A comprehensive description of oocyte developmental stages in Pacific halibut, Hippoglossus stenolepis

Abstract

Accurate characterization of oocyte development is essential to understanding foundational aspects of reproductive biology and successful management of Pacific halibut (Hippoglossus stenolepis). Here this study provides complete histological descriptions for eight oocyte developmental stages in addition to postovulatory follicles and demonstrates the potential for oocyte size frequency distribution to act as a proxy for ovarian developmental stage and future maturity assessments. Importantly, it provides the first histological evidence that Pacific halibut have a group‐synchronous ovarian developmental pattern with determinate fecundity and support for their batch‐spawning strategy.

Full text

BRIEF COMMUNICATION
A comprehensive description of oocyte developmental stages
in Pacific halibut, Hippoglossus stenolepis
Teresa Fish
1,2
| Nathan Wolf
1
| Bradley P. Harris
1
| Josep V. Planas
2
1
Fisheries, Aquatic Science and Technology
Laboratory, Alaska Pacific University,
Anchorage, Alaska, 99508
2
International Pacific Halibut Commission,
Seattle, Washington
Correspondence
Teresa Fish, Fisheries, Aquatic Science and
Technology Laboratory, Alaska Pacific
University, 4101 University Drive, Anchorage,
AK, 99508, USA.
Email: tfish@alaskapacific.edu
Funding information
Funds to support T.F. and for sample collection
and processing were provided by International
Pacific Halibut Commission. Additional support
for T.F. was provided by Alaska Education Tax
Credit funds contributed by the At-Sea
Processors Association and the Groundfish
Forum.
Abstract
Accurate characterization of oocyte development is essential to understanding foun-
dational aspects of reproductive biology and successful management of Pacific hali-
but (Hippoglossus stenolepis). Here this study provides complete histological
descriptions for eight oocyte developmental stages in addition to postovulatory folli-
cles and demonstrates the potential for oocyte size frequency distribution to act as a
proxy for ovarian developmental stage and future maturity assessments. Importantly,
it provides the first histological evidence that Pacific halibut have a group-
synchronous ovarian developmental pattern with determinate fecundity and support
for their batch-spawning strategy.
KEYWORDS
developmental stage, histology, maturity, oocyte, Pacific halibut, reproduction
Understanding the species-specific components of fish reproductive
biology (e.g., age at maturity, fecundity, spawning strategy) is founda-
tional for effective stock management. These indices vary by species
(Kennedy et al., 2014; Núñez et al., 2015; TenBrink &
Wilderbuer, 2015) and can dramatically alter our perception of stock
status (Morgan, 2008). This is especially true for long-lived fish spe-
cies, such as Pacific halibut (Hippoglossus stenolepis), as lifetime contri-
butions to stock recruitment continue for many seasons after
reproductive maturity is reached. For example, changes in reproduc-
tive performance, as deduced from female maturity estimates, exert a
strong influence on spawning biomass estimates and, consequently,
on the stock assessments of the Pacific halibut (Stewart &
Hicks, 2020).
Currently, assessments of female Pacific halibut reproductive matu-
rity involve visual macroscopic inspection of ovaries in the field
(Stewart & Hicks, 2020). While convenient, this approach has yet to be
corroborated by more definitive analysis methods and lacks the speci-
ficity required to provide information on many species-specific
components of reproductive biology. As accurate characterizations of
reproductive development are essential to fisheries management, an
evaluation of the reliability of the current macroscopic staging methods
using more precise assessment techniques is of utmost importance.
Histological analysis of oocyte developmental stages repre-
sents an important initial step to evaluating current maturity
assessment methods (West, 1990). Moreover, when providing
oocyte size-frequency distributions, histological analyses may also
offer alternative methods for characterizing fish reproductive
phases and offer additional information on reproductive parame-
ters, such as fecundity type and spawning pattern. Histological
examinations have successfully characterized ovarian development
in many flatfishes, including California halibut Paralichthys
californicus (Lesyna & Barnes, 2016) and Atlantic halibut
Hippoglossus hippoglossus (Neilson et al., 1993). Furthermore, fram-
ing this characterization using universally descriptive terminology
for teleost oogenesis (Grier et al., 2009) will facilitate future com-
parative examinations (Brown-Peterson et al., 2011).
Received: 19 June 2020 Accepted: 18 September 2020
DOI: 10.1111/jfb.14551
FISH
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2020 The Authors. Journal of Fish Biology published by John Wiley & Sons Ltd on behalf of Fisheries Society of the British Isles.
1880 J Fish Biol. 2020;97:1880–1885.wileyonlinelibrary.com/journal/jfb

Pacific halibut represent an important economic and cultural
resource in the Gulf of Alaska and the rest of the northeastern Pacific
Ocean. In this region, spawning occurs between November and March
along the continental slope and in depressions on the continental
shelf (St-Pierre, 1984). Spawning is generally thought to occur annu-
ally after fish reach reproductive maturity (Stewart & Hicks, 2020;
St-Pierre, 1984; Thompson, 1914), but this assumption has received
mixed support in the literature (Bell, 1981; Novikov, 1964; Seitz
et al., 2005; Vernidub, 1936). Early investigations of Pacific halibut
oocyte size (Kolloen, 1934; Thompson, 1914, 1916) documented a
developing cohort of oocytes within the ovary immediately after
spawning, thus supporting the premise of annual spawning. Nonethe-
less, a detailed histological characterization of ovarian development in
female Pacific halibut to be used in histology-based reproductive
phase maturity classification has not been conducted to date.
Here, this study presents comprehensive histological descriptions
of oocyte developmental stages and documents postovulatory follicles
(POFs) in Pacific halibut. In addition, it details the range of oocyte
diameters among developmental stages and explores differences in
the size-frequency distributions of oocytes in ovarian tissue at differ-
ent developmental stages to investigate the relation between oocyte
size and oocyte developmental stages. This work provides the most
precise assessment of developmental stage for the species to date,
documents the spawning strategy and offers a foundation for more
specific assessments of Pacific halibut reproductive biology in the
future.
TABLE 1 Description of oocyte developmental stages of Pacific halibut, Hippoglossus stenolepis, associated growth phases (modified from
Brown-Peterson et al., 2011 and Grier et al., 2009), and postovulatory follicles (POFs)
Pacific halibut oocyte histology Oocyte diameters (μm)
Growth phase
(acronym)
Developmental stage
(acronym) Description Photo
Sample
size
Mean
± S.D.
Range
(min–max)
Primary growth
(PG)
One nucleolus (PGon) Oocytes are small, angular and compact with
a single large nucleolus. Cytoplasm
granules stain dark purple.
51 116
±89
36–381
Perinucleolar (PGpn) Oocytes are larger and rounder than PGon.
Nuclei develop and flatten around the
nucleus. Cytoplasm granules stain light
purple.
55 235
±92
103–479
Cortical alveolar (CA) First cortical alveoli appear as white stain in
the periphery of the oocyte.
237 445
±80
195–664
Secondary
growth (SG)
Primary vitellogenesis
(Vtg1)
Yolk globules first appear at the periphery,
stain pink and fill inwards occupying up to
one-third of the cytoplasm.
663 544
±69
362–750
Secondary
vitellogenesis (Vtg2)
Yolk globules transition from only the
periphery of the ooplasm and fill inwards
to the nucleus.
341 686
±91
465–910
Tertiary vitellogenesis
(Vtg3)
Yolk globules completely fill the ooplasm to
the central nucleus and coalesce into
larger yolk globules.
500 1171
± 216
706–1644
Oocyte
maturation
(OM)
Germinal vesicle
migration (GVM)
The nucleus begins to migrate through a
cytoplasm fully filled with large yolk
globules.
302 1271
± 257
811–1769
Periovulatory (PO) Nucleus no longer visible and the yolk
globules coalesce into a central yolk mass.
Oocyte is still within the follicle wall.
54 2037
± 270
1600–2811
Postovulatory follicle
(POF)
Collapsed empty follicle wall remaining after
a periovulatory oocyte is expelled.
FISH ET AL.1881
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To conduct this study, the authors collected approximately
30 female Pacific halibut each month from September 2017 through
August 2018 (n= 356) from the Portlock region in the Gulf of Alaska
using longline fishing vessels specifically chartered for sampling. Indi-
vidual sampling trips occurred over 1–4 day periods. The authors
focused collection efforts on larger (≥90 cm) individuals to increase
the probability of sampling postpubescent fish (Clark et al., 1999;
Loher & Seitz, 2008). Once fish were on-board the fishing vessel,
approximately 1 cm
3
tissue was excised from the central area of the
ovary and fixed in 10 ml of 10% buffered formalin from each fish.
Ovarian tissue samples were sent to an independent laboratory to be
processed for histology where two series of 4 μm thick Paraffin sec-
tions, separated by approximately 500 μm, were mounted on two
slides and stained with haematoxylin and eosin.
Ovarian follicles in mounted and stained ovarian tissue sections
were examined visually under 1×–10×magnification (Leica DM LB2
and Leica M80 microscopes, Leica Camera Inc., Allendale, NJ, USA).
Oocyte developmental stages were categorized following the univer-
sal oocyte stages described by Brown-Peterson et al. (2011) with the
exception that the germinal vesicle breakdown and hydration stages
were combined into a single periovulatory stage (PO; Table 1). In addi-
tion, the early growth phases detailed by Grier et al. (2009) are used
to describe primary growth (PG) oocytes. Individual female develop-
mental stages were assigned based on the most advanced oocyte
developmental stage present in the ovarian tissue (Table 1).
To assess potential differences in oocyte diameter among oocyte
developmental stages, 3–5 of the most advanced stage oocytes (i.e.,
largest) from each fish were photographed at 1×–10×magnification
using a microscope-mounted camera (Zeiss Axiocam ERc 5s or Leica
IC80 HD; Carl Zeiss Microscopy LLC, White Plains, NY, USA and Leica
Camera Inc., Allendale, NJ, USA, respectively) and a minimum of
50 oocytes sectioned through the middle of the nucleus from each
developmental stage were measured (Table 1). Area measurements
(μm
2
) of photographed ovarian follicles were conducted using
ImagePro Premier 9.1 (Media Cybernetics Inc., Rockville, MD, USA) by
tracing the perimeter of the oocyte and extracting the oocyte area. All
measured areas were converted to mean diameters for further analy-
sis in ImagePro Premier 9.1 by calculating the mean length of diame-
ters measured at 2 degree intervals passing through the traced oocyte
centroid. Differences in the mean rank oocyte diameter among devel-
opmental stages were analysed using the Kruskal–Wallis rank sum
test, followed by Dunn's post hoc test. All statistical analyses were
conducted in R version 3.5.2.
Potential differences in the size distribution of oocytes present in
fish from different developmental stages were examined following
methods detailed in Guzmán et al. (2017). Briefly, a 161 mm
2
image of
mounted ovarian tissue was scanned at 9600 dpi (Epson Perfection
V800 photo scanner; Epson US, Hillsboro, OR, USA) from a sub-
sample of five fish of each observed female developmental stage,
from cortical alveolar (CA) to PO, and from the three fish classified in
the PG perinucleolar (PGpn) stage (Table 1). Using these scanned
images, the areas of all oocytes sectioned through the nucleus within
a9,25or64mm
2
region of interest were measured. In all cases, the
smallest region in which at least 18 oocytes could be measured was
used. Area measurements and diameter conversions were performed
using the methods described earlier. To facilitate the visualization of
the results, oocyte areas of each developmental stage were natural
log (ln) adjusted, then summed across 50 μm diameter bins. Results
are presented as proportional occupancy of each ln-adjusted oocyte
developmental stage.
The present study provides histological descriptions of eight
oocyte developmental stages [PG one nucleolus (PGon), PGpn, CA,
primary, secondary and tertiary vitellogenesis (Vtg1-3, respectively),
germinal vesicle migration (GVM) and PO] as well as POFs in Pacific
halibut (Table 1). The results represent the first description of oocyte
development stages in Pacific halibut. Oocyte diameters were mea-
sured from each of the eight oocyte developmental stages and ranged
from 36 μm for the smallest oocyte in the PGon stage to 2811 μm for
the largest oocyte in the PO stage (Table 1). In all cases, mean oocyte
diameters increased with progression through developmental stages
and significant differences in mean rank diameters were observed
among all developmental stages (Kruskal–Wallis rank sum test:
χ
2
= 1906.3, df =7,P< 0.001; Dunn's post hoc test, P< 0.025) except
between PGon and PGpn and between Vtg3 and GVM (Figure 1a). No
atretic oocytes were identified in the present study. The presence of
POFs and atretic oocytes will be further investigated in a future study
evaluating seasonal or temporal changes in maturity in female Pacific
halibut.
Explorations of oocyte size distributions in fish at different female
developmental stages showed that Pacific halibut follow a pattern
typical of fish species with group-synchronous ovarian development
with determinate fecundity (Ganias, 2013; Lubzens et al., 2010). Fish
in early developmental stages displayed unimodal distributions of
oocyte diameters, which became increasingly right-skewed from
PGpn to CA (Figure 1b). This mode –generally up to 500 μm in diame-
ter –shares similar morphological characteristics to corresponding PG
oocytes described in other fish species (Grier et al., 2009; Selman
et al., 1993) and was present in all female developmental stages
(Figure 1b). Fish in more advanced developmental stages displayed
bimodal oocyte size distributions, with increasing separation between
the two modes with progressing developmental stage (Figure 1b). This
second mode developed as Vtg1 oocytes further separated in size
from the previtellogenic cohort at the Vtg2 developmental stage
(Figure 1b). At the Vtg3 developmental stage, a hiatus was present
between the two modes, effectively separating the previtellogenic
mode (oocytes <350 μm in diameter) and the larger or leading cohort
of vitellogenic oocytes (> 500 μm in diameter). At the GVM and PO
developmental stages, oocytes in the leading cohort continued to
increase in size (> 1200 μm in diameter) up until hydration
(c. 2000 μm in average). The selective recruitment of oocytes from
early into later (i.e., GVM and PO) female developmental stages, as
shown by the gradual transition from unimodal to bimodal size distri-
butions leading to two different populations of oocytes in the more
advanced stages of ovarian development, is evidence for a group-
synchronous ovarian developmental pattern with determinate fecun-
dity in this species (Ganias, 2013; Lubzens et al., 2010). Furthermore,
1882 FISH ET AL.
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although the most advanced oocytes in the GVM developmental stage
represented a unique, leading mode, the leading oocytes in the PO
developmental stage showed a larger range of sizes that included
GVM and PO oocytes at different steps of hydration (Figure 1b).
These observations suggest that, despite the lack of information of
the temporal progression of these events and the relatively small sam-
ple size in the PO developmental stage, GVM oocytes may be
recruited for final maturation, hydration and subsequent ovulation in
batches, supporting the notion that Pacific halibut, like its Atlantic
congener (Haug & Gulliksen, 1988), is a batch spawner, as suggested
by a previous report of the spawning behaviour of one tagged Pacific
halibut female (Seitz et al., 2005).
The histological description of oocyte developmental stages
produced by this work provides an important and necessary
(a) Primary
Growth (PG)
Oocyte stage
Secondary
Growth (SG)
Oocyte
Maturation (OM)
PGpn
n = 271 (3)
CA
n = 196 (5)
Vtg1
n = 228 (5)
Vtg2
n = 318 (5)
Vtg3
n = 258 (5)
GVM
n = 170 (5)
PO
n = 129 (5)
f
e
e
d
c
b
PO
GVM
CA
Vtg3
Vtg2
Vtg1
PGpn
PGon
90
30
10
3
1
90
30
10
3
1
90
30
10
3
1
90
30
10
In oocyte area (% of total)
3
1
90
30
10
3
1
90
30
10
3
1
90
30
10
3
1
0 500 1000 1500 2000
0 500 1000
Diameter (μm)
1500 2000
a
a
(b)
FIGURE 1 Description of changes in
Pacific halibut mean oocyte diameters with
(a) developmental stage and (b) oocyte size
distribution with individual fish
developmental stage. Vertical broken lines
separate the growth stages at the midpoint
between mean oocyte developmental stage
diameters. In (a), mean oocyte diameters are
indicated by black circles, one- and two
standard deviations are indicated by wide
black and grey bars, respectively; and
minimum and maximum values are indicated
by narrow grey bars, with different letters
(a–f) indicating statistical significance
(P< 0.025) among groups. In (b) the
distribution of oocyte diameters is
represented as the proportional occupancy of
ln(oocyte area) for each of the observed
female developmental stages (defined as the
most advanced stage of oocyte present in the
sample population; PGpn, PGca, etc.), with
nindicating the total number of oocytes
measured from a sub-set of females (number
of individual fish shown in parentheses)
FISH ET AL.1883
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framework for characterizing reproductive phase and allows for
future explorations of reproductive development in Pacific halibut.
The authors propose future research to evaluate seasonal changes
in ovarian developmental stages, as defined here, as well as repro-
ductive phases by investigating POFs and atresia observed in con-
junction with the developmental stages. After a thorough
investigation of reproductive phases in Pacific halibut, the authors
further recommend future research in the area of assessing the
accuracy of current macroscopic reproductive maturity staging
methods, with special attention to the potential identification of
skip-spawning females and their assignment to particular macro-
scopic maturity stages. The authors believe that their results on
oocyte menstruation and size-frequency distributions presented
here will be important to explore potentially time- and cost-
efficient alternatives to histological oocyte evaluations for repro-
ductive assessments in this commercially important species.
Oocyte measurement methods have previously been demon-
strated as effective reproductive phase indicators in Atlantic hali-
but (Neilson et al., 1993), and newer methods (Friedland
et al., 2005; Thorsen & Kjesbu, 2001; Witthames et al., 2009) with
improved detection, cost and efficiency could potentially be
applied to Pacific halibut. Although fecundity at size or age of
Pacific halibut is a knowledge gap, it should be noted that although
mean oocyte diameter has been linked to oocyte density for use in
calculating fecundity (Thorsen & Kjesbu, 2001), the analysis would
require additional oocyte diameter explorations to calibrate whole
mount oocytes with the histology-derived oocyte diameters
presented here.
ACKNOWLEDGEMENTS
This study and T.F. were supported by funding from the International
Pacific Halibut Commission. The authors thank setline survey special-
ists and Joan Forsberg from the International Pacific Halibut Commis-
sion for their help with biological sample collection and microscope
set-up and image collection, respectively, Corina Cabrera and Elena
Eberhardt from Alaska Pacific University for assistance collecting and
assuring data quality, and Dr. Jose Guzmán from the School of Aquatic
and Fishery Sciences, University of Washington for assistance and
clarification on oocyte reproductive stage assessments.
CONFLICT OF INTEREST
All authors declare that they have no conflict of interest.
AUTHOR CONTRIBUTIONS
T.F. contributed to the study design, collected the samples, analysed
and interpreted the results, and was the primary manuscript author.
N.W. assisted with analysis, interpretation and presentation of the
results and assisted T.F. in writing the manuscript. B.P.H. assisted with
the presentation of the results and edited the manuscript.
J.V.P. conceived the study, supervised data collection, analysis and
interpretation, and edited the manuscript. All authors read and
approved the final manuscript.
COMPLIANCE WITH ETHICAL STANDARDS
Pacific halibut collections were conducted under a letter of acknowl-
edgement from National Oceanographic and Atmospheric Administra-
tion (NOAA) Fisheries and biological samples were obtained following
guidelines for the euthanasia of finfish from the American Veterinary
Medical Association (AVMA, 2020).
ORCID
Teresa Fish https://orcid.org/0000-0002-1310-7216
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How to cite this article: Fish T, Wolf N, Harris BP, Planas JV.
A comprehensive description of oocyte developmental stages
in Pacific halibut, Hippoglossus stenolepis.J Fish Biol. 2020;97:
1880–1885. https://doi.org/10.1111/jfb.14551
FISH ET AL.1885
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