Tubulointerstitial Nephritis Antigen-Like 1 Is Expressed in ...

BIOLOGY OF REPRODUCTION 81, 948–955 (2009) Published online before print 8 July 2009. DOI 10.1095/biolreprod.109.078162

Tubulointerstitial Nephritis Antigen-Like 1 Is Expressed in Extraembryonic Tissues and Interacts with Laminin 1 in the Reichert Membrane at Postimplantation in the Mouse1 Tadashi Igarashi,3 Yumiko Tajiri,3 Masahiro Sakurai,4 Eimei Sato,4 Dan Li,5 Kuniaki Mukai,5 Makoto Suematsu,5 Emiko Fukui,3 Midori Yoshizawa,3 and Hiromichi Matsumoto2,3 Laboratory of Animal Breeding and Reproduction,3 Division of Animal Science, Faculty of Agriculture, Utsunomiya University, Utsunomiya, Japan Laboratory of Animal Reproduction,4 Graduate School of Agricultural Science, Tohoku University, Sendai, Japan Department of Biochemistry and Integrative Medical Biology,5 School of Medicine, Keio University, Tokyo, Japan INTRODUCTION

Tubulointerstitial nephritis antigen-like 1 (Tinagl1, also known as adrenocortical zonation factor 1 [AZ-1] or lipocalin 7) has been cloned from mouse adrenocortical cells and is known to be closely associated with zonal differentiation of adrenocortical cells. In cell culture systems, TINAGL1 is a matricellular protein that interacts with both structural matrix proteins and cell surface receptors. However, the physiological roles of TINAGL1 and regulation of its expression are still not clearly understood. In the present study, the expression and localization of TINAGL1 in peri-implantation mouse embryos was examined. During preimplantation, the expression of both Tinagl1 mRNA and TINAGL1 protein was increased just prior to implantation. In blastocysts, TINAGL1 expression was localized to the trophectoderm. Using a progesterone-treated, delayed-implantation model, TINAGL1 was found to be upregulated in implantationcompetent blastocysts after estrogen treatment. During postimplantation, TINAGL1 expression was restricted to extraembryonic regions. Marked expression was detected in the Reichert membrane on Embryonic Days 6.5 (E6.5) and E7.5. Colocalization of laminin 1 and TINAGL1 was also examined. Using an anti-LAMA1 antibody, colocalization of LAMA1 and TINAGL1 was observed in postimplantation embryos. Colocalization was also detected in the Reichert membrane. Immunoprecipitation analysis determined that LAMA1 and TINAGL1 interact in embryos on E7.5. These results demonstrate that after implantation, TINAGL1 is an extraembryonic tissue-specific protein. In particular, TINAGL1 is a novel component of the Reichert membrane that interacts with laminin 1. These results suggest that TINAGL1 most likely plays a physical and physiological role in embryo development at postimplantation.

The development of the mammalian embryo includes dynamic changes, such as fertilization, cell division, establishment of cell polarity, compaction to form a morula, and lineage differentiation to form a blastocyst [1]. After hatching from the zona pellucidae, blastocysts gain implantation competency. The blastocyst is composed of distinct cell types; the pluripotent inner cell mass (ICM) generates future cell lineages of the embryo proper, whereas the outer epithelial trophectoderm (TE) makes the first physical and physiological connection with the maternal uterus for implantation. The invasive trophoblasts of mouse blastocysts adhere, spread, and migrate on extracellular matrix (ECM) substrates [2–5] and penetrate the three-dimensional ECM structures [6]. The TE of the implantation-competent blastocyst alters its functional programming via changes in cell surface molecules. In contrast to the ECM, extracellular proteins that do not contribute directly to the formation of structural elements in vertebrates but serve to modulate cell-matrix interactions and cell function are categorized as matricellular proteins [7]. The mouse ortholog of tubulointerstitial nephritis antigen-like 1 (Tinagl1, also known as adrenocortical zonation factor 1 [AZ1] or lipocalin 7) has been cloned from mouse adrenocortical cells and is known to be tightly linked with zonal differentiation of adrenocortical cells [8]. A recent study has shown that the secretory protein TINAGL1 is a novel matricellular protein that interacts with both structural matrix proteins and cell surface receptors [9]. Further, on the basis of its colocalization and binding ability with laminin 1 and collagens, TINAGL1 was found to be a component of the basal lamina [9]. It has been reported that TINAGL1 expression was upregulated after differentiation of murine F9 embryonal carcinoma cells by all-trans-retinoic acid and dibutyryl cAMP into parietal endodermlike cells [10]. The parietal endoderm arises from the ICM in the blastocyst as a result of differentiation events and produces large quantities of ECM proteins to form the Reichert membrane, which separates the yolk cavity from the maternal tissue [11, 12]. The Reichert membrane contains laminin and collagen IV [13]. Therefore, there is a possibility that TINAGL1 is associated with embryonic functions during peri-implantation. Our present investigation focused on whether Tinagl1 is involved in embryonic development. The expression and localization of Tinagl1 in preimplantation and postimplantation mouse embryos were examined first. The expression of both mRNA and protein was increased in the blastocyst during the preimplantation stage. At postimplantation, TINAGL1 was expressed in extraembryonic tissues. It has been reported that TINAGL1 binds to laminin 1 in cell culture system [9].

developmental biology, early development, embr yo, extraembryonic tissue, implantation, laminin, Reichert membrane, Tinagl1, trophoblast

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Supported in part by the Japan Society for the Promotion of Science grant 17780207 to H.M., and Young Scientist’s Research at Utsunomiya University (H.M.). 2 Correspondence: Hiromichi Matsumoto, Laboratory of Animal Breeding and Reproduction, Division of Animal Science, Faculty of Agriculture, Utsunomiya University, 350 Minemachi, Utsunomiya, Tochigi 321-8505, Japan. FAX: 81 28 649 5431; e-mail: [email protected] Received: 9 April 2009. First decision: 3 May 2009. Accepted: 23 June 2009. Ó 2009 by the Society for the Study of Reproduction, Inc. eISSN: 1259-7268 http://www.biolreprod.org ISSN: 0006-3363

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ABSTRACT

DISTRIBUTION OF TINAGL1 IN THE MOUSE EMBRYO

Therefore, we hypothesized that TINAGL1 interacts with laminin 1 in the extraembryonic tissue to support postimplantation embryonic development. Our results demonstrated that TINAGL1 is colocalized with laminin 1 in extraembryonic tissue during the postimplantation period. Furthermore, we demonstrated that TINAGL1 is a novel component of the Reichert membrane that interacts with laminin 1. MATERIALS AND METHODS Animals, Treatments, and Collection of Embryos

Real-Time RT-PCR The total RNA extraction from each of the 10 embryos and subsequent DNA synthesis were performed using Cells-to-cDNA II (Ambion, Austin, TX). The real-time RT-PCR was performed using an SYBR green PCR Kit (Qiagen, Hilden, Germany) on an ABI 7500 Real Time PCR System (Applied Biosystems, Foster City, CA), according to the manufacturer’s instructions. Levels of Tinagl1 relative to those of H2afz were determined. Primer sets for Tinagl1, as well as control genes for expression of H2afz, were designed as follows: Tinagl1 5 0 -CCATCCTCCACTGTCATGAAT-3 0 (forward) and 5 0 CAAAGGCAGTGGGTAGCAC-3 0 (reverse); and H2afz 5 0 -CTCAGGACTC TAAATATTCCTAACAGC-3 0 (forward) and 5 0 -TGTTTTTCACAGAGATA CAGTCCAC-3 0 (reverse). Primers were validated by RT-PCR and agarose gel electrophoresis prior to use. Real-time RT-PCR was performed with a 25-ll reaction volume using cDNA from 1.7 embryos. Samples were cycled for 15 sec at 948C, 30 sec at 578C, and 35 sec at 728C for 45 cycles. Data were analyzed using 7500 SDS Software with the DDCt method (Applied Biosystems). Levels of Tinagl1 mRNA were analyzed using a one-way factorial ANOVA, followed by the Fisher protected least-significant differences test. All experiments were repeated three times.

Western Blot Analysis Embryos were collected and placed in 23 SDS sample buffer (0.5 M TrisHCl at pH 6.8, 10% 2-mercaptoethanol, and 20% glycerol). Lysates obtained from 50 embryos were applied to each lane and separated by SDS-PAGE, followed by transfer to Immobilon membranes (Millipore, Billerica, MA). Rabbit polyclonal antibody to TINAGL1 was prepared as described previously [9]. The membranes were incubated with antibodies against TINAGL1 (1:1000) at 48C overnight and with horseradish peroxidase (HRP)-labeled goat anti-rabbit antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) at room temperature for 1 h. The HRP activity was visualized using ECL Plus (GE Healthcare Bio-Sciences KK, Tokyo, Japan). All experiments were repeated three times, with similar results.

Immunohistochemical Analysis To investigate the expression and cellular distribution of TINAGL1, immunohistochemical analysis was performed as described previously [9, 14, 15], with slight modifications. In brief, preimplantation embryos were fixed in 3.7% formaldehyde in Ca2 þ- and Mg2 þ-free Dulbecco PBS at room temperature for 30 min, permeabilized in 0.25% Tween-20 in PBS for 5

min, and then incubated overnight at 48C with rabbit polyclonal antibody against TINAGL1. After several washes with PBS containing 0.5% Triton X100 and 0.5% bovine serum albumin (BSA), embryos were incubated with fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit antibody (MP Biomedicals, Solon, OH) for 1 h at room temperature. Nuclei were labeled with Hoechst 33258 (5 lg/ml; Molecular Probes, Eugene, OR) for 30 min at room temperature. They were viewed under an Axiovert 200MOT-LSM microscope with an ApoTome optical sectioning device (Carl Zeiss Inc., Oberkochen, Germany), with section thickness of 1.4 lm. TINAGL1 labeled with FITC appears green, and nuclei stained with Hoechst 33342 appear blue. Images shown are representative of at least 30 embryos obtained from more than five different animals for each stage. All experiments were repeated at least three times. For postimplantation embryos, Zamboni-fixed, 20-lm sections from frozen tissues were examined. The sections were fixed in cold acetone on ice for 10 min, immersed in 3% hydrogen peroxide in methanol for 10 min, treated with 1% BSA in PBS for 30 min, and then incubated overnight at 48C with rabbit polyclonal antibody to TINAGL1. After washing, sections were incubated sequentially with biotinylated goat anti-rabbit antibody (Zymed Laboratories Inc., San Francisco, CA) and HRP-conjugated streptavidin (Zymed Laboratories). Reactions were visualized using 3amino-9-ethyl carbazole (Zymed Laboratories) as a chromogen, and the sections were subsequently counterstained with hematoxylin. Reddish deposits indicate the sites of immunoreactive TINAGL1. Images shown are representative of at least six sections obtained from more than three different animals for each stage. All experiments were repeated at least three times. To investigate colocalization of LAMA1 (laminin a1 chain) and TINAGL1, immunofluorescence analysis was performed. After staining the whole-mount samples or frozen 10-lm sections obtained from snap-frozen tissues for TINAGL1, the samples were incubated sequentially with rat antimouse LAMA1 monoclonal antibody (Millipore), donkey anti-rat immunoglobulin G (IgG) antibody conjugated with Alexa Fluor 594 dye (Molecular Probes), and Hoechst 33342 and were viewed under an Axiovert 200MOTLSM microscope. LAMA1 and TINAGL1 labeled with Alexa Fluor 594 dye and FITC appear red and green, respectively. Nuclei stained with Hoechst 33342 were stained blue. Images shown are representative of at least 30 embryos or six sections obtained from more than three different animals for each stage. All experiments were repeated at least three times.

Biacore Analysis and Immunoprecipitation TINAGL1 and laminin interactions were assessed by surface plasmon resonance (Biacore X instrument; GE Healthcare Bio-Sciences KK) and immunoprecipitation using whole E7.5 embryos. Tissue lysates were prepared using lysis buffer (150 mM NaCl, 50 mM Tris-HCl [pH 8.0], 1% Nonidet P40, and protease inhibitor cocktail [Sigma, St. Louis, MO]). Laminin in TINAGL1-binding protein was detected using surface plasmon resonance with a Biacore X instrument as described previously [16], with slight modifications. As the running buffer, HBS-EP (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20) containing 0.1% Surfactant P20 (GE Healthcare Bio-Sciences KK) was used. The anti-TINAGL1 antibody was immobilized on sensor chip CM5 according to the manufacturer’s instructions. Proteins were diluted to 50 lg/ml with running buffer containing NSB Reducer (GE Healthcare Bio-Sciences KK). To detect the TINAGL1-protein complex, 50 ll of this solution was injected at a flow rate of 5 ll/min at 258C (first step). To confirm the presence of laminin within the complex, 50 ll of anti-mouse LAMA1 monoclonal antibody was injected at a flow rate of 5 ll/min at 258C (second step). As negative controls for proteins and the antibody, BSA and normal rat serum were injected according to the manufacturer’s instructions. The biosensor surface was regenerated with 5 ll of 10 mM glycine-HCl (pH 2.0) at a flow rate of 5 ll/min. All experiments were repeated three times. Immunoprecipitation was carried out using a Catch and Release reversible immunoprecipitation system (Millipore) according to the manufacturer’s instructions. For anti-mouse LAMA1 monoclonal antibody immunoprecipitation, 500 lg of tissue lysate was used, and immunoprecipitation was performed overnight. Immunoprecipitates were separated by SDS-PAGE and transferred to Immobilon membranes (Millipore). Membranes were incubated sequentially with rabbit polyclonal antibody to TINAGL1 (1:2000), peroxidase-labeled monoclonal mouse anti-rabbit light chain-specific IgG (Jackson ImmunoResearch Laboratories) (1:5000), biotinylated goat antimouse IgG (1:50 000; Zymed Laboratories), and HRP-streptavidin conjugate (1:50 000; Zymed). The HRP activity was visualized using 3,3 0 -diaminobenzidine tetrahydrochloride (Zymed Laboratories). All experiments were repeated twice.


ICR mice were purchased from CLEA Japan Inc. (Tokyo, Japan) or Japan SLC Inc. (Shizuoka, Japan) and bred in our animal care facility. The experimental procedures were performed in accordance with the instructions of the Guide for the Care and Use of Laboratory Animals published by Utsunomiya University. Female ICR mice were mated with fertile males to induce pregnancy (Day 1 ¼ vaginal plug). Preimplantation embryos were collected on Days 1–4 of pregnancy. Delayed implantation was induced according to previously described methods [14]. In brief, mice were ovariectomized on the morning (0800–0900 h) of Day 4 of pregnancy and maintained on daily injections of progesterone (P4; 2 mg/mouse) from Day 5 to Day 7. To terminate delayed implantation and to initiate implantation, P4-primed delayed-implanting pregnant mice were injected with estradiol-17b (E2; 25 ng/mouse). Dormant blastocysts were collected 18 or 23 h after the last P4 injection, whereas activated blastocysts were collected 18 or 23 h after the last P4 and E2 injections. Preimplantation embryos at the 1-cell, 2-cell, 4-cell, 8-cell, compacting 10to 20-cell, and morula and blastocyst stages were collected on days 1 (1000 h), 2 (1000 h), 2 (2300 h), 3 (1000 h), 3 (1400 h), and 3 (1800 h), and 4 (1000, 1800, and 2300 h), respectively. Postimplantation embryos were examined on Embryonic Day 4.5 (E4.5; Day 5 of pregnancy), E5.5, E6.5, and E7.5.

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FIG. 1. Tinagl1 mRNA expression levels in preimplantation mouse embryos determined by real-time RT-PCR. Expression of Tinagl1 mRNA in the embryos during preimplantation development is represented as an nfold increase (Fold). Bars indicate mean 6 SEM values from triplicate measurements of duplicate samples and are represented relative to blastocysts at 1000 h on Day 4 of pregnancy. Letters above bars indicate significant differences among days of pregnancy (P , 0.05). ND, not determined; 16-cell, compacting 10- to 20-cell; BL, blastocysts on Day 4.

Trophoblast Outgrowth Trophoblast outgrowth assay was performed as described previously [2], with slight modifications. Dishes were precoated with 10 lg/ml fibronectin. Blastocysts were collected at 1900–2000 h on Day 4 and cultured in CMRL1066 medium supplemented with 2 mg/ml sodium lactate, 60 lg/ml sodium pyruvate, 1% fetal calf serum, 20 lg/ml penicillin, and 20 lg/ml streptomycin at 378C in an environment of 5% CO2 in air for 48 h. To investigate the expression and localization of TINAGL1 and LAMA1, immunohistochemical analysis was performed as described above.

RESULTS Expression of Tinagl1 mRNA and TINAGL1 Protein in Preimplantation Embryos Real-time RT-PCR was performed to determine the relative levels of Tinagl1 mRNA. No Tinagl1 mRNA expression was detected from the one-cell to morula stages, whereas Tinagl1 mRNA was expressed in the blastocyst, and expression increased prior to implantation at 2300 h on Day 4 of pregnancy (Fig. 1). To determine the relative levels of TINAGL1 protein, Western blot analysis was performed, and a 52 3 103 M band was present. As shown in Figure 2, expression of TINAGL1 protein was detected in embryos during the preimplantation stage of development. Protein expression also increased in the blastocyst prior to implantation. Localization of TINAGL1 in Embryos During Preimplantation Development To investigate the expression and cellular distribution of TINAGL1 in embryos, immunohistochemical analysis was

FIG. 2. Western blot analysis of TINAGL1 expression in mouse embryos during peri-implantation. To detect TINAGL1, 50 embryos were applied to each lane. BL, blastocysts on Day 4.

FIG. 3. Distribution of TINAGL1 in embryos during preimplantation development. Immunofluorescence localization shows TINAGL1 in green and nuclei in blue. A) One-cell stage. B) Two-cell stage. C) Four-cell stage. D) Eight-cell stage. E) Compacting 10- to 20-cell stage. F) Morula. G) Negative control. Bar ¼ 30 lm.

performed (Figs. 3 and 4). Immunofluorescence staining for TINAGL1 was observed at all stages examined, confirming the presence of TINAGL1 during development throughout preim-


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blastocoele site of the TE just prior to implantation (Fig. 4, C and D). Upregulation of TINAGL1 in Implantation-Competent (Activated) Blastocysts FIG. 4. Differential localization of TINAGL1 at the trophectoderm in blastocysts prior to implantation. Immunofluorescence localization shows TINAGL1 in green and nuclei in blue. A) Blastocyst prior to hatching at 1800 h on Day 4 of pregnancy. B) Hatched blastocyst at 1800 h on Day 4. C and D) Hatched blastocysts at 2300 h on Day 4. E) Negative control. Bar ¼ 30 lm.

plantation. In embryos up to the eight-cell stage, TINAGL1 was uniformly distributed in the cytoplasm (Fig. 3, A–D). In contrast, the distribution of TINAGL1 changed after compaction of embryos. In compacting 10- to 20-cell embryos and compacted morulae, TINAGL1 was primarily expressed in the outer cells (Fig. 3, E and F). In blastocysts, TINAGL1 expression was localized to the TE, and this localization persisted in blastocysts after hatching from the zona pellucidae (Fig. 4, A and B). In mice, the first attachment reaction between the blastocyst TE and the uterine luminal epithelium occurs around midnight on Day 4 of pregnancy. In hatched blastocysts at 2300 h on Day 4 of pregnancy, TINAGL1 expression became restricted to the surface only at the

Ovariectomy on the morning of Day 4, prior to preimplantation estrogen secretion, results in blastocyst dormancy and delayed implantation [14, 17]. The condition of delayed implantation can be maintained by continued P4 treatment but is terminated upon injection of estrogen, leading to implantation-competent (activated) blastocysts and subsequent implantation. The attachment reaction between the blastocyst and uterus occurs around 24 h after E2 injection. To compare the distribution of TINAGL1 protein in dormant and implanting blastocysts, P4-treated dormant and P4 plus estrogen-treated activated blastocysts were examined. In dormant blastocysts, TINAGL1 expression was very low to undetectable at 18 and 23 h after the last P4 injection (Fig. 5, A and D). TINAGL1 expression was distinct in the TE after E2 injection (Fig. 5, B, C, E, and F). At 18 h after E2 injection, TINAGL1 was expressed in the cytoplasm of the TE or was restricted to the surface only at the blastocoele site of the TE (Fig. 5, B and C). In contrast, expression was restricted to the surface at the blastocoele site of the TE at 23 h (Fig. 5, E and


FIG. 5. Upregulated expression of TINAGL1 in E2-activated implanting blastocysts. A–C) Blastocysts collected at 18 h after the last injection of P4 or P4 plus E2. D–F) Blastocysts collected at 23 h after the last injection. A and D) Dormant blastocysts. B, C, E, and F) Activated blastocysts. G) Negative control on activated blastocyst. Bar ¼ 30 lm.

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FIG. 6. TINAGL1 expression is specific to extraembryonic regions in postimplantation embryos. A) E4.5. B) E5.5. C) E6.5. D) E7.5. E) Negative control on E5.5. ExE, extraembryonic ectoderm; EPC, ectoplacental cone; EE, embryonic ectoderm; RM, Reichert membrane. Bars ¼ 100 lm.

" FIG. 7. Distribution of LAMA1 and TINAGL1 in mouse embryos during peri-implantation. Immunofluorescence localization shows LAMA1, TINAGL1, and nuclei in red, green, and blue, respectively. A) Hatched blastocysts at 2300 h on Day 4. B) E4.5. C) E5.5. D) E6.5. E) E7.5. F) Negative control on E4.5. Bars ¼ 50 lm.


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detectable expression in the embryonic tissues derived from the ICM (Fig. 6, B–D). Colocalization of LAMA1 with TINAGL1 in Postimplantation Embryos FIG. 8. Detection of the TINAGL1-LAMA1 complex in embryos on E7.5. A) Sensorgram readings of TINAGL1-laminin interaction using a Biacore X instrument. An anti-TINAGL1 antibody was coupled to Biacore sensor chips and exposed to embryonic tissue lysate to detect TINAGL1-protein complex (first step). The presence of LAMA1 within the complex was then confirmed using an antibody for LAMA1 (second step). All experiments were repeated three times, with similar results. RU, resonance units. B) Relative response of LAMA1 antibody as a percentage of the value of tissue lysate against immobilized anti-TINAGL1 antibody on the sensor chip described in A. Values are means 6 SEM. All experiments were repeated three times. C) Immunoprecipitation with an anti-LAMA1 antibody. Proteins extracted from embryonic tissue were immunoprecipitated with an anti-LAMA1 antibody, then Western blotted with an antibody against TINAGL1. All experiments were repeated two times, with similar results.

TINAGL1 binds to laminin 1 [9]. Therefore, TINAGL1 may be associated with laminin in peri-implantation embryos. To address this issue, immunofluorescence double staining was performed to examine the distribution of LAMA1 and TINAGL1. Just prior to implantation, in hatched blastocysts at 2300 h on Day 4 of pregnancy, LAMA1 was expressed in both TE and ICM, whereas TINAGL1 was localized to the surface at the blastocoele site of the TE (Fig. 7A). In contrast, as shown in Figure 7, B–E, LAMA1 was colocalized with TINAGL1 in extraembryonic tissue. On E4.5, LAMA1 was expressed in the TE (Fig. 7B). The expression was restricted to extraembryonic regions on E5.5 (Fig. 7C). On E6.5 and E7.5, expression of LAMA1 was detected in the Reichert membrane (Fig. 7, D and E).

F). Collectively, the results suggest upregulation of TINAGL1 in E2-activated blastocysts undergoing implantation.

TINAGL1 Interactions with LAMA1 in the Reichert Membrane

Expression of TINAGL1 During the Postimplantation Period Immunohistochemical analysis was performed on postimplantation embryos to examine the expression and cellular distribution of TINAGL1 (Fig. 6). In postimplantation blastocysts on E4.5 (Day 5 of pregnancy), TINAGL1 protein was localized in the TE (Fig. 6A). With progression of implantation and postimplantation embryonic development, TINAGL1 expression was restricted to extraembryonic regions. Expression was noted in the extraembryonic ectoderm, but not in the ectoplacental cone, on E5.5 (Fig. 6B). On E6.5 and E7.5, strong expression was detected in the Reichert membrane (Fig. 6, C and D). In contrast, there was no

Because TINAGL1 and LAMA1 were colocalized in the Reichert membrane, potential interactions of LAMA1 with TINAGL1 were examined. Using an anti-TINAGL1 antibody coupled to Biacore sensor chips, Biacore analysis showed elevated signals, indicating a TINAGL1-protein complex in tissue lysates from embryos on E7.5 (first step; Fig. 8A). To confirm the presence of laminin within the complex (second step), anti-LAMA1 antibody was injected. Elevated signals showed the presence of a TINAGL1-LAMA1 complex in embryos on E7.5 (Fig. 8A). The relative resonance unit for the TINAGL1-LAMA1 complex in the TINAGL1-protein complex was 16.2% 6 1.4% (mean 6 SEM; Fig. 8B). Immunoprecipitation with LAMA1 for tissue lysates from embryos on E7.5, followed by Western blotting to detect


FIG. 9. Localization of TINAGL1 and LAMA1 in outgrowth of blastocysts. Immunofluorescence localization shows TINAGL1, LAMA1, and nuclei in green, red, and blue, respectively. A) TINAGL1. B) LAMA1. C) TINAGL1, LAMA1, and nuclei. D) Merged image of C with brightfield imaging. Bars ¼ 100 lm.

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TINAGL1, demonstrated interaction between TINAGL1 and LAMA1 (Fig. 8C). Collectively, these results along with immunohistochemical analysis demonstrate that TINAGL1 binds with laminin in the Reichert membrane. Distinct Localization of TINAGL1 and LAMA1 in Cultured Blastocyst Outgrowths

DISCUSSION TINAGL1 is a matricellular protein that interacts with both structural matrix proteins and cell surface receptors, and it is involved in the regulation of intracellular signaling that determines cellular functions [9]. The present investigation demonstrated that TINAGL1 is expressed in peri-implantation embryos. At the blastocyst stage, the expression of TINAGL1 was upregulated by E2 prior to implantation. From the blastocyst stage to postimplantation development, expression of TINAGL1 was localized to extraembryonic tissues. In particular, TINAGL1 was expressed in the Reichert membrane and interacted with LAMA1. These results suggest that TINAGL1 is associated with postimplantation development in mouse embryos. The results of this study demonstrated that TINAGL1 is linked to LAMA1, and it is thus a component of the Reichert membrane. The laminins are heterotrimeric glycoproteins, each composed of LAMA (a chain), LAMB (b chain), and LAMC (c chain) [18]. Laminin 1 (LAMA1, LAMB1, and LAMC1) and laminin 10 (LAMA5, LAMB1, and LAMC1) are the only laminins known to be expressed in the early embryo. Although laminin 1 is a major laminin found at early stages of embryogenesis in both embryonic and extraembryonic basement membranes, laminin 10 is required for later embryonic developmental events, such as neurulation and organogenesis [18]. A deficiency in only one gene of laminin 1 results in embryonic lethality; for example, a targeted mutation of Lama1 resulted in lack of a Reichert membrane and embryonic lethality by E7, whereas Lamb1deficient or Lamc1-deficient embryos lacked basement membranes and did not survive beyond E5.5 [19–21]. LAMA1 is also a component of laminin-3 (LAMA1, LAMB2, and LAMC1). However, Lamb2-deficient mice develop massive proteinuria due to failure of the glomerular filtration barrier [22], suggesting that the role of LAMA1 is contribution of the basement membrane during early postimplantation development. Therefore, our results using an anti-LAMA1 antibody suggest that TINAGL1 is integrated with laminin 1 in the Reichert membrane. During postimplantation development, appropriate maintenance of the blastocyst cavity allows the Reichert membrane to form under the trophoblast cell layer. The Reichert membrane allows efficient diffusion of gases and nutrients between the


As shown in Figure 7B, TINAGL1 was colocalized with LAMA1 in the TE on E4.5. Invasive trophoblasts in mouse embryos adhere, spread, and migrate on ECM substrates. Therefore, TINAGL1 may be associated with adherence, spread, or migration of trophoblast cells. To address this issue, we examined the localization of TINAGL1 and LAMA1 in the outgrowth of the blastocysts. As shown in Figure 9, TINAGL1 was localized around the ICM, whereas TINAGL1 was not detectable in trophoblast outgrowths. In contrast, LAMA1 was expressed in the trophoblastic cells and ICM. In outgrowth trophoblast, higher expression of LAMA1 was detected in outer cells compared with cells around the ICM. These results suggest that the role of TINAGL1 is distinct from the differentiation of trophoblastic cells associated with laminin 1 in implanting blastocysts.

maternal blood in the yolk sac placenta and the embryo, but it protects the embryo from contact with the maternal immune system. It is likely that the morphogenesis of these structures requires stabilizing interactions between the trophoblast cells and the surrounding decidual cells [23]. Laminin 1 and collagen IV form part of the basement membrane and the Reichert membrane [24–26]. Although the lack of just one laminin 1 component causes embryonic lethality by E7 [19– 21], collagen IV is dispensable for the initiation of early development during the postimplantation period [27]. Indeed, Col4a1/b-deficient embryos have been reported to develop up to E9.5 with varying degrees of growth retardation, although lethality resulted between E10.5 and E11.5 because of structural deficiencies in the basement membranes and, ultimately, failure of the integrity of the Reichert membrane [27]. In a previous study, TINAGL1 was demonstrated to bind with both laminin 1 and collagen IV [9]. Therefore, one of the physiological functions of TINAGL1 combined with laminin 1 may be to stabilize the Reichert membrane to support early postimplantation embryonic developmental events. During preimplantation development, TINAGL1 protein was expressed from the one-cell to the blastocyst stage, whereas expression of both protein and mRNA was increased in the blastocysts. Expression was stimulated by E2 prior to implantation. In P4-treated dormant blastocysts, TINAGL1 expression was very low to undetectable. Therefore, TINAGL1 is not likely necessary to maintain blastocyst status. In contrast, expression of Tinagl1 mRNA increased in blastocysts just prior to implantation at 2300 h on Day 4 of pregnancy, whereas TINAGL1 protein was localized to the surface of the blastocoele site of the TE at same time. In implanted E4.5 blastocysts (Day 5 of pregnancy), TINAGL1 protein was expressed in the TE. These results suggest that Tinagl1 mRNA upregulated by E2 is translated in implanted blastocysts and expressed in the TE. In implanted E4.5 blastocysts, TINAGL1 was colocalized with LAMA1 in the TE. However, differential expression was observed in the outgrowth blastocysts. Although TINAGL1 was localized around the ICM, LAMA1 was expressed in the trophoblast cells. In particular, the expression in outer trophoblast cells was higher than in cells around the ICM. It has been reported that Laminin 1 promotes trophoblast cell migration and decreases spreading [28]. These results suggest that TINAGL1 expression in the implanting blastocysts is not involved in cell migration of trophoblasts. Furthermore, upregulated expression of TINAGL1 by E2 prior to implantation may not be necessary for early postimplantation development because there was no expression of TINAGL1 in trophoblasts of outgrowth blastocysts. TINAGL1 expressed after E5.5 is most likely used for formation of the Reichert membrane to support embryonic development during postimplantation. Although Tinagl1 mRNA expression was increased in blastocysts, the expression of mRNA was undetected from the one-cell to the morula stage. In contrast, the expression of TINAGL1 protein was constant before blastulation. These results indicate the stage-specific differential expression pattern of Tinagl1 in embryos during preimplantation development. The gene expression profiling during preimplantation development revealed the distinctive patterns of maternal RNA degradation and embryonic gene activation, including two major transient waves of de novo transcription [29]. The first wave corresponds to zygotic genome activation (ZGA). The second wave, mid-preimplantation gene activation (MGA), contributes dramatic morphological changes during late preimplantation development. Therefore, expression of Tinagl1 mRNA is not associated with ZGA, whereas TINAGL1


expression is related to postimplantation development. Constant protein expression from the one-cell to the morula stage suggests that maternally expressed TINAGL1 protein exists in the cytoplasm. TINAGL1 was primarily expressed in the outer cells during compaction and was localized to the TE in blastocysts. TINAGL1 expressed during preimplantation could be involved in polarization and morphogenesis of the blastocyst. In conclusion, our study has shown that the expression of TINAGL1 is localized to extraembryonic tissues. In particular, TINAGL1 is a novel member of the Reichert membrane that interacts with laminin 1. In the uterus, a network of blood sinuses surrounding the embryo provides nutrients and oxygen by diffusion through the parietal yolk sac and the Reichert membrane. Therefore, these results suggest that TINAGL1 is associated in part with the yolk sac placenta to support embryonic development. These results may help elucidate the role of the extracellular matrix and its binding proteins in the Reichert membrane for embryonic development during postimplantation. Determining the definitive role of TINAGL1 during development will require further studies involving deletion of this gene.

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