**2.1 EVs derived from the ovary**

The ovarian follicle is the basic unit of female ovary, which contains an oocyte that is surrounded by two-layer granulosa cells (GCs, including membrane/mural granulosa and cumulus granulosa), stromal cells (theca cells), and a liquid called follicular fluid (FF). FF offers a suitable micro-environment for follicle growth and oocyte development (oogenesis) in the ovary, so their relative stability of composition is indispensable. Similar to gap junctions between oocytes and somatic cells which mediated cell-to-cell communication, EVs in GCs, somatic cells, and FF mediate signal transmission and cell communication [7, 8], suggesting that EVs are carriers for molecular transfer in the ovary.

The first EVs in FF was reported in equine, which contains microRNAs (miRNAs) and proteins, and they could be taken by surrounding GCs *in vitro* and *in vivo* [9]. MiRNAs are involved in RNA silence as post-transcriptional regulators [10]. There are numerous miRNAs indirectly regulating follicular growth and oocyte maturation in human FF [7]: (1) miR-10b, miR-21-5p, miR-31, miR-95, miR-99b-3p, miR-134, miR-135b, miR-140-3p, and miR-190b participate in WNT signaling through glycoprotein signaling molecular WNTs potentially regulate follicle formation, growth, and ovulation/luteinization [11]. (2) miR-203, miR-218 regulate MAPK (ERK1/2) pathway which activation is crucial for oocyte meiotic resumption, GCs proliferation and cumulus expansion [12]. (3) miR-489, miR-493, miR-503, miR-542-5p, miR-654-3p, miR-874, miR-886-5p, miR-887 as mediators of TGFβ. (4) miR-337-5p, miR-339-3p, miR-370, miR-449a, miR-455-5p, miR-483-5p as mediators of ErbB, etc. In addition, as we know, the expansion of cumulus-oocyte complex (COC) is a key process of ovulation. A study showed that bovine FF EVs taken in cumulus could increase gene expression of cumulus expansion (PTGS2, PTX3, and TNFAIP6) and support the expansion process *in vitro* [13].
