| Japanese flounder, Paralichthys olivaceus, is popular among consumers in China because of growth fast and delicate meat quality, and it is regarded as one of the most economically important marine species. The female flounder grows significantly faster and bigger than the male one. Breeding and culturing all-female population of flounder is a promising approach to boost production and improve the farmer’s economic benefits. Mitotic gynogenesis is an effective technique to produce not only all-female flounder, but also all-homozygous diploids fish in just one generation. All-homozygous diploids, namely doubled haploid(DH) can be used to generate clonal lines by a second round of gynogenesis. DH and clonal fish have important significance for theory research and practical application for gynogenesis breeding. However, using DH fish as material has been affected due to the low induced ratio and reproductive disorder which can not ovulate during the breeding season. At present, only a few studies which mainly concentrated in the describing phenotype of reproductive capacity have been done, but the specific reasons about DH sterility are not clear. Therefore, this study attempted to induce DH flounder through improving homozygosity of parent by meiotic gynogenesis. At the same time, in order to revealing the cause of sterile DH flounder from the angle of endocrinology, we determined the gonadotropin and sex steroid hormone levels of sterile DH flounder and fertile flounder. Furthermore, proteomic and transcriptome were used to identify the important genes and proteins involved in gonadal development. This can help to explore the molecular mechanism of homozygous sterility in DH flounder, and provide useful information for finding the key genes and proteins and study its function. The main results were as follows:(1) To determine the relations between the induction efficiency of DH and parental homozygosity, 6 meiotic gynogenetic females(G1-G6) which experienced once meiotic gynogenesis and 6 common females(C1-C6) were selected for production mitotic gynogenesis in our experiments. The results found that the homozygosity of gynogenesis fish was very significant higher than common fish in that they have more homozygous loci(P < 0.01). Homozygosity of gynogenesis parental fishes was 0.70 that was significant higher than the common fishes. There are five lines(C1, G1, G2, G4, G5) produced all the homozygotic fishes, two lines(G3, G6) produced 90% homozygotes and 10% heterozygotes. It was not expected that the G3 and G6 lines produced the heterozygotes other than C1, this suggest that the homozygosity is not the promise of full homozygotes in progenies. For induction rates of mitotic gynogenesis, the individuals with higher homozygosity have the higher induction rate,the induction rate of gynogenesis parental fishes reaches 34.86% CHR(converted hatching rate) and 19.18% CNR(converted normality rate). Positive significant correlations between CHR, CNR and parent’s homozygosity were found, the Pearson’s correlation value were 0.920(P = 0.000 < 0.01) and 0.925(P = 0.000 < 0.01) respectively. Generally, both the CHR and CNR of meiotic gynogenesis are significantly higher than mitotic gynogenesis.(2) Dysgenesis is a universal phenomenon in DH fish. With the purpose of revealing DH sterile mechanism from the perspective of endocrinology, the changes of several hormone levels were determined by enzyme linked immunosorbent assay and the gonad development was observed by histological section. The results showed that the accumulated amounts of Luteinizing hormone(LH), follicle stimulating hormone(FSH) and estradiol(E2) for DH fishes were significantly lower than the controls. However, the contents of testosterone(T) and 17, 20β-dihydroxy-4-pregnen-3-one(17α, 20β-DHP) in DH were strikingly higher than controls. In histology, the volume of gonads for DH fishes is smaller and the gonadosomatic index(4.0%) was significantly lower in comparison with controls(10.1%), the oocytes developed mostly at the stage II and stage III corresponds with the staged IV and staged V oocytes for controls. The above results imply that the abnormal hormone levels in DH fishes may lead to the abnormalities of the gonad in development.(3) To obtain a better understanding of the molecular mechanisms of female sterility, Proteome was utilized to compare the proteins differentially expressed in gonad of fertile and sterile Japanese flounder DH fishes. We screened the differential expressed proteins between fertile and infertile DH Japanese flounder, overall 42 proteins were identified out of 1600 validated reproducible protein spots by two-dimensional gel electrophoresis and MALDI-TOF MS. Comparing to fertile DH samples, 11 proteins exhibited a higher abundance in sterile fishes, whereas 29 proteins showed a lower abundance, 2 proteins were solely expressed in fertile fishes but not expressed in sterile fishes. Among these candidate proteins, Vitellogenin 1(Vtg1), Scaffold attachment factor B2(SAFB2), Nucleobindin 1(NUCB1) are down-regulated proteins in gonads of infertile fishes compared to fertile controls. In addition, several calcium-binding proteins such as EF-hand calcium-binding domain-containing protein 3(EFCAB3), EF-hand calcium-binding domain-containing protein 4B(EFCAB4B), Basonuclin 2(BNC2) are also found to be down-regulated in infertile fishes. These candidate proteins will provide us an important clue for further analysis of the molecular mechanisms of homozygotic infertility.(4) RNA-Seq was used to analyze the differential expression of genes between the fertile female DH Japanese flounder and sterile ones. The aim is to screen for major genes that result in sterility in DH and provide a molecular basis for the intensive study of the gonad development in teleost. The Japanese flounder transcriptome was generated from fertile and sterile DH fish in same family using the Illumina sequencing platform. After sequencing, assembly and annotation,we obtained 52474 contigs of which 60.7 % shared homologies with existing sequences. 1225 differentially expressed unigenes were discovered including 492 up-regulated unigenes and 733 down-regulated unigenes. The Gene Ontology and KEGG analyses showed that the significantly different up-regulated genes, such as CYP11A1, CYP11B2, CYP17A1, CYP21A1, HSD3β and PRLR, were principal correlated with sterol metabolic process, steroid biosynthetic process, and C21-steroid hormone biosynthetic. The significantly different down-regulated genes were primary associated with immune response, vitamin D metabolic process, antigen processing and presentation, hemoglobin import, renal absorption, transcytosis, lipoprotein transport, proteolysis and so on. Using deep sequencing technology, we conducted a comprehensive search of gene expressed in fertile and sterile gonads of female DH Japanese flounder. These significantly different expression genes will provide further insights not only into DH reproductive dysfunction, but also the oogenesis in Japanese flounder and molecular mechanism of piscine oocytes mature process.In conclusion, it may be a feasible method to obtain the DH genetic materials using the meiotic gynogenesis as parental fish in flounder. We can see that some important genes and proteins involved in regulating the process of sexual sterol hormones were low expressed levers in sterile DH flounder, such as CYP11A1 CYP11B2, CYP17A1, CYP21A1 and SAFB2, we also found that LH, FSH and E2 which play important roles in gonad development were significantly lower than fertile DH flounder in the breeding season. This may be the primary cause that gonads can not develop normally and DH fish oocyte can not mature. |
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