Research Summary

Dr. Craig V. Sullivan


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The simple model for vitellogenic oocyte growth illustrated in the upper left has become outdated, as we have discovered multiple Vtgs and Vtg receptors with disparate functions during oocyte growth and maturation.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


image010.gifWe 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.

image019.gifIn 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.



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