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Endocrinologie | Open | Published:

L’aromatase testiculaire: Du gène a la proteine

The testicular aromatase: From the gene to the protein

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.

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Correspondence to S. Carreau.

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Mots clés

  • ARNm
  • Gène
  • Aromatase
  • Testicule
  • Mammifères

Key words

  • Aromatase gene
  • P450arom mRNA
  • estrogens
  • testis
  • mammals