Partial formation of sperm dimorphism from spermatocytes of the cottoid fish, Hemilepidotus gilberti in cell culture.

Polymorphism of sperm is considered to be significant for the reproductive strategy in some animal species. The phenomenon is thought to occur in the species-specific stage of spermatogenesis, but how the identical germ cells are differentiated towards polymorphic sperm remains unknown. We here performed a germ cell culture in the cottoid fish, Hemilepidotus gilberti, whose sperm exhibit dimorphism with fertilizable eusperm and unfertilizable parasperm. In the culture, germ cells, which were obtained with an identical morphology, a spherical shape of 5-7 microm in diameter, differentiated into smaller spherical cells with a single nucleus, a moving flagellum and localized mitochondria. In addition, large retroflex-shaped cells with two elongated nuclei were also observed in the cell culture. Germ cells that had each morphological feature were histologically also observed in some cysts of the spermatogenetic testis, suggesting that the former type of cell corresponded to developing eusperm and the latter corresponded to developing parasperm. When BrdU was incorporated into germ cells in the culture, it was detected in both cells with eusperm-like and those with parasperm-like morphologies. These findings suggest that DNA-duplicating spermatocytes are potent to autonomously progress a part of spermatogenesis to form dimorphic sperm.

Significance of egg-jelly substances in the internal fertilization of the newt, Cynops pyrrhogaster.

Japanese newt, Cynops pyrrhogaster, undergoes internal fertilization as do most urodeles. In this study, we focused on the roles of egg-jelly in fertilization of C. pyrrhogaster and characterized the substances associated with those roles. When dry sperm were directly inseminated onto the egg, normal fertilization occurred without the presence of water. Egg-jelly extract (JE) prepared with Steinberg's salt solution contained the activity for the initiation of sperm motility. A substance of about 50 kDa in JE was significant for this activity; an inactive form of the substance probably exists in JE. Strong activity to induce acrosome reaction was detected in JE. It was inhibited by the treatment of JE with WGA, suggesting that carbohydrate in JE may be important for the induction of the acrosome reaction. This study suggests that two significant processes of fertilization are regulated by substances in the egg-jelly of the newt, C. pyrrhogaster.

Requirement of protamine for maintaining nuclear condensation of medaka (Oryzias latipes) spermatozoa shed into water but not for promoting nuclear condensation during spermatogenesis.

Protamine is an arginine-rich basic protein found in the sperm nuclei of many vertebrates, but its actual roles in spermatozoa remain to be elucidated. In this study, we investigated the physiological roles of protamine by examining protamine-less spermatozoa produced in vitro in the presence of the transcriptional inhibitor actinomycin D. Even under inhibited transcription, medaka spermatocytes underwent meiosis and differentiated into spermatozoa with a condensed nucleus and an elongated flagellum. Using a newly produced anti-medaka protamine antibody, we confirmed the absence of protamine protein in the spermatozoa differentiated in the presence of actinomycin D. These findings clearly indicate that sperm nuclear condensation in medaka is independent of protamine. Since medaka spermatozoa are shed into water upon natural fertilization, we also investigated the roles of protamine by comparing the differences between the nuclear morphology of protamine-equipped and protamine-less spermatozoa immersed in water. The nuclei without protamine more rapidly swelled than did those with protamine and completely broke down within 10 min, whereas more than 80% of the sperm nuclei with protamine resisted the disruption under similar conditions. These findings strongly suggest that a physiological role of protamine in medaka spermatozoa is to protect the ejaculated spermatozoa against the disruption by low osmotic pressure until arrival at the eggs for successful fertilization.

Sperm surface heparin/heparan sulfate is responsible for sperm binding to the uterine envelope in the newt, Cynops pyrrhogaster.

The sperm-binding properties of egg envelopes are investigated in the newt, Cynops pyrrhogaster. Sperm binding was only seen on the uterine envelope when acrosome-reacted sperm were inseminated. No acrosome-intact sperm bound to the envelopes. By scanning electron microscopic observation, acrosome-reacted sperm were revealed to bind to a seat-like structure present on the surface of the uterine envelope. Sperm binding to the uterine envelope was inhibited by treatment of eggs with heparin or heparan sulfate, or treatment of acrosome-reacted sperm with heparinase prior to insemination. A molecule with a molecular mass of 75 kDa was purified from the uterine envelope by affinity chromatography with heparin-Sepharose. These results indicated that sperm binding was mediated by heparin-like molecules expressed on the surface of acrosome-reacted sperm and the 75 kDa molecule was present as a constituent of uterine envelopes.

Initiation of sperm motility in the newt, Cynops pyrrhogaster, is induced by a heat-stable component of egg-jelly.

Sperm motility in amphibians is thought to be initiated by a decrease in environmental osmolarity. However, fertilisation in the newt, Cynops pyrrhogaster, is achieved in an environment without osmotic change. We show here that sperm motility initiating activity is present in jelly layer extract (JE). JE was gel-filtrated and a single peak with sperm motility initiating activity was detected in the fraction corresponding to about 50 kDa. The activity was strengthened by heat treatment of JE at 100 degrees C for 30 min. This suggests that JE includes the inactive form of sperm motility inducing substance (SMIS) in addition to active substance. Thus JE was fractionated before and after the heat treatment. When JE was fractionated first and then each fraction was heated, the activity was detected in the fraction both above 500 kDa and below 500 kDa. When heat-treated JE was fractionated, the activity was detected only in the fraction below 500 kDa. These results suggest that JE includes the inactive form of SMIS of more than 500 kDa in molecular weight. A regulatory mechanism for the initiation of sperm motility in C. pyrrhogaster is proposed according to the results of the present study.

Fibroblast Growth Factor May Regulate the Initiation of Oocyte Growth in the Developing Ovary of the Medaka, Oryzias latipes.

The distribution of fibroblast growth factor (FGF) was investigated in developing and matured ovaries of the medaka, Oryzias latipes. In the fry, FGF localized in the cytoplasmic region of all oocytes in the ovary at the pre-vitellogenic stage. Before the initiation of vitellogenesis, it disappeared in the cytoplasmic region and newly appeared around each oocyte, and then it localized around all oocytes in the ovary at the vitellogenic stage. Interestingly, the change in FGF distribution was orderly occurring from the posterior to anterior region of the ovary. In the adult, FGF was detected by immunofluorescence staining around the oocytes. These results suggest that FGF plays a significant role in the initiation of oocyte development through follicle cells, and the expression of FGF is rigidly regulated in the developing ovary of O. latipes.

Distributions of fibroblast growth factor and the mRNA for its receptor, MFR1, in the developing testis of the medaka, Oryzias latipes.

The distribution of fibroblast growth factor (FGF) and of the mRNA for its receptor encoded by MFR1 were examined in the testis of the medaka, Oryzias latipes. FGF was detected in the region of somatic cells, especially in Sertoli cells in the matured testis by immunostaining. The staining was also detected in some of the type A spermatogonia. The distribution of FGF in the testis of the fry, in which spermatogenesis had not been initiated, was different from that in the adult. Signals of mRNA were observed in the same region as FGF in the matured testis. Staining was also detected in the testis of the fry in which spermatogenesis had been initiated. Therefore, the role of FGF in the testis of the fry in the early developmental stages may be different from that of the adult: These results indicate that FGF regulates the activation of somatic cells in the testis through the MFR1 receptor, and that it might play a significant role in the initiation and the progression of spermatogenesis in the testis of O. latipes.

Establishment of in vitro spermatogenesis from spermatocytes in the medaka, Oryzias latipes.

Spermatocytes of the teleost, Oryzias latipes, at meiotic prophase were cultured without contact with somatic cells. They began to divide, progressing through the meiotic divisions and differentiating into round spermatids within 48 h. The chromosome number in both the primary and secondary spermatocytes at metaphase was n = 24. In spermatids, a single flagellum was formed and the release of residual bodies was observed in vitro. The size and shape of the flagellum were the same as those seen in vivo. The expression of protamine mRNA was detected in round spermatids. This result suggests that gene expression, as well as morphological change, is regulated by the progression of spermatogenesis in cell culture. Furthermore, when the eggs of O. latipes were inseminated with germ cells cultured for 10 days, normal embryos developed and hatched out. These results suggest that the spermatocytes of O. latipes develop into fertile sperm in cell culture.

Hymenolepis nana: immunity against oncosphere challenge in mice previously given viable or non-viable oncospheres of H. nana, H. diminuta, H. microstoma and Taenia taeniaeformis.

When mice, previously given oral inoculation with viable oncospheres of the heterologous cestode species (Hymenolepis diminuta, H. microstoma, Taenia taeniaeformis) and the homologous one (H. nana), were challenged with oncospheres of H. nana 4 days after the primary inoculation, they showed strong and complete resistance to H. nana challenge, respectively. However, the resistance was not evoked in mice given either infective eggs of Toxocara canis or non-viable oncospheres of all cestode species examined. Congenitally athymic nude mice given viable oncospheres did not show any resistance to H. nana either. Eosinophil infiltration around cysticercoids of H. nana in the intestinal villi appeared to be more prominent in mice previously given viable oncospheres of H. diminuta than in mice given non-viable oncospheres or PBS only. Some of the eosinophils in the villus harboring cysticercoid(s) of H. nana invaded the epithelia in the former, whereas all eosinophils remained in the lamina propria in the latter. There was almost no eosinophil infiltration in nude mice. Microscopic observations revealed that oncospheres of H. diminuta, which require beetles as the intermediate host like H. microstoma, could invade the mouse intestinal tissue. Therefore, it is strongly suggested that the strong cross resistance to H. nana in mice, induced by oncospheres of all heterologous cestode species, is thymus-dependent and due to oncospheral invasion into the intestinal tissue of mice.

Vaccination against Taenia taeniaeformis infection in rats using a recombinant protein and preliminary analysis of the induced antibody response.

Primary screening of a cDNA expression library of Taenia taeniaeformis oncospheres in lambda gt11 bacteriophage was carried out using rabbit anti-T, taeniaeformis oncosphere serum affinity-purified from oncosphere pellets. From approximately 1.6 x 10(5) plaques, 21 single clones that were positive with the affinity-purified antibodies were isolated. Sibling analysis revealed that 17 clones out of the 21 could be assigned to five different antigen families. Only family 1 was strongly recognized by a serum prepared in a rabbit against a partially purified host-protective oncosphere antigen fraction. The fragments of lambda DNA were inserted into a pGEX plasmid vector that encodes glutathione S-transferase (GST) of Schistosoma japonicum. Clones designated TtO-18, -49.53 (family 1), 46 (family 2), 15 (family 3), 40 (family 4) and 66 (family 5) were established as subclones in pGEX-1 plasmid vectors which produced GST fusion proteins. All GST fusion proteins were soluble and recognized by anti-GST and anti-TtO sera. Three vaccination experiments with these fusion proteins using specific-pathogen-free Wistar rats revealed that all three fusion proteins of family 1 were exclusively effective against T. taeniaeformis oncosphere challenge with approximately 95% and 91% reductions in cystic metacestode and total metacestode recoveries, respectively. Rats vaccinated with fusion proteins of family 1 produced antibodies which reacted with a 21-kDa oncosphere antigen component which appeared to be a major oncosphere stage-specific antigen.

Stage-specific antigens of Hymenolepsis microstoma recognized in BALB/c mice.

Antigenicity of eggs (oncospheres), cysticercoids and adults (with immature segments only) of the bile duct tapeworm Hymenolepsis microstoma was analysed using immunoblotting techniques and indirect immunofluorescent antibody (IFA) techniques with immune sera of BALB/c mice (i) infected with different doses of cysticercoids, (ii) during patent or prepatent infection with the lumen phase of the parasite or (iii) sensitized with live or dead eggs. Antibody responses detected by IFA test and immunoblotting showed that antigenicity of eggs (oncospheres) differed from that of cysticercoids and adults. Single worm infections were sufficient to stimulate antibody responses. Mice which had patent infection showed strong antibody responses to all three (egg (oncosphere), cysticercoid, adult) antigens, while mice given two prepatent infections showed some antibody responses to cysticercoid and adult antigens only. Although the normal intermediate hosts of this parasite are arthropods, antibodies to some major egg (oncosphere) antigens were produced in mice given eggs of this parasite orally, either through inoculation of eggs or ingestion of faeces contaminated with eggs. Antibodies were not produced in mice dosed with non-viable eggs. The results are consistent with the hypothesis that cestode parasites express phase- (or stage-) specific antigens.

Hymenolepis diminuta and H. nana: cross immunity against the lumen phase in BALB/c mice.

When BALB/c mice initially given cysticercoids of Hymenolepis diminuta orally (Day 0) were challenged with eggs or cysticercoids of H. nana, almost all the mice became completely resistant to H. nana challenges from Day 30 onward, and no luminal adults of H. nana were established. There was a tendency for the number of tissue cysticercoids recovered 4 days after egg challenge in immunized mice to be much less than that in control mice (P less than 0.001, Student's t test). However, when these cysticercoids recovered from immune group mice were inoculated into uninfected mice, they matured in the lumen. Thus, the cross immunity to H. nana challenge evoked by an initial prepatent infection with H. diminuta appeared to be directed not against the tissue phase but against the lumen phase of H. nana. When BALB/c mice initially given eggs of H. nana were challenged with H. diminuta, they became resistant to H. diminuta from Day 15 onward. When the mice given eggs of H. nana were treated with a cestocide, praziquantel, at the beginning of the expected luminal development of H. nana and experienced a tissue phase only before challenge with H. diminuta, they showed no resistance to H. diminuta. Thus, the cross immunity to H. diminuta challenge evoked by an initial patent infection with H. nana appeared to be due to the immunogens of the lumen phase of H. nana but not those of the tissue phase. The cross immunity may be, therefore, essentially evoked by the lumen phase of these two phylogenetically closely related species and not by or against the tissue phase of H. nana.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in surface antigens of Hymenolepis nana during differentiation and maturation in mice.

The surface antigens of oncosphere, cysticercoid, adult scolex and adult strobila (other than scolex) of Hymenolepis nana differ critically from one another. When the oncosphere of H. nana undergoes differentiation and development into the mature tapeworm, the infected mouse first produces anti-oncosphere antibody, followed by anti-cysticercoid, anti-adult scolex and finally anti-strobila (other than scolex region) antibodies of IgG, IgM and IgA isotypes as detected by indirect immunofluorescent antibody test. The parasite changed its surface antigens throughout its differentiation and maturation, and all developmental stages were recognized by the infected mouse host. However, there appeared no further changes in surface antigens during aging after maturation. The antibody responses were always delayed compared with the differentiation and maturation of the parasite.

Induction and inhibition of in vitro oocyte maturation and production of steroids in fish follicles by forskolin.

The effects of forskolin (FK) on in vitro oocyte maturation and production of steroids were examined in Oryzias latipes. When oocytes within preovulatory follicles were preincubated in the presence of FK for 2-10 hr, they matured normally after additional incubation for 10-20 hr in plain culture medium. Naked (follicle cell-free) oocytes did not mature under these conditions. FK stimulated dose-dependent production of steroids (estradiol-17 beta, E2, and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, 17 alpha,20 beta-diOHprog) and cAMP in follicle (granulosa) cells. On the other hand, exposure to FK within 2 hr after 17 alpha,20 beta-diOH prog stimulation caused reversible inhibition of gonadotropin (PMS)- or 17 alpha,20 beta-diOH prog-induced maturation of the intrafollicular oocytes in vitro. FK also significantly inhibited the 17 alpha,20 beta-diOHprog-induced maturation of naked oocytes, suggesting the existence of adenylate cyclase in fish oocytes. These data indicate that in Oryzias latipes, FK induces oocyte maturation by stimulating follicular production of maturation-inducing steroid (MIS), probably 17 alpha,20 beta-diOH prog, via an increase in cAMP, and that it may inhibit oocyte maturation by increasing ooplasmic cAMP and some inhibitory interaction between the granulosa cells and the oocyte through intercellular communication.

Immunocytochemical demonstration of steroid hormones in the granulosa cells of the medaka, Oryzias latipes.

Immunocytochemical identification of steroid hormones was performed in granulosa cells isolated from large ovarian follicles of the medaka. The granulosa cells recovered from daily spawning females, 8.5 h after the onset of light, reacted with anti-progesterone and anti-17 alpha-hydroxyprogesterone antibody, while the granulosa cells treated 2.5 h after the onset of light did not. After 6 h stimulation with gonadotropin (PMS) in vitro, the isolated granulosa cells reacted with anti-progesterone antibody. The granulosa cells examined 8.5 h after the onset of light did not react with anti-estradiol or anti-estriol antibody. These results demonstrate that, in Oryzias latipes, progestins (progesterone and 17 alpha-hydroxyprogesterone) are synthesized in the granulosa cells which are directly stimulated by gonadotropin.

On the effects of cyanoketone on gonadotrophin- and steroid-induced in vitro maturation of Oryzias oocytes.

Effects of cyanoketone (CK) on the gonadotrophin- and steroid-induced maturation in vitro of Oryzias latipes oocytes were examined. In oocyte covered with intact follicle or granulosa cell layers, CK inhibited gonadotrophin- and pregnenolone-induced maturation but not 20 beta-OH-progesterone-induced maturation. However, when follicle cells were removed completely from the oocytes, CK conspicuously lacked inhibitory effect on pregnenolone-induced oocyte maturation. It was ascertained by using purified pregnenolone that pregnenolone-induced maturation was not due to contamination with other maturation-inducing steroids. It was also observed immunocytochemically that a purified pregnenolone stimulated in vitro production of progestins in the granulosa cells, and that CK inhibited this stimulation. These results indicate that pregnenolone, like gonadotrophin, induces maturation of intrafollicular oocytes of O. latipes by stimulation of production of such steroids as progesterone in follicle cells (granulosa cells), while it can directly induce maturation of oocytes not surrounded by follicle cells.