1. Introduction
Cells release different types of extracellular vesicles (EVs) in the extracellular microenvironment [
1]. They affect recipient cells directly through the transfer of bioactive cargo (mRNA, proteins, and lipids) or indirectly through affecting the cellular epigenome [
2,
3]. EVs and exosomes have been isolated from various types of cells and biological fluids such as saliva [
4], blood plasma [
5], and urine [
6]. Concerning reproductive fluids, they can be obtained from the uterine [
7], seminal [
8], follicular [
9], and oviductal fluids [
10]. Given their ubiquitous role, it has been proposed that EVs and exosomes isolated from reproductive samples are closely related to gamete and embryo development [
11,
12]. In 2013, Al-Dossary et al. dubbed the exosomes derived from the oviductal fluid as “oviductosomes” [
13] based on their site of origin. There is markedly increasing interest in understanding oviduct-derived EVs for their potential physiological roles in the reproductive process, such as sperm capacitation, oocyte maturation, and embryo development [
10,
14,
15].
Our recent studies have demonstrated that canine in vitro oviductal cell-derived extracellular vesicles (OC-EVs) affect not only the viability, proliferation rate, and gene/protein expression of cumulus cells [
16] but, also, exert regulatory functions on cumulus–oocyte complexes (COCs) by enhancing oocyte development via the EGFR/MAPK signaling pathway [
17]. Moreover, the proteins derived from oviductal EVs could regulate the physiological functions of gamete and embryos [
18,
19].
Previous proteomic research demonstrated the protein composition of the oviductal fluid. Those proteomes have been suggested as a potential tool for understanding reproductive physiology [
20,
21,
22,
23,
24]. However, few systematic studies are unraveling the molecular content of the OC-EVs to understand their possible roles in gamete/oocyte/embryo development in the canine reproductive system. Therefore, this study would provide valuable information regarding the protein content and its molecular function with the signaling pathway in OC-EVs.
The proteomic content of oviductal EVs has been studied in different species. For example, mouse oviductal fluid contains plasma membrane Ca
2+-ATPase 4a and epididymal sperm adhesion molecule 1, molecules that play an essential role in sperm capacitation and fertility [
15,
25]. Similarly, in bovines, 319 proteins were identified in EVs from the oviduct; several of these proteins were involved in fertilization and embryo development [
10].
In this study, we aimed to describe the proteome of canine OC-EVs. This endeavor is essential, given the unique reproductive characteristics of bitches compared with other mammals: at ovulation, the oocyte is in prophase I and will undergo maturation into a metaphase II in the oviductal canal after a period of 48–72 h [
26,
27]. Therefore, understanding the protein composition of canine OC-EVs can provide valuable information for the establishment of a successful in vitro maturation system.
Therefore, our efforts in this research were directed towards characterizing canine OC-EV protein compositions by employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) and its potential physiological relevance following a functional analysis of the resultant set of proteins. This comprehensive study in canine species will form a platform to suggest the potential role of EVs in canine oocyte development and bring new insight into the EV contributions to establishing stable assisted reproductive techniques in canine reproduction.
4. Discussion
Canine oocyte maturation possesses a unique event in which ovarian follicles release immature prophase I oocytes, requiring an additional 48–72 h to undergo maturation in the oviductal canal [
26,
27,
32]. The interaction between the oviduct secretome and oocytes is pivotal to the meiotic and cytoplasmic maturation of the oocytes. Hence, as a continuation of our previous studies, we characterized the OC-EVs and analyzed their protein contents to better understand their involvement in the oocyte maturation process in this unique species. The current results dig into the pathways controlled through the OC-EVs to regulate the canine oocyte maturation and early embryo development. Several studies have been reported on the molecular cargo of the oviductal EVs in different species; however, there is a lack of information in canine species.
In bovines, a mass spectrometry analysis identified 319 proteins in the oviductal EVs, where 97 proteins were exclusively expressed in in vivo EVs, 47 proteins were expressed only in vitro, and 175 proteins were common [
10]. A functional analysis of the resultant proteins revealed essential pathways involved in sperm–oocyte binding and fertilization [
10]. Additionally, a mass spectrometry and DAVID functional annotation clusters analysis identified 336 clusters of proteins in bovine oviductal EVs (170 were differentially abundant across the estrous cycle) that suggested the involvement of the proteins in metabolism and gamete–oviduct interactions [
39]. Furthermore, a shotgun proteomics and bioinformatics analysis identified the proteome of bovine oviductal fluid and revealed 266 secreted proteins (109 (41%) of them were shared for both in vivo and in vitro conditions). Our LC-MS/MS results showed a total of 1038 proteins in the three biological samples, sharing 398 common proteins. In fact, the qualitative proteomics of EV cargoes are highly variable in both biological and technical replicates, with a higher incidence among the former. We observed 40% identical proteins in our three samples, a number within the range (35–60%) of overlapped peptide lists from pairs of technical replicates [
40]. Tiruvayipati et al. [
41] recently reported that only 17% could be detected as common proteins within the same cell line, which is very smaller than what we detected (~40%). Moreover, they found that there is an average variance (i.e., relative standard deviation) between the quantitative protein analysis within the same line up to 47%. Furthermore, the issue of interbiological (47%) and intrabiological variations (45%) was recently highlighted within the urinary-derived EVs [
42]. Indeed, LC-MS is regarded as a highly complex analytical technique, and the proteomics experiments based on this technique can be subject to a large variability despite recent advances in technological and computational tools [
43]. Therefore, future studies with larger sample sizes are required to facilitate more accurate estimations among biological variations and to reduce the biological variability among the samples.
The current pathway analysis results indicated the involvement of the proteins in cell growth, metabolism, immunomodulation, and extracellular matrix components. A functional analysis revealed the possible relations of the proteins to the local immune system, gametes maturation, fertilization, and early embryo development [
44]. Several studies unveiled the molecular contents of oviductal EVs and were reviewed in Almiñana and Bauersachs [
45]. In felines, EVs contain three-fold more proteins than in bovines and are enriched in proteins related to energy metabolism, membrane modification, and reproductive function. A total of 1511 protein groups were identified through ultraperformance liquid chromatography and tandem mass spectrometry (UPLC-MS/MS) [
46]. Notably, a comprehensive analysis of bovine oviduct EVs revealed significant differences in hundreds of differentially expressed genes in frozen and fresh oviduct epitheliums [
19].
Oviduct EVs exert physiological actions on different spatial levels (
Figure 6). Lee et al. [
16] indicated that oviduct EVs upregulated the EGFR/MAPK signaling pathway in the canine cumulus cells on the level of oocyte maturation,. Moreover, oviduct EVs enhanced oocyte maturation and cumulus cell viability and proliferation, as well as reduced the production of reactive oxygen species and apoptotic rates. Additionally, according to our previous studies [
16,
17,
28], we found that oviduct cells exposed to progesterone significantly improved the oocyte and cumulus cell development via the EGFR and MAPK(ERK)1/3 signaling pathways. Therefore, we assumed that progesterone would partially modify the protein content of EVs in this study. In the current study, the OC-EVs analysis identified several proteins that belong to ERK/MAPK signaling (
Figure 5), such as MAPK1, ERK1/2, P38 MAPK, RAS, and HSP27. These pathways are involved in oocyte and cumulus proliferation and expansion [
17,
35,
36,
37,
38]. Paradoxically, canine oviduct EVs at high concentrations might perturb oocyte maturation through targeting the TGFβ pathway via mir-375 [
47]. On the embryonic level, oviduct EVs transferred mRNA and microRNA (miRNA) and altered the bovine embryo transcriptome [
19]. In a murine model, supplementing an embryo transfer medium with oviduct EVs improved birth rates by preventing apoptosis and promoting differentiation [
48]. On the oviduct level, a juxtracrine effect of oviduct EVs on the surrounding oviduct cells is also possible. An in vitro model showed that a culture with EVs derived from the oviductal mesenchymal cell line increased the number of ciliated cells in the Mullerian epithelial cell line, suggesting a juxtracrine/paracrine effect of oviduct cells in modulating their cell functions [
49]. On the sperm level, as previously mentioned, EVs regulate sperm functions and capacitation [
13,
15,
46,
50], while there is scant information about the effects of oviduct EVs on canine sperm functions. A recent report showed that using dog oviduct EVs improved their post-thaw motility and prevented a premature acrosome reaction of red wolf spermatozoa [
51].
Notably, several proteins were detected as associated with the actin cytoskeleton (
Table S3), such as actin, cofilin, transgelin, and lamin. The cofilin-actin pathway is essential for meiotic development and cytokinesis during oocyte maturation [
52]. A previous study suggested that actomyosin-cofilin pathways regulate meiotic spindle migration and cytokinesis during bovine oocyte maturation [
53]. During cytokinesis, the intermediate filament vimentin (
Table S3) contributes to the cleavage furrow, crucial for normal cell division. A slight distortion in the normal regulation of vimentin and other intermediate filament assembly/disassembly is associated with cytokinetic failure, aneuploidy, and binucleation, resulting in cell cycle distortion and cellular senescence [
54,
55].
Additionally, some metabolic enzymes were also detected, such as pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase (
Table S3), which are key players in glucose metabolism in the cell [
56]. A Gene Ontology analysis (
Table S4) and IPA (
Table S5) showed that OC-EVs contain proteins associated with different biological processes and canonical pathways involved in carbohydrate, lipid, and protein metabolism. Similarly, recent findings suggest that EVs regulate metabolism in COCs and/or embryos [
11,
23,
39]. Besides, proteins associated with the pathways involved in embryonic development [
57,
58], such as actin, cyclin-dependent kinases, and several intermediate filaments, were also detected (
Table S5).
The results also showed that OC-EVs contain numerous ribosome and RNA-binding proteins and other proteins involved in the process of protein synthesis, which may possess different RNAs to regulate gene expression and RNA degradation. They might transfer ribosomal constituents to the COCs and/or embryos (
Figure 3) [
59]. Moreover, protein processing in the rough endoplasmic reticulum is a fundamental process needed for cell survival in which the synthesis, folding, post-translational modification, transport, and sorting of proteins and some lipids occur [
60,
61]. Several proteins associated with rough endoplasmic reticulum functions were detected in the isolated EVs, such as endoplasmin (HSP90), 40S ribosomal protein S26, and 60S ribosomal protein L13a (
Tables S1 and S2).
A functional analysis of the OC-EVs revealed processes related to cell death and survival (
Table 1), indicating the possible role of EVs in regenerative effects on damaged cells of the oviductal canal, including the oocytes and/or the embryos [
62]. Notably, studying the proteomics of oviductal EVs highlights the possible effects on embryonic development. Several studies reported the positive effects of oviduct-EVs on embryonic development in different species. In bovines, oviduct epithelial cell-derived EVs increased the embryo cell number (trophectoderm and inner cell mass) and the post-vitrification survival, in addition to the alteration of essential transcripts expression [
14,
63], rendering them superior quality. Moreover, in vitro-produced embryos were able to uptake in vivo oviduct EVs during the culture and increased the blastocyst rate, prolonged the embryo survival, and improved the embryo quality, and this was confirmed through the functional proteomics analysis [
10].
Collectively, the extensive characterization of the protein cargo of OC-EVs revealed proteins that are associated with oocyte maturation and embryo development competence. Additionally, they may be associated with a variety of signaling processes that occur between the oocyte and cumulus cells, as well as cell death and survival. Our findings provide a strong basis for highlighting the potential function of OC-EVs as a paradigm for establishing a reliable system for in vitro oocyte maturation, in vitro fertilization, and the in vitro culture of preimplantation embryos in canine species.