Takashi Iwamatsu

Production of transgenic fish: introduction and expression of chicken δ-crystallin gene in medaka embryos

To produce a model of transgenic fish, recombinant plasmids containing chicken delta-crystallin gene were microinjected into the oocyte nucleus of a small teleost, medaka (Oryzias latipes). About 50% of the microinjected oocytes developed to 7-day-old embryos. By Southern blotting delta-crystallin gene was detected in 4 of 8 embryos, and, by Western blotting, delta-crystallin polypeptides in 5 of 16. In 1 of 6 examined histologically, delta-crystallin DNA was detected in all the tissues, and delta-crystallin polypeptides, in many of the tissues including the lens. Thus, the exogenous gene and/or its products were detected in 10 of 30 embryos examined. This is the first report of successful production of transgenic fish.

The Wave Pattern of Free Calcium Release Upon Fertilization in Medaka and Sand Dollar Eggs

A transient rise in the concentration of Ca2+ in the cortex upon fertilization was demonstrated in medaka eggs injected with aequorin. Detection of the aequorin luminescence with an ultra‐high sensitivity photonic microscope system revealed a wave of increased Ca2+ concentration starting at the site of sperm entry (animal pole) and being propagated along the cortex of the egg toward the antipode. The wave traversed the entire egg surface within 2–3 min. The peak value of the aequorin luminescence, and therefore the peak value of the Ca2+ transient, was generally higher at the site of sperm entry than in other regions. The peak values of the luminescence (and therefore of the Ca2+ concentration in the cortex) remained fairly constant during propagation of the wave. Microinjection of Ca2+ into the cortex also induced a Ca2+ wave. When the egg was stimulated by microinjection of Ca2+ at the equatorial region, the Ca2+ wave was propagated at a fairly constant speed over the egg surface, except at the region near the vegetal pole where the wave was retarded. Simultaneous recording of the Ca2+ wave and the wave of cortical change (breakdown of cortical alveoli) in eggs during fertilization revealed that the Ca2+ wave preceded the wave of cortical change.

Development of the steroidogenic capacity of medaka (Oryzias latipes) ovarian follicles during vitellogenesis and oocyte maturation.

Developmental changes in the steroidogenic capacity of medaka, Oryzias latipes, ovarian follicles at 12 different stages during vitellogenesis and oocyte maturation were examined using 18-hr incubations. Medaka were acclimated to conditions of 26 degrees on a lighting regime of 14 hr light and 10 hr dark. Under these conditions, females usually spawn daily within 1 hr of the onset of light. The process of vitellogenesis and oocyte maturation occurs within 72 hr, the breakdown of the germinal vesicle (GVBD) and ovulation being completed at 6 and 1 hr, respectively, before the expected time of spawning. Vitellogenic follicles between 32 and 16 hr before spawning produced large amounts of estradiol-17 beta spontaneously and in response to partially purified chum salmon gonadotropin (SGA) or pregnant mares serum gonadotropin (PMSG). However, postvitellogenic follicles between 12 and 4 hr before spawning showed very little evidence of estradiol-17 beta production. By contrast, basal concentrations of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHprog) remained very low in follicles during vitellogenesis and were elevated in those collected during oocyte maturation; there was a close relationship between the medium concentration of 17 alpha,20 beta-diOH-prog and the percentage GVBD in the oocytes. 17 alpha,20 beta-DiOHprog production in response to PMSG was very low in follicles during early and mid-vitellogenesis and increased in those collected at 28 hr before spawning, a time which coincided with the first acquisition of the ability of the follicles to undergo maturation in response to gonadotropin. These results clearly demonstrate that a distinct shift from the secretion of predominantly estradiol-17 beta to the secretion of 17 alpha,20 beta-diOHprog occurs in the medaka ovarian follicle immediately prior to oocyte maturation. Considering the potency of 17 alpha,20 beta-diOHprog for the induction of oocyte maturation in vitro, these results further suggest that 17 alpha,20 beta-diOHprog is a naturally occurring steroidal mediator of oocyte maturation in the medaka.

Influence of follicular development on steroid production in the medaka (Oryzias latipes) ovarian follicle in response to exogenous substrates.

Changes in the capacity of medaka, Oryzias latipes, ovarian follicles to convert exogenous 17 alpha-hydroxyprogesterone (17 alpha-OHprog) or testosterone to testosterone, estradiol-17 beta, and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHprog) were examined using 18-hr incubations. Under a constant long photoperiod (14 hr light-10 hr dark) at 26 degrees medaka spawn daily within 1 hr of the onset of light. Under these conditions, vitellogenesis and oocyte maturation of individual follicles occur within 72 hr, allowing accurate determination of the time of oocyte maturation and ovulation. In the absence of substrates, vitellogenic follicles isolated between 28 and 16 hr before spawning produced increased amounts of estradiol-17 beta, while postvitellogenic follicles between 14 and 8 hr produced a large amount of 17 alpha,20 beta-diOHprog. However, under the same conditions, testosterone levels were very low in follicles from all stages of development. The capacity of follicles to produce estradiol-17 beta in response to 17 alpha-OHprog or testosterone increased as follicles developed from the early to late vitellogenic stage, but declined during oocyte maturation. Maximum estradiol-17 beta production was observed in follicles at 20 hr before spawning. In contrast, the conversion of 17 alpha-OHprog to 17 alpha,20 beta-diOHprog was low in vitellogenic follicles and increased in follicles isolated immediately prior to or during oocyte maturation, with maximum 17 alpha,20 beta-diOHprog production occurring in follicles isolated at 14 hr before spawning. These results demonstrate a distinct shift in the activities of steroidogenic enzymes from C17-20 lyase, 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD), and aromatase to 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) occurring in the medaka ovarian follicles immediately prior to oocyte maturation.

Time Sequence of Early Events in Fertilization in the Medaka Egg

The time sequence of early events in fertilization was examined in eggs of the medaka Oryzias latipes. The mean time after insemination required for sperm attachment to the egg surface through the micropyle depended on sperm concentrations. It was 3 ± 1 sec with a range from 1 to 6 sec after insemination when concentration of spermatozoa was high (about 2 × 108/ml at 23°–25°C). The mean time from sperm attachment until cessation of its movement on the egg surface was 4 ± 1 sec with a range from 1 to 9 sec. Small cortical alveoli at the animal pole region within 15 μm of the sperm attachment point began to undergo exocytosis 9 ± 0.3 sec (range 5–16 sec) after sperm attachment. The velocity at which the exocytosis wave propagated increased from the earliest initiation point of exocytosis up to the 100 μm area, and became constant at about 12 μm/sec from 100 μm to 500 μm from the sperm attachment point. The present results suggest that at the time of fertilization in the fish egg, exocytosis of small cortical alveoli in the area about 15 μm away from the sperm attachment point occurs simultaneously.

Sex Reversal in the Medaka Oryzias latipes by Brief Exposure of Early Embryos to Estradiol-17β

Abstract To induce sex reversal of male to female, freshly-fertilized eggs of the S-rR strain medaka (Oryzias latipes) were immersed in saline containing estradiol-17β (E2) in different concentrations for various durations until hatching. Results of the present experiment showed that the immersion duration in 1 μg/ml E2 to induce 100% reversal of sex differentiation in the genotypic males was enough only for one day (24 hr) post-fertilization (dpf) and that treatment with E2 for 1 dpf resulted in a dose-dependent manner with the maximum sex reversal of 100% at 1 μg/ml. To ascertain early developmental periods efficacious for inducing sex reversal, additional brief immersion treatments of eggs with E2 were further performed individually for four different early developmental periods (Stages 4–9, 10–12, 13–15 and 16–18) within 1 dpf. As a result, induction of sex reversal was observed in all these short immersion periods without any restricted efficacy. Between both experimental and control groups treated with or without E2 for 1 dpf, differences in the number of germ cells in a gonad were compared in newly-hatched fry. It was found that gonads of the genotypic males (XY) treated with E2 revealed the female type which contained many germ cells with much dividing activity. These data suggest that a possible switch mechanism that exogenous E2 could trigger to change the genetic cascades involved in sex determination upon fertilization exists in early developmental stages.

Cytoplasmic Ca2+ release induced by microinjection of Ca2+ and effects of microinjected divalent cations on Ca2+ sequestration and exocytosis of cortical alveoli in the medaka egg

Intracellular release of Ca2+ by microinjection of Ca2+ was analyzed by measuring the luminescence of aequorin loaded in eggs of the medaka (Oryzias latipes). Microinjection of Ca2+ into the cortical cytoplasm induced propagative waves of cytoplasmic Ca2+ release and exocytosis of cortical alveoli initiated at the injection point. The Ca2+ wave was initiated with a time lag after some was sequestered at the region of the microinjection. Microinjection of Mg2+ or Mn2+ failed to trigger Ca2+ release and exocytosis. When the aequorin-loaded eggs were inseminated after microinjection of Mg2+, Mn2+, or Co2+ into a restricted region of the vegetal hemisphere, the wave of Ca release was propagated through the injected region toward the vegetal pole, but neither Ca sequestration (fall in Ca-aequorin luminescence) nor exocytosis occurred at the area of cortex where the eggs were injected with these divalent cations. These results suggest that a significant period is required to induce Ca2+ release from cytoplasmic stores by the increased Ca2+ concentration and that both the phenomena of Ca2+ release and Ca sequestration are involved in the process of exocytosis.

CHANGES IN THE CHORION AND SPERM ENTRY INTO THE MICROPYLE DURING FERTILIZATION IN THE TELEOSTEAN FISH, ORYZIAS LATIPES

Specific antibodies against the major chorionic glycoproteins (ZI1 ‐2 and ZI3) of unfertilized eggs were used to analyze the differences in the chorion and its surrounding constituents before and after fertilization. The glycoproteins in the inner layers of the chorion and its surrounding material were specifically stained by both of the antibodies. Thirty and 60 min after activation, the thickness of the chorions inner layers was already reduced and the micropylar canal was closed. At the same time, the broadly diluted mucous area (DMA) of glycoproteins on the outermost layer of the chorion in unfertilized eggs was modified to a thin, compact layer. When unfertilized eggs were treated with trypsin, the inner third portion of the micropylar canal closed and the glycoproteins in the DMA were digested. The incidence of sperm entry into the micropyle of these eggs was extremely reduced. These results suggest that in medaka eggs, the chorionic glycoproteins in the DMA on the chorion surface, which have an affinity for spermatozo, play an important role in sperm guidance into the micropyle.

Effect of 5-hydroxytryptamine on steroidogenesis and oocyte maturation in pre-ovulatory follicles of the medaka Oryzias latipes

The effect of 5‐hydroxytryptamine (5‐HT) on steroidogenesis and oocyte maturation in pre‐ovulatory follicles of the medaka Oryzias lalipes was examined using in vitro culture system. The earliest breakdown of the germinal vesicle of intrafollicular oocytes occurred about 17 hr after the beginning of incubation in the presence of 5‐HT at concentration of 10 ng/ml or more. 5‐HT induced oocyte maturation in a dose‐dependent manner. Cyanoketone inhibited this stimulation. The concentration of 5‐HT required to induce oocyte maturation corresponded to that required to enhance the production (secretion) of estradiol‐17β and 17α,20β‐dihydroxy‐4‐pregnen‐3‐one by pre‐ovulatory follicle cells. At a concentration of 1 μg/ml, the follicle had to be exposed to 5‐HT for at least 4 hr for oocyte maturation accompanied by ovulation to occur. These results indicate that 5‐HT induces in vitro maturation of medaka oocytes by stimulating 17α,20β‐dihydroxy‐4‐pregnen‐3‐one production by pre‐ovulatory follicular cells.

Fine structure of the storage micropocket of spermatozoa in the ovary of the guppy Poecilia reticulata

Abstract To investigate the internal fertilization of the guppy Poecilia reticulata, the present electron microscopic observations were focused on the morphology of the sperm storage site in females. In the ovary of the mature female guppy, many spermatozoa were found in a synaptic knob-shaped micropocket (SSP) as the sperm storage (probably sperm entry) site on the follicle surface which was the expanded blind alley of a small tract extending from the ovarian cavity. Oocytes in the developmental stage of oil droplet formation already showed the attachment of the terminal end of the small tract opening into the ovarian cavity. The lateral wall of the tract attaching to the follicle surface consisted of epithelial cells fast jointed with tight junctions and desmosomes. The thick lateral wall of SSP was constructed with complex epithelial cell layers, and the terminal bottom was comprised of a single layer of epithelial cells on the surface of the follicular layer, which consisted of a very thin thecal cell layer, basement membrane, and granulosa cell layer. The vitellus was enclosed by the follicular layer and thin chorion, in which the micropyle was absent. In fully-grown oocytes, the germinal vesicle containing comparative short chromosomes did not always locate in the vicinity of the storage SSP of spermatozoa.