Purification of GP-83, a glycoprotein secreted by the human epididymis and conjugated to mature spermatozoa

Epididymal secretions are critical for mammalian spermatozoa to acquire both forward motility and an ability to recognize and penetrate oocytes. Previous studies identified two glycoproteins, GP-83 and GP-39, which were secreted by the human epididymis and may be related to maturation of sperm function. In this study, GP-83 was purified from human seminal fluid by DEAE-ion exchange, gel filtration chromatography and preparative gel elution. The isoelectric point (pI) of purified GP-83 was 6.57. Monospecific antiserum to GP-83 was induced in male New Zealand rabbits and confirmed on immunoblots. GP-83 was found in fluid, tissue and sperm extracts of corpus and cauda epididymis, but not in the caput. Immunohistochemical localization identified GP-83 in the luminal contents and in the supranuclear region and cell membrane of principal cells of the corpus and cauda epididymis. GP-83 was found on the anterior acrosome in ejaculated spermatozoa, and shifted to the equatorial region after capacitation and the acrosome reaction.     

Late postnatal development and differentiation of the ductus epididymidis in a dasyurid marsupial (Antechinus stuartii)

Summary The general histology and ultrastructural features of the developing ductus epididymidis were examined in the brown marsupial mouse, Antechinus stuartii, from April, when males were sexually immature, until August, when the adult males were involved in mating activities, just prior to the annual male die-off. Samples were also examined 3 and 6 months after the August die-off period in males kept in isolation from conspecifics during the prebreeding and breeding periods. In April, tubule diameter and epithelial height were largest in the caput and least in caudal segments but the reverse was observed thereafter. Epithelial height increased in caput segments in August and remained high in the post die-off samples. However, caput epithelial height and tubule diameters were low compared with the remainder of the duct from July until February. Luminal shape in caudal segments (10, 11 and 12) changed in June from circular to a narrow slit, and the epithelium became variable in height. The epididymal epithelium was undifferentiated with few cytoplasmic organelles in April. Differentiation occurred mostly from May to June in associaion with an increased abundance of cytoplasmic organelles, increasing prostatic weight and rising plasma androgen levels. Differentiated principal and basal cells were found in caput and corpus regions in May and in caudal segments in June in association with the de novo development of a brush border of microvilli. Few clear cells were seen in caput and corpus regions of the duct in May but they, and mitochondria-rich cells, were common throughout the duct from June. Development of the unusual structural features of the cauda epididymidis preceded the arrival of spermatozoa in June. The presence of degenerating spermatozoa and cytoplasmic droplets in the cauda at this time suggested that it was not yet capable of supporting sperm viability. There was no evidence to suggest that the presence of spermatozoa has a stimulatory effect on the epididymis. Intact sperm were observed throughout the duct from July. Free cytoplasmic droplets, which showed some evidence of degeneration, collected in large masses in the distal corpus/ proximal cauda epididymidis of adult males between aggregates of spermatozoa. Epididymal differentiation appeared complete by mid-July; few ultrastructural changes occurred after this time. Recruitment of spermatozoa into the epididymis ceased by August and was associated with a rapid decline in sperm content in the proximal caput segments. In the November and February samples, spermatozoa were present only in distal corpus and proximal cauda segments. As in some eutherian mammals, differentiation of the epididymis in A. stuartii occurs in a descending fashion from caput to cauda. Development is linked to the onset of fluid and androgen production from the testis, which is essential for developing and maintaining a suitable caudal environment for storage and survival of spermatozoa.     

Maturation antigen of the mouse sperm flagellum: II. Origin from holocrine cells of the distal caput epididymis

During epididymal transit, the mouse sperm flagellum acquires a surface glycoprotein (SMA4) from epididymal fluid that functions as a sperm antiagglutinin. To determine the origin of this molecule, testes and epididymides of male mice were sectioned for light microscopy and stained with wheat germ agglutinin (WGA)-peroxidase, a probe that has been used previously to examine the biology of SMA4. WGA reactivity was localized to the cytoplasm in a small population of cells in the distal caput epididymis. Testis cells and principle cells of the caput were nonreactive with WGA, while stereocilia were stained on principle cells in the corpus and cauda. The WGA-positive cells in the distal caput were identified as holocrine cells on the basis of morphology, distribution, and PAS + reaction. At high magnification, intense WGA reactivity was due to the presence of numerous apical granules in the cytoplasm. The location of the cells in distal caput coincided exactly with the region of tubule in which sperm first acquired SMA4 on their flagellae. These data suggest that holocrine cells near the junction of caput and corpus epididymis are the source of the sperm antiagglutinin SMA4.     

Posttesticular development of spermatozoa of the tammar wallaby (Macropus eugenii)

Tammar wallaby spermatozoa undergo maturation during transit through the epididymis. This maturation differs from that seen in eutherian mammals because in addition to biochemical and functional maturation there are also major changes in morphology, in particular formation of the condensed acrosome and reorientation of the sperm head and tail. Of spermatozoa released from the testes, 83% had a large immature acrosome. By the time spermatozoa reached the proximal cauda epididymis 100% of sperm had condensed acrosomes. Similarly 86% of testicular spermatozoa had immature thumb tack or T shape head-tail orientation while only 2% retained this immature morphology in the corpus epididymis. This maturation is very similar to that reported for the common brush tail possum, Trichosurus vulpecula. However, morphological maturation occurred earlier in epididymal transit in the tammar wallaby. By the time spermatozoa had reached the proximal cauda epididymis no spermatozoa had an immature acrosome and thumbtack orientation. Associated with acrosomal maturation was an increase in acrosomal thiols and the formation of disulphides which presumably account for the unusual stability of the wallaby sperm acrosome. The development of motility and progressive motility of tammar wallaby spermatozoa is similar to that of other marsupials and eutherian mammals. Spermatozoa are immotile in the testes and the percentage of motile spermatozoa and the strength of their motility increases during epididymal transit. During passage through the caput and corpus epididymis, spermatozoa first became weakly motile in the proximal caput and then increasingly progressively motile through the corpus epididymis. Tammar wallaby spermatozoa collected from the proximal cauda epididymis had motility not different from ejaculated spermatozoa. Ultrastructural studies indicated that acrosomal condensation involved a complex infolding of the immature acrosome. At spermiation the acrosome of tammar wallaby spermatozoa was a relatively large flat or concave disc which projected laterally and anteriorly beyond the limits of the nucleus. During transit of the epididymal caput and proximal corpus the lateral projections folded inwards to form a cup like structure the sides of which eventually met and fused. The cavity produced by this fusion was lost as the acrosome condensed to its mature form as a small button-like structure contained within the depression on the anterior end of the nucleus. During this process the dorsal surface of the immature acrosome and its outer acrosomal membrane and overlying plasma membrane were engulfed into the acrosomal matrix. This means that the dorsal surface of the acrosomal region of the testicular tammar wallaby sperm head is a transient structure. The dorsal acrosomal surface of the mature spermatozoon appears ultrastructurally to be the relocated ventral surface of the acrosomal projections which previously extended out beyond the acrosomal depression on the dorsal surface of the nucleus of the immature spermatozoon.     

GP-83 and GP-39, two glycoproteins secreted by human epididymis are conjugated to spermatozoa during maturation

Surface glycoconjugates of spermatozoa are modified during epididymal maturation, which is closely related to the development of sperm function. In addition, recognition of surface glycoconjugates is one of very critical events in sperm-oocyte interaction. The binding of carbohydrate-specific lectins to the human sperm surface during epididymal maturation has been investigated. However, the glycoproteins responsible for lectin binding in sperm maturation are not well documented. This study used wheat germ agglutinin (WGA), peanut agglutinin (PNA) and concanavalin A (Con-A) to identify sperm maturation-related glycoproteins in human epididymis. Histochemical localization revealed that the binding sites of WGA, PNA and Con-A were mainly in the principal cells and luminal contents of the human epididymis, but not in the interstitial regions. Each lectin displayed a fairly distinct regional localization. On Western blots probed with WGA and Con-A, glycoproteins of 83 kDa (GP-83) and 39 kDa (GP-39) were identified in the sperm extracts, epididymal fluid and tissue extracts of the corpus and cauda epididymides, but not in the caput. PNA identified GP-83 in the same manner as WGA and Con-A, but did not recognize GP-39. These results suggest that lectin-binding glycoproteins GP-83 and GP-39 found on mature spermatozoa may be secreted by the principal cells of corpus and cauda epididymis, and conjugated to spermatozoa during their transit in human epididymis.     

Maturation antigen of the mouse sperm flagellum. I. Analysis of its secretion,association with sperm,and function

We report here recent findings on the sperm maturation antigen SMA4, which is secreted by holocrine cells of the distal caput epididymis and binds to the flagellar surface of mouse sperm during epididymal transit. Washed sperm from the caput and corpus epididymides of mice were examined by immunofluorescence and SDS-PAGE using wheat germ agglutinin, which binds specifically to SMA4 as a primary probe. Results indicate that sperm first exhibit WGA reactivity on their flagellae in the region of the distal caput, and that the appearance of WGA receptors is due to the binding of a 54-Kd glycoprotein (SMA4) to the cell surface. Extracts of epididymis containing SMA4 were tested for their ability to bind to the surfaces of caput and corpus sperm. Caput sperm surfaces bound SMA4 in & temperature-independent manner, and binding occurred in the presence of enzyme inhibitors., suggesting a nonenzymatic process. Biochemical studies revealed that SMA4 contains disulfide bonds which stabilize it on the sperm surface and restrict its mobility. Terminal carbohydrate residues of the molecule are sialic acids. The addition of SMA4 to caput sperm flagellae prevented tail-to-tail agglutination, normally seen when caput sperm are diluted into saline; and SMA4 was able to disperse clumps of agglutinated caput sperm. The data suggest that a primary function of SMA4 is to prevent tail-to-tail agglutination of sperm during storage in the epididymis.     

Actin polymerisation during morphogenesis of the acrosome as spermatozoa undergo epididymal maturation in the tammar wallaby (Macropus eugenii)

In the tammar wallaby ( Macropus eugenii ), post-testicular acrosomal shaping involves a complex infolding and fusion of the anterior and lateral projections of the scoop-shaped acrosome into a compact button-like structure occupying the depression on the anterior end of the sperm nucleus. The present study has generated cytochemical and histological evidence to demonstrate that the occurrence of actin filaments (F-actin, labelled by Phalloidin-FITC) in the acrosome of tammar wallaby spermatozoa is temporally and spatially associated with the process of acrosomal shaping in the epididymis, through a pool of monomeric actin (G-actin, labelled by Rh-DNase I) present in the acrosome throughout all stages of epididymal maturation. F-actin was not detected in the acrosome of testicular spermatozoa, but was found in the infolding and condensing acrosome of caput and corpus epididymal spermatozoa. When the spermatozoa completed acrosome shaping in the cauda epididymidis, F-actin disappeared from the acrosomal area. The strong correlation between the occurrence of F-actin and the events of acrosomal shaping suggested that the post-testicular shaping of the acrosome might depend on a precise succession of assembly and disassembly of F-actin within the acrosome as the spermatozoa transit the epididymis. Thus, actin filaments might play a significant role in the acrosomal transformation, as they are commonly involved in morphological changes in somatic cells.     

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.     

Epithelial cells of the epididymis show regional variations with respect to the secretion or 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 the 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.     

Andrology: Evaluation of motility, fertilizing ability and embryonic development of murine epididymal sperm after coculture with epididymal epithelium

Murine sperm from the caput, corpus and cauda epididymis werecocultured with epididymal epithelial cells of their own regionor more distal regions, in the presence and absence of androgens(testosterone and dihydrotestosterone). Epitheial cell cultureswere used 3 or 10 days after preparation in a complex tissueculture medium (Chang's) as plated tubules. The coculture studiesinvolving spermatozoa and oocytes with epithelial cells werecarried out in T6 medium. Motility of caput spermatozoa wasmaintained for 24 h in the presence of day 3 corpus and caudaepithelial cells and hormones but not under other conditions.Likewise, the motility of corpus spermatozoa was maintainedfor 24 h in the presence of day 3 cauda epithelial cells andhormones but not other conditions. Fertilization of zonaintactoocytes by epididymal spermatozoa was not affected by theircoculture for 24 h with epithelial cells but fertilization ratesfor zona-free oocytes were increased for caput spermatozoa coculturedwith more distal epithelial cells. Fertilization rates for bothzona-intact and zona-free oocytes were increased for corpusspermatozoa cocultured with more distal cauda epithelial cells.The developmental capacity of embryos derived from caput spermatozoawas not significantly increased by coculture with epithelialcells but those derived from corpus spermatozoa cocultured withcauda epithelial cells were signilicantly increased. We concludethat the presence of more distal epithelial cells of the mouseepididymis maintains motility in culture, increases the abilityof caput and corpus spermatozoa to fertilize zona-free oocytesand increases the developmental capacity of embryos formed fromcorpus spermatozoa. These observations demonstrate the functionof epididymal regions in the maturation of murine spermatozoafor fertilization and embryo development.     

Maturation of spermatozoa in the epididymis of the Chinese hamster

Chinese hamster spermatozoa gain their ability to move when they descend from the testis to the distal part of the caput epididymis, but it is not until they enter the corpus epididymis that they become capable of fertilizing eggs. The maturation of the spermatozoa proceeds as they further descend the tract and perhaps continues even in the vas deferens. During transit between the distal caput and proximal cauda epididymides, small membrane-limited vesicles (and tubules) appear on the plasma membrane over the acro somes of the spermatozoa. The number of vesicles appearing on the sperm brane reaches a maximum when the spermatozoa are in the proximal cauda epididymis. It declines sharply in the distal cauda epididymis. Spermatozoa in the vas deferens are free of the vesicles. The origin, chemical nature, and functional role of the vesicles that appear on the sperm surface during epididymal transit must be the subject of further investigation.     

Lysosomal integral membrane proteins exhibit region and cell type specific distribution in the epididymis of the adult rat.

The epididymis, a post-testicular site required for maturation and storage of spermatozoa, is actively involved in exocytic and endocytic events, two phenomena likely to depend on the integrity of the lysosomal system. To study the lysosomal system of the epididymis, five monoclonal antibodies, previously characterized as recognizing five distinct lysosomal integral membrane proteins (LIMPs 1-5), were used as molecular probes of lysosome distribution in cells lining the epithelium. Immunocytochemical localization of LIMPs, using biotin-streptavidin immunoperoxidase methodology, was performed on frozen sections of adult rat epididymides and in cell cultures prepared from either the caput or cauda epididymis. In frozen sections, a heterogeneous distribution of the different LIMPs along the length of the epididymis was observed. For example, the distribution of LIMP 1 (35-50 K) was detected in all cells of the caput and quite dramatically in clear cells of the distal caput, corpus, and cauda epididymis, but specifically not in the principal cells of the distal caput, corpus, and cauda. In contrast, LIMP 2 (64-71 K) was present in all cells of the epididymis, except clear cells. LIMPs 4 and 5 (93 K and 93 K) were detected in all epididymal cells, including the clear cells. Finally, whereas the regional and cell type distribution of LIMP 3 (74 K) in the epididymis was identical to that of LIMPs 4 and 5, the nature of the vesicles immunostained was distinct. In cultured cells, the general immunostaining patterns observed in vivo were maintained during the duration of the primary cultures for all five LIMPs. Our results begin to address the molecular heterogeneity of the lysosomal system along the length of the epididymis, and may suggest in part a basis for underlying structural and functional characteristics of the epididymis leading to the sequential maturation of sperm.     

Lysosomal integral membrane proteins exhibit region and cell type specific distribution in the epididymis of the adult rat

The epididymis, a post-testicular site required for maturation and storage of spermatozoa, is actively involved in exocytic and endocytic events, two phenomena likely to depend on the integrity of the lysosomal system. To study the lysosmal system of the epididymis, five monoclonal antibodies, previously characterized as recognizing five distinct lysosomal integral membrane proteins (LIMPs 1–5), were used as molecular probes of lysosome distribution in cells lining the epithelium. Immunocytochemical localization of LIMPs, using biotin-streptavidin immunoperoxidase methodology, was performed on frozen sections of adult rat epididymides and in cell cultures prepared from either the caput or cauda epididymis. In frozen sections, a heterogeneous distribution of the different LIMPs along the length of the epididymis was observed. For example, the distribution of LIMP 1 (35–50 K) was detected in all cells of the caput and quite dramatically in clear cells of the distal caput, corpus, and cauda epididymis, but specifically not in the principal cells of the distal caput, corpus, and cauda. In contrast, LIMP 2 (64–71 K) was present in all cells of the epidiyms, except clear cells. LIMPs 4 and 5 (93 K and 93 K) were detected in all epididymal cells, including the clear cells. Finally, whereas the regional and cell type distribution of LIMP 3 (74 K) in the epididymis was identical to that of LIMPs 4 and 5, the nature of the vesicles immunostained was distinct. In cultured cells, the general immunostaining patterns observed in vivo were maintained during the duration of the primary cultures for all five LIMPs. Our results begin to address the molecular heterogeneity of the lysosomal system along the lenght of the epidiymis, and may suggest in part a basis for underlying structural and functional characteristics of the epididymis leading to the sequential maturation of sperm.     

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.     

Cytochemical study of spermiogenesis and mature spermatozoa in the lizard Tropidurus itambere (Reptilia, Squamata)

The present study was undertaken to detect essential components of spermatozoa by ultrastructural and cytochemical analyses of testis and epididymis of the lizard Tropidurus itambere at different points of its annual reproductive cycle. Cytochemical investigations of spermiogenesis have not been performed so far in Squamata and are scarce for lower vertebrates. Essential components are: 1) polysaccharides, identified by PAS staining, abundantly present in Sertoli cell elongations, acrosomal vesicles and the acrosome of sperm cells; 2) glycoconjugate variations, labeled by different lectins and used to investigate cell modifications during spermiogenesis and found in mature spermatozoa in the female's seminal receptacle; 3) basic proteins, present in large quantities in spermatozoa in the subacrosomal cone, the pericentriolar material, the midpiece dense bodies, the peripheral fibers of the axoneme, and the fibrous sheath of the flagellum; 4) the final reaction product of acid phosphatase activity in several stages of acrosome development, specifically in the clear zone and epinuclear electron-lucent region of spermatozoal acrosomes, as well as in very active lysosomes found during the quiescent period of the reproductive cycle; 5) lipids, abundantly present in the cytoplasm of Leydig cells during the quiescent period. The cytochemical methods show that the ultrastructurally complex acrosome is also biochemically heterogeneous, with specific layers rich in glycoproteins, basic proteins or acid phosphatase. These different components may play a role during sperm penetration into the ovule. Basic proteins are largely responsible for structures surrounding the axoneme to provide resistance to the flagellum. In the quiescent period, acid phosphatase activity is involved in the elimination of superfluous sperm cells, whereas lipids in Leydig cells are used for hormone synthesis which starts at this time point to initiate a new reproductive cycle. Variations in lectin staining revealing glycoconjugates show that spermatozoa undergo post-testicular maturation up to their storage in the female.     

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.     

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.     

Autometallographic demonstration of zinc ions in rat sperm cells

An in-vitro technique for autometallographic (AMG) demonstration of chelatable zinc in electroejaculated sperm cells and spermatozoa from the epididymis is presented and the localization of zinc ions in rat spermatozoa is described. Sperm cells from caput epididymis showed zinc staining in all parts of the tail and a sparse, dispersed staining in the acrosome. Spermatozoa from cauda epididymis showed heavy staining in the acrosome but no staining in the tail, or post-acrosomal part of the sperm head. This distinct acrosomal AMG staining was also found in ejaculated spermatozoa, but additionally a segmentation of the tail was seen based on differences in staining intensity. The membrane penetrating chelator diethyldithiocarbamate (DEDTC) was found to block the AMG staining whereas calcium-EDTA, known not to pass through cell membranes, did not influence the staining, proving that the detected zinc ions are intracellularly located. Two different approaches for demonstrating the presence of a chelatable zinc pool at electron microscope levels are presented, and the ultrastructural presence of AMG grains located in the acrosome and in the mitochondria of the midpiece is demonstrated. It is postulated that an exchange of zinc ions takes place between the epididymal epithelium and the sperm cells as they pass along the epididymal duct.      

Spermiation and sperm maturation in the marmoset

The scanning and transmission electron microscopes were used to examine the processes of spermiation and sperm maturation in the marmoset. We observe that the heads of late spermatids are embedded in the apical aspect of the large sleeve-like columnar portion of Sertoli cells. As spermiogenesis progresses, spermatids become associated with numerous small apical Sertoli cell extensions. These finger-like processes undergo a sequence of changes during spermiation. Spermatozoa from the caput, corpus, and cauda epididymides were examined. In caput epididymis of marmoset, the apical segment of the spermatozoa extends well beyond the rostral edge of the nucleus and folds back on itself. In sagittal sections, the acrosome exhibits a distinct hook shape. In the corpus, the distinctive hook-shaped apical segment of the acrosome is observed in some spermatozoa, but the apical extension is significantly smaller or projects out only slightly beyond the nucleus. In cauda epididymis, the extension is absent. A similar acrosomal hook has been reported in the pigtailed monkey, which is an Old World species. We suggest that changes in acrosome structure during sperm maturation may be fairly widespread among primates.     

A cytochemical study on surface charges and lectin-binding sites in epididymal and ejaculated spermatozoa of Macaca fascicularis

Changes in electronegative and electropositive surface charges and in lectin receptors (concanavalin A and wheat germ agglutinin) were investigated on sperm plasma membranes of the monkey (Macaca fascicularis) during epididymal transit and after ejaculation. Electronegative charges at pH 1.8, which were uniformly distributed on the whole plasma membrane of caput epididymal spermatozoa, increased mainly on the postacrosomal cap and the tail during epididymal passage. Electropositive charges at pH 9 were simultaneously found on the whole cell surface of caput epididymal spermatozoa with a stronger labeling on the acrosomal apex, the postacrosomal cap, and the tail. These charges disappeared during passage through the epididymis corpus. The surface distribution of lectin receptors varied inversely during epididymal transit with an increase in concanavalin A receptors and a decrease in wheat germ agglutinin receptors. These data show that changes in the monkey sperm plasma membrane during epididymal maturation occur in the distal corpus of the epididymis.