谷氨酰胺通过激活HSF1/HSP70通路改善绵羊冻融精液质量

doi:10.11843/j.issn.0366-6964.2025.05.021

摘要/Abstract

摘要：

旨在探讨谷氨酰胺在绵羊精子冷冻保护过程中的作用机制。本研究使用6头2~4岁健康的成年湖羊精液，检测冷冻解冻后的精子活力、质膜和顶体完整性、线粒体膜电位、ATP含量、α-酮戊二酸脱氢酶活性和异柠檬酸脱氢酶活性及精子HSP70、HSF1及pHSF1蛋白表达。结果表明，在冷冻稀释液中添加谷氨酰胺能够显著提高绵羊精子总活力(P < 0.05)。5 mmol·L^-1谷氨酰胺组解冻后精子活力、质膜和顶体完整性、线粒体膜电位、ATP含量、α-酮戊二酸脱氢酶和异柠檬酸脱氢酶活性均显著高于对照组(P < 0.05)。谷氨酰胺显著提高了解冻后精子HSP70和pHSF1的表达(P < 0.05)，HSF1的抑制剂DTHIB抑制了这一过程，并显著降低了解冻后精子的总活力(P < 0.05)，通过外源补充HSP70蛋白可以抵消DTHIB的抑制作用，并显著降低DTHIB处理导致的精子总活力降低(P < 0.05)。本研究通过检测绵羊解冻后精液的各质量参数，发现谷氨酰胺可通过上调冷冻-解冻精子中HSF1蛋白的磷酸化水平、提高HSP70蛋白的表达、保护线粒体功能，进而改善绵羊精子的冷冻保存效果。

关键词: 精子冷冻, 谷氨酰胺, 热休克蛋白70, 热休克因子1

Abstract:

This study aimed to explore the mechanism of glutamine action during the cryopreservation process of sheep sperm. Semen samples were collected from 6 healthy adult Hu sheep aged 2-4 years. Frozen-thawed sperm motility, plasma membrane and acrosome integrity, mitochondrial membrane potential, ATP content, α-ketoglutarate dehydrogenase (α-KGDH) activity, isocitrate dehydrogenase (IDH) activity, and the expression levels of HSP70, HSF1, and phosphorylated HSF1 (pHSF1) proteins were evaluated. The results demonstrated that the addition of glutamine to the freezing extender significantly improved the total motility of sheep sperm (P < 0.05). The 5 mmol·L^-1 glutamine group exhibited significantly higher frozen-thawed sperm motility, plasma membrane and acrosome integrity, mitochondrial membrane potential, ATP content, α-KGDH activity, and IDH activity compared to the control group (P < 0.05). Glutamine significantly upregulated the expression of HSP70 and pHSF1 in frozen-thawed sperm (P < 0.05). The HSF1 inhibitor DTHIB suppressed this process and significantly reduced the total motility of frozen-thawed sperm (P < 0.05). Exogenous supplementation of HSP70 protein counteracted the inhibitory effect of DTHIB and significantly alleviated the reduction in sperm total motility induced by DTHIB treatment (P < 0.05). In this study, various quality parameters of frozen-thawed semen from sheep were examined and found that glutamine could improve the cryopreservation of sheep sperm by upregulating the phosphorylation level of HSF1 protein, increasing the expression of HSP70 protein, and protecting the mitochondrial function in frozen-thawed sperm, thereby enhancing the cryopreservation effect of sheep sperm.

Key words: sperm cryopreservation, glutamine, heat shock protein 70, heat shock factor 1

中图分类号:

S826.3

张健, 海尔汗, 张建军, 李博渊, 张家新. 谷氨酰胺通过激活HSF1/HSP70通路改善绵羊冻融精液质量[J]. 畜牧兽医学报, 2025, 56(5): 2219-2229.

ZHANG Jian, HAI Erhan, ZHANG Jianjun, LI Boyuan, ZHANG Jiaxin. Glutamine Enhances Frozen-Thawed Semen Quality in Sheep via Activation of the HSF1/HSP70 Signaling Pathway[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(5): 2219-2229.

图/表 6

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活力参数 Motility parameter	谷氨酰胺浓度/(mmol·L^-1) Gln concentration
活力参数 Motility parameter	0	2	5	10	20
总活力/% TM	59.23±3.74^c	63.87±2.13^bc	72.90±1.35^a	68.30±0.96^ab	66.50±1.20^b
前向运动/% PM	40.20±0.49^b	43.10±1.79^b	48.33±1.48^a	40.77±1.31^b	35.43±0.83^c
平均运动速率/(μm·s^-1)VAP	62.42±3.43^c	64.08±5.11^c	88.19±0.55^a	79.74±0.77^b	67.34±0.62^c
直线运动速率/(μm·s^-1)VSL	52.86±1.03^c	52.32±2.07^c	67.13±0.06^a	68.40±0.66^a	62.20±0.54^b
曲线运动速率/(μm·s^-1)VCL	104.14±1.92^c	112.96±2.30^b	152.39±1.59^a	116.64±1.00^b	86.34±1.29^d

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