Expression characteristics of Hsd3b7 in the gonads of Paralichthys olivaceus.

Steroidogenesis is an important biological process for gonadal differentiation and development. In mammals, 3ß-hydroxysteroid dehydrogenase 7 (HSD3B7) could convert 3ß-hydroxy of 7α-hydroxycholesterol into a ketone and form 7α-hydroxy-4-cholesten-3-one, which may affect steroidogenesis. However, in fish, the study of Hsd3b7 is still lacking. In this study, Hsd3b7 was identified in the olive flounder Paralichthys olivaceus, an important mariculture fish. According to bioinformatics analysis, Hsd3b7 belongs to a Rossmann-fold NAD(P)(+)-binding protein and can interact in a predictable manner with Hsd17b2, -3, and - 4, which play a role in steroidogenesis. In the adult flounder, Hsd3b7 was expressed in various tissues, at particularly high level in male muscle. The expression levels of Hsd3b7 at gonadal development stages I-V initially increased and then decreased, with an inflection point in the ovary at stage III and in the testis at stage IV. At stage III, the expression level of Hsd3b7 was significantly higher in the ovary than in the testis (P < 0.01). The results of in situ hybridization (ISH) revealed that it was mainly expressed in oocytes of phases I-IV or around oocytes of phases IV-V in the ovaries and around spermatid lobules at stages IV-V in the testes. Three regulatory sites of SRY-box transcription factor 9 (Sox9), a transcription factor involved in steroidogenesis and gonadal differentiation, were predicted in the promoter of Hsd3b7. After intraperitoneal injection with the recombination flounder Sox9a, the expression of Hsd3b7 was significantly up-regulated (P < 0.01). During the flounder gonadal differentiation, 17ß-estradiol (E2, 5 µg/g feed) and 17α-methyltestosterone (T, 5 µg/g feed) were used to obtain the phenotypic female or male flounder, and the results showed that in the E2 group, Hsd3b7 expression was highest at 2 cm TL, the primordial gonad stage, which was significantly higher than that at 12 cm TL (P < 0.05). In the T group, Hsd3b7 expression level was also highest at 2 cm TL and significantly higher than at 10 and 12 cm TL (P < 0.05). Moreover, Hsd3b7 was detected to be localized mainly around oogonia and spermatogonia during the differentiated gonads with ISH. These findings first introduce the expression characteristics of Hsd3b7 and the effect of Sox9a on its expression, which contribute to our understanding of the function of Hsd3b7 in fish gonads.

Expression of scp3 and dazl reveals the meiotic characteristics of the olive flounder Paralichthys olivaceus†.

Olive flounder Paralichthys olivaceus is an important cultured marine fish. We found that the meiosis marker scp3 and its intrinsic regulator dazl were mainly expressed in the gonads. During the ovarian differentiation, scp3 signal was detected first in pre-meiotic oogonia at 60-mm total length (TL) and then in primary oocytes at 80- and 100-mm TL, with a sharp increase in scp3 expression level observed at 80- and 100-mm TL. Dazl signal was detected in primordial germ cells at 30-mm TL and oogonia at 60-mm TL, but no significant change of expression was observed. During the testicular differentiation period, scp3 and dazl expression remained at low levels, and scp3 signal was weakly detected in spermatogonia at 80-mm TL, whereas dazl signal was not found. During the ovarian developmental stages, the highest expression levels of scp3 and dazl were detected at stages I and II, respectively, and strong signals of scp3 and dazl were detected in primary oocytes and oocytes at phases I and II. In the testis, the high expression of scp3 and dazl was detected at stages II-IV and II-III, respectively. Scp3 signal was weakly observed in pre-meiotic spermatogonia at stages I and II and strongly detected in primary spermatocytes at stages III-V. Dazl was detected in the nuclei of spermatogonia and spermatids at stages II-IV. Furthermore, scp3 expression in the ovary could be promoted by 17α-ethynylestradiol and tamoxifen, whereas dazl expression could be downregulated by tamoxifen.

Detection of Haemophilus influenzae by loop-mediated isothermal amplification coupled with nanoparticle-based lateral flow biosensor assay.

BACKGROUND: Haemophilus influenzae was the most aggressive pathogen and formed a major cause of bacterial meningitis and pneumonia in young children and infants, which need medical emergency requiring immediate diagnosis and treatment. However, From isolation to identification of H. influenzae, the traditional diagnose strategy was time-consuming and expensive. Therefore, the establishment of a convenient, highly sensitive, and stable detection system is urgent and critical. RESULTS: In this study, we used a combined method to detect H. influenzae. Six specific primers were designed on the basis of outer membrane protein P6 gene sequence of H. influenzae. The reaction condition such as the optimum temperature was 65℃, and the optimum reaction time was 30 min, respectively. Through the loop-mediated isothermal amplification (LAMP) in combination with nanoparticle-based lateral flow biosensor (LFB), the sensitivity of LAMP-LFB showed 100 fg was the lowest genomic DNA templates concentration in the pure cultures. Meanwhile, the specificity of H. influenzae-LAMP-LFB assay showed the exclusive positive results, which were detected in H. influenzae templates. In 55 clinical sputum samples, 22 samples were positive with LAMP-LFB method, which was in accordance with the traditional culture and Polymerase Chain Reaction (PCR) method. The accuracy in diagnosing H. influenzae with LAMP-LFB could reach 100%, compared to culture and PCR method, indicating the LAMP-LFB had more advantages in target pathogen detection. CONCLUSIONS: Taken together, LAMP-LFB could be used as an effective diagnostic approach for H. influenzae in the conditions of basic and clinical labs, which would allow clinicians to make better informed decisions regarding patient treatment without delay.

A Rapid Detection of Haemophilus influenzae Using Multiple Cross Displacement Amplification Linked With Nanoparticle-Based Lateral Flow Biosensor.

Haemophilus influenzae is a major human pathogenic bacterium, resulting in a series of diseases, such as pneumonia, bacteremia, meningitis. However, it is hard to diagnose H. influenzae quickly. In this study, the multiple cross displacement amplification (MCDA) and nanoparticle-based lateral flow biosensor (LFB) (MCDA-LFB) were combined to detect H. influenzae, which has been proven to be reliable, rapid, and not complicated. On the basis of H. influenzae outer membrane protein P6 gene, 10 specific primers were designed. The best MCDA condition was 61°C for 1 h. The sensitivity of H. influenzae-MCD-LFB assay showed, in the pure cultures, the minimum concentration of genomic DNA templates was 100 fg. The specificity of H. influenzae-MCD-LFB assay showed only H. influenzae templates were detected, and no cross-reactivity was found in non-H. influenzae isolates and other Haemophilus species. In 56 sputum samples, with MCDA-LFB method and PCR detection, 21 samples were positive, which was in consistent with the traditional culture method. The accuracy of diagnosis of MCDA-LFB, in comparison with the traditional culture method and PCR detection, can reach 100%, indicating that the MCDA-LFB assay gains an advantage over the cultured-based method for target pathogen detection. In conclusion, the MCDA-LFB assay is suitable for the sensitive, rapid, and specific detection of H. influenzae, which might be used as a potential diagnostic tool for H. influenzae in basic and clinical laboratories.

Sexual dimorphism of DNA and histone methylation profiles in the gonads of the olive flounder Paralichthys olivaceus.

DNA methylation and histone methylation are two types of the most important epigenetic modifications. However, research on their differential expression in gonads of male and female fish is limited. In this study, we examined the characteristics of DNA methylation and tri-methylation of lysine 4 of histone H3 (H3K4me3) modification profiles in the gonads of the wild-type and meio-gynogenetic olive flounders Paralichthys olivaceus. Enzyme-linked immunosorbent assay (ELISA) analysis revealed that the global DNA methylation level was higher in the testis than in the ovary. Real-time quantitative PCR (qPCR) results indicated that maintenance DNA methyltransferase gene dnmt1 and de novo DNA methyltransferase gene dnmt3a are highly expressed in the ovary, while DNA demethyltransferase genes tets are highly expressed in the testis. The inconsistency of DNA methylation and methyltransferase genes in the gonads might associate with the differential distribution in the testis. 5-mC mainly located in the spermatids of the testis was shown with immunohistochemistry (IHC). Furtherly, dnmt3a and tets are mainly located in spermatocytes and oocytes with in situ hybridization (ISH) analysis. As for H3K4me3, total level is higher in the ovary detected with western blot assay. IHC results showed that the signals of H3K4me3 in Sertoli cells of the testis were stronger than those in spermatocytes and spermatids. Methyltransferase gene kmt2b and demethylase genes kdm5a and kdm5c also exhibit much higher expression in the testis with qPCR, and ISH stronger signals of kmt2b and kdm5s were detected in spermatocytes. These results implied that DNA methylation and H3K4me3 are involved in the flounder sex differences and gametogenesis.

Effects of busulfan on somatic cells after inhibiting germ cells in the gonads of the young olive flounder Paralichthys olivaceus.

Busulfan is widely used in some species to inhibit germ cell proliferation. This study was conducted to evaluate effects of busulfan on germ and somatic cells in gonads of olive flounder, Paralichthys olivaceus, one of the most economically important mariculture fish species. After intraperitoneal injection with 80 (80B) or 120 (120B) mg/kg busulfan, both gonads were atrophied, and ovaries were discolored with adhesion to the visceral mass. Histological results indicated that germ cells in the gonads were detached, and there was a larger nucleus size and smaller cytoplasmic volume in spermatogonia. Numbers of oocytes and somatic cells in the ovary were both less (P < 0.05), while in the testis, numbers of spermatogonia and somatic cells were markedly lesser and greater, respectively (P < 0.05). In ovaries of the flounder treated with 80B and 120B, relative abundance of vasa and cyp19a1a mRNA transcripts was very small in the cytoplasm of oocytes, while the cyp19a1a transcript was still present in theca cells. In the testis of flounder treated with 80B and 120B, abundance of vasa was markedly less (P < 0.05) with there being very little vasa in spermatogonia and disruption of the spermatogonium structure. In the 80B treatment group, amh was in lesser abundance with there being very little amh in spermatogonia, however, with the 120B treatment there was a large amh abundance in spermatogonium with there being disruption of structure of these germ cells and Sertoli cells. Busulfan, therefore, might inhibit the development of spermatogonia in the flounder testis.

Sex-Dependent RNA Editing and N6-adenosine RNA Methylation Profiling in the Gonads of a Fish, the Olive Flounder (Paralichthys olivaceus).

Adenosine-to-inosine (A-to-I) editing and N6-methyladenosine (m6A) are two of the most abundant RNA modifications. Here, we examined the characteristics of the RNA editing and transcriptome-wide m6A modification profile in the gonads of the olive flounder, Paralichthys olivaceus, an important maricultured fish in Asia. The gonadal differentiation and development of the flounder are controlled by genetic as well as environmental factors, and the epigenetic mechanism may play an important role. In total, 742 RNA editing events were identified, 459 of which caused A to I conversion. Most A-to-I sites were located in 3'UTRs, while 61 were detected in coding regions (CDs). The number of editing sites in the testis was higher than that in the ovary. Transcriptome-wide analyses showed that more than one-half of the transcribed genes presented an m6A modification in the flounder gonads, and approximately 60% of the differentially expressed genes (DEGs) between the testis and ovary appeared to be negatively correlated with m6A methylation enrichment. Further analyses revealed that the mRNA expression of some sex-related genes (e.g., dmrt1 and amh) in the gonads may be regulated by changes in mRNA m6A enrichment. Functional enrichment analysis indicated that the RNA editing and m6A modifications were enriched in several canonical pathways (e.g., Wnt and MAPK signaling pathways) in fish gonads and in some pathways whose roles have not been investigated in relation to fish sex differentiation and gonadal development (e.g., PPAR and RNA degradation pathways). There were 125 genes that were modified by both A-to-I editing and m6A, but the two types of modifications mostly occurred at different sites. Our results suggested that the presence of sex-specific RNA modifications may be involved in the regulation of gonadal development and gametogenesis.

Amh dominant expression in Sertoli cells during the testicular differentiation and development stages in the olive flounder Paralichthys olivaceus.

The olive flounder Paralichthys olivaceus, an important marine fish, shows gender differences in growth. The mechanism on its gonadal differentiation direction affected with exogenous factors still needs to be clarified. The anti-Müllerian hormone (amh) gene is involved in fish testicular differentiation and maintenance. The aim of this study was to investigate the expression of the flounder amh in tissues and the gonads. The quantitative expression analysis results showed that it was highly expressed in the testis, especially in the testis at stages I - IV (P < 0.05). Also, amh was detected in Sertoli cells surrounding spermatogonia and peripheral seminiferous lobule of the testis with in situ hybridization (ISH) and immunohistochemistry (IHC). During the differentiation period, the amh expression in the testis of the tamoxifen treatment group (100 ppm) was higher than that in the ovary of the 17ß-estradiol (E2, 5 ppm) group, and the expression levels of amh during process of the male differentiation in the tamoxifen group were higher than those in the 17ɑ-methyltestosterone (MT, 5 ppm) group (P < 0.05). ISH results also exhibited that amh was expressed in the somatic cells that surrounded the germ cells of juvenile flounder similar to adult ones. Furthermore, the flounder gonads in the tamoxifen group maintained more germ cells and somatic cells than those in the MT group from 20 to 80 mm total length (TL). Especially, at 60 and 80 mm TL, the numbers of germ and somatic cells in the tamoxifen group were significantly higher than those in the MT group (P < 0.05). In summary, amh might initiate the process of testicular differentiation, and is involved in the early development and maintenance of testis.

Characteristics and sex dimorphism of 17ß-hydroxysteroid dehydrogenase family genes in the olive flounder Paralichthys olivaceus.

Sex steroid hormones play important roles in fish sex differentiation, gonadal development and secondary sexual characteristics. Olive flounder Paralichthys olivaceus is a valuable commercial marine fish species and has marked sexual dimorphism. However, the mechanisms of action of sex hormones in flounder sex are still unclear. In this study, a total of ten Hsd17b family genes, including Hsd17b3, -4, -7, -8, -9, -10, -12a, -12b, -14 and -15, were identified in the flounder, which encoded critical enzymes acting on sex steroid synthesis and metabolism. Hsd17b genes were distributed on eight chromosomes. Hsd17b12a and -12b were located on chromosomes 19 and 7, respectively. It was speculated that these two genes were just highly similar rather than different transcripts derived from the same gene. According to the results of domain and motif analyses, they all belonged to the SDR superfamily and contained conserved Hsd17b motifs TGxxxGxG, PGxxxT, NNAG and YxxxK. Analysis of amino acid sequences predicted that Hsd17b1, -4, -7, -12a and -14 were hydrophilic proteins. The stability of Hsd17b1, -3 and -12b proteins was predicted to be low. The various Hsd17b family genes differed in tissue expression pattern, and Hsd17b10, -12a and -12b were highly expressed in the flounder ovary. Moreover, throughout gonadal development, Hsd17b3 was highly expressed in the testis, and Hsd17b1, -12a and -12b were highly expressed in the ovary, suggesting that they might play an important role in testosterone synthesis in the testis or estrogen synthesis in the ovary. Activities of Hsd17b3 at stages I-V were all significantly higher in the testis than in the ovary (P < 0.05, P < 0.01). Transfection analysis in HEK293T cells showed that Hsd17b1 and -3 were located in both the cytoplasm and nucleus. Additionally, after challenging fish with tamoxifen, Hsd17b3 expression level in the testis decreased significantly (P < 0.01), and in the ovary no significant change was observed. Moreover, the expression of Hsd17b1 in the ovary was significantly upregulated after injection with flutamide (P < 0.05). These findings introduce the characteristics of the flounder Hsd17b in subfamily, which contribute to our understanding of the regulation of sex steroid hormone synthesis in fish gonadal development.

SOX2 participates in spermatogenesis of Zhikong scallop Chlamys farreri.

As an important transcription factor, SOX2 involves in embryogenesis, maintenance of stem cells and proliferation of primordial germ cell (PGC). However, little was known about its function in mature gonads. Herein, we investigated the SOX2 gene profiles in testis of scallop, Chlamys farreri. The level of C. farreri SOX2 (Cf-SOX2) mRNA increased gradually along with gonadal development and reached the peak at mature stage, and was located in all germ cells, including spermatogonia, spermatocytes, spermatids and spermatozoa. Knockdown of Cf-SOX2 using RNAi leaded to a mass of germ cells lost, and only a few spermatogonia retained in the nearly empty testicular acini after 21 days. TUNEL assay showed that apoptosis occurred in spermatocytes. Furthermore, transcriptome profiles of the testes were compared between Cf-SOX2 knockdown and normal scallops, 131,340 unigenes were obtained and 2,067 differential expression genes (DEGs) were identified. GO and KEGG analysis showed that most DEGs were related to cell apoptosis (casp2, casp3, casp8), cell proliferation (samd9, crebzf, iqsec1) and spermatogenesis (htt, tusc3, zmynd10, nipbl, mfge8), and enriched in p53, TNF and apoptosis pathways. Our study revealed Cf-SOX2 is essential in spermatogenesis and testis development of C. farreri and provided important clues for better understanding of SOX2 regulatory mechanisms in bivalve testis.

Expression characteristics and functional analysis of Krüppel-like factor 4 in adductor muscle and mantle of Zhikong scallop Chlamys farreri.

Krüppel-like factor 4 (KLF4) is an important transcription factor involving in formation and maintenance of muscles in mammals. However, no data are available on KLF4 function in shellfish muscles which play vital roles in the movement, stress response, and physiology in shellfish. In the present study, we revealed that the Klf4 mRNA of Zhikong scallop Chlamys farreri was expressed in most tissues, which has high level in adductor muscle, mantle, kidney, and testis. Positive signals of the Klf4 mRNA and protein were visible in all skeletal muscle fibers of adductor muscle, and all the cells of C. farreri mantle. Furthermore, the knockdown of Klf4 mRNA in adductor muscle and mantle by means of in vivo RNA interference led to some different phenotypes, including disordered arrangement of muscle fibers in adductor muscle and mantle, abnormal structures of skeletal muscles, and reduced muscle fibers under endepidermis of mantle. Our findings demonstrated that Klf4 plays important roles in maintenance of muscle functions in C. farreri adductor muscle and mantle, and suggested that its regulatory way in skeletal muscle may be different from the smooth muscle in shellfish.

A novel role of Krüppel-like factor 4 in Zhikong scallop Chlamys farreri during spermatogenesis.

Krüppel-like factor 4 (KLF4) is a kind of zinc finger transcription factor, which is involved in terminal differentiation of epithelial cells and reprogramming of somatic cells to induced pluripotent stem (iPS) cells in mammals. In the present study, we identified a full-length cDNA of Klf4 in Zhikong scallop Chamys farreri (Cf-Klf4) and found that Cf-Klf4 presented a sexual dimorphic expression characteristic in C. farreri gonads. Cf-Klf4 expression was significantly higher in testes than in ovaries from growing stage to mature stage detected by quantitative real-time PCR, and was located in male gametes, except for spermatozoa during spermatogenesis through in situ hybridization and immunohistochemistry, while no positive signal was visible in female gametes during oogenesis. Furthermore, the knockdown of Cf-Klf4 in testes by means of in vivo RNA interference led to an obviously developmental retardance, lower gonadosomatic index, less male gametes and more apoptotic spermatocytes. Interestingly, we found that two out of eight scallops showed a hermaphroditic phenotype characteristic of male-to-female sex reversal when the Klf4 mRNA and protein levels were knocked down in males. These results verified that Klf4 plays an important role in testis functional maintenance and is necessary in spermatogenesis of C. farreri.

Piwi1 is essential for gametogenesis in mollusk Chlamys farreri.

Piwi (P-element induced wimpy testis) is an important gene involved in stem cell maintenance and gametogenesis in vertebrates. However, in most invertebrates, especially mollusks, the function of Piwi during gametogenesis remains largely unclear. To further understand the function of Piwi during gametogenesis, full-length cDNA of Piwi1 from scallop Chlamys farreri (Cf-Piwi1) was characterized, which consisted of a 2,637 bp open reading frame encoding an 878-amino acid protein. Cf-Piwi1 mRNA was mainly localized in the spermatogonia, spermatocytes, oogonia, oocytes of early development and intra-gonadal somatic cells. Additionally, the knockdown of Cf-Piwi1 by injection of Cf-Piwi1-dsRNA (double-stranded RNA) into scallop adductor led to a loss of germ cells in C. farreri gonads. Apoptosis was observed mainly in spermatocytes and oocytes of early development, as well as in a small number of spermatogonia and oogonia. Our findings indicate that Cf-Piwi1 is essential for gametogenesis in the scallop C. farreri.

Different expression of sox17 gene during gametogenesis between scallop Chlamys farreri and vertebrates.

SOX17, a member of SRY-related high-mobility-group box (SOX) family, involves in endoderm formation, angiogenesis and carcinogenesis, and its expression characteristics are different in spermatogenesis among several vertebrates. In this study, we cloned a full-length cDNA sequence of sox17 from Zhikong scallop Chlamys farreri, and determined its expression characteristics in gonad at mRNA and protein levels. The cDNA sequence was 2802 bp in length, predicted to encode a protein of 511 amino acids and contained a conservative HMG-box of SOX family, while lacked the C-terminal region of SOX17 comparing to vertebrates. Semi-quantitative RT-PCR showed that C. farreri sox17 (Cf-sox17) mRNA exhibited a different tissue distribution, and the transcript abundance was the highest in the gonads. In situ hybridization determined that the Cf-sox17 mRNA was located in various germ cells in testis and ovary. Similar result of Cf-SOX17 protein was also observed by immunohistochemical detection. The location in gonad is different from that of mammals and fish in which SOX17 is only located in some specific germ cells. Our finding revealed a different characteristic of sox17 expression in gametogenesis between scallop and vertebrates, which implied that Cf-sox17 may involve in gametogenesis of bivalves and the function may differ from that in vertebrates.

Characteristics of 17ß-hydroxysteroid dehydrogenase 8 and its potential role in gonad of Zhikong scallop Chlamys farreri.

17ß-Hydroxysteroid dehydrogenases (17ß-HSDs) are important enzymes catalyzing steroids biosynthesis and metabolism in vertebrates. Although studies indicate steroids play a potential role in reproduction of molluscs, little is known about the presence and function of 17ß-HSDs in molluscs. In the present study, a full-length cDNA encoding 17ß-HSD type 8 (17ß-HSD8) was identified in the Zhikong scallop Chlamys farreri, which is 1104bp in length with an open reading frame of 759bp encoding a protein of 252 amino acids. Phylogenetic analysis revealed that the C. farreri 17ß-HSD8 (Cf-17ß-HSD8) belongs to the short chain dehydrogenase/reductase family (SDR) and shares high homology with other 17ß-HSD8 homologues. Catalytic activity assay in vitro demonstrated that the refolded Cf-17ß-HSD8 expressed in Escherichia coli could effectively convert estradiol-17ß (E2) to estrone (E1), and weakly catalyze the conversion of testosterone (T) to androstenedione (A) in the presence of NAD(+). The Cf-17ß-HSD8 mRNA was ubiquitously expressed in all tissues analyzed, including gonads. The expression levels of Cf-17ß-HSD8 mRNA and protein increased with gametogenesis in both ovary and testis, and were significantly higher in testis than in ovary at growing stage and mature stage. Moreover, results of in situ hybridization and immunohistochemistry revealed that the mRNA and protein of Cf-17ß-HSD8 were expressed in follicle cells and gametes at all stages except spermatozoa. Our findings suggest that Cf-17ß-HSD8 may play an important role in regulating gametogenesis through modulating E2 levels in gonad of C. farreri.