Dynamic testicular adhesion junctions are immunologically unique. I. Localization of p120 catenin in rat testis

In the seminiferous epithelium, morphologically diverse junctions mediate inter-Sertoli and Sertoli-germ cell adhesive contact, but the molecular composition of such junctions is not well known. At prototypical adherens junctions, proteins termed catenins bind to the intracellular domain of classic cadherins and regulate the strength of adhesion. Using a panel of monoclonal antibodies (5A7, 8D11, and 15D2), p120 catenin (p120) was localized in postnatal and adult rat testis cryosections and touch preparations by immunofluorescence. Immunoprecipitation of testis homogenates showed that at least four p120 isoforms were expressed from Postnatal Day 7 through adulthood. Both inter-Sertoli and Sertoli-germ cell junctions were p120-positive, however, individual p120 monoclonals were localized to specific junctions. The 5A7 and 8D11 antibodies colocalized with beta-catenin and plectin at inter-Sertoli and Sertoli-spermatocyte junctions. At inter-Sertoli junctions, p120 was juxtaposed to but did not colocalize with f-actin. Thus, p120 is likely a component of inter-Sertoli desmosome-like junctions. In contrast, the 15D2 monoclonal antibody specifically immunostained Sertoli-round spermatid and inter-Sertoli cell junctions in a dynamic pattern. From the time that round spermatids form to their differentiation into elongate spermatids, Sertoli-round spermatid 15D2 immunostaining cycled from a single mass to a curvilinear pattern, and finally to punctate structures scattered throughout the epithelium. This localization and stage-specific immunostaining pattern indicated that 15D2 recognized Sertoli-round spermatid desmosome-like junctions. Between Sertoli cells, 15D2 immunostained newly formed junctions (at Postnatal Days 21 through 43), but not mature junctions in the adult. From these data, we conclude that p120 is a component of most, if not all, desmosome-like junctions, and that desmosome-like junctions between different cell types contain a unique molecular composition.     

Protocadherin alpha3 acts at sites distinct from classic cadherins in rat testis and sperm

The testis expresses a variety of cadherin superfamily members including classic cadherins and protocadherins. This report describes the first localization of a protocadherin protein in testis and sperm. After cloning rat cDNAs for protocadherin alpha3 and alpha4, isoform-specific polyclonal antibodies were generated against protocadherin alpha3. Western blotting of rat testis showed that protocadherin alpha3 was solubilized completely by Triton X-100, in contrast to the adhesion junction components N-cadherin, beta-catenin, and p120 catenin. Corroborating this data, protocadherin alpha3 was immunolocalized to the spermatid acrosomal area, intercellular bridge, and flagellum, but not classic cadherin-based adhesion junctions. Acrosome-associated protocadherin alpha3 was first detected at step 8 of spermiogenesis, and this association remained on cauda epididymal sperm. Acrosome immunostaining was reduced, but present, in acrosome-reacted sperm. Spermatid intercellular bridges became positive for protocadherin alpha3 coincident with the appearance of plectin, occurring at spermiogenic steps 8 to 9, and elongate spermatid bridges remained positive throughout spermatogenesis. The developing flagellum was uniformly immunostained for protocadherin alpha3 up to approximately spermiogenic step 17. Subsequently, flagellar immunostaining was confined to the principal piece, and this pattern continued in cauda epididymal sperm. These data show that protocadherin alpha3 performs functions unique from classic cadherins in spermatogenesis and suggest a role for protocadherin alpha3 in organizing germ cell-specific structures including the intercellular bridge, flagellum, and acrosome.     

Localization of testis-variant histones in rat testis chromatin.

Nucleosome core particles and oligonucleosomes were isolated by digesting rat testis nuclei with micrococcal nuclease to 20% acid-solubility, followed by fractionation of the digest on a Bio-Gel A-5m column. The core particles thus isolated were characterized on the basis of their DNA length of 151 +/- 5 base-pairs and sedimentation coefficient of 11.4S. Analysis of the acid-soluble proteins of the core particles indicated that histones TH2B and X2 are constituents of the core particles, in addition to the somatic histones H2A, H2B, H3 and H4. The acid-soluble proteins of the oligonucleosomes comprised all the histones, including both the somatic (H1, H2A, H2B, H3, H4 and X2) and the testis-specific ones (TH1 and TH2B). It was also observed that histones TH1 and H1 are absent from the core particles and were readily extracted from the chromatin by 0.6 M-NaCl, which indicated that both of them are bound to the linker DNA.     

Localization of epoxide-metablozing enzymes in rat testis

Antibodies raised against rat hepatic epoxide hydrolase (EC 3.3.2.3) and glutathione S-transferases (EC 2.5.1.18) B, C and E were used to determine the presence and localizations of these epoxide-metabolizing enzymes in testes of sexually immature and mature Wistar and Holtzman rats. Unlabeled antibody peroxidase-antiperoxidase staining for each enzyme was readily detected in rat testes at the light microscopic level. Although significant strain-related differences were not apparent, staining intensity for certain enzymes differed markedly between Leydig cells and seminiferous tubules. Leydig cells of immature and mature rats were stained much intensely for epoxide hydrolase and glutathione S-transferase B and E than were seminiferous tubules, whereas Sertoli cells, spermatogonia, spermatocytes and spermatids, as well as Leydig cells, were stained intensely by the anti-glutathione S-transferase C. Age-related differences in staining for glutathione S-transferase B were not obvious, while the anti-glutathione S-transferase C stained seminiferous tubules more intensely in immature rats, and antibodies to expoxide hydrolase and glutathione S-transferases C and E stained Leydig cells much more intensely in mature rats. These observations thus demonstrate that testes of both sexually immature and mature rats contain epoxide hydrolase and glutathione S-transferases. Except for glutathione S-transferase C in immature rats, Leydig cells appear to contain much higher levels of enzymes than do seminiferous tubules. During sexual maturation, the testicular level of glutathione S-transferase B appears to remain constant, while levels of epoxide hydrolase and glutathione S-transferases C and E increase within Leydig cells and the level of glutathione S-transferase C decreases within seminiferous tubules.     

Localization of gamma-butyrobetaine hydroxylase in the rat testis

The activity of gamma-butyrobetaine hydroxylase in testes is comparable on a protein basis to the amount of activity found in liver and accounts for a significant amount of the total activity found in the rat. The enzyme is localized to the seminiferous tubules of the testes. In attempting to further localize this enzyme, activity remained the same when reported on a protein basis in cryptorchid testes as compared to controls, and activity was found in bovine sperm cells obtained from the caput epididymis. These results are consistent with the localization of this enzyme in both the Sertoli cells and the germ cells.     

Molecular linkage between cadherins and actin filaments in cell-cell adherens junctions.

The cell-cell adherens junction is a site for cadherin-mediated cell adhesion where actin filaments are densely associated with the plasma membrane through its well-developed plasmalemmal undercoat. Recent research has focused on the molecular linkage between cadherins and actin filaments in the undercoat of adherens junctions in order to understand the functions of these undercoat-constitutive proteins in the regulation and signal transduction of cadherin-based cell adhesion.     

Two immunologically distinct Yc-type subunits are present in rat lung glutathione S-transferases.

Two types of 25 000-Mr subunits are present in rat lung glutathione S-transferase I (pI 8.8). These subunits, designated Yc and Yc', are immunologically and functionally distinct from each other. The homodimers YcYc (pI 10.4) and Yc'Yc' (pI 7.6) obtained by hybridization in vitro of the two subunits of glutathione S-transferase I (pI 8.8) were isolated and characterized. Results of these studies indicate that only the Yc subunits express glutathione peroxidase activity and cross-react with the antibodies raised against glutathione S-transferase B (YaYc) or rat liver. The Yc' subunits do not express glutathione peroxidase activity and do not cross-react with the antibodies raised against glutathione S-transferase B of rat liver. The amino acid compositions of these two subunits are also different. These two subunits can also be separated by the two-dimensional gel electrophoresis of glutathione S-transferase I (pI 8.8) of rat lung.     

Tubulobulbar complexes are intercellular podosome-like structures that internalize intact intercellular junctions during epithelial remodeling events in the rat testis

Tubulobulbar complexes are actin-related double-membrane projections that resemble podosomes in other systems and form at intercellular junctions in the seminiferous epithelium of the mammalian testis. They are proposed to internalize intact junctions during sperm release and during the translocation of spermatocytes through basal junction complexes between neighboring Sertoli cells. In this study we probe apical tubulobulbar complexes in fixed epithelial fragments and fixed frozen sections of rat and mouse testes for junction molecules reported to be present at apical sites of attachment (ectoplasmic specializations) between Sertoli cells and spermatids. The adhesion molecules nectin 2 (PVRL2), nectin 3 (PVRL3) and alpha 6 integrin (ITGA6) are present in the elongate parts of tubulobulbar complexes and concentrated at their distal ends. Tubulobulbar complexes contain cortactin (CTTN), a key component of podosomes, and vesicles at the distal ends of tubulobulbar complexes that contain junction molecules are related to early endosome antigen (EEA1). N-cadherin (CDH2), a protein reported to be present at ectoplasmic specializations, is not localized to these unique junctions or to tubulobulbar complexes but, rather, is primarily concentrated at desmosomes in basal regions of the epithelium. Our results are consistent with the conclusion that tubulobulbar complexes are podosome-like structures that are responsible for internalizing intact intercellular junctions during spermatogenesis.     

Localization of ornithine decarboxylase in rat testicular cells and epididymal spermatozoa

We have employed a monospecific, polyclonal antibody to ornithine decarboxylase (ODC) for the immunocytochemical localization of ODC in freshly isolated testicular cells, epididymal spermatozoa, and cultured Sertoli cells. Antigenically detectable material was present in the cytoplasm of all cell types tested and was highly concentrated in the acrosomal vesicle of round spermatids and in the acrosome region of epididymal spermatozoa. The specific enzymatic activity of ODC, as measured biochemically, was much higher in the interstitial cells than in the other testicular cell types, and no ODC activity was detected in the epididymal spermatozoa or in the Sertoli cells after 5 days in culture. These studies showed that, while all testicular cell types studied contained ODC-like immunoreactive molecules, only testicular germ cells and interstitial cells exhibited detectable ODC activity.     

Characterization of estrogen binding in the developing rat testis. Ontogeny of the testicular cytoplasmic estrogen receptor.

The properties and physical characteristics of a steroid binding component present in the immature (7 to 35 day) rat were investigated and found to be different from those of the 17 beta-estradiol receptor in the mature rat testis. These properties include a binding capacity of 483 fmol estradiol/mg protein, a Ka at equilibrium of 4.23 x 10(7)M-1, and broad steroid specificity as shown by interaction with several steroids; no binding was observed with diethylstilbestrol. The component, found in blood and several tissues including the testis, migrated as a 4.6S peak on sucrose gradients. This 4.6S component, which interacted with an anti-alphafetoprotein antiserum, decreased with age and was not detectable in the testis after day 21 or in the serum after day 25. These data suggest that this component is alphafetoprotein. Ontogenic appearance of the testicular cytoplasmic 17 beta-estradiol receptor in the developing rat was further elucidated. Sucrose gradient sedimentation analysis of cytosols revealed an 8S binding component that was first detectable at 23 days. Specific binding (fmol [3H]-estradiol/testis) was relatively low in neonates, rose to 59 fmol during the third week, and increased dramatically to 333 fmol at 7 weeks; binding rose only gradually after maturity. The receptor was tissue specific and steroid specificity studies demonstrated that only diethylstilbestrol and other estrogens were effective in competing with 17 beta-estradiol for binding sites. The Ka at equilibrium was determined as 3 x 10(10)M-1 and the binding sites were saturable in an in vitro system. The receptor did not interact with anti-alphafetoprotein antiserum as indicated by sucrose gradient studies. These data demonstrate the developmental appearance of the testicular cytoplasmic estradiol receptor in the immature rat.     

The Sertoli cell occluding junctions and gap junctions in mature and developing mammalian testis.

Special occluding junctions between Sertoli cells near the base of the seminiferous epithelium are the structural basis of the blood-testis permeability barrier. In micrographs of thin sections, multiple punctate pentalaminar contacts between apposed membranes are observed in the junctional regions.In freeze-fractured mature testis, the junctional membranes exhibit up to 40 parallel circumferentially oriented rows of intramembrane particles preferentially associated with the B-fracture face, but with complementary shallow grooves on the A-face. Short rows of particles may remain with the A-face resulting in discontinuities in the B-face particle rows. In addition, elongate aggregations of particles of uniform size (～70 A) arranged in one or more closely packed rows are occasionally found adjacent to the linear depressions on the A-face of the Sertoli junction. These are interpreted as atypical gap junctions.In immature testis, occluding junctions are absent but typical gap junctions are common. These gradually disappear. In the second postnatal week, linear arrays of particles appear on the B-face. Initially meandering and highly variable in direction, these gradually adopt a consistent orientation parallel to the cell base. The establishment of the blood-testis barrier appears to be correlated with this reorganization of the intramembrane particle rows. Sertoli junctions were shown to be resistant to hypertonic solutions that rapidly dissociate junctions of other epithelia.Sertoli junctions thus differ from other occluding junctions in their (1) basal location, (2) large number of parallel particle rows, (3) absence of anastomosis between rows, (4) preferential association of the particles with the B-face, (5) intercalation of atypical gap junctions, (6) unusual resistance to dissociation by hypertonic solutions.     

Receptor-mediated endocytosis of testicular transferrin by germinal cells of the rat testis

Summary The present study examines events of the Sertoli cell iron delivery pathway following the secretion of diferric testicular transferrin (tTf) into the adluminal compartment of the rat seminiferous epithelium. The unidirectional secretion of tTf by Sertoli cells was verified, in vivo, and it was shown that this protein is internalized by adluminal germ cells. It was further determined by Scatchard analysis that this internalization was mediated by high affinity transferrin binding sites on the surface of round spermatids, numbering 1453/cell and displaying a K_d=0.6×10^-9 M. Northern blot analysis of RNA isolated from adluminal germ cells, namely spermatocytes, round spermatids and elongating spermatids, indicated that these cells expressed Tf receptor mRNA and ferritin mRNA in levels inversely related to their stage of maturation. Finally it was determined that following binding and internalization in round spermatids, Tf became associated with the endosomal compartment and was recycled back to the cell surface. This study illustrates the immediate fate of tTf once it is secreted by the Sertoli cell. Thus, diferric tTf binds of Tf receptor on the surface of adluminal germ cells, is internalized by receptor-mediated endocytosis and the apo Tf-Tf receptor complex is recycled back to the cell surface where apotTf is released into the adluminal fluid.     

Exocrine function of testis with germinal testicular tumour.

Semen collected immediately before or soon after therapeutic unilateral orchidectomy from 86 men with germinal tumours of the testis was analysed. The mean sperm count was 65.6 +/- SEM 10.3 X 10(6) per ejaculate. This was significantly different from that in a control population, in whom the mean was 165.2 +/- 12.9 X 10(6) per ejaculate. The difference appeared to be due to a subpopulation of 32 patients with counts of 20 X 10(6) per ejaculate; the distribution of counts in the remaining patients was similar to that in the controls. The cause of this abnormality of exocrine function is unknown: it may either be due to the tumour or its treatment, or both, or, alternatively, it may predate the events of the malignant transformation, possibly even acting as an inducing or promoting factor.     

Spermatogenesis and testis development are normal in mice lacking testicular orphan nuclear receptor 2

Early in vitro cell culture studies suggested that testicular orphan nuclear receptor 2 (TR2), a member of the nuclear receptor superfamily, may play important roles in the control of several pathways including retinoic acids, vitamin D, thyroid hormones, and ciliary neurotrophic factor. Here we report the surprising results showing that mice lacking TR2 are viable and have no serious developmental defects. Male mice lacking TR2 have functional testes, including normal sperm number and motility, and both male and female mice lacking TR2 are fertile. In heterozygous TR2(+/-) male mice we found that beta-galactosidase, the indicator of TR2 protein expression, was first detected at the age of 3 weeks and its expression pattern was restricted mainly in the spermatocytes and round spermatids. These protein expression patterns were further confirmed with Northern blot analysis of TR2 mRNA expression. Together, results from TR2-knockout mice suggest that TR2 may not play essential roles in spermatogenesis and normal testis development, function, and maintenance. Alternatively, the roles of TR2 may be redundant and could be played by other close members of the nuclear receptor superfamily such as testicular orphan receptor 4 (TR4) or unidentified orphan receptors that share many similar functions with TR2. Further studies with double knockouts of both orphan nuclear receptors, TR2 and TR4, may reveal their real physiological roles.     

Localization of glycoprotein glycosyltransferases in the Golgi apparatus of rat and mouse testis

Golgi fractions prepared from rat testis have been shown to be enriched in the following glycoprotein glycosyltransferases: N-acetylglucosaminyltransferase, 47-fold, galactosyltransferase, 33-fold, and N-acetylglucosaminide fucosyltransferase, 15-fold. Appreciably lower transferase levels were obtained in other subcellular fractions. In the mouse, Golgi fractions were prepared from testis homogenates, testis cell suspensions and partially purified testis germinal cells; these fractions were also enriched in the above glycoprotein glycosyltransferases. Electron microscopic analysis indicated that a major portion of the total transferase activity was located in the Golgi apparatus of both rat and mouse testis although these experiments could not rule out the possible presence of some transferase activity in other organelles.