Abstract
Introduction
Female reproductive aging is accompanied by progressive declines in ovarian mitochondrial function and steroidogenic capacity. We previously identified widespread age-associated alterations in alternative splicing in the mouse ovary, including isoform remodeling in genes involved in mitochondrial function, with particularly pronounced changes in granulosa cells. Among these candidates, Ndufs4, which encodes an accessory subunit of mitochondrial respiratory Complex I, exhibited increased expression of a short isoform in aged ovaries relative to a canonical long isoform. Because mitochondrial function is integral to steroid hormone biosynthesis, we investigated whether expression of the age-associated Ndufs4 isoform alters steroidogenic function in primary ovarian granulosa cells.
Methods
Female C57BL/6J mice were stimulated with PMSG, and ovaries were collected 46 h later. Primary granulosa cells were isolated, cultured, and transfected with in vitro-transcribed mRNA encoding the canonical Ndufs4 long isoform, the age-associated Ndufs4 short isoform, or mTagBFP2 control using Lipofectamine MessengerMAX. Following transfection, cells were stimulated with 8-Br-cAMP to activate cAMP-dependent steroidogenic signaling. Steroidogenic acute regulatory protein (StAR) abundance was assessed by quantitative immunofluorescence, and progesterone secretion into conditioned media was measured by ELISA.
Results
Expression of the age-associated short Ndufs4 isoform was associated with reduced StAR protein abundance compared with the canonical isoform, consistent with impaired regulation of a key mediator of mitochondrial cholesterol transport during steroidogenesis. Progesterone production following 8-Br-cAMP stimulation was also reduced in cells expressing the short isoform. Cells expressing the canonical long isoform produced 4.9 ng progesterone/well, whereas cells expressing the age-associated short isoform produced 2.7 ng/well, corresponding to a 45% reduction in progesterone production.
Conclusion
These preliminary findings suggest that overexpression of an age-associated Ndufs4 isoform attenuates mouse granulosa cell steroidogenic responsiveness. Reduced StAR abundance together with diminished cAMP-stimulated progesterone production support a functional link between age-associated Ndufs4 isoform remodeling and impaired granulosa cell steroidogenesis. Ongoing studies will define the isoform-specific effects of Ndufs4 and determine how age-associated transcript remodeling contributes to declining ovarian function during reproductive aging.