MicroRNAs (miRNAs) play important roles in many developmental processes, including cell differentiation and apoptosis. Transition of proliferative ova

Expansion of the cumulus complex surrounding the oocyte is critical for ovulation of a fertilizable egg. The ovulation inducing surge of luteinizing hormone leads to an increased expression of genes such as prostaglandin-endoperoxide synthase 2 (Ptgs2), pentraxin-related protein 3 (Ptx3), and tumor necrosis factor alpha-induced protein 6 (Tnfaip6) that support cumulus expansion. Factors released by mural granulosa and cumulus granulosa cells into the follicular fluid induce paracrine signaling within the follicular compartment. The follicular fluid that separates these distinct granulosa cell types is an enriched fluid containing numerous proteins, nucleic acids, and other macromolecules. Extracellular vesicles (EVs) are also present however no physiologically relevant functions of follicular EVs have yet been demonstrated. In our study, the effect of follicular EVs on COC expansion and relevant gene expression was assayed. Follicular EVs were isolated using ultracentrifugation from follicular fluid of small (3-5 mm) and large antral (>9 mm) bovine follicles, then characterized by nanoparticle tracking analysis, electron microscopy and western blot analysis. To test for bioactivity, mouse and bovine COC were cultured with follicular EVs. Cumulus expansion and Ptgs2, Ptx3, and Tnfaip6 gene expression were measured following COC maturation culture. The results demonstrated that follicular EVs can support both measurable cumulus expansion and increased gene expression.

The ribonuclease III endonuclease, Dicer1 (also known as Dicer), is essential for the synthesis of the 19–25 nucleotide noncoding RNAs known as micro-RNAs (miRNAs). These miRNAs associate with the RNA-induced silencing complex to regulate gene expression posttranscriptionally by base pairing with 3′untranslated regions of complementary mRNA targets. Although it is established that miRNAs are expressed in the reproductive tract, their functional role and effect on reproductive disease remain unknown. The studies herein establish for the first time the reproductive phenotype of mice with loxP insertions in the Dicer1 gene (Dicer1^fl/fl) when crossed with mice expressing Cre-recombinase driven by the anti-müllerian hormone receptor 2 promoter (Amhr2^Cre/+). Adult female Dicer1^fl/fl;Amhr2^Cre/+ mice displayed normal mating behavior but failed to produce offspring when exposed to fertile males during a 5-month breeding trial. Morphological and histological assessments of the reproductive tracts of immature and adult mice indicated that the uterus and oviduct were hypotrophic, and the oviduct was highly disorganized. Natural mating of Dicer1^fl/fl;Amhr2^Cre/+ females resulted in successful fertilization as evidenced by the recovery of fertilized oocytes on d 1 pregnancy, which developed normally to blastocysts in culture. Developmentally delayed embryos were collected from Dicer1^fl/fl; Amhr2^Cre/+ mice on d 3 pregnancy when compared with controls. Oviductal transport was disrupted in the Dicer1^fl/fl;Amhr2^Cre/+ mouse as evidenced by the failure of embryos to enter the uterus on d 4 pregnancy. These studies implicate Dicer1/miRNA mediated posttranscriptional gene regulation in reproductive somatic tissues as critical for the normal development and function of these tissues and for female fertility.

MicroRNAs (miRNAs) mediate posttranscriptional gene regulation by binding to the 3′ untranslated region of messenger RNAs to either inhibit or enhance

Cells respond to inflammatory disease states by releasing exosomes containing highly specific protein and RNA cargos, but how inflammation alters cargo specificity and secretion of exosomes is unknown. We show that increases in exosome secretion induced by either viral infection or LPS/ATP exposure result from inflammasome activation and subsequent caspase-1–dependent cleavage of the trafficking adaptor protein RILP. This cleaved form of RILP promotes the movement of multivesicular bodies toward the cell periphery and induces selective exosomal miRNA cargo loading. We have identified a common short sequence motif present in miRNAs that are selectively loaded into exosomes after RILP cleavage. This motif binds the RNA binding protein FMR1 and directs miRNA loading into exosomes via interaction with components of the ESCRT (endosomal sorting complex required for transport) pathway. These results indicate that inflammasome-mediated RILP cleavage, and sequence-specific interactions between miRNAs and FMR1, play a significant role in exosome cargo loading and enhanced secretion during cellular inflammatory responses.

Exosomes and microvesicles (i.e., extracellular vesicles: EVs) have been identified within ovarian follicular fluid and recent evidence suggests that EVs are able to elicit profound effects on ovarian cell function. While existence of miRNA within EVs has been reported, whether EV size and concentration as well as their cargos (i.e., proteins and RNA) change during antral follicle growth remains unknown. Extracellular vesicles isolated from follicular fluid of small, medium and large bovine follicles were similar in size, while concentration of EVs decreased progressively as follicle size increased. Electron microscopy indicated a highly purified population of the lipid bilayer enclosed vesicles that were enriched in exosome biomarkers including CD81 and Alix. Small RNA sequencing identified a large number of known and novel miRNAs that changed in the EVs of different size follicles. Ingenuity Pathway Analysis (IPA) indicated that miRNA abundant in small follicle EV preparations were associated with cell proliferation pathways, while those miRNA abundant in large follicle preparations were related to inflammatory response pathways. These studies are the first to demonstrate that EVs change in their levels and makeup during antral follicle development and point to the potential for a unique vesicle-mediated cell-to-cell communication network within the ovarian follicle.