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Laboratory of Animal Breeding,3 Graduate School of Agriculture and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan
Department of Obstetrics and Gynecology,4 University of Pennsylvania Medical Center, University of Pennsylvania, Philadelphia, Pennsylvania 19104
Research Unit in Reproductive Medicine,5 Hospital de Ginecobstetricia "Dr. Luis Castelazo Ayala," Instituto Mexicano del Seguro Social, Mexico D.F. 10101, Mexico
Korea Research Institute of Bioscience and Biotechnology,6 Taejon, 305-333, Korea
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cytokines, early development, embryo, growth factors implantation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and epidermal growth factor [8]. These factors can influence embryonic development and implantation in an autocrine/paracrine manner [9]. The detailed molecular mechanisms governing embryo implantation and endometrial receptivity are still poorly understood. Progranulin, also known as acrogranin [10, 11], originally was purified from guinea pig testes. Subsequently, other investigators independently characterized this protein as the epithelin/granulin precursor [12] or as teratoma-derived adipogenic cell line 1246 (PC cell)–derived growth factor [13]. Progranulin, a growth factor in its own right, has been reported in various studies as having a molecular weight of 88 or 68 kDa [14, 15]. It is the parent molecule from which the approximately 6-kDa, cysteine-rich granulin/epithelin growth modulators are derived. Progranulin is highly expressed in the hypothalamus of newborn male rats [16] and is involved in sexual differentiation of the rat brain [17]. In the early blastocyst, this protein is present only in the trophectoderm [18] and is thought to be involved in embryonic and neonatal development. In vitro-culture experiments have demonstrated that progranulin can regulate the emergence of the trophectoderm in the developing mouse blastocyst as well as the subsequent growth and function in the embryonic epithelium [18]. Progranulin also may play proliferative and developmental roles during gastrulation and embryonic development of the epidermis, nervous system, and blood vessels, as well as during spermatogenesis [19]. However, little is known about the presence and potential functions of progranulin during the peri-implantation period in the mouse. Recently, transcript changes in mouse blastocysts during pre- and postadhesion periods were analyzed using cDNA microarray technology, and Grn transcript was found to be strongly expressed in pre- and postadhesion blastocysts. Transcription levels of Grn were higher in postadhesion blastocysts than in preadhesion blastocysts [20].
Based on the expression patterns of Grn transcript in pre- and postadhesion blastocysts, we hypothesized that progranulin plays a role in regulating the embryonic implantation process in the mouse. Therefore, we examined the expression patterns of progranulin and Grn mRNA in mouse embryos during the peri-implantation period. The potential functions of progranulin in regulating implantation processes—hatching, adhesion, and trophoblast outgrowth—also were examined.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female (age, 6 wk) and male (age, 8 wk) ICR and CF-1 mice were purchased from SLC (Shizuoka, Japan) and Charles River Laboratories (Wilmington, MA), respectively. These mice were maintained at a controlled temperature (25°C) under a constant photoperiod (14L:10D), and food and water were given ad libitum. After 4 days of acclimatization, female mice in estrus were mated with males of the same strain (1:1). The following morning, females were inspected, and if a copulatory plug was found, that day was designated as Day 0.5 of pregnancy. These female mice were killed humanely by cervical dislocation or by CO2 asphyxiation on Day 3.5 of pregnancy. The protocols for mouse experimentation were approved by the respective animal care committees at the University of Tokyo and University of Pennsylvania.
Blastocyst Culture and Collection
Development of early blastocysts flushed with Dulbecco modified Eagle medium (DMEM; Sigma, St. Louis, MO) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma), 100 U/ml of penicillin, and 40 µg/ml of streptomycin (DMEM plus 10% FBS) was analyzed using a transfilter assay as described previously [20, 21]. Briefly, early blastocysts on Day 3.5 of pregnancy were placed onto a polyethylene terephthalate track-etched membrane (pore size, 3.0 µm; Becton Dickinson and Company, San Jose, CA) that had been positioned over a uterine stromal cell monolayer and cocultured in DMEM plus 10% FBS under 95% air and 5% CO2. At 24 h after initiation of the in vitro culture, blastocysts that did not hatch from the zona pellucida were designated as preadhesion blastocysts. At 36 h after initiation of the coculture, blastocysts that hatched from the zona pellucida and adhered were designated as postadhesion blastocysts. When trophoblast cells had grown outward and the primary giant trophoblast cells became visible around attached blastocysts after 48 h of coculture, these blastocysts were designated as outgrowth embryos [22]. Early blastocysts, pre- and postadhesion blastocysts, and outgrowth embryos were collected and subjected to RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR) analysis or fixed in 4% paraformaldehyde/PBS at 4°C overnight for whole-mount in situ hybridization analysis.
For progranulin analysis, early blastocysts also were cultured in DMEM plus 3% FBS under 95% air and 5% CO2. Embryos at different developmental stages were designated as described above. These embryos were collected and lysed with the solution containing 1% NP-40, 0.1% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 0.1% SDS, 1 mM EDTA, 10 mM PMSF, 4 µg/ml of aprotinin, and 1 µg/ml of leupeptin for immunoblot analysis or were fixed in 4% paraformaldehyde/ PBS at 4°C overnight for whole-mount immunohistochemistry analysis. In addition, embryo culture media were collected at 24, 36, and 48 h after the initiation of culture, concentrated, and subjected to Western blot analysis.
RNA Extraction and RT-PCR
Total RNAs from staged blastocysts (200 blastocysts each) were extracted using Isogen Reagent (Nippon Gene, Toyama, Japan) according to the manufacturer's protocol. The RNA extracted from each sample then underwent RT into cDNA using oligo(dT) 12–18 primers and SuperScript II (Gibco BRL Life Technologies, Rockville, MD) according to the protocol suggested by the manufacturer, and these RT products (50 µl/stage) were used as templates for PCR analysis.
Levels of Grn mRNA in stage-specific, peri-implantation blastocysts were examined by PCR. Primers used for Grn mRNA detection were 5'-gct gtg aag aca gag tgc att g-3' (forward) and 5'-tcc agg gta ctt gga gga tac c-3' (reverse) [20]. The PCR mixture consisted of 1 µl of RT product, 1 µl of 10x PCR buffer, 0.4 µl each of forward and reverse primers (10 pM), 0.2 µl of dNTP mixture (10 mM), 0.3 µl of MgCl2 (50 mM), 6.6 µl of ddH2O, and 0.1 µl of Taq DNA polymerase (5 U/µl; Invitrogen, Carlsbad, CA). The PCR was performed under the following conditions: 94°C for 5 min, followed by 35 cycles of 94°C for 45 seconds, 57°C for 45 seconds, and 72°C for 1 min. The PCR product was analyzed by electrophoresis on 1% agarose gel stained with ethidium bromide. The cDNA fragment was extracted from the agarose gel using a QIAquick Gel Extraction Kit (Qiagen, Tokyo, Japan) and then cloned into pGEM T-easy vector (Promega, Madison, WI), and the nucleotide sequences were determined by DNA sequencing (ABI-PRISM, Foster City, CA). Glyceraldehyde 3-phosphate dehydrogenase (G3PDH) mRNA was used as an internal control.
Whole-Mount In Situ Hybridization
Preparation of Grn cRNA probe and whole-mount in situ hybridization were performed as described previously [20]. In brief, the Grn cDNA-pGEM T-easy construct was digested with the restriction endonucleases SacII and SalI for sense and antisense cRNA probes, respectively. Sense and antisense cRNA probes were labeled using the digoxigenin-labeling kit (Roche Molecular Biomedicals, Mannheim, Germany) according to the manufacturer's protocol. For whole-mount in situ hybridization, blastocysts were prehybridized and then incubated in the hybridization buffer containing 1 µg/ml of digoxigenin-labeled sense or antisense Grn cRNA probe overnight. After serial washing and RNase treatment, blastocyst samples were blocked and incubated with alkaline phosphatase-conjugated antidigoxigenin antibody (1:2000 dilution; Roche Molecular Biochemicals) in the blocking buffer at room temperature for 1 h. After intense washing, signal was visualized through the use of nitro blue tetrazolium (Promega) and 5-bromo-4-chloro-3-indolyl-phosphate (Promega) until the color developed to the desired extent. All procedures were performed in a 96-well plate (Nuclon, Roskilde, Denmark) except for the experiment with outgrowth embryos, which was performed in a four-well plate (Nuclon).
Whole-Mount Immunohistochemistry
Blastocyst samples were collected as described above. Fixed blastocysts were washed three times with the blocking buffer containing 20 mM Tris-HCl (pH 7.4), 20 mM glycine, 130 mM NaCl, and 10 mg/ml of BSA. After 10 min of incubation in the blocking buffer plus 0.1% Triton X-100, these blastocysts were then blocked with the blocking buffer supplemented with 10% normal goat serum at room temperature for 60 min, followed by incubation in the blocking buffer plus 10% normal goat serum containing 10 µg/ml of affinity-purified rabbit anti-mouse progranulin immunoglobulin (Ig) G at room temperature for 60 min. This antibody was raised against the mouse progranulin peptide, MVAGLEKIOARQTTC (1618 kDa; unpublished data). After washing several times with the blocking buffer, blastocysts were incubated with the secondary antibody, goat anti-rabbit IgG conjugated with rhodamine (1:100 dilution; Chemicon, Temecula, CA) for 60 min. After washing in PBS, signal was viewed under a confocal laser-scanning microscope (Olympus Optical Co. Ltd., Tokyo, Japan). Nonimmune rabbit IgG (10 µg/ml) was used as a negative control. All procedures were performed in a 96-well plate except for the experiment with outgrowth embryos, which was performed in a four-well plate.
Generation of Recombinant Progranulin
Mammalian expression vector pcDNA3-His (Invitrogen) was used for the generation of recombinant progranulin [21]. A full-length mouse Grn cDNA was amplified from mouse preadhesion blastocyst cDNA using PCR with 5'-gg ggt acc atg gca tgg gtc ctg atg agc tgg ct-3' (forward) and 5'-ccg ctc gag tta cag tag cgg tct tgg gac-3' (reverse). The PCR fragments were digested with Kpn1 and Xho1 restriction enzymes and ligated into the Kpn1 and Xho1 sites of the pcDNA3-His plasmid. Identification and orientation of the construct with the Grn cDNA was confirmed by DNA sequencing (ABI-PRISM). The plasmid was transfected into NIH3T3 cells using TransFast Transfection Reagent (Promega) following the manufacturer's instructions. After 48 h, these cells were subjected to selection for stably transfected cells using 400 µg/ml of G418. Selection was continued until monolayer colonies formed. The transfectants were transferred and maintained in DMEM supplemented with 10% FBS and 400 µg/ml of G418. After the transfectants were grown to 90% confluency, the medium was discarded, and fresh DMEM medium containing 1.5% FBS, 400 µg/ml of G418, and 15 mM of sodium butyrate was added. One day later, the culture medium was collected. Purification of the recombinant progranulin was performed by means of Talon Metal Affinity Resin (BD Biosciences Clontech, Palo Alto, CA) according to the protocol suggested by the manufacturer. The recombinant progranulin was filter-sterilized (pore size, 0.22 µm) and adjusted to 1 mg/ml. To determine the bioactivity of purified recombinant progranulin on preimplantation development, mouse conceptuses (n = 30) at the 8-cell stage were cultured for 48 h in DMEM plus 0.3% FBS containing 0.00, 0.01, 0.1, or 1 µg/ml of recombinant protein or 20 µg/ml of rabbit anti-mouse progranulin peptide IgG. In addition, the recombinant progranulin was deglycosylated using peptide N-glycosidase F (PNGase F; Prozyme, San Leandro, CA) according to the manufacturer's instructions and later subjected to Western blot analysis.
Western Blot Analysis
Protein samples of lysates, media, and fresh buffer itself from the blastocyst culture as well as recombinant proteins were analyzed by 10% SDS-PAGE [21]. Briefly, after electrophoresis, the fractionated proteins were transferred to a polyvinylidene fluoride membrane (NEN Life Science Products, Inc., Boston, MA), followed by blocking with 5% (w/v) skim milk in the solution containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% (v/v) Tween 20 (TBST) as well as 10% normal donkey serum at room temperature for 1 h. The membrane was incubated overnight with 10 µg/ml of rabbit anti-mouse progranulin peptide IgG or rabbit anti-His antibody (1:300; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The membranes were then washed three times with TBST and incubated with the secondary antibody, donkey anti-rabbit IgG antibody conjugated with horseradish peroxidase (Amersham Biosciences, Piscataway, NJ) at room temperature for 1 h. The immune complexes were then visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL).
Blastocyst Hatching Assay
Initially, 63 early blastocysts (n = 7 per progranulin dose) were cultured for 48 h in DMEM plus 3% FBS containing 0.00, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, or 2 µg/ml of recombinant progranulin preparations. The effect of progranulin on blastocyst hatching, adhesion, and outgrowth was examined at 24, 36, and 48 h after initiation of the in vitro culture. It was determined that the recombinant progranulin at a concentration of 0.2 µg/ ml was optimal for blastocyst hatching, adhesion, and/or outgrowth.
To investigate the effect of recombinant progranulin on mouse blastocyst hatching, early blastocysts flushed from the uterus with DMEM on Day 3.5 of pregnancy were washed three times with fresh DMEM. Blastocysts (200 total, 10 blastocysts/well of a 96-well plate) were cultured in DMEM (80 µl/well) supplemented with 1% nonessential amino acids, 2 mM glutamine, 10 mg/ml of BSA, 100 U/ml of penicillin, and 40 µg/ml of streptomycin. These blastocysts were divided into four treatment groups: no further treatment (control group, n = 50, five wells), incubation with 0.2 µg/ml of recombinant progranulin (progranulin group, n = 50, five wells), treatment with 100 µg/ml of rabbit anti-mouse progranulin peptide IgG (antiprogranulin group, n = 50, five wells), and incubation with normal rabbit IgG (normal rabbit IgG group, n = 50, five wells) at 37°C for 60 h under 95% air and 5% CO2. At 24, 36, 48, and 60 h after initiation of in vitro culture, the hatching was observed under an inverted phase-contrast microscope (Olympus Optical Co. Ltd.). Conceptuses were classified as hatched blastocysts when they were completely free from the zona pellucida. The numbers of blastocysts undergoing hatching under each condition were recorded, and the ratios of hatched blastocysts relative to the total number of embryos were calculated.
Blastocyst Adhesion and Outgrowth Assay
Blastocyst adhesion and outgrowth assays were performed in 96-well plates containing DMEM supplemented with 3% FBS, 100 U/ml of penicillin, and 40 µg/ml of streptomycin (the medium). In brief, early mouse blastocysts flushed from the uterus on Day 3.5 of pregnancy were washed three times with the same solution. A total of 350 blastocysts (10 blastocysts/well) were cultured in the medium only (control group, n = 100), in the medium plus 0.2 µg/ml of recombinant progranulin (progranulin group, n = 100), in the medium plus 100 µg/ml of rabbit anti-mouse progranulin peptide IgG (antiprogranulin group, n = 100), or in the medium plus the normal rabbit IgG (normal rabbit IgG group, n = 50) under 95% air and 5% CO2. At 24, 36, and 48 h after initiation of the in vitro culture, the outgrowth was observed under an inverted phase-contrast microscope, whereas the adhesion was examined at 48 h after the initiation of culture. Blastocyst adhesion was examined once, because gentle pipetting was required to determine whether the embryo would detach from the bottom of the well. Definitions for embryo adhesion [20, 21] and outgrowth [22] have been published previously. The numbers of blastocysts undergoing adhesion and outgrowth were recorded, and the ratios of blastocysts with adhesion and outgrowth relative to the total number of embryos were calculated. The number of blastocysts undergoing hatching also was recorded, and the ratios of blastocysts with adhesion and outgrowth relative to the hatched blastocysts were determined.
For determination of the outgrowth area of blastocysts, plastic Petri dishes (Falcon 1008; Falcon Labware, Oxnard, CA) were precoated with 50 µg/ml of fibronectin (Roche Diagnostics, Indianapolis, IN) prepared in Hanks balanced salt solution (HBSS; Sigma) and incubated overnight at 37°C as small (5-µl) drops under a layer of light mineral oil. The substratum was then washed three times with HBSS containing 4 mg/ml of BSA to remove unbound protein or peptides. Then, potassium simplex optimized medium containing essential and nonessential amino acids (KSOM/ aa; EmbryoMax, Phillipsburg, NJ), with either 80 µg/ml of affinity-purified antiprogranulin antibody [10] or nonspecific rabbit IgG, was added and pre-equilibrated for 2 h in a three-gas incubator before introducing one embryo in each drop. These embryos were blastocysts that had been flushed from uteri on Day 3.5 of pregnancy and cultured for 72 h in KSOM/aa under light mineral oil (Fisher Scientific, Pittsburgh, PA) to allow spontaneous hatching. Using an inverted microscope (Nikon, Melville, NY), the outgrowths were photographed individually each day for 5 days. To improve the quality of the images, the contrast of each image was enhanced with Photoshop (Adobe Systems, Inc., San Jose, CA), and the area of the outgrowth was measured with NIH ImageJ software (http://rsb.info.nih.gov/ij/).
Bromodeoxyuridine Incorporation Assay
Collected early blastocysts were placed in 20-µl drops of KSOM/aa and incubated under mineral oil (Sigma) at 37°C in a humidified atmosphere containing 5% CO2 [18]. Two different concentrations of affinity-purified antiprogranulin antibody (20 and 40 µg/ml) [10] were tested. Medium alone and nonspecific rabbit IgG (40 µg/ml) were used as a control as described previously [18]. Each experimental group consisted of 20 embryos that were cultured for 14 h. In another culture group, the embryos were removed from the antibody after 14 h, washed through several drops of the medium, and then cultured in a fresh drop of antibody-free medium for another 14 h to see the reversible effect and to corroborate that the antibody does not have a direct toxic effect on the embryos.
The embryos that were cultured in the presence of two different concentrations of affinity-purified antiprogranulin antibody (20 and 40 µg/ ml), KSOM/aa medium alone, or nonspecific rabbit IgG (40 µg/ml) for 14 h and were analyzed to detect DNA synthesis as described previously [23]. Embryos were labeled with 10 µM bromodeoxyuridine (BrdU) for 45 min. Following the incubation in BrdU, the embryos were washed with PBS containing 0.3% BSA (PBS/BSA) and then fixed with 3.7% paraformaldehyde. The DNA was denatured by incubating the embryos with 2 N HCl at 37°C for 1 h, and the sample was then neutralized by the addition of 0.1 M borate buffer (pH 8.5) for 15 min. The incorporated BrdU was detected as described previously [24]. Briefly, after washing with PBS/ BSA, the embryos were incubated with BrdU monoclonal antibody (Boehringer-Mannheim) for 45 min, washed, and then incubated with an anti-mouse IgG antibody conjugate with Texas Red (Jackson ImmunoResearch Laboratories, West Grove, PA) for 45 min. The embryos were then washed with PBS/BSA and mounted with VectaShield (Vector Laboratories, Burlingame, CA). Fluorescence was detected using a Leica TCS 4D laser-scanning confocal microscope.
Isolation of Inner Cell Mass Cells from Blastocysts by Immunodissection
Embryos were collected, and the zonae pellucidae were removed with acid Tyrode solution [25]. The zona pellucida-free blastocysts were incubated at room temperature for 10–15 min in 10% anti-mouse T-cell antiserum (Thy-1; Accurate Chemical and Scientific Corp., Westbury, NY) diluted in collection medium (MEM containing 3 mg/ml polyvinylpyrrolidone [MEM/PVP] plus 1 mg/ml of BSA). After washing in MEM/PVP medium, blastocysts were incubated in 1:10 dilution of guinea pig complement (Sigma) at 37°C for 15 min; during this step, the trophectodermal cells were evenly lysed and swollen. The inner cell masses (ICMs) were released from the lysed trophectoderm during the wash in collection medium [26]. Finally, individual ICMs were placed in 4-µl drops of KSOM/ aa and incubated under mineral oil at 37°C in a humidified atmosphere containing 5% CO2. Two different concentrations of affinity-purified antiprogranulin antibody (20 and 40 µg/ml) were tested. Medium alone and nonspecific rabbit IgG (40 µg/ml) were used as a control, and they were cultured for 14 h.
Statistical Analysis
The RT-PCR intensity was determined using Photoshop. Two-way analysis of variance was employed, followed by Duncan multiple-range tests to determine differences among treatment groups (STATISTICA; Statsoft, Tulsa, OK). Results are shown as the mean ± SEM, and a value of P < 0.05 was considered to be statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Levels of mouse Grn mRNA in early blastocysts, pre- and postadhesion blastocysts, and outgrowth embryos were examined by RT-PCR. High levels of Grn mRNA were present in blastocysts during the peri-implantation period (Fig. 1). Whole-mount in situ hybridization analysis with the digoxigenin-labeled antisense cRNA probe revealed that Grn mRNA was expressed in mouse peri-implantation blastocysts, localized in both ICM and trophectoderm (Fig. 2). Signal specificity was confirmed using a sense cRNA probe as control, which yielded no signal (Fig. 2).
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Progranulin Localization in Peri-implantation Blastocysts
The expression and localization of progranulin in peri-implantation mouse blastocysts were investigated by indirect immunostaining. This procedure revealed that progranulin appeared on the surface (Fig. 3, inset) of trophoblasts at the stages of early blastocysts and pre- and postadhesion blastocysts, but not in the ICM (Fig. 3). In outgrowth embryos, however, progranulin was present in both the ICM and trophectoderm, including trophoblast giant cells (Fig. 3). Specificity of the immunostaining was confirmed using normal rabbit IgG as a negative control.
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Production and Secretion of Progranulin into Culture Media
Pre- and postadhesion blastocysts, outgrowth embryos, and culture media were collected as described in Materials and Methods. Immunoblotting was performed with the rabbit anti-mouse progranulin IgG. Bands of 68 and 88 kDa were detected in pre- and postadhesion blastocysts. However, only the 88-kDa form was found in outgrowth embryos and the surrounding media at 24, 36, and 48 h of in vitro blastocyst culture. No band was detected in the fresh medium (DMEM plus 3% FBS) (Fig. 4). These data indicate that peri-implantation embryos synthesized and secreted progranulin into the surrounding medium.
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Recombinant progranulin was effectively produced from NIH3T3 cells that had been transfected with the pcDNA3-His plasmid containing a full length of mouse Grn cDNA (Fig. 5A). Similar to the results obtained previously [18], blastocoelic cavity formation was stimulated by the addition of recombinant progranulin but inhibited by the antibody (data not shown). An 88-kDa form of recombinant progranulin was produced, from which a 68-kDa form was generated by deglycosylation (Fig. 5, B and C). The value of 68 kDa represented the approximate molecular weight predicted from the mouse Grn cDNA sequence, indicating that carbohydrate contributed approximately 20 kDa to the 88-kDa form found in blastocysts, recovered from blastocyst culture media, or expressed as a recombinant protein.
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Regulation of Progranulin on Blastocyst Hatching
The effect of progranulin on mouse blastocyst hatching was examined using an in vitro model. At 24 h of in vitro culture, blastocyst hatching was observed in groups of embryos subjected to normal IgG, to no factor or to progranulin, whereas at this time, no blastocyst hatching was observed in the group of blastocysts treated with rabbit anti-mouse progranulin IgG. The number of blastocysts hatched in the antiprogranulin group did appear to increase over time, but not to the degree observed with controls. At 36, 48, and 60 h of in vitro culture, the number of blastocysts hatched in the group treated with the antiprogranulin did not increase, whereas hatching did increase in the other groups of blastocysts (P < 0.01) (Fig. 6A). No significant difference was observed between control and normal IgG groups (Fig. 6A). The hatching rate of blastocysts in the progranulin-treated group was significantly higher than in the control group at 36, 48, and 60 h of in vitro culture (P < 0.01) (Fig. 6B). These data indicate that progranulin regulated the degree and/or timing of blastocyst hatching and that the addition of exogenous progranulin promoted blastocyst hatching.
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Effect of Progranulin on Blastocyst Adhesion and Outgrowth
Blastocyst adhesion was examined at 48 h after initiation of the in vitro culture. As shown in Figure 7A, the number of adhering embryos in a group of blastocysts treated with the rabbit anti-mouse progranulin IgG was lower than that in the normal IgG and control groups (P < 0.01) (Fig. 7A, left). The number of adhering embryos in the group of blastocysts treated with recombinant progranulin was higher than that in the control group (P < 0.05) (Fig. 7A, right). The ratio of blastocyst adhesion in the group of blastocysts treated with recombinant progranulin or the anti-progranulin antibody was similar to those calculated from the total number of blastocysts within a treatment (Table 1). However, adhesion was not found to be promoted by the additional progranulin treatment when the numbers of adhering embryos relative to those of hatched blastocysts were calculated. These results indicate that progranulin was not as effective on blastocyst adhesion and outgrowth as it was on blastocyst hatching.
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The effects of progranulin on mouse blastocyst outgrowth were examined using the in vitro blastocyst-culture system. Outgrowth among any treatment groups was not seen at 24 h after initiation of the in vitro culture. Minimal numbers of trophoblast outgrowth were observed in normal IgG, control, and progranulin-treated groups at 36 h of culture. A substantial increase in the number of embryos with blastocyst outgrowth was found by 48 h of culture. In the group of blastocysts treated with rabbit anti-mouse progranulin IgG, trophoblast outgrowth was not observed until 48 h of in vitro culture (P < 0.01) (Fig. 7B, left). Addition of recombinant progranulin increased the number of embryos exhibiting outgrowth at 36 and 48 h (P < 0.01) of in vitro culture (Fig. 7B, right). No differences were found between control and normal IgG groups (Fig. 7B, left). The rate of blastocyst outgrowth in the group of embryos treated with the antiprogranulin antibody was significantly lower than that in the other treatment groups (P < 0.01), whereas a significant difference in the rate of blastocyst outgrowth was not found between control and progranulin-treated groups (Table 1). Furthermore, the area of embryo outgrowth determined using an in vitro culture increased over a 5-day culture period. However, when embryos were incubated with the rabbit anti-mouse progranulin IgG, an increase in the area of blastocyst outgrowth was not observed (Fig. 8).
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Inhibition of DNA Synthesis in Blastocysts Cultured with Antiprogranulin
We have reported previously [18], and have confirmed in the present study, that progranulin stimulates trophectoderm epithelial cell proliferation. When DNA synthesis was assayed by the incorporation of BrdU, the trophoblast, but not the ICM, DNA synthesis was inhibited by high concentrations of the antiprogranulin antibody (Fig. 9, A and B). This effect was reversible; when the antibody was removed by washing, the treated embryos progressively incorporated BrdU (Fig. 9C). Immunosurgery was then used to isolate ICMs, which also were treated with the antibody as described in Figure 9, A–C. Under these conditions, the antibody had no effect on the ICM cultures, indicating that progranulin is specific for the trophectoderm (Fig. 9, D and E).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previously, we established an in vitro blastocyst-culture system to research mouse blastocyst implantation processes [20]. Using this culture system, the transcription levels of pre- and postadhesion mouse blastocyst mRNA were analyzed using cDNA microarray technology, and we found that Grn transcript levels were higher in postadhesion blastocysts than in preadhesion embryos. Levels of Grn mRNA detected in the present study confirmed those in our previous investigation [20]. Expression and localization studies of progranulin in peri-implantation blastocysts revealed that progranulin was localized to the trophoblasts of early blastocysts, pre- and postadhesion blastocysts, and the ICM and trophectoderm of outgrowth embryos. These results provide the evidence that progranulin is associated with implantation or likely plays a role during related events in the mouse.
Preimplantation mammalian blastocysts secrete progranulin into the surrounding medium, which in turn stimulates the development of blastocysts in an autocrine manner [18]. In the present study, the observations in which peri-implantation blastocysts at each developmental stage also synthesized and secreted progranulin into the surrounding medium indicated that progranulin acted in an autocrine manner on peri-implantation blastocysts. Instead of a single, 68-kDa form as previously reported [18], peri-implantation blastocysts also produced an 88-kDa form of progranulin, which was the form recovered from medium conditioned by these embryos. In the present study, NIH3T3 cells also generated recombinant progranulin with a size of 88 kDa as detected by Western blot analysis (Fig. 5). When this protein was digested with peptide N-glycosidase F, it showed an apparent molecular weight of 68 kDa, which was similar to the molecular mass of the polypeptide predicted by the cDNA sequence. These results indicate that the 88-kDa form of progranulin found in blastocyst lysates and their culture media was glycosylated. Previously, progranulin, purified from PC cell-conditioned medium, was found as an apparent 88-kDa protein, digestion of which with peptide N-glycosidase F yielded an apparent 68-kDa protein component [13]. This result and the observations from the present study suggest that progranulin produced by the blastocysts is the 88-kDa glycoprotein, containing approximately 20 kDa of carbohydrates. Also of interest, we previously found that normal renal tissue expresses a 68-kDa form of progranulin, whereas cancerous renal tissues express both 68- and 88-kDa forms [27]. The fact that highly proliferative cells, such as tumorigenic PC cells, renal carcinoma cells, and invasive trophoblasts, express an 88-kDa form of progranulin is intriguing and suggests that the highly glycosylated 88-kDa form may be a marker for the proliferative or invasive states of these cells.
Blastocyst hatching from the zona pellucida is a critical step for successful implantation. In addition to endogenous progranulin production, exogenous progranulin significantly promoted blastocyst hatching (Fig. 6B), whereas the rabbit anti-mouse progranulin IgG decreased the number of conceptuses progressing to the hatching stage. In addition to the cavitation study, bioactivity of recombinant progranulin had been determined by examining the rate of embryo hatching in DMEM supplemented with 3% FBS and 0.2 µg/ml of recombinant progranulin in the presence or absence of 100 µg/ml of rabbit anti-mouse progranulin IgG or normal rabbit IgG, with results similar to those described in the present study (data not shown). Our experimental results described herein revealed that progranulin was an important factor in promoting blastocyst hatching, possibly by accelerating the onset of this process. However, clarification of the definitive roles that progranulin plays in blastocyst hatching, degree, and/or timing await further investigations.
Trophectoderm adhesion to the uterine endometrium is the limiting step of implantation and subsequent placentation. In addition to blastocyst outgrowth, blastocyst adhesion appeared to be promoted by progranulin. This conclusion is based on the observation that blastocyst adhesion was lowered by the use of its antibody (P < 0.01) but enhanced by the addition of exogenous progranulin (P < 0.05). When the rate of blastocyst adhesion and outgrowth relative to the number of blastocysts that had been hatched was calculated (Table 1), the rates of blastocyst adhesion and outgrowth were not as high as those observed originally (Fig. 7). These results suggest that rather than being direct effects of progranulin on the enhancement of blastocyst adhesion and outgrowth, the rate of blastocyst adhesion and outgrowth promoted by progranulin could have resulted from the effect of progranulin on promotion of the onset of blastocyst hatching.
These studies also shed light on the mechanisms dictating the differential response to progranulin signaling in the two compartments of the blastocyst. Although Grn mRNA could be detected in the ICM and trophectoderm, the protein was detected only in trophoblasts. In the BrdU incorporation assay, DNA synthesis in the trophoblast, but not in the ICM, was inhibited by use of the progranulin antibody. Two possible reasons may account for this differential inhibition of DNA synthesis by the antibody. First, the ICM could be secluded from the antibody by virtue of the trophoblast epithelial layer. Alternatively, the ICM could be insensitive to the effects of the antiprogranulin. Isolation of ICM by immunosurgery and subsequent incubation with the progranulin antibody revealed that progranulin was specific for the trophectoderm. These results suggest that the progranulin receptor is expressed only in the trophectoderm, not in the ICM, at the blastocyst stage. Future studies leading to identification and localization of the progranulin receptor in the blastocyst will provide additional insights concerning the actions of progranulin on the trophectoderm and ICM cell of the blastocyst.
Taken together, the present study revealed, to our knowledge for the first time, that progranulin is involved in the implantation processes of mouse embryos. However, the precise molecular mechanisms by which progranulin, either alone or together with other factors, regulates the processes of hatching and subsequent adhesion and blastocyst outgrowth await further investigation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Kazuhiko Imakawa, Laboratory of Animal Breeding, Graduate School of Agriculture and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. FAX: 81 3 5841 8180; akaz{at}mail.ecc.u-tokyo.ac.jp
Received: 14 January 2005.
First decision: 12 February 2005.
Accepted: 10 May 2005.
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 P. C. Hanington, D. R. Barreda, and M. Belosevic A Novel Hematopoietic Granulin Induces Proliferation of Goldfish (Carassius auratus L.) Macrophages J. Biol. Chem., April 14, 2006; 281(15): 9963 - 9970. [Abstract] [Full Text] [PDF]
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