The light-dependent daily cycle of ovulation in the oviparous medaka fish, Oryzias latipes (Atherinomorpha: Beloniformes: Adrianichthyidae) artificially pregnant with developing embryos.

In the oviparous medaka fish, Oryzias latipes, mature spermatozoa that were artificially introduced into the ovarian cavity retaining ovulated eggs could internally fertilize these eggs. This enabled us to examine the effect of ovarian gestation on the ovulation cycle. Most freshly ovulated eggs could be internally fertilized in the ovarian cavity. Yet eggs ovulated 24 h after single insemination remained unfertilized in the ovarian cavity. Artificially pregnant females persisted in a daily cycle of ovulation, which occurred shortly before the onset of light under the present reproductive conditions. Females continuously ovulated a certain number of eggs despite ovarian gestation, that is, the presence of embryos within the ovarian cavity. Repeated cycles of ovulation led to crowding in the ovarian cavity because the group of fertilized eggs, with their hardened egg envelope (chorion or zona radiata), plugged the genital orifice. The development of fertilized eggs was retarded and ceased around the initiation stage of blood circulation, but when they were transferred from the ovarian cavity into regular saline, they regained their ability to develop normally up to hatching. These results show that in oviparous female medaka, ovarian gestation exerted little effect on the time of ovulation and the number of ovulated eggs.

Shift of the Vegetal Pole Area of Full-Grown Oocytes Toward the Ovulatory Site of the Ovary in the Medaka Fish, Oryzias latipes (Beloniformes: Adrianichthyidae).

This study presents novel findings on the dynamics of growing oocytes in the ovary of the medaka fish, Oryzias latipes. In the ovary of mature females, all follicles are anchored tightly to the abdominal ovarian rete via fibrous follicular stalks on the follicle surface. The follicular stalks lie at the end of the follicle opposite the site of its attachment to the ovarian wall. Various lengths of the follicular stalks reflect the spatial arrangement of follicles in the ovary. Herein, a line that connects the follicular stalk to the opposite side of the follicle toward or attaching to the ovarian wall is called the follicle axis. The animal-vegetal axis in late stage III oocytes is already recognizable as a line that connects the center of the oocyte nucleus and the vegetal pole area; this is ascertainable by the morphological landmark of a compact distribution of granulosa cells or rudimentary attachment filaments. In growing oocytes later than stage V, the beginning of vitellogenesis, the tufted attachment filaments are located on a discrete region of the vegetal pole area. Our observations reveal that during growth, oocytes are arranged randomly between the animal-vegetal axis and the follicle axis, whereas the vegetal pole area of full-grown oocytes in preovulatory follicles turns close to the inner surface of the ovarian wall, from which mature oocytes subsequently ovulate into the ovarian lumen. It is suggested that in the O. latipes ovary, mature oocytes always transpose the vegetal pole area to the ovulatory site of the ovarian wall prior to ovulation. The expulsion of mature oocytes from the vegetal pole appears to be the regular mode of ovulation in O. latipes.

Effects of co-administration of estrogen and androgen on induction of sex reversal in the medaka Oryzias latipes.

To investigate how estrogen and androgen affect each other in inducing sex reversal in the medaka, O. Iatipes, 17ß-estradiol (E2) and 17α-methyldihydrotestosterone (MDHT) were co-administered by a convenient method for hormonal treatment, in which freshly fertilized eggs were immersed for 24 h in saline containing either or both of the two sex steroids in different concentrations and/or ratios. The minimal concentrations of E2 and MDHT sufficient to induce the maximal rate of sex reversal from male to female and from female to male were 500 ng/ml and 2.5 ng/ml, respectively, both of which were referred to as the most efficacious dose (MED), and each equivalent for the inducing potency in sex reversal. E2 and MDHT, when simultaneously administered at MED, greatly suppressed each other to induce each corresponding sex reversal. Thus, the present experimental results indicate that E2 and DMHT are antagonists that induce corresponding sex reversal, and suggest that genotypic sex in the medaka might be modified through an unknown factor of common affinity to both sex steroids, by which the pathway of differentiation of either sex could be switched at the early stages of development far before gonadal sex differentiation.

Reproductive role of attaching filaments on the egg envelope in Xenopoecilus sarasinorum (Adrianichthidae, teleostei).

Eggs of Xenopoecilus sarasinorum possess two distinct types of filaments on the surface of the egg envelope (chorion), long, attaching filaments restricted to the vegetal pole and weak, nonattaching filaments around the animal pole (micropyle). Both types are formed during oogenesis. After mature eggs were spawned through the urogenital pore, they were fertilized and hung in an abdominal concavity of the female. Oviposition never took place in the presence of embryos in the concavity because of the retardation of oogenesis. The loosely tangled tips of the attaching filaments that are retained within the ovarian cavity plug the urogenital pore by forming a hard complex with the epithelial cells. Into this plug structure that fuses with the inner wall of the urogenital pore, capillaries are provided. Within 5 days after the initiation of hatching, this plug degenerates and is released from the urogenital pore. Thus, in female X. sarasinorum, the reproductive cycle seems to be regulated by the physiological function ofthe plug structure formed by the attaching filaments in response to the presence of developing embryos.

Oviposition cycle in the oviparous fish Xenopoecilus sarasinorum.

To clarify the reproduction of the oviparous teleost Xenopoecilus sarasinorum, changes in oocyte composition and oviposition cycle were investigated. After release, a batch of spawned eggs hung from the urogenital pore by attaching filaments (36.3+/-0.8 in number, n=31; about 4.3-7.8 mm in length, 5-8 microm in diameter) on the chorion (egg envelope) in the vegetal pole region. Females accommodated a cluster of fertilized eggs in a belly concavity until the embryos hatched. Hatching of embryos took place from 18-19 days after oviposition (25 degrees C). Between 0-2 days following hatching, the attaching filaments disappeared from the urogenital pore. Between 3 and 4 days following hatching, most of the females spawned again. The growth of oocytes proceeded slowly throughout the period when the egg cluster was carried in the belly, and no ovulation occurred during this period. If the current brood was accidentally lost, the day of the next oviposition was sooner. This might imply that carrying embryos in the belly affects endocrine activity, as in viviparous reproduction.

Sex reversal in medaka treated in vitro with 17alpha-methyldihydrotestosterone during oocyte maturation.

Using the S-rR strain of the medaka Oryzias latipes, we examined the effect of a non-aromatizable androgen on sex determination. Intrafollicular immature oocytes isolated before breakdown of the germinal vesicle were incubated in the presence of 17alpha-methyldihydrotestosterone (MDHT) for about 10 h during their maturational period. At the end of incubation, mature oocytes were rinsed and then artificially inseminated in regular saline. The fertilized eggs were then allowed to develop in tap water, and the fry were reared on a regular powdered diet until adulthood. Sex reversal of female to male was observed in a manner dependent on the dose of MDHT. In the solvent control group in which intrafollicular oocytes were matured in medium containing no exogenous androgen, no sex reversal was observed. The present finding, that the sex of medakas can be reversed by a single in vitro exposure of immature oocytes to androgen during the preovulatory period, suggests the existence in the oocyte of a sex determinant sensitive to sex steroids. This method for controlling the sex of eggs before fertilization may establish sex-determined eggs as potent material for investigating the mechanism of sex determination in the medaka.

Testosterone content of developing eggs and sex reversal in the medaka (Oryzias latipes).

To understand the effect of testosterone on sex differentiation, the quantities of testosterone (T) and estradiol-17beta (E2) in developing eggs of medaka (Oryzias latipes) were measured by radioimmunoassay, and the influence on sex differentiation of treating embryos with exogenous androgens was also examined. Endogenous T of eggs dispersed into the environmental water at spawning, and precipitously declined to a minimum level during incubation for 2 days post-fertilization (dpf). It did not significantly increase during development. The E2 content of fertilized eggs increased when eggs were incubated in medium containing exogenous T at the concentrations of 100 and 500 ng/ml, but not in low concentrations of 10 ng/ml or less. The presence of 500 ng/ml 17alpha-methyltestosterone (MT) in the incubation medium also induced an increase in the E2 content of embryos. Exposure of embryos to exogenous 1 ng/ml T that corresponded with the level of T in eggs shortly after fertilization was enough to induce sex reversal of genotypic females to functional males. The co-existence of T and aromatase inhibitor in incubation medium inhibited not only the T-induced increase in the embryonic E2 content, but also the estrogenic effect of T in causing the paradoxical sex reversal from genotypic males to phenotypic females. However, treatment of embryos with the non-aromatizable androgen, 17alpha-methyldihydrotestosterone, induced no detectable increase in the E2 content of embryos, but still brought about sex reversal of genotypic males into females. This contradictory result suggests that the conversion of androgens to E2 may not always be the cause for induction of paradoxical sex reversal by T treatment. Consequently, these results on sex reversal induced by treatment of embryos with exogenous androgens suggest that endogenous T of developing medaka embryos may not act as the natural andro-inducer, and that genotypic sex can be modified by exogenous sex steroids at early developmental stages long before gonadal differentiation in the medaka.

Sex reversal in the medaka Oryzias latipes by brief exposure of early embryos to estradiol-17beta.

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-17beta (E2) in different concentrations for various durations until hatching. Results of the present experiment showed that the immersion duration in 1 microg/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 microg/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.

In vivo fertilization and development of medaka eggs initiated by artificial insemination.

Fertilization and development in the ovarian cavity of oviparous fish, Oryzias latipes, were examined using the S-rR strain in which the sex genotype can be easily distinguished by the body color of the fish. Mature eggs were fertilized within the ovarian cavity after a sperm suspension was artificially introduced with a small bore-pipette through the urinogenital opening. Three batches of eggs ovulated within 48 hrs were fertilized and began to develop in the ovarian cavity, while eggs ovulated 72 hrs post-insemination (PI) were no longer fertilized. These observations indicate that ovulation occurs irrespective of the existence of developing embryos within the ovarian cavity. All embryos developing in the ovarian cavity were, however, retarded and ceased development before the stage of initiation of blood circulation at room temperature. These embryos developed normally and hatched after they were transferred from the ovarian cavity into regular saline 48 or 72 hrs PI. When these individuals matured sexually, their sex differentiation was found to be normal, and sex reversal was not observed.

Estradiol-17beta content in developing eggs and induced sex reversal of the medaka (Oryzias latipes).

To clarify the effect of exogenous estradiol-17beta (E2) on sex differentiation, the E2 content of developing eggs of Oryzias latipes was measured by radioimmunoassay. Endogenous E2 was present in lower concentrations in ovulated, mature eggs in the ovarian cavity than in intrafollicular oocytes. The E2 content of eggs precipitously declined to a minimum level by 2 days post-fertilization. The E2 content of eggs was affected by 24 hr of incubation in medium containing exogenous E2 at concentrations above 10 ng/ml. Short (24 hr) exposure of fertilized eggs in the early developmental stage to exogenous E2 at concentrations of 10 ng/ml induced sex reversal of some genotypic males to functional females. However, endogenous E2 levels in fertilized eggs might not influence sexual differentiation in embryogenesis. The present results suggest the possibility that concentrations of exogenous E2 higher than that of endogenous E2 triggers a priming step in the cascade of sex differentiation toward the female, and this effect is maintained.

Experimental hybridization among Oryzias species. II. Karyogamy and abnormality of chromosome separation in the cleavage of interspecific hybrids between Oryzias latipes and O. javanicus.

In interspecific hybridization between Oryzias latipes and O. javanicus, all hybrid embryos failed to develop and died before hatching. Cytological examination of fertilization and early development was performed to discover the cause of lethal development. When O. latipes eggs were inseminated by sperm of O. javanicus, the cortical reaction was induced normally. Chromosomal material in the fertilized eggs was visualized using the DNA-specific fluorochrome Hoechst. The spermatozoon was capable of penetrating into the egg cytoplasm through the micropyle, and the sperm nucleus transformed to the male pronucleus. The female pronucleus that formed after extrusion of the second polar body migrated towards the male pronucleus. The female and the male pronuclei underwent DNA synthesis and encountered each other in the center of the blastodisc, fused with one another and formed a zygote nucleus before breakdown of the nuclear envelope. Metaphase chromosomes with electron dense chromatin regions were abnormally divided into each blastomere in cleavage. The abnormally separating chromatin masses were also labeled by BrdU. The abnormal separation resulting in partial loss of fragmented chromatin might be a cause of abortive development in the interspecific hybrids between O. latipes and O. javanicus.

Electron microscopic observations of karyogamy in the fish egg.

In the initial step of pronuclear association in fertilized fish eggs, the female and male pronuclei (containing large nucleolus-like bodies) were juxtaposed in the center of the blastodisc and formed nucleoplasmic projections along adjacent surfaces. After contact of the pronuclei, small internuclear bridges joining them were formed by fusion at several regions of the nuclear envelope projections. No specific site of fusion or breakdown of nuclear envelopes was recognized in the pronuclei during karyogamy. In the advanced stage, clumps of condensing chromatin appeared in the nucleoplasm of the newly fused pronuclei. The number and diameter of the internuclear bridges increased gradually by progressive fusion in many regions, finally yielding a spherical zygote nucleus. Following complete formation of the zygote nucleus, the pronuclear envelope began to break down concomitantly with shrinkage of the nucleoplasm, which was highly convoluted around the entire nuclear surface. The nucleoplasm containing chromosomes then mingled with the perinuclear cytoplasm.

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

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.