The RHOX Homeodomain Proteins Regulate the Expression of Insulin and Other Metabolic Regulators in the Testis*

Background: RHOX5 is a transcription factor important for spermatogenesis. Results: Using Rhox5-null mice, we demonstrate that RHOX5 regulates key metabolism genes, including insulin (Ins), resistin (Retn), and adiponectin (Adipoq) in specific cell types and developmental stages in the testis. Conclusion: RHOX5 is a crucial regulator of metabolism genes in the testis. Significance: This work provides a framework for understanding why metabolism defects cause male infertility. Defects in cellular metabolism have been widely implicated in causing male infertility, but there has been little progress in understanding the underlying mechanism. Here we report that several key metabolism genes are regulated in the testis by Rhox5, the founding member of a large X-linked homeobox gene cluster. Among these Rhox5-regulated genes are insulin 2 (Ins2), resistin (Retn), and adiponectin (Adipoq), all of which encode secreted proteins that have profound and wide-ranging effects on cellular metabolism. The ability of Rhox5 to regulate their levels in the testis has the potential to dictate metabolism locally in this organ, given the existence of the blood-testes barrier. We demonstrate that Ins2 is a direct target of Rhox5 in Sertoli cells, and we show that this regulation is physiologically significant, because Rhox5-null mice fail to up-regulate Ins2 expression during the first wave of spermatogenesis and have insulin-signaling defects. We identify other Rhox family members that induce Ins2 transcription, define protein domains and homeodomain amino acid residues crucial for this property, and demonstrate that this regulation is conserved. Rhox5-null mice also exhibit altered expression of other metabolism genes, including those encoding the master transcriptional regulators of metabolism, PPARG and PPARGC1A, as well as SCD1, the rate-limiting enzyme for fatty acid metabolism. These results, coupled with the known roles of RHOX5 and its target metabolism genes in spermatogenesis in vivo, lead us to propose a model in which RHOX5 is a central transcription factor that promotes the survival of male germ cells via its effects on cellular metabolism.

[1]  M. Wilkinson,et al.  Rapid Evolution of a Homeodomain: Evidence for Positive Selection , 1997, Journal of Molecular Evolution.

[2]  R. Kaur,et al.  GATA Factors and Androgen Receptor Collaborate To Transcriptionally Activate the Rhox5 Homeobox Gene in Sertoli Cells , 2008, Molecular and Cellular Biology.

[3]  D. Goulis,et al.  Metabolic syndrome and reproduction: I. Testicular function , 2008, Gynecological endocrinology : the official journal of the International Society of Gynecological Endocrinology.

[4]  D. Fleenor,et al.  Prolactin induction of insulin gene transcription: roles of glucose and signal transducer and activator of transcription 5. , 2001, Endocrinology.

[5]  K. Moley,et al.  The effects of type 1 diabetes on the hypothalamic, pituitary and testes axis , 2012, Cell and Tissue Research.

[6]  H. Rootwelt,et al.  Essential fatty acid deficiency induces fatty acid desaturase expression in rat epididymis, but not in testis. , 2007, Reproduction.

[7]  P. B. Jensen,et al.  The Nkx6.1 homeodomain transcription factor suppresses glucagon expression and regulates glucose-stimulated insulin secretion in islet beta cells. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[8]  K. Moley,et al.  Insulin Rescues Impaired Spermatogenesis via the Hypothalamic-Pituitary-Gonadal Axis in Akita Diabetic Mice and Restores Male Fertility , 2012, Diabetes.

[9]  C. Cheng,et al.  Drug transporter, P-glycoprotein (MDR1), is an integrated component of the mammalian blood-testis barrier. , 2009, The international journal of biochemistry & cell biology.

[10]  A. Atkinson,et al.  Insulin dependant diabetes mellitus: implications for male reproductive function. , 2007, Human reproduction.

[11]  M. D. Leibowitz,et al.  Differential effects of rexinoids and thiazolidinediones on metabolic gene expression in diabetic rodents. , 2001, Molecular pharmacology.

[12]  M. Meistrich,et al.  Pem homeobox gene promoter sequences that direct transcription in a Sertoli cell-specific, stage-specific, and androgen-dependent manner in the testis in vivo. , 2003, Molecular endocrinology.

[13]  G. Zhou,et al.  Insulin- and Mitogen-activated Protein Kinase-mediated Phosphorylation and Activation of Peroxisome Proliferator-activated Receptor γ* , 1996, The Journal of Biological Chemistry.

[14]  J. Chirgwin,et al.  Characterization of the two nonallelic genes encoding mouse preproinsulin , 2005, Journal of Molecular Evolution.

[15]  L. Laurent,et al.  The RHOX homeobox gene cluster is selectively expressed in human oocytes and male germ cells. , 2013, Human reproduction.

[16]  B. Ahrén,et al.  Temporal and dietary fat content-dependent islet adaptation to high-fat feeding-induced glucose intolerance in mice. , 2007, Metabolism: clinical and experimental.

[17]  R. Slater.  Purification of RNA , 1992 .

[18]  D. Hales,et al.  Testosterone inhibits cAMP-induced de Novo synthesis of Leydig cell cytochrome P-450(17 alpha) by an androgen receptor-mediated mechanism. , 1987, The Journal of biological chemistry.

[19]  P. Chatelain,et al.  Differentiating effects of somatomedin-C/insulin-like growth factor I and insulin on Leydig and Sertoli cell functions. , 1988, Reproduction, nutrition, developpement.

[20]  C. R. Bagnell,et al.  Senescence-associated phenotypes in Akita diabetic mice are enhanced by absence of bradykinin B2 receptors. , 2006, The Journal of clinical investigation.

[21]  S. Woods,et al.  Insulin as an adiposity signal , 2001, International Journal of Obesity.

[22]  M. Wilkinson,et al.  Regulation of the Rhox5 Homeobox Gene in Primary Granulosa Cells: Preovulatory Expression and Dependence on SP1/SP3 and GABP1 , 2005, Biology of reproduction.

[23]  Jianzhi Zhang,et al.  Remarkable expansions of an X-linked reproductive homeobox gene cluster in rodent evolution. , 2006, Genomics.

[24]  M. Rao,et al.  Isolation of Sertoli, Leydig, and spermatogenic cells from the mouse testis. , 2011, BioTechniques.

[25]  Cynthia Kenyon,et al.  Timing Requirements for Insulin/IGF-1 Signaling in C. elegans , 2002, Science.

[26]  C. Lin,et al.  Effects of glucose concentration on in vitro fertilization in BALB/c mice. , 2003, Reproduction in domestic animals = Zuchthygiene.

[27]  P. Halban,et al.  Dominant negative pathogenesis by mutant proinsulin in the Akita diabetic mouse. , 2003, Diabetes.

[28]  J. Chudek,et al.  The adipose tissue--a novel endocrine organ of interest to the nephrologist. , 2002, Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association.

[29]  R. Brinster,et al.  The germline stem cell niche unit in mammalian testes. , 2012, Physiological reviews.

[30]  James A. MacLean,et al.  Rhox: A New Homeobox Gene Cluster , 2005, Cell.

[31]  M. Wilkinson,et al.  Alternatively Spliced T-cell Receptor Transcripts Are Up-regulated in Response to Disruption of Either Splicing Elements or Reading Frame* , 2007, Journal of Biological Chemistry.

[32]  P. Oliveira,et al.  Diabetes and the impairment of reproductive function: possible role of mitochondria and reactive oxygen species. , 2008, Current diabetes reviews.

[33]  S. Brichard,et al.  Insulin and insulin-like growth factor 1 antagonize the stimulation of ob gene expression by dexamethasone in cultured rat adipose tissue. , 1997, The Biochemical journal.

[34]  M. Wilkinson,et al.  The Rhox5 homeobox gene regulates the region-specific expression of its paralogs in the rodent epididymis. , 2012, Biology of reproduction.

[35]  K. Moley,et al.  Paternal effect on embryo quality in diabetic mice is related to poor sperm quality and associated with decreased glucose transporter expression. , 2008, Reproduction.

[36]  J. Toppari,et al.  Novel expression of resistin in rat testis: functional role and regulation by nutritional status and hormonal factors , 2004, Journal of Cell Science.

[37]  Seung-Min Yang,et al.  The Rhox Homeobox Gene Cluster Is Imprinted and Selectively Targeted for Regulation by Histone H1 and DNA Methylation , 2011, Molecular and Cellular Biology.

[38]  M. Wilkinson,et al.  Androgen-induced Rhox homeobox genes modulate the expression of AR-regulated genes. , 2010, Molecular endocrinology.

[39]  T. Franke,et al.  PI3K/Akt: getting it right matters , 2008, Oncogene.

[40]  D. Sakkas,et al.  Effects of glucose and fructose on fertilization, cleavage, and viability of mouse embryos in vitro. , 1993, Biology of reproduction.

[41]  J. Issa,et al.  DNA demethylation-dependent AR recruitment and GATA factors drive Rhox5 homeobox gene transcription in the epididymis. , 2012, Molecular endocrinology.

[42]  Luís Rato,et al.  Effect of insulin deprivation on metabolism and metabolism-associated gene transcript levels of in vitro cultured human Sertoli cells. , 2012, Biochimica et biophysica acta.

[43]  C. Geserick,et al.  New androgen response elements in the murine pem promoter mediate selective transactivation. , 2001, Molecular endocrinology.

[44]  R. Sharpe,et al.  Development and function of the adult generation of Leydig cells in mice with Sertoli cell-selective or total ablation of the androgen receptor. , 2005, Endocrinology.

[45]  M. Dym,et al.  Spermatogenic cells of the prepuberal mouse: isolation and morphological characterization , 1977, The Journal of cell biology.

[46]  M. Wilkinson,et al.  Expression of the pem homeobox gene in Sertoli cells increases the frequency of adjacent germ cells with deoxyribonucleic acid strand breaks. , 2002, Endocrinology.

[47]  L. Morris,et al.  Identification of a tandem duplicated array in the Rhox α locus on mouse chromosome X , 2006, Mammalian Genome.

[48]  G. Weir,et al.  Mechanism of insulin Gene Regulation by the Pancreatic Transcription Factor Pdx-1 , 2005, Journal of Biological Chemistry.

[49]  B. Yandell,et al.  Loss of stearoyl–CoA desaturase-1 function protects mice against adiposity , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[50]  M. Romano,et al.  Insulin modifies the proliferation and function of chicken testis cells. , 2001, Poultry science.

[51]  F. Berenbaum,et al.  Differentiation regulates interleukin-1beta-induced cyclo-oxygenase-2 in human articular chondrocytes: role of p38 mitogen-activated protein kinase. , 2002, The Biochemical journal.

[52]  D. Fawcett,et al.  The normal development of the blood‐testis barrier and the effects of clomiphene and estrogen treatment , 1973, The Anatomical record.

[53]  M. Wilkinson,et al.  The Pem homeobox gene: rapid evolution of the homeodomain, X chromosomal localization, and expression in reproductive tissue. , 1996, Genomics.

[54]  M. Welsh.  Glucose regulation of insulin gene expression. , 1989, Diabete & metabolisme.

[55]  B. Conne,et al.  An essential role for insulin and IGF1 receptors in regulating sertoli cell proliferation, testis size, and FSH action in mice. , 2013, Molecular endocrinology.

[56]  L. Sussel,et al.  Transcription Factor Occupancy of the Insulin Gene in Vivo , 2003, The Journal of Biological Chemistry.

[57]  M. Wilkinson,et al.  Pem Homeobox Gene Regulatory Sequences That Direct Androgen-dependent Developmentally Regulated Gene Expression in Different Subregions of the Epididymis* , 2002, The Journal of Biological Chemistry.

[58]  J. MacLean.  The role of Rhox homeobox factors in tumorigenesis. , 2013, Frontiers in bioscience.

[59]  A. Kralli,et al.  PGC-1, a versatile coactivator , 2001, Trends in Endocrinology & Metabolism.

[60]  M. Alves,et al.  In vitro cultured human Sertoli cells secrete high amounts of acetate that is stimulated by 17β-estradiol and suppressed by insulin deprivation. , 2012, Biochimica et biophysica acta.

[61]  C. Macleod,et al.  The Pem homeobox gene is X-linked and exclusively expressed in extraembryonic tissues during early murine development. , 1994, Developmental biology.

[62]  O. Madsen,et al.  Differential expression of the two nonallelic proinsulin genes in the developing mouse embryo. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[63]  A. Sinclair,et al.  The Rhox Homeobox Gene Family Shows Sexually Dimorphic and Dynamic Expression During Mouse Embryonic Gonad Development1 , 2008, Biology of reproduction.

[64]  M. Griswold.  The central role of Sertoli cells in spermatogenesis. , 1998, Seminars in cell & developmental biology.

[65]  C. Macleod,et al.  Normal reproductive and macrophage function in Pem homeobox gene-deficient mice. , 1998, Developmental biology.

[66]  J. Orth,et al.  Evidence from Sertoli cell-depleted rats indicates that spermatid number in adults depends on numbers of Sertoli cells produced during perinatal development. , 1988, Endocrinology.

[67]  A. Butte,et al.  Coordinated reduction of genes of oxidative metabolism in humans with insulin resistance and diabetes: Potential role of PGC1 and NRF1 , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[68]  H. Rootwelt,et al.  Expression and Regulation of Δ5-Desaturase, Δ6-Desaturase, Stearoyl-Coenzyme A (CoA) Desaturase 1, and Stearoyl-CoA Desaturase 2 in Rat Testis , 2003 .

[69]  Luís Rato,et al.  Hormonal control of Sertoli cell metabolism regulates spermatogenesis , 2012, Cellular and Molecular Life Sciences.

[70]  K. Hayashi,et al.  Regulated Expression of Rhox8 in the Mouse Ovary: Evidence for the Role of Progesterone and RHOX5 in Granulosa Cells1 , 2013, Biology of reproduction.

[71]  M. Wilkinson,et al.  Rhox homeobox gene cluster: recent duplication of three family members , 2006, Genesis.

[72]  T. Matsuoka,et al.  Identification of a Novel PDX-1 Binding Site in the Human Insulin Gene Enhancer* , 2004, Journal of Biological Chemistry.

[73]  J. Toppari,et al.  Novel expression and direct effects of adiponectin in the rat testis. , 2008, Endocrinology.

[74]  M. Carey,et al.  A mechanism for coordinating chromatin modification and preinitiation complex assembly. , 2006, Molecular cell.

[75]  Rui Zhang,et al.  Rapid evolution and copy number variation of primate RHOXF2, an X-linked homeobox gene involved in male reproduction and possibly brain function , 2011, BMC Evolutionary Biology.

[76]  P. Cohen,et al.  Mechanism of activation of protein kinase B by insulin and IGF‐1. , 1996, The EMBO journal.

[77]  Jonathan E. M. Keebler,et al.  A murine specific expansion of the Rhox cluster involved in embryonic stem cell biology is under natural selection , 2006, BMC Genomics.

[78]  J. Toppari,et al.  Paracrine regulation of mammalian oocyte maturation and male germ cell survival. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[79]  M. Lazar,et al.  Resistin and obesity-associated insulin resistance , 2002, Trends in Endocrinology & Metabolism.

[80]  R J Schwartz,et al.  Identification of Novel DNA Binding Targets and Regulatory Domains of a Murine Tinman Homeodomain Factor, nkx-2.5(*) , 1995, The Journal of Biological Chemistry.

[81]  J. Baker,et al.  Effects of an Igf1 gene null mutation on mouse reproduction. , 1996, Molecular endocrinology.

[82]  L. Russell,et al.  Postnatal development of the Sertoli cell barrier, tubular lumen, and cytoskeleton of Sertoli and myoid cells in the rat, and their relationship to tubular fluid secretion and flow. , 1989, The American journal of anatomy.

[83]  M. Wilkinson,et al.  The Rhox genes. , 2010, Reproduction.

[84]  R. Sauer,et al.  Specificity of minor-groove and major-groove interactions in a homeodomain-DNA complex. , 1995, Biochemistry.

[85]  M. Wilkinson,et al.  Deletion mutagenesis of large (12-kb) plasmids by a one-step PCR protocol. , 2001, BioTechniques.

[86]  D. Sibley,et al.  The Blood-Testis Barrier and Its Implications for Male Contraception , 2012, Pharmacological Reviews.

[87]  P. Sassone-Corsi,et al.  Specialized rules of gene transcription in male germ cells: the CREM paradigm. , 2004, International journal of andrology.

[88]  Michael Lehrke,et al.  The Many Faces of PPARγ , 2005, Cell.

[89]  C. Geserick,et al.  OTEX, an androgen-regulated human member of the paired-like class of homeobox genes. , 2002, The Biochemical journal.

[90]  S. Ackerman,et al.  The RHOX5 Homeodomain Protein Mediates Transcriptional Repression of the Netrin-1 Receptor Gene Unc5c* , 2008, Journal of Biological Chemistry.

[91]  A. Mosley,et al.  Glucose regulation of insulin gene expression requires the recruitment of p300 by the beta-cell-specific transcription factor Pdx-1. , 2004, Molecular endocrinology.

[92]  G. Bouma,et al.  New Candidate Genes Identified for Controlling Mouse Gonadal Sex Determination and the Early Stages of Granulosa and Sertoli Cell Differentiation1 , 2010, Biology of reproduction.

[93]  S. Abe,et al.  IGF-I, IGF-II and insulin promote differentiation of spermatogonia to primary spermatocytes in organ culture of newt testes. , 1999, The International journal of developmental biology.

[94]  C. Geyer,et al.  Identification and characterization of Rhox13, a novel X-linked mouse homeobox gene. , 2008, Gene.

[95]  M. Wilkinson,et al.  Site-directed mutagenesis of large (13-kb) plasmids in a single-PCR procedure. , 2000, BioTechniques.

[96]  M. Wilkinson,et al.  Two novel human X-linked homeobox genes, hPEPP1 and hPEPP2, selectively expressed in the testis. , 2002, Gene.

[97]  M. Wilkinson,et al.  Pem: a testosterone- and LH-regulated homeobox gene expressed in mouse Sertoli cells and epididymis. , 1996, Developmental biology.

[98]  A. Bhardwaj,et al.  Regulation and Function of the Rhox5 Homeobox Gene , 2007, Annals of the New York Academy of Sciences.

[99]  P. Scherer,et al.  ACRP30/adiponectin: an adipokine regulating glucose and lipid metabolism , 2002, Trends in Endocrinology & Metabolism.

[100]  D. Accili,et al.  Testis determination requires insulin receptor family function in mice , 2003, Nature.