Ad4BP/SF-1 regulates cholesterol synthesis to boost the production of steroids

Housekeeping metabolic pathways such as glycolysis are active in all cell types. In addition, many types of cells are equipped with cell-specific metabolic pathways. To properly perform their functions, housekeeping and cell-specific metabolic pathways must function cooperatively. However, the regulatory mechanisms that couple metabolic pathways remain largely unknown. Recently, we showed that the steroidogenic cell-specific nuclear receptor Ad4BP/ SF-1, which regulates steroidogenic genes, also regulates housekeeping glycolytic genes. Here, we identify cholesterogenic genes as the targets of Ad4BP/SF-1. Further, we reveal that Ad4BP/SF-1 regulates Hummr, a candidate mediator of cholesterol transport from endoplasmic reticula to mitochondria. Given that cholesterol is the starting material for steroidogenesis and is synthesized from acetyl-CoA, which partly originates from glucose, our results suggest that multiple biological processes involved in synthesizing steroid hormones are governed by Ad4BP/SF-1. To our knowledge, this study provides the first example where housekeeping and cell-specific metabolism are coordinated at the transcriptional level.This work was supported by Grants 16H05142 (K.M.), 17H06427 (K.M.), 16K08593 (T.B.), and 17J03270 (M.I.) from the Japan Society for the Promotion of Science (JSPS) KAKENHI; The Uehara Memorial Foundation (K.M.); Takeda Science Foundation (T.B.); The Shin-Nihon of Advanced Medical Research (T.B.).Supplementary information accompanies this paper at https://doi.org/10.1038/s42003-018-0020-z

Differential lactate and cholesterol synthetic activities in XY and XX Sertoli cells

SRY, a sex-determining gene, induces testis development in chromosomally female (XX) individuals. However, mouse XX Sertoli cells carrying Sry (XX/Sry Sertoli cells) are incapable of fully supporting germ cell development, even when the karyotype of the germ cells is XY. While it has therefore been assumed that XX/Sry Sertoli cells are not functionally equivalent to XY Sertoli cells, it has remained unclear which specific functions are affected. To elucidate the functional difference, we compared the gene expression of XY and XX/Sry Sertoli cells. Lactate and cholesterol metabolisms, essential for nursing the developing germ cells, were down-regulated in XX/Sry cells, which appears to be caused at least in part by the differential expression of histone modification enzymes SMCX/SMCY (H3K4me3 demethylase) and UTX/UTY (H3K27me3 demethylase) encoded by the sex chromosomes. We suggest that down-regulation of lactate and cholesterol metabolism that may be due to altered epigenetic modification affects the nursing functions of XX/Sry Sertoli cells.This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 21249018 and 16H05142 (K. Mo.), Ministry of Education, Culture, Sports, Science, and Technology, Japan (MEXT) KAKENHI Grant Number 22132002 (K. Mo.), the Uehara Memorial Foundation, and Takeda Science Foundation (T.B.)

Heterogeneity of ovarian theca and interstitial gland cells in mice.

It has been established that two developmentally and functionally distinct cell types emerge within the mammalian testis and adrenal gland throughout life. Fetal and adult types of steroidogenic cells (i.e., testicular Leydig cells and adrenocortical cells) develop in the prenatal and postnatal period, respectively. Although the ovary synthesizes steroids postnatally, the presence of fetal-type steroidogenic cells has not been described. We had previously established transgenic mouse lines in which fetal Leydig cells were labeled with an EGFP reporter gene by the FLE (fetal Leydig enhancer) of the Ad4BP/SF-1 (Nr5a1) gene. In the present study, we examined the reporter gene expression in females and found that the reporter gene is turned on in postnatal ovaries. A comparison of the expressions of the EGFP and marker genes revealed that EGFP is expressed in not all but rather a proportion of steroidogenic theca and in interstitial gland cells in the ovary. This finding was further supported by experiments using BAC transgenic mice in which reporter gene expression recapitulated endogenous Ad4BP/SF-1 gene expression. In conclusion, our observations from this study strongly suggest that ovarian theca and interstitial gland cells in mice consist of at least two cell types

Intrinsic Function of the Aryl Hydrocarbon (Dioxin) Receptor as a Key Factor in Female Reproduction

Dioxins exert a variety of adverse effects on organisms, including teratogenesis, immunosuppression, tumor promotion, and estrogenic action. Studies using aryl hydrocarbon receptor (AhR)-deficient mice suggest that the majority of these toxic effects are mediated by the AhR. In spite of the adverse effects mediated by this receptor, the AhR gene is conserved among a number of animal species, ranging from invertebrates to vertebrates. This high degree of conservation strongly suggests that AhR possesses an important physiologic function, and a critical function is also supported by the reduced fertility observed with AhR-null female mice. We demonstrate that AhR plays a crucial role in female reproduction by regulating the expression of ovarian P450 aromatase (Cyp19), a key enzyme in estrogen synthesis. As revealed by in vitro reporter gene assay and in vivo chromatin immunoprecipitation assay, AhR cooperates with an orphan nuclear receptor, Ad4BP/SF-1, to activate Cyp19 gene transcription in ovarian granulosa cells. Administration to female mice of an AhR ligand, DMBA (9,10-dimethyl-1,2-benzanthracene), induced ovarian Cyp19 gene expression, irrespective of the intrinsic phase of the estrus cycle. In addition to elucidating a physiological function for AhR, our studies also suggest a possible mechanism for the toxic effects of exogenous AhR ligands as endocrine disruptors

Aristaless related homeobox gene, Arx, is implicated in mouse fetal Leydig cell differentiation possibly through expressing in the progenitor cells.

Development of the testis begins with the expression of the SRY gene in pre-Sertoli cells. Soon after, testis cords containing Sertoli and germ cells are formed and fetal Leydig cells subsequently develop in the interstitial space. Studies using knockout mice have indicated that multiple genes encoding growth factors and transcription factors are implicated in fetal Leydig cell differentiation. Previously, we demonstrated that the Arx gene is implicated in this process. However, how ARX regulates Leydig cell differentiation remained unknown. In this study, we examined Arx KO testes and revealed that fetal Leydig cell numbers largely decrease throughout the fetal life. Since our study shows that fetal Leydig cells rarely proliferate, this decrease in the KO testes is thought to be due to defects of fetal Leydig progenitor cells. In sexually indifferent fetal gonads of wild type, ARX was expressed in the coelomic epithelial cells and cells underneath the epithelium as well as cells at the gonad-mesonephros border, both of which have been described to contain progenitors of fetal Leydig cells. After testis differentiation, ARX was expressed in a large population of the interstitial cells but not in fetal Leydig cells, raising the possibility that ARX-positive cells contain fetal Leydig progenitor cells. When examining marker gene expression, we observed cells as if they were differentiating into fetal Leydig cells from the progenitor cells. Based on these results, we propose that ARX acts as a positive factor for differentiation of fetal Leydig cells through functioning at the progenitor stage

Overlapped expression of EGFP driven by BAC-Ad4BP and mCherry by mFLE.

<p>Ad4BP-BAC-EGFP transgenic mice were crossed with mFLE-mCherry transgenic mice to generate double transgenic mice (n = 3). Fluorescence views of the adult ovary are shown (A-C). The ovaries were subjected to immunofluorescence with the antibodies for EGFP (green in D and G) and mCherry (red in E and H). Merged views of EGFP and mCherry are shown in F and I, which are further stained with DAPI (blue). Arrows in F and I indicate theca cells (t) or the interstitial gland (ig). Insets are enlarged views of the areas enclosed by rectangles. t, theca cells; ig, interstitial gland. Scale bars for A-C = 500 μm and those for D-I = 100 μm.</p

Distribution of EGFP-positive cells in mFLE-EGFP gonads.

<p>mFLE-EGFP transgenic mouse ovaries at P7 (A, n = 4), P14 (B, n = 2), P21 (C, n = 4), and adult stage at P42 or P56 (D, F, G-I, M-O, n = 4), and testes at E18.5 (E) and adult stage at P42 or P56 (J-L, P-R) were sectioned and subjected to immunofluorescence with antibodies for EGFP (green), Ad4BP/SF-1 (green in E, and red in others), and 3β-HSD (red). Merged images for Ad4BP/SF-1 and 3β-HSD (E, F), EGFP and Ad4BP/SF-1 (I, L), and EGFP and 3β-HSD (O, R) are shown. A-F, I, L, O, and R were further stained with DAPI (blue). Arrows in E indicate Sertoli cells (Se) or Leydig cells (Le). Arrows in F, I, L, O, and R indicate theca cells (t) or the interstitial gland (ig). Closed white arrowheads in G-L indicate cells double positive for EGFP and Ad4BP/SF-1, while those in M-R indicate cells double positive for EGFP and 3β-HSD. Open arrowheads in G-L indicate single positive cells for Ad4BP/SF-1, while those in M-R indicate single positive cells for 3β-HSD. Insets are enlarged views of the areas enclosed by rectangles. t, theca cells; ig, interstitial gland; gr, granulosa cells; Le, Leydig cell; Se, Sertoli cell; tc, testicular cord; is, interstitial space. Scale bars = 100 μm.</p

Expression of EGFP induced by Ad4BP-BAC-EGFP.

<p>The ovary (A), testis (B), adrenal gland (C), pituitary (D), and VMH (E), in which endogenous <i>Ad4BP/SF-1</i> is expressed, were prepared from adult Ad4BP-BAC-EGFP transgenic mice (n = 3). EGFP expression was observed under a fluorescent microscope. The testis (F-H), adrenal gland (I-K), pituitary (L-N), VMH (O-Q), and spleen (R-T) were sectioned, followed by immunofluorescence with antibodies for EGFP (green in F, I, L, O, and R) and Ad4BP/SF-1 (red in G, J, M, P, and S). Merged views of EGFP and Ad4BP/SF-1 are shown in H, K, N, Q, and T, which are further stained with DAPI (blue). Insets are enlarged views of the areas enclosed by rectangles. st, seminiferous tubule; adc, adrenal cortex; ess, endothelial cell of splenic sinus. Scale bars in A-E = 500 μm and those in F-T = 100 μm.</p

Distribution of EGFP-positive cells in Ad4BP-BAC-EGFP ovary.

<p>The adult ovaries at P42 or P56 were prepared (n = 3) and subjected to immunofluorescence with the antibodies for EGFP (green in A-C and J-L), Ad4BP/SF-1 (red in D-F), and 3β-HSD (red in M-O). Merged views for EGFP and Ad4BP/SF-1 are shown in G, H and I, while those for EGFP and 3β-HSD are shown in P, Q, and R; these are stained simultaneously with DAPI (blue). Arrows in G, H, P and Q indicate theca cells (t) or the interstitial gland (ig). Insets are enlarged views of the areas enclosed by rectangles. f, follicle; t, theca cells; ig, interstitial gland; cl, corpus luteum. Scale bars = 100 μm.</p

Expression of EGFP induced by FLE in fetal and postnatal gonads.

<p>The gonads (ovary and testis) of mFLE-EGFP transgenic mice were prepared in E15.5 (A and B, n = 2), P7 (C and G, n = 4), P14 (D and H, n = 2), P21 (E and I, n = 4), and adult stage at P42 or P56 (F and J, n = 4). Whole views of the gonads are shown. B is a fluorescence view of A. The ovaries are delineated by broken lines. Scale bars = 500 μm.</p