Sperm capacitation is an important physiological prerequisite for fertilization [1], as sperm must be able to capacitate and undergo the acrosome reaction to bind with zona pellucida and interact with oocyte. A too-high or too-rapid induction of capacitation can lead to sperm death and prevent fertilization [2]. Seminal plasma (SP), as an essential medium for sperm, contains proteins, amino acids, enzymes, fructose, and other carbohydrates, lipids, major minerals, and trace elements that not only transport, protect and support sperm to reach an oocyte but also prevent sperm capacitation and interact with female reproductive tract [[3], [4], [5]]. In addition, it has been demonstrated that SP-derived extracellular vesicles (EVs) can maintain sperm function in humans [6] and boar [7] by fusing to the sperm membrane.

EVs are small membrane vesicles that contain protein, DNA, and RNA from the cells that secrete them [8] and are found in most porcine biofluids [9]. Numerous researches have indicated that EVs participate in intercellular communication by selectively transferring their components to target cells [10]. EVs can be classified into two main subtypes: exosomes (diameter ranging from 30 to 150 nm) and microvesicles (diameter ranging from 100 to 1000 nm) [11]. SP-derived EVs are secreted by prostate (prostasomes), epididymis (epididymosomes), seminal vesicles, testis, and other gonads [12], which have been implicated in sperm-fertilizing capacitation [[13], [14], [15]], sperm motility modulation [16], protection against oxidative stress [17], and sperm damage caused by female immune response [18]. Because sperm is a transcriptionally quiescent cell, post-transcriptional mechanisms are critical for sperm biology [19,20], and the transcript profiles of mature mammalian sperm mainly consist of untranslated mRNAs during round spermatid stage [21]. However, recent research revealed that RNA profiles between fresh and capacitated boar sperms are different [22], implying that SP-derived EVs may participate in sperm RNA profiles remodeling during sperm capacitation by conveying post-transcriptional regulatory factors, such as microRNAs (miRNAs).

MiRNAs are a class of small non-coding RNAs (sncRNAs) that regulate gene expression post-transcriptionally, affecting mRNA stability and translation [23], and are involved in mammalian spermatogenesis [24], sperm viability maintenance [25], sperm apoptosis [26], and fertility [27]. However, limited research has been conducted on the role of miRNAs from SP-derived EVs in regulating sperm functions. Due to unique miRNA profiles of SP-derived EVs from various origins [12], miRNAs in SP-derived EVs are currently mainly recognized as biomarkers of many pathological states, including azoospermia [28] and prostate cancer [29].

We previously demonstrated that miR-21-5p is the most abundant miRNA in boar SP-derived EVs and can specifically inhibit pig Vinculin (VCL) gene [30], which encoded an actin filament (F-actin)-binding protein that involve in cell-matrix adhesion and cell-cell adhesion [31]. Recent research has shown that VCL protein is implicated in sperm capacitation by forming a protein complex [32]. However, the function of EV-delivered miR-21-5p on sperm capacitation remains unclear. Therefore, we have first co-incubated sperm with SP-derived EVs, then SP-derived EVs were traced and expression of miR-21-5p and pre-miR-21-5p were evaluated in sperm. Subsequently we transfected miR-21-5p into boar sperm, and then expression of miR-21-5p, mRNA and protein expression of VCL of sperm, sperm capacitation rate were evaluated after induced capacitation. In addition, we have localized the VCL protein within the sperm during capacitation and sperm capacitation rate were evaluated after treated sperm with VCL protein inhibitor (Chrysin). Chrysin is a flavonoid that participates in multiple biological and physiological processes and is abundant in edible plants such as passion flowers and mushrooms [33,34]. Recent research revealed that Chrysin could attenuate the activation of VCL protein, in turn inhibiting the F-actin crosslinking responsible for actin bundling [35]. Finally, we evaluated expression of miR-21-5p, mRNA and protein expression of VCL, and acrosome integrity of sperm under liquid storage conditions after miR-21-5p transfection.