Haptoglobin is a Sertoli cell product in the rat seminiferous epithelium: its purification and regulation

Using multiple HPLC steps, a protein of 67 kDa (estimated by gel permeation HPLC) was purified from Sertoli cell-enriched culture medium that consisted of two dissimilar subunits of 9 (alpha chain) and 24 (beta chain) kDa on SDS-polyacrylamide under reducing conditions. Direct protein sequence analysis of the 9-kDa subunit revealed a sequence of NH2-VELGNDATDIEXD, which is identical to the alpha subunit of the rat haptoglobin (Hp). Hp is a 67-kDa tetrameric serum acute-phase protein consisting of two alpha and two beta subunits (alpha2beta2) of 8.5 kDa and 24.5 kDa, respectively. Using a 351-bp cDNA coding for Hp for northerns and two Hp primers for RT-PCR, we have demonstrated the expression of Hp in Sertoli and Leydig cells, germ cells, and the testis, but not in the epididymis. In contrast to the hepatic haptoglobin, an acute-phase protein whose steady-state mRNA level increased by as much as fivefold during induced inflammation, the testicular homolog reduced by fourfold within 24 hours following induced inflammation, suggesting that this gene is regulated differently in the testis and in the liver. Moreover, the testicular steady-state Hp mRNA level increased steadily after birth during maturation, suggesting its involvement in spermatogenesis. Using primary Sertoli cell cultures in vitro, it was found that the Sertoli cell Hp expression was not regulated by either FSH, testosterone, estradiol, dexamethasone, interleukin-1beta (IL-1beta), IL-6, interferon-gamma (INF-gamma), transforming growth factor-beta (TGF-beta), lymphocyte inhibitory factor (LIF), or germ-cell-conditioned medium (GCCM). Since transferrin secreted by Sertoli cells is an important molecule in maintaining the crucial iron level necessary for spermatogenesis, the identification of haptoglobin as a Sertoli and germ cell product adds a new member to the growing family of metal transporters in the testis that are likely to play an important role in iron metabolism in the testis.     

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.     

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.     

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.     

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.     

The additional predictive value contributed by quantitative chromatin pattern description as compared to DNA ploidy level measurement in 257 superficial bladder transitional cell carcinomas

The purpose of the present study was to demonstrate the post-translational modifications of sperm plasma membrane proteins by fatty acid acylation during sperm maturation in the epididymis. Rat epididymal spermatozoa were incubated at 37 degrees C with various concentrations (100 microCi and 1 mCi) of [9-10(n)3H]palmitic acid in a medium containing Tyrode's solution supplemented with sodium bicarbonate, sodium pyruvate and sodium lactate. The incorporation of [3H]palmitate in vitro was determined in epididymal spermatozoa and an attempt was made to identify the lipid-linked proteins of purified plasma membranes of maturing epididymal spermatozoa by autoradiography. The studies demonstrated that [3H]palmitate was covalently linked to a subset of membrane cytoskeleton proteins of maturing rat spermatozoa. The pattern of incorporation of lipid was a maturation-associated phenomenon as caput spermatozoa incorporated more radioactivity than did caudal spermatozoa. The labelled proteins appeared to be membrane-bound since 82% of radioactivity was associated with membrane fractions. Autoradiograms of SDS-PAGE gels of labelled caput sperm extract showed three prominent palmitate-incorporating protein bands of about 70, 56 and 36 kDa and few minor bands. Most of these proteins were present in the membrane fraction of caput spermatozoa. Labelled gels of both the sperm extracts and of purified membranes showed resistance to hydroxylamine treatment, suggesting that there are amide bonds between lipid and proteins. The higher incorporation of labelled palmitate by immature spermatozoa of the caput epididymis compared with mature spermatozoa from the cauda epididymis and the addition of palmitate to plasma membrane proteins of caput epididymal spermatozoa suggest that fatty acylation is a post-translational modification of sperm membrane proteins.     

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.     

Sertoli cell-specific expression of rat androgen-binding protein in transgenic mice: effects on somatic cell lineages

The Sertoli cells of many species produce an androgen binding protein (ABP) which carries testicular androgens to the epididymis and is thought to play a role in sperm maturation. In the present report we analyzed the morphological modifications present in Leydig, Sertoli, and peritubular cells of the testis of young adult male mice transgenic for ABP gene, which overproduce ABP in testis. By in situ hybridization we demonstrated that ABP is specifically produced by Sertoli cells. Using light and electron microscopy, we detected scattered alterations of the seminiferous tubule cells which include cell degeneration and vacuolization. Leydig and Sertoli cells present morphological signs of hyperfunctioning compensatory mechanisms which include increased amounts of lipid droplets probably due to the existence of a stimulated steroid synthesis that in turn could be a consequence of the decreased unbound testosterone and/or a direct paracrine effect of ABP. Peritubular cells also present numerous signs of hyperstimulation.     

Effect of zinc on the epithelial lining of mice epididymis--a light microscopic study

Intraperitoneal zinc chloride was administered at 7.5 micrograms/g body weight and 15 micrograms/g body weight to 10-12 weeks old Swiss albino mice for 5 consecutive days. Control animals were given normal saline. The testis and epididymis were dissected and examined under the light microscope. Micrographs of the testes appeared normal in both treated and nontreated animals. However the group of animals treated with the higher dosage of zinc chloride showed evidence of rupture and collapse of the epididymal epithelial lining. The testes were not affected probably because of (a) known higher testicular concentration of metallothioneins which can bind the zinc and consequently detoxify the metal and (b) "stratified" epithelium comprising of spermatogenic and Sertoli cells.     

Immunochemical distribution and immunohistochemical localization of 20beta-hydroxysteroid dehydrogenase in neonatal pig tissues

Immunochemical distribution of 20beta-hydroxysteroid dehydrogenase (HSD) in neonatal pig tissues was investigated by Western blot analysis of the proteins reacting with anti-20beta-HSD antibody. 20beta-HSD was present in all organs investigated: brain, lung, thymus, submandibular gland, heart, liver, kidney, spleen, adrenal gland, testis, epididymis, prostate, vas deferens and seminal vesicle. In particular, high concentrations of 20beta-HSD were detected in the testis, followed by the kidney and liver, by the [125I]-protein A binding method. Immunohistochemical localization of the enzyme was achieved in paraffin sections of the testis, kidney, liver, epididymis, and vas deferens by the streptoavidin-biotin complex method. In the testis, very strong immunostaining was found only in interstitial Leydig cells, whereas the cells in seminiferous tubules, such as Sertoli cells and spermatogenic cells, were entirely negative. In the kidney, strong immunostaining was detected in epithelial cells of Henle's loop. The immunoreactive proteins were also localized in the hepatic lobules of the liver, tall columnar cells of the ductus epididymidis of the epididymis, and mucosal epithelium cells and muscularis of the vas deferens. These observations indicate that tissue distribution of 20beta-HSD is similar to that of carbonyl reductase in the human and rat. However, the specific and abundant expression of 20beta-HSD in testicular Leydig cells of the neonatal pig, which are concerned with the synthesis of androgens, suggests that 20beta-HSD has a very important physiological role in testicular function during the neonatal stage.     

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.     

Expression of inhibin alpha-subunit in horse testis

Inhibin is believed to play roles in the pituitary secretion of FSH and in the paracrine regulation of testicular function. Although it has been generally accepted that inhibin is produced in Sertoli cells, there was a recent evidence for the localization of inhibin in Leydig cells of primates, rat and sheep. However, there is no report on the expression of inhibin in the adult horse testis. Therefore, using immunohistochemistry, western blotting and in situ hybridization techniques, the present study examined inhibin alpha-subunit (Ih-alpha) expression in the adult horse testis. For the detection of Ih-alpha protein, we used anti-porcine Ih-alpha antibody in immunohistochemistry and western blotting. Furthermore, digoxigenin-labeled complementary RNA probes were prepared to detect intracellular messenger RNA (mRNA) of Ih-alpha. Immunostainings for Ih-alpha were found not only in Leydig cells but also in Sertoli cells. The intensity in Leydig cells was stronger than in Sertoli cells. Immunoreactivities for Ih-alpha were found at approximately 46 kDa, 56 kDa and 90 kDa in the homogenates from testicular interstitial tissues. The bands at 56 kDa and 90 kDa agree with previous report, but not at 46 kDa. Signals for mRNA of Ih-alpha by in situ hybridization were detected in Leydig cells and in the basal region of seminiferous epithelium including Sertoli cells. These results suggest that Ih-alpha is expressed in Leydig cells and Sertoli cells of horse testis, and the expression level should be higher in Leydig cells than Sertoli cells.     

Developmental expression of testis messenger ribonucleic acids in the rat following propylthiouracil-induced neonatal hypothyroidism

Propylthiouracil- (PTU) induced transient neonatal hypothyroidism increases adult rat testis weight 80-100%; this effect involves prolongation of Sertoli cell proliferation. To gain insight into developmental effects of PTU on the testis, we used Northern analysis to examine chronological expression of Sertoli cell mRNA in postnatal rat testes from rats that were untreated (controls) or were given PTU from birth to Day 25. Treated rats showed prolonged early expression of genes associated with dividing Sertoli cells such as MIS (Müllerian inhibiting substance) and c-erbA alpha (thyroid hormone receptor). Expression of several other Sertoli cell mRNAs (androgen-binding protein [ABP], clusterin, and inhibin-beta B) was delayed, as was that of hemiferrin, a spermatid-specific mRNA. Temporal expression patterns for other mRNAs (sulfated glycoprotein [SGP]-1, transferrin, and inhibin-alpha) were similar in control and treated animals. Additionally, thyroid hormone replacement in PTU-treated animals decreased MIS and c-erbA alpha mRNA expression to control levels. The altered developmental pattern of expression of a number of major Sertoli cell genes reflects a prolonged mitogenesis and delayed maturation of Sertoli cells in neonatally hypothyroid animals. Furthermore, our results suggest that thyroid hormone may directly potentiate molecular events associated with cessation of Sertoli cell proliferation and maturation during early testis development.     

Is sperm motility maturation affected by static magnetic fields?

Kinematic parameters were evaluated in mouse epididymal extracts to monitor maturation of sperm movement in animals exposed to static magnetic fields using the Sperm-Class Analyzer computerized image analysis system. For this purpose, animals were exposed to a field of 0.7 T generated by a permanent magnet over 10 or 35 days for either 1 or 24 hr/day. The values of the motion endpoints were similar in animals used as controls and in those exposed to the nonionizing radiation, whatever the period of exposure or daily dosage. Changes in motility were observed in all groups: the percentage of total motile and progressive motile spermatozoa increased during passage through the epididymis, with major changes between the caput and corpus epididymides, and the pattern of swimming changed clearly towards more rapid and straighter trajectories. The processes of initiation of sperm motility and maturation of displacement patterns were not then affected by magnetic treatment. Moreover, it appears that sperm production is unaffected because no changes were observed in testicular or epididymal weights after exposure to static magnetic fields.     

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.