Effects of sheep follicular fluid-derived exosomes and miR-148a on theca cell function in vitro

Exosomes facilitate intercellular communication by transporting proteins, nucleic acids, and other biomolecules, impacting recipient cell functions. In follicular development, exosomes from follicular fluid, secreted by granulosa cells, oocytes, and theca cells (TCs), are essential for follicle health. TCs are key in this process, influencing both development and hormonal output. However, the impact of sheep follicular fluid exosomes on TCs proliferation and the roles of the miRNAs they carry remain unclear. This study aims to investigate these effects, examining how exosomes and their miRNAs influence TCs development and hormone secretion through high-throughput sequencing.

In this study, exosomes were isolated from sheep follicular fluid by ultracentrifugation, and their integrity was confirmed by determining the particle size distribution by nanoparticle tracking analysis (NTA) and detecting marker proteins such as CD63 and TSG101 by Western blot. Subsequently, an in vitro isolation and culture system for sheep theca cells (TCs) was established, and the cells were treated with 200 µg/mL of exosomes. The functional effects of exosomes were assessed by EdU proliferation assay and ELISA for steroid hormone secretion. The exosome small RNAs were extracted and sequenced, and the 20 miRNAs with the highest expression abundance were screened, and the target genes were predicted using TargetScan8.0 and miRDB, and the predicted genes were analysed by GO and KEGG enrichment. For miR-148a, its mimic and inhibitor were synthesised and transfected into TCs to verify its effects on cell proliferation and hormone secretion; the targeting relationship between miR-148a and transforming growth factor β2 (TGFβ2) was verified by combining with a dual luciferase reporter system, and TGFβ2 was further knocked down by siRNA to evaluate its role in the proliferation of TCs.

The results showed that exosomes with a particle size distribution of 30-150 nm were successfully obtained by ultracentrifugation, which expressed CD63 and TSG101 with good integrity. Screening of exosome concentration showed that 200 µg/mL of exosomes significantly increased the proliferation rate and the secretion level of steroid hormones in TCs. Small RNA sequencing results showed that 130 miRNAs were identified, and the top 20 high-abundance miRNAs predicted 37,343 target genes. GO and KEGG analyses showed that these target genes were significantly enriched in follicle development-related signalling pathways, such as PI3K-AKT, MAPK, Rap1 and Ras. Functional experiments demonstrated that miR-148a mimics could significantly promote TCs proliferation, but had no significant effect on steroid hormone secretion. Dual luciferase and siRNA experiments showed that miR-148a directly targeted the 3'UTR of TGFβ2. Knock-down of TGFβ2 by transfecting its siRNA significantly enhances the proliferation rate of TCs, indicating that miR-148a promotes TC proliferation by down-regulating TGFβ2.

The above findings indicate that sheep follicular fluid exosomes can significantly enhance follicular membrane cell proliferation and steroid hormone secretion. The miR-148a carried in exosomes can promote the proliferation of TCs by inhibiting the expression of its target gene TGFβ2. This study will help to reveal the molecular mechanism of exosomes in follicular development and provide new perspectives for improving reproductive efficiency and genetic improvement in sheep.