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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 ovarian development. Joe Kunkel, John Bohannon, Rahul Sharma, and Joe Zydlewski UMass Amherst Biology Department. NOAA/CMER Project. NMFS: Frank Almeida, Woods Hole

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Joe Kunkel, John Bohannon, Rahul Sharma, and Joe Zydlewski UMass Amherst Biology Department

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  1. 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

  2. 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?

  3. 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.

  4. 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

  5. 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?

  6. 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.

  7. Cod Lipovitellin Purification • Heat stable cod Lv • Large Stokes Radius • monomeric ~100 kDa

  8. IEP of cod serum and anti-cod-Lvs fs • 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

  9. anti-cod-Lv reacts with other Gadid Lv! • QIEP: Quantitative Immunoelectrophoresis • Anti-cod-Lv1 • cod Vg = haddock Vg? • cod fast Vg =slow Vg?

  10. Atlantic Cod NMF study: define the I/D boundary maturation • All ………… 713 • DEL 94-12 ... 504 • AL 95-02 … 63 • Isabel 96-01 .. 146 • Stages: I D R S T • Sample cod serum & ovary to find the Immature/Developing(I/D) boundary. IDRST drst DRST drst DRST

  11. 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

  12. 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!

  13. 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!

  14. 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

  15. Kinemage 3-D plot • Immature • Developing • Ripe • Spent • Resting • Males

  16. Kinemage 3-D plot • Immature • Developing • Ripe • Spent • Resting • Males

  17. Kinemage 3-D plot • Immature • Developing • Ripe • Spent • Resting • Males

  18. Kinemage 3-D plot • Immature • Developing • Ripe • Spent • Resting • Males

  19. Kinemage 3-D plot • Immature • Developing • Ripe • Spent • Resting • Males (3 of 100)

  20. Developing Ovaries, H&E, low X *

  21. 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

  22. Odd cod found in Cape Cod Bay • 3 similarly deformed cod suggest oddities could be found in the biochemical realm as well.

  23. 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

  24. 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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