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Pregnancy |
a Prince Henry's Institute of Medical Research, Clayton, Victoria 3168, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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female reproductive tract, gene regulation, implantation, pregnancy, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, we used the mouse as a model and searched for unrecognized molecules important in the early stage of implantation. In the mouse on Day 4.5 of pregnancy (vaginal plug = Day 0), the uterus undergoes dramatic morphological changes in association with cell proliferation and differentiation, leading to the acquisition of a receptive state [4, 5]. Uterine remodeling at this time is marked by an increase in vascular permeability at implantation sites [6]. We hypothesized that the proliferation and differentiation of endometrial cells at this time are associated with up- or downregulation of a number of genes, many of which are still unknown [1]. To identify those uterine genes potentially critical for uterine receptivity, we used the technique of RNA differential display (DDPCR) [7, 8] and compared the mRNA expression pattern of implantation and interimplantation sites on Day 4.5 of pregnancy [9–11].
One of the genes identified as being differently regulated at the two sites was proprotein (prohormone) convertase 6 (PC6), a member of a serine proteinase family responsible for processing precursor proteins to their active forms by selective proteolysis. Although Wong et al. [12] recently reported the uterine expression of PC6 mRNA in the mouse and indicated its involvement during decidualization, the roles of PC6 in the uterus and particularly during embryo implantation have not been well studied. In the present investigation, we examined the uterine expression of PC6 mRNA in mice during early pregnancy, in particular at implantation and interimplantation sites at the time of implantation. The influence of the embryo on the uterine expression of PC6 mRNA was also investigated using pseudopregnant mice. In addition, we examined the multiple transcripts of PC6 mRNA at the implantation site and compared the uterine transcript profile with that in the intestine, where PC6 expression was previously investigated [13].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Swiss O/B mice were housed and handled according to the Monash University animal ethics guidelines on the care and use of laboratory animals. All experimentation was approved by the Institutional Animal Ethics Committee at the Monash Medical Centre. Adult female mice (6–8 wk old) were mated with fertile males of the same strain to produce normal pregnant animals or were mated with vasectomized males to produce pseudopregnant mice. The morning of finding a vaginal plug was designated as Day 0 of pregnancy. Uterine tissues were collected from nonpregnant mice, pregnant mice on Days 3–11, and pseudopregnant mice on Day 4.5. Other organs were also collected from nonpregnant mice. Tissues were snap-frozen in liquid nitrogen for Northern analysis or fixed in 4% buffered formalin (pH 7.6) for in situ hybridization. All experiments were repeated in three animals.
For nonpregnant, pseudopregnant, and Day 3.5 pregnant mice, the entire uterus was collected. For Day 4.5 pregnant mice, implantation sites were made visible by injection of a Chicago blue dye solution (1% in saline, 0.1 ml/mouse) into the tail vein 5 min before the animals were killed. Implantation sites were separated from interimplantation sites, and both sites were retained. For pregnant mice on Day 5.5 onward, implantation and interimplantation sites were visualized without dye injection. For nonpregnant mice, the uterus was also collected from different stages of the estrous cycle: metestrus, diestrus, proestrus, and estrus. The stages of the cycle were determined by analysis of vaginal smears [14]. To induce deciduoma, nonpregnant mice were ovariectomized and treated with estradiol and progesterone as described by Finn and Pope [15], and uteri were collected 48 h after the administration of oil stimulus.
DDPCR, Reamplification of cDNAs, and Subcloning
DDPCR was performed as previously published [10] and as described originally by Liang and Pardee [7, 8]. Uterine mRNA expression was compared between implantation and interimplantation sites on Day 4.5 of pregnancy with 80 polymerase chain reaction (PCR) primer combinations (20 random 5' primers combined with 4 oligo-dT anchored 3' primers) [10]. After the DDPCR analysis, the differential display pattern was further verified by Northern blotting analysis, and cDNAs from confirmed bands were subcloned into pGEM-T vector (Promega, Madison, WI) and sequenced.
Northern Analysis
Northern analysis was performed as previously described [10]. RNA (15 µg) was denatured with dimethylsulfoxide and glyoxal and analyzed following electrophoresis through a 1.2% agarose gel and transfer to positively charged nylon membranes (Hybond-N+; Amersham, Piscataway, NJ). Radiolabeled cDNA probes were generated by random primer labeling of 25 ng cDNA with [32P]deoxy-CTP (50 µCi/reaction). Hybridization was at 42°C overnight. To determine lane-to-lane loading variation, each blot was also probed with a mouse cDNA probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or 18S rRNA. Between hybridizations, blots were stripped by incubation at 80°C for 3 h in 1 mM EDTA/0.1% SDS followed by rinsing in H2O. Each Northern analysis was repeated in three animals. The optical density of the hybridization signals was quantified and analyzed as previously described [10].
Reverse Transcription PCR and Cloning
For reverse transcription PCR (RT-PCR), 1 µg DNA-free total RNA was reverse transcribed at 46°C for 1–1.5 h in a 20-µl reaction mixture using 100 ng random hexanucleotide primers and avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim, Nunawading, Vic., Australia) with the cDNA synthesis buffer. The following PCR was performed in a total volume of 40 µl with 1–1.5 µl of the RT reaction buffer, 1x PCR buffer, 20 µM dNTPs, 10 pmol forward and reverse primers, and 2.5 units of Taq DNA polymerase (Boehringer Mannheim). The PCR was performed in three stages as follows: 1 cycle of incubation for 5 min at 95°C, 1 min at 52–60°C, and 2 min at 72°C; 32 cycles of denaturation for 45 sec at 95°C, annealing for 50 sec at 52–60°C, and extension for 1 min at 72°C; and a final incubation for 5 min at 72°C. An annealing temperature of 52–60°C was optimal for all PCRs. PCR products were analyzed on 1.5% agarose gels stained with ethidium bromide. Bands of interest were cut from the gels, purified with the Qiaquick gel extraction kit (Qiagen Pty Ltd., Clifton Hill, Vic., Australia), cloned into a pGEM-T easy vector (Promega) according to the instructions of the manufacturer, and sequenced by the institute's automated sequencer using the ABI Prism BigDye terminator cycle sequencing ready reaction kit.
The RT-PCR cloned cDNA fragments representing different regions of two known isoforms of mouse PC6 (PC6A and PC6B) are summarized in Table 1, together with the primers used, the size of each PCR product, and the nucleotide ranges represented. The location of these fragments on PC6A and PC6B mRNA is illustrated in Figure 1. Fragment MUSPC6-fragD (656 base pairs [bp]) (Table 1 and Fig. 1) was further digested with HindII, and the two resultant fragments (177 bp and 479 bp) were referred to as MUSPC6-fragDA (177 bp) and MUSPC6-fragDB (479 bp), respectively. These cDNA fragments each represent a unique protein domain of PC6 [13, 16]: MUSPC6-fragA, the signal peptide domain; MUSPC6-fragDA, the propeptide domain; MUSPC6-fragDB, the subtilisin-like catalytic domain; MUSPC6-fragG, the Homo-B domain; MUSPC6-fragH, the common Cys-rich domain present in both the A and B isoforms; MUSPC6-fragI, the C-terminal of the A isoform, thereby specific to the A isoform only; MUSPC6-fragK, the unique Cys-rich domain of the B isoform; and MUSPC6-fragM, the unique transmembrane domain of the B isoform.
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In Situ Hybridization
Sense and antisense digoxygenin (DIG)-labeled RNA probes against PC6 (clone 9.5, Table 2) were generated using the DIG RNA Labeling kit (Boehringer Mannheim) according to the manufacturer's instructions. Five-micrometer sections of formalin-fixed, paraffin-embedded tissues were subjected to in situ hybridization as described previously [9]. All sections were counterstained with Mayer hematoxylin.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We utilized the RNA differentiated display technique and compared the pattern of uterine gene expression in implantation and interimplantation sites on Day 4.5 of pregnancy. Ten bands showing differential expression were detected on DDPCR gels [10]. One of these bands (band 9) is fully analyzed here.
On the DDPCR gel, this band was much more intense at implantation than at interimplantation sites in all three animals tested (data not shown). To verify that this band indeed represents a gene(s) that is differentially expressed between the two sites, the cDNA products were extracted from the DDPCR gel, reamplified, cloned into the pGEM-T vector, and used for Northern blots with the cloned inserts as probes. Among the 10 clones analyzed, 2 identical clones (clones 9.5 and 9.10) explicitly detected differential expression of mRNA between the two sites on the Northern blot; expression was much higher at implantation than interimplantation sites (data not shown). This finding confirmed that clones 9.5 and 9.10 contained cDNA representing the original expression pattern of band 9 on the DDPCR gel. The remaining clones contained another two different DNA species, but their expression could not be confirmed by Northern blot analysis.
Sequence Analysis and GenBank Database Comparison
Band 9 resulted from the DDPCR amplification of Day 4.5 implantation site mRNA with the following two primers: 5' primer GTGACGTAGG and 3' primer T12MA [10]. After confirmation that clone 9.5 contained the cDNA representing band 9, the nucleotide sequence of this clone was determined (Table 2). It has 158 nucleotides and contained the unique and expected primer sequence of GTGACGTAGG at the 5' end and the reverse complementary sequence of T12MA at the 3' end (underlined in Table 2). This finding confirmed that the cDNA in clone 9.5 was definitely the direct PCR product amplified from the specific primers applied during DDPCR amplification.
When compared with other sequences in the GenBank database, the sequence of clone 9.5 showed 98% identity to mouse serine protease PC6 (accession D12619; 2994 bp), 98% identity to mouse PC5 (accession L14932; 2848 bp), and 98% identity to human PC6 isoform A (HPC6A, accession U56387; 2844 bp). Mouse PC5 and PC6 represent different nomenclatures for the same gene (Pcsk5) product and are 90% homologous to HPC6A cDNA. A detailed comparison of clone 9.5 with mouse PC6 cDNA (Table 2) revealed that clone 9.5 aligned to nucleotides 254–411 of PC6 cDNA.
RT-PCR Cloning and Detection of PC6 Expression During Early Pregnancy
To further establish the identity of clone 9.5 and verify that PC6 mRNA was indeed differentially expressed between the two sites, two additional cDNA fragments of PC6, MUSPC6-fragA and MUSPC6-fragB (Table 1 and Fig. 1) representing the immediate up- and downstream regions of PC6 cDNA from clone 9.5, were cloned by RT-PCR and used as cDNA probes on the Northern blots. As expected, a pattern of mRNA expression similar to that when clone 9.5 was used as a probe was observed between implantation and interimplantation sites (data not shown). This finding allowed conclusive identification of clone 9.5 and revealed that PC6 mRNA is upregulated in implantation sites on Day 4.5 of pregnancy.
Subsequently, MUSPC6-fragB was used as the cDNA probe for additional Northern blots to systematically determine the expression pattern of this gene in the uterus in relation to the time of implantation and early pregnancy. Total RNA from the uterus of nonpregnant mice (estrus) and pregnant mice at the initial stage of implantation (Day 4.5 of pregnancy) through to fully established implantation and placentation (Day 10.5 of pregnancy) was analyzed (Fig. 2). Very low expression was observed in nonpregnant mice and in mice at Day 3.5 of pregnancy. However, from Day 4.5 to Day 6.5 of pregnancy, a dramatic upregulation of this gene occurred only at the implantation sites (Figs. 2 and 3). Beyond Day 6.5 of pregnancy, the expression level decreased and returned to nonpregnant levels. Thus, upregulation of PC6 mRNA is very transient between Day 4.5 and Day 6.5 of pregnancy and is implantation site specific.
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At implantation sites during the active embryo implantation period, the uterus expresses multiple transcripts of PC6 of sizes approximately 2.2, 3.5, 5.5, and 10 kilobases (kb), with the 3.5-kb transcript being most abundant. This finding is in accord with the report of multiple PC6 mRNA species in the brain and intestine [13, 16].
PC6 mRNA Expression During the Estrous Cycle
The influence of the estrous cycle on the expression of PC6 mRNA in the nonpregnant uterus was examined by Northern analysis. Total RNA was examined for 16 individual mice, 4 mice/stage, at metestrus, diestrus, proestrus, and estrus. In all cases, expression was very low at estrus and proestrus and increased slightly during metestrus and diestrus (data not shown). However, at all stages of the estrous cycle, expression was much lower than that at implantation sites in pregnant mice. In addition, treatment of ovariectomized mice with estradiol or progesterone did not result in any significant upregulation of PC6 mRNA in the uterus (data not shown). These results indicate that the observed upregulation in implantation sites is not a direct result of the influence of the ovarian hormones during pregnancy but may require embryonic signals.
Effects of the Embryo on Uterine Expression of PC6
To determine whether the presence of embryos in the uterus is essential for the observed changes in PC6 mRNA expression during early pregnancy, total RNA was isolated from mice on Day 4.5 of normal pregnant and from pseudopregnant mice, and the mRNA expression patterns were compared by Northern analysis (Fig. 4). In pseudopregnant mice, the mRNA levels were equivalent to those at interimplantation sites in pregnant animals and were much lower than levels at implantation sites (Fig. 4). This finding indicates that the observed increase in PC6 mRNA expression at the implantation sites during early pregnancy requires the presence of an embryo in the uterus.
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In Situ Hybridization of PC6 mRNA in the Uterus
In situ hybridization was used to determine the cell types expressing PC6 mRNA in the uterus. No significant positive staining was detected in nonpregnant uteri or in pregnant uteri before Day 3.5 of pregnancy (Fig. 5A). Positive signals were only detected between Days 4.5 and 6.5 of pregnancy at implantation sites (Fig. 5, C–F and H); throughout this period, the interimplantation sites were always negative (Fig. 5B). Close examination of the staining pattern at implantation sites revealed that the signal was localized in the decidual cells at the antimesometrial pole of the uterus (Fig. 5, C, E, and F). The identity of decidual cells was previously confirmed by immunohistochemical staining for the decidualization marker desmin [10]. The mesometrial side of the uterus always had fewer positively stained cells than did the antimesometrial pole (Fig. 5, C and E), probably reflecting the fact that the decidual reaction is initiated at the antimesometrial pole and extends to the mesometrial pole 2 days later [17]. No positive signals were detected in any cells in the uterus by Day 8.5 of pregnancy (data not shown). Thus, expression of PC6 mRNA is transient and specific to the early stages of decidualization. Positive but less intense staining was also detected in some decidual cells in oil-induced deciduoma (Fig. 5I), suggesting that PC6 is associated with mouse decidualization induced not only by embryos but also by other stimuli, such as oil.
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Tissue Distribution of PC6 mRNA
Northern analysis was performed to investigate the distribution of PC6 mRNA expression in a range of mouse tissues (Fig. 6). When equal amounts of total RNA were compared, the implantation sites on Day 5.5 showed the highest level of expression (>10-fold increase over expression in other organs). Among the 11 other tissues tested, only intestine, ovary, testis, and brain had detectable expression.
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Comparison of PC6 mRNA Profiles Between the Uterus and Intestine and Evidence of a Uterus- and Pregnancy-Specific Transcript
PC6 was previously studied in the brain and intestine [13, 16]; therefore, intestine was chosen as the positive control tissue for the present study. However, the profile of the mRNA transcripts detected by Northern blotting differed between the uterus and the intestine (Figs. 2 and 6). Both tissues show multiple transcripts ranging from 2.2 kb to 10 kb, but a 6.5-kb transcript is present in the intestine and a 5.5-kb transcript is present in the uterus (Figs. 2 and 6). The 6.5-kb transcript in the intestine encodes a PC6 isoform designated mouse PC6B (GenBank accession D17583, 5208 bp), which has an extremely large Cys-rich motif [13]. This isoform of PC6 is membrane bound, whereas the previously documented PC6 with an mRNA transcript of 3.5 kb is soluble [18]. Hereinafter, the soluble form of PC6 is referred to as PC6A and the membrane-bound isoform is referred to as PC6B. At the cDNA level, PC6A and PC6B are generated via alternative splicing of the same primary transcript [13]. At the protein level, PC6 has five domains (sequentially from N- to C-terminal): signal peptide, propeptide, subtilisin-like catalytic domain, homo B domain, and cysteine (Cys)-rich domain [13]. The first four domains are identical in PC6A and PC6B isoforms, the differences being only in the C-terminal end.
To confirm the presence of a unique 5.5-kb transcript at uterine implantation sites and to determine whether it is different from the intestinal 6.5-kb transcript, cDNA fragments encoding individual domains of mouse PC6 protein (both isoforms) were subjected to RT-PCR, cloned (Table 2), and used as cDNA probes on Northern blots containing RNA from uterine tissues of nonpregnant (estrus) mice, from implantation and interimplantation sites on Days 4.5 and 5.5 of pregnancy, and from the intestine (Fig. 7). All of the multiple bands observed previously in the uterus and intestine were detected in the intestine (Fig. 7a) when the following fragments were used as probes: MUSPC6-fragA (representing the signal peptide domain), -fragDA (propeptide domain); -fragDB (subtilisin-like catalytic domain); -fragG (Homo-B domain); and -fragH (the common Cys-rich domain present in both the A and B isoforms). In all cases, a 5.5-kb uterus-specific transcript was present at the implantation sites, but the 6.5-kb PC6B isoform-specific transcript was detected only in the intestine (Fig. 7a). Thus, cDNA sequences represented by these probes are common to all of the multiple mRNA transcripts, and the protein domains represented by these cDNAs are common to all possible isoforms of the PC6 protein. These results also confirm that the mRNA transcript profiles are different between the uterus and the intestine. When MUSPC6-fragI, an A isoform-specific cDNA fragment, was used as a probe, only the 3.5-kb transcript was detected in all tissues (Fig. 7b), indicating that only this transcript contains this A isoform-specific cDNA sequence. This result confirms the known difference between the A and B isoforms at the cDNA level. When the two B isoform-specific cDNA fragments, MUSPC6-fragK and MUSPC6-fragM, were used as probes, only the 6.5-kb transcript was detected and it was detected only in the intestine (Fig. 7c).
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These results confirm that the 5.5-kb uterus-specific transcript is different from the 6.5-kb transcript in the intestine. This 5.5-kb uterus-specific transcript may encode a previously unidentified isoform of PC6 that is different from the previously described A and B isoforms.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Furthermore, Northern analysis demonstrated that the level of PC6 mRNA at implantation sites during early pregnancy is substantially higher than than in other organs, including intestine and brain where PC6 expression has previously been reported. The observed upregulation at implantation sites is not solely a direct result of the influence of the ovarian hormones; it requires other factors, possibly of embryonic origin. The in situ hybridization studies localized PC6 mRNA in the decidual cells, and decidualization requires the presence of an embryo or other stimulus applied to a hormone-primed uterus. PC6 mRNA was detected in cells in the developing decidua at the antimesometrial side adjacent to the implanting embryo on Days 4.5–6.5. To a lesser extent, artificially decidualized uterus also showed positive staining for PC6 mRNA. These data clearly suggest a physiological role for PC6 in the process of decidualization in the mouse.
Endometrial decidualization is a crucial and complex process involving massive proliferation and differentiation of the endometrial stroma with localized increase in vascular permeability and edema [5]. Decidualization begins in the stromal region immediately surrounding the mucosal crypt where the embryo is implanting, and the mesometrial decidua forms at the mesometrial pole 2 days later. The fully transformed antimesometrial decidual cells are large, polyhedral, and closely packed and have large quantities of glycogen, lipids, and intermediate filaments [5]. The proposed functions of decidualization include supplying nutrition to the growing embryo until the formation of a proper placenta, protecting the mother from the invasive nature of the embryo, and protecting the embryo from the maternal immune system. Although the mechanism of decidualization is still not well understood, a number of gene products have been implicated in this process, e.g., insulin-like growth factor (IGF) 1 and IGF binding protein 4, cyclin D3, transforming growth factor (TGF) ß, epidermal growth factor (EGF) family growth factors including TGF
, neu differentiation factor, heparin-binding EGF, amphiregulin, betacellulin, and epiregulin [3, 5, 19, 20]. The complexity and importance of the decidual process have been further illustrated by animal models that lack the ability to exhibit uterine decidualization, with resultant failure of implantation. These models include mice lacking leukemia-inhibitory factor [21], prostaglandin synthase 2 [22], EGF receptor (EGFR) [23], interleukin 11 [24], and Hoxa-10 [25].
PC6 is a member of the proprotein convertase (PC) family of serine proteases, which are structurally related to bacterial subtilisins and yeast kexin [26, 27]. Seven mammalian PCs have been identified so far: PC1/3, PC2, furin/PACE, PC4, PC5/6, PACE4, and PC7/PC8/LPC. Some of these PCs have isoforms that are generated via alternative splicing [28]. PCs cleave precursor polypeptides at basic residues (usually arginines) within the general motif of (K/R) - (X)n - (K/R)
(where n = 0, 2, 4, or 6 and X is any amino acid and usually is not a cysteine) [27] to generate bioactive peptides and proteins. Included among their substrates is a wide variety of molecules that are translated as inactive proproteins and must be cleaved as the initial step in activation. Such molecules include propeptide hormones, proneuropeptides, precursors of growth factors, cell surface receptors, and viral surface glycoproteins [26, 27, 29].
PC6 is expressed in many endocrine and nonendocrine tissues, but its expression is highest in the adrenal gland and the gut [16, 30]. In the present study, we found that the expression of PC6 at implantation sites is substantially higher than that in the gut. According to the known functions of PCs, the observed expression pattern of PC6 in the uterus may determine the cascade of events leading to the tissue-specific production of biologically active peptides and proteins essential for implantation. Among the precursors proposed (by virtue of their amino acid sequences) to be cleaved by PC6 are a number of molecules known to be expressed in the endometrium and with likely roles at implantation. These molecules include integrins, members of the TGFß superfamily, pro-IGFI and -II, and pro-EGFs [27]. Given the highly restricted temporal and spatial expression pattern of PC6 in the decidual cells, likely substrates for PC6 are the pro-TGFßs, pro-IGFs, and some of the pro-EGFs, which are particularly involved in decidualization. However, the exact substrate(s) must be experimentally determined. EGFR signaling in the stroma is essential for uterine stroma proliferation and decidualization [23, 31]. Therefore, we suggest that PC6 plays a key role in the activation of EGF-related growth factors in decidualization. Nevertheless, unknown factors playing essential roles in decidualization cannot be excluded as substrates for PC6.
In this study, we also identified a unique transcript of PC6 mRNA in the uterus. Multiple transcripts of PC6 mRNA were detected in the uterus as is consistent with previous findings in the brain and intestine [13, 16]. In addition, we identified expression of a unique transcript of 5.5 kb at implantation sites in the uterus, and this transcript is different from the 6.5-kb transcript in the intestine. Given that the intestinal 6.5-kb transcript is an alternatively spliced variant of PC6 mRNA and encodes a unique isoform of PC6 protein (the B isoform), it is possible that the uterus-specific 5.5-kb transcript is another differentially spliced mRNA variant and may encode a previously unidentified isoform of PC6. Further work is needed to clone the precise mRNA of this new transcript and to determine its significance at the protein level and its role in implantation.
Before the initiation of this study, apart from one brief reference [32], PC6 had not been described in the uterus nor had it been associated with the process of implantation. Just prior to the submission of this report, expression of PC6 was reported by Wong et al. [12] in the mouse uterus during implantation. Overall, our data confirm those of that study but more clearly illustrate cell-specific expression of PC6 in the mouse uterus during implantation and, in particular, its differential expression between implantation and interimplantation sites. In addition, in the present study we demonstrated that ovarian hormones do not directly regulate the uterine expression of PC6. Moreover, the exceptionally high level of implantation-associated uterine expression of PC6 mRNA (>10-fold higher than that in any other organ) indicates an important role for PC6 during implantation. We also examined the multiple transcripts of PC6 mRNA in the uterus and demonstrated that the uterine transcript of 5.5–6.5 kb is different from that found in the intestine, suggesting that an additional isoform of PC6 different from the membrane-bound PC6B is uniquely expressed in the uterus at implantation. In contrast, Wong et al. did not examine the uterine isoform in detail but simply stated that both the soluble and membrane-bound forms of PC6 were expressed during implantation.
The unique expression of PC6 presented suggests an important role for PC6 during implantation in the mouse. Wong et al. [12] proposed that PC6 might participate in the regulation of tissue inhibitor of metalloproteinase 3 during implantation. However, more data are needed to support this hypothesis. Future studies will use specific antibodies (when they become available) to confirm the observed expression pattern of PC6 at the protein level. Future studies are also needed to establish the specific substrate and hence the precise role of PC6 during decidualization and implantation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Gui-Ying Nie, Prince Henry's Institute of Medical Research, PO Box 5152, 246 Clayton Rd., Clayton, Vic 3168, Australia. FAX: 61 3 9594 6125; guiying.nie{at}med.monash.edu.au
3 Current address: Department of Obstetrics and Gynecology, Mie University School of Medicine, Mie, Japan
Received: 28 April 2002.
First decision: 20 May 2002.
Accepted: 22 August 2002.
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