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BOR - Papers in Press, published online ahead of print July 9, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.017525
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BIOLOGY OF REPRODUCTION 69, 1593–1599 (2003)
DOI: 10.1095/biolreprod.103.017525
© 2003 by the Society for the Study of Reproduction, Inc.

Female Reproductive Tract

Temporal Regulation of Gene Expression During the Expected Window of Receptivity in the Rhesus Monkey Endometrium1

William C. Okulicz2,3,4, and Christopher I. Ace3

Departments of Obstetrics/Gynecology3 Physiology,4 University of Massachusetts Medical School, Worcester, Massachusetts 01655


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Progesterone has been shown to regulate a number of genes and gene networks in the primate endometrium. This action of progesterone is essential to provide an appropriate milieu for embryo-endometrial communication that can lead to implantation and the successful initiation of pregnancy. A temporal regulation of endometrial genes is most likely required to achieve an appropriate state of receptivity in the primate endometrium. Using simulated menstrual cycles in the rhesus monkey, endometrial tissue was harvested at days that encompass the expected window of receptivity (4–10 days after the estradiol surge) and subsequently converted to cycle day-specific cDNA populations. Using differential display reverse transcriptase-polymerase chain reaction, 12 cDNA fragments were isolated and sequenced whose mRNA levels were elevated during this time frame. The temporal expression patterns of these mRNAs were confirmed by semiquantitative polymerase chain reaction. Two of these fragments exhibited high homology to previously characterized human genes: 1) secretory leukocyte protease inhibitor, also known as antileukoprotease, an endometrial neutrophil elastase inhibitor with antibacterial and anti-inflammatory properties; and 2) syncytin, also known as endogenous retrovirus W envelope protein, a highly fusogenic membrane glycoprotein that induces formation of giant syncytia and is believed to be important in decidual and placental development. The temporal regulation of these genes by progesterone supports their likely role in the orchestration of molecular and cellular events that are required to achieve a state of receptivity in the primate endometrium.

female reproductive tract, implantation, menstrual cycle, progesterone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The endometrium has been the focus of numerous studies because of its critical roles in menstruation, embryo implantation, and pregnancy in women. Endometrial tissue undergoes massive structural and biochemical changes during the menstrual cycle, and it is the changing pattern of estradiol (E) and progesterone (P) secretion that primarily governs regulation of growth, differentiation, shedding, and reconstruction that is necessary for continued reproductive competence [1–5]. This sequential secretion of ovarian E and P during the primate menstrual cycle is essential for the orderly regulation of gene expression that leads to a ‘receptive’ endometrium that can allow embryo implantation.

The precise molecular mechanisms that result in the temporal and spatial control of gene expression in the primate endometrium, leading to a state of receptivity (window of implantation), are largely unknown. P action has, however, been shown to be essential for proper endometrial maturation and the maintenance of pregnancy. These effects of P are expected to be mediated primarily through its cognate receptor [6, 7]. These features of P action on the endometrium most likely involve a cascade of signal transduction pathways that invoke the regulation of ligands, receptors, cytokines, and growth factors. Inadequate (low) P levels during the secretory (or luteal) phase cause impaired endometrial receptivity and subsequent infertility, and these studies further underscore the importance of P in the process of endometrial receptivity [8–10]. Successful embryo implantation is a precisely coordinated multistep process that involves many factors, including the evolving importance of maternal-embryo communication [11].

A number of factors have been identified that may be involved in endometrial maturation and subsequent embryo implantation in the human [12–15] and different animal models [16–18]. Overall, these studies suggest that a single factor is unlikely to be solely responsible for the endometrial preparation required for receptivity. It is more likely that temporal control of expression of a number of genes ultimately leads to an appropriate uterine environment for implantation.

Our laboratory has been primarily interested in P-dependent regulation of endometrial response in the rhesus monkey, a model for human endometrial function. Our previous studies have identified a number of known or novel genes that are likely to play roles in endometrial maturation during the secretory phase [19–24]. Although the mRNA expression of these and other genes has been shown to be induced by P in the endometrium (see above), many P-regulated genes have yet to be identified and characterized in the context of their temporal regulation. In this article, we describe the use of differential display reverse transcription-polymerase chain reaction (DDRT-PCR) to identify P-dependent genes from a temporal (cycle day-specific) series of endometrial cDNA populations spanning the expected window of receptivity in the rhesus monkey. Our results show that several known and other unknown genes and gene fragments display a pattern of restricted expression during this period that is consistent with their role(s) in the regulation of endometrial receptivity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal Model—Rhesus Monkey

The development and use of artificial menstrual cycles in the rhesus monkey were first described by Hodgen [25]. These studies showed that simulation of the menstrual cycle by the timed insertion and removal of silastic implants of E or P was sufficient to allow the endometrium to support implantation and eventual delivery (in vitro fertilization and surrogate transfer). Our previously published studies [21, 26–28] describe in detail the protocols for creation of adequate cycles. Briefly, mature female rhesus monkeys (Macaca mulatta) were housed in individual cages and ovariectomized at least 2 mo before initiation of artificial menstrual cycles. Silastic implants containing E or P were placed subcutaneously in the intrascapular area under ketamine anesthesia (10 mg/kg). The following protocol for placement or removal of the implants was used to create adequate secretory phases: basal E levels (70–100 pg/ml of serum) were maintained with a single 3.0-cm implant throughout the cycle; the E surge was created by sequential insertion of three 3.0-cm E implants on Days 10–12, followed by their removal on Day 13 (E peak); each of two P implants of 3.0-cm length were inserted sequentially on Day 13 and Day 16 followed by their sequential removal on Day 23 and Day 25 to simulate the luteal phase. Inadequate secretory phases were created by the placement of one 1.5-cm silastic capsule of P on Day 13 and the removal of three E capsules (one E implant remains). This protocol has been shown to result in appropriate P levels characteristic of an inadequate secretory phase in the rhesus monkey [21]. All protocols used in these studies were approved by the Institutional Animal Care and Use Committee.

Tissue Collection and RNA Preparation

Tissue was collected by endometrectomy at hysterotomy as previously described [19, 28]. Tissue harvesting was performed before insertion or removal of implants from three to four individual animals for each of Days 14, 17, 21, 23, and 26 of the menstrual cycle. All samples were subsequently stored at -80°C until further processing. Total RNA was isolated and poly(A)+-enriched RNA was prepared using oligo d(T) spin columns as previously described [23]. The integrity of all total mRNA samples was examined by visual inspection of intact 28S and 18S ribosomal RNA bands on agarose gels before further processing (15–20 analyses). Samples were treated with DNase I to ensure the removal of traces of genomic DNA.

Synthesis and Amplification of cDNA Populations

One microgram of poly(A)+ RNA from each of three to four animals for each time point (Days 14, 17, 21, 23 and 26) was pooled just before cDNA synthesis (a total of 3–4 µg per time point). The Superscript Choice System (Life Technologies, Rockville, MD) was used for first-strand cDNA synthesis using 1 µg of oligo(dT)T7 primer (5'-AAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGCGCT15-3') and 100 ng of random hexamers per reaction. After second-strand synthesis, 1 µg of EcoRI adaptors per reaction were ligated to the cDNA termini to enable subsequent PCR amplification of the cDNA populations as previously described [20, 21, 23]. Complimentary DNA populations were purified in Qiaquick spin columns (Qiagen, Venlo, The Netherlands) and amplified in 100-µl PCRs containing 0.5 µm of LINK-CUA primer, 0.25 mM dNTPs, 1.5 mM MgCl2, 1x buffer, and 2 U of Taq polymerase in a thermal cycler (94°C, 1 min; 50°C, 1 min; 72°C, 2 min) for 30 cycles. The LINK-CUA primer (5'-CUACUACUACUAAATTCGCGGCCGCGTCGAC-3') is complementary to the EcoRI adaptor [29]. Following amplification of the cDNA populations, one fiftieth (40 ng) was used as template in PCR reactions (for conditions see Semiquantitative PCR Analysis below) using primers specific for human 18S ribosomal RNA (Ambion, Inc., Austin, TX), a housekeeping gene commonly used to normalize the cDNA populations. This analysis, together with inspection of the cDNA product (smears) on agarose gels, showed that all five cDNA populations were correctly synthesized and amplified. The cDNA fragments ranged in size from 200 base pairs (bp) to >1 kilobase (kb) and contained equivalent amounts of 18S ribosomal RNA (Fig. 1).



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  		FIG 1. A) Ethidium bromide-stained gel of polymerase chain reaction (PCR)-amplified cDNA populations from Days 14, 17, 21, 23, and 26 of adequate artificial menstrual cycles in the rhesus monkey. B) Semiquantitative PCR of 18S ribosomal RNA in the above amplified cDNA populations. M denotes 100-base pair marker of a DNA ladder

DDRT-PCR, Cloning, and Sequencing

DDRT-PCR was performed using the RNAimage kit (GenHunter Corp., Nashville, TN). Two nanograms of cDNA were amplified in 20-µl reactions containing 1x buffer, 2.0 µM dNTPs, 20 Ci/mmol alpha-(33P) ATP, 0.2 µM H-T11N oligo dT primer (5'-AAGCTTTTTTTTTTTN-3'), 0.2 µM H-APN arbitrary primer (5'-AAGCTTNNNNNNN-3'), and 0.5 U of Taq polymerase (Qiagen). Different combinations of oligo dT and arbitrary primers were used in separate reactions to generate different sequence profiles from which fragments could be isolated. The latter combinations that exhibited expression patterns of interest were subsequently repeated for reproducibility, and those that were reproducible were chosen for further analysis. Fragment 1 was generated with HT11G and H-AP8, fragment 5 with H-T11G and H-AP5, and fragments 9, 10, 11, and 12 with H-T11A and H-AP7 (RNAimage kit, GenHunter Corp.). Reactions were performed in a PTC-200 thermal cycler (MJ Research, Waltham, MA) at 94°C for 1 min, 40°C for 2 min, and 72°C for 1 min for 40 cycles and analyzed by denaturing polyacrylamide gel electrophoresis, omitting the fixing stage. The autoradiogram and gel were aligned by needle punctures, and individual bands were carefully excised from the gel with a razor blade. Gel slices attached to filter paper were eluted by boiling in 100 µl of water for 10 min and spun to remove debris, and the supernatant was precipitated with glycogen. DNA fragments thus isolated were reamplified as described above except with 250 µM dNTPs in the absence of radiolabel. Products were directly cloned into the plasmid vector pCR 2.1-TOPO (Invitrogen, Carlsbad, CA), and sequenced (University of Massachusetts Medical School Nucleic Acid Facility, Worcester, MA). Homology searches were performed against GenBank entries using NCBI BLAST programs (National Center for Biotechnology Information, Bethesda, MD).

Semiquantitative PCR Analysis

Once the sequence of the DDRT-PCR fragments was determined, specific amplimers were designed for each (see below) and used in PCR reactions with the five cDNA populations (Days 14, 17, 21, 23, and 26) as a template to confirm their expression patterns. One fiftieth of the amplified cDNA populations (40 ng) was analyzed in 100-µl reactions containing 0.5 µM fragment-specific primers, 0.25 mM dNTPs, 1.5 mM MgCl2, 1x buffer, and 2 U of Taq polymerase in a thermal cycler (94°C, 1 min; 48–55°C, 1 min; 72°C, 2 min). PCR reactions were performed for 24–45 cycles to determine the linear range of amplification for each primer set. Products were analyzed by agarose gel electrophoresis, and comparative evaluation was performed by densitometric analysis (QuantiScan; Biosoft Inc., Ferguson, MO) of photographed gels. Amplimer sequences and linear cycle length were as follows: fragment 1, 5'-TCGTCAGAATCGACGTGGTA-3' and 5'-ACTCCTTCCTGATGGTGTGC-3'(25 cycles); fragment 5, 5'-TGGGGACTAGGAAGGACTGA-3' and 5'-TGTAATTCGTCACAG-GAACCA-3' (30 cycles); fragment 9, 5'-AGCTTAACGAGGGAGGAAGC-3' and 5'-AAAGGACCTGGACCACACAAG-3' (30 cycles); fragment 10, 5'-CGAGGAGCAACATCAGAGG-' and 5'-GCTGGGATTACAGGCTTGA-3' (35 cycles); fragment 11, 5'-AACGAGGGGGTAAACATGAA-3' and 5'-AGTCATCGCTAGCAAAAAGC-3' (30 cycles); and fragment 12, 5'-GTCATCGCTGGGAAGGTAAA-3' and 5'-CAGGGTCCAAAGAGGAGTAACA-3' (35 cycles). Some differences in temporal expression patterns between the differential display results and the subsequent semiquantitative PCR (SQ-PCR) verification are likely a result of the difference in primers used in each method (i.e., arbitrary versus gene-specific primers, respectively).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Because of the small amounts of material harvested by endometrectomy, we adopted an amplification protocol for our cDNA populations that we had used previously [21, 30]. cDNA smears could be observed in all preparations following amplification (Fig. 1A). We estimate that after one round of amplification our original cDNA amount has increased approximately 70-fold based on the increase of 18S levels (data not shown). Figure 1B shows that equivalent levels of the 18S ribosomal RNA housekeeping gene are present in all amplified cDNA populations under nonsaturating PCR conditions (14 cycles, determined empirically).

We next used our cDNA populations together with DDRT-PCR to determine if we could identify temporal-specific gene expression patterns during an adequate secretory phase. Initially, 12 bands (fragments) that exhibited putative temporal-specific expression were excised, eluted, cloned, and sequenced. Because false-positive results can occur with the use of DDRT-PCR [22, 23, 31] and due to limiting amounts of tissue available for Northern blots, we designed fragment-specific PCR primers from our sequence data to confirm the regulation of all 12 selected fragments by semiquantitative- (SQ) PCR. SQ-PCR analysis using our five DNA populations confirmed the temporal nature of expression for six of the above fragments (fragments 1, 5, 9, 10, 11, and 12). Six bands were determined to be false positive because they were detected at equivalent levels in all populations (data not shown). Figures 2 and 3 show DDRT-PCR (upper panel) and SQ-PCR (lower panel) profiles for fragments 1, 5, 10, and 11 and fragments 9 and 12, respectively. Fragments 1, 9, and 12 were expressed on Days 17, 21, and 23 with little if any detection on Days 14 and 26. Fragment 10 was expressed predominantly on Day 17 and decreased on Days 21 and 23 to undetectable levels on Day 26. Fragment 5 expression increased on Day 17 to a steady level through Day 26, whereas fragment 11 was only detectable on Days 23 and 26.



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  		FIG 2. Differential display acrylamide gel (upper panel) and primer-specific polymerase chain reaction analysis (agarose gel, lower panel) showing the temporal expression of endometrial fragments 1 (A), 5 (B), 10 (C), and 11 (D) spanning Days 14–26 of adequate artificial menstrual cycles in the rhesus monkey



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  		FIG 3. Differential display acrylamide gel (upper panel) and primer-specific polymerase chain reaction analysis (agarose gel, lower panel) showing the temporal expression of endometrial fragments 9 (A) and 12 (B) during Days 14–26 of adequate artificial menstrual cycles in the rhesus monkey

Nucleic acid sequence data showed that fragments 5, 10, and 11 were homologous to as yet uncharacterized BAC or PAC clones on human chromosomes 20, 16, and 10, respectively (Table 1). Fragment 1 exhibited 96% identity to human predicted protein KIAA1096 mRNA [32], which is similar to BAT2 [33]. Homology searches of GenBank databases identified fragment 9 as human antileukoprotease mRNA (also called secretory leukocyte protease inhibitor [SLPI]) (94%) [34–37] and fragment 12 as a human endogenous retrovirus W (HERV-W) envelope mRNA (also called syncytin) (92%) [38]. Table 1 also shows that although the other fragments did not display the temporal specificity expected, each was up-regulated during the secretory phase and exhibited high nucleic acid sequence homology to as yet uncharacterized human BAC/PAC clones and one previously identified zinc finger protein: fragment 8 was 99% identical to human RINZF mRNA.


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  		TABLE 1. Homology of endometrial, temporally expressed cDNA fragments to NCBI databases

We have previously described the construction of two cDNA libraries from both adequate and inadequate secretory-phase endometria (PcDNA and IcDNA, respectively) [21]. IcDNA was isolated from endometrium that does not support implantation due to the low levels of circulating P. We expect that this latter cDNA population lacks or is deficient in some of the P-dependent mRNAs encoding genes important for correct endometrial maturation. With this in mind, we tested both PcDNA and IcDNA for the presence of the two genes we have positively identified as being expressed during the window of receptivity, namely, syncytin and SPLI. Figure 4 shows the results of gene-specific SQ-PCR using adequate and inadequate libraries as template. Lanes 5 and 6 show that the control housekeeping gene 18S ribosomal RNA is expressed at comparable levels in both PcDNA and IcDNA under nonsaturating conditions. Interestingly, although syncytin was readily detected in adequate endometrium, we could not detect any expression in IcDNA (lanes 3 and 4), consistent with it being a gene that requires high levels of P for its expression. Lanes 1 and 2 show that SLPI was also detected in PcDNA and that its expression in inadequate endometrium appeared to be elevated (3-fold to 5-fold). This is consistent with our hypothesis that this is a P-dependent gene whose expression is increased at low levels of P and decreased by high levels of P (autologously down-regulated by P), as we have noted previously with other P-dependent genes [21, 22].



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  		FIG 4. Semiquantitative polymerase chain reaction analysis of secretory leukocyte protease inhibitor (lanes 1–2), syncytin (lanes 3–4), and control 18S ribosomal RNA (lanes 5–6) in adequate (P) and inadequate (I) endometrial cDNA populations.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Using DDRT-PCR, we have identified six cDNA fragments whose mRNA expression is temporally up-regulated during the expected window of endometrial receptivity (Days 21–23) in an adequate (normal) menstrual cycle. Three of these fragments represent sequences that have not yet been characterized. One fragment represents a predicted protein human KIAA1096 (based on the open reading frame) [39]. This sequence is similar to BAT2, which shares similarities with some transcriptional regulatory proteins containing zinc finger motifs and proline or glutamine-rich regions [33]. BAT2 also contains motifs typical of the integrin receptor family [12]. BAT2 has been mapped within the class III region of the major histocompatibility complex, which encodes genes involved in immune function or major histocompatibility complex-associated disease susceptibility [33].

Fragment 9 was identified as human antileukoproteinase or SLPI [34]. This protein is a neutrophil elastase inhibitor that also has antibacterial and anti-inflammatory properties. In addition to its antiprotease activity, SLPI has been shown to regulate intracellular enzyme synthesis, epithelial cell growth by activation, and repression of distinct growth-regulatory genes and mediate normal wound healing [36, 37]. King et al. [40] have shown by immunohistochemical analysis that SLPI is expressed in glandular epithelium of human endometrium from the mid to late secretory phase, suggesting a direct or indirect regulation by P. These studies are in agreement with our results that show increased expression of this gene from Day 17 to 23 of adequate secretory phases in the rhesus monkey. Leukocytes infiltrate the endometrium before menstruation and may in part be responsible for the rise in secretory SLPI during the secretory phase. SLPI has been found to have antibacterial effects, and one region of the molecule has 37% homology with defensins, a family of antibacterial proteins [40, 41]. SLPI also inhibits the NF kappa B signal transduction pathway involved in inflammatory response [42]. The most likely role of SLPI is as a natural antibiotic and anti-inflammatory molecule. Infection ascending through the cervix could pose a threat to the implanting and developing conceptus [40].

Fragment 12 was identified as the envelope gene (also called syncytin) of the recently characterized human endogenous defective retrovirus HERV-W [35, 38]. Syncytin is a highly fusogenic membrane glycoprotein that appears to be expressed specifically in the placenta. The protein induces the formation of giant syncytia and can mediate fusion of cytotrophoblasts into the syncytiotrophoblast layer, which is essential for pregnancy maintenance [43, 44]. Endogenous viral syncytin appears to have been sequestered to serve an important physiological role during pregnancy and placental morphogenesis [38]. Our results are the first to describe the expression of this gene in the primate endometrium. Its potential role in the nonpregnant uterus, however, remains to be elucidated. We speculate that syncytin could be a P-induced endometrial decidualization factor (it induces syncytia or multinucleated cells). Interestingly, the expression of HERV-K (closely related to HERV-W) is stimulated in cultured human tumor cells by sequential E and P treatment, most likely mediated by the presence of a P receptor binding site situated in its long terminal repeat [45]. Since differentiation of P-induced endometrial stromal cells into decidual cells is essential for embryo implantation and placentation, expression of syncytin during the window of receptivity of the primate menstrual cycle is likely to be important in preparation of the endometrium for successful implantation.

Overall, we envisage that a coordinated, steroid-induced activation and repression of many genes during the changeover from E to P dominance during the menstrual cycle is important for correct endometrial maturation. We propose a general working model of P-dependent gene regulation during the primate secretory phase as shown in Figure 5 based on our data and data in the literature. During a normal secretory phase, the early phase of P regulation likely involves both inhibition (Fig. 5, A and B) and induction (Fig. 5, C and D) of hormone-dependent genes that are sensitive to the initial rise in serum P level. As the secretory phase proceeds, rising titers of serum P lead to continued induction of P-dependent genes as exemplified by fragment 5 in our results (Fig. 5C). Although in this study we did not isolate any E-dependent fragments repressed by P (Fig. 5A), our laboratory has previously reported genes and gene fragments that clearly fit this profile [20–22].



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  		FIG 5. A schematic representation together with examples of relative gene expression patterns in the endometrium during the primate secretory phase. A) Progesterone (P) repression of estradiol (E)-dependent genes. B) Autologous down-regulation of P-dependent genes, e.g., fragment 10. C) P-induction of genes during the secretory phase, e.g., fragment 5. D) P-induction of gene expression during the window of receptivity, e.g., fragment 12 (syncytin). E) A composite cascade of gene expression patterns A–D. The associated panel (B–D) for each fragment shows portions of the relevant reverse transcription-polymerase chain reaction gel

Superimposed on the above two regulatory actions of P is an apparent autologous down-regulation of some but not all P-dependent genes, as exhibited by fragment 10 (Fig. 5B). Several laboratories have shown that some genes are only activated just before and during potential implantation in the human and other animal models [12, 46–52]. This scenario of a sharply restricted window of P-dependent gene regulation (activation) is also included in our working model (Fig. 5D) and further supported by our present data. For example, both fragment 9 (SLPI) and fragment 12 (syncytin) appear to fall into this latter category of genes that are up-regulated during the window of endometrial receptivity. However, when expression of SLPI and syncytin was compared in adequate (high P level) versus inadequate (low P level) secretory phase endometria, syncytin was not detectable in inadequate cDNA, whereas SLPI expression was (3-fold to 5-fold) greater in inadequate endometrium. This suggests that SLPI may in fact be a gene whose expression is increased at low levels of P and subsequently decreased by increasing levels of P and therefore may be autologously down-regulated by P, as we have noted with other P-dependent genes [21, 22].

This working model summarizes our hypothesis that waves of P-dependent gene regulation, a cascade, are central to proper maturation of the primate endometrium (Fig. 5E). All levels of regulation are considered important because of their potential to be linked. That is, preceding gene expression patterns may control and direct subsequent expression that allows full endometrial maturation. It is anticipated that refinements or additions to this working model will result from future studies in our laboratory and others.


   ACKNOWLEDGMENTS
 
The authors thank Drs. Christopher Longcope and Janet Tast and Charlene Franz for their help and support of this work.


   FOOTNOTES
 
1 This work was supported in part by grants from the National Institute of Child Health and Human Development (NICHD) (HD 31620) and the Indo-US Program on Contraceptive and Reproductive Health Research. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the NICHD. Back

2 Correspondence: William C. Okulicz, Departments of Obstetrics/Gynecology and Physiology, University of Massachusetts Medical School, 55 Lake Ave. North, Worcester, MA 01655. FAX: 508 856 5933; william.okulicz{at}umassmed.edu Back

Received: 24 April 2003.

First decision: 9 May 2003.

Accepted: 30 June 2003.


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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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