joe kunkel, john bohannon, rahul sharma, and joe zydlewski umass amherst biology department

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

Cod Vitellogenesis

• Purification of Cod Lv

• Antiserum production and validation

• New slime and serum collection at sea

• Estradiol induction in aquarium held cod

• Serum analysis of NMFS samples

• Correlation serum Vg with ovarian histology

Winter Flounder Lipovitellin

• Heat stability of Lv!• Monomeric.• Labile is nicked!

• Single peptide = stability?• Lipoprotein = stability?

NOAA/CMER Project

• Request for Vitellogenin measurement in the Atlantic cod, Gadus morhua

• NMFS contact: Frank Almeida, NMF, Woods Hole

• Albatross IV Fall & Spring Bottom Survey

ET50 of cod maturation

• Fall and Spring Bottom Survey

• Leg IV and V cover Georges Bank and Gulf of Maine

• Fall categorization of immature vs developing is a problem in establishing ET50 of maturation.

Most fish Lipovitellin is heat stable!

• Heat stable Lv: hagfish, red fish, Atlantic 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 stabile• Large Stokes Radius

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

anti-cod-Lv reacts with other Gadid Lv!

• QIEP: Quantitative Immunoelectrophoresis

• Anti-cod-Lv1

• cod Vg = haddock Vg?

• cod fast Vg = slow Vg?

Cod historical average maturation.

• 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!

Atlantic Cod inherited by CMER study

• All ………… 713• DEL 94-12 ... 504 • AL 95-02 … 63• Isabel 96-01 .. 146

• Serum and ovary samples from females; plus only a few male sera.

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

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)

Developing Ovaries, H&E, low X

Cod developing follicles, H&E

• cortical granules, 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 (eg. > 47 cm.)

• cod Vg is not secreted in mucous

• Lv of most fish are heat stable

• some ‘odd’ cod males may produce Vg

The End

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.