Sol et a I.: Gonadal development and changes in plasma reproductive steroids in Pleuronectes vetulus 
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Table 1 
Classification scheme for female ovarian developmental 
stages, modified from histological criteria outlined in 
Johnson et al. (1991). 
Stage 
Features 
Regressed 
Primary and secondary oocytes 
Previtellogenic 
Vacuolated secondary oocytes with 
clear peripheral vacuoles; zonal ra- 
diata present 
Vitellogenic 
Yolked oocytes present 
Vitellogenic with 
Yolked globules coalescing; hydrated 
hydrated oocytes 
oocytes present, no postovulatory fol- 
licles (POFs) 
Spawning 
Hydrated oocytes with POFs 
Spawned out 
Many POFs visible, yolked oocytes 
undergoing resorption, and inflam- 
matory infiltrate, beta or gamma 
atretic follicles or macrophage aggre- 
gates (or both) generally present 
ing to the criteria modified from Johnson et al. (1991). 
Testicular developmental stage was classified as 
modified from Billard ( 1992). GSI was calculated by 
using the formula GSI = (gonad weight (g) /gutted 
weight (g)) x 100. 
only spermatogonia; in early recrudescence, primary 
and secondary spermatocytes begin to appear; in the 
late recrudescent stage spermatogonia, spermato- 
cytes (primary and secondary), and spermatids are 
evident, but no spermatozoa; in early spermiogen- 
esis primarily spermatocytes (primary and second- 
ary), spermatids, and early sperm production are 
evident; in the spawning stage predominately ma- 
ture sperm fill the tubular space and sperm ducts; 
in the spawned-out testis, the tubular space and sperm 
ducts are largely empty and few sperm remain. 
Size at sexual maturation 
The size of English sole captured in this study ranged 
from 212 to 455 mm for females and from 195 to 345 
mm total length for males. Histological analyses of 
the gonads showed that a majority of the female sole 
<260 mm TL were immature (regressed-previ- 
tellogenic), and failed to reach vitellogenesis (Table 
2). Male sole <230 mm reached spermatogenesis but 
most did not fully mature and spawn (Table 3). Fe- 
male sole <260 mm were excluded from further sta- 
tistical analyses, but no size limit was used for male 
sole. 
Proportions of fish undergoing 
gonadal maturation and spawning 
Statistical analysis 
Mean (±SE) GSI and plasma reproductive steroid 
levels were calculated by month and gonadal matu- 
ration stage. Analysis of variance (AN OVA) and sub- 
sequent multiple comparison testing by Fisher’s pro- 
tected least significant difference (PLSD) test (95% 
Cl), were used to test for changes in these factors 
during the reproductive cycle (Dowdy and Wearden, 
1991). The data was normalized by log transforma- 
tion prior to statistical comparisons. 
Results 
Classification of testis 
The testicular developmental stages for male English 
sole are shown in Figure 1, A-F. The testis consists 
of tubules of reproductive cells in various stages of 
development. Testicular development is categorized 
into six stages: regressed (Fig. 1A), early recrudes- 
cence (Fig. IB), late recrudescence (Fig. 1C), early 
spermiogenesis (Fig. ID), spawning (Fig. IE), and 
spawned out (Fig. IF). Each stage is characterized 
as follows: in the regressed stage, tubules contain 
Female Figure 2 shows the proportion of female sole 
at different ovarian developmental stages by month. 
Even among adult sole (>260 mm), regressed females 
were found all year, and the mean proportion of the 
regressed animals each month was 24% (±11%, SD). 
Developing females were found as early as July; 14% 
of the female sole sampled in July were vitellogenic. 
The proportion of vitellogenic sole generally increased 
until January (78%), then decreased in the follow- 
ing months; by March only 11% of the sole sampled 
were vitellogenic. Spawned out females were found 
as early as October (1%), and the proportion in- 
creased through March (31%). 
Male Figure 3 shows the proportion of male sole at 
different testicular developmental stages categorized 
by month of capture. Animals collected in July con- 
sisted of regressed males (12%) and early develop- 
ing males (25% each were at early and late recru- 
descence), and males in early spermiogenesis (38%). 
Spawning males were found as early as October 
(16%), and the proportion generally increased until 
February (91%). By March, 41% were spawning and 
36% were spawned out. Unlike females, in which a 
certain proportion of fish did not develop during the 
reproductive cycle, almost all males collected did 
