Deciphering dynamic interactions between spermatozoa and the ovarian microenvironment through integrated multi-omics approaches in viviparous Sebastes schlegelii.

The ovarian microenvironment plays a crucial role in ensuring the reproductive success of viviparous teleosts. However, the molecular mechanism underlying the interaction between spermatozoa and the ovarian microenvironment has remained elusive. This study aimed to contribute to a better understanding of this process in black rockfish (Sebastes schlegelii) using integrated multi-omics approaches. The results demonstrated significant upregulation of ovarian complement-related proteins and pattern recognition receptors, along with remodeling of glycans on the surface of spermatozoa at the early spermatozoa-storage stage (1 month after mating). As spermatozoa were stored over time, ovarian complement proteins were progressively repressed by tryptophan and hippurate, indicating a remarkable adaptation of spermatozoa to the ovarian microenvironment. Before fertilization, a notable upregulation of cellular junction proteins was observed. The study revealed that spermatozoa bind to ZPB2a protein through GSTM3 and that ZPB2a promotes spermatozoa survival and movement in a GSTM3-dependent manner. These findings shed light on a key mechanism that influences the dynamics of spermatozoa in the female reproductive tract, providing valuable insights into the molecular networks regulating spermatozoa adaptation and survival in species with internal fertilization.

Detection of complex chromosome rearrangements using optical genome mapping.

Chromosomal structural variations (SVs) are a main cause of human genetic disease. Currently, karyotype, chromosomal microarray analysis (CMA), and fluorescent in situ hybridization (FISH) form the backbone of current routine diagnostics (CRD). These methods have their own limitations. CRD cannot identify cryptic balanced SVs and complex SVs even if these techniques were performed either simultaneously or in a sequential manner. Optical genome mapping (OGM) is a novel technology that can identify several classes of SVs with higher resolution, but studies on the applicability of OGM and its comparison with CRD are inadequate for difficult and complicated chromosomal SVs are lacking. Herein, seven patients with definite complicated SVs involving at least two breakpoints (BPs) were recruited for this study. The results of BPs and SVs from OGM were compared with those from CRD. The results showed that all BPs of five samples and partial BPs of two samples were detected by OGM. The undetected BPs were all close to the repeat-rich gap region. Besides, OGM also detected additional SVs including a cryptic balanced translocation, two additional complex chromosomal rearrangement (CCR). OGM yielded the additional information, such as the orientation of acentric fragments, BP positions, and genes mapped in the BP region for all the cases. The accuracy of additional SVs and BPs detected by OGM was verified by FISH panel and next-generation sequencing and Sanger sequencing. Taken together, OGM exhibit a better performance in detecting chromosomal SVs compared to the CRD. We suggested that OGM method should be utilized in the clinical examination to improve the efficiency and accuracy of genetic disease diagnosis, supplemented by FISH or karyotyping to compensate for the SVs in the repeat-rich gap region if necessary.

Development of CRISPR Cas9, spin-off technologies and their application in model construction and potential therapeutic methods of Parkinson's disease.

Parkinson's disease (PD) is one of the most common degenerative diseases. It is most typically characterized by neuronal death following the accumulation of Lewis inclusions in dopaminergic neurons in the substantia nigra region, with clinical symptoms such as motor retardation, autonomic dysfunction, and dystonia spasms. The exact molecular mechanism of its pathogenesis has not been revealed up to now. And there is a lack of effective treatments for PD, which places a burden on patients, families, and society. CRISPR Cas9 is a powerful technology to modify target genomic sequence with rapid development. More and more scientists utilized this technique to perform research associated neurodegenerative disease including PD. However, the complexity involved makes it urgent to organize and summarize the existing findings to facilitate a clearer understanding. In this review, we described the development of CRISPR Cas9 technology and the latest spin-off gene editing systems. Then we focused on the application of CRISPR Cas9 technology in PD research, summarizing the construction of the novel PD-related medical models including cellular models, small animal models, large mammal models. We also discussed new directions and target molecules related to the use of CRISPR Cas9 for PD treatment from the above models. Finally, we proposed the view about the directions for the development and optimization of the CRISPR Cas9 technology system, and its application to PD and gene therapy in the future. All these results provided a valuable reference and enhanced in understanding for studying PD.

Transcriptome sequencing reveals ovarian immune response and development during female sperm storage in viviparous black rockfish (Sebastes schlegelii).

Black rockfish (Sebastes schlegelii) is a viviparous teleost fish whose spermatozoa were transferred into the female ovary cavity and stored for up to five months and then fertilized with the matured eggs. There is no clarity about the molecular characteristics of ovarian follicles during female sperm storage in Sebastes schlegelii. In this study, histological observation, transcriptomic analysis and hormone level detection were performed in ovaries at stages of pre-mating (PRM), post-mating (POM) and pre-fertilization (PRF). Histological observation displayed that oocytes developed from the primary growth (PG) stage to the mature stage during the three stages. Furthermore, somatic cells around the oocyte were proliferated and spermatozoa were found near the layer of epithelial cells. Transcriptomic analysis showed that there were 437 and 747 differentially expressed genes (DEGs) in ovarian comparison of PRM-vs-POM and POM-vs-PRF, respectively. GO enrichment and KEGG analysis revealed that lots of DEGs from PRM-vs-POM were linked to immune-related pathways, such as antigen processing and presentation, immune response, and complement and coagulation cascade. Meanwhile, seven DEGs associated with immune response were differentially expressed after spermatozoa treatment in ovarian tissue in vitro. While the DEGs from POM-vs-PRF were mostly enriched in the pathways related to homeostasis maintenance and cellular junction and metabolism. In addition, we found increased estrogen (E2) and 11-ketotestosterone (11-KT) level and decreased testosterone level in ovarian follicles during the sperm storage period by ELISA, suggesting that sex hormones are involved in the dynamic change of ovarian follicles. In total, this study could provide new hints for understanding the immune adaption and developmental signatures of ovarian follicles post copulation in black rockfish and other viviparous fish.

Is the six-minute walk test still reliable compared to cardiopulmonary exercise test for exercise capacity in children with congenital heart disease?

Objectives: We aimed to assess the validity of the six-minute walk test (6MWT) to reflect the functional capacity of children with congenital heart disease (CHD), evaluate a possible correlation between the 6MWT distance with cardiopulmonary exercise test (CPET) variables, as well as to find a cutoff value to stratification the physical fitness in this population. Methods: We enrolled 459 children with CHD, 6-18 years old, who performed a complete CPET and 6MWT on the same day in a cross-sectional observational study. Correlations between variables of CPET and six-minute walking distance (6MWD) were analyzed and cutoff values of 6MWD were identified for the classification of the physical fitness in the population. Results: The mean distance ambulated during the 6MWT was 578 ± 65 m, 590 ± 65 m for boys, and 562 ± 62 m for girls (p < 0.001). Both VO2max and %predicted VO2max showed a correlation with the 6MWT distance (r = 0.35, p < 0.001 and r = 0.51, p < 0.001, respectively), and an inverse correlation was found between VE/VCO2 slope and the 6MWT distance (r = -0.31; p < 0.001). There appeared to be a linear association between 6MWD and VO2max up to a 6MWD of approximately 600 m. We divided the population into 4 subgroups (boys <130 cm; boys ≥130 cm; girls <130 cm; girls ≥130 cm), and get the cutoff values (554 m, 617 m, 549 m, 587 m) respectively equivalent to 80% of predicted VO2max. The 6MWT distances of another 102 patients were applied for external verification of the cutoff values. Conclusions: Our study provided evidence on when a 6MWT should be considered as a convincing complementary test in the pediatric population with CHD and explored the classification of exercise tolerance using a 6MWD value. The cut-off values for 6MWD may be qualified as an intervention target for exercise rehabilitation.

Expression Analysis of ZPB2a and Its Regulatory Role in Sperm-Binding in Viviparous Teleost Black Rockfish.

Black rockfish is a viviparous teleost whose sperm could be stored in the female ovary for five months. We previously proposed that zona pellucida (ZP) proteins of black rockfish play a similar sperm-binding role as in mammals. In this study, SsZPB2a and SsZPB2c were identified as the most similar genes with human ZPA, ZPB1 and ZPB2 by Blastp method. Immunohistochemistry showed that ovary-specific SsZPB2a was initially expressed in the cytoplasm of oocytes at stage III. Then it gradually transferred to the region close to the cell membrane and zona pellucida of oocytes at stage IV. The most obvious protein signal was observed at the zona pellucida region of oocytes at stage V. Furthermore, we found that the recombinant prokaryotic proteins rSsZPB2a and rSsZPB2c could bind with the posterior end of sperm head and rSsZPB2a was able to facilitate the sperm survival in vitro. After knocking down Sszpb2a in ovarian tissues cultivated in vitro, the expressions of sperm-specific genes were down-regulated (p < 0.05). These results illustrated the regulatory role of ZP protein to the sperm in viviparous teleost for the first time, which could advance our understanding about the biological function of ZP proteins in the teleost.

Identification and functional characterization of Pomstna in Japanese flounder (Paralichthys olivaceus).

Myostatin (MSTN) as a negative regulator of muscle growth has been identified in Japanese flounder. Yet, most fish experienced the teleost specific genome duplication and possess at least two mstn genes. In current study, the second mstn gene named Pomstna is identified in Japanese flounder. Pomstna is clustered with other mstn2 of teleosts and owned highly conserved TGF-beta domain. In addition to muscle, Pomstna also highly expressed in brain and spleen. Using the primarily cultured muscle cells of Japanese flounder, we found that Pomstna could inhibit the proliferation and differentiation of muscle cells in vitro. As a ligand of TGF-beta signaling pathway, Pomstnb could regulate the expression of p21 and myod by activating the TGF-beta signaling pathway. Different from the function of Pomstnb, Pomstna could not activate the TGF-beta signaling pathway in vitro. During the differentiation of PoM cells, the expression of Pomstnb decreased significantly but the expression of Pomstna showed no change. Our study suggests that Pomstna could negatively regulate the growth and differentiation of muscle like Pomstnb yet through a different regulatory mechanism than Pomstnb. The present study suggests that muscle proliferation and differentiation were regulated by mstn not only through the TGF-beta signaling pathway but also other unknown mechanisms.

Molecular Mechanism Based on Histopathology, Antioxidant System and Transcriptomic Profiles in Heat Stress Response in the Gills of Japanese Flounder.

As an economically important flatfish in Asia, Japanese flounder is threatened by continuously rising temperatures due to global warming. To understand the molecular responses of this species to temperature stress, adult Japanese flounder individuals were treated with two kinds of heat stress-a gradual temperature rise (GTR) and an abrupt temperature rise (ATR)-in aquaria under experimental conditions. Changes in histopathology, programmed cell death levels and the oxidative stress status of gills were investigated. Histopathology showed that the damage caused by ATR stress was more serious. TUNEL signals confirmed this result, showing more programmed cell death in the ATR group. In addition, reactive oxygen species (ROS) levels and the 8-O-hDG contents of both the GTR and ATR groups increased significantly, and the total superoxide dismutase (T-SOD) activities and total antioxidant capacity (T-AOC) levels decreased in the two stressed groups, which showed damage to antioxidant systems. Meanwhile, RNA-seq was utilized to illustrate the molecular mechanisms underyling gill damage. Compared to the control group of 18 °C, 507 differentially expressed genes (DEGs) were screened in the GTR group; 341 were up-regulated and 166 were down-regulated, and pathway enrichment analysis indicated that they were involved in regulation and adaptation, including chaperone and folding catalyst pathways, the mitogen-activated protein kinase signaling (MAPK) pathway and DNA replication protein pathways. After ATR stress, 1070 DEGs were identified, 627 were up-regulated and 423 were down-regulated, and most DEGs were involved in chaperone and folding catalyst and DNA-related pathways, such as DNA replication proteins and nucleotide excision repair. The annotation of DEGs showed the great importance of heat shock proteins (HSPs) in protecting Japanese flounder from heat stress injury; 12 hsp genes were found after GTR, while 5 hsp genes were found after ATR. In summary, our study records gill dysfunction after heat stress, with different response patterns observed in the two experimental designs; chaperones were activated to defend heat stress after GTR, while replication was almost abandoned due to the severe damage consequent on ATR stress.

Genome-wide identification and analysis of scavenger receptors and their expression profiling in response to Edwardsiella tarda infection in Japanese flounder (Paralichthys olivaceus).

The scavenger receptors (SRs) gene family, as one of pattern recognition receptors, participates in the innate immune response in diverse lineages. However, the systematic identification, characteristics and functions of SRs family are lacking in teleost. Here, we identified all 19 SRs family members in Japanese flounder (Paralichthys olivaceus) based on the genome and transcriptome data. Phylogenetic and Ka/Ks analysis demonstrated that these SRs genes were divided into five classes and all exhibited pronounced purified selection pressures. Whole genome duplication event was found in colec12, scarb2, and lamp1. Gene structure, functional domain and motif distribution analyses indicated that SRs within the different subfamilies are severely conservative. SRs genes showed diverse expression patterns in the embryogenesis and unchanged tissues. The regulations of 14 SRs genes in blood, gill and kidney after E. tarda infection suggested their roles in innate immune response. Meanwhile, ten SRs genes were differentially expressed after E. tarda stimulation in macrophages in vitro. Then we proved that PoSCARA3 could suppress the activity of NF-κB and AP-1 in HEK 293T cells by dual-luciferase assays. In summary, this study provided valuable basis for further functional characterization and immune functions of SRs genes in P. olivaceus.

Intensive masculinization caused by chronic heat stress in juvenile Cynoglossus semilaevis: Growth performance, gonadal histology and gene responses.

The sea temperature has been observed to chronically increase during the past decades, leaving unpredictable influences to the marine biological resources. Thus, it is of vital significance to study the biological responses of ocean inhabited organisms with the artificially stimulated heat stress environment. Cynoglossus semilaevis provides us with an ideal model to study the influence of chronic heat stress on the sexual differentiation in marine teleosts for its genetic sex determination (GSD) + environmental effected (EE) sex determination system. In this study, the comparative experiment was conducted employing heated seawater (HT group) and ambient seawater (CT group) to cultivate juvenile C. semilaevis respectively. Significant differences were exhibited in growth performance and a delayed germ cell development effect was found in pseudomales formed under chronic heat stress. Using transcriptome analysis, the transcription profile of 55 days post fertilization (dpf) and 100 dpf juveniles' gonads were studied. A total of 47 libraries were constructed with an average mapping rate of 94.63% after assembling. GO and KEGG enrichment were proceeded using DEGs screened out between (1) pseudomale gonads at 55 dpf and 100 dpf in HT and CT group (2) pseudomale and female gonads at 55 dpf and 100 dpf in HT and CT group. Terms and pathways involved in steroid stimulation, reproduction ability, germ cell proliferation et al. were shed light on. The expression pattern of 29 DEGs including amh, hsp90b1, pgr et al. were also provided to supplement the results of functional enrichment. Weighted gene co-expression networks analysis (WGCNA) was constructed and hspb8-like, histone H2A.V were exhibited to play vital roles in the heat-induced masculinization. Our findings facilitate the understanding for transcriptional variations in intensive masculinization cause by chronic heat stress of C. semilaevis and provide referable study of the influences on the teleosts in elevated sea temperature.

Long-term exposure to environmental relevant triclosan induces reproductive toxicity on adult zebrafish and its potential mechanism.

Triclosan (TCS) is widely used in personal care products and has become a contaminant ubiquitously found in the aquatic environment. It is reported exposure to triclosan can cause serious toxic effects on aquatic animals. However, the molecular mechanisms about long-term exposure to TCS-induced reproductive toxicity are not well elucidated. In the present study, adult zebrafish were exposed to TCS (2, 20 and 200 µg/L) for 150 days, and then the reproductive capacity assessment, steroid hormone and VTG quantitative measurement, histopathology observation and RNA sequencing analysis were performed to investigate the effects of TCS on its reproduction. The results indicated that long-term exposure to TCS causes the regulation disorder of the endocrine system, resulting in a reduction of the number of normal germ cells, and ultimately a decrease in the hatching rate and survival rate of offspring. This study revealed the toxic effects and contributed to our deep understanding about the potential disease of TCS exposure in the aquatic environment.

Functional differentiation of two lhx8 paralogs and possible regulatory role of lhx8a in Japanese flounder (Paralichthys olivaceus).

Lhx8, belonging to the LIM-Homebox family, is involved in the tooth, nervous system, and primordial follicles development in mammals. However, little is known about the regulatory roles of lhx8 in teleosts. In this study, two lhx8 duplicates were identified in Paralichthys olivaceus, termed Polhx8a and Polhx8b, respectively. Bioinformatic analysis showed that Polhx8a was more likely to be a teleost-specific paralog. According to expression analysis, Polhx8a transcripts were almost exclusively concentrated in the oocytes, while Polhx8b was weakly expressed in the spleen, gill, and some facial organs, indicating sub-functionalization of this gene pair during evolution. Furthermore, Polhx8a mRNA level elevated from perinucleolar oocyte (PNO) stage to vitellogenic oocyte (VO) stage transition and changed after exogenous hormone stimulation, proving that Polhx8a was involved in the oocyte development and could be regulated by sex hormones. Yeast two-hybrid, bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (co-IP) experiments captured the positive protein interactions between PoLhx8a and the other two oocyte-specific transcription factors: PoFigla and PoNobox. After knocking down lhx8a in embryos or adult ovaries in vivo, the expression of oocyte-associated genes was significantly down-regulated (P < 0.05). Our findings suggest the evolution and functional differentiation of lhx8 genes, and shed light on the potential role of lhx8a in protein interactions and gene regulation in teleosts.

Molecular characterization and functional analysis of cyp11a and cyp11b in black rockfish (Sebastes schlegelii).

The cyp11 includes cyp11a and cyp11b in most mammals and teleosts, encoded cholesterol side chain lyase and 11ß-hydroxylase, respectively. It is essential in steroid hormone synthesis. However, studies on the regulation of cyp11 are limited, especially in teleosts. In this study, the molecular characterization and function of cyp11a and cyp11b of black rockfish was investigated. Both of them showed high homology with other teleost counterparts by phylogenetic analysis. The expression of cyp11a and cyp11b exhibited a clear sexually dimorphic pattern, with a higher expression level in testis than that of in ovaries. During the different developmental stages (40 dpf, 80 dpf, 190 dpf, 360 dpf, 720 dpf), the expression of cyp11a was earlier than cyp11b. In situ hybridization results showed that cyp11a and cyp11b were mainly expressed in oogonia and oocytes of the ovary. They were located in spermatogonia and interstitial compartment in the 1.5-year-old gonads, and spermatocytesgonia and the peritubular myoid cell of the testis in the 2.5-year-old gonads. To explore the distinct roles of cyp11a and cyp11b in gonads, oestrogen and androgens were used to stimulate the primary testicular and ovarian cells. The expressions of cyp11a and cyp11b were tested under different dose of 17α-methyltestosterone (17α-MT) and 17ß-estradiol (E2). The results showed cyp11a was significantly increased at 10-6  mol ml-1 of 17α-MT and 10-8  mol ml-1 of E2 in ovary and 10-10  mol ml-1 of 17α-MT and E2 in testis, while cyp11b was significantly decreased after 17α-MT and E2 treatment. These results indicated that cyp11a and cyp11b were likely to have different functions, and also implied they might play an important roles in the differentiation of gonads and the synthesis of steroids in black rockfish.

The association between the pattern of change in N-terminal pro-B-type natriuretic peptide and short-term outcomes in children undergoing surgery for congenital heart disease.

OBJECTIVES: This study aimed to determine whether changes in perioperative N-terminal pro-B-type natriuretic peptide (NT-proBNP) are associated with short-term outcomes in children undergoing surgery for congenital heart disease (CHD). METHODS: We retrospectively included 873 consecutive children with CHD after cardiac surgery. NT-proBNP concentrations were collected from each child prior to and at 1, 12, 36 and 72 h after surgery. The patients had postsurgical follow-ups at 30, 90 and 180 days. The end point was postoperative composite adverse events. RESULTS: The patients were classified into 3 groups using joint latent class mixture time-to-event models: (i) relatively stable (86.7%), (ii) decreasing (7.2%) and (iii) increasing (6.1%). In total, 257 (29.4%) adverse events occurred. The joint latent class mixture time-to-event models showed that increasing NT-proBNP was strongly associated with adverse events, with adjusted hazard ratio of 2.33 (95% confidence interval 1.52-3.60). Multinomial logistic regression showed that the variables associated with the pattern of change were age, weight at surgery, mode of delivery and cardiopulmonary bypass time. CONCLUSIONS: The pattern of dynamic postsurgical changes in NT-proBNP may facilitate outcome stratification and identification of a high risk for adverse events.

Evaluation of Physical Fitness in Children With Congenital Heart Diseases Versus Healthy Population.

We aimed to evaluate and compare the physical fitness of children with different severity of the cardiac defect with their healthy peers. We included 182 children after cooperation for congenital heart disease (CHD) and 129 healthy children as controls, 6-18 years old, who performed a complete cardiopulmonary exercise test (CPET) in a cross-sectional observational study. The CHD patients were further subdivided into three subgroups according to diagnostic characteristics and surgical methods: simple CHD group (SCHD), complex CHD group (CCHD)and total cavopulmonary connection group (TCPC). Physical fitness was compared between groups using generalized linear model and multiple linear regression analysis. In comparison with age and gender adjusted healthy controls, children in SCHD, CCHD, and TCPC subgroups had accordingly graded down values of peak oxygen consumption (VO2max: 44.86 ± 5.41 vs. 39.91 ± 5.59 vs. 36.95 ± 5.92 vs. 32.04 ± 5.38 mL/kg/min, P < 0.05) and %predicted VO2max (1.01 ± 0.14 vs. 0.91 ± 0.13 vs. 0.83 ± 0.15 vs. 0.72 ± 0.13, P < 0.05) for each group. The proportion of %predicted VO2max above 80% in the CHD and the control group were 60.4% and 96.1% (P < 0.001). The VO2max decreased by 0.98 mL/kg/min per year in CHD children, and the corresponding %predicted reference diminished by 2.0%. Children with TCPC had the highest mean decrease of VO2max and %predicted VO2max per year of age (1.23 mL/kg/min, corresponding 3.0%). Although the mean overall physical fitness of children with simple and complex defect after biventricular correction were significantly different from healthy population, they were close to normal (>80%predicted VO2max). However, TCPC group had much lower exercise capacity.

Cloning and molecular characterization of PRL and PRLR from turbot (Scophthalmus maximus) and their expressions in response to short-term and long-term low salt stress.

The pituitary hormone prolactin (PRL) regulates salt and water homeostasis by altering ion retention and water uptake through peripheral osmoregulatory organs. To understand the role of PRL and its receptor (PRLR) in hypoosmoregulation of turbot (Scophthalmus maximus), we characterized the PRL and PRLR gene and analyzed the tissue distribution of the two genes and their gene transcriptional patterns in the main expressed tissues under long-term and short-term low salt stress. The PRL cDNA is 1486 bp in length, incorporating an ORF of 636 bp with a putative primary structure of 211 residues. And the PRLR cDNA is 2849 bp in length, incorporating an ORF of 1944 bp with a putative primary structure of 647 residues. The deduced amino acid sequences of these two genes shared highly conserved structures with those from other teleosts. Quantitative real-time PCR results showed that PRL transcripts were strongly expressed in the pituitary and very weakly in brain, but were hardly expressed in other tissues. PRLR transcripts were most abundant in the kidney, to a lesser extent in the gill, intestine, brain, and spleen, and at low levels in the pituitary and other tissues examined. The expression of PRL in the pituitary increased after short-term or long-term low salt stress, and the highest expression level appeared 12 h after stress (P < 0.05). And there is no significant difference between both low salt group (5 ppt and 10 ppt) at each sampling point. The variation of PRLR expression in gill under short-term low salt stress is similar to that of PRL gene in pituitary, with highest value in 12 h (P < 0.05). However, the expression under long-term low salt stress was significantly higher than control group even than 12 h group under 5 ppt (P < 0.05). The expression of PRLR in the kidney increased first and then decreased after low salt stress, and the highest value also appeared in 12 h after stress and there was no significant difference between the salinity groups. After long-term low salt stress, the expression level also increased significantly (P < 0.05), but it was flat with 24 h, which was lower than 12 h. The variation of PRLR expression in the intestine was basically consistent with that in the kidney. The difference was that the expression level of 24 h after stress in the 5 ppt group was significantly higher than that of the 10 ppt group (P < 0.05). After a comprehensive analysis of the expression levels of the two genes, it can be found that the expression level increased and peaked at 12 h after short-term low salt stress, indicating that this time point is the key point for the regulation of turbot in response to low salt stress. This also provides very important information for studying the osmotic regulation of turbot. In addition, our results also showed that the expression of PRLR was stable in the kidney and intestine after long-term low salt stress, while the expression in the gill was much higher than short-term stress. It suggested that PRL and its receptors mainly exert osmotic regulation function in the gill under long-term low salt stress. At the same time, such a result also brings a hint for the low salt selection of turbot, focusing on the regulation of ion transport in the gill.

Genome-wide identification of nonvisual opsin family reveals amplification of RPE-retinal G protein receptor gene (RGR) and offers novel insights into functions of RGR(s) in Paralichthys olivaceus (Paralichthyidae, Teleostei).

Opsins play important roles in the image-forming visual pathways and numerous biological systems such as the biological clock and circadian rhythm. However, the nonvisual opsins involved in nonimage forming process are not clear to date. The aim of this study was to characterize nonvisual opsins in Paralichthys olivaceus. A total of 24 nonvisual opsin genes were identified. Expressions of these genes in eye, brain, heart, testis, and fin were investigated by quantitative real-time polymerase chain reaction (qRT-PCR). Testis contained a surprisingly large number of nonvisual opsins including Opn4m2, Tmt2a, Tmt3b, Opn3, RRH, Opn7a, and Opn7b. Syntenic and phylogenetic analyses confirmed that the RGRa and RGRb originated from the teleost-specific genome duplication (TSGD). qRT-PCR results demonstrated high RGRa and RGRb expression in the eye, while the expression levels in the brain, heart, testis, and fin were relatively weak. In situ hybridization results presented here revealed the presence of both RGRa and RGRb mRNA-positive signals in the ganglion cell layer but absence in the intracellular compartment of retinal pigment epithelium (RPE) and Müller glial cells. Therefore, we hypothesized that RGRa and RGRb had undergone subfunctionalization in P. olivaceus after TSGD. In conclusion, this study provides novel insights into the evolutionary fates of the RGR genes, still, further studies need to be done to explore the mechanism about the lack of RGR genes' expression in RPE.

Molecular characterization and expression profiles provide new insights into GATA5 functions in tongue sole (Cynoglossus semilaevis).

GATA5 is a member of the GATA transcription factor family, which serves essential roles in varieties of cellular functions and biological processes. In this study, we have accomplished the molecular cloning, bioinformatic analysis and preliminary function study of C. semilaevis GATA5. The full-length cDNA nucleotide sequence is 1955 bp, with a coding sequence of 1167 bp, which encodes a polypeptide of 388 amino acids. Homology, phylogenetic, gene structure and synteny analysis showed that C. semilaevis GATA5 was highly conserved among vertebrates. Tissue distribution pattern exhibited that C. semilaevis GATA5 was significantly expressed in heart, intestine, liver, kidney and gonad, with a sexual dimorphic feature observed in testis and ovary. Embryonic development expression profiles showed that C. semilaevis GATA5 transcripts increased at the blastula stage, and peaked at the heat-beating period. Strong signals were detected at spermatids of male testis and stage III oocytes of female ovary by ISH. The expression of C. semilaevis GATA5 was regulated by 17α-MT and E2 after hormone stimulation to the ovary. Together, all the results pointed out that GATA5 might play a vital role during gonadal maturation and the reproductive cycle of C. semilaevis. This study lays the foundation for further researches on the sex control breeding in tongue sole.

A chromosome-level genome of black rockfish, Sebastes schlegelii, provides insights into the evolution of live birth.

The black rockfish (Sebastes schlegelii) is a teleost in which eggs are fertilized internally and retained in the maternal reproductive system, where they undergo development until live birth (viviparity). In the present study, we report a chromosome-level black rockfish genome assembly. High-throughput transcriptome analysis (RNA-seq and ATAC-seq) coupled with in situ hybridization (ISH) and immunofluorescence reveal several candidate genes for maternal preparation, sperm storage and release, and hatching. We propose that zona pellucida (ZP) proteins retain sperm at the oocyte envelope, while genes in two distinct astacin metalloproteinase subfamilies serve to release sperm from the ZP and free the embryo from chorion at prehatching stage. We present a model of black rockfish reproduction, and propose that the rockfish ovarian wall has a similar function to the uterus of mammals. Together, these genomic data reveal unprecedented insights into the evolution of an unusual teleost life history strategy, and provide a sound foundation for studying viviparity in nonmammalian vertebrates and an invaluable resource for rockfish ecological and evolutionary research.

Two cyp17 genes perform different functions in the sex hormone biosynthesis and gonadal differentiation in Japanese flounder (Paralichthys olivaceus).

P450c17, a key enzyme in the steroid generation pathway, plays an important role in the production of sex steroid and cortisol. In this study, two cyp17 gene isoforms, Pocyp17-I and Pocyp17-II were isolated from Paralichthys olivaceus gonads. Domain architecture analysis of Pocyp17-I and Pocyp17-II revealed that they had three regions important to enzymatic function. Structural analysis showed that Pocyp17-I and Pocyp17-II had 8 and 9 exons respectively, and the difference was caused by the insertion of an extra intron (intron1) in the latter. Quantitative real-time polymerase chain reaction results indicated that the expression of these two genes showed sexually dimorphism that Pocyp17-I and Pocyp17-II were highest expressed in testis and ovary, respectively. The in situ hybridization analysis of gonads indicated that Pocyp17-I and Pocyp17-II mRNA were both detected in oocytes, spermatocytes and Sertoli cells. After injection of androgen and estrogen (17α-methyltestosterone, 17ß-estradiol) of different concentrations, the expression level of Pocyp17-I decreased significantly (P < 0.01), whereas estrogen had no influence on Pocyp17-II, but androgen upregulated the expression of Pocyp17-II (P < 0.05). Moreover, Pocyp17-I expression level was down-regulated significantly by NR0b1 but up-regulated by NR5a2 (P < 0.05), whereas Pocyp17-II expression level was down-regulated significantly by NR0b1 and NR5a2 (P < 0.05). All these results demonstrated that there were differences in expression patterns, feedback actions of sex hormones and transcriptional regulations between cyp17-I and cyp17-II, which revealed that cyp17-I and cyp17-II might perform different functions in sex hormones biosynthesis and gonadal differentiation in Japanese flounder.