As known to us, follicular follicle in the ovary provides a unique microenvironment for oocyte development, such as interactions between follicular somatic cells and oocytes, and it is crucial to study the components of follicular fluid to elucidate the mechanism of oocyte maturation. It has been found that exosomes (EXs) are an essential carrier for signals transduction of interaction between follicular somatic cell and oocyte in follicular fluid media according to some reports19,20. In this paper, HFF exosomes can be successfully isolated with an average size of 75 nm, and checked by using transmission electron microscopy, which is similar to these reports21 − 23.
For understanding the molecular mechanism of oocyte maturation, it is crucial to reveal the axis of follicular somatic cells-EXs-oocyte by study the expressed genes in the PFF EXs on the RNA level. It was also reported that identified potential miRNA targets using bioinformatics analysis, which is very important to porcine oocyte growth in follicles24,25. Especially, follicle size is closely related to oocyte development according to previous reports26,27, and the miRNA sequencing experiment using the total 12 PFF exosomes samples(3 repeats each group with different size follicle sample) was carried out according to above methods. The results firstly show that about 99% of all valid reads are with group A and B, and about 99% of all valid reads are with group C and D from four six samples could be successfully mapped to the porcine non-coding RNAs database as indicated in table 1 for further analysis. Secondly, these 8 miRNA genes of 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) expression is significantly enhanced with porcine follicle growth from < 3 mm to 5 mm, and miR-10a-5p expression is almost 8 times higher than group A sample as shown in Fig. 1. It is also found that miR-10a family expression is closely related to reproductive system function, which miR-10a and miR-10b, are expressed at basal levels in GCs but are highly expressed in theca and stroma cells within the ovary, and they could repress proliferation and induce apoptosis in human, mouse and rat granulosa cells, at least partly through repressing BDNF by directly binding to its 3′ UTR, and the miR-10 family and the TGF-β pathway form a negative feedback loop in GCs28. With porcine follicle further growth to 5–8 mm, these 6 miRNA genes of PFF EXs (miR-194a-5p, miR-7137-3p,miR-182, miR-146b, miR-4332 and miR-9793-5p) expression is significantly enhanced compared to control group(< 3 mm), and miR-194a-5p expression is almost 4 times higher than group A sample as shown in Fig. 2. Exosomes is carriers to follicles from surrounding somatic cells body blood to influence oocyte maturation, which immune stimulus could enhance vasodilatation in order to promote EXs function29,30. It is also indicated that miR-194a family expression play important roles in immunology response and function, such as flounder immune response31 and zebrafish32.
Until follicle grow to 8 mm, these 9 miRNA genes of 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) expression is significantly reduced compared to control group(< 3 mm), and miR-46a-5p and miR-202a-5p gene expression is almost 3 times lower than group A sample as shown in Fig. 3. It is proved that miR-202a-5p gene is greatly expressed in bovine un-maturated oocytes33, and miR-199a-5p gene is greatly expressed in GV stage occyte34. Among these group B, C and D, It is found that these different expressed miRNAs are choose and all clustered in Figs. 4, 5 and 6 for further analysis, which is indicates some are closely related to oocyte development proved by those published papers28,35,36. Based on the above miRNAs significant expression analysis, ten relevant miRNAs (miR-10a-5 ps, 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 are choose to take for further target genes analysis. Currently, there is no gold standard for miRNA gene target analysis37,38, we use Miranda and RNA software to predict the targeting regulation for further GO analysis and pathways according to reported papers39,40.
Those found miRNAs(miR-125b, let-7d-5p,miR-200b, miR-26a and miR-92a) expressed in PFF exosomes from different size follicles, which is very closely related to oocyte development, which is reported by previous published papers17,41. The gene number and significance about neurotransmitter secretion changes greatly as shown in Fig. 7–8, which the life activity of the hypothalamus-pituitary-ovarian endocrine axis is very vigorous during porcine follicular growth process42. The result also show that many targeting genes involves in regulation of FSH secretion in Fig. 7–8, which is proved by many reports about FSH controlling the follicular development in different size follicles43,44. Therefore, it can be indicated that the functions of many targeting gene by these miRNAs mainly focus on oocyte development by FSH simulating porcine follicular growth form small stage (Group A), middle stage (Group B) to matured stage (Group C and D). The results show that the pathways mainly related to the biosynthesis pathway of TGF-beta signaling pathway, Primary bile acid biosynthesis, Nicotinate and nicotinamides metabolism as shown in Fig. 9, which these potential target genes are closely related to the oocyte development and follicle growth. It was also found that many targeting genes by these miRNAs mainly involved in TGF-beta related signaling pathway, which may play a significant role during early stages of porcine oocytes nuclear and cytoplasmic maturation, and these investigated transcripts may be also recommended as the markers of porcine oocytes with high capability in further embryo development45,46.
However, these potential genes GO and pathway are predicted; the associations observed in previous published papers are limited. Some research papers also show that each miRNA can have many gene targets and the gene targets may be different based on cell type 47, and the exact influence of a miRNA on gene expression may be physiologically indispensable but difficult to identify statistically48. In conclusion, this study provides new insights into the global transcriptome changes and the abundance of specific transcripts in porcine oocytes in correlation with follicle size.
Therefore,our study has shown that differently expressed miRNAs of PFF EXs in the different size follicles with group B, C and D compared to the control (group A), which has different targeting cluster genes(miR-125b, let-7d-5p,miR-200b, miR-26a and miR-92a) in porcine oocyte maturation according to the GO and Pathway analysis, which could be used as biomarkers for understanding oocyte maturation process and choosing oocyte with high quality for further research. Moreover, there are still some questions about porcine oocyte development mechanism related to MiRNA in EXs as following. What is the relation of these different expressed miRNAs contained by exosomes and oocyte maturation in details? Which type’s miRNAs can genuinely promote follicle growth and oocyte maturation and how to control the network about oocyte cytoplasmic and nuclear maturation mechanisms?