The features of sperm maturation in the epididymis of a marsupial,the brushtailed possum Trichosurus vulpecula

Possum spermatozoa undergo a distinctive process of maturation in the epididymis, as shown by change in the properties of the sperm surface, by modification of their morphology and by their increasing capacity for progressive motility. Modification of the sperm surface over the head and tail is demonstrated by the different affinities of sperm from successive regions of the epididymis for FITC-conjugated wheat germ agglutinin and concanavalin A, and for cationic ferric oxide colloidal particles. Changes in sperm head morphology are caused by (1) a dramatic reshaping and consolidation of the acrosome in which excess plasma membrane overlying it is sloughed as a cluster of vesicles, (2) a reorientation of the nucleus almost parallel to the axis of the tail and (3) distal movement of the droplet from its initial envelopment of the nucleus to an eccentric position on the anterior segment of the midpiece. Spermatozoa released from the testis and caput epididymidis are essentially immotile or exhibit only lazy uncoordinated movements, whereas many from the corpus and most from the more distal regions of the epididymis display an energetic, progressive motility imparted by a rapid and stiff tail beat of narrow arc. This maturation of the capacity for motility is accompanied by an enhanced stability of the dense fibers and sheath, which become more resistant to the disruptive action of SDS and DTT, and by changes in the ultrastructure of the sperm tail. These include modification of the matrix of the mitochondria and also an unusual differentiation of the midpiece into two distinct segments. The anterior segment is defined by profuse peri-mitochondrial stacks of membranes which develop as spermatozoa pass through the epididymis. These membranes, although prominent in mature spermatozoa fixed in situ, appear sparse and disorganised in spermatozoa fixed after 15 to 30 minutes of active motility in physiological medium, suggesting their possible utilisation in motile spermatozoa. The posterior segment is characterised by a thick peri-mitochondrial cytoplasmic sleeve, by spirally arranged parallel fibrous bands immediately beneath the plasma membrane and, subsequently, as spermatozoa pass into the lower corpus epididymidis, by rows of flask-shaped surface invaginations which develop between the spiral bands. Despite broad similarities in the features of sperm maturation in this marsupial and in eutherian mammals, there are distinct differences in the structural organisation of their spermatozoa, particularly in the sperm head. Until more is known of the details of fertilisation in marsupials the significance of these differences will remain unclear.     

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.     

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.     

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.     

Results of microsurgical vasoepididymostomy: role of epididymis in sperm maturation

One-hundred-and-ninety patients with obstructive azoo-spermiacaused by bilateral epididymal blockage have been followed for4 years after undergoing ’specific tubule‘ vasoepididymostomy.When anastomosis was required in the corpus epididymidis, the’patency‘ rate was 78% and the overall pregnancyrate was 56%. The pregnancy rate for ’patent‘ caseswas 72%, indicating that a high fertility rate can be obtainedwith spermatozoa that have not passed through the full lengthof corpus epididymidis. By contrast, with vasoepididymostomyto the caput epididymidis there was a 73% ’patency‘rate, but the overall pregnancy rate was only 31%. The pregnancyrate for ’patent‘ cases was 43%. Spermatozoa fromthe corpus epididymidis have a higher rate of fertility thanspermatozoa from the caput epididymidis, but spermatozoa fromproximal areas of the corpus have no less fertility than spermatozoafrom the distal corpus epididymidis. The most remarkable observationis that in almost half the cases, spermatozoa which have neverjourneyed beyond the caput epididymidis seem to be capable ofcausing pregnancy     

ILA 147 immunoreactivity of the bull spermatozoa membrane during epididymal maturation

A cytochemical quantitative study was carried out to detect immunostaining of bull spermatozoa during epididymal maturation using an ILA 147 monoclonal antibody and a standard immunoperoxidase method. This antibody recognizes a bovine panleukocyte determinant. Microdensitometric measurements were made on spermatozoa collected from different sites of the male genital tract (caput, corpus and cauda epididymidis and ductus deferens). On the basis of ILA 147 staining, at least 5 subpopulations of sperm cells from each site of the genital tract were found. These subpopulations showed: 1) immunostaining in the acrosome domain and in the cytoplasmic droplet in a proximal position; 2) immunostaining in the acrosome domain and in the distal cytoplasmic droplet; 3) immunostaining in the acrosomal region and lack of a cytoplasmic droplet; 4) immunostaining only in the cytoplasmic droplet; 5) lack of a cytoplasmic droplet and absence of staining of the head. The possible relationship between the presence of ILA 147 on the sperm head and the maturation process was evaluated, and we suggest that the most significant changes in ILA 147 expression occur in the corpus epididymidis. The absence of immunocytochemical staining in some spermatozoa may be related to plasma membrane damage due to spontaneous peroxidative damage of lipids.     

Cytochemical and ultrastructural characteristics of the stallion epididymis (Equus caballus)

The epididymis of stallion castrated during the breeding and non breeding seasons were subdivided into six regions and their ultrastructural and cytochemical characteristics were studied in order to provide a better understanding of the structure-function relationship of this androgen target organ. Even when the stallion has been postulated to be a seasonal breeder, our results do not show significant ultrastructural or cytochemical differences in both seasons. The pseudostratified epithelium is composed mainly of principal and basal cells and intraepithelial lymphocytes. The principal cells show morphological features of protein or glycoprotein secretion, especially in the caput epididymidis. Although PAS, CFH and AB positive substances were found throughout the epididymis, the reactivity was maximal in the caput region. This positive reaction can be ascribed to acidic glycoproteins. In stallion tissues, 4.0 acetylated sialic acid occurs in relatively high amount and is possible that the acid glycoprotein observed in our material have also this characteristic. The principal cells of the distal caput and corpus epididymides also display morphological hallmarks of absorptive and anabolic activity. These results are consistent with the histological reactions that demonstrate that the enzymes involved in active transport showed the strongest reaction in the corpus region. The acid phosphatase reaction was also strongest in these segments. In the cauda region, where the spermatozoa are stored ready for ejaculation, morphological signs of metabolic activity were also observed, but less notorious than in the more proximal segments. Resorption of non ejaculated spermatozoa was also observed in this region. It is difficult to evaluate the functional meaning of the spermatophagy in the epididymis because the images of sperm phagocytosed by epithelial cells were seen only in one or two cases. The chemical composition of the epididymal fluids changes along the length of this organ, concomitantly with the sperm maturation process, and it is possible to assume that some of these changes are a result of the secretory and absorptive activities of the principal cells.     

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.     

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.     

Variation in intramembranous particle distribution in the cell membrane of the sperm head of the plains rat and western chestnut mouse (Pseudomys Spp.)

Freeze-fracture studies were used to investigate the size, density, and distribution of intramembranous particles (IMPs) in the plasma membrane of the sperm head of two species of Australian hydromyine rodents Pseudomys australis and P. nanus both of which are characterized by having, in addition to a dorsal hook, two long ventral hooks that extend from the antero-concave surface. It was found that on the P face of the plasma membrane over the dorsal hook of caput and upper corpus spermatozoa a paracrystalline arrangement of small, approximately 7nm, IMPs were interspersed with a few large, 9 to 12 nm, IMPs. In the cauda epididymidis the small particles became randomly arranged. On the P face of the plasma membrane over the ventral hooks of caput sperm, and in the postacrosomal region, a much higher density of large, 9 to 12 nm, IMPs was present. In the ventral hooks of cauda sperm the IMPs were increased even further and were arranged in short, variably orientated, ridges; this did not appear to be evident in the plasma membrane over the postacrosomal region. It suggests a change in distribution of the IMPs in the cell membrane over the ventral hooks, but not over the postacrosomal region, during epididymal transit of the spermatozoa. This may indicate that the plasma membrane of these two regions of the head of ejaculated spermatozoa has intrinsic differences in its structure and function.     

Maturation of human spermatozoa (from selected epididymides of prostatic carcinoma patients) with respect to their morphology and ability to undergo the acrosome reaction

Spermatozoa were recovered form three regions of the epididymisof six prostatic carcinoma patients. After washing and incubatingfor 3 h in Ham's F-10 medium, with or without 5 µM A23187for the last 30 min, spermatozoa were tested for vitality byhypotonic swelling and permeated with methanol to detect theacrosome with peanut agglutinin. Whereas the extent of spontaneousacrosome reactions was similar for spermatozoa from all regionsof the duct, 17 and 28% of spermatozoa from all regions of theduct, 17 and 28% of spermatozoa from the corpus and cauda epididymidisrespectively, responded to stimulation by A23187 with acrosomereactions but there was no stimulation by A23187 of spermatozoafrom the efferent ducts. The percentage of morphologically normalspermatozoa increased stepwise towards the distal regions, withabnormalities being mostly enlarged heads in more proximal regions:they were largely absent form the cauda epididymidis. Spermhead swelling was similarly observed in cynomolgus monkey spermatozoafrom the caput epididymidis but not the more distal regions.These forms were not observed when spermatozoa were fixed beforesmearing, indicating that they were artefacts of sperm preparation.The changes in the susceptibility of non-fixed epididymal spermatozoato produce morphological artefacts and the gain in their acrosomalresponse to ionophore demonstrate maturational changes of spermatozoain the human epididymis.     

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.     

Effect of hepatocyte growth factor on sperm motility

PROBLEM: Hepatocyte growth factor (HGF) exists abundantly in seminal plasma and its receptor, c-met, is expressed on spermatozoa. Considering its motogenic activity, we speculated that HGF might affect the movement ability of spermatozoa. METHODS: Recombinant HGF was added to washed spermatozoa and their movements were analyzed using a computer-assisted sperm analyzer. The concentration of HGF in the seminal plasma of infertile patients (n = 83) was measured by ELISA, and the data were compared with their hormonal profile and semen parameters. RESULTS: The HGF physiological concentration (1 ng/mL) maintained the motility of sperm after a long incubation, though the difference was not statistically significant. Recombinant HGF did not affect the linearity or frequency of movement, which suggested that it does not evoke the hyperactivation of spermatozoa. The concentration of HGF in seminal plasma did not correlate with any clinical parameter of the patients. CONCLUSIONS: These findings contradict the theory that HGF controls the movement of sperm. The main role of this axis in the male reproductive system might be maturation in the epididymis.     

Morphometric analysis of intact sperm heads and of sperm nuclei in the mouse

A morphometric analysis of mouse sperm and of their nuclei was undertaken to investigate their respective post-testicular maturation. Sperm were collected from the testis, caput and cauda epididymidis, and their corresponding nuclei were isolated. Results indicate that the post-testicular maturation of sperm is distinct from that of nuclei. The size of intact sperm heads increases in the caput followed by a subsequent decrease in the cauda. In contrast, sperm nuclei decrease progressively in size. In general, a greater magnitude and number of alterations in intact heads and nuclei occur while in transit from the testis to the caput than during passage to the cauda epididymis. These results suggest that the period immediately following their release from the testis is crucial to the complete morphological maturation of sperm heads and nuclei. © 1993 Wiley-Liss, Inc.     

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.     

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.     

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.     

Influence of abdominal temperature on epididymal function in the rat and rabbit

The effects of body temperature on sperm maturation and sperm storage in the epididymis have been studied in rats and rabbits by surgical reflection of the epididymis into the abdomen without disturbing its continuity with a functional scrotal testis. Rats so treated remained fertile during a period of 13 months, as judged by a sequence of normal litters born to their female cage mates, and cryptepididymal rabbits remained fertile for four months or more according to the individual, as evidenced by eggs fertilized and litters born after artificial insemination or natural mating. By contrast, spermatozoa confined by ligatures within an abdominal cauda epididymidis never retain their potential for motility for more than a brief period (5 days in rat; 8–10 days in rabbit), as compared with several weeks in contra-lateral scrotal controls. It is uncertain whether their early death at abdominal temperatures resulted primarily from a suppressive effect of the temperature on the spermatozoa themselves, or on the caudal epithelium. Because of an increasing presence of apparently immature spermatozoa in the ejaculate of the first cryptepididymal rabbit between 7 and 11 months postoperatively, the rate of sperm transport through the epididymis of seven cryptepididymal rabbits was estimated by labelling with tritiated thymidine. As judged by the time of appearance of labelled spermatozoa in the ejaculate, transposition of the rabbit epididymis to the abdomen ultimately causes a progressive increase in the rate of sperm transport through it from a normal of nine days to two to five days in the cryptepididymal state. Thus, although maturation may eventually be compromised to some degree because of an increased rate of sperm passage, and sperm storage is inhibited, abdominal temperature apparently does not alter the essential biochemical nature of the epididymal milieu required for maturation. These results are reviewed in light of the suggestion (Bedford, '78) that the cauda epididymidis has been the prime mover in scrotal evolution.     

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.