Studies On The Mechanism Of Feminization Of Southern Catfish, Silurus Meridionalis Chen

Southern catfish (Silurus meridionalis, Chen), which is endemic to China and widely distributed in the Yangtze River basin, is an important economic fish in China.The fries obtained by artificial propagation were all female, while the sex ratio of the feral catfish was1:1. Previous studies from our group have demonstrated that the water, photoperiodicity, rearing temperature after hatching and artificial insemination were not the reason for the feminization of Southern catfish. But the effects of other environmental factors such as hatching temperature, rearing density and feed to Southern catfish have not been reported. Studies of some key genes involved in sex determination and differentiation of Southern catfish, such as Dmrtl, Foxl2, Sfl and Cypl9ala, shown that these genes play important roles in the Southern catfish gonad differentiation of, and feminization was probably caused by high estrogen levels in Southern catfish. Whether the expression of these genes mentioned above had changed during the feminizing of Southern catfish was not well studied. The role of transcription factors in controlling the estrogen level by regulating the Cypl9a1a expression had not been demonstrated. For over ten years, the reason for feminization of Southern catfish had not been elucidated, which is not only a serious impediment to study the sex determination and differentiation of Southern catfish, but also affects the development of the Southern catfish aquaculture.In order to elucidate this phenomenon, three expriments were set as follows:1. Hatching temperatures:16℃,19℃and22℃were set for the hatching of fertilized eggs.2. Feeding density:The fries were randomly divided into two groups for feeding individually and in groups, respectively.3. Feed:The fries were randomly divided into two groups. One of the groups was fed with tubificidae during the fisrt two months after hatching while the other always fed with artificial diet.Gonads of90and120dah (days after hatching) Southern catfish were dissected and genders of these fish were preliminarily judged by the gonad shape. The gonads were fixed in Bouin’s solution respectively and then embeded in paraffin and sectioned at5-μm thickness. Statistical analysis of sex ratio was performed by microscopic observations of the sections. According to the previous studies on the morphology and microstructure of Southern catfish, the gender of gonads was identified. The ovaries were light-flesh-colored and translucent. Histological observation showed that the cross-section of ovary was ellipse, and the size of the oocytes varied, cytoplasm basophilic, nuclear staining was not obvious. Looking from the exterior, the testies was in a flat, translucent, long and narrow shape; Microscopic observation showed that the testis was strip-shaped with a large number of spermatogonia in the ampulla at this time. It was found that the Southern catfish which fed with tubificidae were all females, while the sex ratios in all other treatment groups were about1:1. The feminization of the Southern catfish by artificial propagation may be attributed to the feeding of tubificidae during the early development stage.Expression of the Dmrtl (two alternatively spliced variants Dmrt1a and Dmrt1b), Sfl, Foxl2and Cyp19a1a mentioned above in the feminization process of the Southern catfish was examined by Real-time PCR. The results showed that, in contrast to Foxl2and Cypl9a1a, the expression levels of Dmrt1a and Dmrt1b in the20dah gonads of Southern catfish fed with tubificidae were higher than that fed with artificial feed, while Sfl expression level showed no significant differences. In the90dah gonads, the expression levels of Dmrt1a, Dmrt1b and Sfl in testis were higher than those in ovary, while the expression levels of Foxl2and Cypl9a1a in testis were lower than those in ovary.These results indicated that the expressions of transcription factors and Cypl9a1a in Southern catfish were greatly affected by feeding with tubificidae. It was concluded that the increased expression level of Cypl9a1a and in turn the high level of estrogen resulted in the feminization of Southern catfish.To study the transcriptional regulation of Cyp19a1a, the1.5kb promoter obtained by Genome walking was subcloned into the pGL3-Basic vector. Promoter analysis using HEK293cells revealed that Cypl9a1a transcription was activated by Sfl and Dmrt1b in a dose-dependent manner (10-250ng) when used alone. Suppressed luciferase activity was observed in the promoter assays when Dmrtla was used alone. In addition, the luciferase assays also showed that Sfl-activated Cypl9ala promoter activity was further enhanced by Dmrt1b, while suppressed by Dmrt1a when cotransfected with Sfl, Therefore, it was speculated that transcription factors Dmrt1a、 Dmrtlb and Sfl play an essential role in controlling the estrogen level by regulating the Cyp19ala expression in Southern catfish. Based on the results of feed experiment, environmental estrogens may increase the endogenous estrogen level by affecting the transcription factors and the transcriptions of Cypl9a1a, and in turn lead to the feminization of Southern catfish.In addition, the XY Nile tilapia (Oreochromis niloticus) fries were fed with tubificidae. It was found that both the gonads of treatment and control group developed normally. We speculate that sex reversal in Nile tilapia can not be induced by the concentrations of environmental estrogens in tubificidae.In summary, our study shows that the feminization of Southern catfish by artificial propagation is caused by tubificidae. Environmental estrogens derived from tubificidae may increase the endogenous estrogen level, and then lead to the feminization of Southern catfish.