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

This study investigates the presence of vitellogenin (Vg) in serum and slime of Atlantic cod, as well as its relation to ovarian development. It explores the need for a maturation assay for Atlantic cod and examines the purification of cod lipovitellin (Lv). The study also discusses the heat stability of cod Lv and the possibility of "odd" cod males producing Vg.

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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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