Skip to main content
Download PDF
  • Endocrinologie
  • Published:

L’aromatase testiculaire: Du gène a la proteine

The testicular aromatase: From the gene to the protein

Andrologie volume 8, pages 191–198 (1998)Cite this article

Resume

La conversion irréversible des androgènes en estrogènes est assurée par l’aromatase, complexe enzymatique constitué de deux entités protéiques: un cytochrome P450 aromatase, spécifique de la liaison du stéroïde et de la réaction d’aromatisation, et une réductase ubiquitaire. L’aromatase est présente dans le réticulum endoplasmique de nombreuses cellules stéroïdogènes de vertébrés, et notamment les gonades. C’est une protéine unique de 503 acides aminés chez l’homme appartenant à la superfamille des cytochromes P450; elle est codée par un gène d’au moins 75 kb comportant 17 exons, dont 8 non codants soumis à un épissage alternatif. Le développement de la RT-PCR quantitative a permis de dénombrer dans le testicule du rat adulte, les transcrits de l’aromatase, non seulement dans les cellules de Leydig et de Sertoli mais aussi dans les cellules germinales à différents stades de leur maturation. Les taux d’ARNm du P450arom décroissent de 50% dans les cellules germinales entre le stade spermatocyte pachytène et spermatide ronde, et deviennent très faibles dans les spermatozoïdes.

En outre, en fonction du degré de maturation du testicule, le nombre de copies de l’ARNm varie, en particulier dans la cellule de Sertoli adulte où le taux est 20 fois inférieur à celui des cellules immatures alors que dans les cellules de Leydig la quantité d’ARNm augmente sensiblement avec l’âge. Très récemment nous avons rapporté l’existence de deux transcrits non-codants (perte du domaine de liaison à l’hème) dans les spermatocytes pachytènes et les spermatides rondes résultant d’un épissage alternatif impliquant la partie 3′ des pré-ARNm de l’aromatase. L’aromatase a été par ailleurs localisée par immunohistochimie dans les cellules de Leydig chez l’homme, chez le rat, le porc, le bélier et l’étalon. Nous avons confirmé l’existence de l’aromatase dans les cellules de Leydig, mais nous l’avons aussi mise en évidence sur coupes testiculaires, dans les cellules situées à proximité de la lumière des tubes séminifères (plus précisément dans les spermatides allongées et les spermatozoïdes). Après incubation avec de l’androstènedione tritiée, la présence d’une protéine biologiquement active, non seulement dans les cellules de Leydig mais aussi dans les cellules germinales (activité enzymatique 2–5 fois plus forte dans les spermatozoïdes que dans les cellules germinales plus jeunes), a été confirmée. Les effets précis de l’aromatase testiculaire et des estrogènes restent à explorer de manière approfondie dans la mesure où l’on ne connaît pas bien les cibles moléculaires de ces hormones. En résumé, la présence du cytochrome P450 aromatase dans les cellules somatiques et les cellules germinales du rat mâle confirment donc l’existence d’une source supplémentaire d’estrogènes dans le testicule de certains mammifères ce qui serait en faveur d’un rôle physiologique nouveau pour ces hormones sexuelles femelles dans la régulation des fonctions testiculaires.

Abstract

The cytochrome P450 aromatase (P450arom) is the terminal enzyme responsible for the formation of estrogens from androgens and is present in the endoplasmic reticulum membrane of various tissues. This microsomal protein associated with the ubiquitous reductase catalyzes the aromatization of the A-ring of C19 androgens to estrogens via 3 hydroxylations. P450arom is involved in development, reproduction, sexual differentiation and behaviour, but also in bone and lipid metabolisms, brain functions and diseases such as breast and testicular tumours. Aromatase belongs to the cytochrome P450 gene superfamily and is encoded by 9 exons from a single gene over 75 kb long. Moreover, this gene also includes 8 non-coding exons, all of them are in 5′ under alternative splicing control, after their transcription downstream an equivalent number of tissue-specific promoters. Several sizes of mRNA have been described out of this gene, which could also be submitted to differential regulation. The encoded-protein has a molecular weight around 55kDa and is composed of 503 amino acids in human. In adult rat somatic and germ cells we have amplified, using RT-PCR, specific transcripts for P450arom which showed 100% homology with the corresponding fragments of the rat ovary cDNAs. In purified Leydig cells and pachytene spermatocytes, the amount of P450arom mRNA is 10 fold higher than in Sertoli cells. According to the stage of germ cell maturation, the level of P450arom mRNA transcript decreases, being more elevated in younger than in mature germ cells. By contrast, the aromatase activity in the microsomal fractions is 2–4 fold greater in spermatozoa when compared to the two other enriched-germ cell preparations studied. In parallel by Western blotting, we revealed a 55 kDa protein in a mixed germ cells preparation and we have been able to immunolocalize the P450arom on mature rat testicular slices, namely in Leydig cells and elongated spermatids. These data demonstrate the presence of a functional cytochrome P450arom in the male rat Leydig cells and germ cells. Consequently, the existence of an additional source of estrogens within the genital tract of the mammalian testis is obvious and therefore, a new physiological role for these female hormones in the regulation of testicular functions should be taken into account.

References

  1. ALMADHIDI J., SERALINI G.E., FRSNEL J., SILBERZAHN P., GAILLARD J.L. Immunohistochemical localization of cytochrome P450 aromatase in equine gonads. J. Histochem. Cytochem., 1995; 43: 571–577.

    PubMed  CAS  Google Scholar 

  2. BILINSKA B., LESNIAK M., SCHMALZ B.: Are ovine Leydig cells able to aromatize androgens? Reprod. Fert. Dev., 1997; 9: 193–199.

    Article  CAS  Google Scholar 

  3. BULUN S.E., ROSENTHAL I.M., BRODIE A.M., et al.: Use of tissue-specific promoters in the regulation of aromatase cytochrome P450 gene expression in human testicular and ovarian sex cord tumors, as well as in normal fetal and adult gonads. J. Clin. Endocrinol. Metab., 1993; 77: 1616–1621.

    Article  PubMed  CAS  Google Scholar 

  4. CARANI C., QIN K., SIMONI M., FAUSTINI-FUSTINI M., SERPENTE S., BOYD J., KORACH K.S., SIMPSON E.R.: Effect of testosterone and estradiol in a man with aromatase deficiency. N. Engl. J. Med., 1997; 337: 91–95.

    Article  PubMed  CAS  Google Scholar 

  5. CARREAU S.: Germ cells-Sertoli cells interactions and Leydig cell function. In: Dufau M.L., Fabbri A., Isidori A., eds. Cell and Molecular Biology of the Testis. Frontiers in Endocrinology, 1994; 5: 137–148.

  6. CARREAU S., LEVALLET J.: Activité aromatase dans les cellules germinales mâles: quelle signification fonctionnelle? Andrologie, 1997, 7(3): 305–315.

    Article  Google Scholar 

  7. CHOI I., COLLANTE W.R., SIMMEN R.C.M., SIMMEN F.A.: A developmental switch in expression from blastocyst to endometrial/placental-type cytochrome P450 aromatase genes in the pig and horse. Biol. Reprod., 1997; 56: 688–696.

    Article  PubMed  CAS  Google Scholar 

  8. CONLEY A.J., CORBIN C.J., HINSHELWOOD M.M., LIUZ, SIMPSONE R., FORD J.J., HARADA N: Functional aromatase expression in porcine adrenal gland and testis. Biol. Reprod., 1996; 54: 497–505.

    Article  PubMed  CAS  Google Scholar 

  9. EDDY E.M., WASHBURN T.F., BUNCH D.O., et al.: Targeted disruption of the estrogen receptor gene in male mice causes alteration of spermatogenesis and infertility. Endocrinology, 1996; 137: 4796–4805.

    Article  PubMed  CAS  Google Scholar 

  10. FOUCAULT P., CARREAU S., KUCZYNSKI W. et al.: Human Sertoli cells in vitro Lactate estradiol-17β and transferrin production. J Androl., 1992; 13: 361–367.

    PubMed  CAS  Google Scholar 

  11. HARADA N., YAMADA K., SAITO K., KIBE N., DOHMAE S., TAKAGI Y.: Structural characterization of the human estrogen synthetase (aromatase) gene. Biochem. Biophys. Res. Commun., 1990; 166: 365–372.

    Article  PubMed  CAS  Google Scholar 

  12. HESS R.A., BUNICK D., BAHR J.M.: Sperm, a source of estrogen. Environ. Health Perspect., 1995; 103: 59–62.

    Article  PubMed  Google Scholar 

  13. HESS R.A., BUNICK D., LEE K.H., et al.: A role for estrogens in the male reproductive tract. Nature, 1997a; 390: 509–512.

    Article  PubMed  CAS  Google Scholar 

  14. HESS R.A., GIST D.H., BUNICK D., et al.: Estrogen receptor (α & β) expression in the excurrent ducts of the adult male rat reproductive tract. J. Androl., 1997b; 18: 602–611.

    PubMed  CAS  Google Scholar 

  15. HICKEY G.J., KRASNOW J.S., BEATTIE W.G., RICHARDS J.S.: Aromatase cytochrome P450 in rat ovarian granulosa cells before and after luteinization: adenosine 3′5′-monophosphate-dependent and independent regulation. Cloning and sequencing of rat aromatase cDNA and 5′genomic DNA. Mol. Endocrinol., 1990; 4: 1–12.

    Article  Google Scholar 

  16. INKSTER S., YUE W., BRODIE A.: Human testicular aromatase: immunocytochemical and biochemical studies. J. Clin. Endocrinol. Metab., 1995; 80: 1941–1947.

    Article  PubMed  CAS  Google Scholar 

  17. JANULIS L., BAHR J.M., HESS R.A., BUNICK D.: P450 aromatase messenger ribonucleic acid expression in male rat germ cells: detection by reverse transcription- polymerase chain reaction amplification. J. Androl., 1996a; 17: 651–658.

    PubMed  CAS  Google Scholar 

  18. JANULIS L., HESS R.A., BUNICK D. et al.: Mouse epididymal sperm contains active P450 aromatase which decreases as sperm traverse the epididymis. J. Androl., 1996b; 17: 111–116.

    PubMed  CAS  Google Scholar 

  19. KORACH K.S.: Insights from the study of animals lacking functional estrogen receptor. Science, 1994; 266: 1524–1527.

    Article  PubMed  CAS  Google Scholar 

  20. KORACH K.S., COUSE J.F., CURTIS S.W., et al.: Estrogen receptor disruption: molecular characterization and experimental and clinical phenotypes. Rec. Prog. Horm. Res., 1996; 51: 159–188.

    PubMed  CAS  Google Scholar 

  21. KORACH K.S., LINDZEY J.: Alterations in male reproduction in estrogens receptor knock out mice. Andrologie, 1998; sous presse.

  22. KUIPER G.G.J.M., CARLSSON B., GRANDIEN K., et al.: Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors α and β. Endocrinology, 1997; 138: 863–870.

    Article  PubMed  CAS  Google Scholar 

  23. KUROSUMI M., ISHIMURA K., FUJITA H., OSAWA Y.: Immunocytochemical localization of aromatase in rat testis. Histochemistry, 1985; 83: 401–404.

    Article  PubMed  CAS  Google Scholar 

  24. KWON S., HESS R.A., BUNICK D., et al.: Rooster testicular germ cells and epididymal sperm contain P450 aromatase. Biol. Reprod., 1995; 53: 1259–1264.

    Article  PubMed  CAS  Google Scholar 

  25. LEPHART E.D., PETERSON K.G., NOBLE J.F., GEORGE F.W., McPHAUL M.J. The structure of cDNA clones encoding the aromatase P-450 isolated from a rat Leydig cell tumor line demonstrates differential processing of aromatase mRNA in rat ovary and a neoplastic cell line. Mol. Cell. Endocrinol., 1990; 70: 31–40.

    Article  PubMed  CAS  Google Scholar 

  26. LEVALLET J., CARREAU S.: Expression in vitro du gène de l’aromatase dans les cellules testiculaires du rat. C. R. Acad. Sc. (Paris), 1997; 320: 123–129.

    CAS  Google Scholar 

  27. LEVALLET J., BILINSKA B., MITTRE H., GENISSEL C., FRESNEL J., CARREAU S.: Expression and immunolocalization of functional cytochrome P450 aromatase in mature rat testicular cells. Biol. Reprod., 1998a; 58: sous presse.

  28. LEVALLET J., DELARUE B., MITTRE H., CARREAU S.: Alternative splicing events in the coding region of the P450 aromatase gene in male rat germ cells. J. Molec. Endocrinol. 1998b; 20: sous presse.

  29. LI H., PAPADOPOULOS V., VIDIC B., DYM M., CULTY M.: Regulation of rat testis gonocyte proliferation by platelet-derived growth factor and estradiol: identification of signaling mechanisms involved. Endocrinology, 1997; 138: 1289–1298.

    Article  PubMed  CAS  Google Scholar 

  30. LUBAHN D.B., MOYER J.S., GOLDING T.S., et al.: Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene. Proc. Natl. Acad. Sci. USA, 1993; 90: 11162–11166.

    Article  PubMed  CAS  Google Scholar 

  31. MAHENDROO M.S., MEANS G.D., MENDELSON C.R., SOMPSONE R.: Tissue- specific expression of human P-450 arom. J. Biol. Chem., 1991; 266: 11276–11281.

    PubMed  CAS  Google Scholar 

  32. MEANS G.D., MAHENDROO M.S., CORBIN C.J., et al.: Structural analysis of the gene encoding human aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. J. Biol. Chem., 1989; 264: 19385–19391.

    PubMed  CAS  Google Scholar 

  33. MEANS G.D., KILGORE M.W., MAHENDROO M.S., MENDELSON C.R., SIMPSON E.R.: Tissue-specific promoters regulate aromatase cytochrome P450 gene expression in human ovary and fetal tissues. Mol. Endocrinol., 1991; 5: 2005–2013.

    Article  PubMed  CAS  Google Scholar 

  34. NAKAZUMI H., SASANO H., MAEHARA I., et al.: Estrogen metabolism and impaired spermatogenesis in germ cell tumors of the testis. J. Clin. Endocrinol. Metab., 1996; 81: 1289–1295.

    Article  PubMed  CAS  Google Scholar 

  35. NAKHALA A.M., MATHER J.P., JANNE O.A., BARDIN C.W.: Estrogen and androgen receptors in Sertoli, Leydig, myoid and epithelial cells: effects of time in culture and cell density. Endocrinology, 1984; 115: 121–118.

    Article  Google Scholar 

  36. NELSON D.R., KOYMANS L., KAMATAKI T., et al.: P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics, 1996; 6: 1–42.

    Article  PubMed  CAS  Google Scholar 

  37. NITTA H., BUNICK D., HESS R.A. et al.: Germ cells of the mouse testis express P450 aromatase. Endocrinology, 1993; 132: 1396–1401.

    Article  PubMed  CAS  Google Scholar 

  38. PAPADOPOULOS, V., CARREAU S., SZERMANJOLY E. et al.: Rat testis 17β- estradiol: identification by gas chromatography-mass spectrometry and age related cellular distribution. J. Steroid Biochem., 1986; 24: 1211–1216.

    Article  PubMed  CAS  Google Scholar 

  39. PAPADOPOULOS V., JIA M.C., CULTY M., HALL P.F., DYM M.: Rat Sertoli cell aromatase cytochrome P450: regulation by cell culture conditions and relationship to the state of cell differentiation. In Vitro Cell. Dev. Biol., 1993; 29A: 943–949.

    Article  CAS  Google Scholar 

  40. SIMPSON E.R., EVANS, C.T., CORBIN J.C., POWELL F.E., LEDESMA D.B., MENDELSON C.R.: Sequencing of cDNA inserts encoding aromatase cytochrome P450. Mol. Cell. Endocrinol., 1987; 52: 267–272.

    Article  PubMed  CAS  Google Scholar 

  41. SIMPSON E.R., MAHENDROO M.S., MEANS G.D. et al.: Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocrine Rev., 1994; 15: 342–355.

    CAS  Google Scholar 

  42. SIMPSON E.R., MICHAEL M.D., AGARWAL V.R., HINSHELWOOD M.M., BULUN S.E., ZHAO Y.: Expression of the CYP 19 (aromatase) gene: an unusual case of alternative promoter usage. FASEB J., 1997; 11: 29–36.

    PubMed  CAS  Google Scholar 

  43. SHARPE R.M.: Do males rely on female hormones. Nature, 1997; 390: 447–448.

    Article  PubMed  CAS  Google Scholar 

  44. SHETTY G., KRISHNAMURTHY H., KRISHNAMURTHY H.N., BHATNAGAR S., MOUDGAL R.N.: Effect of estrogen deprivation on the reproductive physiology of male and female primates. J. Steroid Biochem. Molec. Biol., 1997; 61 (3–6): 157–166.

    Article  PubMed  CAS  Google Scholar 

  45. ZHOZU M., BULUN S.E., ZHAO Y., SIMPSON E.R.: Expression of a novel exon 1 of the aromatase gene: exon 1.6. The Endocrine Society, 1997, 79th anual meeting, P1-417.

  46. STEIMKAMPF M.P., MENDELSON C.R., SIMPSON E.R.: Effects of epidermal growthfactor and insulin-like growth factor-1 on the levels of mRNA encoding aromatase cytochrome P450 of human ovarian granulosa cells. Mol. Cell. Endocrinol., 1988; 59: 93–99.

    Article  Google Scholar 

  47. TERASHIMA M., TODA K., KAWAMOTO T., et al.: Isolation of a full-lenght cDNA encoding mouse aromatase P450. Arch. Biochem. Biophys., 1991; 285: 231–237.

    Article  PubMed  CAS  Google Scholar 

  48. TODA K., TERASHIMA M., KAWAMOTO T. et al.: Structural and functional characterization of human aromatase P-450 gene. Eur. J. Biochem., 1990; 193: 559–565.

    Article  PubMed  CAS  Google Scholar 

  49. TODA K, SHIZUTA Y.: Molecular cloning of a cDNA showing alternative splicing of the 5′-untranslated sequence of mRNA for human aromatase P450 gene. Eur. J. Biochem., 1993; 213: 559–565.

    Article  Google Scholar 

  50. TOPPARI J., LARSEN J.C., CHRISTIANSEN P., et al.: Male reproductive health and environmental xenoestrogens. Environ. Health Perspect., 1996; 104: 741–803.

    Article  PubMed  CAS  Google Scholar 

  51. TSUBOTA T., NITTA H., OSAWA Y., et al.: Immunolocalization of steroidogenic enzymes P450scc 3β-HSD P450c17 and P450arom in the hokkaido brown bear (Ursus arctos yesoensis) testis. Gen. Comp. Endocrinol., 1993; 92: 439–444.

    Article  PubMed  CAS  Google Scholar 

  52. TSUBOTA T., HOWELL-SKALLA L., NITTA H., et al.: Seasonal changes in spermatogenesis and testicular steroidogenesis in the male blackbearUrsus americanus. J. Reprod. Fertil., 1997; 109: 21–7.

    Article  PubMed  CAS  Google Scholar 

  53. YOUNG J., BULUN S.E., AGARWAL V., et al.: Aromatase expression in a feminizing adrenocortical tumor. J. Clin. Endocrinol. Metab., 1996; 81: 3173–3176.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratoire de Biochimie-IRBA, CNRS EP 9, Université de Caen, Esplanade de la Paix, 14032, CAEN Cédex

    S. Carreau & J. Levallet

Authors
  1. S. Carreau
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J. Levallet
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Carreau, S., Levallet, J. L’aromatase testiculaire: Du gène a la proteine. Androl. 8, 191–198 (1998). https://doi.org/10.1007/BF03034484

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF03034484

Mots clés

Key words

Basic and Clinical Andrology

ISSN: 2051-4190