Research Summary
Teleost oocyte growth & maturation: Multiple
vitellogenins and their receptors
We aim to discover
the fundamental cellular mechanisms by which oocytes grow and mature in fishes.
Oocyte growth involves massive accumulation of proteins, lipids, and other
factors necessary to build and sustain a new life. Little is known about
mechanisms of oocyte lipidation, but we have learned much about the deposition
of yolk proteins. They are derived from a circulating precursor protein,
vitellogenin (Vtg), that is produced by the liver in response to estrogen,
secreted into the bloodstream, and taken up by growing oocytes via
receptor-mediated endocytosis. Vitellogenin is then cleaved by an enzyme
(cathepsin D) into a suite of yolk proteins that are stored in yolk granules,
globules or platelets. These yolk proteins include a large lipoprotein (lipovitellin,
Lv) made up of a heavy chain (LvH) and a light chain (LvL), and three smaller
proteins, phosvitin (Pv) which is made up largely of polyserine residues that
are phosphorylated and carry calcium in freshwater teleosts, beta’-component
(beta’c), which contains neither lipid nor phosphorus but has numerous
disulfide bonds and is likely to be important in establishing Vtg structure,
and a final small yolk protein derived from the C-terminal end of Vtg (Ct) that
is similar to beta’c. Complete Vtgs possess all of these yolk protein domains.
The simple model for
vitellogenic oocyte growth illustrated in the upper left has become outdated,
as we have discovered multiple Vtgs and Vtg receptors with disparate functions
during oocyte growth and maturation. Fully-grown
oocytes undergo final maturation, which is defined by resumption of meiosis and
cytoplasmic maturation including yolk protein hydrolysis, clearing of the
ooplasm, and coalescence of the lipid droplets.
The hydrolysis of yolk proteins yields free amino acids (FAA), which
osmotically drive oocyte hydration and acquisition of proper egg buoyancy. Along with key collaborators in Japan, Dr. Akihiko
Hara (Faculty
of Fisheries, Hokkaido University) and Dr. Takahiro Matsubara (Hokkaido National Fisheries
Research Institute), we have been investigating the fundamental
physiological mechanisms of oocyte growth and maturation and embryonic and
larval nutrition that depend on Vtgs. We
discovered that most advanced teleosts (Acanthomorpha) possess two
paralogous complete forms of Vtg (VtgAa and VtgAb) that are disparately
processed during oocyte maturation. With
the exception of a small apoprotein that may be important in delivery of lipid
to late stage larvae, all of the yolk proteins derived from VtgAa are
completely cleaved into FAA during final maturation, generating the osmotic
gradient that drives oocyte hydration (and egg buoyancy) and providing a pool
of diffusible FAA nutrients required for early embryonic nutrition. The major
portion of Lv derived from the other complete Vtg, VtgAb, remains largely intact
and is utilized as a large lipoprotein nutrient during later embryonic and
larval development. To date, the
disparate processing of dual complete Vtgs has been observed by us in barfin
flounder, red sea bream, mosquitofish, grey mullet, white perch and striped
bass. These observations have been integrated into the model shown at right,
which explains how the differential processing of dual complete forms of Vtg
provides for proper oocyte hydration and egg buoyancy while delivering the
special nutrients required by early embryos and later stage embryos and larvae.
During oocyte growth,
the Vtgs are stored in the same yolk compartment as cathepsin D, which cleaves
the Vtgs into yolk proteins, and cathepsins B or L, which are responsible for
the cleavage of yolk proteins into FAA during ocyte maturation. This latter cleavage of yolk proteins is
triggered by activation of a vacuolar ATPase that acidifies the yolk
compartment and activates cathepsins B or L.
Our studies of white perch, and research by other investigators, have
shown that activation of this ATPase occurs in response to the
maturation-inducing steroid hormone, a progestin (20beta-S in white
perch). However, until now there has
been no satisfactory explanation of how yolk proteins derived from VtgAa are
subject to extensive proteolysis during oocyte maturation whereas the major
yolk protein (Lv) derived from VtgAb remains largely intact. Our working hypothesis is that the two forms
of Vtg must bind to different forms of Vtg receptor (Vtgr) to be delivered into
different endosomal compartments that differ in cathepsin content or degree of
acidification during oocyte maturation.
This hypothesis was based, in part, on our discovery of an unusual form
of Vtg, VtgC, that lacks Pv, beta’c and Ct yolk protein domains and that does
not undergo proteolysis into smaller yolk proteins during oocyte growth or into
FAA during oocyte maturation. The VtgC
appears to be present in all teleost taxa.
We utilized a novel Vtgr-binding assay along with ligand blotting
procedures to discover that the VtgC lacks an oocyte receptor, which emphasizes
the link between receptor binding and the proteolytic fate of Vtgs. The VtgC must enter the oocyte in the fluid
phase in association with receptor-mediated endocytosis of the other Vtgs or
with other micropino-cytotic activity of the oocyte. The binding assay and ligand blotting
procedures were utilized to discover three Vtgr’s, the largest of which
(>> 200 kDa) preferentially binds VtgAa and the smallest of which (~97
kDa) preferentially binds VtgAb and is likely to be the ‘classical’ Vtgr that
we previously cloned as cDNA and fully characterized in white perch. The third receptor of intermediate size (~150
kDa) is promiscuous, binding both VtgAa and VtgAb, albeit with apparently low
affinity, and, based on recent findings for other lipoprotein receptors, could
possibly be involved in some signaling function, such as monitoring the
relative availability of the two forms of complete Vtg in the circulation. In addition, we have utilized affinity
chromatography to isolate a fraction of ovarian membrane proteins that bind to
VtgAa and VtgAb, and have submitted the fraction to mass spectrometry
procedures (ESI-LC/MS/MS), generating peptide spectra that were mapped to sequences
present in a striped bass ovarian transcriptome library that we constructed
using next generation high through-put sequencing of expressed sequence
tags. Three Vtgrs were discovered, one
of which is the ‘classical’ Vtgr and the other two are completely novel forms
of lipoprotein receptor. We are now
working to establish identity between these novel Vtgrs and the VtgAa receptor
and promiscuous Vtgr. Our findings have
led us to develop a new model of oogenesis shown above at left wherein VtgAa
and VtgAb, by virtue of their binding to different forms of Vtgr, are delivered
to different endosomal compartments where they are subject to different degrees
of proteolysis during oocyte maturation.
Reproduction of aquaculture broodstocks: The egg quality
problem
Spawning striped bass in egg quality
trials
We utilize the findings of our
research on reproductive physiology to solve problems in aquaculture,
especially those encountered during induced maturation and spawning of fishes.
A major problem with reproduction of striped bass is variable and often poor
egg quality. At present, only about half
of the female broodfish in the National Program for Genetic Improvement and
Selective Breeding for the Striped Bass Industry can be successfully spawned
with high fertility. We are testing the
hypothesis that the multiple Vtg system is dysfunctional in fish producing poor
quality eggs. This hypothesis arises
from our observations that oocyte hydration, egg buoyancy, early embryonic
survival and late larval vigor and survival all are impaired to some extent in
low quality spawns. Other investigators
have shown that, in related species, the expression and activation of cathepsins
(D, B and L) and the deposition of specific yolk proteins are impaired in low
quality spawns yielding poorly buoyant eggs.
Our approach to testing the ‘multiple Vtg’ hypothesis has been to
collect fully grown oocytes (biopsies) and ovulated eggs from females producing
high and low quality spawns as assessed by evaluations of egg fertility, early
embryo survival, hatching rates, and larval development and survival until
first feeding. The samples of oocytes
and eggs are extracted and the yolk is subjected to electrophoretic separation
(SDS-PAGE) to reveal distinct yolk protein bands. The bands are first subjected to Western
blotting procedures using our extensive panel of antisera against teleost Vtgs
and yolk proteins, or are subjected to phosphoprotein staining, to tentatively
identify them as LvH, LvL, Pv, beta’c or Ct components derived from the
different forms of Vtg. They are then
excised from the gel and submitted to mass spectrometry (ESI-LC/MS/MS), which
provides peptide spectra that can be mapped back to the parent Vtg (VtgAa,
VtgAb, and VtgC) sequences deduced from their full-length cDNAs, which we have
cloned and sequenced. Special mass spectrometry procedures (e.g. iTRAQ) can
later be used to identify the ratios of Vtg-type specific yolk proteins (e.g.
LvHAa versus LvHAb) present in individual gel bands. In this way, we will develop a detailed
picture of the deposition of different forms of Vtg into striped bass oocytes
and of the maturational processing of yolk in this species and will be able to
detect any differences in these processes between good and poor quality
spawns. This project is being executed
in collaboration with Dr. Norm Glassbrook (NCSU Genomic
Sciences Laboratory).
Working with
Drs. Jennifer Schaff (NCSU Genomic Sciences Laboratory) and Robert Chapman (Marine Genomics Project, S.C.
Hollings Marine Laboratory), we have developed an ovarian
transcriptome library for striped bass that includes about half of the
genes expected to be expressed in the ovary (> 11,000 contiguous cDNA
sequences) and we are working to develop a more detailed whole organism
transcriptome for the closely related white perch, which should include
information on virtually all genes (~25,000) expressed in the ovary at any
maturational stage. In addition, we have
utilized differential display procedures to identify ~88 genes that are
differentially expressed during various stages of oocyte growth. We plan to utilize these new resources to
develop cDNA microarrays that can be utilized to simultaneously visualize
differences in expression of thousands of genes in good and poor quality
oocytes and eggs. This more global
evaluation of the ovarian transcriptome as it relates to egg quality should
provide invaluable insights into which physiological processes are impaired in
females producing poor quality eggs, and it should also be informative as to
which husbandry processes might be modified to improve egg quality.
Domestication and DNA marker-assisted selective breeding
of superior striped bass and white bass broodstocks
A central focus of
our aquaculture research is selective breeding of an improved cultivar for the
hybrid striped bass industry, a major component of finfish aquaculture in the
United States. We pioneered the
commercial production of hybrid striped bass in North Carolina farm ponds in
the late 1980s and this industry has since spread nationwide. We have established genetically diverse
broodstocks of the parent species (striped bass and white bass), developed
reliable techniques for their hatchery propagation, and domesticated both
species over several generations (striped bass, 4 generations; white bass, 8
generations). Our current efforts are directed at discovering the degree to
which important production traits, such as growth rate or body conformation,
have a genetic basis or have been altered by the process of domestication.
Because genetic contributions to fish “performance” differ between life history
stages, these studies have involved all phases of aquaculture production. They
included rearing of larvae in nursery ponds for ~30 days until they are
recovered as juveniles, growout of the fish in tanks or ponds for 9-12 months
until they are recovered as subadults, and growth of these advanced fingerlings
to market-size. Aquaculture ponds are notoriously variable environments and
effects of environmental variation between ponds have the potential to mask
genetic contributions to fish performance. Accordingly, we conduct “common
garden” performance evaluations in which progeny from different parental
crosses are reared communally in the same pond(s). We utilize microsatellite
DNA markers to unambiguously identify offspring produced by each parental pair
involved in a series of crosses designed to provide information on the genetic
basis of specific traits. The broodstock
development efforts are conducted in cooperation with Dr. Andy McGinty and
staff at the NCSU Pamlico Aquaculture Field Laboratory and are being executed
in collaboration with Dr. Adam Fuller and associates (USDA-ARS 
Stuttgart
National Aquaculture Research Center).
Working with
our collaborator, Dr. Caird
Rexroad III (National
Center for Cool and Cold Water Aquaculture) we have developed about 500 new
microsatellite DNA markers as the first step toward mapping the striped bass genome and discovering
markers linked to genes that actually regulate fish performance (quantitative
trait loci, QTL) and that can be used to predict performance in the selective
breeding program. The current effort is
to develop a medium density linkage map of the striped bass genome using
parents and progeny from two striped bass families utilized in the type of
performance trials discussed above. This
effort includes collaboration with Dr. Rexroad, Dr.
Charlene Couch (NCSU Department of
Genetics), and Drs.
Kimberly Reece and Jan
Cordes (Virginia Institute of Marine Science, Aquaculture Genetics and Breeding
Technology Center). The new linkage
map, an array of the microsatellite markers in which distances are measured by
the rate of recombination between them, will be similar to but more detailed
than the one for the closely related European sea bass pictured at the
right. The map will provide signposts
for more detailed mapping of the striped bass genome and discovery of QTL
associated with these signposts; and, as in the case of the sea bass, allelic
variation at some microsatellite loci may prove to be associated with important
production traits of striped bass and could be targeted in the breeding
program. The ultimate goal of these
efforts is to be able to select future broodstock based on knowledge of their
relevant genotypes without being totally dependant on lengthy and costly
rearing trials to select future breeders.
In addition to the research on striped
bass genetics and breeding, we are working to release to industry a special
NCSU line of domesticated white bass (NCSU-WB1) that has been genetically
well-characterized and certified free of the pathogen, viral hemorrhagic
septicemia (VHS), and for which procedures for broodstock husbandry and
nutritional conditioning for spawning are well established. The genetic characterization of the line
involves extensive genotyping of 2 year classes of the fish (7th filial
generation) at the striped bass microsatellite loci discussed above in order to
obtain information on genetic diversity (e.g. allelic diversity, heterozygosity,
inbreeding coefficient) as a benchmark for further selective breeding. This information is essential to detect and
avoid inbreeding in the ongoing selection program, which could result in
depression of fitness and performance traits.
Another goal of this work is to establish a microsatellite DNA
genotyping system for distinguishing our NCSU-WB1 line of white bass from other
captive or wild stocks so that any unauthorized adulteration or distribution of
the line can be detected and avoided. The white bass genetics work is being
performed in collaboration with Dr. Charlene Couch (NCSU Department of
Genetics). Certification of the NCSU-WB1
line as VHS free, which involves our collaboration with Dr. Andy Goodwin
(University of Arkansas at Pine Bluff Aquaculture/Fisheries Center of
Excellence and Fish Disease and Diagnostics Program) is important as a
requirement for its widespread distribution to industry, and the line tested
free of VHS in 2007. In recent years,
VHS, which is an exceptionally virulent pathogen with a broad host range, has
spread through the Great Lakes region and it is critical that the further
spread of this dangerous pathogen be avoided.
As the Great Lakes, formerly the major source of white bass broodfish
for hybrid striped bass production, are now closed to broodstock collections,
there is increasing pressure on the hybrid striped bass industry to develop or
adopt domesticated broodstock. This
development will require that the requisite procedures for successful
reproduction of the domesticated fish be available. While we have previously shown that the
NCSU-WB1 line can be used to produce hybrid striped bass with egg fertility and
progeny survival and performance rates equivalent to those obtained using wild
broodfish, there remains uncertainty as to the earliest age at which the
NCSU-WB1 fish can be reproduced successfully and the length of time that the
fish need to be fed expensive conditioning diets prior to spawning. We are working to eliminate these
uncertainties in current breeding trials.
Development and distribution of the NCSU-WB1 line of white bass and the
research described above is being conducted in collaboration Dr. Ron Hodson
(NCSU Emeritus Professor), the Striped
Bass Growers Association and several of its individual members (Carolina
Fisheries, CastleHayne Fisheries, Keo Fish Farms), and the Stuttgart National
Aquaculture Research Center, and it is a central activity of the National
Program for Genetic Improvement and Selective Breeding for the Hybrid Striped
Bass Industry.