RESUMEN
N6-methyladenosine (m6A) plays a crucial role in many bioprocesses across species, but its function in granulosa cells during oocyte maturation is not well understood in animals, especially domestic animals. We observed an increase in m6A methyltransferase-like 3 (METTL3) in granulosa cells during oocyte maturation in Haimen goats. Our results showed that knockdown of METTL3 disrupted the cell cycle in goat granulosa cells, leading to aggravated cell apoptosis and inhibition of cell proliferation and hormone secretion. Mechanistically, METTL3 may regulate the cell cycle in goat granulosa cells by mediating Aurora kinase B (AURKB) mRNA degradation in an m6A-YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) manner and participating in AURKB transcription via the Cyclin D1 (CCND1)-Retinoblastoma protein (RB)-E2F transcription factor 1 (E2F1) pathway. Overall, our study highlights the essential role of METTL3 in granulosa cells during oocyte maturation in Haimen goats. These findings provide a theoretical basis and technical means for understanding how RNA methylation participates in oocyte maturation through granulosa cells.
Asunto(s)
Cabras , Metiltransferasas , Animales , Femenino , Metiltransferasas/genética , Metiltransferasas/metabolismo , Cabras/metabolismo , Aurora Quinasa B , Ciclina D1/genética , Ciclo CelularRESUMEN
RNA N6-methyladenosine (m6A) is essential for many bioprocesses in many species, but its role in goat testis development remains elusive, especially alkB homolog 5 (ALKBH5), one of the m6A demethylases. To this end, nine healthy Haimen goats of different ages were chosen randomly to provide testes. The results showed that the expression level of ALKBH5 was increased significantly (P < 0.05) in the 9-month group compared with the 0-day and 3-month groups, and ALKBH5 was located in goat spermatocytes with the highest expression level compared with Leydig cells and Sertoli cells. Thus, pcDNA3.1-ALKBH5 was constructed to explore the influences of the ALKBH5 increase in goat spermatogonial stem cells (SSC) in vitro. The results showed that the expression level of ALKBH5 in SSC transfected with pcDNA3.1-ALKBH5 (OE_ALKBH5) was significantly increased (P < 0.001) compared with that in SSC transfected with pcDNA3.1-EGFP (EGFP). With ALKBH5 overexpression in SSC, flow cytometry analysis showed that cells at G1 phase were significantly reduced (P < 0.01), while cells at S phase significantly increased (P < 0.01), and cell apoptosis was inhibited. Accordingly, the mRNA degradation of CCND1, CCNE1, and BCL2 was suppressed with ALKBH5 overexpression in SSC after treatment with actinomycin D. Furthermore, the mRNA levels of pluripotency maintenance- and cell differentiation-associated genes were changed between the two groups. Overall, the results indicated the crucial role of ALKBH5 during Haimen goat testis development. The results of this study provide a theoretical basis and technical means for RNA methylation participating in goat testis development.