Skip to main content
  • Steroides Sexuels et Lignee Spermatogenetique
  • Published:

Activité aromatase dans les cellules germinales mâles: quelle signification fonctionnelle?

Germ cell aromatase: any relevant physiological role?

Resume

La capacité de la gonade mâle à convertir les androgènes en oestrogènes est bien établie: cette transformation est catalysée par l'aromatase composée d'une glycoprotéine enzymatique microsomiale, le cytochrome P450 aromatase (P450arom) et d'une réductase ubiquitaire. Dans les cellules testiculaires du rat mature, le P450arom a été immunolocalisé principalement dans les cellules de Leydig mais également dans les cellules germinales chez la souris, l'ours brun et le coq alors que chez l'homme, le porc et le cheval, l'aromatase est principalement localisée dans les cellules de Leydig. L'activité aromatase a été mesurée dans les cellules de Leydig du rat immature et mature, mais aussi dans les cellules de Sertoli: cette activité varie en fonction de l'âge. Par RT-PCR, nous avons amplifié un fragment de 289 pb du messager de l'aromatase qui présente 100% d'homologie avec la séquence de l'aromatase d'ovaire de rat, et qui est détectable non seulement dans les cellules de Leydig et de Sertoli mais aussi dans les spermatocytes pachytènes, les spermatides rondes et les spermatozoïdes testiculaires. Avec un anticorps anti-cytochrome P450arom humain nous avons démontré l'existence d'une protéine de 55 kDa dans les tubes séminifères et dans une suspension hétérogène de cellules germinales (spermatocytes pachytènes et spermatides rondes). Nous avons comparé la quantité d'ARNm du cytochrome P450arom dans les cellules germinales avec celle des cellules de Leydig et de Sertoli à l'aide de la RT-PCR quantitative. Dans les cellules de Leydig purifiées du rat âgé de 90 jours, la quantité d'ARNm codant pour l'aromatase est de 36 × 10−3 amoles/gmg d'ARN et 10 fois supérieure à celui des cellules de Sertoli. Dans les cellules germinales, le taux d'ARNm dans les spermatocytes pachytènes est de 367 × 10−3 amoles/μg d'ARN et cette valeur est 3 fois supérieure à celle des spermatides rondes; dans les spermatozoïdes ce taux est inférieur à 10−3 amoles/μg d'ARN. Après incubation avec de l'androstènedione tritiée, les activités aromatases des fractions microsomiales sont de 0,62; 0,46 et 2,96 pmoles/mg de protéine/h respectivement dans les spermatocytes, les spermatides et les spermatozoïdes (pour les cellules de Leydig cette activité est de 5 pmoles). En conclusion, nous avons démontré la présence d'une aromatase fonctionnelle dans les cellules germinales du rat mâle, la maturation du messager (comparaison taux d'ARNm et activité enzymatique) semble être progressive depuis le stade spermatocyte pachytène jusqu'au spermatozoïde ce qui est en accord avec les données rapportées chez la souris. En somme, les observations rapportées dans la littérature et nos résultats, plaident fortement en faveur d'un rôle nouveau mais encore mal connu pour les oestrogènes testiculaires dans le développement du tractus génital mâle puis la mise en place et le maintien de la spermatogenèse. Ainsi compte tenu des anomalies rencontrées dans la spermatogenèse chez des animaux déficients en récepteur aux oestrogènes et chez l'homme porteur de tumeurs testiculaires, les oestrogènes (et/ou le rapport androgènes/oestrogènes) pourraient représenter un nouveau marqueur de l'infertilité et/ou de l'hypofertilité masculine.

Abstract

The ability of the male gonad to convert androgens into estrogens is well known. According to age, aromatase activity has been already measured in immature and mature rat Leydig cells as well as in Sertoli cells. Recently, in different studies, a cytochrome P450arom has even been immunolocalized not only in Leydig cells but also in germ cells of mouse, brown bear and rooster whereas in pig, ram and human the aromatase is mainly present in Leydig cells. Our purpose was to investigate the testicular cell distribution of cytochrome P450arom mRNA in adult rat using RT-PCR. With 2 highly specific primers located on exons 8 and 9, we have been able to amplify a 289 bp aromatase fragment not only in Leydig cells and Sertoli cells but more importantly in highlyenriched preparations of pachytene spermatocytes, round spermatids and testicular spermatozoa. These amplified products showed 100% homology with the corresponding fragment of the rat ovary cDNA. In parallel, using an anti-human cytochrome P450arom antibody we have demonstrated the presence of a 55 kDa protein in seminiferous tubules and crude germ cell (pachytene spermatocytes and round spermatids) preparation of the mature rat. After incubation with tritiated androstenedione, the aromatase activities in the microsomal fractions were 3.12±0.19 pmoles/mg/h in the testis, 1.25±0.13 in the seminiferous tubules and 1.53±0.15 in the crude germ cells. In purified testicular spermatozoa the aromatase activity was 2.96±0.69 pmoles/mg/h and found to be 5-fold higher when compared to that of either purified pachytene spermatocytes or round spermatids. Using a quantitative RT-PCR method with a standard cDNA 29 bp shorter, we have compared the amount of cytochrome P450arom mRNA in mature rat Leydig cells and Sertoli cells. In purified Leydig cells from 90 day-old rats the P450arom mRNA level was: 36.2±3.4×10−3 amoles/μg RNA whereas in Sertoli cells the mRNA level was 10 fold lower. In pachytene spermatocytes, round spermatids and testicular spermatozoa the P450arom mRNA levels were re pectively 367.2±76.6, 117.6±22.0 and <1×10−3 amole/μg RNA.

In conclusion we have demonstrated that the P450 aromatase is present not only in Sertoli cells and Leydig cells from mature rat testis but a biologically active aromatase exists also in germ cells (pachytene spermatocytes, round spermatids and spermatozoa). The existence of an additional source of estrogens within the genital tract of the male is now well documented and that suggests a putative role for these hormones during the male germ cell development.

References

  1. ALMADHIDI J., SERALINI, G.E., FRESNEL 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. BELLVE A.R., CAVICCHIA J.C., MILLETTE C.F. et al.: Spermatogenic cells of the perpubertal mouse. J. Cell. Biol., 1977, 74: 68–85.

    Article  PubMed  CAS  Google Scholar 

  3. BENAHMED M.: Growth factors and cytokines in the testis. Dans: Comhaire F.H., ed. Male infertility. Chapman & Hall Medical, 1995: 55–96.

  4. BILINSKA B., LESNIAK M, SCHMLZ B.: Are ovine Leydig cells able to aromatize androgens? Reprod. Fert. Dev., sous presse.

  5. BOITANI C., RITZEN E.M., PARVINEN M.: Inhibition of rat Sertoli cell aromatase by factor(s) secreted specifically at spermatogenic stages VII and VIII. Mol. Cell. Endocrinol., 1981, 23: 11–22.

    Article  PubMed  CAS  Google Scholar 

  6. BOUJRAD N., HOCHEREAU de REVIERS M.T., CARREAU S.: Evidence for a germ cell control of Sertoli cell function in three models of germ cell depletion in adult rat. Biol. Reprod., 1995, 53: 1345–1352.

    Article  PubMed  CAS  Google Scholar 

  7. CARREAU S.: L'Androgen Binding Protein (ABP) chez le rat, le bélier, le taureau et l'homme: analyse comparée. Dans: Forest M.G., Pugeat M., eds. Protéines de liaison des hormones stéroïdes. INSERM/J. Libbey, Eurotext Ltd, 1986, 149: 293–303.

  8. CARREAU S., PAPADOPOULOS V., DROSDOWSKY M.A.: Stimulation of adult rat Leydig cell aromatase activity by a Sertoli cell factor. Endocrinology, 1988, 122: 1103–1109.

    PubMed  CAS  Google Scholar 

  9. 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.

  10. CARREAU S.: Paracrine control of human Leydig cell and Seroli cell functions. Folia Histochem Cytobiol, 1996, 34 (3): 111–119.

    PubMed  CAS  Google Scholar 

  11. CHOMCZYNSKI P., SACCHI N.: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 1987, 162: 156–159.

    Article  PubMed  CAS  Google Scholar 

  12. CONLEY A.J., CORBIN C.J., HINSHELWOOD M.M. et al.: Functional aromatase expression in porcine adrenal gland and testis. Biol. Reprod., 1996, 54: 497–505.

    Article  PubMed  CAS  Google Scholar 

  13. COOKE P.S., YOUNG P., HESS R.A., CUNHA G.R.: Estrogen receptor expression in developing epididymis, efferent ductules, and other male reproductive organs. Endocrinology, 1991, 128: 2874–2879.

    PubMed  CAS  Google Scholar 

  14. De JONG F.H., HEY A.H., Van Der MOLEN H.J.: Oestradiol-17β and testosterone in rat testis tissue: effect of gonadotrophins, localization and productionin vitro. J. Endocrinol., 1974, 60: 409–419.

    Article  PubMed  Google Scholar 

  15. DORRINGTON J.H., ARMSTRONG D.T.: Follicle stimulating hormone stimulates oestradiol-17β synthesis in cultured Sertoli cells. Proc. Natl. Acad. Sci. USA, 1975, 72: 2677–2681.

    Article  PubMed  CAS  Google Scholar 

  16. 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 

  17. FOUCAULT P., DROSDOWSKY M.A., CARREAU S.: Germ cell and Sertoli cell interactions in human testis: evidence for stimulatory and inhibitory effects. Human Reprod., 1994, 9: 2062–2068.

    CAS  Google Scholar 

  18. FREE M.J., JAFFE R.A.: Collection of rete testis fluid from rats without previous efferent duct ligation. Biol. Reprod., 1979, 20: 269–278.

    Article  PubMed  CAS  Google Scholar 

  19. HENDRY W.F., PARSLOW J.M., STEDRONSKA J.: Exploratory scrotomy in 168 azoospermic males. Br. J. Urol., 1983, 55: 785–791.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  Google Scholar 

  21. 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 region. Mol. Endocrinol., 1990, 4: 1–12.

    Article  Google Scholar 

  22. HYDERS, M., STANCEL G.M., LOOSE-MIRCHELL D.S.: Presence of an estradiol response region in the mousec-fos oncogene. Steroids, 1991, 56: 498–504.

    Article  Google Scholar 

  23. 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 

  24. 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., 1996, 17: 111–116.

    PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  26. KUMARI G.L., ALLAG I.S., DAS R.P., DATTA J.K.: Regional differences in steroidogenesis and hormone levels in the epididymis and vas deferens of adult rats. Int. J. Androl., 1980, 3: 267–281.

    Article  PubMed  CAS  Google Scholar 

  27. 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 

  28. 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 

  29. LEJEUNE H., SANCHEZ P., SAEZ J.M.: Enhancement of long-term testosterone secretion, 3β-hydroxysteroid dehydrogenase, cytochrome P450 side chain cleavage, 17a hydroxylase and aromatase mRNAs in normal human Leydig cells by coculture with human Sertoli cell-enriched preparations. 8th European Testis Workshop, de Panne, 1994, A 36.

  30. LEJEUNE H., JEGOU B., CARREAU S., SAEZ J.M.: Régulation paracrine et autocrine des fonctions testiculaires. In: Belaish J., Drosdowsky M.A., Vermeulen A., Eds. Endocrinologie sexuelle de l'homme. Doin-Paris, 1996, 7: 77–103.

  31. Le MAGUERESSE B., JEGOU B.: Possible involvement of germ cells in the regulation of oestradiol-17β and ABP secretion by immature rat Sertoli cells (in vitro studies). Biochem. Biophys. Res. Commun., 1986, 141: 861–869.

    Article  PubMed  Google Scholar 

  32. LEVALLET J., MITTRE H., SERALINI G.E., LEYMARIE P., CARREAU S.: L'aromatase testiculaire chez le rat: apport de la RT PCR dans l'étude de la localisation cellulaire de l'enzyme. Ann Endocrinol., 1995, 56 (4): 280.

    Google Scholar 

  33. LEVALLET J., MITTRE H., CARREAU S.: Germ cells and somatic cells of adult rat testis express functional P450 aromatase. Mol. Cell. Endocrinol. (soumis pour publication).

  34. LEVALLET J., CARREAU S.: Expression du gène de l'aromatase dans les cellules testiculaires du rat. C.R. Acad. Sc. (soumis pour publication).

  35. LUBAHN D.B., MOYER J.S., GOLDING T.S., COUSE J.F., KORACH K.S., SMITHIES O.: 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 

  36. MARIE E., VENDRELY E., de REVIERS M., CARREAU S.: Stéroides plasmatiques et intratesticulaires: existe-t-il des correlations avec la spermatogenèse chez l'homme fertile et infécond. Ann Endocrinol., 1995, 56 (4): 337.

    Google Scholar 

  37. MEISTRICH M.L., LONGTIN J., BROCK W.A., GRIMES S.R., MACE M.L.: Purification of rat spermatogenetic cells and preliminary biochemical analysis of these cells. Biol. Reprod., 1981, 25: 1065–1077.

    Article  PubMed  CAS  Google Scholar 

  38. MARTEL C., MELNER M.H., GAGNE D., SIMARD J., LABRIE F.: Widespread tissue distribution of steroid sulfatase, 3\-hydroxysteroid dehydrogenase/isomerase (3\-HSD), 17b-HSD, 5a-reductase and aromatase activities in the Rhesus monkey. Mol. Cell. Endocrinol., 1994, 104: 103–111.

    Article  PubMed  CAS  Google Scholar 

  39. NAKAZUMI H., SASANO H., MAEHARA I., OZAKI M., TEZUKA F., ORIKASA S.: 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 

  40. NAKHALA A.M., MATHER J.P., JANNE O.A., BARDIN C.W.: Estrogen and androgen receptors in Sertoli, Leydig, Myoid and epitheliad cells: effects of time in culture and cell density. Endocrinology, 1984, 115: 121–128.

    Google Scholar 

  41. 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 

  42. PAPADOPOULOS V., CARREAU S., DROSDOWSKY M.A.: Effects of phorbol ester and phospholipase C on LH-stimulated steroidogenesis in purified rat Leydig cells. FEBS Lett., 1985, 188: 312–316.

    Article  PubMed  CAS  Google Scholar 

  43. PAPADOPOULOS V., CARREAU S., SZERMANJOLY E., DROSDOWSKY M.A., DEHENNIN L., SCHOLLER R.: Rat testis 17\-estradiol: identification by gas chromatography — mass spectrometry and age-related cellular distribution. J. Steroid Biochem., 1986, 24: 1211–1216.

    Article  PubMed  CAS  Google Scholar 

  44. PAPADOPOULOS V., DROSDOWSKY M.A., CARREAU S.: On the existence of two Leydig cell populations in aged human testis. Ann. N.Y. Acad. Sci., 1987, 513: 356–359.

    Article  Google Scholar 

  45. 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 

  46. PAYNE A.H., KELCH R.P., MUSICH S.S., HALPERN M.E.: Intratesticular site of aromatization in the human. J. Clin. Endocrinol. Metab., 1976, 42: 1081–1087.

    PubMed  CAS  Google Scholar 

  47. POMERANTZ D.K.: Effects of in vivo gonadotropin treatment on estrogen levels in the testis of the immature rat. Biol. Reprod., 1979, 21: 1247–1255.

    Article  PubMed  CAS  Google Scholar 

  48. POMERANTZ D.K.: Developmental changes in the ability of follicle stimulating hormone to stimulate estrogen synthesis in vivo by the testis of the rat. Biol. Reprod., 1980, 23: 948–954.

    Article  PubMed  CAS  Google Scholar 

  49. PUDNEY J., CANICK J.A., CLIFFORD N.M., KNOPP J.B., CALLARD G.V.: Location of enzymes of androgen and estrogen biosynthesis in the testis of the ground squirrel. Biol. Reprod., 1985, 33: 971–980.

    Article  PubMed  CAS  Google Scholar 

  50. ROMMERTS F.F.G., de JONG F.H., BRINKMANN A.O., Van Der MOLEN H.J.: Development and cellular localization of rat testicular aromatase activity. J. Reprod. Fert., 1982, 65: 281–288.

    Article  CAS  Google Scholar 

  51. SAEZ J.M., SANCHEZ P., BERTHELON M.C., AVALLET O.: Regulation of pig Leydig cell aromatase activity by gonadotropins and Sertoli cells. Biol. Reprod., 1989, 41: 813–820.

    Article  PubMed  CAS  Google Scholar 

  52. SANGER F., NICKLEN S., COULSON A.R.: DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sc. USA, 1977, 74: 5463–5467.

    Article  CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

  54. THOMPSON E.A. Jr., SIITERI P.K.: Utilization of oxygen and reduced nicotinamide adenine dinucleotide phosphate by human placental microsomes during aromatization of androstenedione. J. Biol. Chem., 1974, 249: 5364–5372.

    PubMed  CAS  Google Scholar 

  55. TSAI-MORRIS C.H., AQUILANO D.R., DUFAU M.L.: Cellular localization of rat testicular aromatase activity during development. Endocrinology, 1985, 116: 38–46.

    Article  PubMed  CAS  Google Scholar 

  56. TSUBOTA T., NITTA H., OSAWA Y. et al.: Immunolocalization of steroidogenic enzymes, P450scc, 3\-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 

  57. VERHOEVEN G., CAILLEAU J.: Testicular peritubular cells secrete a protein under androgen control that inhibits induction of aromatase activity in Sertoli cells. Endocrinology, 1988, 123: 2100–2110.

    PubMed  CAS  Google Scholar 

  58. VORNBERGER W., PRINS G., MUSTO N.A., SUAREZ-QUIAN C.A.: Androgen receptor distribution in rat testis: new implications for androgen regulation of spermatogenesis. Endocrinology, 1994, 134: 2307–2316.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Carreau, S., Levallet, J. Activité aromatase dans les cellules germinales mâles: quelle signification fonctionnelle?. Androl. 7, 305–315 (1997). https://doi.org/10.1007/BF03034548

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF03034548

Mots clés

Key words