Conceptus-Derived Prostaglandins Regulate Endometrial Function in Sheep¹

Abstract

In sheep, the trophectoderm of the elongating conceptus secretes interferon tau (IFNT) and prostaglandins (PGE2, PGF2alpha, PGI2). The PGs are derived from PG synthase 2 (PTGS2), and inhibition of PTGS2 in utero prevents conceptus elongation. IFNT increases expression of many genes in the endometrial epithelia that regulate conceptus elongation. This study tested the hypothesis that PGs secreted by the conceptus regulate endometrial functions that govern conceptus elongation. Cyclic ewes received intrauterine infusions of control vehicle or early pregnancy levels of IFNT, PGE2, PGF2alpha, or PGI2 from Days 10–14 postestrus. Expression levels of endometrial GRP, IGFBP1, and LGALS15, whose products stimulate trophectoderm cell migration and attachment, were increased by PGE2, PGI2, and IFNT. All PGs and IFNT increased expression of the HEXB protease gene, but only IFNT increased the CST6 protease inhibitor gene. Differential effects of PGs were observed for expression of the CTSL protease gene and its inhibitor, CST3. IFNT, PGF2alpha, and PGI2 increased ANGPTL3 expression, but only IFNT and PGE2 increased HIF1A expression, both of which regulate angiogenesis. For glucose transporters, IFNT and all PGs increased SLC2A1 expression, but only PGs increased SLC2A5 expression, whereas endometrial SLC2A12 and SLC5A1 expression levels were increased by IFNT, PGE2, and PGF2alpha. Infusions of all PGs and IFNT increased the amino acid transporter SLC1A5, but only IFNT increased SLC7A2 expression. In the uterine lumen, only IFNT increased glucose levels, and only PGE2 and PGF2alpha increased total amino acids. These results indicate that PGs and IFNT from the conceptus coordinately regulate endometrial functions important for growth and development of the conceptus during the peri-implantation period of pregnancy.

Introduction

Maternal support of blastocyst growth and development into an elongated conceptus (embryo/fetus and associated membranes) is critical for pregnancy recognition signaling, implantation, and establishment of pregnancy in ruminants [1, 2]. After hatching from the zona pellucida on Day 8, the blastocyst develops into an ovoid or tubular form by Days 11–12 and is then termed a conceptus [1, 3]. The conceptus begins to elongate on Day 12 and reaches up to 14 cm or more in length by Day 16. Conceptus elongation requires substances secreted from endometrial luminal epithelium (LE), glandular epithelium (GE), and superficial GE (sGE) [4, 5]. During early pregnancy in ruminants, functions of the endometrium are regulated primarily by progesterone from the ovarian corpus luteum (CL) and by factors secreted from the conceptus, including interferon tau (IFNT) [6–9]. IFNT, secreted by mononuclear trophectoderm cells of the conceptus during elongation, exerts antiluteolytic effects on the endometrium. In both sheep and cattle, IFNT silences expression of estrogen receptor alpha 1 (ESR1), which prevents up-regulation of oxytocin receptor (OXTR) expression in the endometrial LE and sGE, which is required for the endometrium to generate oxytocin-dependent luteolytic pulses of prostaglandin F2α (PGF2α) in response to oxytocin from the CL and/or posterior pituitary in nonpregnant ewes. In this way, IFNT maintains CL function for continued production of progesterone, the unequivocal hormone of pregnancy that stimulates and maintains endometrial functions necessary for conceptus survival and growth in the uterus [9].

The sequential and combinatorial effects of progesterone and IFNT on the endometrium result in specific changes in the intrauterine milieu necessary for blastocyst growth and conceptus elongation [2, 8, 10]. In the endometrial LE/sGE, progesterone induces expression of many genes between Days 10 and 12 post-estrus/mating that encode enzymes (prostaglandin G/H synthase and cyclooxygenase 2 or PTGS2 and 11-beta-hydroxysteroid dehydrogenase or HSD11B1), adhesion proteins (galectin-15 or LGALS15, insulin-like growth factor binding protein one or IGFBP1), a protease (cathepsin L or CTSL), protease inhibitors (cystatin C or CST3 and CST6), a cell proliferation factor (gastrin releasing peptide or GRP), glucose transporters (SLC2A1, SLC5A1), and a cationic amino acid (arginine, lysine, and ornithine) transporter (SLC7A2). In uterine GE, progesterone induces expression of several genes between Days 12 and 14−16, including a cell proliferation factor (GRP) and a glucose transporter (SLC5A11). In the endometrial epithelia of the ovine uterus, IFNT increases expression of a number of the progesterone-induced genes that encode enzymes (HSD11B1), secreted proteins (CST3, CTSL, GRP, IGBP1, LGALS15) as well as transporters for glucose (SLC2A1 and SLC5A11), and amino acids (SLC7A2) [2, 8, 10, 11]. Thus, IFNT induces or increases expression of many genes in the endometrium that regulate conceptus growth and development during the peri-implantation period of pregnancy, in addition to eliciting antiluteolytic effects that maintain the CL and thus progesterone production.

Our previous studies in sheep support the concept that PGs from the conceptus and/or endometrium up-regulate IGFBP1 and HSD11B1 in endometrial LE/sGE on Day 12 of pregnancy [12, 13]. Development of a Day 11 ovoid conceptus into the elongated and filamentous form is coincident with onset of PTGS2 expression in the conceptus and endometrium and increased amounts of PGs in the uterine lumen [14–17]. From Days 12 to 16 of early pregnancy, sheep conceptuses secrete predominantly PGE2, PGF2α, and PGI2 [15, 17, 18], and Day 14 sheep conceptuses produce more PGs than the endometrium [19]. Of particular importance, in utero inhibition of PTGS2 during early pregnancy in sheep inhibits blastocyst growth into an elongated conceptus [20]. Given that membrane receptors for PGE2, PGF2α, and PGI2 are expressed in the endometrial epithelia and conceptuses of sheep and cattle [20–23], it is likely that the effects of PGs at the conceptus-uterine interface are manifest via autocrine, paracrine, or perhaps intracrine actions. The present study tested the hypothesis that PGs secreted by the elongating conceptus regulate endometrial functions that govern conceptus elongation.

Materials and Methods

Experimental Design

Mature Rambouillet ewes (Ovis aries) were observed for estrus (designated as Day 0) in the presence of a vasectomized ram and used in experiments only after exhibiting 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 Research and Teaching and approved by the Institutional Animal Care and Use Committee of Texas A&M University.

As described previously [20, 24], ewes (n = 30) were checked daily for estrus (Day 0) and subjected to a mid-ventral laparotomy and implanted with two Alzet 2ML1 osmotic pumps on Day 10 post-estrus, using a surgical approach [25, 26]. The pumps provided a constant infusion of control (CX) vehicle, recombinant ovine IFNT (101 μg), PGE2 (251 ng), PGF2α (409 ng), or PGI2 (1483 ng) into the lumen of each uterine horn each day (n = 5 ewes/treatment). Recombinant ovine IFNT was prepared for intrauterine infusion as described previously [27]. The amount of recombinant ovine IFNT was based on published estimates of IFNT production by Day 14 ovine conceptuses, which is approximately 600 ng per hour or 14.4 μg per day [28]. Intrauterine infusion of that amount of IFNT mimics effects of the conceptus on endometrial expression of hormone receptors and IFNT-stimulated genes during early pregnancy in ewes [20, 29]. PGs were purchased from Cayman Chemical Company (Ann Arbor, MI), and the amount infused into the uterus was based on their production by Day 14 conceptuses [20].

At necropsy on Day 14, the uterine lumen was flushed with 20 ml of 10 mM Tris (pH 7.2). The volume of the uterine flushing was measured and recorded and then clarified by centrifugation (3000 × g at 4°C for 15 min). The supernatant was carefully removed with a pipette, divided into aliquots, frozen in liquid nitrogen, and stored at −80°C. Several sections (∼0.5 cm) from the mid-portion of each uterine horn 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, St. Louis, MO). The remaining endometrium was physically dissected from myometrium, frozen in liquid nitrogen, and stored at −80°C for subsequent RNA extraction.

Analyses of Glucose and Amino Acids

Uterine flushing samples were analyzed for glucose, using a fluorometric method involving hexokinase and glucose-6-phosphate dehydrogenase, using methods described previously [30, 31]. Amino acids in the extract were determined by fluorometric HPLC methods involving pre-column derivatization with o-phthalaldehyde, using methods described previously [31, 32]. The amounts of total recoverable glucose and amino acids in the uterine lumen were determined by using the total volume of the uterine flushing recorded at necropsy.

Total RNA Isolation and Real-Time PCR Analysis

Using methods described previously [20, 24], total RNA was isolated from endometria and reverse transcribed and analyzed by real-time PCR using an ABI prism 7900HT system with Power SYBR Green PCR Master Mix (Applied Biosystems, Foster, CA). Specific oligonucleotide primers were designed by Oligo 5 software (Molecular Biology Insights, Inc.) (see Supplemental Table S1; available online at www.biolreprod.org). Primer specificity and efficiency (−3.6 > slope > −3.1) were confirmed using a test amplification run. Each individual sample test was run in triplicate under the following conditions: 50°C for 2 min; 95°C for 10 min; and 95°C for 15 sec and 60°C for 1 min for 40 cycles. A dissociation curve was generated at the end of amplification to ensure that a single product was amplified. PCR without template or template substituted with total RNA was used as a negative control to verify experimental results. The threshold line was set in the linear region of the plots above baseline noise, and threshold cycle values were determined as the cycle number at which the threshold line crossed the amplification curve. Ovine GAPDH was used as the reference gene.

In Situ Hybridization Analysis

A partial cDNA for ovine GRP was amplified by RT-PCR using the specific primers and PCR conditions described previously [33]. A partial cDNA for ovine SLC2A12 was amplified by RT-PCR using total RNA isolated from endometria of Day 16 pregnant ewes and methods described previously [33]. The sense primer (5′-ATTTTGTCCTCCTGCCTCC-3′) and antisense primer (5′-CCGTGAGTTCCTCTGTTG-3′) were derived from the Bos taurus SLC2A12 mRNA coding sequence (GenBank accession no. NM_001011683) and amplified a 424-bp product. Partial ovine cDNAs were cloned into pCRII, using Cloning kit (Invitrogen, Carlsbad, CA), and sequences were verified using an ABI PRISM Dye Terminator cycle sequencing kit and ABI PRISM automated DNA sequencer (Perkin-Elmer Applied Biosystems, Foster City, CA).

Localization of GRP and SLC2A12 mRNAs in the ovine uterus was determined by radioactive in situ hybridization analysis using methods described previously [34]. After hybridization, washing, and ribonuclease A digestion, slides were dipped in NTB-2 liquid photographic emulsion (Kodak, Rochester, NY) and exposed at 4°C for 10 days based on the intensity of radioactive signal of slides placed on Kodak MR film for 16 h. All slides were exposed to photographic emulsion for the same period. Slides were developed in Kodak D-19 developer, counterstained with Gill hematoxylin (Fisher Scientific, Fairlawn, NJ), and dehydrated through a series of graded alcohol concentrations to xylene, and then coverslips were affixed with Permount (Fisher Scientific). Images of representative fields were recorded under brightfield or darkfield illumination, using an Eclipse 1000 model photomicroscope (Nikon Instruments Inc., Lewisville, TX) fitted with a Nikon DXM1200 digital camera.

Statistical Analysis

All quantitative data were subjected to least squares ANOVA, using the General Linear Models procedures of Statistical Analysis System software (SAS Institute Inc., Cary, NC). For analysis of real-time PCR data, the threshold cycle values of the target mRNA were analyzed for effects of treatment, with the GAPDH values used as a covariate. In all analyses, error terms used in tests of significance were identified according to the expectation of the mean squares for error. Significance (P < 0.05) was determined by probability differences of least squares means.

Results

Expression of Genes in the Endometrium Related to Elongation and Implantation of the Conceptus

Relative to the that in CX, infusion of IFNT, PGE2 and PGI2 increased (P < 0.05) the abundance of GRP mRNA, whose product is a candidate cell proliferation and migration factor (Table 1). Moreover, IFNT and all PGs increased (P < 0.05) expression of IGFBP1 and LGALS15, which mediate trophectoderm migration and attachment. IFNT and all PGs stimulated (P < 0.05) expression of EFNA1, which is implicated in blastocyst attachment and spreading. Infusion of IFNT up-regulated (P < 0.05) expression of the protease CTSL and its inhibitor CST3. By comparison, CTSL expression was increased (P < 0.05) by PGE2 and PGI2, whereas CST3 expression was increased (P < 0.05) only by PGF2α and PGI2. Another protease inhibitor, CST6, was increased (P < 0.05) by IFNT but not by any of the PGs. Furthermore, IFNT and all PGs augmented (P < 0.05) expression of another protease, HEXB.

Table 1.

Effects of treatment on the abundance of endometrial mRNA for selected genes.

Gene symbola	Approved gene name	IFNT vs. CXb	PGE2 vs. CXb	PGF2α vs. CXb	PGI2 vs. CXb
Genes associated with cellular proliferation, migration, and attachment
EFNA1	Ephrin-A1	+2.7*	+1.6†	NE	+1.9*
GRP	Gastrin-releasing peptide	+7.8*	+2.6*	−2.0*	+3.6*
IGFBP1	Insulin-like growth factor binding protein 1	+9.2*	+4.0*	+3.8*	+6.5*
LGALS15	Lectin, galactoside-binding, soluble, 15	+11.6*	+5.6*	NE	+8.0*
Genes associated with endometrial remodeling and placentation
CTSL	Cathepsin L	+2.2*	+1.7*	NE	+1.7*
CST3	Cystatin C	+2.6*	NE	+1.9*	+1.9*
CST6	Cystatin 6	+2.1*	NE	NE	NE
HEXB	Hexosaminidase B (beta polypeptide)	+2.6*	+1.4‡	+1.8†	+3.3*
Genes associated with angiogenesis
ANGPTL3	Angiopoietin-like 3	+1.6†	NE	+2.4*	+1.5‡
HIF1A	Hypoxia inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor)	+1.5*	+1.6*	NE	−1.4†
HIF2A	Hypoxia inducible factor 2, alpha subunit (basic helix-loop-helix transcription factor)	+2.1*	+1.5†	+1.5*	NE
Genes associated with transport of glucose
SLC2A1	Solute carrier family 2, (facilitated glucose transporter), member 1	+2.9*	+1.7*	+1.7*	+1.7*
SLC2A5	Solute carrier family 2 (facilitated glucose/fructose transporter), member 5	NE	+2.2*	+1.8*	+2.1*
SLC2A12	Solute carrier family 2 (facilitated glucose transporter), member 12	+2.8*	+1.9†	+1.8†	NE
SLC5A1	Solute carrier family 5, (sodium/glucose cotransporter), member 1	+2.3*	+1.6*	+2.3*	NE
SLC5A11	Solute carrier family 5, (sodium/glucose cotransporter), member 11	+2.7*	+2.0*	NE	+1.6†
Genes associated with transport of neutral and cationic amino acids
SLC1A5	Solute carrier family 1 (neutral amino acid transporter), member 5	+2.0*	1.6‡	+2.3*	+2.0*
SLC7A2	Solute carrier family 7, (cationic amino acid transporter, y+ system), member 2	+3.2*	NE	NE	NE

^a

Official gene symbols were abbreviated.

^b

Data are presented as fold change;

^*

P < 0.01,

^†

P < 0.05,

^‡

P < 0.10 for the comparison; and NE indicates no effect of treatment (P > 0.10).

With respect to mediators of angiogenesis, HIF1A was stimulated (P < 0.05) by IFNT and PGE2, whereas HIF2A expression was increased (P < 0.05) by IFNT, PGE2, and PGF2α. By comparison, ANGPTL3 expression was increased (P < 0.05) by IFNT, PGF2α, and PGI2. Regarding transporters of glucose, infusion of IFNT and all PGs increased (P < 0.05) SLC2A1 expression; however, the abundance of SLC2A5 was increased (P < 0.05) only by PGs. Endometrial SCL2A12 and SLC5A1 levels were increased (P < 0.05) by IFNT, PGE2, and PGF2α, whereas SLC5A11 expression was increased (P < 0.05) by IFNT, PGE2, and PGI2. All infused PGs and IFNT increased (P < 0.05) SLC1A5 mRNA abundance; however, only IFNT increased (P < 0.05) the abundance of SLC7A2.

In situ hybridization analysis found that GRP mRNA was present predominantly in the upper and middle GE of the ovine endometrium and increased in response to IFNT, PGE2, and PGI2 compared to that in CX ewes (Fig. 1). Interestingly, IFNT increased GRP mRNA also in the endometrial LE. SLC2A12 mRNA was present in both the LE and GE and was stimulated by intrauterine infusions of IFNT, PGE2, and PGF2α compared to that in CX ewes (Fig. 1).

Fig. 1.

In situ localization of GRP and SLC2A12 mRNAs in the uteri of cyclic ewes infused with vehicle CX, IFNT, PGE2, PGF2α, or PGI2. LE, luminal epithelium; GE, glandular epithelium; S, stroma. All representative photographs are shown at a 560 μm width of field.

Glucose and Amino Acids in the Uterine Lumen

The amount of glucose was 5.5-fold greater (P < 0.01) in the uterine flushing fluids from ewes infused with IFNT than in those from CX ewes (Fig. 2). In contrast, infusion of PGs did not alter (P > 0.10) the amount of glucose in the uterine lumen. As summarized in Table 2, IFNT and all PGs increased (P < 0.05) the levels of beta-alanine, whereas IFNT, PGE2, and PGF2α increased (P < 0.05) levels of asparagine, glutamic acid, and taurine. Levels of histidine, leucine, and phenylalanine were higher (P < 0.05) in uterine flushing samples from ewes infused with all PGs but not IFNT. Only IFNT and PGI2 increased (P < 0.05) the amount of recoverable arginine. In contrast, IFNT and PGI2 reduced (P < 0.05) tyrosine and valine. Furthermore, IFNT, PGE2, and PGI2 decreased (P < 0.05) glutamine. Relative to that in CX ewes, the total amount of amino acids was greater (P < 0.05) in the uterine flushing samples from ewes infused with PGE2 or PGF2α but not PGI2 or IFNT (Table 2).

Fig. 2.

Recoverable glucose in the uterine lumen of ewes infused with vehicle as a control (CX), IFNT, PGE2, PGF2α, or PGI2. The quantity of glucose in each uterine flush was determined by fluorometric analysis. Data are presented as total nmol with SEM. Differences relative to the CX are denoted. *P < 0.01.

Table 2.

Effects of treatment on recoverable amino acids in uterine flushing samples (nmol: means ± SEM).

	Treatmenta
Component	CX	IFNT	PGE2	PGF2α	PGI2	SEM
Ala	249	253	234	269	176	32
Arg	104	134*	126	114	130§	8
Asn	20	31*	39†	31‡	25	3
Asp	29	34	39†	26	18§	3
β-Ala	20	38*	37†	33‡	38§	3
Cit	67	83	92	93	68	14
Gln	255	142*	148†	273	99§	18
Glu	192	261*	271†	253‡	118§	16
Gly	6077	6082	7081	8097‡	6525	422
His	15	22	35†	29‡	19	3
Ile	28	31	39†	29	27	3
Leu	163	162	185†	193‡	198§	5
Lys	24	33*	58†	17	13§	3
Met	5	6	8†	6	7	1
Orn	7	9	10	8	6	1
Phe	45	49	51†	51‡	51§	2
Ser	358	478	836†	379	622	108
Tau	257	461*	495†	456‡	333	42
Thr	2178	2970	3086	2919	2837	495
Trp	62	51*	65	83‡	90§	3
Tyr	98	81*	99	89	71§	4
Val	51	38*	70†	42‡	28§	3
Total no. of amino acids	10 303	11 449	13 104†	13 489‡	11496	920

^a

Differences (P < 0.05) are denoted as *IFNT vs. CX; ^†PGE2 vs. CX, ^‡PGF2α vs. CX, and ^§PGI2 vs. CX.

Discussion

Results of the present study strongly support the hypothesis that PGs synthesized and secreted by the conceptus act in paracrine alone or in concert with IFNT and progesterone to coordinately regulate endometrial functions that govern conceptus growth and elongation during the peri-implantation period of pregnancy in sheep. Furthermore, the present study provides novel insights into how conceptus-derived PGs and IFNT regulate endometrial functions. In ruminants, conceptus growth and elongation clearly depend on the uterine milieu, because hatched blastocysts and trophoblastic vesicles do not elongate in vitro but do so when transferred into the uterus [4, 35]. The ovine endometrium synthesizes and secretes a number of factors, including IGFBP1 and LGALS15, that stimulate trophectoderm migration and attachment, which are essential cellular processes involved in conceptus growth and elongation [13, 33, 36, 37]. Both IGFBP1 and LGALS15 are induced in the endometrial LE/sGE by ovarian progesterone between Days 10 and 12 post-estrus/mating, and their expression in the endometrium is further stimulated by IFNT from the conceptus [13, 38]. The present study provides novel evidence that expression of both genes in the endometrial LE and sGE, as well as that of EFNA1 and GRP, is increased by one or more PGs secreted by the conceptus. In the sheep uterus, EFNA1 is up-regulated in the endometrium between Days 9 and 12 of pregnancy [36]. EFNA1 is expressed in epithelia of human and mouse endometria and is implicated in blastocyst attachment and spreading [39, 40]. GRP is expressed predominantly in the upper and middle endometrial GE, and its expression is increased substantially by IFNT [33]. Derivatives of GRP are hypothesized to stimulate trophectoderm cell proliferation and migration based on its biological activity in other cell types [41]. Results of the present study confirm that IFNT increases GRP expression and provides novel evidence that PGE2 and PGI2 increase GRP expression in GE of the ovine uterus. Collectively, available results indicate that IFNT and PGs from the conceptus increase expression of genes in the endometrial epithelia, whose products reciprocally act on the trophectoderm to stimulate proliferation, migration and/or attachment to the uterine LE.

The orchestrated actions of proteases and protease inhibitors govern endometrial remodeling and regulate uterine receptivity for conceptus implantation in many species [42–46]. In the present study, IFNT and PGs differentially regulated CTSL and CST3, whereas only IFNT stimulated CST6. In the sheep uterus, the lysosomal cysteine protease CTSL and its inhibitor CST3 are up-regulated in the endometrial LE/sGE between Days 10 and 12 post-estrus/mating and are abundantly expressed in those epithelia between Days 12 and 20 of pregnancy [47]. Cathepsins have biological roles in degrading extracellular matrix, catabolism of intracellular proteins, and processing of prohormones [42]. CST6 (cystatin E/M) is another cysteine proteinase inhibitor that is up-regulated in the endometrium between Days 9 and 12 of pregnancy in sheep [36]. In the ovine uterus, HEXB expression is up-regulated between Days 9 and 12 of pregnancy and is affected by products of the conceptus [36], and progesterone increases the amount of HEXB in the uterine lumen of sheep, pigs, and horses [48]. The present study provides novel evidence that HEXB is stimulated by IFNT as well as by all PGs secreted by the ovine conceptus. HEXB encodes the beta subunit of a hexosaminidase that catalyzes hydrolysis of glycoproteins, glycosaminoglycans, and glycolipids [49]. The proteases and protease inhibitors in the uterine lumen likely have a biological role in modification of proteins on the endometrial epithelia and conceptus trophectoderm and processing of proteins in uterine lumen before uptake by the conceptus.

In the sheep uterus, blood flow increases approximately 2-fold between Days 10 and 20 of pregnancy [50], which is concomitant with up-regulation of endometrial genes associated with angiogenesis, including HIF1A, HIF2A, VEGFA, CITED2, and ANGPTL3 [36, 50–52]. The present study found that IFNT and PGE2 increased endometrial HIF1A and HIF2A expression, whereas IFNT, PGF2α, and PGI2 stimulated ANGPTL3 expression. The onset of HIF1 expression in the endometrial LE/sGE on Day 12 of pregnancy is temporally associated with PTGS2 expression and an increase in PGs in the uterine lumen [16, 17, 52, 53]. In human endometria, PGE2 increased HIF1A mRNA and protein abundance via the PTGER2 receptor [54]. ANGPTL3 is up-regulated between Days 9 and 12 of pregnancy in the ovine uterus [36]. Collectively, results of the present study indicate that IFNT from conceptus and PGs differentially regulate expression of proangiogenic factors to increase uterine blood flow during early pregnancy in sheep. Increased uterine blood flow is considered crucial to enhancing the availability of micronutrients for transport into the uterine lumen.

The microenvironment of the uterine lumen is critical for the conceptus' peri-implantation survival and development [55]. In the uterine lumen, the amount of recoverable glucose and select amino acids (arginine and glutamine) substantially increase between Days 10 and 16 of pregnancy in sheep [31]. Glucose and amino acids are major sources of energy for developing conceptuses [31], and glucose and select amino acids (arginine and leucine) stimulate hypertrophy, hyperplasia, and migration of trophectoderm cells [56]. The selective delivery of essential nutrients (amino acids and glucose) from maternal serum into the uterus is governed by a number of specific transporters expressed in endometrial epithelia [36, 57–59]. Results of the present study confirm the fact that IFNT increases expression of glucose transporters (SLC2A1, SLC5A1, SLC5A11) in the endometrium of the ovine uterus [20, 59]. Novel findings of the present study are that PGs increase expression of a number of facilitative (SLC2A1, SLC2A5, SLC2A12) and sodium-dependent (SLC5A1, SLC5A11) glucose transporters in the endometrium. Of particular note, all PGs but not IFNT increased SLC2A5 expression in the endometrium. SLC2A5 is up-regulated in the endometrium between Days 9 and 12 of pregnancy and by early P4 in a sheep model of accelerated blastocyst development [36]. Another novel finding of the present study was that IFNT and all infused PGs increased the glucose transporter SLC2A12 in ovine endometrium. SLC2A12 was originally identified in MCF-7 breast cancer cells and breast tumors, which have high glucose requirements [60–62]. Although not previously reported in the sheep uterus, SLC2A12 is expressed in the mouse uterus, oocyte, and at all stages of preimplantation embryos [63, 64]. In situ localization studies revealed that SLC2A12 is specifically expressed in endometrial LE/sGE of sheep uterus and is increased by IFNT, PGE2, and PGI2. SLC1A5 transports neutral (e.g., glutamine, alanine, cysteine, serine, and threonine) and acidic amino acids (glutamate and aspartate), whereas SLC7A2 primarily transports arginine [57, 58], and SLC1A5 is predominantly expressed by the endometrial LE/sGE during early pregnancy in sheep [57]. With respect to amino acid transport, IFNT and all PGs increased expression of SLC1A5, but only IFNT stimulated SLC7A2 in the endometrium. Our results indicate that only IFNT increased the amount of recoverable glucose and select amino acids in the uterine lumen, whereas PGE2 and PGF2α increased the total amount of recoverable amino acids. Based on available results, it appears that IFNT alone is able to increase the expression of all studied glucose transporters to a larger extent than individual PGs. Further, it is likely that conceptus IFNT and PGs have additive or synergistic effects on the transports of glucose and various amino acids into the uterine lumen to support growth and development of the elongating conceptus.

In the present study, we found that IFNT and PGs differentially regulated expression of a number of progesterone-induced and IFNT-stimulated genes (CST3, CTSL, IGFBP1, GRP, HIF2A, LGALS15, SLC2A1, SLC5A11, SLC7A2) that are specifically expressed in the endometrial LE/sGE and/or GE of sheep uterus [8, 9]. However, the molecular mechanism whereby IFNT regulates expression of those genes in the endometrial epithelia is not known. Receptors for IFNT (IFN-alpha subunit receptor 1 or 2 [IFNAR1 and IFNAR2]) are most abundant on endometrial epithelia, but the canonical Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway mediating effects of IFNT in the stroma and GE is not active in the endometrial LE/sGE due to expression of IFN regulatory factor 2 (IRF2), a potent transcriptional repressor of classical type I IFN-stimulated genes including STAT1 and STAT2 [65–67]. Our recent study supports the concept that PTGS2-derived PGs are part of the noncanonical pathway of IFNT action within the endometrium [20]. Indeed, receptors for PGE2, PGF2α, and PGI2 are expressed in the LE, GE, and stroma of the ovine uterus during early pregnancy [20, 23], but the cellular pathways mediating the paracrine effects of conceptus PGs on endometrial function are not known in the sheep. In human uterine decidua, the stimulatory effects of PGs on IGFBP1 expression are mediated by the cAMP/protein kinase A (PKA) signaling pathway [68]. In human endometrial carcinoma cells, the PKC pathway regulates IGFBP1 expression [69]. Membrane receptors for PGE2 and PGI2 are coupled to adenylate cyclase and generate cAMP, whereas stimulation of membrane receptors for PGF2α results in activation of phospholipase C and consequent elevation in calcium levels [70, 71]. Future studies should examine potential interactive effects of IFNT and PGs from the conceptus on endometrial gene expression and functions and ascertain the cellular signaling pathways used by individual PGs within the various cell types in the uterine endometrium.

In summary, results of the present study established that IFNT and PTGS2-derived PGs from the conceptus act in a paracrine manner on the endometrium and differentially regulate gene expression and functions that are important for conceptus growth and development. These results emphasize the importance of PGs along with IFNT during early pregnancy in ruminants. Indeed, PGE2 and PGI2 are critical regulators of blastocyst implantation, decidualization, and uterine angiogenesis during pregnancy in mice, rats, hamsters, mink, and humans [72–74]. Additionally, our studies suggest that feed additives or in vitro embryo culture additives that decrease conceptus or endometrial production of certain PGs or affect PG receptor expression or function may be detrimental to establishment of pregnancy. Indeed, pregnancy rates were substantially reduced in heifers that received meloxicam, a partially selective inhibitor of PTGS2, on Day 15 after insemination [75].

Acknowledgment

The authors thank all members of the Laboratory for Uterine Biology and Pregnancy and Kendrick LeBlanc at Texas A&M University for assistance and for management of the sheep used in this study.

Author notes

¹

Supported by AFRI competitive grants 2009-01722 and 2012-67015-30173 from the USDA National Institute of Food and Agriculture.