High level of endostatin in epididymal epithelium: protection against primary malignancies in this organ?

Rete testis and epididymis are rare locations for primary tumors or metastasis. Assuming that this may be related to expression level of angiogenic inhibitors, we focused our study on the expression pattern of collagen 18/endostatin. In situ hybridization and immunohistochemistry for collagen 18 and endostatin were carried out on sections of human rete testis and epididymis as well as on epididymal adenoma and human testicular tissue with or without carcinoma in situ (CIS). In situ hybridization revealed strong expression of collagen 18 mRNA in rete testis, efferent ducts and epididymal duct. Immunostaining showed collagen 18 in epithelium and basement membrane as well as in blood vessels of rete testis. Further, in both efferent ducts and epididymal duct, collagen 18 was mainly localized in the basement membrane of these ducts and of the blood vessel wall. Endostatin immunostaining was localized in the epithelium of rete testis, efferent ducts and epididymal duct. This pattern of endostatin staining was absent in epididymal adenoma tissue while tumor associated blood vessels exhibited strong endostatin staining. No endostatin staining was detectable in normal germinal epithelium and CIS cells while Leydig cells exhibited strong endostatin staining. High endostatin expression in epididymis may protect this organ against tumor development. Gene therapeutic strategies providing high expression of endostatin in normal epithelia may be useful to prevent tumor development.     

Dye permeability and alkaline phosphatase activity of testicular capillaries in the postnatal rat

Summary Staining of testicular and epididymal tissues after intravenous, intraperitoneal or subcutaneous administration of a number of dyes was investigated in rats at different stages of postnatal development. After light green injections heavy staining of both testis and epididymis was visible to the naked eye in neonatal animals up to the age of 10 days, while in rats over 15 days old no appreciable staining of the testis could be seen, although the caput epididymis was strongly coloured. From 3–8 hours after subcutaneous acriflavine administration, the nuclei in the blood vessel walls of the testis, as well as the nuclei in the rete testis, tubuli efferentes and caput epididymis, fluoresced in all age groups. The nuclei of the interstitial and tubular cells were stained intensely until the age of 5 days. Thereafter the intensity gradually diminished until the age of 20 days, when no nuclear fluorescence was visible in the seminiferous tubules and even the interstitial nuclei fluoresced weakly or not at all.The histochemical alkaline phosphatase activity of the testicular capillaries was studied by Gomori's method, using fresh and postfixed cryostat sections from postnatal rat testes. The testicular capillaries exhibited appreciable activity at the age of 10 days.On the basis of the present and previous observations on the permeability of the testicular capillaries, the existence of a blood-testis barrier in the puberal and adult rat testis is suggested.Development of the blood-testis barrier and the alkaline phosphatase activity of the testicular capillaries are suggested to reflect general vascular maturation at the beginning of puberty in the rat.Supported by grants from Yrjö Jahnsson's Foundation and P. O. Klingendahl Foundation.     

Expression of scatter factor/hepatocyte growth factor is regionally correlated with the initiation of sperm motility in murine male genital tract: Is scatter factor/hepatocyte growth factor involved in initiation of sperm motility?

Based upon findings that the scatter factor/hepatocyte growth factor (SF/HGF) has strong mitogenic and motogenic properties, and that the sperm cell acquires its fertilizing capacity and motility in the distal parts of mammalian epididymis, the present study was conducted to investigate the role of SF/HGF in initiation of sperm cell motility. This was investigated by determining the expression of SF/HGF in various regions of the murine male genital tract by scatter and cell tracking assays using MDCK epithelial cells, Western blot procedure, and the immunohistochemical procedure using paraffin sections of various regions of the male genital tract. The findings from all these assays indicate that SF/HGF is differentially expressed in various parts of the male genital tract with slight or no expression in the testes, caput epididymis, and vas deferens, and with the highest expression in cauda and corpus (distal) epididymis followed by expression in the corpus (proximal) epididymis. This region-specific SF/HGF expression pattern coincides with the pattern of acquiring the fertilizing capacity and motility by the sperm cell during its transit through the male genital tract. However, wherever SF/HGF was expressed in the male genital tract, its molecular weight was slightly higher (Mr, 82 kD), compared to the SF/HGF expressed in various other somatic tissues (Mr, 78 kD), indicating that the genital tract SF/HGF may be a different molecular species that shares some immunoreactive epitopes with the somatic cell SF/HGF. Incubation of immotile sperm from caput epididymis with the purified human placental SF/HGF of 78 kD initiated motility in 5–15% of sperm population. These results strongly suggest that the SF/HGF-like activity is expressed in the male genital tract in a region-specific manner, and this activity may have a role in initiation of sperm motility acquired during its transit through the epididymis in mammals. © 1994 Wiley-Liss, Inc.     

Computer-assisted visualization of the rat epididymis: a methodological study based on paraffin sections autometallographically stained for zinc ions

A concept for the computer-assisted visualization of tubular organs is presented. Unmarked histological zinc-stained serial sections from the epididymis of the Wistar rat were aligned to demonstrate the concept. Virtual images were made through the aligned sections and served as controls for the alignment process. Animation of the serial sections and the virtual images revealed new information about the structure of the organ under investigation. The analysis was used to upgrade the anatomical knowledge of rat epididymis by describing how the epididymal duct runs through the structure. The proximal parts of the epididymis contain large communicating septa of connective tissue dividing the caput and the upper part of the corpus epididymidis into segments. The tortuousness was high in the caput with many turns within a small area of the epididymis, whereas longer loops were found in the lower part of the corpus and cauda epididymidis. The tube of the vas deferens was found to become an integrated part of the ductal system in the cauda epididymidis, although it was histologically easy to distinguish from the epididymal duct. The total number of cross-sections of the ductus epididymidis in the 2254, 15-mu m-thick, tissue sections analysed was 104 700, giving a minimum length of the ductal system of 1.5 m. © 1998 Chapman & Hall     

Glutathione conjugation in male reproductive system: studies on glutathione-S-transferase of bull and boar epididymis

Male reproductive organs are extremely sensitive to the negative influence of toxic environmental factors as well as drugs, and until now not many attempts have been made at studying the detoxication enzymes and the relationship between the activity of those enzymes and spermatozoa fertility. In the present work we studied cytosolic glutathione-S-transferases (GST, EC 2.5.1.18) from different parts (head, corpus and tail) of bull and boar epididymis. We isolated two molecular forms of GST from each part of epididymis, characterized their biochemical properties and examined the mechanism of the catalyzed reaction. On the basis of their substrate specificity and isoelectric point, the isoforms were found to belong to the near neutral GST class mi. All examined GST forms exhibited higher affinity towards GSH than towards 1-chloro-2,4-dinitrobenzene (CDNB) and bull epididymis GST forms showed biphasic Lineweaver-Burk double reciprocal curves in the presence of GSH as a variable substrate. Boar epididymis anionic GST had the -SH groups both in the GSH and the CDNB binding place, whereas the cationic GST form--arginine residues in the CDNB binding place. Bull epididymis GST forms contained neither thiol nor arginine residues essential for catalytic activity.     

Circadian rhythm of lactate dehydrogenase in rat testis

Activity of total lactate dehydrogenase (LDH) and of the isozyme X (LDH X or C4) have been determined at 2 hr intervals during 24 hr cycles in testis of adult rats maintained since birth in a photoperiod of 14 hr light: 10 hr dark. LDH X activity of epididymal sections (caput, corpus and cauda) from the same animals was also determined. Total LDH and LDH X activities in testis exhibited circadian rhythms with different timing. LDH X in the three portions of epididymis showed diurnal variations similar to those in testis. Rats subjected to constant light or constant dark presented marked modifications of LDH X profiles, indicating that the photoperiod plays a synchronizer role. While total soluble proteins did not show variations in testis of rats exposed to the photoperiod, a circadian rhythm was demonstrated in animals maintained in constant light or dark.     

Lactate dehydrogenase activity of rat epididymis and spermatozoa: effect of constant light

During its passage through the epididymis, the gamete undergoes a process of "maturation" leading to the acquisition of its fertilizing ability. The epididymis displays regional variations in the morphology and metabolic properties of its epithelium which are relevant for the progressive development of mature sperm characteristics. The epididymis has spontaneous peristaltic contractions and receives sympathetic innervation that is modulated by melatonin, a hormone synthesized and released by the pineal gland. Constant lighting disrupts melatonin synthesis and secretion. We have studied the effect of constant light on lactate dehydrogenase (LDH; EC 1.1.1.27) and its isozyme C4 activities and protein content in whole epididymis, epididymal tissue and in spermatozoa from caput and cauda segments. Animals were exposed from birth to an illumination schedule of 14 h light:10 h dark (group L:D). At 60 days of age one group of animals was submitted to constant light over 50 days (group L:L). In order to test the fertilizing ability, the rats of each group were mated with soliciting estrous females. The percentage of pregnancies in females mated with males maintained in L:L was remarkably lower than those in females mated with males maintained in the L:D photoperiod (44% and 88% respectively). Constant light increased protein concentration and LDH activity in caput as well as in cauda of total epididymis. On the contrary, in epididymal tissue, the protein content decreased in both epididymal sections compared with controls. When enzymatic activity was expressed in Units per spermatozoa, constant light induced a significant reduction of total LDH and LDHC4 in caput and cauda spermatozoa while LDH activity of epididymal tissue was not affected. In spite of the decrease in LDH per sperm cell when rats were exposed to constant light, in total epididymis (epididymis tissue plus sperm cells content) and in spermatozoa, values of enzyme activities expressed per weight unit were higher than those of controls. This is explained by the increase in the amount of stored spermatozoa, both in caput and cauda, produced by exposure of animals to constant light. Our results confirm that in rats, chronic exposure to constant light promotes a reduction of fertilizing ability and indicates that continuous lighting reduces the total LDH and LDHC4 activities, possibly due to moderate aging of spermatozoa within the duct by lengthening of the sperm transit through the epididymis.     

Antimicrobial activity of human EPPIN, an androgen-regulated, sperm-bound protein with a whey acidic protein motif

The role of epididymal sperm-binding proteins in reproductive tract immunity is now well recognized in addition to their role in sperm maturation. Spermatozoa acquire forward motility and fertilizing ability during their passage through the epididymis, where they acquire a wide variety of proteins belonging to different classes. Previously, we demonstrated that EPPIN (epididymal protease inhibitor), an androgen-regulated, sperm-binding protein containing protease-inhibitory motifs, is expressed specifically in the testis and epididymis. In the present study, we investigated the antibacterial activity of EPPIN against Escherichia coli and the mechanism of antimicrobial action. EPPIN exhibited dose- and time-dependent antibacterial activity that was relatively insensitive to salt. However, EPPIN lost its antibacterial activity completely on reduction and alkylation of its cysteines, indicating the importance of disulfide bonds for its activity. EPPIN permeabilized the outer and inner membranes of E. coli, which is consistent with its ability to induce striking morphological alterations of E. coli membranes as shown by scanning electron microscopy. EPPIN did not cause disruption of eukaryotic membranes in the rat erythrocyte hemolytic assay. The present results indicate that EPPIN has a role in the innate immune system of human epididymis.     

Cofactor activity of dihydroflavin mononucleotide and tetrahydrobiopterin for murine epididymal indoleamine 2,3-dioxygenase

Dihydroflavin mononucleotide (FMNH2) and tetrahydrobiopterin (BH4) serve as cofactors for indoleamine 2,3-dioxygenase isolated from mouse epididymis. The optimal pH was between 7 and 8, and FMNH2-dependent activity was 4 to 5-fold higher than activity with methylene blue as the electron donor. Using FMNH2 with a FMN reductase system, the enzyme exhibited higher efficiency and specificity for L-Trp (an apparent Km of 1 X 10(-5)M and an apparent Vmax of 182 nmol/min/mg of protein). The apparent Km and Vmax for D-Trp were 6.2 X 10(-5)M and 31 nmole/min/mg, respectively. Consequently, these observations appear to present the first evidence for a flavin-dependent mammalian dioxygenase.     

Comparison of the N-acetylhexosaminidase components in the ram testis and epididymis by isoelectric focusing

1. Several enzymic components, with both N-acetyl-beta-glucosaminidase and N-acetyl-beta-galactosaminidase activity, have been demonstrated in the ram testis and epididymis by isoelectric focusing. 2. The component (I), which predominates in the testis and caput of the epididymis, is isoelectric at pH6.2+/-0.1, whereas the predominant component (II) in the epididymal isthmus and cauda is isoelectric at pH7.0+/-0.1. 3. The total activity and the relative proportions of the enzymic components vary in the different sections of the epididymis. 4. Although their pH optima are slightly different, the appropriate K(m) values for components I and II for the hydrolysis of 4-methylumbelliferyl-N-acetyl-beta-glucosaminide and N-acetyl-beta-galactosaminide and the ratios of the maximal velocities towards the two substrates are very similar.     

Immature rat epididymal epithelial cells grown in static primary monolayer culture on permeable supports

Summary N-acetylglucosaminidase (NAG), acid phosphatase (ACP) and alkaline phosphatase (AKP) were localised histochemically in fixed cells from the 37-day-old rat epididymis grown in static monolayer culture for 2–8 days. ACP and NAG were cytosolic enzymes found in perinuclear positions, whereas staining of AKP was consistent with a membranous position. These enzymes were also examined in frozen tissue sections of the epididymis, from rats of the equivalent age, where NAG had intense activity in both supra- and infra-nuclear cytoplasm and ACP was more active apically. For the first time AKP was localised along basolateral membranes of the epithelium and in the lumen of the mid-caput region. The monolayer in culture was of principal cells only and they maintained their polarity and ultrastructural characteristics, but the height of the cells was reduced compared to that obtained in situ.     

Characterization of epidermal growth factor receptor in testis, epididymis and vas deferens of non-human primates.

The presence of the epidermal growth factor receptor (EGFR) in testis, epididymis and vas deferens of monkeys was demonstrated using a polyclonal antibody (RK2) raised against a peptide-specific sequence of the intracellular domain of the human EGFR. Immunoblotting of membrane preparations revealed a specific band at approximately 170 kDa corresponding to those of controls, A431 and monkey liver cells. Cryostat sections were stained by biotin-streptavidin peroxidase immunocytochemistry. The liver showed positive staining along the basolateral membranes of the hepatocytes lining the sinusoids. The testis showed positive staining indicating the presence of EGFR in Leydig cells, Sertoli cells and peritubular cells. In the epididymis, immunostaining of the EGFR was observed on both the basolateral and the luminal borders of the epididymal epithelium. Immunofluorescence studies revealed a similar pattern of EGFR distribution in the epididymis and indicated that the luminal immunostaining was vesicular. In the vas deferens, positive immunostaining was detected in a pattern very similar to that observed in the epididymis. There was no positive staining in the interstitium of the epididymis or in the smooth muscle cell layers of the vas deferens. The sections of all tissues treated with pre-immune serum were negative. These results suggest that EGF in the primate testis may act at the level of somatic cells. In addition, the basolateral and luminal EGFR staining in the epididymis and vas deferens suggest that these cells respond to an EGF, or EGF-like, source both at the basal, luminal or at both sides of the cells, or that these tissues serve as sites of EGF transcytosis across the epithelium.     

Seasonal changes in the chemical composition of red deer (Cervus elaphus) semen

During reproductive quiescence, apart from a rapid decrease in total protein content, there was a 2–3 fold decrease in the activity of certain enzymes (glutamic-oxaloacetic transaminase (GOT), alkaline phosphatase, acrosin inhibitors) in the seminal plasma of red deer stags. The activity of the enzymes of the spermatozoa, hyaluronidase and acrosin, increased in the seminal plasma, probably due either to ageing of the spermatozoa, accumulated in the epididymis, or to changes in the permeability of cell membranes.These phenomena were accompanied by an increased concentration of albumins originating in blood, and by a noticeable disappearance of globulin fractions. This would point to a change in the permeability of the tissue barriers in the deer stag reproductive system, and to a decreased rate of protein synthesis in particular sections of this system, especially in the epididymis, during reproductive quiescence.     

Effects of prenatal exposure to a 900 MHz electromagnetic field on 60-day-old rat testis and epididymal sperm quality

We investigated the effects of exposure in utero to a 900 megahertz (MHz) electromagnetic field (EMF) on 60-day-old rat testis and epididymis. Pregnant rats were divided into control (CG; no treatment) and EMF (EMFG) groups. The EMFG was exposed to 900 MHz EMF for 1 h each day during days 13 ? 21 of pregnancy. Newborn rats were either newborn CG (NCG) or newborn EMF groups (NEMFG). On postnatal day 60, a testis and epididymis were removed from each animal. Epididymal semen quality, and lipid and DNA oxidation levels, apoptotic index and histopathological damage to the testis were compared. We found a higher apoptotic index, greater DNA oxidation levels and lower sperm motility and vitality in the NEMFG compared to controls. Immature germ cells in the seminiferous tubule lumen, and altered seminiferous tubule epithelium and seminiferous tubule structure also were observed in hematoxylin and eosin stained sections of NEMFG testis. Nuclear changes that indicated apoptosis were identified in TUNEL stained sections and large numbers of apoptotic cells were observed in most of the seminiferous tubule epithelium in the NEMFG. Sixty-day-old rat testes exposed to 900 MHz EMF exhibited altered sperm quality and biochemical characteristics.     

Profiling and imaging proteins in the mouse epididymis by imaging mass spectrometry

Different aspects of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) have been used as discovery tools to obtain global and time-correlated information on the local proteomic composition of the sexually mature mouse epididymis from both qualitative and semiquantitative points of view. Tissue sections and laser captured microdissected cells and secretory products were analyzed by MALDI-MS and from the recovered protein profiles, over 400 different proteins were monitored. Over 50 of these, some of which have been identified, displayed regionalized behavior from caput to cauda within the epididymis. Combining the information obtained from high-resolution imaging mass spectrometry and laser captured microdissection experiments, numerous proteins were localized within the epididymis at the cellular level. Furthermore, from the signal intensities observed in the different protein profiles organized in space, semiquantitative information for each protein was obtained.     

Simultaneous Formalin Fixation and Edta Decalcification, With Carbowax Embedding for Preservation of Acid Phosphatase

Carbowax serial sections from pubic symphyses of female mice, fixed and decalcified in a 10% formalin-5% Versenate solution for 18 hr at 4 C, pH 5.2, were incubated for 30 min with Burstone's simultaneous coupling reagent (pH 5.2); substrate: naphthol AS-TR and the diazonium salt, fast red violet L.B. All sections were counterstained with 1% methyl green at pH 4.0 in a phospho-citrate buffer. Inhibition by 0.01 M NaF, 0.0002 M CuCl₂, 10% tartaric acid and 0.01 M NaCN, as well as substrate-deficient and heat-inactivated controls, demonstrated conclusively that acid phosphatase was functionally preserved. Strong enzymatic activity was exhibited by osteoclasts, chondroclasts and free multinucleated giant cells. In addition, megakaryocytes, histiocytes, plasma cells, and monocytes exhibited moderate activity. The results demonstrated the technique to be consistently reproducible.     

胎儿附睾的组织化学观察

本研究用光镜观察了在６例不同胎龄的胎儿及２例新生儿的附睾各段内ＡＫＰ、ＡＣＰ和ＳＤＨ在胎儿期附睾起始段的活性比其它各段都强,并且以３３周时为最强;至新生儿期,ＡＣＰ和ＳＤＨ在胎儿期附睾起始段的活性比其它各段都强并以３３周时最强,至新生儿期,ＡＣＰ和ＳＤＨ的组织化学活性及ＰＡＳ反应,并进行了半定量评估。结果显示：ＡＫＰ只在新生儿时期有微弱的活性;ＡＣＰ和ＳＤＨ的酶活性都比３３周时弱。ＰＡＳ阳生反应首先出现在６月龄的附睾各段,随后逐渐增强,至３３周时,附睾体、尾段的反应最为强烈,此后至新生儿期则轻微地减弱。根据文献对上述不同胎龄胎儿及新生儿附睾内酶活性及ＰＡＳ反应的变化进行了讨论。     

Changes in the specific activity of N-acetyl-beta-D-hexosaminidase in the rat epididymis with age.

The specific activity of the lysosomal enzyme N-acetyl-beta-D-hexosaminidase was determined in the caput and cauda of the epididymis of rats as a function of age. The activity peaks at six weeks of age in both parts of the epididymis but is higher in the cauda. The results indicate a relationship between changes occurring in sperm in the epididymis and the lysosomal enzymes.