蒙古羊双羔性状基因ADAMTS-1作用机制研究

doi:10.16431/j.cnki.1671-7236.2023.08.020

摘要/Abstract

摘要： 【目的】以产单、双胎蒙古羊为动物模型初步探究双羔主效基因含Ⅰ型血小板结合蛋白基序的解聚蛋白样金属蛋白酶(ADAMTS-1)的作用机制,为蒙古羊高效繁育及其相关研究提供理论依据。【方法】选取具有产单、双胎记录的雌性蒙古羊各12只,采用自然发情方式,通过阴道黏膜涂片和公羊爬跨确定雌性蒙古羊的发情期和间情期,分别取产双羔发情期(DE)、产双羔间情期(DD)、产单羔发情期(SE)、产单羔间情期(SD)蒙古羊卵巢,利用HE染色的方式观察卵巢有腔卵泡形态差异。取蒙古羊卵巢颗粒细胞进行培养,运用免疫荧光技术确定ADAMTS-1表达位置。通过实时荧光定量PCR技术检测ADAMTS-1、孕酮受体(PGR)、丝裂原活蛋白激酶4(MAPK4)、雄激素受体 (AR)、丝氨酸蛋白酶抑制剂2(SERPINE2)、多功能蛋白聚糖蛋白聚糖(Versican)在不同组别中的mRNA相对表达水平,并通过Western blotting技术检测ADAMTS-1前体蛋白(pro-ADAMTS-1)和成熟蛋白(MP-ADAMTS-1)在不同组别中的蛋白相对表达水平。【结果】通过HE染色结果可以确定产单、双胎蒙古羊卵巢组织内有腔卵泡形态无表观差异,运用免疫荧光方法检测到ADAMTS-1在蒙古羊卵巢颗粒细胞上表达。通过荧光定量PCR及Western blotting结果可知,DE组蒙古羊中ADAMTS-1在卵巢组织中基因及蛋白表达量显著高于其他3组(P＜0.05),而在SE与SD组无显著差异(P＞0.05);SE组蒙古羊卵巢组织内AR和PGR基因表达量最高,且显著高于其他3组(P＜0.05),DE组显著高于DD组(P＜0.05),且在同一发情阶段产单羔蒙古羊卵巢组织内AR、PGR基因表达量显著高于产双羔蒙古羊卵巢组织;SE组蒙古羊卵巢内MAPK4基因表达量最高,且显著高于其他3组(P＜0.05),其他3组间无显著差异(P＞0.05);DD组蒙古羊卵巢组织内Versican、SERPINE2基因表达量最高,且显著高于DE和SD组(P＜0.05);SE组这两个因子显著高于SD组(P＜0.05)。【结论】ADAMTS-1于蒙古羊卵巢颗粒细胞中稳定表达,ADAMTS-1对蒙古羊双羔性状的排卵机制具有重要的正向调控作用,而其他多种激素受体以及胞内关键信号基因也与卵巢内卵泡的发育、排出密切相关。

关键词: 蒙古羊; ADAMTS-1; 双羔性状; 卵巢

Abstract: 【Objective】 Using single and twin Mongolian sheep as animal models,the mechanism of the main effect gene of double lambs containing type Ⅰ platelet-binding protein motif of disintegrin-like metalloproteinase (ADAMTS-1) was preliminarily explored to provide a theoretical basis for efficient breeding and related research in Mongolian sheep.【Method】 Twelve female Mongolian sheep with records of single and double births were selected,and use natural estrus methods to determine the estrus and interestrus periods of female Mongolian sheep through vaginal mucosal smears and male sheep crawling.The ovaries of Mongolian sheep during the stages of double lambing estrus (DE),double lambing dioestrum (DD),single lambing estrus (SE),and single lambing dioestrum (SD) were collected,and the morphological differences of ovarian antral follicles were observed using HE staining.Granulosa cells from the ovary of Mongolian sheep were cultured and the location of ADAMTS-1 expression was determined by immunofluorescence.The relative mRNA expression of ADAMTS-1,progesterone receptor (PGR),mitogen-activated protein kinase 4 (MAPK4),androgen receptor (AR),serine protease inhibitor 2 (SERPINE2) and multifunctional proteoglycan proteoglycan (Versican) were determined by Real-time quantitative PCR in different groups.And the relative protein expression of ADAMTS-1 precursor protein (pro-ADAMTS-1) and mature protein (MP-ADAMTS-1) in different groups were detected by Western blotting.【Result】 There were no apparent differences in the morphology of the luminal follicles within the ovarian tissue from single and twin Mongolian sheep,as determined by the results of HE staining,and ADAMTS-1 was detected on granulosa cells in the ovary of Mongolian sheep using immunofluorescence.The results of fluorescence PCR and Western blotting showed that the gene and protein of ADAMTS-1 in ovarian tissue of Mongolian sheep in DE group were significantly higher than those in the other three groups (P＜0.05),while there was no significant difference between SE and SD groups (P>0.05). The expression of AR and PGR genes in ovarian tissue of Mongolian sheep in SE group were the highest and significantly higher than those in the other 3 groups (P＜0.05),and that in DE group was also significantly higher than that in DD group (P＜0.05).Moreover,the expression of AR and PGR genes in ovary of single lambs at the same diestrus stage was significantly higher than in ovary of double lambs.The expression of MAPK4 gene in ovarian tissue of Mongolian sheep in SE group were the highest and significantly higher than that the other three groups (P＜0.05),while there was no significant difference among the other three groups (P＞0.05).The expression of Versican and SERPINE2 genes in ovarian tissue of Mongolian sheep in DD group were the highest and significantly higher than those in DE and SD groups (P＜0.05),these two factors in SE group were significantly higher than that in SD group (P＜0.05).【Conclusion】 ADAMTS-1 was stably expressed in granulosa cells of ovary in Mongolian sheep,and ADAMTS-1 had an important positive regulatory role on ovulation mechanism of double lamb trait in Mongolian sheep,while various other hormone receptors and intracellular key signaling genes were also closely related to follicle development and expulsion in ovary.

Key words: Mongolian sheep; ADAMTS-1; double lamb trait; ovary

中图分类号:

Q953.3

赵梦瑶, 陈晓良, 李秀男, 苏乐德, 田瑛, 菅瑞珍, 杨燕燕, 李海军. 蒙古羊双羔性状基因ADAMTS-1作用机制研究[J]. 中国畜牧兽医, 2023, 50(8): 3229-3238.

ZHAO Mengyao, CHEN Xiaoliang, LI Xiunan, SU Lede, TIAN Ying, JIAN Ruizhen, YANG Yanyan, LI Haijun. Study on the Mechanism of the Double Lamb Trait Gene ADAMTS-1 in Mongolian Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(8): 3229-3238.

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