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BOR - Papers in Press, published online ahead of print October 12, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.042655
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BIOLOGY OF REPRODUCTION 74, 344–351 (2006)
DOI: 10.1095/biolreprod.105.042655
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Cloning of the Bovine Antiapoptotic Regulator, BCL2-Related Protein A1, and Its Expression in Trophoblastic Binucleate Cells of Bovine Placenta1

Koichi Ushizawa 4, Toru Takahashi 4, Kanako Kaneyama 3 4, Misa Hosoe 4, and Kazuyoshi Hashizume 2 5

Reproductive Biology and Technology Laboratory,4 Developmental Biology Department, National Institute of Agrobiological Sciences, Ibaraki 305-8602, Japan Department of Veterinary Medicine,5 Faculty of Agriculture, Iwate University, Iwate 020-8550, Japan

ABSTRACT

This report studied the identification and sequence of a full-length cDNA for the bovine BCL2 antiapoptotic family member, BCL2-related protein A1 (BCL2A1), and its localized and quantitative expression in the placenta to clarify the regulatory mechanism of trophoblast cell proliferation and differentiation during implantation and placental development. We cloned a full-length bovine BCL2A1 cDNA with 725 nucleotides and an open-reading frame corresponding to a protein of 175 amino acids. The predicted amino acid sequence shared 78% homology with human BCL2A1. All BCL2 homology domains (BH1, BH2, BH3, and BH4) in bovine BCL2A1 were conserved as well as in other mammalian BCL2A1. In the placentomes, in situ hybridization demonstrated that the BCL2A1 was limited in binucleate cells expressing various pregnancy-specific molecules like placental lactogen. BCL2-associated X protein (BAX) was also expressed in binucleate cells. Quantitative real-time RT-PCR detection exhibited a high-level expression of BCL2A1 in the conceptus at Day 21 of gestation, and it was expressed and increased in the extraembryonic membrane, cotyledon, and intercotyledon from implantation to term. BAX expression intensity increased with progression of gestation and remained elevated in postpartum. Caspase-3 protein (CASP3) and mRNA (CASP3) were detected from late gestation to postpartum in placenta as well as in the results of TUNEL detection. We believe that the apoptosis of binucleate cells may be regulated by the balance of the BCL2A1 and BAX. BCL2A1 genes produced a BCL2A1 protein in the mammalian cell-expression system. This molecule is a new candidate for antiapoptotic maintenance of the binucleate cells that support placental functions throughout gestation in bovine.

apoptosis, gene regulation, implantation, placenta, trophoblast

INTRODUCTION

BCL2-related protein A1 (BCL2A1) is a member of the BCL2 family, an intracellular membrane-associated protein that suppresses apoptosis [1]. BCL2A1 was first cloned from mouse hematopoietic cell lineage, as a granulocyte macrophage-colony stimulating factor inducible, BCL2-related gene [2]. Three independent approaches were used to clone its human homologue from fetal liver, activated endothelium, and a myeloid leukemia [3–5]. In normal human tissues, BCL2A1 is induced by inflammatory cytokines, such as interleukin-1ß and vascular endothelial growth factor [3–6].

The BCL2 family of apoptotic regulators is characterized by the presence of BCL2 homology (BH) domains and can be subdivided into three groups: antiapoptotic proteins (BCL2, BCL2A1, BCL2L1, BCL2L2, and MCL1), proapoptotic proteins (BAX, BAK1, and BOK), and proapoptotic BH3 domain-only family members (BAD, BIK, BLK, BID, BCL2L11, PMAIP1, and BBC3) [7, 8]. The BH3 domain is a motif of 9 to 16 residues forming an amphipathic helix that is necessary for death induction and dimerization among BCL2 family members [7]. The BH4 domain is near the N-terminus [9], and is composed of the motif of 6 to 8 residues [7]. The antiapoptotic family members commonly belong in the BH4 domain [9]. In humans and mice, BCL2A1 conserves the BH1, BH2, and BH4 domains, and to a lesser degree the BH3 domain, and it is functionally defined as an inhibitory antiapoptotic protein [2–4].

Expression of some antiapoptotic BCL2 family members through the placenta has been reported in humans, monkeys, rats, and mice [10–14]. However, BCL2A1 expression has been reported primarily in bone marrow, lymphoid organs, peripheral leukocytes, and lungs [3–5, 15], and there has been no report of BCL2A1 appearing in the placenta [4]. Using microarray analysis, we found that the expressed sequence tag (EST) that corresponds to the BCL2A1 gene was expressed in the bovine extraembryonic membrane during the implantation period [16, 17]. There is no prior report regarding a gene responsible for regeneration and maintenance of the trophoblast cell during gestation in bovine. The proliferation, differentiation, and death in the trophoblast cell and endometrial epithelium are essential processes to implantation and placental growth [12, 18]. The purposes of this study are 1) to clone a full-length bovine BCL2A1 mRNA, 2) to identify its expression, 3) to examine the regulation mechanism of trophoblast maintenance during gestation, and 4) to investigate the translation of BCL2A. In our current research, we cloned a full-length bovine BCL2A1 cDNA to investigate the localization and quantitative expression of mRNA in bovine placenta. Simultaneously, we investigated the expression of BAX, a proapoptotic member of the BCL2 family, and caspase-3 mRNA (CASP3), A BCL2 family cascade protein and an apoptosis execution factor in the caspase family, comparing it with BCL2A1. The balance of BAX and BCL2A1 may regulate the apoptosis [19] and CASP3 may execute the apoptosis [20, 21]. In addition, we detected the apoptosis in placenta of each stage by TUNEL and immunohistochemistry of CASP3. These results were compared with the expression of BCL2A1, BAX and CASP3. Finally, we investigated the translation of the cloned bovine BCL2A1 and sugar chain addition of the recombinant protein in the HEK293 cell. This paper is the first report confirming that BCL2A1 is present in the mammalian trophoblast.

MATERIALS AND METHODS

Animals and Tissues

The Japanese Black cows were housed and fed in the Institute ranch at least 6 mo after they had been purchased from a commercial market at about 6 mo old, following the general procedure of the National Institute of Agrobiological Sciences for the use of animals. We artificially inseminated on the day of estrus (designated as Day 1 of gestation), and collected tissue samples from conceptuses, extraembryonic membranes, and placenta on Days 14–16, 20–21, 27–28, 56–64, 144–149, 245–252, and postpartum (286–294). Although we separated extraembryonic tissues into two portions, cotyledon (COT; villous trophoblast), and intercotyledon (ICOT; extravillous trophoblast, the areas between cotyledonary villi), on Days 27–28, it was difficult to separate COT and ICOT, so the COT contained only a very few villi. On Days 14–16 and 20–21, it was also difficult to separate the trophoblastic and embryonic portions. Tissues from two different cows on Day 14 and from another cow on Day 16 of gestation were our Day 14 conceptus (Day 14CON, n = 3). Tissues from one cow on Day 20 and two different cows on Day 21 of gestation were our Day 21 fetal membrane (Day 21CON, n = 3). Tissues from two different cows on Day 27 and one cow on Day 28 of gestation were our Day 27 extraembryonic membrane (Day 27EEM; removed from embryo, n = 3). We collected placentomal tissues on Days 56, 58, and 64 (n = 3) and designated them Day 60COT and Day 60ICOT. We designated samples from Days 144, 148, and 149 (n = 3), and Days 245 (two samples) and 258 (one sample, total n = 3) as Day 150COT, Day 150ICOT, Day 250COT, and 250ICOT. We also collected three samples of the postpartum COT and ICOT on Days 286, 290, and 294 (n = 3). The samples were snap-frozen and stored at –80°C until RNA extraction. Placentomes of Day 56, Day 120, and postpartum were fixed in 10% formalin (Sigma-Aldrich), embedded in paraffin wax, and stored at 4°C for histological examination with in situ hybridization. All procedures for these animal experiments were carried out in accordance with the guidelines and ethics approved by the Animal Ethics Committee of the National Institute of Agrobiological Sciences for the use of animals.

Cloning a Full-Length BCL2A1 cDNA

We isolated a new, full-length bovine BCL2A1 cDNA based on microarray data [16] (GenBank accession no. BP112244/BF045739 on Gene Expression Omnibus microarray platform accession no. GPL1221) from bovine cotyledonary tissue using the 3'-rapid amplification of cDNA ends (RACE) method. Briefly, we isolated a complete RNA from a bovine placentome on Day 64 of gestation using ISOGEN (Nippon Gene). We performed 3'-RACE using a 3'-full RACE core set (Takara) with a specific forward primer (5'-AGCCAGGAGAAGATGACTGACA-3'). The primers were designed from bovine EST BP112244. We sequenced the 3'-RACE products using an ABI Prism 370 automatic sequencer (Applied Biosystems) after cloning into a pGEM-T Easy vector (Promega).

Reverse Transcription-PCR

We used RT-PCR (detailed in a previous report [22]) to study the distribution of bovine BCL2A1, BCL2, BAX, and CASP3 expression in tissue. We used bovine glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) as a control for the PCR. We placed total RNA in a total reaction mixture for reverse transcription and to synthesize template cDNA, using the oligo (dT) primer and Superscript II reverse transcriptase (Invitrogen) at 42°C for 50 min. Each PCR contained 1 µl cDNA template (1 µg/µl), 1 µl forward primer (20 µM), 1 µl reverse primer (20 µM), 2 µl deoxynucleotide triphosphate mixture (dNTP; 2 mM), 1.2 µl MgCl2 (25µM), 2 µl 10x PCR buffer II, 11.7 µl autoclaved milliQ water, and 0.1 µl AmpliTaq gold DNA polymerase (Applied Biosystems). Amplification conditions included denaturation at 95°C for 30 sec and extension at 72°C for 1 min. We performed 27 cycles for BCL2A1, BCL2, and BAX samples and 36 cycles for the CASP3 sample. We set the annealing temperature at 58°C for 30 sec, performing a single denaturation step at 95°C for 10 min before the first PCR cycle and a final extension step at 72°C for 10 min after the last PCR cycle. PCR products were analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining. We designed the primer encoding for the bovine BCL2A1 sequence according to the sequence illustrated in Figure 1. We used GenBank accession nos. U92434, U92569, and AY575000 to design the primers of bovine BCL2, BAX, and CASP3. Table 1 lists the designated primers. All the primers were commercially synthesized (Tsukuba Oligo Service).


Figure 1
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  		FIG. 1. Nucleotide and deduced amino acid sequences of bovine BCL2A1. The asterisks indicate the termination codon. The polyadenylation signal is underlined with a solid line


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  		TABLE 1. Oligonucleotide primers used for RT-PCR

In Situ Hybridization

We used a full-length cDNA of bovine BCL2A1, BAX, CASP3, and placental lactogen gene (PL) as templates for hybridization probe synthesis. We prepared digoxigenin (DIG)-labeled antisense- and sense-complementary RNA probes as described in previous studies [23]. For hybridization, we cut placentomes into 7-µm-thick sections. Using recommended protocols, we performed in situ hybridization using the automated Ventana HX System Discovery and RiboMapKit and BlueMapKit (Ventana). Briefly, we hybridized sections using DIG-labeled probes in RiboHybe (Ventana) hybridization solution at 69°C for BCL2A1, 67°C for BAX, 65°C for CASP3, and 66°C for PL, for 6 hours. After hybridization, we washed the sections three times in RiboWash (Ventana; 65°C, 6 min) and fixed them in RiboFix (Ventana; 37°C, 10 min). We then detected hybridization signals using monoclonal antidigoxin biotin conjugate (Sigma). After preparation, we observed the hybridized glasses with a Nikon ECLIPSE E800 photomicroscope (Nikon).

Real-Time RT-PCR

Using real-time RT-PCR, we quantitatively confirmed gene expression of bovine BCL2A1, BAX, and CASP3 in each stage of gestation. Details of the real-time RT-PCR procedures have been described in previous reports [24]. Briefly, we reverse-transcribed 50 ng total RNA into cDNA for 30 min at 48°C using MultiScribe reverse transcriptase with an oligo (dT) primer, dNTP mixture, MgCl2 and RNase inhibitor. We used the Primer Express computer software program (Applied Biosystems) to design the primer pairs and oligonucleotide probes labeled with a fluorescent reporter FAM or VIC dye at the 5' end and a fluorescent quencher TAMRA dye at the 3' end. Table 2 lists the primers and probes for each gene. Each PCR contained 1 µl cDNA template (200 ng/µl), 0.5 µl forward primer (50µM), 0.5µl reverse primer (50µM), 0.5 µl dATP (10 mM), 0.5 µl dCTP (10 mM), 0.5 µl dGTP (10 mM), 0.5 µl dUTP (20 mM), 5.5µl MgCl2 (25µM), 2.5µl 10x TaqMan PCR buffer A, 11.6 µl autoclaved milliQ water, 0.25 µl Ampli Erace UNG and 0.15 µl AmpliTaq gold DNA polymerase (Applied Biosystems). The thermal cycling conditions included initial sample incubation at 50°C for 2 min and at 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and at 60°C for 1 min. The cycle threshold values (CT) indicated the quantity of the target gene in each sample, and we determined them in real time using the ABI Prism 7700 sequence detector (Applied Biosystems). We ran triplicate quantitative analysis for each cDNA template, and determined the relative difference in the initial amount of each mRNA species (or cDNA) by comparing the CT values. We generated the standard curves for each gene by serial dilution of plasmid-containing BCL2A1, BAX, CASP3, or GAPDH cDNA to quantify mRNA concentrations. The ratio of BCL2A1, BAX, or CASP3 mRNA to GAPDH mRNA was calculated to adjust for variations in the RT-PCR reaction.


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  		TABLE 2. Oligonucleotide primers and TaqMan probes used for real-time RT-PCR analysis

TUNEL Staining

TUNEL analysis was done on paraffin-embedded sections (7 µm) by an apoptosis detection system, the In Situ Cell Death Detection Kit, POD (Roche Diagnostics). Briefly, sections were deparaffinized and rehydrated with xylene and a graded series of alcohols (100%, 90%, 80%, and 70%) for 2 min each. This was followed by a 20-min incubation of sections with proteinase K (Roche; 15 µg/ml in 10 mM Tris/HCl, pH 7.5) at room temperature. After two washes with PBS, sections were incubated with 0.3% hydrogen peroxide (H2O2) in methanol for 30 min to quench endogenous peroxidase (POD) activity. Sections were then rinsed twice in PBS and reacted with 50 µl of the TUNEL reaction mixture (Roche) for 60 min in a humidified chamber at 37°C. The sections were then rinsed three times in PBS and incubated for an additional 30 min with 50 µl of the Converter-POD (Roche) followed by 10 min with 3,3'-diaminobenzidine (DAB; Roche). This procedure ensures the detection of TUNEL-labeled cells. For positive controls, sections were treated with DNase 1 to induce DNA strand breaks, or peroxidase blocking solution was excluded. Negative controls were achieved by omitting terminal deoxynucleotidyl transferase (TdT; Roche). The percentage of TUNEL-labeled binucleate cells was examined on 3.36 mm2 x four fields of each three sections in each gestation period.

Immunocytochemistry of CASP3

Paraffin sections were deparaffinized and rehydrated with xylene and a graded series of alcohols, and then incubated with proteinase K (Roche) for 20 min at room temperature. After being washed twice with PBS, sections were incubated with 0.3% H2O2 in methanol for 30 min to quench endogenous POD activity. Following two washes with PBS, sections were incubated in 10% goat serum for 30 min at room temperature. Sections were incubated for 2 h at room temperature in PBS containing a mouse monoclonal anti-CASP3 antibody (Santa Cruz Biotechnology) 1:100 dilution. Sections were washed with PBS and peroxidase-labeled polymer conjugated to goat anti-mouse IgG (DAKO ChemMate Envision; DAKO Cytomation) for 1 h at room temperature and color was developed using DAB solution (Roche). Nuclei were stained in the hematoxylin as a counterstain. As a negative control, the primary antibody was omitted from the reaction. The percentage of CASP3 positive binucleate cells was also examined on 3.36 mm2 x four fields of each three sections in each gestation period.

Production of BCL2A1 Recombinant Protein

The bovine BCL2A1 sequence encoding the mature BCL2A1 protein region containing the FLAG epitope tag sequence was inserted into the pFLAG-CMV-4 vector (Sigma). For transient transfection, the constructed plasmid was transfected into HEK 293 cells using FuGENE 6 (Roche). Stably transfected HEK 293 cells were adapted to a suspension culture in a spinner flask using 293 SFM II medium (Invitrogen, Gibco) and then cultured in an atmosphere of 5% CO2 in air at 37°C for 3 days. We separated the medium by centrifugation and stored it at –30°C.

Western Blot Analysis

The proteins in the conditioned media were loaded on each lane, separated by SDS-PAGE, and electrophoretically transferred onto a polyvinylidene difluoride membrane [25]. We then performed Western blotting using the method of Towbin et al. [26]. Briefly, the membrane was blocked in 10% skim milk overnight and incubated with the anti-FLAG M2 (Sigma; 1µg/ml in 1% skim milk in Tris-buffered saline [TBS]) for 1 h at room temperature. We then incubated it with anti-mouse IgG, conjugated with alkaline phosphatase (Sigma; diluted 1:3000) for 1 h at room temperature. Immunopositive bands were stained using NBT (Bio-Rad) and BCIP (Bio-Rad).

RESULTS

Cloning of Bovine BCL2A1 cDNA

We cloned a full-length cDNA for bovine BCL2A1 from bovine placentome and identified the 725-bp open-reading-frame (ORF) cDNA as bovine BCL2A1 (Fig. 1). The protein sequence region (CDS) was composed of 528 bp. The 3' untranslated region contains one AATAAA polyadenylation signal start, 21 bases upstream from the poly (A) addition site [27]. The amino acid sequence deduced from a full-length bovine BCL2A1 cDNA is 175 amino acids, and the BCL2A1 protein deduced from the BCL2A1 nucleotide sequence has an expected size of 20044.8 daltons. We found no N-glycosylation or secretory signal sequence in bovine BCL2A1 (Fig. 2). The predicted amino acids sequence of bovine BCL2A1 exhibited similarity of 78% to human BCL2A1 (NM_004049), 70% to rat BCL2A1 (NM_133416), 65% to mouse BCL2A1A (NM_009742), and 65% to chicken BCL2A1 (NM_204866) (Fig. 2). BH1, BH2, BH3, and BH4 domains were well conserved (Fig. 2). We submitted this mRNA sequence to the DNA Data Bank of Japan (DDBJ accession No. AB195549).


Figure 2
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  		FIG. 2. Comparison of amino acid sequence of bovine BCL2A1 with BCL2A1 of other species. Residues identical to BCL2A1 are shown in black boxes. Amino acid sequence alignments were performed with assistance from Clustal W 1.83 on the DDBJ website. BH1, BH2, BH3, and BH4 refer to the BCL2 homology domain in the BCL2 family

Expression of BCL2A1 mRNA in Bovine Placentome

Figure 3 depicts the tissue distribution of BCL2A1, BCL2, BAX, and CASP3 amplified with RT-PCR. All tissues examined expressed BCL2A1, BCL2, BAX, and CASP3 mRNA except BCL2A1 mRNA in endometrium (Day 0 of estrous cycle). In placenta (cotyledon) of each stage, BCL2 mRNA appeared to be slightly lower than in other tissue. CASP3 mRNA was not amplified in the PCR of 27 cycles, but its expression was amplified in 36 cycles. In the placenta, its amplification was still fair during gestation close to term.


Figure 3
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  		FIG. 3. Expression of bovine BCL2A1, BCL2, BAX, and CASP3 mRNA in bovine tissues. RT-PCR used tissues from heart, liver, lung, kidney, spleen, and endometrium. Endometrial tissue was from Day 0 of estrous cycle. Cotyledonary tissue at Days 60, 150, and 250 of gestation served as placental samples. GAPDH expression in each tissue is shown as standard data

We determined BCL2A1, BAX, CASP3, and PL mRNA localization by in situ hybridization in the bovine placentome on Days 60 and 120 of gestation and postpartum (Fig. 4). DIG-labeled BCL2A1, BAX, CASP3, and PL antisense RNA specifically detected the mRNA transcript in both placentomes. The BCL2A1 expression was found in trophoblastic binucleate cells in the cotyledon and the intercotyledonary membrane, similar to PL expression (Fig. 4). No BCL2A1 mRNA was found in the maternal tissues (caruncle and intercaruncle in endometrium). However, BAX mRNA appeared in binucleate cells as well as in other trophoblastic cells and the endometrium. The CASP3 expression was mainly observed in the binucleate cells and the other trophoblastic cells of the postpartum placenta. We detected no significant signal with the sense probe.


Figure 4
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  		FIG. 4. Localization of BCL2A1, BAX, CASP3, and PL in the bovine placentome during gestation. First line, Day 60 of gestation; second line, higher magnification of the first line, except CASP3, which is a postpartum material; third line, Day 120 of gestation; fourth line (bottom), postpartum. BCL2A1 (A–D), BAX (E–H), CASP3 (I–L) and PL (M–P) mRNA were detected using in situ hybridization. Seven-microgram sections of bovine placentome were individually hybridized with a DIG-labeled antisense cRNA probe. Bar = 100 µm on the top and bottom, and 20 µm in the middle. COT, Cotyledonary area; ICOT, intercotyledonary area; CAR, caruncular area

Figure 5 presents the results of quantitative real-time RT-PCR analysis of BCL2A1, BAX, and CASP3 mRNA in conceptus, extraembryonic membrane, cotyledon, and intercotyledon during gestation. We detected BCL2A1 mRNA expression in all examined tissues except in Day 14 CON. Expression intensity increased with the progress of gestation in all tissues. When comparing COT and ICOT, the intensities in COT were always higher, but the only significant differences were on Day 250 and postpartum. BAX mRNA expression increased after Day 28EEM. The expression intensities were significant in COT and ICOT on Day 250 and postpartum when compared to those in other gestation days. On Day 21, the expression level was temporarily higher and decreased on Day 28. On Day 14, expression was barely detected, but it was stable. Expression ratio (mean ± SD) between BCL2A1 and BAX of COT in Figure 5 was 13.3 ± 2.2, 4.1 ± 2.4, 1.1 ± 0.7, and 0.5 ± 0.1 on Days 60, 150, 250, and postpartum, respectively. The ratio on Day 60 was significantly different from that found at all other gestational stages (P < 0.05). The value on Day 150 was also significant from that found postpartum (P < 0.05). There was no difference between the value on Day 250 and the postpartum value. In the COT, BCL2A1 and BAX ratios decreased with the progress of the gestation period, and reached the lowest levels at postpartum. CASP3 expression was weakly but constantly detected in the COT and ICOT during gestation; however, it increased remarkably in postpartum COT. On Day 21, CASP3 expression, as well as BAX expression, temporarily increased.


Figure 5
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  		FIG. 5. Quantitative real-time RT-PCR analysis of bovine BCL2A1 (A), BAX (B), and CASP3 (C) mRNA in bovine trophoblast-related tissue. Total RNA was extracted from conceptus (CON) cotyledon containing extraembryonic membrane (EEM), cotyledon (COT), and intercotyledonary fetal membrane (ICOT) on Days 14, 21, 27, 60, 150, and 250 of gestation, as well as postpartum. The expressions of these mRNA were normalized to the expression of GAPDH measured in the same RNA preparation. Values are means ± SD. Values with different letters are significantly different (P < 0.05)

The Detection of Apoptosis in Bovine Placentome

The placentomal apoptosis signal was detected by TUNEL and CASP3 immunohistochemistry. The TUNEL-positive cells in the trophoblast were clearly found on Day 120, and more intensive expression was presented in the trophoblast cells in postpartum. As shown in Table 3, TUNEL-positive binucleate cell number clearly increased from Day 60 to postpartum. Faint CASP3 staining was found in placentome during gestation, but clear positive cells were detected in the postpartum COT, especially the binucleate cell and other trophoblast cells (Fig. 6, D and H). The CASP3-positive binucleate cells were also increased with the progress of gestation, and the largest number was detected in postpartum.


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  		TABLE 3. Percentage of morphologically apoptotic, TUNEL, and CASP3 positive trophoblastic binucleate cells in 3.36 mm x 4 fields (mean ± SD) in each stage of gestation


Figure 6
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  		FIG. 6. TUNEL detection and CASP3 immunostaining in the bovine placentome on Day 60 of gestation (upper line), Day 120 of gestation (second line) and postpartum (third and bottom lines). The bottom line shows higher magnification of the third line. Seven-microgram sections of bovine placentome were individually stained with TUNEL (A–D) and CASP3 (E–H). Bar = 100 µm (top to third line), 20 µm (bottom line). The arrow shows the stained binucleate cells

Translation of BCL2A1 cDNA in HEK 293 Cells

The cloned BCL2A1 cDNA was translated in the HEK 293 cells and confirmed using Western blotting (Fig. 7). We detected an intense band with approximately 21 kDa in the culture media. Bovine BCL2A1 recombinant protein had predicted 20-kDa molecules from sequence data, and the FLAG epitope tag addressed about 1 kDa [28], clearly demonstrating that the protein lacked glycosylation. The sugar chain was not detected in the recombinant protein.


Figure 7
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  		FIG. 7. Production of recombinant BCL2A1 proteins. We collected conditioned media from HEK 293 cells, which were transiently transfected with each gene, and placed the protein on each lane. The proteins were separated by SDS-PAGE, and specific proteins were detected by Western blot analysis using an anti-FLAG tag. Only the vector was transfected into HEK 293 cells. MW marker, Molecular weight marker; NC, negative control

DISCUSSION

In this research, we cloned a bovine BCL2A1 cDNA, a member of the bovine BCL2 family, from placenta. Expression of this gene was limited to binucleate cells of trophoblastic tissues. The gene contained a 725-nucleotide ORF and 528-bp CDS, and the deduced amino acid sequence from a full-length bovine BCL2A1 cDNA was 175. The gene was primarily expressed in trophoblast cells on Day 21; the expression increased in middle gestation, then declined in postpartum. The expression profile coincided with the time of implantation and progress of gestational changes in the trophoblast function in bovine [29, 30]. These data support the speculation in the present study that suitable maintenance may be dependent on BCL2A1 for trophoblast. Endometrial epithelial cells are required to coordinate the fetal-maternal interface during gestation and promote an appropriate implantation and placental growth.

The deduced bovine BCL2A1 protein was similar to those of other mammalian BCL2A1 (Fig. 2). However, the N-glycosylation site (Asn-X-Ser/Thr) appeared at residue 128–130 in humans, rats, and mice, but not in bovine [2–4]. This suggested that the bovine BCL2A1 has no sugar chain. The translation data in HEK 293 in the current study supports this assumption. The cysteine residue positions are not conserved in each mammalian BCL2A1. Bovine BCL2A1 has five cysteine residues at Cys-55, –107, –115, –165, and 169, and human BCL2A1 also has five cysteine residues, although Cys-55 and –165 were the only common portions. Both rat and mouse BCL2A1 had only one cysteine residue, and their positions differed completely. They kept no relative position to those of bovine BCL2A1 (Fig. 2). These data suggest that bovine BCL2A1 frames a specific three-dimensional structure, and that its protein is functionally different from the BCL2A1 of the other species tested, human, rat, and mouse.

The BH4 domain is a member of the antiapoptotic BCL2 family [9], and the presence of a glutamine-rich protein in the BH4 domain is a unique feature of BCL2A1 [31]. We believe that the glutamine residues are essential for the cell proliferation activity and the apoptosis mechanism of BCL2A1 [31]. The BH4 domain of bovine BCL2A1 also contains three glutamines, as does the human BCL2A1 (Fig. 2). We anticipate that the BH4 domain may have activity similar to humans [31].

BCL2A1 mRNA expression was higher than that of BCL2 mRNA in placenta (Fig. 3). Both BCL2 families play a role in preventing blood cell death [32, 33]. In bovine placenta, BCL2A1 is an important molecule for maintaining trophoblast function during gestation. We believe that placental BCL2A1 may play a similar role in trophoblast cell function in various species because only BCL2 expression was detected in syncytiotrophoblasts in human placenta, which are known as multinucleate cells [34]. However, no data are available on BCL2A1 expression in human placenta [4]. BCL2A1 mRNA was expressed in binucleate cells in trophoblasts, and kept throughout gestation (Figs. 4 and 5). The bovine binucleate cells maintained this expression throughout gestation [35]. Other proapoptotic BCL2 families like BAX and BAK have an opposite function to that of BCL2A1. We detected expression of the proapoptotic gene, BAX, in various tissues (Fig. 3). Specifically, the BAX appeared in the binucleate cells of the cotyledon or intercotyledon, as depicted in Figure 4. We detected the BAX mRNA in the embryonic membrane from before implantation and throughout gestation, and the binucleate cells may be the main source. We found both BCL2A1 and BAX throughout gestation in binucleate cells. Because the cell population is kept at a constant 20% throughout gestation, the contradictory data suggest that new binucleate cells replace old ones during gestation in bovine placenta [36]. Therefore, both apoptotic and antiapoptotic signals appeared in the same cell. The higher BAX expression on Day 21 may indicate that various cells are dying and only the cells necessary for implantation and formation of a placenta survive (Fig. 5). Bovine binucleate cells maintain gestation, and BCL2A1 is part of this process. Additionally, we examined CASP3 and CASP3 expression in placenta during gestation. A slight expression and change were found during gestation, except in the implantation period, and then it was distinct in postpartum (Figs. 4, 5, and 6). A reverse correlation was detected between CASP3 and BCL2A1, and CASP3 expression correlated with BAX (Fig. 5). TUNEL detection results correlated to CASP3 expression, especially in the the highest TUNEL positive signal was detected in binucleate cells postpartum (Fig. 6). The percentages of TUNEL- and CASP3-positive binucleate cells resembled each other and were 0.7% at Day 60, 4% at Day 120, and 13% postpartum (Table 3). Increased CASP3 and BAX expression was found on Day 21 (Fig. 5). This may be related to the progress of implantation as reported in other species [18, 37]. In the real-time PCR of COT, the BCL2A1/BAX expression ratio decreased postpartum, in contrast to numbers of TUNEL- and CASP3 positive binucleate cells, which increased (Table 3). We anticipated that the binucleate cell maintenance and/or apoptosis would be regulated by the balance of the BCL2A1 and BAX expression levels. However, BAX may also be regulated to balance with other antiapoptotic BCL2 family members, because a reduced BCL2A/BAX ratio was found. Apoptosis of endometrial and trophoblast cells may be affected by trophoblastic cell factors, because interferon-tau (IFNT) induced proapoptotic protein BAX [38]. More detailed studies are needed to clarify the regulation of the maintenance of endometrial and trophoblastic cells in the implantation interface in bovine. Recently BCL2, a key molecule for trophoblast cell viability, was found in syncytotrophoblast of monkey [12]. However, it is still difficult to generalize the role of BCL2A1 for trophoblast cell survival and apoptosis in various species. The present study suggests that it participation in participates of the fetal-maternal interface in bovine.

Bovine binucleate cells produce various specific molecules such as PL, prolactin-related proteins (PRPs), pregnancy-associated glycoproteins (PAGs) and steroids [28, 39–42]. We observed BCL2A1 expression in the binucleate cells similar to temporary PL expression. During gestation, BCL2A1 expression increased with gestation progress and peaked around the middle of gestation, then gradually declined to postpartum. This profile coincides with PL and PRP expression [29, 30, 35, Fig. 4]. However, binucleate cells expressed not only PL and BCL2A1, but also BAX. BCL2A1 expression is confined in the binucleate as well as PL, especially in the early stage of gestation. These data suggest an antiapoptotic function for BCL2A1 in the bovine trophoblast cell. The expression in the early period of gestation is a surprise, and higher and continuous expression of BCL2A1 provides clear evidence that BCL2A1 regulates binucleate cell function during gestation. The number of dead binucleate cells quickly declined close to term in bovine [43], and the pattern was similar to that of BCL2A1 expression. A complex relationship may be needed for cell survival and the proliferation and production of various molecules such as PLs, PAGs, prostanoids, and steroids [44]. However, the regulatory variations between BCL2A1 and PL require future study. HEK293 cell systems provided a BCL2A1 cDNA clone for our study that produced and secreted a 20-kDa protein (Fig. 7). Therefore, the identified gene sequence of bovine BCL2A1 may be translated to protein in vivo, and the antiapoptotic function may participate in determining the fate of trophoblastic cells in bovine.

In conclusion, we have identified the BCL2A1 gene and its encoding BCL2A1 protein as members of the bovine BCL2 family. BCL2A1 was expressed in bovine trophoblastic binucleate cells in placentome and was similar to bovine PL, PRPs, and some PAGs. BAX was also expressed in binucleate cells from early in gestation to term, with peak expression in the middle of gestation, and BCL2A1 and BAX may participate in the maintenance of gestation through the regulation of the population of binucleate cells. The BCL2A1 molecule is a new candidate for antiapoptotic maintenance of binucleate cells in bovine.

ACKNOWLEDGMENTS

The authors would like to thank Prof. Yukio Tsunoda of Kinki University and Dr. Tomoyuki Tokunaga of the National Institute of Agrobiological Sciences for their financial support to K.U.

FOOTNOTES

1 Supported by grants from the Bio-oriented Technology Research Advancement Institution (BRAIN); a grant-in-aid (HC-04–2261–1) from the Ministry of Agriculture, Forestry, and Fisheries of Japan; a grant-in-aid from Ito-zaidan (Tokyo, Japan); and a grant (Hoga-kenkyu 16658105; Kiban-kenkyu C 17580284) from the Ministry of Education, Culture, Sport, Science, and Technology of Japan. Back

2 Correspondence: Kazuyoshi Hashizume, Department of Veterinary Medicine, Faculty of Agriculture, Iwate University, 3–18–8 Ueda, Morioka, Iwate 020-8550, Japan. FAX: 81 19 621 6212; kazuha{at}iwate-u.ac.jp Back

3 Current address: Department of Technology, National Livestock Breeding Center, 1 Odakurahara, Odakura, Nishigo, Fukushima 961-8511, Japan. Back

Received: 8 April 2005.

First decision: 26 April 2005.

Accepted: 12 October 2005.

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T. Takahashi, O. Yamada, M. J Soares, and K. Hashizume
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