Jin-Yong Chung

Maternal pancreatic β cells undergo functional and structural changes to adapt to increased metabolic demands during pregnancy. Lactogen signaling via the prolactin receptor (PRLR) contributes to these adaptations by increasing β-cell mass, insulin transcription, and glucose-stimulated insulin secretion. In other lactogen-responsive tissues such as the mammary glands and specific hypothalamic nuclei, gestation induces epigenetic changes, some of which persist long after birth. We have previously found that PRL treatment in islets regulates the expression of epigenetic modifiers. However, whether lactogen signaling in β cells mediates epigenetic changes to regulate chromatin accessibility has not been examined. Therefore, our objective was to determine whether PRLR signaling alters chromatin accessibility of β cells to facilitate transcriptional regulation. Using single-cell assay for transposase-accessible chromatin using sequencing (scATAC), we identified differentially accessible regions (DARs) in β cells that had 718 overrepresented motifs following PRL treatment of murine islets. Validating this approach, these included motifs bound by established PRLR signaling effectors such as the STAT family of transcription factors (TFs). Using RNA-sequencing we identified transcriptional changes in 41 TFs whose motifs were overrepresented in DARs, including several previously linked to PRLR signaling within β cells, including Myc, Mafb, and Esr1. Importantly, we also identified TFs not previously associated with PRLR signaling, including OVOL2, an established regulator of epigenetic landscape within cells. OVOL2 is a TF involved in epithelial to mesenchymal transition inhibition and energy homeostasis with unknown roles in pancreatic β cells. Here, we establish that OVOL2 acts as a negative regulator of lactogen-dependent effects on β-cell proliferation, establishing a novel regulator of PRLR signaling.

Retinal pigment epithelium (RPE) is essential for the survival of retinal photoreceptors. To study retinal degeneration, sodium iodate (NaIO₃) has been used to cause oxidative stress-induced RPE death followed by photoreceptor degeneration. However, analyses of RPE damage itself are still limited. Here, we characterized NaIO₃-induced RPE damage, which was divided into three regions: periphery with normal-shaped RPE, transitional zone with elongated cells, and center with severely damaged or lost RPE. Elongated cells in the transitional zone exhibited molecular characteristics of epithelial–mesenchymal transition. Central RPE was more susceptible to stresses than peripheral RPE. Under stresses, SIRT6, an NAD⁺-dependent protein deacylase, rapidly translocated from the nucleus to the cytoplasm and colocalized with stress granule factor G3BP1, leading to nuclear SIRT6 depletion. To overcome this SIRT6 depletion, SIRT6 overexpression was induced in the nucleus in transgenic mice, which protected RPE from NaIO₃and partially preserved catalase expression. These results demonstrate topological differences of mouse RPE and warrant further exploring SIRT6 as a potential target for protecting RPE from oxidative stress-induced damage.

Styrene is the precursor of polystyrene. Human exposure to styrene could occur in occupational and residential settings and via food intake. Styrene is metabolized to styrene-7,8-oxide by cytochrome P450 enzyme. In the present study, we investigated the cytotoxicity mediated by styrene and styrene-7,8-oxide in TM3 testicular Leydig cells in vitro. We first monitored the nuclear fragmentation in Leydig cells after exposure to styrene or styrene-7,8-oxide. Hoechst 33258 cell staining showed that styrene exposure in TM3 Leydig cells did not exhibit nuclear fragmentation at any concentration. In contrast, nuclear fragmentation was seen in styrene-7,8-oxide-exposed cells. These results indicate that cytotoxicity-mediated cell death in Leydig cells is more susceptible to styrene-7,8-oxide than to styrene. Following styrene treatment, procaspase-3 and XIAP protein levels did not show significant changes, and cleaved (active) forms of caspase-3 were not detected. Consistent with the western blot results, the active forms of caspase-3 and XIAP proteins were not prominently altered in the cytoplasm of cells treated with styrene. In contrast to styrene, styrene-7,8-oxide induced cell death in an apoptotic fashion, as seen in caspase-3 activation and increased the expression of XIAP proteins. Taken together, the results obtained in this study demonstrate a fundamental idea that Leydig cells are capable of protecting themselves from cytotoxicity-mediated apoptosis as a result of styrene exposure in vitro. It remains unclear whether the steroid-producing function, i.e., steroidogenesis, of Leydig cells is also unaffected by exposure to styrene. Therefore, further studies are needed to elucidate the endocrine disrupting potential of styrene in Leydig cells.

Oxidative stress of the retinal pigment epithelium (RPE) is a major risk factor for age-related macular degeneration (AMD). As a dry AMD model via oxidative stress, sodium iodate (NaIO₃), which is primarily toxic to the RPE, has often been used at a high dose to cause RPE death for studying photoreceptor degeneration. Thus, characterization of RPE damage by a low dose of NaIO₃ is still limited. To quantify RPE damage caused by NaIO₃ in mice, we recently developed a morphometric method using RPE flat-mounts. Here, we report that NaIO₃ has a narrow range of dose–effect correlation at 11–18 mg/kg body weight in male C57BL/6J mice. We evaluated the usefulness of our quantification method in two experimental settings. First, we tested the effect of NF-κB inhibition on NaIO₃-induced RPE damage in male C57BL/6J mice. IKKβ inhibitor BAY 651942 suppressed upregulation of NF-κB targets and protected the RPE from oxidative stress. Second, we tested sex-specific differences in NaIO₃-induced RPE damage in C57BL/6J mice using a low dose near the threshold. NaIO₃ caused more severe RPE damage in female mice than in male mice. These results demonstrate the usefulness of the quantification method and the importance of fine-tuning of the NaIO₃ dose. The results also show the therapeutic potential of IKKβ inhibition for oxidative stress-related RPE diseases, and reveal previously-unrecognized sex-specific differences in RPE susceptibility to oxidative stress.

Previous studies reported that, with aging, Leydig cell intracellular antioxidants are reduced in concentration and intracellular ROS levels increase, suggesting that oxidant/antioxidant imbalance may contribute to the reduced testosterone production that characterizes the aging cells. As yet, little is known about how the Leydig cell oxidant/antioxidant environment is regulated. Sirt1, an enzyme that deacetylates transcription factors, and the transcription factor Nrf2, have been shown to be associated with cellular response to oxidative stress. We hypothesized that Sirt1 and/or Nrf2 might be involved in regulating the oxidant/antioxidant environment of Leydig cells, and therefore, the testosterone production. We found that Sirt1 and Nrf2 are present in the Leydig cells of Brown Norway rats, though reduced in aged cells. In MA-10 cells in which Sirt1 or Nrf2 were suppressed by nicotinamide (NAM) or ML385, respectively, or in which siRNAs were used for knockdown of Sirt1 or Nrf2, increased ROS levels and decreased progesterone production occurred. In rat Leydig cells, inhibition of Sirt1 by culturing the cells with NAM resulted in increased ROS and reduced testosterone production, and subsequent removal of NAM from the culture medium resulted in increased testosterone production. Activation of rat Leydig cells Sirt1 with honokiol or of Nrf2 with sulforaphane resulted in the maintenance of testosterone production despite the exposure of the cells to oxidizing agent. These results, taken together, suggest that Sirt1 and Nrf2 are involved in maintaining the Leydig cell oxidant/antioxidant environment, and thus in maintaining steroid production.

The Leydig cells of the mammalian testis produce testosterone (T) in response to luteinizing hormone (LH). In rats and men with reduced serum T levels, T replacement therapy (TRT) will raise T levels, but typically with suppressive effects on sperm formation. The rate-determining step in T formation is the translocation of cholesterol to the inner mitochondrial membrane, mediated by protein–protein interactions of cytosolic and outer mitochondrial membrane proteins. Among the involved proteins is cholesterol-binding translocator protein (TSPO) (18 kDa TSPO). We hypothesized that in contrast to TRT, the administration of the TSPO agonist N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide (FGIN-1-27), by stimulating the ability of the Leydig cells to produce T, would result in the elevation of serum T levels while maintaining intratesticular T concentration and therefore without suppression of spermatogenesis. Age-related reductions in both serum and intratesticular T levels were seen in old Brown Norway rats. Both exogenous T and FGIN-1-27 increased serum T levels. With exogenous T, serum LH and Leydig cell T formation were suppressed, and intratesticular T was reduced to below the concentration required to maintain spermatogenesis quantitatively. In contrast, FGIN-1-27 stimulated Leydig cell T formation, resulting in increased serum T without reductions in intratesticular T concentrations or in testicular sperm numbers. FGIN-1-27 also significantly increased serum and intratesticular T levels in rats made LH-deficient by treatment with the gonadotropin-releasing hormone antagonist cetrorelix. These results point to a possible approach to increasing serum T without negative effects on spermatogenesis, based upon stimulating T production by the Leydig cells themselves rather than administering T exogenously.

Background: Cholesterol import into the mitochondria of steroid‐producing cells is the rate‐determining step in steroidogenesis. Numerous studies have provided evidence that the cholesterol‐binding translocator protein (18 kDa TSPO) plays an important role in cholesterol translocation into mitochondria and that it also might act on cholesterol homeostasis. Several TSPO‐specific ligands have been shown to increase steroid production in vitro and in vivo.Objectives: The present study assessed the effects of the TSPO drug ligand FGIN‐1‐27 on cholesterol accumulation and lipid droplet formation in relationship to steroid formation.Materials and Methods: Using MA‐10 and primary Leydig cells, immunocytochemical and molecular methods were used to examine cholesterol accumulation, the formation of lipid droplets, and steroid formation in response to LH and FGIN‐1‐27. Additionally, we determined the effects of Tspo knockout by CRISPR/Cas9, and of siRNA knockdowns of Tspo and Plin2 (Perilipin 2; also known as adipose differentiation‐related protein, ADFP) on LH‐ and FGIN‐1‐27‐induced steroidogenesis.Results: In response to LH and FGIN‐1‐27, cultured MA‐10 cells and primary Leydig cells increased steroid formation, cholesterol accumulation, and lipid droplet formation. Cholesterol accumulation in the lipid droplets also was increased in Tspo knockout cells. Knockout of Tspo or its knockdown in MA‐10 cells resulted in reduced progesterone formation in response to both LH and FGIN‐1‐27, as did knockdown of Plin2. Steroid production also was inhibited by the cholesteryl ester hydrolase inhibitor diethylumbelliferyl phosphate.Discussion and Conclusion: These results support the conclusion that FGIN‐1‐27 stimulates steroid formation by increasing TSPO‐mediated cholesterol translocation into the inner mitochondria for steroidogenesis, as well as into the cytosol for lipid droplet formation. FGIN‐1‐27 also increased steroid formation at least in part by inducing the conversion of cholesteryl ester located in lipid droplets to cholesterol, thus making available more substrate for steroid formation.

The initial steps of steroidogenesis occur in the mitochondria. Dynamic changes in the mitochondria are associated with their fission and fusion. Therefore, understanding the cellular and molecular relationship between steroidogenesis and mitochondrial dynamics is important. The hypothesis of the present study is that mitochondrial fission and fusion are closely associated with steroid hormone synthesis in the testicular Leydig cells. Steroid hormone production, induced by dibutyryl cyclic-adenosine monophosphate (dbcAMP) in Leydig cells, was accompanied by increased mitochondrial mass. Mitochondrial elongation increased during the dbcAMP-induced steroid production, whereas mitochondrial fragmentation reduced. Among the mitochondrial-shaping proteins, the level of Drp1 was altered in response to dbcAMP stimulation. The increase in Drp1 Ser 637 phosphorylation correlated with steroid hormone production in the MA-10 Leydig cells as well as in the primary adult rat Leydig cells. Drp1 was differentially expressed in the Leydig cells during testicular development. Finally, gonadotropin administration altered the status of Drp1 phosphorylation in the Leydig cells of immature rat testes. Overall, mitochondrial dynamics is directly linked to steroidogenesis, and Drp1 plays an important regulatory role during steroidogenesis. This study shows that Drp1 level is regulated by cAMP and that its phosphorylation via PKA activation plays a decisive role in mitochondrial shaping by offering an optimal environment for steroid hormone biosynthesis in the Leydig cells. Therefore, it is suggested that PKA-mediated Drp1 ser 637 phosphorylation is indispensable for steroidogenesis in the Leydig cells, and this phosphorylation results in mitochondrial elongation via the relative attenuation of mitochondrial fission during steroidogenesis.

There have been few studies investigating the association between atopic dermatitis (AD) and prenatal exposure to heavy metals. We aimed to evaluate whether prenatal exposure to heavy metals is associated with the development or severity of AD in a birth cohort study. A total of 331 subjects were followed from birth for a median duration of 60.0 months. The presence and severity of AD were evaluated at ages 6 and 12 months, and regularly once a year thereafter. The concentrations of lead, mercury, chromium, and cadmium in umbilical cord blood were measured by inductively coupled plasma mass spectrometry. Cord blood mononuclear cells (CBMCs) were isolated and stimulated for analysis of cytokine production using ELISA. Heavy metal levels in cord blood were not associated with the development of AD until 24 months of age. However, a positive correlation was observed between the duration of AD and lead levels in cord blood (p=0.002). AD severity was also positively associated with chromium concentrations in cord blood (p=0.037), while cord blood levels of lead, mercury, and cadmium were not significantly associated with AD severity (p=0.562, p=0.054, and p=0.055, respectively). Interleukin-13 production in CBMCs was positively related with lead and chromium levels in cord blood (p=0.021 and p=0.015, respectively). Prenatal exposure to lead and chromium is associated with the persistence and severity of AD, and the immune reaction toward a Th2 polarization.