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Endocrinology Vol. 139, No. 2 485-490
Copyright © 1998 by The Endocrine Society

ARTICLES

The Role of Prolactin in the Development of Reproductive Photorefractoriness and Postnuptial Molt in the European Starling (Sturnus vulgaris)1

Alistair Dawson and Peter J. Sharp

Institute of Terrestrial Ecology (A.D.), Monks Wood, Abbots Ripton, Huntingdon, Cambridgeshire PE17 2LS, United Kingdom; and Roslin Institute (Edinburgh) (P.J.S.), Roslin, Midlothian EH25 9PS, United Kingdom

Address all correspondence and requests for reprints to: Alistair Dawson, Institute of Terrestrial Ecology, Centre for Ecology and Hydrology, Abbots Ripton, Huntingdon, Cambridgeshire PE17 2LS, United Kingdom. E-mail: a.dawson{at}ite.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Seasonal breeding in many birds, including the European starling, is terminated by the development of absolute reproductive photorefractoriness, followed by a postnuptial molt, when photo-induced PRL secretion is at its seasonal maximum. To determine whether this photo-induced increase in PRL secretion has a causal role in the development of photorefractoriness or molt, European starlings were actively immunized against vasoactive intestinal polypeptide (VIP), the PRL releasing hormone in birds, or against PRL, during a photo-induced breeding cycle. In half of the VIP-immunized birds, the photo-induced increase in PRL was completely suppressed. Although these birds became photorefractory, the rate of gonadal regression was markedly slowed. These birds did not molt. In the remaining VIP-immunized birds, the photo-induced increase in PRL was inhibited but not completely suppressed. In these birds, and in those immunized against PRL, gonadal regression was also slowed, but molt progressed as normal. There were no significant differences in concentrations of plasma thyroxine between treatment and control groups, indicating that the effects of immunization on gonadal regression were not mediated by the induction of hypothyroidism. These results are consistent with the view that in the European starling the seasonal photo-induced increase in PRL accelerates gonadal regression during the onset of photorefractoriness but does not itself cause photorefractoriness. Further, the seasonal increase in PRL is required for the induction of the postnuptial molt.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AT TEMPERATE latitudes, seasonal breeding in many birds is terminated by the development of absolute reproductive photorefractoriness, followed by a postnuptial molt (1, 2). Absolute photorefractoriness is the condition in which the gonads regress in summer and early fall when days are longer than those that stimulated gonadal growth in spring. Gonadal recrudescence cannot be induced in birds exhibiting absolute photorefractoriness by a further increase in daylength and generally, the dissipation of this condition requires exposure to short days (1, 2). The development of absolute photorefractoriness involves a central nervous mechanism because it is characterized by a decrease in the activity of GnRH neurons (3, 4, 5, 6), resulting in a decrease in gonadotropin secretion. This occurs at a time when photo-induced PRL secretion is at its seasonal maximum (7, 8, 9). Because PRL is antigonadotropic/gonadal in birds (10, 11, 12) and may play a role in the regulation of molt (13, 14, 15), the seasonal increase in plasma PRL may play a role in the development of reproductive photorefractoriness and postnuptial molt.

This hypothesis has been difficult to investigate because, until recently, there has been no satisfactory method to suppress PRL secretion or block the actions of PRL in photostimulated birds. PRL secretion in birds is under stimulatory rather than inhibitory control as in mammals (16). For this reason, PRL secretion in birds cannot be suppressed by treatment with dopamine agonists such as bromocriptine (17). Two methods are now available to manipulate PRL in photostimulated birds. The first is active immunoneutralization of starling PRL. Recombinant-derived chicken PRL fusion protein has been used successfully in the bantam hen as an immunogen to block a physiological function of PRL, the initiation of incubation behavior (18). The amino acid sequence of chicken PRL is 89% homologous with European starling PRL (19), suggesting that the chicken PRL fusion protein should be suitable as an immunogen to block the biological actions of PRL in the starling.

The second method now available to manipulate PRL in birds stems from the identification of the physiologically relevant avian PRL releasing hormone as vasoactive intestinal polypeptide (VIP) (20, 21). Active immunization against VIP suppresses photo-induced PRL secretion in the turkey (22).

The aim of this study is to determine the role of PRL in the development of absolute photorefractoriness and postnuptial molt using a well established avian model: the European starling (2). The methodology employed involves active immunization against recombinant-derived chicken PRL or VIP during a photo-induced breeding cycle.

PRL has been shown to affect thyroid function in birds (23, 24, 25); hypothyroidism prevents the development of photorefractoriness and molt (26, 27, 28). Concentrations of plasma thyroxine were therefore measured in starlings immunized against PRL and VIP to assess whether any effect on the development of photorefractoriness or molt might be secondary to the induction of hyopothyroidism.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Juvenile starlings were caught from the wild during July and August and were kept in outdoor aviaries with food (chick starter crumbs) and water provided ad libitum. In late August, 40 males were moved indoors with four birds per cage under a light intensity of 400 lux at 18 C. Daylength was 12-h light, 12-h darkness per day, and this was reduced 2 weeks later to 8-h light, 16-h darkness. The experimental treatment began in mid-October, by which time the birds had finished molting into adult plumage and would have been fully photosensitive. Experiments were conducted in accordance with humane practice as specified by the UK’s Animal (Scientific Procedures) Act, 1986.

Antigen preparation
VIP. Two 15-amino acid polypeptides corresponding to the C-terminal and N-terminal sequences of chicken VIP (cVIP) (29) were synthesized on a Biosearch 9500 peptide synthesizer (New Brunswick Scientific Ltd., Watford, Hertsfordshire, UK) using solid-phase t-BOC chemistry, and the reagents and conditions recommended by the suppliers of the instrument. The peptide sequences were RKQMAVKKYLNSLVLT-NH2 (VIPC) and HSDAVFTDNYSRFRK-NH2 (VIPN). These were extended at the amino terminus by the addition of cysteine to allow them to be conjugated through the thiol group of cysteine to the purified protein derivative (PPD) of tuberculin (Central Veterinary Laboratory, Weybridge, Surrey, UK). Conjugation was carried out using sulfosuccinimidyl H-(maleimidomethyl) cyclohexane-1-carboxylate following the manufacturer’s protocol [Pierce and Warrener (UK) Ltd., Chester, UK].

PRL. A recombinant-derived chicken PRL fusion protein (PRL-ß-galactosidase) was prepared as described by March et al. (18).

Treatment groups and immunization protocol
Birds were allocated at random to five groups of eight. One group comprised nonimmunized controls. The other four groups were immunized starlings, with immunization starting with an intramuscular priming vaccination into the pectoral muscle with bacillus Calmelte-Guerin vaccine (0.1 ml, Evans Medical Ltd., Leatherhead, Surrey, UK) followed at 3-week intervals with im injections of VIPC/VIPN-PPD 1:1 (VIP-PPD group), PPD (control immunogen; PPD group), PRL-ß-galactosidase (Prl-Gal group) or ß-galactosidase (control immunogen; Gal group). Each bird was injected with a total of 4 mg conjugate emulsified in Freund’s incomplete adjuvant (Sigma, Poole, UK). To make the emulsions easier to inject, they were homogenized by sonication with an equal volume of physiological saline containing 2% Tween 80 (Sigma). The final volume was 250 µl, which was injected into four sites in the pectoral muscles. The first antigen injection was done 7 weeks before birds being transferred to long days, and this was repeated every 3 weeks until 14 weeks after transfer to long days.

Experimental procedure
Two weeks before transfer to long days, a blood sample was collected from each bird. About 500 µl blood was collected into heparinized capillary tubes after pricking a wing vein. The blood was centrifuged and plasma stored at -20 C until assayed. At week 0, another blood sample was collected from each bird and the birds were laparotomized. They were anaesthetized with an im injection of 70 µl Sagatal (Pentobarbitone Sodium, 60 mg·liter-1; Veterinary Drug Co. plc., York, UK), and an incision was made between the last pair of ribs. The dimensions of the left testis were measured to the nearest 0.5 mm. Testicular volume was calculated as 4/3{pi}a2b, where a is half the diameter at the widest point and b is half the long axis. Daylength was increased from 8-h light, 16-h dark to 18-h light, 6-h dark (mid-winter to mid-summer daylength at 52°N). Further blood samples were taken after 1, 2, 3, 4, 6, 8, 10, and 13 weeks. Testicular dimensions were recorded for all birds after 2, 4, 6, 9, 12, and 16 weeks. The progress of molt of the primary feathers was recorded. The primaries are the flight feathers on the outer part of the wing. The time taken for the primaries to be molted spans the period of molt of all of the other feathers. A dropped primary feathers scored 1, one-quarter, half, three-quarter, and fully regrown feathers scored 2, 3, 4, and 5, respectively, so that when all 9 primary feathers had fully regrown the molt score was 45. Plasma samples were assayed for PRL (except for birds immunized against PRL, in which endogenous antibodies would make results of RIA meaningless), VIP and PRL antibody titers and thyroxine.

PRL RIA
PRL was measured using a recombinant-derived starling PRL RIA as described by Bentley et al. (30). Recombinant-derived starling PRL was used as the standard, for the radiolabeled tracer and to raise the antiserum. The sensitivity of the assay was 0.09 ng/tube. The intraassay and interassay coefficients of variation were 7.5% and 16.3%, respectively. Duplicate 20-µl plasma samples were assayed, and samples with high values were re-assayed at 10 µl.

Measurement of VIP and PRL antibody titers
Plasma was measured for the presence of VIP or PRL antibodies using a modified enzyme-linked immunosorbent protocol. Recombinant-derived starling PRL (50 ng), or chicken VIP (50 ng; Peninsula Laboratories, St. Helens, Merseyside, UK), in 50 µl 0.1 M carbonate buffer (pH 9.5) was added to each well in 96-well microtiter plates and allowed to dry. Each well was then filled with 200 µl block solution, and the plates left for at least 2 h. The block solution, required to eliminate nonspecific binding, was made by adding 5 g dried skimmed milk powder to 100 ml TBST (5 mmol Tris-HCl·liter-1 pH 7.5, 200 mmol NaCl·liter-1, 0.05% (vol/vol) Tween 20). Wells were aspirated, and 100 µl of starling plasmas, diluted 1:64, were added. After 2 h, the plates were washed five times in TBST, followed by the addition of 100 µl of rabbit antichicken IgG conjugated to horseradish peroxidase (Sigma) diluted 1:2000 in block solution. Peroxide activity was measured using O-phenylenediamine (Sigma) as a substrate. Plates were read using a Multiplate Reader at 490 nm.

Thyroxine RIA
Plasma thyroxine was measured using a RIA as described by Bentley et al. (30). The sensitivity of the assay was 0.2 nmol·liter-1 and 2.0 nmol·liter-1 depressed binding by 50%. The intra and interassay coefficients of variation were 6.3% and 10.6%. respectively. Duplicate 20-µl plasma samples were assayed.

Statistical analyses
Data were normalized by log transformation before analysis. For analysis of changes within groups with time, single factor ANOVA with repeated measures was used, followed, where significant, by Dunnet’s multiple comparison tests, comparing values with those at week 0. For analyses between groups, single factor ANOVA was used, followed by Tukey’s multiple comparison tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Among the birds immunized against VIP-PPD, four molted and four did not. Because this was a clear qualitative difference in a response in which PRL was thought to play a role, these two groups were treated separately in all analyses. They were designated VIP-PPD (a), those that did not molt, and VIP-PPD (b), those that did molt. Immunization against ß-galactosidase had detrimental effects. Four of the Prl-Gal group died and one of the Gal group died. Data refer only to those birds that survived.

VIP and PRL antibody titers (Fig. 1Go)
Antibody titers were measured after 0, 3, and 8 weeks of photostimulation. Of the birds immunized against cVIP-PPD, the VIP-PPD (a) group had consistently higher titers of antibodies that bound cVIP than the VIP-PPD (b) group (0 weeks ns; 3 weeks P < 0.05; 8 weeks P < 0.001). The VIP control birds immunized against the carrier protein PPD, or not immunized, had antibody titers at the detection limit of the assay. The birds immunized against cPrl-Gal had high titers of antibodies that bound stPrl. The nonimmunized PRL control birds had no antibodies against stPrl, but those immunized against Gal alone showed low nonspecific binding.



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  		Figure 1. Titer of antibodies against VIP (top panel) or PRL (lower panel) in European starlings at 0, 3, and 8 weeks after transfer from short days (8-h light/day) to long days (18-h light/day). The group VIP-PPD (a) was four birds immunized against VIP-PPD in which the photo-induced increase in PRL was completely suppressed and which did not molt. The group VIP-PPD (b) was four birds immunized against VIP-PPD but in which the photo-induced increase in PRL was only partially suppressed and in which molt was unaffected. The group Prl-Gal was the four surviving birds (four died) immunized against PRL-ß-galactosidase. The group PPD (n = 8) and Gal (n = 7) were immunized against control immunogens. Controls (n = 8) were not immunized. Each point represents the mean ± SE.

 
Plasma PRL (Fig. 2Go)
Plasma PRL was low before photostimulation in all groups immunized against VIP. In nonimmunized control birds, PRL increased significantly (P < 0.01) 1 week after transfer to long days, reached peak values between 4 and 8 weeks, and then decreased. In the control birds immunized against the carrier protein PPD, plasma PRL increased significantly (P < 0.01) after 1 week, reached a peak at 4 weeks, and then decreased. PRL decreased more rapidly (although not statistically) in these birds than in nonimmunized controls. The reason for this is unclear, but it may have been a nonspecific effect of immunization on PRL release. In the VIP-PPD (a) birds, which had the highest VIP antibody titers, plasma PRL remained low after photostimulation. In the VIP-PPD (b) birds, PRL increased (P < 0.05 after 1 week; P < 0.01 after 2 weeks) but was lower than in the control groups. Values in these birds were consistently significantly (P < 0.05) higher than in the VIP (a) group, from 2 weeks onwards. PRL could not be measured in birds immunized against PRL.



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  		Figure 2. Changes in plasma PRL concentration in starlings transferred from short days (8-h light/day) to long days (18-h light/day) and immunized against VIP (VIP-PPD (a) n = 4; VIP-PPD (b) n = 4), the VIP carrier protein PPD (PPD n = 8) or not immunized (controls n = 8). Each point represents the mean ± SE.

 
Testicular volume and bill color (Fig. 3Go)
In the three control groups (nonimmunized, PPD-, and Gal-immunized) changes in testicular volume were identical. Testicular size increased rapidly during the first 2 weeks of photostimulation, reached the maximum between 2 and 4 weeks, and by 6 weeks the testes had regressed. In the Prl-Gal and VIP-PPD (b) groups, testicular growth rate over the first 2 weeks was the same as in the controls. In the VIP-PPD (a) group, initial testicular growth rate was slower than in the controls (P < 0.01) and slower than in the VIP-PPD (b) group (P < 0.05). Testicular regression was slower in both VIP-PPD groups and the Prl-Gal group than in the controls. At 6 weeks, testicular volume was significantly (P < 0.001) higher in VIP-PPD (a), VIP-PPD (b), and Prl-Gal groups than in any of the control groups. Testicular regression was particularly slow in the VIP-PPD (a). Testicular volume in VIP-PPD (a) birds was significantly greater than in controls at 6, 9 (P < 0.001), and 12 (P < 0.01) weeks, and greater than in VIP(b) at 6 (P < 0.05), 9 (P < 0.001), and 12 (P < 0.01) weeks. By the end of the study, the testes of both VIP-PPD groups and the Prl-Gal group had regressed and were not significantly different from controls.



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  		Figure 3. Changes in volume of the left testis in starlings transferred from short days (8-h light/day) to long days (18-h light/day) and immunized against VIP (VIP-PPD (a) n = 4; VIP-PPD (b) n = 4), the VIP carrier protein (PPD n = 8), PRL (Prl-Gal n = 4), the PRL carrier protein (Gal n = 7) or not immunized (controls n = 8). Each point represents the geometric mean ± SE. Where error bars are not shown, they were smaller than the symbol. To aid comparisons, the data for the nonimmunized controls are shown as a broken line in each panel. Significant differences between treatment groups and the nonimmunized controls are shown: **, P < 0.001; *, P < 0.01. Testicular volume in VIP-PPD(a) birds was significantly greater than in VIP (b) at 6 (P < 0.05), 9 (P < 0.001), and 12 (P < 0.01) weeks.

 
In European starlings, the increase in circulating testosterone as birds become photosensitive causes the bill to change color from black to yellow (31). The bills of all birds were yellow at the beginning of the experiment. They reverted to black during the period of testicular regression. In the VIP-PPD (a) group, the slower rate of testicular regression was reflected in a delay in the return of black coloration.

Molt (Fig. 4Go)
Molt began 6 weeks after transfer to long days and was completed by 18 weeks in all control groups. The Prl-Gal birds and three of the VIP-PPD (b) birds also molted at the same time and rate. One of the VIP-PPD (b) birds (B01) dropped only two primary feathers and regrew one of them, so ending with a molt score of 6. None of the VIP-PPD (a) dropped any feathers.



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  		Figure 4. Progression of molt of primary feathers in starlings transferred from short days (8-h light/day) to long days (18-h light/day) and immunized against VIP (VIP-PPD (a) n = 4; VIP-PPD (b) n = 4), the VIP carrier protein (PPD n = 8), PRL (Prl-Gal n = 4), the PRL carrier protein (Gal n = 7) or not immunized (controls n = 8). Each point represents the mean ± SE. Where error bars are not shown, they were smaller than the symbol. B01 was one of the 4 VIP-PPD (b) birds; it dropped only two primary feathers and regrew one of them, so ending with a molt score of 6.

 
Plasma thyroxine (Fig. 5Go)
The immunization procedures did not result in the development of hypothyroidism. Plasma thyroxine increased immediately following photostimulation. This was marginally significant (P < 0.05) in all groups except the VIP-PPD (a) group, in which it was not quite significant. The magnitude of the increase in the VIP-PPD (a) group was the same as that in the controls. There were no significant differences between any groups.



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  		Figure 5. Changes in plasma thyroxine concentration in starlings transferred from short days (8-h light/day) to long days (18-h light/day) and immunized against VIP (VIP-PPD (a) n = 4; VIP-PPD (b) n = 4), the VIP carrier protein (PPD n = 8), PRL (Prl-Gal n = 4), the PRL carrier protein (Gal n = 7) or nonimmunized (controls n = 8). Each point represents the mean ± SE.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study establishes, for the first time, that the seasonal peak of photo-induced PRL secretion associated with the development of avian photorefractoriness accelerates gonadal regression but is not required for the eventual development of photorefractoriness. This conclusion is consistent with the finding that the extent to which active immunization against VIP suppressed PRL secretion was directly related to the rate of gonadal regression. The extent to which PRL secretion was depressed by active immunization against VIP could be readily explained by the levels of VIP antibody titers. The finding that immunization against chicken PRL also slowed the rate of gonadal regression supports the view that the effects of immunization against VIP on gonadal regression is mediated by the suppression of PRL secretion and not by some other mechanism.

The finding that active immunization against VIP was more effective than active immunization against chicken PRL in slowing the rate of development of photorefractoriness may be explained in two ways. First, the small differences in the amino acid sequence of chicken and starling PRLs may be sufficiently significant to reduce the ability of the chicken PRL antibody generated in the starling to block the biological activity of endogenous starling PRL. Secondly, it is possible that the small amounts of VIP in the hypophysial portal vasculature (32) are easier to immunoneutralize than the relatively larger amounts of PRL in the peripheral circulation (32).

The mechanisms by which the immunomodulated reduction in PRL function slowed the development of photorefractoriness remains to be established. This study eliminates the possibility that the reduction in the circulating PRL or immunoneutralization of endogenous PRL may have compromised thyroid function (see Introduction) rendering the birds hypothyroid. In the starling, hypothyroidism prevents the development of photorefractoriness (28, 33). Because the concentrations of plasma thyroxine in VIP- and PRL-immunized starlings were the same as in control birds, the delaying effect of reduced PRL function on the development of photorefractoriness was not mediated by a reduction in thyroid function.

The site of action of PRL in accelerating the development of photorefractoriness could be at all levels of the hypothalamo-pituitary-gonadal axis. For example, in the chicken, systemic administration of PRL depresses plasma LH (34) and in the turkey, suppresses hypothalamic GnRH-I and -II content (35), suppresses LH ß-subunit gene expression (12), and inhibits the steroidogenic action of LH (10).

The finding that immunosuppression of photo-induced PRL secretion did not prevent the development of photorefractoriness is consistent with other studies in birds. In turkeys (36), starlings (37), and white crowned sparrows (38), a further increase in PRL secretion is superimposed on the long day-induced increase during incubation. This increase is associated with ovarian regression but does not induce photorefractoriness. In all these birds, a further cycle of egg laying and incubation may be induced providing they have not been exposed to long days for too long (36, 37, 38). As in starling, there is also evidence in seasonally breeding mammals that photo-induced seasonal changes in PRL secretion are not essential for gonadal growth and regression (39). For example, in the long day-breeding hamster, suppression of plasma PRL concentration by treatment with bromocriptine during the period following transfer from short to long days, delays but does not prevent testicular recrudescence (40). Although the starling is also a long day-breeder, it has been argued that the termination of breeding, i.e. the development of photorefractoriness, was more analogous to the long day-induced inhibition of breeding in short day-breeding mammals (2). In some short day-breeding mammals, there is evidence, as in the starling, for a modulatory role for PRL in the timing of seasonal gonadal regression. Thus, administration of bromocriptine in spring to suppress photo-induced PRL secretion delays long day-induced gonadal regression in the blue fox (41) and red deer (42).

Molt in birds is dependent upon, and inducible by, thyroid hormones and is inhibited by oestrogen and testosterone (15, 43). The finding that immunosuppression of photo-induced PRL secretion had no effect on the concentration of plasma thyroxine eliminates the possibility that the associated inhibition of the postnuptial molt was secondary to a depression in thyroid function. However, immunosuppression of photo-induced PRL secretion slowed photo-induced testicular regression, suggesting that the associated prolonged maintenance of androgen secretion, marked by the retention of the yellow coloration of the bill, may have suppressed postnuptial molt. This explanation for the inhibition of the postnuptial molt would be entirely satisfactory if the delayed regression of the testes observed in starlings with immunosuppressed PRL secretion was associated with a delayed postnuptial molt. This was not observed. Birds either molted normally or did not molt at all. It is therefore possible that the immunosuppression of photoinduced PRL secretion inhibited the postnuptial molt by removing a direct stimulatory action of PRL. This conclusion is consistent with the observation that the administration of PRL to castrated cockerels readily induces molt (13). But it is difficult to reconcile this finding with the observation that treatment of hens with PRL during a pause in egg laying, induced by short days and starvation (36), partially inhibited the subsequent long day-induced molt. A direct role for PRL in the induction of the postnuptial molt is consistent with the observations of blue fox (41), red deer (42), and hamster (44) showing that treatment with bromocriptine to prevent photo-induced PRL secretion, delays or prevents the spring molt.

In the discussion so far, we have assumed that the effects caused by VIP-immunoneutralization result from suppression of the photo-induced increase in PRL secretion. It remains possible that VIP acts directly, or through some mechanism other than PRL, to control gonadal regression or molt. However, this seems less likely. A direct systemic effect of VIP is unlikely because concentrations of VIP in peripheral circulation are low (32), and although VIP is a putative mediator between the photoperiodic signal and GnRH output (45), encephalic VIP is unlikely to have been neutralized by a peripheral immune response.

In conclusion, these observations demonstrate that the normal rate of gonadal regression during the onset of photorefractoriness in the European starling is dependent upon high concentrations of circulating PRL. They also suggest a role for PRL in the induction of the postnuptial molt. However, photo-induced PRL secretion is not required for the eventual onset of photorefractoriness.


   Acknowledgments
 
We thank Ms. C. Howarth for cloning the starling PRL, Mr. N. S. Huskisson and Mr. M. Quigley (Microchemical Faculty, Babraham Institute, Babraham, Cambridge, UK) for preparation of the VIP-PPD conjugates, Mr. P. Wilson for assistance with the immunization procedure and with the PRL and antibody titer assays, and the National Hormone and Pituitary Program for the gift of NIADDK-ovine PRL-S18.


   Footnotes
 
1 This research was supported by NERC and BBSRC Core Strategic Grants Back

Received July 8, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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