Gestagen is a collective term for endogenous and synthetic progesterone receptor (PR) ligands. In teleost fishes, 17α,20β-dihydroxy-4-pregnen-3-one (DHP) and 17α,20β,21-trihydroxy-4-pregnen-3-one (20β-S) are the predominant progestogens, whereas in other vertebrates the major progestogen is progesterone (P4). Progestins are components of human contraceptives and hormone replacement pharmaceuticals and, with P4, can enter the environment and alter fish and amphibian reproductive health. In this study, our primary objectives were to clone the fathead minnow (FHM) nuclear PR (nPR), to develop an in vitro assay for FHM nPR transactivation, and to screen eight gestagens for their ability to transactivate FHM nPR. We also investigated the ability of these gestagens to transactivate FHM androgen receptor (AR). Fish progestogens activated FHM nPR, with DHP being more potent than 20β-S. The progestin drospirenone and P4 transactivated the FHM nPR, whereas five progestins and P4 transactivated FHM AR, all at environmentally relevant concentrations. Progestins are designed to activate human PR, but older generation progestins have unwanted androgenic side effects in humans. In FHMs, several progestins proved to be strong agonists of AR. Here, we present the first mechanistic evidence that environmental gestagens can activate FHM nPR and AR, suggesting that gestagens may affect phenotype through nPR- and AR-mediated pathways.

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Heme-iron recycling from senescent red blood cells (erythrophagocytosis) accounts for the majority of total body iron in humans. Studies in cultured cells have ascribed a role for HRG1/SLC48A1 in heme-iron transport but the in vivo function of this heme transporter is unclear. Here we present genetic evidence in a zebrafish model that Hrg1 is essential for macrophage-mediated heme-iron recycling during erythrophagocytosis in the kidney. Furthermore, we show that zebrafish Hrg1a and its paralog Hrg1b are functional heme transporters, and genetic ablation of both transporters in double knockout (DKO) animals shows lower iron accumulation concomitant with higher amounts of heme sequestered in kidney macrophages. RNA-seq analyses of DKO kidney revealed large-scale perturbation in genes related to heme, iron metabolism and immune functions. Taken together, our results establish the kidney as the major organ for erythrophagocytosis and identify Hrg1 as an important regulator of heme-iron recycling by macrophages in the adult zebrafish.

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.

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 as mrp-5 mutants are unviable due to their inability to export heme from the intestine to extra-intestinal 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 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 (MAMs), 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-seq show 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.