Unraveling the Transcriptomic Profiles of Large and Small Donkey Follicles.

Background: The diameter of mature follicles in donkeys is several times larger than in cattle and sheep, but the key genes responsible for maintaining follicular development and preventing apoptosis remain unclear.

Methods: This study observed the process of donkey follicular development using ultrasound and analyzed the changes in common reproductive hormones in serum. Granulosa cells (GCs) were collected from large (mature follicles, diameter ≥ 37 mm) and small (atretic follicles, diameter 10-25 mm) follicles for sequencing to screen differentially expressed genes (DEGs) and signaling pathways influencing the development of mature follicles. The roles of selected genes were further validated in in vitro cultured GCs.

Results: Donkey follicles exhibited rapid growth 5-7 days before ovulation, reaching maturity at a diameter of 37 mm. The maximum diameter of ovulatory follicles was approximately 40.7 mm, while non-ovulatory follicles began to undergo atresia when reaching about 25 mm. Serum reproductive hormone levels aligned with follicular developmental status. RNA sequencing identified 3291 DEGs between large and small follicles, with KEGG analysis highlighting enrichment in the PI3K-Akt signaling pathway, focal adhesion, amoebiasis, and cancer pathways. Lentiviral overexpression and interference assays targeting the DEGs and revealed that positively regulates , a key gene in the PI3K-Akt pathway.

Conclusions: The gene in mature donkey follicles regulates in the PI3K-Akt signaling pathway, potentially inhibiting apoptosis in follicular granulosa cells and sustaining follicular development until ovulation. This study provides insights into the mechanisms underlying follicular development in donkeys.