removed by physical dissection, and the remaining intercaruncular endometrium was dissected from the myometrium. Endometrial samples were frozen in liquid nitrogen and stored at −80°C for RNA extraction.
As described previously , ewes were mated at estrus (Day 0) to intact rams and then assigned randomly to receive daily i.m. injections from Days 1.5 through 9 of either corn oil vehicle (CO; n = 6) or 25 mg progesterone (P4; n = 6). All ewes were hysterectomized on Day 9 and the uteri processed as described for study 1. In a complimentary study, ewes were mated and assigned randomly to receive daily i.m. injections of either (1) corn oil vehicle (CO, n = 8) or (2) 25 mg P4 (Sigma Chemical Co.) from Days 1.5 through 12 (n = 7). All ewes were hysterectomized on Day 12 and the uteri processed as described for study 1.
As described previously , cyclic ewes (n = 20) were checked daily for estrus and then ovariectomized and fitted with indwelling uterine catheters on Day 5. Ewes were then assigned randomly (n = 5/treatment) to receive daily i.m. injections of P4 and/or a progesterone receptor (PGR) antagonist (ZK 136,317; generously provided by Dr. Kristof Chwalisz, Schering AG, Germany) and intrauterine (i.u.) infusions of either control (CX) serum proteins and/or recombinant ovine IFNT as follows: (1) 50 mg P4 (Days 5–16) and 200 μg serum proteins (Days 11–16) [P4+CX], (2) P4 and 75 mg ZK 136,317 (Days 11–16) and serum proteins [P4+ZK+CX], (3) P4 and IFNT (2 × 107 antiviral units, Days 11–16) [P4+IFN], or (4) P4 and ZK and IFNT [P4+ZK+IFN]. Steroids were administered i.m. daily in corn oil vehicle. Both uterine horns of each ewe received twice-daily injections of either CX serum proteins (50 μg/horn/injection) or IFNT (5 × 106 antiviral units/horn/injection). Recombinant ovine IFNT was produced in Pichia pastoris and purified as described previously . Serum proteins were prepared for intrauterine injection as described previously . This regimen of P4 and IFNT mimics the effects of P4 and IFNT from the CL and conceptus, respectively, on endometrial expression of hormone receptors and IFNT-stimulated genes during early pregnancy in ewes . All ewes were hysterectomized on Day 17 and uteri processed as described for study 1.
As described previously , cyclic ewes (n = 20) were ovariectomized and fitted with intrauterine catheters on Day 5 postestrus. Ewes were then assigned randomly (n = 5 ewes/treatment) to receive daily i.m. injections of P4 (Sigma) or P4 and PGR antagonist (ZK 136,317; Schering) and intrauterine infusions of either CX serum proteins or recombinant ovine IFNT as follows: (1) 50 mg P4 (Days 5–24) and 200 μg serum proteins (Days 11–24) [P4+CX], (2) P4 and 75 mg of ZK136,317 (Days 11–24) and serum proteins (200 μg) [P4+ZK+CX], (3) P4 and IFNT (2 × 107 antiviral units, Days 11–24) [P4+IFN], or (4) P4 and ZK and IFNT [P4+ZK+IFN]. All ewes were hysterectomized on Day 25 postestrus, and the uteri were then processed as described for study 1. Recombinant ovine IFNT and serum proteins were prepared for intrauterine injection as described for study 4.
As described previously , cyclic ewes (n = 15) were ovariectomized and fitted with intrauterine catheters on Day 5 postestrus. All ewes received daily i.m. injections of 50 mg P4 (Days 5–25) and intrauterine injections of recombinant ovine IFNT (2 × 107 antiviral units/day) from Days 11 through 20. Ewes (n = 5 ewes/treatment) also received daily intrauterine injections of either serum proteins (200 μg) [P4+CX], recombinant ovine CSH1 (200 μg) [P4+CSH1], or recombinant ovine GH (200 μg) [P4+GH] from Day 16 through Day 25. All ewes were hysterectomized on Day 25, and the uteri were then processed as described for study 1. Recombinant ovine CSH1 and ovine GH were prepared in bacteria and purified as described previously .
Using methods described previously , ewes (n = 4) were made unilaterally pregnant. On Day 80 of pregnancy, uterine secretions (e.g., uterine milk) were collected from the nongravid uterine horn of unilaterally pregnant ewes by flushing the uterine horn with 100 ml saline. In addition, samples of allantoic and amniotic fluids were obtained from the conceptus in the gravid uterine horn. Uterine milk, allantoic fluids, and amniotic fluids were clarified by centrifugation and stored at −80°C.
Total cellular RNA was isolated from frozen endometrium using Trizol reagent (Gibco-BRL) according to manufacturer's recommendations. The quantity and quality of total RNA was determined by spectrometry and denaturing agarose gel electrophoresis, respectively.
RT-PCR of ovine GRP and GRPR cDNAs. Partial cDNAs for ovine GRP and GRPR mRNAs were amplified by RT-PCR using total RNA from Day 18 pregnant endometrium, Day 18 conceptus, Day 45 placental cotyledon, and/or Day 45 intercotyledonary placenta. For GRP, the sense primer (5′-GCT GGC CAA GAT GTA CAC G-3′) and antisense primer (5′-CAG TAC AGC TGG GGG TTC C-3′) were derived from the ovine endometrial GRP mRNA (GenBank accession no. S75723). For GRPR, the sense primer (5′-TCA AAG CTG CAC TGA TCT GG-3′) and antisense primer (5′-AGC AGG TAG AGG GCA AAA GG-3′) were derived from the bovine GRPR mRNA coding sequence (GenBank accession no. XM_611960) and amplified a 499-base-pair (bp) product. For ACTB, the sense primer (5′-ATG AAG ATC CTC ACG GAA CG-3′) and antisense primer (5′-GAA GGT GGT CTC GTG AAT GC-3′) amplified a 270-bp product. PCR amplification was conducted using ~50 ng cDNA as follows: (1) 95°C for 5 min; (2) 95°C for 30 sec, 55°C for 1 min (for GRP and ACTB), and 54.2°C for 1 min (for GRPR); and 72°C for 1 min for 35 cycles; and (3) 72°C for 10 min. Partial ovine ACTB, GRP, and GRPR cDNAs were cloned into pCRII using a T/A Cloning Kit (Invitrogen), and their sequences were verified using an ABI PRISM Dye Terminator Cycle Sequencing Kit and ABI PRISM automated DNA sequencer (Perkin-Elmer Applied Biosystems).
Location of GRP and GRPR mRNAs was determined in sections (5 μm) of the ovine uterus by radioactive in situ hybridization analysis as described previously . Briefly, deparaffinized, rehydrated, and deproteinated uterine tissue sections were hybridized with radiolabeled antisense or sense cRNA probes generated from linearized ovine GRP and GRPR partial cDNAs using in vitro transcription with [α-35S]-UTP. After hybridization, washing, and ribonuclease A digestion, slides were dipped in NTB-2 liquid photographic emulsion (Kodak) and exposed at 4°C for 5 days for GRP and 4 wk for GRPR. Slides were developed in Kodak D-19 developer, counterstained with Gills hematoxylin (Fisher Scientific), and then dehydrated through a graded series of alcohol to xylene. Coverslips were then affixed with Permount (Fisher). Images of representative fields were recorded under bright-field and dark-field illumination using a Nikon Eclipse 1000 photomicroscope (Nikon Instruments, Inc.) fitted with a Nikon DXM1200 digital camera.
In studies 5 and 6, the relative abundance of GRP mRNA in the endometrial glands was determined with the Scion Image software (Release beta 4.03, Scion Corporation, NIH) using methods described previously . Briefly, photomicrographs of at least 10 regions of the uterus from each ewe were acquired under dark-field illumination and converted to a TIFF file. The optical intensity for the mRNA hybridization signals in the endometrial glands was determined using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image ). The inter- and intrasection variation in optical intensity value measurements was less than 5%.
Immunocytochemical localization of GRP protein was performed using methods described previously  in cross sections of ovine uteri or uteroplacental tissues with a rabbit anti-porcine GRP polyclonal antibody (catalog T-4354; Bachem Americas Inc.) at a final dilution of 1:1000 (2 μg/ml). Antigen retrieval was performed using Pronase E digestion as described previously . Negative controls included substitution of purified rabbit IgG for the primary antibody at the same final concentration. Sections were not counterstained after developing or before affixing coverslips.
Uterine flushings from cyclic and pregnant ewes in study 1 were concentrated using Centricon-3 columns (Amicon). Protein concentrations for uterine flushings, uterine milk, allantoic fluid, and amniotic fluid were determined using the Bradford protein assay (Bio-Rad) with bovine serum albumin (BSA) as the standard. Proteins were denatured and separated by 15% SDS-PAGE, and Western blot analysis was performed as described previously  using enhanced chemiluminescence detection (SuperSignal West Pico, Pierce) and X-OMAT AR X-ray film (Kodak). Immunoreactive GRP protein was detected using the rabbit anti-porcine GRP polyclonal antibody at a 1:5000 (0.4 μg/ml) final dilution.
All quantitative data was subjected to least-squares analysis of variance using the General Linear Models procedures of the Statistical Analysis System (SAS Institute). Slot-blot hybridization data were corrected for differences in sample loading by using the 18S rRNA data as a covariate. Preplanned orthogonal contrasts were used to determine effects of treatment in study 3 (Day 9 CO vs. Day 9 P4, Day 12 CO vs. Day 12 P4, and Day 9 CO vs. Day 12 CO), studies 4 and 5 (P4+CX vs. P4+IFN, P4+CX vs. P4+ZK+CX, and P4+IFN vs. P4+ZK+IFN), and study 6 (P4+CX vs. P4+GH, P4+CX vs. P4+CSH1). In all analyses, error terms used in tests of significance were identified according to the expectation of the mean squares for error. A P-value of 0.05 or less was deemed a significant effect of treatment. Data are presented as the least-squares means (LSM) with overall standard error of the mean (SEM).
In situ hybridization analyses of the ontogeny and location of GRP mRNA in uteri of cyclic (C) and pregnant (P) ewes (Fig. 1) revealed that GRP mRNA was essentially undetectable in endometria from cyclic ewes between Days 10 and 14 but abundant in both glandular (GE) and luminal (LE) epithelia on Day 16. Similarly, GRP mRNA was not detected in the endometria of Day 10 pregnant ewes but was detected thereafter at increasing levels in GE of upper and middle uterine glands but not in endometrial stroma, myometrium, blood vessels, immune cells, or conceptus trophectoderm. Interestingly, GRP mRNA was detected at low levels in the endometrial LE of one Day 12 pregnant ewe, three Day 14 pregnant ewes, three Day 16 pregnant ewes, but none of the Day 18 or 20 pregnant ewes. As illustrated in Figure 2, GRP mRNA was abundant only in endometrial GE between Days 40 and 120 of gestation. GRP expression was not detected in either cotyledonary or caruncular tissues of placentomes (Fig. 2) but was abundant in endometrial glands outside the capsules of placentomes.
Immunohistochemical analysis indicated that GRP protein was localized predominantly to secretory vesicles within GE and on the LE surface in uteri from Day 16 of the estrous cycle (Fig. 3). In early pregnant ewes, GRP in endometrial GE increased after Day 12 and was abundant by Day 18 of pregnancy. In placentomes, immunoreactive GRP was detected only in GE adjacent to the capsule of placentomes (Fig. 4). The overall abundance of immunoreactive GRP protein paralleled changes in abundance of GRPR mRNA in the endometria.
This study used a sheep model in which exogenous P4 prematurely increases circulating levels of P4 that advances growth and development of blastocysts/conceptuses and thus production of IFNT . As illustrated in Figure 5A, ewes were bred at estrus (Day 0) and then received daily injections of either CO vehicle as a control or 25 mg P4 in CO beginning on Day 1.5 postmating. As shown in Figure 5B, endometrial GRP mRNA abundance was very low and not affected by treatment with early P4 (P > 0.10) on Day 9. However, endometrial GRP mRNA levels were about 23-fold higher (P < 0.01) in P4- than CO-treated ewes on Day 12 and most abundant in glands in the upper area of the endometrium (Fig. 5C). As observed in some Day 12 and Day 14 pregnant ewes, GRP mRNA was also present at lower abundance in LE of uteri from P4-treated ewes on Day 12.
Using a custom ovine endometrial cDNA array, we previously identified GRP as a candidate IFNT-stimulated gene in the ovine endometrium . Therefore, we conducted study 4 (see Fig. 6A) in order to validate that microarray result. Treatment of ewes with P4 for 12 days did not change (P > 0.10, P4+CX vs. P4+ZK+CX) endometrial GRP mRNA abundance (Fig. 6B). GRP mRNA and protein were detected at very low levels in subsets of GE in middle to deep endometrial glands of P4+CX and P4+ZK-treated ewes (Fig. 6C). However, intrauterine infusions of IFNT increased (P < 0.05, P4+CX vs. P4+IFN) GRP mRNA levels by threefold in endometria of P4-treated ewes (Fig. 6B), and GRP mRNA was present in GE of upper and middle glands throughout endometria from those ewes (Fig. 6C). Intrauterine infusions of IFNT did not increase (P > 0.10, P4+CX vs. P4+ZK+IFN) endometrial GRP mRNA levels in P4+ZK-treated ewes (Fig. 6C), indicating a permissive effect of P4 on actions of IFNT.
Several other genes uniquely or predominantly expressed by endometrial glands of ovine uteri include stanniocalcin 1 (STC1), secreted phosphoprotein 1 (SPP1), and uterine milk protein (UTMP) are only up-regulated by long-term exposure of the uterus to P4 [42, 47, 51]. Therefore, study 5 was conducted to determine the effects of prolonged P4 treatment for 20 days and intrauterine infusions of IFNT on GRP expression in ovine uteri (Fig. 7A). GRP mRNA and protein were abundant in endometrial glands of uteri from P4-treated ewes (P4+CX and P4+IFN) but very low to absent in endometrial glands of uteri of ewes receiving the PGR antagonist (P4+ZK+CX and P4+ZK+IFN) (Fig. 7B). Intrauterine infusions of IFNT increased GRP mRNA levels about threefold (P < 0.01) in endometrial glands of P4-treated but not P4+ZK-treated ewes (Fig. 7C).
In addition to being P4-induced genes in endometrial GE of ovine uteri, SPP1, STC1, and UTMP are also stimulated by ovine CSH1 and/or ovine GH [38, 43, 52]. Therefore, study 6 was conducted to determine effects of intrauterine infusion of ovine CSH1 and ovine GH on GRP expression in uterine endometrial glands of P4-treated and IFNT-infused ewes (Fig. 8A). GRP mRNA and protein were abundant in GE of endometrial glands of all ewes but was not different between endometria from ewes infused with ovine CSH1 versus ovine GH (Fig. 8, B and C).
A single protein of ~13 kDa was detected by Western blot analyses of uterine milk proteins and allantoic fluid but not amniotic fluid from Day 80 unilaterally pregnant ewes using a rabbit anti-porcine GRP polyclonal antibody (Fig. 9). Immunoreactive GRP was not detected in uterine luminal fluid from Day 10 through 16 cyclic ewes or Day 10 through 16 pregnant ewes (data not shown).
The major high affinity receptor for GRP1–27 and GRP18-27 is GRPR, although NMBR and BRS3 can bind those forms of GRP with much lower affinity . Although GRPR mRNA could be detected by RT-PCR in DNAse-treated RNA samples from Day 18 conceptuses, GRPR mRNA was not detected in Day 18 endometria, Day 45 placental cotyledons, or Day 45 intercotyledonary placentae (Fig. 10). The partial ovine conceptus GRPR cDNA (GenBank accession no. EU487010) had 99% identity with bovine GRPR mRNA, and the inferred amino acid sequence had 98% identity with bovine GRPR. The abundance of GRPR mRNA in the Day 18 conceptus was very low, whereas all the samples contained ACTB mRNA. As expected from the RT-PCR analyses, GRPR mRNA was not detected in uteri, conceptuses, or placentae of ewes at any stage of gestation by in situ hybridization analysis (data not shown). Further, BRS3 and NMBR mRNAs were not detected in samples of ovine endometrial, conceptus, or placenta by RT-PCR (data not shown).
In the present studies, we found that GRP is predominantly expressed in endometrial glands of the ovine uterus as described previously [7, 15–17]. The ontogeny of GRP expression in the ovine uterus during early pregnancy is correlated with blastocyst growth and elongation, formation of a filamentous conceptus, and growth and development of the fetus and placenta . Collectively, results of the present studies using appropriate physiological models validate GRP as a novel IFNT-stimulated gene in the endometrial glands of the ovine uterus, reveal that IFNT induction of GRP is dependent on P4, and found that exposure of the ovine uterus to P4 for 20 days is required to stimulate GRP expression in endometrial glands. The pattern of IFNT induction followed by P4 stimulation for GRP in the endometrial glands is unique and distinctly different from many other epithelial genes, such as cystatin C (CST3), cathepsin L (CTSL), endothelial PAS domain protein 1 (EPAS1), and lectin, galactoside-binding, soluble, 15 (galectin 15) (LGALS15), because those genes are P4 induced and then stimulated by IFNT in the LE/sGE of the endometrium [38, 54, 55]. In the present studies, exposure of the uterus to P4 for only 12 days did not affect GRP expression in endometrial glands. Similarly, Whitley and coworkers  found that administration of P4 for 10 days did not affect endometrial GRP mRNA levels and that estrogen and/or estrogen with P4 substantially decreased GRP mRNA levels in endometria of long-term ovariectomized ewes. In the ovine uterus, the ability of P4 to induce expression of genes encoding proteins secreted into the uterine lumen is thought to require P4 down-regulation of its own receptor in the endometrial epithelia [32, 56]. Treatment of ewes with anti-progestins, such as the ZK compounds, inhibit P4 from down-regulating PGR in endometrial epithelia of the ovine uterus and that leads to loss of expression of SPP1 , STC1 , and GRP. Likewise, administration of estrogen with P4 up-regulates PGR, which, in turn, ablates induction of genes such as GRP, SPP1, and UTMP in endometrial GE of ewes , which may explain the findings of Whitley and coworkers  that estrogen and/or estrogen with P4 substantially decreased GRP mRNA levels in endometria of long-term ovariectomized ewes. Although the initial up-regulation of GRP mRNA in the superficial GE on Days 12 and 14 of pregnancy can be attributed to IFNT from the conceptus, maintenance of GRP expression in endometrial glands from Days 20 through 120 of pregnancy is almost certainly due to effects of P4 since IFNT is not produced after Days 21 through 25 of gestation . Although the GRP gene from cattle and sheep has not been cloned, preliminary analysis of the 5′ untranslated region of ovine and bovine GRP mRNAs, which represents some of the proximal promoter, did not reveal any conserved PGR binding sites (G. Song and T.E. Spencer, unpublished results). Therefore, P4-regulated growth factors or “progestamedins” from the stroma, such as fibroblast growth factors 7 and 10 and hepatocyte growth factor [58, 59], may act in a paracrine manner on endometrial GE to regulate GRP expression since the endometrial GE have receptors for those growth factors, but lack detectable PGR throughout most of gestation (see  for review). Cloning and analysis of ovine GRP promoter/enhancer regions could be useful in understanding the basis for cell-specific expression of genes as well as novel mechanisms of IFNT and P4 action on the uterus.
Results of the present studies validate GRP as a bona fide IFNT-stimulated gene in the superficial and upper glands of the endometrium. In the present study, temporal alterations in endometrial GRP mRNA and protein paralleled growth and development of the blastocyst into a filamentous conceptus  and production of IFNT by mononuclear trophectoderm that reaches a maximum on Day 16 . Similarly, up-regulation of GRP expression in response to early P4 administration in study 3 can be associated with increased amounts of IFNT in uterine flushings due to advanced formation of a filamentous conceptus in ewes receiving early P4 as compared to tubular blastocysts in CO-treated ewes on Day 12 . The induction of GRP by IFNT was predominantly in the upper and middle uterine glands, which is different from other novel IFNT-stimulated genes, such as LGALS15 and wingless-type MMTV integration site family, member 7A (WNT7A), that are confined to uterine LE and superficial GE [54, 62]. IFNT activates the classical JAK-STAT-IRF1 pathway [50, 63, 64] utilized by other type I IFNs (see ). However, the effects of IFNT likely involves a nonclassical cell signaling pathway because the proximal promoter of ovine and bovine GRP lack binding sites that mediate the classical effects of type I IFNs, including those for STAT1, IRF9, and IRF1 (G. Song and T.E. Spencer, unpublished observation). Potential nonclassical signaling pathways utilized by IFNT to stimulate GRP expression in endometrial glands include MAPK (p38 and ERK1/2) and PI3K-AKT1 signaling pathways (see [66, 67] for review). In the present studies, IFNT stimulation of GRP expression in endometrial glands was dependent on P4, which has also been observed for a number of other nonclassical IFNT-stimulated genes expressed in the endometrial LE/sGE, including CST3, CTSL, EPAS1, and LGALS15 [38, 54, 55, 68]. Although the endometrial glands lack detectable nuclear PGR, the requirement for P4 may be due to production of progestamedins from the stroma with epithelial receptors [32, 58, 59]. Thus, uncovering the regulatory networks and pathways regulating GRP expression would help unravel how genes are expressed in a cell-specific manner within the endometrium and how P4 is permissive to effects of IFNT on ovine endometrial GE.
In the present studies, we detected a 13-kDa form of GRP in uterine luminal fluid or uterine milk from the nongravid uterine horn of Day 80 unilateral pregnant ewes and in allantoic fluid from the conceptus. Similarly, SPP1, STC1, and UTMP are secreted by the endometrial GE and are present in uterine luminal and allantoic fluids during pregnancy [47, 69–72]. In domestic animals, secretory products of the endometrial glands are transported into the allantois via areolae that are specialized structures of the placenta that form over the openings on uterine glands . Indeed, results of the present study and those of others support the idea that GRP protein is synthesized by endometrial GE, secreted into the uterine lumen, absorbed and transported via placental areolae into the fetal circulation, and cleared by the fetal kidneys into the allantoic sac via the urachus. The allantois stores most secreted proteins from endometrial GE, including SPP1, STC1, and UTMP [47, 51, 74]. Interestingly, GRP receptors are expressed in the fetal kidney, and GRP is hypothesized to regulate kidney development in sheep .
All forms of GRP are derived from preproGRP . The 13-kDa immunoreactive GRP-like protein in allantoic fluid and uterine milk of Day 80 pregnant ewes, detected by the rabbit anti-porcine GRP1-27 antibody, is likely proGRP, which is 118 amino acids and has a predicted molecular weight of 12.4 kDa based on the inferred amino acid sequence from an endometrial GRP cDNA from the ovine uterus . If the ovine GRP preproprotein is processed similarly to human and rat GRP  by signal peptidase and then prohormone convertase, it would yield a signal peptide of 23 amino acids, GRP1-27 of ~2.8 kDa with an amidated C-terminus and a C-terminal GRP31-134 of ~9.3 kDa. Endoproteolytic cleavage of GRP1-27 releases a decapeptide designated GRP18-27. Both GRP1-27 and GRP18-27 have C-terminal sequences identical with the frog peptide bombesin or neuromedin-C . The rabbit anti-porcine GRP antibody used for Western blot and immunohistochemical analyses in the present study has 100% cross-reactivity with human, porcine, and ovine GRP1-27 and GRP18-27 but would not detect the C-terminal GRP31-134. In the present studies, immunoreactive proGRP was not detectable in uterine luminal fluid from cyclic or early pregnant ewes, perhaps because it is processed into GRP1-27 and GRP18-27. Indeed, GRP1-27 and GRP18-27 were detected in ovine endometrium and uterine flushings by size chromatography [6, 12].
Although GRP1-27 and GRP18-27 are present in endometrial and uterine luminal fluids, our studies failed to detect significant expression of their receptors (GRPR, NMBR or BRS3) in ovine conceptuses, placentae, or endometria. Similarly, Whitley and coworkers  did not detect BRS3 mRNA in tissues of pregnant ovine uteri (endometrium, myometrium, chorioallantois, or amnion) or nonpregnant uteri but did detect BRS3 mRNA in the pituitary and hypothalamus. Thus, available evidence supports the idea that endometrial gland-derived GRP1-27 or GRP18-27 do not regulate conceptus or placental growth and differentiation but rather may have effects on development of the kidney and perhaps other organs of the embryo/fetus [11, 23]. Although classical N-terminal-derived GRP1-27 and GRP18-27 from the endometrial glands may not influence placental growth and differentiation because of a lack of GRP receptors, derivatives of the C-terminus of proGRP are biologically active, and their activity is mediated by a receptor distinct from GRPR [76–78]. Those studies found that various forms of human proGRP (18–125, 31–125, and 42–98) stimulated proliferation and migration of human cancer cell lines as well as inositol phosphate production and activity of the MAPK cell signaling pathway. Further, each peptide was able to compete with the other for binding to cells, but a GRPR antagonist did not inhibit binding of either peptide . Interestingly, the C-terminal residues of proGRP are well conserved between mammalian proGRPs , and ovine GRP31-134 has about 70% similarity with the C-terminal sequence of human GRP31-125. Therefore, future studies should determine if the C-terminus of proGRP present in the uterine lumen has a biological role in peri-implantation conceptus growth and development.
The authors thank all members of the Laboratory for Uterine Biology and Pregnancy for assistance and management of animals. We also are grateful to Drs. Robert C. Burghardt and Greg A. Johnson for helpful discussions.
1Supported in part by National Institutes of Health grant 5 R01 HD32534 and National Research Initiative Competitive grant 2005-35203-16252 from the USDA Cooperative State Research, Education, and Extension Service.