An oocyte-specific astacin family protease, alveolin, is released from cortical granules to trigger egg envelope hardening during fertilization in medaka (Oryzias latipes).

It has long been hypothesized that in fishes the contents of cortical granules are involved in the hardening of egg envelope following fertilization. We previously purified the egg envelope hardening initiation factor from the exudates released from activated medaka (Oryzias latipes) eggs and tentatively termed this protein alveolin. Alveolin is a member of the astacin metalloprotease family and was proposed to be a protease which hydrolyzes ZPB at one restricted position to allow starting cross-linking with ZPC. Here, we investigated the complete pathway from biosynthesis and accumulation to secretion of alveolin. A single alveolin transcript was detected only in ovarian preparations, confirming the specific expression of alveolin in the ovary. In situ hybridization indicated that the alveolin mRNA is already expressed in the very early previtellogenic oocytes. However, immunocytochemical studies revealed that the appearance of alveolin protein was delayed until the beginning of the vitellogenic stage. The cortical granules isolated from unfertilized eggs contained a high molecular weight form of glycosylated alveolin with a 50kDa relative molecular mass. Hypotonic treatment burst isolated granules in vitro and transformed alveolin to a 21.5kDa form, which is the same size as that of natural alveolin released from eggs upon fertilization. This transformation was inhibited in the presence of leupeptin and 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), suggesting that a serine protease is involved in alveolin activation upon fertilization. Furthermore, the phylogenetic relationship of alveolin with other vertebrate astacin family members was analyzed. The result shows that alveolin and its teleostean homologs make a new group which is separate from either the hatching enzyme, meprin and BMP1/tolloid groups.

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.

How to stay attached-Formation of the ricefish plug and changes of internal reproductive structures in the pelvic brooding ricefish, Oryzias eversi Herder et al. (2012) (Beloniformes: Adrianichthyidae).

Teleost fishes show an enormous diversity of parental care, ranging from no care to viviparity with maternal provisioning of embryos. External brooders carry their developing eggs attached to their bodies. This requires the formation of novel morphological structures to support attachment. The pelvic brooding ricefish Oryzias eversi evolved such a structure, called the "plug." The plug anchors attaching filaments from the fertilized eggs inside the female reproductive system, allowing the female to carry the embryos until hatching. Using histological sections and µ-computed tomography scanning, we show that the plug is formed by several types of interstitial cells, blood capillaries, and collagen fibrils that encapsulate the end of the attaching filaments in the anterior part of the gonoduct. Even 15 days after the loss of the protruding attaching filaments, the plug remains. In addition, the developed plug contains multinucleated giant cells that are derived from fusing macrophages. We thus hypothesize that the ricefish plug, which is vital for egg attachment in O. eversi, evolved due to an inflammatory reaction. We assume that it forms similar to a foreign body granuloma, as a reaction to irritation or injury of the gonoduct epithelium by the attaching filaments. Our study further corroborates that pelvic brooding entails a complex set of adaptations to prolonged egg-carrying in the female reproductive system. During brooding, for instance, ovulation in the ovary is suppressed and the anterior part of the gonoduct is characterized by an intricate, recessed folding.

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.

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.

The fragmented testis method: development and its advantages of a new quantitative evaluation technique for detection of testis-ova in male fish.

A new quantitative evaluation technique, termed the fragmented testis method, has been developed for the detection of testis-ova in genotypic male fish using the medaka (Oryzias latipes). The routine traditional histological method for detection of testis-ova in male fish exposed to estrogens or suspected endocrine-disrupting chemicals has several disadvantages, including possible oversight of testis-ova due to limited sampling of selected tissue sections. The method we have developed here allows for the accurate determination of the developmental stages and the number and the size of testis-ova in a whole testis. Each testis was removed from the fish specimen, fixed with 10% buffered formalin solution, and then divided into small fragments on a glass slide with a dissecting needle or scalpel and aciform forceps in glycerin solution containing a small amount of methylene blue or toluidine blue. If present, all developing testis-ova of various sizes in fragmented testicular tissues were clearly stained and were observable under a dissecting microscope. Testis-ova occurred in controls were ascertained, while spermatozoa were also distinguishable using this method. This proved to be a convenient and cost-effective method for quantitatively evaluating testis-ova appearance in fish, and it may help to clarify the mechanism of testis-ova formation and the biological significance of testis-ova in future studies of endocrine disruption.

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.

Chromosome Formation During Fertilization in Eggs of the Teleost Oryzias latipes.

Upon fertilization, eggs shift their cell cycle from the meiotic to the mitotic pattern for embryogenesis. The information on chromosome formation has been accumulated by various experiments using inhibitors to affect formation and behavior of chromosomes in the cycle of cell proliferation. Based on experimental results on meiosis and early stages of development of the teleost Oryzias latipes, we discuss the roles of the activities of histone H1 kinase, microtubule-associated protein kinase, DNA polymerase, DNA topoisomerase, and other cytoplasmic factors in formation and separation of chromosomes.

Stages of normal development in the medaka Oryzias latipes.

Unfertilized eggs of Oryzias latipes were artificially inseminated and incubated at 26+/-1 degrees C. Careful observation of the process of embryonic development by light microscopy allowed division of the process into 39 stages based on diagnostic features of the developing embryos. The principal diagnostic features are the number and size of blastomeres, form of the blastoderm, extent of epiboly, development of the central nervous system, number and form of somites, optic and otic development, development of the notochord, heart development, blood circulation, the size and movement of the body, development of the tail, membranous fin (fin fold) development, and development of such viscera as the liver, gallbladder, gut tube, spleen and swim (air) bladder. After hatching, development of the larvae (fry) and young can be divided into six stages based on such diagnostic features as the fins, scales and secondary sexual characteristics.

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.

Medaka Oocytes Rotate Within the Ovarian Follicles During Oogenesis: (medaka oocyte/rotation/ovarian follicle/attaching filament/animal-vegetal axis).

The purpose of the current investigation was to ascertain whether medaka oocytes rotate within the follicle. Isolated medaka follicles were incubated in modified L15 Medium for 3 hr at 26°C. During incubation, movement of oocytes within follicles held on slides under a microscope was recorded by a video cassette recorder. Within the follicle, the surface of which was marked with carbon particles, the movement of the intrafollicular oocyte was traced by dislocation of its attaching and non-attaching filaments on the chorion. Pre-vitellogenic oocytes exhibited rotation around the predetermined animal-vegetal axis, accompanied by rotation at a slightly oblique angle to the axis. The velocity of oocyte rotation was about 40-48 µm hr-1 and was similar among oocytes of different stages between the pre-vitellogenic and early vitellogenic phases of oogenesis. Rotation was inhibited by cytochalasin B treatment. Also, it was not observed in oocytes surrounded only by the granulosa cell layer when the thecal cell layer and the basement membrane were removed from the follicle. In oocytes with a thick chorion, rotation was also inhibited by impaling the oocytes with a glass needle at a right angle to the animal-vegetal axis of the oocyte. These results provide evidence that growing medaka oocytes rotate primarily around their animal-vegetal axis and at a slightly oblique angle to the axis. That the rotation of medaka oocytes may depend upon the movement of the granulosa and the thecal cells within the follicles was discussed.

Effects of pH on the Fertilization Response of the Medaka Egg: (pH/Oryzias egg/cortical reaction/sperm penetration/microinjection).

The effects of changing the intra- and extracellular pH on the cortical reaction and sperm penetration into eggs were examined in Oryzias latipes. When eggs were inseminated in a saline adjusted to various pH values with different buffers, fertilization took place normally over a wide range of external pH (pH0 ) from about 6 to 10 with a peak at 7 to 8.5. The primary causes of the reduced fertilization were impaired motility or immobility of the spermatozoa in the acidic saline and the swelling of the chorion in the alkaline saline. A rise in the intracellular pH (pHi ) shortly after commencement of the cortical reaction was found by monitoring the color of a pH indicator micro-injected into the cytoplasm. The experimental results obtained by microinjection of various pH buffers (pH 4-12) suggest that the intracellular membrane fusion of the plasma membrane with the cortical alveolar membrane (CABD) is affected by the acidic pHi while the propagation of the CABD and the intercellular membrane fusion between the plasma membranes of the egg and the spermatozoon are affected by the acidic pH0 .

Exocytosis of Cortical Alveoli and Its Initiation Time in Medaka Eggs Induced by Microinjection of Various Agents: (cortical alveolus exocytosis/inositol 1, 4, 5-trisphosphate/microinjection/medaka egg).

Various agents were microinjected into the cortical cytoplasm at the animal pole of unfertilized eggs of Oryzias latipes under Ca-free conditions. The agents that triggered a wave of the cortical alveolus exocytosis were Ca2+ , inositol, 1, 4, 5-trisphosphate (IP3 ), Ca-ionophore A23187, cGMP, GMP, GTP and guanosine 5'-0-(2-thio-triphosphate)(GTP-γ-s), while CAMP, ATP, gnanosine 5'-0-(2-thio-triphosphate)(GDP- ß-s), inositol monophosphate (IMP) and inositol triphosphate (ITP) were ineffective. Ca2+ , IP3 and A231 87 induced the propagative exocytosis after a time lag (5-8 sec), irrespective of the presence of Co2+ . The time lag was shorter than that (13-28 sec) following microinjection of cGMP or GTP, while were not effective in the presence of Co2+ . The present data suggest that (1) free cytoplasmic Ca2+ participates in both an early and a late step in exocytosis, and (2) cGMP or GTP acts on an early step before initiation of Ca2+ release during exocytosis in the medaka egg.

Evidence for the Presence of a Low Molecular Weight Factor in Sera from Various Animals that Induces in vitro Maturation of Fish Oocytes: (serum factor/Oryzias oocyte/maturation).

Studies were made on whether sera from various animals or serum components could induce in vitro maturation of medaka oocytes in the absence of any exogenous hormone. When intra-follicular oocytes were preincubated in medium containing serum of some mammals or chickens, and were then washed, they matured during further incubation in the same medium without the serum. In contrast, no remarkable stimulatory effect on oocyte maturation was recognized in sera from some other mammals and from reptiles, amphibian and fish. A dialysate of rabbit serum collected using a cellulose membrane (mol. wt. retention 3,500) also induced in vitro maturation of intra-follicular oocytes. This activity was stable on treatment with proteolytic enzymes or heat-treatment. Small molecular weight fractions with this activity were obtained by filtering the dialysate through Sephadex G-10. The oocytes matured by the serum factor were capable of developing normally beyond the stage of embryonic body formation. From these results, it is concluded that the serum of higher vertebrates contains a low molecular weight factor that stimulates in vitro nuclear and cytoplasmic maturation of intrafollicular fish oocytes.

INHIBITORY EFFECT OF RABBIT SERUM FACTOR ON IN VITRO OOCYTE MATURATION OF THE TELEOST FISH, ORYZIAS LATIPES.

The present study indicates that a factor in rabbit serum inhibits the in vitro steroid- and gonadotropin-induced maturation of oocytes of the teleost fish, Oryzias latipes. Such inhibitory activity could not be recognized in the serum of this fish or in the fluids from mammalian follicles. Passage of the serum inhibitor through a cellulose membrane indicated that it has a molecular weight of less than 3,500. The inhibitory activity on steroid-induced oocyte maturation was not destroyed by heating, by repeated freezing and thawing or by treatment with proteolytic enzymes, lipase or amylases. However, its activity could be removed by extraction with charcoal but not with ethyl ether or toluene. The inhibitory action of the heat-stable and dialyzable serum factor was reversible. The factor appears to exert its inhibitory effect upon the oocyte itself in an early step of maturation, rather than upon the follicle cells.

SCANNING ELECTRON MICROSCOPIC STUDY ON THE SURFACE CHANGE OF EGGS OF THE TELEOST, ORYZIAS LATIPES, AT THE TIME OF FERTILIZATION.

The surface change of the egg of the teleost, Oryzias latipes, during fertilization was observed with a scanning electron microscope. The microvilli of the outer surface of the unfertilized egg show a slight difference in density between the animal and vegetal pole areas. In the initial step of the breakdown of cortical alveoli (CA), several small holes or gapes are formed at the apical part of the CA membrane, becoming a large aperture from which the alveolar contents are discharged. The formation of microvilli is observed on the inner surface of the exposed cavity left by the CA, starting from the periphery of the aperture and propagating throughout the whole inner surface in accompaniment with the release of the alveolar contents. After the completion of CA breakdown, the CA membrane cannot be distinguished from the original egg plasma membrane.

INITIATION OF CLEAVAGE IN FISH EGGS BY INJECTION OF FLAGELLA OR MICROTUBULES OF SEA URCHIN SPERMATOZOA.

Flagella and their microtubules obtained from sea urchin (Hemicentrotus pulcherrimus) spermatozoa were injected into unfertilized eggs of the medaka (Oryzias latipes) with a micropipette. Upon activation, some of the eggs began the first cleavage with three or more irregular blastomeres, and developed to the morula stage. It is suggested that sperm flagellar microstubule material is one of the cleavage initiation substances.

FINE STRUCTURE OF LOACH OOCYTES DURING MATURATION IN VITRO.

The morphological changes during in vitro maturation of Misgurnus anguillicaudatus oocyte are described. The process of oocyte maturation can be divided into three provisional stages based on morphological events. Fully-grown, immature oocytes are opaque yellowish-white. The morphological characteristics of their ooplasm are the existence of annulate lamellae, a mass of long mitochondria and an electron dense layer beneath the vitelline surface. Three hr after a 1 hr exposure to corticosterone, these structures disappear and the cortical ooplasm becomes semi-transparent. In this stage of the maturation process (Stage I), the germinal vesicle, without a nucleolus, moves toward the animal pole, and scattered cytoplasmic inclusions approach the vitelline surface. Six hr after exposure to the hormone (Stage II), the whole ooplasm becomes semi-transparent and large yolk platelets are seen in the animal pole region. Tubular endoplasmic reticula develop throughout the ooplasm and some cortical alveoli (CA) become aligned beneath the vitelline surface. Nine hr after exposure to the hormone (Stage III), the oocyte chorion separates from the follicle cells. Most CA align beneath the vitelline surface and cytoplasm accumulates in the cortical region of the animal hemisphere.