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Biology of Reproduction 64, 992-1000 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Monocyte Chemotactic Protein-1 and -2 Messenger Ribonucleic Acids in the Ovine Uterus: Regulation by Pregnancy, Progesterone, and Interferon-{tau}1

Eric Asselin3,a, Greg A. Johnsona, Thomas E. Spencera, and Fuller W. Bazer2,a

a Center for Animal Biotechnology and Genomics and Department of Animal Science, Texas A&M University, College Station, Texas 77843-2471

ABSTRACT

Endometrial leukocytes may play important roles during pregnancy. Because chemokines are regulators of immune cell activity and trafficking, this study determined if mRNAs for monocyte chemotactic proteins (MCP) were present in the ovine uterus and regulated by progesterone (P) and/or recombinant ovine interferon tau (roIFN-{tau}). Uteri of normal cycling and pregnant ewes (experiment 1) and uteri of ovariectomized ewes receiving intrauterine infusions of IFN-{tau} and/or i.m. injections of P (experiment 2) were used to detect MCP-1 and MCP-2 mRNA. In experiment 1, slot-blot hybridization analysis of endometrial total RNA revealed that MCP-1 and MCP-2 mRNA levels did not change during the estrous cycle but increased between Days 13 and 19 of pregnancy. Using in situ hybridization, MCP-1 and MCP-2 mRNA were localized to immune cells in the subepithelial compact stroma. Histomorphological studies and in situ hybridization for major basic protein (MBP) indicated that MCP-positive immune cells were eosinophils. In experiment 2, treatment with P and roIFN-{tau} increased (P < 0.05) the number of MCP-1- and MCP-2-expressing eosinophils in the endometrium compared to ewes treated with P alone. Injection of the P receptor antagonist (ZK 137,316) inhibited effects of P and/or roIFN-{tau} to recruit eosinophils expressing MCP-1 and MCP-2 mRNAs. Endometrial production of MCPs by eosinophils during early pregnancy may play a role(s) in central implantation and/or placentation in ewes that is crucial for successful establishment of pregnancy.

cytokines, gene regulation, other hormones, placenta, pregnancy, progesterone, reproductive immunology, trophoblast, uterus

INTRODUCTION

Monocyte chemotactic proteins (MCPs) are members of the cellular chemoattractant (CC) chemokine family that were first identified in tumors as chemoattractants for macrophages [1]. Monocyte chemotactic protein-1 recruits monocytes/macrophages, T cells, basophils, mast cells, and natural killer (NK) cells for infiltration into sites of inflammation [2, 3]. Monocyte chemotactic protein-1 may stimulate the respiratory burst and Ca2+ uptake required to transform monocytes into activated macrophages [4] and increase the ability of monocytes to inhibit growth of certain tumors [5]. This chemokine is produced by fibroblasts [6], endothelial cells [7], lymphocytes, monocytes, and eosinophils [8, 9]. In the female reproductive tract, MCP-1 expression has been found in decidua and endometrial cells [10–12], as well as the mouse uterus [13]. Monocyte chemotactic protein-1 production by unidentified immune cells increases in response to prostaglandin (PG)F2{alpha} during luteolysis in the corpus luteum (CL) of ruminants [14, 15]. Monocyte chemotactic protein-2 is a CC chemokine that is functionally related to MCP-1 because it specifically attracts monocytes and T lymphocytes [3]. In contrast to MCP-1, MCP-2 is chemotactic for eosinophils [3, 16, 17] and has been detected in porcine luteal cells [18].

In the ovary, MCP-1 is associated wtih structural and functional luteolysis in humans [19], rodents [20, 21], and ruminants [14, 15, 22]. It has been shown that increased MCP-1 production in response to PGF2{alpha} induces infiltration of macrophages into the CL which may be involved in structural regression [14, 15]. Luteolysis must be prevented in pregnant females as the production of progesterone (P) by the CL is obligatory for the establishment and maintenance of pregnancy. In ruminants, interferon-tau (IFN-{tau}) is the pregnancy recognition signal produced by the conceptus between Days 11 and 23 of pregnancy [23–26]. Interferon;ch{tau} acts on the endometrium in concert with P to prevent development of the luteolytic mechanism, thereby prolonging the functional life span of the CL.

During the estrous cycle, the density of macrophages and T lymphocytes in the ovine and bovine uterus do not change [27]. However, during early pregnancy, the number of CD45R+ lymphocytes increases in both endometrium [28] and uterine and jugular venous blood [29, 30]. It has been postulated that these are NK cells that produce factors to enhance establishment of pregnancy [30]. Monocyte chemotactic protein-1 and MCP-2 are chemotactic for and may recruit NK cells to the ruminant uterus [31] during early pregnancy. Therefore, the objective of this study was to determine 1) if abundance of mRNAs for MCP-1 and MCP-2 in ovine endometrium change in response to days of the estrous cycle and/or pregnancy and 2) the effects of P and IFN-{tau} on endometrial levels of MCP-1 and MCP-2 mRNAs.

MATERIALS AND METHODS

Animals

Mature ewes of primarily Rambouillet breeding were observed daily for estrous behavior using vasectomized rams and assigned randomly to treatments after exhibiting at least two estrous cycles of normal duration (16–18 days). All experimental and surgical procedures complied with the Guide for Care and Use of Laboratory Animals and were approved by the Institutional Agricultural Animal Care and Use Committee of Texas A&M University (Animal Use Protocols 7-286 and AG-239).

Experimental Design and Treatments

Experiment 1 At estrus (Day 0), ewes were assigned randomly to cyclic or pregnant status. Ewes assigned to the pregnant status were bred at estrus and at 12 h and 24 h postestrus with intact rams. Fifty-two ewes were ovariohysterectomized (n = 4 ewes/day) on Days 1, 3, 5, 7, 9, 11, 13, and 15 of the estrous cycle or Days 11, 13, 15, 17, and 19 of pregnancy. In cyclic ewes and in pregnant ewes on Days 11 to 17, the uterine lumen was flushed with 20 ml sterile saline at hysterectomy. Pregnancy was confirmed by the presence of an apparently normal conceptus in uterine flushings (Days 11–17) or by histology in the uterine lumen (Day 19).

Experiment 2 As described previously by Johnson et al. [32], 20 cyclic ewes were fitted with uterine catheters on Day 5 of the estrous cycle (Day 0 = estrus) and assigned randomly (n = 5 ewes per treatment) to receive daily i.m. injections of P and/or ZK136,317, a P receptor (PR) antagonist (Schering AG, Berlin, Germany) from Days 5 to 24 and intrauterine injections of protein from Days 11 to 24 as follows: 1) 50 mg P and 100 µg control serum proteins (CX) (P-CX); 2) P and 75 mg ZK and CX proteins (P + ZK-CX); 3) P and recombinant ovine (ro)IFN-{tau} (2 x 107 antiviral units); and 4) P and ZK and roIFN-{tau} (P + ZK-IFN). The uterine horns of each ewe received twice daily (0700 and 1900 h) injections of either CX proteins or roIFN-{tau} (50 µg protein per horn per injection). The P was administered at 0700 h in a total volume of 1 ml corn oil vehicle. All ewes were hysterectomized on Day 30.

Tissue Collection and Processing

At hysterectomy, several sections (~0.5 cm) from the ipsilateral uterine horn were fixed in fresh 4% paraformaldehyde in PBS (pH 7.2). After 24 h, fixed tissues were changed to 70% ethanol for 24 h and then dehydrated and embedded in Paraplast-Plus (Oxford Labware, St. Louis, MO). The remaining endometrium was physically dissected from myometrium, frozen in liquid nitrogen, and stored at -80°C for RNA extraction.

Reverse Transcriptase-Polymerase Chain Reaction

Total endometrial RNA (400 ng) from a Day 17 pregnant ewe was used for preparation of first-strand cDNA using reverse transcriptase (RT) as described by Asselin et al. [33]. Expression of the MCP-1 gene was determined by amplification of a 471-base pair (bp) region (140–611 bp) of the bovine MCP-1 gene sequence [34]. Amplification was carried out using the MCP-1 antisense (AS) downstream sequence 5'-CAGAGGAAAGAATTTGCCCA-3' and the sense (S) upstream sequence 5'-CGCCTGCTGCTATACATTCA-3'. For major basic protein (MBP), primers were chosen from homologous regions of the known human [35] and rat [36] MBP sequences and a 495-bp region was amplified from the rat MBP sequence (53–548 bp): (AS) 5'-ATCCTGCCTCCAATCCAGAC-3' and (S) 5'-CCCTCTACTTCTGGCTCTTC-3'. Polymerase chain reaction (PCR) cycling conditions were 30 sec at 94°C, 30 sec at 55°C, and 30 sec at 72°C for 35 cycles, followed by a 10-min extension at 72°C. After amplification, MCP-1 and MBP cDNAs were cloned into the pCR 2.1cloning vector (Invitrogen Cloning Kit, Carlsbad, CA) and transformed into competent Escherichia coli DH5{alpha} cells. The inserts were digested with EcoRI enzyme, purified from a 1% agarose gel and subcloned into the pCR II plasmid (Invitrogen). The cDNAs were sequenced in both directions.

Analysis of RNA

Isolation of RNA Total cellular RNA was isolated from endometrial samples using the Trizol reagent (Gibco-BRL, Grand Island, NY) according to manufacturer's recommendations.

Preparation of AS cRNA probes Radiolabeled riboprobes were synthesized for slot-blot hybridization analyses by in vitro transcription using a MAXIscript Kit (Ambion, Austin, TX) according to manufacturer's recommendations. Antisense ovine MCP-1 and MBP cRNA probes were generated by linearizing DNA templates with HindIII and in vitro transcription with T7 polymerase. Antisense human MCP-2 probe [37] was generated by linearizing the DNA templates with HindIII and in vitro transcription with T3 polymerase. Plasmid template for 18S rRNA (pT718S; Ambion) was used as a control.

Slot-blot hybridization analysis Due to the large number of endometrial total RNA samples, steady-state levels of mRNA were assessed in endometrial total RNA samples using slot-blot hybridization. For each ewe, denatured total cellular RNA (20 µg) was analyzed using radiolabeled AS cRNA probes as described previously [32]. To correct for total RNA loading differences, a duplicate RNA slot membrane was hybridized with radiolabeled AS 18S rRNA cRNA (pT718S; Ambion). Following washing, nonspecific hybridization was removed by RNase A digestion [38]. The radioactivity associated with each slot was quantified by electronic autoradiography using an Instant Imager (Packard Instrument Company, Meridian, CT).

In Situ Hybridization Analysis

Monocyte chemotactic protein-1, MCP-2, and MBP mRNAs were localized in uterine tissue sections by in situ hybridization analysis as described previously [32]. Deparaffinized, rehydrated, and deproteinated uterine tissue sections (5–7 µm) were hybridized with radiolabeled AS or S ovine MCP-1, MCP-2, or MGP cRNA probes generated from linearized plasmid templates using in vitro transcription with [{alpha}-35S]UTP (3000 Ci/mmol; Amersham, UK). Washed and RNAse-digested slides were intially exposed to Kodak BioMax x-ray film for 16 h to estimate the length of exposure to film emulsion. Autoradiographs of slides were prepared using Kodak NTB-2 liquid photographic emulsion [38]. Slides were stored at 4°C for 1–2 wk, developed in Kodak D-19 developer, counterstained with Harris modified hematoxylin (Fisher Scientific, Fairlawn, NJ), dehydrated through a graded series of alcohol to xylene, and coverslipped.

Histomorphological Studies

Uterine tissue sections (5 µm) were deparaffinized and rehydrated as described above. Hematoxylin/eosin staining was carried out using a standard procedure [39]. Briefly, sections were stained with Harris hematoxylin, rinsed in water, and quickly dipped in 1% acidic alcohol (70% ethanol; 0.25 N HCl). Sections were then washed in distilled deionized water, stained with eosin (1%) for 1 min, and dehydrated through a graded series of alcohol to xylene and coverslipped.

Photomicroscopy and Digital Imaging

Photomicrographs of in situ hybridization and histomorphological slides were taken using a Zeiss Axioplan 2 photomicroscope (Carl Zeiss, Inc.) fitted with a Hamamatsu chilled 3CCD color camera (Hamamatsu Corporation, Bridgewater, NJ). Digital images were captured and/or assembled using Adobe Photoshop 4.0 (Adobe Systems, Seattle, WA) and a MacIntosh PowerMac G3 computer (Apple Computer, Cupertino, CA). Black-and-white prints were electronically printed using a Kodak DS8650 color printer.

Statistical Analyses

Data were subjected to least-squares ANOVA using the general linear models procedures of the Statistical Analysis System [40]. In experiment 1, effects of day on steady-state levels of endometrial mRNA were examined by regression analysis within status (cyclic or pregnant). Data from Days 11, 13, and 15 were examined by two-way ANOVA considering variation due to day postestrus, status (cyclic vs. pregnant), and their interaction. In experiment 2, orthogonal contrasts were used to detect specific effects of treatment [41]. In both experiments, slot-blot hybridization data (total counts) were normalized for differences in sample loading using the 18S rRNA data as a covariate in ANOVA. All tests of significance were performed using the appropriate error terms according to the expectations of the mean squares [41]. Data are presented as least-square means (LSM) total counts with SE.

RESULTS

Monocyte Chemotactic Protein-1 and MCP-2 mRNAs in the Endometrium During the Estrous Cycle and Pregnancy

Monocyte chemotactic protein-1 mRNA was not detected in endometrium of cyclic ewes (Fig. 1A). In contrast, endometrial MCP-1 mRNA increased linearly (P < 0.05) between Days 13 and 19 in pregnant ewes. Similarly, levels of MCP-2 mRNA increased linearly (P < 0.05) during early pregnancy (Fig. 1B). In situ hybridization analysis revealed that MCP-1 and MCP-2 mRNAs were expressed exclusively by endometrial immune cells located immediately beneath the luminal epithelium (Fig. 2). During early pregnancy, the presence of MCP-1- and MCP-2-positive cells was first observed on Day 13. Cell numbers increased on Day 15 and were maximal on Days 17 and 19.



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  		FIG. 1. Slot-blot analysis of steady-state levels of MCP-1 (A) and MCP-2 (B) mRNA in endometrium of cyclic and pregnant ewes (n = 3 to 4 ewes/day). Slot blots were quantitated by electronic autoradiography, and data are expressed as LSM total counts with SE. The MCP-1 and MCP-2 mRNA were not detected during the estrous cycle. However, both MCP;ch1 and MCP-2 mRNA levels increased during early pregnancy (day x status, P < 0.05), and within the pregnant ewes, an effect of day was detected (P < 0.05)



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  		FIG. 2. In situ localization of MCP-1 (A) and MCP-2 (B) mRNA in the endometrium of cyclic and pregnant ewes. Cross-sections of the uterine wall from cyclic and pregnant (Px) ewes were hybridized with {alpha}-35S-labeled antisense or sense (d17Pxs) ovine MCP-1 and human MCP-2 cRNA probes. Developed slides were couterstained lightly with hematoxylin and photomicrographs taken under brightfield or darkfield illumination. All photomicrographs are shown at a magnification of x260. LE, Luminal epithelium; GE, glandular epithelium; sc, stratum compactum; ss, stratum spongiosum

Histomorphology of Cells

In order to identify the immune cells with detectable MCP-1 and MCP-2 mRNA, sections of uterine tissue were stained with hematoxylin and eosin (Fig. 3). These immune cells exhibited bilobed nuclei and cytoplasmic granules that stained red, indicative of eosinophils. Furthermore, in situ hybridization analysis showed that MBP mRNA was localized specifically to MCP-1 mRNA and MCP-2 mRNA positive cells (Fig. 4). The MBP mRNA positive cells were not detected in cyclic ewes. The MBP mRNA positive cells were first detected on Day 13 of pregnancy, and maximal numbers were on Days 17 and 19 of pregnancy. The MBP mRNA positive cells were localized specifically in stratum compactum beneath the luminal epithelium.



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  		FIG. 3. Uterine tissue sections from Day 17 (A–C) and Day 19 (D–F) pregnant ewes were stained with Harris's hematoxylin and eosin. Representative photomicrographs of sectioned uteri are shown. Eosinophils exhibited bilobed nuclei and cytoplasmic granules that stained red. Photomicrographs are at a magnification of x260 (A and D), x327 (B and E), and x671 (C and F). E, Eosinophils; L, luminal epithelium; cG, stratum compactum (superficial or shallow) glandular epithelium; sG, stratum spongiosum (deep) glandular epithelium; S, stroma; TE, conceptus trophectoderm



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  		FIG. 4. In situ localization of MBP mRNA in endometrium of cyclic and pregnant ewes. Cross-sections of the uterine wall from cyclic and pregnant (Px) ewes were hybridized with {alpha}-35S-labeled antisense or sense (d17Pxs) ovine MBP cRNA probes. Developed slides were couterstained lightly with hematoxylin, and photomicrographs were taken under brightfield or darkfield illumination. All photomicrographs are shown at a magnification of x260. LE, Luminal epithelium; GE, glandular epithelium; sc, stratum compactum; ss, stratum spongiosum

Effect of P and roIFN-{tau} on MCP-1 and MCP-2 mRNAs

In experiment 2, P-treated ewes had higher (P < 0.05) levels of endometrial MCP-1 (Fig. 5A) and MCP-2 (Fig. 5B) mRNAs compared to P + ZK-treated ewes. In ewes receiving P alone, intrauterine administration of roIFN-{tau} increased the abundance (P + CX vs. P + roIFN-{tau}, P < 0.05) of endometrial MCP-1 and MCP-2 mRNAs. The addition of ZK also inhibited the combined effects of P and roIFN-{tau} (P + ZK-roIFN-{tau}). In situ hybridization revealed that MCP-1 mRNA positive cells were localized in a pattern similar to that for early pregnant ewes and were present only in P-treated ewes regardless of intrauterine protein treatment (Fig. 6). To confirm that these immune cells were eosinophils, MBP mRNA, as well as MCP-2 mRNA, was determined to be expressed exclusively by MCP-positive immune cells (data not shown).



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  		FIG. 5. Slot-blot analysis of steady-state levels of MCP-1 and MCP-2 mRNA in endometrium of ewes treated with P alone or P + roIFN-{tau}. Blots were hybridized with antisense radiolabeled cRNA probes generated from an ovine (MCP-1) and human (MCP-2) cDNA and then digested with RNAse A. Slot blots were quantitated by electronic autoradiography, and data are expressed as LSM total counts with SE. Columns with different superscripts are different (P < 0.05), indicating stimulatory effects of P and P + roIFN-{tau} treatments



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  		FIG. 6. In situ localization of MCP-1 mRNA in endometrium of ewes treated with P alone or P + roIFN-{tau}. Cross-sections of the uterine wall from treated ewes were hybridized with {alpha}-35S-labeled antisense or sense (P-Cxs) ovine MCP-1 cRNA probes. Developed slides were couterstained lightly with hematoxylin, and photomicrographs taken under darkfield illumination. All photomicrographs are shown at a magnification of x260. LE, Luminal epithelium; GE, glandular epithelium; sc, stratum compactum; ss, stratum spongiosum

DISCUSSION

Results of the present study are the first to show that eosinophils contain mRNAs for both MCP-1 and MCP-2 and that the abundance of these mRNAs increases in eosinophils within the uterus during early pregnancy in ewes. Eosinophils are not present in the stratum compactum stroma of cyclic ewes but are attracted into this region of the uterus after Day 11 of pregnancy. The respective roles of IFN-{tau} and P in this process are uncertain. Interferon-{tau} may be responsible for activation of expression of MCP-1 and MCP-2 genes or for affecting chemotaxis of eosinophils into the uterus. The MCP mRNA positive cells had staining patterns typical of eosinophils in which a bilobed nucleus was surround by cytoplasmic granules that stained positive with eosin. Further, these immune cells expressed MBP mRNA that encodes an eosinophil granule-associated protein [42]. Using a partial ovine MBP cDNA cloned by RT-PCR, in situ hybridization anlayses demonstrated that MBP mRNA was colocalized to the same cells that expressed MCP-1 and MCP-2 mRNAs. This evidence indicates that eosinophils express both MCP-1 and MCP-2 mRNA in uteri of early pregnant ewes.

Monocyte chemotactic protein-1 is produced by human eosinophils [8, 9] and is chemotactic for monocyte/macrophages, T lymphocytes, and NK cells, all of which are present in the ruminant uterus during early pregnancy. Monocyte chemotactic protein-1, along with MCP-2, may recruit these cells from the bloodstream into the uterine endometrium. The regulation of granulocyte chemotactic protein- 2 (GCP-2) expression in the bovine uterus by IFN-{tau} during pregnancy may be involved in regulation of inflammation and angiogenesis [43]. In different types of immune cells expression of MCPs is regulated by IFN-{gamma} [44–46], although IFN-{gamma} expression in the ovine uterus has not been reported. Although MCP-1 and GCP-2 are not chemotactic for eosinophils [1, 17], MCP-2 is chemotactic for eosinophils [1, 3, 16]. Results of the present study suggest that eosinophils expressing MCP-1 and MCP-2 are attracted to the ovine uterus only during pregnancy. Progesterone may influence this, either directly or by up-regulation of another chemokine, and IFN-{tau} may act to either recruit more eosinophils or to increase MCP-1 and MCP-2 production by resident eosinophils. Increases in MCP-2 may be involved in a positive feedback loop to recruit more eosinophils.

The infiltration of leukocytes into the endometrium of ewes during early pregnancy is a complex process and may involve various mediators. Prostaglandins, particularly PGE2, increase permeability of blood vessels. In ewes, levels of PGE2 increase during early pregnancy [47, 48], because cyclooxygenase-2 (COX-2), a rate-limiting enzyme in the conversion of arachidonic acid to PGs, increases during early pregnancy in the ovine uterus [49]. Prostaglandin E2 is also an effective immunomodulator in NK cells [50, 51], and roIFN-{tau} stimulates PGE2 and COX-2 gene expression in bovine endometrial cells in vitro [52–54]. The increase in COX-2 protein in the human endometrium also coincides with leukocyte accumulation in the uterus [11]. Thus, chemokines and PGs may act in concert locally during the peri-implantation period to stimulate infiltration of leukocytes into the uterus in response to effects of P and IFN-{tau}.

Interestingly, MCP-1 is expressed in CL of cows [22] and sheep [14, 15]. In ewes, MCP-1 mRNA is present in unidentified immune cells in CL in response to PGF2{alpha} [14]. During luteolysis, the numbers of monocytes/macrophages [55, 56] increase in CL [15, 20], and MCP-1 may recruit these cells. Eosinophils also increase in ovine CL in response to PGF2{alpha}, and luteal cells produce a specific chemoattractant for eosinophils [57]. Given that MCP-1 is localized to immune cells in both the endometrium (eosinophils) and possibly CL [14], similar mechanisms, perhaps involving P and PGF2{alpha}, may exist in both the ovary and uterus. A specific chemoattractant for eosinophils may be produced, and in turn, eosinophils may be activated to produce MCP-1 to attract other immune cells into the tissue. In the rat uterus, an eosinophil chemotactic factor has been described [58, 59] and such a factor may be a candidate in the recruitment of eosinophils to the uterus of ruminants.

In conclusion, results of the present study suggest that eosinophils are attracted to the pregnant ovine uterus and may produce growth factors or cytokines favorable to the establishment of pregnancy. Increased levels of MCP-1 mRNA in eosinophils suggests that other immune cells attracted into the endometrium during early pregnancy may also influence establishment of pregnancy. Monocyte chemotactic protein-2 may also be important in the recruitment of additional eosinophils. The finding that both MCP-1 and MCP-2 mRNAs are expressed by eosinophils supports the hypothesis that the immune and reproductive systems are closely linked during the pregnancy recognition period.

ACKNOWLEDGMENTS

The authors thank Drs. Els Van Coillie and Ghislain Opdenakker (Laboratory of Molecular Immunology, University of Leuven) for generously providing human MCP-2 cDNA; Dr. Shawn W. Ramsey and Mr. Todd Taylor of the Texas A&M University Sheep and Goat Center for care and management of ewes; Dr. Robert C. Burghardt for assistance with photomicrography; and Dr. Kristof Schawlisz (Schering AG) for provision of the ZK137,316 antagonist compound.

FOOTNOTES

First decision: 25 October 2000.

1 Supported by a Medical Research Council of Canada (MRC) fellowship (E.A.) and, in part, by NIH grants HD32534 (F.W.B.), F32-HD08501 (G.A.J.), and P30 ES09106. Back

2 Correspondence: Fuller W. Bazer, Department of Animal Science, Texas A&M University, 442 Kleberg Center, College Station, TX 77843-2471. FAX: 979 862 2662; fbazer{at}cvm.tamu.edu Back

3 Current address: Department de Chimie-Biologie, Universite du Quebec-Trois-Rivieres, Trois-Rivieres, PQ, Canada G9A 5H7. Back

Accepted: November 2, 2000.

Received: September 19, 2000.

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