ABSTRACT
Junctional devices in Sertoli cells conform the blood-testis barrier and play a key role in maturation and differentiation of germ cells. The spacial distribution of ectoplasmic specializations of Sertoli cells was studied by beta-actin immunolabelling, using laser confocal and transmission electron microscopy. For confocal microscopy, beta-actin immunolabelling of ectoplasmic specializations was studied over the background of either prosaposin or glutaredoxin immunolabelling of the Sertoli cytoplasm. Labelling was found near the basal lamina, surrounding early spermatocytes (presumably in leptotene-zygotene) or at one of two levels in the seminiferous epithelium: (1) around deep infoldings of the Sertoli cell cytoplasm, in tubular stages before spermiation, and (2) in the superficial part of the seminiferous epithelium, in tubular stages after or during spermiation. For transmission electron microscopy, beta-actin immunolabelling of ectoplasmic specializations was also used. Ectoplasmic specializations were found at two different levels of the seminiferous epithelium. We also used freeze fracture to analyze the characteristics of tubulo-bulbar complexes, a known component of apical ectoplasmic specializations. Also, these different approaches allowed us to study the complex arrangement of the actin cytoskeleton of Sertoli cells branches, which surround germ cells in different stages of the spermatogenic cycle. Our results show a consistent labelling for beta-actin before, during and after the release of spermatozoa in the tubular lumen (spermiation) suggesting a significant role of the actin network in spermatic cell differentiation. In conclusion, significant interrelations among the beta-actin network, the junctional complexes of the blood-testis barrier and the ectoplasmic specializations were detected at different stages of the seminiferous cycle.
Subject(s)
Animals , Male , Rats , Actins/metabolism , Sertoli Cells/metabolism , Cytoskeleton/metabolism , Cytoplasm/metabolism , Testis/metabolism , Blood-Testis Barrier/metabolism , Cells, Cultured , Sertoli Cells/ultrastructure , Cytoskeleton/ultrastructure , Rats, Wistar , Testis/cytology , Testis/ultrastructureABSTRACT
Cellular and subcellular organization and distribution of actin filaments have been studied with various techniques. The use of fluorescence photo-oxidation combined with phalloidin conjugates with eosin has allowed the examination of the precise cellular and subcellular location of F-actin. Correlative fluorescence light microscopy and transmission electron microscopy studies of F-actin distribution are facilitated with this method for morphological and physiological studies. Because phalloidin-eosin is smaller than other markers, this method allows the analysis of the three-dimensional location of F-actin with high-resolution light microscopy, three-d serial sections reconstructions, and electron tomography. The combination of selective staining and three-dimensional reconstructions provide a valuable tool for revealing aspects of the synaptic morphology that are not available when conventional electron microscopy is used. By applying this selective staining technique and three-dimensional imaging, we uncovered the structural organization of actin in the postsynaptic densities in physiological and pathological conditions.
Subject(s)
Humans , Animals , Actins/metabolism , Eosine Yellowish-(YS)/pharmacology , Eosine Yellowish-(YS)/metabolism , Photooxidation , Central Nervous System/metabolism , Central Nervous System/ultrastructure , Staining and Labeling/methods , Fluorescent Dyes/pharmacology , Phalloidine/pharmacology , Imaging, Three-Dimensional/methods , Models, Molecular , Actin Cytoskeleton/metabolism , Actin Cytoskeleton/ultrastructure , Microscopy, Fluorescence/methods , Oxidation-Reduction , PhotonsABSTRACT
Ultrastructural synaptic changes of retinal origin in the pars dorsalis lateral geniculate nuclei (dLGN) after enucleation have been studied in this laboratory, showing a filamentous hypertrophy with maximal expression at 4-6 days post-lesion in monkeys (Pecci Saavedra et al., 1970, 1971). The aim of this work was to elucidate the nature of the newly formed filament in dLGN in post-enucleated rats. Male Wistar rats were fixed with 4 paraformaldehyde plus 0.25 glutaraldehyde in 0.1M phosphate buffer, through the abdominal aorta after 3, 5, and 7 days postenucleation. Sections obtained were incubated with antibodies to the phosphorylated portion of the 160 Kd neurofilaments (1:3000) and anti-GFAP (1:25000). There was an increase in 160 Kd neurofilament staining in axons and degenerating nerve endings in dLGN, as well as a typical astroglial immunostained reaction. Our results show that the newly formed neurofilaments after deafferentation are of the 160 Kd type, commonly present in normal axons.