# Hormonal changes during meiotic maturation and ovulation in the brook trout (Salvelinus fontinalis)

Frederick W. Goetz, Alexis Fostier, Bernard Breton, Bernard Jalabert

► To cite this version:

Frederick W. Goetz, Alexis Fostier, Bernard Breton, Bernard Jalabert. Hormonal changes during meiotic maturation and ovulation in the brook trout (Salvelinus fontinalis). Fish Physiology and Biochemistry, 1987, 3 (4), pp.203-211. 10.1007/BF02180281. hal-02728152

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# Hormonal changes during meiotic maturation and ovulation in the brook trout (*Salvelinus fontinalis*)

Frederick W. Goetz<fnref n="1" />, Alexis Y. Fostier<fnref n="2" />, Bernard Breton<fnref n="2" />, Bernard Jalabert<fnref n="2" />

**Keywords:** estradiol-17β, 17α, 20β-dihydroxy-4-pregnen-3-one, gonadotropin, vitellogenesis, ovarian maturation

## Abstract

The plasma levels of estradiol-17β (E2), 17α, 20β-dihydroxy-4-pregnen-3-one (17,20-P) and gonadotropin (GTH) were measured in brook trout (*Salvelinus fontinalis*) during the period from the end of vitellogenesis to postovulation. Blood samples were taken according to specific stages of maturation, including germinal vesicle breakdown (GVBD) and ovulation. E2 levels were quite high (~ 45 ng/ml) at the end of vitellogenesis (and prior to GVBD) and dropped precipitously by GVBD (~ 2 ng/ml). They remained low through ovulation and postovulation. 17,20-P levels were low prior to GVBD (~ 0.7 ng/ml) and increased dramatically at GVBD (~ 148 ng/ml). The levels of 17,20-P remained high at ovulation (~ 142 ng/ml) and then dropped significantly within 24 h to approximately half of the ovulatory values. They decreased even further by 7 days postovulation. GTH levels rose gradually through GVBD and ovulation from a postvitellogenic level of approximately 3 ng/ml to a 7 day postovulatory value of approximately 10 ng/ml. The overall results; 1) decrease in estradiol prior to GVBD, 2) increase in 17,20-P at GVBD and 3) gradual GTH rise through GVBD and ovulation, are similar to those reported for other salmonids.

## Introduction

Over the last five years there have been many papers describing the changes in circulating steroids and gonadotropins in fish during the period from vitellogenesis until ovulation. Most of these investigations have been conducted on salmonids and major trends in the results of these studies appear reasonably similar. Circulating estradiol (vitellogenic hormone) is relatively high prior to final oocyte maturation and drops precipitously prior to germinal vesicle breakdown (GVBD). On the other hand, the levels of 17α, 20β-dihydroxy-4-pregnen-3-one (17,20-P: maturational hormone) are low prior to final maturation and begin to increase just prior to GVBD. They generally increase dramatically

during GVBD and may increase even further following ovulation. Gonadotropin levels increase gradually prior to ovulation and generally continue to increase postovulation. Although this general pattern of hormonal changes is very similar from one salmonid species to another, there are some subtle differences. The most evident of these is in the absolute level of steroids and gonadotropins measured. For example, reported peak levels of 17,20-P have varied from approximately 60 ng/ml in amago salmon (Young *et al.* 1983) to between 300 and 400 ng/ml in rainbow trout (Fostier *et al.* 1986; Scott *et al.* 1983; Springate *et al.* 1984). Also, the rate and duration of steroid changes have varied between species.

For several years, we have been investigating the

<footnote n="1">¹University of Notre Dame Department of Biological Sciences Notre Dame, Indiana 46556, USA</footnote>

<footnote n="2">²Laboratoire de Physiologie des Poissons Institut National Recherche Agronomique Campus de Beaulieu Rennes cedex, France</footnote>
hormonal control of oocyte final maturation and
ovulation in the brook trout (*Salvelinus fontinalis*)
using an *in vitro* incubation system. One of the ob-
jectives of the present study was to determine the
changes in circulating steroid and gonadotropin
levels prior to and during final maturation so that
they could be compared to results from other sal-
monid species and to our past *in vitro* results. Com-
pared to other salmonids, there have only been a
few reports concerning hormonal changes in mem-
bers of the genus *Salvelinus* (Kagawa *et al.* 1981;
Tam *et al.* 1986). Since this species is now being used
in the assay of acidic environmental conditions
(Tam and Payson 1986), it is important to describe
the basic biology of this species; particularly
reproduction.

A second important objective of this study was to
critically observe the hormone levels, especially
17,20-P, at the time of GVBD and ovulation.
Although a large number of papers have reported
on hormonal changes during the reproductive sea-
son, only a few of these have critically correlated
the changes with specific maturational stages (e.g.
germinal vesicle position, GVBD and ovulation). In
many past studies, blood samples have been taken
on a chronological basis which frequently will not
allow for strict correlations between hormone levels
and specific maturational stages. In the present
study, samples were obtained according to the at-
tainment of specific stages such as GVBD or ovula-
tion. We were particularly interested to see if there
was a consistent change in 17,20-P levels between
GVBD and ovulation. Past investigations in this
laboratory have studied the possible interactions of
prostaglandins in brook trout ovulation (Goetz *et
al.* 1982; Cetta and Goetz 1982; Goetz and Cetta
1983) and future investigations are directed at
studying the possible hormonal regulation of
prostaglandin synthesis by steroids and gonado-
tropins. Thus, it would be helpful to know more
about the basic hormonal events that occur in this
species during final maturation and ovulation.

Materials and methods

Animals

Female brook trout (300-400 g) were purchased
during the reproductive season from private fish
hatcheries in northern Wisconsin. They were held
under natural photoperiods in tanks supplied with
continuously flowing wellwater at 12.5°C. Oocyte
samples were obtained from fish by gentle pressure
applied to the abdomen as previously described
(Cetta and Goetz 1982). These oocytes were treated
in a fixative (Goetz and Bergman 1978) to deter-
mine germinal vesicle position within the oocyte. At
the time of the first blood sample (October 4, 1984),
fish contained ovaries in which the germinal vesicle
was approximately 4/5 the distance from the center
to the egg membrane. A total of nine fish were used
in the investigation and they were serially blood
sampled, relative to maturational stage, as des-
cribed below.

Blood sampling

Initial blood samples were taken from fish prior to
any extensive cytoplasmic clearing and before the
germinal vesicle had reached the egg membrane.
Following this, the ovaries of these fish were pe-
riodically monitored to determine the time of
GVBD, and a second blood sample was taken at
this time. Obtaining fish exactly at the time of
GVBD is extremely difficult. As a result, of the 9
fish in the experiment only 6 were sampled at exact-
ly this period. When it was determined that a fish
had undergone GVBD, it was examined at least
every 6 h to determine when ovulation occurred.
This was accomplished by applying slight pressure
to the abdomen to observe the extrusion of ovu-
lated eggs. A blood sample was taken when eggs
could be easily extruded from the ovipore and the
abdomen had softened. Although this ovulatory
criterion may be considered somewhat arbitrary,
we feel that, as a result of past experiments in-
volving similar sampling, in which fish were actual-
ly killed at each period (Cetta and Goetz 1982), this
time represents a point at which fish are just fully
ovulated or nearly so. This would not be considered
the beginning of ovulation which is extremely dif-
ficult to ascertain in salmonids without actually
killing the fish. Serial blood samples were also
taken 24 h and 7 days postovulation. In all post-
ovulatory samples, fish still contained ovulated
eggs in the body cavity. Very little spontaneous
oviposition could be observed up to 7 days postovu-
lation.

At each sampling time, a 1.0 ml sample of blood
was removed from the caudal vasculature using
a 1.0 ml syringe fitted with a 23 gauge needle.
Syringes were pre-rinsed in a 1,000 IU/ml heparin
solution. Blood was centrifuged at 1,500 g for 20
minutes at 4°C after which the plasma was removed
and stored at -20°C until assayed.

Hormone assay

For steroid assays, 400 µl of plasma was extracted
twice with 2.0 ml of a mixture of ethyl acetate:cyclohexane (1:1) and the extracts separated on
LH-20 according to the methods described by Fostier and Jalabert (1986). Radioimmunoassays
(RIA) of 17,20-P and estradiol-17β were performed
as described by Fostier et al. (1981) and Jalabert
and Fostier (1984). Cross-reactivities of various
steroids with the antisera used in the RIAs have
been described (Fostier and Jalabert 1986; Jalabert
and Fostier 1984).

Gonadotropin was measured by RIA using an
antibody directed against salmon gonadotropin.
The assay and its characteristics have been des-
cribed previously (Breton *et al.* 1978).

Analysis and statistics

RIA data was analyzed using a logit-log transfor-
mation and all potency estimates for steroids were
corrected for the procedural loss of material during
extraction and separation. Potencies given in the
text and figures are expressed as the mean ± SEM.
Potency estimates at various stages were analyzed
using a one-way ANOVA followed by Tukey's mul-
tiple comparison test.

Results

GVBD and ovulation

The first blood sample was taken on Oct. 4 (1984) at which time all fish contained oocytes with sub-peripheral germinal vesicles. The first fish to undergo GVBD did so on Oct. 7 while the last underwent GVBD on Oct. 28. The time from GVBD to ovulation varied between 1 and 2 days. For 6 fish, the exact time of GVBD could be determined (i.e. we were able to follow the germinal vesicle as it dispersed). For 5 of these fish we were reasonably sure that an ovulatory sample was taken within 6 h of the time of ovulation. For an additional 3 fish we were not able to determine the exact time of GVBD but were able to determine that they had undergone GVBD but had not yet ovulated. These fish were followed at 6 h intervals and we were reasonably certain that an ovulatory blood sample was taken from them within 6 h of ovulation. Hence, the sample size of 8 at ovulation and at 24 h postovulation (Figs. 1-3). One of the 8 fish died between the 24 h postovulatory sample and the 7 day postovulatory sample, hence the sample size of 7 for the last sample.

Estradiol-17β

Circulating estradiol levels were high (45.1 ± 5.8 (SEM) ng/ml) in fish containing oocytes with intact, subperipheral germinal vesicles (Fig. 1). At the time of GVBD the levels had dropped to 2.3 ± 0.5 ng/ml and remained at approximately this level through 7 days postovulation. Pre-GVBD levels were significantly (P < 0.05) higher than the mean levels at all other stages, but no other significant differences were observed.

17α, 20β-Dihydroxy-4-pregnen-3-one

Females sampled prior to GVBD had very low cir-
culating 17,20-P levels (0.7 ± 0.1 ng/ml-Fig. 2). At
GVBD the levels had increased significantly (P <
0.05) to a mean of 147.8 ± 22.9 ng/ml and at ovu-
<figure><img src="image_2.png" /><figcaption>Fig. 1. Circulating estradiol-17β levels in brook trout at different stages of maturation and ovulation. Stage 1 = prematurational; 2 = GVBD; 3 = ovulation; 4 = 24 h postovulation; 5 = 7 days postovulation. All values are means ± SEM for N fish. * indicates significant difference from all other means at P < 0.05.</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>Fig. 3. Circulating gonadotropin levels in brook trout at different stages of maturation and ovulation. Stages as in Fig. 1. All values are means ± SEM for N fish. * indicate significant differences at P < 0.05.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>Fig. 2. Circulating 17,20-P levels in brook trout at different stages of maturation and ovulation. Stages as in Fig. 1. All values are means ± SEM for N fish. Bars with similar * designation are not significantly different. All other comparisons significantly different at P < 0.05.</figcaption></figure>

<table><caption>Table 1. 17,20-P levels in brook trout females serially sampled at GVBD and within 6 h of ovulation</caption><thead><tr><th rowspan="2">Fish</th><th colspan="2">17,20-P (ng/ml)</th></tr><tr><th>GVBD</th><th>Ovulation</th></tr></thead><tbody><tr><td>1</td><td>92.5</td><td>217.5</td></tr><tr><td>2</td><td>124.1</td><td>149.3</td></tr><tr><td>3</td><td>202.0</td><td>150.5</td></tr><tr><td>4</td><td>85.1</td><td>111.4</td></tr><tr><td>5</td><td>218.7</td><td>97.9</td></tr></tbody></table>

lation the levels remained at approximately this level (142.0 ± 15.7 ng/ml). Of five fish in which a blood sample had been obtained exactly at GVBD and within 6 h of ovulation, the 17,20-P levels rose in 3 between GVBD and ovulation, and dropped in 2 of the fish (Table 1).

The levels of 17,20-P measured 24 h postovulation (65.2 ± 11.7 ng/ml) were significantly (P < 0.05) lower than those measured at GVBD or ovulation but were still significantly (P < 0.05) higher than pre-maturational levels. By seven days postovulation, 17,20-P levels (16.5 ± 8.9 ng/ml)
<table><caption>Table 2. Peak values of circulating 17,20-P in salmonids during natural reproduction</caption><thead><tr><th>Species</th><th>17,20-PG<br>(ng/ml)</th><th>Stage</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="4">Rainbow trout</td><td>320 (X)</td><td>GVBD</td><td>Fostier et al. (1986)</td></tr><tr><td>300-400</td><td>4 days prior to<br>ovulation</td><td>Scott et al. (1983)</td></tr><tr><td>400 (X)</td><td>ovulation</td><td>Springate et al. (1984)</td></tr><tr><td>113 (X)</td><td>postovulation</td><td>Liley et al. (1986)</td></tr><tr><td>Brook Trout</td><td>148/142</td><td>GVBD/ovulation</td><td>present paper</td></tr><tr><td>Chum Salmon</td><td>332 (X)</td><td>spawning population*</td><td>Ueda et al. (1984)</td></tr><tr><td>Coho Salmon</td><td>270</td><td>4 days prior to<br>ovulation</td><td>Van der Kraak et al. (1984)</td></tr><tr><td rowspan="3">Atlantic Salmon</td><td>70-80</td><td>GVBD</td><td>Fitzpatrick et al. (1986)</td></tr><tr><td>120-280</td><td>ovulation</td><td>Wright and Hunt (1982)</td></tr><tr><td>68 (X)</td><td>GVBD but prior<br>to ovulation</td><td>So et al. (1985)</td></tr><tr><td>Amago Salmon</td><td>50-70</td><td>GVBD/ovulation</td><td>Young et al. (1983)</td></tr><tr><td>Sockeye Salmon</td><td>74 (X)</td><td>GVBD-presumed</td><td>Truscott et al. (1986)</td></tr><tr><td>Masu Salmon</td><td>30-70</td><td>2-4 days prior to<br>ovulation</td><td>Yamauchi et al. (1984)</td></tr></tbody></table>

were not significantly different from prematurational ones, however at this time the levels were extremely variable ranging from a high of 51.9 ng/ml to a low of 0.7 ng/ml.

## Gonadotropin

Gonadotropin (GTH) levels rose gradually from a pre-GVBD level of 3.3 ± 0.4 ng/ml to a postovulatory (7 days) level of 9.9 ± 1.6 ng/ml (Fig. 3). GTH levels measured at 7 days postovulation were significantly (P < 0.05) higher than those measured prior to and during GVBD and ovulation. They were not significantly different from the levels measured 24 h postovulation (8.5 ± 0.9 ng/ml). In addition, although the mean GTH levels at GVBD (5.5 ± 0.5 ng/ml) and ovulation (5.4 ± 0.6 ng/ml) were higher than those prior to GVBD, this difference was not significant.

## Discussion

Levels of circulating steroids and gonadotropins during meiotic maturation and ovulation have been

reported in a number of species in the Salmonidae. A summary of some of these results, pertinent to this discussion, are given in Tables 2 and 3. From the results given for 17,20-P (Table 2), it appears that the absolute values of 17,20-P measured at GVBD or ovulation are greatest in rainbow trout in which levels are generally between 300 and 400 ng/ml and much lower in several other species including amago salmon (~60 ng/ml), masu salmon (30-70 ng/ml) and sockeye salmon (74 ng/ml). Thus, at levels of approximately 145 ng/ml, brook trout appear to be intermediate between these species. The absolute level of steroid measured may be a result of the degree of domestication of the trout stock (Liley et al. 1986). Liley et al. (1986) observed that the maximum level of 17,20-P measured in wild rainbow trout females was approximately 113 ng/ml just following ovulation. This is considerably lower than that reported for domestic rainbow stocks (see Table 2). However, the wild trout were able to spawn which is not usually the case in studies on domestic rainbow trout. Social stimuli may also have an effect on the absolute level of hormones (Liley et al. 1986). In our study on brook trout, females were held together but isolated from males. In addition, the tanks used to hold females

<footnote>* sample contained ovulated and nonovulated females; (X) = mean value.</footnote>
<table><caption>Table 3. Circulating levels of estradiol-17β at two stages of maturation in various species of salmonids</caption><thead><tr><th rowspan="2">Species</th><th colspan="2">Estradiol<br>(ng/ml)</th><th rowspan="2">Reference</th></tr><tr><th>Premat.*</th><th>OV or POV**</th></tr></thead><tbody><tr><td rowspan="6">Rainbow trout</td><td>~ 20</td><td>~ 2</td><td>Fostier et al. (1986)</td></tr><tr><td>~ 18</td><td>~ 2</td><td>Scott et al. (1983)</td></tr><tr><td>~ 60</td><td>30-2.8 (spawning)</td><td>van Bohemen and Lambert<br>(1981)</td></tr><tr><td>~ 50</td><td>~ 2</td><td>Scott and Sumpter (1983)</td></tr><tr><td>~ 48</td><td>~ 2</td><td>Scott et al. (1982)</td></tr><tr><td>~ 7</td><td>~ 2</td><td>Breton et al. (1983)</td></tr><tr><td rowspan="2">Brown Trout</td><td>~ 45</td><td>~ 2</td><td>present paper</td></tr><tr><td>~ 40</td><td>~ 5</td><td>Tam et al. (1986)</td></tr><tr><td rowspan="3">Coho Salmon</td><td>~ 16</td><td>1-2</td><td>van der Kraak et al. (1984)</td></tr><tr><td>3-6</td><td>2-3</td><td>Sower and Schreck (1982)</td></tr><tr><td>13-18</td><td>~ 3</td><td>Fitzpatrick et al. (1986)</td></tr><tr><td>Chum Salmon</td><td>~ 13</td><td>~ 5***</td><td>Ueda et al. (1984)</td></tr><tr><td>Atlantic Salmon</td><td>34</td><td>0.9</td><td>Stuart-Kregor (1981)</td></tr><tr><td>Sockeye Salmon</td><td>~ 21</td><td>~ 1</td><td>Truscott et al. (1986)</td></tr><tr><td>Masu Salmon</td><td>4-25</td><td>~ 1</td><td>Yamauchi et al. (1984)</td></tr><tr><td>White-spotted Char</td><td>~ 13</td><td>~ 2</td><td>Kagawa et al. (1981)</td></tr></tbody></table>

* time period varied from several days to one month prior to final maturation

** OV = ovulation and POV = postovulation

*** sample contained ovulated and nonovulated females

did not contain any gravel substrate and the females did not spontaneously release eggs.

The observation that 17,20-P levels were significantly elevated in brook trout at GVBD further supports a role for this steroid in the induction of meiotic maturation in this species. Past studies on this species have demonstrated that 17,20-P is the most potent maturational steroid when tested *in vitro* (Duffey and Goetz 1980), and its synthesis by brook trout ovarian tissue has been tentatively demonstrated (Theofan and Goetz 1983). The results presented here also clearly demonstrate that in brook trout 17,20-P levels are elevated at GVBD and ovulation and that there is not a consistent increase or decrease in the circulating levels of this progestagen between these two periods. In coho salmon there was also no consistent change in the level of 17,20-P from GVBD to ovulation (Fitzpatrick et al. 1986), while in Atlantic salmon, Truscott et al. (1985) suggested that there was a slight decrease from the time just following GVBD until ovulation, though it is doubtful that this was sta-

tistically significant. Regardless, in all investigations that have been reported so far, it cannot be precisely determined how long, following GVBD, that an ovulatory sample was taken. In several cases (Fostier et al. 1981; Fitzpatrick et al. 1986) serial samples were taken from fish at two or three day intervals and, therefore, 48-72 h may have elapsed between GVBD and ovulation. In other investigations the sampling periods were even wider apart or sampling was not done serially, making it impossible to determine how long, before the ovulatory sample, that the fish had undergone GVBD. Although we cannot say that our ovulatory sample was taken during the time of ovulation, because of our sampling regime it is possible to state that it was taken within 6 h of ovulation and this appears to be much closer to the time of GVBD than in any other investigation yet reported. Of course, the circulating levels may not accurately reflect synthetic changes that are occurring in the follicle, and these may be important in the stimulation of ovulation. Another interesting observation was that in brook
trout, 17,20-P levels quickly dropped following ovulation in fish that still retained ovulated eggs in the body cavity. In rainbow trout, the levels of 17,20-P also drop following ovulation (Fostier et al. 1981) but since they are at least twice as high at ovulation as in brook trout it appears to take a longer time for them to decrease.

As Table 3 indicates, estradiol levels are relativ-
ely high prior to GVBD in all salmonids while at the
time of ovulation they are generally low; 1-2
ng/ml. It has been suggested that the fall in estra-
diol is a consequence of a shift in the steroidogenic
pathway prior to GVBD (Fostier and Jalabert 1986)
and physiologically the decrease may be indirectly
involved in the control of oocyte final maturation
(Jalabert and Fostier 1984a,b). There appears to be
quite a range of prematurational values for estra-
diol levels, though this may be related more to the
exact time at which the sample was taken rather
than a species difference in absolute concentra-
tions. In brook trout the estradiol levels are very
high, even shortly before GVBD. For example, in
several fish that underwent GVBD within 9 days
following the first blood sample, the mean was 37.7
ng/ml. In contrast, from the literature it appears
that estradiol levels in rainbow trout at a similar
period are lower; in the range of 10-20 ng/ml
(Fostier et al. 1986).

The seasonal changes in estrogen levels in brook
trout have recently been reported (Tam *et al.* 1986).
Although these levels cannot be strictly related to
the stages reported here since the sampling was not
done on a stage basis, it does appear that near the
end of vitellogenesis the levels reach a maximum of
approximately 40 ng/ml (Tam *et al.* 1986). This
value is nearly the same as the one we obtained for
fish just prior to GVBD; presumably at a similar
stage. In addition, our post-GVBD value of ap-
proximately 2.0 ng/ml, is also similar to those
reported for postovulatory brook trout (Tam *et al.*
1986). From the data presented here it would be
assumed that in brook trout the estradiol levels
remain high until just prior to GVBD; inferring that
the drop is closely tied to final maturation.
However, Tam and coauthors (1986) reported that,
of brook trout obtained from two hatcheries, there
was a decrease in estrogen in brook trout obtained

from one of the hatcheries even though none of
them had ovulated. Although it was not explicitly
stated, it could be inferred from their figures that
the decrease in this case was from approximately 40
ng/ml to 25 ng/ml. It could be that these fish were
undergoing GVBD at just about the time that blood
sampling was conducted and this was the reason for
the decrease.

In the present study, gonadotropin levels in-
creased gradually during GVBD and ovulation and
reached peak values at seven days postovulation.
This has also been observed in several other
salmonids (Crim et al. 1975; Fostier et al. 1981;
Scott et al. 1983). It should be pointed out that even
though levels were greatest at 7 days postovulation,
they were also quite variable at this time, ranging
from a high of 15.1 ng/ml to a low of 4.0 ng/ml.
In rainbow trout, GTH levels continue to increase
following ovulation and can reach values as high as
100 ng/ml in fish still holding eggs in the body cavi-
ty and 50 ng/ml in fish spent immediately following
ovulation (Jalabert and Breton 1980). Truscott et
al. (1986) also reported GTH values of 90 ng/ml in
post-spawned sockeye salmon. It is possible that
higher values would have been observed in some
brook trout females if sampling was continued. In
addition, in rainbow trout, GTH levels decline
somewhat following ovulation and then increase
even further following this decline (Fostier et al.
1981). Of the 7 females sampled at 7 days postovu-
lation, GTH levels had begun to decline in 3 and
this might indicate that sampling may have occured
in a transient postovulatory trough. In comparison
to rainbow trout, the absolute values of GTH mea-
sured were lower in brook trout but this may have
been the result of using a RIA based on salmon
GTH. Using a RIA based on salmon gonadotropin,
Crim et al. (1975) also reported an increase in plas-
ma GTH in brook trout through the time of ovula-
tion. Although it is difficult to exactly compare the
two studies, it appears that the GTH levels mea-
sured were slightly higher in the earlier study by
Crim et al. (1975) compared to the present one.
## Acknowledgements

The authors gratefully acknowledge the technical assistance of Odile Marcuzzi and Elisabeth Sambroni.

This material is based upon work partially supported by a NATO Grant for International Collaboration (#85/0147) to FWG and AYF.

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