Tadashi Andoh

Multiple Vitellogenins (Vgs) in Mosquitofish (Gambusia affinis): Identification and Characterization of Three Functional Vg Genes and Their Circulating and Yolk Protein Products

Abstract The objectives of this study were to characterize multiple forms of vitellogenin (Vg) in mosquitofish (Gambusia affinis) and to discover the fate of each Vg during its processing into product yolk proteins. Two Vg preparations, with apparent masses of 600 kDa (600 Vg) and 400 kDa (400 Vg), were isolated from the plasma of fish treated with estradiol-17β (E2) by various chromatographic procedures. Immunological analyses verified the presence of two different Vg proteins (600 VgA and 600 VgB) in the 600 Vg preparation and of a single protein in the 400 Vg preparation. Three major yolk proteins (Yps) with apparent masses of 560, 400, and 28 kDa were observed in extracts of ovarian follicles from vitellogenic females. Immunological analyses demonstrated that the 400 Vg underwent no change in native mass after being incorporated into oocytes. The 600 Vgs gave rise to a 28 kDa β′-component and a native 560 kDa Yp, which was heterodimeric in structure, consisting of two types of complexes between phosvitin (Pv) and lipovitellin (Lv) heavy- and light-chains. Full-length cDNAs encoding the 600 VgA, 600 VgB, and 400 Vg were isolated from a liver cDNA library of E2 treated fish. Similar to the zebrafish vg3 gene, the 400 Vg cDNA lacked a Pv domain and was classified as an incomplete or phosvitinless (C-type) Vg. The deduced primary structures of 600 VgA and 600 VgB were complete, and these were categorized as type A and type B Vgs, respectively, according to our recent classification scheme. This is the first report on the characterization of three functional Vg genes and their circulating and yolk protein products in any vertebrate species.

Sperm Attractant in the Micropyle Region of Fish and Insect Eggs

ABSTRACT In some animals, such as fish, insects, and cephalopods, the thick egg coat has a narrow canal—a micropyle—through which spermatozoa enter the eggs. In fish, there is no indication that spermatozoa are attracted by eggs from a distance, but once spermatozoa come near the outer opening of the micropyle, they exhibit directed movement toward it, suggesting that a substance exists in this defined region to attract spermatozoa. Since Coomassie Blue (CB) binds preferentially to the micropyle region in flounder, herring, steelhead, and other fish, it probably stains this sperm guidance substance. This substance—a glycoprotein based on lectin staining—is bound tightly to the surface of the chorion, but can be removed readily by protease treatment. Although fertilization in fish (flounder) is possible after removal of this substance, its absence makes fertilization inefficient, as reflected by a drastic reduction in fertilization rate. The sperm “attraction” to the micropyle opening is species specific and is dependent on extracellular Ca2+. Eggs of some insects, including Drosophila, have distinct micropyle caps with CB affinity, which also may prove to assist sperm entry. Our attempts to fertilize fly eggs in vitro were not successful.

Chemical and physical guidance of fish spermatozoa into the egg through the micropyle

Abstract Eggs of teleost fish, unlike those of many other animals, allow sperm entry only at a single site, a narrow canal in the eggs chorion called the micropyle. In some fish (e.g., flounder, herring, and Alaska pollock), the micropyle is a narrow channel in the chorion, with or without a shallow depression around the outer opening of micropyle. In some other fish (e.g., salmon, pufferfish, cod, and medaka), the micropyle is like a funnel with a conical opening. Eggs of all the above fish have a glycoprotein tightly bound to the chorion surface around the micropyle. This glycoprotein directs spermatozoa into the micropylar canal in a Ca2+-dependent manner. This substance, called the micropylar sperm attractant or MISA, increases fertilization efficiency and is essential in herring. In flounder, salmon, and perhaps medaka, fertilization is possible without MISA, but its absence makes fertilization inefficient because most spermatozoa swim over the micropyle without entering it. The mechanism underlying sperm-MISA interactions is yet to be determined, but at least in herring the involvement of Ca2+ and K+ channel proteins, as well as CatSper and adenylyl cyclase, is very likely. In some other fish (e.g., zebrafish, loach, and goldfish), the chorion around the micropyle is deeply indented (e.g., zebrafish and loach) or it has radially or spirally arranged grooves around the outer opening of the micropyle (e.g., goldfish). MISA is absent from the eggs of these fish and sperm entry into micropylar canal seems to be purely physical. Summary Sentence In fish, sperm entry into the egg through the micropyle is guided either chemically or physically depending on the species

The use of poly-L-lysine to facilitate examination of sperm entry into pelagic, non-adhesive fish eggs.

The fish egg is surrounded by a thick envelope called the chorion. The fertilizing spermatozoon enters the egg through a canal-like structure in the chorion, the micropyle. Examination of micropyle at fertilization is difficult if eggs are large and have no distinct landmarks surrounding the micropyle, or if they are positively buoyant in water. Eggs of many commercially important fishes (e.g., flounder, sea bream and eel) are buoyant in water or only slightly adhere to solid objects (e.g., sands, rock and water plants), which makes observation of spermatozoa at fertilization difficult. Here, we report that such eggs can be firmly attached to plastic and glass dishes that have been previously coated with poly-L-lysine. These adhering eggs can be fertilized and develop normally on the dishes. Observations of micropyles of three fish species, before and after sperm entry are presented.

Multiple molecular forms of glucagon and insulin in the kaluga sturgeon, Huso dauricus

Five molecular forms of glucagon and two molecular forms of insulin were characterized from the kaluga sturgeon. Substitutions occurred at two to thirteen internal amino acid residues among the five molecular forms of glucagons, indicating that these glucagons were encoded by five distinct genes. The amino acid sequences of two insulins from the kaluga sturgeon were identical to those of paddlefish insulin-II and Russian sturgeon insulin except that kaluga sturgeon insulin-I had an extension of five residues at the B-chain N-terminus. This is the first demonstration that more than two molecular forms of glucagon have been characterized from a single animal species.

Development of a widely applicable immunoassay for insulin in marine teleosts that regulates cross-reactivity using biotinylation and inhibits interference by plasma components

Amino acids are important insulinotropins in fish, and their effects vary between amino acids and fish species. Insulin levels are indicative of growth efficiency and stress levels in fish; however, interspecies comparisons of insulin levels are hampered by the difficulty of measuring insulin concentration in each fish. We developed a widely applicable competitive immunoassay using biotinylated yellowtail (Seriola quinqueradiata) insulin for measuring insulin in marine teleosts, including yellowtail and red seabream (Pagrus major), which are the most common species raised by aquaculture in Japan. Amino acid sequence substitution was limited at the ninth residue of the A-chain (A9) between these two species, and analysis of the primary structures of insulins from six phylogenetically far teleosts suggested that the sequences of yellowtail and red seabream insulins are identical to those of many teleosts, except the A9 residue. However, A9 is known to be an epitope that confers cross-reactive differences on insulin. We solved this problem through immunoreactive invalidation of this residue by biotinylation. The binding-inhibition curves of yellowtail and red seabream insulins were identical following the use of this technique. However, yellowtail and red seabream plasma was found to contain components that interfere with immunoassays. This problem was solved by the extraction of plasma using equal volume of acid-ethanol in yellowtail and by cooling at 0°C during the cross-reaction between the ligand and antibody in red seabream. Serially diluted plasma samples from both species exhibited linearity after these treatments. In a recovery test using plasma with added yellowtail insulin, the average recovery varied from 96.2% to 109.4%. A post-feeding rise in insulin was confirmed by this immunoassay in yellowtail, and peak of the rise was 39.8±7ng/ml at 1h postfeeding from 3.9±1.1ng/ml at 0h. This indicates that this assay is sufficient for measuring the baseline concentration of plasma insulin after starvation and is a useful indicator of nutritional status in yellowtail, as in other teleosts. This immunoassay demonstrated high performance and resisted interference from plasma components; consequently, it constitutes a useful tool for the interspecies evaluation of insulinotropins and represents a widely applicable insulin immunoassay for many teleosts.

Effects of different green light intensities on the growth performance and endocrine properties of barfin flounder Verasper moseri

We previously reported that the somatic growth of barfin flounder, Verasper moseri, was effectively stimulated by the green light compared to the blue and red lights. Herein, we report the effects of different green light intensities on the growth and endocrine system of the fish. Fish were reared in a dark room with light from a light-emitting diode (LED) at a peak wavelength of 518nm under controlled photoperiod (10.5:13.5h, light:dark cycle; 06:00-16:30, light) with three levels of photon flux density (PFD)-2 (low), 7 (medium), or 21 (high) μmol·m-2·s-1 at the water surface. The average water temperature was 10.2°C, and the fish were fed until satiety. The fish reared under high PFD of green light showed the highest specific growth rates, followed by the medium PFD group. Under high PFD, the fish showed the highest amount of melanin-concentrating hormone mRNA in their brains and insulin in plasma, while the lowest amount of growth hormone was observed in their pituitary glands. These results suggest that the green light stimulated the growth of barfin flounders in a light intensity-dependent manner in association with their central and peripheral endocrine systems. However, when the fish were reared in an ordinary room where they received both ambient and green LED lights, the fish under LED and ambient light grew faster than those under ambient light only (control). Moreover, no difference was observed in the specific growth rate of the fish reared under the three different green LED light intensities, suggesting that the growth was equally stimulated by the green light within a certain range of intensities under ambient light.