RESUMO
Retinoid X receptors (RXRs) can form homo- or heterodimers with orphan receptors involved in multiple intertwined signaling pathways. However, there is limited study on the formation of sex phenotypes and the regulation of steroidogenesis by RXRs in fish. Here, in Paralichthys olivaceus, we first indicated that PPARγ::RXRα was predictably a transcription factor for steroidogenesis genes, and Foxl2 and Dmrt1 were also transcription factors for rxrs and their partner receptor genes. When the flounder fry were exposed to LG100268 (LG, RXRs agonist, 50 mg/kg diet), the percentage of males increased from 50% to 71.4%. Before histological differentiation of the flounder ovary (3 cm TL) and testis (6 cm TL), the transcripts of rar ß and rar γ (P < 0.05) were activated, and the steroidogenesis gene Hsd3b1 was down-regulated (P < 0.05). The ratios of testosterone (T)/17ß-estradiol (E2) were all greatly increased (P < 0.05), and the ratio of 11-ketotestosterone (11-KT)/E2 was elevated at 3 cm TL. Moreover, LG was used to treat the cultured gonads in vitro (10 µM) and the fish with intraperitoneal injection in vivo (12 mg/kg body weight), respectively. LG was able to up-regulate rxr γ, rar γ, and ppar δ, and Hsd3b1 was significantly up-regulated (P < 0.05). The ratios of 11-KT/E2 in the culture medium and in the ovaries of the fish were decreased. Furthermore, the recombinant flounder Foxl2 protein was able to significantly down-regulate ppar γ (P < 0.05) and tr ß (P < 0.01) in the ovaries in vitro, and the result of the Dmrt1 in the testes was opposite to that of the Foxl2, probably indicating a feedback loop between RXRs' partner receptors and Foxl2/Dmrt1. These findings introduce for the first time the mode of action of RXRs on the flounder steroidogenesis and provide important data to learn the potential function of RXRs in fish sex differentiation and the potential role of RXRs in aquatic animals in the presence of water pollutants.
Assuntos
Linguado , Masculino , Animais , Feminino , Receptores X de Retinoides/genética , Linguado/genética , Regulação da Expressão Gênica no Desenvolvimento , Gônadas/metabolismo , Ovário/metabolismoRESUMO
As a promising biotechnology, fish germ cell transplantation shows potentials in conservation germplasm resource, propagation of elite species, and generation of transgenic individuals. In this study, we successfully transplanted the Japanese flounder (P. olivaceus), summer flounder (P. dentatus), and turbot (S. maximus) spermatogonia into triploid Japanese flounder larvae, and achieved high transplantation efficiency of 100%, 75-95% and 33-50% by fluorescence tracking and molecular analysis, respectively. Eventually, donor-derived spermatozoa produced offspring by artificial insemination. We only found male and intersex chimeras in inter-family transplantations, while male and female chimeras in both intra-species and intra-genus transplantations. Moreover, the intersex chimeras could mature and produce turbot functional spermatozoa. We firstly realized inter-family transplantation in marine fish species. These results demonstrated successful spermatogonial stem cells transplantation within Pleuronectiformes, suggesting the germ cells migration, incorporation and maturation within order were conserved across a wide range of teleost species.
Assuntos
Linguados/fisiologia , Espermatogônias/fisiologia , Transplante de Células-Tronco/veterinária , Animais , Movimento Celular , Proliferação de Células , Marcadores Genéticos , Masculino , Poliploidia , Processos de Determinação Sexual , Especificidade da Espécie , Transplante de Células-Tronco/métodosRESUMO
Dynein axonemal light intermediate chain 1 (dnali1) is an important part of axonemal dyneins and plays an important role in the growth and development of animals. However, there is little information about dnali1 in fish. Herein, we cloned dnali1 gene from the genome of olive flounder (Paralichthys olivaceus), a commercially important maricultured fish in China, Japan, and Korea, and analyzed its expression patterns in different gender fish. The flounder dnali1 DNA sequence contained a 771 bp open reading frame (ORF), two different sizes of 5' untranslated region (5'UTR), and a 1499 bp 3' untranslated region (3'UTR). Two duplicated 922 nt fragments were found in dnali1 mRNA. The first fragment contained the downstream coding region and the front portion of 3'UTR, and the second fragment was entirely located in 3'UTR. Multiple alignments indicated that the flounder Dnali1 protein contained the putative conserved coiled-coil domain. Its expression showed sexually dimorphic with predominant expression in the flounder testis, and lower expression in other tissues. The gene with the longer 5'UTR was specifically expressed in the testis. The highest expression level in the testis was detected at stages IV and V. Transient expression analysis showed that the 922 bp repeated sequence 3'UTR of dnali1 down-regulated the expression of GFP at the early stage in zebrafish. The flounder dnali1 might play an important role in the testis, especially in the period of spermatogenesis, and the 5'UTR and the repetitive sequences in 3'UTR might contain some regulatory elements for the cilia.
Assuntos
Dineínas/genética , Proteínas de Peixes/genética , Linguado/metabolismo , Testículo/metabolismo , Regiões 3' não Traduzidas , Animais , Sequência Conservada , Dineínas/química , Dineínas/metabolismo , Feminino , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Linguado/genética , Masculino , Fases de Leitura Aberta , Domínios Proteicos , Caracteres SexuaisRESUMO
Olive flounder (Paralichthys olivaceus) is a commercially important flatfish species cultured in East Asia. Female flounders generally grow more rapidly than males, therefore control of the sex ratio seems to be a proposed way to increase production. However, the sex determination gene and sex determination mechanism have yet been elucidated. The brain is an important organ that is involved in gonadal development. To explore the sex differences of gene expression in the brain before and during the flounder gonadal differentiation, we used messenger RNA (mRNA)-seq technology to investigate transcriptomes of male and female brains. Between female and male brains, 103 genes were differentially expressed before ovarian differentiation, 16 genes were differentially expressed before testicular differentiation, and 64 genes were differentially expressed during gonadal differentiation. According to annotation and Kyoto Encyclopedia of Genes and Genomes information, the differentially expressed genes (DEGs) were involved in circadian rhythm, circadian rhythm-fly, circadian entrainment, dopaminergic synapse, calcium signaling, glutamatergic synapse, taste transduction, herpes simplex infection, long-term depression, retrograde endocannabinoid signaling, and the synaptic vesicle cycle pathways. MicroRNA (miRNA)-seq was performed during the gonadal differentiation and the target genes of miRNAs were predicted. Integrated analysis of mRNA-seq and miRNA-seq showed that 29 of the 64 DEGs were regulated by the differentially expressed miRNAs during the gonadal differentiation. Our study provides a basis for further studies of brain sex differentiation and the molecular mechanism of sex determination in olive flounder.
Assuntos
Encéfalo/metabolismo , Estradiol/farmacologia , Linguado/crescimento & desenvolvimento , Linguado/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Gônadas/crescimento & desenvolvimento , Metiltestosterona/farmacologia , Caracteres Sexuais , Diferenciação Sexual/efeitos dos fármacos , Animais , Sequência de Bases , Feminino , Masculino , MicroRNAs/genética , RNA Mensageiro/genética , RNA-Seq/métodos , TranscriptomaRESUMO
Germ cells undergo a series of cellular changes including differentiation, mitosis, meiosis and maturation, and eventually develop into a large number of functional gametes. The available data regarding teleost gametogenesis in seasonal batch spawners is limited. In this study, we investigated spermatogenesis with special attention on spermatogonia differentiation using heat-induced masculine juveniles of genetically female Japanese flounder. Meanwhile, the Nanos2 expression had been detected by immunohistochemistry for analysis of spermatogonial stem cells (SSCs) distribution. Spermatogonia began mitosis at 35 dph, and basement membrane firstly appeared and gradually surrounded and separated the spermatogonia (type A) into single and paired status. At this period, the spermatogonia continuously maintained mitotic proliferation. As a result, the number of spermatogonia including isolated and clusters (2-8 spermatogonia) significantly increased in the presumptive testes. From 85 dph to 120 dph, with the mitosis of spermatogonia, germline acinar-clusters formed. In the clusters, type A spermatogonia differentiated into type B, and multi-spermatogonia surrounded by several sertoli cell formed cystsï¼which represented the formation of lobular precursors. After that, type B spermatogonia began meiosis, which indicated the initiation of spermatogenesis. In adult testes, most type A spermatogonia distributed in the peripheral region and a few clung to a basement membrane in the internal germinal epithelium. Various spermatogenic cysts with germ cells in different developmental stages existed in a testicular lobules, moreover the germ cells in earlier stages resided in the distal termini, and the advanced stages were adjacent to the spermatic duct of testes. Therefore, the testes of Japanese flounder belonged to an intermediated distribution of SSCs, which might contribute greatly to multiple spermiation during breeding season. These findings would improve the understanding the mechanisms of SSCs differentiation and testicular development, and may be of great value in future studies of the spermatogenesis regulation.