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Urinary oestradiol and testosterone levels from novel male mice approach values sufficient to disrupt early pregnancy in nearby inseminated females

Department of Psychology, Neuroscience and Behaviour, McMaster University, Hamilton, Ontario, L8S 4K1, Canada

Correspondence should be addressed to D deCatanzaro; Email: decatanz{at}mcmaster.ca

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Previous research has established that exposure to novel male mice can disrupt intrauterine implantation of fertilised ova in inseminated females and that much of this effect is mediated by factors in the male urine. The present studies were designed to examine whether the steroid content of male urine is sufficient to account for this effect. Pregnancy was terminated by exogenous 17ß-oestradiol administered intranasally on days 2–4 after insemination in doses as low as 0.14 µg/day. Enzyme immunoassay indicated that male mouse urine reliably contains unconjugated 17ß-oestradiol and testosterone. A small but significant increase in the amount of urinary oestradiol was observed in males housed nearby previously inseminated females as opposed to those housed in isolation. This influence was absent in the sire and absent in novel males when the sire was also present. The quantity of active steroids in novel male urine approaches the level sufficient to account for the disruption of implantation in nearby inseminated females.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
In many mammals, urine carries pheromones, being deposited discretely as a social response and influencing the behaviour and reproductive state of conspecifics (Bruce 1960, Reynolds 1971, Hurst 1990). One established phenomenon is the Bruce effect, disruption of intrauterine implantation in inseminated females by exposure to novel males or their urine (Bruce 1960, Dominic 1965, deCatanzaro et al. 1996). Several studies indicate that this effect depends upon the urine chemistry of the novel males impinging on olfactory mechanisms in previously inseminated females (Parkes & Bruce 1962, Marchlewska-Koj 1981, Brennan et al. 1999, Zacharias et al. 2000). When applied directly to females, urine from males housed nearby other females is more potent than that from isolated males (deCatanzaro et al. 1999), which could correspond to androgen increases in males after exposure to females (Bliss et al. 1972, Macrides et al. 1975, Batty 1978, Bernstein et al. 1983).

Oestradiol and testosterone have been found in urine and faeces of both sexes in mice, while oestrone conjugate levels are relatively low in female excretions and undetectable in males (Muir et al. 2001, Vella & deCatanzaro 2001, deCatanzaro et al. 2003, 2004, Beaton & deCatanzaro 2005). More than any other known substance, exogenous androgens and oestrogens, especially 17ß-oestradiol, can disrupt intrauterine implantation (Burdick & Whitney 1937, Harper 1969, deCatanzaro et al. 1991, 2001). Accordingly, the present studies were designed to compare quantities of oestradiol in male urine to those sufficient to terminate pregnancy through direct nasal administration. Pregnancy outcome was examined following varied doses of exogenous 17ß-oestradiol given via nasal route in a small quantity of ethanol (deCatanzaro et al. 2001). Behavioural observations were conducted to document the contact of inseminated female mice with novel male urine in conditions matching those optimising the effect in this laboratory (see Table 1, extracting simple conditions from deCatanzaro et al. 1996, 2000, Spironello & deCatanzaro 1999, Zacharias et al. 2000). Urine was collected daily and non-invasively from novel males that were in olfactory and limited tactile contact with inseminated females and then analysed through enzyme immunoassay for testosterone and oestradiol. Measures from novel males were compared when they were housed alone or when they were in contact with inseminated females. Measures from sires were compared to those from novel males.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

Behavioural observation during male-induced pregnancy disruption
A two-tiered apparatus, as previously used (Table 1) and described (e.g. deCatanzaro et al. 1996), exposed an inseminated female (lower compartment measuring 30 x 21 x 13 cm) to two novel male mice (in two separate upper compartments each measuring 15 x 21 x 13 cm) with excretions passing through stainless steel wire grid (squares of 0.5 cm²) that also allowed limited behavioural interaction between the female and each male. An opaque Plexiglas (P + A Plastics Inc, Hamilton, Ontario, Canada) barrier separated males to prevent aggression. Each animal had independent access to food and water. The inseminated females and novel males were placed in this apparatus at the start of the dark phase of the lighting cycle on day 1 of pregnancy. They were remained there for 120 h and then were returned to normal housing. A total of six sets of animals (each with one female and two males) were examined. A Sony model TVR33 digital video camera equipped with an infrared camera-mounted spotlight was used to observe subjects during repeated h sessions during days 1–5 of pregnancy. Sessions started at about 1 , 4 and 6 h after the commencement of the dark phase of the lighting cycle. Video recordings were observed, with counts taken of discrete instances of female nasal contacts to a male’s nasal region, male’s genital region, a distinguishable male urine droplet or to the grid separation. Counts were also taken at instances of the female licking the grid and the apparatus walls. Counts were also taken of female nasal grooming, involving licking the fore-paws then rubbing the snout with them and of simple snout rubbing gestures.

On day 5, samples of animals’deposits on the apparatus walls were taken. For each cage, a cotton swab was dipped in 1 ml ethanol, then repeatedly wiped against the female’s compartment wall and a second swab was similarly wiped against the compartment wall of a stimulus male in the upper level. After the ethanol had evaporated, the cotton swab was rinsed in 1 ml ethanol, which was then extracted and analysed via enzyme immunoassay for 17ß-oestradiol as described below.

Intranasal steroid administration
Inseminated females, each housed alone in a standard cage, were anaesthetised lightly under isofluorane gas on each of days 2, 3 and 4 after sperm plug detection at 4 h in the dark phase of the lighting cycle. As the animal became immobilised, a micropipette tip was gently introduced into one nostril and administered a vehicle of 20 µl 100% ethanol with varied steroid content. Doses were 0.00, 0.07, 0.10, 0.14, 0.19, 0.24, 0.28, 0.43, 0.86 and 1.30 µg 17ß-oestradiol; sample sizes were 25, 9, 9, 9, 9, 8, 7, 6, 8 and 4 respectively. Otherwise females were undisturbed, then monitored for birth.

Non-invasive daily urinary steroid measurement in isolated males
Six males each were housed in a urinary collection apparatus made of clear Plexiglas (P + A Plastics Inc), measuring 15 x 21 x 13 (height) cm. There was a stainless steel wire-grid floor with open squares measuring 0.71 cm², raised approximately 1 cm above a clean flat Teflon-coated stainless steel surface, permitting excretions to collect on that surface. Daily urinary sampling commenced after 2 days acclimation and was conducted daily at the start of the dark phase of the lighting cycle. When housed alone, most male mice reliably delivered a small pool of urine around the onset of the dark phase in sufficient quantity for analysis. For some mice on some days, collection of a sufficient sample required monitoring up to 5 h after the onset of the dark phase. Sampling of about 500 µl fresh urine/day per subject was achieved without handling the subjects by lifting the apparatus away from the stainless steel floor, then aspirating urine from pools with a 1 cm³ syringe and 26 gauge needle with care to prevent contamination by faeces (deCatanzaro et al. 2004, Beaton & deCatanzaro 2005). Clean collection surfaces were substituted before new sample collection. The samples were stored at –20 °C.

Urinary sampling from novel males exposed to inseminated females
Each male was housed in one compartment of the urinary collection apparatus made of clear Plexiglas (P + A Plastics Inc), with two compartments each measuring 15 x 21 x 13 (height) cm divided by a vertical wire-grid partition (deCatanzaro et al. 2004). The apparatus had a stainless steel wire-grid floor raised above a clean Teflon-coated stainless steel surface as described above, with a strip of silicone below separating excretions from the compartments. Continuous access to food and water was provided in a corner of each compartment opposite to the grid wall, with the food source beyond a wire grid in a small closet to prevent food particles from contaminating excretions. Initially, each male was alone in the apparatus. After 2 days acclimation, collections of urine began just after commencement of the dark phase of the lighting cycle. When housed in social situations including an inseminated female, urine collection from males could be challenging, as they deposited urine in discrete droplets, most often emitted next to the grid separating the male from the female. However, usually there was a small pool deposited at the onset of the dark phase of the lighting cycle and subsequent droplets were collected to gain a sufficient sample for analysis within 6 h of that point. Each cage was placed above a clean tray and then urine was collected as described above. Immediately after this collection, 29 of the males had a previously inseminated female, on day 1 of pregnancy, placed in the opposite compartment of the apparatus. The other 17 males remained in the apparatus alone. After 3 days, each cage was again placed above a clean tray and the urine samples were again collected commencing just after the start of the dark phase of the lighting cycle.

Urinary collection from sires and/or novel males exposed to inseminated females
A urinary collection apparatus was divided into three rectangular compartments by vertical wire grid. One compartment measured 30 x 9 x 15 (height) cm. Two equal adjacent square compartments measured 15 x 15 x 15 cm such that each compartment had a 225 cm² interface through grid with each other compartment. Wire grid between the two square compartments had squares of 0.25 cm² to prevent aggression between males in these compartments, while that between each square compartment and the rectangular one had squares of 1 cm² to allow males to have limited interactions with the inseminated female. Each compartment had an outset closet away from the grid walls that provided continuous access to food and water. All compartments had wire-grid floor with squares of 0.5 cm² raised 1 cm above a Teflon-coated collection pan as described above. On day 1 of pregnancy, inseminated females were each placed in the rectangular compartment of an apparatus. They were randomly assigned to conditions, where (1) the sire, (2) a novel male or (3) both the sire and the novel male were placed in the adjacent compartments for days 1–8 of gestation, with the third compartment being empty in the first two conditions. Twenty males were prepared in each condition. Urine samples were obtained from the males after commencement of the dark phase of the lighting cycle on each day and were taken for 8 successive days. A clean tray was substituted just before each collection began.

Assay procedures
ELISA methods were previously validated (Munro et al. 1991, Muir et al. 2001, deCatanzaro et al. 2004). For 17ß-oestradiol, the interplate coefficient of variation (CV) was 8.4% at 30% bound and 4.1% at 70% bound and the intraplate CV was 8.7%. For testosterone, the interplate CV was 6.7% at 30% bound and 3.4% at 70% bound and the intraplate CV was 7.1%. Creatinine, 17ß-oestradiol and testosterone were obtained from Sigma Chemical. Antibodies to 17ß-oestradiol, testosterone and corresponding horseradish peroxidase conjugates were obtained from the Department of Population Health and Reproduction at the University of California, Davis, USA. Cross reactivities for anti-17ß-oestradiol are: 17ß-oestradiol 100%, oestrone 3.3%, progesterone 0.8%, testosterone 1.0%, androstenedione 1.0% and all other measured steroids <0.1%. Cross reactivities for anti-testosterone are: testosterone 100%, 5-dihydrotestosterone 57.4%, androstenedione 0.27%, androsterone and DHEA, cholesterol, 17ß-oestradiol, progesterone and pregnenolone <0.05%. The assay was carried out on NUNC (Roskilde, Denmark) Maxisorb plates, which were first coated with 50 µl antibody stock diluted 1:10 000 in a coating buffer (50 mmol/l bicarbonate buffer, pH 9.6) and stored for 12–14 h at 4 °C. Wash solution (0.15 mol/l NaCl solution containing 0.5 ml Tween 20 per litre) was added to each well to rinse away any unbound antibody and then 50 µl phosphate buffer/ well was added. The plates were incubated at room temperature for 2 h for 17ß-oestradiol and 30 min for testosterone before adding standards, samples or controls. For oestradiol, urine samples were diluted 1:8 in phosphate buffer before adding to the plate. For testosterone, urine was diluted 1:4 each in phosphate buffer. For each hormone, two quality control urine samples at 30 and 70% binding (the low and high ends of the sensitive range of the standard curve) were prepared. For all assays, 50 µl oestradiol or testosterone horse-radish peroxidase was added to each well, with 20 µl standard, sample or control for oestradiol or 50 µl standard, sample or control for testosterone. The plates were incubated for 2 h at room temperature. The plates were then washed and 100 µl substrate solution of citrate buffer, H₂O₂ and 2,2'-azinobis (3-ethylbenzthiazoline-6-sulphonic acid) was added to each well and the plates were covered and incubated, while shaking at room temperature for 30–60 min. The plates were then read with a single filter at 405 nm on the microplate reader (Bio-Tek Instruments Inc, Winnooski, VT, USA, EL 312E or ELX 808). Blank absorbance was subtracted from each reading to account for non-specific binding. In all assays, optical densities were obtained, standard curves were generated, a regression line fit and samples interpolated into the equation to get a value in picograms per well.

Due to variations in fluid intake and output, concentration of urine in experimental samples was adjusted for creatinine. Standard creatinine values of 100, 50, 25,12.5, 6.25 and 3.12 µg/ml were used with distilled water as zero. All urine samples were diluted 1:50 urine:phosphate buffer (0.1 mol/l sodium phosphate buffer, pH 7.0 containing 8.7 g NaCl and 1 g BSA/l). Using Dynatech Immulon (VWR International, Mississauga, Ontario, Canada) flat bottom plates, 50 µl/well of standard were added with 50 µl distilled water, 50 µl 0.75 M NaOH and 50 µl 0.4 M picric acid. The plate was then shaken and incubated at room temperature for 30 min. The plate was measured for optical density on a plate reader with a single filter at 490 nm. Standard curves were generated, regression lines were fit and the regression equation was applied to the optical density for each sample. Steroid measures were adjusted for creatinine by dividing the obtained value by the measure of creatinine per millilitre of urine for the particular sample.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Videotaped samples of the behaviour of novel males and inseminated females during days 1–5 of gestation (conditions match those of Table 1) indicated that males and females both showed behaviour exploring the new environment during the initial day, including many contacts between males and females. Through the next few days, novel males became increasingly agitated and remained so throughout the exposure, repeatedly depositing small drops of urine in the vicinity of females. Cage walls become progressively covered with males’ milky-white emissions. Females were observed repeatedly to investigate rather than avoid novel males and their urine and other excretions. Averages of 2.1 ± 0.5 nasal–nasal contacts, 2.4 ± 0.8 female nasal contacts with male genitals, 0.2 ± 0.1 female nasal contacts with recently deposited identifiable male urine droplets, 42.4 ± 12.1 female nasal contacts with the wire grid, 0.5 ± 0.1 instances of the female licking the cage walls and 2.2 ± 0.7 instances of the female licking the wire grid were observed per individual per hour over the 5-day exposure. In addition, the females were observed to groom their nasal regions on an average of 8.2 ± 1.0 times/h and to rub the nasal region 1.9 ± 0.9/h; many of the latter gestures occurred just after the female had sniffed some part of a male. Progressively over the time of exposure, all female nasal regions became distinctively yellowed from urine contact. Analyses of swabs from the apparatus walls showed a clear presence of 17ß-oestradiol, with readings of 3.22 ± 0.69 ng/ml ethanol solvent from the males’ compartments and 2.31 ± 0.69 ng/ml ethanol solvent from the females’ compartments.

Figure 1 shows pregnancy outcome from inseminated females administered varied doses of 17ß-oestradiol dissolved in ethanol to the nasal area under light isofluorane gas daily on days 2–4 of pregnancy. There was a full disruption of all pregnancies at doses over 0.25 µg/day, except for one subsequently cannibalised pup produced by one female at 0.43 µg/day, with a probabilistic disruption of pregnancy at lower doses, still reaching statistical significance at 0.14 µg/day, ²(1) = 4.03, P<0.05.

View larger version (13K): [in this window] [in a new window]   Figure 1 The number of pups delivered by inseminated females after they had received varied intranasal doses of 17ß-oestradiol in 20 µl ethanol daily on days 2–4 of pregnancy under light isofluorane gas anaesthesia. Where two or more values fall at the same point, points are slightly offset so that all data can be shown. Values near zero are zero. The inset shows the percentage of females that were parturient comparing subjects given 0.0, 0.14 and 0.19 µg/day.

View larger version (22K): [in this window] [in a new window]   Figure 2 Creatinine-adjusted values for 17ß-oestradiol (E2) and testosterone (T) in urine of six young adult males (80–100-days old) in 21 successive daily samples. These males were individually housed and without sexual experience and measures were taken non-invasively without handling animals.

View larger version (11K): [in this window] [in a new window]   Figure 3 Mean ( ± S.E.M.) creatinine-adjusted values for 17ß-oestradiol in urine of novel males taken when males were either alone or in the presence of previously inseminated females. In one condition, males were measured when alone, then re-measured alone; others were first measured alone, then after exposure to inseminated females across a wire grid during days 1–3 of pregnancy.

View larger version (21K): [in this window] [in a new window]   Figure 4 Daily mean ( ± S.E.M.) creatinine-adjusted values for 17ß-oestradiol and testosterone in urine of novel males and sires. All males were in the presence of inseminated females and repeated urinary samples were collected during days 1–8 of gestation. In the two panels on the left, sires of pregnancies are compared to novel males when each is housed alone with the inseminated female, but across a wire grid. In the panels on the right, the sire, a novel male and an inseminated female coexisted in a single apparatus with compartments separated by wire mesh grid.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

Data presented in Fig. 1 show that administration of three acute intranasal doses of 140 ng exogenous 17ß-oestradiol on days 2–4 of gestation (total intranasal dose of 420 ng) was sufficient to produce a significant pregnancy loss. Even lower doses may be effective when given systemically (deCatanzaro et al. 1991, 2001). In urine from novel males alone with females, removing adjustment for creatinine, our data indicate a range of individual sample values for males alone with females of 2–20 ng/ml 17ß-oestradiol and 3–160 ng/ml testosterone. The quantity of urine produced by male house mice depends upon age, housing and social conditions (Drickamer 1995). Dominant adult males were measured at 5.4 ml of urine/day (Drickamer 1995); outbred novel males in the present study were older on an average and probably larger than those in that study and they have been housed alone and had repeated mating experience, suggesting resemblance to dominant males (Brain 1975). Conceivably, daily urinary volume could be enhanced by arousal-induced polydipsia in novel males (Levine & Levine 1989), which might also help to account for some of the observed variation in creatinine values. Our Bruce effect paradigm involves exposure of each female to two males over 5 days (see Table 1), so we estimate that each female is potentially exposed to an average in the range of 300–450 ng oestradiol and 1800–2100 ng testosterone from male urine alone. Additional quantities of both steroids have been found in male faeces, where values of 6.7 ± 0.7 ng/ g oestradiol and 4.3 ± 0.4 ng/g testosterone were measured (Muir et al. 2001). We also found substantial quantities of oestradiol in the excretions deposited by the males on the apparatus walls. These are values for 17ß-oestradiol and testosterone alone, while other oestrogens could summate with this as could weaker effects of androgens, which convert metabolically to oestrogens (Harper 1969, deCatanzaro et al. 1991, 2001). A series of acute oestrogen and androgen exposures may be more important for disrupting implantation than the total cumulative dose. Precise comparison of steroid exposure from males and exogenous intranasal exposure is limited. Timing of exposure differs; exposure to male excretions is chronic over 5 days, whereas the intranasal oestradiol administration was constrained to three acute doses by the need to minimise handling and anaesthetic administration.

While implantation requires some oestrogenic action at the uterus (Harper 1992) and there may be a natural peak in oestradiol post-implantation (deCatanzaro et al. 2004), small elevations of oestrogens above optimal levels during implantation can clearly end pregnancy. Exogenous oestrogens at this point are established to alter the rate of flow of fertilised ova through the fallopian tubes (Burdick & Whitney 1937). They could also act at the hypothalamus, where minute amounts of oestrogens can induce oestrus (Pfaff 1980). No other pure natural constituents of male urine have been established to have such potent effects at such low doses. Brevicomin and thiazole, putative pheromones isolated from male urine (Jemiolo et al. 1986, Schwende et al. 1986), do not induce pregnancy loss when applied to inseminated female mice (Brennan et al. 1999). Many other evidences suggest a fairly direct role of androgens and oestrogens in the Bruce effect. Castrated novel males cannot disrupt pregnancy unless given replacement testosterone (Bruce 1965, Rajendren & Dominic 1988, Vella & deCatanzaro 2001). Exogenous antibodies to oestrogens can mitigate the Bruce effect (deCatanzaro et al. 1995a) as well as restraint stress-induced pregnancy disruption (deCatanzaro et al. 1994). Removal of androgen-dependent male glands, the preputials and vesicular-coagulating complex, does not diminish male capacity to disrupt pregnancy (deCatanzaro et al. 1996, Zacharias et al. 2000), but castration induces a loss in this capacity over 6 weeks in conjunction with urinary steroid decline (Vella & deCatanzaro 2001). Males that have just mated to two or more ejaculations do not induce the Bruce effect (Spironello & deCatanzaro 1999), corresponding to an established refractory period of a few days following repeated ejaculation (McGill & Blight 1963) and diminished plasma testosterone levels following ejaculation (Bliss et al. 1972, Batty 1978). Reduction in urinary oestradiol of novel males through a combination of an aromatase-inhibiting drug and a phytoestrogen-free diet attenuates male capacity to disrupt pregnancy (Beaton & deCatanzaro 2005).

Behaviour between novel males and females is important for transmission of urine from male to female (Hurst 1990, deCatanzaro et al. 1995b, 1996, 2000, deCatanzaro & Murji 2004). Sires tend to be passive in the presence of females that they have inseminated (deCatanzaro & Storey 1989, deCatanzaro & Murji 2004), but novel males become extremely agitated and direct urine at inseminated females, attempting to mount and intromit if not separated (deCatanzaro et al. 1996). Intense intermale aggression, often producing death if not stopped, results between novel males if they are not separated by barriers in these conditions (deCatanzaro et al. 1996, 2000). Whether or not males compete, most females in these circumstances lose pregnancy after exposure to significant quantities of novel male urine. Greater numbers of novel males (deCatanzaro et al. 1996) and males of a distinct strain (Parkes & Bruce 1962, Spironello & deCatanzaro 1999) produce a more robust effect. The effect requires female contact with male excretions, as it is not significant when males are housed below females, and it is diminished by manipulations such as additional wire grids that reduce female olfactory and tactile contact with the males themselves (deCatanzaro et al. 1996, deCatanzaro & Murji 2004). Females investigate rather than avoid novel males and the sire’s presence can actively (through aggression) and passively (when confined) prevent contacts between novel male and female and mitigate the Bruce effect (deCatanzaro & Murji 2004).

There is a longstanding suggestion that imprinting upon the odours of the sire and olfactory identification of novel males may be critical for the Bruce effect (Thomas & Dominic 1988, Kaba et al. 1994, Brennan et al. 1999). Mice have mechanisms of recognising conspecifics at some level (Bakker 2003, Brennan 2004) and female mice are better able to discriminate between signals of individuality than are male mice (Baum & Keverne 2002). Odours of individuality in mice are influenced by the major histocompatibility complex (Singh et al. 1987, Singer et al. 1997). Urinary proteins extracted from male urine can disrupt implantation when applied to inseminated females (Marchlewska-Koj 1981), but this may be due to appended smaller molecules. High molecular weight (HMW) proteins from male urine do not readily block pregnancy unless combined with low molecular weight (LMW) constituents from either familiar or unfamiliar males (Peele et al. 2003). These LMW constituents do not appear as bands using gel electrophoresis, suggesting that they are at an extremely low concentration or that they are not proteins. Female exposure to LMW urinary constituents alone from familiar or unfamiliar males results in a pregnancy disruption of 50–62% of subjects vs 100% for strange male HMW recombined with strange male LMW constituents (Brennan & Peele 2003, Peele et al. 2003). We suggest that androgens and oestrogens, especially 17ß-oestradiol at 272.4 Da, have very low atomic weight in comparison to HMW proteins and unlike large proteins, they are lipophilic and readily absorbed into the system via nasal or cutaneous exposure. A contribution of factors of individual recognition may still be supported by some influence of HMW proteins and the fact that novel males of a different strain can induce a greater Bruce effect than the female’s strain (Parkes & Bruce 1962, Marsden & Bronson 1965, Spironello & deCatanzaro 1999). The female endogenous adrenocortical androgens and oestrogens are implicated in the -vulnerability of intrauterine implantation to diverse stressors in various mammals (Harper 1969, deCatanzaro & MacNiven 1992, deCatanzaro et al. 1994). The female’s recognition of novelty plus the direct consequences of the assertive behaviour of novel males could stimulate female adrenocortical activity, which could synergise with exogenous androgens and oestrogens in the novel males’ excretions to produce the Bruce effect.

	Acknowledgements

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
This research was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) awarded to D deCatanzaro. We thank Arun Gupta, Krishna Nadella, Abid Hidayat, Ravjit Ahluwalia, Michelene Palumbo, Sandra Rodrigue and Cameron Muir for their assistance. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

	Footnotes

 
Received 15 September 2005
First decision 17 November 2005
Revised manuscript received 28 February 2006
Accepted 19 May 2006

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