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Biology of Reproduction 65, 240-246 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Effects of Matrix Proteins on the Expression of Matrix Metalloproteinase-2, -9, and -14 and Tissue Inhibitors of Metalloproteinases in Human Cytotrophoblast Cells During the First Trimester1

Ping Xua, Yan-ling Wanga, Yun-shang Piaoa, Su-xia Baia, Zhi-jie Xiaoa, Ya-li Jiaa, Shu-yi Luoa, and Lin-zhi Zhuang2,a

a State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China

ABSTRACT

The activity of matrix metalloproteinases (MMPs) specifies the ability of the trophoblast cell to degrade extracellular matrix (ECM) substrates. Usually the process of normal human placentation involves a coordinated interaction between the fetal-derived trophoblast cells and their microenvironment in the uterus. In this study, the effects of ECM proteins on the expression of MMP-2, -9, and -14 (membrane-type MMP-1); and the production of tissue inhibitors of metalloproteinase (TIMP) types -1, -2, and -3 have been investigated. Cytotrophoblast cells at 9 or 10 wk of gestation were cultured on various ECM coated dishes under serum-free conditions. Gelatin zymography analysis showed that cells grown on fibronectin (FN), laminin (LN), and vitronectin (VN) secreted more MMP-9 (about 1.5- to 3-fold more) than cells cultured on collagen I (Col I), whereas the secretion of MMP-9 by cells cultured on collagen IV (Col IV) was only half that by the cells on Col I. Northern Blot analysis gave the same results as zymography, indicating that expression of the MMP-9 gene in cytotrophoblast cells can be affected by matrix proteins. There was no significant difference in the expression of MMP-2 either at protein or mRNA levels among the cells cultured on the different matrix substrates. The expression of MMP-14 was regulated in a manner similar to that of MMP-2. Using ELISA, we detected higher levels of TIMP-1 in the culture medium of cells grown on VN, LN, and FN compared with that grown on Col I. But the expression of TIMP-3 mRNA was remarkably inhibited by VN, and ECM proteins had no effect on TIMP-1 and TIMP-2 mRNA expression. It was also observed that cultured cytotrophoblast cells expressed the corresponding receptors for the tested matrix proteins, such as integrins {alpha}1, {alpha}5, {alpha}6, ß1, and ß4. Furthermore, the adhesiveness of cytotrophoblast cells on Col I, Col IV, FN, and LN was increased by 62%, 45%, 21%, and 22%, respectively, when compared with adhesiveness on VN. Isolated cytotrophoblast cells remained stationary when cultured on dishes coated with Col I and Col IV, but they assumed a more motile morphology and aggregated into a network when cultured on LN and VN. These data indicate that human trophoblast cells interact with their microenvironment to control their behavior and function.

ECM, implantation/early development, placenta, trophoblast

INTRODUCTION

Human implantation and subsequent placental development require the invasion of trophoblast cells into the maternal endometrium. In general, trophoblast cells must cross a variety of maternal cell layers and their associated basement membranes, as well as the interstitial matrix [1–3]. The invasive ability of trophoblast cells is mediated by matrix degradation enzymes, matrix metalloproteinases (MMPs), among which the type IV collagenases (MMP-2 and MMP-9) play major roles [4, 5]. Both MMP-2 and MMP-9 are able to degrade basement membrane collagen IV (Col IV), fibronectin (FN), laminin (LN), elastin, entactin, and proteoglycans as well as gelatin (denatured Col IV) [6–8]. MMP-14 is a membrane-type MMP that has been reported to be involved in catalyzing the conversion of pro-MMP-2 into an active form [9]. Therefore, MMP-14 may function as an important plasma membrane-associated regulator of the activity of MMP-2. Some studies on the regulation of MMP expression in human trophoblast cells have primarily focused on the role of hormones and cytokines such as hCG and interleukin-1ß [10, 11]. The effects of extracellular matrix proteins on MMP expression have not been well elucidated.

As the trophoblast cells penetrate the decidual stroma, they are confronted with various matrix proteins, including collagens, glycoproteins, and proteoglycans [12, 13]. Human trophoblast cells are known to adhere to both LN [14, 15] and FN [16] in vitro; it has been suggested that LN and FN may play important roles in controlling protease secretion [17, 18]. The role of vitronectin (VN) in trophoblast invasion has been examined primarily in the mouse model. VN promotes mouse blastocyst adhesion and migration in vitro [19], but its effect in the human trophoblast has not yet been determined.

For controlled cellular invasion, it is essential that the balance between protease activation and inhibition be maintained [20–22]. MMP activity is inhibited by a group of endogenous tissue inhibitors of metalloproteinases (TIMPs). It has been reported that TIMPs are also produced by human trophoblast cells [23–25].

In the present study, the effects of several different extracellular matrix (ECM) proteins (Cols I and IV, FN, LN, and VN) on the expression and production of MMP-2, MMP-9, MMP-14, TIMP-1, TIMP-2, and TIMP-3 in cultured human trophoblast cells from first trimester placenta were investigated. In addition, we also examined the cell surface expression of integrins, which are receptors of ECM proteins, and studied the effects of ECM proteins on the behavior and adhesion patterns of cultured cytotrophoblast cells.

MATERIALS AND METHODS

Treatment of Culture Wells with Matrix Proteins

The wells of 24- or 96-well culture plates (Corning, Corning, NY) were coated with various matrix proteins, including Col I, Col IV, FN (Sigma Chemical Co., St. Louis, MO), LN, and VN (Life Technologies, Grand Island, NY). The coating conditions were based on the suppliers' instructions. All the matrix proteins were coated at a depth of 4 µg/cm2 onto each culture well and the plates were left at room temperature overnight.

Isolation and Cultivation of Human Cytotrophoblast Cells

Cytotrophoblast cells were isolated and cultured as previously described [26]. Briefly, human chorionic villi tissues were obtained from patients who had therapeutic termination of pregnancy at 9 or 10 wk of gestation. Informed consent was provided by the patients and the project was approved by the Institutional Human Research Committee. The tissues were digested with 0.25% trypsin and 15 IU/ml DNase I (Sigma) at 7–9°C. The cells were plated at 1–2 x 105 cells/well in 24-well plates coated with different matrix proteins with serum-free FD medium (Ham F12/Dulbecco modified Eagle medium [DMEM] 1:1; Gibco BRL, Gaithersburg, MD) supplemented with 1 ng/ml epidermal growth factor (EGF; Collaborative Research Inc., Lexington, MA), 10 µg/ml insulin, 0.1% BSA, 1.75 mM Hepes (Sigma), and 2 mM glutamine (Dongfeng Chemical Co., Shanghai, China); and kept in 5% CO2 at 37°C.

Cell Adhesion Assay

The cell adhesion assay was performed as described by Yang et al. [27]. Briefly, the cytotrophoblast cells (1 x 105/well) were seeded onto 96-well plates precoated with matrix proteins as described above. The cells were incubated for 2 h at 37°C and were gently washed with warm PBS three times. The adherent cells were fixed with 4% paraformaldehyde in PBS for 15 min. After washing, the cells were stained with 2% Giemsa for 30 min and then treated with methanol. The optical density was measured at 630 nm, which represents the relative number of cells adhering to the substrate.

Enzyme-Linked Immunosorbent Assay

The secreted TIMP-1 in the media from cytotrophoblast cells cultured on the different matrix proteins was quantified by a commercially available sensitive ELISA assay kit (Amersham Life Science, Little Chalfont, Buckinghamshire, UK). The harvested culture media were standardized according to the protein content of cell lysates, which was measured by the method of Bradford [28]. About 10–20 µl of medium per sample was applied for assay.

Gelatin Zymography

The presence of MMP-2 and MMP-9 in the media was demonstrated by gelatin zymography [29]. The harvested culture media were standardized according to the protein content of cell lysates, which was measured by the method of Bradford [28]. Thus, 10–20 µl medium, equivalent to 6 µg protein of cell lysates, was loaded to each lane for zymography. The sample was electrophoresed under nonreducing conditions in a 10% polyacrylamide gel containing 1 mg/ml gelatin (DIFCO Laboratories, Detroit, MI). After electrophoresis, the gels were washed at room temperature for 1 h in 2.5% Triton X-100, 50 mM Tris-HCl pH 7.5, and then incubated at 37°C overnight in a buffer containing 150 mM NaCl, 5 mM CaCl2, and 50 mM Tris-HCl pH 7.6. Thereafter, gels were stained with 0.1% (w/v) Coomassie Brilliant Blue R-250 in 30% (v/v) isopropyl alcohol and 10% glacial acetic acid for 60 min, and destained in 10% (v/v) methanol and 5% (v/v) glacial acetic acid. Semiquantification of the bands was performed with a densitometer.

Immunocytochemistry

The primary antibodies used were purchased from Chemicon International (Temecula, CA): anti-integrins {alpha}1 (AB 1934, dilution 1:200), {alpha}5 (MAB 1986, dilution 1:100), {alpha}6 (MAB 1972, dilution 1:100), ß1 (MAB 1951, dilution 1:200), ß4 (MAB 1964, dilution 1:200), and {alpha}vß3 (MAB 1976, dilution 1:200).

Cytotrophoblast cells were cultured in 8-well chamber slides (Nalge Nunc International Corp., Naperville, IL). After 48 h of culture, the cells were fixed in 4% paraformaldehyde for 15 min at 4°C, then washed twice in PBS. After washing, the cells were blocked in 10% goat serum for 20–30 min at room temperature and then incubated with primary antibodies overnight at 4°C. Subsequently, slides were washed in PBS and incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (diluted 1:100 in PBS containing 1% BSA; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at 37°C for 30 min. Slides were then washed and mounted with an aqueous mounting medium. The labeled cells were visualized using a Leica TCS NT confocal system (Leica, Germany). Immunocytochemical controls consisted of omission of primary antibody.

Complementary DNA Probes for Northern Blot Analysis

The cDNA plasmids for human MMP-2 and MMP-9 were generously provided by Dr. Karl Tryggvason (Karolinska Institute, Stockholm, Sweden) [30, 31]. According to the cDNA sequences described originally by Sato et al. [32], Stetler-Stevenson et al. [33], and Apte et al. [34], the specific fragments for human MMP-14 (nt 210–630), TIMP-2 (nt 640–1029), and TIMP-3 (nt 61–590) were amplified by reverse transcription-polymerase chain reaction from extracted human placenta total RNA, and the fragments were then cloned onto pBlueScript KS(+) (Stratagene, La Jolla, CA), pT-Adv (Clontech Laboratories Inc, Palo Alto, CA), and pGEM-T (Promega Corporation, Madison, WI) vectors, respectively, to obtain the recombinant plasmids. A 476-base pair (bp) TIMP-1 cDNA (nt 225–700) was amplified the same way. The primer is forward: 5'-GCG TTA TGA GAT CAA GAT GAC C-3', backward: 5'-AGG CTT CAG CTT CCA CTC C-3'. The fragment was confirmed by sequencing and then cloned into pGEM-T (Promega) and digested out by EcoRI (Promega) for labeling.

Northern Blot Analysis

Total cellular RNA was extracted from cultured cytotrophoblast cells using TRIzol Reagent (Gibco) according to the manufacturer's instructions. For Northern blot analysis, RNA (20 µg) was separated on formaldehyde-agarose gels (1% agarose, 2.2 M formaldehyde, and 1x MOPS [3-[N-morpholino] propanesulfonic acid]) and then transferred to nylon membranes (Amersham Life Science). The cDNA probes were labeled with [{alpha}-32P]dCTP using the Nick Translation system (Gibco). The nylon membranes were prehybridized for 4 h at 60°C in prehybridization buffer (0.2 M sodium phosphate pH 7.4, 0.1 mM EDTA, 7% [w/v] SDS, 1% [w/v] BSA, and 15% [v/v] formamide) and further hybridized for 24 h at 60°C in the fresh prehybridization buffer containing 10% (w/v) dextran sulfate. After hybridization, the membranes were washed and then exposed to x-ray film (Fuji Photo Film Co., Tokyo, Japan) for 24–96 h at -80°C. The signals were quantified by densitometric scanning and normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, which was detected by human GAPDH cDNA probe.

Statistics

Statistical analysis was performed by one-way ANOVA and significant differences among groups were subsequently established by the Student-Newman-Keuls method. Values of P < 0.05 were accepted as significant.

RESULTS

Expression of Integrins by Cultured First-Trimester Cytotrophoblast Cells

After 48 h in culture, cytotrophoblast cells were fixed and stained with anti-integrin antibodies as described above. As shown by confocal immunofluorescence micrography, cells stained with antibodies against integrins {alpha}1, {alpha}5, {alpha}6, ß1, ß4, and {alpha}vß3 demonstrated intense staining on the cell surface, indicating that the cells used in this study expressed these integrins (Fig. 1, A–F).



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  		FIG. 1. Immunofluorescent confocal micrographs of expression of integrins by human cytotrophoblast cells cultured for 48 h. Cells were fixed and stained as described in Materials and Methods. A) Integrin {alpha}1, B) integrin {alpha}5, C) integrin {alpha}6, D) integrin ß1, E) integrin ß4, and F) integrin {alpha}vß3

Cytotrophoblast Cell Adhesion to ECM Proteins

Cell attachment is one of the functions mediated by matrix proteins. In this study, the effects of matrix proteins on cytotrophoblast cell attachment were quantified 2 h after cell plating. The highest attachment rate was obtained with Cols I and IV, followed by FN and LN. The lowest attachment rate was exhibited by cells on VN (Fig. 2).



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  		FIG. 2. Adhesion of human cytotrophoblast cells to various matrix proteins. Each value represents the mean ± SD of triplicate values from three to four separate experiments. Bars without the same letter are significantly different from each other (P < 0.05)

Behavior of Cytotrophoblast Cells Cultured on Different Matrix Proteins

Cytotrophoblast cells attached to and spread on Col I, Col IV, FN, LN, and VN after 12 h of culture. The cells maintained a dispersed monolayer and no significant difference in behavior was seen among the cells cultured on the different matrix proteins. After 48 h of culture, cells grown on Cols I and IV formed an even epithelial monolayer, and some cells became elongated on Col IV. In contrast, the cells on LN and VN formed a network of multicellular aggregates with large zones that were devoid of cells, whereas cells grown on FN had smaller zones devoid of cells (Fig. 3).



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  		FIG. 3. Phase-contrast microscopy of cytotrophoblast cells grown on different matrix proteins. Cells were plated and cultured on collagen I (A), collagen IV (B), fibronectin (C), laminin (D), and vitronectin (E) and photographed after 48 h of culture (magnification x100)

Secretion of MMP-2, MMP-9, and TIMP-1 by Cytotrophoblast Cells Cultured on Different Matrix Proteins

Cytotrophoblast cells at 9–10 wk were cultured on specific matrix proteins for 48 h. Zymographic analysis showed that cells cultured on FN, LN, and VN secreted more MMP-9 (about 1.5- to 3-fold more) than cells cultured on Col I. The production of MMP-9 by cells cultured on Col IV was much less than that of the cells on Col I (P < 0.05). However, the production of MMP-2 remained unchanged among the cells cultured on the different matrixes (Fig. 4).



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  		FIG. 4. Secretion of MMP-2 and MMP-9 by human cytotrophoblast cells cultured on different matrix proteins for 48 h. A) Gelatin zymography of a representative experiment. B) Densitometric analysis of gelatin zymography; bars represent the relative amounts of MMP-2 and MMP-9. Data are means ± SD from three separate experiments. Bars without the same letter are significantly different from each other (P < 0.05)

Using an ELISA assay, we found that TIMP-1 production was the highest in cytotrophoblast cells on VN after 48 h of culture, and was approximately twofold higher than that of the cells on Col I. There were slight increases in TIMP-1 production by the cells on LN and FN when compared with that by the cells grown on Col I (P < 0.05; Fig. 5).



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  		FIG. 5. Secretion of TIMP-1 by human cytotrophoblast cells cultured on different matrix proteins for 48 h. Levels of TIMP-1 in the media were determined by ELISA. Data are means ± SD from at least three separate experiments. Bars without the same letter are significantly different from each other (P < 0.05)

Expression of MMP-2, MMP-9, MMP-14, TIMP-1, TIMP-2, and TIMP-3 mRNA in Cytotrophoblast Cells Cultured on Different Matrix Proteins

To examine whether matrix proteins can affect the mRNA transcripts of MMP-2, MMP-9, MMP-14, TIMP-1, TIMP-2, and TIMP-3 in cytotrophoblast cells, total RNA was extracted from cells cultured for 48 h. The Northern blot results for MMP-2 and MMP-9 were consistent with the results from zymography. The highest levels of MMP-9 mRNA were detected in the cells grown on LN and VN (approximately twofold higher than the cells on Col I). A slight increase was also observed in MMP-9 mRNA expression in the cells grown on FN when compared with those on Col I (P < 0.05). It is interesting that the amount of MMP-9 mRNA in the cells grown on Col IV was only half of that on Col I, and the expression of TIMP-3 in the cells grown on VN decreased to 60% of that on Col I. There was no difference in the expression of MMP-2, MMP-14, TIMP-1, and TIMP-2 mRNA by the cells cultured on various matrix proteins (Figs. 6 and 7).



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  		FIG. 6. A) Autoradiogram of a representative Northern blot showing MMP-14, MMP-2, and MMP-9 mRNA expression by human cytotrophoblast cells cultured on different matrix proteins for 48 h. B) Densitometric analysis of autoradiograms. The relative amounts of a) MMP14, b) MMP-2, and c) MMP-9 mRNA were standardized by GAPDH mRNA. Data are expressed as the ratio change, means ± SD from three separate experiments. Bars without the same letter are significantly different from each other (P < 0.05)

DISCUSSION

Human implantation and subsequent placental development requires a series of complex and coordinated interactions between the fetal-derived trophoblast cells and the maternal uterus. As the trophoblast cells migrate through the decidual stroma, they are confronted with various basement membranes and matrix substrates. In our current study, we found that the trophoblast cells exhibited different degrees of attachment to the matrix proteins. This difference may account for the striking variation in morphology and migration behavior seen after cytotrophoblast cells were cultured on these substrates for 48 h. In our observation, when the cells were cultured for 12 h after plating on the different matrix proteins, they maintained a dispersed monolayer and they were all confluent (data not shown). After 48 h, cells plated on Cols I and IV still remained stationary and maintained a uniform monolayer. In contrast, large zones devoid of cells were seen on LN and VN, and smaller zones devoid of cells were seen on FN that had had the same inoculation as the cells plated on Cols I and IV. We suggest that this phenomenon may be caused by cell movement and aggregation to form networks. These observations are not in agreement with those reported by Burrows et al. [16] who observed that trophoblast cells remained sessile on LN and became migratory on FN. This difference may be due to the absence of serum in our cell culture system, because attachment factors in serum will affect cell adhesion and spreading.

Emonard et al. [17] found that when LN binds to normal or malignant human trophoblast cells it stimulates type IV collagenase secretion. Bischof et al. [18] also reported that binding of human trophoblast cells to FN and LN could induce MMP secretion. Our results, using both zymography and Northern blot, have shown for the first time that the expression of MMP-9 mRNA and protein by trophoblast cells cultured on FN and LN was much higher than that of cells cultured on Cols I and IV. These results indicate that matrix proteins can affect MMP-9 gene expression as well as its protein secretion in human trophoblast cells. In addition, we also determined that VN has an even stronger effect on the expression of MMP-9 in human cytotrophoblast cells. Our previous work [35] has shown that the expression of MMP-9 increases with the development of the placenta during early pregnancy. It has been demonstrated that the invasiveness exhibited in vitro by human trophoblast cells depends on the production of MMP-9 [5]. Furthermore, Morgan et al. [36] found that human trophoblast cell lines, which secreted MMP-9, had high invasive ability, whereas the BeWo cell line, which produces mainly MMP-2 and little MMP-9, was noninvasive. Therefore, it is likely that the expression of MMP-9 is critical for cell invasion. Thus, determining the regulation of MMP-9 expression in human trophoblast cells is important for understanding the mechanism of trophoblast invasion during placentation. The results presented in this paper suggest that different matrix proteins exert different effects on the expression of MMP-9 in human trophoblast cells. As all these matrix proteins exist in maternal uterine tissue [12, 13], they undoubtedly have an important influence on the invasive property of the trophoblast cells. Moreover, cytotrophoblast cells may also secrete LN, FN, and VN during the first trimester [35]. It is possible that these matrix proteins may regulate the expression of MMP-9 in trophoblast cells by an autocrine mechanism.

Werb et al. [37] have reported that a monoclonal antibody against the FN receptor, or covalently immobilized FN-derived peptides containing the arg-gly-asp (RGD) sequence, induced MMP gene expression in fibroblasts. Experiments with a human melanoma cell line have revealed that stimulation of the {alpha}vß3 integrin (VN receptor) enhanced gelatinase A (MMP-2) production [38]; however, the mechanisms of action of ECMs on the production of MMPs in human trophoblast cells is not completely understood. It has been reported that human trophoblast cells expressed the integrins that bind Cols I and IV, FN, LN, and VN [16, 39, 40]. Our results have also shown that cultured cytotrophoblast cells were able to express the integrin subunits {alpha}1, {alpha}5, {alpha}6, {alpha}vß3, ß1, and ß4 on the cell surface. This suggests that the effects of ECM proteins on the expression of MMP-9 in human cytotrophoblast cells may be mediated by these cell surface receptors, which are capable of transducing positive and negative signals from ECM proteins to the cell interior to affect gene activity.

The present study shows that there is no significant difference in the expression of MMP-2 among cells cultured on different matrix protein-coated dishes. This may be due to the different transcriptional control of MMP-2 and MMP-9 expression [31]. There is a phorbol ester responsive element (AP-1 site) present in the promoter region of MMP-9 and other MMPs, whereas no such element exists in the promoter region of MMP-2. Indeed, in several different systems studied, MMP-2 tends to be constitutively expressed rather than to be inducible [41–43]. In this study, similar to that with MMP-2, the levels of MMP-14 and TIMP-2 mRNA remained stable in the trophoblast cells that were cultured on the different ECM proteins. It is suggested that the MMP-14–TIMP-2 complex acts as a cell surface receptor for pro-MMP-2, thus anchoring the proenzyme for activation by the complex [44–46]. Therefore, the coordinated expression of MMP-2, MMP-14, and TIMP-2 enables the trophoblast cells to gain proper matrix degradation activity to invade the uterine stroma.

The results from the ELISA assay in our study have shown that TIMP-1 production was enhanced by LN, FN, and VN, which is similar to the regulation of MMP-9. It is known that TIMP-1 preferentially binds and inhibits MMP-9 in both the latent and active forms [47]. Therefore, this coordinate regulation of MMP-9 and TIMP-1 production in human cytotrophoblast cells could be a mechanism by which the extent of ECM protein degradation is limited. From our study, the matrix proteins may regulate TIMP-1 production at the posttranscriptional level, because they did not affect the expression of TIMP-1 mRNA. TIMP-3 is another inhibitor of MMPs, and its expression is in accord with that of MMP-9 in the first trimester human placenta [48]. The expression regulation mechanism seems different for TIMP-1 and TIMP-3. VN showed a stronger inhibitory effect on TIMP-3 mRNA expression when compared with other matrix proteins, which may contribute to VN's stimulation of cell migration.

Trophoblast cell invasion into the decidua is a complex and dynamic process that includes cell adhesion to matrix proteins, the degradation of matrixes, and cell motility. In our observations, trophoblast cells show a striking variation in adhesion characteristics and morphology when they are plated on different matrix proteins; moreover, these cells exhibit different expression profiles for MMPs and TIMPs. From these observations an overall conclusion we can make is that the interaction of trophoblast cells and the uterine ECM may transmit necessary signals (positive and negative) to the cells, which is very important for the cells to gain proper access to the uterine stroma and to result in subsequent, successful placentation.



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  		FIG. 7. A) Autoradiogram of a representative Northern blot showing TIMP-1, TIMP-2, and TIMP-3 mRNA expression by human cytotrophoblast cells cultured on different matrix proteins for 48 h. B) Densitometric analysis of the autoradiograms. The relative amounts of a) TIMP-1, b) TIMP-2, and c) TIMP-3 mRNA were standardized by GAPDH mRNA. Data are expressed as the ratio change, means ± SD from three separate experiments. Bars without the same letter are significantly different from each other (P < 0.05)

ACKNOWLEDGMENTS

We thank Dr. Karl Tryggvason for the generous gifts of MMP-2 and MMP-9 cDNA probes (Karolinska Institute, Stockholm, Sweden), Dr. Jennie P. Mather (Raven, Biotechnologies), and Dr. Alison Holloway (University of Toronto, Toronto, ON, Canada) for suggestions on the manuscript.

FOOTNOTES

First decision: 30 September 1999.

1 This study was supported by the ninth-five National Pan-Deng project, the Knowledge Innovation Program of the Chinese Academy of Sciences, and grant RF 94025 #30 from the Rockefeller Foundation. Back

2 Correspondence: Lin-zhi Zhuang, State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 19 Zhongguancun Lu, Beijing 100080, China. FAX: 86 10 62529248; zhuanglz{at}panda.ioz.ac.cn Back

Accepted: February 26, 2001.

Received: August 27, 1999.

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