Activities and androgenic regulation of kreb cycle enzymes in the epididymis and vas deferens of rhesus monkey

The activities of nine enzymes of the TCA cycle were estimated in the initial segment, caput, corpus and cauda segments of epididymis and vas deferens of adult rhesus monkey and expressed as units per mg DNA. These enzymes were also estimated in epididymal segments and vas deferens of castrated and castrated-androgen replaced monkeys as well. Results indicated higher activities of most of the enzymes in vas deferens as compared to epididymal segments. All the enzymes showed marked reduction in epididymis and vas deferens after castration, the effect being much more pronounced in the epididymis, than in the vas. Androgen replacement in castrated monkeys stimulated most of the enzymes markedly in epididymis and in the vas deferens as compared to their castrated values. The response of cauda and vas deferens to exogenous androgen treatment was however moderate, as compared to the other epididymal segments. The studies indicate that energy metabolism in the epididymis (as well as in the vas deferens) is strictly androgen dependent and the energy charge of these target organs is likely to fall appreciably after castration, which may in turn affect many energy dependent processes of these organs (e.g. absorption, secretion of specific substances etc.) which have been considered important for sperm maturation and survival.     

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

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

Surveillance of multiresistant bacteria: justification, role of the laboratory, indicators, and recent French data]

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.     

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 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 [corrected] +/- 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 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.     

Segment-specific morphological changes in aging Brown Norway rat epididymis

In aging Brown Norway rats, both spermatogenesis and steroidogenesis decrease. Little is known about changes in the epididymis during aging. However, since the two major components entering the epididymis from the testis change, we hypothesized that epididymal histology would be affected by advancing age. The epididymides of Brown Norway rats ranging in age from 3 to 24 mo were prepared for light and electron microscopy. Striking quantitative and qualitative changes were noted. There was an age-dependent increase in the thickness of the basal membrane and in the number of halo cells. There were also major segment-specific changes in the appearance of cells along the epididymis with age. At 12 mo, basal cells in the initial segment emitted pseudopods into the basement membrane. By 18 mo, in the caput epididymidis, clear cells were filled with lysosomes; these cells frequently showed bulging protrusions into the lumen. In the corpus epididymidis, the cytoplasm of principal cells had numerous large lysosomes both below and above the nucleus; apical cells were usually occupied by one giant membranous lysosome. In the proximal cauda, clear cells became filled with dense lysosomes, and principal cells presented large clear vacuoles; debris from spermatozoa was found in the larger vacuoles. In summary, aging of the epididymis was accompanied by the emergence of characteristic features of aging and activation of the immune system. Furthermore, there were many cell- and segment-specific changes. Finally, these changes were not related to the presence of spermatozoa, often preceding their disappearance, thus indicating that there may be an intrinsic mechanism of aging in epididymal epithelial cells.     

Rat epididymis-specific sperm maturation antigens. I. Evidence that the 26 kD 4E9 antigen found on rat caudal epididymal sperm tail is derived from a protein secreted by the epididymis

Monoclonal antibody 4E9, which was raised against a partially purified detergent extract of rat caudal epididymal sperm, recognizes the tail of sperm from the cauda, but not from caput epididymidis, as well as epithelial cells in a restricted region of the distal caput/corpus epididymidis and proteins in epididymal fluid from corpus and cauda epididymidis. The antigen is apparently a glycoprotein, since it is retained on a Ricinus communis agglutinin I lectin column. Epididymal fluid antigens have apparent M(rs) of 38-26 kD, whereas the membrane-associated form of the molecule has an M(r) of 26 kD. Immunocytochemical data and Western immunoblot data suggest that the membrane antigen is derived from the fluid antigen, which, in turn, is secreted by the epididymal epithelium. Characterization of the membrane antigen indicates that it is tightly associated with the sperm surface, behaving as though it is an integral membrane protein. The antigen persists on ejaculated sperm.     

Effect of chronic experimental poisoning with benzene and ethylene vapors on the circulatory system

To examine the content and the composition of free amino acids in the intraluminal fluid of rat epididymis, the fluids were obtained by light pressure on the dissected tissues. The amount of the total free amino acids in the pressed fluid from the caput epididymis was significantly higher than those of the cauda epididymis and the testis. Glu and Gln were predominant amino acids in the caput, and their amounts occupied more than half of the total ninhydrin reactive compounds. Such a high concentration of Glu and Gln was not observed either in the cauda or in the testis. Castration decreased Glu and increased Gln in amount. Testosterone treatment to castrated animals did not restore Glu and Gln contents in the pressed fluid from the caput epididymis to the level observed in intact rats completely. Therefore, it was assumed that a large amount of Glu in the caput was due to many factors; secretion and metabolism of epithelial cells of the gland which might be regulated by androgen, inflow of rete testis fluid, and sperm metabolism of amino acids in the epididymis. The results obtained from the caput epididymis to which the efferent duct of the testis was ligated also supported this interpretation.     

Testosterone secretion by the early fetal pig testes in organ culture

The alkaline phosphatase activity was measured in testicular fluid and in epididymal plasma from caput and cauda epididymidis in boars with normal sperm production and in boars in which the number of spermatozoa passing from the testis to the epididymidis was reduced. The testicular fluid and the epididymal plasma from caput epididymidis contained low amounts of alkaline phosphatase in comparison with epididymal plasma from the cauda. This applies to both groups of boars e.g. boars with normal as well as with totally lacking or lowered sperm production. As no fluid resorption takes place between caput and cauda the distal part of the epididymidis must be the main production site for alkaline phosphatase. The production there is not related to the presence of spermatozoa in the duct. In the caput, on the other hand, it seems that the level of alkaline phosphatase in some way is influenced by the sperm supply to the duct.     

Shedding of a rat epididymal sperm protein associated with infertility induced by ornidazole and alpha-chlorohydrin

The protein composition of epididymal fluid and sperm extracts of rats treated with the nitroimidazole compound ornidazole was investigated by two-dimensional gel electrophoresis. Epididymal luminal fluid from the corpus and cauda regions of male animals rendered infertile by ornidazole treatment contained a prominent protein (contraception-associated protein 1, CAP1) with a molecular mass of approximately 25 kDa and an isoelectric point (pI) of 5.8; it was not found in fluids, but was present in sperm, from fertile vehicle-fed rats. Infrared matrix-assisted laser desorption/ionization mass spectrometry indicated that the molecular mass of CAP1 was 20420+/-120 daltons. Analysis of 17 amino acids demonstrated 49% homology to a diuretic hormone from an insect (Acheta domesticus). Densitometric quantitation of CAP1 on silver-stained gels indicated its presence in greater amounts in cauda than in corpus fluid from treated animals, whereas fluid from the rete testis lacked CAP1. In vitro incubations of tissue from the caput, corpus, and cauda epididymidal regions with [35S]methionine gave no hint that CAP1 was a secretion product of the epididymal epithelium. The absence of CAP1 from luminal fluid obtained from the sperm-depleted corpus epididymidis of efferent duct-ligated ornidazole-fed rats suggested a spermatozoal origin. CAP1 was present in spermatozoa from the caput epididymidis but not from the rete testis in control animals. Less CAP1 was present in detergent extracts of cauda sperm from ornidazole-treated rats than in sperm from control animals, suggesting a contraceptive-related displacement of protein from sperm to fluid. The association of ornidazole- and alpha-chlorohydrin-induced infertility with the presence of CAP1 in epididymal fluid, probably originating from spermatozoa, suggests a critical role for this protein in fertilization.     

Histochemical studies on sialic acids in the epididymis during post-natal development of the rat

A variety of sialic acids contained in the rat epididymis during post-natal development were examined by means of lectin and carbohydrate histochemistry. Epididymides from male Sprague-Dawley rats on post-natal days 14, 21, 30, 39, 49, 56 and 70 were fixed in Bouin's fluid and embedded routinely in paraffin wax. Hydrated sections were subjected either to the lectin methods using biotinylated Sambucus sieboldiana lectin or Maackia amurensis lectin or to the selective periodate oxidation-phenylhydrazine-thiocarbohydrazide-silver protein-physical development technique with or without saponification. The results revealed that sialic acids appeared in the epididymal epithelium at day 14, followed by particular distribution patterns corresponding to cell differentiation during days 21-39. High-level O-acetylation of sialic acids was observed in the principal cells of the initial segment and proximal caput after day 39. These results suggest that sialic acids with different linkages and O-acetylation become adult in distribution at the 'differentiation' period under the influence of androgen, before spermatozoa reach the epididymal lumen. Such carbohydrates may be correlated, at least in part, with sperm-binding sialoproteins, which increase dramatically during the window between days 21 and 39.     

Autoradiographic distribution and physiological regulation of 2-[125I]iodomelatonin binding in rat epididymis

Autoradiographic study was conducted to localize 2-[125I]iodomelatonin binding in the rat epididymis. In the peripubertal (6 weeks old), postpubertal (8 weeks old) and adult (3 months old) rats, intense specific 2-[125I]iodomelatonin labelling of the corpus epididymidis was observed. The intensity of 2-[125I]iodomelatonin binding in the distal epididymal segment was significantly decreased in orchidectomized rats but the effect could be reversed with testosterone replacement. The intensity of 2-[125I]iodomelatonin binding in the distal rat epididymal segment did not show any diurnal rhythmicity when mid-light period and mid-dark period levels were compared, and was unaffected by constant lighting. Our data suggest androgen-dependent expression of 2-[125I]iodomelatonin binding sites, independent of light-induced changes in circulating melatonin, in the rat corpus epididymidis. A novel role of melatonin and its receptor in the regulation of the functions of rat corpus epididymidis is strongly implicated.     

Classification and quantification of abnormal sperm along the epididymal tract. Comparison between adult and aged hamsters

The types and averages of abnormal sperm were studied in the epididymis of adult and aged golden hamsters. Abnormal spermatozoa represent 14.6-19.6% of the total of spermatozoa in adults, 31.7-42.1% in middled-aged hamsters, and 39.3-50% in advanced-aged hamsters. Twelve abnormal shapes were found, with the lack of an acrosome, the lack of a head, and the coiling of the tail, being the most frequent in the three age groups. An important increase in the number of coiled spermatozoa was found in the corpus and proximal cauda of the epididymis, but a decrease was observed in the distal cauda. Our data suggest that the epididymis produces secondary defects in spermatozoa running from the proximal caput to the middle zone of the duct, but that many of these spermatozoa are eliminated in the distal cauda. Such a result is mainly found in aged animals, in which a higher percentage of abnormal sperm from secondary origin is found with respect to adults.     

Assessment of protein synthesis and secretion by rat seminiferous and epididymal tubules in vivo

In vivo microperifusion and micropuncture were used to study tubule protein synthesis and proluminal secretion by the male reproductive tract in vivo. Seminiferous and caput and cauda epididymal tubules were perifused for 3 h. with [35S]-methionine. Perifused interstitial fluid (IF), lumen fluid (LF), and tubule extract (TE) were collected. Proteins were separated by SDS-PAGE, and autoradiograms were developed. Trichloroacetic acid precipitable proteins in each fluid were determined and a protein synthesis index (PSI) was calculated. PSI values demonstrated that the cauda epididymis synthesized less protein in vivo than did either seminiferous or caput tubules. Seminiferous tubules synthesized and secreted into the tubule lumen a relatively constant panel of proteins. Epididymal tubules synthesized and secreted proteins in a region-specific manner. In the caput epididymis the most prominent secreted bands were consistent with the heavy and light chains of epididymal clusterin. In the cauda epididymis, the most prominent synthesized and secreted protein was a 25 kDa protein consistent with the protein D. The above approach to studying protein synthesis and secretion will allow direct study of the physiological and pathophysiological effects on this important epithelial function in vivo.     

Effect of the colony-stimulating factor-1 null mutation, osteopetrotic (csfm(op)), on the distribution of macrophages in the male mouse reproductive tract

Macrophages are found throughout the male reproductive tract and its accessory glands. Mice homozygous for a null mutation (csfm(op)) in the gene for the mononuclear phagocytic growth factor colony-stimulating factor-1 (CSF-1) have a significantly lower density of macrophages, defined by the mononuclear phagocytic antigen F4/80, in the testis, cauda and caput epididymis, prostate, seminal vesicles, and vas deferens. These data indicate that CSF-1 is the major growth factor regulating the occurrence of macrophages in male reproductive tissues. The residual macrophages were correctly located in the tissue except in the caput epididymis, where they failed to take up positions adjacent to the tubular epithelium. Restoration of circulating CSF-1 concentrations in csfm(op)/csfm(op) males totally restored F4/80+ cell density in the testis and caput and cauda epididymis and partially restored their density in the vas deferens and seminal vesicles but failed to affect density in the prostate. This failure to correct all populations with circulating CSF-1 suggests the requirement for local synthesis of CSF-1 at appropriate developmental stages and/or its expression in a cell surface-associated form. The absence of macrophages in the testis and epididymis of csfm(op)/csfm(op) mice correlates with dysfunction in these tissues, suggesting that macrophages play important nonimmunological roles in these tissues.     

Mice with mottled mutation--a model for defective copper metabolism in humans

Androgen binding protein (ABP) has been shown to be secreted by Sertoli cells and to be actively taken up by the efferent ducts and proximal caput epididymidis and, yet, to be present at high concentrations in epididymal fluids. In the present study, ABP was immunolocalized by light microscopy in epithelial cells of the efferent ducts and epididymis of adult rats and during postnatal development and by electron microscopy in specific organelles within these cells. In adults, the efferent ducts actively endocytosed Sertoli cell-derived ABP. In the epididymis, principal cells displayed a variable staining reminiscent of a checkerboardlike pattern, with cells being intensely, moderately, or weakly reactive throughout their cytoplasm or unreactive. In the electron microscope, reactive cells displayed a labeling of their Golgi apparatus and secretory vesicles indicative of an epididymal-secreted form of ABP. However, labeling was also noted over endosomes of principal cells, but only of the initial segment and intermediate zone, which, along with labeling of coated pits and vesicles, indicated that ABP was also endocytosed by principal cells of these regions. The postnatal study revealed that principal cells attained an adultlike staining pattern indicative of secretion in a region-specific manner at different ages, suggesting that ABP secretion is regulated by different factors. Ligation of the efferent ducts of 15-day-old animals revealed no reaction along the entire epididymis in animals sacrificed at later ages, suggesting the importance of luminal testicular factors in its regulation during development. In addition, as in the adult, ABP was also endocytosed by principal cells, but only in the initial segment and intermediate zone. Taken together, the present results indicate that secretion of ABP occurs along the entire epididymis, whereas endocytosis is region specific. The functional role of ABP in the epididymis in relation to sperm maturation is discussed.     

The practical utility of performing peri-ictal SPECT in the evaluation of children with partial epilepsy

The distribution of membrane filipin sterol complexes (FSC) in the plasma membrane of the acrosomal region (PMAR) of rabbit sperm from epididymis and testis, in normal and hypercholesterolaemic rabbits, was examined at ultrastructural level. Membrane FSG were quantitatively analysed on freeze fracture replicas of filipin-treated cells. Cauda epididymal sperm shows a significant increase in filipin sterol complexes concentration in PMAR of hypercholesterolaemic animals compared to normal rabbits. Hypercholesterolaemic animals had 0.53 +/- 0.08 FSC micron-2 in the marginal segment of PMAR and 0.26 +/- 0.03 FSC micron-2 for normal animals. In the principal piece we found 0.70 +/- 0.07 FSC micron-2 for hypercholesterolaemic and 0.43 +/- 0.03 FSC micron-2 for control animals. We also counted 0.58 +/- 0.04 FSC micron-2 in the equatorial segment of PMAR for hypercholesterolaemic and 0.38 +/- 0.03 FSC micron-2 for normal animals respectively. The FSC concentration of testicular sperm, like sperm from corpus and caput of epididymis in hypercholesterolaemic animals, did not differ from the controls. Cholesterol, phospholipids and cholesterol:phospholipid ratio in caudal epididymal sperm from treated males did not differ from controls. Only the sphingomyelin concentration decreases in cauda epididymal sperm from hypercholesterolaemic males compared to controls. The results presented in this paper suggest that the lipidic domains in PMAR of hypercholesterolaemic rabbits changes when the gametes go through the epididymis.     

Sound-induced activation of auditory cortices in cochlear implant users with post- and prelingual deafness demonstrated by positron emission tomography

Lactoferrin has been for the first time purified from the porcine cauda epididymal fluid as a 70 kDa protein. Both Western and Northern blot analyses show that lactoferrin is synthesized in the regions from the distal caput to the cauda epididymis and secreted into the luminal fluid. Lactoferrin is first secreted as a 75 kDa glycoprotein and its carbohydrate moieties are gradually digested to form 70 kDa protein in the cauda epididymis. Lactoferrin has already bound to the surface of the epididymal sperm because the anti-lactoferrin antiserum induces the mature sperm tail-to-tail agglutination. These results strongly suggest new physiological functions of lactoferrin on the sperm maturation in the epididymis.     

Induction of programmed death/apoptosis androgen-dependent mouse mammary tumor cell line (Shionogi Carcinoma 115) by androgen withdrawal

Shionogi Carcinoma 115 (SC 115) cells are a cloned cell line derived from androgen-dependent mouse mammary tumor. They can grow in serum-free culture if a physiological level of androgen is present in the medium, but can not proliferate in culture without testosterone. In the present study, the mechanism of cell death in SC 115 cells after androgen withdrawal was examined. Based upon the temporal sequence of DNA fragmentation, morphologic changes and loss of cell viability, androgen withdrawal induces programmed cell death (apoptosis) of SC 115 cells in serum-free culture. Northern blot analysis was used to identify a series of genes whose expression per cell is enhanced during the recruitment of cells from a nonproliferative (i.e. G0) state into G1 (i.e.,cyclins D1 and C), from G1 into the S phase of the cell cycle (i.e., cdk2), and during the programmed cell death pathway (i.e. testosterone repressed prostatic message-2 (TRPM-2), transforming growth factor-beta1 (TGF-beta1) and glucose regulated 78 kilodalton protein (GRP-78). Expression of TRPM-2, TGF-beta1, GRP-78, and calmodulin genes increases, but that of cyclins C and D1, and cdk2 genes decreases during programmed cell death of SC 115 cells. These results demonstrate that androgen-dependent SC 115 cells undergo programmed cell death induced by androgen withdrawal, and that this death does not require proliferation or progression into G1 of the proliferative cell cycle. SC 115 cells should be a good model for investigating programmed death of hormone-dependent cancer.