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.