In Vivo and In Vitro Matured Oocytes From Mice of Advanced Reproductive Age Exhibit Alternative Splicing Processes for Mitochondrial Oxidative Phosphorylation

Abstract

The mean age of women seeking infertility treatment has gradually increased over recent years. This has coincided with the emergence of in vitro maturation (IVM), a method used in assisted reproductive technology for patients with special requirements. However, when compared with conventional in vitro fertilization, IVM is associated with poor embryonic development potential and low live birth rates, thus limiting the widespread application of this technique. In this study, we performed RNA-sequencing transcriptomic assays and identified a total of 2,627 significant differentially expressed genes (DEGs) between IVM oocytes and in vivo matured oocytes from mice of advanced reproductive age. Next, Kyoto Encyclopedia of Genes and Genomes pathway analysis was used to identify the potential functions of the DEGs. The most significantly enriched pathway was oxidative phosphorylation (OXPHOS). In addition, we constructed a protein-protein interaction network to identify key genes and determined that most of the hub genes were mtDNA-encoded subunits of respiratory chain complex I. Antioxidant supplementation lead to an increase in ATP production and reduced the gene expression profile of the OXPHOS pathway in the IVM group. Moreover, alternative splicing (AS) events were identified during in vivo or in vitro oocyte maturation; data showed that skipped exons were the most frequent type of AS event. A number of genes associated with the OXPHOS pathway exhibited alterations in AS events, including Ndufa7, Ndufs7, Cox6a2, Ndufs5, Ndufb1, and Uqcrh. Furthermore, the process of IVO promoted the skipping of exon 2 in Ndufa7 and exon 3 in Ndufs7 compared with the IVM oocytes, as determined by semi−quantitative RT−PCR. Collectively, these findings provide potential new therapeutic targets for improving IVM of aged women who undergo infertility treatment.

Deregulation of oxidative phosphorylation pathways in embryos derived in vitro from prepubertal and pubertal heifers based on whole-transcriptome sequencing

“…The OXPHOS pathway is a significant source of ATP production in mitochondria and depends on five multisubunit respiratory chain protein complexes that are directly involved in oocyte maturation [ 34 ], fertilization [ 35 ] and preimplantation development of embryos [ 36 , 37 ]. In the current study, we found that the first and fifth complexes of the respiratory chain had the highest number of DEGs.…”

“…Moreover, NDUFA3 is located in the inner mitochondrial membrane and communicates with the previously mentioned core subunits ND1, ND3 and NDUFS8 and with the supernumerary subunits NDUFA8 and NDUFA13. Qin et al [ 37 ] reported that the expression of NDUFA3 was downregulated in in vivo matured oocytes compared to in vitro matured oocytes, which are generally considered to have lower developmental competence [ 37 ]. In contrast, in our study, we demonstrated higher expression of NDUFA3 in blastocysts derived from oocytes collected from pubertal heifers than in those derived from prepubertal heifers, which in turn accounts for their better developmental competence.…”

“…This phenomenon has a crucial role in embryogenesis in the process of supporting rapid cell proliferation [ 66 ]. Qin et al [ 37 ] demonstrated the downregulation of UQCRC1 in in vitro matured oocytes from mice, which had higher levels of ROS than in vivo matured oocytes [ 37 ]. We presume that the lower expression of the UQCRC1 gene in blastocysts derived from prepubertal heifers oocytes compared to pubertal heifers oocytes could also lead to a higher level of ROS, which we demonstrated in a previous study in those blastocysts [ 21 ], and at the same time reinforce the Warburg effect.…”

“…However, in our study, only one of the DEGs involved in OXPHOS, ATPase H+/K + transporting non gastric alpha2 subunit ( ATP12A ), which belongs to the cation transport ATPase (P-type), was demonstrated to have higher expression in blastocysts derived from oocytes collected from prepubertal heifers than in those derived from pubertal heifers. Qin et al [ 37 ] demonstrated higher expression of ATP12A and ROS levels in in vitro matured (IVM) oocytes compared to in vivo matured (IVO) oocytes derived from mice [ 37 ]. We also indicated higher expression of ATP12A in blastocysts derived from oocytes collected from prepubertal heifers than in blastocysts derived from oocytes collected from pubertal animals, which correlated with previously demonstrated higher expression of ROS in those blastocysts, as confirmed by immunofluorescence staining [ 21 ].…”