| Rainbow trout(Onchorynchus mykiss), belonging to the salmoniformes, salmonidae, Oncorhynchus, is one of the FAO recommended high quality fish to the world breeding objects, and is also the main introduced and cultured species in China. Due to its advantages of triploid female rainbow trout in aquaculture, such as gonad abortion, good muscle quality, it has become the general trend that the female triploid rainbow trout is developed as the main species in trout aquaculture.It is generally known that salmonidae fish is able to survive and reproduce even with a set or two sets of extra chromosomes, which does not exist in other vertebrates. The testes in tetraploid and triploid rainbow trout have the same structure and function as that of diploid. However, triploid female rainbow trout is an exception, since its ovarian is linear in early stage and lacks of primary oocyte. Therefore, the differences between sex differentiation and development mechanism in triploid female rainbow trout have become the focus of research. Although sex determination and differentiation related genes in diploid rainbow trout ha ve been identified, such as sd Y, Dmrt1, Cyp19a1 a, Foxl2 and Sox9, but the specific gonad phenotype and expression pattern of sex control genes in triploid female rainbow trout have not been studied thoroughly, especially the characteristics of germ cells differentiation in early stage ovarian due to retardation of development. The aim of this study was to reveal the mechanism of gonads abortion and natural sex reversal in triploid female rainbow trout. In order to obtain the results, the specific candidate genes which played an important regulatory role in gonad development in triploid female rainbow trout were researched as key objects. The histological evidence of somatic and germ cell differentiation and steroid hormone expression patterns were also determined. In addition, the gonad phenotypic development of triploid female has been controlled through the induction of exogenous hormones to verify the cascade regulation relationships among the genes and the interaction effects between sex cells reshape and the decline rules in sex steroid hormones. The main results were as follows:(1)The differentiation characteristics in ovary of diploid and triploid rainbow trout are basically identical. The diploid and triploid ovary differentiation began at 84 dpf and 98 dpf, respectively. But the ovary differentiation in triploid rainbow trout existed obstacle, which showed slow development in early stage and fewer oogonium and follicular cells. During the period of 154~574 dpf, large number of oocytes were in diploid ovary, while the oocytes were significantly decreased in triploid female rainbow trout ovarian. The ovary was linear, and a part of the female germ cells began to dedifferentiation during the later development. At 964 dpf, triploid female gonad began to reconstruction and redifferentiation. The sperm cell-like clusters appeared to distribute in gonads, which were slightly smaller than oogonium and similar to the germ cell in early stage of testis development in diploid trout.(2)During the period of 334~964 dpf, the contents of E2 and T in serum were gradually increasing in diploid rainbow trout. The E2 levels in serum of female rainbow trout were between 50~650 pg/ml, which was five to eight times higher than that of male. The T levels in serum of male rainbow trout were between 20~180 pg/ml, which was ten to twenty-five times higher than that of female. However, the E2 level increased at first and then decreased in triploid females with the peak value of 110 pg/ml(574 dpf), but always failed to reach the E2 level in diploid. The T level in triploid females gradually increased and almost reached the diploid male level in later development. With the development of gonad in triploid females, the oocyte degraded seriously, leading to blocked estrogen metabolic pathways. The amounts of T converting to E2 were reduced greatly when the testis sertoli cells appeared.(3)During the period of 154~964 dpf, RT-PCR was carried out to detect the differential expression of gonad specific genes in female triploid rainbow trout. And the diploid female and male rainbow trout were used as the control. The results indicated that the gene expression patterns of Cyp19a1 a, Foxl2, sdY, Dmrt1, Amh and Sox9 were obtained in the gonads of diploid and triploid rainbow trout. In triploid female gonad, Cyp19a1 a and Foxl2 expressions decreased after the first increase, which were significantly lower than in ovarian of diploids(P<0.05). The expression of Cyp19a1 a and Foxl2 were significantly up-regulated(P<0.05) only during the period of 154~574 dpf, but they are significantly higher than that in the whole growth period in the diploid testis(P<0.05). The expressions of sd Y and Dmrt1, Amh and Sox9 were continuously up-regulated, in triploid females showed a trend of decrease after the first increases, which were expressed significantly higher than in diploid during the period of 574~964 dpf(P<0.05). The expression of sdY and Dmrt1 was significantly lower than that in diploid testis in the early development(P<0.05), but reached maximum at 574 dpf. And the expression of sd Y was significantly higher than that of diploid male(P<0.05). Although Amh and Sox9 expressions in triploid female were more hundreds of times of diploid female in the period of 574 ~964 dpf, but their expressions were still significantly lower than that in diploid testis( P <0.05).(4)Triploid female rainbow trout had experienced the stagnation of the ovaries, oocyte degradation, germ cells dedifferentiate gonads restructuring and sex reversal. The E2 level in serum of triploid female rainbow trout was highest in the larval stage. During this stage, Cyp19a1 a and Foxl2 were continuously up-regulated, while the expression level of sd Y, Dmrt1, Amh and Sox9 were lower, and the T level was relatively lower. As the development into adulthood(784-964 dpf), estrogen alone had been unable to induce continuous high expression of Foxl2 and the expression of Cyp19 a also decreased sharply. During this period, sdY, Amh and Sox9 were continuously up-regulated, and sdY expression even reached the level of diploid male at the same tim, while Foxl2 and Cyp19a1 a expression were sharply decreased to tiny. The estradiol levels decreased at the same period, while testosterone levels increased continuously and even reached the levels of male diploid.(5)This study is the first design experiments on triploid female rainbow trout. The exogenous androgen and estrogen were used here at the usual dosage in rainbow trout aquaculture. ATD group successfully induced testis differentiation in triploid female rainbow trout and its development mode was consistent with diploid male. The testis in ATD group developed slowly but had no effect on the natural physiological response. MET treatment could not induce the same results as in diploid male-like processing, and MET genetic for female gonad treatment were also always at the lower level of differentiation, which seriously hampered the development of the gonads in any direction. Therefore, this study suggested that the dual role of ATD induction and gonad reconstruction triggered the sex reverse of female to male and the testis development. The estrogen treatment during the dedifferentiation and redifferentiation of gonad in female triploid rainbow trout could not effectively promote or maintain the ovary developmen t.(6)This study was aimed to understand the characteristics of the triploid female gonad differentiation of rainbow trout and those involved candidate gene expression profiles on the basis of the female-to-male biological model, to explore the effects of masculine treatment on the trace expression of genes in diploid when the genome is enlarged, and to verify the regulatory mechanism of gonad specific genes in the process of oocytes dedifferentiation and redifferentiation. The overall expression patterns showed that Cyp19a1 a and Foxl2 were all suppressed by ATD in the whole development process. These two typical female genes didn’t express effectively even in the late development. Because the female genes were down regulated or suppressed, the ovary was abortive and the oocytes dedifferentiated to be the primary germ cell. With the up-regulated expression of sd Y and Dmrt1, the primary germ cell was stimulated to redifferentiate into male germ cells and the testis development was activated under the synergy of Sox9 and Amh. During the triploid female differentiation process, MET could not inhibit the expression of Cyp19a1 a and Foxl2. In contrast, MET induced the up-regulated expression of these tow genes and also other male specific genes at the same time. The exogenous estradiol treatment destroyed the cascade control of Foxl2 and Cyp19a1 a and always inhibited the expression of Cyp19a1 a during the period of gonad dedifferentiation and redifferentiation in triploid female. Due to the degradation or disappear of the follicular layer, the absolute amounts of female genes decreased sharply, which led to the synthesis deficiency of endogenous estrogen. As cyp19a1 a expression was at a low level originally and the exogenous estrogen was severely suppressed, the dedifferentiation of the oocytes was aggravated. The exogenous estrogen even could promote the expression of male specific genes in the differentiation period. The results indicated that there was a feedback regulation relationship between estrogen and Foxl2 and Cyp19a1 a in the female differentiation pathway.To sum up, this study got the following conclusions:(1) The ovary differentiation of female triploid rainbow trout was activated by Cyp19a1 a and Foxl2 expression, and was obviously regulated by estrogen. In the gonadal abortive period, the combined regulation of sdY and Dmrt1 will lead to disorder expression of Cyp19a1 a and Foxl2 in the female pathway. The significant up-regulation of Sox9 and Amh will have an inhibition effect on the expression of sdY and Dmrt1 during the transition period.(2) The deficiency of endogenous estrogen in female triploid rainbow trout was the key factor to cause the failure of the ovary development.(3) The regulatory mechanism of rainbow trout feminization pathways was destroyed, leading to the decline of endogenous estrogen synthesis ability, which caused the failure of ovarian development and eventually the natural male-like reversal phenomenon.(4) Oocytes differentiation has inhibition effect on Cyp19a1 a expression and the synthesis of estradiol.(5) The aromatase inhibitors could destroy the differentiation of gonads somatic cell and germ cell, significantly up-regulate the expression of male specific genes and promote the female to male differentiation. |
PDF Full Text Request