Akio Shimizu

Shift of Chloride Cell Distribution during Early Life Stages in Seawater-Adapted Killifish, Fundulus heteroclitus

Abstract The shift of chloride cell distribution was investigated during early life stages of seawater-adapted killifish (Fundulus heteroclitus). Chloride cells were detected by immunocytochemistry with an an-tiserum specific for Na+, K+-ATPase in whole-mount preparations and paraffin sections. Chloride cells first appeared in the yolk-sac membrane in the early embryonic stage, followed by their appearance in the body skin in the late embryonic stage. Immunoreactive chloride cells in the yolk-sac membrane and body skin often formed multicellular complexes, as evidenced by the presence of more than one nucleus. The principal site for chloride cell distribution shifted from the yolk-sac membrane and body skin during embryonic stages to the gill and opercular membrane in larval and later developmental stages. Our observations suggest that killifish embryos and newly-hatched larvae could maintain their ion balance through chloride cells present in the yolk-sac membrane and body skin until branchial and opercular chloride cells become functional.

Endocrine profiles during the short reproductive cycle of an autumn-spawning bitterling, Acheilognathus rhombea ☆

When autumn-spawning bitterling, Acheilognathus rhombea (known as the kanehira bitterling in Japan) were held under a constant short photoperiod (10 hr light-14 hr dark) at 25 degrees during their natural breeding season, the females spawned regularly at intervals of about 5 days. During each 5-day period, the oocytes passed through three distinct physiological stages: vitellogenesis (3 days), maturation (1 day), and spawning (1 day). During the maturational stage, germinal vesicle breakdown (GVBD) was first observed at 18 hr and was complete by 21 hr. Ovulation occurred at 24 hr. Steroid measurements made on plasmas collected from fish throughout their reproductive cycle showed that estradiol-17 beta levels were about 12 ng/ml at the beginning of the vitellogenic stage and about 5 ng/ml during the other stages. Testosterone levels were about 2 ng/ml during the vitellogenic stage, but fell to about 0.5 ng/ml during maturation and spawning. Progesterone levels were very low throughout the cycle and showed little variation. Both 17 alpha-hydroxyprogesterone and 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one levels were both very low (0.5 ng/ml) up until 12 hr on Day 4 (maturation), at which time they increased rapidly. The levels reached peaks of 4 and 7 ng/ml, respectively, at 18 hr and reached basal levels again at 8 hr the following day. GtH-like glycoprotein levels also began to increase at 12 hr on Day 4, but peaked later, at the time of ovulation (24 hr).

Effect of photoperiod and temperature on gonadal activity and plasma steroid levels in a reared strain of the mummichog (Fundulus heteroclitus) during different phases of its annual reproductive cycle

Mummichog, a spring and summer spawning teleost, were exposed to various photoperiod and temperature conditions to investigate the environmental regulation of the annual reproductive cycle. In early spring, latter phases of gonadal development (vitellogenesis in females and active spermatogenesis in males) were effectively accelerated by warm temperature (16 degrees C) regardless of the photoperiod (11L or 16L), although internal factor(s) may be concerned with triggering the initiation of the development. In late summer, intense gonadal regression which leads to the termination of the spawning period was accelerated by a short day length (</=13L) in both yearlings and underyearlings. In underyearlings, however high water temperature and internal factor(s) may also be concerned. In early autumn, early phases of gonadal development (growth of cortical alveolus stage oocytes in females and basal spermatogenesis in males) were induced by moderate or lower temperatures (</=22 degrees C) even under a short day length (11L). From the middle of autumn to early winter, this fish is probably in the refractory period, and did not progress to the latter phases even under adequate temperature and long day length conditions (22 degrees C-16L). Mummichog showed a probable circa-annual rhythm of gonadal activity under constant temperature and photoperiod conditions (22 degrees C-16L): this rhythm may be the basis of the trigger of the gonadal development prior to the spawning period, the termination of the spawning period in underyearlings, and the occurrence of the refractory period during autumn. Plasma concentrations of estradiol-17beta in females and testosterone in males correlated well with the gonadal development during early spring and the regression during late summer. However, there was no correlation between plasma steroid levels and the degree of progress during the early phases of gonadal development in autumn, suggesting other factor such as direct action of GtH(s) or mediating substance other than sex steroids for these phases.

Molecular characterization, tissue distribution, and mRNA expression profiles of two Kiss genes in the adult male and female chub mackerel (Scomber japonicus) during different gonadal stages

Kisspeptins, encoded by the Kiss1 gene, have emerged as key modulators of reproduction in mammals. In contrast to the placental mammals, some teleosts express two Kiss genes, Kiss1 and Kiss2. In the present study, full-length cDNAs of Kiss1 and Kiss2 in the chub mackerel were cloned and sequenced. Chub mackerel Kiss1 and Kiss2 cDNAs encode 105 and 123 amino acids, respectively. A comparison of the deduced amino acid sequences of chub mackerel Kiss1 and Kiss2 with those of other vertebrate species showed a high degree of conservation only in the kisspeptin-10 region (Kp-10). The Kp-10 of chub mackerel Kiss1 (YNFNSFGLRY) and Kiss2 (FNFNPFGLRF) showed variations at three amino acids. Tissue distribution analysis using quantitative real-time PCR (qRT-PCR) revealed that the Kiss1 and Kiss2 transcripts were expressed in different tissues of adult chub mackerel. In addition, their levels in the adipose tissue exhibited sexually dimorphic expression. Further, to have a basic understanding on the involvement of Kiss1 and Kiss2 in the seasonal gonadal development, their relative mRNA expression profiles in the brain, pituitary, and gonads at different gonadal stages were analyzed using qRT-PCR. Kiss1 and Kiss2 levels in the brain showed a differential expression profile between male and female fish. In males, Kiss1 and Kiss2 levels gradually decreased from the immature stage to spermiation and reached a minimal level during the post-spawning period. In contrast, Kiss1 levels in the brain of females did not vary significantly among the different gonadal stages. However, Kiss2 levels fluctuated as that of males, gradually declining from the immature stage to the post-spawning period. The pituitary Kiss1 levels did not show significant fluctuations. However, Kiss1 levels in the gonads were highly elevated during spermiation and late vitellogenesis compared to the immature and post-spawning period. These results suggest the possible involvement of two Kiss genes in the brain and Kiss1 in the gonads of chub mackerel during seasonal gonadal development.

Immunocytochemical applications of specific antisera raised against synthetic fragment peptides of mummichog GtH subunits: examining seasonal variations of gonadotrophs (FSH cells and LH cells) in the mummichog and applications to other acanthopterygian fishes.

Two distinct types of gonadotrophs, FSH (GtH I) cells and LH (GtH II) cells, were immunocytochemically identified from mummichog (Fundulus heteroclitus; Cyprinodontiformes, Acanthopterygii) pituitary using antisera raised against synthetic fragment peptides of FSHbeta and LHbeta. Both cell types were abundant during the spawning period (spring and early summer) and decreased in number during the post-spawning immature period. The number of FSH cells increased again during the early phases of gonadal development (cortical alveoli accumulation in the oocytes and basal spermatogenesis) in early winter, whereas the number of LH cells did not. Only FSH cells were abundant during the latter phases of gonadal development (vitellogenesis and active spermatogenesis) in early spring. These observations suggest that both GtHs have important yet different roles for reproduction in this species. Antisera against the conservative region of the FSHbeta and the LHbeta subunits immunostained FSH cells and LH cells, respectively, also in red seabream (Pagrus major; Perciformes, Acanthopterygii) and small mouth bass (Micropterus dolomieu; Perciformes, Acanthopterygii), suggesting the possibility of their use for other acanthopterygian fishes.

Sexually Dimorphic Expression of Gonadotropin Subunits in the Pituitary of Protogynous Honeycomb Grouper (Epinephelus merra): Evidence That Follicle-Stimulating Hormone (FSH) Induces Gonadal Sex Change

Recent studies have suggested that the hypothalamic-pituitary-gonadal axis is involved in gonadal sex change in sex-changing teleosts. However, its underlying mechanism remains largely unknown. In this study, we focused on the distinct roles of two gonadotropins (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), in the protogynous hermaphrodite teleost, honeycomb grouper (Epinephelus merra). First, we investigated the expression pattern of mRNAs for GTH subunits (cga, fshb, and lhb) in the pituitaries from fish at the different sexual phases. Real-time RT-PCR analyses showed that fhsb mRNA levels in the female pituitary were low. However, fshb transcripts increased dramatically in association with testis development. In contrast, levels of cga and lhb mRNAs did not significantly vary during sex change. In addition, immunohistochemical observations of Fshb- and Lhb-producing cells in the pituitary, through the use of specific antibodies for detections of teleost GTH subunits, were consistent with sexually dimorphic expression of Fshb. In order to identify the role of GTH in gonad of honeycomb grouper, we treated females with bovine FSH (50 or 500 ng/fish) or LH (500 ng/fish) in vivo. After 3 wk, FSH treatments induced female-to-male sex change and up-regulated endogenous androgen levels and fshb transcripts, whereas LH treatment had no effect on sex change. These results suggest that FSH may trigger the female-to-male sex change in honeycomb grouper.

Appearances and chronological changes of mummichog Fundulus heteroclitus FSH cells and LH cells during ontogeny, sexual differentiation, and gonadal development

To examine relationships between gonadal stages and the initial appearance and subsequent development of gonadotrophs, hatched larvae of the mummichog Fundulus heteroclitus were reared until first maturity under suitable conditions for maturation (20 degrees C-16L). Evident FSH cells generally appeared 1-2 weeks after hatching (wah), around or slightly before the morphological sex differentiation which occurred at 2 wah. During this period, 3beta-hydroxysteroid dehydrogenase positive cells also appeared in the gonads. While FSH cells existed throughout the early phases of gonadal development such as cortical alveoli formation and basic spermatogenesis, LH cells appeared later (6-12 wah), after the commencement of the early gonadal development. Both FSH cells and LH cells were abundant at 36 wah when the fish had attained full maturity. These results indicate the possibility that FSH is responsible for gonadal differentiation by inducing steroidogenesis in the gonads, implying the importance of FSH on the early phases of gonadal development. These results also suggest cooperation of FSH and LH in later phases of gonadal development such as yolk globule accumulation and active spermatogenesis. The mode of changes in the abundances of the gonadotrophs according to the gonadal development was somewhat different from previously observed changes during the annual reproductive cycle in adult mummichog. Possible complementary roles of the two GTHs in vitellogenesis and spermatogenesis may be involved in the difference by providing flexibility to the controlling mechanism.

Changes in the expression of pituitary gonadotropin subunits during reproductive cycle of multiple spawning female chub mackerel Scomber japonicus.

The endocrine regulation of reproduction in a multiple spawning fish with an asynchronous-type ovary remains largely unknown. The objectives of this study were to monitor changes in the mRNA expression of three gonadotropin (GtH) subunits (GPα, FSHβ, and LHβ) during the reproductive cycle of the female chub mackerel Scomber japonicus. Cloning and subsequent sequence analysis revealed that the cDNAs of chub mackerel GPα, FSHβ, and LHβ were 658, 535, and 599 nucleotides in length and encoded 117, 115, and 147 amino acids, respectively. We applied a quantitative real-time PCR assay to quantify the mRNA expression levels of these GtH subunits. During the seasonal reproductive cycle, FSHβ mRNA levels remained high during the vitellogenic stages, while GPα and LHβ mRNA levels peaked at the end of vitellogenesis. The expression of all three GtH subunits decreased during the post-spawning period. These results suggest that follicle-stimulating hormone (FSH) is involved in vitellogenesis, while luteinizing hormone (LH) functions during final oocyte maturation (FOM). Both GPα and FSHβ mRNA levels remained high during the FOM stages of the spawning cycle and increased further just after spawning. Thus, FSH synthesis may be strongly activated just after spawning to accelerate vitellogenesis in preparation for the next spawning. Alternatively, LHβ mRNA levels declined during hydration and then increased after ovulation. This study demonstrates that chub mackerel are a good model for investigating GtH functions in multiple spawning fish.

Cloning and characterization of estrogen receptor α in mummichog, Fundulus heteroclitus

Abstract Developmental exposure to 17β-estradiol (E2) induced the death of embryos and fry, malformations, sex reversal, and incomplete ossification of vertebrae and cranial bones in the cyprinodont fish, the mummichog (Fundulus heteroclitus). To clarify the mechanism by which exogenous estrogens caused these developmental effects, we determined the sequence of an estrogen receptor (ER) coding region, encoded by 620 amino acid residues. This region shared 80% identity to that of ERα of medaka (Oryzias latipes). Northern blot analysis showed that two ERα mRNAs with 5.5 and 4 kb were expressed in the liver. These mRNAs were strongly induced by E2 stimulation. The 4 kb mRNA was expressed 8 h after treatment, whereas the 5.5 kb mRNA was not induced until 12 h after E2 stimulation. Vitellogenin (VTG) was expressed 8 h after E2 stimulation in the male liver. Receptor binding assays using the protein of F. heteroclitus ERα (fhERα) ligand binding domain showed that alkylphenols bind to fhERα with a higher affinity (50 times or more) as compared with the human ERα. The present results demonstrate that the fhERα has a sequence very similar to that of medaka, and the mRNA for this receptor was induced by E2-stimulation, followed subsequently by VTG expression. Furthermore, alkylphenols bind to fhERα more efficiently than to human ERα.

Immunological Characterization and Distribution of Three GnRH Forms in the Brain and Pituitary Gland of Chub Mackerel (Scomber japonicus)

The presence of three gonadotropin-releasing hormone (GnRH) forms in the brain of the chub mackerel, Scomber japonicus, namely, salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II), and seabream GnRH (sbGnRH), was confirmed by combined high performance liquid chromatography (HPLC) and time-resolved fluoroimmunoassay (TR-FIA). Immunocytochemical localization of the three GnRH forms in the brain was Investigated by using specific antisera, to elucidate possible roles of each GnRH form in reproduction in this species, and double immunolabeling was used to localize GnRH-ir (immunoreactive) fibers Innervating the pituitary. sGnRH-ir neurons were localized in the ventral olfactory bulb and terminal nerve ganglion region. Further, sGnRH-ir fibers were found in different regions of the brain, with prominent fibers running in parallel in the preoptic area (POA) without entering the pituitary. cGnRH-II-ir cell bodies were observed only in the midbrain tegmentum region, with a wide distribution of fibers, which were dense in the midbrain tegmentum and spinal cord. SbGnRH-ir cell bodies were localized in the nucleus preopticus of the POA, with fibers in the olfactory bulb, POA, and hypothalamus. Among the three GnRH forms, only SbGnRH-ir fibers innervated the pituitary gland from the preoptic-hypothalamic region, targeting follicle stimulating hormone (FSH) and luteinizing hormone (LH)-producing cells in the proximal pars distalis, as demonstrated by double immunocytochemistry. The localization of the GnRH-ir system was similar in male and female fish. These results demonstrate that multiple GnRH forms exist in the brain of the chub mackerel and suggest that they serve different functions, with SbGnRH having a significant role in reproduction in stimulating FSH- and LH-producing cells, and sGnRH and cGnRH-II serving as neurotransmitters or neuromodulators.