Preliminary studies of freshly collected halibut (Hippoglossus hippoglossus) spcrm by light microscopy indicated that sperm motility, i.e. the percentage of motile sperm and the duration of fonvard progressive movement, was optimized under conditions of osmotic pressure ranging between 400 and 1100 mOsmol/kg and pH for 6.5-8.5. The quality of sperm, stored in vitro on ice, deteriorated rapidly within a few hours, a charactenstic particularly evident in sperm after freezing and thawing tests. Studies of several cryoprotectants indicated that halibut sperm could be successfully preserved frozen when diluted with 3 parts extender (sucrose: 150 mM, CaCI, : 1 . 7 mM, MgSO,: 7 mM, glycine: 86mM and Tris: 30mM at pH: 8), 10% of 1-2 propanediol and one part sperm (3:l). Additional studies of the motility of seawater-activated halibut sperm were conducted by dark field microscopy under stroboscopic illumination. These observations indicated that fast forward-moving sperm motility, which lasts 60-70 s, is correlated with propagating waves on the full length of the sperm flagellum with an initial beat frequency of 45-50 Hz which abruptly drops to about 10 Hz after 60-80 S. The beat frequency is blocked by KCN (5mM) and NaN, (1.2mM), on intact sperm. The flagellum beat frequency of demembranatedlreactivated sperm is dependent on the ATP concentration in the reactivation solution.

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Atlantic halibut (Hippoglossus hippoglossus) collected off Newfoundland first mature at about 80 cm fork length (FL) for males and about 115–120 cm FL for females. Captive Newfoundland halibut did not release milt or eggs or have detectable levels of estradiol-17β or 11-ketotestosterone until exceeding 80 cm (males) and 115–120 cm (females). Estradiol-17β and testosterone increased to highest levels in females during gonadal recrudescence before spawning. Lower levels were observed in spawning fish. Vitellogenin (VTG) levels were highest in spawning fish. A sudden drop in estradiol-17β and VTG preceded release of the first batch of eggs. Estradiol-17β, testosterone, and VTG fluctuated with release of successive batches of eggs. Male halibut started to mature during fall and early winter, as indicated by increased testosterone and 11-ketotestosterone and abdominal swelling. Milt was first released in January and February when testosterone and 11-ketotestosterone were near maximum levels. Hence, rising levels of plasma sex steroids and VTG in fall indicate that reproductive activity is underway 1–2 mo before any noticeable swelling of the abdomen. Individual maturing halibut can be sexed by rising levels of estradiol-17β and VTG (females) and 11-ketotestosterone (males) in late fall and early winter.

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Gonadotropin (GTH) was purified from the pituitaries of the Pacific chinook salmon using a combination of stepwise ethanol precipitation and concanavalin-A affinity chromatography. The ct and /3 subunits were dissociated and fractionated by C-I 8 reverse-phase high-performance liquid chromatography with a 0.1 '10 trifluoroacetic acid/acetonitrile gradient. An enriched cDNA library was screened for the j-GTH gene( s) using two synthetic oligonucleotides based on partial protein data. A positive, full-size clone (E3) was identified and sequenced. It contains 657 base pairs and codes for a 142-amino-acid precursor protein. The mature salmon /3-GTH (1 19 amino acids) is structurally homologous to human luteinizing hormone and chorionic gonadotropin. The effect of testosterone implantation on pituitary GTH and /3-GTH mKNA was examined with radioimmunoassay and Northern blot analysis. There was a corresponding increase in both the pituitary GTH and mRNA levels.The pituitary glycoprotein hormones are a family of structurally related, subunit-containing proteins. This family includes luteinizing hormone, follicle-stimulating hormone and thyroid-stimulating hormone. In addition, the placenta produces a related horrnbne, chorionic gonadotropin. These proteins consist of two non-identical, non-covalently linked subunits, designated as CI and j chains. While all these hormones share the same CI subunit, the a chains are the hormone-specific subunits [1].In teleosts gonadotropin (GTH) is an anterior pituitary hormone required for seasonal reproductive development of fish gonads [2]. Unlike the chorionic gonadotropin, where the protein structure as well as the cDNA of the /3 subunit is known [3, 41, little information is available on the structure of fish GTH. Discrepancy exists concerning the number and sizes of fish GTH hormones as well as their biological activities [2].The pituitary concentration of GTH rises and falls annually in association with the gonadal development [5]. A number of studies have shown that androgenic and estrogenic steroid hormones markedly influence the production of GTH in the fish pituitary gland [6]. Investigations on the control of fish pituitary hormones at the molecular level is limited at the present time. As a first step to study the molecular biology of fish GTH, we have purified the salmon GTH and its subunits and cloned the j-GTH mRNA isolated from the pituitary of Pacific chinook salmon. The effect of testosterone on j-GTH mRNA level was examined.Corresporzdele,ice fo C. L. Hew, Department of Clinical Biochemistry, University of Toronto, 100 College Street, Toronto, Ontario, Canada M 5G 1 L5Abbreviations. GTH, gonadotropin; j?-GTH, subunit of gonadotropin; HPLC, high-performance liquid chromatography; SDS-PAGE, sodium dodecyl sulfate/polyacrylamide gel electrophoresis; ConA. concanavalin A; LH, luteinizing hormone; CG, chorionic gonadotropin. MATERIALS AND METHODS Collection of experimental materialsPituitary glands of fall chinook Pacific salmon, Oncorlzynchus tsclzawytscha were collect...