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Molecular forms of esterases were resolved in non-denaturing conditions by using two-dimensional gel electrophoresis with isoelectric focusing in the first dimension and a time-dependent polyacrylamide gradient gel electrophoresis (PAGGE) in the second dimension. This procedure was used to analyse sequential changes in esterase composition along the excurrent genital duct of the mouse and to initiate a specific identification of the androgen-regulated molecular forms. Almost all the 68 variants (pH 3.9-6.4 and 50-300 kDa) revealed by alpha-naphtyl acetate from the fluids of the three parts of the epididymis (caput, corpus, cauda) and vas deferens, could be assigned to the carboxylesterase group as shown by their action on various substrates and sensitivity to inhibitors. Some of these variants co-migrated with those in the serum and testis, whereas other enzyme forms made their first appearance in the caput (13), in the corpus (26) and in the vas deferens (3). The major changes occurred between the caput and the corpus of the epididymis. Only a few acidic spots were not revealed after neuraminidase digestion. Castration of mice (4 weeks) resulted in inhibition of the activity of 34 esterase forms, and thus abolished most of the regional differences in the excurrent duct system. By re-initiating or repressing the synthesis of regional esterase variants, testosterone supplementation (2 and/or 4 weeks) of castrated animals restored the normal esterase pattern in the three epididymal parts, but not in the vas deferens. The major effect of efferent duct ligation (4 weeks) was the emergence in the corpus and cauda of the epididymis of two variants found in the caput of uncastrated mice.

The fine structure, nature and fate of the components of the nucleolus were studied in young (steps 1, 2), intermediate (steps 3, 4, 5) and mature spermatids (steps 6, 7, 8) of man and monkey, by use of several cytochemical techniques (alcoholic PTA; sodium tungstate: EDTA; HAPTA; nuclease-gold complexes; NOR silver staining). As controls, comparative ultrastructural and cytochemical observations of the nucleolus in spermatids and Sertoli cells were made in the same sections of seminiferous tubules. In the young spermatids of the two species studied, the nucleolar masses exhibited identical features. Segregation of the nucleolar components took place in the nuclei of step 1 spermatids. No typical fibrillar center was observed. In spermatids at steps 1 and 2, the nucleolar masses appeared to be made up of two fibrillar components of equal density, one spherule-shaped, the other forming cords, both surrounded by clusters of 15-20 nm-diameter granules. Alcoholic PTA and sodium tungstate yielded a selective positive contrast of the two fibrillar components whereas EDTA and RNase-gold reacted with the peripheral granular material. Treatment with RNase-gold and DNase-gold complexes resulted in preferential labeling at the periphery of the fibrillar components. After NOR silver staining, numerous small silver grains were localized over the fibrillar cords, suggesting the persistence of specific acidic non-histone proteins. On the contrary, the spherule was never stained. In intermediate spermatids, when the nucleolar components were dissociated, scattered clusters of granules stained by EDTA and HAPTA remained in the entire nucleoplasm. Nucleolar disintegration was accompanied by dispersion of argyrophilic material.(ABSTRACT TRUNCATED AT 250 WORDS)

Two-dimensional electrophoresis with time-dependent polyacrylamide gradient gel electrophoresis (PAGGE) in the second dimension was applied to the separation of native molecular forms of esterases from serum and testis of four strains of mice (C57BL/6J, Swiss OF1, F1 hybrid derived from these two populations and Tfm). In Phast System, a modified pH 3-9 gradient, a linear 8-25% gel gradient and a migration time corresponding to 300 Vh, were found to provide the best conditions for esterase analysis. About 70 esterase-active fractions could be separated with good reproducibility. The variants were characterized by their pI (3.9-7.35), their relative mobility and the visual estimation of their susceptibility towards neuraminidase and different esterase inhibitors. In the two tissues, the distribution of the esterase variants corresponded to a 50-500 kDa molecular mass range of calibration proteins, but most of the serum and testis-specific isoforms were confined to the 59-72 kDa range. All serum variants contained a terminal N-acetylneuraminic acid residue, whereas only the testicular esterases in common with those in serum appeared sensitive to neuraminidase. Cholinesterases with a low relative mobility and carboxylesterases with a high relative mobility were detected in serum, while carboxylesterases accounted for the greatest part in the testis which also contained cholinesterases and acetylesterases. Minor interspecies differences were found between C57BL/6J and Swiss OF1 esterases. The expression of two variants which differed between these two species seemed intermediate for the hybrid originating from these two populations. Two new spots were detected in the two-dimensional map of esterases from the strain bearing the Tfm mutation.

Esterase isozymes were studied in mouse epididymis of two inbred strains (C57BL, DBA/2) and in a natural population (Swiss OF1), by using vertical polyacrylamide gel electrophoresis and staining with alpha or beta-naphthyl acetate as a substrate. Eighteen (C57BL), 17 (DBA/2) or 16 (Swiss OF1) epididymal isozymes were separated; four were common to the testis, and five to both the testis and the serum. The use of different inhibitors showed that carboxylesterase activities account for the greater part of the total epididymis non-specific esterase activity. This comparative study revealed minor interspecies variations since only two isozymes were not expressed in the same manner in the three populations examined. Among the nine isozymes which appeared solely in the epididymis, the profiles varied between tissues and fluids as well as between the proximal part in which sperm maturation occurs and the distal part where sperm storage takes place. The variations proceeded from the relative activity of isozymes and the presence or absence of some of them; two characterized the proximal part and one the distal part in the three species. By comparing testis and epididymal tissues and fluids, it is suggested that the isozymes found in epididymal fluids originated from the testis, the epididymal epithelium or both. In addition to this epididymal secretory function, the lack in the fluid of the distal part of one isozyme identified in the testis, and two in the proximal part may also provide evidence for its reabsorptive function.

The in vitro synthesis and secretion of newly formed proteins were investigated in the principal cells of small pieces of the head and body of mouse epididymis inserted into a modified Rose chamber. Our ultrastructural observations revealed that the cells resumed their normal ultrastructural appearance only after 3 h. Therefore, after preliminary 3-hour culture, the pieces were incubated with tritiated leucine at intervals ranging from 5 min to 6 h. The incorporation of radioactive leucine measured by scintillation counting demonstrated that each of the anatomical segments studied secreted a significant amount of proteins into the culture medium.

The cytology of epithelial cells of the mouse epididymis was studied under light and electron microscopy. Four principal areas--the initial segment, the head, the body and the tail--were distinguished according to the morphological features of the main cells. Transitional areas separating these segments have been described also; one of the segments, intermediate between the head and the body, was longer. Five other cell types and their sites were noted: "apical" cells, corresponding to the "narrow" cells of Sun and Flickinger (1980), were located in the initial to the intermediate segment; clear cells were scattered in the epithelium from the intermediate segment to the tail; basal cells were sporadically distributed along the entire duct. Halo and dark cells were observed also.

In Monkey spermatids at different steps of spermiogenesis, the use of DNase-gold complex showed, at the ultrastructural level, a labeling over the chromatin and concomitantly over the chromatoid body, centriole associated body and annular chromatoid body. The results obtained with the DNase-gold complex containing either DNA or actin led to discuss the nature of the substances revealed by the labeling in the cytoplasmic structures.

At 2 weeks of age, 11 isoenzymes were expressed and similar banding patterns on vertical polyacrylamide gel electrophoresis (PAGE), stained by alpha- or beta-naphthyl acetate as a substrate, were obtained for tissues or fluids from the proximal and the distal parts of the mouse epididymis. After this period, the emergence of new bands or the disappearance of certain others led to a regional differentiation which appeared progressively in tissues and fluids, earlier in the distal part than in the proximal part. The changes occurring during epididymal differentiation affected the isoenzymes specific to the epididymis more than those common to testis and serum. Castration of adult mice induced a decrease in esterase activity and changes in the number of isoenzymes, leading to the loss of regional specificity of the banding patterns. The dedifferentiation process modified the electrophoretic profiles of the distal part only. Androgen replacement restored the regional specificity of cytosol banding patterns after 2 weeks of treatment and the normal intensity of bands after 4 weeks. Some differences in the fluid isoenzymes nevertheless persisted. The androgen-dependence of esterase isoenzymes can be attributed to circulatory hormones rather than to androgens from the testis via the rete testis as shown by efferent ductule ligation which did not modify the epididymal esterase profiles.

The proteins of epididymal tissues and fluids recovered from different regions of the mouse epididymis from a natural population and an inbred line were examined by polyacrylamide gel electrophoresis under denaturing conditions. Two epididymal specific peptides on the order of 88 and 20 Kilodaltons (Kd), undetected in serum and testicular extracts, were identified in the initial segment, caput, corpus and cauda. Another specific 30 Kd peptide was localized in the cauda tissue and fluid. Castration caused the disappearance of the three specific epididymal bands and of a non-specific 34 Kd band. In contrast, a new band appeared at 14.5 Kd. Testosterone propionate administration only restored the three specific epididymal bands and had no effect on the 14.5 peptide. Variations in the staining intensity of the four bands, which were suppressed in castrated animals, were observed after ductuli efferentes ligation.

The subcellular distribution of esterases was studied in mouse epididymis by using 5-bromo-indoxyl-acetate as a substrate. In all the cells of the duct, a low level of esterase activity was detected except in one of the five segments of the head--segment IV; in one of the three types of apical cells--the "prominent cells"; and in the "clear cells" scattered in the middle and distal parts. In these cells, the intensity of the reaction was high. The reaction product was consistently found in the endoplasmic reticulum and was more abundant in cells showing a high level of activity than in others. In cells with low esterase activity, the reaction was mainly restricted to this organelle. In highly active cells, the spectrum of subcellular locations was selectively enlarged and esterase was demonstrated in almost all cell compartments, including the cell membrane, nuclear envelope, mitochondria, lytic structures, and, more rarely in the Golgi apparatus or microvilli. These locations were dependent on cell type. A weak enzyme activity also appeared on mature spermatozoa.

Rapid cryo-fixation of mouse spermatozoa followed by freeze substitution, thin section and freeze-fracture demonstrates the finer detail possible with quick-freeze as compared to chemical fixation. As seen in thin sections the unit membrane is composed of 2-4 nm size particles and the cytoplasm, mitochondria, and axonemal filament components all appear rich in fine structure detail. The filamentous structure of the post-acrosomal sheath and its connection with the plasmalemma is presented and compared to previous studies on this structure. Freeze-fracture data demonstrates 7-9 nm size, plasmalemmal, PF-face particles most heavily concentrated in the region just ahead of the striated ring. The outer acrosomal EF-face contains linear arrays of 7-9 nm size EF-face particles. The inner acrosomal membrane contains scattered, 7-9 nm size PF-face particles. The inner and outer nuclear membranes also contain scattered, 7-9 nm size particles. The results of this study present data which supports and extends previous studies on mouse spermatozoa. The results are discussed in terms of the advantages of cryo-fixation and freeze-substitution compared to conventional fixation in the preservation of fine structure detail.

The proteins of epididymal tissues and fluids recovered from five regions of the human epididymis were separated by polyacrylamide gel electrophoresis under denaturing conditions. Among the 60 peptides identified, eight appeared to be expressed solely in the epididymal duct when compared to serum and testis proteins. Three of these (92, 47 and 24 Kd) showed a degree of regional specificity in fluids. The 92 Kd peptide was found in the caput and proximal corpus, the 47 Kd in the distal corpus and cauda and the 24 Kd in the caput of the epididymis. Three of the specific epididymal proteins (39, 30, 26 Kd) displayed a remarkable analogy to those found in man and monkey in other conditions and which are present at the sperm surface in the epididymis cauda.

A scanning electron microscopic study was carried out on long-headed sperm from normal human semen with the purpose of establishing morphometrical standards for this atypical variety which is difficult to differentiate from the normal type in the spermocytogram. Subjective discrimination between the two populations (with elongated and normal heads) was confirmed by multidimensional analyses. They also made statistical identification of thin-headed spermatozoa (an intermediate form between the two preceding types) possible. Moreover, through the analyses an elongation index for distinguishing the elongated type from light microscopic observations was able to be calculated. Statistical data showed that the acrosome accounted for 30% of the elongated head, the postacrosomal cap and the posterior nuclear space--both bounded by furrows--occupied 9% and 6% respectively, whereas the posterior part of the head, covered by a voluminous cytoplasmic sheath, took up 56%. This lengthening of the head was accompanied by an elongation of the middle piece which thus became the second most important discriminating factor between the two populations.

Spermatozoa from four epididymal regions and from the ejaculate of the monkey, Macaca fascicularis, always present the same particle organization for a given sample. The plasmalemma contains homogeneously distributed, 7-8 nm size, PF-face, particles throughout the head, midpiece and principal piece. A unique, particle arrangement occurs in the plasmalemmal, PF-face in spermatozoa from the proximal levels of the body of the epididymis only. Square arrays of 4-6 nm size, PF-face plasmalemmal particles occur in this region of the epididymis at the peripheral edge of the spermatozoon acrosomal region only. Throughout other regions of the epididymis the plasmalemma contains only the homogeneously distributed, 7-8 nm size, PF-face particles. Ejaculate spermatozoa exhibit a similar particle organization to that of spermatozoa found in the distal portions of the epididymis. However, ejaculate spermatozoa of one animal from the eight used in this study exhibited a series of 7-8 nm size, linearly arranged, plasmalemmal, PF-face particles situated just proximal to the striated ring. This array of particles continued further rostral on the lateral aspect than on the flattened medial aspect of the spermatozoon. Spermatozoa from the tail of the epididymis and from ejaculates of the other seven animals lacked such specialization. The above data is discussed in terms of the population of particles generally, the significance of the presence of linearly arranged particles proximal to the striated ring, and the possible significance of the square arrays of particles present transiently in one region of the epididymis only. A correlation is made between these square arrays and similarly arrayed particles found in rat and boar epididymal spermatozoa.

The ultrastructure of the principal cells of the mouse epididymis was studied using osmium impregnation techniques which have the advantage that the endoplasmic reticulum (ER) content displays a positive reactivity after glutaraldehyde fixation whereas the Golgi condensing vacuoles are negative. In the proximal part (caput) of the epididymis, the Golgi apparatus formed a large supranuclear area filled with electronluscent secretory vacuoles while, in the medial (corpus) and distal (cauda) parts, dictyosomes were small and sparse with few secretory vacuoles. In all the principal cells of the caput, the supranuclear ER cisternae were heterogeneously impregnated. In the corpus and cauda, the ER appeared as an extensive continuous network of canaliculi and saccules which were fenestrated when surrounding mitochondria. The ER content was homogeneously stained but impregnation intensity varied from cell to cell. In the apex of the caput cells, numerous impregnated or electronluscent vesicles were seen in close apposition to the plasma membrane, while in the corpus and cauda some Golgi vacuoles and extensions of ER canaliculi were observed in the terminal webb region. Thus, in the epididymal caput, osmium impregnation suggested that two distinct secretory pathways were functioning continuously. The first corresponded to the transport of proteins to the cell membrane by the Golgi condensing vacuoles. The second might only affect the small impregnated vesicles of the ER, through which proteins bypassed the Golgi apparatus and were exported towards the lumen. In the corpus and cauda, the network organization of the ER and the association of fenestrated cisternae with mitochondria (also found in absorptive epithelial cells) supported the view of a predominant absorptive function in these epididymal parts.

Regional differences in the proximal part of mouse epididymis were reported to provide a morphological baseline for studies on functional zonation of this part that is critical in sperm maturation. Macroscopical, histological, ultrastructural, and histochemical observations permitted us to subdivide this part into five segments, characterized by epithelial height, nuclear position, cytological and histochemical features of principal cells. Segment I corresponded to the initial segment previously described in rodents. Segment II differed from segment I by endoplasmic reticulum (ER) and dictyosomes aspect in principal cells, apical alkaline phosphatase and Ca2+-dependent ATPase activities. Segment III was characterized by spermatozoa package, high content of cells in multivesicular bodies, mitochondria shape, complex interdigitating membranes, and strong periodic acid-Schiff (PAS)-positive cell border. Segments IV and V presented the same cytological features but differed by their esterase activity. In the principal cells of each segment, dense spherical concretions were scattered in ER caveolae. Cells with apical nuclei were classified into two groups. The cells of the first group presented the same morphological and histochemical features as the adjacent principal cells and were scattered in the five segments ("apical cells"). The cells of the second group differed from the others by their goblet shape, a dense cytoplasm, and a high mitochondria succinate-D activity. They presented different cytological and histochemical features depending on their localization in segments I ("narrow cells"), II ("prominent cells"), or III, IV, V ("mitochondria goblet-cells"). The possible relationships between epithelium structure and epididymal functions were herein discussed.