Abstract

Peri-implantation conceptus (embryo/fetus and associated extraembryonic membranes) growth and development are primarily regulated by secretions from the uterus. This study investigated the effects of progesterone on preimplantation conceptus development and endometrial galectin 15 (LGALS15). Ewes received daily injections of either corn oil (CO) vehicle or 25 mg progesterone (P4) from 36 h postmating to hysterectomy. Treatment with P4 increased blastocyst diameter by 220% on Day 9 and advanced time of elongation of blastocysts to a filamentous conceptus on Day 12. Effects of P4 treatment on blastocyst development were blocked by administration of RU486, a progesterone receptor antagonist. Consistent with early elongation of blastocysts, interferon tau (IFNT) protein was about 50-fold greater in uterine flushes from Day 12 in ewes receiving P4 compared with those receiving CO. Expression of cathepsin L (CTSL) and radical S-adenosyl methionine domain containing 2 (RSAD2), both IFNT-stimulated genes, was increased in endometria of Day 12 P4-treated ewes. LGALS15 mRNA, expressed only in the endometrial luminal epithelium and superficial glands, was detected between Days 9 and 12 and was more abundant in ewes receiving P4 than in those receiving CO on both Days 9 and 12. RU486 treatment ablated P4 induction of LGALS15 mRNA in the endometrial epithelia. LGALS15 protein in uterine flushings was not different on Day 9 but tended to be greater in P4-treated ewes than in those receiving CO on Day 12. The advanced development of blastocysts in P4-treated ewes is hypothesized to involve early induction of specific genes in the endometrial epithelia, such as LGALS15, and undoubtedly components of uterine histotroph.

Introduction

Maternal support of conceptus (embryo/fetus and associated membranes) growth and development is critical for pregnancy recognition signaling and implantation [1, 2]. In sheep, morula-stage embryos enter the uterus on Days 4 to 5 and the blastocyst, formed by Day 6, contains a blastocoele or central cavity surrounded by a monolayer of trophectoderm [3, 4]. After hatching from the zona pellucida on Day 8, blastocysts develop into a tubular form by Day 11, elongate to 10 cm or more in length by Day 14, and reach 25 cm or more in length by Day 17. Factors supporting growth of pre- and peri-implantation blastocysts and elongating conceptuses are thought to be obtained primarily from secretions of the uterus, collectively referred to as histotroph. This hypothesis is supported by results from studies of asynchronous uterine transfer of embryos and trophoblast vesicles [5, 6] and from studies of uterine gland knockout (UGKO) ewes [7, 8]. The endometrial luminal (LE) and glandular (GE) epithelia are the main sources of uterine secretions, collectively referred to as histotroph.

During early pregnancy, endometrial functions are regulated primarily by progesterone from the corpus luteum and hormones from the placenta [912]. In sheep, pregnancy recognition and establishment involves elongation of the spherical blastocyst to a filamentous conceptus between Days 12 and 16 and production of interferon tau (IFNT) by the conceptus [13, 14]. IFNT is antiluteolytic and acts on the endometrium to inhibit development of the luteolytic mechanism, thereby maintaining corpus luteum (CL) function and ensuring continued production of P4 [15]. Progesterone acts on the uterus to stimulate and maintain endometrial functions necessary for conceptus growth, implantation, placentation and development to term [911, 16]. The concentrations of P4 in early pregnancy clearly affect embryonic survival during early pregnancy [17]. Increasing concentrations of P4 from Days 2 to 5 or Days 5 to 9 enhanced conceptus development and size on Day 14 in heifers [18] and Day 16 in cows [19], while animals with lower concentrations in the early luteal phase had retarded embryonic development [20, 21] and decreased production of interferon tau (IFNT) from bovine conceptuses [20]. In both lactating dairy cows and heifers, there is a strong positive association between early luteal phase plasma P4 concentrations and embryonic survival rate [2224]. Indeed, P4 supplementation of cattle after artificial insemination increased embryonic survival [17, 2528]. However, the mechanisms through which pre-implantation progesterone regulates blastocyst survival and growth are not well investigated, but presumably are mediated by secretions from the endometrium. In sheep, progesterone acts on the endometrium to induce a number of genes that encode for proteins secreted into the uterine lumen, including galectin 15 (LGALS15) and secreted phosphoprotein one (SPP1 or osteopontin) [29, 30]. These proteins are hypothesized to regulate conceptus survival, growth, and adhesion during implantation [4, 31].

This study tested the hypotheses that pre-implantation conceptus growth and survival in the ovine uterus can be stimulated by progesterone and involves increases in expression of specific endometrial genes. The objectives were to determine effects of early progesterone on blastocyst growth and endometrial expression of secreted proteins. Results indicate that progesterone stimulation of blastocyst growth is manifest after shedding of the zona pellucida and is associated with earlier expression of LGALS15 by endometrial epithelia.

Materials and Methods

Animals

Mature Suffolk-type 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.

Experimental Design

Study 1

At estrus, ewes (n = 5) were mated to intact Suffolk rams. Ewes received daily injections of 25 mg progesterone (P4) in corn oil (CO) vehicle from Days 1.5 to 6. On Day 6, the uterine lumen was flushed with 20 ml sterile saline and morphology of the blastocyst(s) examined by light microscopy.

Study 2

At estrus, ewes were mated to intact Suffolk rams and then assigned randomly to receive daily i.m. injections from Days 1.5 to 9 of either corn oil vehicle (CO; n = 6) or 25 mg progesterone (P4; n = 6). All ewes were hysterectomized on Day 9. The uterine lumen was flushed with 20 ml sterile saline. If pregnant, the morphology of the blastocyst(s) was examined by light microscopy, fixed in 4% (wt/vol) paraformaldehyde in PBS (pH 7.2), and diameter measured and images recorded using a Nikon SMZ800 microscope with camera.

At hysterectomy, sections (∼ 0.5 cm) from the mid-portion of each uterine horn ipsilateral to the CL were fixed in fresh 4% paraformaldehyde. After 24 h, fixed tissues were changed to 70% (vol/vol) ethanol for 24 h, dehydrated through a graded series of alcohol to xylene, and then 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 or protein 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 further analysis.

Study 3

At estrus (Day 0), ewes were mated to intact Suffolk rams and assigned randomly to receive daily intramuscular (i.m.) injections of either: (a) corn oil vehicle from Days 1.5 to 12 (CO, n = 8); (b) 25 mg progesterone (P4; Sigma Chemical Co., St. Louis, MO) from Days 1.5 to 12 (P4, n = 7); or (c) 25 mg progesterone (P4; Days 1.5 to 8, n = 5) and then P4 and 75 mg of RU486 (Sigma), a progesterone receptor (PGR) antagonist, from Days 8 to 12 (P4+RU). Blood samples were collected daily from CO- and P4-treated ewes beginning on Day 0 via jugular venipuncture. All ewes were hysterectomized on Day 12, and the uterine lumen flushed with 20 ml sterile saline. If pregnant, the morphology of the blastocyst(s) was examined by light microscopy, fixed in 4% paraformaldehyde, and images of blastocysts captured using a Nikon SMZ800 microscope with camera. The volume of the uterine flush was recorded, clarified by centrifugation (3000 g for 30 min at 4°C), aliquoted and frozen at −80°C. The uteri were then processed as described for study 2.

RNA Isolation

Total cellular RNA was isolated from frozen ipsilateral endometrium of pregnant ewes only (Studies Two and Three) using Trizol reagent (Gibco-BRL, Bethesda, MD) according to manufacturer's recommendations. The quantity and quality of total RNA were determined by spectrometry and denaturing agarose gel electrophoresis, respectively.

Slot Blot Hybridization Analysis

Steady-state levels of LGALS15, CTSL, and RSAD2 mRNA in endometria were assessed by slot blot hybridization using methods described previously [32]. Briefly, radiolabeled antisense and sense cRNA probes were generated by in vitro transcription using linearized plasmid templates containing partial cDNAs, RNA polymerases, and [α-32P]-UTP. Denatured total endometrial RNA (20 μg) from each ewe in Studies One and Two was hybridized with radiolabeled cRNA probes. To correct for variation in total RNA loading, a duplicate RNA slot blot membrane was hybridized with radiolabeled antisense 18S cRNA (pT718S; Ambion, Austin, TX). Following washing, the blots were digested with ribonuclease A and radioactivity associated with slots was quantified using a Typhoon 8600 MultiImager (Molecular Dynamics, Piscataway, NJ). Data are expressed as relative units (RU).

In Situ Hybridization Analysis

Location of CTSL, RSAD2, LGALS15, SPP1 and SERPIN (ovine uterine serine proteinase inhibitor or uterine milk protein) mRNAs in the ovine uterus were determined by radioactive in situ hybridization analysis using methods described previously [32]. Radiolabeled antisense and sense cRNA probes were generated by in vitro transcription using linearized partial plasmid cDNA templates, RNA polymerases, and [α-35S]-UTP. Deparaffinized, rehydrated, and deproteinated uterine tissue sections were hybridized with radiolabeled antisense or sense cRNA probes. 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 5 days. Slides were developed in Kodak D-19 developer, counterstained with Gill's hematoxylin (Fisher Scientific, Fairlawn, NJ), 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 brightfield or darkfield illumination using a Nikon Eclipse 1000 photomicroscope (Nikon Instruments Inc., Lewisville, TX) fitted with a Nikon DXM1200 digital camera.

Immunohistochemistry

Immunocytochemical localization of immunoreactive PGR and LSGAL15 protein in the ovine uterus was performed using methods described previously [29, 33]. PGR protein was detected using a primary mouse monoclonal antibody against human PGR (MA1–411; Affinity Bioreagents, Golden, CO) at a final concentration of 0.25 μg/ml and a Vectastain ABC anti-mouse kit (Vector Laboratories, Inc., Burlingame, CA). LGALS15 protein was detected using rabbit anti-ovine galectin 15 antibody (kindly provided by Dr. Els Meeusen, Australia) [34] at a final dilution of 1:10000 and a Vectastain ABC anti-rabbit kit. For both PGR and LGALS15, antigen retrieval was performed using boiling citrate buffer as described previously [33]. Negative controls included substitution of the primary antibody with mouse IgG (PGR) or rabbit IgG (LGALS15). Immunoreactive protein was visualized using diaminobenzidine tetrahydrochloride (Sigma) as the chromagen. Sections were dehydrated and coverslipped affixed with Permount.

Western Blot Analyses

Protein content of concentrated flushes was determined using a Bradford protein assay (Bio-Rad, Hercules, CA) with BSA as the standard. Uterine flush proteins (30 μg) were denatured and separated by 15% SDS-PAGE for both IFNT (study 1) and LGALS15 (studies 1 and 2). Western blot analyses were conducted as described previously [35] using enhanced chemiluminescence detection. Immunoreactive IFNT was detected using primary rabbit anti-ovine IFNT serum at a 1:5000 dilution. Rabbit anti-ovine IFNT serum was generated by immunizing rabbits with recombinant ovine IFNT [36].

Slot Blot Quantification of Proteins in Uterine Flushings

Uterine flushings (30 μg for IFNT and 20 μg for LGALS15) were diluted to 200 μl final volume with Tris buffered saline (TBS). Nitrocellulose membranes (Schleicher and Shuell), presoaked with TBS, was loaded into the slot blot apparatus backed by Whatman filter paper. The wells were subsequently washed with 200 μl TBS prior to addition of the diluted sample, and then washed again with 200 μl TBS. The membrane was allowed to air dry and then blocked in 5% (wt/vol) milk/TBS-tween (TBST) for 1 h at room temperature. The membrane was incubated in either primary rabbit anti-ovine IFNT serum at 1:1000 dilution (30 μg protein per slot) or rabbit anti-ovine LGALS15 serum [34] at 1:2500 dilution (20 μg protein per slot) in 2.5% milk/TBST overnight at 4°C. The blot was then washed for 30 min in TBST followed by incubation with goat anti-rabbit IgG horseradish peroxidase conjugate at 1:20000 diluted in 2.5% milk/TBST for 1 h at room temperature. The blot was washed again for 30 min in TBST and immunoreactive proteins were detected using enhanced chemiluminescence. Blots were imaged and quantified using a Typhoon 8600 Variable Mode Imager (Amersham Biosciences Corp., Piscataway, NJ). The total amounts of IFNT and LGALS15 protein were calculated based on the amount of uterine flush protein loaded into each well, the concentration of protein in the uterine flush, and the recovered volume of the uterine flush recorded at surgery.

Radioimmunoassay

Blood samples were allowed to clot for 1 h at room temperature. Serum was then collected following centrifugation (3000 x g for 30 min at 4°C) and stored at −20°C for hormone analysis. Concentrations of progesterone in serum were determined according to manufacturer's specifications using an antiserum highly specific for progesterone (DSL-3900 ACTIVE Progesterone Coated-Tube Radioimmunoassay Kit, Diagnostic Systems Laboratories, Webster, TX). The RIA used rabbit anti-progesterone immunoglobulin coated tubes and iodinated progesterone. The primary anti-serum cross-reacts 6.0%, 2.5%, 1.2%, 0.8%, 0.48%, and 0.1% with 5α-Pregnane-3,20-dione, 11-Deoxycorticosterone, 17α-Hydroxyprogesterone, 5β-Pregnane-3,20-dione, 11-Deoxycortisol, and 20α-Dihydroprogesterone, respectively. The progesterone standard curve (1.0–190.8 nmol/L) was provided in the assay kit. The intra-assay variation was 10.1%. Assay results were calculated using the AssayZap Version 3.1 program (Biosoft, Ferguson, CA).

Photomicroscopy

Photomicrographs of in situ hybridization and immunocytochemistry slides were taken using a Nikon Eclipse E1000 photomicroscope (Nikon Instruments, Melville, NY). Digital images were captured using a Nikon DXM 1200 digital camera and assembled using Adobe Photoshop 7.0 (Adobe Systems, Seattle, WA). Images of embryos were captured using a Nikon SMZ800 microscope with camera.

Statistical Analyses

Data from radioimmunoassay, slot blot hybridization, and protein slot blot analyses were subjected to least-squares analysis of variance using the General Linear Models procedures of the Statistical Analysis System (SAS Institute, Cary, NC). Slot blot hybridization data were corrected for differences in sample loading by using the 18S rRNA data as a covariate. Protein slot blot data was corrected for differences in conceptus number by using them as a covariate. Data are presented as the least-squares means (LSM) with overall standard error (SEM).

Results

Circulating Progesterone

Circulating concentrations of progesterone in study 3 were determined by radioimmunoassay of serum from jugular venous blood (Figure 1). Progesterone increased (linear, P < 0.01) after Day 3 and reached maximal levels by Day 11 in CO ewes. Exogenous treatment of ewes with P4 beginning at 36 h postmating increased concentrations of progesterone in serum after Day 1, and they remained higher in P4 than CO ewes to Day 12 (P < 0.0001, day x treatment).

Fig. 1

Concentrations of progesterone in serum of ewes treated daily with either corn oil (CO) vehicle or progesterone (P4) from 36 h postmating to Day 12. Concentrations of progesterone were higher in P4 than CO ewes after Day 1 (day x treatment, P<0.01). Data is presented as LSM with overall SEM

Fig. 1

Concentrations of progesterone in serum of ewes treated daily with either corn oil (CO) vehicle or progesterone (P4) from 36 h postmating to Day 12. Concentrations of progesterone were higher in P4 than CO ewes after Day 1 (day x treatment, P<0.01). Data is presented as LSM with overall SEM

Early Progesterone Enhances Blastocyst Development

In all studies, ewes assigned to P4 treatment group received P4 early beginning 36 h postmating (Day 1.5). In study 1, embryos, recovered on Day 6 from P4 ewes were morphologically classified as either morulae or blastocysts, with 4 of 5 possessing an intact zona pellucida (data not shown). In study 2, hatched blastocysts were recovered on Day 9 from both CO- and P4-treated ewes (Figure 2). Of particular interest was the fact that blastocyst diameters were 220% greater (P < 0.02) in P4 (636 ± 64 μm) than CO (282 ± 64 μm) ewes. In study 3, the blastocysts recovered from CO-treated ewes were spherical to slightly tubular in morphology on Day 12 (Figure 2). In contrast, blastocysts recovered from P4-treated ewes were elongated and filamentous. No blastocysts were recovered from ewes receiving P4+RU treatment. Therefore, early P4 treatment enhanced blastocyst growth and development after hatching from the zona pellucida, but did not affect time of blastocyst hatching.

Fig. 2

Effects of early exogenous progesterone treatment on blastocyst morphology on Days 9 and 12 of pregnancy. In study 2, ewes were treated daily with corn oil (CO) vehicle or progesterone (P4) from 36 h postmating. On Day 9, blastocyst diameter was greater (P < 0.02) for ewes receiving P4 (636 ± 64 μm) than CO (282 ± 64 μm). In study 1, ewes were treated daily with CO or P4 from 36 h postmating to Day 12 or P4 from 36 h postmating to Day 8, followed by P4 and RU486 (P4+RU) from Days 8 to 12. Elongated and filamentous conceptuses were recovered from P4-treated ewes, while only spherical to early tubular blastocysts were recovered from CO-treated ewes. No blastocysts were recovered from P4+RU treated ewes. Bar = 400 μm

Fig. 2

Effects of early exogenous progesterone treatment on blastocyst morphology on Days 9 and 12 of pregnancy. In study 2, ewes were treated daily with corn oil (CO) vehicle or progesterone (P4) from 36 h postmating. On Day 9, blastocyst diameter was greater (P < 0.02) for ewes receiving P4 (636 ± 64 μm) than CO (282 ± 64 μm). In study 1, ewes were treated daily with CO or P4 from 36 h postmating to Day 12 or P4 from 36 h postmating to Day 8, followed by P4 and RU486 (P4+RU) from Days 8 to 12. Elongated and filamentous conceptuses were recovered from P4-treated ewes, while only spherical to early tubular blastocysts were recovered from CO-treated ewes. No blastocysts were recovered from P4+RU treated ewes. Bar = 400 μm

Immunolocalization of PGR Protein

PGR protein was most abundant in endometrial LE and GE with lower levels in the stroma of Day 9 CO-treated ewes (Figure 3). For P4-treated ewes on Day 9 (study 2), PGR protein was markedly lower in the endometrial cells, particularly in the epithelia. In Day 12 CO-treated ewes (study 3), PGR protein was either very low or not detectable in the nuclei of the endometrial LE. In P4-treated ewes, PGR abundance was markedly reduced as compared with CO ewes, particularly in the endometrial LE and GE. In P4+RU-treated ewes, PGR protein was higher in endometrial stroma and GE, but not different in the LE.

Fig. 3

Effects of CO, P4, or P4 and RU486 on expression of progesterone receptor (PGR) protein in endometria from Day 9 (study 2) and Day 12 (study 3) ewes. Immunoreactive PGR protein was detected using a mouse monoclonal antibody against human PGR. For the IgG control, normal mouse IgG was substituted for the primary antibody. Sections were not counterstained. LE, luminal epithelium; GE, glandular epithelium; S, stroma. All photomicrographs are shown at the same width of field (420 μm)

Fig. 3

Effects of CO, P4, or P4 and RU486 on expression of progesterone receptor (PGR) protein in endometria from Day 9 (study 2) and Day 12 (study 3) ewes. Immunoreactive PGR protein was detected using a mouse monoclonal antibody against human PGR. For the IgG control, normal mouse IgG was substituted for the primary antibody. Sections were not counterstained. LE, luminal epithelium; GE, glandular epithelium; S, stroma. All photomicrographs are shown at the same width of field (420 μm)

IFNT and IFNT-stimulated Genes

As illustrated in Figure 4, immunoreactive IFNT (17 kDa) was detected in uterine flushings from all P4-treated ewes on Day 12, but was markedly lower or absent in uterine flushings from Day 12 CO ewes. IFNT was not detected in uterine flushings from either CO or P4 ewes on Day 9 (study 2) as well as on Day 12 if ewes received P4+RU treatment (study 3). As determined by protein slot blot analyses, relative total amounts of IFNT protein in uterine flushings from Day 12 ewes in study 3 were approximately 155-fold greater (P < 0.0002) in P4 (12 126 ± 2 763 relative units) compared with CO ewes (77 ± 10 relative units).

Fig. 4

Western blot analysis of IFNT in uterine flushings from Day 12 ewes. In study 3, ewes were treated daily with CO or P4 from 36 h postmating to Day 12 or P4 from 36 h postmating to Day 8, followed by P4 and RU486 (P4+RU) from Days 8 to 12. Proteins in uterine flushings were analyzed by 15% SDS-PAGE (30 μg/lane) and Western blot analysis using rabbit antiovine IFNT serum. IFNT was detected in uterine flushings from all ewes treated with P4. Representative results from 3 ewes per treatment are presented

Fig. 4

Western blot analysis of IFNT in uterine flushings from Day 12 ewes. In study 3, ewes were treated daily with CO or P4 from 36 h postmating to Day 12 or P4 from 36 h postmating to Day 8, followed by P4 and RU486 (P4+RU) from Days 8 to 12. Proteins in uterine flushings were analyzed by 15% SDS-PAGE (30 μg/lane) and Western blot analysis using rabbit antiovine IFNT serum. IFNT was detected in uterine flushings from all ewes treated with P4. Representative results from 3 ewes per treatment are presented

Cathepsin L (CTSL) is expressed only in the endometrial LE and superficial GE in response to both P4 and IFNT [37, 38]. Endometrial CTSL mRNA levels were not different (P > 0.10) on Day 9 in study 2. In study 3, endometrial CTSL mRNA levels were about 2-fold higher (P < 0.05) in P4 than CO ewes and about 4-fold higher in P4 than P4+RU ewes on Day 12 (Figure 5A). As expected, CTSL mRNA was detected only in the endometrial LE and superficial GE of uteri from CO and P4 ewes in both studies (Figure 5C).

Fig. 5

Effects of treatments with CO, P4, or P4 and RU486 on endometrial CTSL and RSAD2 mRNA. A) Steady-state levels of CTSL mRNA in the endometria from Day 9 (study 2) and Day 12 (study 3) ewes, as determined by slot blot analysis, increased only in endometria of P4-treated ewes on Day 12 (*, P < 0.05). Data are presented as relative units (RU) with SEM. B) Steady-state levels of RSAD2 mRNA in the endometrium of Day 12 ewes (study 3) increased only in endometria from P4-treated ewes on Day 12 (*, P < 0.05). C) In situ localization of CTSL and RSAD2 mRNAs in endometria of Day 9 (study 2) and Day 12 ewes (study 3) are presented. Cross-sections of uteri were hybridized with radiolabeled antisense or sense ovine cRNA probes for each respective gene. LE, luminal epithelium; S, stroma; sGE, superficial glandular epithelium. All representative photomicrographs are shown at the same width of field (630 μm)

Fig. 5

Effects of treatments with CO, P4, or P4 and RU486 on endometrial CTSL and RSAD2 mRNA. A) Steady-state levels of CTSL mRNA in the endometria from Day 9 (study 2) and Day 12 (study 3) ewes, as determined by slot blot analysis, increased only in endometria of P4-treated ewes on Day 12 (*, P < 0.05). Data are presented as relative units (RU) with SEM. B) Steady-state levels of RSAD2 mRNA in the endometrium of Day 12 ewes (study 3) increased only in endometria from P4-treated ewes on Day 12 (*, P < 0.05). C) In situ localization of CTSL and RSAD2 mRNAs in endometria of Day 9 (study 2) and Day 12 ewes (study 3) are presented. Cross-sections of uteri were hybridized with radiolabeled antisense or sense ovine cRNA probes for each respective gene. LE, luminal epithelium; S, stroma; sGE, superficial glandular epithelium. All representative photomicrographs are shown at the same width of field (630 μm)

Radical S-adenosyl methionine domain containing 2 (RSAD2), also known as viperin, is an IFNT-stimulated gene [38]. RSAD2 mRNA was not detected in endometria from Day 9 ewes in study 3 (Figure 5C). Endometrial RSAD2 mRNA levels were about 9-fold higher (P < 0.05) in endometria of P4 versus CO- or P4- versus P4+RU-treated ewes on Day 12 in study 3 (Figure 5B). In situ hybridization analysis localized RSAD2 mRNA primarily to the stratum compactum stroma of the endometrium in Day 12 P4-treated ewes (Figure 5C). Thus, IFNT-stimulated genes are only induced in the endometrium from P4-treated Day 12 ewes with an elongated and filamentous conceptus that secretes IFNT.

LGALS15, SPP1, and Uterine SERPIN

LGALS15, SPP1, and SERPIN are progesterone-induced genes expressed in the endometrial epithelia during early pregnancy [29, 30, 39]. In study 2, steady-state levels of LGALS15 mRNA were about 3-fold higher (P < 0.01) in the endometria of P4- compared with CO-treated ewes on Day 9 (Figure 6A). Similarly, endometrial LGALS15 mRNA levels were approximately 2-fold higher (P < 0.01) in P4- compared with CO-treated ewes on Day 12 in study 3 (Figure 6A). Further, LGALS15 mRNA was approximately 59-fold and 136-fold lower (P < 0.01) in endometria of P4+RU-treated ewes compared with ewes receiving CO or P4, respectively. As expected, LGALS15 mRNA was present in LE and superficial GE of endometrium from Day 9 P4 and Day 12 CO and P4-treated ewes, but absent in endometria from Day 9 CO ewes and Day 12 ewes that received P4+RU treatment (Figure 6B). The melanocytes present in the sub-epithelial stroma of Day 12 CO ewes were not positive for LGALS15 mRNA, but rather diffract light and appeared white under darkfield conditions.

Fig. 6

Effects of treatments with CO, P4, or P4 and RU486 on endometrial LGALS15 mRNA. A) Steady-state levels of LGALS15 mRNA in endometria from ewes on Day 9 (study 2) and Day 12 (study 3), determined by slot blot analyses, indicated increased expression in endometria of P4-treated ewes on Days 9 and 12 (*, P < 0.01) and lower (P < 0.001) expression in endometria of P4+RU-treated ewes compared with P4- or CO-treated ewes. Data are presented as relative units (RU) with SEM. B) In situ localization of LGALS15 mRNA in endometria from Day 9 (study 2) and Day 12 ewes (study 3). Cross-sections of uteri were hybridized with radiolabeled antisense or sense ovine cRNA probes for LGALS15. LE, luminal epithelium; S, stroma; sGE, superficial glandular epithelium. The black melanocytes in the stratum compactum of Day 12 endometria do not express LGALS15 mRNA, but appear white in a darkfield photomicrograph. All representative photomicrographs are shown at the same width of field (420 μm)

Fig. 6

Effects of treatments with CO, P4, or P4 and RU486 on endometrial LGALS15 mRNA. A) Steady-state levels of LGALS15 mRNA in endometria from ewes on Day 9 (study 2) and Day 12 (study 3), determined by slot blot analyses, indicated increased expression in endometria of P4-treated ewes on Days 9 and 12 (*, P < 0.01) and lower (P < 0.001) expression in endometria of P4+RU-treated ewes compared with P4- or CO-treated ewes. Data are presented as relative units (RU) with SEM. B) In situ localization of LGALS15 mRNA in endometria from Day 9 (study 2) and Day 12 ewes (study 3). Cross-sections of uteri were hybridized with radiolabeled antisense or sense ovine cRNA probes for LGALS15. LE, luminal epithelium; S, stroma; sGE, superficial glandular epithelium. The black melanocytes in the stratum compactum of Day 12 endometria do not express LGALS15 mRNA, but appear white in a darkfield photomicrograph. All representative photomicrographs are shown at the same width of field (420 μm)

LGALS15 protein was localized predominantly to endometrial LE and superficial GE in uteri from Day 9 and Day 12 ewes (Figure 7A). In Day 9 ewes (study 2), LGALS15 protein was more abundant at the apical surface of the endometrial LE in P4- compared with CO-treated ewes (as denoted by arrowhead). LGALS15 protein was abundant in endometrial LE and superficial GE of uteri from Day 12 in CO- and P4-treated ewes (study 2). LGALS15 protein was also detected in the LE and superficial GE of the endometria from Day 12 P4+RU-treated ewes, but the immunoreactive protein was concentrated more toward the apical surface of the epithelia rather than uniformly distributed throughout the epithelia as in Day 12 CO- and P4-treated ewes.

Fig. 7

Effects of treatment of ewes with CO, P4, or P4 and RU486 on endometrial LGALS15 protein in the uterus. A) Immunolocalization of LGALS15 protein in endometria from Day 9 (study 2) and Day 12 (study 3) ewes revealed an increase in apical distribution in the luminal epithelium (LE) of P4-treated ewes from Day 9 (denoted by arrowhead). For the IgG control, normal rabbit IgG was substituted for the primary antibody. Representative photomicrographs are shown at the same width of field (420 μm) with the exception of the higher magnification (210 μm width of field) images of Day 9 CO and P4 endometrial LE (upper right). Sections were not counterstained. LE, luminal epithelium; sGE, superficial ductal glandular epithelium; S, stroma. B) Total amounts of LGALS15 protein in uterine flushings from Day 9 ewes (study 2) were not different between CO and P4-treated ewes. Data are presented as relative units (RU) with SEM. C) LGALS15 protein in uterine flushings from Day 12 ewes (study 3) tended to be higher (P < 0.06) for ewes treated with P4 or P4+*RU compared with those treated with CO

Fig. 7

Effects of treatment of ewes with CO, P4, or P4 and RU486 on endometrial LGALS15 protein in the uterus. A) Immunolocalization of LGALS15 protein in endometria from Day 9 (study 2) and Day 12 (study 3) ewes revealed an increase in apical distribution in the luminal epithelium (LE) of P4-treated ewes from Day 9 (denoted by arrowhead). For the IgG control, normal rabbit IgG was substituted for the primary antibody. Representative photomicrographs are shown at the same width of field (420 μm) with the exception of the higher magnification (210 μm width of field) images of Day 9 CO and P4 endometrial LE (upper right). Sections were not counterstained. LE, luminal epithelium; sGE, superficial ductal glandular epithelium; S, stroma. B) Total amounts of LGALS15 protein in uterine flushings from Day 9 ewes (study 2) were not different between CO and P4-treated ewes. Data are presented as relative units (RU) with SEM. C) LGALS15 protein in uterine flushings from Day 12 ewes (study 3) tended to be higher (P < 0.06) for ewes treated with P4 or P4+*RU compared with those treated with CO

The relative amount of LGALS15 protein in uterine flushings was determined in both studies. For Day 9 ewes, total LGALS15 protein in uterine flushings was not different (P > 0.10) in P4- compared with CO-treated ewes (Figure 7B); however, LGALS15 protein tended (P = 0.06) to be more abundant in uterine flushings from P4- compared with CO-treated ewes on Day 12 as well as in uterine flushings from ewes receiving P4 compared with ewes treated with P4+RU (Figure 7C).

SPP1 (osteopontin) and SERPIN mRNAs were not detected in endometria of any ewes in study 1 or study 2 (data not shown).

Discussion

Advancement of development of conceptuses at varying time points by administration of early exogenous progesterone has been described in both cattle [19, 40] and in sheep [41, 42]. In the present study, early exogenous P4 treatment accelerated growth of hatched blastocysts as evidenced by increased blastocyst diameter on Day 9 in P4-treated ewes and the presence of elongated and filamentous conceptuses on Day 12 in uteri from P4-treated ewes. Indeed, blockade of progesterone actions by administration of RU486 from Days 8 to 12 resulted in loss of all embryos. This supports the concept that there is an unequivocal requirement for progesterone actions for maintenance of pregnancy in sheep, as there is in other mammals. Importantly, hatching of the blastocyst from the zona pellucida was not accelerated by exogenous progesterone administration, nor did it prevent transport of embryos from the oviduct into the uterus. Therefore, early P4 effects appear to be manifest after hatching of the blastocyst from the zona pellucida, which normally occurs on Day 8 [4]. Thus, the zona pellucida may act as a barrier to embryotrophic factors in the uterine histotroph, and hatching is required for them to gain access to the blastocyst.

Advancement of blastocyst growth coincided with functional development in the present study, because blastocysts from early P4-treated ewes secreted greater quantities of IFNT on Day 12 than blastocysts recovered from CO-treated ewes. In fact, IFNT production by the blastocyst is developmentally regulated and affected by uterine factors [43, 44]. Further evidence for effects of IFNT was induction of RSAD2 in the stroma and increased CTSL and LGALS15 in LE and sGE of endometria from Day 12 P4-treated ewes. CTSL, LGALS15, and RSAD2 are IFNT-stimulated genes in the ovine uterus [29, 37, 38]. Thus, timely communication between maternal endometrium and conceptus development allowed maintenance of synchrony and establishment of pregnancy in the present study. Since early exposure of the uterus to P4 accelerates development of the luteolytic mechanism, it is essential that P4 stimulate development of the conceptus so that it secretes sufficient quantities of IFNT to abrogate development of the luteolytic mechanism [4, 13]. In dairy cattle, an early increase in circulating concentrations of P4 was highly correlated with increased production of IFNT by the conceptus as well as decreased quantities of circulating prostaglandin metabolite, suggesting that the luteolytic mechanism is inhibited by advanced blastocysts [19, 20].

Our working hypothesis is that the stimulatory effects of P4 on blastocyst survival and growth are mediated by specific effects of P4 on the endometrium. During the pre-implantation period, P4 is essential for blastocyst survival and acts through receptors present in all endometrial cell types. Continuous exposure of the endometrium to P4 for 8–10 days down-regulates PGR expression in endometrial epithelia [45], so that PGR protein is not detectable in endometrial LE and GE in ewes after Days 11 and 13 of pregnancy, respectively [46, 47]. The paradigm of loss of PGR in uterine epithelia immediately before implantation is common across mammals [9, 48]. Thus, induction of several genes in the LE and superficial GE implicated in conceptus implantation, including LGALS15, CST3, and CTSL, and also in the GE, including SPP1, SERPINs, and STC1, requires loss of epithelial cell PGR [29, 30, 37, 38, 4951]. Loss of epithelial PGR is thought to reprogram patterns of gene expression in the endometrial epithelia.

One of the most abundant P4-regulated genes in sheep uteri is LGALS15 [29, 52]. Galectin proteins have a conserved carbohydrate recognition domain that bind beta-galactosides, thereby cross-linking glycoproteins as well as glycolipid receptors on the surface of cells and initiating biologic responses that include cell adhesion, growth, and differentiation [53, 54]. In cyclic and pregnant ewes, induction of LGALS15 mRNA occurs between Days 10 and 12 in endometrial LE and superficial GE [29], which is associated with the decline and loss of PGR from those epithelia [46, 47]. Further, P4 induction of LGALS15 mRNA in endometrial LE and superficial GE is inhibited by a PGR antagonist [29, 38]. Of particular importance, LGALS15 is a novel gene expressed only by LE and superficial GE by intrauterine infusions of IFNT in progestinized ewes [29, 38]. In the present study, LGALS15 mRNA was induced by early exogenous P4 in study 2 (Day 9) and stimulated in study 3 (Day 12). Further, administration of RU486, a PGR antagonist, ablated LGALS15 mRNA expression in endometrial epithelia which supports evidence that LGALS15 is induced by progesterone (via down-regulation of the PGR) and further stimulated by IFNT from the developing conceptus.

In the present study, LGALS15 protein was higher at the apical surface of endometrial LE of Day 9 P4-treated ewes and tended to be more abundant in uterine flushings from Day 12 P4-treated ewes. Although secreted LGALS15 protein was not greater in the uterine flush of Day 9 P4 ewes, LGALS15 protein was more concentrated near the apical surface of the LE of endometria. It is likely that a factor from the conceptus during trophoblast outgrowth and elongation stimulates the secretion of proteins, such as LGALS15, from the endometrial epithelia. Nonetheless, stimulation of blastocyst growth and development in response to early exogenous P4 treatment was strongly associated with increases in endometrial LGALS15 mRNA in studies 2 and 3 and in uterine flush LGALS15 protein in study 3 (Day 12). Available results indicate that LGALS15 protein is synthesized and secreted by endometrial LE and superficial GE into the uterine lumen, where it is absorbed by conceptus trophectoderm [29]. Indeed, LGALS15 protein is detectable on the surface of the trophectoderm and within crystalline structures found inside the trophectoderm [29, 52]. Thus, the amount of LGALS15 protein present in the uterine lumen of Day 12 P4-treated ewes may have been greater than for CO ewes, except that the elongated and filamentous conceptuses of the P4-treated ewes may have imbibed a large amount of the secreted protein. In P4+RU-treated ewes, LGALS15 mRNA was not present in the endometrium, but LGALS15 protein was observed in the endometrial LE and abundant in the uterine flushings. The crystal nature of LGALS15 protein may be responsible for low levels of immunoreactive protein in the LE which do not express LGALS15. Further, LGALS15 is a secreted lectin that presumably binds glycoconjugates that are part of the extracellular matrix and rather stable, potentially further accounting for the low levels of LGALS15 protein observed in cells not expressing the gene. SPP1 and uterine SERPIN are well-characterized progesterone-induced genes that appear specifically in the endometrial glands between Days 14 to 16 (SPP1) and 16 to 18 (SERPIN) of pregnancy [31, 55]. These genes were not responsible for effects of progesterone on pre-implantation blastocyst growth and development, because SPP1 and uterine SERPIN mRNAs were not detected in endometria from any ewes in the present studies.

Collectively, these results illustrate that rate of development of blastocysts in utero is stimulated by actions of P4 on the uterus and support the hypothesis that P4 acts on the endometrium to down-regulate expression of PGR in epithelia and to induce expression of specific genes that encode secreted proteins, such as LGALS15, that stimulate blastocyst growth and development. Indeed, a large number of undefined components of histotroph, including other secreted proteins, amino acids, sugars and ions, may also be regulated by progesterone during the peri-implantation period of pregnancy to stimulate conceptus growth and development [10, 56]. Results of the present study allow further development of a model in which early exogenous P4 treatment can advance conceptus development to allow it to maintain synchrony with the endometrium, as evidenced by advanced onset of IFNT production by the conceptus and induction of IFNT-stimulated genes in the endometrium that are hypothesized to be required for conceptus implantation and establishment and maintenance of pregnancy. Future studies will use genomic and proteomic approaches to identify the mechanisms by which progesterone acts on the endometrium to enhance blastocyst survival and growth after hatching from the zona pellucida.

Acknowledgments

The authors thank Kanako Hayashi, Gwonhwa Song, Greg Johnson, and Frankie White for their technical assistance and other members of the laboratory for their support.

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Author notes

1
Supported by the National Research Initiative Competitive Grant No. 2005-35203-16252 from the USDA Cooperative State Research, Education and Extension Service and NIH Grants 5-R01-HD32534 and 5-P30-ES09106.