Harvesting and separation of two populations of lysosomes from porcine endometrium.

The lysosomes present in homogenates of porcine endometrium epithelium equilibrate in two density regions of Percoll gradients. Patterns with varying proportions between high and low density peaks are observed, when aliquots of a tissue sample are processed with different all-glass Potter-Elvejhem homogenizers. The described constant-tolerance shearing device (CTSD), in contrast, provides homogenate fractions with higher latencies and steady distribution patterns. They are characteristic for each of the six lysosomal markers and the six other structure-bound enzymes measured in gradient fractions of the particulate matter harvested between 600g and 17,000g. The 17,000g sediments of CTSD homogenates contain more than 40% of the total lysosomal enzymatic activities. Recoveries from Percoll gradients are between 93 and 101%. Enrichments in the high density region range from 35-fold (beta-glucosidase) to 82-fold (acid ribonuclease). Both lysosomal populations exhibit latencies between 89 and 94%. Our results indicate that light lysosomes can be artificially generated by inappropriate homogenization, which should be considered in experiments on the formation and maturation of lysosomes.

A hormone pulse induces transient changes in the subcellular distribution and leads to a lysosomal accumulation of the estradiol receptor alpha in target tissues.

An intrauterine pulse-stimulation with estradiol induced changes in the subcellular localization of estrogen receptor alpha in porcine endometrium, as detected with F(ab') fragments of various anti-receptor antibodies covalently linked to nanogold. The low-sterically hindered immunoreagents--recognizing different epitopes within the hormone binding domain--allowed for an efficient immunolabeling of estradiol receptor alpha, detecting it both in the cytoplasm and the nucleus of nonstimulated epithelium cells. In the cytoplasm, the receptor often seemed to be associated with actin filaments and the endoplasmatic reticulum. After the stimulation with estradiol, a predominantly nuclear localization and a labeling of nucleoli was observed. Our immunoelectron microscopy study demonstrates a localization of the receptor in cytoplasmic organelles that increased after the hormone pulse. These organelles exhibited the morphological properties of lysosomes and relocated to the perinuclear area. In analogous cytoplasmic organelles, the presence of cathepsin D was detected via indirect immunogold labeling, justifying their classification as lysosomes. Quantitative examinations revealed that not only the number of lysosomes in the proximity of the nucleus but also their immunostaining for estradiol receptor alpha increased significantly after the hormone pulse. Thus, estradiol induces both the rapid shift of receptor into the nucleus, a slower perinuclear accumulation of lysosomes and an increase of lysosomal ERalpha-immunoreactivity. These results suggest a role for lysosomes in the degradation of receptor shuttling out of the nucleus. This could serve as termination of the estradiol receptor alpha-dependent activation of target cells. This hypothesis is strengthened by the fact that the receptor content in uterine tissue declined drastically few hours after the hormone pulse.

Functional and morphological changes of the thyroid gland following 5 days of pulsatile TRH stimulation in male rats.

The aim of the present study was to evaluate in vivo the selective effects of a small increase in plasma TSH levels on thyroid function, proliferation and morphology. Chronically catheterized male Sprague-Dawley rats were stimulated i.v. over 5 days either with TRH (2 micrograms TRH in 100 microliters 0.9% (w/v) NaCl (TRH-P) or the NaCl carrier alone (P), both given as pulses every 2 h. Control groups were cotreated i.v. with 10 micrograms thyroxine (T4)/100 g body weight per day (TRH-P + T4) starting 2 days before pulsatile stimulation. TSH plasma levels were approximately doubled by TRH-P (P < or = 0.001), T4 plasma levels significantly increased (P < or = 0.001) but tri-iodothyronine plasma levels did not change compared with treatment with P. No significant changes between groups were found in thyroid weight and in intrathyroidal iodine content, but the percentage of 5-bromo-2'-desoxyuridine-labelled thyrocytes as a marker of proliferation in TRH-P-treated animals was significantly increased over P or TRH-P + T4 (P < or = 0.001). Ultrastructural analysis of the thyroid evaluated by electron microscopy revealed a significant increase in the number of lysosomes (P < or = 0.001). The size of the endoplasmic reticulum (ER) in relation to the cytoplasm was significantly increased when treated with TRH-P compared with P or TRH-P + T4 (P < or = 0.001). Post-embedding immunogold staining revealed Tg as a major product within ER cisternae. Immunogold labelling was moderate in controls and higher densities of gold particles were obtained in TRH-P-treated animals (P < or = 0.001). In conclusion, short-term pulsatile TRH stimulation increasing the plasma levels of immunoreactive TSH only twofold is capable of inducing hypertrophy of the thyrocytes by gross ultrastructural changes which are paralleled by an increase in circulating T4. These data underscore the dominant role of TSH on thyroid ultrastructure within the narrow boundaries of normal physiological regulation.

Subcellular localization of estradiol receptor in MCF7 cells studied with nanogold-labelled antibody fragments.

Ultrastructural localization studies of estradiol receptor in hormone-deprived and hormone-stimulated MCF7 cells were done using F(ab') fragments of three different antibodies (#402, 13H2, HT277) covalently linked to nanogold. These ultra-small, non-charged immunoreagents, combined with a size-enlargement by silver enhancement, localized estradiol receptor in both nuclear and cytoplasmic areas of non-stimulated target cells; stimulation with the steroid induced a predominantly nuclear labelling. In the cytoplasm of resting cells, tagging was often observed at or in the proximity of stress fibers. In the nucleus a large proportion of receptor was found inside the nucleolus, specially with the reagent derived from antibody 13H2. We postulate that different accessibilities of receptor epitopes account for the different labelling densities observed at cytoskeletal elements and the nucleoli.

[Transcervical injection into the uterine lumen of castrated swine as a cell and organ biological research design. The Mariensee model]. / Die transcervicale Injektion in das Uteruslumen von kastrierten Schweinen als zell- und organbiologische Versuchsanordnung. Das Mariensee Modell.

The technique and the equipment necessary for the transcervical intrauterine injection in trained, ovariectomized pigs is detailed. Experimental groups of 14-16 week-old animals are recruited from German landrace litters with large numbers (4-9) of females. A silastic tubing containing a crystalline suspension of estradiol in propylene glycol is subcutaneously implanted behind the right ear. Ovariectomy and the detachment of the left uterine horn from the cervix are performed 7-10 days later. The animals are then kept for 6-7 weeks in ventilated stables, heated to 20 degrees C in wintertime, with 12 hr light dark cycle, free access to water and semi-ad libitum supplies of pelleted standard feed. The silastic implant gives rise to plasma levels of 8-12 pg estradiol/ml and still contains crystalline sediments when removed before the experiment. The manipulations for the transcervical, intrauterine instillation of solutions, imitating the reproductive action of the boar, are practiced daily for at least one week. The animals learn quickly to enter a restraining box, which is facilitated by the simultaneous offering of pellets drenched with root beer. The intrauterine injections proceed thereafter without any signs of stress from the animals. Stress is also avoided during slaughter by unexpected electric stunning on leaving the box, followed by exsanguination. Uteri are quickly excised and chilled in crushed ice until processed. One to two grams of endometrium cells can be harvested by curettage from each, treated and untreated (detached) horn. The Mariensee model allows for defined kinetic analyses of treated cells and provides nontreated controls from the same animal. It combines the advantages of in-vivo and in-vitro experiments.

Immunoelectron microscopy in embryos.

Immunogold labeling of proteins in sections of embryos embedded in acrylate media provides an important analytical tool when the resolving power of the electron microscope is required to define sites of protein function. The protocol presented here was established to analyze the role and dynamics of the activated protein kinase C/Rack1 regulatory system in the patterning and outgrowth of limb bud mesenchyme. With minor changes, especially in the composition of the fixative solution, the protocol should be easily adaptable for the postembedding immunogold labeling of any other antigen in tissues of embryos of diverse species. Quantification of the labeling can be achieved by using electron microscope systems capable of supporting digital image analysis.

Immunogold labelling of the cytoplasmic estradiol receptor in resting porcine endometrium.

Serial sections of resting porcine endometrium were analyzed with the monoclonal antibody 13H2 using goat antimouse IgG/5 nm gold as secondary reagent or with either polyclonal antibodies from goat #402 or the rat monoclonal antibody H222, both in combination with protein G/12 nm gold. A modestly higher labelling of nuclei than of cytoplasm was seen only with the monoclonal antibody H222. Polyclonal #402 and monoclonal 13H2 showed fewer attachments over nuclear than over cytoplasmic areas. The highest densities of attachment and of predominantly cytoplasmic labelling were obtained with the monoclonal antibody 13H2. The results confirm the earlier assumption of a restricted accessibility of estradiol receptor in the cytoplasm of resting cells for immunoreagents.

Characterization of microsomal subfractions from porcine endometrium cells.

Crude microsomes from porcine endometrium and three subfractions obtained by a modification of Rothschild's technique were characterized by RNA/protein ratio, marker enzyme activities and morphological appearance. The microsomes were devoid of glucose-6-phosphatase activity. They contained approximately 10% of arylesterase-, approximately 30% of both NADPH-cytochrome reductase- and UDPgalactose-N-acetyl-glucosamine beta-D-galactosyltransferase- and approximately 60% of 5'-nucleotidase activities present in the homogenates. Subfraction I (smooth membranes) had twice the galactosyltransferase activity of Subfraction II (smooth and rough membranes + free ribosomes); both subfractions were rich in 5'-nucleotidase and cytochrome reductase activities. Subfraction III (rough membranes) had very low marker activities but exhibited the highest RNA/protein ratio, which was lowest in I.

Retrieval of estradiol receptor in paraffin sections of resting porcine uteri by microwave treatment. Immunostaining patterns obtained with different primary antibodies.

The unmasking of estradiol receptor in paraffin sections of Bouin's-fixed uterine tissue from ovariectomized gilts was attained with microwave treatment. Immunocytochemistry of the receptor was performed using a polyclonal or five monoclonal antibodies, two of which are commercially available, reacting with different domains of the protein and an amplified-peroxidase system for detection. With five of the antibodies, a predominance of nuclear staining was observed in cells of endometrial glands, while one monoclonal antibody (13H2), reacting with the receptor's domain E, showed a preference for the cytoplasmic receptor. In stroma, all antibodies detected more receptor in nuclei than in cytoplasm. In epithelium, the commercially available antibody H222, our monoclonals 13H2 and HT65, and the polyclonal antibody 402 demonstrated more receptor in cytoplasmic than in nuclear areas. In myometrium, the nuclei from longitudinal and ring muscles were definitely stained with the antibodies. We conclude that the accessibilities of the antibody epitopes of the receptor differ according to the functional uterine cell type.

Ultrastructural evidence for the presence of crystalline structures in pig vaginal epithelial cells.

Terminally differentiated intermediate layers of vaginal epithelial cells from the late follicular phase of the pig show crystalline structures. Analysis of two dimensional images by transmission electron microscopy revealed that these structures are composed of rhomboid subunits. The longer side measures 26 nm whereas the smaller side measures 24 nm. The angles between the sides are 100 degrees and 80 degrees. These structures are not associated with any cellular structures and are not evident in other phases of the oestrous cycle. Structurally, they do not match with Reinke's crystals and are probably regulated by hormones.

Identification of target cells by immunohistochemical detection of covalently rearranged estradiol in rehydrated paraffin sections.

Estradiol is released from the binding niche of the receptor and covalently arrested in the molecular vicinity by the Mannich reaction during target fixation in acetic acid/formaldehyde. The exposed steroid is freely accessible for appropriate antibodies. It can be visualized in sections by the second antibody/enzyme technique in high resolution and without enhancements.

Origin and quantification of cytoplasmic estradiol receptor in resting target cells.

The exhaustive extraction of microsomal estradiol receptor by Surfynol/dithiothreitol/trypsin in low ionic strength buffer was employed for distribution studies on non-stimulated porcine endometrium. It was found that more than half of the cytoplasmic receptor contents were of microsomal origin. "Empty" structures did not interfere with receptor analysis by agargel electrophoresis. The combined yields from homogenate fractions corresponded to those obtained from unfractionated homogenates. Freeze-fracturing of endometrium had a moderate receptor-solubilizing effect.

Assignment of estradiol-17 beta dehydrogenase and of estrone reductase to cytoplasmic structures of porcine endometrium cells.

Homogenates of porcine endometrium contain substantial activity for the dehydrogenation of estradiol-17 beta but little for estrone reduction. Both activities are associated with cytoplasmic structures. The dehydrogenase is characterized by a pH 7.7 optimum, Km 2.2 x 10(-7) mol/l for estradiol and Km 4.4 x 10(-5) mol/l for the cosubstrate NAD+. The corresponding figures for the reductase are pH 6.6, Km 1.1 x 10(-6) mol/l for estrone and Km 2.1 x 10(-5) mol/l for the cosubstrate NADPH. The (mitochondrial/lysosomal) 17,000 x g particulate fraction contains a 52-fold higher dehydrogenase than reductase activity. The (microsomal) 200,000 x g particulate fraction is only 16-fold richer in dehydrogenase. Isopycnic centrifugations of the two fractions in Percoll gradients reveal that estrone reductase and the coequilibrating marker enzyme cytochrome c reductase occur in constant proportions, whereas the dehydrogenase/cytochrome c reductase ratios are different. Both, the kinetic data and the structural assignments speak in favour of individual enzymes catalyzing the dehydrogenation of estradiol and the reduction of estrone. All gradient fractions exhibiting dehydrogenase activity feature small, electrodense vesicles of 0.15-0.20 microns in diameter as a common structural element which might harbour the dehydrogenase.

Structural assignment and extractability of microsomal estradiol receptors.

Two electrophoretically different forms of estradiol receptor can be extracted from crude porcine endometrium microsomes with low ionic strength buffers. Better yields (approximately 50%) of both forms are obtained in the presence of Surfynol 485. Dithiothreitol boosts the solubilization of basic receptor. Together, Surfynol and dithiothreitol have a more than additive effect, amounting to 3-4 times the quantities of receptor extracted with plain buffer. Trypsin more than triples the yields obtained with Surfynol/dithiothreitol, while degrading both receptor forms to a characteristic fragment. Hyaluronoglucosaminidase is somewhat less effective than trypsin. It changes acidic receptor to basic. The proportions of acidic/basic receptor in microsomal subfractions are different. Rough endoplasmic reticulum contains almost exclusively basic receptor. Smooth membranes are rich in acidic receptor. The efficacy of both enzymes is closely related to the proportion of acidic receptor found in Surfynol/dithiothreitol extracts.

Immunogold labelling of estradiol receptor in MCF7 cells.

The distribution of estradiol receptor in serial sections of estradiol-deprived and estradiol-stimulated MCF7 cells was studied by using mouse monoclonal antibodies reacting with different domains of the receptor and goat-antimouse IgG/6 nm gold. In the nucleus and the cytoplasm of estradiol-deprived cells, the receptor was detected by all three monoclonals (13H2, HT 65 and MA1-310). The antibodies 13H2 and MA1-310 detected receptor associated to the microfilament bundles in the cytoplasm. Higher densities of antireceptor attachment to the nuclear areas were accompanied by a reduction in the attachment to the cytoplasm after estradiol stimulation of the cells. The results confirm earlier observations on the presence of cytoplasmic estrogen receptor in estradiol-deprived cells and support the premise of an estradiol-induced translocation of this ligand-dependent transcription regulator.

Immunocytochemistry by electron spectroscopic imaging using well defined boronated monovalent antibody fragments.

Contributing to the rapidly developing field of immunoelectron microscopy a new kind of markers has been created. The element boron, incorporated as very stable carborane clusters into different kinds of peptides, served as a marker detectable by electron spectroscopic imaging (ESI)--an electron microscopic technique with high-resolution potential. Covalently linked immunoreagents conspicuous by the small size of both antigen recognizing part and marker moiety are accessible by using peptide concepts for label construction and their conjugation with Fab' fragments. Due to a specific labeling of the free thiol groups of the Fab' fragments, the antigen binding capacity was not affected by the attachment of the markers and the resulting immunoprobes exhibited an elongated shape with the antigen combining site and the label located at opposite ends. The labeling densities observed with these reagents were found to be significantly higher than those obtained by using conventional colloidal gold methods. Combined with digital image processing and analysis systems, boron-based ESI proved to be a powerful approach in ultrastructural immunocytochemistry employing pre- and post-embedding methods.

Multicellular and aggregative behaviour of Salmonella typhimurium strains is controlled by mutations in the agfD promoter.

A colony morphology type is described in which cells of Salmonella typhimurium form a rigid multicellular network with expression of thin aggregative fimbriae that mediate tight intercellular bonds. Surface translocation of cells on plates and adherence to glass and polystyrene surfaces in biofilm assays are further characteristics of the morphotype. This morphotype (rdar) is normally expressed only at low temperature. However, in two unrelated S. typhimurium strains, spontaneous mutants were found forming rdar colonies independent of temperature. Allelic replacement proved a single point mutation in the promoter region of PagfD in each of the two mutants to be responsible for the constitutive phenotype of a multicellular behaviour. Transcription levels of the two divergently transcribed agf operons required for biogenesis of thin aggregative fimbriae by Northern blot analysis with gene probes for agfA and agfD as well as expression levels of AgfA by Western blotting were compared in the wild type, the constitutive mutants and their respective ompR and rpoS- derivatives. In the wild type the rdar morphotype and expression of thin aggregative fimbriae are restricted to low temperature on plates containing rich medium of low osmolarity, but biogenesis of thin aggregative fimbriae occurs upon iron starvation at 37 degrees C. In the upregulated mutants biogenesis of thin aggregative fimbriae is only abolished at high osmolarity at 37 degrees C and in the exponential phase in broth culture. Control of expression of thin aggregative fimbriae and rdar morphology takes place at the transcriptional level at the agfD promoter. A functional ompR allele is required, however an rpoS mutation abolishes transcription only in the wild type, but has no influence on expression of thin aggregative fimbriae in the constitutive mutants.

Passage of a heterologous protein through ileal enterocytes of newborn piglets: immunolabelling of bovine serum albumin at the light- and electron microscopic level.

The passage of bovine serum albumin through ileum enterocytes of neonatal pigs was studied by light microscopy with indirect immunoperoxidase and by electron microscopy with post-embedding direct immunogold methods. Vacuoles filled with the heterologous protein were seen as early as 10 min after the administration of either bovine serum or solutions of bovine serum albumin by gavage. The sizes of vacuoles increased with time, their electron densities and immunoreactivities were at variance. The formation of albumin-containing vacuoles was independent of the concentration of the solutions fed, ranging from 1 to 7%. Bovine serum albumin becomes discernible in the capillaries at 4 h after feeding. By then, the intact albumin transported through enterocytes amounted to more than 10% of the circulating plasma proteins. Of several thousand enterocytes screened in the whole study only one--from the piglet 4 h after feeding--contained lysosomes.

The 23-kDa light-stress-regulated heat-shock protein of chenopodium rubrum L. is located in the mitochondria.

The 23-kDa nuclear-encoded heat-shock protein (HSP) of Chenopodium rubrum L. is regulated by light at the posttranslational level. Higher light intensities are more effective in inducing the accumulation of the mature protein under heat-shock conditions. Based on this and other properties the protein was considered to belong to the group of small chloroplastic HSPs. However, we have now obtained the following evidence that this 23-kDa HSP is localized in the mitochondria: (i) Immunogold-labelled protein was almost exclusively restricted to the mitochondria in electron microscope thin sections. (ii) Using purified, isolated mitochondria from potato tubers the in-vitro-synthesized translation product of 31 kDa was readily transported into mitochondria where it was processed to the 23-kDa product. (iii) The protein could be detected by Western blotting in a preparation of washed mitochondria of Chenopodium, while under the same conditions no signal could be obtained in a preparation of isolated chloroplasts. (iv) Finally, sequence comparison with the published sequences of mitochondrial proteins by Lenne et al. (1995, Biochem J 311:805-813) and LaFayette et al. (1996, Plant Mol Biol 30:159-169) showed clearly that the 23-kDa protein is considerably more similar to these two proteins than to the group of plastid small HSPs. From these data we infer that mitochondria are involved in the response of the plants to high light stress under heat-shock conditions.