Estrogen-induced and suppressed gene expression during XY sex reversal in a teleost fish, tilapia

To identify the down-stream gene products of estrogen during ovarian differentiation, we performed subtractive hybridization using mRNA derived from normal and estrogen treated XY gonads. The clones obtained after subtractive hybridization screening were re-screened by reverse-Northern analysis. Finally seven up-regulated and three down-regulated gene products were obtained.     

Use of an automatic cell-counting system with LED illumination for enumeration of marine bacteria

ABSTRACT:   The concentration of aquatic bacteria is basic information required to evaluate the status of environments and to assess bacterial contribution to material cycles. However, the standard direct counting method using epifluorescence microscopy (EFM) is tedious and there is variation in the counts among workers. Here an automatic counting system that consists of Bioplorer (BP) and image analysis has been applied to marine bacteria. BP is composed of a light-emitting diode (LED) illuminant, an optical unit, a driving stage and a charge-coupled device camera. In combination with fluorescent labeling and simplified membrane filtration, bacteria are enumerated automatically. The reproducibility, sensitivity and accuracy of the system were tested for natural marine bacteria, in comparison with EFM and flow cytometry (FCM). The counts obtained by BP showed good correlation with those obtained by EFM and FCM methods. The counts were significantly higher in inshore and oceanic samples, indicating high sensitivity with low background noise. Considering its reproducibility, objectivity, ease of use and compact size, BP can be used as a routine tool for counting aquatic bacteria in substitution for EFM or FCM.     

Diurnal rhythm of serum steroid hormone levels in the Japanese whiting,Sillago japonica,a daily-spawning teleost

Ovarian developmental stages and serum steroid hormone levels were examined at six different times of day (0100, 0600, 1000, 1300, 1600, 2000 h) in a marine teleost, the Japanese whiting Sillago japonica, which has an asynchronous-type ovary containing oocytes at various stages of development and spawns every day during a period ranging up to three months. The largest oocytes in the ovaries at the active vitellogenic or post-vitellogenic stages were found between 0100 and 1300 h. Oocyte maturation indicated by germinal vesicle breakdown (GVBD) occurred at 1600 h, and ovulated oocytes were observed in the ovaries collected at 2000 h. These processes were accompanied by a significant daily change in serum steroid hormone levels. The serum level of estradiol-17β showed a peak in fish with mature oocytes sampled at 1600 h. In these fish, the second-largest oocytes in the ovaries were at the initial stage of vigorous vitellogenesis, the secondary yolk stage. Therefore the highest level of serum estradiol-17β was considered to be due to the second-largest oocytes. Testosterone levels remained low and constant throughout the experimental period. The serum levels of 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-diOHprog) peaked at 1600 h at which time all fish had mature oocytes. These results indicate that the Japanese whiting possesses a diurnal rhythm of oocyte development including vitellogenesis, oocyte maturation and ovulation, and further suggest that daily cycles in oocyte growth and maturation which simultaneously take place in an ovary are regulated by diurnal secretions of estradiol-17β and the maturation-inducing steroid, 17α,20β-diOHprog.     

Molecular cloning of the three gonadotropin subunits and early expression of FSHβ during sex differentiation in the nile tilapia, Oreochromis niloticus

Gonadotropin (GTH)α, FSHβ and LHβ cDNAs were cloned from the Nile tilapia. Northern blot analysis detected a single band for each subunit. Preliminary studies indicate that FSHβ is expressed as early as 0 days after hatching (dah) in the fish pituitaries.     

Application of NaLA,a fishing net configuration and loading analysis system,to bottom gill nets

ABSTRACT:   The net-shape and loading analysis system (NaLA) was developed to determine fishing net configuration and load in a previous study. The system has since been applied to general gill nets and aquaculture nets, and its validity has been proven through model experiments in tanks. In this study, the system was applied to estimate the dynamic behavior of a bottom gill net for walleye pollock, to test the system's applicability of the system to gear operations in the field. To obtain in situ data, four bottom gill net operations were performed in February 2004 off the coast of Sawara, Hokkaido, Japan. During operations, vertical displacements of the bottom gill net's float and sinker lines were measured as representative values of gear behavior, and ocean current direction, and speed at the gear position were observed simultaneously. Then, bottom gill net behavior was simulated using NaLA, incorporating observed environmental conditions and gear specifications. The resulting calculated behavior was compared to measured behavior in terms of the relationship between net height and environmental or setting conditions. Agreement between the calculated and measured net behavior was found. Thus, it is believe that our NaLA calculation model has the potential to simulate the dynamic behavior of bottom gill nets in situ .     

Germ cells during gonadal sex differentiation in the teleost

We reported previously that two isoforms of vasa mRNA and protein are present in tilapia (Kobayashi et al. 2002). One (vas-s) lacks a part of the N-terminal region found in the other isoform (vas). Both isoforms are expressed in oocytes through the embryonic stages when PGCs localize in the lateral plate mesoderm. After PGCs localized in the gonadal anlagen, vas-s expression increased and vas expression became undetectable. Expression of both isoforms was observed again after morphological gonadal sex differentiation, irrespective of genotypic sexes. These changes are in accordance with sex differences in number of gonial germ cells during this period, suggesting that molecular sexual dimorphism occurs in germ cells between both sexes during this period.     

Mechanisms of synthesis and action of 17α,20β-dihydroxy-4-pregnen-3-one,a teleost maturation-inducing substance

This article briefly reviews the current status of investigations, mainly based on the amago salmon,Oncorhynchus rhodurus, on the mechanisms of synthesis and action of 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-diOHprog). Pituitary gonadotropin is of primary importance in triggering meiotic maturation in teleost oocytes. However, the maturational action of gonadotropin is not direct, but is mediated by the follicular production of maturation-inducing substance (MIS). It is now well established that 17α,20β-diOHprog is the major MIS of salmonids. Production of this steroid occursvia the interaction of two distinct cell layers, the thecal and granulosa cell layers (2-cell type model). The first step of the stimulating effect of gonadotropin in both layers is the receptor-mediated activation of adenylate cyclase and formation of cAMP. Our findings suggest that the major stimulating action of gonadotropin on 17α,20β-diOHprog biosynthesis is due to the stimulation of 17α-hydroxyprogesterone production by the thecal layer and the selective induction of thede novo synthesis of 20β-hydroxysteroid dehydrogenase in the granulosa layer. 17α,20β-diOHprog acts at the surface of the oocyte. The early steps following 17α,20β-diOHprog action involve the formation of the major cytoplasmic mediator of this steroid, maturation-promoting factor (MPF). It was shown that goldfish MPF induces meiotic maturation inXenopus oocytes andvice versa. The chemical characterization of fish MPF is important for our understanding of the precise mode of maturational action of 17α,20β-diOHprog.     

Specific binding of [3H]17α,20β-dihydroxy-4-pregnen-3-one to oocyte cortices of rainbow trout (Oncorhynchus mykiss)

Specific binding of [³H]17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP) to plasma membranes prepared from defolliculated oocytes of rainbow trout (Onchorhynchus mykiss) was identified and characterized. Binding was rapid and reached equilibrium in 30 min. 17α,20β-DP strongly inhibited [³H] 17α,20β-DP binding in a competitive manner. Scatchard analysis revealed two different binding sites: a high affinity binding site with a K_d of 18 nM and a B_max of 0.2 pmoles/mg protein; and a low affinity binding site with a K_d of 0.5 μM and a B_max of 1 pmoles/mg protein. This binding activity was successfully solubilized with n-heptyl-β-D-thioglucoside. [³H]17α,20β-DP binding to solubilized preparations reached equilibrium in 1h, and was competitively inhibited with 17α,20β-DP and 17α,20β,21-trihydroxy-4-pregnen-3-one. However, Scatchard analysis showed a single binding site with a K_d of 0.3 μM. The reason for the disappearance of the high affinity binding site in solubilized preparations remains unclear. These results demonstrate that a specific binding site for 17α,20β-DP exists in the plasma membrane of rainbow trout oocytes.

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Résumé Une liaison spécifique de le [³H]17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP), avec des membranes plasmiques d'ovocytes défollicularisés de truite arc-en-ciel (Onchorhynchus mykiss), a été identifiée et caractérisée. Sa cinétique est rapide et atteint son équilibre en 30 minutes. Le 17α,20β-DP inhibe fortement, et de manière compétitive, la liaison de la [³H] 17α,20β-DP. Une étude de Scatchard a mis en évidence deux sites diffŕents de liaison: un site de forte affinité, de K_d 18 nM et de B_max 0,2 pmoles/mg de protéine; et un site de faible affinité, de K_d 0,5 μM et de B_max 1 pmoles/mg de protéine. L'activité de liaison a été solubilisée, avec succés, par le n-heptyl-β-D-thioglucoside. Dans la fraction soluble, la liaison de le [³H]17α,20β-DP atteint un équilibre en 1h.; et elle est complétement inhibiée par la 17α,20β-DP et le 17α,20β,21-trihydroxy-4-pregnen-3-one. Cependant, une étude de Scatchard ne permet de déceler qu'un seul site de liaison, de K_d 0,3 μM. La disparition du site de liaison de forte affinité dans la fraction soluble reste inexpliquée. Ces résultats démontrent l'existence d'un site spécifique de liaison du 17α,20β-DP dans les membranes plasmiques des ovocytes de truite arc-en-ciel.

     

Steroidogenic shift is a critical event for ovarian follicles to undergo final maturation

The steroidogenesis in the granulosa-thecal layers of fish ovarian follicles undergo a distinct shift from the production of estradiol-17β (E₂) to 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP, maturation-inducing hormone, MIH) prior to meiotic maturation. This review attempts to explain the underlying mechanisms of steroidogenic shift.