Potent Influence of Exogenous Melatonin on In Vitro Oocyte Maturation in the Longchin Goby, Chaenogobius annularis.

Effects of changes in photoperiod on the reproductive events in fish are suggested to be mediated mainly via the action of melatonin (MEL). Changing levels of plasma MEL throughout the day and year are suggested to influence the hypothalamus-pituitary-gonadal axis in fish. Therefore, in this study, we aimed to investigate the effects of MEL on oocyte maturation and germinal vesicle breakdown (GVBD) in the marine fish, Chaenogobius annularis, in vitro. Oocytes at three different stages (pre-, mid-, and late-vitellogenesis) were incubated with (a) only MEL (5, 10, 50, 100, 500, and 1,000 pg/mL) and (b) 50 pg/mL of 17α,20ß-dihydroxy-4-pregnen-3-one (17α20ßP), maturation-inducing hormone (MIH) of this species, and MEL (4-h incubation before addition of MIH). Any single MEL treatment did not significantly induce GVBD. However, treatment with 50 pg/mL MEL or MIH significantly induced GVBD. These results suggest that preincubation with MEL accelerates the effect of MIH on longchin goby oocyte maturation.

In Vitro Sex Steroid Metabolism in Red Spotted Grouper, Epinephelus akaara during Oocyte Maturation.

We studied steroid metabolites produced from red-spotted grouper ovarian follicles during maturation. Oocytes with 350-500 µm diameter were in vitro incubated in the presence of [3H] 17α-hydroxyprogesterone as a precursor. Steroid metabolites were extracted from incubated media and oocytes. The extracts were separated and identified using thin layer chromatography, high performance liquid chromatography and gas chromatography-mass spectrometry. The identified metabolites were androstenedione (A4), testosterone (T) and estrone (E1). The metabolites of A4 was dominant in all size of oocytes and it was the highest in 480 µm diameter oocytes. The metabolites of two progestins, 17α,20ß-dihydroxy-4-pregnen-3-one and 17α,20α-dihydroxy-4-pregnen-3-one were detected in the oocytes less than 480 µm diameter although they were not identified definitely. In the oocytes of 480 µm diameter, metabolite of progestin was the highest, and germinal vesicle (GV) was still in the middle of cytoplasm. In the oocytes of 500 µm diameter, GV was began to migrate and the major metabolites were A4 and E1. The metabolite of E1 was detected in all size of oocytes and it was higher than that of E2. These results suggest that oocytes of 480 µm diameter are the transitional stage involving steroidogenic shift to final oocyte maturation and potential function of E1 during maturation process.

Mature and Bi-Sexual Phase Gonad Occurrence in Cultured Red Spotted Grouper, Epinephelus akaara.

This study reports the presence of mature and bi-sexual phase gonads in red spotted grouper, Epinephelus akaara after less than a year of cultivation in a commercial indoor tank and a net cage. In December 2018, juveniles were placed in an indoor tank and cultured for five months. In June 2019, the fish were transferred to a net cage and cultured until September. The rearing temperatures ranged from 19.86°C-24.65°C in the indoor tank and 21.86°C-27.65°C in the net cage. During the net cage culture period, specimens were randomly selected for histological gonad examination. The highest gonadosomatic index (GSI) value was measured in July (3.38±2.53), and dramatically decreased in August (0.44±0.21) and September (0.42±0.30). In July, some mature fish showed signs of vitellogenic stage oocyte development (vitellogenic and oil droplet stage oocytes), but immature fish were in an early developmental stage containing peri-nucleolus stage (PNS) oocytes. Bi-sexual phase gonads containing spermatocytes and spermatids were observed in the lumen and several PNS oocytes. By August and September, most specimens showed early-stage ovary development. However, mature testis (in August) and bi-sexual phase gonads (in September) were also observed. These results provide evidence for early puberty and hermaphroditism in the red spotted grouper.

Thermal stress induces a distinct transcriptome profile in the Pacific oyster Crassostrea gigas.

Oysters are frequently subjected to heat stress during tidal emersion/immersion cycles in their habitats due to attachment on the rocky shore. To understand the effect of temperature elevation on the whole transcriptome over time, the Pacific oyster Crassostrea gigas was exposed to seawater temperature 32°C for 72h from the control 20°C. RNA-seq identified differentially expressed stress responsive transcripts upon thermal stress in the gill tissues of C. gigas. The primary effect of heat stress appears to be significantly induced transcription of molecular chaperones, including members of the heat shock protein (hsp) families, while genes typically associated with protein metabolism, such as those involved in protein degradation (e.g. ATP-dependent proteolysis pathway) and biosynthesis (e.g. ribosomal protein genes), were repressed. In particular, several hsp70 isoforms and a small hsp20 maintained prolonged mRNA expressions for 72h. This study provides preliminary insights into the molecular response of C. gigas to heat stress and suggests a basis for future studies examining molecular adaptation or thermotolerance metabolism in the Pacific oyster.

Histological Analysis of Early Gonadal Development and Sex Differentiation in Chameleon Goby, Tridentiger trigonocephalus.

This study describes the developmental process of gonads in chameleon goby, Tridentiger trigonocephalus from the stage of hatching to 100 days after hatching (DAH). Based on histological observation, the primordial germ cells were observed in mesentery between mesonephric duct and gut at 15 DAH (total length, TL: 6.8±0.2 mm). At 20 DAH (TL: 7.9±0.1 mm), the primordial gonad began to protrude into peritoneal cavity and developed between mesonephric duct and gut. Initial ovarian differentiation was identified by the presence of ovarian cavity and oogonia in the gonads at 55 DAH (TL: 21.1±1.3 mm). Testicular differentiation started at 65 DAH (TL: 23.7±0.9 mm) with appearance of spermatogonial cells in the gonads. These findings indicate that sex differentiation in T. trigonocephalus occurs earlier in females than males, suggesting that this species can be classified as an undifferentiated gonochorist.

RAG-1 and IgM Genes, Markers for Early Development of the Immune System in Olive Flounder, Paralichthys olivaceus.

Fish larvae are immediately exposed to microbes from hatching to maturation of their lymphoid organs, therefore effective innate mechanisms is very important for survival. However, the knowledge of the development of immune system in fish is limited and in demand now. In vertebrates, recombination-activating gene 1 (RAG-1) and immunoglobulin M (IgM) have been considered as very useful markers of the physiological maturity of the immune system. In this study, the expression of the both genes was assessed throughout the early developmental stages of olive flounder larvae (5-55 dph) and used as markers to follow the development of immune system. RAG-1 and IgM mRNA expression was detectable at 5 dph and remained so until 55 dph. These patterns of expression may suggest that the olive flounder start to develop its function around 5 dph. Tissue distribution was found that both genes mRNAs are only expressed in the immune-related organ such as spleen, kidney and gill. The early detection of IgM mRNA led to the investigation of its presence in oocytes. Both RAG-1 and IgM mRNA transcripts were detected in unfertilized oocytes, suggesting that they are maternally transferred. The biological significance of such a phenomenon remains to be investigated.

Expression of Vimentin Intermediate Filament for Vascular Development in Olive Flounder (Paralichthys olivaceus).

Cardiovascular system is the primary organ to develop and reach a functional state, which underscores the essential role of the vasculature in the developing embryo. The vasculature is a highly specialized organ that functions in a number of key physiological works including the carrying of oxygen and nutrients to tissues. It is closely involved in the formation of heart, and hence it is essential for survival during the hatching period. The expression of genes involved during vascular development in the olive flounder (Paralichthys olivaceus) in the days after hatching is not fully understood. Therefore, we examined the expression patterns of genes activated during the development of flounder. Microscopic observations showed that formation of blood vessels is related to the expression of the vimentin gene. Also, the temporal expression patterns of this vimentin-like gene in the developmental stages and in the normal tissues of olive flounder. The purpose of this study was to examine the expression patterns of vimentin in normal tissues of the olive flounder and during the development of the vascular system in newly hatched olive flounders and HIF-1 plays a vital role in the formation of blood vessels during development. Vimentin expression was strong at the beginning of the development of blood vessels, and was present throughout all developmental stages. Our findings have important implications with respect to the roles of vimentin and HIF-1 in the development and evolution of the first blood vessels in olive flounder. Further studies are required to elucidate the vimentin-mediated hypoxic response signal transduction and to decipher the functional role of vimentin in developmental stages.

Expression Analysis of Cathepsin F during Embryogenesis and Early Developmental Stage in Olive Flounder (Paralichthys olivaceus).

Cathepsins are members of the multigene family of lysosomal cysteine proteinases and have regulated function in several life processes. The potential role of cathepsin F cysteine gene was expected as protease in the yolk processing mechanism during early developmental stage, but expression analysis was unknown after fertilization. The alignment analysis showed that amino acid sequence of cathepsin F from olive flounder liver expressed sequence tag (EST) homologous to cathepsin F of other known cathepsin F sequences with 87-98% identity. In this study, we examined the gene expression analysis of cathepsin F in various tissues at variety age flounder. Tissue distribution of the cathepsin F mRNA has been shown to be ubiquitous and constitutive pattern regardless of age in each group, although derived from cDNA library using liver sample. The mRNA level of cathepsin F more increased as developmental proceed during embryogenesis and early developmental stage, especially increased in the blastula, hatching stage and 3 days post hatching (dph). As a result, it may suggest that the proteolysis of yolk proteins (YPs) has been implicated as a mechanism for nutrient supply during early larval stages in olive flounder.

Expression Analysis of Visual Arrestin gene during Ocular Development of Olive Flounder (Paralichthys olivaceus).

Olive flounder (Paralichthys olivaceus) is one of the commercial important flatfish species in Korea. The ocular signal transduction pathway is important in newly hatched flounders because it is closely involved in the initial feeding phase thus essential for survival during the juvenile period. However, the study of gene expression during ocular development is incomplete in olive flounder. Therefore we examined the expression analysis of specifically induced genes during the development of the visual system in newly hatched flounders. We searched ocular development-involved gene in the database of expressed sequence tags (ESTs) from olive flounder eye and this gene similar to arrestin with a partial sequence homology. Microscopic observation of retinal formation corresponded with the time of expression of the arrestin gene in the developmental stage. These results suggest that arrestin plays a vital role in the visual signal transduction pathway of the retina during ocular development. The expression of arrestin was strong in the ocular system during the entirety of the development stages. Our findings regarding arrestin have important implications with respect to its biological role and evolution of G-protein coupled receptor (GPCR) signaling in olive flounder. Further studies are required on the GPCR-mediated signaling pathway and to decipher the functional role of arrestin.

The Expression Analysis of Complement Component C3 during Early Developmental Stages in Olive Flounder (Paralichthys olivaceus).

Fish larvae are immediately exposed to microbes from hatching to maturation of their lymphoid organs, therefore effective innate mechanisms is very important for survival in such an environment. The key component of innate immune system, C3 is central protein of all activation pathways of the complement system, leading to inflammatory reactions, such as opsonisation, chemotaxis, and cell lysis of pathogens. Although, innate mechanisms is essential for survival in the early stage of development, little is known about defence mechanisms. In this study, the alignment analysis showed that amino acid sequence of C3 from olive flounder liver EST homologous to other known C3 sequences with 73-99% identity. Also, we examined the tissue distribution of olive flounder C3 and analyzed expression pattern from the fertilized egg until 28 days post hatching. As a result, olive flounder C3 mRNA was expressed only in the liver and the mRNA level more increased as developmental proceed during the early stage. These results may suggest that olive flounder C3 plays an important function in the early immune response of olive flounder larvae.

Expression of Perforin Gene for Early Development of Nephrons in Olive Flounder (Paralichthys olivaceus).

The innate immune system is the only defense weapon that invertebrates have, and it is the fundamental defense mechanism for fish. The innate immune response is important in newly hatched flounders because it is closely involved in the initial feeding phase, which is why it is essential for survival during the juvenile period. The expression analysis of genes involved in the innate immune response in the olive flounder (Paralichthys olivaceus) in the days after hatching is incomplete. Therefore, we have begun to examine the expression patterns of genes specifically induced during the development of the innate immune system in newly hatched flounders. Microscopic observation showed that pronephron formation corresponded with the expression of perforin-encoding gene. These results suggest that perforin plays a vital role in the innate immunity of the kidney during developmental stages. Perforin expression was strong at the start of the development of the innate immune response, and continued throughout all the development stages. Our findings have important implications with respect to perforin's biological role and the evolution of the first defense mechanisms in olive flounder. Further studies are required to elucidate the perforin-mediated innate immunity response and to decipher the functional role of perforin in developmental stages.

Stage and Tissue Specific Expression of Four TCR Subunits in Olive Flounder (Paralichthys olivaceus).

TCR subunits are members of membrane-bound receptors which allow the fast and efficient elimination of the specific fish pathogens have regulated function in adaptive immunity. Sequence structure of TCR subunits have been reported for various teleosts, but the information of each TCR subunit functional characterization through expression analysis in fish was unknown. In this study, we examined the gene expression of TCR subunits in the early developmental stages and observed transcript levels in various tissues from healthy adult olive flounder by RT-PCR. The mRNA expression of alpha subunit was already detected in the previous hatching step. But the transcripts of another TCR subunit were not observed during embryo development and increased after hatching and maintained until metamorphosis at the same level. It was found that all TCR subunits mRNAs are commonly expressed in the immune-related organ such as spleen, kidney and gill, also weak expressed in fin and eye. TCR alpha and beta subunit were expressed in brain, whereas gamma and delta were not expressed same tissue. The sequence alignment analysis shows that there are more than 80% sequence homology between TCR subunits. Because it has a high similarity of amino acid sequence to expect similar in function, but expression analysis show that will have may functional diversity due to different time and place of expression.

Reproductive Cycle of Chameleon Goby, Tridentiger trigonocephalus in the Southern Coastal Waters of Korea.

The objective of this study was to characterize the reproductive cycle of the chameleon goby, T. trigonocephalus. Gonadal development was investigated using a histological method. Specimens were collected monthly, from April 2009 to March 2010. The gonadosomatic index (GSI) of females began to increase in April, reaching the maximum in May, and declined sharply in August. In males, the GSI began to increase in April and reaching the maximum in July. The annual reproductive cycle of T. trigonocephalus can be divided into four successive stages in females: the growing (November-March), maturing (April-May), ripe and spawning (June-July), and recovery (August-October) stages. Males passed through growing (November-March), maturing (April-June), ripe and spermiation (July-August), and recovery (September-October) stages. These results indicate the spawning season is from June to July. The relationship between fecundity (Fc) and body length (BL) was Fc=86.1511BL(2.6506). Fecundity was ranged from 3,448-9,654 eggs in a BL of 4.8-7.2 cm and it was increased as BL increased.

Changes in Plasma Sex Steroid and Cortisol Levels during Annual Reproductive Cycle of Ribbed Gunnel, Dictyosoma burgeri.

We investigated the changes in plasma sex steroid hormones, testosterone (T), estradiol-17ß (E2), 17,20ß -dihydroxy-4-pregnen-3-one (17α20ßP), 11-ketotestosterone (11KT) and cortisol levels from ribbed gunnel, Dictyosoma burgeri in associated with annual reproductive cycle. The gonadosomatic index (GSI) of females increased from November, peaked in February and decreased rapidly from March. The GSI of males also increased from November, peaked in January and then decreased gradually. In females, E2 levels increased and remained high from December to February. The levels of T showed a similar tendency and correlated (rs =0.898, p<0.01) with E2 levels. The levels of 17α20ßP increased rapidly in February (4.78±1.01 ng/ml) and peaked in July (5.08±0.65 ng/ml). Cortisol level was peaked in March and correlated with 17α20ßP levels (rs =0.696, p<0.01). In males, the levels of T was peaked in January and then decreased rapidly. The levels of 11KT were remained high from October to January. On the other hand, the levels of 17α20ßP fluctuated during reproductive cycle. These results suggest that plasma sex steroids in ribbed gunnels have annual periodicity, and that cortisol may involve in maturation of females.

Differential Expression Patterns of Crystallin Genes during Ocular Development of Olive Flounder (Paralichthys olivaceus).

Olive flounder Paralichthys olivaceus is one of the most widely cultured fish species in Korea. Although olive flounder receive attention from aquaculture and fisheries and extensive research has been conducted eye morphological change in metamorphosis, but little information was known to molecular mechanism and gene expression of eye development- related genes during the early part of eye formation period. For the reason of eyesight is the most important sense in flounder larvae to search prey, the screening and identification of expressed genes in the eye will provide useful insight into the molecular regulation mechanism of eye development in olive flounder. Through the search of an olive flounder DNA database of expressed sequence tags (EST), we found a partial sequence that was similar to crystallin beta A1 and gamma S. Microscopic observation of retinal formation correspond with the time of expression of the crystallin beta A1 and gamma S gene in the developmental stage, these result suggesting that beta A1 and gamma S play a vital role in the remodeling of the retina during eye development. The expression of crystallin beta A1 and gamma S were obviously strong in eye at all tested developing stage, it is also hypothesized that crystallin acts as a molecular chaperone to prevent protein aggregation during maturation and aging in the eye.

The Morphological Study of Wild and Farmed Olive Flounder (Paralichthys olivaceus): The Role of Indirect Selection within and between Populations.

This study was conducted to analyze the difference of body types within and between wild and farmed populations of olive flounder Paralichthys olivaceus using measured records of morphological traits. The results showed that surveyed traits and standard deviation were 1,355±742 g of body weight, 48.01±7.79 cm of total length, and 40.96±6.80 cm of body length. Also body height, body shape index and condition factor were 17.19±3.43 cm, 9.99±0.74 and 11.16±1.54, respectively. As result of least squares mean and standard error for each trait assumed in this study, those of farmed population showed significantly higher than those of wild population in all traits, exclusively in total length and body length (p<0.01). Particularly, the values of the body height and the body weight of the farmed population were higher than those of the wild population in the same total length. And the phenotypic correlation coefficients of the body weight, the total length, the body length and the body height showed strong positive correlation in all populations. These result suggested that morphological differences exist in farmed and wild flounder. Therefore, introduction of wild flounder is essential for the future production to improve the body type of farmed flounder, and parental fish should be chosen by considering selection of commercially important traits in the production process.

Effects of BPA and DES on longchin goby (Chasmichthys dolichognathus) in vitro during the oocyte maturation.

This study was performed in order to assess whether bisphenol (BPA) and diethylstilbestrol (DES) had agonistic or antagonistic effects on oocyte maturation using marine fish. We tested the effects of these chemicals on in vitro maturation, germinal vesicle breakdown (GVBD), assay using oocytes from the longchin goby, Chasmichthys dolichognathus. During the maturation process, low concentrations of BPA and DES triggered GVBD depending on the stage of oocyte development; BPA at 0.044 nM and DES at 0.037, 0.37, and 3.73 nM induced GVBD in 0.82-0.88 mm diameter oocytes (germinal vesicle located near the center of oocytes). In 0.76-0.80 mm diameter oocytes (fully vitellogenic oocytes), BPA induced GVBD at relatively higher concentrations (4.38, 43.8, and 438 nM). In 0.86-0.90 mm diameter oocytes, BPA and DES had no observable effect on GVBD at the concentrations tested. Oocytes with diameters between 0.82 and 0.88 mm appeared to be more sensitive to these chemicals. Moreover, our results showed that BPA and DES did not inhibit GVBD.