Key Laboratory of Animal Genetics, Breeding and Reproduction of Ministry of Agriculture and Rural Affairs, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, China.
Yunnan Animal Science and Veterinary Institute, Kunming, China.
Anim Genet. 2023 Apr;54(2):189-198. doi: 10.1111/age.13287. Epub 2023 Jan 11.
ALAS1 is a member of the α-oxoamine synthase family, which is the first rate-limiting enzyme for heme synthesis and is important for maintaining intracellular heme levels. In the ovary, ALAS1 is associated with the regulation of ovulation-related mitochondrial P450 cytochromes, steroid metabolism, and steroid hormone production. However, there are few studies on the relationship between ALAS1 and reproductive traits in goats. In this study, a mutation located in the promoter region of ALAS1 (g.48791372C>A) was found to be significantly (p < 0.05) associated with the kidding number of Yunshang black goats. Specifically, the mean kidding number in the first three litters and the kidding numbers of all three litters were significantly (p < 0.05) higher in individuals with the CA genotype or AA genotype than in those with the CC genotype. To further investigate the regulatory mechanism of ALAS1, the expression of ALAS1 in goat ovarian tissues with different genotypes was verified by real-time quantitative PCR. The results showed that the expression of ALAS1 was significantly higher in the ovaries of individuals with AA genotype than those with AC and CC genotypes (p < 0.01), and the expression trend of transcription factor ASCL2 was consistent with ALAS1. Additionally, the ALAS1 g.48791372C>A mutation created a new binding site for the transcription factor ASCL2. The luciferase activity assay indicated that the mutation increased the promoter activity of ALAS1. Overexpression of the transcription factor ASCL2 induced increased expression of ALAS1 in goat granulosa cells (p < 0.05). The opposite trend was shown for the inhibition of ASCL2 expression. The results of real-time quantitative PCR, EdU and Cell Counting Kit-8 assays indicated that the transcription factor ASCL2 increased the proliferation of goat granulosa cells by mediating the expression of ALAS1. In conclusion, the transcription factor ASCL2 positively regulated the transcriptional activity and expression levels of ALAS1, altering granulosa cell proliferation and the kidding number in goats.
ALAS1 是α-氧胺合酶家族的成员，它是血红素合成的第一个限速酶，对维持细胞内血红素水平很重要。在卵巢中，ALAS1 与排卵相关的线粒体 P450 细胞色素、类固醇代谢和类固醇激素产生的调节有关。然而，关于 ALAS1 与山羊繁殖性状的关系的研究较少。在这项研究中，发现位于 ALAS1 启动子区域的一个突变（g.48791372C>A）与云尚黑山羊的产羔数显著相关（p<0.05）。具体来说，CA 基因型或 AA 基因型个体的第一至三胎产羔数和三胎总产羔数均显著高于 CC 基因型个体（p<0.05）。为了进一步研究 ALAS1 的调控机制，通过实时定量 PCR 验证了不同基因型山羊卵巢组织中 ALAS1 的表达。结果表明，AA 基因型个体的卵巢中 ALAS1 的表达显著高于 AC 和 CC 基因型个体（p<0.01），转录因子 ASCL2 的表达趋势与 ALAS1 一致。此外，ALAS1 g.48791372C>A 突变在转录因子 ASCL2 上创造了一个新的结合位点。荧光素酶活性测定表明，该突变增加了 ALAS1 的启动子活性。转录因子 ASCL2 的过表达诱导山羊颗粒细胞中 ALAS1 的表达增加（p<0.05）。ASCL2 表达被抑制时则呈现相反的趋势。实时定量 PCR、EdU 和细胞计数试剂盒-8 检测结果表明，转录因子 ASCL2 通过调节 ALAS1 的表达来增加山羊颗粒细胞的增殖。总之，转录因子 ASCL2 正向调节 ALAS1 的转录活性和表达水平，改变颗粒细胞的增殖和山羊的产羔数。
