Eri Shiraishi

High temperature causes masculinization of genetically female medaka by elevation of cortisol

In poikilothermic vertebrates, sex determination is sometimes influenced by environmental factors such as temperature. However, little is known about the molecular mechanisms underlying environmental sex determination. The medaka (Oryzias latipes) is a teleost fish with an XX/XY sex determination system. Recently, it was reported that XX medaka can be sex‐reversed into phenotypic males by high water temperature (HT; 32–34°C) treatment during the sex differentiation period. Here we report that cortisol caused female‐to‐male sex reversal and that metyrapone (an inhibitor of cortisol synthesis) inhibited HT‐induced masculinization of XX medaka. HT treatment caused elevation of whole‐body levels of cortisol, while metyrapone suppressed the elevation by HT treatment during sexual differentiation. Moreover, cortisol and 33°C treatments inhibited female‐type proliferation of germ cells as well as expression of follicle‐stimulating hormone receptor (fshr) mRNA in XX medaka during sexual differentiation. These results strongly suggest that HT induces masculinization of XX medaka by elevation of cortisol level, which, in turn, causes suppression of germ cell proliferation and of fshr mRNA expression. Mol. Reprod. Dev. 77: 679–686, 2010.

Estrogen rescues masculinization of genetically female medaka by exposure to cortisol or high temperature

Medaka (Oryzias latipes) is a teleost fish with an XX/XY sex determination system. Recently, it was reported that XX medaka can be sex‐reversed into phenotypic males by exposure to high water temperature (HT) during gonadal sex differentiation, possibly by elevation of cortisol, the major glucocorticoid produced by the interrenal cells in teleosts. Yet, it remains unclear how the elevation of cortisol levels by HT causes female‐to‐male sex reversal. This paper reports that exposure to cortisol or HT after hatching inhibited both the proliferation of female‐type germ cells and the expression of ovarian‐type aromatase (cyp19a1), which encodes a steroidogenic enzyme responsible for the conversion of androgens to estrogens, and induced the expression of gonadal soma‐derived growth factor (gsdf) in XX gonads during gonadal sex differentiation. In contrast, exposure to either cortisol or HT in combination with 17β‐estradiol (E2) did not produce these effects. Moreover, E2 completely rescued cortisol‐ and HT‐induced masculinization of XX medaka. These results strongly suggest that cortisol and HT cause female‐to‐male sex reversal in medaka by suppression of cyp19a1 expression, with a resultant inhibition of estrogen biosynthesis. This mechanism may be common among animals with temperature‐dependent sex determination. Mol. Reprod. Dev. 79: 719–726, 2012.

High-Performance Pulsed-Power Generator Controlled by FPGA

The high reliability, high repetition rate, high performance, and compactness of pulsed-power generators are required for industrial applications. Also, the control of a pulsed-power generator becomes more complicated with increasing functions. An all-solid-state pulsed-power generator can be controlled by using a field-programmable gate array (FPGA). The pulsed-power generator consists of a charger, a magnetic pulse compression circuit, and a controller using the FPGA. The performance characteristics of the pulsed-power generator, such as the variable firing interval from shot to shot and the diagnosis of incorrect operation, are easily achieved by rewriting the programming of the Verilog hardware description language on the FPGA.

Identification of two teleost homologs of the Drosophila sex determination factor, transformer-2 in medaka (Oryzias latipes)

Transformer-2 (Tra2), an RNA-binding protein, is an important regulator in Drosophila sex determination. In vertebrates, however, the role of Tra2 homologues is not known. We identified two teleost homologues of Tra2, which we named Tra2a and Tra2b, in medaka (Oryzias latipes). Furthermore, we demonstrated that both Tra2 mRNAs were predominantly expressed in germ cells of both sexes before the onset of sex differentiation, suggesting that both Tra2 homologues might be involved in the sex differentiation in medaka.

Nanosecond Pulsed Electric Field Suppresses Development of Eyes and Germ Cells through Blocking Synthesis of Retinoic Acid in Medaka (Oryzias latipes)

Application of nanosecond pulsed electric fields (nsPEFs) has attracted rising attention in various scientific fields including medical, pharmacological, and biological sciences, although its effects and molecular mechanisms leading to the effects remain poorly understood. Here, we show that a single, high-intensity (10–30 kV/cm), 60-ns PEF exposure affects gene expression and impairs development of eyes and germ cells in medaka (Oryzias latipes). Exposure of early blastula stage embryos to nsPEF down-regulated the expression of several transcription factors which are essential for eye development, causing abnormal eye formation. Moreover, the majority of the exposed genetic female embryos showed a fewer number of germ cells similar to that of the control (unexposed) genetic male at 9 days post-fertilization (dpf). However, all-trans retinoic acid (atRA) treatment following the exposure rescued proliferation of germ cells and resumption of normal eye development, suggesting that the phenotypes induced by nsPEF are caused by a decrease of retinoic acid levels. These results confirm that nsPEFs induce novel effects during embryogenesis in medaka.

Single Nanosecond Pulsed Electric Field Effects on Embryonic Development of the Medaka Fish

The effects of nanosecond pulsed electric field (ns PEF) on the egg development of the medaka fish are investigated. A pulsed power modulator using a magnetic pulse compression circuit was employed to generate pulses of 0.5-20 kV pulses. Fertilized eggs of strain d-rR medaka were used. The ages of the experimental eggs were new laid, 24-h postfertilization, and 48-h postfertilization. Propidium iodide and fluorescein isothiocyanate (FITC)-dextran conjugate, and a fluorescence microscope were used to study the effects of ns PEF on the blastomeres and the extent of egg structure damage. The FITC was injected to experimental eggs by a microinjection system, which can deliver nanoliters of FITC without producing any damage to the egg structure. Each egg was set at the middle of a 2- or 4-mm cuvette, and a single pulse was applied. The control eggs were sham treated. After the pulse application, the eggs were observed under the fluorescence microscope until they hatched or died. By applying low electric field pulses (10 kV/cm), the effects were minimal and temporary. These eggs could recover and grow as fishes. By increasing the electric field (20 kV/cm), embryos showed abnormal growth with deformed body structure or missing organs. For higher electric fields (over 30 kV/cm), immediate extended damage were observed. The research was motivated by its application in regenerative medicine to control embryonic-stem-cell differentiation and proliferation.

Influence of 60 ns pulsed electric fields on embryonic stem cells

Influence of pulsed electric fields on mice embryonic stem cells was investigated. The pulsed power system used in this experiment had a pulse width of 60 ns, with an electric field varying from 10 to 30 kV/cm, a current varying from 200 to 600 amperes and a repetition rate of 1 pulse per second. Results show that the pulsed electric field suppresses ES cell proliferation, while the undifferentiated marker expression is retained during proliferation.

Single nanosecond pulsed power induced structural modifications of medaka fish embryo

The effects of nanosecond pulsed electric field on the medaka fish egg development in low and high conductivity mediums are investigated. A pulsed power modulator using a magnetic compression circuit (MPC) was employed to generate 0.5 to 20 kV pulses with 50 nanosecond pulse duration. Input voltage and current were measured by using a high voltage probe, a current monitor, and an oscilloscope. Fertilized eggs of strain d-rR medaka were used. The ages of the experimental eggs were 1, 6, and 48 hours post fertilization (hrPF). Fluorescein isothiocyanate (FITC)-Dextran conjugates with different molecular weights and a florescence microscope were used to study the effects of the pulsed electric field on the blastomeres and the extend of eggs structure damage. The FITCs were injected to experimental eggs by a micro-injection system, which can deliver nano-liters of FITC without producing any damage to egg structure. In group A, 10 ng of FITC was injected to blastomere of eggs at 1 hrPF. In group B, 30 ng of FITC was injected to yolk sphere at 1 hrPF. Group C eggs were kept as control. After injection, each egg was set at the middle of a 2 mm or 4 mm cuvette and a single electric pulse was applied. The control eggs were sham treated. After pulse application the eggs were observed under the fluorescent microscope until they hatched or died. By applying low electric field pulses (up to 10 kV/cm) in 17 mS/cm medium (PBS), the eggs showed small size temporary pore formation. These eggs could recover and grew as fish. By increasing the electric field (10 to 20 kV/cm) more FITC flow from blastomere were observed, embryos showed abnormal growth with deformed body structure or missing eyes. For higher electric fields (over 30 kV/cm) immediate extended damage were observed.