动物营养学报 >

2022 , Vol. 34 >Issue 5: 3286 - 3296

DOI: https://doi.org/10.3969/j.issn.1006-267x.2022.05.054

研究论文 RESEARCH PAPER

α-酮戊二酸/酮戊二酸受体1系统对雄性动物睾酮分泌和精子生成的影响

  • 徐畅 ,
  • 张姹 ,
  • 谭成全 ,
  • 习欠云 ,
  • 王声会 ,
  • 江青艳 ,
  • 束刚
展开
  • 1. 华南农业大学动物科学学院, 农业部华南动物营养与饲料科学观测实验站, 广东省动物营养调控重点实验室, 广州 510642;
    2. 广东温氏种猪科技有限公司, 云浮 527400
徐畅(1996—),男,安徽马鞍山人,硕士研究生,从事动物营养调控研究。E-mail:18855034725@163.com

收稿日期: 2021-10-24

  网络出版日期: 2022-05-14

基金资助

广东省特支计划本土创新创业团队（青年）（2019BT02N630）；广东省重点领域研发计划项目（2019B020218001）；京基智农横向课题

收起

Effects of α-Ketoglutaric Acid/Oxoglutarate Receptor 1 System on Testosterone Secretion and Spermatogenesis of Male Animals

  • XU Chang ,
  • ZHANG Cha ,
  • TAN Chengquan ,
  • XI Qianyun ,
  • WANG Shenghui ,
  • JIANG Qingyan ,
  • SHU Gang
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  • 1. Guangdong Provincial Key Laboratory of Animal Nutrition Control, Scientific Observing and Experimental Station of Animal Nutrition and Feed Science in South China, Ministry of Agriculture, College of Animal Science, South China Agricultural University, Guangzhou 510642, China;
    2. Guangdong Wenshi Breeding Swine Technology Co., Ltd., Yunfu 527400, China

Received date: 2021-10-24

  Online published: 2022-05-14

Supported by

 

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摘要

本试验旨在研究α-酮戊二酸（AKG）/酮戊二酸受体1（OXGR1）系统对雄性动物睾酮分泌和精子生成的影响。首先采用免疫荧光检测OXGR1在睾丸组织中的定位。然后以酮戊二酸受体1敲除（OXGR1 knockout，OXGR1KO）小鼠为模型，采用酶联免疫吸附剂测定（ELISA）法检测血清睾酮含量，运用苏木精-伊红（HE）染色分析睾丸曲细精管空腔面积、睾丸组织形态差异，采用实时荧光定量PCR检测睾丸生精标志基因mRNA表达水平。在急性试验中，选取16只10周龄雄性小鼠，按体重随机分成4组，分别注射0、1、5、10 mg/kg AKG 6 h后检测血清睾酮含量和小鼠攻击性，再选取10周龄雄性小鼠和OXGR1KO小鼠各12只，并按体重随机分成4组，每组注射5 mg/kg AKG 6 h后检测血清睾酮含量。在离体试验中，用不同浓度AKG处理睾丸间质细胞（TM3细胞）24 h后，检测TM3细胞上清液睾酮含量；并使用200 μmol/L AKG处理TM3细胞检测细胞内瞬时钙离子浓度。在长期试验中，选取8周龄雄性小鼠和OXGR1KO小鼠各12只，并按体重随机分成4组，分别为WT组、WT+2% AKG组、OXGR1KO组和OXGR1KO+2% AKG组，试验28 d后检测小鼠血清睾酮含量和睾丸曲细精管空腔面积。结果表明：1） OXGR1特异性分布在睾丸间质细胞。2） OXGR1KO可显著或极显著降低小鼠血清睾酮含量和β-微管蛋白Ⅲ（Tubb3）、性别决定基因盒9（Sox9） mRNA相对表达水平（P<0.05或P<0.01）。3）腹腔急性注射5 mg/kg AKG 6 h后，可显著提高小鼠血清睾酮含量（P<0.05），增加小鼠攻击性。4）离体研究表明，AKG以剂量依赖方式极显著上调睾丸间质细胞（TM3）上清液睾酮含量（P<0.01），且200 μmol/L AKG可极显著促进TM3细胞瞬时钙离子释放（P<0.01）。5）长期饮水添加2% AKG 4周可极显著提高小鼠血清睾酮含量（P<0.01），极显著减少睾丸曲细精管空腔面积（P<0.01），而OXGR1KO可显著逆转AKG对血清睾酮含量和曲细精管形态变化的影响（P<0.05）。综上所述，AKG作为重要的生物活性代谢中间产物，可通过OXGR1提高雄性动物血清睾酮含量，影响曲细精管发育和精子生成。

关键词： α-酮戊二酸; 酮戊二酸受体1; 睾酮; 空腔面积

本文引用格式

徐畅 , 张姹 , 谭成全 , 习欠云 , 王声会 , 江青艳 , 束刚 . α-酮戊二酸/酮戊二酸受体1系统对雄性动物睾酮分泌和精子生成的影响[J]. 动物营养学报, 2022 , 34(5) : 3286 -3296 . DOI: 10.3969/j.issn.1006-267x.2022.05.054

Abstract

This experiment was conducted to investigate effects of α-ketoglutarate acid (AKG)/oxoglutarate receptor 1 (OXGR1) system on testosterone secretion and spermatogenesis of male animals. Firstly, the localization of OXGR1 in testicular tissue was detected by immunofluorescence. Then, OXGR1 knockout (OXGR1KO) mice were adopted to test the serum testosterone content by enzyme linked immunosorbent assay (ELISA) method, the hematoxylin-eosin (HE) staining was used to analyze the differences of testicular seminiferous tubule lumen area and testicular tissue morphology, and the mRNA expression level of testicular spermatogenesis marker genes were detected by real-time quantitative PCR. Then, in the acute test, sixteen 10-week-old mal mice were randomly divided into 4 groups according to the body weight, and the serum testosterone content and aggressiveness of the mice were detected after injection of 0, 1, 5 and 10 mg/kg AKG for 6 h, respectively; then twelve 10-week-old male mice and OXGR1KO mice were randomly divided into 4 groups according to the body weight, and the serum testosterone content was detected after 6 h injection of 5 mg/kg AKG in each group. In vitro test, the leydig cells (TM3) cells were treated with different concentrations of AKG for 24 h, and the content of testosterone in supernatant of TM3 cells was detected; the TM3 cells were treated with 200 μmol/L AKG to detect the intracellular calcium ion concentration. In long-term test, twelve 8-week-old male mice and OXGR1KO mice were selected and randomly divided into 4 groups according to the body weight:WT group, WT+2%AKG group, OXGR1KO group and OXGR1KO+2%AKG group, after 28 days of experiment, the serum testosterone content and testicular seminiferous tubule lumen area were detected. The results showed as follows:1) the OXGR1 was specifically distributed in leydig cells of testis. 2) OXGR1KO could significantly reduce the serum testosterone content and the mRNA relative expression of class Ⅲ beta-tubulin (Tubb3) and sex-determining region Y box 9 (Sox9) (P<0.05 or P<0.01). 3) After acute intraperitoneal injection of 5 mg/kg AKG for 6 h, the serum testosterone content of mice was significantly increased (P<0.05), and the aggressiveness of mice was increased. 4) The in vitro study showed that AKG could significantly increase the testosterone content in supernatant of TM3 cells in a dose-dependent manner (P<0.01), and 200 μmol/L AKG could significantly promote the release of instantaneous intracellular calcium of TM3 cells (P<0.01). 5) Long-term drinking water supplemented with 2%AKG for 4 weeks could significantly increase the serum testosterone content (P<0.01), significantly decrease the lumen area of testicular seminiferous tubules (P<0.01), while OXGR1KO could significant reversal AKG-induced serum testosterone content and seminiferous tubule morphology change (P<0.05). In conclusion, AKG, as an important bioactive metabolic intermediate, can increase the serum testosterone content of male animals through OXGR1 and affect the development of seminiferous tubules and spermatogenesis.

Key words： AKG; OXGR1; testosterone; lumen area

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