利用鱼类性逆转技术创制黄颡鱼XX雄鱼的方法

杨天毅; 熊阳; 丹成; 郭稳杰; 刘汉勤; 桂建芳; 梅洁

doi:10.7541/2018.107

利用鱼类性逆转技术创制黄颡鱼XX雄鱼的方法

杨天毅¹,
熊阳¹,
丹成¹,
郭稳杰¹,
刘汉勤²,
桂建芳^1,3,
梅洁^1, ,

1.
华中农业大学水产学院，武汉 430070
2.
武汉百瑞生物技术有限公司，武汉 430070
3.
中国科学院水生生物研究所，淡水生态与生物技术国家重点实验室，武汉 430072

基金项目: 国家现代农业产业技术体系特色淡水鱼体系项目(CARS-46)资助

详细信息

作者简介:
杨天毅(1991—)，男，土家族，湖北恩施人；硕士研究生；主要从事鱼类遗传育种研究。E-mail： 229001655@qq.com

通信作者:
梅洁，博士，教授；E-mail： jmei@mail.hzau.edu.cn

中图分类号: S962.1
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出版历程
- 收稿日期: 2018-01-07
- 修回日期: 2018-05-31
- 网络出版日期: 2018-07-16
- 发布日期: 2018-08-31

PRODUCTION OF XX MALE YELLOW CATFISH BY SEX-REVERSAL TECHNOLOGY

1.
College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China
2.
Wuhan Bairui Biological Technology Co. Ltd, Wuhan 430070, China
3.
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China

Funds: Supported by the China Agriculture Research System (CARS-46)

Corresponding author:

摘要

摘要: 目前黄颡鱼(Pelteobagrus fulvidraco Richardson)生产上使用的母本非常混杂, 成为影响黄颡鱼产业发展的一个突出问题。建立一个优良性状稳定的全雌家系对于全雄黄颡鱼和杂交黄颡鱼的生产尤为重要, 而将XX雌性黄颡鱼逆转为XX雄性黄颡鱼是创制全雌家系中的关键一步。黄颡鱼性染色体连锁分子标记的开发为鉴定XX雄鱼提供了技术支撑。研究使用不同浓度的17α-甲基睾酮(MT)和芳香化酶抑制剂来曲唑(LZ)处理黄颡鱼鱼苗54d (7—60日龄)。61日龄测量并统计各组鱼的存活率、体长及体重。解剖观察性腺结构, 结合性腺组织切片和性别分子标记分析各组实验鱼中XX鱼性腺发育情况。结果显示: 2种药物对黄颡鱼的存活率影响较小, 与对照组没有显著差异; MT处理的XX性腺为空腔状精小囊结构, 不能逆转为功能性精巢; 适当剂量的LZ可以将XX雌性黄颡鱼性逆转成正常的XX雄鱼, 诱导效果随着剂量的增加而增强。用完全逆转的XX生理雄鱼分别与XX雌鱼和YY雌鱼交配, 能够正常繁殖并具备较好的繁殖能力。研究建立了一种使用芳香化酶抑制剂来曲唑创制黄颡鱼XX雄鱼的方法, 为全雄黄颡鱼的品种改良及新品种的培育奠定了基础。
- 黄颡鱼 /
- 甲基睾丸酮 /
- 芳香化物酶抑制剂 /
- 性逆转 /
- XX雄鱼
Abstract: One of the serious problems in aqua-cultural yellow catfish is that the background of female parents is confused. It is particularly important to establish all-female families for the all-male and hybrid yellow catfish production, in which sex-reversal of XX female to XX male is a key step. Development of sex chromosome-linked markers provides a technical support for the identification of XX males in yellow catfish. In this study, different concentrations of 17α-methyltestosterone (MT) and aromatase inhibitor letrozole (LZ) were used for 54 days to treat yellow catfish (7 to 60 days post fertilization). The survival rate, body length, and body weight of each group were measured at 61 days after fertilization (61 days post fertilization). Gonadal structure was observed and histologically analyzed in XX individuals that were identified by sex-linked markers. As a result, MT and LZ treatments had no significant effects on the survival rate when compared with the control group. In addition, the XX gonad treated by MT showed seminal vesicles and could not be reversed into functional testis. However, appropriate dose of LZ could induce the sex-reversal of XX female to normal XX male, and the induction effect was enhanced with the increase of dose as well. The XX male could normally mate with XX female and YY female, which demonstrated appropriate reproductive ability. The present study proposed a reliable method to create XX male using the aromatase inhibitor LZ, which lays the foundation for the breed improvement of all-male yellow catfish and cultivation of new varieties of yellow catfish.
- Yellow catfish /
- 17α-Methyltestosterone /
- Aromatase inhibitors /
- Sex-reversal /
- XX male

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图 1 利用PCR反应鉴定黄颡鱼遗传性别的示意图

Figure 1. The genetic sex of yellow catfish was identified by polymerase chain reaction (PCR) method with the sex-linked markers

下载: 全尺寸图片幻灯片

图 2 61日龄对照组正常卵巢和精巢结构

A. Bouin’s液固定的XX卵巢形态; B. XX卵巢切片HE染色图片; C. Bouin’s液固定的XY精巢形态; D. XY精巢切片HE染色图片;NO: 正常卵巢; RH: 生殖孔; PO: 初级卵母细胞; NT: 正常精巢; MG: 生殖突; SG: 精原细胞; SC: 精母细胞

Figure 2. The phenotype and histology of normal ovary and testis at 61 days post fertilization in the control group

A. The XX ovary fixed by Bouin’s solution; B. Histological analysis of XX ovary; C. The XY testis fixed by Bouin’s solution; D. Histological analysis of XY testis; NO: normal ovary; RH: reproduction hole; PO: primary oocyte; NT: normal testis; MG: male genital swelling; SG: spermatogonium; SC: spermatocyte

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图 3 61日龄MT处理组XX黄颡鱼性腺结构

A. Bouin’s液固定的MT₂₅组XX不良精巢形态; B. MT₂₅组XX不良精巢切片HE染色图片; C. Bouin’s液固定的MT₅₀组XX不良精巢形态; D. MT₅₀组XX不良精巢切片HE染色图片; DT: 不良精巢; SV: 精小囊

Figure 3. Dysplastic testis after MT treatment_{at 61 days post fertilization}

A. Dysplastic testis of MT₂₅ group fixed by Bouin’s solution; B. Histological analysis of dysplastic testis of MT₂₅ group; C. Dysplastic testis of MT₅₀ group fixed by Bouin’s solution; D. Histological analysis of dysplastic testis of MT₅₀ group; DT: dysplastic testis; SV: seminal vesicle

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图 4 61日龄LZ处理组XX黄颡鱼性腺结构

A-B. XX不良精巢(无精小叶)的Bouin’s液固定和切片HE染色图片; C-E. XX不良精巢(部分精小叶)的Bouin’s液固定和切片HE染色图片; D. 无精小叶部位的切片; E. 有精小叶部位的切片; F-G. XX精巢(完整精小叶)的Bouin’s液固定和切片HE染色图片; SG: 精原细胞; SC: 精母细胞; SZ: 精子; MG: 生殖突; SV: 精小囊

Figure 4. The phenotype and histology of XX gonad after LZ treatment at 61 days post fertilization

A-B. The phenotype and histology of dysplastic testis without seminiferous lobule; C-E. The phenotype and histology of dysplastic testis with part of seminiferous lobule; F-G. The phenotype and histology of XX normal testis; SG: spermatogonium; SC: spermatocyte; SZ: spermatozoon; SV: seminal vesicle; MG: male genital swelling

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图 5 一龄XX生理雄鱼性腺结构

A. 对照组XY精巢; B. 对照组XY精巢切片; C. XX生理雄鱼精巢; D. XX生理雄鱼精巢切片; NT: 正常精巢; SZ: 精子

Figure 5. Gonad structure of one-year-old XX physiological male

A. XY testis of the control group; B. Histological analysis of the XY testis in the control group; C. Testis of XX physiological male; D. Histological analysis of the XX physiological male testis; NT: normal testis; SZ: spermatozoon

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图 6 子代性别分子鉴定

Figure 6. The sex genotype of offspring was identified by PCR method with the sex-linked markers

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表 1 61日龄时不同浓度MT和LZ处理组的存活率、体长及体重统计

Table 1 Effects of various doses of MT and LZ on survival rate, total length, and weight at 61 days post fertilization (n=30)

实验组 Experimental group	存活率 Survival (%)	体长Total length (cm)	体重 Weight (g)
0 (Control)	97.10±0.91	5.89±0.23^a	3.71±0.39^a
MT₂₅	94.26±0.87	4.32±0.09^b	1.57±0.05^c
MT₅₀	95.05±2.41	4.34±0.12^b	1.54±0.13^c
LZ₁₀₀	95.06±2.40	5.08±0.17^ab	2.30±0.24^bc
LZ₃₀₀	96.36±1.05	5.58±0.67^a	3.14±1.10^ab
LZ₁₀₀₀	94.50±0.91	5.21±0.03^a	2.64±0.29^abc
注: 表中同列数据中不同肩注字母表示有显著差异(P<0.05), 相同字母或无字母表示差异不显著(P>0.05); 下同 Note: In the same line, different letter superscripts mean significant differences (P<0.05), and the same letter superscripts or no letter superscripts mean no significant differences (P>0.05); the same applies below

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表 2 61日龄不同浓度MT和LZ处理组的XX基因型黄颡鱼的性腺结构统计

Table 2 The effects of various doses of MT and LZ on the gonad of XX yellow catfish at 61 days post fertilization (n=15)

实验组 Experi-mental group	XX与XY 性别比 Sex ratio of XX to XY	不良精巢 (无精小叶) Dysplastic testis without seminiferous lobule (%)	不良精巢 (部分精小叶) Dysplastic testis with part of seminiferous lobule (%)	精巢(完整精小叶) Normal testis with full of seminiferous lobule (%)
0 (Control)	0.67±0.73	0.00±0.00^b	0.00±0.00^b	0.00±0.00^b
MT₂₅	0.84±0.32	100.00±0.00^a	0.00±0.00^b	0.00±0.00^b
MT₅₀	1.05±0.51	100.00±0.00^a	0.00±0.00^b	0.00±0.00^b
LZ₁₀₀	1.33±0.76	94.47±4.86^a	5.57±5.32^b	0.00±0.00^b
LZ₃₀₀	0.79±0.62	25.00±26.63^b	55.00±3.61^a	20.00±18.02^ab
LZ₁₀₀₀	0.72±0.25	0.00±0.00^b	64.30±8.28^a	35.70±5.57^a

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表 3 XX生理雄鱼受精率统计

Table 3 The fertilization rate of XX physiological male

实验组 Experimentalgroup	受精率 Fertilization rate (%) XX♀	受精率 Fertilization rate (%) YY♀
Ctr-XY♂	0.91±0.02^a	0.88±0.03^a
LZ-XX♂	0.80±0.05^b	0.77±0.04^b

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利用鱼类性逆转技术创制黄颡鱼XX雄鱼的方法

作者简介: 杨天毅(1991—)，男，土家族，湖北恩施人；硕士研究生；主要从事鱼类遗传育种研究。E-mail： 229001655@qq.com

通信作者: 梅洁，博士，教授；E-mail： jmei@mail.hzau.edu.cn

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PRODUCTION OF XX MALE YELLOW CATFISH BY SEX-REVERSAL TECHNOLOGY

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作者简介:
杨天毅(1991—)，男，土家族，湖北恩施人；硕士研究生；主要从事鱼类遗传育种研究。E-mail： 229001655@qq.com

通信作者:
梅洁，博士，教授；E-mail： jmei@mail.hzau.edu.cn