Endocytosis of transcobalamin in male rabbit germ cells: electron microscope radioautography study.

The binding of 125I-iodinated transcobalamin to a suspension of isolated rabbit germ cells was studied by Scatchard plot. The number of binding sites was evaluated to about 1000 per cell, and its association constant (Kass) in order of 14.6 l/nmole. The distribution to structures related to endocytosis was determined by ultrastructural histomorphometric studies. Both coated and uncoated structures were present regardless of maturation stage. The number of coated vesicles was at its highest in the initial maturation steps, whereas the number of uncoated vesicles was highest in the final maturation steps. The endocytosis of 125I-iodinated transcobalamin by the suspension of germ cells was studied by electron microscope radioautography. The tracer was mostly detected over the plasma membrane, coated vesicles and multivesicular bodies of germ cells. The grains were observed mainly over spermatocytes and round spermatids; 31.6% and 32% of these cells, respectively, were labeled. In contrast the tracer was detected in only 8.11% of elongated spermatids. In conclusion, iodinated transcobalamin is internalized in rabbit germ cells by receptor-mediated endocytosis. This phenomenon was predominant in the early stages of germ cell maturation.

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 demonstrated recently to be present on germ cells from the rat and on membrane-enriched fractions from rat germ cells. The present study was undertaken 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 epithelium with either an intact or a bypassed blood testis barrier, were exposed to culture medium containing 12,000 cpm/ml [3H] delta 6-testosterone (2.5 pg) photoaffinity-labeled ABP, purified from rat testis. The follow-up of labeled ABP/germ cell interactions was based on qualitative and quantitative transmission electron microscopy autohistoradiography. Attention was focused on adluminal germ cells from pachytene spermatocytes to mature spermatids, which are normally present above the Sertoli cell tight junctions. Our observations revealed the presence in rat germ cells of structures related to specific endocytosis, namely coated pits and vesicles which stained positively with anticlathrin antibodies. When exposed to the [3H] delta 6-testosterone-ABP complex, adluminal germ cells showed marked labeling of these endocytic organelles. Preincubation either with excess unlabeled ABP or pretreatment by EDTA reduced the labeling significantly. Once internalized, ABP was found to be confined to the endocytic and nuclear compartments. The nuclear labeling was high in primary spermatocytes and round spermatids but was absent in elongated spermatids with condensed chromatin, in which transcriptional activity had almost stopped. In contrast, at later steps of spermiogenesis, the cytoplasm became heavily labeled, especially in the postnuclear part of elongated spermatids and in residual bodies about to be phagocytozed by Sertoli cells. Experiments using ligated seminiferous tubules, with an intact blood-testis barrier, clearly showed that ABP was captured at the basal part of Sertoli cells, transported up to the adluminal compartment and delivered to germ cells which finally internalized the protein. Experiments using largely opened 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 incubated in teh presence of Sertoli cells, the labeling observed at the surface of the germ cell line, especially over coated pits, was mostly found facing thin Sertoli cell processes, suggesting the possible existence of a specific mechanism for the presentation of ABP by the Sertoli cells to adluminal germ cells.(ABSTRACT TRUNCATED AT 400 WORDS)