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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

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slide1

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
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
Winter Flounder Lipovitellin
  • Heat stability of Lv!
  • Monomeric.
  • Labile is nicked!
  • Single peptide = stability?
  • Lipoprotein = stability?
noaa cmer project
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
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
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
Cod Lipovitellin Purification
  • Heat stabile
  • Large Stokes Radius
iep of cod serum and anti cod lvs
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
slide9

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
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
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
Atlantic Cod inherited by CMER study

maturation

  • 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
Inheritedvs 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
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
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
slide16
Kinemage 3-D plot
  • Immature
  • Developing
  • Ripe
  • Spent
  • Resting
  • Males
slide17
Kinemage 3-D plot
  • Immature
  • Developing
  • Ripe
  • Spent
  • Resting
  • Males
slide18
Kinemage 3-D plot
  • Immature
  • Developing
  • Ripe
  • Spent
  • Resting
  • Males
slide19
Kinemage 3-D plot
  • Immature
  • Developing
  • Ripe
  • Spent
  • Resting
  • Males
slide20
Kinemage 3-D plot
  • Immature
  • Developing
  • Ripe
  • Spent
  • Resting
  • Males (3 of 100)
cod developing follicles h e
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
Odd cod found in Cape Cod Bay
  • 3 similarly deformed cod suggest oddities could be found in the biochemical realm as well.
conclusions
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

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.