Our project on the Atlantic cod, Gadus morhua, received funding in the Fall of 1996 and I am reporting the progress made toward its goals in its first quarter.

Progress Outline:

1. Existing-Data Gathering: A tissue and database on cod was transferred from NMF.
2. Existing-Data analysis: Existing data on tissue and serum are suggestive.
3. Serum Sample Analysis: A start has been made and conclusions drawn.
4. New Sample Gathering: Necessary contacts have been initiated.
5. Equipment Acquisition: Equipment has been purchased and is being configured.
6. Personnel Identification: Two student trainees were identified and roles established.
7. Cooperation with NOAA: Almost all requested samples have been supplied.

1. Existing-Data Gathering: Joe Kunkel received from Frank Almeida Atlantic cod serum samples, tissue samples, tissue sections and an associated database that had been prepared by the National Marine Fisheries Staff from three trips to Georges Bank. Part of our proposal was to try to make sense of the existing tissue and serum samples. There were several special samples we acquired from NMF which have a bearing on our proposed research. A protease inhibitor, aprotinin, was injected into several fish a half hour prior to projected bleeding in order to inhibit proteolytic cleaving of the yolk serum precursor Vitellogenin (Vg) during the bleeding process. This provided us with one of the types of Vg source that we had proposed to produce. Another set of codfish males had been injected with estradiol and maintained in NMF holding tanks until bled. We had also proposed such an estradiol injected male as starting material for Vg purification. These two sources will be processed to see if the protocols used produced Vg in sufficient quantity for our purposes.
2. Existing-Data analysis: The data files on Length, Total Weight, Gutted Weight and Gonadal Weight were processed to produce Gonadal Somatic Indices vs body length plots which could be used to predict which samples were of interest to inspect more closely. A separate plot of data for fish which we had gonadal tissue sections prepared was also made and used to identify potentially interesting serum samples.
3. Serum Sample Analysis: The serum samples of potential interest were run on SDS-PAGE gels 10A-13C by Corrie Perlroth to evaluate if we could recognize the Vg peptides directly without immunology. It is essential to characterize the macromolecular properties of both Vg and its processed counterpart in the egg, Lipovitellin (Lv).
4. New Sample Gathering: Contact with the Gloucester Lobster Fisherman's Association was instituted through the Gloucester Harbormaster to find out if they could help us in obtaining fresh/live cod roe. They are willing to do the collection for free as a gesture to help the industry.
5. Equipment Acquisition: The required equipment for the analysis of the ovarian tissue samples was assembled and configuration of the equipment has begun. An ACI 200 MHz Pentium Pro computer was purchased to allow the image analysis to proceed efficiently. A 4 Gbyte disk drive was purchased for installation on the Biology marlin SPARCstation. This will provide adequate storage for the high resolution true color images of the cod gonads that will be used to characterize the reproductive state of the female and correlate with her Vg titer.
6. Personnel Identification: Two potential student participants and trainees for the cod project were identified and their roles established. Corrie Perlroth is an undergraduate Biology Major who has been trained by Ruth Hartling and myself to carry out the SDS-PAGE analysis of serum samples. All SDS-PAGE analyses completed to date have been done by her. Robert Alperin-Lee is an undergraduate who plans to participate in the nascent Biology BS/MS program and will use the cod project as a focus of his Masters project/thesis. His past experience as a biochemical technician and specificly with column chromatography make him ideally suited for helping in purifying the cod Vg or Lv. He has set up a gel filtration column and is in the process of calibrating it for use in our analysis of male vs female serum proteins. This is the first step in deciding whether our estradiol injected male serum contains sufficient Vg for use in antibody production.
7. Cooperation with NOAA: Cooperation with NOAA was said to be absolutely essential for this project. Frank Almeida of the Woods Hole National Marine Fisheries Laboratory arranged the collection of many of the necessary biological materials using their usual resources for collecting Atlantic cod on the Georges Bank. The following requested biological samples were provided (or not provided):

(1) Vitellogenic female serum and male serum (but not mucus) . Some serum from fish injected with aprotinin 30 min. prior to bleeding was provided. Female serum for characterization and potential purification of Vg with ancillary data enabling us to predict high natural Vg titer. Male serum was provided from large adult males to provide a large volume to be used in preparative amounts in developing the purification process and for potential adsorption of non-female specific antibodies from the crude antiserum and for quenching curves during the calibration procedure. (Large scale mucus collection from both vitellogenic females and males was not obtained and not yet needed.)

(2) Spawned eggs (unfertilized). (Not obtained and we are now trying to source some.)

(3) Ovaries sampled and fixed for histological examination. Obtained.

(4) Individually identified specimen records. Complete data records from individual fish for a multivariate information database were obtained.

(5) Immature or spent males and females for injection of estradiol-17ß. Serum from estradiol injected males was obtained.

In conclusion, we feel we are in most respects on task in our search for an antigen to make an antiserum to cod Vg.