The appearance of basal cells in the developing murine epididymis and their temporal expression of macrophage antigens

This work demonstrates similarities between epididymal basal cells and macrophages in the mouse. Light microscopic studies of the postnatal development of the murine epididymis showed that basal cells were not present before days 12, 14 and 16 in the cauda, caput and corpus epididymis, respectively. An increase in cell number per unit length of tubule perimeter was demonstrated in all segments between days 20 and 27, when testicular fluid and spermatozoa start entering the epididymis. In the adult, there were more basal cells per unit perimeter in the cauda than caput or corpus epididymis. Conspicuous and consistent expression by basal cells of antigens detected by antibodies against tissue-fixed macrophages (F4/80) and mature macrophages (Mac-1) occurred only after they became established within the epithelium. Basal cells in the cauda epididymis did not display either antigen in the adult, although they persisted in the caput region. Such developmental patterns are compatible with the hypothesis that basal cells play a role in immune defence against sperm autoantigens.     

Secretion of glycosidases in human epididymal cell cultures

The dynamics of glycosidase secretion was evaluated in human epididymal cell culture. Epithelial cells from caput, corpus, and cauda epididymis were isolated from tissue obtained from patients undergoing therapeutic orchidectomy due to prostatic carcinoma. The activities of alpha-glucosidase, N-acetylglucosaminidase, beta-glucuronidase, and alpha-mannosidase were analyzed in conditioned culture media. Glycosidase activity was significantly higher in corpus and/or cauda than in caput epididymis. There was a time-dependent increase in enzyme activities that was maximal between 10 and 14 days of culture in all epididymal regions. Epididymal glycosidases are secreted by cultured epithelial cell from human epididymis with an increase toward the distal regions of this organ, which may be related to the dynamics of sperm maturation. Cultures from different epididymal regions may represent a valuable tool to study of human epididymal function.     

The ultrastructure of dog epididymis

Summary The ultrastructure of the epididymal duct and ductuli efferentes in the dog has been studied by electron microscopy. The epididymidis can be separated into the classical divisions of caput, corpus and cauda epididymidis on the basis of general morphology and ultrastructure. The ductuli efferentes have a low epithelium with pronounced cilia at the apices of cells and appear to provide primarily a transport role for spermatozoa. In the epididymis proper the caput region is characterized by an extremely large Golgi apparatus with large numbers of lysosomes and nuclear inclusions. Secretory activity appears to be most common in the corpus region. Absorption and secretion are most active in the first two segments while in the cauda epideidymidis the long-term storage of spermatozoa in the lumen is associated with many dense crystalline bodies formed in the epithelial cells within the Golgi apparatus and possibly deriving from absorbed macromolecular material from the lumen. The theory of whole sperm cell resorption by the epididymal duct is not supported by this study.     

Postnatal changes in the expression of claudin-11 in the testes and excurrent ducts of the domestic rabbit (Oryctolagus cuniculus domesticus)

We examined the expression of claudin-11 (CLDN11) in the testes and male reproductive tracts of rabbits. The rabbit CLDN11 cDNA sequences were nearly identical with human, mouse, and bovine CLDN11. The levels of CLDN11 mRNA and protein (22 kDa) were markedly increased in the testis during adult development. On postnatal day (PND) 10, CLDN11 was colocalized with ZO-1 at the lateral contacts between adjacent Sertoli cells and was perpendicular to the basal lamina. In adult testis on PND 180, CLDN11 was codistributed with ZO1, and the pattern of immunoreactivity consisted of wavy linear tracts parallel to the basal lamina, which was different according to the spermatogenic stage. These results suggest that CLDN11 participates in inter-Sertoli cell tight junctions (TJs) at the blood-testis barrier in adult rabbits. CLDN11 was also found in the basal regions of Sertoli cells adjacent to the basal lamina in adult testis, suggesting that CLDN11 also participates in the adhesion between Sertoli cells and the basal lamina. CLDN11 mRNA and protein expressions were decreased in the adult epididymis compared with those in immature animals. In adults, CLDN11 mRNA levels were relatively high in the efferent duct, followed by those in the vas deferens, proximal corpus, and distal cauda, although low levels were observed in the initial segment and caput. On PND 10, CLDN11 immunoreactivity was identified at the apicolateral contacts between adjacent epithelial cells in the epididymis and vas deferens. In adults, CLDN11 was found in the nonciliated cells in the efferent duct and at the lateral contacts in the epithelial cells in the epididymal segments. In the caput, CLDN11 was found at the apicolateral contacts between adjacent epithelial cells, but expression was weak to negligible in the corpus of the vas deferens. CLDN11 may play an important role in TJs and cell adhesion in immature rabbit excurrent duct epithelia. In adult rabbits, CLDN11 in efferent duct epithelium and epididymal epithelium may contribute to the specific environment for sperm maturation.     

Two-dimensional gel electrophoretic analysis of rat sperm membrane interaction with cauda epididymal fluid

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to analyze the polypeptide composition of rat cauda epididymal fluid, blood serum and membrane-enriched fractions of caput, corpus, and cauda epididymal spermatozoa. Several polypeptides were found in both cauda fluid and blood serum, and in both cauda fluid and epididymal spermatozoa. Prominent cauda epididymal fluid polypeptides that were associated with caput, corpus, and cauda sperm membranes were 32 and 33 kDa. Passage of spermatozoa from the caput to the cauda epididymidis was characterized by the loss of three glycopolypeptides of 32, 30 and 29 kDa, and by the addition of a 37-kDa glycopolypeptide. Incubation of intact caput, corpus and cauda spermatozoa with cauda epididymal fluid revealed major changes in the polypeptide maps of the incubation fluid and the membrane-enriched fractions of caput and corpus, but not cauda spermatozoa. The incubation of cauda fluid with caput and corpus sperm cells was characterized by a loss of several polypeptides and the addition of a 24-kDa glycopolypeptide. The most striking change in spermatozoa incubated with cauda epididymal fluid was the addition of two glycopolypeptides of 32 and 33 kDa to the polypeptide maps of caput sperm cells. These data demonstrate that rat spermatozoa undergo surface modifications during epididymal maturation and that these modifications can be influenced by epididymal fluid.     

Glutathione peroxidase activity in cell cultures from different regions of human epididymis

AIM: To study the secretory activity and androgen regulation of glutathione peroxidase (GPx) in epithelial cell cultures from human epididymis. METHODS: Tissue was obtained from patients undergoing therapeutic orchidectomy for prostatic cancer. Epithelial cell cultures were obtained from the caput, corpus and cauda epididymides. Enzymatic activity was measured in conditioned media by colorimetric methods in absence or presence of 1, 10 or 100 nmol/L testosterone. The effect of 1 micromol/L flutamide was also evaluated. RESULTS: GPx activity was higher in cultures from corpus and cauda than caput epididymidis. The presence of different concentrations of testosterone increase enzyme activity in cell cultures from all epididymal regions. Addition of flutamide reverses the androgen dependent increase of GPx activity. CONCLUSION: GPx activity is secreted from human epididymal cells in a region dependent manner and is regulated by androgens.     

Gamma-glutamyl transpeptidase activity in the epididymis during fetal and postnatal development in rats.

The histochemical and biochemical distributions of gamma-glutamyl transpeptidase (gamma-GT) were investigated in the epididymis of rats during fetal and postnatal development. In the epididymal homogenates, gamma-GT activity was detected on the fifth day after birth. A sharp increase was observed after 30 days of life in the caput homogenates. Moderate levels of the enzyme were found in the cauda epididymis. Gamma-GT is histochemically detected from the 15th day of gestation in Wolffian ducts and in 17- to 18-day-old fetuses in newly differentiated epididymal tubules. Enzyme activity, was associated with the plasma membranes (apical, lateral, and basal), was preponderant on the apical part of the epithelial cells. During the first 15 days of the postnatal life, the histochemical reaction intensities were identical from the caput to the cauda epididymidis. From the 18th day onwards, enzyme activity decreased in the corpus and in the cauda, while gamma-GT increased in the caput epididymidis, and a strong activity was found on the apical surface of epithelial cells. Weak or moderate gamma-GT activity of spermatozoa in the caput tubules, increasing steadily from caput to cauda epididymidis, suggests that gamma-GT may be related to the functional maturation of spermatozoa.     

Stage and region-specific localization of lipocalin-type prostaglandin D synthase in the adult murine testis and epididymis

Lipocalin-type prostaglandin D synthase in semen has been associated with male fertility, although this relationship is not well defined. To gain insight into potential mechanisms, the objective of the present study was to immunocytochemically localize lipocalin-type prostaglandin D synthase within the testis, efferent ducts, and 4 segments of mouse epididymis. In the testis, immunoperoxidase staining was localized within the Sertoli cells only at stages VI-VIII of the spermatogenic cycle, which is just prior to spermiation. Intense staining was also evident throughout the interstitial tissue, including Leydig cells. The entire epithelium of the efferent ducts, including ciliated and nonciliated cells, was immunoreactive. A distinct pattern of immunostaining for lipocalin-type prostaglandin D synthase was observed in different regions of epididymis, suggesting a possible role in sperm maturation. Staining for lipocalin-type prostaglandin D synthase was strikingly absent in the initial segment. In caput epididymidis, staining was evident throughout the cell cytoplasm of principal cells with some cells more intensely stained than adjacent ones. In the corpus region, overall staining intensity decreased and appeared to be concentrated in the apical region of principal cells, but some cells were completely unreactive. Reaction product in the cauda region was heavily concentrated on microvilli and within the epididymal lumen. In all epididymal regions, expression of lipocalin-type prostaglandin D synthase was specific to epithelial principal cells; no immunoreactivity was apparent in other cell types. The specific localization of lipocalin-type prostaglandin D synthase within the testicular interstitial tissue, Sertoli cells, and principal cells of caput epididymidis strongly suggests that this protein plays an integral role in both the development and maturation of sperm.     

Erratum

During passage of hamster spermatozoa through the epididymis their maturation is shown to involve changes in the sperm head, midpiece (mitochondria) and tail. The sum of these changes results in a dramatic increase in the fertilizing potential of the spermatozoa. When comparable numbers of spermatozoa from the caput or corpus epididymis were injected into one uterine horn of mature females, following ovulation induction, and spermatozoa from the cauda epididymis were injected into the contralateral horn, no fertilization was observed with caput epididymal spermatozoa, 1.7% of oocytes were fertilized by corpus epididymal spermatozoa, whereas 79.5% fertilization was obtained with cauda epididymal spermatozoa. Total sperm numbers increased from caput to corpus to cauda [28.3 ± 12.2, 40.6 ± 20.8, 144 ± 62 million, respectively]. The percentage of progressively motile spermatozoa increased from 27.9 ± 6.4 to 33.8 ± 4.8 to 70 ± 10.7 during this passage. Viability, measured by exclusion of the dye, propidium iodide, was significantly less in spermatozoa from the cauda than from the proximal or mid-caput epididymis. The percentage of the live cells that were stained intensely by rhodamine-123 (a measure of mitochondria1 membrane potential) increased during epididymal passage from 22.8 ± 7.8% in the proximal caput epididymis to 57.2 ± 16.5% in the cauda epididymis. Staining with acridine orange (a measure of DNA packaging in the sperm head) indicated an increase in chromatin condensation in cauda epididymal spermatozoa, when compared to those obtained from the caput or corpus.     

Investigation of epididymal sperm maturation in the golden hamster

During passage of hamster spermatozoa through the epididymis their maturation is shown to involve changes in the sperm head, midpiece (mitochondria) and tail. The sum of these changes results in a dramatic increase in the fertilizing potential of the spermatozoa. When comparable numbers of spermatozoa from the caput or corpus epididymis were injected into one uterine horn of mature females, following ovulation induction, and spermatozoa from the cauda epididymis were injected into the contralateral horn, no fertilization was observed with caput epididymal spermatozoa, 1.7% of oocytes were fertilized by corpus epiddymal spermatozoa, whereas 79.5% fertilization was obtained with cauda epididymal spermatozoa. Total sperm numbers increased from caput to corpus to cauda [28.3 ± 12.2, 40.6 ±20.8, 1434 ±62 mihon, respectively]. The percentage of progressively motile spermatozoa increased from 27.9 ±6.4 to 33.8 ± 4.8 to 70 ± 10.7 during this passage. Viability, measured by exclusion of the dye, propidium iodide, was significantly less in spermatozoa from the cauda than from the proximal or mid-caput epididymis. The percentage of the live cells that were stained intensely by rhodamine-123 (a measure of mitochondrial membrane potential) increased during epididymal passage from 22.8 ±7.8% in the proximal caput epididymis to 57.2 ± 16.5% in the cauda epididymis. Staining with acridine orange (a measure of DNA packaging in the sperm head) indicated an increase in chromatin condensation in cauda epididymal spermatozoa, when compared to those obtained from the caput or corpus.     

I. Abnormalities in cells of the testis, efferent ducts, and epididymis in juvenile and adult mice with beta-hexosaminidase A and B deficiency

Beta-hexosaminidase (Hex) is a lysosomal enzyme that exists as two major isoenzymes: Hex A (subunit structure, alphabeta) and Hex B (betabeta). The presence of Hex in the testis and epididymis suggests important roles for the enzyme and its substrates in male fertility and reproductive functions. Disruption of the Hexb gene encoding the beta-subunit of Hex has led to the generation of a mouse model of human Sandhoff disease that survives to adulthood, enabling us to analyze the effects of Hex A and Hex B deficiency on epithelial cellular morphology of the male reproductive tract. At 1 and 3 months of age, the testes, efferent ducts, and epididymides of Hex-deficient (Hexb -/-) and wild-type (Hexb +/+) mice were perfuse fixed and analyzed by routine light and electron microscopy (LM and EM, respectively) as well as with immunocytochemistry employing antibodies to lysosomal proteins. In the testis, the morphological appearance and topographical arrangement of the cell types of the seminiferous epithelium of Hexb -/- mice were similar to those of wild-type animals at both ages. Both Sertoli and germ cells appeared to be unaffected. However, at both ages, myoid cells and macrophages showed an increased number of lysosomes in their cytoplasm as compared with the number seen in controls. The epithelial cells of the efferent ducts also showed an accumulation of lysosomes that increased with age as compared with controls. Principal cells of the entire epididymis revealed an increase in the size and number of lysosomes at 1 month of age as compared with those of controls, and by 3 months, these lysosomes often filled the supranuclear and basal regions of the cells. Narrow cells of the distal initial segment and intermediate zone, normally slender cells showing several lysosomes, became greatly enlarged and entirely filled with lysosomes in Hexb -/- mice. Clear cells of the caput, corpus, and cauda regions also showed a progressive increase in the size and number of lysosomes with age as compared with controls; the clear cells of the mutant mice were often enlarged and at times bulged into the lumen. Some basal cells of each epididymal region in Hexb -/- mice were similar to controls at 1 and 3 months, showing few lysosomes, while others showed an accumulation of lysosomes. Lysosomes of all affected epithelial cells were of varying sizes, but many large ones were present, apparently resulting from lysosomal fusion. Although pale stained, their identification as lysosomes was confirmed by EM immunocytochemistry with anti-cathepsin D and anti-Hex A antibodies. Predominantly in the proximal initial segment, large, pale cellular aggregates were noted in the LM analysis at the base of the epithelium, which by EM analysis were identified as belonging to two different cell types, narrow cells and halo cells. Taken together, these data reveal an increase in the size and number of lysosomes in all epithelial cell types lining the efferent ducts and entire epididymis as well as in myoid cells and macrophages of the testis. In the light of data showing epididymal defects restricted predominantly to the initial segment in Hexa -/- (Hex A-deficient) mice, our data on the Hexb -/- mice demonstrate a major role for Hex that can be fulfilled by either Hex A or Hex B in the epididymis.     

大鼠精子在附睾成熟过程中肉毒碱量变化的研究

肉毒碱被认为是与精子在附睾中成熟有关的成熟因子。本文报告测定大鼠附睾头部,体部及尾部精子中的肉毒碱。结果表明精子在循附睾头、体、尾运行过程中,精子中肉毒碱含量逐步增加,在附睾体一头部,附睾尾一头部的精子内的肉毒碱量呈现十分显著性差异。结果提示附睾体部或体一头部交界部位可能是精子在附睾中成熟的关键部位。     

Origin of the luminal fluid proteins of the rat epididymis

Using a combined microperfusion and high resolution gel electrophoresis technique, the origin of the epididymal fluid proteins of the rat has been investigated. Some proteins originate from the testis, others are secreted by the epididymis or are released by spermatozoa. Of particular interest is a 32 000 dalton protein found to be actively secreted by the caput epithelium in situ and concenrated in the lumen. The cauda epididymidis contained the highest concentration of this protein. Radioactive labelling of the sperm surface proteins revealed that this protein was present on the surface of the mature cauda but not on the immature caput or corpus sperm, suggesting its acquisition by the sperm surface during epididymal transit. Another sperm surface protein of interest (MW 40 000) is present only on the plasma membrane of the cauda but not on that of the caput or corpus sperm. Since this protein was not identified in the epididymal perfusates or luminal fluids, its presence may result from some modification events taking place in the sperm membrane during maturation.     

c-mos原癌基因在小鼠附睾的表达

目的研究原癌基因c-mos在小鼠附睾的表达及c-Mos蛋白在小鼠附睾上皮细胞的定位。方法采用半定量RT-PCR和间接免疫荧光的方法分别检测c-mos mRNA和蛋白在附睾不同区域的表达,并通过免疫电镜对c-Mos蛋白在附睾上皮细胞内进行精确定位。结果c-mos mRNA表达量在小鼠附睾头部最高,体部最低;仅在附睾头部管腔面检测到c-Mos蛋白表达;电镜观察见c-Mos蛋白定位在附睾上皮细胞的顶部胞质内。结论c-mos原癌基因在小鼠附睾的区域特异性表达以及c-Mos蛋白在附睾上皮细胞的定位,提示c-mos基因可能在精子成熟过程中发挥调节功能。     

Localization and expression of spermadhesin PSP-I/PSP-II subunits in the reproductive organs of the boar

The epithelial localization and expression of the spermadhesin PSP-I and PSP-II subunits were determined in the testis, ductus epididymes (caput, corpus and cauda), seminal vesicles and bulbourethral glands of mature boars, using immunohistochemical, western blotting and RT-PCR methods. Immunohistochemistry showed positive labelling for PSP-I and PSP-II antibodies in the epithelium of seminal vesicles in all males tested. Positive immunolabelling, but with variable intensity, was also present in the epididymal epithelium (caput, corpus and cauda), although varying largely among segments and boars. Immunoreactivity was nearly or completely absent in the seminiferous epithelium and the bulbourethral gland, although SDS-PAGE and western blotting revealed the presence of PSP-I and PSP-II immunoreactive bands in all the tissue extracts, including the testis and the bulbourethral gland. mRNA amplification by RT-PCR using primers specific for PSP-I and PSP-II showed a trend similar to that observed for western blotting, i.e. intensity variation between tissues (even between segments of the same epididymis) and among boars. Our results indicate that the seminal vesicles are the main source of PSP-I and PSP-II spermadhesins, although epididymal segments, testis and the bulbourethral gland also participate in the expression of both proteins.     

The testicular and epididymal luminal amino acid microenvironment in the rat

Concentrations of amino acids were measured in arterial and testicular venous blood, and in fluids from the seminiferous tubule, rete testis, and the caput, corpus, and cauda epididymidis. There were no significant differences in the concentrations of any amino acids between arterial and testicular venous blood, whereas there were significant differences between arterial/venous blood and testicular interstitial fluid. The predominant amino acids measured within seminiferous tubule fluid (STF) and rete testis fluid (RTF) were glycine, alanine, glutamate, and glutamine. RTF contained approximately equal concentrations of basic and total amino acids, but 17 times higher acidic amino acids and 1.2 and 1.3 times lower uncharged polar and nonpolar amino acids, respectively, compared to STF. The concentration of total amino acids within caput fluid reached over 50 nmol/L, but then declined to approximately 50% and 0.1% of caput for corpus and cauda, respectively. The predominant amino acids measured within epididymal luminal fluids were glutamate and taurine; glutamate contributed to approximately 90% of the total amino acids measured in caput fluid. The presence of glutamate and taurine within the epididymal lumen is due primarily to a direct contribution from the epididymal epithelium, as measured using the split-drop stopped-flow microperfusion technique. Several other amino acids within the lumen also originate from the epididymal epithelium. Amino acids contribute approximately 20%, 9%, and 2% of the total osmolality of caput, corpus, and cauda fluid, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hormonal regulation of bovine secretory proteins derived from caput and cauda epididymal epithelial cell cultures

The goal of this study was to investigate the effect of hormones (testosterone, dihydrotestosterone [DHT], and hydrocortisone) on the protein secretion of caput and cauda epididymal epithelial cells cultured in principal cell medium (PCM). A confluent monolayer of caput and cauda epididymal epithelial cells was obtained from serum-containing PCM in the presence or absence of hormones after 7 days of culture at 38.5 degrees C (5% CO(2) in air). The protein secretion of epididymal epithelial monolayers incubated in serum-free PCM for 3 days was examined. The secreted proteins were separated by 2-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE). A comparison of the different protein patterns showed 61 spots, of which 11 were secreted only in the presence of hormones, 3 appeared to show hormone-related changes, and 25 were region-specific. Most of these secreted proteins were low-molecular-weight acidic proteins. To obtain evidence of the epididymal origin of the secreted proteins, proteins present in caput and cauda epididymal plasma were analyzed. In conclusion, our data indicate that hormones influence the synthesis of a number of caput and cauda epididymal proteins. Some of these proteins could be important for improving our understanding of spermatozoa maturation and storage and their acquisition of fertilizing ability.     

Purification and Localization of Acidic Epididymal Glycoprotein (AEG): A Sperm Coating Protein Secreted by the Rat Epididymis

A protein designated acidic epididymal glycoprotein (AEG) was purified from rat epididymis using ion exchange chromatography on DEAE-Sephadex, gel filtration in Sephadex G-75 and affinity chromatography on Concanavalin-A Sepharose. AEG is a major secretory product of the epididymis making up 2–3 per cent of total soluble protein. Antibody to AEG was raised in rabbits and purified by affinity chromatography on AEG-Sepharose. Quantitation of AEG in cytosol using "rocket" immunoelectrophoresis showed AEG to increase in epididymal segments from caput to cauda. Ligation of the midcorpus decreased AEG in the cauda. Localization of AEG using an immunoperoxidase method revealed that it is secreted largely by the epithelium of caput and corpus beginning with the region distal to the initial segment. It appears to be secreted by a specific cell type, probably the so-called "principal" cell. Specific staining of AEG was also noted in "clear" cells in the cauda. Spermatozoa become coated with AEG as they leave the initial segment and remain so during passage through the cauda.