Running title: Measuring testicular retinoic acid pulses Summary sentence: Pulses of RA are generated within testis tubules at Stages VIII-IX of the cycle of the seminiferous epithelium. Key words: testis, retinoic acid, spermatogenesis, spermatogonia

The asynchronous cyclic nature of spermatogenesis is essential for continual sperm production and is one of the hallmarks of mammalian male fertility. While various mRNA and protein localization studies have indirectly implicated changing retinoid levels along testis tubules, no quantitative evidence for these changes across the cycle of the seminiferous epithelium currently exists. This study utilized a unique mouse model of induced synchronous spermatogenesis, localization of the retinoid signaling marker STRA8 and sensitive quantification of retinoic acid concentrations to determine whether there are fluctuations in retinoid levels at each of the individual stages of germ cell differentiation and maturation to sperm. These data show that processive pulses of retinoic acid are generated during spermatogonial differentiation, and are the likely trigger for cyclic spermatogenesis and allow us, for the first time, to understand how the cycle of the seminiferous epithelium is generated and maintained. In addition, this study represents the first direct quantification of a retinoid gradient controlling cellular differentiation in a postnatal tissue.

[1]  M. Mark,et al.  Role of retinoic acid receptor (RAR) signaling in post-natal male germ cell differentiation. , 2015, Biochimica et biophysica acta.

[2]  Mark K. Adams,et al.  The Retinaldehyde Reductase Activity of DHRS3 Is Reciprocally Activated by Retinol Dehydrogenase 10 to Control Retinoid Homeostasis* , 2014, The Journal of Biological Chemistry.

[3]  P. Treuting,et al.  Inhibition of Retinoic Acid Biosynthesis by the Bisdichloroacetyldiamine WIN 18,446 Markedly Suppresses Spermatogenesis and Alters Retinoid Metabolism in Mice* , 2014, The Journal of Biological Chemistry.

[4]  S. Higashijima,et al.  Visualization of an endogenous retinoic acid gradient across embryonic development , 2013, Nature.

[5]  M. Griswold,et al.  Turning a Spermatogenic Wave into a Tsunami: Synchronizing Murine Spermatogenesis Using WIN 18,4461 , 2013, Biology of reproduction.

[6]  Y. Saga,et al.  Retinoic acid signaling in Sertoli cells regulates organization of the blood-testis barrier through cyclical changes in gene expression , 2012, Development.

[7]  L. Wolpert,et al.  Gradients of signalling in the developing limb. , 2012, Current opinion in cell biology.

[8]  N. Isoherranen,et al.  A sensitive and specific method for measurement of multiple retinoids in human serum with UHPLC-MS/MS , 2012, Journal of Lipid Research.

[9]  G. Duester,et al.  Alcohol and aldehyde dehydrogenases: retinoid metabolic effects in mouse knockout models. , 2012, Biochimica et biophysica acta.

[10]  J. L. Napoli.  Physiological insights into all-trans-retinoic acid biosynthesis. , 2012, Biochimica et biophysica acta.

[11]  A. Palou,et al.  Lipid metabolism in mammalian tissues and its control by retinoic acid. , 2012, Biochimica et biophysica acta.

[12]  Y. Nabeshima,et al.  Retinoic acid metabolism links the periodical differentiation of germ cells with the cycle of Sertoli cells in mouse seminiferous epithelium , 2012, Mechanisms of Development.

[13]  P. Dollé,et al.  Involvement of retinol dehydrogenase 10 in embryonic patterning and rescue of its loss of function by maternal retinaldehyde treatment , 2011, Proceedings of the National Academy of Sciences.

[14]  M. Griswold,et al.  Inhibiting vitamin A metabolism as an approach to male contraception , 2011, Trends in Endocrinology & Metabolism.

[15]  K. Palczewski,et al.  Vitamin A Deficiency Results in Meiotic Failure and Accumulation of Undifferentiated Spermatogonia in Prepubertal Mouse Testis1 , 2011, Biology of reproduction.

[16]  M. Griswold,et al.  Retinoic Acid Availability Drives the Asynchronous Initiation of Spermatogonial Differentiation in the Mouse1 , 2010, Biology of reproduction.

[17]  M. Griswold,et al.  The key role of vitamin A in spermatogenesis. , 2010, The Journal of clinical investigation.

[18]  M. Kane,et al.  Ethanol elevates physiological all‐trans‐retinoic acid levels in select loci through altering retinoid metabolism in multiple loci: a potential mechanism of ethanol toxicity , 2010, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[19]  D. Bok,et al.  Retinoid X receptor beta (RXRB) expression in Sertoli cells controls cholesterol homeostasis and spermiation. , 2008, Reproduction.

[20]  G. Duester.  Retinoic Acid Synthesis and Signaling during Early Organogenesis , 2008, Cell.

[21]  Ying Li,et al.  Expression of Stimulated by Retinoic Acid Gene 8 (Stra8) in Spermatogenic Cells Induced by Retinoic Acid: An In Vivo Study in Vitamin A-Sufficient Postnatal Murine Testes1 , 2008, Biology of reproduction.

[22]  D. Johnston,et al.  Stage-specific gene expression is a fundamental characteristic of rat spermatogenic cells and Sertoli cells , 2008, Proceedings of the National Academy of Sciences.

[23]  M. Kane,et al.  Quantitative profiling of endogenous retinoic acid in vivo and in vitro by tandem mass spectrometry. , 2008, Analytical chemistry.

[24]  A. Mushegian,et al.  RDH10 is essential for synthesis of embryonic retinoic acid and is required for limb, craniofacial, and organ development. , 2007, Genes & development.

[25]  P. Chambon,et al.  Prepubertal testis development relies on retinoic acid but not rexinoid receptors in Sertoli cells , 2006, The EMBO journal.

[26]  J. Rossant,et al.  Retinoid Signaling Determines Germ Cell Fate in Mice , 2006, Science.

[27]  David C Page,et al.  Retinoic acid regulates sex-specific timing of meiotic initiation in mice. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[28]  K. Loveland,et al.  Expression of Nuclear Transport Importins beta 1 and beta 3 Is Regulated During Rodent Spermatogenesis1 , 2006, Biology of reproduction.

[29]  P. Chambon,et al.  Retinoic acid metabolism and signaling pathways in the adult and developing mouse testis. , 2006, Endocrinology.

[30]  M. Kane,et al.  Quantification of endogenous retinoic acid in limited biological samples by LC/MS/MS. , 2005, The Biochemical journal.

[31]  D. Wolgemuth,et al.  Role of retinoid signaling in the regulation of spermatogenesis , 2004, Cytogenetic and Genome Research.

[32]  Emily Gale,et al.  Opposing FGF and Retinoid Pathways Control Ventral Neural Pattern, Neuronal Differentiation, and Segmentation during Body Axis Extension , 2003, Neuron.

[33]  A. Meyer,et al.  Hindbrain patterning revisited: timing and effects of retinoic acid signalling. , 2001, BioEssays : news and reviews in molecular, cellular and developmental biology.

[34]  J. Goltz,et al.  The cAMP-dependent Protein Kinase Site (Ser312) Enhances Dorsal Nuclear Import through Facilitating Nuclear Localization Sequence/Importin Interaction* , 1998, The Journal of Biological Chemistry.

[35]  M. Griswold,et al.  Testicular synchrony: evaluation and analysis of different protocols. , 1992, Biology of reproduction.

[36]  M. Griswold,et al.  Expression of Receptors during the Cycle of the Seminiferous Epithelium a , 1991, Annals of the New York Academy of Sciences.

[37]  M. Meistrich,et al.  Stage-synchronized seminiferous epithelium in rats after manipulation of retinol levels. , 1991, Biology of reproduction.

[38]  L. Russell,et al.  Histological and Histopathological Evaluation of the Testis , 1990 .