Late Germ Cells Research Articles

Expression of cellular retinol-binding protein and lecithin-retinol acyltransferase in developing rat testis.

Vitamin A deficiency in mammals results in the loss of germ cells on the adluminal side of the blood-testis barrier, suggesting a need for vitamin A that would be supplied by the surrounding Sertoli cells. Cellular retinol-binding protein (CRBP) and lecithin-retinol acyltransferase (LRAT) are two proteins found in Sertoli cells that are known to be involved in vitamin A trafficking. To clarify the role of these two proteins in the delivery of vitamin A to developing germ cells, we have examined changes in their cell-specific expression during the onset of puberty in the rat. In adult rats, Sertoli cell expression of CRBP varies with the cycle of the seminiferous epithelium. Here, we demonstrate that differences in the intensity of CRBP immunoreactivity are detectable in Sertoli cells of different tubules as early as postnatal Day 4, prior to the onset of meiosis. This indicates that variable expression of CRBP by Sertoli cells is established independently of late germ cells and may anticipate the cyclical variation seen in the adult. We further demonstrate that the specific activity of LRAT in rat testis increases tenfold between postnatal Days 20 and 35. This increase is attributable to the appearance of post-meiotic germ cells: the LRAT activity of microsomes prepared from a round spermatid-enriched cell fraction from post-pubertal rat testis could account for the majority of the LRAT activity observed in the whole testis. The presence of LRAT activity within adluminal germ cells suggests that they receive vitamin A as retinol and synthesize the retinyl esters that have been shown to be present in mature sperm.

Endocytosis of Human Sex Steroid‐binding Protein in Monkey Germ Cells

We investigate in this study the hypothesis of human sex steroid-binding protein hSBP internalization into germ cells in a primate model. Human SBP was purified from late-pregnancy serum and labeled either with colloidal gold particles (18 nm) or with [3H]delta 6-testosterone by photoaffinity treatment. The germ cells were isolated from sexually mature monkey testis or caput epididymis (Macaca fascicularis) by mechanical means and cell suspensions (4 x 10(6) per 100 microliters culture medium) were incubated in presence of hSBP-gold complex (60 ng/100 microliters) or hSBP-[3H]delta 6-testosterone complex (66 ng/100 microliters, 20,000 cpm) for 2, 5, 15, 45, and 60 min. The samples were processed for electron microscopy followed by autoradiographic treatment for the radiolabeled samples. Localization of the label occurred over the whole germ cell lineage whichever tracer was used. Spermatogonia, spermatocytes, spermatids, testicular and epididymal spermatozoa exhibited specific binding sites over the plasma membrane associated with clathrin-like coated pits and vesicles. At 34 degrees C, intracellular localization of the labeled ligand was found within coated vesicles, in early and late endosomes. In addition, in early spermatogenic cells, labeled ligand was detected in the nuclei and/or associated with the nuclear envelope whereas in late spermatids and residual bodies, the labeling was accumulated in multivesicular, prelysosomal structures. Quantitative analysis of the "labeled cells/total cells" ratio exhibited a negative correlation to the maturation steps, epididymal spermatozoa being the least labeled. The cellular distribution is similar with one or the other protein in the same spermatogenic cells. Unlabeled hSBP treatment prior to labeled hSBP reduced significantly the internalization. Lowering the temperature to 4 degrees C prevented endocytosis and enhanced membrane binding. EDTA pretreatment strongly decreased hSBP internalization and modified the early endocytic steps, namely, the pinching off of the coated vesicles. It is concluded that monkey germ cells are able to internalize the human sex steroid-binding protein through specific endocytic organelles. This endocytosis leads to the labeling of the nuclei in the early spermatogenic cells and of the multivesicular bodies in the late germ cells. This strongly suggests that steroid-binding proteins may be required for spermatogenesis in acting at the germ cell lineage level either by themselves or by serving as steroid transmembrane carriers.

The blood-testis barrier in the toad (Bufo arenarum Hensel): a freeze-fracture and lanthanum tracer study.

Intercellular junctions between Sertoli cells in the toad testis were studied by freeze-fracture and electron-opaque intercellular markers. These junctional specializations are characterized in thin sections by a series of focal fusions on the outer leaflets of both adjacent cell plasmalemmas, associated with bundles of fine filaments in the subjacent Sertoli cell cytoplasms. However, the wide subsurface cisterna of the endoplasmic reticulum, a component constantly associated with Sertoli cell junctions in mammals, is absent in the toad. The intravascularly injected lanthanum hydroxide, used as a tracer compound, gains access to the seminiferous tubules and surrounds spermatogonia and leptotene spermatocytes, but is persistently excluded from germ cells in later stages of development. This indicates that, as is the case in the mammalian testis, a permeability barrier to lanthanum is established which isolates all germ cells beyond leptotene spermatocytes. Freeze-fracture reveals the characteristic occluding junctions between Sertoli cells, but a variation in their geometric patterns was clearly observed in different regions of the toad seminiferous epithelium. The membrane-fractured faces of Sertoli cells embracing differentiating spermatids exhibit a deep junctional complex: up to 50 rows of particles between adjacent Sertoli cells separate these late germ cells from the periphery of the seminiferous tubules. Sertoli cells surrounding early germ cells generally exhibit, instead, a discontinuous, poorly developed network of interconnected rows of particles with few widely spaced strands. This seems to permit the percolation of the intercellular marker in areas of the seminiferous epithelium containing spermatogonia and leptotene spermatocytes.

Vasectomy in rhesus monkeys. IV. Electron microscopic studies of the seminiferous epithelium.

AbstractStudies were undertaken to assess the fine structural effects of vasectomy upon the rhesus monkey testis, using 27 monkeys subjected to one of three surgical techniques: control sham operation (COS), unilateral silk vasoligation (USV), or unilateral clasp vaso‐occlusion (UCV). The monkeys were sacrificed from 1 to 66 weeks after surgery; tissues were fixed in glutaral‐dehyde in collidine buffer and processed for electron microscopy (EM) using routine techniques. No alterations were noted in the seminiferous epithelium of any COS animal; only focal lesions were found in vasectomized animals. These changes correlated with length of time post‐vasectomy, a finding consistent with earlier light microscopic studies (Heidger et al., '78). Even at the longest post‐operative periods studied, spermatogenesis appeared in most animals to be normal. One animal exhibited areas in which the seminiferous epithelium consisted of Sertoli cells and spermatogonia, only. The seminiferous epithelium of approximately one‐third of other animals, both on the vasectomized and contra‐lateral sides, exhibited such alterations as development of extensive infoldings and duplication within the basal lamina, and presence within the basal Sertoli cell cytoplasm of late spermatids and sperm tails. These alterations were observed in both UCV and USV animals. The presence of such late germ cells in the basal Sertoli cell cytoplasm was noted by light microscopy; however, lu‐minal spermiophages encountered in one animal at the light microscopic level were not detected in our EM study, which underscores the limitations of EM as a survey technique. All UCV animals developed spermatic granulomas of the vas, whereas only 3 of 13 USV animals developed such granulomas. It does not appear that the alterations exhibited by the animals in this study would necessarily dispose toward impaired spermatogenesis following vasovasostomy.