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BOR - Papers in Press, published online ahead of print December 11, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.011874
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BIOLOGY OF REPRODUCTION 68, 1455–1462 (2003)
DOI: 10.1095/biolreprod.102.011874
© 2003 by the Society for the Study of Reproduction, Inc.

Embryo

A Novel Role of Luteinizing Hormone in the Embryo Development in Cocultures1

S. Mishraa, Z.M. Leia, and Ch.V. Rao2,a

a Division of Research, Department of Obstetrics, Gynecology and Women's Health, University of Louisville Health Sciences Center, Louisville, Kentucky 40292


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bovine oviductal epithelium contains LH receptors, which function in the increase of synthesis of oviductal glycoprotein (OGP). As with cocultures of embryos with oviductal epithelial cells, OGP is thought to promote early embryonic growth and development. These findings led us to test the hypothesis that LH treatment of cocultures further increases embryo development through OGP mediation. Coculture of >=10 two-cell bovine embryos with bovine oviductal epithelial cells increased the development of the embryos into blastocysts. Treatment of these cocultures with hCG, used as a surrogate for LH because of its stability and purity, further increased embryo development. The hCG effect is dose dependent and hormone specific and requires the dimer conformation and the presence of LH receptors in oviductal epithelial cells. The inhibition of OGP synthesis and prevention of protein kinase A activation blocked the hCG effect in cocultures. Reverse transcription polymerase chain reaction and indirect immunofluorescence with laser scanning confocal microscopy demonstrated the presence of LH receptors in bovine oocytes, embryos, and blastocysts. However, embryo LH receptors may not have played any role in the beneficial hCG effects in cocultures. These findings suggest that elevated periovulatory LH levels may promote preimplantation embryo development in oviducts. These results have important implications for assisted reproductive technologies in which cocultures are used to improve pregnancy rates.

early development, embryo, human chrionic gonadotropin, luteinizing hormone, oviduct


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oviducts are dynamic organs that support gamete transport, maturation, fertilization, early embryonic growth and development, and the timely transport of embryos for implantation in the uterus. Oviducts are targets of estradiol and progesterone produced in response to FSH and LH stimulation of ovaries [1]. These steroid hormones are delivered to oviducts, perhaps by local and systemic routes [2]. Oviducts are also regulated by a wide variety of other agents, including locally synthesized molecules [3–21]. The local synthesis of these molecules may be controlled by circulating hormones, so that they can work in an additive, synergistic, or antagnostic manner to regulate different oviductal functions [8, 13, 16, 20, 22, 23]. LH, a hormone released from the anterior pituitary gland in response to GnRH stimulation from the hypothalamus, is among the circulatory hormones that can regulate oviductal functions [8, 13, 16, 19, 20]. LH regulates indirectly by increasing ovarian synthesis of steroid hormones and directly by activating the LH receptors, which have been demonstrated in oviducts of several species [8, 13, 16, 19, 24–27]. LH receptor activation results in upregulation of cyclooxygenases 1 and 2, 5-lipoxygenase, oviductal glycoprotein (OGP), endothelin 1, and endothelin receptor types A and B, which play important roles in different oviductal functions [8, 13, 16, 20]. Bovine oviduct contains LH receptors, and their activation results in an increased synthesis of OGP [16, 28], which binds to embryos to increase their development [29–33]. We hypothesized that LH treatment of cocultures with oviductal epithelial cells would further increase embryonic development into blastocysts. Human CG, a structural and functional homolog of LH, was used to test the above hypothesis. The results demonstrate that hCG treatment increases two-cell embryo development in cocultures through OGP mediation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

The following were obtained from various colleagues: monoclonal antibody to bovine OGP (1H10) from Dr. Yutaka Sendai (Research Institute for the Functional Peptides, Yamagata, Japan); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA from Dr. Russell Prough (University of Louisville Health Sciences Center); polyclonal antibody raised against a synthetic N-terminus 15- to 38-amino acid sequence of LH receptors from Dr. Patrick Roche (Mayo Clinic, Rochester, MN); highly purified hCG (CR-127; 14 900 IU/mg), human LH (AFP-0264B; 4015 IU/mg), human FSH (AFP-87929B, 1683 IU/mg), human thyroid-stimulating hormone (TSH, AFP-4314C; 15 IU/mg), {alpha} subunit (AFP8492A) and ß subunit (AFP8494A) of hCG from NIDDK National Hormone & Pituitary Program and Dr. A.F. Parlow (Torrance, CA). The rest of the materials were purchased from various sources. Trizol, horseradish peroxidase-labeled anti-rabbit IgG, antibiotic-antimycotic solution (10 000 U/ml penicillin, 10mg/ml streptomycin and 25 µg/ml fungizone), Percoll, Ca2+, Mg2+ and phenol-free 10x Hanks balanced salt solution (HBSS), and medium 199 with glutamate and insulin were from Gibco BRL (Grand Island, NY). Fetal bovine serum (FBS) was from Atlanta Biologicals (Norcross, GA). Streptavidin-horseradish peroxidase conjugate, monoclonal antibodies to epithelial cell membrane antigen (EMA) and vimentin were from Dako (Carpenteria, CA). MasterAmp reverse transcription polymerase chain reaction (RT-PCR) kits were from Eipicenter Technologies Corp. (Madison, WI). The dNTPs, DNase-I and random prime labeling kits were from Promega Corp. (Madison, WI). Collagenase (type IA-S; 320 U/mg solid) and anti-rabbit IgG fluorescein isothiocyanate (FITC) conjugate were from Sigma (St. Louis, MO). The 4',6'-diamidino-2-phenylindole (DAPI) was from Molecular Probes (Eugene, OR), the isoquinoline-sulfonamide (H-89) and bisindolylmaleimide (Bis) were from Calbiochem (San Diego, CA), and the two-cell bovine embryos were from BOMED (Bovine Oocyte Maturation and Embryo Development, Madison, WI). The embryos were shipped overnight in a special container with an insulated thermoregulated package with battery pack and charging unit to maintain the temperature at 39°C (Minitube of America, Verona, WI). Upon receipt, the embryos were washed twice in medium 199 containing 10% FBS pre-equilibrated for 2 h at 38.6°C in 5% CO2 and 95% air. Only healthy looking compact two-cell embryos were used in the experiments.

Collection of Bovine Ovaries and Oviducts

Late follicular phase ovaries and oviductal ampullary segments were collected from a local slaughter house, immediately placed either in ice cold PBS (ovaries) or in medium 199, pH 7.4, containing 10% FBS, 10 µg/ml insulin, 2% antibotic-antimycotic mixture (oviducts), and brought to the laboratory. The oocytes were aspirated from 2- to 5-mm follicles and washed twice in 10 mM Hepes containing 125 mM NaCl, 3 mM KCl, 2 mM NaHCO3, 0.4 mM Na2HPO4, 10 mM sodium lactate, 2 mM Ca2+, and 0.5 mM Mg2+. The oocytes were then gently vortexed for 90 sec and pipetted several times through a glass Pasteur pipette to dislodge and remove cumulus cells. Then cumulus-free oocytes were then washed in medium 199 and processed for indirect immunofluorescence and laser scanning confocal microscopy. The oviducts were processed for cell dispersion.

Cell Dispersion

The ampullary segments were flushed with 10 ml of Ca2+, Mg2+, and phenol red-free HBSS and then cut open longitudinally to expose the epithelial cell layer [13, 16]. The tissues were then digested for 45 min at 37°C in a shaking water bath in capped sterile tubes containing 5 ml of 1% collagenase in medium 199. The digested tissues were first strained through a 250-µm pore size nylon mesh while gently scraping the mucosal folds. The filtrate was then passed through a 30-µm pore size nylon mesh. The epithelial cells retained on the mesh were backwashed with 20 ml of 1x HBSS containing a 2% antibiotic-antimycotic mixture. The backwash solutions containing enriched epithelial cells were centrifuged for 30 min at 1500 x g in 50–60% Percoll solution to pellet contaminating red blood cells and cellular debris. The epithelial cells present in the upper Percoll layer were removed and placed in a 10% antibiotic-antimycotic mixture. About 95% of the cells were immunostained with an anti-EMA antibody, which binds to epithelial cells, and the rest were immunostained with an anti-vimentin antibody, which binds to fibroblasts. Cell viability, which was determined by trypan blue exclusion, was >95%.

Coculture

Oviductal epithelial cells (0.6 x 106 cells/well) were cultured for 48 h in 24-well culture plates in medium 199 containing 10% FBS in a humidified 5% CO2 atmosphere in a CO2 incubator [34, 35]. The monolayers were washed twice in medium 199 to remove unattached cells. Where oligodeoxynucleotide (ODN) treatments were indicated, these compounds were added with 10 µg/ml lipofectamine to the fresh replacement medium. After 24 h, various hormones and test agents were added. After an additional 24 h, the medium was replaced with fresh medium of the corresponding composition, and groups of 10–15 two-cell embryos were randomly transferred in microdrops. The embryos were covered with 25–30 µl of mineral oil and cultured for another 5 days, with medium changes every 48 h with corresponding fresh medium. The treatments were maintained until the end of the culture. In some experiments (Fig. 1), two-cell embryos were placed in pre-equilibrated 100-µl culture drops of medium 199 containing 10% FBS with or without hCG and were covered with mineral oil. The development of two-cell embryos into blastocysts was monitored daily with an inverted light microscope. Only those embryos containing >36 cells and a cavity were counted as blastocysts.



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  		FIG. 1. Effect of culturing two-cell bovine embryos in the presence or absence of oviductal epithelial cells and 10 ng/ml hCG. The number of embryos used per culture varied as shown on the x-axis. Letters indicate significant differences (P < 0.05) compared with corresponding coculture without hCG (a), compared with one embryo cultured in medium (b), compared with 10 embryos cultured in medium (c), and compared with 10 embryos cocultured without hCG (d)

Design and Synthesis of Phosphorothioate ODNs

Twenty-four-base pair (bp) (LH receptor) and 27-bp (OGP) sequences spanning the translation initiation ATG codons of porcine LH receptor and bovine OGP genes were designed using a designer PCR computer program (Research Genetics, Huntsville, AL). The phosphorothioate ODN sequences were synthesized using a Pharmacia LKB Gene Assembler Special automated DNA synthesizer with a standard phosphoramidate chemical procedure and were desalted on NAP-10 columns. The antisense and sense sequences were as follows: LH receptor antisense, 5'-AGC GCC AGG GAC CGC CGT CTC ATG-3'; LH receptor sense, 5'-CAT GAG ACG GCG GTC CCT GGC GCT-3'; OGP antisense, 5'-CAG ATC CCG AGG CAG GAT TGA GGC AGG-3'; OGP sense, 5'-GTC TAG GGC TCC GTC CTA ACT CCG TCC-3'.

Immunocytochemistry

Cell monolayers were fixed in 3% paraformaldehyde for 30 min and processed for immunostaining for LH receptors or OGP by an avidin-biotin immunoperoxidase method [8]. Dilutions of 1:400 for LH receptor antibody and 1:500 for OGP antibody were used. Nonspecific IgG was used in procedural controls.

Reverse Transcription Polymerase Chain Reaction

Total RNA was isolated by the Trizol method from about 50 oocytes and early embryos. Then RT and 40 cycles of PCR were performed in the same tubes with bovine LH receptor or GAPDH primers using MasterAmp RT-PCR kits. The PCR products were electrophoresed in 2% NuSieve agarose gels and stained with ethidium bromide. A 123-bp DNA ladder was included in an adjacent lane to determine the sizes of the amplified fragments. The expected sizes of these amplified fragments were 458 bp for LH receptors and 374 bp for GAPDH.

The sequences of primers used for the LH receptor and GAPDH amplification were as follows: LH receptor, 5'-GCC TGA CAT CAA GGA GAA GC-3' (forward) and 5'-CAG GGA AAT CAG CGT TGT CC-3' (reverse); GAPDH, 5'-TGG ACT CCA CGA CGT ACT CA-3' (forward) and 5'-CTC TCT GCT CCT CCT CTT CG-3' (reverse).

Indirect Immunofluorescence and Laser Scanning Confocal Microscopy

Oocytes and embryos were fixed for 30 min in 3% paraformaldehyde in PBS and permeabilized by exposure to Tyrode solution, pH 2.5 [36]. They then were incubated overnight at 4°C with a 1:50 dilution of LH receptor antibody followed by 2 h of incubation with FITC-conjugated secondary antibody. Oocytes and embryos were then washed thoroughly in PBS, and nuclei were stained with 300 nM DAPI for 15 min, rinsed several times with PBS, mounted in fluoromount G, and scanned by a Leica 4D TCS laser confocal microscope. Nonspecific IgG and primary antibody omission were used as procedural controls. The binding of FITC-labeled secondary antibody gave green fluorescence, and DAPI stained the nuclei blue.

Statistical Analysis

All the experiments were performed in triplicate and were repeated three times with cells isolated from different oviducts. The data from all the experiments were pooled for the calculation of means and SEMs. An ANOVA with a Duncan multiple range test was used for determining the significance of differences between experimental groups [37].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Embryo Cultures

Two-cell embryos have an inherent low capacity to develop into blastocyts. This capacity, under different culture conditions, was investigated first. The development of two-cell embryos in culture medium was modest regardless of embryo number, until there were about five embryos in the culture (Fig. 1). At 10 embryos/culture, however, the development increased (P < 0.05). The addition of highly purified hCG had no effect, regardless of embryo number.

Coculture of two-cell embryos with oviductal epithelial cells had no effect on their development until 10 embryos were used per culture (P < 0.05). The addition of hCG to cocultures increased embryo development when >=5 embryos were used per culture (P < 0.05). These data formed the basis for coculturing 10–15 embryos with oviductal epithelial cells in the remaining experiments.

Effect of hCG in Cocultures

The addition of 0.5–1.0 ng/ml hCG had no effect on two-cell embryo development in cocultures (Fig. 2). However, embryo development increased following the addition of 10 ng/ml hCG, but a further increase in hCG concentrations had no effect beyond that achieved with 10 ng/ml (P < 0.05; Fig. 2). Only LH, but not FSH, TSH, or isolated {alpha} and ß subunits of hCG, mimicked hCG in increasing embryo development in cocultures (Fig. 3).



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  		FIG. 2. Dose dependency of hCG effect on the development of two-cell bovine embryos cultured with oviductal epithelial cells. Asterisks indicate significant differences (P < 0.05) compared with 0 ng/ml hCG



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  		FIG. 3. Hormone specificity of the hCG effect on two-cell bovine embryo development during cocultures with oviductal epithelial cells. Asterisks indicate significant differences (P < 0.05) compared with no hormone addition.

Requirement of LH Receptors for the hCG Effect in Cocultures

Even though LH and hCG, and other protein hormones, work by activating their receptors, the receptor requirement in the context of cocultures was investigated by targeted destruction of LH receptor mRNA by an antisense ODN approach. With this approach, antisense ODN recognizes receptor mRNA and forms a duplex that is hydrolyzed by RNase H, thus blocking gene expression. For these experiments, phosphorothioate LH receptor ODNs, which resist degradation by nucleases [38], were synthesized from an LH receptor sequence beginning at the ATG translation initiation codon. Treatment of oviductal epithelial cells with LH receptor antisense but not sense ODN resulted in a dramatic reduction in LH receptor immunostaining (Fig. 4). Although the addition of LH receptor ODNs alone had no effect, cotreatment with antisense, but not sense, ODN blocked the hCG effect on embryo development in cocultures (Fig. 5).



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  		FIG. 4. LH receptor immunostaining in oviductal epithelial cells cultured for 7 days with or without 5 µM LH receptor antisense or sense ODNs. Nonspecific IgG was used as the procedural control. x300.



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  		FIG. 5. Requirement of LH receptors for the hCG effect on the development of two-cell bovine embryos cocultured with oviductal epithelial cells. Asterisks indicate significant differences (P < 0.05) compared with no additions

Role of OGP in Cocultures

Because LH/hCG receptor activation increases OGP synthesis [16, 28], the hypothesis that OGP mediates the hCG effect in cocultures was tested using an antisense strategy. The ODNs were synthesized from a bovine OGP sequence beginning at the ATG translation initiation codon.

As expected, oviductal epithelial cells showed OGP immunostaining (Fig. 6). This staining decreased to procedural control levels after treatment with the OGP antisense ODN. Treatment with the OGP sense ODN, however, had no effect.



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  		FIG. 6. OGP immunostaining in oviductal epithelial cells cultured for 7 days with ot without 5 µM OGP antisense and sense ODNs. Nonspecific IgG was used as the procedural control. x300

The ODNs were then used to determine the mediatory role of OGP in the hCG effect in cocultures. Although the addition of OGP ODNs alone had no effect, cotreatment with antisense, but not sense, ODN blocked the hCG effect on embryo development (Fig. 7).



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  		FIG. 7. Requirement of OGP for hCG to increase the development of two-cell bovine embryos cocultured with oviductal epithelial cells. Asterisks indicate significant differences (P < 0.05) compared with no additions

Signaling in hCG Action in Cocultures

Cyclic AMP/protein kinase A (PKA) signaling is involved in increasing OGP synthesis following LH/hCG receptor activation [16, 28]. Because the OGP response is required for the hCG effect, we tested the involvement of cAMP/PKA signaling itself in the hCG effect in cocultures. Although H-89, a PKA inhibitor, alone had no effect, cotreatment blocked the hCG effect in cocultures (Fig. 8). However, Bis, a PKC inhibitor, had no effect either alone or when combined with hCG.



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  		FIG. 8. Signaling in LH action to increase development of two-cell bovine embryos cocultured with oviductal epithelial cells. Asterisks indicate significant differences (P < 0.05) compared with no additions

LH Receptors in Oocytes, Embryos, and Blastocysts

The presence of LH receptor mRNA and protein was investigated by nonquantitative RT-PCR and indirect immunofluorescence and laser scanning confocal microscopy, respectively. The RT-PCR resulted in amplification of an LH receptor fragment of expected size from bovine oocytes, embryos, and oviducts used for a positive control (Fig. 9).



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  		FIG. 9. Ethidium bromide-stained gel showing the RT-PCR product of LH receptors (A) and GAPDH (B). Lane 1: 123-bp DNA ladder; lane 2: oocytes; lane 3: embryos; lane 4: oviduct

Indirect immunofluorescence and laser scanning microscopy revealed the presence of LH receptor protein in oocytes, 8- and 16-cell embryos, and blastocysts (Fig. 10). The immunofluorescence was absent in zona pellucida but was visibly concentrated on the oocyte cell surface and diffuse inside the cell (Fig. 10). The immunofluorescence increased and became patchy in 8-cell embryos, was uniform and diffuse in 16-cell embryos, and became patchy, diffuse, and granular in blastocysts (Fig. 10).



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  		FIG. 10. Indirect immunofluorescence and laser scanning confocal microscopy for LH receptors in bovine oocyte (A), 8-cell embryo (B), 16-cell embryo (C), and blastocyst (D). Procedural controls were run on oocytes (E) and blastocysts (F). x400


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Culture of two-cell embryos in medium alone resulted in slow growth. This growth significantly increased when 10 embryos/culture were used, which suggests that crosstalk becomes important when the threshold embryo number is reached. The crosstalk probably involves embryo-derived tropic factors that can act in an autocrine and paracrine manner to stimulate their own growth and development. There is also crosstalk between embryos and oviduct epithelial cells, and it requires >=10 embryos. This crosstalk may involve embryo-derived products that act on oviductal epithelial cells to stimulate their synthesis and release of embryotropic factors. The factors from both embryos and oviductal epithelial cells may decrease apoptosis and/or increase cell proliferation, both of which can result in an increased development of embryos [39, 40].

Although addition of hCG had no effect on embryos cultured in medium, it increased embryonic development in cocultures with >=5 embryos. The dependence of hCG effect on embryo number in the presence of oviductal epithelial cells suggests a collaboration between hCG and embryotropic factors to further enhance embryo development. The hCG effect was dose dependent. The lack of further increase in embryo development with 100 and 1000 ng/ml hCG suggests that higher hCG concentrations may induce a release of another set of factors that counter the effects of those released by low hCG concentration.

LH, which binds to the same receptors as hCG [41], mimicked hCG. The isolated {alpha} and ß subunits of hCG, which do not bind to LH receptors [41], had no effect, suggesting that the dimer hCG conformation is required. FSH and TSH had no effect even though oviducts contain FSH receptors [42]. This lack of effect suggests that FSH plays a role different from that of LH in oviductal functions.

Even though LH and hCG, and other hormones, function by activating their receptors, the receptor requirement in the context of cocultures was investigated by using LH receptor phosphorothioate ODNs. The antisense ODN was designed to inhibit LH receptor synthesis, whereas the sense ODN served as a control. In agreement with previous studies [8, 13, 16, 19, 24, 25], we found that oviductal epithelial cells contain LH receptors. Receptor immunostaining dramatically decreased following treatment with LH receptor antisense, but not sense, ODN. Human CG failed to increase embryo development in oviductal epithelial cells in which LH receptor synthesis was inhibited. This finding suggests that receptors are required for hCG to exert its effects in cocultures.

In view of the finding that LH treatment increases OGP synthesis [16, 28], which probably increases early embryonic growth and development [29–33], we tested the OGP requirement in cocultures treated with hCG by using an antisense approach. The phosphorothioate ODNs were synthesized from a bovine OGP sequence. The antisense, but not sense, OGP ODN treatment resulted in a dramatic decrease in OGP immunostaining, indicating a complete inhibition of its synthesis. Human CG failed to increase embryo development in oviductal epithelial cells in which OGP synthesis was inhibited, indicating that OGP mediated the hCG effect in cocultures. The OGP mediation does not necessarily exclude the potential contribution of other factors because they may work only when OGP is present. Thus, in its absence, these factors will not be effective in promoting embryo development.

The cAMP/PKA signaling pathway mediates LH and hCG action to increase OGP levels [16, 28]. Because OGP mediates hCG action, prevention of PKA activation should also block the hCG effect in cocultures, as does inhibition of OGP synthesis. As expected, H-89, which is a selective inhibitor of PKA activation, blocked the hCG effect. The H-89 effect is specific; Bis, which inhibits PKC, was ineffective.

Although there is some evidence that mammalian oocytes and preimplantation embryos contain LH receptors [43, 44], to our knowledge this is the first study demonstrating the presence of these receptors in bovine oocytes, embryos, and blastocysts. We do not know what roles these receptors might play in embryo development, but they probably played no role in the beneficial effect of hCG in cocultures. This hypothesis is based on the finding that hCG addition to embryos cultured in medium alone had no effect.

Our data support numerous studies demonstrating the slow development of embryos when cultured in medium alone [45–48], the stimulatory effect of oviductal epithelial cells in cocultures [34, 35, 49–55], and the interembryonic cooperation in promoting their own growth and development and in determining the responsiveness to external regulatory factors [47, 56]. The important new finding that emerged from our study was that LH and hCG treatment of cocultures can further increase preimplantation embryo development.

The present findings have important physiological and clinical implications. The physiological implication is that elevated periovulatory serum LH levels may promote preimplantation embryo development in the oviduct. The clinical implication is that hCG treatment of cocultures with reproductive tract epithelial cells, which contain LH/hCG receptors, may further increase pregnancy rates achieved using assisted reproductive technologies [53, 57–59].

This is the first study to demonstrate that not only bovine oviductal epithelial cells but also bovine oocytes, embryos, and blastocysts contain LH receptors. Activation of the oviductal epithelial cell receptors in cocultures resulted in increased development of embryos into blastocysts. This effect was dose dependent and hormone specific, required the presence of its receptors, used the cAMP/PKA signaling pathway, and was mediated by OGP.


   FOOTNOTES
 
1 This work was supported by NIH grant 1 R01 HD 31971. Back

2 Correspondence. FAX: 502 852 0881; cvrao001{at}gwise.louisville.edu Back

Received: 26 September 2002.

First decision: 22 October 2002.

Accepted: 1 November 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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