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. 2024 Jan 3:102:skae020.
doi: 10.1093/jas/skae020.

Roles of Y-27632 on sheep sperm metabolism

Yu Fu  1 , Wenzheng Shen  1 , Haiyu Bai  1 , Zhiyu Zhang  1 , Zhikun Cao  1 , Zibo Liu  1 , Chao Yang  1 , Shixin Sun  1 , Lei Wang  1 , Yinghui Ling  1   2 , Zijun Zhang  1   2 , Hongguo Cao  1   2
Affiliations

Roles of Y-27632 on sheep sperm metabolism

Yu Fu et al. J Anim Sci. .

Abstract

To investigate the effect of Y-27632 on low-temperature metabolism of sheep sperm, different concentrations of Y-27632 were added to sheep semen at 4 °C in this experiment to detect indicators such as sperm motility, plasma membrane, acrosome, antioxidant performance, mitochondrial membrane potential (MMP), and metabolomics. The results showed that the addition of 20 µM Y-27632 significantly increased sperm motility, plasma membrane integrity rate, acrosome integrity rate, antioxidant capacity, MMP level, significantly increased sperm adenosine triphosphate (ATP) and total cholesterol content, and significantly reduced sperm Ca2+ content. In metabolomics analysis, compared with the control group, the 20 µM Y-27632 group screened 20 differential metabolites, mainly involved in five metabolic pathways, with the most significant difference in Histidine metabolism (P = 0.001). The results confirmed that Y-27632 significantly improved the quality of sheep sperm preservation under low-temperature conditions.

Keywords: Y-27632; low temperature; metabolism; semen preservation; sheep.

Plain language summary

Sheep semen preservation and artificial insemination is an important reproductive technology that supports the large-scale and intensive development of the sheep farming industry. Under low-temperature condition, sperm metabolic activity slows down or pauses, energy consumption decreases, thereby prolonging sperm preservation time and motility. During the process of sperm preservation, sperm are susceptible to cold shock damage, which affects the quality of sperm preservation. Y-27632 is a rho-associated cooled-coil kinase (ROCK) inhibitor that competes with ATP to inhibit the kinase activity of ROCK-I and ROCK-II. However, the study of Y-27632 used in sheep semen preservation and its protective mechanism is less. In this study, we used the ROCK inhibitor Y-27632 and the ROCK activator arachidonic acid (AA) for low-temperature preservation of sheep semen and related metabolic regulation mechanisms. This experiment confirmed that Y-27632 played a significant protective role by regulating sperm metabolism and protecting sperm plasma membrane in sheep.

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Figures

Figure 1.
Figure 1.
Experimental design and workflow diagram of Y-27632 regulating sperm metabolism in sheep.
Figure 2.
Figure 2.
Morphological characteristics of the plasma membrane and acrosome of sheep sperm. (A) The sperm plasma membrane of the Y-27632 control group on the 12th day. (B) Sperm plasma membrane of Y-27632 group on the 12th day. (C) The sperm plasma membrane of the AA control group on day 6. (D) Sperm plasma membrane of AA group on the 6th day. Under hypotonic conditions, the sperm morphology is characterized by a curved tail for sperm with intact plasma membrane and a straight tail for sperm with damaged plasma membrane. (E) The sperm acrosome of the Y-27632 control group on day 12. (F) The sperm acrosome of Y-27632 group on the 12th day. (G) The sperm acrosome of the AA control group on day 6. (H) The sperm acrosome of AA group on the 6th day. Isothiocyanate fluorescein-labeled peanut agglutinin (FITC-PNA) stained sperm morphology.
Figure 3.
Figure 3.
The effects of 20 µM Y-27632 (day 18) and 100 µM AA (day 5) on the antioxidant capacity of sheep sperm. (A) SOD activity in 20 µM Y-27632 group. (B) SOD activity in 100 µM AA group. (C) CAT content in 20 µM Y-27632 group. (D) The CAT content in 100 µM AA group. (E) T-AOC level in 20 µM Y-27632 group. (F) T-AOC level in 100 µM AA group. (G) MDA level in 20 µM Y-27632 group. (H) MDA level in 100 µM AA group. (I) ROS level in 20 µM Y-27632 group. (J) ROS level in 100 µM AA group. *P < 0.05, **P < 0.01.
Figure 4.
Figure 4.
The effects of 20 µM Y-27632 (day 18) and 100 µM AA (day 5) on the energy metabolism level of sheep sperm. (A) MMP levels in 20 µM Y-27632 group. (B) MMP levels in 100 µM AA group. (C) ATP content in 20 µM Y-27632 group. (D) ATP content in 100 µM AA group. *P < 0.05, **P < 0.01.
Figure 5.
Figure 5.
The MMP levels of sheep sperm in the 20 µM Y-27632 group (day 18) and the 100 µM AA group (day 5) using JC-1. (A) The high MMP level on the 18th day in Y-27632 control group. (B) The high MMP level on the 18th day in 20 µM Y-27632 group. (C) High MMP level on day 5 in AA control group. (D) High MMP level on day 5 in 100 µM AA group. (E) Low MMP level on the 18th day in Y-27632 control group. (F) Low MMP level on the 18th day in 20 µM Y-27632 group. (G) Low MMP level on day 5 in AA control group. (H) Low MMP level on day 5 in 100 µM AA group. JC-1 fluorescence staining of sperm mitochondria, with red indicating high MMP level and green indicating low MMP level.
Figure 6.
Figure 6.
The effect of 20 µM Y-27632 (day 18) and 100 µM AA (day 5) on Ca2+ level and TC content of sheep sperm. (A) Ca2+ level in the 20 µM Y-27632 group. (B) Ca2+ level in the 100 µM AA group. (C) TC content in the 20 µM Y-27632 group. (D) TC content in the 100 µM AA group. *P < 0.05, **P < 0.01.
Figure 7.
Figure 7.
The effect of 20 µM Y-27632 (day 18) on the ultrastructure of sheep sperm through transmission electron microscopy. (A) Sperm plasma membrane in the control group. (B) Sperm plasma membrane in 20 µM Y-27632 group. (C) Sperm axoneme in the control group. (D) Sperm axoneme in 20 µM Y-27632 group. (E) Sperm acrosome in the control group. (F) Sperm acrosome in the 20 µM Y-27632 group. (G) Sperm mitochondria in the control group. (H) Sperm mitochondria in the 20 µM Y-27632 group. Abbreviations: AC, acrosome; MS, mitochondrial sheath; ODF, outer dense fiber; AX, axoneme; MT, mitochondrion.
Figure 8.
Figure 8.
Metabolomics analysis of 20 µM Y-27632 on sheep sperm (day 18). (A) Permutation test plot of sheep sperm. (B) Volcanic map for screening different metabolites of sheep sperm. (C) Differential metabolite cluster heat map of sheep sperm. (D) KEGG enrichment analysis of differential metabolites of sheep sperm. (E) Metabolic pathway of sheep sperm.
Figure 9.
Figure 9.
The regulatory mechanism of ROCK pathway inhibitor Y-27632 and ROCK pathway activator arachidonic acid (AA) on sperm metabolism in sheep at 4 °C. (A) Y-27632 regulatory mechanism. (B) AA regulatory mechanism. Abbreviations: G-6-P, glucose-6-phosphate; PPP, pentose phosphate pathway; NADP+, nicotinamide adenine dinucleotide phosphate; His, histidine; Ser, serine; Thr, threonine; Ala, alanine; Asp, aspartate; Pro, proline; TCA, tricarboxylic acid cycle; NADH, nicotinamide adenine dinucleotide; NAD, nicotinamide adenine dinucleotide; αKG, α-ketoglutaric acid; ΔΨm, mitochondrial membrane potential; ROS, reactive oxygen species; SOD, superoxide dismutase; CAT, catalase; LPO, lipid peroxide; MDA, malondialdehyde; GSH, glutathione; AA, arachidonic acid.
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