SPERMATOGENIC CELLS DO INTERNALIZE SERTOLI ANDROGEN-BINDING PROTEIN -A TRANSMISSION ELECTRON-MICROSCOPY AUTORADIOGRAPHIC STUDY IN THE RAT

Specific binding sites for androgen-binding protein (ABP) have been de monstrated recently to be present on germ cells from the rat and on me mbrane-enriched fractions from rat germ cells. The present study was u ndertaken to test if such receptors could lead to the activation of a specific internalization pathway as in other cells. Isolated rat germ cells and in situ rat germ cells, maintained within the seminiferous e pithelium with either an intact or a bypassed blood testis barrier, we re exposed to culture medium containing 12,000 cpm/ml [H-3]Delta(6)-te stosterone (2.5 pg) photoaffinity-labeled ABP, purified from rat testi s. The follow-up of labeled ABP/germ cell interactions was based on qu alitative and quantitative transmission electron microscopy autohistor adiography. Attention was focused on adluminal germ cells from pachyte ne spermatocytes to mature spermatids, which are normally present abov e the Sertoli cell tight junctions. Our observations revealed the pres ence in rat germ cells of structures related to specific endocytosis, namely coated pits and vesicles which stained positively with anticlat hrin antibodies. When exposed to the [H-3]as-testosterone-ABP complex, adluminal germ cells showed marked labeling of these endocytic organe lles. Preincubation either with excess unlabeled ABP or pretreatment b y EDTA reduced the labeling significantly. Once internalized, ABP was found to be confined to the endocytic and nuclear compartments. The nu clear labeling was high in primary spermatocytes and round spermatids but was absent in elongated spermatids with condensed chromatin, in wh ich transcriptional activity had almost stopped. In contrast, at later steps of spermiogenesis, the cytoplasm became heavily labeled, especi ally in the postnuclear part of elongated spermatids and in residual b odies about to be phagocytozed by Sertoli cells. Experiments using lig ated seminiferous tubules, with an intact blood-testis barrier, clearl y showed that ABP was captured at the basal part of Sertoli cells, tra nsported up to the adluminal compartment and delivered to germ cells w hich finally internalized the protein. Experiments using largely opene d seminiferous tubules allowing ABP to bypass the blood-testis barrier led to a delay in adluminal germ cell labeling compared with that in isolated germ cells. In all experiments in which germ cells were incub ated in the presence of Sertoli cells, the labeling observed at the su rface of the germ cell line, especially over coated pits, was mostly f ound facing thin Sertoli cell processes, suggesting the possible exist ence of a specific mechanism for the presentation of ABP by the Sertol i cells to adluminal germ cells. In summary, the present study shows f or the first time, to our knowledge, that germ cells have the ability to bind and internalize ABP either directly from a fluid phase, such a s culture medium, and from the Sertoli cell cytoplasm, which may repre sent the physiological situation in vivo. This internalization uses a receptor-mediated endocytosis pathway, and the intracellular site of A BP accumulation varies throughout the spermatogenesis. This strongly s uggests that Sertoli-secreted ABP may be required for some steps of sp ermatogenesis, acting on germ cells either by itself or by serving as a steroid transmembrane carrier.