Jean-Luc Courtens

Study in vitro of the phagocytic function of Sertoli cells in the rat

SummaryAspects of the interaction between residual bodies/cytoplasts from elongated spermatids (RB/CES) and Sertoli cells were studied in vitro. Highly enriched Sertoli cells (91%; experiment A), very highly enriched Sertoli cells (>96%; experiment B), as well as peritubular cells were isolated from testes of 20-day-old rats by means of hypotonic treatment. Isolated Sertoli cells and peritubular cells were also prepared from 45-day-old rats (experiment C). RB/CES were isolated by centrifugal elutriation from testes of rats aged 90–120 days. The kinetics of adhesion of RB/CES to Sertoli cells were similar in all experiments. FSH accelerated binding of RB/CES but markedly reduced the number of RB/CES phagocytosed. Co-culture of the highly enriched Sertoli cells from experiments A and C with isolated peritubular cells did not change the kinetics of adhesion of RB/CES. However, when the contamination of Sertoli cells by peritubular cells was at a minimum (experiment B), addition of peritubular cells induced a slight but significant stimulation of the binding of RB/CES. Transmission electron microscopy revealed the following events within 24 h of co-culture: adhesion of the RB/CES to microvilli of Sertoli cells; internalization of RB/CES; lysis of the membrane of RB/CES; total digestion. Therefore, FSH and peritubular cells modulate the interaction in vitro between Sertoli cells and RB/CES, and the different steps of residual body disposal can be reproduced in co-culture. The co-culture model described in this study provides a useful system for the study of phagocytic activity by Sertoli cells.

A cytochemical study of nuclear changes in boar, bull, goat, mouse, rat, and stallion spermatids.

En bloc staining with alcoholic phosphotungstic acid for lysine detection was applied to the testis of boar, bull, goat, mouse, rat, and stallion. Known changes in spermatid nucleoproteins were reflected in modification of the stainability of the nuclei over five successive periods of spermiogenesis. Species-specific differences occur in the way the chromatin is condensed, the presence and development of a peripheral layer of dense chromatin, the way lysine-rich nucleoproteins are lost from nuclei, and the staining abilities of nuclei of old spermatids. The observed staining pattern adds information about the precise timing of nucleoprotein changes during spermiogenesis and also about the heterogeneous distribution of nucleoproteins in nuclei of spermatids during several steps of differentiation.

Nuclear reorganization in ram spermatids.

Nuclear changes as a function of ram spermatid differentiation have been studied using electron microscopy, cytochemistry, and treatment of isolated nuclei with chemical and mechanical agents. In round nuclei, chromatin consists of intertwined knobby fibers about 210 and 120 A in diameter. In flattening nuclei, these fibers progressively change into smooth filaments at least 25–33 A thick. This change is interpreted as reflecting somatic histone removal known to occur in these nuclei (M. Loir and M. Lanneau, Exp. Cell. Res. 115 , 231, 1978). The packaging of chromatin is carried out by aggregation of the smooth filaments into large (about 300 A) contorted threads which finally coalesce to form a homogeneous mass. In flattened nuclei a small fraction of chromatin (basal knobs) does not undergo complete packaging and retains both cytochemical properties and resistance to disruption similar to those of chromatin in flattening nuclei. Correlations of changes in nuclear morphology, ultrastructure, and resistance to chemical and physical degradation with changes in nucleoproteins suggest that the cystine-containing spermatid-specific proteins could play an important role in chromatin-structure reorganization, shaping and increasing stabilization of the nucleus. The sperm-specific protein promotes terminal packaging and stabilization of chromatin. Other nuclear components considered in this study are: the RNP-containing structures, the nuclear posterior space, and the redundant nuclear envelope; the last two structures are possibly related to the transfer of proteins leaving the chromatin for the cytoplasm.

Modifications in the plasma membranes of epididymal ram spermatozoa during maturation and incubation in utero

Ram spermatozoa from corpus and cauda epididymis have been examined for the surface distribution of concanavalin A binding sites and electropositive and electronegative charges before and after incubation in utero under conditions able to induce capacitation. The electronegative charges at pH 1.8 which are uniformly distributed on the heads of corpus or cauda epididymal spermatozoa increase on the flagella of cauda epididymal spermatozoa. These charges are reduced to a great extent after incubation in utero. Electropositive charges at pH 9 are abundant on the postacrosomal area of both corpus and cauda epididymis spermatozoa. In addition, they appear on the apical ridge of cauda epididymis spermatozoa and do not change markedly after in utero incubation of cauda epididymal sperm. In contrast, the membrane of Corpus epididymal spermatozoa become heavily electropositive after in utero incubation, possibly due to acrosome leakages. Concanavalin A binding, which is important on corpus and cauda epididymal spermatozoa, greatly decreases in most cells from cauda epididymis after in utero incubation. The possible relations with the appearance of fertilizing ability are evoked.

Abnormal spermiogenesis in bulls treated with ethylene dibromide: an ultrastructural and ultracytochemical study

Spermatid differentiation was studied by electron microscopy in bulls treated with a single peritesticular injection of ethylene dibromide (270 mg/testis) and castrated 3, 4, and 5 days after injection, and in bulls treated orally with 10 doses (4 mg/kg body wt/dose) of the compound given on alternate days and castrated 6 days after the last dose. In both cases the morphogenesis of spermatids was impaired. The organelles most affected were the nucleus, the perinuclear substance, the perinuclear ring, and the acrosome. The differentiation of many spermatid nuclei seemed to be arrested or slowed down in comparison with the differentiation of the adjacent cytoplasm. Bromine was ultracytochemically localized at the affected sites of the cells. The action of ethylene dibromide seems to be a direct alkylating one on several cysteine-rich spermatid organelles. Earlier-than-spermatids germ cells were also affected by the prolonged oral treatment.

Ultrastructural detection of basic nucleoproteins: Alcoholic phosphotungstic acid does not bind to arginine residues

Nuclei of old spermatids and spermatozoa are not detectable with alcoholic phosphotungstic acid when they contain lysine-free, arginine-rich nucleoproteins. Addition of NH2 groups to these nuclei, as well as the staining abilities of partially proteolysed nuclei, indicates that amino groups are targets for the stain. Unmasking and or selective blocking of guanidyl groups does not modify the staining pattern. Successful arginine detection with naphthoquinone in phosphotungstic acid-treated, basic nucleoprotein of ram spermatozoa, demonstrates that alcoholic phosphotungstic acid does not bind to guanidyl groups. The comparison of staining abilities of nuclei and of nucleoproteins extracted from spermatozoa of rat, mouse, ram, boar, bull, and goat, indicates that lysine is detected.

OSMIUM AMMINE : REVIEW OF CURRENT APPLICATIONS TO VISUALIZE DNA IN ELECTRON MICROSCOPY

This review has been collectively written. The contribution of the authors is mentioned for each part. References have been grouped at the end of the review. The objective of this review is to outline the principle of the method for electron microscopy, to emphasize the major applications and recent developments of this technique for DNA detection and finally to compare this technique with some other methods of DNA detection.

A cytochemical and immunocytochemical study of DNA distribution in spermatid nuclei of mouse, rabbit, and bull.

SummaryDNA distribution in mouse, rabbit and bull spermatids was analyzed by electron microscopy, after using a Feulgen-like HCl-osmium ammine procedure, and after immunocytochemistry with anti-DNA antibodies. In addition, nucleic acids were visualized with the intercalating dye ethidium bromide and phosphotungstic acid. The parts of DNA displaying a beta helix configuration (possibly A-T rich parts) were identified by epifluorescence microscopy after staining with Hoechst 33258. In all 3 species, young spermatid nuclei were seen to have large areas poor in DNA, as well as DNA-rich areas, which were mostly concentrated into a peripheral layer close to the acrosome and into one or several masses, displaying species-specific locations. These DNA-rich areas were stained with Hoechst 33258. Elongating spermatid nuclei contained homogeneously distributed DNA, and this was evident following both immunocytochemistry and nucleic acid histochemistry in all 3 species. However, the distribution appeared more heterogeneous after the Feulgen-like procedure, and was accompanied by a disappearance of Hoechst-fluorescence. In fully elongated spermatids, all nuclear areas stained with Hoechst 33258, while the 3 other techniques labeled either all or species-specific parts of the condensed chromatin. The reasons for these variable reactions are discussed in terms of technique specificities, DNA configuration and nucleoprotein moiety replacements.