Science fish eggs
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As one of the 17 Sustainable Development Goals (SDGs) set forth by the United Nations, “conservation and sustainable use of ocean and marine resources” is a global challenge. In recent years, due to the decrease of natural fishery product resources because of the changes in the marine environment and the increase of global demand for fishery products, efficient and sustainable development of aquaculture technology for increasing fishery products is demanded.
In our lab, under the keyword "Science fish eggs", we are developing efficient fish resource survey techniques and biotechnology using fish, in addition to seed production technology development and breeding tests using advanced technology based on physiological basic research on fish egg formation. Through the research, we hope to contribute to the SDGs 2 ZERO HUNGER and 14 LIFE BELOW WATER.
The United Nations has designated the decade starting in 2021 as the "Decade of Ocean Science," with the aim of contributing to SDGs. Ocean science, as defined by the UN, includes the field of fisheries.
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When environmental and physiological factors are arranged, the “gonadotropin-releasing hormone” is produced from the hypothalamus, and the “gonadotropin” is produced from the pituitary gland. “Gonadotropin” acts on the cell layer that surrounds the oocyte (follicular cell layer), producing sex steroid hormone and maturation-inducing hormone, which promotes egg growth and egg maturation, leading to egg laying.
As shown above, various hormones are involved. If sexual maturation and spawning do not occur, hormones are artificially administered depending on the stage of maturation that is stopped. This is called artificial maturation.
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When administering hormones, it is important to know whether they are male or female, what stage of maturity they are in, and whether the maturation has progressed after hormone administration. “Female sex hormones” released from the follicle cells and the “oogenesis-related protein” released from the liver can be used as markers for determining sex and maturity.
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“Hormones” and “oogenesis-related protein” of fish can be detected and measured by blood tests. For example, the common female sex hormone in fish is a steroid hormone called estradiol 17β (E2), which is the same female sex hormone found in the blood of women. In this way, many steroid hormones are common to vertebrates, and commercially available kits can be used to measure the hormones. On the other hand, protein-related hormones such as gonadotropin and oogenesis-related proteins partially differ in structure depending on the species, and we have to develop detection and measurement methods for each species respectively. While there are various methods for detecting and measuring steroids and proteins in the blood, one of the cheaper and more common methods is an immunochemical measurement method using an antigen-antibody reaction.
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The first step o developing an immunochemical detection and measurement method is to prepare a highly pure protein of interest as an "immune antigen." The process of increasing the purity of a target protein from a sample containing multiple proteins is called "purification."
Example of purification of oogenesis related protein:
The yolk protein precursor is called vitellogenin (vitellogenin: Vg). The figure on the left is a flowchart of the purification of three types of Vg from the blood of a mullet (Mugil cephalus) to which female hormones have been administered. For example, in the case of VgA, we first use one POROS HQ of the ion-exchange column, followed by a negative affinity column combining the hydroxyapatite column and VgB acid, and finally one Superose 6 of the gel filtration column, which is purified through a total of four-column chromatographies.
Example of the chromatogram of gel filtration column:
Gel filtration column is a method to separate based on the differences in the molecules of protein. The figure on the left is a VgA elution chromatogram with the vertical axis indicating the absorbance value and the horizontal axis indicating the elution fraction number. Generally speaking, if you get one symmetrical peak, we think it is highly purified.
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When rabbits are injected with purified fish proteins, the rabbits recognize them as foreign substances and produce antibodies. This antibody binds to the injected protein but not to the others. A chromogenic/luminescent substrate is attached to the antibody, and the amount of binding is detected and measured as color or luminosity.
Immunochemical assay systems for yolk protein precursors:
The figure on the left shows the standard curve (luminance on the vertical axis and VtgAa concentration on the horizontal axis) and the serum dilution series (Male serum ○, serum containing VtgAa ●) of the chemoluminescence immunoassay system for the yolk protein precursor (synonymous with VtgAa = VgA) of mullet (Mugil cephalus). The higher the VtgAa, the higher the luminosity value, and antibodies don′t react with other contaminating seroproteins (male serum).
Example of measurement of yolk protein precursors:
The figure on the left shows the immunochemical measurement of three types of Vtg using mullet serum at various stages of sexual maturity. The vertical axis is Vtg concentration, and the horizontal axis is gonad weight per body weight (gonad body index: GSI). Early in sexual maturation, Vtg levels are low. When GSI exceeds 10% and Vtg levels tend to decrease as spawning approaches. Continuous monitoring of Vtg as a blood marker for maturity and egg maturation can predict the timing of spawning and hormone administrations.
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