Effect of interfering donkey Zfy gene in vivo on expression of spermatogenesis related genes

doi:10.6048/j.issn.1001-4330.2024.05.026

Xinjiang Agricultural Sciences ›› 2024, Vol. 61 ›› Issue (5): 1277-1283.DOI: 10.6048/j.issn.1001-4330.2024.05.026

• Prataculture·Animal Husbandry Veterinarian • Previous Articles Next Articles

Effect of interfering donkey Zfy gene in vivo on expression of spermatogenesis related genes

LI Mengyu¹(), ZHANG Zhidong², LYU Yihang¹, SUN Yujiang³, ZHENG Xinbao⁴^,⁵, XIAO Haixia⁴^,⁵, JIA Bin¹()

1. College of Animal Science and Technology, Shihezi University, Shihezi Xinjiang 832003, China
2. Ili Kazakh Aotonomous Prefecture Animal Husbandry Station, Yining Xinjiang 835100, China
3. Qingdao Agricultural University, Qingdao Shandong 266169, China
4. Institute of Animal Science, Xinjiang Academy of Animal Sciences, Urumqi 830000, China
5. Key Laboratory of Livestock and Poultry Resources (Sheep & Goat) Evaluation and Utilization, Ministry of Agriculture and Rural Affairs, Urumqi 830000, China

Received:2023-09-30 Online:2024-05-20 Published:2024-07-09
Correspondence author: JIA Bin
Supported by:
"Integration and Demonstration of Key Technologies for Efficient Donkey Breeding"(2020B01002-2-1);2019 Agricultural Major Application Technology Innovation Project of Shandong Province(SD2019XM008);Major Science and Technology Project of Xinjiang Uygur Autonomous Region "Herbivorous Livestock Efficient Breeding Technology Research and Demonstration"

体内干扰Zfy 基因对驴精子发育相关基因表达的影响

李梦雨¹(), 张志东², 吕毅航¹, 孙玉江³, 郑新宝⁴^,⁵, 肖海霞⁴^,⁵, 贾斌¹()

1.石河子大学动物科技学院,新疆石河子 832003
2.伊犁哈萨克自治州畜牧总站,新疆伊宁 835100
3.青岛农业大学,山东青岛 266169
4.新疆畜牧科学院畜牧研究所,乌鲁木齐 830000
5.农业农村部畜禽资源(羊)评价利用重点实验室,乌鲁木齐 830000

通讯作者: 贾斌
作者简介:李梦雨(1996-),女,新疆人,硕士研究生,研究方向为动物遗传育种,(E-mail)1132629130@qq.com
基金资助:
新疆维吾尔自治区重点研发计划项目“驴高效繁殖关键技术集成与示范”(2020B01002-2-1);山东省农业重大应用技术创新项目(SD2019XM008);新疆维吾尔自治区科技重大专项“草食家畜高效繁殖技术研究与示范”

Abstract

Abstract:

【Objective】To explore the effect of interfering with the Zfy gene in vivo on the expression of Zfy protein in donkey testis and its epididymis tissues, as well as the expression of genes related to sperm development.【Methods】Two healthy male donkeys were selected. An empty vector was injected into one testicle as the control group, and a Zfy interference vector was injected into the other testicle as the interference group. Testis and epididymis tissues were collected and processed into frozen sections, fixed in fixative solution, and stored in liquid nitrogen. The expression of green fluorescent protein in tissues was observed using frozen sections. The expression of Zfy protein in the interference and control groups was observed using immunohistochemistry. The impact of Zfy interference on the expression levels of sperm development-related genes SYCP3, STRA8, TNP2, and FAS in the testis and epididymis was detected using RT-qPCR.【Results】The experimental results showed that the green fluorescent protein was mainly expressed in round sperm cells and part of long sperm cells in convoluted spermatic tubules, indicating the successful transfer of the interference vector. The expression level of Zfy protein in the interference group was significantly down-regulated, and interference with Zfy gene led to a significant decrease in SYCP3 gene expression in the testis and a significant decrease in TNP2 and FAS gene expression in the epididymis. However, there was no significant difference in the expression level of the STRA8 gene between the testis and epididymis.【Conclusion】The Zfy gene in donkeys is mainly expressed in round sperm cells. Interference with the Zfy gene significantly inhibits the expression of Zfy protein, SYCP3 gene in the testis, and TNP2 and FAS gene expression in the epididymis.

Key words: donkey; gene interference; Zfy gene; immunohistochemistry; round sperm cell

摘要：

【目的】研究体内干扰Zfy基因对驴睾丸、附睾组织中Zfy蛋白表达以及精子发育相关基因表达的影响。【方法】选取健康种公驴,将一侧睾丸注射空载体作为对照组,另一侧睾丸注射Zfy干扰载体作为干扰组。采集睾丸、附睾组织,分别制作冰冻切片、固定液中固定及液氮中保存。使用冰冻切片观察载体绿色荧光蛋白在组织中的表达情况。使用免疫组化和HE染色观察干扰组和对照组Zfy蛋白表达情况。通过RT-qPCR检测干扰Zfy基因对睾丸、附睾中精子生成相关基因SYCP3、STRA8、TNP2、FAS表达水平的影响。【结果】绿色荧光蛋白主要在驴曲细精管中的圆形精子细胞和部分长形精子细胞中表达,干扰载体成功转入;干扰组驴睾丸Zfy蛋白表达量显著下调;干扰Zfy基因使驴睾丸中SYCP3基因表达量显著下调、附睾中TNP2和FAS基因表达量显著下调、而睾丸和附睾中STRA8基因表达量没有明显差异。【结论】驴Zfy基因主要在驴圆形精子中表达,干扰Zfy基因可显著抑制驴睾丸中Zfy蛋白表达、SYCP3基因表达和附睾中TNP2、FAS基因表达。

关键词: 驴, 基因干扰, Zfy基因, 免疫组化, 圆形精子细胞

CLC Number:

S822

LI Mengyu, ZHANG Zhidong, LYU Yihang, SUN Yujiang, ZHENG Xinbao, XIAO Haixia, JIA Bin. Effect of interfering donkey Zfy gene in vivo on expression of spermatogenesis related genes[J]. Xinjiang Agricultural Sciences, 2024, 61(5): 1277-1283.

李梦雨, 张志东, 吕毅航, 孙玉江, 郑新宝, 肖海霞, 贾斌. 体内干扰Zfy 基因对驴精子发育相关基因表达的影响[J]. 新疆农业科学, 2024, 61(5): 1277-1283.

Figures/Tables 5

Fig.1 Frozen section of asinus testis 48h after injected with RNAi vector Note:A,B is slices white light,C,D is a blue fluorescent photo unter the same field of view,E,F is a green fluorescent photo under the same field of view. All magnifications are 100 times

Fig.2 HE and HE of Equus asinus testis Note:A,C,E,G is the control group,B,O,F,H is experimental group,E,F,G,H is immunohistochemical group

Fig.3 The result of Zfy protein exoression in Equus asinus testis

Fig.4 The mRNA expression level of special genes in injected testis

Fig.5 The mRNA expression level of special genes in epididymis

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Effect of interfering donkey Zfy gene in vivo on expression of spermatogenesis related genes

体内干扰Zfy 基因对驴精子发育相关基因表达的影响

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