Michio Yoneda

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.

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.

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.

Increased expression of kisspeptin and GnRH forms in the brain of scombroid fish during final ovarian maturation and ovulation

BackgroundKisspeptins (Kiss) are prime players in the control of reproductive function through their regulation of gonadotropin-releasing hormone (GnRH) expression in the brain. The experimental scombroid fish, chub mackerel (Scomber japonicus) expresses two kiss (kiss1 and kiss2) and three gnrh (gnrh1, gnrh2, and gnrh3) forms in the brain. In the present study, we analyzed expression changes of kiss and gnrh mRNAs in the brain and corresponding GnRH peptides in the brain and pituitary during final ovarian maturation (FOM) and ovulation.MethodsFemale fish possessing late vitellogenic oocytes were injected with GnRH analogue to induce FOM and ovulation. Fish were observed for daily spawning activities and sampled one week post-injection at germinal vesicle migration (GVM), oocyte hydration, ovulation, and post-ovulatory time periods. Changes in relative mRNA levels of kiss and gnrh forms in the brain were determined using quantitative real-time PCR. Changes in GnRH peptides in the brain and pituitary were analyzed using time-resolved fluoroimmunoassay.ResultsBoth kiss1 and kiss2 mRNA levels in the brain were low at late vitellogenic stage and increased significantly during the GVM period. However, kiss1 mRNA levels decreased during oocyte hydration before increasing again at ovulatory and post-ovulatory periods. In contrast, kiss2 mRNA levels decreased at ovulatory and post-ovulatory periods. Levels of gnrh1 mRNA in the brain increased only during post-ovulatory period. However, levels of gnrh2 and gnrh3 mRNAs were elevated during GVM and then, decreased during oocyte hydration before increasing again at ovulatory period. During post-ovulatory period, both gnrh2 and gnrh3 mRNA levels declined. Peptide levels of all three GnRH forms in the brain were elevated during GVM and oocyte hydration; their levels were significantly lower during late vitellogenic, ovulatory, and post-ovulatory periods. In contrast, pituitary GnRH peptide levels did not show any significant fluctuations, with the GnRH1 peptide levels being many-fold higher than the GnRH2 and GnRH3 forms.ConclusionThe results indicate increased expression of multiple Kiss and GnRH forms in the brain and suggest their possible involvement in the regulation of FOM and ovulation in captive female chub mackerel.

Age validation, growth and annual reproductive cycle of chub mackerel Scomber japonicus off the waters of northern Kyushu and in the East China Sea

The age and growth of chub mackerel Scomber japonicus collected from the East China Sea and the northern waters off Kyushu between June 2000 and June 2001 were determined by observing the otolith surface after dipping it in xylene. The translucent and opaque zones on the otolith surface were identified, and the number of translucent zones was counted. Monthly changes in the frequency of fish with translucent zones on the otolith margin, and in the marginal increments, indicated that the translucent zones were formed between April and June. The seasonal pattern of annulus formation on the otolith became clear by observing the otoliths of fish with known ages, and the otolith formation in wild fish was consistent with that of fish with known ages. The mean gonadosomatic index of male and female fish was high from March to May, and spawning females were observed from mid-March to mid-May. The estimated ages were 1–5 years for males and 1–6 years for females. The von Bertalanffy growth parameters did not significantly differ between male and female. The model was obtained as FLt=406×{1−exp[−0.372×(t+1.68)]

Age validation and growth variability of Japanese flounder Paralichthys olivaceus off the Pacific coast of northern Japan

This study examined age and growth of Japanese flounder Paralichthys olivaceus off the Pacific coast of northern Japan, and determined whether the growth patterns of male and female fish in northern (40–41°N) and southern (37–38°15′N) waters differ. In total 8095 specimens were collected between January 1999 and December 2005. Zonation consisting of opaque and translucent bands on the otolith was evident. Within each opaque band a thin and clear check (ring mark) was observed in all specimens examined. Monthly change in the frequency of appearance of a ring mark on the outer margin of the otolith indicates that ring marks form between July and August. The von Bertalanffy growth model showed a sexual dimorphism in growth, as females grew faster and reached a larger size than males. The growth patterns obtained by tracking the observed total length for monthly collections showed a rapid increase in total length between August and October. Spatial variation in the growth pattern of male and female fish between northern and southern waters was evident, as southern fish were significantly larger than northern counterparts during 1.25–3.00 years post hatch.

Sexual maturation, annual reproductive cycle, and spawning periodicity of the shore scorpionfish, Scorpaenodes littoralis

The ovarian structure, sexual maturation, annual reproductive cycle, and spawning periodicity of the shore scorpionfish, Scorpaenodes littoralis, in Uchiura Bay, central Japan, were examined using specimens collected between May 1995 and March 1998 and fishes reared in laboratory. The ovarian stroma and blood vessels run longitudinally through the center of each ovarian lobe. The ovarian peduncles radiate from the central stroma. During the spawning season, gelatinous material is secreted from the epithelia of both the ovarian peduncle and ovarian wall, and the epithelia show morphological changes accompanying the ovarian maturation cycle. The minimum standard length at maturity was 55.2 mm for males and 40.2 mm for females. Males with mature testes were collected from March to November. Females in the mature or post spawning stages were collected between May and October, when the mean gonadosomatic indices were also high. This indicates that the spawning season of this species occurs between May and October. Four successive types of oocytes were grouped in the mature ovary, comprised of mature, late and early vitellogenic and previtellogenic oocytes respectively, suggesting that this species is a multiple spawner. Four captive females spawned repeatedly at intervals of 2–8 days over a prolonged period (4–8 months); a 2-day spawning interval was the most common for all females. This suggests that female S. littoralis have a 48-h spawning cycle in captivity.

Identification and distribution of three gonadotropin-releasing hormone (GnRH) isoforms in the brain of a clupeiform fish, Engraulis japonicus.

To gain a better understanding of the reproductive endocrinology of a primitive order clupeiform fish (Japanese anchovy, Engraulis japonicus), cDNAs encoding three gonadotropin-releasing hormone (GnRH) isoforms were isolated from the brain, and their distribution was analyzed using insitu hybridization (ISH). The three GnRH isoforms include GnRH1 (herring GnRH), GnRH2 (chicken GnRH-ll) and GnRH3 (salmon GnRH), and their full-length cDNAs encode 88, 86, and 89 deduced amino acids (aa), respectively. Alignment analysis of Japanese anchovy GnRH isoforms showed lower identities with other teleost fish. The major population of GnRH1 neurons was localized in the ventral telencephalon (VT) and nucleus preopticus (NPO) of the preoptic area (POA) with minor population in the anterior olfactory bulb (OB). GnRH2 neurons were restricted to the midbrain tegmentum (MT), specific to the nucleus of the medial longitudinal fasciculus (nMLF). GnRH3 neurons were localized in the olfactory nerve (ON), ventral OB, and transitional area between OB and ON. Interestingly, GnRH1 neurons were also localized in the olfactory bulb, in addition to its major population in the preoptic area. These results indicate the differential distribution of three GnRH isoforms expressed in the brain of the Japanese anchovy.

Growth and maturation variability of female John Dory ( Zeus faber ) in the East China Sea in relation to thermal gradients

This study explored whether the growth and sexual maturity of female John Dory (Zeus faber) differed in relation to hydrographic conditions at sample sites in the East China Sea. John Dory were collected around the margin of the continental shelf from the north-east to south-west waters of the East China Sea, and seasonal changes in spatial distribution were unclear. These probably reflected physiological constraints on salinity, as John Dory are distributed only in waters of high salinity. Thermal gradients along latitudinal lines were evident within sample sites. The northern population was subject to lower temperatures than the southern population during all sampling periods. There were no significant differences in length-age relationships between sampling locations, although the northern population had better body condition than their southern counterparts for a given length. However, northern John Dory matured earlier than their southern counterparts with the same relative condition factor.

Temperature-induced variation in sexual maturation of Japanese anchovy Engraulis japonicus

Temperature is one of the most influential factors for the sexual maturation of fishes, but understanding of the extent to which temperature affects the maturational schedules is limited in multiple-spawning fishes over a protracted season. This study examined the effect of temperature on sexual maturation of Japanese anchovy Engraulis japonicus siblings under high and low temperature regimes on different birthdates. The maturation probability differed between the two temperature regimes. Specimens in high temperature regimes matured at much smaller size and younger age than their counterparts. Also, a significant difference in the maturation probability between sexes was found at low temperatures, but not at high temperatures. Our findings show that temperature affects the maturational schedules of siblings of Japanese anchovy, suggesting that the size and age at sexual maturation could differ among cohorts, even in a given sampling location and/or year.