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In vivo oocyte hydration in Atlantic halibut (Hippoglossus hippoglossus); proteolytic liberation of free amino acids, and ion transport, are driving forces for osmotic water influx

Roderick Nigel Finn^1,*, Gunn C. Østby¹, Birgitta Norberg² and Hans Jørgen Fyhn¹

¹ Department of Zoology, University of Bergen, Allégaten 41, N-5007 Bergen, Norway and
² Institute of Marine Research, Austevoll Aquaculture Station, N-5392 Storebø, Norway

View larger version (40K): [in a new window]   Fig. 1. Photomicrographs of oocytes and eggs observed in biopsies taken from Atlantic halibut, including a water-hardened egg following its incubation in sea water. The inset (below) shows three stages of hydrating oocytes in a typical biopsy. Oocytes are partially dissected and spread out in FO medium for clarity. Oogonia (black arrows on the biopsy plate), were clear, round orbs lacking yolk platelets (mean diameter 0.21±0.08 mm). The pre-hydrated oocytes (1) appeared dark in the biopsy under transmitted light due to diffraction and scattering caused by the yolk platelets. Hydrating oocytes (2) showed increased transparency with increased size, as a result of yolk platelet coalescence. The ovulated eggs (3) were fully separated from their follicular layers, and were highly transparent. The water-hardened egg shows the newly formed perivitelline space (PVS) and blastodisc resulting from cytoplasmic streaming to the animal pole upon water activation. The eggs were not fertilised.

View larger version (15K): [in a new window]   Fig. 2. Size-frequency distribution of oocytes in an ovary biopsy of Atlantic halibut (N=498, female A). PH oocyte, pre-hydrated oocytes; H oocyte, group of oocytes early in hydration; OV eggs, ovulated eggs.

View larger version (21K): [in a new window]   Fig. 3. Ovulation intervals of two female Atlantic halibut (female A, open circles; female B, closed circles) determined from wet mass (means ± S.D.) of oocytes at the time of biopsy sampling. Curves are fitted by eye, and the ovulation intervals calculated as the t (h) between the appearance of ovulated eggs in sequential biopsies.

View larger version (28K): [in a new window]   Fig. 4. Atlantic halibut oocyte water content (% wet mass), diameter and dry mass as a function of oocyte wet mass during final maturation (shaded area).

View larger version (19K): [in a new window]   Fig. 5. Osmotic pressure (means ± S.D., vertical error bars) of yolk and ovarian fluid (shaded area denotes the total range of values) as a function of oocyte wet mass (means ± S.D. of the pooled sample used for osmometry, horizontal error bars) (N=5–20) during final oocyte maturation of Atlantic halibut.

View larger version (37K): [in a new window]   Fig. 6. Atlantic halibut individual oocyte protein content (A), free amino acid content (FAA) (B) and electrophoretic protein profiles (C), as functions of oocyte and egg wet mass during final maturation (shaded area). The gel in C (7.5 % homogeneous SDS-PAGE gel, Coomassie Blue stained; molecular mass standards indicated at left) is organised relative to oocyte and egg wet mass with reference to the protein and amino acid data. The molecular masses of the oocyte and egg protein bands are identified to the right of the gel. PH oocyte, pre-hydrated oocytes; H oocyte, hydrating oocytes; OV egg, ovulated egg; WH egg, water-hardened egg.

View larger version (57K): [in a new window]   Fig. 7. Atlantic halibut oocyte content of (A) indispensable and (B) dispensable free amino acids as a function of oocyte and egg wet mass during final oocyte maturation in the two females (A, open circles; B, closed circles). Leu, leucine; Lys, lysine; Val, valine; Ile, isoleucine; Thr, threonine; Arg, arginine; Met, methionine; Phe, phenylalanine; Trp, tryptophan; Ala, alanine; Ser, serine; Gln, glutamine; Glu, glutamate; Gly, glycine; Asn, asparagine; Asp, aspartate; Pro, proline; Tyr, tyrosine; Tau, taurine.

View larger version (22K): [in a new window]   Fig. 8. Amino acid profiles for the pre-hydrated oocytes (PH oocyte) and ovulated eggs (OV egg) of Atlantic halibut. Values are means ± S.D. (N=25 for PH oocyte, N=24 for OV egg). Amino acid abbreviations as in Fig. 7.

View larger version (26K): [in a new window]   Fig. 9. Atlantic halibut individual oocyte contents (nmol) of chloride (Cl–), potassium (K+), inorganic phosphate (Pi) and sodium (Na+) as a function of oocyte and egg wet mass during final oocyte maturation (shaded area). OV egg, ovulated egg; WH egg, water-hardened egg.

View larger version (33K): [in a new window]   Fig. 10. Solute concentrations (mmol kg–1 H2O) in the hydrating oocytes of Atlantic halibut as calculated from solute contents (nmol oocyte–1, Figs 6, 9) and water content (mg oocyte–1, from Fig. 4) as a function of oocyte and egg wet mass. The curves are polynomials. Data for NH4+ ions refer to the present oocyte material, as published in Terjesen et al. (2001). solutes, sum of measured solutes; FAA, free amino acids.

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