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Mise en évidence des transcrits du cytochrome P450 aromatase dans les spermatozoïdes humains éjaculés

Expression of P450 aromatase transcripts in ejaculated human spermatozoa

Resume

La conversion irréversible des androgènes en estrogènes est liée à l’existence d’un complexe enzymatique microsomial: le cytochrome P450 aromatase. Chez les mammifères, la présence de cette protéine dans le testicule est depuis longtemps établie. En effet, les ARNm codant pour le cytochrome P450 aromatase (P450arom), ainsi qu’une activité enzymatique ont été détectés non seulement dans les cellules somatiques du testicule (cellules de Sertoli et cellules de Leydig), mais également dans les cellules germinales de nombreuses espèces. Chez l’homme, la présence intracellulaire d’estrogènes et la mise en évidence d’un récepteur pour ces hormones sont autant de données permettant d’envisager les spermatozoïdes comme des cellules cible et/ou des cellules source d’estrogènes. Le but de notre travail a donc été d’étudier la possibilité pour les spermatozoïdes humains de synthétiser des estrogènes en recherchant d’une part, la présence de transcrits codant pour le P450arom et d’autre part, en mesurant les taux intracellulaires d’estradiol. Des ARNr 18 S et 28 S ont étès mis en évidence dans 10 des 14 échantillons de spermes étudiés provenant de patients normospermes. A l’aide d’amorces spécifiques, nous avons détecté par RT-PCR (transcription inverse associée à une polymérisation en chaîne) l’ARNm de l’aromatase dans trois échantillons, alors que tous expriment l’ARNm codant pour la GAPDH. Une “semi-nested-PCR” nous a alors permis de confirmer la présence de transcrits de l’aromatase dans les échantillons où le signal n’était pas visible en première PCR. Les taux intracellulaires d’estrogènes dans le spermatozoïde humain varient de 47 à 222 fmol/éjaculat et sont en accord avec ceux précédemment publiés. En conclusion, nous rapportons pour la première fois la présence de messagers codant pour le P450arom dans le spermatozoïde humain, et la possibilité de leur traduction en protéine biologiquement active.

Abstract

The conversion of androgens into estrogens is catalyzed by the cytochrome P450 aromatase. The presence of this protein within the mammalian testis has now been clearly established, as mRNA coding for aromatase and P450 aromatase activity have been detected in somatic cells (Sertoli cells and Leydig cells) and in germ cells in the testis of various spcies. Conversely, very limited data on aromatase in testicular cells are available in men. It has been shown that human spermatozoa are able to synthesize not only androgens but also, to a lesser extent, estrogens. It has also been reported that the sperm membrane contains an estrogen-receptor-related protein that is able to bind steroids. The ability of human spermatozoa to convert androgens into estrogens has been studied using two approaches: (1) detection of specific aromatase (P450arom) transcripts and (2) measurement of endogenous estradiol. Total RNA was extracted from individual ejaculates of normospermic patients using a guanidium isothiocyanate-isoamylic acid method. The presence of 18 and 28 S rRNA was detected on 1.5% agarose gel containing ethidium bromide in 10/14 patients. Three responses were observed when sperm mRNA was used as template in reverse transcription-polymerase chain reaction (RT-PCR) with P450arom specific primers: 1) presence of the expected 293 bp PCR product (3/14), 2) detection of a weak signal by UV absorbance (6/14) and 3) no visible staining (5/14). However, all samples expressed transcripts for a housekeeping gene, human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Sequence alignments from PCR products of spermatozoa and granulosa cells with the published human P450arom sequence are identical except for certain unidentified bases. We confirmed the presence of P450 aromatase transcripts by nested-PCR in patients without a positive P450arom mRNA signal on the first PCR. Intracellular estradiol concentrations in human spermatozoa are in the range of 47–222 fmol per ejaculate. These data demonstrate, for the first time, the presence of P450arom gene specific PCR products likely coding for a biologically active enzyme in ejaculated human spermatozoa.

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Correspondence to S. Carreau.

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Prix de DEA décerné par la Société d’Andrologie de Langue Française.

Présentation au XVIIème Congrès de la SALF, 7–8 décembre 2000, Bordeaux.

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Lambard, S., Galeraud-Denis, I. & Carreau, S. Mise en évidence des transcrits du cytochrome P450 aromatase dans les spermatozoïdes humains éjaculés. Androl. 11, 36 (2001). https://doi.org/10.1007/BF03034508

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Mots clés

  • Aromatase
  • spermatozoïde humain
  • estrogènes
  • RT-PCR

Key words

  • Aromatase
  • human spermatozoa
  • estrogens
  • RT-PCR
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