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Low density lipoprotein receptor-related protein-2/megalin (LRP-2) is a receptor belonging to the low density lipoprotein receptor family that mediates endocytosis and lysosomal degradation of a variety of ligands including apolipoprotein J (Apo J)/clusterin/SGP-2. LRP-2 has been shown to be expressed regionally in the adult rat epididymis. In this study, we describe the pattern of expression of LRP-2 in the efferent ducts and epididymis during postnatal development of the rat and examine the role of testicular luminally derived substances on its expression. The expression of LRP-2 was analyzed immunocytochemically in tissues of normal animals ranging in age from postnatal day 7-90 and in 15-day-old efferent-duct-ligated animals sacrificed at later ages. In the efferent ducts, LRP-2 expression, appearing as a dense band on the apical surface of the nonciliated epithelial cells, was noted as early as day 7, well before the entry of sperm, Sertoli-cell-derived secretory products, and high levels of androgens. Efferent duct ligation studies further revealed that expression under this condition was comparable to controls at all later ages examined, suggesting that the factor regulating its expression was not a luminally derived testicular substance. In normal untreated animals, LRP-2 expression was not apparent at any of the ages examined in the proximal initial segment of the epididymis. By comparison, the distal initial segment, although having no LRP-2 expression from 7-15 days, showed expression in principal cells by day 21 which intensified at days 29 and 39. However, by day 49 and at later ages (56 and 90), LRP-2 immunoreactivity over principal cells became spotty or with weak or moderate reactivity in some cells and none in others. LRP-2 expression in the intermediate zone, proximal caput, corpus, and cauda regions also appeared in principal cells by day 21, intensified at days 29 and 39 and persisted as such at all later ages examined, correlating with high levels of androgens shown to occur by day 39. Although LRP-2 expression in the distal caput region was evident in principal cells at days 21 and 29, it became spotty with weak, moderate, or absent reactivity over principal cells at all later ages. These data suggest that LRP-2 expression is under the influence of both stimulatory and region-specific inhibitory factors. Analysis of 15-day-old efferent-duct-ligated animals at all later ages examined revealed that there was no change in LRP-2 expression along the entire epididymis, suggesting that both the stimulatory and inhibitory factors are not luminally derived testicular substances. The observed pattern of LRP-2 expression in all regions of the epididymis, except the distal caput region, was similar to that described for Apo J internalization by principal cells during postnatal development, showing a correlation between LRP-2 expression and its ligand, Apo J. In summary, LRP-2 expression in the epididymis undergoes region-specific changes during postnatal development and appears to be influenced by both stimulatory and inhibitory factors.

A well-characterized cell-free assay that reconstitutes Golgi transport is shown to require physically fragmented Golgi fractions for maximal activity. A Golgi fraction containing large, highly stacked flattened cisternae associated with coatomer-rich components was inactive in the intra-Golgi transport assay. In contrast, more fragmented hepatic Golgi fractions of lower purity were highly active in this assay. Control experiments ruled out defects in glycosylation, the presence of excess coatomer or inhibitory factors, as well as the lack or consumption of limiting diffusible factors as responsible for the lower activity of intact Golgi fractions. Neither Brefeldin A treatment, preincubation with KCl (that completely removed associated coatomer) or preincubation with imidazole buffers that caused unstacking, activated stacked fractions for transport. Only physical fragmentation promoted recovery of Golgi fractions active for transport in vitro. Rate-zonal centrifugation partially separated smaller transport-active Golgi fragments with a unique v-SNARE pattern, away from the bulk of Golgi-derived elements identified by their morphology and content of Golgi marker enzymes (N-acetyl glucosaminyl and galactosyl transferase activities). These fragments released during activation likely represent intra-Golgi continuities involved in maintaining the dynamic redistribution of resident enzymes during rapid anterograde transport of secretory cargo through the Golgi in vivo.

The epithelial cells lining the cauda epididymidis and vas deferens are active in endocytosis and have an abundance of lysosomes and a well-characterized secretory apparatus. However, little is known about the nature of lysosomal proteins contained within lysosomes, the types of receptors on the cell surface, and the types of proteins secreted by these cells. In the present study, cathepsins A, D, B, and sulfated glycoprotein (SGP)-1, well-characterized lysosomal proteins, as well as SGP-2, a secretory protein and low-density lipoprotein receptor-related protein-2 (LRP-2), an endocytic receptor, were immunolocalized at the light-microscopic level within epithelial cells of the cauda epididymidis and vas deferens. Principal cells showed numerous intensely reactive lysosomes for cathepsins A, D, and SGP-1 in all regions of the cauda and vas deferens and for cathepsin B only in the cauda epididymidis. Basal cells were intensely reactive for cathepsin A, unreactive for cathepsins D and B, and weakly reactive for SGP-1 in the cauda region. In the vas deferens, these cells were intensely reactive for cathepsin A and SGP-1 and unreactive for cathepsin B; in the case of cathepsin D, basal cells were weakly reactive in the proximal vas deferens but intensely reactive in the middle and distal vas deferens. Clear cells, present in the cauda region and proximal vas deferens, were intensely reactive for cathepsin A, weakly reactive for SGP-1, and unreactive for cathepsins D and B, while narrow cells found mainly in the proximal vas deferens were intensely reactive for cathepsins A, D, and SGP-1 and unreactive for cathepsin B. Thus, the expression of different lysosomal enzymes in the cauda epididymidis and vas deferens is not only cell- but also region-specific, suggesting differences in the type of substrates internalized by these cells. SGP-2, a secretory protein, showed a checkerboardlike staining pattern in the cytoplasm of principal cells of the cauda epididymidis, while the cytoplasm of all principal cells were intensely reactive in the vas deferens. This type of reaction, as well as staining of sperm, suggests that SGP-2 is secreted into the lumen, where it functions in relation to sperm. The endocytic receptor LRP-2 was noted only on the apical surface of principal cells of the cauda and vas deferens and in spherical structures indicative of endosomes suggestive of their role in the uptake of various ligands, including SGP-2, for which it has a high binding affinity. Thus SGP-2 in the cauda and vas deferens is not only secreted but endocytosed by principal cells, suggestive of an active turnover in the lumen. In summary, the epithelial cells of the cauda and vas deferens show marked differences in expression of lysosomal proteins, SGP-2, and LRP-2 suggestive of differences in their functional activity while sperm are stored and protected in these regions.

Steroidogenesis and spermatogenesis decrease in aging Brown Norway rats. We therefore hypothesized that there must be accompanying morphological changes taking place in the seminiferous tubules of the aging testis. The testes of Brown Norway rats ranging in age from 3 to 24 months were prepared for light and electron microscopy. To assess the integrity of the blood-testis barrier with age, a lanthanum nitrate study was done. The normal seminiferous tubules present in rats at 3 and 12 months of age were largely replaced at 24 months by fully regressed tubules that were virtually devoid of germ cells and contained large intercellular spaces. An electron-microscopic study of these regressed tubules showed a complete loss of cyclical variations of the organelles of the Sertoli cells. The nucleus was more irregularly shaped and was present at various levels in the epithelium. The endoplasmic reticulum was a loose, vesiculated network that was unlike the elaborate, tubular, anastomotic network noted in young animals. The lysosomes were large, oddly-shaped, and contained lipidic inclusions, in contrast to the distinct membrane-bound lysosomes and dense core bodies found in the young animals. Adjacent Sertoli cell processes encompassed large, empty intercellular spaces, possibly occupied previously by germ cells. The typical Sertoli-Sertoli junctions of the blood-testis barrier in the young animal were rarely seen at 24 months and were replaced by focal contact points, usually between three Sertoli cell processes. In the aged animals, lanthanum nitrate permeated the basal and adluminal compartments, extending between Sertoli cell processes and entering the intercellular spaces and lumen. In summary, during aging, there is a breakdown of the blood-testis barrier, and there are striking changes in the appearance of Sertoli cells. These results suggest a possible intrinsic limitation that prevents stem cells from renewing themselves, whether because of a degeneration of immunological origin or because of a lack of Sertoli cell support.

Glutathione S-transferases (GSTs) are dimeric proteins grouped into five classes based on the degree of amino acid homology of their subunits. They are involved in cellular detoxification through the catalyzation of the conjugation of reduced glutathione with various electrophilic substances. In the present study, the distribution of Ya and Yc subunits from the alpha family, Yb1 and Yo subunits of the mu class, and the Yf subunit of the pi class were examined with light microscope immunocytochemistry in Bouin-fixed, paraffin-embedded tissue of different regions of the cauda epididymidis and vas deferens. In the cauda, principal cells showed high levels of expression of Ya, Yc, and Yo subunits, while in the vas deferens, staining decreased to moderate levels for the Ya and Yo subunits and to low levels for the Yc subunit. While Yf was maintained at low levels in principal cells of all cauda and vas deferens regions, Yb1 expression was more erratic, presenting a checkerboard-like staining pattern in the proximal vas deferens and showing moderate cytoplasmic but intense nuclear reactivity in all other regions. Basal cells in the cauda were intensely reactive for Yf, while in the vas deferens, they became unreactive. Conversely, basal cells were unreactive for Ya in the cauda and proximal vas deferens, while in the middle and distal vas deferens, they became moderately reactive. In the case of Yb1 and Yo, some basal cells were reactive while others appeared unreactive in all cauda and vas deferens regions. Yc elicited the display of both reactive and unreactive basal cells in the cauda regions, and while the cells were moderately reactive in the proximal vas deferens, they became intensely reactive in the middle and distal vas deferens. In summary, both principal and basal cells show varying degrees of GST expression in the different regions of the cauda and vas deferens, suggesting that these cells are subjected to a complex, changing environment of substrates. Furthermore, while expression often differs from principal to basal cells, the absence of reactivity of a given GST in one cell type is usually compensated for by expression in the other cell type in any given region of the cauda or vas deferens. Taken together, the data suggest that ample protection from harmful circulating electrophiles can be provided for sperm during their storage in the cauda and vas deferens. In addition, since principal cells of the vas deferens are involved in steroid synthesis, the presence of GSTs in these cells may also serve to bind steroids, or this presence may be involved in steroid isomerization.

Principal cells show marked structural differences in the proximal, middle, and distal regions of the vas deferens, reflective of diverse functional activities. In the present study, we performed electron microscopy to examine the structural features of principal cells using glutaraldehyde-fixed, Epon-embedded material, while functional parameters were examined using light microscopic immunocytochemistry on Bouin-fixed, paraffin-embedded material. In the proximal region, the cuboidal principal cells resembled those of the cauda epididymidis, but few clear cells and occasional narrow cells were present. In the middle region, principal cells often contained blebs of their apical cytoplasm containing vesicular and tubular profiles. These blebs extended far from the cell surface and appeared to be liberated into the lumen, suggesting an apocrine type of secretion. In the distal region, dilated intercellular spaces containing numerous membranous profiles of different shapes and sizes were noted between adjacent principal cells and overlying basal cells. The use of an anti-aquaporin-1 antibody revealed an intense reaction over the endothelial cells of numerous vascular channels in the lamina propria. Taken together, these observations suggested water transport from the lumen of the vas deferens via the dilated spaces to underlying vascular channels, the function of which may be to concentrate sperm. The infranuclear cytoplasm of principal cells of this region showed whorls of smooth endoplasmic reticulum (sER). Large intracytoplasmic cavities were found within the sER aggregates, and these contained membranous profiles that appeared to peel off from the surrounding sER elements. Various images of such cavities closely juxtaposed to the lateral plasma membrane suggested that the membranous profiles of the intercellular spaces were derived from them. Use of anti-3beta-hydroxysteroid dehydrogenase antibody revealed an intense reaction over principal cells of the vas deferens, as well as over the blebs in the lumen of the vas deferens, which is indicative of the steroid synthesis performed by these cells. The release of sER membranous profiles into the dilated spaces and the presence of blebs in the lumen may represent a means of transporting steroids that are destined for different sites out of the principal cells. Steroids in the blebs would be ultimately destined for utilization by luminal sperm, while those steroids in the dilated spaces are designed for utilization by muscle layers of the lamina propria. In summary, principal cells of the vas deferens appear to be involved in synthesis and secretion of steroids and in eliminating water from the lumen of the vas deferens.

Androgen binding protein (ABP) has been shown to be secreted by Sertoli cells and to be actively taken up by the efferent ducts and proximal caput epididymidis and, yet, to be present at high concentrations in epididymal fluids. In the present study, ABP was immunolocalized by light microscopy in epithelial cells of the efferent ducts and epididymis of adult rats and during postnatal development and by electron microscopy in specific organelles within these cells. In adults, the efferent ducts actively endocytosed Sertoli cell-derived ABP. In the epididymis, principal cells displayed a variable staining reminiscent of a checkerboardlike pattern, with cells being intensely, moderately, or weakly reactive throughout their cytoplasm or unreactive. In the electron microscope, reactive cells displayed a labeling of their Golgi apparatus and secretory vesicles indicative of an epididymal-secreted form of ABP. However, labeling was also noted over endosomes of principal cells, but only of the initial segment and intermediate zone, which, along with labeling of coated pits and vesicles, indicated that ABP was also endocytosed by principal cells of these regions. The postnatal study revealed that principal cells attained an adultlike staining pattern indicative of secretion in a region-specific manner at different ages, suggesting that ABP secretion is regulated by different factors. Ligation of the efferent ducts of 15-day-old animals revealed no reaction along the entire epididymis in animals sacrificed at later ages, suggesting the importance of luminal testicular factors in its regulation during development. In addition, as in the adult, ABP was also endocytosed by principal cells, but only in the initial segment and intermediate zone. Taken together, the present results indicate that secretion of ABP occurs along the entire epididymis, whereas endocytosis is region specific. The functional role of ABP in the epididymis in relation to sperm maturation is discussed.

The effects of vitamin E deficiency on the rat testis and epididymis were examined in a light- and electron-microscopic analysis. Various groups of animals were made vitamin E-deficient, beginning at postnatal day 10, via their lactating mothers, until day 21, when they were separated from their mothers. The groups were maintained thereafter on either a vitamin E-deficient or a normal diet (controls). The vitamin E-deficient animals of group A, sacrificed at day 42, revealed testes that were normal in appearance, with a full complement of germ cells when compared to their controls (group B). Group C, however, sacrificed at day 48, revealed major abnormalities in the testes, unlike both their controls (group D) and normal, untreated animals (group E). Spermatogenesis was incomplete; the most advanced cell type was predominantly step-7 spermatids. However, many of these cells, as well as earlier spermatids, appeared to undergo degeneration, evidenced by large pale areas in their nuclei, disrupted acrosomes, and a cytoplasm with uncharacteristic organelles. Multinucleated cells, characterized by their chromatoid bodies as spermatids, were often seen in the seminiferous tubule lumen. Sertoli cells were normal in appearance, except for numerous, large lipid droplets in their basal region, at stages I-VIII; in appropriate controls (group D), such droplets were absent at these stages. These lipid inclusions presumably represented the final breakdown products of the late spermatids, which were phagocytosed by Sertoli cells between days 42 and 48. However, numerous germ cells, often recognized as round spermatids, and multinucleated cells were noted in the epididymal lumen, which indicates that such cells were spared from Sertoli cell phagocytosis. These data suggest that vitamin E plays a key role in the maintenance and survival of spermatids. In the epididymis, vitamin E deficiency resulted in principal, narrow, and apical cells that showed a poorly developed secretory and endocytic apparatus at days 42 (group A) and 48 (group C), unlike those of normal, untreated animals (group E). On the other hand, clear cells of groups A and C showed a highly developed endocytic apparatus in the cauda region only, whereas in the caput and corpus regions, endocytic apparatuses were small and undifferentiated, unlike those of group E. Thus, in the epididymis, vitamin E plays a role in the structural differentiation of principal cells along the entire epididymis, whereas, in the case of clear cells, its role is region-specific. Readministration of vitamin E to the diet restored a normal appearance to both the testis and the epididymis, which indicates that the effects on these tissues are reversible. Taken together, these data indicate that vitamin E plays important roles in maintaining the viability of the spermatid population and in allowing epithelial epididymal cells to acquire their fully differentiated structural appearance.

beta-hexosaminidase is an essential lysosomal enzyme whose absence in man results in a group of disorders, the G(M2) gangliosidoses. beta-hexosaminidase activity is many times higher in the epididymis than in other tissues, is present in sperm, and is postulated to be required for mammalian fertilization. To better understand which cells are responsible for beta-hexosaminidase expression and how it is regulated in the male reproductive system, we quantitated the mRNA expression of the alpha- and beta-subunits of beta-hexosaminidase and carried out immunocytochemical localization studies of the enzyme in the rat testis and epididymis. beta-hexosaminidase alpha-subunit mRNA was abundant and differentially expressed in the adult rat testis and epididymis, at 13- and 2-fold brain levels, respectively. In contrast, beta-subunit mRNA levels in the testis and epididymis were 0.3- and 5-fold brain levels. During testis development from 7-91 postnatal days of age, testis levels of alpha-subunit mRNA increased 10-fold and coincided with the appearance of spermatocytes and spermatids in the epithelium; in contrast, beta-subunit mRNA was expressed at low levels throughout tests development. In isolated male germ cells, beta-hexosaminidase alpha-subunit expression was most abundant in haploid round spermatids, whereas the beta-subunit mRNA was not detected in germ cells. Within the epididymis both alpha- and beta-subunit mRNA concentrations were highest in the corpus, with 1.5-fold and 9-fold initial segment values, respectively. Light microscopic immunocytochemistry revealed that beta-hexosaminidase was localized to Sertoli cells and interstitial macrophages in the testis. In the epididymis, beta-hexosaminidase staining was most intense in narrow cells in the initial segment, principal cells in the caput and proximal corpus, and clear cells throughout the duct. Electron microscopic immunocytochemistry revealed that beta-hexosaminidase was predominantly present in lysosomes in Sertoli and epididymal cells. The cellular and regional specificity of beta-hexosaminidase immunolocalization suggest an important role for the enzyme in testicular and epididymal functions.

Apical and narrow cells of the initial segment and intermediate zone of the adult rat epididymis were glutaraldehyde fixed and Epon embedded for routine light (LM) and electron (EM) microscopic analysis and Bouin fixed and paraffin embedded for LM immunocytochemical analysis in order to examine their structural features, distribution, and functions. The goblet-shaped apical cells comprised 10.7 +/- 1.0% of the total epithelial population in the proximal initial segment but only 1.3 +/- 0.5% in the intermediate zone. In the EM, these cells presented numerous mitochondria, few C-shaped vesicles, and a pale round or oblong nucleus located in the upper half of their cytoplasm. The slender elongated narrow cells increased from 2.8 +/- 0.3% in the proximal initial segment to 6.3 +/- 0.4% in the intermediate zone. In an EM analysis, these cells presented numerous C-shaped vesicles and mitochondria and a small flattened nucleus located in the upper half of their cytoplasm. The structural features of both these cell types differed not only from each other but also from the neighboring principal and basal cells of each region. Of the various antibodies examined to lysosomal proteins, narrow and apical cells expressed high levels of cathepsin D, while beta-hexosaminidase A was expressed at high levels in narrow cells but only moderately in apical cells. Apical cells were intensely reactive for the Yf subunit of glutathione S-transferase (GST)-P, whereas no reaction was seen in narrow cells; the Yo subunit of GST was localized within both cell types but only in the proximal initial segment. Narrow cells exclusively expressed carbonic anhydrase II. Selective differences in the immunolocalization of these various proteins were also noted between these two cell types and principal and basal cells. The localization of cathepsin D and beta-hexosaminidase A within narrow and apical cells suggests these cells may be involved in the degradation of specific proteins within their lysosomes, whereas the presence of GSTs may aid in protecting spermatozoa from a changing environment of harmful electrophiles. Localization of carbonic anhydrase II exclusively within narrow cells suggests that these cells may modify the pH of the lumen resulting in the quiescence of sperm motility in the proximal end of the epididymis. Together, the data indicate that apical and narrow cells differ not only from each other but also from principal and basal cells in their structure and relative distribution. They also express different proteins within the distinct epididymal regions, indicating that they perform different functions.

Background: Glutathione S-transferases (GSTs) are a family of isozymes that catalyze the conjugation of glutathione to various electrophilic compounds. Recently, the Yf subunit of GST-P, found in high concentrations in the epididymis, has been immunolocalized within epithelial principal and basal cells of this tissue. In order to determine which factor(s) were involved in the regulation of expression of this protein, adult rats were orchidectomized and treated with or without testosterone implants, ligated or hypophysectomized., Methods: The epididymides were fixed by perfusion with Bouin's fixative and examined with an anti-Yf antibody using light microscope immunocytochemistry., Results: In normal untreated animals, principal cells were reactive in the distal initial segment, intermediate zone and caput epididymidis, but unreactive in all other epididymal regions. Ligation of the efferent ducts up to day 14 had no effect on expression of the Yf protein in principal cells of any region. The staining pattern was comparable to that observed in normal untreated animals suggesting that luminally derived testicular factors were not involved in regulation of Yf expression. Following orchidectomy, principal cells became unreactive by day 14 in all epididymal regions. However, administration of testosterone to orchidectomized animals prevented the loss of reactivity seen in principal cells of the distal initial segment, intermediate zone, and caput epididymidis, suggesting that expression of the Yf protein in principal cells of these regions is regulated by circulating testosterone. In contrast, basal cells after each experimental treatment including hypophysectomy maintained the exact staining pattern observed in normal untreated animals, i.e., they were intensely reactive in the proximal initial segment, distal caput, corpus, and proximal cauda regions, suggesting that expression of the Yf protein in these cells does not require factors derived from the testis or pituitary gland. However, a change in the shape and arrangement of basal cells was noted following each treatment. These cells transformed from flattened hemispherical cells showing thin elongated lateral processes to large bulbous, dome-shape cells closely packed together and showing only a few short lateral processes., Conclusions: In basal cells, expression of the Yf protein is not regulated by testicular or pituitary factors, whereas expression in principal cells is regulated by circulating testosterone. Due to the decrease in size of the epididymal tubules after each treatment, it is postulated that the shape and arrangement of basal cells in normal untreated animals are governed in part by the volume and pressure exerted upon the tubules by luminal fluids and spermatozoa derived from the testis.

RNA-binding proteins that bind to the 3' untranslated region of mRNAs play important roles in regulating gene expression. Here we examine the association between the 70 kDa poly (A) binding protein (PABP) and stored (RNP) and polysomal mRNAs during mammalian male germ cell development. PABP mRNA levels increase as germ cells enter meiosis, reaching a maximum in the early postmeiotic stages, and decreasing to a nearly nondetectable level towards the end of spermatogenesis. Most of the PABP mRNA is found in the nonpolysomal fractions of postmitochondrial extracts, suggesting that PABP mRNA is either inefficiently translated or stored as RNPs during spermatogenesis. Virtually all of the testicular PABP is bound to either polysomal or nonpolysomal mRNAs, with little, if any, free PABP detectable. Analysis of several specific mRNAs reveals PABP is bound to both stored (RNP) and translated forms of the mRNAs. Western blot analysis and immunocytochemistry indicate PABP is widespread in the mammalian testis, with maximal amounts detected in postmeiotic round spermatids. The presence of PABP in elongating spermatids, a cell type in which PABP mRNA is nearly absent, suggests that PABP is a stable protein in the later stages of male germ cell development. The high level of testicular PABP in round spermatids and in mRNPs suggests a role for PABP in the storage as well as in the subsequent translation of developmentally regulated mRNAs in the mammalian testis.

Glutathione S-transferases (GSTs) are dimeric proteins that come from a multigene family. They can be grouped into five classes (alpha, mu, pi, sigma, theta) based on the degree of amino acid homology of their subunits. These GST isozymes are able to catalyze the conjugation of glutathione with a wide variety of electrophiles, thereby protecting important cellular constituents from electrophilic attack. In the present study, the distribution of the Ya and Yc subunits from the alpha family, as well as the Yb1 and Yb2 subunits from the mu gene family was examined using immunocytochemistry in the adult rat testis and epididymis. The results of these four GST subunits were also compared with two other subunits, the Yf and Yo proteins, which have already been investigated in our laboratory [Veri et al. (1993), J. Androl., 14:23-44; Veri et al. (in press), J. Androl.]. In the testis, Leydig cells were intensely stained for all six subunits. Within the seminiferous epithelium, Sertoli cells were reactive only for antibodies raised against the Ya, Yb1 and Yf subunits. Among germ cells, all spermatogonia, spermatocytes and step 1-15 spermatids were virtually unreactive for each of the six GSTs. However, moderate to intense staining was seen over steps 16-19 spermatids with the anti-Yo and anti-Ya antibodies, and intense staining over step 19 spermatids with the anti-Yb1 and anti-Yb2 antibodies. In the rete testis, Yf, Yo, Yb1, and Yb2 subunits were intensely reactive over the epithelial cells with weak staining for Yc and no staining for Ya antibodies. Interestingly, in the efferent ducts the Yc, Yb1, and Yf proteins were intensely reactive over ciliated cells, whereas only the Yc protein was intensely reactive over nonciliated cells. In the epididymis, immunoreactivity varied among the principal and basal cells of a given epididymal region for each GST antibody. In the case of principal cells, several of the GSTs showed a similar immunostaining pattern along the tubule. Although not identical in intensity of reaction, the Yc, Yb1, Ya and Yo proteins showed an increase in staining intensity from the proximal to distal segments of the epididymis. In contrast, the Yb2 protein was intensely expressed only in the distal caput with weak levels throughout the rest of the epididymis. The Yf reactivity was strongest from the distal initial segment to the distal caput and unreactive in the corpus and proximal cauda epididymidis.(ABSTRACT TRUNCATED AT 400 WORDS)

The epididymal junctional complex between adjacent principal cells is composed of apically located gap, adherens and tight junctions. Tight junctions between adjacent epithelial cells lead to the formation of the blood-epididymal barrier. The objectives of this study were to examine the structure of the epididymal junctional complex in the different regions of the epididymis and to review the regulation of epithelial cadherin in the rat epididymis. Changes in the structure of the junctional complex, at the level of the electron microscope, were evident when comparing the initial segment to other regions of the epididymis. In the initial segment, the tight junction spanned a considerable length of the apical plasma membrane but had few desmosomes. In the other regions of the epididymis, the span of merging plasma membranes was considerably reduced, but in these regions, numerous desmosomes were present in the apical region. Several examples of what appeared to be a loss of portions of the plasma membrane of adjacent principal cells were evident along the entire epididymis. Such images as the invagination of a portion of the lateral plasma membrane of one principal cell into another, constriction of the invaginated area and eventual detachment leading to the formation of annular junctions suggest that there is a turnover of plasma membranes. The formation of cellular junctions involves the interactions of cell adhesion proteins followed by the addition of junctional proteins which assemble into tight and gap junctions. Epithelial cadherin (E-Cad), a calcium-dependent cell adhesion protein, was localized to the principal cells of the epididymis. Immunocytochemistry at the level of the electron microscope showed that E-Cad was present between the lateral plasma membranes of adjacent principal cells, both in the region of the junctional complex and in the deeper lying areas. E-Cad was also present in annular junctions located in close proximity to the junctional complex, indicating that these structures were related to the plasma membrane. E-Cad mRNA levels are regulated during postnatal epididymal development. In the caput-corpus epididymidis, E-Cad mRNA concentrations increase to peak at 42 days of age. This is well correlated with the conversion of testosterone to dihydrotestosterone in the epididymis. In the cauda epididymidis, however, E-Cad mRNA concentrations do not increase as a function of age, indicating that this protein is regulated in a segment-specific manner.(ABSTRACT TRUNCATED AT 400 WORDS)

The objective of this study was to determine the cellular and subcellular distribution of small nuclear ribonucleoprotein particles (snRNPs) in the adult rat testis in relation to the different cell types at the various stages of the cycle of the seminiferous epithelium. The distribution of snRNPs in the nucleus and cytoplasm of germ cells was quantitated in an attempt to correlate RNA processing with morphological and functional changes occurring during the development of these cells. Light-microscopic immunoperoxidase staining of rat testes with polyclonal anti-Sm and monoclonal anti-Y12 antibodies localized spliceosome snRNPs in the nuclei and cytoplasm of germ cells up to step 10 spermatids. Nuclear staining was intense in Sertoli cells, spermatogonia, spermatocytes, and in the early steps of round spermatid development. Although comparatively weaker, cytoplasmic staining for snRNPs was strongest in mid and late pachytene spermatocytes and early round spermatids. Quantitative electron-microscopic immunogold labeling of Lowicryl embedded testicular sections confirmed the light-microscopic observations but additionally showed that the snRNP content peaked in the cytoplasm of midpachytene spermatocytes and in the nuclei of late pachytene spermatocytes. The immunogold label tended to aggregate into distinct loci over the nuclear chromatin. The chromatoid body of spermatids and spermatocytes and the finely granular material in the interstices of mitochondrial aggregates of spermatocytes were found to be additional sites of snRNP localization and were intensely labeled. This colocalization suggests that these dense cytoplasmic structures may be functionally related. Anti-U1 snRNP antibodies applied to frozen sections showed the same LM localization pattern as spliceosome snRNPs. Anti-U3 snRNP antibodies applied to frozen sections stained nucleoli of germ cells where pre-rRNA is spliced.

Background: Glutathione S-transferases (GSTs) are a family of isozymes that catalyze the conjugation of glutathione with various toxic electrophilic compounds. GSTs are composed of several classes based on the degree of sequence homology of their subunits. The Yo subunit, a member of the mu class, is expressed at high levels in the testis and epididymis. The purpose of this study was to immunolocalize the GST-Yo in these tissues during development., Methods: The testes and epididymides of rats aged 7, 15, 21, 28, 39, 42, 45, 49, and 56 days were fixed in Bouin's fixative, and immunostained for light microscopic analysis., Results: In the testis the cytoplasm of all germ cells was unreactive until day 39. At that time, step 18 spermatids appeared moderately reactive, while the few observed step 19 spermatids were intensely reactive as were their residual bodies. The presence of residual bodies indicates that spermiation takes place as early as day 39; however, the number of step 19 spermatids is low at this age. A progressive increase in the size of the tubule and number of elongating spermatids was seen between days 42 and 49. In addition, by day 49, a weak staining was observed in steps 12-15, moderate in steps 16-17, and intense in steps 18-19 spermatids. In terms of the intensity of staining, cell types stained, size of the tubules, and number of elongating spermatids, no difference was noted between day 49, 56, and adult animals. Thus Yo protein expression in germ cells reached maturity by day 49. The epithelial cells of the rete testis were intensely reactive at day 7 and remained so throughout development. In contrast, while the epithelial cells of the efferent ducts at day 7 were intensely reactive, they were weakly reactive by day 39 and remained so at later ages. Along the entire epididymis, the columnar epithelial cells showed a moderate apical/supranuclear reaction from day 7 to 28. By day 39 principal cells of the initial segment became weakly reactive, while those in the caput and corpus were moderately stained, a situation seen at later ages including adults. Only by day 49 did principal cells of the proximal cauda become moderately stained as seen in adult animals. Thus the expression of the Yo protein in the principal cells of the proximal cauda may be regulated by different factors than those of the caput and corpus epididymidis. Alternatively, the expression of the Yo subunit in principal cells of the proximal cauda may develop later since this region would be the last to receive luminally derived testicular products. In the initial segment, the decrease in staining of principal cells at day 39 may be due to an inhibiting factor emanating from the testis. Spermatozoa appeared in the lumen of each epididymal region well after the expression of Yo had reached its adult staining pattern indicating that they are not a factor., Conclusions: Overall these results suggest that the expression of GST-Yo in the various cells of the testis and epididymis are controlled by different factors during postnatal development.

Glutathione S-transferases (GSTs) are a family of isozymes that catalyze the conjugation of glutathione (GSH), a tripeptide found in all mammalian cells; this function plays a protective role, as the addition of GSH to an electrophile generally forms a less toxic product. The pi class of GSTs contains homodimers of the Yf subunit, also known as Yp or rat subunit 7; this subunit is found in high concentrations in the testis and epididymis. The objective of the present study was to localize immunocytochemically the Yf subunit in the testis and in the various regions of the epididymis using light, electron, and confocal microscopy. In the testis, immunoperoxidase staining was localized exclusively to Sertoli and Leydig cells. The low cuboidal epithelial cells of the rete testis and the sparse ciliated cells of the ductuli efferents were also immunoreactive. A distinct pattern of immunostaining for the Yf subunit was observed in the different regions of the epididymis. The proximal area of the initial segment showed intense reactivity localized to epithelial basal cells. Basal cells in the middle area of the initial segment were also reactive, as were a second unidentified population of cells located in the apical region of the epithelium. The epithelium, including both principal and basal cells, in the distal initial segment, intermediate zone, and proximal caput epididymidis showed a weak, moderate, or strong degree of reactivity, respectively. In the distal caput epididymidis, however, principal cells showed a checkerboard-like pattern of immunoreactivity, with some cells being intensely stained or faintly stained, whereas others were unreactive. Strikingly, in the corpus and proximal cauda epididymidis, intense immunostaining was localized exclusively over the epithelial basal cells. As viewed in the light and confocal microscope, the intensely stained basal cells showed extensive processes that covered most of the base of the epididymal tubule. Upon quantitation of the immunogold labeling density (the number of gold particles/microns2) in principal and basal cells of the different regions of the epididymis, we observed a sharp decline in immunogold labeling of principal cells coupled with a dramatic increase in labeling of basal cells as we progressed along the tissue, particularly in the transition from the caput to the corpus epididymidis. This study constitutes the first demonstration of a protein that is selectively expressed in epithelial basal cells of the corpus and proximal cauda epididymidis.(ABSTRACT TRUNCATED AT 400 WORDS)

Recently, a cDNA that coded for an enteric smooth muscle gamma-actin (SMGA) that was expressed in post-meiotic mouse testicular cells was identified. To determine the cellular location(s) of the protein encoded by this cDNA, this SMGA was probed for by immunocytochemistry in the cells of the seminiferous epithelium with two different monoclonal antibodies (Mabs), B4 and HUC 1-1, known to be muscle actin selective. As a control, we also examined the immunoreactivity of a third Mab, C4, that reacts with all non-muscle and muscle vertebrate isoactins. Using light and electron microscopy, a progressive increase in immunolabeling was observed with the muscle selective HUC 1-1 Mab over a loose actin filamentous network distributed throughout the cytoplasm of steps 4-16 spermatids. Thereafter, the labeling decreased such that at step 17 spermatids, only cytoplasmic labeling in the tail of the spermatids was observed. No labeling of this network was noted with the C4 or B4 Mabs. However, myoid cells enveloping seminiferous tubules and smooth muscle cells of interstitial blood vessels demonstrated comparable intense labeling with each of the three Mabs. The C4 Mab intensely labeled actin filaments of the Sertoli-Sertoli and Sertoli-spermatid ectoplasmic specializations. Also well labeled were numerous actin filaments found in the apical Sertoli cell processes encapsulating the heads of late step 19 spermatids at stage VII of the cycle of the seminiferous epithelium. In addition, actin filamentous bundles enveloping tubulobulbar complexes of the late spermatids within the Sertoli cell apical processes were intensely labeled. The actin filaments in the Sertoli apical processes and surrounding the tubulobulbar complexes were also strongly immunolabeled with the HUC 1-1 Mab. The C4 Mab but not the B4 or HUC 1-1 Mabs, recognized actin in the subacrosomal space of steps 4-18 spermatids. This study suggests that there are muscle isoforms of actin within the cytoplasm of developing spermatids and within apical processes of Sertoli cells.

The initial segment of the epididymis of rats, fixed with glutaraldehyde, was postfixed with reduced osmium, a technique that clearly delineates the membranes of cisternae of the endoplasmic reticulum (ER) and the various elements of the Golgi apparatus, or with tannic acid to enhance the coats of vesicles and ribosomes on ER cisternae. The material was also treated to demonstrate various phosphatase activities (NADPase, TPPase, CMPase, G-6-Pase) or impregnated with osmium tetroxide. In osmium-impregnated material, the Golgi apparatus of the epithelial principal cells of the initial segment appeared in the light microscope as a branching, anastomosing ribbon forming a large network in the supranuclear region. In the electron microscope, ER were of two types: the heavily granulated, flattened, rough ER seen in the infranuclear and juxtanuclear regions and the distended, tubular, sparsely granulated ER, showing only few ribosomes, seen interlaced with the Golgi ribbon in the supranuclear region and at the apical pole of the cell. Of particular interest in this cell was the fact that the sparsely granulated ER approximated the Golgi stack on both its cis- and trans-faces. On the cis-face of the Golgi stack, the sparsely granulated ER cisternae showed the usual finger- or bud-like protrusions directed toward the cis element of the Golgi stack and around which numerous small 80 nm vesicles or membranous tubules were clustered. The Golgi stack consisted of the following elements in a cis-trans axis: the cis osmiophilic element, a first saccule slightly dilated, saccules two to four (S2-S4), which were NADPase-positive, and saccules five to seven and the eight Golgi element, which were TPPase-positive. On the trans-aspect of the Golgi stacks, several (up to four) CMPase-positive trans-Golgi networks were observed often in close apposition to the sparsely granulated ER cisternae. One of the trans-Golgi networks showed a "peeling-off" configuration, i.e. part of it was closely apposed to the overlying Golgi element of the stack, whereas the remaining part was separated from the stack by a space occupied by a cisterna of sparsely granulated ER. The other trans-Golgi networks were completely separated from the stack and were often seen sandwiched between sparsely granulated ER cisternae. Thus, ER cisternae showed extensive areas of close apposition but no continuity with the trans-Golgi networks. Although the saccules of the Golgi stacks showed NADPase and/or TPPase activity, the trans-Golgi networks displayed CMPase activity, thus facilitating their identification from the closely associated unreactive sparsely granulated ER cisternae.(ABSTRACT TRUNCATED AT 400 WORDS)

The present view is that glucocorticoid hormones bind to their cytoplasmic receptors before reaching their nuclear target sites, which include specific DNA sequences. Although it is believed that cytoplasmic sequestration of steroid receptors and other transcription factors (such as NFKB) may regulate the overall activity of these factors, there is little information on the exact subcellular sites of steroid receptors or even of any other transcription factors. Tritiated (3H)-dexamethasone 21-mesylate (DM) is an affinity label that binds covalently to the glucocorticoid receptor (GR), thereby allowing morphological localization of the receptor at the light and electron microscope levels as well as for quantitative radioautographic (RAG) analysis. After injection of 3H-DM into the testis, a specific radioautographic signal was observed in Leydig cells, which correlated with a high level of immunocytochemically demonstrable GR in these cells at the light-microscope level. To localize the 3H-DM binding sites at the electron microscope (EM) level, the testes of 5 experimental and 3 control adrenalectomized rats were injected directly with 20 microCi 3H-DM; control rats received simultaneously a 25-fold excess of unlabeled dexamethasone; 15 min later, rats were fixed with glutaraldehyde and the tissue was processed for EM RAG analysis combined with quantitative morphometry. The radioautographs showed that the cytosol, nucleus, smooth endoplasmic reticulum (sER), and mitochondria were labeled. Since the cytosol was always adjacent to tubules of the sER, the term sER-rich cytosol was used to represent label over sER networks, which may also represent cytosol labeling due to the limited resolution of the radioautographic technique. Labeling was highest in sER-rich cytosol and mitochondria, at 53% and 31% of the total, respectively. Cytosol (exclusively of all organelles) and nucleus showed comparatively weak labeling, at 9% and 7%, respectively. This study thus clearly establishes with the electron microscope, the localization of glucocorticoid binding sites using DM. It remains to be determined whether or not these DM binding sites represent bona fide glucocorticoid receptors or nonreceptor proteins that bind DM. Whereas the functional significance of the subcellular distribution of DM is not known, the labeling of the cytosol may represent localization of the steroid and GR in their traditional compartment. The steroid antagonistic properties of DM may have prevented the DM-GR complexes from translocating to the nucleus. However, the significant labeling of the sER-rich cytosol and mitochondria was unexpected and raises intriguing questions that are being addressed in current studies.

Cytochemical studies with over 40 different mammalian cell types have indicated that NADPase activity is associated with the Golgi apparatus and/or lysosomes of all cells. In the majority of cases, NADPase is restricted to saccular elements comprising the medial region of the Golgi stack and an occasional lysosome. There is often weak NADPase activity in other Golgi compartments such as the trans Golgi saccules and/or elements of the trans Golgi network. In some cells, however, strong NADPase activity is found within these latter compartments, either exclusively in trans Golgi saccules or elements of the trans Golgi network, or in combination with medial Golgi saccules and each other including (1) medial Golgi saccules + trans Golgi saccules, (2) medial Golgi saccules + trans Golgi saccules + trans Golgi network, or (3) trans Golgi saccules + trans Golgi network. In some rare cases, no NADPase activity is detectable in either Golgi saccules or elements of the trans Golgi network, but it is observed in an occasional lysosome or throughout the lysosomal system of these cells. It is unclear at present if these variations in the distribution of NADPase across the Golgi apparatus, and between the Golgi apparatus and lysosomal system, are due to differences in targeting mechanisms or to the existence of "bottlenecks" in the natural flow of NADPase along the biosynthetic pathway toward lysosomes. While no clear pattern in the association of strong NADPase activity with lysosomes was apparent relative to the ultrastructural distribution of NADPase activity in Golgi saccules or elements of the trans Golgi network, the results of this investigation suggested that cells having NADPase localized predominantly toward the trans aspect of the Golgi apparatus (in trans Golgi saccules or elements of the trans Golgi network or both) have few NADPase-positive lysosomes. The only exception is hepatocytes which were classified as predominantly trans but had noticeable NADPase activity within medial Golgi saccules and elements of the trans Golgi network as well, and highly reactive lysosomes. Other cells showing highly reactive lysosomes including (1) Kupffer cells of liver and those forming the proximal convoluted tubules of the kidney, both of which also had strong NADPase activity within medial and trans Golgi saccules and elements of the trans Golgi network, (2) Leydig cells of the testis and interstitial cells of the ovary, which also showed strong NADPase activity within medial Golgi saccules, and (3) macrophages from lung, spleen and testis, and Sertoli cells from the testis all of which showed no Golgi associated NADPase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

The presence and distribution of glucocorticoid receptors in the rat testis were examined by using 2 approaches: in vivo quantitative radioautography and immunocytochemistry. Radioautographic localization was made possible through the availability of a glucocorticoid receptor affinity label, dexamethasone 21-mesylate, which binds covalently to the glucocorticoid receptor, thereby preventing dissociation of the steroid-receptor complex. Adrenalectomized adult rats were injected with a tritiated (3H) form of this steroid into the testis and the tissue was processed for light-microscope radioautography. Silver grains were observed primarily over the Leydig cells of the interstitial space and to a lesser extent, over the cellular layers which make up the seminiferous epithelium, with no one cell type showing preferential labeling. To determine the specificity of the labeling, a 25- or 50-fold excess of unlabeled dexamethasone was injected simultaneously with the same dose of (3H)-dexamethasone 21-mesylate. In these control experiments, a marked reduction in label intensity was noted over the Leydig as well as tubular cells. Endocytic macrophages of the interstitium were non-specifically labeled, indicating uptake of the ligand possibly by fluid-phase endocytosis. A quantitative analysis of the label confirmed the presence of statistically significant numbers of specific binding sites for glucocorticoids in both Leydig cells and the cellular layers of the seminiferous epithelium; 86% of the label was found over Leydig cells, and only 14% over the cells of the seminiferous epithelium. These binding data were confirmed by light-microscope immunocytochemistry using a monoclonal antibody to the glucocorticoid receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclophosphamide is a widely used anticancer and immunosuppressive drug that affects fertility in men. In a previous study, we found that chronic, daily treatment of male rats with low doses of cyclophosphamide had no apparent effect on the pituitary-gonadal axis, whereas it had time- and dose-dependent effects on male reproductive organ weights, the hematologic system, and on pregnancy outcome. To determine whether cyclophosphamide induces morphological changes within the male reproductive system, a detailed qualitative and quantitative evaluation of changes in the histology of the testis and epididymis was undertaken. Adult male Sprague-Dawley rats were gavage-fed for 1, 3, 6, and 9 wk with saline (control), 5.1 (low dose) or 6.8 (high dose) mg/kg/day of cyclophosphamide; the testes and epididymides were prepared for light and electron microscopy. At the light microscopic level, the orderly process of spermatogenesis in the seminiferous tubules was not affected at any time point with either dose of the drug. A number of time-dependent drug-induced changes in the histology of the epididymis, however, were apparent: 1) an increase in the relative number and a change in the distribution of halo cells in the caput epididymidis, 2) an increase in the number and size of clear cells in the caput and/or cauda epididymidis, and 3) an increase in the size of clear cells in both the caput and cauda epididymides; these changes were time dependent. At the electron microscopic level, there was a dose-dependent, two- to threefold increase in the number of spermatozoa with abnormal flagellar midpieces in the lumen of both the caput and cauda epididymides. Although the 9 plus 2 axonemal complex and the 9 outer dense fibers were present and appeared normal, the close approximation of these two structures was lost in these abnormal spermatozoa. Such abnormal flagellar midpieces were also found in the testes of control and treated rats. Electron microscopic examination of the testis revealed that both Sertoli and Leydig cells were normal in appearance. The type and timing of the effects of cyclophosphamide on the histology of the testis and epididymis suggest that the drug could be affecting germ cells by 1) inducing changes in the developing spermatozoa in the testis, some of which are seen microscopically in the epididymal lumen, and/or 2) affecting epididymal morphology and function.

The present study was performed to demonstrate the binding, mode of uptake, pathway and fate of iodinated human chorionic gonadotropin ([125I]hCG) by Leydig cells in vivo using electron microscope radioautography. Following a single injection of [125I]hCG into the interstitial space of the testis, the animals were fixed by perfusion with glutaraldehyde at 20 minutes, 1, 3, 6 and 24 hours. The electron microscope radioautographs demonstrated a prominent and qualitatively similar binding of the labeled hCG on the microvillar processes of the Leydig cells at 20 minutes, 1, 3, and 6 hours. The specificity of the [125I]hCG binding was determined by injecting a 100-fold excess of unlabeled hormone concurrently with the labeled hormone. Under these conditions, the surface, including the microvillar processes of Leydig cells, was virtually unlabeled, indicating that the binding was specific and receptor-mediated. In animals injected with labeled hCG and sacrificed 20 minutes later, silver grains were also seen overlying the limiting membrane of large, uncoated surface invaginations and large subsurface vacuoles with an electron-lucent content referred to as endosomes. A radioautographic reaction was also seen within multivesicular bodies with a pale stained matrix. At 1 hour, silver grains appeared over dense multivesicular bodies and occasionally over secondary lysosomes, in addition to the structures mentioned above, while at 3 and 6 hours, an increasing number of secondary lysosomes became labeled. At 24 hours, binding of [125I]hCG to the microvillar processes of Leydig cells persisted but was diminished, although a few endosomes, multivesicular bodies and secondary lysosomes still showed a radioautographic reaction. No membranous tubules that were seen in close proximity to, or in continuity with, endosomes and multivesicular bodies were observed to be labeled at any time interval. Likewise, an attempt to correlate silver grains with small coated or uncoated pits, the stacks of saccules of the Golgi apparatus and other Golgi-related elements including GERL, proved unsuccessful, since these structures were mostly unlabeled. These in vivo experiments thus demonstrate the specific binding of [125I]hCG to the plasma membrane of Leydig cells predominantly on their microvillar processes, and the subsequent internalization of the labeled hCG to secondary lysosomes. In addition, binding and internalization of hCG persisted for long periods of time.

The apical region of nonciliated cells of the ductuli efferentes of the rat contains tubular coated pits (TCP) connected to the apical plasma membrane, apical tubules (AT) which occasionally show a partial coat, and endosomes which are often continuous with one or more apical tubules. To investigate the formation and fate of TCP and AT, a quantitative analysis was performed on the labeling indices of these structures at various time intervals (0.5-120 min) after a single injection of a tracer, cationic ferritin (CF), into the lumen of the rete testis. The labeling indices of both TCP and AT exhibited similar cyclical patterns, first reaching a peak at 25 min, then dropping to a minimum at 35 min, then rising to a second peak at 60 min. Since TCP were well labeled at 30 sec while AT were not, the tracer must rapidly enter TCP and thence AT. However, since tracer was virtually absent from the lumen by 30 min, it was not possible to reconcile the second peak of labeling index of TCP and AT by this mechanism. In another experiment, rats were injected once as before, injected again at 30 min, and then sacrificed at 30 min following the second injection. The results from this experiment showed that the labeling index of TCP and AT did not drop but was similar to that of the 60-min peak after a single injection. The interpretation is that there was recycling of tracer, which had already migrated from TCP to AT to endosomes, back to the apical plasma membrane via apical tubules. Moreover, when rats were injected once, injected again at 30 min, and sacrificed 3 min following the second injection, the labeling index for TCP and AT was significantly higher (P less than .05) than at the 30-min time interval after a single injection, indicating that recycled apical tubules were functionally capable of binding further CF. Morphological observations on images of transition between TCP and AT and the fact that AT were often found connected to endosomes suggest that TCP detach from the cell surface to give rise to AT, which in turn fuse to form endosomes. The kinetic analysis demonstrates in quantitative terms that a portion of the AT, which fuses to form endosomes, recycles back to the apical plasma membrane and contributes to the formation of new TCP.

Upon release from the seminiferous epithelium, spermatoza show a small droplet of cytoplasm attached to the neck region. During transit of spermatozoa in the caput epididymidis, this cytoplasmic droplet migrates along the middle piece of the flagellum. In the corpus epididymidis, the droplet shows a lateral displacement, while in the cauda epididymidis it detaches from the spermatozoon. In the electron microscope, cytoplasmic droplets attached to spermatozoa were seen to contain numerous, short, straight or C-shaped, flattened membranous elements referred to as lamellae, small vesicles, and small particles (35-nm diameter) with a diffuse wall showing no apparent unit membrane. The lamellae were stacked closely on one another or arranged in a loose array. Structurally as well as cytochemically, with different cytochemical markers, the lamellae and vesicular elements failed to show any evidence of being components of the Golgi apparatus or elements of the endoplasmic reticulum. The lamellae, vesicular elements, and 35-nm particles were also seen free in the lumen of the corpus epididymidis but were especially prominent in the cauda epididymidis at a time when droplets were being released from spermatozoa. The lumen of the epididymis, as spermatozoa passed from the caput to the cauda epididymidis, was also noted to acquire progressively a flocculent background material. The epididymal epithelium is composed predominantly of principal and clear cells. The endocytic activity of clear cells was examined in rats at different time intervals after a single injection of cationic ferritin into the lumen of the cauda epididymidis. At 2 min the tracer was bound to the microvilli of these cells and was also observed within large coated and uncoated pits, subsurface coated vesicles, and numerous subsurface small uncoated vesicular membranous elements (150-200-nm diameter). At 5 min, in addition to the above structures, the tracer was present in endosomes, while at 15 and 30 min, pale and dense multivesicular bodies appeared labeled, respectively. At 1 and 2 hr, but more so at 6 hr large dense membrane-bound bodies identified cytochemically as secondary lysosomes became labeled. All of the above endocytic structures were also seen to contain the 35-nm particles, flattened or vesicular membranous profiles, and a fine flocculent background material reminiscent of those seen free in the lumen or found in cytoplasmic droplets attached to spermatozoa. (ABSTRACT TRUNCATED AT 400 WORDS)

Transitional cells line the intermediate region of rat seminiferous tubules situated between the rete testis and the seminiferous epithelium proper. These tall elongated cells orient themselves in a downstream direction and converge on one another distally in the lumen of the rete testis where they form a distinct papillalike structure through which a narrow patent lumen is apparent. In addition to widely dispersed Golgi apparatus and mitochondria, these cells contain an abundance of microtubules, cisternae of endoplasmic reticulum, and a distinct lobulated nucleus showing clumps of chromatin and a prominent nucleolus. The endocytic activity of these cells was examined by employing adsorptive (cationic ferritin, concanavalin A ferritin) and fluid-phase tracers (native ferritin, horseradish peroxidase-colloidal gold complex, and concanavalin A ferritin in presence of alpha methyl-D-mannoside). Such tracers were injected separately into the lumen of the rete testis, and the animals were killed at 2, 5, 15, and 30 min and 1, 2, and 6 hr after injection. At 2 min, both adsorptive and fluid-phase tracers were found within coated and uncoated pits of the apical plasma membrane of these cells as well as in large, subsurface, uncoated spherical, C-shaped, and tubular membranous elements. At 5 min the tracers were seen in endosomes of different sizes; while at 15 min and 30 min, pale and dense multivesicular bodies of small and large sizes, respectively, were labeled. At 1-hr and longer time intervals secondary lysosomes became labeled. While both fluid-phase and adsorptive tracers followed the same pathway and fate, binding to the apical and lateral plasma membranes of the transitional cells and to the membrane delimiting coated and uncoated pits was observed only with the adsorptive tracers. These results demonstrate that the transitional cells are actively involved in both fluid-phase and adsorptive endocytosis, which may play an important role in modifying the composition of the luminal fluid. The transitional cells of the distal zone of the intermediate region rest on an elaborate basement membrane (BM) complex which includes a thin BM immediately underlying these cells, a thick distal layer of BM, and strands of BM spanning the distance between the two in the form of a loose anastomotic network. Use of antisera against heparan sulfate proteoglycan, laminin, and type IV collagen revealed the presence of all three components within all areas of the BM complex. In the meshes of the anastomotic BM network, extracellular vesicles were observed.(ABSTRACT TRUNCATED AT 400 WORDS)

The existence of two classes of spermatogonial stem cells in the rat testis, i.e., reserve type A0 spermatogonia and renewing, types A1-A4 spermatogonia, postulated by Clermont and Bustos-Obregon ('68), was reexamined in a quantitative analysis of type A spermatogonia in both whole mounts of tubules and in radioautographed sections of testes from animals killed at various times, up to 26 days, after one or multiple injections of 3-H-thymidine. The cell counts obtained from whole mounts of tubules revealed that the number of isolated type A0 cells per unit area of limiting membrane remained constant throughout the cycle of the seminiferous epithelium. Paired type A0 spermatogonia also remained unchanged in number per unit area of basement membrane from stage I to stage VIII of the cycle. The low mitotic index of type A0 spermatogonia (0.1%) indicated that these cells were not actively involved in the production of spermatogonia or spermatocytes during each cycle of the seminiferous epithelium and thus were considered as reserve stem cells. The type A1 spermatogonia, which are formed during stage I of the cycle, remained resting until stage IX, when they undertook a series of four successive divisions resulting in the production of new type A1 and Intermediate-type spermatogonia. An analysis of the labeling indices of type A spermatogonia obtained from cell counts in radioautographed testicular sections after a single or multiple 3-H-thymidine injections indicated that the percentages of labeled type A cells corresponded to the percentages of type A1-A4 at each stage, whereas the percentages of unlabeled type A cells corresponded to the percentages of type A0 spermatogonia obtained from counts of cells in whole mounts. This confirmed that type A0 cells were generally non-proliferative throughout the cycle of the seminiferous epithelium while the type A1-A4 spermatogonia underwent complete renewal during each cycle. The present results thus support the concept of the existence of two classes of spermatogonial stem cells in rats.