Gonadotropin Regulation of Retinoic Acid Activity in the Testis

  • Seyedmehdi Nourashrafeddin Department of Obstetrics, Gynecology and Reproductive Sciences, School of Medicine, University of Pittsburgh, Pittsburgh, USA. AND Institute for Family Health, Vali-e-Asr Reproductive Health Research Center, Tehran University of Medical Sciences, Tehran, Iran. AND School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.
  • Batool Hosseini Rashidi Institute for Family Health, Vali-e-Asr Reproductive Health Research Center, Tehran University of Medical Sciences, Tehran, Iran.
Keywords: LH and FSH, Retinoic acid, Rhesus monkey, Spermatogenesis


Initiation of spermatogenesis in primates is triggered at puberty by an increase in gonadotropins; i.e., follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Prior to puberty, testis of the monkey contains only undifferentiated germ cells. However, sermatogonial differentiation and spermatogenesis may be initiated prior to puberty after stimulation with exogenous LH and FSH. Retinoic acid (RA) signaling is considered to be a major component that drives spermatogonial differentiation. We were interested in evaluating the relative role of LH and FSH, either alone or in combination, in regulating the retinoic acid signaling in monkey testis. Sixteen juvenile male rhesus monkeys (Macaca mulatta) were infused with intermittent recombinant single chain human LH (schLH) or recombinant human FSH (rhFSH) or a combination of both for 11 days. We then analyzed the expression of the several putative RA signaling pathway related genes; i.e. RDH10, RDH11, ALDH1A1, ALDH1A2, CYP26B1, CRABP1, CRABP2, STRA6, STRA8 in the testis after 11 days of stimulation with vehicle, LH, FSH and combination LH/FSH using quantitative real-time PCR (qPCR). The qPCR results analysis showed that administration of gonadotropins affected a significant change in expression of some RA signaling related genes in the monkey testis. The gonadotropins, either alone or in combination dramatically increased expression of CRABP2 (p≤0.001), whereas there was a decrease in ALDH1A2 expression (p≤0.001). Moreover, combined gonadotropin treatment led to the significant decrease in CRABP1 expression (p≤0.05). These findings are the first evidence that the activity of retinoic acid signaling in the monkey testis is regulated through gonadotropins (LH/FSH) levels.


Amory JK, Arnold S, Lardone MC, Piottante A, Ebensperger M, Isoherranen N, Muller CH, et al. Levels of the retinoic acid synthesizing enzyme aldehyde dehydrogenase-1A2 are lower in testicular tissue from men with infertility. Fertil Steril 2014;101:960-6.

Amengual J, Zhang N, Kemerer M, Maeda T, Palczewski K, Von Lintig J. STRA6 is critical for cellular vitamin A uptake and homeostasis. Hum Mol Gene 2014;23:5402-17.

Anderson EL, Baltus AE, Roepers-Gajadien HL, Hassold TJ, de Rooij DG, van Pelt AM, et al. Stra8 and its inducer, retinoic acid, regulate meiotic initiation in both spermatogenesis and oogenesis in mice. Proc Natl Acad Sci U S A 2008;105:14976-80.

Arnold SL, Kent T, Hogarth CA, Schlatt S, Prasad B, Haenisch M, et al. Importance of ALDH1A enzymes in determining human testicular retinoic acid concentrations. Lipid Res 2015;56:342-57.

Bookout AL, Cummins CL, Mangelsdorf DJ, Pesola JM, Kramer MF. High-throughput real-time quantitative reverse transcription PCR. Curr Protoc Mol Biol 2006;Chapter 15:Unit 15.8.

Bouillet P, Sapin V, Chazaud C, Messaddeq N, Decimo D, Dolle P, et al. Developmental expression pattern of Stra6, a retinoic acid-responsive gene encoding a new type of membrane protein. Mech Dev 1997;63:173-86.

Budhu AS, Noy N.) Direct channeling of retinoic acid between cellular retinoic acid-binding protein II and retinoic acid receptor sensitizes mammary carcinoma cells to retinoic acid-induced growth arrest. Mol Cell Bio 2002;22:2632–41.

Chomcyznski P, Sacchi N. Single-step method off RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156-9.

Chung SS, Sung W, Wang X, Wolgemuth DJ. Retinoic acid receptor alpha is required for synchronization of spermatogenic cycles and its absence results in progressive breakdown of the spermatogenic process. Dev Dynam 2004;230;754-66.

Chung SS, Wolgemuth DJ. Role of retinoid signaling in the regulation of spermatogenesis. Cytogenet Genome Res 2004;105:189-202.

Childs AJ, Cowan G, Kinnell HL, Anderson RA, Saunders PT. Retinoic Acid signalling and the control of meiotic entry in the human fetal gonad. PLoS One 2011;6:e20249.

Clark BJ, Wells J, King SR,Stocco DM. The purification, cloning, and expression of a novel luteinizing hormone-induced mitochondrial protein in MA-10 mouse Leydig tumor cells. Characterization of the steroidogenic acute regulatory protein (StAR). J Biol Chem 1994;269:28314-22.

Clermont Y. Kinetics of spermatogenesis in mammals: seminiferous epithelium cycle and spermatogonial renewal. Physiol Rev 1972;52:198-236.

Clermont Y. Two classes of spermatogonial stem cells in the monkey (Cercopithecus aethiops). Am J Anatomy 1969;126:57-71.

Clermont Y, Antar M. Duration of the cycle of the seminiferous epithelium and the spermatogonial renewal in the monkey Macaca arctoides. Am J Anat 1973;136:153-65.

DeFalco T, Potter SJ, Williams AV, Waller B, Kan MJ, Capel B. Macrophages Contribute to the Spermatogonial Niche in the Adult Testis. Cell Rep 2015;12:1107-19.

De Rooij DG, van Alpen MMA, van de Kant HJG.

Duration of the cycle of the seminiferous epithelium, its stages in the rhesus monkey (Macaca mulatta). Biol Reprod 1986;35:587-91.

Dong D, Ruuska SE, Levinthal DJ, Noy N. Distinct roles for cellular retinoic acid-binding proteins I and II in regulating signaling by retinoic acid. J Biol Chem 1999;274:23695-8.

Ehmcke J, Wistuba J, Schlatt S. Spermatogonial stem cells: questions, models and perspectives. Hum Reprod Update 2006;12:275-82.

Fan X, Molotkov A, Manabe S, Donmoyer CM, Deltour L, Foglio MH, et al. Targeted disruption of Aldh1a1 (Raldh1) provides evidence for a complex mechanism of retinoic acid synthesis in the developing retina. Mol Cell Biol 2003;23:4637-48.

Ford SL, Reinhart AJ, Lukyanenko Y, Hutson JC, Stocco DM. Pregnenolone synthesis in immature rat Sertoli cells. Mol Cell Endocrinol 1999;157:87-94.

Gely-Pernot A, Raverdeau M, Célébi C, Dennefeld C, Feret B, Klopfenstein M, et al. Spermatogonia differentiation requires retinoic acid receptor gamma. Endocrinology 2012;153:438–49.

Gregory CW, DePhilip RM. Detection of steroidogenic acute regulatory protein (stAR) in mitochondria of cultured rat Sertoli cells incubated with follicle-stimulating hormone. Biol Reprod 1998;58:470-4.

Griswold MD, Bishop PD, Kim KH, Ping R, Siiteri JE, Morales C. Function of vitamin A in normal and synchronized seminiferous tubules. Ann N Y Acad Sci 1989;564:154-72.

Heckert LL, Griswold MD. Expression of follicle-stimulating hormone receptor mRNA in rat testis and Sertoli cells. Mol Endocrinol 1991;5:670-7.

Hogarth CA, Arnold S, Kent T, Mitchell D, Isoherranen N, Griswold MD. Processive pulses of retinoic acid propel asynchronous and continuous murine sperm production. Biol Reprod 2015;92:37.

Hogarth CA, Amory JK, Griswold MD. Inhibiting vitamin A metabolism as an approach to male contraception. Trends Endocrinol Metab 2011;22:136-44.

Honecker F, Rohlfing T, Harder S, Braig M, Gillis AJ, Glaesener S, et al. Proteome analysis of the effects of all-trans retinoic acid on human germ cell tumor cell lines. J Proteomics 2014;96:300-13.

Jørgensen A, Nielsen JE, Blomberg Jensen M, Græm N, Rajpert-De Meyts E. Analysis of meiosis regulators in human gonads: a sexually dimorphic spatio-temporal expression pattern suggests involvement of DMRT1 in meiotic entry. Mol Hum Reprod 2012;18:523-34.

Lampron C, Rochette-Egly C, Gorry P, Dolle P, Mark M, Lufkin T, et al. Mice deficient in cellular retinoic acid binding protein II (CRABPII) or in both CRABPI and CRABPII are essentially normal. Development 1995;121:539-48.

Le Bouffant R, Guerquin MJ, Duquenne C, Frydman N, Coffigny H, Rouiller-Fabre V, et al. Meiosis initiation in the human ovary requires intrinsic retinoic acid synthesis. Hum Reprod 2010;10:2579-90.

Li H, Palczewski K, Baehr W, Clagett-Dame M. Vitamin A deficiency results in meiotic failure and accumulation of undifferentiated spermatogonia in prepubertal mouse testis. Biol Reprod 2011;84:336-41.

Liu RZ, Garcia E, Glubrecht DD, Poon HY, Mackey JR, Godbout R. CRABP1 is associated with a poor prognosis in breast cancer: adding to the complexity of breast cancer cell response to retinoic acid. Mol Cancer 2015;14:129.

Mangelsdorf DJ, Umesono K, Evans RM. The Retinoid Receptors. In: Sporn MB, Roberts AB, Goodman DS, eds. The Retinoid, Biology, Chemistry, and Medicine. 2nd ed. New York: Raven Press 1994;319-49.

McLean DJ, Friel PJ, Pouchnik D, Griswold MD. Oligonucleotide microarray analysis of gene expression in follicle-stimulating hormone-treated rat Sertoli cells. Mol Endocrinol 2002;16:2780–92.

Mic FA, Haselbeck RJ, Cuenca AE, Duester G. Novel retinoic acid generating activities in the neural tube and heart identified by conditional rescue of Raldh2 null mutant mice. Development 2002;129:2271–82.

Molotkova N, Molotkov A, Sirbu IO, Duester G. Requirement of mesodermal retinoic acid generated by Raldh2 for posterior neural transformation. Mech Dev 2005;122:145-55.

Morales C, Griswold MD. Retinol-induced stage synchronization in seminiferous tubules of the rat. Endocrinology 1987;121:432-4.

Noy N. Retinoid-binding proteins: mediators of retinoid action. Biochem J 2000;348:481-95.

Noy N, Blaner WS. Interactions of retinol with binding proteins : studies with rat cellular retinol-binding protein and with rat retinol-binding protein. Biochemistry 1991;30:6380-6.

Plant TM. Neuroendocrine control of the onset of puberty. Front Neuroendocrinol 2015;38:73-88.

Plant TM, Marshall GR. The functional significance of FSH in spermatogenesis and the control of its secretion in male primates. Endocr Rev 2001;22:764-86.

Ramaswamy S, Plant TM, Marshall GR. (Pulsatile stimulation with recombinant single chain human luteinizing hormone elicits precocious sertoli cell proliferation in the juvenile male rhesus monkey (Macaca mulatta). Biol Reprod 2000;63:82-8.

Ramaswamy S, Marshall GR, Pohl CR, Friedman RL,

Plant TM. Inhibitory and stimulatory regulation of testicular inhibin B secretion by luteinizing hormone and follicle-stimulating hormone, respectively, in the rhesus monkey (Macaca mulatta). Endocrinology2003;144:1175-85.

Rajan N, Kidd GL, Talmage DA, Blaner WS, Suhara A, Goodman DS. Cellular retinoic acid-binding protein messenger RNA: levels in rat tissues and localization in rat testis. J Lipid Res 1991;32:1195-204.

Raverdeau M, Gely-Pernot A, Féret B, Dennefeld C, Benoit G, Davidson I, et al. Retinoic acid induces sertoli cell paracrine signals for spermatogonia differentiation but cell autonomously drives spermatocyte meiosis. Proc Nat Acad Sci USA 2012;109:16582-7.

Schrans-Stassen BH, van de Kant HJ, de Rooij DG, van Pelt AM. Differential expression of c-kit in mouse undifferentiated and differentiating type a spermatogonia. Endocrinology 1999;140:5894-900.

Simorangkir DR, Ramaswamy S, Marshall GR, Roslund R, Plant TM. Sertoli cell differentiation in rhesus monkey (Macaca mulatta) is an early event in puberty and precedes attainment of the adult complement of undifferentiated spermatogonia. Reproduction 2012;143:513-22.

Simorangkir DR, Ramaswamy S, Marshall GR, Pohl CR, Plant TM. A selective monotropic elevation of FSH, but not that of LH, amplifies the proliferation and differentiation of spermatogonia in the adult rhesus monkey (Macaca mulatta). Hum Reprod 2009;24:1584-95.

Snyder EM, Small C, Griswold MD. Retinoic acid availability drives the asynchronousinitiation of spermatogonial differentiation in the mouse. Biol Reprod 2010;83:783-90.

Thatcher JE, Isoherranen N. The role of CYP26 enzymes in retinoic acid clearance. Expert Opin Drug Metab Toxicol 2009;5:875-86.

Theodosiou M, Laudet V, Schubert M. From carrot to clinic: an overview of the retinoic acid signaling pathway. Cell Mol Life Sci 2010;67:1423-45.

Tong MH, Yang QE, Davis JC, Griswold MD. Retinol dehydrogenase 10 is indispensible for spermatogenesis in juvenile males. Proc Natl Acad Sci USA 2013;110:543-8.

Vo HP, Crowe DL. Transcriptional regulation of retinoic acid responsive genes by cellular retinoic acid binding protein-II modulates RA mediated tumor cell proliferation and invasion. Anticancer Res 1998;18:217-24.

Wood MA, Mukherjee P, Toocheck CA, Walker WH. Upstream stimulatory factor induces Nr5a1 and Shbg gene expression during the onset of rat Sertoli cell differentiation. Biol Reprod 2011;85:965-76.

Zar JH. Multiple comparisons. In: Zar JH, editor. Biostatistical Analysis. Englewood Cliffs, NJ: PrenticeHall, 1974:151-62.

Zheng WL, Bucco RA, Schmitt MC, Wardlaw SA, Ong DE. Localization of cellular retinoic acid-binding protein (CRABP) II and CRABP in developing rat testis. Endocrinology 1996;137:5028-5.

Zhou Q, Li Y, Nie R, Friel P, Mitchell D, Evanoff RM, et al. Expression of stimulated by retinoic acid gene 8 (Stra8) and maturation of murine gonocytes and spermatogonia induced by retinoic acid in vitro. Biol Reprod 2008;78:537-45.

How to Cite
Nourashrafeddin S, Hosseini Rashidi B. Gonadotropin Regulation of Retinoic Acid Activity in the Testis. Acta Med Iran. 56(1):34-2.