RÉSUMÉ
Mpv17 (mitochondrial inner membrane protein MPV17) deficiency causes severe mitochondrial DNA depletion syndrome in mammals and loss of pigmentation of iridophores and a significant decrease of melanophores in zebrafish. The reasons for this are still unclear. In this study, we established an mpv17 homozygous mutant line in Nile tilapia. The developing mutants are transparent due to loss of iridophores and aggregation of pigment granules in the melanophores and disappearance of the vertical pigment bars on the side of the fish. Transcriptome analysis using skin of fish at 30 dpf (days post fertilization) revealed that the genes related to purine (especially pnp4a) and melanin synthesis were significantly downregulated. However, administration of guanine diets failed to rescue the phenotype of the mutants. In addition, no obvious apoptosis signals were observed in the iris of the mutants by TUNEL staining. Significant downregulation of genes related to iridophore differentiation was detected by qPCR. Insufficient ATP, as revealed by ATP assay, α-MSH treatment and adcy5 mutational analysis, might account for the defects of melanophores in mpv17 mutants. Several tissues displayed less mtDNA and decreased ATP levels. Taken together, these results indicated that mutation of mpv17 led to mitochondrial dTMP deficiency, followed by impaired mtDNA content and mitochondrial function, which in turn, led to loss of iridophores and a transparent body color in tilapia.
RÉSUMÉ
Relaxin 3 is a neuropeptide that plays a crucial role in reproductive functions of mammals. Previous studies have confirmed that rln3a plays an important role in the male reproduction of tilapia. To further understand the significance of its paralogous gene rln3b in male fertility, we generated a homozygous mutant line of rln3b in Nile tilapia. Our findings indicated that rln3b mutation delayed spermatogenesis and led to abnormal testes structure. Knocking out rln3b gene resulted in a decrease in sperm count, sperm motility and male fish fertility. TUNEL detection revealed a small amount of apoptosis in the testes of rln3b-/- male fish at 390 days after hatching (dah). RT-qPCR analysis demonstrated that mutation of rln3b gene caused a significant downregulation of steroid synthesis-related genes such as cyp17a1, cyp11b2, germ cell marker gene, Vasa, and gonadal somatic cell marker genes of amh and amhr2. Furthermore, we found a significant down-regulation of hypothalamic-pituitary-gonadal (HPG) axis-related genes, while a significantly up-regulation of the dopamine synthetase gene in the rln3b-/- male fish. Taken together, our data strongly suggested that Rln3b played a crucial role in the fertility of XY tilapia by regulating HPG axis genes.
Sujet(s)
Hypogonadisme , Spermatogenèse , Testicule , Tilapia , Animaux , Mâle , Tilapia/génétique , Hypogonadisme/génétique , Spermatogenèse/génétique , Testicule/métabolisme , Relaxine/génétique , Relaxine/métabolisme , Fécondité/génétique , Mobilité des spermatozoïdes/génétique , Mutation , Protéines de poisson/génétique , Protéines de poisson/métabolismeRÉSUMÉ
Relaxin3 (rln3) has been associated with various emotional and cognitive processes, including stress, anxiety, learning, memory, motivational behavior, and circadian rhythm. Notably, previous report revealed that Rln3a played an indispensable role in testicular development and male fertility in Nile tilapia (Oreochromis niloticus). However, the underlying molecular mechanisms remain largely unknown. We found that Rln3a is expressed exclusively in the diencephalon* (Di*) of the brain. Deficiency of Rln3a resulted in a significant increase in serum dopamine level and an upregulation of gene expression of gnrh1 and kisspeptin2. To further elucidate the role of Rln3a in fish fertility, we collected two different regions of Di* and hypothalamus (Hyp) tissues for subsequent RNA-seq analysis of both wild-type (rln3a+/+) and rln3a-/- male tilapia. Upon the transcriptomic data, 1136 and 755 differentially expressed genes (DEGs) were identified in the Di* and Hyp tissues, respectively. In Di*, the up-regulated genes were enriched in circadian rhythm, chemical carcinogenesis, while the down-regulated genes were enriched in type II diabetes mellitus, dopaminergic synapse, and other pathways. In Hyp, the up-regulated genes were enriched in circadian rhythm, pyrimidine metabolism, while the down-regulated genes were enriched in type I diabetes mellitus, autoimmune thyroid disease, and other pathways. Subsequently, the results of both qRT-PCR and FISH assays highlighted a pronounced up-regulation of core circadian rhythm genes, cry1b and per3, whereas genes such as clocka, clockb, and arntl exhibited down-regulation. Furthermore, the genes associated with dopamine biosynthesis were significantly increased in the Hyp. In summary, the mutation of rln3a in male tilapia resulted in notable changes in circadian rhythm and disease-linked signaling pathways in the Di* and Hyp. These changes might account for the fertility defects observed in rln3a-/- male mutants in tilapia.
Sujet(s)
Encéphale , Cichlides , Fécondité , Animaux , Mâle , Cichlides/génétique , Cichlides/métabolisme , Encéphale/métabolisme , Fécondité/génétique , Relaxine/génétique , Relaxine/métabolisme , Protéines de poisson/génétique , Protéines de poisson/métabolismeRÉSUMÉ
Steroidogenic acute regulatory protein (Star) plays an essential role in the biosynthesis of corticosteroids and sex steroids by mediating the transport of cholesterol from the outer to the inner membrane of mitochondria. Two duplicated Star genes, namely star1 and star2, have been identified in non-mammalian vertebrates. To investigate the roles of star genes in fish steriodogenesis, we generated two mutation lines of star1-/- and star1-/-/star2-/- in Nile tilapia (Oreochromis niloticus). Previous studies revealed that deficiency of star2 gene caused delayed spermatogenesis, sperm apoptosis and sterility in male tilapia. Our present data revealed that mutation of star genes impaired male fertility. Disordered seminiferous lobules and spermatic duct obstruction were found in the testis of both types of mutants. Moreover, significant decline in semen volume, sperm abnormality and impaired fertility were also detected in star1-/- and star1-/-/star2-/- males. In star1-/- male fish, lipid accumulation, up-regulation of steroidogenic enzymes, and significant decline of androgens were found. Additionally, hyperplasic interrenal cells, elevated steroidogenic gene expression level and decline of serum glucocorticoids were detected in star1 mutants. Intriguingly, either 11-KT or cortisol supplementation successfully rescued the impaired fertility of the star1-/- mutants. Taken together, these results further indicate that Star1 might play critical roles in the production of both 11-KT and glucocorticoids, which are indispensable for the maintenance of male fertility in fish.
Sujet(s)
Cichlides , Fécondité , Glucocorticoïdes , Mutation , Phosphoprotéines , Testostérone , Animaux , Mâle , Cichlides/génétique , Cichlides/métabolisme , Testostérone/métabolisme , Testostérone/sang , Testostérone/analogues et dérivés , Fécondité/génétique , Phosphoprotéines/génétique , Phosphoprotéines/métabolisme , Glucocorticoïdes/métabolisme , Protéines de poisson/génétique , Protéines de poisson/métabolisme , Testicule/métabolismeRÉSUMÉ
In mammals, glial cell derived neurotrophic factor (GDNF) plays a critical role in the self-renewal and maintenance of spermatogonial stem cells (SSCs) in testis and oogenesis in ovary, whilst retinoic acid (RA), the key factor of meiosis initiation, can downregulate its expression. Unlike mammals, two Gdnf replication genes are widely present in teleost fishes, however, our understanding of them is still poor. In the present study, two paralogous gdnf from Nile tilapia (Oreochromis niloticus), namely as Ongdnfa and Ongdnfb, were characterized, and then their cellular expression profiles in testis and ovary and responsiveness to RA treatment at the tissue and cellular levels were investigated. In phylogenetic tree, the Gdnfa and Gdnfb from teleost fishes were clustered into two different subclasses, respectively, and then clustered with the homologs from cartilaginous fish and tetrapods, suggesting that OnGdnfa and OnGdnfb are orthologous to GDNF and paralogous to each other. Ongdnfa is expressed in Sertoli cells and Leydig cells in testis and oocytes in ovary. The expression pattern of Ongdnfb is similar to Ongdnfa. In the ex vivo testicular organ culture, RA down-regulated the expression of Ongdnfa, whereas up-regulated the expression of Ongdnfb (P < 0.05), suggesting that they have differential responsiveness to RA signaling. RA treatment of the cultured cells derived from adult Nile tilapia testis which have the expression of RA receptors (RAR), Ongdnfa and Ongdnfb further confirmed the above result. Collectively, our study suggests that Ongdnfa and Ongdnfb have non-germline expression patterns in testis and germline expression patterns in ovary; furthermore, they have differential responsiveness to RA signaling, implying that they might have differential biological functions. This study broadens and enriches our understanding of fish GDNF homologs and lays foundation for the study of their biological functions in the future.
Sujet(s)
Cichlides , Facteur neurotrophique dérivé des cellules gliales , Ovaire , Testicule , Trétinoïne , Animaux , Trétinoïne/pharmacologie , Facteur neurotrophique dérivé des cellules gliales/génétique , Facteur neurotrophique dérivé des cellules gliales/métabolisme , Mâle , Femelle , Cichlides/génétique , Cichlides/métabolisme , Testicule/métabolisme , Testicule/effets des médicaments et des substances chimiques , Ovaire/métabolisme , Ovaire/effets des médicaments et des substances chimiques , Phylogenèse , Régulation de l'expression des gènes/effets des médicaments et des substances chimiques , Protéines de poisson/génétique , Protéines de poisson/métabolismeRÉSUMÉ
Hedgehog (Hh) signaling is crucial in cardiovascular development and maintenance. However, the biological role of Patched1 (Ptch1), an inhibitory receptor of the Hh signaling pathway, remains elusive. In this study, a Ptch1 ortholog was characterized in Nile tilapia (Oreochromis niloticus), and its function was investigated through CRISPR/Cas9 gene knockout. When one-cell embryos were injected with CRISPR/Cas9 targeting ptch1, the mutation efficiency exceeded 70%. During 0-3 days post fertilization (dpf), no significant differences were observed between the ptch1 mutant group and the control group; at 4 dpf (0 day after hatching), about 10% of the larvae showed an angiogenesis defect and absence of blood flow; from 5 dpf, most larvae exhibited an elongated heart, large pericardial cavity, and blood leakage and coagulation, ultimately dying during the 6-8 dpf period due to the lack of blood circulation. Consistently, multiple differentially expressed genes related to angiogenesis, blood coagulation, and heart development were enriched in the ptch1 mutants. Furthermore, Smoothened (Smo) antagonist (cyclopamine) treatment of the ptch1 mutants greatly rescued the cardiovascular disorders. Collectively, our study suggests that Ptch1 is required for cardiovascular development and vascular integrity via Smo signaling, and excessive Hh signaling is detrimental to cardiovascular development.
Sujet(s)
Cichlides , Animaux , Cichlides/métabolisme , Protéines Hedgehog/génétique , Protéines Hedgehog/métabolisme , Transduction du signal , Techniques de knock-out de gènes , Mutation , Récepteur Smoothened/génétiqueRÉSUMÉ
Sex is determined by multiple factors derived from somatic and germ cells in vertebrates. We have identified amhy, dmrt1, gsdf as male and foxl2, foxl3, cyp19a1a as female sex determination pathway genes in Nile tilapia. However, the relationship among these genes is largely unclear. Here, we found that the gonads of dmrt1;cyp19a1a double mutants developed as ovaries or underdeveloped testes with no germ cells irrespective of their genetic sex. In addition, the gonads of dmrt1;cyp19a1a;cyp19a1b triple mutants still developed as ovaries. The gonads of foxl3;cyp19a1a double mutants developed as testes, while the gonads of dmrt1;cyp19a1a;foxl3 triple mutants eventually developed as ovaries. In contrast, the gonads of amhy;cyp19a1a, gsdf;cyp19a1a, amhy;foxl2, gsdf;foxl2 double and amhy;cyp19a1a;cyp19a1b, gsdf;cyp19a1a;cyp19a1b triple mutants developed as testes with spermatogenesis via up-regulation of dmrt1 in both somatic and germ cells. The gonads of amhy;foxl3 and gsdf;foxl3 double mutants developed as ovaries but with germ cells in spermatogenesis due to up-regulation of dmrt1. Taking the respective ovary and underdeveloped testis of dmrt1;foxl3 and dmrt1;foxl2 double mutants reported previously into consideration, we demonstrated that once dmrt1 mutated, the gonad could not be rescued to functional testis by mutating any female pathway gene. The sex reversal caused by mutation of male pathway genes other than dmrt1, including its upstream amhy and downstream gsdf, could be rescued by mutating female pathway gene. Overall, our data suggested that dmrt1 is the only male pathway gene tested indispensable for sex determination and functional testis development in tilapia.
Sujet(s)
Processus de détermination du sexe , Tilapia , Animaux , Femelle , Mâle , Régulation de l'expression des gènes au cours du développement , Gonades/métabolisme , Ovaire/métabolisme , Processus de détermination du sexe/génétique , Différenciation sexuelle/génétique , Testicule/métabolisme , Tilapia/génétiqueRÉSUMÉ
The recycling of electronic waste, i.e., waste Printed Circuit Boards (WPCBs), provides substantial environmental and economic advantages. In fact, the concentration of valuable precious and base metals in WPCBs is even higher compared to those found in mined ores. Nevertheless, it is still challenging to selectively extract precious metals with low concentrations from the pregnant leaching solution, due to the co-deposition of base metals, like Cu, which have higher concentrations. In this research, stepwise recovery of precious metals and copper directly from WPCBs thiosulfate leaching solution was facilitated by the Ti cathode coated with MoS2 (MoS2/Ti). The in-situ enrichment of Au(S2O3)23- and Ag(S2O3)23- at the surface of MoS2 enables the high efficiency and selectivity of electrodeposition, which has been confirmed through COMSOL Multiphysics simulations and visualization. As a result, the first-step electrodeposition at 0.6 V recovered 92.44 % Au and 98.18 % Ag without any co-deposition of Cu. Subsequently, the second-step recovery employed a constant current of 0.03 A, achieving 100 % recovery of copper within 12 h. Furthermore, this study optimized the reduction potential, NH3·H2O concentration, and S2O32- concentration for the stepwise electrodeposition process. These findings provide valuable insights for establishing a closed loop circular economy in the electronics industry.
RÉSUMÉ
Oogenesis is a complex process regulated by precise coordination of multiple factors, including maternal genes. Zygote arrest 1 (zar1) has been identified as an ovary-specific maternal gene that is vital for oocyte-to-embryo transition and oogenesis in mouse and zebrafish. However, its function in other species remains to be elucidated. In the present study, zar1 was identified with conserved C-terminal zinc finger domains in Nile tilapia. zar1 was highly expressed in the ovary and specifically expressed in phase I and II oocytes. Disruption of zar1 led to the failed transition from oogonia to phase I oocytes, with somatic cell apoptosis. Down-regulation and failed polyadenylation of figla, gdf9, bmp15 and wee2 mRNAs were observed in the ovaries of zar1-/- fish. Cpeb1, a gene essential for polyadenylation that interacts with Zar1, was down-regulated in zar1-/- fish. Moreover, decreased levels of serum estrogen and increased levels of androgen were observed in zar1-/- fish. Taken together, zar1 seems to be essential for tilapia oogenesis by regulating polyadenylation and estrogen synthesis. Our study shows that Zar1 has different molecular functions during gonadal development by the similar signaling pathway in different species.
Sujet(s)
Protéines d'oeuf , Protéines de poisson , Tilapia , Animaux , Femelle , Souris , Cichlides/génétique , Cichlides/métabolisme , Protéines d'oeuf/métabolisme , Oestrogènes , Facteurs de clivage et de polyadénylation de l'ARN messager/génétique , Ovogenèse/génétique , Polyadénylation , Tilapia/génétique , Tilapia/métabolisme , Facteurs de transcription/génétique , Danio zébré/métabolisme , Protéines de poisson/métabolismeRÉSUMÉ
The Nile tilapia (Oreochromis niloticus), with a system of XX/XY sex determination, is a worldwide farmed fish with a shorter sexual maturation time than that of most cultured fish. Tilapia show a spawning cycle of approximately 14 days and can be artificially propagated in the laboratory all year round to obtain genetically all female (XX) and all male (XY) fry. Its genome sequence has been opened, and a perfect gene editing platform has been established. With a moderate body size, it is convenient for taking enough blood to measure hormone level. In recent years, using tilapia as animal model, we have confirmed that estrogen is crucial for female development because 1) mutation of star2, cyp17a1 or cyp19a1a (encoding aromatase, the key enzyme for estrogen synthesis) results in sex reversal (SR) due to estrogen deficiency in XX tilapia, while mutation of star1, cyp11a1, cyp17a2, cyp19a1b or cyp11c1 affects fertility due to abnormal androgen, cortisol and DHP levels in XY tilapia; 2) when the estrogen receptors (esr2a/esr2b) are mutated, the sex is reversed from female to male, while when the androgen receptors are mutated, the sex cannot be reversed; 3) the differentiated ovary can be transdifferentiated into functional testis by inhibition of estrogen synthesis, and the differentiated testis can be transdifferentiated into ovary by simultaneous addition of exogenous estrogen and androgen synthase inhibitor; 4) loss of male pathway genes amhy, dmrt1, gsdf causes SR with upregulation of cyp19a1a in XY tilapia. Disruption of estrogen synthesis rescues the male to female SR of amhy and gsdf but not dmrt1 mutants; 5) mutation of female pathway genes foxl2 and sf-1 causes SR with downregulation of cyp19a1a in XX tilapia; 6) the germ cell SR of foxl3 mutants fails to be rescued by estrogen treatment, indicating that estrogen determines female germ cell fate through foxl3. This review also summarized the effects of deficiency of other steroid hormones, such as androgen, DHP and cortisol, on fish reproduction. Overall, these studies demonstrate that tilapia is an excellent animal model for studying reproductive endocrinology of fish.
Sujet(s)
Cichlides , Tilapia , Animaux , Mâle , Femelle , Tilapia/génétique , Tilapia/métabolisme , Androgènes , Hydrocortisone , Cichlides/métabolisme , Oestrogènes/métabolisme , Différenciation sexuelle/génétiqueRÉSUMÉ
The proliferation of spermatogonia directly affects spermatogenesis and male fertility, but its underlying molecular mechanisms are poorly understood. In this study, Smoothened (Smo), the central transducer of Hedgehog signaling pathway, was characterized in medaka (Oryzias latipes), and its role and underlying mechanisms in the proliferation of spermatogonia were investigated. Smo was highly expressed in spermatogonia. In ex vivo testicular organ culture and a spermatogonial cell line (SG3) derived from medaka mature testis, Smo activation promoted spermatogonia proliferation, while its inhibition induced apoptosis. The expression of glioma-associated oncogene homolog 1 (gli1) and regulator of cell cycle (rgcc) was significantly upregulated in SG3 after Smo activation. Furthermore, Gli1 transcriptionally upregulated the expression of rgcc, and Rgcc overexpression rescued cell apoptosis caused by Smo or Gli1 inhibition. Co-immunoprecipitation assay indicated that Rgcc could interact with cyclin-dependent kinase 1 (Cdk1) to regulate the cell cycle of spermatogonia. Collectively, our study firstly reveals that Smo mediates the proliferation of spermatogonia through Gli1-Rgcc-Cdk1 axis. In addition, Smo and Gli1 are necessary of the survival of spermatogonia. This study deepens our understanding of spermatogonia proliferation and survival at the molecular level, and provides insights into male fertility control and reproductive disease treatment.
Sujet(s)
Oryzias , Animaux , Mâle , Spermatogonies/métabolisme , Protéine à doigt de zinc GLI1/génétique , Protéine à doigt de zinc GLI1/métabolisme , Protéine-kinase CDC2/génétique , Protéine-kinase CDC2/métabolisme , Prolifération cellulaire , Protéines Hedgehog/métabolismeRÉSUMÉ
Amur catfish (Silurus asotus) is an ecologically and economically important fish species in Asia. Here, we assembled the female and male Amur catfish genomes, with genome sizes of 757.15 and 755.44 Mb, respectively, at the chromosome level using nanopore and Hi-C technologies. Consistent with the known diploid chromosome count, both genomes contained 29 chromosome-size scaffolds covering 98.80 and 98.73 % of the complete haplotypic assembly with scaffold N50 of 28.87 and 27.29 Mb, respectively. The female (n = 40) and male (n = 40) pools were re-sequenced. Comparative analysis of sequencing and re-sequencing data from both sexes confirmed the presence of an XX/XY sex determination system in Amur catfish and revealed Chr5 as the sex chromosome containing an approximately 400 kb Y-specific region (MSY). Gene annotation revealed a male-specific duplicate of amhr2, namely amhr2y, in MSY, which is male-specific in different wild populations and expressed only in the testes. Amur catfish shared partially syntenic MSY and amhr2y genes with the southern catfish (S. meridionalis, Chr24), which were located on different chromosomes. High sequence divergence between amhr2y and amhr2 and high sequence similarity with amhr2y were observed in both species. These results indicate the common origin of the sex-determining (SD) gene and transition of amhr2y in the two Silurus species. Accumulation of repetitive elements in the MSY of both species may be the main driver of the transition of amhr2y. Overall, our study provides valuable catfish genomic resources. Moreover, determination of amhr2y as the candidate SD gene in Amur catfish provides another example of amhr2 as the SD gene in fish.
Sujet(s)
Poissons-chats , Animaux , Femelle , Mâle , Poissons-chats/génétique , Génome/génétique , Génomique/méthodes , Chromosomes , Annotation de séquence moléculaireRÉSUMÉ
The normal development of lens fiber cells plays a critical role in lens morphogenesis and maintaining transparency. Factors involved in the development of lens fiber cells are largely unknown in vertebrates. In this study, we reported that GATA2 is essential for lens morphogenesis in Nile tilapia (Oreochromis niloticus). In this study, Gata2a was detected in the primary and secondary lens fiber cells, with the highest expression in primary fiber cells. gata2a homozygous mutants of tilapia were obtained using CRISPR/Cas9. Different from fetal lethality caused by Gata2/gata2a mutation in mice and zebrafish, some gata2a homozygous mutants of tilapia are viable, which provides a good model for studying the role of gata2 in non-hematopoietic organs. Our data showed that gata2a mutation caused extensive degeneration and apoptosis of primary lens fiber cells. The mutants exhibited progressive microphthalmia and blindness in adulthood. Transcriptome analysis of the eyes showed that the expression levels of almost all genes encoding crystallin were significantly down-regulated, while the expression levels of genes involved in visual perception and metal ion binding were significantly up-regulated after gata2a mutation. Altogether, our findings indicate that gata2a is required for the survival of lens fiber cells and provide insights into transcriptional regulation underlying lens morphogenesis in teleost fish.
Sujet(s)
Cécité , Cichlides , Facteur de transcription GATA-2 , Microphtalmie , Tilapia , Animaux , Cécité/génétique , Cichlides/génétique , Microphtalmie/génétique , Mutation , Tilapia/génétique , Danio zébré/génétique , Facteur de transcription GATA-2/génétiqueRÉSUMÉ
Although CRISPR/Cas9 has been used in gene manipulation of several fish species in vivo, its application in fish cultured cells is still challenged and limited. In this study, we established an integrated CRISPR/Cas9 plasmid system and evaluated its efficiency of gene knock-out or knock-in at a specific site in medaka (Oryzias latipes) in vitro and in vivo. By using the enhanced green fluorescent protein reporter plasmid pGNtsf1, we demonstrate that pCas9-U6sgRNA driven by endogenous U6 promoter (pCas9-mU6sgRNA) mediated very high gene editing efficiency in medaka cultured cells, but not by exogenous U6 promoters. After optimizing the conditions, the gene editing efficiencies of eight sites targeting for four endogenous genes were calculated, and the highest was up to 94% with no detectable off-target. By one-cell embryo microinjection, pCas9-mU6sgRNA also mediated efficient gene knock-out in vivo. Furthermore, pCas9-mU6sgRNA efficiently mediated gene knock-in at a specific site in medaka cultured cells as well as embryos. Collectively, our study demonstrates that the genetic relationship of U6 promoter is critical to gene editing efficiency in medaka cultured cells, and a simple and efficient system for medaka genome editing in vitro and in vivo has been established. This study provides an insight into other fish genome editing and promotes gene functional analysis.
RÉSUMÉ
Mammalian Nanog is critical in pluripotency acquisition and maintenance. Nonetheless, a recent report from zebrafish (Danio rerio) suggests that Nanog is not required for embryonic cells which is not like the mammalian homologs, but is necessary for the proper formation of the extra-embryonic yolk syncytial layer (YSL). However, whether its biological function in other fishes is conservative remains to be investigated. Our previous work shows that Nanog from Nile tilapia (Oreochromis niloticus) (termed as Ong thereafter) displays differential spatiotemporal expression patterns from the other teleost fishes including zebrafish. In this study, Ong co-expression with Pou5f3 (another core pluripotent transcription factor), transcriptional regulation and its biological functions during embryonic development and in the survival and proliferation of embryonic cells were investigated. At the blastula stage, both Ong and Pou5f3 were highly expressed in embryonic cells and co-located in the nucleus. After that, the expression of both Ong and Pou5f3 began to decrease at the gastrula stage (24 haf) and then exhibited a differential expression profile at the segmentation stage (28-36 haf). Ong disappeared in embryonic cells and was limited to YSL, whilst Pou5f3 was highly expressed in embryonic cells even some with obvious cytoplasmic distribution. Luciferase assay indicated that Ong was negatively regulated by Pou5f3 and positively regulated by androgen and itself. Ong depletion in fertilized one-cell embryos through CRISPR/Cas9 led to blastula blockage or death, and the survival and proliferation of blastula-derived embryonic cells in vitro failed. Collectively, Ong has similar expression and biological function to Pou5f3 at the blastula stage, which is similar to mammalian homolog but different from zebrafish homolog. These data suggest that the expression patterns and functions of Nanog are not conservative in fishes and vary from species to species. This study enriches our understanding about Nanog and its evolution.
Sujet(s)
Cichlides , Danio zébré , Animaux , Cichlides/génétique , Cichlides/métabolisme , Régulation de l'expression des gènes au cours du développement , Blastula , Protéines de poisson-zèbre/génétique , Protéines de poisson-zèbre/métabolisme , Développement embryonnaire/génétique , Mammifères/métabolisme , Protéine homéotique Nanog/génétique , Protéine homéotique Nanog/métabolismeRÉSUMÉ
Background: Steroid hormones play an essential role in many reproductive processes of vertebrates. Previous studies revealed that teleost-specific Cyp17a2 (cytochrome P450 family 17 subfamily a 2) might be required for the production of cortisol in the head-kidney and 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) in ovary during oocyte maturation. However, the role of Cyp17a2 in male reproduction remains to be largely unknown. The aim of this study was to explore the essentiality of cyp17a2 gene in male steroidogenesis, spermatogenesis, and male fertility. Methods: A homozygous mutation line of cyp17a2 gene was constructed in tilapia by CRISPR/Cas9 gene editing technology. The expression level of germ cell and meiosis-related genes and steroidogenic enzymes were detected by qRT-PCR, IHC, and Western blotting. EIA and LC-MS/MS assays were used to measure the steroid production levels. And sperm quality was examined by Sperm Quality Analyzer software. Results: In this study, cyp17a2 gene mutation resulted in the significant decline of serum DHP and cortisol levels. On the contrary, significant increases in intermediate products of cortisol and DHP were found in cyp17a2-/- male fish. The deficiency of cyp17a2 led to the arrest of meiotic initiation in male fish revealing as the reduction of the expression of germ cell-related genes (vasa, piwil, oct4) and meiosis-related genes (spo11 and sycp3) by 90 dah. Afterwards, spermatogenesis was gradually recovered with the development of testis in cyp17a2-/- males, but it showed a lower sperm motility and reduced fertility compared to cyp17a2+/+ XY fish. Deletion of cyp17a2 led to the abnormal upregulation of steroidogenic enzymes for cortisol production in the head-kidney. Moreover, unaltered serum androgens and estrogens, as well as unchanged related steroidogenic enzymes were found in the testis of cyp17a2-/- male fish. Conclusion: This study proved that, for the fist time, Cyp17a2 is indispensable for cortisol and DHP production, and cyp17a2 deficiency associated curtailed meiotic initiation and subfertility suggesting the essentiality of DHP and cortisol in the male fertility of fish.
Sujet(s)
Cichlides , Animaux , Femelle , Mâle , Cichlides/métabolisme , Chromatographie en phase liquide , Hydrocortisone/métabolisme , Mobilité des spermatozoïdes , Sperme , Spectrométrie de masse en tandem , FéconditéRÉSUMÉ
Oogenesis is a complex developmental process responsible for the production of eggs from oogonia in fish and other animals. However, transcriptional regulation underlying oogenesis is not fully understood. In the present study, we demonstrated in the teleost fish Nile tilapia that the Sox transcription factor family member Sox3 was involved in regulating oocyte growth during oogenesis. Fluorescence in situ hybridization showed that Sox3 expression was enriched in growing oocytes of ovary but could not be detected in testes. CRISPR/Cas9-mediated homozygous mutation in the Sox3 gene disrupted oocyte growth. Further analysis revealed that Sox3 mutation caused a decrease in the contents of neutral lipids in oocytes and estradiol-17 beta (E2) production. RNA-seq-based transcriptome profiling and RT-qPCR analysis in ovaries demonstrated that the expression levels of genes involved in E2 production, lipid accumulation, and yolk formation were significantly downregulated following Sox3 mutation. Altogether, our findings indicate that Sox3 is required for oocyte growth in Nile tilapia and provides insights into transcriptional regulation underlying oogenesis in teleost fish.
Sujet(s)
Cichlides , Animaux , Femelle , Cichlides/génétique , Hybridation fluorescente in situ , Ovogenèse/génétique , Ovocytes/métabolisme , OvaireRÉSUMÉ
The bromodomain (BRD) proteins specifically recognize the N-acetyllysine motifs, which is a key event in the reading process of epigenetic marks. BRDs are evolutionarily highly conserved. Over recent years, BRDs attracted great interest because of their important roles in biological processes. However, the genome-wide identification of this family was not carried out in many animal groups, in particular, in teleosts. Moreover, the expression patterns were not reported for any of the members in this family, and the role of the BRD family was not extensively studied in fish reproduction. In this study, we identified 16 to 120 BRD genes in 24 representative species. BRDs expanded significantly in vertebrates. Phylogenetic analysis showed that the BRD family was divided into eight subfamilies (I-VIII). Transcriptome analysis showed that BRDs in Nile tilapia (Oreochromis niloticus) exhibited different expression patterns in different tissues, suggesting that these genes may play different roles in growth and development. Gonadal transcriptome analysis showed that most of the BRDs display sexually dimorphic expression in the gonads at 90 and 180 dah (days after hatching), including 21 testis-dominated genes (brdt, brd4a and brd2b, etc.), and nine ovary-dominated genes (brd3b, brd2a and kat2a, etc.). Consistent with transcriptomic data, the results of qRT-PCR and fluorescence in situ hybridization showed that brdt expression was higher in the testis than in the ovary, suggesting its critical role in the spermatogenesis of the tilapia. Male fish treated with JQ1 (BET subfamily inhibitor) displayed abnormal spermatogenesis. The numbers of germ cells were reduced, and the expression of steroidogenic enzyme genes was downregulated, while the expression of apoptosis-promoting genes was elevated in the testis tissue of treated fish. Our data provide insights into the evolution and expression of BRD genes, which is helpful for understanding their critical roles in sex differentiation and gonadal development in teleosts.