860 
Fishery Bulletin 96(4), 1 998 
(Johnson and Casillas, 1991; Fargo and Tyler, 1994). 
However, no information exists on other reproduc- 
tive steroids involved in oocyte development and 
spawning in female English sole, or on changes in 
reproductive parameters in male English sole dur- 
ing gonadal recrudescence. This study was designed 
to gain a better understanding of reproductive pro- 
cesses in both female and male English sole. This 
information can be used to assess the impact of en- 
vironmental degradation on their reproductive de- 
velopment and to develop effective techniques for flat- 
fish mariculture. It may also be useful in evaluating 
the reproductive status of English sole stocks that 
are a fisheries resource off the northwest coast of 
the United States. 
Wild populations of female and male English sole 
were sampled during the 1992-93 and 1994-95 
spawning cycles at two residential sites as well as at 
a known spawning ground in Puget Sound, Wash- 
ington. Several reproductive parameters, including 
gonadosomatic index (GSI), histologically determined 
stages of gonadal development, and steroid hormones 
(female: plasma testosterone [T] and E2; male: T and 
1 1-ketotestosterone [11KT] ) were monitored through- 
out the spawning season. Additionally, 17a, 20p- 
dihydroxy-4-pregnen-3-one (17a, 20(3-P), a potent 
maturation inducing steroid (MIS) in many teleosts 
(see review by Scott and Canario, 1987), was tested 
as the potential MIS in English sole. 
Materials and methods 
Chemicals 
Tritium-labeled steroids were purchased from 
DuPont NEN (USA), and Amersham (UK). The anti- 
bodies for E2 and T were purchased from G. 
Niswender (Colorado State University, United 
States). The 11KT antibody was a gift from Y. 
Nagahama (Japan), and the 17a, 20P-P antibody was 
a gift from A. P. Scott (United Kingdom). The stan- 
dards for the steroids were purchased from Stera- 
loids, Inc. (USA). 
Collection of samples 
The sampling times for fish were chosen to coincide 
with the period of gonadal recrudescence and spawn- 
ing in English sole (Ketchen, 1947; Harry, 1959; 
Johnson et al., 1991). Maturing female (>200 mm 
total length (TL)) and male sole ( >190 mm TL) were 
collected from Tulalip Bay (October through Janu- 
ary) and University Point (January through March) 
by otter trawl during the 1992-93 (October- 
March) and 1993-94 (October) spawning cycles. Ad- 
ditional sole were collected monthly at Pilot Point 
from February 1994 to December 1995. Tulalip Bay 
and Pilot Point (residential sites) were chosen for 
their large populations of English sole, and Univer- 
sity Point was chosen because sole migrate to this 
area for spawning (Johnson et al., 1991; Collier et 
al., 1992). 
Fish were kept in holding tanks aboard the re- 
search vessel for approximately one hour before sam- 
pling was carried out. The sole were weighed (to the 
nearest g) and measured (to the nearest mm), and 
blood ( 1 mL) was collected from the caudal vein with 
a heparinized syringe. Blood samples were centri- 
fuged for 10 minutes at 800 x g, and plasma was col- 
lected and stored in triplicate bullet vials (250 pL 
each vial) for subsequent analysis of reproductive 
steroids (females: E2,T, and 17a, 20(3-P; males: 11KT, 
and T). Immediately following blood collection, the 
fish were sacrificed by severing the spinal cord. The 
gonads were removed and weighed, and samples of 
gonadal tissue were collected and preserved in 
Dietrich’s fluid (Gray, 1954) for histological exami- 
nation. The abdominal contents were removed and 
the gutted carcass was weighed. Plasma samples 
were frozen and stored at -80 C until steroid analy- 
ses could be performed. 
Sample analyses 
Plasma T, E2, and 11KT levels were determined by 
radioimmunoassay (RIA) as described by Sower and 
Schreck (1982), and 17a, 20(3-P levels were deter- 
mined by RIA as described by Scott et al. ( 1982). Each 
RIA (E2, 11KT, and T, and 17a, 20(3-P) consisted of 
duplicates of standards (ranging from 0 to 2.0 ng / 
mL), samples, and quality control samples (pooled 
plasma from male or female English sole). Detection 
limits of the assays (20-80% binding range of the 
standard curve) were 0.006-0.3 ng/mL plasma for 
E2; 0.009-0.4 ng/mL for 11KT; 0.002-0.13 ng/mL for 
T; and 0.003-0.08 ng/mL plasma for 17a, 20(3-P. For 
each assay, samples with steroid concentrations out- 
side the detection limits of the assay were retested 
by adjusting the sample volume. All samples were 
corrected for dilution factor. Mean (±SE) of quality 
control samples were 4.4 ±0.55 ng/mL for E2 (n=15); 
0.89 ±0.12 ng/mL for 11KT (n= 6); and 0.29 ±0.04 ng/ 
mL for T (rc=16). The steroid 17a, 20(3-P was not de- 
tected in quality control samples. 
Gonadal tissues collected for histological exami- 
nation were embedded in paraffin, sectioned, stained 
with Harris hematoxylin and eosin-phloxine (Luna, 
1968) and examined by light microscopy. Ovarian 
developmental stage (Table 1) was classified accord- 
