Serum and slime vitellogenin in the Atlantic cod, Gadus morhua,
and its relation to ovariandevelopment
Joe Kunkel, John Bohannon, Rahul Sharma, and Joe Zydlewski
UMass Amherst Biology Department
NOAA/CMER Project
• NMFS: Frank Almeida, Woods Hole
• Need for ET50 maturation assay for Atlantic cod, Gadus morhua
• Albatross IV Fall & Spring Bottom Survey
• Is Vg secreted in slime?
Cod maturation problem?
• Fall and Spring Bottom Survey random samples
• Leg IV and V cover USA cod stocks on Georges Bank and Gulf of Maine
• A problem exists in establishing the ET50 of cod maturation
• Visual inspection of the ovary leads to errors.
Develop and Validate aCod Maturation Assay
• Purification of Cod Lv
• Antiserum production and validation
• New slime and serum collection at sea
• Vg induction by Estradiol in aquarium held cod
• Serum analysis of ‘inherited’ samples
• Correlating serum Vg with ovarian histology
Fish Lipovitellin purification strategy
• Heat stability of Winter flounder Lv (Hartling, 1997)
• Labile Lv is nicked!• Large monomer (94 kDa).
• Single peptide = stability?• Lipoprotein = stability?
Most fish Lipovitellin is heat stable!
• Heat stable Lv: hagfish, red fish, American shad, cod, red hake, tautog, plaice, winter flounder, zebra fish.
• Heat labile Lv: chicken, cockroach, silk moth, gypsy moth, frog, spiny dog fish.
Cod Lipovitellin Purification
• Heat stable cod Lv• Large Stokes Radius• monomeric ~100 kDa
IEP of cod serum and anti-cod-Lvs
• Both anti-cod-Lvs react with two forms of cod Vg: fast & slow
• The fast and slow forms of cod Lv are found variably in serum samples
f s
anti-cod-Lv reacts with other Gadid Lv!
• QIEP: Quantitative Immunoelectrophoresis
• Anti-cod-Lv1
• cod Vg = haddock Vg?
• cod fast Vg = slow Vg?
Atlantic Cod NMF study: define the I/D boundary
• All ………… 713• DEL 94-12 ... 504 • AL 95-02 … 63• Isabel 96-01 .. 146
IDRST drst DRST drst DRST
• Stages: I D R S T• Sample cod serum
& ovary to find the Immature/Developing (I/D) boundary.
Inherited vs new samples
• All ……….. 1305• DEL 94-12 ... 504 • AL 95-02 … 63• Isabel 96-01 .. 146• AL 98-02 ….. 75 • AL 98-11 ….. 43 • AL 99-03 ….. 227 • GL 99-01 ….. 73 • AL 99-10 ….. 174
Biggest Gadus morhua in decade?
• AL 99-03, Leg V.• 227 cod sampled by
J. Kunkel & J. Burnette.• Station 317 cod #1: 141cm
28 kg spent female. • sample of slime, blood,
otoliths and ovary.
• No Vg in serum!
Cod in NMF Woods Hole Aquarium
• Short otter trawl cod.• Two weeks acclimation. • 10 ug Estradiol
injection.• Mucous scraped.• Tail vein bled.
• No Vg in mucous!
QIEP of Vg in Estradiol injected cod
• Slime contains no Vg
• Vg titer in serum starts to decline in 3rd week post injection
• Sex needs to be determined at end of experiment
• 5 sequential experiments
• Kinemage 3-D plot
• Immature
• Developing
• Ripe
• Spent
• Resting
• Males
• Kinemage 3-D plot
• Immature
• Developing
• Ripe
• Spent
• Resting
• Males
• Kinemage 3-D plot
• Immature
• Developing
• Ripe
• Spent
• Resting
• Males
• Kinemage 3-D plot
• Immature
• Developing
• Ripe
• Spent
• Resting
• Males
• Kinemage 3-D plot
• Immature
• Developing
• Ripe
• Spent
• Resting
• Males (3 of 100)(3 of 100)
Developing Ovaries, H&E, low X
Cod developing follicles, H&E• cortical granules,
basophilia, nucleoli, thin chorion
• small yolk granules, large cortical granules, 2 chorion layers
• growing yolk granules dominate cortex
• large yolk granules, growing follicle, thick chorion
Odd cod found in Cape Cod Bay
• 3 similarly deformed cod suggest oddities could be found in the biochemical realm as well.
Conclusions
• anti-cod-Lv is not a litmus for maturation, but could be used to delimit lower reaches of maturation range (e.g. ... > 47 cm.)
• cod Vg is not secreted in mucous
• Lv of most fish are heat stable
• some ‘odd’ cod males may produce Vg
Thanks to:NMFS partners: Frank Almeida, Jay Burnette.
CMER: John Boremann and Kevin Friedland.
Shipmates: Nancy McHugh, Linda Despres, John Galbraith, Nina Shepherd, Holly Yachmetz, Jason Link. (+ countless crew)
UMass students and colleagues: Ruth Hartling, Ray Moniz, Gerda Kunkel, and Umass fall ‘99 Histology 523 Honors Students.
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