Distribution pattern of HSP60 immunoreactivity in the testicular tissue of infertile men.
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Biopsy↗
Biomedical subjects
J Seitz
Publications and source records attributed to J Seitz.
Developmental expression of heat shock protein 60 (HSP60) in the rat testis and ovary.
Heat shock protein 60 (HSP60), a member of the chaperonin family, has an essential role in mediating correct folding of nuclear encoded proteins imported to mitochondria. We have investigated immunocytochemical expression of HSP60 in developing fetal, newborn, postnatal, and pubertal testis and ovary, and in the adult ovary of the rat. In the fetal gonads, HSP60 was expressed in the germ cells organized into sex cords and in the developing Leydig cells of the testis. In the pubertal testis, Leydig cells were strongly, spermatogonia and premeiotic spermatocytes moderately labeled, spermatids unlabeled. In the postnatal ovary, oocytes at all stages of folliculogenesis were positive for HSP60. In the pubertal ovary, glandular theca cells, and in the mature ovary, also the cells of the corpora lutea exhibited intense cytoplasmic labeling. At the electron microscopic level, immunogold particles were localized in the mitochondrial matrix, and in the Western blot analysis the antibody detected one single band of 60 kDa. Anti-HSP60 labeling in male and female sex steroid producing cells and their progenitors seems to be coordinated with the functional differentiation of these endocrine cells of the gonad. In the oocytes, a key element required for proper folding of imported mitochondrial proteins seems to be constitutively expressed throughout folliculogenesis. However, the data suggest that in the male germ cells mitochondrial chaperonin HSP60 is either not needed during the haploid phase of spermatogenesis or its level becomes extensively reduced and therefore undetectable by the methods used in the study.
Animals↗
Macrophage migration inhibitory factor production by Leydig cells: evidence for a role in the regulation of testicular function.
Macrophage migration inhibitory factor (MIF), described originally as a product of activated T lymphocytes, recently has been found to be released by monocytes/macrophages and the anterior pituitary gland. Immunohistochemical studies of the adult rat testis using an affinity-purified polyclonal antimurine MIF antibody demonstrated strong staining for MIF in Leydig cells and their putative precursors. Peritubular myoid cells and the seminiferous epithelium were negative for MIF staining; however, a weak reaction around the heads of elongated spermatids also was observed. The expression of MIF messenger RNA and protein in whole rat testis was demonstrated by Northern blot and Western blot analyses, respectively. Both MIF messenger RNA and protein immunoreactivity in Leydig cells was observed in testes obtained from long term hypophysectomized rats. Significant concentrations of intracellular MIF were detected in lysates of the TM3 Leydig cell line (7.23 +/- 2.6 pg/microgram protein), and testicular interstitial fluid contained 14.7 +/- 1.6 ng/ml MIF protein, as measured by MIF-specific enzyme-linked immunosorbent assay. To gain insight into the possible biological role of MIF in the testis, cultures of adult rat seminiferous tubules and purified Leydig cells were incubated together with recombinant murine MIF (rMIF). Neither rMIF (50 ng/ml) nor a neutralizing anti-MIF antiserum was found to affect basal or LH-stimulated Leydig cell steroidogenesis in vitro. However, a dose-dependent decrease in the secretion of inhibin by the seminiferous tubules was observed at rMIF concentrations ranging from 10-100 ng/ml. Taken together, these data indicate that Leydig cells produce MIF in vivo and suggest an important regulatory role for this newly discovered mediator of testicular function.
Animals↗
Instructive induction of prostate growth and differentiation by a defined urogenital sinus mesenchyme.
Instructive influences of fetal mesenchyme were examined in heterotypic tissue recombinants consisting of urogenital sinus mesenchyme (UGM) from male and female rats and distal ductal tips from adult rat prostate. Tissues were grown under the renal capsule of male hosts for periods up to 28 days. Resultant growths exhibited typical prostate histology. Expression of lobe-specific proteins for the ventral (prostatic steroid binding protein [PSBP]) lateral (seminal vesicle secretion II [SVS II]), and dorsal prostate (secretory transglutaminase [TGase]) were examined by immunocytochemistry. Male or female UGM combined with terminal segments of the ventral or dorsal prostate and immunolabeled with antibodies to lobe-specific proteins demonstrated expression of all three secretory products. The pattern of staining was consistent with a compound inductive response from the UGM. Unique to this study was our ability to use a defined mesenchymal tissue (female ventral mesenchymal pad [VMP]). This tissue is specifically associated with ductal branching morphogenesis and cytodifferentiation of the ventral prostate. Distal ductal tips from the dorsal lobe of the adult male prostate when recombined with female VMP and grown in vivo exhibited transformation of secretory phenotype, and the epithelium expressed mRNAs for PSBP. Immunocytochemistry of serial sections did not demonstrate labeling for TGase in the new epithelial growth. Ultrastructural analysis of the heterotypic recombinants indicated that the epithelium had similar characteristics to those of normal ventral prostate. Early stages of the mesenchymal-epithelial interactions resulted in dedifferentiation of the adult epithelium to solid cords of stratified cells. These findings illustrate the potent instructive capacity of a defined fetal UGM to influence development and cytodifferentiation of adult prostate epithelium.
Animals↗
Immunocytochemical localization of seminal proteins in salivary and lacrimal glands of the rat.
Antibodies against 10 different secretory proteins from the accessory sex glands of the male rat were used for immunohistochemical studies of salivary and lacrimal glands from intact and castrated rats, at the light- and electron-microscopic levels. In the parotid gland, secretory acinar cells showed immunoreactivity with antibodies against prostatic binding protein, cystatin-related peptide and acid phosphatase (isoenzyme pI 8.0; 5.6) typical of ventral prostate, and seminal vesicle secretion VI. Western blotting analysis indicated that immunoreactivity against prostatic binding protein was attributable to a subunit, presumably C3. Acid phosphatase pI 5.6 showed a molecular weight of 66 kDa, which is at variance with the prostatic form. Immunoreactivity for secretory transglutaminase, derived from the coagulating gland, was restricted to myoepithelial and stromal cells. In castrated animals, the immunoreactivity of acinar cells was reduced to the background level, whereas stromal transglutaminase immunoreactivity was unaltered. The distribution pattern of immunoreactivity for the proteins mentioned was almost identical in the lacrimal gland. Significant differences were however observed in the immunoreactivity of the inframandibular gland, where serous glandular cells were non-immunoreactive for seminal proteins, with the exception of acid phosphatase isoenzyme pI 8.0. Granules present in the convoluted granular ducts were immunoreactive particularly for acid phosphatase (isoenzyme pI 5.6) but much less for cystatin-related peptide; immunoreactivity was reduced after castration. The straight portion of the inframandibular duct system was immunoreactive for transglutaminase, but no influence of castration was visible. The distribution of immunoreactivity for seminal proteins present in the salivary and lacrimal glands and the pronounced androgen-dependence of their expression point to functional relationships of the respective proteins at both glandular sites.
Androgens↗
Distribution of rat hepatic steroid 5 alpha-reductase 1 as shown by immunohistochemistry.
Peptide fragments of rat liver 5 alpha-reductase were synthesized according to the published primary sequence data. The peptide were used (i) in free form and (ii) linked to keyhole limpet hemocyanin (KLH), respectively, for immunization of rabbits. Resulting antisera showed positive reaction with the respective peptide-antigen in an ELISA. In Western blot experiments the antisera specifically recognized a 26,000 mol wt protein in extracts from female and male rat liver. All antisera, as well as isolated immunoglobulins, showed inhibition of 5 alpha-reductase activity in microsomes prepared from rat liver. Positive immunohistochemical reactions were observed in the perinuclear cytoplasm of hepatocytes. No reaction was seen in Kupffer- and connective tissue cells. Acinar heterogeneity of the immunoreaction was seen in the male rat liver with a gradient from the portal canal to the central vein. After androgen-deprivation a considerable increase in immunoreactivity was seen in male rat liver cells, indicating androgen-dependent suppression of the enzyme in male liver.
3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
[Juvenile nasopharyngeal angiofibroma and antro-choanal polyp: role of MRT in the differential diagnosis].
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Angiofibroma↗
Expression of mitochondrial heat shock protein 60 in distinct cell types and defined stages of rat seminiferous epithelium.
Changes in the level of the gene transcript of heat shock protein (hsp)60, a mitochondrial chaperonin, during the cycle of rat seminiferous epithelium and its cellular localization were studied. The seminiferous epithelium showed a cell type-specific expression of hsp60. Immunostaining of adult rat testis revealed localization in Sertoli and Leydig cells. In germ cells, mitochondria of spermatogonia and early primary spermatocytes were immunoreactive for hsp60. Mitochondria of all other germ cell types were completely negative for hsp60. Stage-specific expression of hsp60 was determined from pooled segments of stage-specific microdissected tubules by a combination of Western blotting and polymerase chain reaction (PCR). High concentrations of hsp60 were found in stages I-V and IX-XIV, and low levels were detected in the other stages, i.e., VI-VIII. In stages with high hsp60 expression, spermatogonia divide mitotically, whereas in stages lacking mitosis, the hsp60 level was much weaker. In seminiferous epithelium, two different types of mitochondria are present. Therefore, immunoelectron microscopy was used to differentiate these two morphologically distinct types of mitochondria. The crista type of mitochondria (e.g., in Sertoli cells and spermatogonia) reacted with the antibody against hsp60, whereas hsp60 was negative in so-called "condensed"-type mitochondria found in midpachytene spermatocytes and more advanced germ cells. It could be shown for the first time that expression of the hsp60 gene is regulated during the cycle of the seminiferous epithelium. The results indicate that the gene product is primarily needed during the initial steps of spermatogenesis in which most of the cell divisions occur, while its expression during the differentiation of spermatids and sperm is obviously not necessary. The presence of hsp60 in stages with mitotic activity suggests a very active mitochondrial protein import and protein assembly machinery that generates further mitochondria for the dividing cells.
Animals↗
Mitochondrial differentiation during meiosis of male germ cells.
In male germ cells mitochondria undergo dramatic morphological changes during spermatogenesis, at least three different types of mitochondrion being present. The usual cristae type of mitochondrion in spermatogonia, preleptotene and leptotene spermatocytes develops, via an intermediate form in zygotene spermatocytes, to the condensed form with almost no cristae which is typical of pachytene spermatocytes and early spermatids. In cell culture experiments in which isolated preparations of meiotic germ cells were used, it was shown that condensed mitochondria in pachytene spermatocytes cultured in Earle's minimal essential medium dedifferentiated to the intermediate type, while Sertoli cell-conditioned medium (SC-CM) was able to maintain the condensed structure. SC-CM was also able to induce conversion of the intermediate type to the condensed type in isolated zygotene spermatocytes. Preliminary biochemical characterization showed the involvement of one or several proteinaceous factors > 10 kDa (PMMF: paracrine mitochondria maturation factor) that were protease (subtilisin)- and heat-sensitive. Three mitochondrial proteins served as markers for germ cells in different phases of maturation. The chaperonin hsp60 was detectable in the orthodox-type mitochondria of spermatogonia and primary spermatocytes (leptotene and zygotene). An ATP- dependent mitochondrial matrix enzyme -- the Lon-protease -- appeared in the orthodox and intermediate forms of mitochondria in leptotene and zygotene spermatocytes. Sulphydryl oxidase is present in the condensed mitochondria of pachytene spermatocytes and early spermatids.
Animals↗
Hormonally induced changes in apocrine secretion of transglutaminase in the rat dorsal prostate and coagulating gland.
Coagulating gland and dorsal prostate of the rat are peculiar in secreting transglutaminase, a protein-cross linking enzyme that is released in an apocrine fashion. To elucidate whether or not the intracellular pathway and the unusual extrusion mechanism proceed constitutively or were differentially regulated, transglutaminase immunoreactivity was studied both at the light and electron microscopic levels. In addition, ultrastructural morphometry and scanning densitometry were applied to quantitate hormone-dependent distribution of transglutaminase. Coagulating glands and dorsal prostate, respectively, from sexually active rats were compared to those from sexually inactive, castrated, estradiol-treated or testosterone-substituted castrated animals. In intact, sexually active animals, no labeling of the cisternae of rough endoplasmic reticulum was seen, but instead the hyaloplasm was labeled. In the supranuclear portions of the cells an increase in labeling density of the hyaloplasm subjacent to the plasma membrane was found, whereas no labeling of either Golgi stacks or vesicles was observed. Apical blebs projecting into the acinar lumen were densely labeled. In castrated animals, epithelium showed a reduction of rough endoplasmic reticulum, loss of secretory blebs, and a decrease in cell size. Morphometric analysis of immunolabeling of coagulating gland epithelium from experimental animals resulted in a highly significant reduction of labeling of the hyaloplasm and apical blebs which was reversed by testosterone supplementation of castrated animals. After estrogen treatment, the reduction in immunolabeling was less pronounced, but morphology of apical blebs was obviously changed. Results from scanning densitometry of Western blots correlated with quantitative immunoelectron microscopical findings. Northern blot analysis using a secretory transglutaminase cDNA probe showed characteristic changes at the RNA levels. Our results indicate that apocrine secretion of transglutaminase in rat coagulating gland and dorsal prostate is a hormonally controlled process, where androgen deprivation results in impaired biosynthesis and release of transglutaminase, whereas estradiol treatment only partially inhibits secretion, but changes morphological features of the glandular epithelium, especially apocrine bleb formation.
Animals↗
Tissue-type transglutaminase expression in the Dunning tumor.
Transglutaminases with different functions and tissue distribution patterns can be distinguished by specific antibodies and by inhibition of enzyme activity in the presence of guanosine triphosphate (GTP). The most common form is the so-called tissue-type transglutaminase that is apparently involved in membrane stabilization processes, e.g. during apoptosis, and can be inhibited by incubation with GTP at low calcium concentrations. A secretory transglutaminase that cannot be inhibited by GTP is synthesized in an androgen-dependent manner in the dorsal prostate of the rat, the site suggested to represent the origin of the Dunning tumor used as an experimental model in prostate cancer research. Here we studied the expression of transglutaminases in different Dunning tumor lines--mainly in the highly differentiated H subline--and characterized the enzyme both biochemically and immunocytochemically. A very high enzyme activity was found only in the less well differentiated HI-F tumor line. Immunohistochemical reactions and Western blot analysis showed that there is no secretory transglutaminase present in any of the Dunning tumor lines studied. Transglutaminase activity of the Dunning tumor results from the so-called tissue-type enzyme that is non-organ specific. The absence of a secretory form of transglutaminase does not support the contention of a prostatic origin of the Dunning tumor.
Animals↗
Sulfhydryl oxidase immunoreactivity in seminiferous tubules of infertile men.
Sulfhydryl oxidase (SOx) immunoreactivity was investigated in the seminiferous epithelium of human biopsy material from the testes of 33 adult men with disturbed fertility. SOx immunoreactivity was expressed in normal seminiferous epithelium in type-A spermatogonia (27 +/- 4% of all spermatogonia) (n = 4), in spermatocytes and round spermatids. Mature spermatozoa as well as Sertoli cells were unlabelled. Within the interstitium, Leydig cells were immunopositive. In biopsies of oligozoospermic men showing hypospermatogenesis (n = 24), an increase in labelled spermatogonia up to more than 90% was observed in biopsies, where seminiferous epithelia revealed only spermatogonia and Sertoli cells. Within the group of oligozoospermic patients there was a significant increase of labelled spermatogonia from 43 +/- 13% (greater than 20 mill/ejaculate) (n = 7) to 55 +/- 16% (less than 20 and greater than 10 mill/ejaculate) (n = 6) to 68 +/- 8% (less than 5 mill/ejaculate) (n = 11) and a significant (P = 0.01) decrease of score count from 7.0 +/- 2.7 to 2.0 +/- 1.8. In this group the increase of labelled spermatogonia was correlated with sperm concentrations in the ejaculate (correlation coefficient: r = -0.6). In biopsies of azoospermic patients showing maturation arrest at the level of spermatocytes or spermatids (n = 5) the percentage of labelled spermatogonia was within the range of 24% to 59%.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Arguments against the prostatic origin of the R-3327 Dunning H tumor.
The Dunning tumor, originally described as a carcinoma of the rat dorsal prostate, has for long been used as an experimental model of prostatic cancer. We have recently presented a number of morphological findings that are incompatible with the prostatic origin of the H-subline of the Dunning tumor. In this paper, biochemical and immunohistochemical markers of rat prostate and mammary gland are studied in the R-3327 Dunning H tumor. Pieces of the H tumor were inoculated in male or lactating female rats. The electrophoretic protein pattern of Dunning tumor extracts was more similar to that of the mammary gland than the dorsolateral prostate. Proteins selectively appearing after metabolic labeling in Dunning tumors grown in lactating rats corresponded to labeled proteins in mammary glands from the same animals. Secretory proteins typical of the lateral prostate (SVS II) and dorsal prostate (transglutaminase) could not be detected immunohistochemically in the Dunning tumor. Western blot studies of tumor extracts and slot blot analysis of RNA preparations from the tumor confirmed the absence of SVS II and prostate specific transglutaminase from the Dunning tumor. On the other hand, the presence of mammary gland proteins such as milk fat globule membrane proteins, lactoperoxidase and lactalbumin were detected in the Dunning tumor by immunohistochemistry and Western blotting, but were absent from the dorsolateral prostate. Transferrin-mRNA, expressed in the male urogenital tract and also in the liver and other tissues, was detected in the mammary gland and Dunning tumor, but not in the dorsolateral prostate. The absence of mammary gland secretory beta-casein in the Dunning tumor was related to the elevated Ha-ras oncogene expression in the tumor, previously reported to suppress casein expression. The findings clearly demonstrate that the prostate cannot be the origin of the Dunning tumor, presently being used in prostatic cancer research. The designation prostatic adenocarcinoma for this tumor is therefore invalid. Furthermore, the data support our view that mammary gland might be the origin of the Dunning tumor, although the derivation from the bulbourethral or the parotid glands cannot strictly be excluded.
Adenocarcinoma↗
SVS II--an androgen-dependent actin-binding glycoprotein in rat semen.
Rat seminal vesicles and the lateral prostate secrete a glycoprotein designated as SVS II in an androgen-dependent manner. SVS II, which has a M(r) of 49,000 and a pI of 10.5, is an actin-binding protein. G- and F-actins cosediment with SVS II at a ratio of 2:1 (actin:SVS II). SVS II affects the kinetics of actin polymerization in the same way as do barbed end capping proteins. Interaction with actin is specific for the skeletal and cardiac muscle isoforms and there is no corresponding interaction with cytoplasmic actins. The binding site is close to the C-terminus of actin. Monospecific polyclonal antibodies directed against the N-terminus of actin cross-react with SVS II, but there is no cross-reaction by a monoclonal antibody directed against a C-terminal epitope on actin. Recent sequence analysis of SVS II shows a sequence of about 14 residues that is repeated 13 times between residues 86 and 298. The consensus sequence based on these repeats is homologous to residues 10 to 25 of actin; this may account for the immunological cross-reactivity. Like actin, SVS II binds and inhibits the activity of DNase I, but SVS II has no effect on the ATPase activity of myosin subfragment 1. Thus, SVS II is an actin-binding protein which retains some properties of actin itself.
Actins↗
Purification and characterization of transglutaminases from the genital tract of the male rat.
In the genital tract of the male rat two different forms of the enzyme transglutaminase (TGase) could be identified and characterized. The coagulating gland and the dorsal prostate secrete a glycosylated and acylated TGase with a molecular weight of 65,000 dalton and pI value of 8.7. This secretory form was purified to homogeneity using preparative isoelectric focusing and gel filtration on a Superdex 200 column. Running fast protein liquid chromatographic gel filtration on a Superose 12 column in the presence of calcium ions, high-molecular-weight aggregates were physically formed which could only be eluted using drastic conditions (0.1 M sodium hydroxide). In the presence of 10 mM EDTA this tendency to aggregate was greatly diminished. Utilizing a Superdex 200 column for gel filtration, the secretory TGase was even eluted as a monomeric protein. Testicular TGase was isolated by ion-exchange fast protein liquid chromatography on a Mono Q and by gel filtration on a Superdex 200 column. This enzyme represents a tissue-type TGase with a molecular weight of 82,000 dalton and pI value of 5.25. Hydrophobic interaction chromatography on a phenyl-Superose column showed no further enrichment of the GTP-binding form of transglutaminase.
Animals↗
Purification and molecular characterization of a secretory transglutaminase from coagulating gland of the rat.
A transglutaminase (TGase, EC 2.3.2.13) was isolated from the secretion of rat coagulating gland (CGS-TGase). The protein consists of a single polypeptide chain and has a molecular mass of 65 kDa. During purification the net charge changes from pI 7.6 in the crude extract to pI 8.5-8.7 for the purified protein. Nearly equal numbers of glutamyl- and lysyl-residues were detected by amino acid analysis. The protein therefore represents an appropriate substrate of autocatalytic crosslinking. The total number of cysteine residues is 18-19, six of which being present in free form. One of the thiol groups is essential for the enzymic activity. The protein core is glycosylated with mannosyl residues and in addition substituted with saturated acyl residues and phosphoinositol. The phosphoinositol anchor was demonstrated by use of a specific antibody. Removal of the acyl- and glycosyl-residues or of the total anchor group results in autoaggregation and decrease of enzymic activity. In contrast to tissue-type TGases, Ca2+ dependent enzymic activity of CGS-TGase is not inhibited by GTP. The secretory TGase shows no immunological cross-reactivity to tissue-type enzyme or blood factor XIII.
Amino Acids↗
A new staining method for the detection of activities of H2O2-producing oxidases on gels and blots using cerium and 3,3'-diaminobenzidine.
Cerium chloride (CeCl3) was used to trap the hydrogen peroxide generated by several oxidases on native gels and blots. The pale yellow color of cerium perhydroxide formed is converted to a brown-black precipitate by the subsequent reaction with 3,3'-diaminobenzidine. The suitability of this method for the detection of the activity of several oxidases on gels and on blots under nondenaturing conditions, employing different electrophoretic systems and resolving techniques, is demonstrated. Moreover, this method has proven to be highly suitable for the assessment of the substrate and stereospecificity of oxidases, the determination of their molecular weights, and the isoelectric points of isoforms.
3,3'-Diaminobenzidine↗
Immunohistochemical distribution of sulfhydryl oxidase in the human testis.
Sulfhydryl oxidase (SOx) is an enzyme that catalyzes the oxidation of sulfhydryl compounds. It is present in mitochondria of certain testicular cells at specific stages of functional activation. In the mature human testis moderate SOx immunoreactivity is found in Leydig cells, and lacking in Sertoli and in peritubular cells. The Adark spermatogonia usually contain immunoreactive mitochondria, while in Apale spermatogonia immunoreactivity is mostly low. In stage V of spermatogenesis, Apale spermatogonia were found containing immunoreactive material. Leptotene (stages IV and V) and zygotene (stage VI) primary spermatocytes display a moderate immunoreaction. It is strongest in pachytene spermatocytes of stages I-IV, decreases in stage V, and is low during diakinesis and in secondary spermatocytes. Late spermatids usually show a stronger immunoreactivity than early spermatids. At stage V of spermatogenesis the late spermatids contain only few immunoreactive particles. Spermatozoa are free of SOx-immunoreactive mitochondria. In residual bodies small amounts of SOx-immunoreactive particles are seen. Compared to rat and hamster testis, SOx immunoreactivity of the human testis is less clearly stage-dependent and it is not confined to certain germ cell stages. As deduced from the findings in patients with spermatogenic disorders, the SOx immunoreactivity of spermatogonia in human testis seems to be of diagnostic relevance.
Adult↗