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Edited by Michael Marletta, University of California, Berkeley, CA; received June 23, 2021; accepted December 21, 2021
Multidrug Resistance Proteins (MRPs) are typically implicated in cancer biology. Here, we show that MRP9 and MRP5 localize to mitochondrial-associated membranes and play a concerted role in maintaining mitochondrial homeostasis and male reproductive fitness. Our work fills in significant gaps in our understanding of MRP9 and MRP5 with wider implications in male fertility. It is plausible that variants in these transporters are associated with male reproductive dysfunction.
Multidrug Resistance Proteins (MRPs) are transporters that play critical roles in cancer even though the physiological substrates of these enigmatic transporters are poorly elucidated. In Caenorhabditis elegans, MRP5/ABCC5 is an essential heme exporter because mrp-5 mutants are unviable due to their inability to export heme from the intestine to extraintestinal tissues. Heme supplementation restores viability of these mutants but fails to restore male reproductive deficits. Correspondingly, cell biological studies show that MRP5 regulates heme levels in the mammalian secretory pathway even though MRP5 knockout (KO) mice do not show reproductive phenotypes. The closest homolog of MRP5 is MRP9/ABCC12, which is absent in C. elegans, raising the possibility that MRP9 may genetically compensate for MRP5. Here, we show that MRP5 and MRP9 double KO (DKO) mice are viable but reveal significant male reproductive deficits. Although MRP9 is highly expressed in sperm, MRP9 KO mice show reproductive phenotypes only when MRP5 is absent. Both ABCC transporters localize to mitochondrial-associated membranes, dynamic scaffolds that associate the mitochondria and endoplasmic reticulum. Consequently, DKO mice reveal abnormal sperm mitochondria with reduced mitochondrial membrane potential and fertilization rates. Metabolomics show striking differences in metabolite profiles in the DKO testes, and RNA sequencing shows significant alterations in genes related to mitochondrial function and retinoic acid metabolism. Targeted functional metabolomics reveal lower retinoic acid levels in the DKO testes and higher levels of triglycerides in the mitochondria. These findings establish a model in which MRP5 and MRP9 play a concerted role in regulating male reproductive functions and mitochondrial sufficiency.
Author contributions: I.G.C., P.K., D.B., and I.H. designed research; I.G.C., P.K., P.W., J.Y., J.D.P., M.A.K., and I.H. performed research; I.G.C., P.K., J.L., J.D.P., M.A.K., D.B., and I.H. contributed new reagents/analytic tools; I.G.C., P.K., J.L., P.W., J.Y., J.D.P., M.A.K., D.B., and I.H. analyzed data; and I.G.C. and I.H. wrote the paper.
Competing interest statement: I.H. is the President and Founder of Rakta Therapeutics Inc. (College Park, MD), a company involved in the development of heme transporter-related diagnostics.
This article is a PNAS Direct Submission.
This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2111617119/-/DCSupplemental.
RNAseq data have been deposited in NCBI Gene Expression Omnibus (GSE176740) (94).
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