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Pregnancy |
State Key Laboratory of Reproductive Biology,3 Institute of Zoology, Chinese Academy of Sciences,Beijing 100080, China
Department of Obstetrics and Gynecology,4 Second College of China Medical University, Shenyang 110004, China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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embryo, implantation, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A successful implantation is precisely regulated by various factors derived from both the embryo and the maternal uterus. Any failure of the normal controlling mechanism during this process leads to serious diseases, such as abortion and preeclampsia, due to incomplete implantation, and hydatidiform mole and even choriocarcinoma, due to overimplantation. In humans, 75% of the lost pregnancies are due to failure of implantation and therefore are not clinically recognized as pregnancies. Failed implantation is also a major limiting factor in assisted reproduction [3]. Therefore, understanding the mechanisms of implantation will help to reduce infertility rates, enhance reproductive health, and improve contraceptive design.
Underlying the morphological and functional changes in the uterus during implantation are the alterations in gene expression. Previously, selected individual candidate genes or gene families were analyzed using one-by-one approaches. Recently, powerful high-throughput technologies have developed to simultaneously provide overall information about molecular events. Several novel molecular markers relevant to uterine receptivity and embryo implantation were reported in mice using the technology of gene chips [4, 5]. However, the results obtained in mice are not always pertinent to primates [6]. Indeed, it is well known that embryo implantation in mice requires coordinative production of both estrogen and progesterone, while in primates, progesterone alone is sufficient for promoting endometrium maturity and blastocyst implantation [7].
Recently, much effort has been made to elucidate the mechanisms involved in establishment of endometrium receptivity [8–10]. Using microarray technology, Riesewijk et al. [8], Carson et al. [9], and Kao et al. [10] described the gene expression profiling during the transition of human endometrium toward the receptive phase. The studies provided groups of genes differentially expressed in the normal human endometrium during the stage of the implantation window in the absence of embryo implantation. The genes were believed to be involved in the preparation of the endometrium for implantation of the embryo. However, little is known about the genes differentially induced when embryonic implantation occurs in primates. The genes are likely to be expressed temporally and spatially and should reflect the dynamic maternal-fetal dialog at the implantation site during the implantation window.
Due to ethical considerations, it is almost impossible to obtain specimens of human endometrium at the implantation site at the very beginning of pregnancy. However, the rhesus monkey (Macaca mulatta) is an appropriate model given its close evolutionary relationship to human beings and evidence of analogous processes during pregnancy [7]. For example, the females have a relatively regular menstrual cycle of 26–35 days during the mating season (October–March) and pinopodes appear on the uterine luminal surface at the midsecretory phase as a specific marker of endometrial receptivity for embryonic implantation [11]. In the present study, we have used the rhesus monkey to study the nature of events associated with blastocyst-endometrium interaction during the early stage of implantation.
The endometrium at the implantation site was obtained in the pregnant rhesus monkeys on the ninth day after ovulation when the blastocyst adheres to the uterine luminal epithelium and just begins to implant into the endometrium. The suppression subtractive hybridization (SSH) was then set up to screen the critical genes that were differentially expressed at the very beginning of embryonic implantation in rhesus monkeys.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult rhesus monkeys were housed at Fujian Experimental Center of Non-Human Primate, Fujian Institute of Planned Parenthood, Fuzhou, China. The animals were caged individually under a photoperiod regime of 12L:12D and were fed ad libitum. The menses of the females were observed and recorded carefully for at least two menstrual cycles, and those with regular menstrual cycles of approximately 27–30 days were selected for study. The ovulation day was estimated according to the menstrual cycle, based on the evidence that the secretory stage usually lasts for 14 days after ovulation. During the 9th through 19th days of the menstrual cycle, one female monkey was allowed to cohabit with one male of proven fertility. On the 9th and 11th days after ovulation, a hysterectomy was performed on the female monkey. The uterus was rapidly dissected as the ventral and dorsal parts. The endometrium at the implantation site and a nonimplantation site were each snap frozen and stored in liquid nitrogen. The biopsies, including intact maternal-fetal interfaces, were immediately fixed in 4% paraformaldehyde buffer at 4°C for 16 h. The fixed tissues were then gradually dehydrated in ethanol and embedded in paraffin. Sections of 6 µm thickness were collected on Super Frost+ glass slides (Menzel-Gläser, Germany). The project was approved by the Local Ethical Committee in the State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences.
Isolation of Total RNA
Total RNAs from the endometrium at the implantation site and nonimplantation site of the rhesus monkey were isolated, using TRIzol reagent (Gibco BRL, Grand Island, NY) according to the manufacture's instructions. To remove the genomic DNA, the RNA samples were treated with DNase I (Promega, Madison, WI) for 15 min at 37°C, then extracted with phenol:chloroform:isopropyl alcohol (25:24:1) and finally concentrated by ethanol precipitation. The RNA was quantified by spectrophotometry and then stored at –70°C.
Suppression-Subtractive Hybridization (SSH)
Due to the limitation in the tissue amount of rhesus monkey implantation site, SSH was performed using the PCR-Select cDNA Subtraction System in combination with the SMART PCR cDNA Synthesis and the Advantage cDNA PCR Systems (Clontech Inc., Palo Alto, CA). In brief, total RNAs from the implantation and nonimplantation sites were synthesized into double-stranded cDNAs according to the manufacture's instructions. The first-strand cDNA synthesis reaction included 1 µg total RNA, the CDS synthesis primer, the SMART II oligonucleotide, and 200 U Superscript II reverse transcriptase. The product was diluted with 40 µl TE buffer (10 mM Tris, 1 mM EDTA, pH 7.6). A 2-µl aliquot was mixed with Advantage cDNA polymerase mix and polymerase chain reaction (PCR) primers to synthesize the double-strand cDNA with PCR. The cycling conditions were 95°C for 1 min followed by 17 cycles of 95°C for 15 sec, 65°C for 30 sec, and 68°C for 6 min on Perkin-Elmer GeneAmp PCR system (PE) 480. The PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Valencia, CA). Then the double- strand cDNAs were digested by RsaI and precipitated by NH4OAc. For the subtraction reaction, the cDNA of the implantation site was used as tester cDNA to subtract that of the nonimplantation site as the driver. The condition of the primary PCR was 94°C for 25 sec, followed by 27 cycles of 94°C for 10 sec, 66°C for 30 sec, and 72°C for 90 sec on PE 2400. One microliter of one-tenth diluted primary PCR product was added into a new PCR tube for a second round of PCR reaction. The secondary PCR condition was 94°C for 10 sec, followed by 10 cycles of 68°C for 30 sec and 72°C for 90 sec. The PCR products generated by SSH were cloned into pGEM T-easy plasmid vector (Promega) and transformed into JM109 competent cells (Promega).
Evaluation of Subtraction Efficiency
To evaluate the efficiency of the subtraction, the relative amount of glyceraldehye-3-phosphate dehydrogenase (GAPDH) cDNA present in the subtracted and unsubtracted cDNA populations after SSH was examined by PCR amplification using the GAPDH 5' and 3' primers provided in the PCR-Selected cDNA Subtraction System (Clontech Inc.).
Dot Blot Analysis
The transformed JM109 cells grew as white/blue colonies on the LB plate. Three hundred and seventy-six white colonies were randomly selected and grown in 100 µl LB medium in standard 96-well plates. Then 1 µl of each bacterial culture was used to amplify the cDNA inserts in the recombinant plasmids carried by the bacteria using PCR according to the instruction of PCR-Select Differential Screening Kit (Clontech Inc.). A five-microliter aliquot of each PCR product was denatured by adding an equal amount of 0.6 N NaOH, and 2 µl of the mixture was dotted onto Hybond N nylon membrane (Amersham Pharmacia Biotech, Grand Island, NY). Ninety-four cDNA fragments, including two negative controls provided by the kit, were dotted on one membrane to make a blot, and two identical blots were made for every 94 colonies. Altogether, four pairs of blots were prepared and subsequently cross-linked by ultraviolet radiation. The cDNAs from the implantation and nonimplantation sites were 32P- labeled using the reagents in the PCR-Select Differential Screening kit (Clontech Inc.) in the presence of [
-32P]-dCTP (Yahui Biotehnology Inc., Beijing, China). The labeled probes were purified by NICK column (Amersham Pharmacia Biotech) and the specific activity of each probe was estimated using a scintillation counter. The probes with specific activity of >107 cpm were used for further hybridization. The blots were prehybridized with hybridization-buffer (6x SSC, 5x Denhart, 0.5% SDS, and 100 µg/ml sheared salmon sperm DNA) for 2 h at 72°C. Hybridizations were performed overnight at 72°C in the hybridization buffer containing 32P-labeled cDNA probes. The blots were washed and adjusted to autoradiography (Kodak BioMax MS film, Eastman Kodak Co., Rochester, NY) for 24–96 h at –80°C. The dot blot analysis was repeated three times with three independent sets of blots. The clones that hybridized to the cDNA probe of the implantation site but not to that of the nonimplantation site were selected for further analysis.
DNA Sequencing and Analysis
DNA Sequencing was performed using an ABI 3700 DNA Analyzer (Perkin-Elmer Applied Biosys., Foster City, CA) in the United Gene Holdings, Ltd. (Shanghai, China). The sequences obtained were compared against GenBank/EMBL database using the on-line computer BLAST program.
Reverse Northern Blot Analysis
Half a microliter of the synthesized double-strand cDNA of the implantation site and the nonimplantation site were electrophoresed on 1.2% agarose gel and vacuum transferred to Hybond N+ nylon membrane (Amersham Pharmacia Biotech). Based on the result of the sequencing analysis, the differential clones that represented various genes were used as the probes and labeled with 32P. Membranes were prehybridized in hybridization buffer (6x SSC, 5x Denhart, 0.1% SDS, and 100 µg/ml sheared salmon sperm DNA) for 2
4 h at 68°C. Hybridizations were performed at 68°C overnight in the hybridization buffer containing specific radioactive probe. Membranes were washed and adjusted to autoradiography (Kodak BioMax MS film, Eastman Kodak Co.) overnight at –80°C.
Semiquantitative Reverse Transcription-PolymeraseChain Reaction
One microgram of total RNAs from the implantation site and the nonimplantation site were reverse transcribed in a 20 µl reaction mixture with oligo(d)T primers (Promega) by SuperScript reverse transcriptase as specified by the manufacturer (Gibco BRL). One microliter aliquot of the product was PCR amplified with specific primers (SBS Genetech, Beijing) that were designed according to the cDNA sequences of the differential clones. The primer sequences and PCR conditions are listed in Table 1. The cycle numbers of the PCR reactions ranged from 24 to 32 according to the abundance of various transcripts, to make sure that the amplifications were carried on within the exponential phase. The relative amounts of the amplified products were normalized with the density of GAPDH that had been amplified with the same template. All the PCR products were then subcloned into the pGEM T easy vector (Promega) and verified by sequencing.
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Immunohistochemistry
Paraffin sections were deparaffinized, rehydrated, and then retrieved in 10 mM citrate buffer (pH 6.0) at 95°C for 15 min. After immersion in 1% hydrogen peroxide, the sections were incubated with rabbit polyclonal antibody against cytokeratin 18 (CH18-0059; 1:200; Zymed Laboratories, Inc., South San Francisco, CA) at 4°C overnight. The negative control was assessed by replacing the primary antibodies with the nonimmune rabbit serum at the same concentration. Final visualization was achieved using DAKO Envision Kits (DAKO Cytomation, Glostrup, Denmark). Counterstaining with hematoxylin was carried out before slides were mounted. The assessment of staining intensity was evaluated under an Olympus microscope.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Altogether, five of eight monkeys were proven pregnant on gestational Day 9–11. On Day 9, the implantation site appeared as a small distinct hematosed spot on the endometrium usually located on the upper one third of the ventral or dorsal side of the uterus with a diameter of approximately 0.5 mm. A relatively smaller hematosed spot was also present on the corresponding position of the opposite side (Fig. 1A). On Day 11, the diameter of the hematosed spot increased to about 1 mm. The endometrium several millimeters away from the hematosed spot was designated as the nonimplantation site.
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Using immunostaining for cytokeratin, we further characterized the histological structure of the implantation site and nonimplantation site in rhesus monkeys. It was shown that more extensive and elongated epithelial glands as well as loosened stroma were notably observed in the endometrium of the implantation site compared with that in the nonimplantation site (Fig. 1, B–E). On Day 11, the intact maternal-fetal interface consisted of the implanting embryo and the invaded maternal endometrium. Lacuna existed among the trophoblast layer, and large amounts of epithelial plaque were observed under the uterine luminal epithelium (Fig. 1F).
Suppression-Subtractive Hybridization
Total RNAs from the implantation and nonimplantation sites were synthesized into double-strand cDNAs using SMART PCR cDNA Synthesis kit and amplified with the Advantage cDNA PCR kit. Based on the results of the preliminary experiment, the optimal cycle number of PCR reaction was 17 cycles when the synthesized double-strand SMART cDNA was shown as a smear of 0.5–7 kilobases (kb) on the 1.2% agarose gel. After digestion by RsaI, the smear moved to 0.5–3.5 kb.
A subtracted cDNA library was constructed to enrich the transcripts that were differentially expressed in the endometrium of the implantation site. The subtraction efficiency was evaluated by detecting the housekeeping gene (GAPDH) in both the subtracted and nonsubtracted cDNA pools with PCR amplification. The PCR product of GAPDH appeared to be detectable on the agarose gel after amplification for 28 cycles and 23 cycles in the subtracted and the nonsubtracted cDNA pools, respectively. The data indicated that the amount of GAPDH transcript was reduced by over 30-fold after subtraction (Fig. 2). After ensuring that the GAPDH gene had been extensively removed in the subtracted pools, the subtracted cDNAs were cloned into a pGEM-T easy vector and transformed into JM109 competent cells. Three hundred and seventy-six clones were selected at random and the inserts were reamplified by PCR. The amplified cDNAs were dotted onto Hybond N+ membranes and hybridized separately with 32P-labeled unsubtracted cDNAs from the implantation site or nonimplantation site, and positive signals were detected in 82 of 376 (Fig. 3). All the positive clones were analyzed by DNA sequencing and then compared against the GenBank/EMBL databases using the online Blast system. Of the 82 differential clones, it was determined that 79 clones corresponded to 36 different genes that shared more than 90% homology to the known genes of the humans or the rhesus monkeys, 2 clones showed more than 93% homology to one human EST, and 1 clone was homologous to human DNA sequence at 96%, which might represent a novel gene (Table 2). Among the differentially expressed genes, the most notable one was S100A10, which appeared as 15 differential clones out of the 79. Ten out of the 79 clones represented secreted frizzled-related protein 4 (sFRP4) gene. All the 38 different cDNA sequences have been submitted to the GenBank database as rhesus monkey EST (see Table 2 for the accession number). The cDNA fragment of rhesus monkey GAPDH gene was also cloned (452 base pairs [bp]) and sequenced, which showed 98% nucleotide sequence homology to that of human GAPDH gene (Accession No. BC029618). The sequence was also submitted to the GenBank database (Accession No. BQ427389).
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Reverse Northern Blot Analysis
Reverse Northern blot analysis was used to confirm the results of SSH. The cDNA fragments representing 38 different genes were separately radiolabeled as probes. After normalization with the hybridization signal of GAPDH, 33 genes showed strong expression in the endometrium of the implantation site; however, they exhibited weak expression in that of the nonimplantation site (Fig. 4). Five genes (corresponding to clone numbers 1C4, 2B8, 3C6, 1D11, 1A1, 3H3) exhibited no signals in either site.
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Semiquantitative Reverse Transcription-PolymeraseChain Reaction
Expression of six differential genes (S100A10, sFRP4, IGFBP-7, 2C12, 4E7, 3B6) in rhesus monkey endometria was determined by semiquantitative reverse transcription- polymerase chain reaction (RT-PCR) using specific sets of primers. All the genes exhibited strong expression in the implantation site; however, they presented no or faint expression in the nonimplantation site (Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The rhesus monkey is a seasonal breeder, with menstrual cycles occurring from October to March in the Northern Hemisphere. In the Fujian Experimental Center for Non- Human Primates, the fertility rate of caged monkeys is usually no more than 30%. In the present work, through meticulous estimation of the ovulation day and extension of the time to cohabitate with a proven male, the pregnancy rate increased significantly to more than 60% (5/8). Although dye staining of the implantation site has been widely used in mice, this approach rarely worked in the rhesus monkeys to our knowledge. Based on the evidence that the endometrial capillary permeability will increase when blastocyst implantation occurs [2], the obvious hematosed spot on the upper one third of the endometrium was deemed to be the implantation site. We have previously observed the normal uterine endometria at different stages of the menstrual cycle and found no such hematosed spot. Furthermore, we studied the histological structure of the rhesus monkey endometria at both the implantation site and the nonimplantation site. The marked alterations in the structure of the epithelial glands and stroma were notably observed in the endometrium of the deemed implantation site. Meanwhile, we observed the intact maternal-fetal interface, including the implanting blastocyst in the specimen of gestational Day 11, which was consistent with that reported by Enders et al. [17]. All the data helped us to confirm the veracity of our specimens. As a matter of fact, it is difficult to determine pregnancy status by either B-type ultrasound or hormone assay before gestational Day 15 in the rhesus monkey. The methods mentioned in this study could vastly improve the accuracy and success of pregnancy determination.
Due to the quantity limitation in the specimen of the implantation sites, we combined SMART PCR cDNA synthesis technology with SSH to screen the genes differentially expressed in the implantation site, then confirmed the SSH results by reverse Northern blot analysis [18, 19] and semiquantitative RT-PCR. Data from literature suggested the viability of this combination [13–16, 18, 19]. With the SMART PCR cDNA synthesis system, the pool of double- strand cDNA can be exponentially amplified in a full-length form, maintaining the complexity and the relative abundance of the original mRNA population, which is suitable for studies of differential gene expression [13, 19]. In this laboratory, we used PCR reactions to compare the expression levels of several genes between SMART PCR-amplified cDNAs and the corresponding total RNAs and confirmed the same expression pattern between the two pools. Therefore, the SMART-amplified cDNA pool could replace the original RNA pool in gene screening and subsequent reverse Northern blot analysis.
The genes differentially expressed in the implantation site were screened from 376 clones, which were randomly selected from the subtracted cDNA library. In this way, some new genes and those showing homology to various human genes were identified. It is still likely that we have missed some other genes that might be important for embryo implantation. Much more effort will be needed to screen more genes. On the other hand, the implanted blastocyst was possibly included in the specimen of the implantation site endometrium. Due to the enrichment of the low-abundant message by SSH, some genes that were expressed by the blastocyst when implantation occurred might also be cloned in this experiment. In situ hybridization will be necessary to locate the genes in detail.
Among the genes screened from the subtracted cDNA library of the rhesus monkey implantation site, the most notable one was S100A10, which appeared as 15 clones out of 76 different ones. The S100A10 protein is a member of the S100 family, which is also known as calpactin I or p11, and functions as one of the mediators in the calcium-dependent signaling pathway. Two copies of S100A10 could combine with two copies of annexin II, forming the annexin II heterotetramer (AIIt). Thus, the S100A10 protein was considered as the ligand of annexin II. Kassam et al. [20] revealed that AIIt stimulated the activation of plasminogen by facilitating the tissue plasminogen activator (t-PA)-dependent conversion of plasminogen to plasmin. Therefore, S100A10 was also named as the receptor of t-PA. During the process of gestation, AIIt was found to be exposed on the surface of syncytiotrophoblast (STB) cells [21, 22], while tPA mRNA was located in the epithelial glandular cells of the rhesus monkey implantation site on Days 15– 16 postovulation [23]. On gestational Day 9 in the rhesus monkey, there was only a small number of STB cells present on the surface of the implanting blastocyst that act as the forerunner of adhesion and penetration in the uterine epithelium. If S100A10 was expressed by these primary STB cells, a much higher expression level would be expected, which could lead to a high frequency occurrence of the gene in the subtracted library. On the other hand, as the mediator of calcium-dependent signaling pathway, the specific expression of S100A10 in the implantation site may suggest that some of the cell events occurred at the beginning of trophoblast adhesion and invasion is calcium dependent. Further study is being carried out in this laboratory to demonstrate the location of S100A10 on the maternal- fetal interface during the process of implantation in the rhesus monkey.
The highly frequent appearance (10 clones in 76) of the gene encoding sFRP4 in the subtracted library of the implantation site is also interesting. The sFRP4 belongs to the sFRP family, the members of which are regulators of the Wnt signaling pathways [24]. Wnt pathways are involved in the control of gene expression during various cell behaviors, including adhesion and polarization. In addition, they often work in combination with other signaling pathways [25]. The sFRPs are a group of secreted glycoproteins, structurally resembling Wnt receptors-frizzled (Fz) proteins while lacking the transmembrane domains. They have been shown to inhibit Wnt action by competitively binding to Fz proteins [24]. In humans, frpHE, the homologous gene of sFRP4, was found to be expressed in the endometrial stroma at the proliferative stage but was hardly detectable in the secretory or menstrual endometrium. The expression pattern suggested that frpHE might be regulated by steroid hormones during the menstrual cycle [26]. Fujita et al. [27] reported that sFRP4 was upregulated in rat decidual cells during pregnancy by using the SSH method. With Northern blot analysis, they found that sFRP4 expression in rat endometrium was hardly detected on gestational Day 6 when implantation occurred, while the expression level peaked on Day 12. The data are inconsistent with our results and may be due to the sensitivity of different methods used in the experiment. As mentioned above, the SSH method itself could enrich the differentially expressed genes and thus allow the low abundant message to be detected. Researchers from several laboratories have demonstrated the involvement of Wnt signaling pathways in cell events during the menstrual cycles and pregnancy. The deletion of wnt-2 or wnt-7b led to abnormal development of placenta and death of embryos before parturition [28, 29]. Homozygous null mutation in one of the Wnt receptors, Fz5, resulted in embryo death due to defects of angiogenesis of the yolk sac [30]. As the natural antagonist of Fz proteins, the specific expression of sFRP-4 in the rhesus monkey implantation site might suggest its involvement in modulating stromal cell proliferation and decidualization.
The other interesting molecule is insulin-like growth factor binding protein 7 (IGFBP-7), which is also named as IGFBP-rP1, tumor adhesion factor (TAF), prostacyclin stimulating factor (PSF), or mac25. Being a member of the IGFBP superfamily, IGFBP-7 is structurally homologous to the other members but presents a lower affinity for insulin- like growth factors (IGFs) [31]. Recent studies on IGFBP- 7 were focused on the area of oncology. It was revealed that IGFBP-7 could act as a potential tumor suppressor and might possess antiproliferation capabilities [31–33]. For instance, Sprenger et al. [33] found that IGFBP-7 could inhibit growth of the immortalized or malignant human prostate epithelial cells in soft agar as well as the tumor formation in nude mice by inducing cell apoptosis. Moreover, IGFBP-7 was able to stimulate prostacyclin production in vascular endothelial cells, thereby controlling the vascular permeability [34]. On the other hand, evidence has indicated the involvement of IGFBP-7 in the functions of endometrium. Degeorges et al. [35] found that IGFBP-7 was present in the endometrial glandular epithelium at both secretory and proliferative phases, whereas it was largely negative in the stromal cells. More recently, Dominguez et al. [36] demonstrated that IGFBP-7 was extremely upregulated in human uterine endometrial stromal cells during the receptive stage, and thus proposed that the protein would be the biochemical marker for endometrial receptivity. The data were somewhat consistent with our results obtained in the rhesus monkey. We propose that the differentially expressed IGFBP-7 might be involved in increasing the vascular permeability in the implantation site. Meanwhile, there is follistatin-like motif located near the amino terminal of IGFBP-7 protein, which can mediate the combination and cooperation of IGFBP-7 with some other growth factors. This may be one of the pathways for IGFBP-7 to be involved in the regulation of fetal-maternal cross talk during implantation. In placenta, IGFBP-7 presented at a low level in amnionic and chorionic cells and was negative in trophoblasts. Oh et al. [37] also reported a low expression level of IGFBP-7 in placenta using Northern blot analysis. However, there was scarce data in the literature regarding the temporal and spatial changes of IGFBP-7 at the maternal-fetal interface during different stages of pregnancy, especially at the beginning of implantation. These issues are under study in this laboratory.
There were some other interesting genes differentially expressed at the rhesus monkey implantation site that have not been investigated to date. To the best of our knowledge, this is the first investigation on the gene expression pattern in the implantation site during the very early stage of implantation in the rhesus monkey using SSH technology. With the differentially expressed cDNA fragments in hand, further studies are being carried out, attempting to localize the genes at the fetal-maternal interface and determine their roles in embryonic implantation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Yan-Ling Wang, State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 25 Bei Si Huan Xi Road, Haidian, Beijing 100080, China. FAX: 86 10 62529248; wangyl{at}panda.ioz.ac.cn
Received: 18 April 2003.
First decision: 16 May 2003.
Accepted: 2 January 2004.
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