ࡱ> =;67>y bjbjEE 8''t&NN))))))))8)*<)]*,",,,---[[[[[[[$_8b[)1--11[)),,v]III1l),), WI1[IIRaRS,Qȉ5)@EUSV]0]sSfb$G`b<Sb)S-.I>/|/---[[H`---]1111b---------N W(: Author s institutions: 1Center for Animal Genomics, Veterinary Faculty, University of Ljubljana, Gerbi eva 60, SI-1000 Ljubljana, Slovenia 2University of Zagreb, School of Medicine, Dept. Histology and Embryology, `alata 3, 10000 Zagreb, Croatia 3Centre for Functional genomics and Bio-Chips, Institute of Biochemistry, Medical Faculty, University of Ljubljana, Zaloka 4, SI-1000 Ljubljana, Slovenia Title: Initiation of steroidogenesis precedes expression of cholesterolgenic enzymes in the fetal mouse testes Authors: 1Bdefeld T., 2Jezek D., 3Rozman D., and 1Majdic G. Short title: Steroidogenesis and cholesterol production in the fetal mouse testis Key words: Mouse, testis, fetal, human, 3beta-hydroxysteroid dehydrogenase, StAR, cyp51, NADPH cytochrome P450 reductase, immunocytochemistry Corresponding author: Gregor Majdic Center for Animal Genomics Veterinary Faculty, University of Ljubljana Gerbi eva 60, SI-1000 Ljubljana Slovenia Phone: +386 1 4779210, Fax: +386 1 2832243 Email:  HYPERLINK "mailto:gregor.majdic@vf.uni-lj.si" gregor.majdic@vf.uni-lj.si Abstract Sexual differentiation is a carefully regulated process that ultimately results in a development of the male or female phenotype. Proper development of the male phenotype is dependent upon the action of testosterone, produced by Leydig cells, and antimullerian hormone, produced by Sertoli cells. Leydig cells start to produce testosterone around day 12.5 in the fetal mouse testis, and continue to produce high levels of testosterone throughout the gestation. In the present study we examined whether expression of two enzymes, lanosterol 14-demethylase (cyp51) and cytochrome P450 NADPH reductase, involved in the cholesterol production, occurs simultaneously with proteins required for the production of steroid hormones. Immunocytochemical staining with antibodies against lanosterol 14-demethylase , cytochrome P450 NADPH reductase, steroidogenic acute regulatory protein (StAR) and 3beta-hydroxysteroid dehydrogenase I (3-HSD I) was used to determine the ontogeny of expression of these four proteins. As expected, 3-HSD I and StAR proteins were detected on day 12.5 p.c., while expression of cyp51 and NADPH cytochrome P450 reductase appeared one day later, on day 13.5. Thereafter, the expression of all four proteins remained strong throughout the gestation. In human fetal testes, all four proteins were expressed in the Leydig cells at all ages studied, although very young samples from the time of initial Leydig cell development were not examined as they were not available. Results of this study suggest that initial steps of steroid hormone production in murine Leydig cells are dependent on exogenously derived cholesterol. Later on (from day 13.5 onwards), murine and human Leydig cells are able to synthesize cholesterol and are therefore not dependent on exogenous cholesterol resources. Introduction Mammalian gonads develop as genital ridges on the ventral side of the mesonephros and these subsequently develop into testis or ovary, depending on the presence or absence of Y chromosome and Sry gene  ADDIN EN.CITE George199446465<style face="normal" font="default" charset="238" size="100%">Sex determination and differentiation</style>Ross2005414117Ross, A. J.Capel, B.Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA.Signaling at the crossroads of gonad developmentTrends Endocrinol MetabTrends Endocrinol Metab19-25161AnimalsFemaleGerm CellsGonads/cytology/embryology/*growth & developmentHumansLeydig Cells/physiologyMaleMiceRatsSertoli Cells/physiologySex CharacteristicsSignal Transduction/*physiology2005Jan-Feb15620545http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15620545 (George & Wilson, 1994; Ross & Capel, 2005). Shortly after differentiation, testes start to produce hormones necessary for the proper development of male secondary sexual organs. Antimullerian hormone (AMH), produced by Sertoli cells, is responsible for the regression of the mullerian ducts that would otherwise develop into oviducts, uterus and upper part of vagina  ADDIN EN.CITE Josso2001343417Josso, N.di Clemente, N.Gouedard, L.Unite de Recherches sur l'Endocrinologie du Developpement (INSERM), Ecole Normale Superieure, Departement de Biologie, 1 rue Maurice-Arnoux, 92120 Montrouge, France. josso@wotan.ens.frAnti-Mullerian hormone and its receptorsMol Cell EndocrinolMol Cell Endocrinol25-321791-2AnimalsBone Morphogenetic Protein Receptors, Type IFemaleGene Expression Regulation/genetics/*physiology*GlycoproteinsGranulosa Cells/cytology/metabolismGrowth Inhibitors/biosynthesis/pharmacology/*physiologyHumansMaleMullerian Ducts/abnormalities/drug effectsProtein-Serine-Threonine Kinases/*metabolismReceptors, Peptide/*metabolismSertoli Cells/cytology/metabolismSex Differentiation/drug effects/physiologySignal Transduction/physiologyTesticular Hormones/biosynthesis/pharmacology/*physiologyTransforming Growth Factor beta/biosynthesis/pharmacology/*physiology2001Jun 2011420127http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11420127 (Josso et al., 2001) while steroid hormone testosterone is responsible for proper development and masculinization of secondary male sexual organs  ADDIN EN.CITE George199446465<style face="normal" font="default" charset="238" size="100%">Sex determination and differentiation</style>(George & Wilson, 1994). Female sexual organs develop in the absence of hormones as ovaries remain hormonally inactive throughout the fetal life and the female reproductive tract develops also in the complete absence of gonads  ADDIN EN.CITE George199446465<style face="normal" font="default" charset="238" size="100%">Sex determination and differentiation</style>(George & Wilson, 1994). Sertoli cells are the first cells that develop in the fetal testis, followed shortly by Leydig cells. Leydig cells appear in the interstitial tissue around day 12.5 p.c. in mice  ADDIN EN.CITE Habert2001121217Habert, R.Lejeune, H.Saez, J. M.INSERM-INRA U 418, Universite Paris 7, 2 Place Jussieu, 75251, Paris, France.Origin, differentiation and regulation of fetal and adult Leydig cellsMol Cell EndocrinolMol Cell Endocrinol47-741791-2AdultAnimalsAutocrine Communication/*physiologyCell DifferentiationFollicle Stimulating Hormone/metabolismHumansLeydig Cells/*cytology/*metabolismMaleParacrine Communication/*physiologyRodentiaSex Differentiation/*physiologySteroids/*biosynthesisTestis/*embryology2001Jun 2011420130http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11420130 (Habert et al., 2001). The exact origin of these cells is not yet clear and they might derive either from mesenchymal like stem cells (perycites and vascular smooth muscle cells) or stem cells from neural crest  ADDIN EN.CITE Buehr1993181817Buehr, M.Gu, S.McLaren, A.MRC Mammalian Development Unit, London, UK.Mesonephric contribution to testis differentiation in the fetal mouseDevelopmentDevelopment273-811171AnimalsCell Differentiation/physiologyCells, CulturedComparative StudyEmbryonic Induction/*physiologyFemaleMaleMesonephros/cytology/*physiologyMiceTestis/cytology/*embryology1993Jan8223251http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8223251 Davidoff2004303017Davidoff, M. S.Middendorff, R.Enikolopov, G.Riethmacher, D.Holstein, A. F.Muller, D.Institute of Anatomy, University of Hamburg, Germany. davidoff@uke.uni-hamburg.deProgenitor cells of the testosterone-producing Leydig cells revealedJ Cell BiolJ Cell Biol935-441675AnimalsCell Differentiation/*physiologyCell Lineage/physiologyIntermediate Filament Proteins/metabolismLeydig Cells/cytology/*metabolismMaleMiceMice, TransgenicMuscle, Smooth, Vascular/cytology/growth & development/metabolismNerve Tissue Proteins/metabolismNeuroglia/cytology/metabolismNeurons/cytology/metabolismOrganogenesis/physiologyPericytes/cytology/metabolismRatsRats, WistarStem Cells/cytology/*metabolismTestis/cytology/*growth & development/metabolismTestosterone/*biosynthesisTime FactorsWound Healing/physiology2004Dec 615569711http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15569711 (Buehr et al., 1993; Davidoff et al., 2004). Early markers of differentiating Sertoli and Leydig cells are expression of AMH and steroidogenic enzymes, respectively. Testosterone synthesis from cholesterol requires four steroidogenic enzymes, of which 3-hydroxisteroid dehydrogenase/(5-(4 isomerase (3-HSD) is thought to be constitutively expressed in Leydig cells and is therefore useful marker for these cells. Greco and Payne  ADDIN EN.CITE Greco1994323217Greco, T. L.Payne, A. H.Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor 48109-0278.Ontogeny of expression of the genes for steroidogenic enzymes P450 side-chain cleavage, 3 beta-hydroxysteroid dehydrogenase, P450 17 alpha-hydroxylase/C17-20 lyase, and P450 aromatase in fetal mouse gonadsEndocrinologyEndocrinology262-813513-Hydroxysteroid Dehydrogenases/geneticsAnimalsAromatase/geneticsCholesterol Side-Chain Cleavage Enzyme/geneticsEnzymes/*geneticsFemaleFetus/*metabolism/physiology*Gene Expression RegulationGonads/*embryologyMaleMiceMice, Inbred C57BLRNA, Messenger/metabolismSex Determination (Analysis)Steroid 17-alpha-Hydroxylase/geneticsSteroids/*biosynthesis1994Jul8013361http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8013361 (1994) studied the expression of 3-HSD I, P450scc, P450c17 and P450arom mRNA using RT-PCR in fetal testes and ovaries from C57BL6/J mice. They reported the expression of P450scc, 3-HSD I and P450c17 mRNA on day 13.5 p.c. but there are no other reports about early expression of proteins involved in production of steroid hormones in early fetal mouse gonads, although Gondos (1980) reported that testosterone production in the fetal mouse testes starts around day 12.5 13.0 p.c. The initial steps in the biosynhesis of steroid hormones represent a transport of cholesterol to the outer mitochondrial membrane, followed by translocation of cholesterol across the outer mitochondrial membrane and intermembrane space to reach cytochrome P450scc, which resides on the inner mitochondrial membrane. StAR (steroidogenic acute regulatory protein) is a phosphoprotein that is obligatory for this transport and it is thought that StAR connects both mitochondrial membranes, forming a hydrophobic tunnel through which cholesterol could cross hydrophilic intermembrane space  ADDIN EN.CITE Clark1994282817Clark, B. J.Wells, J.King, S. R.Stocco, D. M.Department of Biochemistry and Molecular Biology, Texas Tech University Health Sciences Center, Lubbock 79430.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 ChemJ Biol Chem28314-2226945Amino Acid SequenceAnimalsBase SequenceCell LineCercopithecus aethiopsCholic AcidsCloning, MolecularDetergentsElectrophoresis, Polyacrylamide Gel*Gene ExpressionLeydig Cell Tumor/*metabolismLuteinizing Hormone/*pharmacologyMiceMitochondria/drug effects/*metabolismMolecular Sequence DataMolecular WeightNeoplasm Proteins/*biosynthesis/chemistry/isolation & purificationOligodeoxyribonucleotidesPeptide Fragments/chemistry/isolation & purification*PhosphoproteinsProtein BiosynthesisProtein Structure, SecondaryRecombinant Proteins/biosynthesis/chemistry/isolation & purificationRestriction MappingTransfectionTumor Cells, Cultured1994Nov 117961770http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=7961770 Stocco2001424217Stocco, D. M.Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA.Tracking the role of a star in the sky of the new millenniumMol EndocrinolMol Endocrinol1245-54158AnimalsCholesterol/metabolismGene Expression RegulationHumansModels, MolecularPhosphoproteins/chemistry/genetics/*physiologySteroids/biosynthesis2001Aug11463850http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11463850 (Clark et al., 1994; Stocco, 2001). In situ hybridization studies have shown the presence of StAR mRNA in the fetal mouse gonads as early as day 10.5 p.c. in both sexes. During subsequent development, StAR mRNA expression persisted in the fetal testis but was absent from the fetal ovaries  ADDIN EN.CITE Clark1995161617Clark, B. J.Soo, S. C.Caron, K. M.Ikeda, Y.Parker, K. L.Stocco, D. M.Department of Cell Biology and Biochemistry, Texas Tech University Health Science Center, Lubbock 79430, USA.Hormonal and developmental regulation of the steroidogenic acute regulatory proteinMol EndocrinolMol Endocrinol1346-55910AnimalsBase SequenceCyclic AMP/pharmacologyEmbryo/*metabolismFemaleGene Expression Regulation, DevelopmentalLuteinizing Hormone/metabolismMaleMiceMolecular Sequence DataOrgan SpecificityPregnancyProteins/genetics/*metabolismRNA, Messenger/*metabolismResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.Steroids/*metabolismTesticular NeoplasmsTumor Cells, Cultured1995Oct8544843http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8544843 (Clark et al., 1995). Cholesterol is an obligatory precursor for all steroid hormones and has to be either synthesized by the steroidogenic cells or delivered to them by blood or surrounding cells  ADDIN EN.CITE Azhar2003242417Azhar, S.Leers-Sucheta, S.Reaven, E.Geriatric Research, Education and Clinical Center, VA Palo Alto Heath Care System, Palo Alto, California 94304, USA. salman.azhar@med.va.govCholesterol uptake in adrenal and gonadal tissues: the SR-BI and 'selective' pathway connectionFront BiosciFront Bioscis998-10298Adrenal Glands/*metabolismAnimalsAntigens, CD36/*physiologyCholesterol, HDL/*metabolismGonads/*metabolismHumans*Membrane ProteinsOrgan Specificity/physiology*Receptors, Immunologic*Receptors, LipoproteinReceptors, ScavengerScavenger Receptors, Class BSignal Transduction/*physiology2003Sep 112957864http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12957864 </EndNote>(Azhar et al., 2003). Lanosterol 14a-demethylase (cyp51) is a member of the cytochrome P450 superfamily involved in early steps of cholesterol biosynthesis. As cholesterol is an integral part of cell membranes and is involved in many other functions in the cells, cyp51 is present in most, if not all, mammalian cells  ADDIN EN.CITE Choudhary2003111117Choudhary, D.Jansson, I.Schenkman, J. B.Sarfarazi, M.Stoilov, I.Department of Pharmacology, Surgical Research Center, University of Connecticut Health Center, Farmington 06030, USA.Comparative expression profiling of 40 mouse cytochrome P450 genes in embryonic and adult tissuesArch Biochem BiophysArch Biochem Biophys91-1004141Aging/*genetics/physiologyAnimalsBase SequenceComparative StudyCytochrome P-450 Enzyme System/classification/*geneticsEmbryo/enzymologyGene Expression Profiling/*methodsGene Expression Regulation, Developmental/*geneticsGene Expression Regulation, Enzymologic/*geneticsMaleMiceMice, Inbred BALB CMolecular Sequence DataOligonucleotide Array Sequence Analysis/methodsOrgan SpecificityResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.Reverse Transcriptase Polymerase Chain Reaction/methodsSequence AlignmentSequence Analysis, DNA/methods2003Jun 112745259http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12745259 (Choudhary et al., 2003). However, its expression is stronger in steroidogenic tissues such as gonads and adrenal glands in adult animals  ADDIN EN.CITE Stromstedt1996434317Stromstedt, M.Rozman, D.Waterman, M. R.Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA stromsm@ctrvax.vanderbilt.eduThe ubiquitously expressed human CYP51 encodes lanosterol 14 alpha-demethylase, a cytochrome P450 whose expression is regulated by oxysterolsArch Biochem BiophysArch Biochem Biophys73-813291Amino Acid SequenceAnimalsBase SequenceCell LineCholesterol/*metabolismCytochrome P-450 Enzyme System/*biosynthesis/geneticsDNA, ComplementaryEscherichia coli/genetics*Gene Expression Regulation, EnzymologicHumansMolecular Sequence DataOxidoreductases/*biosynthesis/geneticsRatsTissue Distribution1996May 18619637http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8619637 Majdic20005517Majdic, G.Parvinen, M.Bellamine, A.Harwood, H. J., Jr.Ku, W. W.Waterman, M. R.Rozman, D.Veterinary Faculty, Clinic of Reproduction, Cesta v Mestni log 47a, 1000 Ljubljana, Slovenia.Lanosterol 14alpha-demethylase (CYP51), NADPH-cytochrome P450 reductase and squalene synthase in spermatogenesis: late spermatids of the rat express proteins needed to synthesize follicular fluid meiosis activating sterolJ EndocrinolJ Endocrinol463-741662AnimalsCholestenes/metabolismCytochrome P-450 Enzyme System/*analysisImmunoblottingImmunohistochemistryLeydig Cells/enzymologyLiver/enzymologyMaleNADPH-Ferrihemoprotein Reductase/*analysisOxidoreductases/*analysisRatsRats, Sprague-DawleyRats, WistarResearch Support, Non-U.S. Gov'tSpermatids/*enzymology*SpermatogenesisSqualene Synthetase/*analysis2000Aug10927636http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10927636 (Stromstedt et al., 1996; Majdic et al., 2000), but there are no reports about the cyp51 expression, or expression of any of the other enzymes involved in cholesterologenesis, during early stages of fetal gonadal development. NADPH cytochrome P450 reductase is another enzyme required for cholesterol production, as it is necessary partner in several enzymatic reactions during transformation of acetate to cholesterol. In the present study, immunoexpression of cyp51, NADPH cytochrome P450 reductase, StAR and 3-HSD I proteins was studied in murine gonads during fetal development with the aim to determine whether cholesterol is produced endogenously or derived exogenously during initial steps of steroid hormone production. Materials and Methods Animals and tissue recovery C57BL/6 mice were bred in standard conditions. Females were paired with males and checked every morning for the presence of copulatory plug. The morning on the day when plug was found was designated as day 0.5 p.c. Pregnant females were euthanized by CO2 followed by cervical dislocation on different days of pregnancy. Fetuses were dissected and either whole fetuses or fetal gonads were fixed in Bouins solution overnight (whole fetuses) or 3-4 hours (isolated gonads) and subsequently processed into paraffin wax using standard procedures. All animal experiments were done according to ethical principles and in accordance with EU directive (86/609/EEC). Animal experiments were approved by the Veterinary commission of Slovenia. Sex determination Mice fetuses 11.5, 12.0 and 12.5 days old were genotyped to determine sex. At the time of dissection, amnions were carefully removed and digested in the thermostatic shaker in 200 ml of PCR DNA buffer (Promega, Madison, WI, USA) containing 0.15 mg of Proteinase K (Sigma, Taufkirchen, Germany) at 55 C overnight. Three microliters of lysate were used for PCR reaction containing primers for the Sry gene as described before  ADDIN EN.CITE Luo1994353517Luo, X.Ikeda, Y.Parker, K. L.Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiationCellCell481-90774Adrenal Glands/*embryologyAnimalsAnimals, NewbornApoptosisBase SequenceCell MovementCloning, MolecularCorticosterone/bloodCorticotropin/bloodDNA-Binding Proteins/*genetics/physiologyFallopian Tubes/embryologyFemaleGerm Cells/cytologyGonads/*embryologyMaleMiceMice, Knockout/geneticsMolecular Sequence DataReceptors, Cytoplasmic and Nuclear/*genetics/physiologyResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.Sex Differentiation/*geneticsTranscription Factors/*genetics/physiology1994May 208187173http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8187173 (Luo et al., 1994). Immunocytochemistry Sections 7 m thick were mounted on the slides coated with 3-aminopropyl triethoxysilane (TESPA, Sigma) and dried overnight at 42C. Before incubation with the primary antibody, sections were dewaxed in xylene, rehydrated in graded ethanols, washed in water and 0.01M PBS containing 0.05% Tween 20 (Svanova Biothech AB, Uppsala, Sweden) . Endogenous peroxidases were blocked by incubating the sections for 20 minutes in 1% H2O2 in PBS-Tween 20 at room temperature, followed by 5 minute wash in PBS-Tween 20. For StAR immunostaining, antigen retrieval was performed in 0.01M Na-citrate pH 6.0 (Sigma) before incubation with primary antiserum. Sections were boiled in the microwave for 20 minutes and left undisturbed for additional 20 minutes, followed by a 5 minute wash in PBS-Tween 20. All sections were blocked in normal goat serum diluted 1:5 in PBS-Tween 20 (Chemicon Temecula, CA, USA) for 30min. Rabbit polyclonal antibodies against the human CYP51 protein (gift from Mike Waterman, Vanderbilt University, Nashville, TN, USA), rabbit polyclonal antibodies against NADPH cytochrome P450 reductase (Abcam, Cambridge, UK), rabbit polyclonal antibodies against 3-HSD I (gift from Ian Mason, University of Edinburgh, Edinburgh, Scotland) and rabbit polyclonal antibodies against StAR (gift from Dale Hales, Northwestern University, Chicago, IL, USA) were used at dilutions of 1:50, 1:500, 1:1000 and 1:200, respectively  ADDIN EN.CITE Doody1990313117Doody, K. M.Carr, B. R.Rainey, W. E.Byrd, W.Murry, B. A.Strickler, R. C.Thomas, J. L.Mason, J. I.Department of Obstetrics and Gynecology, Cecil H. and Ida Green Center for Reproductive Sciences, University of Texas, Southwestern Medical Center, Dallas 75235.3 beta-hydroxysteroid dehydrogenase/isomerase in the fetal zone and neocortex of the human fetal adrenal glandEndocrinologyEndocrinology2487-9212653-Hydroxysteroid Dehydrogenases/*analysisAdrenal Glands/*embryology/enzymologyAdrenocorticotropic Hormone/pharmacologyCells, CulturedElectrophoresis, Polyacrylamide GelEnzyme Induction/drug effectsHumansHydrocortisone/biosynthesis/secretionImmunoblottingIsomerases/*analysisMolecular WeightMultienzyme Complexes/*analysis/biosynthesisProgesterone Reductase/*analysis/biosynthesisSteroid Isomerases/*analysis/biosynthesisTissue Distribution1990May2158427http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=2158427 Ronen-Fuhrmann1998404017Ronen-Fuhrmann, T.Timberg, R.King, S. R.Hales, K. H.Hales, D. B.Stocco, D. M.Orly, J.Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Israel.Spatio-temporal expression patterns of steroidogenic acute regulatory protein (StAR) during follicular development in the rat ovaryEndocrinologyEndocrinology303-151391AnimalsCOS CellsChorionic Gonadotropin/pharmacologyFemale*Gene Expression RegulationMitochondria/metabolismOvarian Follicle/*metabolismPhosphoproteins/analysis/*geneticsRNA, Messenger/analysisRatsRats, Sprague-Dawley1998Jan9421428http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9421428 Majdic20005517Majdic, G.Parvinen, M.Bellamine, A.Harwood, H. J., Jr.Ku, W. W.Waterman, M. R.Rozman, D.Veterinary Faculty, Clinic of Reproduction, Cesta v Mestni log 47a, 1000 Ljubljana, Slovenia.Lanosterol 14alpha-demethylase (CYP51), NADPH-cytochrome P450 reductase and squalene synthase in spermatogenesis: late spermatids of the rat express proteins needed to synthesize follicular fluid meiosis activating sterolJ EndocrinolJ Endocrinol463-741662AnimalsCholestenes/metabolismCytochrome P-450 Enzyme System/*analysisImmunoblottingImmunohistochemistryLeydig Cells/enzymologyLiver/enzymologyMaleNADPH-Ferrihemoprotein Reductase/*analysisOxidoreductases/*analysisRatsRats, Sprague-DawleyRats, WistarResearch Support, Non-U.S. Gov'tSpermatids/*enzymology*SpermatogenesisSqualene Synthetase/*analysis2000Aug10927636http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10927636 (Doody et al., 1990; Ronen-Fuhrmann et al., 1998; Majdic et al., 2000). Sections were incubated with antibodies diluted in PBS-Tween 20 containing normal goat serum (5:1 v/v) overnight at 4C (for all four antisera). The following day sections were washed twice in PBS-Tween 20 (5 minutes each wash), incubated for 30 minutes with 1:100 dilution of goat anti-rabbit IgG (Dako, Glostrup Denmark) in PBS-Tween 20 and washed again in PBS-Tween 20 twice for 5 minutes. For detection of bound antibodies, sections were incubated with a 1:500 dilution of rabbit peroxidase-antiperoxidase complex (Jackson Immunochemicals, West Grove, PA, USA) in 0.05M Tris, pH 7.4 for 30min and then washed twice in PBS-Tween 20 (5 minutes each). Color reaction product was developed by incubating sections in a solution of 0.05% (w/v) 3, 3-triaminobenzendine tetrahydrochloride (Sigma) in 0.05M Tris-HCl, pH 7.4 and 0.01% hydrogen peroxide. After 5-30 min, sections were washed twice in distilled water, counterstained with hematoxyline, dehydrated in graded ethanols, cleared in xylene and coversliped using Pertex mounting medium (Medite, Burgdorf, Germany). Specificity of the antisera was evaluated by using normal rabbit serum instead of primary antisera. Photomicrographs were taken on Nikon microphot FXA microscope. Results Mouse gonads 3-hydroxysteroid dehydrogenase I (3-HSD I) No immunopositive cells were detected in the fetal gonads on days 11.5 p.c and 12.0 p.c.. On day 12.5 p.c., several immunopositive cells were found in the fetal testes (Fig. 1a) while no immunoexpression was found in the fetal ovary (not shown). Immunoexpression of 3-HSD I remained strong in the testes throughout the gestation (Fig. 1c  13.5 p.c., 1e  18.5 p.c.). Steroidogenic acute regulatory protein (StAR) No StAR immunoexpression was detected at 11.5 p.c. and 12.0 p.c., and few scattered positive cells were found on day 12.5 p.c (Fig. 1b) in the mouse fetal testis. On day 13.5 p.c., expression increased and several strongly immunopositive cells were found in the fetal testes (Fig. 1d) while no immunopositive cells were detected in the fetal ovary at 12.5 or 13.5 p.c. (not shown). Similar to 3-HSD I, StAR immunoexpression in the testis remained strong throughout the fetal period (Fig. 1f, 18.5 p.c.). Lanosterol 14-demethylase (cyp51) Cyp51 protein was absent in the fetal gonads on days 11.5 p.c. to 12.5 p.c. (Fig 2a) but appeared in the fetal testis on day 13.5 (Fig 2c). Thereafter, immunoexpression remained strong in the fetal Leydig cells (Fig 2e) and was absent in the fetal ovaries (not shown). NADPH cytochrome P450 reductase NADPH cytochrome P450 reductase protein was not detected in the fetal gonads on days 11.5 p.c., 12.0 p.c. and 12.5 p.c. (Fig 2b) but appeared in the fetal testis on day 13.5 (Fig 2d). Thereafter, immunoexpression remained strong in the fetal Leydig cells (Fig 2f) and was absent in fetal ovaries (not shown). Discussion Sexual differentiation is a carefully regulated process that requires precise temporal and spatial development of different cell types in the developing gonads  ADDIN EN.CITE George199446465<style face="normal" font="default" charset="238" size="100%">Sex determination and differentiation</style>Ross2005414117Ross, A. J.Capel, B.Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA.Signaling at the crossroads of gonad developmentTrends Endocrinol MetabTrends Endocrinol Metab19-25161AnimalsFemaleGerm CellsGonads/cytology/embryology/*growth & developmentHumansLeydig Cells/physiologyMaleMiceRatsSertoli Cells/physiologySex CharacteristicsSignal Transduction/*physiology2005Jan-Feb15620545http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15620545 (George & Wilson, 1994; Ross & Capel, 2005). Hormones produced by the fetal testis are essential for the development of all male secondary sexual organs. Therefore, proper development and differentiation of testicular cells is crucial for the normal development of the male phenotype. In contrast, the fetal development of the female phenotype is largely hormone independent as even in the absence of ovaries, mullerian ducts develop into the oviducts, uterus and upper portion of vagina  ADDIN EN.CITE George199446465<style face="normal" font="default" charset="238" size="100%">Sex determination and differentiation</style>(George & Wilson, 1994). In the present study, immunoexpression of four different proteins involved in the production of steroid hormones or their precursor, cholesterol, was studied during gonadal development in the mouse fetuses with the aim to establish whether the newly developed Leydig cells posses enzymes for steroidogenesis and production of cholesterol or are dependent on exogenously derived cholesterol for the production of testosterone. Cholesterol is an essential part of the cell membranes, has important roles as a signaling molecule during the embryonic development and is a precursor for the steroid hormone synthesis. Requirement for cholesterol during the embryonic development is clearly demonstrated by embryonic lethality of a knockout mice model lacking squalene synthase, enzyme involved in the production of cholesterol  ADDIN EN.CITE Tozawa1999444417Tozawa, R.Ishibashi, S.Osuga, J.Yagyu, H.Oka, T.Chen, Z.Ohashi, K.Perrey, S.Shionoiri, F.Yahagi, N.Harada, K.Gotoda, T.Yazaki, Y.Yamada, N.Department of Metabolic Diseases, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.Embryonic lethality and defective neural tube closure in mice lacking squalene synthaseJ Biol ChemJ Biol Chem30843-827443AnimalsCholesterol/biosynthesisCrosses, GeneticEmbryonic and Fetal DevelopmentFarnesyl-Diphosphate Farnesyltransferase/*deficiency/*geneticsFemale*Fetal DeathGene Expression Regulation, EnzymologicGenotypeGestational AgeHeterozygoteLipoproteins/bloodLiver/enzymologyMaleMiceMice, KnockoutNeural Tube Defects/enzymology/*geneticsRNA, Messenger/geneticsReceptors, LDL/deficiency/genetics/physiologyTestis/enzymology1999Oct 2210521476http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10521476 (Tozawa et al., 1999). Most cells in the mammalian body have the ability to synthesize cholesterol from its precursor acetate. However, exogenously derived cholesterol is equally important for the proper development and function of the mammalian body and carefully regulated balance between exo- and endogenously derived cholesterol is essential for proper function of cells  ADDIN EN.CITE Azhar2003242417Azhar, S.Leers-Sucheta, S.Reaven, E.Geriatric Research, Education and Clinical Center, VA Palo Alto Heath Care System, Palo Alto, California 94304, USA. salman.azhar@med.va.govCholesterol uptake in adrenal and gonadal tissues: the SR-BI and 'selective' pathway connectionFront BiosciFront Bioscis998-10298Adrenal Glands/*metabolismAnimalsAntigens, CD36/*physiologyCholesterol, HDL/*metabolismGonads/*metabolismHumans*Membrane ProteinsOrgan Specificity/physiology*Receptors, Immunologic*Receptors, LipoproteinReceptors, ScavengerScavenger Receptors, Class BSignal Transduction/*physiology2003Sep 112957864http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12957864 </record></Cite></EndNote>(Azhar et al., 2003). Cyp51 or lanosterol 14-demethylase is involved in late steps of cholesterol synthesis  ADDIN EN.CITE <EndNote><Cite><Author>Stromstedt</Author><Year>1996</Year><RecNum>43</RecNum><record><rec-number>43</rec-number>17Stromstedt, M.Rozman, D.Waterman, M. R.Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA stromsm@ctrvax.vanderbilt.eduThe ubiquitously expressed human CYP51 encodes lanosterol 14 alpha-demethylase, a cytochrome P450 whose expression is regulated by oxysterolsArch Biochem BiophysArch Biochem Biophys73-813291Amino Acid SequenceAnimalsBase SequenceCell LineCholesterol/*metabolismCytochrome P-450 Enzyme System/*biosynthesis/geneticsDNA, ComplementaryEscherichia coli/genetics*Gene Expression Regulation, EnzymologicHumansMolecular Sequence DataOxidoreductases/*biosynthesis/geneticsRatsTissue Distribution1996May 18619637http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8619637 </url></related-urls></urls></record></Cite></EndNote>(Stromstedt et al., 1996). Cyp51 transforms lanosterol into 4,4,-dimethyl 5a-cholesta, 8,14,24-diene-3b-ol, also called follicular fluid meiosis activating sterol (FF-MAS) as some studies suggested that this sterol acts as a meiosis activating substance at least in vitro  ADDIN EN.CITE Byskov1997272717Byskov, A. G.Yding Andersen, C.Hossaini, A.Guoliang, X.Juliane Marie Center for Children, Women, and Reproduction, University Hospital of Copenhagen, Denmark.Cumulus cells of oocyte-cumulus complexes secrete a meiosis-activating substance when stimulated with FSHMol Reprod DevMol Reprod Dev296-305463AnimalsCells, CulturedChorionic Gonadotropin/pharmacologyFemaleFollicle Stimulating Hormone/*pharmacologyGranulosa Cells/drug effects/secretionHumansLuteinizing Hormone/pharmacologyMeiosis/*drug effectsMiceOocytes/drug effects/*secretion1997Mar9041132http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9041132 Byskov1999474717Byskov, A. G.Andersen, C. Y.Leonardsen, L.Baltsen, M.Laboratory of Reproductive Biology, Juliane Marie Center for Children, Women and Reproduction, University Hospital of Copenhagen, DK-2100 Copenhagen, Denmark. agb.lrb@notes.rh.dkMeiosis activating sterols (MAS) and fertility in mammals and manJ Exp ZoolJ Exp Zool237-422853AnimalsCells, CulturedCholestadienols/chemistry/*metabolismFemaleFertility/*physiologyFollicular Fluid/metabolismHumansMaleMeiosis/*physiologyOocytes/cytology/physiologySignal TransductionSpermatogenesis/physiologyTestis/metabolism1999Oct 1510497322http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10497322 (Byskov et al., 1997; Byskov et al., 1999). Cyp51 is strongly expressed in the adult gonads and adrenal glands. Interestingly, very strong expression of Cyp51 was detected in postmeiotic germ cells, suggesting an important role of this enzyme in germ cells during spermatogenesis  ADDIN EN.CITE Stromstedt19986617Stromstedt, M.Waterman, M. R.Haugen, T. B.Tasken, K.Parvinen, M.Rozman, D.Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.Elevated expression of lanosterol 14alpha-demethylase (CYP51) and the synthesis of oocyte meiosis-activating sterols in postmeiotic germ cells of male ratsEndocrinologyEndocrinology2314-211395AnimalsCytochrome P-450 Enzyme System/*genetics/metabolism*Gene ExpressionHumansL-Lactate Dehydrogenase/metabolismMaleMeiosis/*physiologyOocytes/*cytologyOxidoreductases/*genetics/metabolismRNA, Messenger/analysisRatsResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.Spermatozoa/*enzymologySqualene Synthetase/geneticsSterols/*biosynthesisTestis/cytology/enzymologyTissue Distribution1998May9564839http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9564839 Cotman2004292917Cotman, M.Jezek, D.Fon Tacer, K.Frangez, R.Rozman, D.Laboratory for Medical Genetics, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, SI-1000 Ljubljana, Slovenia.A functional cytochrome P450 lanosterol 14 alpha-demethylase CYP51 enzyme in the acrosome: transport through the Golgi and synthesis of meiosis-activating sterolsEndocrinologyEndocrinology1419-261453Acrosome/*enzymologyAnimalsCattleCell FractionationCytochrome P-450 Enzyme System/*metabolismEjaculationGolgi Apparatus/*metabolismLiver/enzymologyMaleMeiosis/*physiologyMiceMice, Inbred CBANADPH-Ferrihemoprotein Reductase/metabolismOxidoreductases/*metabolismSheepSpermatids/enzymologySpermatozoa/enzymologySterols/biosynthesisTestis/cytology/enzymology2004Mar14630712http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=14630712 (Stromstedt et al., 1998; Cotman et al., 2004). This increased expression of Cyp51 could be connected with the remodeling of cell membranes in the developing spermatids, although accumulation of Cyp51 product T-MAS (testes meiosis activating substance) suggest additional roles for this sterol, possibly as a signaling molecule. NADPH cytochrome P450 reductase is an electron transferring enzyme, required for the activity of several enzymes involved in cholesterol production, including cyp51. The presence of NADPH cytochrome P450 reductase is thought to be a rate limiting step in several enzymatic reactions during cholesterol synthesis and the activity of this enzyme is absolutely necessary for the production of cholesterol. In the fetal gonads, we found immunoexpression of cyp51 together with expression of NADPH cytochrome P450 reductase on day 13.5 p.c. in the fetal testis and this expression persisted throughout fetal development. In the fetal testes, cyp51 and NADPH cytochrome P450 reductase were present in the interstitial, presumably Leydig cells, suggesting that de novo cholesterol biosynthesis is at least partially utilized for steroidogenesis in the fetal Leydig cells. Interestingly, the expression of both StAR and 3-HSD I proteins (as shown in this study) and the production of testosterone (Gondos, 1980) starts earlier, already on day 12.5 p.c. suggesting that initial steps of testosterone production in the fetal Leydig cells is dependent on exogenously derived cholesterol, what would be in agreement with previous studies, suggesting that plasma lipoproteins are the main source of cholesterol in steroidogenic cells  ADDIN EN.CITE Andersen1978232317Andersen, J. M.Dietschy, J. M.Relative importance of high and low density lipoproteins in the regulation of cholesterol synthesis in the adrenal gland, ovary, and testis of the ratJ Biol ChemJ Biol Chem9024-3225324Adrenal Glands/drug effects/*metabolismAnimalsCholesterol/*biosynthesisFemaleHumansKineticsLipoproteins, HDL/*blood/pharmacologyLipoproteins, LDL/*blood/pharmacologyMaleOrgan SpecificityOvary/drug effects/*metabolismRatsTestis/drug effects/*metabolism1978Dec 25214438http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=214438 Hou1990333317Hou, J. W.Collins, D. C.Schleicher, R. L.Department of Biochemistry, Emory University, Decatur, Georgia.Sources of cholesterol for testosterone biosynthesis in murine Leydig cellsEndocrinologyEndocrinology2047-551275Aminoglutethimide/pharmacologyAnimalsCholesterol/*metabolismCholesterol, HDL/pharmacologyHydroxymethylglutaryl CoA Reductases/metabolismIntracellular Membranes/metabolismLeydig Cells/*metabolismLovastatin/pharmacologyMaleMiceMice, Inbred C57BLSterols/metabolismTestosterone/*biosynthesis1990Nov2226298http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=2226298 Azhar1998252517Azhar, S.Nomoto, A.Leers-Sucheta, S.Reaven, E.Geriatric Research, Education, and Clinical Center, VA Palo Alto Health Care System, CA 94304, USA.Simultaneous induction of an HDL receptor protein (SR-BI) and the selective uptake of HDL-cholesteryl esters in a physiologically relevant steroidogenic cell modelJ Lipid ResJ Lipid Res1616-28398Amino Acid SequenceAnimalsAntigens, CD36/*biosynthesis/geneticsBase SequenceBiological Transport, Active/drug effectsBucladesine/pharmacology*Carrier ProteinsCaveolin 1*CaveolinsCell Membrane/metabolismCholesterol Esters/*metabolismDNA Primers/geneticsFemaleGene Expression/drug effectsGranulosa Cells/drug effects/metabolism/ultrastructureLipoproteins, HDL/*metabolismLuteal Cells/drug effects/metabolism/ultrastructureMembrane Proteins/metabolismMicroscopy, ElectronModels, BiologicalMolecular Sequence DataRNA, Messenger/genetics/metabolism*RNA-Binding ProteinsRatsRats, Sprague-Dawley*Receptors, ImmunologicReceptors, Lipoprotein/*biosynthesisReceptors, ScavengerScavenger Receptors, Class BSteroids/*biosynthesis1998Aug9717722http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9717722 (Andersen & Dietschy, 1978; Hou et al., 1990; Azhar et al., 1998). However, in our study we only used immunocytochemistry to determine the presence of the cyp51 and NADPH cytochrome P450 reductase proteins. As immunocytochemistry has a limited detection potential, it cannot be excluded at the present that both proteins were expressed at low levels also at an earlier age, but below detection limits of immunocytochemistry. One possibility to get a more conclusive answer would be to examine the mRNA expression using quantitative RT PCR, although the presence of the mRNA does not necessarily indicate the presence of bioactive proteins. The only conclusive answer could therefore be derived from measuring enzyme activity directly, but this would be almost impossible to conduct precisely, as specific dissection of fetal gonads (with exclusion of all surrounding tissue including nearby adrenal cells) on day 12.5 p.c. is almost impossible without microscopic dissector, but preparation of tissue for such dissection would interfere with enzyme activity. As antibodies against both NADPH cytochrome P450 reductase and cyp51 were shown before by western blot and immunocytochemistry (REFERENCE) to readily detect the respective proteins, this study nevertheless suggest that the significant amounts of NADPH cytochrome P450 reductase and cyp 51 proteins in the fetal mouse testis are only present from day 13.5 p.c. onwards. Both StAR and 3-HSD I proteins were detected in our study at 12.5 p.c., immediately after the formation of testicular cords and Sertoli cell differentiation (reviewed in  ADDIN EN.CITE <EndNote><Cite><Author>Swain</Author><Year>1999</Year><RecNum>50</RecNum>5017Swain, A.Lovell-Badge, R.Division of Developmental Genetics, Medical Research Council (MRC), National Institute for Medical Research, London NW7 1AA, UK.Mammalian sex determination: a molecular dramaGenes DevGenes Dev755-67137AnimalsCell DifferentiationCell LineageDNA-Binding Proteins/geneticsGerm Cells/growth & developmentGonads/*embryologyMaleMammals/*embryologyMiceMorphogenesis*Nuclear ProteinsReceptors, Peptide/geneticsReceptors, Retinoic Acid*Repressor ProteinsSertoli Cells/physiology*Sex Determination (Genetics)Sex-Determining Region Y ProteinTestis/embryologyTranscription Factors/*genetics1999Apr 110197976http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10197976 Swain & Lovell-Badge, 1999). This is interesting, as in the rat fetuses, Leydig cells develop about 1 - 2 days later then Sertoli cells and several studies suggested that Sertoli cells regulate Leydig cell differentiation  ADDIN EN.CITE Lejeune1992484817Lejeune, H.Skalli, M.Chatelain, P. G.Avallet, O.Saez, J. M.INSERM U-307, Hopital Debrousse, Lyon, France.The paracrine role of Sertoli cells on Leydig cell functionCell Biol ToxicolCell Biol Toxicol73-8383AnimalsCell Communication/*physiologyHormones/*physiologyLeydig Cells/*physiologyMaleSertoli Cells/*physiology/secretion1992Jul-Sep1446260http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1446260 Racine1998494917Racine, C.Rey, R.Forest, M. G.Louis, F.Ferre, A.Huhtaniemi, I.Josso, N.di Clemente, N.Institut National de la Sante et de la Recherche Medicale, Ecole Normale Superieure, Departement de Biologie, Montrouge, France.Receptors for anti-mullerian hormone on Leydig cells are responsible for its effects on steroidogenesis and cell differentiationProc Natl Acad Sci U S AProc Natl Acad Sci U S A594-9952AnimalsCell Differentiation/physiologyCells, CulturedGene Transfer Techniques*GlycoproteinsGrowth Inhibitors/*physiologyLeydig Cells/cytology/*physiologyMaleMiceMice, TransgenicReceptors, Peptide/*physiology*Signal TransductionSteroids/*metabolismTesticular Hormones/*physiology1998Jan 209435237http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9435237 (Lejeune et al., 1992; Racine et al., 1998). The present study together with previous report of testosterone production in the fetal mouse testis on day 12.5 p.c. ( Gondos, 1980) implicate that delay between Leydig and Sertoli cells appearance in the developing testis is much shorter in mice than in rat. The fetal mouse testis starts to produce testosterone and antimullerian hormone almost simultaneously, unlike in rats where there is at least one day difference in the onset of expression of AMH and steroidogenic enzymes  ADDIN EN.CITE Tran1987454517Tran, D.Picard, J. Y.Campargue, J.Josso, N.Immunocytochemical detection of anti-mullerian hormone in Sertoli cells of various mammalian species including humanJ Histochem CytochemJ Histochem Cytochem733-43357AnimalsAnimals, DomesticAnimals, NewbornChildChild, PreschoolEndoplasmic Reticulum/metabolismFetusFluorescent Antibody Technique*Glycoproteins*Growth InhibitorsHistocytochemistryHumansInfantInfant, NewbornMaleMicroscopy, Electron/methodsRatsSertoli Cells/*metabolism/ultrastructureTesticular Hormones/*analysis1987Jul3295030http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=3295030 Munsterberg1991373717Munsterberg, A.Lovell-Badge, R.Laboratory of Eukaryotic Molecular Genetics, MRC National Institute for Medical Research, London, UK.Expression of the mouse anti-mullerian hormone gene suggests a role in both male and female sexual differentiationDevelopmentDevelopment613-241132Amino Acid SequenceAnimalsBase SequenceBlotting, NorthernCattleCloning, MolecularFemaleGene Expression/*physiologyGenes/*genetics*GlycoproteinsGranulosa Cells/physiologyGrowth Inhibitors/*geneticsHumansMaleMiceMolecular Probe TechniquesMolecular Sequence DataMullerian Ducts/*physiologySequence Homology, Nucleic AcidSertoli Cells/physiologySex Differentiation/*geneticsTesticular Hormones/*genetics1991Oct1782869http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1782869 Majdic1998363617Majdic, G.Saunders, P. T.Teerds, K. J.MRC Reproductive Biology Unit, Edinburgh, United Kingdom.Immunoexpression of the steroidogenic enzymes 3-beta hydroxysteroid dehydrogenase and 17 alpha-hydroxylase, C17,20 lyase and the receptor for luteinizing hormone (LH) in the fetal rat testis suggests that the onset of Leydig cell steroid production is independent of LH actionBiol ReprodBiol Reprod520-55823-Hydroxysteroid Dehydrogenases/*biosynthesisAnimalsAnimals, NewbornCytoplasm/enzymologyFemaleFetus/metabolismImmunohistochemistryLeydig Cells/*metabolismMalePregnancyRatsRats, WistarReceptors, LH/*biosynthesisSteroid 17-alpha-Hydroxylase/*biosynthesisTestis/cytology/embryology/*enzymology1998Feb9475409http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9475409 (Tran et al., 1987; Munsterberg & Lovell-Badge, 1991; Majdic et al., 1998), suggesting that Sertoli cell regulation of Leydig cell development in mice must be much quicker process than in rat. In conclusion, in the present study we demonstrated early expression of two proteins required for the production of steroid hormones, StAR and 3-HSD I, suggesting almost simultaneous development of Sertoli and Leydig cells in the fetal mice testes. As expression of cyp51 and NADPH cytochrome P450 reductase appeared only one day later, these results suggest that early steroid hormone production is mostly dependent on exogenously derived cholesterol. Acknowledgement The authors are grateful to Mike Waterman, Ian Mason and Dale Hales for their generous gifts of the antibodies. This work was done with support from Slovenian Ministry of Higher education and science research programme P4-0053. Tomaz Bdefeld is supported by the fellowship from foundation Stein. References  ADDIN EN.REFLIST Andersen, J. M. & Dietschy, J. M. (1978) Relative importance of high and low density lipoproteins in the regulation of cholesterol synthesis in the adrenal gland, ovary, and testis of the rat. 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Molecular Reproduction and Development, 46, 296-305. Choudhary, D., Jansson, I., Schenkman, J. B., Sarfarazi, M. & Stoilov, I. (2003) Comparative expression profiling of 40 mouse cytochrome p450 genes in embryonic and adult tissues. Archives in Biochemistry and Biophysics, 414, 91-100. Clark, B. J., Soo, S. C., Caron, K. M., Ikeda, Y., Parker, K. L. & Stocco, D. M. (1995) Hormonal and developmental regulation of the steroidogenic acute regulatory protein. Molecular Endocrinology, 9, 1346-1355. Clark, B. J., Wells, J., King, S. R. & Stocco, D. M. (1994) 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). Journal of Biological Chemistry, 269, 28314-28322. Cotman, M., Jezek, D., Fon Tacer, K., Frangez, R. & Rozman, D. (2004) A functional cytochrome p450 lanosterol 14 alpha-demethylase cyp51 enzyme in the acrosome: Transport through the golgi and synthesis of meiosis-activating sterols. Endocrinology, 145, 1419-1426. Davidoff, M. S., Middendorff, R., Enikolopov, G., Riethmacher, D., Holstein, A. F. & Muller, D. (2004) Progenitor cells of the testosterone-producing leydig cells revealed. Journal of Cell Biology, 167, 935-944. Doody, K. M., Carr, B. R., Rainey, W. E., Byrd, W., Murry, B. A., Strickler, R. C., Thomas, J. L. & Mason, J. I. (1990) 3 beta-hydroxysteroid dehydrogenase/isomerase in the fetal zone and neocortex of the human fetal adrenal gland. Endocrinology, 126, 2487-2492. George, F. W. & Wilson, J. D. (1994) Sex determination and differentiation. In: The physiology of reproduction. (eds. E. Knobil and J. Neill), pp. 3-28. Raven Press, New York. Gondos, B. (1980) Development and differentiation of the testis and male reproductive tract. In: Testicular development, structure and function. (eds. A. Steinberger and B. Steinberger), pp. 3-20. 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L. & Payne, A. H. (1994) Ontogeny of expression of the genes for steroidogenic enzymes p450 side-chain cleavage, 3 beta-hydroxysteroid dehydrogenase, p450 17 alpha-hydroxylase/c17-20 lyase, and p450 aromatase in fetal mouse gonads. Endocrinology, 135, 262-268. Habert, R., Lejeune, H. & Saez, J. M. (2001) Origin, differentiation and regulation of fetal and adult leydig cells. Molecular and Cellular Endocrinology, 179, 47-74. Hou, J. W., Collins, D. C. & Schleicher, R. L. (1990) Sources of cholesterol for testosterone biosynthesis in murine leydig cells. Endocrinology, 127, 2047-2055. Josso, N., di Clemente, N. & Gouedard, L. (2001) Anti-mullerian hormone and its receptors. Molecular and Cellular Endocrinology, 179, 25-32. Lejeune, H., Skalli, M., Chatelain, P. G., Avallet, O. & Saez, J. M. (1992) The paracrine role of sertoli cells on leydig cell function. Cell Biology and Toxicology, 8, 73-83. Luo, X., Ikeda, Y. & Parker, K. L. (1994) A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell, 77, 481-490. Majdic, G., Parvinen, M., Bellamine, A., Harwood, H. J., Jr., Ku, W. W., Waterman, M. R. & Rozman, D. (2000) Lanosterol 14alpha-demethylase (cyp51), nadph-cytochrome p450 reductase and squalene synthase in spermatogenesis: Late spermatids of the rat express proteins needed to synthesize follicular fluid meiosis activating sterol. Journal of Endocrinology, 166, 463-474. Majdic, G., Saunders, P. T. & Teerds, K. J. (1998) Immunoexpression of the steroidogenic enzymes 3-beta hydroxysteroid dehydrogenase and 17 alpha-hydroxylase, c17,20 lyase and the receptor for luteinizing hormone (lh) in the fetal rat testis suggests that the onset of leydig cell steroid production is independent of lh action. Biology of Reproduction, 58, 520-525. Munsterberg, A. & Lovell-Badge, R. (1991) Expression of the mouse anti-mullerian hormone gene suggests a role in both male and female sexual differentiation. Development, 113, 613-624. Racine, C., Rey, R., Forest, M. G., Louis, F., Ferre, A., Huhtaniemi, I., Josso, N. & di Clemente, N. (1998) Receptors for anti-mullerian hormone on leydig cells are responsible for its effects on steroidogenesis and cell differentiation. Proceedings of the National Academy of Sciences of the U S A, 95, 594-599. Ronen-Fuhrmann, T., Timberg, R., King, S. R., Hales, K. H., Hales, D. B., Stocco, D. M. & Orly, J. (1998) Spatio-temporal expression patterns of steroidogenic acute regulatory protein (star) during follicular development in the rat ovary. Endocrinology, 139, 303-315. Ross, A. J. & Capel, B. (2005) Signaling at the crossroads of gonad development. Trends in Endocrinology and Metabolism, 16, 19-25. Stocco, D. M. (2001) Tracking the role of a star in the sky of the new millennium. Molecular Endocrinology, 15, 1245-1254. 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Y., Campargue, J. & Josso, N. (1987) Immunocytochemical detection of anti-mullerian hormone in sertoli cells of various mammalian species including human. Journal of Histochemistry & Cytochemistry, 35, 733-743.  Figure legends: Figure 1: No 3-HSD I and StAR immunopositive cells were detected in fetal testes on day 12.0 p.c. Few 3-HSD I (a) and StAR (b) immunopositive cells (arrows) were detected in fetal testes on day 12.5 p.c.. Expression of both proteins increased one day later (c  3-HSD I, d  StAR; day 13.5 p.c.) and remained strong throughout gestation (e  3-HSD I, f  StAR; day 18.5 p.c.). Insert - sections incubated with normal rabbit serum. Bar = 50 m. Figure 2: No cyp51 (a) or NADPH cytochrome P450 reductase !˻ֻػۻּڼ "% !# $136˹˹hC096mH sH h<6mH sH h1hlU6mH sH h)}(6mH sH hh)}(6mH sH h1hlU5mH sH hlUmH sH I  dgddw0d^`0gddw   $&BCtbtVTLhqmH sH UhiRhdw5mH sH #h<]B*CJOJQJ^JaJph#hdwB*CJOJQJ^JaJphhdw5B*CJ\aJphh<]mH sH hphdwmH sH hdw5mH sH hdwmH sH ha4mH sH hXg5mH sH jhlUUmH sH h1hlU5mH sH hlUmH sH h)}(6mH sH hh)}(6mH sH  |}~h]hgdZ &`#$gdF\z 0^`0gd10d^`0gddwdgddw (b) immunopositive cells were detected in fetal testes on day 12.0 p.c. First immunopositive cells appeared on day 13.5 (c cyp51, d NADPH cytochrome P450 reductase) and thereafter, strong immunoexpression persisted throughout the development (e cyp51, f NADPH cytochrome P450 reductase; day 18.5 p.c.). Insert sections incubated with normal rabbit serum. Bar = 50 m.     PAGE  PAGE 20 C~ľľľįľh10JmHnHuh e h e0Jjh e0JUh[IRjh[IRUh4hdwmH sH h4hcmH sH hcmH sH hdwmH sH  0^`0gd1h]hgdZ 6P1h:p ,'. 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