Epithelial cells of the epididymis show regional variations with respect to the secretion of endocytosis of immobilin as revealed by light and electron microscope immunocytochemistry.

The localization of immobilin, a glycoprotein known to be present and to immobilize spermatozoa in the lumen of the epididymis, was investigated using light and electron microscope immunocytochemistry. In the light microscope, a distinct immunoperoxidase reaction product was observed in the lumen over the brush border of the epithelial nonciliated cells of the efferent ducts, while only a faint reaction was seen over their supranuclear region. In the proximal area of the initial segment of the epididymis no immunoperoxidase staining was observed either over epithelial cells or in the lumen. In the middle area of the initial segment, several epithelial principal cells became intensely immunostained but the majority were unstained; a weak reaction appeared in the lumen. In the distal area of the initial segment, more principal cells became immunostained, and while some were intensely reactive, others were moderately or weakly stained or unreactive. In the intermediate zone and proximal caput epididymidis, the principal cells showed the maximal immunoreactivity with all principal cells being reactive; staining in the lumen also reached its maximal reactivity in these areas. Immunostaining of principal cells gradually decreased along the epididymal duct and disappeared in the cauda epididymidis, however, an intense reaction persisted in the lumen. In the distal area of the cauda epididymidis, clear cells were reactive. In the electron microscope, immunogold labeling of reactive principal cells of the middle and distal areas of the initial segment, intermediate zone, and caput epididymidis was detected over cisternae of endoplasmic reticulum, stacks of Golgi saccules, and spherical electron lucent (200-400 nm in diameter) vesicles. The latter were present on the trans face of the Golgi stack, in the vicinity of th Golgi apparatus, and close to the apical cell surface; they are considered as secretory vesicles involved in the secretion of immobilin. In the distal area of the cauda epididymidis, epithelial clear cells showed an intense immunogold labeling over their endocytic apparatus. Immunogold labeling in the lumen of the epididymis was found over a fine flocculent material dispersed between the sperm. This material was especially abundant in the cauda epididymidis and did not appear to be bound to the surface of the sperm. The present results suggest that principal cells of the epididymis are involved in the secretion of immobilin, but that a differential secretory pattern exists between epididymal segments with maximal secretory activity occurring in the intermediate zone and proximal caput epididymidis, while no secretion takes place in the cauda epididymidis. Excess immobilin appears to be endocytosed for degradation by clear cells of the cauda epididymidis.     

Developmental expression of immobilin in the rat epididymis

Background: Immobilin is a protein secreted by principal cells of the distal initial segment, intermediate zone and caput epididymidis of adult rats, which serves to immobilize spermatozoa. In the distal cauda, epithelial clear cells are involved in its endocytosis. The objective of this study was to correlate the developmental events in the maturation of the epdidymis with the timing of immobilin secretion and endocytosis in order to evaluate the testicular or epididymal factors which may influence or regulate immobilin expression. Methods: Our approach was to follow and compare the developmental expression of immobilin by light microscope immunocytochemistry in control and efferent duct ligated rats of different postnatal ages. Results: Coincident with the morphological maturation of the principal cells by postnatal day 39, immobilin displayed the characteristic secretory immunostaining pattern found in adults. This adult-like expression occurred despite the absence of spermatozoa in the lumen but was coincident with high levels of circulating and luminal androgens. In contrast, immobilin secretion in rats whose efferent ducts were ligated at day 15 was weak to non-existent in the principal cells of the caput epididymidis at day 28 and remained so into adulthood, indicating that principal cells of this region of the epididymis are dependent either directly or indirectly upon testicular factors present in the lumen for immobilin expression. However, secretion of immobilin in the principal cells of the distal initial segment was unaffected by ligation and unlike the case in control rats high levels of immobilin also continued to be secreted into adulthood by the principal cells of the proximal initial segment. Thus in the distal initial segment immobilin secretion is not regulated by luminal factors originating from the testis, while in the proximal initial segment the normal suppression of immobilin that occurs by postnatal day 39 is. Despite ligation, endocytosis of immobilin by clear cells of the distal cauda epididymidis occurred by day 49, indicating that luminal testicular factors are not essential for stimulating the uptake of immobilin by these cells. Conclusions: The results taken together suggest that there are stimulatory and inhibitory luminal testicular factors involved in the regional development of immobilin secretion in the epididymis. There are also immobilin secreting regions in the epididymis, whose secretory development is independent of luminal testicular factors. © 1994 Wiley-Liss, Inc.     

Developmental expression of sulfated glycoprotein-2 in the epididymis of the rat

Background: Sulfated glycoprotein-2 (SGP-2), also designated as clusterin, is a protein secreted by the epididymis and which binds to spermatozoa. In adult rats it is secreted at high levels by principal cells of the distal initial segment, intermediate zone and caput epididymidis, and at relatively lower levels by principal cells of the corpus and cauda epididymidis. The objective of this study was to correlate the developmental events in the maturation of the epididymis with the timing of SGP-2 expression in order to evaluate the testicular or epididymal factors which may regulate it. Methods: Our approach was to follow and compare the developmental expression of SGP-2 by immunocytochemistry in normal untreated control rats and rats whose efferent ducts were ligated on day 15 and examined at different postnatal ages thereafter. Results: In control animals, SGP-2 expression in principal cells of the distal initial segment, intermediate zone, and caput and distal cauda epididymidis, as characterized in normal 90-day-old adult animals, was attained between postnatal days 39 and 49. However, only by postnatal day 56 did SGP-2 display in the corpus and proximal cauda the characteristic secretory pattern found in adult rats. In contrast, in efferent duct ligated rats examined at postnatal day 64, SGP-2 was absent in principal cells of the corpus and proximal cauda epididymidis but continued to be secreted by the distal initial segment, intermediate zone, and caput and distal cauda epididymidis. Furthermore, unlike the case in control rats, SGP-2 was secreted at high levels by the principal cells of the proximal initial segment. Thus during normal postnatal development, in the proximal initial segment, the production of SGP-2 is suppressed by luminal factors originating from the testis, while in the distal initial segment, intermediate zone, and caput epididymidis, it is unaffected by these factors. On the other hand, the production of SGP-2 in the corpus and proximal region of the cauda epididymidis is normally stimulated by luminal factors originating from the testis, while in the distal cauda, it is unaffected by these factors. Conclusions: Our results thus show a differential regulation of SGP-2 expression in principal cells of the proximal versus distal regions of the epididymis and even within subdivisions of each region. In some regions of the epididymis, SGP-2 production appears to be unaffected by luminal factors originating from the testis, while in other regions it is either inhibited or stimulated by these factors. © 1994 Wiley-Liss, Inc.     

Permeability of the diaphragmatic mesothelium: The ultrastructural basis for “stomata”

The ultrastructure of the hamster efferent ducts and epididymis was studied and the results were correlated with previously published data on the composition of luminal fluid obtained by micropuncture. Samples of the efferent ducts and parts of the epididymis designated initial segment, caput, corpus, proximal cauda, distal cauda, and “epididymal vas” were prepared. The efferent ducts contained principal cells characterized by a profusion of apical vesicles and numerous very large vacuoles that were distributed throughout the cytoplasm. Ciliated cells had few vesicles and vacuoles. Occasional cells contained many particles resembling glycogen. In the epididymis, the following trends were observed. The height of the epithelium and the size of the principal cells declined from initial segment to distal cauda. Apical vesicles and vacuoles with a light content were extremely numerous in principal cells of the initial segment and decreased progressively in the more distal regions. In the initial segment, basal and perinuclear rough endoplasmic reticulum was abundant and was distended with a material that resembled newly synthesized protein. Further distally in the epididymis cisternae of the rough endoplasmic reticulum were narrow and contained little intracisternal material. Light cells containing many vesicles, vacuoles, and lysosome-like structures were very prominent in the caudal segments. The epithelium of the epididymal vas had features intermediate between cauda epididymidis and ductus deferens. The cytoplasmic droplet in luminal sperm began to migrate caudally between the caput and corpus epididymidis and reached the posterior extremity of the middle piece in the distal cauda. Some degenerating sperm were observed in the lumen of the distal segments of the epididymis. The abundance of cytoplasmic vesicles and vacuoles in principal cells of the efferent ducts and initial segment of the epididymis correlated with the site of greatest fluid absorption as determined by micropuncture studies, suggesting that these structures are involved in absorption of fluid from the lumen. Between the caput and distal cauda epididymal segments, where absorption of sodium and potassium but not of fluid occurred, there were few vesicles and vacuoles in principal cells, but the “light” cells were large and numerous and contained many vacuoles. The principal cells of the initial segment were best equipped with rough endoplasmic reticulum to synthesize a protein.     

Developmental expression of the Yf subunit of glutathione S-transferase P in epithelial cells of the testis,efferent ducts,and epididymis of the rat

Background: Glutathione S-transferases (GSTs) are a family of isozymes that catalyze the conjugation of the tripeptide, glutathione, to various electrophilic compounds. The major GST in the pi class is GST-P, a homodimer of the Yf subunit, also known as Yp or rat subunit 7. This subunit is found in high concentrations in the epididymis and has recently been immunolocalized within epithelial principal and basal cells of the epididymis. Methods: In the present study we examine in groups of animals fixed in Bouin's fixative for light microscopy and in 4% paraformaldehyde and 0.5% glutaraldehyde in phosphate buffer for electron microscopy, the pattern of immunostaining for the Yf subunit of GST-P in the testis, efferent ducts and epididymis at various ages after birth. Results: In the epididymis, on postnatal days 7 and 15, an immunoperoxidase reaction was localized exclusively to the apical and supranuclear regions of the undifferentiated columnar epithelial cells of the entire epididymis. By day 21, a dramatic change had taken place. In the initial segment, intermediate zone and proximal caput epididymidis, the columnar cells showed a distinct checkerboard-like staining pattern with cells ranging from being intensely reactive to unreactive. In contrast, principal cells of the distal caput, corpus, and proximal cauda epididymidis were weakly reactive. By day 28 the ratio of reactive to unreactive cells in the initial segment, intermediate zone, and proximal caput epididymidis was higher. By day 39, the differentiated columnar epithelial cells, referred to as principal cells, took on their adult staining pattern in the proximal and middle areas of the initial segment as well as the corpus and proximal cauda epididymidis where they were slightly reactive; in the distal initial segment they were strongly reactive. At day 49, principal cells in the intermediate zone and proximal caput became intensely reactive, while showing a distinct checkerboard-like staining pattern in the distal caput; similar observations were made for tissues taken from 56 and 90-day-old animals. Basal cells also showed a variable staining pattern in the different epididymal regions as a function of age. At day 21, when they first appeared, they were unreactive except for an occasional reactive cell in the corpus region. At day 28, only in the corpus epididymidis were many basal cells seen to be reactive. By day 39 the more numerous basal cells of the corpus and proximal cauda epididymidis were intensely reactive and remained so into adulthood. In these regions, basal cells appeared as dome-shaped cells (days 21, 28, 39), but then gradually flattened out and exhibited processes (days, 49, 56, adults) which collectively appeared to envelop the base of each tubule in a mesh-like network. The change in basal cell shape in each region coincided with the arrival of fluid and spermatozoa into the lumen (corpus day 49, proximal cauda day 56). In other epididymal regions, basal cells at day 28 were mostly unreactive. However, there was a gradual increase in the number of reactive basal cells of these regions between day 39 and 56. Conculusions: The present results thus demonstrate a dramatic change in the immunostaining pattern for the y f subunit of GAS-p during postanatal development for both principal and basal cells along the epididymis. Such results suggest that different factors play a role in the regulation of the expression of the y f protein, not only in different epididymal regions, but also in different cell types during postanatal development. © 1994 Wiley-Liss, Inc.     

Morphology of the epithelium of the extratesticular rete testis,ductuli efferentes and ductus epididymidis of the adult male rabbit

The fine structure of the epithelium lining the extratesticular rete testis, ductuli efferentes and ductus epididymidis of the rabbit has been investigated. In the ductuli efferentes the epithelium is composed of two cell types, principal cells and ciliated cells. The latter cell type is distinguished from principal cells by the presence of cilia projecting into the lumen and the position of the nucleus in the apical half of the cell. Principal cells in this segment are characterized by micropinocytotic vesicles on the surface plasma membrane and a variety of small dense bodies scattered throughout the cytoplasm. In the ductus epididymidis basal cells replace ciliated cells as the second cell type, but differences between various segments of the epididymis are related to the fine structure of the principal cells. In the proximal caput epididymidis (Nicander's region 1) the principal cells are tall with long microvilli. They typically contain a small Golgi apparatus and a cluster of dense bodies adjacent to the nucleus. In the distal caput epididymidis (Nicander's regions 2–5) the apical cytoplasm of principal cells is filled with numerous micropinocytotic vesicles and large multivesicular bodies; these features are interpreted as signs of absorptive activity. The multivesicular bodies are absent from the cytoplasm of principal cells in the corpus epididymidis (Nicander's region 6) and, instead, numerous elements of smooth endoplasmic reticulum, a large Golgi apparatus, lipid droplets and dense bodies characterize principal cells in this segment. Towards the proximal cauda epididymidis (Nicander's region 7), the number of dense bodies (lysosomes) in the cytoplasm increases considerably. In the globose cauda (Nicander's region 8), the principal cells are reduced in height, and in addition to the features described in region 7, are characterized by a concentric array of rough endoplasmic reticulum in the basal cytoplasm. These observations are discussed in relation to the role of the epididymis in promoting the maturation and survival of spermatozoa.     

The differential absorptive activity of epithelial cells of the rat epididymus before and after castration

Horseradish peroxidase introduced into the lumen of the rat epididymis was taken up by the columnar cells of the epithelium by five minutes and more so after longer periods. The apical cells and particularly the clear cells in the caput and cauda epididymidis, respectively, showed significantly greater endocytotic activity than the principal cell in both locations. Within 14 days after castration, however, such differences in absorptive activity among the various cell types were essentially obscured because of increased endocytosis by the androgen-deficient principal cells. The results are discussed briefly in terms of the function of different epithelial cell types and secretory/absorptive activity in the epididymis.     

Immunohistochemical localization of 54,000 dalton sialoglycoprotein in the epididymal duct of the germ cell-free W/Wv mouse

A monoclonal antibody T21 specifically recognizes the mouse epididymal sialoglycoprotein of 54,000 dalton (SGP54). The localization of SGP54 was studied in the epididymal duct of germ cell-free WBB6F1W/Wv mutant mice (W/Wv mice) by avidin biotin complex (ABC) immunohistochemistry using T21. None of the testis cells showed immunoreaction. No spermatozoa were present in the epididymal duct lumen. The duct luminal fluid was stained weakly in the proximal corpus epididymidis, and strongly in the cauda epididymidis. Degenerated cells appeared in the duct lumen. The degenerated cells located at the corpus epididymidis showed strong immunostaining in the cytoplasmic region, while the degenerated cells located at the cauda epididymidis showed weak immunostaining. Immunoreaction was also detected between and on microvilli along the epididymis, the intensity being very strong at the distal caput and proximal corpus epididymidis. Invaginations and coated vesicles at the luminal surface of the principal cells were frequently immunostained at the corpus epididymidis. Giant inclusions frequently occurred in the principal cells of the distal caput and corpus epididymidis, with these being very intensely immunostained. These inclusions are ultrastructurally confirmed to be giant multivesicular bodies reported by ABE et al. (1984) in the mouse with the efferent duct cutting. These results suggest that the majority of excess SGP54 are absorbed by the principal cells at the distal caput to corpus epididymidis and catalyzed in the giant multivesicular bodies.     

Postnatal differentiation and development of the rat epididymis: A stereological study

Postnatal development and differentiation of the rat epididymis was studied in the rat from 15 to 120 days of life using stereological techniques. Both the relative volume (volume density) and absolute volume of the epithelial, interstitial, and luminal compartments in the initial segment, caput, corpus, and cauda epididymides were determined. In all segments the volume density of the epithelial compartment increased between days 15 and 30 before falling to adult values at 45 days in the initial segment (0.476 ± 0.031), at 60 days in the caput (0.258 ± 0.028) and at 90 days in the corpus (0.245 ± 0.007) and cauda (0.140 ± 0.004). The relative volume of the interstitium decreased, whilst that of the lumen increased over the same period with adult values being achieved earlier in the proximal segments than in the distal segments. In contrast to volume fraction the absolute volume of all compartments in all segments increased from day 15 to day 90. Between 90 and 120 days the absolute volumes of compartments in the initial segment and caput showed little volume change. All compartments in the corpus and cauda showed significant increases in volume over the same period. A similar pattern of development was observed with respect to the surface area of both the luminal and basement membrane aspects of the epithelium; surface area per unit volume (surface density) in all segments reached adult values at approximately 60 days, whilst the increase in absolute area of the surfaces ceased at 90 days in the initial segment and caput and continued to 120 days in the corpus and cauda. The total length of the epididymal tubule showed the same pattern with no increase in length apparent in the initial segment and caput after 90 days. Length however continued to increase in the corpus and cauda between 90 and 120 days. Tubule and luminal diameter reached their definitive values in the initial segment on day 60 but continued to increase until day 90 in the distal segments. Epithelial height increased between 15 and 30 days in all segments; in the caput, epithelial height was greatest on day 30 before decreasing slightly; and in the corpus and cauda maximal epithelial height was observed on day 45 decreasing to stable values on day 60 and 90, respectively. All the data is consistent with a proximal to distal differentiation and development of the epididymis with growth continuing in all segments after the establishment of the definitive architecture. Growth of the distal segments continued after its cessation in the proximal segments. © 1994 Wiley-Liss, Inc.     

Fine structure of the testis and epididymis of rats treated with cyproterone acetate

Adult male rats were administered the antiandrogen, cyproterone acetate, for 4, 8 or 12 weeks, and the histology and fine structure of the testis and several parts of the epididymis were studied. After treatment for 8 or 12 weeks, the testes of treated animals displayed a great reduction in the abundance of late spermatids. Necrotic cells, many of which were identified as cap-phase spermatids, were present in the seminiferous epithelium. Sertoli cells contained many large lipid droplets and lysosome-like structures with a content of cellular debris, including parts of spermatids. Leydig cells of treated rats were smaller than those of control animals at all the intervals studied. Sperm were absent from the lumen of the middle segment, or caput epididymidis, of severely affected specimens. In the terminal segment, or cauda epididymidis, the microscopic appearance varied in different regions. In the proximal part of the cauda epididymidis, the lumen was usually clear of sperm. The epithelium was tall and the light cells were very large and distended with many dense bodies resembling lysosomes. In contrast, in the distal part of the cauda epididymidis, the lumen was filled with sperm and debris, which appeared to be derived from germ cells. It is suggested that the light cells of the epididymal epithelium may have a role in clearing the lumen in the proximal part of the cauda epididymidis, in which they are particularly large and numerous. The results suggest that in the presence of cyproterone acetate, germ cells develop up to cap-phase spermatids and then begin to undergo degeneration and death. This alteration may have an important role in the antifertility effect of the drug, but changes in the epididymis may contribute also.     

Short-term effects of androgen withdrawal on the structure of different epithelial cells in the rat epididymis

Rat spermatozoa are highly dependent on the milieu of the normal epididymis for their maturation and survival, and die within a few days after androgenic support of the epididymal epithelium is withdrawn. The immediate changes in the ultrastructural organization of the epithelial cells of the rat epididymis, 2, 4, 6 and 14 days following castration have been monitored by morphometric analysis of localized regions of the caput and cauda epididymidis. While castration results in greater endocytosis by principal cells (Moore and Bedford, '79), many of their early structural changes following androgen withdrawal (disappearance of vesicles from the cell apex, reduction in rough endoplasmic reticulum, a drop in the volume of the Golgi cisternae and increase in lysosome content) seem indicative of inhibition of a secretory function. By contrast with the regressive response of the principal cell, the ultrastructure of clear cells in the cauda and of apical cells in the caput region appeared unchanged up to 14 days after castration. The implications of this evidence for specialized functions, and the suggestion of a differential androgen dependence among major cell types of the epididymal epithelium, are discussed briefly.     

Short-term effects of androgen withdrawal on the structure of different epithelial cells in the rat epididymis.

Rat spermatozoa are highly dependent on the milieu of the normal epididymis for their maturation and survival, and die within a few days after androgenic support of the epididymal epithelium is withdrawn. The immediate changes in the ultrastructural organization of the epithelial cells of the rat epididymis, 2, 4, 6 and 14 days following castration have been monitored by morphometric analysis of localized regions of the caput and cauda epididymidis. While castration results in greater endocytosis by principal cells (Moore and Bedford, '79), many of their early structural changes following androgen withdrawal (disappearance of vesicles from the cell apex, reduction in rough endoplasmic reticulum, a drop in the volume of the Golgi cisternae and increase in lysosome content) seem indicative of inhibition of a secretory function. By contrast with the regressive response of the principal cell, the ultrastructure of clear cells in the cauda and of apical cells in the caput region appeared unchanged up to 14 days after castration. The implications of this evidence for specialized functions, and the suggestion of a differential androgen dependence among major cell types of the epididymal epithelium, are discussed briefly.     

The influence of progestin and androgen on the fine structure of the male reproductive tract of the rat. II. Epididymis and sex accessory glands

Young adult male rats were administered medroxyprogesterone (Provera, Upjohn) alone and in combination with testosterone, as has been done to inhibit male fertility. The histology and fine structure of several segments of the epididymis, the ventral prostate, and the seminal vesicle were studied at intervals after treatment for up to 16 weeks. The epididymides of treated animals weighed less than those of control rats. Microscopic alterations in the epididymis were similar in rats treated with Provera alone and in those animals that received Provera and testosterone, but the changes varied with the segment of the epididymis. In the middle segment in the caput epididymidis, the normally abundant luminal sperm were absent but the epithelium retained its normal ultrastructural features. In the terminal segment in the cauda epididymidis, different changes were observed in the proximal and distal portions. In the proximal cauda epididymidis, the lumen was small, irregular in outline, and virtually devoid of sperm. The light cells of the epididymal epithelium in the proximal cauda contained extremely large numbers of dense bodies resembling lysosomes, which occupied most of the supranuclear and basal cytoplasm. In contrast, in the distal part of the cauda epididymidis, the epithelium had a normal appearance but the lumen was filled with debris, sperm, and spherical masses of cytoplasm that were apparently derived from germ cells. It is suggested that the clearing of the lumen of the proximal cauda epididymidis may reflect the greater activity of light cells of the epididymal epithelium in that region. Although alterations in spermatogenesis may be most important in the antifertility effect of progestin and androgen, these alterations in epididymal sperm and epithelium may also play a role. The weights of the prostate and seminal vesicles of rats treated with Provera (1 mg/100 g/day) were greatly reduced compared to those of control rats. Although there was considerable variation, in many specimens treated with Provera alone the epithelium of the prostate showed a change from a columnar to a cuboidal or squamous shape, and there was a reduction in the size and abundance of organelles involved in the formation of secretions. The microscopic structure of the seminal vesicle of rats treated with Provera was less severely affected than the prostate. Although the seminal vesicle epithelium of Provera-treated rats was generally not as tall as in control animals, the cells possessed parallel cisternae of rough endoplasmic reticulum, secretory vacuoles, and an active-appearing Golgi apparatus, suggesting that they continued to be able to form secretions in the presence of Provera. The weights of the sex accessory glands were maintained at control levels by the administration of testosterone, 100 μg/100 g/day, along with the Provera. A normal fine structure was present in the epithelium of both the prostate and seminal vesicle of rats administered this amount of testosterone in addition to Provera. Lower doses of testosterone (15 or 30 μg/100 g/day) were insufficient to maintain normal weight or ultrastructure of the sex accessory glands in the presence of Provera.     

Immunocytochemical localization of sulfated glycoprotein-1 (SGP-1) and identification of its transcripts in epithelial cells of the extratesticular duct system of the rat

The localization of sulfated glycoprotein-1 (SGP-1) in the extratesticular duct system was analyzed using an affinity purified antibody raised against the protein in conjunction with light (LM) and electron (EM) microscope immunocytochemistry. In the LM an intense immunoperoxidase reaction product was observed over the cytoplasm of Sertoli cells as well as over the tails of late spermatids. The rete epithelial cells and noncilliated cells of the efferent ducts also showed an intense uniform reaction over their entire cytoplasm. In the EM, immunogold labeling was noted over the entire endocytic apparatus of these cells including coated pits, endosomes, multivesicular bodies, and secondary lysosomes. Since there was no labeling of the luminal contents including sperm along the epididymis, it was concluded that the Sertoli-derived SGP-1 must dissociate from the sperm and be taken up by epithelial cells at the level of the rete testis and efferent ducts. In all regions of the epididymis, except the cauda, the principal cells showed, in the LM, an intense reaction over bodies of various shapes and sizes in their supranuclear region; this corresponded in the EM to a strong immunogold labeling of secondary lysosomes. No labeling was noted, however, over coated pits, endosomes, or pale multivesicular bodies, suggesting that SGP-1 was not being endocytosed from the lumen. Similar observations were noted for the epithelial clear cells along the entire epididymis. In the cauda epididymidis, principal cells presented a weak immunolabeling of their secondary lysosomes. Northern blot analysis revealed a strong 2.6 Kb band corresponding to the mRNA of SGP-1 in the efferent ducts and all regions of the epididymis with the exception of the cauda. Coincident with the mRNA expression of SGP-1 it was found that small clusters of gold particles representing anti SGP-1, presumably membrane bound, were associated with the Golgi apparatus as well as in close proximity to secondary lysosomes. There was, however, no evidence for the secretion of SGP-1 into the lumen. These results suggest that SGP-1 is synthesized by the epithelial cells of the male duct system and ferried by small vesicles derived from the Golgi apparatus to secondary lysosomes. Because SGP-1 has recently been shown to have substantial sequence similarity to prosaposin, it may be speculated that SGP-1 is instrumental in the degradation of membrane glycolipids present within secondary lysosomes of epithelial cells of the extratesticular duct system.     

Immunocytochemical localization of sulfated glycoprotein-1 (SGP-1) and identification of its transcripts in epithelial cells of the extratesticular duct system of the rat.

The localization of sulfated glycoprotein-1 (SGP-1) in the extratesticular duct system was analyzed using an affinity purified antibody raised against the protein in conjunction with light (LM) and electron (EM) microscope immunocytochemistry. In the LM an intense immunoperoxidase reaction product was observed over the cytoplasm of Sertoli cells as well as over the tails of late spermatids. The rete epithelial cells and nonciliated cells of the efferent ducts also showed an intense uniform reaction over their entire cytoplasm. In the EM, immunogold labeling was noted over the entire endocytic apparatus of these cells including coated pits, endosomes, multivesicular bodies, and secondary lysosomes. Since there was no labeling of the luminal contents including sperm along the epididymis, it was concluded that the Sertoli-derived SGP-1 must dissociate from the sperm and be taken up by epithelial cells at the level of the rete testis and efferent ducts. In all regions of the epididymis, except the cauda, the principal cells showed, in the LM, an intense reaction over bodies of various shapes and sizes in their supranuclear region; this corresponded in the EM to a strong immunogold labeling of secondary lysosomes. No labeling was noted, however, over coated pits, endosomes, or pale multivesicular bodies, suggesting that SGP-1 was not being endocytosed from the lumen. Similar observations were noted for the epithelial clear cells along the entire epididymis. In the cauda epididymidis, principal cells presented a weak immunolabeling of their secondary lysosomes. Northern blot analysis revealed a strong 2.6 Kb band corresponding to the mRNA of SGP-1 in the efferent ducts and all regions of the epididymis with the exception of the cauda. Coincident with the mRNA expression of SGP-1 it was found that small clusters of gold particles representing anti SGP-1, presumably membrane bound, were associated with the Golgi apparatus as well as in close proximity to secondary lysosomes. There was, however, no evidence for the secretion of SGP-1 into the lumen. These results suggest that SGP-1 is synthesized by the epithelial cells of the male duct system and ferried by small vesicles derived from the Golgi apparatus to secondary lysosomes. Because SGP-1 has recently been shown to have substantial sequence similarity to prosaposin, it may be speculated that SGP-1 is instrumental in the degradation of membrane glycolipids present within secondary lysosomes of epithelial cells of the extratesticular duct system.     

Structural features and functions of principal cells of the intermediate zone of the epididymis of adult rats

Background: In the present study, principal cells of the intermediate zone of the epididymis, an area situated between the initial segment and proximal caput, were observed to present morphological features distinct from those of principal cells of other regions. Methods: The epididymides of adult rats were fixed by perfusion with glutaraldehyde and embedded in Epon. Administration of fluid phase tracers was performed in the case of several animals. Localization of anti-SGP-2 and anti-immobilin antibodies in conjunction with light (LM) and electron (EM) microscope immunocytochemistry was also performed. Results: In the LM and EM, the most distinctive feature of many principal cells of this zone was the presence of apically located vacuoles referred to as giant endosomes due to their large size and because they readily incorporated tracers introduced into the lumen of the epididymal duct and were acid phosphatase-negative, Giant endosomes, containing electron-dense granular patches, appeared to form by the progressive fusion of small, medium, and large endosomes. In the supranuclear region, multivesicular bodies (MVBs) and lysosomes were present. Although smaller in size than the giant endosomes, MVBs and lysosomes contained the electron-dense patches. It is suggested from morphological images that giant endosomes fragment into smaller units corresponding to MVBs which gradually transform into lysosomes. Experiments using anti-SGP-2 and anti-immobilin antibodies revealed gold particles over the Golgi apparatus and secretory vesicles (150–300 nm) of principal cells of this zone as well as the luminal contents indicative of secretion of these proteins. Interestingly, giant endosomes were also immunolabeled with both antibodies as were stereocilia, coated pits and vesicles, and endosomes of various sizes; lysosomes were minimally labeled. These results suggest that principal cells of the intermediate zone endocytose as well as secrete SGP-2 and immobilin. The internalized SGP-2 and immobilin may correspond to that secreted further upstream and that, possibly due to their short half-life and terminated function, are removed from the lumen of the duct. Principal cells of this zone secrete these proteins possibly to replenish that lost by endocytosis. Conclusions: Principal cells of the intermediate zone contain giant endosomes. The presence of such large structures suggests that the early events in endocytosis is a slower process in principal cells of this zone as compared to other regions. The fact that these cells both secrete and endocytose SGP-2 and immobilin adds to the complexity of our understanding of how principal cells function along the length of the epididymis. © 1995 Wiley-Liss, Inc.     

Ultrastructural changes in the efferent duct and epididymis of men with obstructive infertility

Ultrastructural changes in the efferent duct and in different regions of the epididymis in men with obstructive azoospermia were compared with corresponding tissues collected from men of proven fertility who underwent castration due to malignancy of the prostate. Major degenerative changes were seen in the efferent duct and the caput epididymidis of men with obstruction at the caput epididymidis which may have been induced by fluid pressure due to defective absorption of testicular fluid in the caput epididymidis. These degenerative changes included decrease in tubular and lumen diameter of the caput and the cauda epididymides, decrease in height of the stereocilia, reduction in-rough endoplasmic reticulum and Golgi material, and presence of lipofuscin and osmiophilic dense bodies. The degenerative changes were less when the site of obstruction was in the cauda epididymidis since fluid reabsorption would continue to take place normally in the caput epididymidis. In men who had undergone vasoepididymostomy (VEA), the ejaculated spermatozoa showed a high percentage of morphological abnormalities which may have occurred due to adverse effects of long-term obstruction on spermatogenesis. © 1993 Wiley-Liss Inc.     

Structural differentiation of the epithelial cells of the testicular excurrent duct system of rats during postnatal development

The light and electron microscopic appearance of the various epithelial cells lining the efferent ducts and different regions of the epididymis were examined in rats on postnatal days 21, 39, 49, 56, and 90 to determine the role of androgens and/or spermatozoa, as well as other possible factors, on the structural differentiation of these cells. Five conclusions may be drawn from the observations made. First, on day 21 epithelial cells of all regions are structurally undifferentiated. Second, it was not until day 49 that nonciliated cells of the efferent ducts resembled those of adult animals, suggesting that more than one factor, such as androgens, testicular products, and/or spermatozoa, is needed for their full structural differentiation. Third, principal cells of the epididymis become structurally differentiated by day 39, i. e., these cells contained an elaborate Golgi apparatus, endoplasmic reticulum cisternae, and numerous 200–400 nm electron lucent secretory vesicles, as well as a full complement of endocytic organelles; this occurred in spite of the absence of spermatozoa in the epididymal lumen. The differentiation of these epididymal cells may be under the influence of androgens, which are known to be high at this time, but may also be due to specific secretions from Sertoli cells secreted directly into the efferent ducts. Fourth, clear cells of the cauda epididymidis are fully differentiated by day 39. The presence of degenerating germ cells in the lumen of the cauda epididymidis and various cellular debris, as well as high androgen levels, may be factors causing the differentiation of the cells of this region. Finally, clear cells of the corpus and cauda epididymidis only become fully differentiated by day 49, at a time when spermatozoa appear in the lumen, despite high levels of androgens at day 39; this observation indicates that the presence of spermatozoa in the lumen may be a necessary factor in causing their differentiation. Overall, these results suggest that a combination of different factors are necessary for the structural differentiation of the various epithelial cell types of the different regions of the epididymis. © 1992 Wiley-Liss, Inc.     

Structural differentiation of the epithelial cells of the testicular excurrent duct system of rats during postnatal development.

The light and electron microscopic appearance of the various epithelial cells lining the efferent ducts and different regions of the epididymis were examined in rats on postnatal days 21, 39, 49, 56, and 90 to determine the role of androgens and/or spermatozoa, as well as other possible factors, on the structural differentiation of these cells. Five conclusions may be drawn from the observations made. First, on day 21 epithelial cells of all regions are structurally undifferentiated. Second, it was not until day 49 that nonciliated cells of the efferent ducts resembled those of adult animals, suggesting that more than one factor, such as androgens, testicular products, and/or spermatozoa, is needed for their full structural differentiation. Third, principal cells of the epididymis become structurally differentiated by day 39, i.e., these cells contained an elaborate Golgi apparatus, endoplasmic reticulum cisternae, and numerous 200-400 nm electron lucent secretory vesicles, as well as a full complement of endocytic organelles; this occurred in spite of the absence of spermatozoa in the epididymal lumen. The differentiation of these epididymal cells may be under the influence of androgens, which are known to be high at this time, but may also be due to specific secretions from Sertoli cells secreted directly into the efferent ducts. Fourth, clear cells of the cauda epididymidis are fully differentiated by day 39. The presence of degenerating germ cells in the lumen of the cauda epididymidis and various cellular debris, as well as high androgen levels, may be factors causing the differentiation of the cells of this region. Finally, clear cells of the corpus and cauda epididymidis only become fully differentiated by day 49, at a time when spermatozoa appear in the lumen, despite high levels of androgens at day 39; this observation indicates that the presence of spermatozoa in the lumen may be a necessary factor in causing their differentiation. Overall, these results suggest that a combination of different factors are necessary for the structural differentiation of the various epithelial cell types of the different regions of the epididymis.