Michiya Matsuyama

Diurnal rhythm of oocyte development and plasma steroid hormone levels in the female red sea bream, Pagrus major, during the spawning season

Abstract Diurnal maturation rhythm in the female red sea bream, Pagrus major , which spawns every day during the spawning season and has an asynchronous-type ovary containing oocytes at various stages of development, was studied by histological investigation of the ovary. Plasma levels of estradiol-17β and testosterone corresponding to the ovarian maturation rhythm were also investigated. The fish had a diurnal ovarian maturation rhythm in which the most advanced oocytes in the ovary finished germinal vesicle breakdown at 07.00 h, arrived at a mature state at 10.00 h, and ovulation began at 13.00 h. Spawning mainly occurred between 18.00 and 19.00 h. Plasma estradiol-17β levels reached a peak of about 1200 pg/ml in fish with pre-mature stage oocytes at 07.00 h, followed by a sharp drop in ovulated fish at 13.00 h (to about 200 pg/ml); the levels showed a tendency to increase from the time of postovulation to the next germinal vesicle breakdown. Testosterone levels decreased (to about 20 pg/ml) at ovulation time (13.00 h) in parallel with the estradiol-17β levels, but were largely unchanged (70–110 pg/ml) throughout the rest of the ovarian maturation cycle. These results are discussed in relation to other studies on the role of these steroid hormones responsible for the ovarian maturation of teleosts.

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.

Expression of the DMRT gene and its roles in early gonadal development of the Japanese pufferfish Takifugu rubripes

DMRT1, a gene containing the Doublesex/Mab-3 DNA-binding motif (DM domain), encodes a transcription factor that regulates early differentiation of Sertoli cells in the testes of vertebrates. Here, we describe the pattern of expression of six DMRT genes (DMRT1, 2a, 2b, 3, 4, and 5) during gonadal development in the Japanese pufferfish, Takifugu rubripes, for the first time. Conventional histological analysis showed that a primordial gonad is present 2 weeks after hatching and that sexual differentiation had occurred by 6 weeks after hatching, as determined by the formation of cavities in the ovaries. RT-PCR analysis showed that the DMRT1 transcript was abundant in the testes, but not in the ovaries, of 3-month-old fish. DMRT3 transcript was also detected in the testes, but not in the ovaries, indicating that the patterns of gonadal expression of DMRT3 are very similar to DMRT1. The other four DMRT genes examined did not exhibit sex-specific expression patterns. In situ hybridization analysis revealed that, after gonadal differentiation was complete, DMRT1 was expressed in the Sertoli cells that were in proximity to proliferating spermatogonia. These results indicate that DMRT1 involved in gonadal development rather than sex differentiation and that its expression correlates with the proliferation of spermatogonia in T. rubripes. The expression profile of DMRT1 in T. rubripes was similar to that in the teleost species Medaka.

Diurnal rhythm of serum steroid hormone levels in the Japanese whiting, Sillago japonica, a daily-spawning teleost

Ovarian developmental stages and serum steroid hormone levels were examined at six different times of day (0100, 0600, 1000, 1300, 1600, 2000 h) in a marine teleost, the Japanese whiting Sillago japonica, which has an asynchronous-type ovary containing oocytes at various stages of development and spawns every day during a period ranging up to three months. The largest oocytes in the ovaries at the active vitellogenic or post-vitellogenic stages were found between 0100 and 1300 h. Oocyte maturation indicated by germinal vesicle breakdown (GVBD) occurred at 1600 h, and ovulated oocytes were observed in the ovaries collected at 2000 h. These processes were accompanied by a significant daily change in serum steroid hormone levels. The serum level of estradiol-17β showed a peak in fish with mature oocytes sampled at 1600 h. In these fish, the second-largest oocytes in the ovaries were at the initial stage of vigorous vitellogenesis, the secondary yolk stage. Therefore the highest level of serum estradiol-17β was considered to be due to the second-largest oocytes. Testosterone levels remained low and constant throughout the experimental period. The serum levels of 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-diOHprog) peaked at 1600 h at which time all fish had mature oocytes. These results indicate that the Japanese whiting possesses a diurnal rhythm of oocyte development including vitellogenesis, oocyte maturation and ovulation, and further suggest that daily cycles in oocyte growth and maturation which simultaneously take place in an ovary are regulated by diurnal secretions of estradiol-17β and the maturation-inducing steroid, 17α,20β-diOHprog.

Reproductive parameters of the chub mackerel Scomber japonicus estimated from human chorionic gonadotropin‐induced final oocyte maturation and ovulation in captivity

Final oocyte maturation and ovulation of captive chub mackerel Scomber japonicus with fully yolk-accumulated occytes were induced by a single injection of human chorionic gonadotropin. Reproductive parameters, including spawning frequency and batch fecundity, which are required to estimate spawning biomass in pelagic fish by the daily egg production method, were analyzed. Germinal vesicle migration (GVM) occurred at 18–24 h post-injection, and the hydration and ovulation of oocytes were completed at 30 and 36 h post-injection, respectively. The results of the maturation process suggest that fish with GVM-stage ovaries captured in the daytime from the field are capable of spawning on the night following their capture. The oocytes used in the oocyte sizefrequency distribution method for batch fecundity estimates should be at late GVM and more advanced stages. The results of sequential artificial insemination showed that the quality of ovulated eggs held in the ovarian lumen rapidly deteriorated as time progressed after ovulation. This indicates that the fertilization window for the ovulated eggs of chub mackerel lasts only a few hours, and spawning behavior should be performed within a few hours after ovulation in the wild population.

Jack mackerel Trachurus japonicus juveniles use jellyfish for predator avoidance and as a prey collector

Juveniles of carangid fishes including jack mackerel Trachurus japonicus are known to associate with jellyfishes. The function of this association behavior was studied through rearing experiments and underwater visual observations. Association behavior of jack mackerel with moon jellyfish in experimental tanks was more frequent in the presence compared to the absence of predators (chub mackerel Scomber japonicus). In the experimental tanks, the presence of jellyfish, however, did not mitigate predation by these predators. Although jack mackerel did not feed on the jellyfish itself, they frequently fed on the captured prey (Artemia nauplii) whilst in the gut cavity of the jellyfish. Underwater observations of giant jellyfish Nemopilema nomurai off Kyoto and Fukui prefectures revealed that approximately 30% of these jellyfish were accompanied by jack mackerel juveniles with body sizes ranging 10–45 mm standard length (SL). Considering that jack mackerel juveniles found in subtidal rocky reefs ranged 40–120 mm SL, we considered that jack mackerel from 10 to 45 mm SL associate with jellyfish as a hiding place as well as a food collector, until they find a suitable reef habitat when they attain approximately 40 mm SL.

Juvenile bisexuality in the red sea bream,Pagrus major

SynopsisThe histology of the gonad of the red sea bream,Pagrus major, was examined in order to study the early gonadal development, sexual maturation and sex ratio in a natural population. A total of 1,117 fish between the ages of 4 months and 8 years were examined. Gonads of 4-month-old fish were either sexually undifferentiated with a central cavity, or ovarian in form. Gonads of 12- and 18-month-old fish were ovaries or bisexual gonads, while those of 2-year-old fish were ovaries, bisexual gonads or testes. Fish aged between 3 and 8 years had ovaries or testes, except for a few bisexual gonads found in 3- and 4-year-old fish. The chronological appearance of females, hermaphrodites and males in that order, and histological evidence, suggested that the testis originates from the ovary via a bisexual gonad in the juvenile stage. The sex ratio of females to males at the age of 2 years and over was about 1:1, suggesting that hermaphroditic red sea bream appear in about 50% of the juvenile population. The sexual pattern in this species, therefore, is concluded to be gonochorism with a bisexual juvenile stage.

Daily spawning and development of sensitivity to gonadotropin and maturation-inducing steroid in the oocytes of the bambooleaf wrasse, Pseudolabrus japonicus

The cycle of oocyte development of the bambooleaf wrasse, Pseudolabrus japonicus, was studied to elucidate the endocrinological mechanism of oocyte maturation in a marine teleost. A single female reared with two males spawned every day for 17 days in captivity, indicating that this species is a daily spawner. Ovarian histology revealed that germinal vesicle migration of the largest oocytes progressed from 12:00 to 3:00 h, and germinal vesicle breakdown (GVBD) was completed at 6:00 h. Ovulation and spawning occurred between 6:00 and 9:00 h. The effectiveness of human chorionic gonadotropin (HCG) and 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P), which is one of the most potent steroidal inducers of GVBD in bambooleaf wrasse oocytes, in inducing final oocyte maturation was examined at eight different times of the day. The responsiveness of the oocyte to HCG and steroid differed at different times of the day. The GVBD could be induced by HCG but not 17,20β-P at 9:00 h. Between 12:00 and 18:00 h, not only HCG but also 17,20β-P induced GVBD. Both GVBD and ovulation spontaneously occurred between 0:00 and 6:00 h without any hormonal treatment. These results clearly showed that the oocyte of the bambooleaf wrasse possessed a diurnal maturation cycle. Responsiveness of oocytes to HCG appeared earlier than responsiveness to 17,20β-P. This suggests that sensitivity to 17,20 β-P is induced by gonadotropic hormone (GTH).

Identification, characterization, and expression profiles of two subtypes of kisspeptin receptors in a scombroid fish (chub mackerel)

The kisspeptin receptor (Kiss1R) is a cognate receptor for kisspeptin (Kiss), and this Kiss-Kiss1R system has been shown to regulate seasonal reproduction in vertebrates. Our previous study found the chub mackerel (Scomber japonicus) brain expresses both kiss1 and kiss2 and exhibits sexually dimorphic changes during the seasonal reproductive cycle. The present study cloned two subtypes of kissr from the chub mackerel brain, and their signal transduction pathways to Kiss1 and Kiss2 were characterized in a mammalian cell line. Results of identification showed that kissr1 and kissr2 mRNAs encode 369 and 378 deduced amino acids, respectively, and share 52% similarity in amino acid sequences. In vitro functional analysis revealed that chub mackerel Kiss receptor signals are also preferentially transduced via the protein kinase C (PKC) rather than protein kinase A (PKA) pathway. Synthetic chub mackerel Kiss1-15 and Kiss2-12 peptides showed the highest potency for the activation of KissR1 and KissR2, respectively, stronger than their corresponding Kiss-10 peptides. Tissue distribution analyses indicated that both genes are highly expressed in the brain and that only kissr2 mRNA is expressed in the pituitary of both sexes. Unexpectedly, both kissr1 and kissr2 mRNAs were detected only in the testes. Seasonal expression changes showed higher expression levels of both kissr1 and kissr2 mRNAs in the brain of females during the early vitellogenic period; however, no significant differences were found in the brain of males. Pituitary kissr2 mRNA levels showed no significant variations. In the testes, the kissr1 mRNA expression level increased dramatically at spermiation compared with the immature and post-spawning periods. However, kissr2 mRNA levels in the testes did not vary significantly at different testicular stages. These results suggest that both kissr1 and kissr2 likely participate in the seasonal ovarian development of females, and thus in males, we propose a paracrine or autocrine role for kissr1 in testicular development.

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.