As is already known, ovarian follicles provide a unique microenvironment for oocyte development and allow for interactions between follicular somatic cells and oocytes. Given this, it is crucial to study the components of follicular fluid to better elucidate their role in the mechanism of oocyte maturation. Previous work has shown that exosomes (EXs) are an essential carrier for the signal transduction occurring between follicular somatic cells and oocytes in follicular fluid21,22. Here, PFF exosomes were successfully isolated and confirmed using transmission electron microscopy. Our findings were similar to those of other reports23.
To better understand the molecular mechanism behind oocyte maturation, it is crucial to reveal the axis of follicular somatic cells-EXs-oocyte by studying the expressed genes in the PFF EXs at the level of the RNA. Past work has used bioinformatics analysis to identify potential miRNA targets which were very important to growth of porcine oocytes in follicles24,25. In particular, follicle size has been shown to be closely related to oocyte development26,27.
Mature miRNAs are produced by a series of nuclease-mediated cleavage processes of longer primary transcripts. These miRNAs are then assembled into RNA-induced silencing complexes to identify target genes, which is done by base complementary base pairing. The degree of complementarity guides the silencing complexes to their target genes, whereby they degrade or block the translation of their target genes. Given this, we conducted an miRNA sequencing experiment using a total of 12 PFF exosome samples (three repeats per group, with groups composed of different follicle sizes) according to previously described methods. First, our results showed that approximately 99% of all valid reads were with groups A-D. As indicated in Table 1, we successfully mapped 12 samples to the porcine non-coding RNA database for further analysis. Our results also showed that 8 miRNA genes obtained from PFF EXs (miR-10a-5p, miR-200b, miR-429, miR-192, miR-141, miR-221-3p, miR-425-5p, miR-7-5p and miR-92a) had significantly enhanced expression in porcine follicles with diameters <3 mm to 5 mm. Moreover, miR-10a-5p expression was almost 8 times higher than that in group A (Figure 2). According to these results, it can be indicated that miR-10a-5p mainly limited expressed in small follicles(contained immature oocytes), but these miRNAs(miR-10a-5p, miR-200b, miR-429, miR-192, miR-141, miR-221-3p, miR-425-5p, miR-7-5p and miR-92a) enhanced the expression with follicle growth(from 5mm diameter follicles to previous ovulation stage) , which is very important to elucidate the mechanism of porcine oocyte maturation.
Our results also indicated that miR-10a family expression was closely related to reproductive system function. More specifically, miR-10a and miR-10b were both expressed at basal levels in granulosa cells, but were highly expressed in theca and stroma cells within the ovary. This indicated they could repress proliferation and induce apoptosis in human, mouse, and rat granulosa cells. This would partly occur by BDNF repression through direct binding to its 3′ UTR, with the miR-10 family and the TGF-β pathway forming a negative feedback loop in GCs28. With porcine follicles of larger diameter (5-8 mm), there was significantly enhanced expression of six miRNA genes obtained from PFF EXs (miR-194a-5p, miR-7137-3p,miR-182, miR-146b, miR-4332 and miR-9793-5p) when compared to the control group (<3 mm). Of these, miR-194a-5p expression was almost four times higher than that of group A (Figure 3). Exosomes act as carriers to follicles from the surrounding somatic cells as well as the body and blood to influence oocyte maturation. Moreover, immune stimulus has been shown to enhance vasodilatation to promote exosomal function29,30. Past work has also shown that miR-194a family expression plays an important role in immunological responses and function, including the flounder31 and zebrafish32 immune responses. Further results shown that miR-194a-5p is highly related to oocyte maturation of follicles growing stage, which can induce immune reaction to enhance ovary local blood circulation to provide more nutrients.
When follicles grow to a diameter of 8 mm, 9 miRNA genes obtained from PFF EXs (miR-202-5p, miR-199a-5p, miR-21-5p, miR-26b-5p, miR-26a, miR-29c, miR-24-3p, miR-146a-5p, and miR-10b) had significantly reduced expression when compared to the control group (<3 mm). Of these, both miR-46a-5p and miR-202a-5p gene expression levels were almost three times lower than those of group A (Figure 4). It has been shown that the miR-202a-5p gene is highly expressed in bovine un-maturated oocytes33, while miR-199a-5p gene is highly expressed in germinal vesicle stage occytes34. From groups B, C, and D, these selected, differentially expressed miRNAs were all clustered in Figures 5, 6, and 7 for further analysis. This analysis confirmed earlier work, indicating that some were closely related to oocyte development35. Based on the significant expression of the above miRNAs, we selected ten relevant miRNAs—miR-10a-5ps, miR-200b, miR-141, miR-92a, miR-221-3p, miR-21-5p, miR-26a, let-7d-5p, miR-125b and miR-99a-5p—related to porcine oocyte development for further target genes analysis. Currently, there is no gold standard for miRNA gene target analysis36,37. Given this, we used Miranda and RNA software to predict the targeting regulation for further GO and pathway analyses according to previously reported papers38,39.
This analysis found selected miRNAs (miR-125b, let-7d-5p, miR-200b, miR-26a, and miR-92a) were expressed in PFF exosomes obtained from different-sized follicles. These miRNAs were also very closely related to oocyte development, which has been previously reported40. The gene number and their significance regarding neurotransmitter secretion changed greatly (Figures 8-9). It is known that the activity of the hypothalamus-pituitary-ovarian endocrine axis is very vigorous during processes related to porcine follicular growth41. These results also show that many targeted genes were involved in the regulation of FSH secretion (Figures 7-8), This corroborates previous work, which has shown how FSH controls the follicular development in different-sized follicles42,43. Therefore, we concluded that the functions of many of these miRNA-targeted genes were primarily focused on oocyte development by FSH simulation. This effect occurs through the porcine follicular growth stage, from its early (group A), middle (group B), and matured (groups C and D) stages. Collectively, these results showed that important pathways identified from this work included those related to the biosynthesis of TGF-beta signaling, primary bile acid biosynthesis, and both nicotinate and nicotinamides metabolism (Figure 10). Importantly, these potential target genes are all closely related to oocyte development and follicle growth. Past work has also shown that many of these miRNA-targeted genes were mainly involved in TGF-beta-related signaling. This pathway may play a significant role during the early stages of porcine oocytes nuclear and cytoplasmic maturation.
It should be noted that while these are potential genes predicted from our GO and pathway analyses, their associations as observed in previous published papers are limited. Some past work has shown that each miRNA can have many gene targets and that these gene targets may be different and based on cell type45. Moreover, the exact influence of one miRNA on gene expression may be physiologically indispensable, but difficult to identify statistically46. In conclusion, this study provides new insights into the global transcriptome changes and the abundance of specific transcripts in porcine oocytes that have been correlated with follicle size.
Taken together, our study has identified differently expressed miRNAs obtained from PFF EXs in different-sized follicles (groups B, C, and D) when compared to the control (group A). Using our GO and pathway analyses, we have identified different targeting cluster genes—miR-125b, let-7d-5p, miR-200b, miR-26a and miR-92a—important to porcine oocyte maturation. These miRNAs could be used as biomarkers for a better understanding of the oocyte maturation process and allow the selection of high-quality oocytes for further research. Despite this work, there remain many open questions about porcine oocyte development mechanisms related to exosomal miRNAs. For instance, what is the detailed relationship between these differentially expressed exosomal miRNAs and oocyte maturation? Which type of miRNA can specifically promote follicle growth and oocyte maturation? How do these miRNAs control the mechanisms regarding oocyte cytoplasmic and nuclear maturation?