Liquid storage of spermatozoa is important for artificial insemination and herd genetic breeding. However, the extended time of storage inducing the rapid decline in spermatozoa quality limits the development of this technology. The molecular mechanisms underlying liquid storage of spermatozoa remain largely unexplored. In this study, the effects of liquid storage on functional quality of spermatozoa were assessed in goat (Capra hircus). The time-dependent decline in spermatozoa motility showed a strong correlation with the significant increase in apoptosis. Moreover, apoptosis-related ultrastructural changes were observed, especially the defects in mitochondria. A significant decrease in mitochondrial membrane potential and changes in the expression of mitochondrial apoptosis-related proteins indicated mitochondrial dysfunction and mitochondrial apoptotic pathway activation. Notably, the abnormally high level of reactive oxygen species (ROS) caused by liquid storage resulted in oxidative damage to mitochondria and accelerated mitochondria-dependent apoptosis, as demonstrated by the addition of ROS scavenger N-acetylcysteine. Furthermore, critical differentially expressed proteins involved in mitochondria-dependent apoptosis and antioxidant defense were identified and profiled by quantitative proteomic analysis, facilitating the understanding of molecular regulation of ROS-induced mitochondria-dependent apoptosis. These outcomes provide insights into the mechanisms underlying liquid storage of goat spermatozoa and enhance the progress of semen storage technology.

The steroidogenic acute regulatory protein (STAR) plays a crucial role in facilitating cholesterol transfer across the inner mitochondrial membrane during the process of steroidogenesis. However, the transcriptional regulation of the bovine STAR gene and its function of progesterone synthesis in luteal cells remain poorly understood. The objective of this study was to analyze the bovine STAR gene structure, identify its active promoter region, and explore its potential roles in progesterone synthesis. Bioinformatics analysis revealed that the bovine STAR gene encodes an 858-bp mRNA transcript, which translates into a protein consisting of 285 amino acids. The phylogenetic tree analysis showed that its genetic distance was closest to that of sheep. Notably, the promoter region of bovine STAR lacks CpG islands, and the core promoter is located within the − 1990/-1 region, which containes potential binding sites for transcription factors such as NF-κB, Sp1, NF-1, and LyF-1. Dual-luciferase reporter assays confirmed the core promoter activity within this region, aligning with the prediction results. Overexpression of the STAR gene in bovine luteal cells significantly enhanced progesterone production and upregulated the expression of steroidogenic enzymes, particularly 3βHSD and CYP11A1. In conclusion, this study identifies the core promoter region of the bovine STAR gene is positioned at -1990/-1. By regulating key steroidogenic enzymes, particularly 3βHSD and CYP11A1, STAR is involved in the synthesis of progesterone in corpus luteum cells.

Exosomes as bilayer membranous vesicles are abundant in seminal plasma and mediate intercellular communication by transferring active biomolecules. Numerous studies have revealed the involvement of exosomes in regulating various biological properties of spermatozoa. However, the beneficial roles of seminal plasma exosomes in maintaining spermatozoon motility and mitochondrial function during liquid storage have not yet been unexplored in goat. In this study, the reduction of ATP content in goat spermatozoa was detected along with the decrease in spermatozoon motility under liquid storage, and the level of oxidative phosphorylation was also decreased. The interaction of exosomes and spermatozoon mitochondria was observed using high pressure freezing/freeze-substitution in combination with transmission electron microscope. Seminal plasma exosomes of goat were isolated and used to incubate with spermatozoa, and the binding and fusing of exosomes with spermatozoa was further validated. Furthermore, the addition of seminal plasma exosomes exhibited an increase in motility and oxidative phosphorylation in liquid-stored spermatozoa. Several mitochondrial functional parameters, including mitochondrial membrane potential, the levels of mitochondrial ROS and intracellular Ca ²⁺ , and the copy number and integrity of mitochondrial DNA, were also improved in spermatozoa after incubating with exosomes. Notably, the level of TFAM protein was increased in exosome-treated spermatozoa, indicating that the enhanced proteins may be delivered by exosomes to spermatozoa. These results suggest that seminal plasma exosomes could improve spermatozoon motility and mitochondrial function by regulating oxidative phosphorylation, which would provide insights into the understanding of protective roles of exosomes in goat spermatozoa during liquid storage.

Liquid storage of semen is a widely used technology for promoting genetic improvement in goat breeding. The short shelf life of spermatozoa greatly limits the application of liquid storage, which urgently needs to explore the underlying regulatory factors. Autophagy as a cellular catabolic process plays critical roles in eliminating damaged material, that thus protects the function and fertilizing ability of spermatozoa. Nevertheless, the regulatory mechanisms of autophagy in goat spermatozoa under liquid storage remain unclear. In this study, the typical morphologic abnormalities and ultrastructural changes in goat spermatozoa, such as plasma membrane swollen and shrunken, acrosome exfoliation, and axoneme exposure, were observed after liquid storage at 4°C. Moreover, assessment of the formation of autophagy in liquid-stored goat spermatozoa was performed by a morphological “gold standard” of electron microscopy. Notably, a large number of vesicles with double-membrane structure indicating autophagosome were found to surround the aberrant spermatozoa, suggesting the activation of autophagy. Several proteins, such as LC3, ATG5, and p62, exhibited differential expression after liquid storage, which further validated the occurrence of autophagy in liquid-stored goat spermatozoa. Furthermore, chloroquine treatment was used to inhibit the autophagy of spermatozoa, which caused a significantly decrease in the quality of liquid-stored spermatozoa, including motility, viability, plasma membrane integrity, and acrosome integrity. Significant increase in ROS and MDA levels of spermatozoa and significant decrease in Ca²⁺ influx and protein tyrosine phosphorylation of spermatozoa were also detected after chloroquine-induced autophagy inhibition. The ultrastructural observation of double-membrane autophagosome provides strong evidences for the activation of autophagy in goat spermatozoa under liquid storage. The inhibition of autophagy mediated by chloroquine indicated that autophagy plays vital roles in the survival of spermatozoa. These results facilitate understanding the activation of autophagy in spermatozoa and provide valuable references for uncovering the underlying regulatory mechanisms of liquid storage of goat spermatozoa.