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Les cellules de la lignée germinale humaine ont la capacité d'internaliser la sex steroid-binding protein humaine (SBPh): Etude par autohistoradiographie en microscopie électronique à transmission (MET)

Germinal cell lineage possess the capacity of sex steroid-binding protein (hSBP) internalization: Transmission electron microscopy (TEM) autohistoradiographic study

Andrologie volume 5, pages 528–543 (1995)Cite this article

Resume

Il a été récemment démontré que les cellules spermatogénétiques de rat sont capables de lier et d'internaliser spécifiquement l'androgen-binding protein de rat (ABPr). De même les cellules spermatogénétiques du macaque, sont capables de lier et d'internaliser spécifiquement la sex steroid-binding protein humaine (SBPh).

Cette étude consiste à tester si des interactions entre les cellules spermatogénétiques et les protéines de liaison des stéroïdes existent également chez l'homme. Les cellules germinales proviennent d'une part des biopsies testiculaires de patients hypofertiles et d'autre part de pulpectomies indiquées pour cancer prostatique.

Les observations en MET révèlent la présence de deux types de structures reliés à l'endocytose dans les cellules spermatogénétiques. Premièrement, les puits et vésicules recouverts de 96 ± 10 nm de diamètre, associés à la membrane plasmique. Deuxièmement, les endosomes précoces (vésicules non recouvertes) de 225 ± 60 nm de diamètre localisés à la périphérie du cytoplasme ainsi que les endosomes tardifs, souvent organisés en corps multivésiculaires (CMV) dans le cytoplasme périnucléaire. Ces deux types de structures de l'appareil endocytaire sont présents à tous les stades de maturation des cellules germinales. Les vésicules non recouvertes sont toujours plus nombreuses que les vésicules recouvertes quel que soit le stade.

Les cellules germinales isolées etin situ maintenues dans l'épithélium séminifère ont été exposées au milieu de culture contenant le complexe SBPh purifiée (à partir de sérum de femme enceinte)-Δ6-testotérone tritiée (80 000 cpm/ml, 30 ng) photomarquée. L'étude des interactions SBPh/cellules germinales est fondée sur les analyses qualitatives et quantitatives du marquage révélé par autohistoradiographie en MET.

Nos résultats montrent que cette incubation provoque un important marquage des cellules spermatogénétiques. La préincubation avec de la SBPh non marquée en large excès, de même qu'un prétraitement par l'EGTA reduit significativement le marquage. Une fois internalisée, la SBPh est localisée au niveau du compartiment endocytaire et spécialement sur les membranes délimitant ce compartiment. Un marquage intranucléaire a été également observé, sauf dans les noyaux condensés des spermatides allongées.

Au total, ce travail montre que les cellules spermatogénétiques humaines possèdent les structures cellulaires caractéristiques d'une activité d'endocytose et ont la capacité de lier et d'internaliser la SBPh à partir du compartiment extracellulaire. Ce résultat confirme ceux obtenus sur des modèles animaux (rat et macaque) et conduit à considérer que l'existence d'interactions entre les cellules spermatogénétiques et les protéines de liaison des stéroïdes constitue un phénomène général et probablement impliqué dans le déroulement de la spermatogénèse. Les mécanismes par lesquels les proteines de liaison des stéroïdes interviennent dans le contrôle de la fertilité chez l'homme restent à élucider.

Abstract

It has been recently demonstrated that rat spermatogenic cells were able to specifically bind and internalize rat androgen-binding protein (rABP) and that monkey spermatogenic cells were, in the same way, able to specifically bind and internalize human sex steroid-binding protein (hSBP).

The present study was undertaken to test if such interactions between spermatogenic cells and steroid-binding proteins do exist in the human. Germ cells were collected from testis biopsies from hypofertile patients and from testis pulpectomies from patients with prostatic cancer. TEM observations revealed the presence of two kinds of structures related to endocytosis in human spermatogenic cells. Firstly: coated pits and vesicles of 96 ±10 nm in diameter, associated with the plasma membrane. Secondly: early endosomes of 225 ± 60 nm in diameter located in the peripheral cytoplasm and late endosomes, often organized into multivesicular bodies (MVB) in the deeper cytoplasm. Both coated and uncoated structures were equally present at all stages and uncoated structures were always more numerous than coated ones. Isolated germ cells and “in situ” germ cells maintained within the seminiferous epithelium were exposed to culture medium containing 80 000 cpm/ml [3H] δ6-testosterone (30 ng) photoaffinity-labelled hSBP purified from human late-pregnancy serum. The follow-up of labelled hSBP/germ cell interactions was based on qualitative and quantitative TEM autohistoradiography. Our observations revealed the presence of a marked labelling of spermatogenic cells. Preincubation either with excess unlabelled hSBP or pretreatment by EGTA reduced the labelling significantly. Once internalized, hSBP was found to be confined to the endocytic compartment and especially with the membrane delimitating this compartment. An intranuclear labelling was also observed which was nevertheless absent from the condensed nuclei of elongated spermatids. This leads to the hypothesis of a specific, probably receptor-mediated, endocytosis of hSBP. This was partly confirmed by our finding that germ cell membrane extracts expressed a specific binding activity for hSBP (0.54 nM and 2 X 7 1010 sites/mg protein).

In summary, the present study shows that human spermatogenic cells do possess active endocytic structures and have the ability to bind and internalize hSBP from the extracellular compartment. This confirm the results obtained in the rat and in the macaca and leads to propose as a general fact that steroid-binding proteins could interact with spermatogenic germ cells and to be required for the achievement of spermatogenesis. The mechanism by which “in fine” steroid-binding protein could be involved in human fertility remains to be discovered.

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Authors and Affiliations

  1. Laboratoire d'Histologie-Embryologie-II, Morphogénèse et Reproduction, Faculté de Médecine de Nancy I, BP 184, 54505, Vandœuvre-lès-Nancy, France

    R. Bedjou

  2. Laboratoire de Biochimie Cellulaire et Moléculaire en Nutrition, Faculté de Médecine de Nancy I, BP 184, 54505, Vandœuvre-lès-Nancy, France

    A. Gerard, F. Felden & J. L. Gueant

  3. Laboratoire de Biochimie Médicale I, Faculté de Médecine de Nancy I, BP 184, 54505, Vandœuvre-lès-Nancy, France

    P. Franck

  4. Service d'Endocrinologie, CHU Brabois (Directeur Pr Leclere), 54500, Vandœuvre-Lès-Nancy, France

    Th Durriez & H. Gerard

  5. Service d'Urologie CHU Brabois (Directeur Pr Mangin), 54500, Vandœuvre-Lès-Nancy, France

    J. Hubert

  6. Clinique Endocrinologique, INSERM U. 307 Hôpital Debrousse, 29, rue Sœur Bouvier, 69322, Lyon Cedex 05, France

    H. Lejeune

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Bedjou, R., Gerard, A., Felden, F. et al. Les cellules de la lignée germinale humaine ont la capacité d'internaliser la sex steroid-binding protein humaine (SBPh): Etude par autohistoradiographie en microscopie électronique à transmission (MET). Androl. 5, 528–543 (1995). https://doi.org/10.1007/BF03034539

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Basic and Clinical Andrology

ISSN: 2051-4190