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BOR - Papers in Press, published online ahead of print June 16, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.030213
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BIOLOGY OF REPRODUCTION 71, 1317–1324 (2004)
DOI: 10.1095/biolreprod.104.030213
© 2004 by the Society for the Study of Reproduction, Inc.

Pregnancy

Expression of Proteasome Subunits Low Molecular Mass Polypeptide (LMP) 2 and LMP7 in the Endometrium and Placenta of Rhesus Monkey (Macaca mulatta) During Early Pregnancy1

Hong-Xing Wang3,4, Hong-Mei Wang3, Qing-Lei Li3, Hai-Yan Lin3,4, Dong Qian3,4, and Cheng Zhu2,3

State Key Laboratory of Reproductive Biology,3 Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China Graduate School of the Chinese Academy of Sciences,4 Beijing 100039, China


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous studies have demonstrated that the ubiquitin-proteasome pathway plays an important role in embryo implantation. Low molecular mass polypeptide (LMP) 2 and LMP7 are the two subunits of 20S proteasome, which are critical for proteasome activity. To further elucidate the roles of LMP2 and LMP7 in embryo implantation during early pregnancy, we cloned partial sequences of the LMP2 and LMP7 genes and studied the spatiotemporal expression of LMP2 and LMP7 in rhesus monkey (Macaca mulatta) uteri on Days 12, 18, and 26 of pregnancy. The results showed that the 349-base pair (bp) LMP2 fragment and the 340-bp LMP7 fragment were 97% and 99% identical, respectively, to those of human homologues. From the statistical results of reverse transcription-polymerase chain reaction, in situ hybridization, and immunohistochemistry, we found that the expression levels of LMP2 and LMP7 significantly increased with the elongation of pregnancy. The LMP2 and LMP7 mRNAs were mainly expressed in the luminal and glandular epithelium on Day 12 of pregnancy. On Days 18 and 26 of pregnancy, strong signals of LMP2 and LMP7 mRNAs were detected in the placental villi, trophoblastic column, and arterial endothelial cells close to the implantation site, and moderate expressions were found in the trophoblastic shell and glandular epithelium. The LMP2 and LMP7 mRNAs were extensively distributed in the stroma on Day 26 of pregnancy. The expression patterns of LMP2 and LMP7 proteins were similar to those of their transcripts, but weak immunostaining of LMP2 and LMP7 proteins was detected in stroma at all stages of pregnancy. These results suggest that LMP2 and LMP7 may be involved in some key processes of trophoblastic invasion, angiogenesis, degradation of the extracellular matrix, immune tolerance, and glandular secretion.

implantation, placenta, pregnancy, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The proteasome is an ATP-dependent, multisubunit, multicatalytic protease that is responsible for the majority of nonlysosomal degradation in eukaryotic cells that recognizes and degrades ubiquitinated proteins, including misfolded, damaged, and other regulatory proteins [1, 2]. The ubiquitin-proteasome pathway (UPP) is involved in regulation of the cell cycle, degradation of some transcription factors, modification of some membrane proteins, assembly of ribosomes, and antigen presentation of major histocompatibility complex (MHC) class I [1, 3, 4]. The 26S proteasome, a 2.5-Mda protease complex, contains the core 20S proteasome together with a 19S regulatory complex bound at both ends [5]. The 20S proteasome is a 700-kDa cylindrical structure that is composed of 28 subunits arranged in four stacked, heptameric rings, with the outer two rings containing seven different {alpha} subunits and the inner two rings containing seven different ß subunits [6, 7]. Each proteolytic ß subunit expressed with an NH2-terminal threonine residue is critical for multiple peptidase activities, including chymotryptic-, tryptic-, and peptidylglutamyl-like activities [8–10].

Two of proteasome subunits with an NH2-terminal threonine residue, low molecular mass polypeptide (LMP) 2 and LMP7, which are induced by interferon-{gamma}, are encoded within the class II region of the MHC, directly adjacent to the transporter associated with antigen presentation (TAP) 1 and TAP2 genes [11]. Several experiments show that incorporation of LMP2 and LMP7 into 20S proteasome is responsible for antigen presentation, and inhibition of proteasome activity affects the capacity of cells to present antigenic peptides [12, 13]. Furthermore, the presence of LMP2 and LMP7 in 20S proteasome produces increased chymotryptic and tryptic activities in vitro and modulates the cleavage-site preferences of the proteasome [14, 15]. The proteasome is localized both in the nucleus and in the cytoplasm of eukaryotic cells, but LMP2 and LMP7 appear to be more intense in the endoplasmic reticulum [16, 17]. Thus, proteasomes in different cells normally differ in subunit composition and functional activities in a way that correlates with the cell's capacity for antigen presentation [1].

Embryo implantation begins when the blastocyst both assumes a fixed position in the uteri and establishes a more intimate relationship with the endometrium [18]. Successful implantation depends on the synchronized development of both invasion of the embryo and receptivity of the endometrium. The process is accompanied by extensive degradation and remodeling of the extracellular matrix (ECM), maternal vascular transformation, and immune tolerance [19]. Despite the fundamental events of embryo implantation being common to many animals, the approach to penetration and the type of placenta vary in different species. Both primate and human adopt the intrusive penetration to the luminal epithelium and form the hemochorial placenta [18]. Thus, the rhesus monkey is an ideal animal model for studying the mechanisms of human implantation. Our previous studies have demonstrated that the UPP is involved in mouse embryo implantation. Lactacystin, a specific inhibitor of proteasome by binding its catalytic, active threonine residues (e.g., LMP2 and LMP7), significantly decreased the numbers of implanted mouse embryos [20]. The UPP is implicated in the generation of MHC class I molecules that play central roles in the establishment of maternal-fetus immune tolerance [21–23]. Interferon-{gamma}, which is a stimulator of antigen presentation, is a key cytokine in regulating a large number of gene expressions and in maintaining immune tolerance between mater and embryo during early pregnancy [24, 25]. The LMP2 and LMP7 are the two key subunits for catalytic activity in proteasome and are required for production of the vast majority of MHC class I ligands [14, 26–28]. Whether LMP2 and LMP7 play crucial roles in embryo implantation, however, remains unclear.

To investigate the roles of the proteasome subunits LMP2 and LMP7 in embryo implantation, we analyzed in the present study the spatiotemporal expression of LMP2 and LMP7 in the rhesus monkey endometrium and placenta during early pregnancy using reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Tissue Collection

Twelve female rhesus monkeys (Macaca mulatta) with normal menstrual cycles and records of pregnancy were obtained from the Center of Medical Primates, Institute of Medical Biology, Chinese Academy of Medical Sciences. Pregnancy predication and sample collection were conducted as previously described [29]. All experimental protocols were approved by the ethical committee of the State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences. The uteri were collected from the rhesus monkeys under ketamine hydrochloride anesthesia on Days 12, 18, and 26 of pregnancy. Four samples were obtained in every stage of pregnancy. The implantation site was located and then cut into two parts. One part was fixed in Bouin solution for 20 h and then embedded in paraffin. Endometrium from the other part was isolated, quickly frozen in liquid nitrogen, and stored at –80°C for RNA extraction.

Reverse Transcription-Polymerase Chain Reaction

Total RNA from the endometrium was extracted with Trizol reagent (Gibco BRL, Grand Island, NY) according to the manufacturer's instructions. The first-strand cDNA was synthesized with Superscript II reverse transcriptase (Gibco BRL) and oligo dT from 2 µg of total RNA. Specific PCR primer pairs from the human gene used in the present study are summarized in Table 1. The ideal ranges of PCR amplification observed were 32 cycles for LMP2 and LMP7 and 28 cycles for ß-actin. In the PCR analysis, 25 µl of the PCR system contained 2 µl of the first-strand cDNA, 200 µmol/L of dNTPs, 2 mmol/L MgCl2, 1 U of Taq polymerase (TaKaRa Corp., China), and 10 pmol of each primer. The parameters for PCR amplification were as follows: denaturation, 45 sec at 94°C; annealing, 45 sec at 58°C for LMP2 and at 57°C for LMP7; and extension, 45 sec at 72°C. Several control reactions were performed to confirm the quality of RT products and to exclude genomic contamination (data not shown). Amplification of ß-actin gene transcripts was used to confirm RNA integrity and efficiency. The PCR products from three individual PCR reactions were separated on 1.2% agarose gel, then excised from the gel and purified using the NucleoTrap Gel Extraction Kit (Clontech, Inc., Palo Alto, CA). The purified products were cloned into pGEM@-T Easy vector (Promega Corp., Madison, WI) and sequenced to confirm sequence identity (Sangon Corp., China).


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  		TABLE 1. Specific primer pairs for PCR

In Situ Hybridization

The digoxin (DIG)-labeled LMP2 and LMP7 cRNA probes were generated according to the manufacturer's protocols. Probe-labeling efficiency was confirmed by dot blot analysis. Several important steps for in situ hybridization were described. Adjacent paraffin sections (thickness, 5 µm) were cut and dried overnight at 40°C. The sections were dewaxed extensively and digested in proteinase K solution (10 µg/ml) for 10 min at 37°C. To preserve mRNA and terminate proteinase K digestion, the sections were fixed in 4% paraformaldehyde. Prehybridization was carried out at 50°C for 2 h in a buffer containing 50% deionized formamide, 10% dextran sulfate (w/v), 0.5% SDS, 1x Denhardt, 10 mmol/L of Tris-HCl (pH 8.0), 10 mmol of dithiothreitol, 2x SSC (1x SSC: 0.15 M sodium chloride and 0.015 M sodium citrate), and 250 µg/ml of sheared salmon sperm DNA (Sigma, St. Louis, MO) without probe in a humid chamber. Slides were then hybridized with 400 ng/ml of the labeled antisense or sense probe in prehybridization buffer overnight at 58°C. To reduce nonspecific binding, slides were completely washed in decreasing concentrations of SSC at 42°C, in buffer A (pH 7.5) (100 mmol/L of Tris, 150 mmol/L of sodium chloride) for 5 min and covered with buffer B, containing 1% blocking reagent (Boehringer Mannheim) in buffer A, for 30 min at room temperature. The slides were then incubated with anti-DIG-alkaline phosphatase antibody (diluted 1:500 in buffer B) for 2 h. The color was developed by nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate (Boehringer-Mannheim) in buffer C (pH 9.5) (100 mmol/L of Tris, 100 mmol/L of sodium chloride, 50 mmol/L of MgCl2). Sense-probe hybridizations were performed as a control for the background level. To remove nonspecific staining, slides were rinsed in 95% ethanol for 30 min and mounted with the Histomount Reagent (Zhongshan Corp.). The results were recorded with a SPOT digital camera system (Diagnostic Instruments, Inc., Sterling Heights, MI), and the digital images were processed by Adobe PhotoShop (Version 7.0; Adobe, San Jose, CA).

Immunohistochemistry

Immunohistochemistry was performed using the Histostain-Plus Kit and diaminobenzidine (DAB; Zhongshan Corp.) as recommended by the manufacturer. Briefly, the paraffin-embedded sections (thickness, 5 µm) were deparaffinized completely. To expose the antigens sufficiently, the slides were immersed in citric acid buffer (10 mmol/L of citrate sodium, 10 mmol/L of citric acid) and boiled in a microwave oven at 92–98°C for 15 min. The sections were cooled to room temperature and then sequentially incubated at room temperature with 3% H2O2 in methanol for 15 min to quench endogenous peroxidase; in normal blocking serum for 30 min; in primary antibody, including rabbit anti-LMP2 immunoglobulin (Ig) G (10 µg/ml) and mouse anti-LMP7 IgG (10 µg/ml) (both from AFFINITI Research Products Ltd., U.K.) for 2 h; in biotinylated secondary antibody for 30 min; and in the AB reagent for 1 h. Intervening PBS washes were performed after incubation when necessary. The sections were stained with DAB and mounted as described above. As a negative control, slides were incubated without primary antibody or with preimmune mouse or rabbit serum.

Statistical Analysis

The quantity of the PCR products was determined by densitometric analysis of the intensities of the bands. The bands were analyzed using MetaView Image Analyzing System (Version 4.50; Universal Imaging Corp., Downingtown, PA). The relative levels of LMP2 and LMP7 mRNAs normalized to ß-actin mRNA were calculated. Signal intensities of the mRNAs and proteins of LMP2 and LMP7 detected by in situ hybridization and immunohistochemistry were quantified by computer-aided laser-scanning densitometry (Personal Densitometer SI; Molecular Dynamics, Inc., Sunnyvale, CA). To make the statistical significance of quantitative difference credible, at least three slides from each of four animals from each group were examined (n = 12). In each specific location on a slide, at least 50 points were randomly selected. The gray level of intercellular substance was considered to be background. Statistical analysis was performed using Statistical Package for Social Science (SPSS for Windows package release 10.0; SPSS, Inc., Chicago, IL). One-way ANOVA was used to evaluate the data from RT-PCR and the average intensity data from in situ hybridization and immunohistochemistry. The general linear model was used to analyze the difference in different locations within a time point and different time points within a location. The level for statistical significance was considered to be P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Temporal Changes of LMP2 and LMP7 mRNA Expression in Rhesus Monkey Endometrium

To detect the expression and variation of LMP2 and LMP7 mRNA in the rhesus monkey endometrium during early pregnancy, LMP2 and LMP7 mRNAs were studied on Days 12, 18, and 26 of pregnancy using RT-PCR. Representative illustrations of PCR products, graphical statistical analysis, and sequence comparison are shown in Figures 1 and 2. As shown in Figures 1A and 2A, a 349-base pair (bp) fragment of the LMP2 gene and a 340-bp band of the LMP7 gene were obtained in each sample. A clear band of ß-actin was also obtained from each sample at all the time points. No specific band was detected in negative controls in the PCR system (data not shown). Densitometry showed (Figs. 1B and 2B) that the expression levels of LMP2 and LMP7 mRNAs increased with the prolongation of pregnancy significantly (P < 0.05). The identity of LMP2 and LMP7 PCR products was confirmed by sequencing. The partial cDNA fragment of the LMP2 gene had 97% sequence identity with the corresponding region of human LMP2 gene (nucleotides 73–421; GenBank accession no. U01025), and 11 point variations were observed in the macaque LMP2 gene fragment compared with its human homologue (Fig. 1C). The partial LMP7 fragment had a high degree of sequence similarity (99%) with the corresponding region of the human homologue (data not shown).



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  		FIG. 1. RT-PCR analysis of LMP2 mRNA in the rhesus monkey endometrium during early pregnancy. A) Representative RT-PCR products of LMP2 and ß-actin (used as an internal control) on Days 12 (D12), 18 (D18), and 26 (D26) of pregnancy. M, DNA marker (bp). B) Statistical analysis of relative level for LMP2 mRNA in the macaque endometrium at different stages of pregnancy. The relative intensity was determined by the ratio of LMP2 mRNA to ß-actin mRNA as measured by densitometry (n = 4). Bar with different letters are significantly different (P < 0.05). C) Comparison of the 349-bp fragment of the rhesus monkey LMP2 gene, as amplified by PCR, with the human homologue



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  		FIG. 2. RT-PCR analysis of LMP7 mRNA in the rhesus monkey endometrium during early pregnancy. A) Agarose-gel electrophoresis of LMP7 and ß-actin (used as an internal control) on Days 12 (D12), 18 (D18), and 26 (D26) of pregnancy. M, DNA marker (bp). B) Relative level for LMP7 mRNA in the macaque endometrium at different stages of pregnancy. The relative intensity was determined by the ratio of LMP7 mRNA to ß-actin mRNA as measured by densitometry (n = 4). Bars with different letters are significantly different (P < 0.05)

Localization of LMP2 and LMP7 mRNAs in the Macaque Endometrium and Placenta During Early Pregnancy

In situ hybridization was performed to demonstrate the spatiotemporal expression of LMP2 and LMP7 mRNAs in the pregnant macaque uterus. The typical structure of the uterus was confirmed by immunohistochemical staining of cytokeratin, actin, and vimentin (data not shown). As shown in Figure 3 and Table 2, the expression patterns of LMP2 and LMP7 mRNAs in the endometrium and placenta were very similar; the average intensities of LMP2 and LMP7 mRNAs at three stages of pregnancy significantly (P < 0.05) increased with the progress of gestation. The LMP2 and LMP7 mRNAs were mainly detected in the luminal and glandular epithelium of endometrium on Day 12 of pregnancy (P < 0.05) (Fig. 3, D12). On Days 18 and 26 of pregnancy, the expression levels of LMP2 and LMP7 mRNAs in the placental villi, cytotrophoblast cells of the trophoblastic column, and arterial endothelial cells near the implantation site were the most abundant (P < 0.05) (Fig. 3, D18 and D26). The LMP2 and LMP7 transcripts were moderately expressed in the trophoblastic shell and glandular epithelium (Fig. 3, D18 and D26). The LMP2 and LMP7 mRNAs in stroma were relatively low on Day 12 of pregnancy (Fig. 3, D12), increased with the progress of pregnancy, and were widely expressed in stroma on Day 26 of pregnancy (P < 0.05) (Fig. 3, D18 and D26). The LMP2 and LMP7 mRNAs were both absent in the myometrium on Days 12, 18, and 26 of pregnancy (Fig. 3, D12 and D26). Control sections hybridized with sense probe of LMP2 or LMP7 showed no specific signal except for a low staining (Fig. 3, Control).



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  		FIG. 3. Localization of LMP2 and LMP7 mRNAs in the uterus and placenta of rhesus monkey on Days 12 (D12), 18 (D18), and 26 (D26) of pregnancy by in situ hybridization. D12: uterus; D18: placenta and uterus; D26: placenta and uterus; Control: control section hybridized with sense probe on D26. A, Arteriole; FMB, fetal-maternal border; L, Lumen of the uterus; LE, luminal epithelium; GE, glandular epithelium; M, myometrium; PV, placental villi; S, stroma; TC, trophoblastic column; TS, trophoblastic shell. Bar = 100 µm; the identical magnification is shown in Control


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  		TABLE 2. Quantification of LMP2 and LMP7 mRNAs at different locations of rhesus monkey uterus and placenta on Days 12 (D12), 18 (D18), and 26 (D26) of pregnancy.a

Immunohistochemical Localization of LMP2 and LMP7 Proteins in the Pregnant Uteri and Placenta

To further examine LMP2 and LMP7 protein localization in the pregnant uteri and placenta, immunohistochemical analysis was performed. As Figure 4 and Table 3 show, the results demonstrated that the localization patterns of LMP2 and LMP7 proteins were similar to those of their mRNAs at three time points. Statistical analysis (Table 3) showed that expressions of LMP2 and LMP7 proteins significantly (P < 0.05) increased with elongation of pregnancy. On Day 12 of pregnancy, strong expressions of LMP2 and LMP7 proteins were found in the luminal and glandular epithelium of endometrium (P < 0.05) (Fig. 4, D12). High levels (P < 0.05) of LMP2 and LMP7 proteins were visualized in the placental villi and cytotrophoblast cells of the trophoblastic column. Moderate LMP2 and LMP7 proteins were detected in the glandular epithelium and arterial endothelial cells situated adjacent to the implantation site on Day 18 of pregnancy (Fig. 4, D18). On Day 26 of pregnancy (Fig. 4, D26), the placental villi, cytotrophoblast cells of the trophoblastic column, and arterial endothelial cells near to the implantation site expressed abundant LMP2 and LMP7 proteins (P < 0.05), and positive LMP2 and LMP7 protein signals were present in the glandular epithelium and trophoblastic shell. Different from expressions of LMP2 and LMP7 mRNAs, however, the expressions of LMP2 and LMP7 proteins in stroma were faint on Days 12, 18, and 26 of pregnancy. Only background signals of LMP2 and LMP7 proteins were detected in myometrium at any time point (Fig. 4, D12 and D26). Control sections substituting the primary antibody with normal rabbit IgG or mouse IgG did not produce any positive immunostaining (Fig. 4, Control).



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  		FIG. 4. Immunohistological staining for LMP2 and LMP7 proteins in the rhesus monkey uterus and placenta on days 12 (D12), 18 (D18), and 26 (D26) early pregnancy. D12: uterus; D18: placenta and uterus; D26: placenta and uterus; Control: control section without primary antibody. A, arteriole; L, Lumen of the uterus; LE, luminal epithelium; GE, glandular epithelium; M, myometrium; PV, placental villi; S, stroma; TC, trophoblastic column; TS, trophoblastic shell. Bar = 100 µm; the identical magnification is shown in Control.


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  		TABLE 3. Quantitive analysis of LMP2 and LMP7 proteins at different locations of rhesus monkey uterus on Days 12 (D12), 18 (D18), and 26 (D26) of pregnancy.a


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Blastocyst implantation and successful establishment of pregnancy require delicate interactions between the embryo and maternal environment. Primate implantation is a very complex process involving a series of morphological, biochemical, and immunological changes in the uteri and placenta [30]. Degradation and remodeling of the ECM as well as cell proliferation and differentiation are necessary for successful implantation. In recent years, proteolytic pathways related to cell proliferation and differentiation such as UPP, have been the focus of study to define the complicated regulatory network of implantation [31]. Results from Bebington et al. [32] suggested that the UPP was critical for the extensive tissue remodeling during early pregnancy [32]. Our previous studies demonstrated that the proteasome-inhibitor lactacystin had an inhibitory effect on mouse embryo implantation, which was achieved by reducing overall proteolysis and MHC class I antigen presentation and degradation of specific proteins [20]. Both LMP2 and LMP7, the two key ß subunits with an NH2-terminal threonine residue, can be modified by lactacystin, which inhibits proteasome activity. Both LMP2 and LMP7 play crucial roles in multiple peptidase activities. Encoded in the class II region of the human MHC, LMP2 and LMP7 have implicated this multisubunit protease in an early step of the immune response, the degradation of intracellular and viral proteins [33]. However, little is known about the roles of LMP2 and LMP7 in embryo implantation. In the present study, we provide the first evidence, to our knowledge, regarding the spatiotemporal expression patterns of LMP2 and LMP7 in macaque endometrium and placenta on Days 12, 18, and 26 of pregnancy, and we explore the idea that LMP2 and LMP7 are involved in early pregnancy.

On Days 12, 18, and 26 of pregnancy, LMP2 and LMP7 mRNAs were detected at three time points by RT-PCR. The expression levels of LMP2 and LMP7 mRNAs increased with the elongation of pregnancy significantly. These results indicate that LMP2 and LMP7 may be involved in some key processes of early implantation.

A series of key events must occur during implantation: apposition of the blastocyst to the uterine epithelium, adhesion of blastocyst to this epithelium, intrusion of placental villi to the endometrium, formation of new vessels, and glandular secretion [18]. Blastocyst, polarized epithelium, and trophoblast are the important sources of a massive secretion of interferon-{gamma} [34]. Strong signals of transcripts and proteins of LMP2 and LMP7 detected in the placental villi, trophoblastic column, and luminal epithelium may be regulated by interferon-{gamma}, which suggest that LMP2 and LMP7 may function in the invasion of placental villi to endometrium. Angiogenesis, the formation of new blood vessels, is a fundamental feature in peri-implantation [35]. Oikawa et al. [36] demonstrated that inhibition of proteasome by lactacystin significantly inhibited angiogenesis and prevented production of plasminogen activator, an important protease that is responsible for the induction of angiogenesis. In the present study, we found that LMP2 and LMP7 were expressed in endothelial cells of arterioles near the implantation site. These results suggest that LMP2 and LMP7 are probably involved in angiogenesis. The presence of LMP2 and LMP7 in glandular epithelium on Days 12, 18, and 26 of pregnancy may relate to the glandular secretion function during early pregnancy. In addition, we found that LMP2 and LMP7 mRNAs were widely detected in stroma on Days 18 and 26 of pregnancy. These results suggest that LMP2 and LMP7 may be involved in regulation of the extensive degradation of ECM, because our previous studies found that inhibition of proteasome significantly decreased expressions of matrix metalloproteinase (MMP)-2 and MMP-9, which are responsible for degradation of the ECM [20].

Implantation and placentation present an immune challenge; the immune tolerance between the mater and fetus is one of the most important factors for successful implantation [37]. The presentation of intracellular proteins to the immune system requires their degradation to small peptides, which then become associated with MHC class I molecules [38]. These MHC class I molecules bind peptides that are generated by degradation of proteins in the cytoplasm, in many cases by a large, multicatalytic, proteolytic particle (i.e., the proteasome). Both LMP2 and LMP7, which are inducible by interferon-{gamma}, alter the catalytic activities of the proteasome and enhance the presentation of at least some antigens [39]. Numerous studies have shown that interferon-{gamma} plays an important regulatory role in early pregnancy, and the effect of interferon-{gamma} on generation of MHC class I molecules is mediated mostly by upregulating the expressions of LMP2 and LMP7 [24, 40]. The transcripts and proteins of LMP2 and LMP7 were widely expressed in rhesus monkey endometrium and placenta. These results hint that LMP2 and LMP7 are responsible for the generation of MHC class I molecules and antigenic peptides during early pregnancy and that the mater and fetus have the same small antigenic peptides, increasing maternal-fetus immune tolerance.

In summary, our investigations identified the expressions of LMP2 and LMP7 mRNAs and proteins in luminal and glandular epithelium, placenta villi, trophoblastic shell, and arterial endothelial cells. These results implicate LMP2 and LMP7 are probably involved in placental villi invasion, degradation of ECM, immune tolerance, glandular secretion, and angiogenesis. The present study will help to elucidate the regulatory role of UPP in embryo implantation.


   ACKNOWLEDGMENTS
 
The authors are grateful to Dr. Genbao Shao and Dr. Lu Qian for assistance with statistical analysis.


   FOOTNOTES
 
1 Supported by the Special Funds for Major State Basic Research Project (G1999055903), the CAS Innovation Program (KSCX3-IOZ-07), and Funds from National Natural Science Foundation of China (30200133). Back

2 Correspondence: Cheng Zhu, State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 25, Bei Si Huan Xi Lu, Beijing 100080, China. FAX: 86 10 62529248; zhuc{at}panda.ioz.ac.cn Back

Received: 26 March 2004.

First decision: 20 April 2004.

Accepted: 3 June 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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