Semen promotes oocyte development in Sebastesschlegelii elucidating ovarian development dynamics in live-bearing fish.

In some vertebrates and invertebrates, semen release factors affecting female physiology and behavior. Here, we report that semen delivered to females is potentially beneficial for promoting oocyte development in a viviparous teleost, Sebastes schlegelii. 88% of mated ovaries develop normally and give birth to larval fish, whereas 61% of non-mated ovaries are arrested in the previtellogenic stage. Semen's significant role (p < 0.0001) in promoting oocyte development may involve remodeling follicular cells and regulating the expression of the extracellular matrix, which facilitates cell communication. Furthermore, the ovarian response to semen may influence the brain, affecting hormone release, follicular cell development and steroid production, and crucial for oocyte growth. This mechanism, which could potentially delay maternal investment in offspring until male genetic input occurs to avoid energy wastage, has not been previously described in teleosts. These findings enhance our understanding of ovarian development in viviparous fish, with broader implications for reproductive biology.

Comparative transcriptome analysis reveals growth and molecular pathway of body color regulation in turbot (Scophthalmus maximus) exposed to different light spectrum.

Fish body color changes play vital roles in adapting to ecological light environment and influencing market value. However, the initial mechanisms governing the changes remain unknown. Here, we scrutinized the impact of light spectrum on turbot (Scophthalmus maximus) body coloration, exposing them to red, blue, and full light spectra from embryo to 90 days post hatch. Transcriptome and quantitative real-time PCR (qRT-PCR) analyses were employed to elucidate underlying biological processes. The results showed that red light induced dimorphism in turbot juvenile skin pigmentation: some exhibited black coloration (Red_Black_Surface, R_B_S), while others displayed lighter skin (Red_White_Bottom, R_W_B), with red light leading to reduced skin lightness (L*) and body weight, particularly in R_B_S group. Transcriptomic and qRT-PCR analyses showcased upregulated gene expressions related to melanin synthesis in R_B_S individuals, notably tyrosinase (tyr), tyrosinase-related protein 1 (tyrp1), and dopachrome tautomerase (dct), alongside solute carrier family 24 member 5 (slc24a5) and oculocutaneous albinism type II (oca2) as pivotal regulators. Nervous system emerged as a critical mediator in spectral environment-driven color regulation. N-methyl d-aspartate (NMDA) glutamate receptor, and calcium signaling pathway emerged as pivotal links intertwining spectral conditions, neural signal transduction, and color regulation. The individual differences in NMDA glutamate receptor expression and subsequent neural excitability seemed responsible for dichromatic body coloration in red light-expose juveniles. This study provides new insights into the comprehending of fish adaptation to environment and methods for fish body color regulation and could potentially help enhance the economic benefit of fish farming industry.

Developmental regulation of the male urogenital papilla in the male marine teleost black rockfish, Sebastes schlegelii (Hilgendorf, 1880)†.

The male external genitalia of the black rockfish (Sebastes schlegelii Hilgendorf, 1880) is a fleshy protrusion known as the urogenital papilla (UGP), which functions to deliver sperm into the female reproductive tract for internal fertilization. It is not known which genes regulate the development of the UGP. The aim of this study was to identify key genes that regulate the development of the UGP in black rockfish and to determine the distribution of androgen receptor gene (ar) in the UGP. A total of 26 adult males and 560 juvenile fish were used in the experiment, in which we divided all normally developing juveniles into normal development and androgen groups. We added methyltestosterone solution (100 µg/l) to the androgen group-treated fish tank, soaked for 2 h per day for 38 days, and sampled 5~10 samples each time every 5 days during the culture process. Gene expression changes related to UGP were analyzed with tissue specificity between control and androgen groups during sex differentiation, adult male maturation, and the copulation stage (September to December) using real-time quantitative polymerase chain reaction. The expression of ar was also localized by two-color in situ hybridization in the UGP region of juvenile fish. Androgen treatment enhanced ar expression levels and the ar signal was stronger in the UGP region of both adult breeding fish and androgen-treated juvenile fish. This study provides insights into the regulation of the external genitalia of black rockfish and presents vital information for the artificial breeding of viviparous fish.

Effect of ovarian fluid on sperm performance in teleost with internal and external fertilization strategies.

Ovarian fluid is essential for successful fertilization by maintaining the viability, motility, and velocity of sperm. The organic compounds and inorganic ions in ovarian fluid significantly influence spermatozoa's motility, velocity, and longevity. However, the effect of ovarian fluid on sperm performance is limited in teleost fish. In this study, the effect of ovarian fluid on sperm performance and its components in external fertilization species (Scophthalmus maximus, turbot) and internal fertilization species (Sebastes schlegelii, black rockfish) was investigated using computer-assisted sperm analysis, high-performance liquid chromatography, and metabolome analysis. The ovarian fluid had a distinct and species-specific effect on both species. In the black rockfish, the ovarian fluid from turbot significantly increased sperm motility (74.07% ± 4.09%), as well as VCL (45 ± 1.67 µm/s), VAP (40.17 ± 1.6 µm/s), and VSL (36.67 ± 1.86 µm/s), and longevity (352 ± 11.31 min) (P < 0.05). In the turbot, only the longevity (71.33 ± 5.69 min) and fertilization rate (65.27% ± 11.59%) showed significantly improvement (P < 0.05). The ovarian fluid was rich in organic compounds, suggesting enrichment in the glycolysis/gluconeogenesis pathways. The results suggest that glycometabolism plays a crucial role in improving sperm performance in teleost with internal fertilization. Thus, incorporating ovarian fluid into the sperm activation medium can enhance artificial fertilization in fish breeding.

Morphology of the urogenital papilla of the male marine teleost Black Rockfish, Sebastes schlegelii (Hilgendorf, 1880), and its role in internal fertilization.

There are few detailed descriptions of the morphology of the male external genitalia, the urogenital papilla (UGP), of the Black Rockfish (Sebastes schlegelii Hilgendorf, 1880). The purpose of this study was to evaluate this organ histologically and to determine the time of development of the UGP in Black Rockfish. Twelve adult males, three adult females and around 500 juveniles were used in the experiment. The juveniles were divided into normal developmental and androgen groups. The androgen group was exposed to methyltestosterone (100 µg/L) for 2 h daily for 38 days. Samples (N = 10 per sampling) were randomly selected for analysis every 5 days from 30 to 116 days after birth. Parameters assessed included the type of epithelium, composition of connective tissue, muscular tissue, and the timing of UGP development. Differences in these parameters between normal developmental and androgen groups were evaluated. The results indicated that the UGP of the adult fish contains the sperm duct and ureter, which have the function of transporting sperm and urine, respectively. The androgen-treated juvenile fish developed the UGP earlier than the normal development group. This study provides a reference for understanding the external genitalia of other viviparous fishes by studying the UGP of the male Black Rockfish.

Rxrs and their partner receptor genes inducing masculinization plausibly mediated by endocrine disruption in Paralichthys olivaceus.

Retinoid X receptors (RXRs) can form homo- or heterodimers with orphan receptors involved in multiple intertwined signaling pathways. However, there is limited study on the formation of sex phenotypes and the regulation of steroidogenesis by RXRs in fish. Here, in Paralichthys olivaceus, we first indicated that PPARγ::RXRα was predictably a transcription factor for steroidogenesis genes, and Foxl2 and Dmrt1 were also transcription factors for rxrs and their partner receptor genes. When the flounder fry were exposed to LG100268 (LG, RXRs agonist, 50 mg/kg diet), the percentage of males increased from 50% to 71.4%. Before histological differentiation of the flounder ovary (3 cm TL) and testis (6 cm TL), the transcripts of rar ß and rar γ (P < 0.05) were activated, and the steroidogenesis gene Hsd3b1 was down-regulated (P < 0.05). The ratios of testosterone (T)/17ß-estradiol (E2) were all greatly increased (P < 0.05), and the ratio of 11-ketotestosterone (11-KT)/E2 was elevated at 3 cm TL. Moreover, LG was used to treat the cultured gonads in vitro (10 µM) and the fish with intraperitoneal injection in vivo (12 mg/kg body weight), respectively. LG was able to up-regulate rxr γ, rar γ, and ppar δ, and Hsd3b1 was significantly up-regulated (P < 0.05). The ratios of 11-KT/E2 in the culture medium and in the ovaries of the fish were decreased. Furthermore, the recombinant flounder Foxl2 protein was able to significantly down-regulate ppar γ (P < 0.05) and tr ß (P < 0.01) in the ovaries in vitro, and the result of the Dmrt1 in the testes was opposite to that of the Foxl2, probably indicating a feedback loop between RXRs' partner receptors and Foxl2/Dmrt1. These findings introduce for the first time the mode of action of RXRs on the flounder steroidogenesis and provide important data to learn the potential function of RXRs in fish sex differentiation and the potential role of RXRs in aquatic animals in the presence of water pollutants.

Acute low-dose phosphate disrupts glycerophospholipid metabolism and induces stress in juvenile turbot (Scophthalmus maximus).

Phosphate, as the main nutrient factor of lake eutrophication brought by pollutants discharged from agriculture and industry, is always considered to be a low-toxicity substance to aquatic animals. But the toxicity mechanism is unclear, and published information is limited. In this study, a 96 h acute stress experiment was conducted on juvenile turbot (Scophthalmus maximus) with 0, 10, and 60 mg/L phosphate solutions. Metabonomic analysis revealed that low-dose phosphate (10 mg/L) disrupted glycerophospholipid, purine, and glycolipid metabolism, as well as the tricarboxylic acid (TCA) cycle in juveniles, even at 96 h of stress, which may lead to cell structure damage and signal recognition disorder between cells. Upregulated key genes in the main glycerophospholipid metabolic pathways, which matched the results of the metabolomic study, were detected. Furthermore, low-dose phosphate (10 mg/L) induced oxidative stress and immunotoxicity in fish, resulting in the raising of relevant genes expression such as cat and sod in liver and kidney. In addition, all phosphate-treated groups had induced lesions on gill tissue, as evidenced by pathological observations. In this study on toxic effects on and mechanism of phosphate in aquatic animals using metabolomics, gene expression, and histopathology, we confirm that acute low-dose phosphate could disrupt glycerophospholipid metabolism and induce stress in juvenile turbot. This can provide advice on the amount of phosphate accumulation for marine fish farming and on protecting species diversity and marine ecosystem from the point of view of phosphate toxicity to marine animals.

Characterization of vasa and dnd homologs in summer flounder, Paralichthys dentatus: Expression analysis and colocalization of PGCs during embryogenesis.

Specification of primordial germ cells (PGCs) is particularly important for germline formation. Many maternal-effect genes such as vasa, dnd, and nanos have been identified. However, the research on distribution patterns of PGCs in marine fish is limited. Vasa has been widely used as a germ cell marker to identify its origination in teleosts because vasa RNA is a component of germ plasm. Dnd is known to be an RNA binding protein that protects germline-specific RNAs from degradation. In this study, we isolated full-length vasa and dnd cDNA from summer flounder to track germ cell origination and their expression patterns by RT-PCR and ISH. The results demonstrated that deduced amino acid sequence of Pdvas and Pddnd shared typically conserved motifs of their homologues and demonstrated high identities with other teleosts. Both vasa and dnd transcripts were exclusively detected in germ cells of the gonads. During embryogenesis, vasa and dnd RNA were located at the cleavage furrows of early cleavage stages, and then through proliferation and migration they eventually moved to a location at the predetermined genital ridge. Phylogenetic analysis revealed that summer flounder belongs to the Euteleostei species, but vasa/dnd transcripts localized at the cleavage furrows was similar to that in zebrafish (Osteriophysans). This suggests that germ cells differentiating at early embryogenesis have no direct relation with phylogenesis. At the same time, we found the spatio-temporal expression pattern of dnd was highly consistent with vasa during this process, which indicated the important function of dnd in keeping the target RNA from being degraded to maintain germ cell fate. These results will provide further understanding of germ plasm localization and PGC differentiation in teleosts, and facilitate germ cell manipulation in marine fishes.

Successful Spermatogonial Stem Cells Transplantation within Pleuronectiformes: First Breakthrough at inter-family Level in Marine Fish.

As a promising biotechnology, fish germ cell transplantation shows potentials in conservation germplasm resource, propagation of elite species, and generation of transgenic individuals. In this study, we successfully transplanted the Japanese flounder (P. olivaceus), summer flounder (P. dentatus), and turbot (S. maximus) spermatogonia into triploid Japanese flounder larvae, and achieved high transplantation efficiency of 100%, 75-95% and 33-50% by fluorescence tracking and molecular analysis, respectively. Eventually, donor-derived spermatozoa produced offspring by artificial insemination. We only found male and intersex chimeras in inter-family transplantations, while male and female chimeras in both intra-species and intra-genus transplantations. Moreover, the intersex chimeras could mature and produce turbot functional spermatozoa. We firstly realized inter-family transplantation in marine fish species. These results demonstrated successful spermatogonial stem cells transplantation within Pleuronectiformes, suggesting the germ cells migration, incorporation and maturation within order were conserved across a wide range of teleost species.

Sexually dimorphic expression and regulatory sequence of dnali1 in the olive flounder Paralichthys olivaceus.

Dynein axonemal light intermediate chain 1 (dnali1) is an important part of axonemal dyneins and plays an important role in the growth and development of animals. However, there is little information about dnali1 in fish. Herein, we cloned dnali1 gene from the genome of olive flounder (Paralichthys olivaceus), a commercially important maricultured fish in China, Japan, and Korea, and analyzed its expression patterns in different gender fish. The flounder dnali1 DNA sequence contained a 771 bp open reading frame (ORF), two different sizes of 5' untranslated region (5'UTR), and a 1499 bp 3' untranslated region (3'UTR). Two duplicated 922 nt fragments were found in dnali1 mRNA. The first fragment contained the downstream coding region and the front portion of 3'UTR, and the second fragment was entirely located in 3'UTR. Multiple alignments indicated that the flounder Dnali1 protein contained the putative conserved coiled-coil domain. Its expression showed sexually dimorphic with predominant expression in the flounder testis, and lower expression in other tissues. The gene with the longer 5'UTR was specifically expressed in the testis. The highest expression level in the testis was detected at stages IV and V. Transient expression analysis showed that the 922 bp repeated sequence 3'UTR of dnali1 down-regulated the expression of GFP at the early stage in zebrafish. The flounder dnali1 might play an important role in the testis, especially in the period of spermatogenesis, and the 5'UTR and the repetitive sequences in 3'UTR might contain some regulatory elements for the cilia.

Germline Specific Expression of a vasa Homologue Gene in the Viviparous Fish Black Rockfish (Sebastes schlegelii) and Functional Analysis of the vasa 3' Untranslated Region.

Germ cells play a key role in gonad development. As precursors, primordial germ cells (PGCs) are particularly important for germline formation. However, the origination and migration patterns of PGCs are poorly studied in marine fish, especially for viviparous economic species. The vasa gene has been widely used as a germ cell marker to identify a germline because vasa RNA is a component of germ plasm. In this study, we described the expression pattern of black rockfish (Sebastes schlegelii) vasa (Ssvas) in gonadal formation and development by in situ hybridization. The results showed that Ssvas failed in localization at the cleavage furrows until the late gastrula stage, when PGCs appeared and migrated to the genital ridge and formed elongated gonadal primordia at 10 days after birth. This study firstly revealed the PGCs origination and migration characteristics in viviparous marine fish. Furthermore, we microinjected chimeric mRNA containing EGFP and the 3'untranslated region (3'UTR) of Ssvas into zebrafish (Danio rerio) and marine medaka (Oryzias melastigma) fertilized eggs for tracing PGCs. We found that, although Sebastes schlegelii lacked early localization, similar to red seabream (Pagrus major) and marine medaka, only the 3'UTR of Ssvas vasa 3'UTR of black rockfish was able to label both zebrafish and marine medaka PGCs. In comparison with other three Euteleostei species, besides some basal motifs, black rockfish had three specific motifs of M10, M12, and M19 just presented in zebrafish, which might play an important role in labeling zebrafish PGCs. These results will promote germ cell manipulation technology development and facilitate artificial reproduction regulation in aquaculture.

Analyses of mRNA-seq and miRNA-seq of the brain reveal the sex differences of gene expression and regulation before and during gonadal differentiation in 17ß-estradiol or 17α-methyltestosterone-induced olive flounder (Paralichthys olivaceus).

Olive flounder (Paralichthys olivaceus) is a commercially important flatfish species cultured in East Asia. Female flounders generally grow more rapidly than males, therefore control of the sex ratio seems to be a proposed way to increase production. However, the sex determination gene and sex determination mechanism have yet been elucidated. The brain is an important organ that is involved in gonadal development. To explore the sex differences of gene expression in the brain before and during the flounder gonadal differentiation, we used messenger RNA (mRNA)-seq technology to investigate transcriptomes of male and female brains. Between female and male brains, 103 genes were differentially expressed before ovarian differentiation, 16 genes were differentially expressed before testicular differentiation, and 64 genes were differentially expressed during gonadal differentiation. According to annotation and Kyoto Encyclopedia of Genes and Genomes information, the differentially expressed genes (DEGs) were involved in circadian rhythm, circadian rhythm-fly, circadian entrainment, dopaminergic synapse, calcium signaling, glutamatergic synapse, taste transduction, herpes simplex infection, long-term depression, retrograde endocannabinoid signaling, and the synaptic vesicle cycle pathways. MicroRNA (miRNA)-seq was performed during the gonadal differentiation and the target genes of miRNAs were predicted. Integrated analysis of mRNA-seq and miRNA-seq showed that 29 of the 64 DEGs were regulated by the differentially expressed miRNAs during the gonadal differentiation. Our study provides a basis for further studies of brain sex differentiation and the molecular mechanism of sex determination in olive flounder.

Visualization of Turbot (Scophthalmus maximus) Primordial Germ Cells in vivo Using Fluorescent Protein Mediated by the 3' Untranslated Region of nanos3 or vasa Gene.

Primordial germ cells (PGCs) as the precursors of germ cells are responsible for transmitting genetic information to the next generation. Visualization of teleost PGCs in vivo is essential to research the origination and development of germ cells and facilitate further manipulation on PGCs isolation, cryopreservation, and surrogate breeding. In this study, artificially synthesized mRNAs constructed by fusing fluorescent protein coding region to the 3' untranslated region (3'UTR) of nanos3 or vasa (mCherry-Smnanos3 3'UTR or mCherry-Smvasa 3'UTR mRNA) were injected into turbot (Scophthalmus maximus) fertilized eggs for tracing PGCs. The results demonstrated that the fluorescent PGCs differentiated from somatic cells and aligned on both sides of the trunk at the early segmentation period, then migrated and located at the dorsal part of the gut where the gonad would form. In the same way, we also found that the zebrafish (Danio rerio) vasa 3'UTR could trace turbot PGCs, while the vasa 3'UTR s of marine medaka (Oryzias melastigma) and red seabream (Pagrus major) failed, although they could label the marine medaka PGCs. In addition, through comparative analysis, we discovered that some potential sequence elements in the3 'UTRs of nanos3 and vasa, such as GCACs, 62-bp U-rich regions and nucleotide 187-218 regions might be involved in PGCs stabilization. The results of this study provided an efficient, rapid, and specific non-transgenic approach for visualizing PGCs of economical marine fish in vivo.

Effects of different light spectra on embryo development and the performance of newly hatched turbot (Scophthalmus maximus) larvae.

Light is a key environmental factor that synchronizes various life stages from embryo development to sexual maturation in fish. For turbot, light spectra have the most influence at the larval and juvenile stages. In the current study, differences in the development of embryos and the performance of newly hatched turbot larvae exposed to five different spectra: full spectrum (LDF), blue (LDB, peak at 450 nm), green (LDG, peak at 533 nm), orange (LDO, peak at 595 nm) and red (LDR, peak at 629 nm), were examined. At 62.8 h post fertilization, a higher number of embryos exposed to short-wavelengths (LDG and LDB) had developed a heartbeat in comparison with embryos exposed to other wavelengths. Larvae exposed to the green spectrum had higher malformation rates than larvae exposed to the other spectra, indicating that larvae exposed to green light may have significantly reduced survival rates. The results of non-specific immunity parameters showed that the mRNA expression levels of cathepsin D (CTSD), cathepsin F (CTSF), catalase (CAT) and metallothionein (MT) in larvae exposed to LDB were significantly higher than those exposed to other spectra, but CAT activity in larvae exposed to LDB was significantly lower than larvae exposed to the other spectra. There was no significant difference in MT activity in larvae exposed to the five different spectra. The mRNA expression level of lysozyme (LZM) in larvae exposed to LDR was significantly higher than other spectra, while there was no significant difference in LZM activity observed in larvae exposed to LDR, LDG, LDB and LDF. The difference of the enzyme activity of total superoxide dismutase (T-SOD) was not significant among larvae exposed to the five spectra. mRNA expression of the heat shock protein 70 (HSP70) was significantly higher in newly hatched larvae exposed to LDB, LDR and LDG, indicating that larvae exposed to LDB, LDG and LDR exhibited a stress response. The mRNA expression level of the insulin-like growth factor-1 (IGF-1) and growth parameters in the newly hatched larvae exposed to the different spectra were not significantly different. The results of the present study indicate that LDO and LDF should be used for embryo incubation and newly hatched larvae when rearing turbot. This study provides a theoretical basis for optimizing the incubation light environment for fertilized turbot eggs, promoting immunity and reducing stress responses in newly hatched larvae.

High temperature increases the gsdf expression in masculinization of genetically female Japanese flounder (Paralichthys olivaceus).

In teleosts, sex is plastic and is influenced by environmental factors. Elevated temperatures have masculinizing effects on the phenotypic sex of certain sensitive species. In this study, we reared genetic XX Japanese flounder at a high temperature (27.5 ±â€¯0.5 °C) and obtained a population of sex-reversal XX males (male ratio, 95.24%). We comparatively analyzed the dynamic characteristics of germ cells and gsdf (gonadal soma-derived factor) expression during sexual differentiation for the experimental (27.5 ±â€¯0.5 °C) and control (18 °C ±â€¯0.5 °C) groups. The results revealed that the germ cell proliferation inhibited and gsdf expression up-regulated in the experimental group, and the gsdf mRNA and proteins expressed in somatic cells that had direct contact with germline stem cells (with Nanos 2 protein expression) including spermatogonia and oogonia by ISH (in situ hybridization) and IHC (immunohistochemistry). In addition, we also overexpressed the gsdf in XX flounders, and the germ cell number of XX flounders bearing gsdf gene significantly decreased and sometimes disappeared completely, which was consistent with the results from high-temperature induction. Therefore, based on all the results, we speculated that the high expression of gsdf might inhibit germ cell proliferation during sex differentiation, and eventually cause sex reversal in the high-temperature induced masculinization of XX Japanese flounder.

Transcriptome Dynamics During Turbot Spermatogenesis Predicting the Potential Key Genes Regulating Male Germ Cell Proliferation and Maturation.

Spermatogenesis is a dynamic developmental process in which spermatogonial stem cells proliferate, differentiate and mature into functional spermatozoa. These processes require an accurate gene regulation network. Here, we investigated the dynamic changes that occur during spermatogenesis through a combination of histological and transcriptome analyses of different developmental stages of the testis. We constructed 18 testis transcriptome libraries, and the average length, N50, and GC content of the unigenes were 1,795 bp; 3,240 bp and 49.25%, respectively. Differentially expressed genes (DEGs) that were related to germ cell proliferation and maturation, such as NANOS3, RARs, KIFs, steroid hormone synthesis-related genes and receptor genes, were identified between pairs of testis at different developmental stages. Gene ontology annotation and pathway analyses were conducted on DEGs with specific expression patterns involved in the regulation of spermatogenesis. Nine important pathways such as steroid hormone biosynthesis related to spermatogenesis were identified. A total of 21 modules that ranged from 49 to 7,448 genes were designed by a weighted gene co-expression network analysis. Furthermore, a total of 83 candidate miRNA were identified by computational methods. Our study provides the first transcriptomic evidence for differences in gene expression between different developmental stages of spermatogenesis in turbot (Scophthalmus maximus).

Spermatogonial stem cells differentiation and testicular lobules formation in a seasonal breeding teleost: The evidence from the heat-induced masculinization of genetically female Japanese flounder (Paralichthys olivaceus).

Germ cells undergo a series of cellular changes including differentiation, mitosis, meiosis and maturation, and eventually develop into a large number of functional gametes. The available data regarding teleost gametogenesis in seasonal batch spawners is limited. In this study, we investigated spermatogenesis with special attention on spermatogonia differentiation using heat-induced masculine juveniles of genetically female Japanese flounder. Meanwhile, the Nanos2 expression had been detected by immunohistochemistry for analysis of spermatogonial stem cells (SSCs) distribution. Spermatogonia began mitosis at 35 dph, and basement membrane firstly appeared and gradually surrounded and separated the spermatogonia (type A) into single and paired status. At this period, the spermatogonia continuously maintained mitotic proliferation. As a result, the number of spermatogonia including isolated and clusters (2-8 spermatogonia) significantly increased in the presumptive testes. From 85 dph to 120 dph, with the mitosis of spermatogonia, germline acinar-clusters formed. In the clusters, type A spermatogonia differentiated into type B, and multi-spermatogonia surrounded by several sertoli cell formed cysts，which represented the formation of lobular precursors. After that, type B spermatogonia began meiosis, which indicated the initiation of spermatogenesis. In adult testes, most type A spermatogonia distributed in the peripheral region and a few clung to a basement membrane in the internal germinal epithelium. Various spermatogenic cysts with germ cells in different developmental stages existed in a testicular lobules, moreover the germ cells in earlier stages resided in the distal termini, and the advanced stages were adjacent to the spermatic duct of testes. Therefore, the testes of Japanese flounder belonged to an intermediated distribution of SSCs, which might contribute greatly to multiple spermiation during breeding season. These findings would improve the understanding the mechanisms of SSCs differentiation and testicular development, and may be of great value in future studies of the spermatogenesis regulation.

Comparative performance of growth, vertebral structure and muscle composition in diploid and triploid Paralichthys olivaceus.

Growth, skeletal structure and muscle composition of cold-shock-induced triploid olive flounder Paralichthys olivaceus were investigated. The average values of total length and total weight of triploids were higher than those of diploids from 5 to 11 months posthatch (mph). The growth difference disappeared after 11 mph. The skeletal structure of flounder at 11 mph was observed by X-ray imaging method. There are four kinds of vertebral deformity including vertebrae fusion, one-sided compression, two-sided compression and vertically shifted. The trunk region (V8-18) and tailing end of the vertebral column were the predominant locations of deformity. In general, the frequencies of vertebral deformities in triploids (60.0%) were higher than those in diploids (33.3%, p < 0.05). Both the number of fish with deformed vertebrae and the average frequencies of deformed vertebrae in triploids were significantly higher than those in diploids (p < 0.05). The muscle tissues of diploid and triploid flounder at 11 mph contain the same types of fatty acid and free amino acid profiles. The number of fatty acids with significant higher contents in diploids and triploids was one and ten, respectively (p < 0.05). The contents of free amino acids showed no difference between triploid and diploid fish.

Identification of type A spermatogonia in turbot (Scophthalmus maximus) using a new cell-surface marker of Lymphocyte antigen 75 (ly75/CD205).

Turbot (Schophthalmus maximus) is one of the most important economic marine flatfish species. However, due to rapid development of the industry, genetic resource recession has brought down the efficiency of aquaculture. Therefore, conservation of the genetic resource is increasingly demanded. Recent research proved that type A spermatogonia possesses the properties of spermatogonia stem cell, and it might provide an ideal solution. Therefore, it is necessary to develop an appropriate molecular marker on type A spermatogonia to further isolate and purify of type A spermatogonia in turbot. In this study, turbot lymphocyte antigen 75 (smly75) gene was identified and its localizations of expressions and the temporal transcription patterns were evaluated qualitatively and semiquantitatively. Investigation in testes of development of spermatogonia showed that smly75 mRNA, contrast with vasa and dnd mRNA, was exclusively localized in type A spermatogonia and not detected in type B spermatogonia, spermatocytes or gonadal somatic cells by in situ hybridization. Thus, the smly75 could be a new and convincing molecular marker on identification of type A spermatogonia. In addition, specifically to development pattern of type A spermatogonia, from 7- to 14- month testes, spermatogonia were dominated and the number of type A spermatogonia was increased, corresponding that smly75 expression was up-regulated gradually, while, in 16 month testes, accompanied by that several spermatogonia differentiated into primary spermatocytes, the smly75 expression down-regulated. Finally, broaden in the whole reproductive cycle, the smly75 transcription significantly variated with the differentiation of germ cells and in accordance with the number of type A spermatogonia. It is suggested that testes from 8 to 14 month old males could be used for further isolation and purification of type A-SG. These results will not only help to better understand type A spermatogonia, but also further facilitate type A spematogonia-mediated germ cell manipulation in turbot.