Dysregulation of alternative splicing patterns in the ovaries of reproductively aged mice

Abstract

Female reproductive aging is marked by a decline in oocyte quality, yet the underlying molecular mechanisms remain incompletely understood. Here, we used long-read direct RNA-sequencing to map transcript isoform changes in mouse ovaries across reproductive age. Comparing young and aged mice under controlled gonadotropin stimulation, we identified widespread alternative splicing changes, including shifts in exon usage, splice site selection, and transcript boundaries. Aged ovaries exhibited increased isoform diversity, favoring distal start and end sites, and a significant rise in exon skipping and intron retention events. Many of these age-biased splicing events altered open reading frames, introduced premature stop codons, or disrupted conserved protein domains. Notably, mitochondrial genes were disproportionately affected. We highlight Ndufs4, a Complex I subunit, as a case in which aging promotes a truncated isoform lacking the canonical Pfam domain. Structural modeling suggests this splice variant could impair Complex I assembly, providing a mechanistic link between splicing and mitochondrial dysfunction in the aging ovary. These findings support the existence of a splicing-energy-aging axis in ovarian physiology, wherein declining mitochondrial function and adaptive or maladaptive splicing changes are intertwined. Our study reveals that alternative splicing is not merely a byproduct of aging but a dynamic, transcriptome-wide regulatory layer that may influence oocyte competence and ovarian longevity. These insights open new avenues for investigating post-transcriptional mechanisms in reproductive aging and underscore the need to consider isoform-level regulation in models of ovarian decline.