Carlos A. Suárez-Quian

Methoxyacetic Acid Disregulation of Androgen Receptor and Androgen-Binding Protein Expression in Adult Rat Testis

Abstract Chemical agents can disrupt the balance between survival and apoptosis during spermatogenesis and thus give rise to reduced counts of spermatozoa (oligospermia). One such agent that produces significant germ cell apoptosis at specific stages of the cycle of the seminiferous epithelium is methoxy acetic acid (MAA), the active metabolite of a commonly used solvent, methoxyethanol. Although MAA gives rise to apoptosis of pachytene spermatocytes, it is not known whether MAA exerts a direct effect on germ cells or whether it also affects other testicular cell types such as the Sertoli cells. In the present investigation, we tested the hypothesis that MAA has direct effects on Sertoli cells in vivo. In MAA-treated rats, stage-specific expression of androgen receptor (AR) protein in Sertoli cells was significantly altered, as determined by AR immunohistochemistry. In MAA-treated animals, high AR expression was found in Sertoli cells coincident with the MAA-induced apoptosis of late-stage pachytene spermatocytes. The altered expression of AR in MAA-treated animals was also seen in seminiferous tubules harvested by laser capture microdissection. In addition to effects on AR expression, androgen-binding protein (ABP) mRNA levels were also altered in a stage-specific manner. Using a different system for mouse Sertoli cell lines TM4 and MSC-1, positive for either AR or ABP, respectively, we found a direct effect of MAA on ABP protein and mRNA expression in the MSC-1 cell but did not detect an effect on AR protein or mRNA expression in TM4 cells. Mouse fibroblasts that express endogenous AR were stably transfected with two AR promoter/reporter systems (MMTV-CAT and probasin-luciferase, respectively). We used these fibroblasts to examine the ability of MAA to potentiate dihydrotestosterone (DHT) activation of AR. Although MAA did not activate AR directly, it did potentiate DHT activation of the AR by 2- to 4-fold. MAA altered the expression level of AR and ABP in vivo and increased AR transcriptional activity in tissue culture cells. The abnormal spermatogenesis generated by MAA is at least partly due to direct effects on Sertoli cells. It is still unclear whether MAA elicits a proapoptotic signal from Sertoli cells or diminishes a prosurvival signal required by germ cells downstream to altering AR and ABP expression in a stage-specific fashion.

Effect of Substrate on the Shape of Sertoli Cells in Vitroa

The importance of the extracellular matrix (ECM) as an essential component of normal cellular function has been recognized only recently. ECM is composed of three basic classes of compounds: collagen molecules; other glycoproteins, including fibronectin and laminin; and glycosaminoglycans.1.2 At present, five genetically distinct types of collagen molecules have been characterized based on their biochemical and antigenic properties. Labeled I to V, each collagen type is generally associated with certain classes of tissue.-2 For example, type I collagen is a component of skin, bone, tendon, and cornea, whereas type IV collagen comprises the lamina densa of typical endothelial and epithelial basal laminae. In all likelihood, the diversity of collagen molecules and their segregation into different microenvironments suggest a specialization in collagen molecules to facilitate their interaction with diverse cell types. In turn, this interaction may regulate cellular activity. The metabolic activity of cultured cells varies with cell shape. In fact, it has been proposed that normal in vitro cell growth requires a delicate balance between cell-cell contact and between cells and ~ubstrate.~ The effect of a collagen substrate in influencing the biochemical activity and shape of cells in vitro is amply documented. Liver parenchyma cells cultured on collagen-coated plates or floating collagen membranes, mimicked morphological and metabolic features of in vivo c e k 4 In response to lactogenic hormones, mammary epithelial cells plated

In vitro models of differentiated Sertoli cell structure and function

SummaryPrimary cultures of Sertoli cells maintained in conventional cultures on plastic culture vessels do not retain many of the structural and functional properties of their in vivo counterparts. Sertoli cell phenotype is better maintained by incorporating certain environmental parameters, intrinsic to the testic, into the Sertoli cell culture system. These environmental parameters include a) high cell density, b) a unique extracellular matrix, c) a semipermeable support between the basal plasma membrane of the cells and blood-derived nutrients in the interstitium, d) chemically distinct microenvironments at the apical and basal surfaces of the cells, and e) cell-to-cell interactions among Sertoli cells and other testicular cell types. Using three variations of Sertoli cell culture we have demonstrated the importance of each of these environmental parameters in obtaining a better Sertoli cell culture model.

Further Observations on the Microfilament Bundles of Sertoli Cell Junctional Complexes

Part of the morphological basis of the blood-testis barrier, which subdivides the seminiferous epithelium into basal and adluminal compartments, is the inter-Sertoli junction. These junctional complexes consist of apposing plasma membranes between adjoining Sertoli cells, a subjacent layer of microfilament bundles, and cisterns of endoplasmic reticulum. Compartmentalization of the seminiferous epithelium presupposes opening and closing of the barrier to permit entry of spermatocytes into the adluminal compartment. Using heavy meromyosin, it was shown that the microfilament bundles contain f-actin, suggesting that they may be contractile, and thus provide the motive force necessary to open and close the This report re-examines the composition of microfilaments contained within inter-Sertoli junctional complexes but suggests an alternative function as to their role. The ultrastructural characterization of microfilaments was carried out on Sertoli cells in viuo and their identity revealed as actin on Sertoli cells in uitro using the S1 fragment of skeletal muscle myosin. Changes in the microfilament bundles at different stages of the seminiferous cycle were observed using NBDphallicidin, a fluorescent probe that binds specifically to f -a~t in .~

The distribution of four lysosomal integral membrane proteins (LIMPs) in rat basophilic leukemia cells

The intracellular distribution of four distinct lysosomal integral membrane proteins (LIMPs), recognized by four monoclonal antibodies, was determined in rat basophilic leukemia (RBL) cells. The monoclonal antibodies were generated against hepatocyte LIMPs and have been characterized previously (Barriocanal et al., 1986a, b). Indirect immunofluorescence microscopy revealed that all four LIMPs are found in secretory vesicles of RBL cells. Ultrastructural immunolocalization, using a pre-embedding peroxidase technique, confirmed these results and also showed the distribution of LIMPs 1 and 4 at the cell surface. The relative, cell surface concentrations of the four LIMPs was determined using a fluorescence activated cell sorter (FACS). In resting RBL cells the concentration of LIMP 1 at the cell surface was highest, followed by LIMP 4. LIMPs 2 and 3 could not be detected at the cell surface. Following stimulation of secretory vesicle exocytosis by A23187, the cell surface concentration of LIMP 4 was increased, whereas the concentration of LIMPs 1-3 remained unchanged. These results are discussed within the context of intracellular sorting during the biogenesis of membrane, secretory vesicle components.

Redistribution of epidermal growth factor receptor as a function of cell density, cell-cell adhesion and calcium in human (A-431) cells

Cell surface expression of the epidermal growth factor (EGF) receptor in several cell lines declines as a function of increased cell density and is associated with diminished responsiveness to EGF. However, the mechanism whereby this density-induced down regulation of receptors occurs has not been discerned. In the present study the distribution of the EGF receptor in A-431 cells as a function of cell density using (1) two polyclonal antibodies raised against peptide specific sequences of the EGF receptor that recognize either the cytoplasmic or extracellular domains of the receptor, respectively, and (2) biotinylated EGF, a specific probe for the cell surface receptor is now investigated. Immunolocalization of the receptor using the polyclonal antibodies or the biotin-EGF revealed that the receptor was homogeneously distributed on the cell surface of individual cells, or in cells plated at low density. In contrast, as cell density increased, prominent EGF immunoreactivity and biotin-EGF staining became limited to the periphery of the cells, at sites of cell-cell apposition, and was characterized by a honeycomb pattern, typical of a basolateral distribution. The effects of low Ca++ treatment, known to cause cells to round up and detach from one another, on EGF receptor distribution in cells at high cell density were then examined. Confocal microscopy of immunostained preparations revealed that incubation of high density cultures in Ca(++)-free media for as little as 10 min restored the homogeneous distribution of the EGF receptor and resulted in strong intracellular staining. Three-dimensional reconstruction of serial optical sections revealed that redistribution of the EGF receptor following low Ca++ treatment involved a heretofore undetected ruffling, an immunostaining pattern characterized by stripes of intense fluorescence signal interspersed with complete absence of fluorescence. Next, cell-cell adhesion was disrupted with antisera to the cell adhesion molecule E-cadherin. Although the antisera caused cells to detach from one another, eventually leading to cell rounding and redistribution of the EGF receptor, the receptor ruffling immunostaining pattern rendered by the low Ca++ treatment was not detected. These results suggest that an association may exist between the plasma membrane EGF receptor distribution, density-induced EGF receptor down regulation, and the growth effects of low Ca++ observed in previous studies.

Differential cell surface expression of four lysosomal integral membrane proteins (LIMPs) in normal rat kidney cells

Four monoclonal antibodies generated against rat, hepatocyte lysosomal integral membrane protein (LIMPs) (Barriocanal et al., 1986a, b) were used as probes to ascertain the distribution of similar proteins in normal rat kidney (NRK) cells. Comparison of immunoprecipitations of LIMPs 1-4 from hepatocytes and NRK cells revealed a marked similarity in the proteins, in both cell types, as determined by SDS-PAGE. Further, the LIMP epitopes recognized by the antibodies are situated intravesicularly. Ultrastructural immunocytochemistry, using pre-embedding peroxidase, revealed that primary and secondary lysosomes in NRK cells are readily stained with all four antibodies, as well as vesicles in the Golgi region. Immunofluorescence microscopy of non-permeabilized NRK cells with antibodies recognizing LIMPs 1 and 4 illustrated a limited but significant punctate staining pattern of the cell surface. Ultrastructural immunoperoxidase indicated these sites to be cell surface localized coated pits and vesicles. However, it is known that all LIMPs are expressed on the cell surface, albeit at different concentrations, although the total number of each LIMP per cell, respectively, is approximately the same (Barriocanal et al., 1987). Treatment of NRK cells with the acidotropic agent NH4Cl decreased the cell surface expression of LIMPs 1, 3 and 4, but had no effect on LIMP 2. Further, the relative diminution of the cell surface expression varied among the four LIMPs. These results are interpreted to suggest that not all lysosomes contain the same integral membrane proteins in their vesicle container.

Localisation du récepteur des androgènes dans le testicule

ResumeLa question de savoir si les cellules de la lignée germinale possèdent ou non des récepteurs aux androgènes reste débattue. Dans ce travail nous avons déterminé la distribution des récepteurs aux androgènes dans le testicule de rat et de souris à laide dun anticorps polyclonal de lapin purifié par affinité, dirigé contre un peptide de 21 acides aminés de la région terminale du récepteur des androgènes (RA), marqué par le système biotine-streptavidine immunoperoxidase.La spécificité de lanticorps est attestée par les points suivants: 1) Présence dune bande spécifique approximativement à 110 kD en Western immunoblot; 2) Elimination spécifique du marquage par pré-adsorption de lanticorps avec le peptide de 21 acides aminés; 3) Mise en évidence dune relation entre lintensité du marquage dans toutes les cellules RA positives en fonction de la dilution des antisérums; 4) Mise en évidence dans tous les tissus connus pour exprimer de façon importante des RA (par exemple lépididyme, la prostate, les vésicules séminales) dun marquage extrêmement intense au niveau du noyau des cellules épithéliales; 5) Obtention dun réaction RA positive avec les anticorps dans les lignées de cellules prostatiques connues pour exprimer les récepteurs aux androgènes; 6) Positivation de cellules COS-7 après leur transfection par un vecteur exprimant le récepteur aux androgènes de rat. De plus la distribution intracellulaire des RA dans les cellules COS-7 est fonction de la présence dandrogènes dans le milieu.Limmunolocalisation des récepteurs aux androgènes montre les points suivants: à lintérieur du compartiment interstitiel chez le rat adulte des RA sont détectés dans quelques cellules de Leydig et dans toutes les cellules musculaires lisses formant la paroi des vaisseaux sanguins, alors que les cellules endothéliales restent négatives. Dans les tubes séminifères les RA sont observés dans tous les noyaux des cellules myoïdes péritubulaires, mais pas dans les couches plus périphériques correspondant aux cellules endothéliales des vaisseaux lymphatiques. Dans les cellules de Sertoli le marquage nucléaire des RA est spécifique du stade de lépithélium séminifère: la positivité du marquage commence à devenir évidente vers le stade IV tardif du cycle, atteint un pic très fort aux stades VII et VIII puis disparaît complètement. Un immunomarquage spécifique a également été mis en évidence dans les noyaux des spermatides allongées stade XI, stade auquel lélongation nucléaire est déjà apparente mais sans que la condensation de la chromatine soit déjà commencée. Dès le début de la condensation de la chromatine des spermatides allongées, limmunomarquage nucléaire disparaît en même temps que le cytoplasme devient positif. Globalement les mêmes observations ont été faites sur le testicule de Souris adulte, excepté le fait que, dans cette espèce, toutes les cellules de Leydig sont très fortement RA positives.Afin de confirmer limmunomarquage des RA nous avons réalisé des études dhybridation in situ dans le testicule de Rat adulte à laide dune sonde antisens ARNc de 236 paires de bases (IADNc de RA nous a été donné par le Docteur Chang, Université du Wisconsin, Madison, WI). Un important signal dhybridation a été observé à la base de lépithélium séminifère, dans la région occupée par les cellules de Sertoli et les spermatogonies. Ceci nous a conduit a réexaminer les résultats de limmunomarquage afin de déterminer si les spermatogonies étaient également positives pour les anticorps anti RA. Nos résultats préliminaires sont en faveur de la présence de RA dans certaines populations spermatogoniales. Au total, ces résultats concordent avec des observations antérieures suggérant que les RA sont présents dans les cellules somatiques du testicule; de ce fait, ce sont ces types cellulaires qui vraisemblablement répondent aux androgènes circulants pour contrôler la spermatogenèse. Cependant nos résultats montrent également un immunomarquage de certains élements de la lignée germinale ce qui réactualise la controverse concernant le fait de savoir si la lignée germinale peut répondre ou non directement aux androgènes.AbstractWhether or not germ cells contain the androgen receptor remains a matter of controversy. In the present study we performed biotinstreptavidin immunoperoxidase using an affinity purified rabbit polyclonal antibody made to a 21 amino acid peptide of the amino terminus of the rat AR to determine androgen receptor (AR) distribution in the rat and mouse testes. Specificity of the antibody was confirmed as follows: 1) Western immunoblots rendered a specific band at approximately 110 kD; 2) preadsorption of the antibody with the 21 amino acid peptide eliminated specifice immunostaining; 3) the intensity of staining in all AR positive cells diminished as a function of antisera dilution; 4) tissues known to express abundant AR (e.g., epididymis, prostate, seminal vesicles) all rendered a robust, nuclear AR immunostaining pattern in the epithelial cells; 5) prostate cell lines known to express AR immunostained positive with the antibody; 6) AR negative COS-7 cells became AR immunopositive when transfected with a vector expressing the rat AR and intracellular AR distribution was a function of androgens. AR immunostaining results revealed the following: Within the interstitial compartment of adult rats, AR was detected in some Leydig cells and all smooth muscle cells forming the walls of blood vessels, but endothelial cells were negative. In the seminiferous tubules AR was observed in all peritubular myoid cell nuelei, but not in the distal layer of Iymphatic endothelial cells. In Sertoli cells, nuclear AR immunostaining was stage specific; moderate AR immunostaining became evident at late stage IV of the cycle, reached a robust peak at stages VII-VIII, and then disappeared completely. Specific AR immunostaining was also discerned in the nuclei of stage XI elongated spermatids, in which nuclear elongation is apparent but chromatin condensation has not yet begun. With onset of chromatin condensation, nuclear AR immunostaining in elongated spermatids was not discerned concomitant with its detection in the cytoplasm. In general, similar observations have now been confirmed in the adult mouse testis, except that an Leydig cells were strongly AR positive. Nucleic acid in situ hybridization studies for AR were performed in adult rat testis using a 236 bp antisense cRNA probe (rat AR cDNA was provided by Dr. C. Chang, U. Wisconsin, Madison, WI) to confirm the AR immunostaining. A prominent hybridization signal at the base of the seminiferous epithelium was observed, in the area occupied by Sertofi and spermatogonia. This led us to re-examine the immunostaining results to determine if spermatogonia were also AR positive. Preliminary results are consistent with the interpretation that AR is present in certain spermatogonial populations. Taken together, these results concur with prior observations suggesting that AR is present in the somatic cells of the testis; thus, it is these cell types that likely respond to circulating androgens to control spermatogenesis. However, they raise anew the controversy of whether germ cells respond directly to androgens.

Synthesis and Properties of an EGF-like Domain (residues 361-406) in the Extreme N-terminal Region of the Mouse EGF Precursor

Various proteins contain EGF-like domains that are not ligands for the EGF receptor. In the present study a cognate polypeptide for residues 361-406 of the mouse EGF precursor was synthesized by the solid-phase method. The product was renatured under oxidative conditions since it probably has an EGF-like array of three cystine disulfide bonds in its native state. HPLC analysis of the renaturation reaction revealed formation of a peak material with no apparent free-SH groups. Accordingly, the HPLC retention time of this product was readily increased by treatment (reduction of disulfides) with dithiothreitol. The renatured 46-mer (PEGF-1) did not displace 125I-EGF bound to rat liver membranes and 125I-PEGF-1 did not exhibit specific binding to membrane preparations from the mouse liver, mammary gland, or kidney, with or without Ca2+ in the binding medium. Although PEGF-1 contains a putative Ca2+ binding motif, specific binding of this cation by the polypeptide could not be demonstrated by electromobility shiff or incubation with 45Ca2+. Immunoassay of PEGF-1 and EGF in fractions obtained following gel filtration of mouse urine revealed multiple peaks of PEGF-1 immunoreactivity with the major peaks eluting at an Mr > 30 kDa. In contrast, virtually all the EGF immunoreactivity eluted at a volume similar to that of 125I-EGF. These data suggest that selective cleavage of the PEGF-1 domain from the precursor does not occur with the proclivity known for that of EGF. Instead, the PEGF-1 probably functions coordinately with other EGF-like domains while tethered to the precursor backbone. Finally, localization of PEGF-1 immunoreactivity occurred only in cell populations of the mouse previously demonstrated as sites for EGF/EGF precursor, which suggests that PEGF-1 is exclusively a domain of the EGF precursor.