Serum and slime vitellogenin in
the Atlantic cod, Gadus morhua,
and its relation to ovarian
development
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.