Hormonal control of uteroglobin secretion in rabbit uterus. Inhibition of uteroglobin synthesis and messenger ribonucleic acid accumulation by oestrogen and anti-oestrogen administration

Investigations were conducted to quantify activity of uteroglobin mRNA and secretion of uteroglobin in rabbit uterus after administration of progesterone and 5alpha-dihydrotestosterone, either alone or concomitantly with oestradiol-17beta and tamoxifen, a non-steroidal anti-oestrogen. Poly(A)-containing mRNA was isolated from the uterine tissue by extraction with phenol/chloroform, precipitation with ethanol and chromatography on oligo(dT)-cellulose. Cell-free translation in vitro of the poly(A)-containing mRNA was carried out in a wheat-germ lysate, and the product isolated by specific immuno-precipitation with anti-uteroglobin antiserum purified by affinity chromatography. Radioimmunoassay was utilized to determine uteroglobin content in the uterine flushings and tissue preparations. When given for 5 days, both progesterone (1mg/kg per day) and 5alpha-dihydrotestosterone (25mg/kg per day) elicited a marked induction of uteroglobin secretion, which was accompanied with accumulation of uteroglobin mRNA in the tissue. Concomitant administration of oestradiol-17beta (50mug/kg per day) or tamoxifen (12.5mg/kg per day) significantly decreased both progesterone- and 5alpha-dihydrotestosterone-induced uteroglobin secretion, with a parallel decrease in the uteroglobin-mRNA activity. The decline in the uteroglobin content of the uterine flushes brought about by oestradiol-17beta or tamoxifen administration was not due to inhibition of secretion of this protein by the endometrial cells, since a simultaneous decrease occurred in the tissue uteroglobin content. After a 5-day pretreatment with progesterone (1mg/kg per day), administration of oestradiol-17beta (50mug/kg per day) during the ensuing 4 days greatly accelerated the decay of the uteroglobin content in the uterine fluid.     

Evidence of a uteroglobin-like protein in epithelial cells of reproductive and non-reproductive tissues of the rabbit.

An antiserum raised in a goat to a uteroglobin-like protein isolated from uterine fluid of oestrous rabbits was used in an immune fluorescence test to localize an antigen present in the reproductive tract of oestrous and pseudopregnant rabbits and mammary gland tissue. The antigen was also present in the vas deferens and seminal vesicle, but not in testis. Non-reproductive tissues, such as lung, small intestine, bladder and thyroid showed specific fluorescent staining which was eliminated or significantly reduced by absorption of the antiserum with a purified uteroglobin preparation.     

Further study of the problem of specific cellular receptors in delayed hypersensitivity]

The work presents the materials obtained as a result of the further study of specific T lymphocyte receptors with the use of so-called receptor antisera, i. e. antisera against the lymphoid cells of mice sensitized with one of the two antigens (Macobacterium tuberculosis or bovine gamma globulin); thus these differed from control antisera against the lymphoid cells of intact mice. These mouse antisera reacted with the lymphoid cells of guinea pigs in experiments of delayed-type hypersensitivity transfer. The cells of sensitized guinea pigs lost their ability to transfer hypersensitivity if, prior to their injection into the recipient guinea pigs, these cells were treated with the above-mentioned mouse antisera, i. e. antisera against the lymphoid cells of mice had a blocking effect on the lymphoid cells of guinea pigs. The blocking action of the antisera proved to be specific: antisera against the lymphoid cells of mice sensitized to bovine gamma gloublin blocked the cells of guinea pigs, also sensitized to bovine gamma globulins, but did not block the cells sensitized to Mycobacterium tuberculosis. The control antisera, taken in the same doses as the factor antisera, did not show a blocking effect on the specific activity of lymphoid cells.     

Evidence for the identity of anti-proteinase pulmonary protein CCSP and uteroglobin

Purified Clara cell secretory protein (CCSP) from rabbit lung was analyzed by SDS gel electrophoresis, and immunoblotting with a specific anti-uteroglobin antibody as well as for its ability to bind [3H]progesterone. The results obtained indicate that proteins CCSP and uteroglobin are identical.     

Cell-free synthesis of preuteroglobin and processing to uteroglobin by polysomes from rabbit endometrium

Polysomes from rabbit endometrium, incubated in a cell-free system in the presence of [35S]methionine, completed polypeptide chains pre-initiated in vivo. Polypeptide chains were analyzed by SDS-gel electrophoresis after immunoprecipitation with anti-uteroglobin. Endometrial polysomes, freed of membranes by detergent treatment, synthesized both mature uteroglobin and preuteroglobin not yet cleaved in vivo. Membrane-bound polysomes synthesized exclusively mature uteroglobin, indicating the processing of preuteroglobin by the membranes bound to the ribosomes.     

Passive protection across subgroups of alphaviruses by hyperimmune non-cross-neutralizing anti-Sindbis serum

Extended hyperimmunization of rabbits with Sindbis (SIN) or Semliki Forest (SF) viruses causes the production of antisera that are cross-reactive with virus-infected cells in antibody-dependent, complement-mediated cytotoxicity assays but that do not cross-neutralize viruses in vitro. C3H/HeJ mice given gamma globulin fractionated from the extended hyperimmune antiserum against SIN, but not control sera, were protected from challenge by 100 LD50 of SF, a virus which is in a different subgroup than SIN. All mice survived if the gamma globulin was given 24 hr before challenge virus and partial protection occurred if the globulin was given 24 hr after the virus. Cobra venom factor treatment of normal C3H mice challenged with SF did not reduce the protection, suggesting that complement was not involved. Methyl palmitate (40 mg/mouse) given before gamma globulin and virus challenge suppressed macrophage activity and reduced the level of protection 23% in females and 70% in males. Silica treatment (3 mg/mouse) reduced the protection equally in both males and females by 92%. In vitro experiments were done to test if it were possible that cross-antibody-dependent cellular cytotoxicity (ADCC) could account for the passive cross-protection observed in this system. Cross-ADCC could be demonstrated in vitro at high dilutions of antiserum (1:25,600). On the basis of the in vitro and in vivo results presented, we suggest that cross-ADCC against SF-infected target cells is one of the likely mechanisms to explain the passive cross-protection observed.     

Production of monoclonal antibodies against avidin

Monoclonal antibodies of the IgG1 subclass were generated against chicken avidin. These antibodies were shown to be as sensitive as polyclonal antiserum in detecting avidin by radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA) methods. Furthermore, the monoclonal antibodies were considerably more specific. Our results with a monoclonal anti-avidin RIA support previous findings that in inflammatory conditions avidin is synthesized also in other organs than the oviduct, although in the liver a major part of the activity detected by polyclonal anti-avidin RIA or biotin-bentonite assay was not due to avidin.     

Antiserum to the milk fat globule membrane. Preparation and capacity to suppress milk secretion

A procedure is described for preparing rabbit antiserum to goat milk fat globule membrane. This membrane is derived from the secretory surface of the lactating cell. Immunoelectrophoresis and enzyme-linked immunosorbent assay showed that antibody development reached maximal levels in about 6–8 weeks. Infusion of 5–10 ml of this antiserum into the lactating mammary gland of goats via the teat canal depressed milk yields temporarily on the infused side to 60–80% of normal. Ordinary serum from rabbit, goat or human did not evoke such a response and rabbit complement was not essential for the effect. Fractionation showed that the globulin fraction of the antiserum contained the milk-suppressing principle. Milk from the antiserum-infused side of the udder showed extensive and tenacious clumping of fat globules on standing 12–24 h. The inhibition of milk flow by antibodies to the secretory membrane resembles a previously observed inhibition following infusion of concanavalin A or its succinyl derivative. Binding of antibodies or lectins which recognize specific surface protein components of the lactating cell appears to be involved in the suppression mechanism. The possible relevance of our findings to autoimmune suppression of exocytosis is noted.     

A modification of the unlabeled antibody enzyme method using heterologous antisera for the light microscopic and ultrastructural localization of insulin, glucagon and growth hormone.

The requirement of using homologous antisera (primary antiserum and peroxidase-antiperoxidase (PAP) complex raised in the same species) in the unlabeled antibody enzyme method has been investigated at the light and electron microscopic level using the localization of insulin, glucagon and growth hormone as model systems. Optimum immunocytochemical staining for all three antigens was observed when sheep or goat antirabbit gamma-globulin (S-ARgammaG or G-ARgammaG) were used to couple rabbit peroxidase-antiperoxidase complex with either guinea pig antisera to insulin (GP-AIS) or glucagon (GP-AGS), or monkey antisera to rat growth hormone (M-ARGH). The cross-reactivity between S-ARgammaG or G-ARgammaG and immunoglobulins in these primary antisera were substantiated by immunoelectrophoresis and radioimmunoassay. S-ARgammaG was shown to produce precipitation arcs with GP-AIS and M-ARGH that were similar to those seen when the latter were reacted with rabbit antiguinea pig gamma-globulin antiserum and goat antimonkey gamma-globulin antiserum, respectively. Radioimmunoassay results revealed that immunoprecipitation of 6-10% as compared to homologous antisera controls yielded excellent staining localization when S-ARgammaG was used for immunocytochemistry. Thus, heterologous antisera (primary antiserum and PAP complex raised in different species) may be used in the unlabeled antibody enzyme method as long as the coupling antiserum shows cross-reactivity with immunoglobulins of the primary antiserum and the PAP complex.     

Characteristics of the purified uteroglobin-like protein from rabbit lung.

A uteroglobin-like protein was prepared from lung extracts of female rabbits by absorption to immobilized anti-uteroglobin immunoglobulin and purified to homogeneity by gel filtration on Sephacryl S-200. The final preparation is indistinguishable from uteroglobin according to its behaviour in Ouchterlony double-diffusion, polyacrylamide gel electrophoresis under denaturing and non-denaturing conditions, ultraviolet spectrum, tryptic peptide analysis, and progesterone-binding properties. Progesterone binding to the lung protein exhibits an affinity similar to that observed with authentic uteroglobin and is equally enhanced by reduction of the protein with dithiothreitol. Competition experiments with non-radioactive steroids demonstrate a similar steroid-specificity for both proteins. Progesterone binding causes a perturbation in the ultraviolet absorbance of tyrosine residues of the lung protein similar to that observed with uteroglobin. These data suggest that the proteins prepared from both sources are biochemically identical.     

Selective immunoprecipitate identification using monoclonal anti-rabbit IgG

A monoclonal mouse antibody directed against rabbit IgG has been conjugated with horseradish peroxidase and used to identify immunoprecipitates which contain rabbit antibodies. By combining a specific rabbit antisera with a general antiserum from another species (e.g., goat antiserum against human serum), immunoprecipitates containing the antigen(s) recognized by the rabbit antibodies have been selectively identified by colorimetric development of peroxidase activity. Since the monoclonal antibody is specific for rabbit IgG and nonprecipitating, the peroxidase conjugate can be included in the agarose with the primary antisera.     

IMMUNOLOGICAL STUDIES OF THE RAT OLFACTORY MARKER PROTEIN1

Abstract— Antisera against the rat olfactory marker protein were prepared by injection of the homogeneous protein into a goat and a rabbit. When the antisera were tested by immunodiffusion against olfactory tissue extracts, many but not all mammalian species cross-reacted against these antisera. Immunoprecipitin titrations with the goat antiserum generally showed higher cross-reactivity against olfactory extracts from species more closely related to the rat. Human olfactory bulb extracts and non-mammalian olfactory tissue extracts did not cross-react with the antisera by either immunodiffusion tests or immunoprecipitin titrations, however, they did cross-react when tested by a competitive binding radioimmunoassay using tritium-labelled purified rat protein and the goat antibody. The olfactory marker protein which is an example of a brain protein specific to one cell, the olfactory chemoreceptor neuron, has a very wide species distribution, being present in rat, mouse, hamster, guinea-pig, sheep, cow, rabbit, pig, dog, man, frog and garfish. Therefore it presumably plays an important and unique role related to the function of this primary chemosensory neuron.     

So-called antireceptor sera

Experiments in delayed type hypersensitivity transfer were carried out with the aim of studying the ability of rabbit antisera against peritoneal exudate cells of rats sensitized with bovine gamma globulin or rabbit kidney tissue antigen to block peritoneal exudate cells of guinea pigs. In the serological test the antisera prepared against the cells of sensitized rats and tentatively named "receptor antisera", reacted not only with the cells of these rats, respectively, but also with guinea pig cells. In hypersensitivity transfer experiments in guinea pigs receptor antisera showed a blocking effect on the transferred cells, making them incapable of transferring hypersensitivity, i. e. rabbit antisera against rat peritoneal exudate cells reacted with guinea pig cells. This interaction was specific: the blocking effect was manifested only when guinea pigs whose cells were used in the transfer were sensitized with the same antigen as the rats against whose cells the receptor antisera had been prepared. The control antisera, taken for the treatment of the transferred cells in the same doses as the receptor antisera, had no blocking effect on the cells.     

Human macromolecular insoluble cold globulin (MICG). II. Immunologic definition of T cell and null cell MICG and the biologic effect of antiserum to MICG.

Thymus cell-derived macromolecular insoluble cold globulin (T-MICG) is a 225,000-dalton protein, selectively synthesized in human T cells. Null cell-derived macromolecular insoluble cold globulin (N-MICG) is a 185,000-dalton protein, synthesized in null cells, and antigenically distinct from T-MICG. Evidence to support these conclusions was provided by using isolated cell preparations that were radiolabeled, lysed in desoxycholate, and precipitated with monospecific antiserum to each component. These studies demonstrated that antiserum to T-MICG precipitated a 225,000 dalton protein from PBL and T cells, but not from B or null cells. Antiserum to N-MICG reacted with a 185,000 dalton protein present in PBL and null cells, but not with lysates from either T or B cells. The plasma membrane distribution of these proteins was shown by absorption of antiserum to T + N-MICG with either isolated T or null cells. Antibody-induced cytotoxicity and immunofluorescence confirmed the cell surface location of T and N-MICG. Divergent biologic effects of these antisera were also noted. Antiserum to T-MICG inhibited T cell rosette formation and the one-way mixed lymphocyte reaction, although anti-N-MICG antiserum had no such effect. The potential importance of these proteins is discussed.     

Specific lysis of GABAergic synaptosomes by an antiserum to glutamate decarboxylase

An antiserum to pure glutamate decarboxylase (GAD) when incubated with rat cortical synaptosomes in the presence of complement caused release of 33-53% of lactate dehydrogenase (LDH) and 22-41% of total GAD. In addition most of the gamma-aminobutyrate (GABA) present was released. Anti-GAD antiserum alone, or complement alone, were without action. The antiserum plus complement had no effect on noradrenaline or choline uptake, and did not release choline acetylase (ChAT). Anti-ChAT serum plus complement released 30-37% of ChAT and 10-13% of LDH. It prevented choline uptake. This serum did not produce GAD release or prevent GABA, choline or noradrenaline uptake. When cortical synaptosomes were exposed to both antisera plus complement, their actions were strictly additive. The data indicate specific lysis of GABAergic and cholinergic synaptosomal sub-populations.     

Immunolocalization of CC10 in Clara cells in mouse and human lung

Two antisera, denoted R41 and R42, were raised against a synthetic peptide from the murine Clara cell-specific protein CC10, and one antiserum, denoted R40, was raised against human recombinant uteroglobin, the human homolog of murine CC10. Purified antigen-specific antisera, denoted R40AP, R41AP, and R42AP were prepared using peptide columns. The purified antisera were characterized by dot blots, immunohistochemistry, and immunoblots. Immunohistochemistry of mouse lung showed specific labeling of Clara cells in distal bronchioles by all three antisera. In human lung, the antiuteroglobin antiserum specifically labeled Clara cells, while the anti-mouse peptide antisera had weak crossreactivity and higher background staining. Electron microscopy revealed immunogold labeling of CC10 granules in Clara cells of mouse lung with all antisera. All antisera also labeled a 5-kDa protein on immunoblots of mouse lung homogenates. The surface epithelium of the alveolar air spaces around the distal bronchioles were CC10 positive suggesting a functional activity for CC10 in the lung parenchyma distal to Clara cells. R40AP immunohistochemical staining of sections of normal human lungs and lungs from patients with surfactant protein B deficiency, bronchopneumonia, and idiopathic alveolar proteinosis illustrate the utility of the anti-human CC10 antibody for diagnostic pathology.     

Positive selection by "panning" of B lymphocytes using unfractionated anti-immunoglobulin antiserum

A simple method allowing a positive selection of mouse B lymphocytes by "panning" has been achieved using rabbit anti-mouse immunoglobulin antisera without further purification. Plastic petri dishes were coated with the gamma-globulin fraction of normal mouse serum and then incubated with excess rabbit anti-mouse gamma globulin antiserum. These plates were then used for the selection of B lymphocytes by incubation of spleen cells in the plates. The adherent cells wee above 95% B lymphocytes as judged by immunofluorescence staining, proliferated in response to bacterial lipopolysaccharide and were not stimulated by concanavalin A.     

Simplified Procedure for Preparation of Specific Antibodies to Gamma Globulins

Antibodies were prepared to rhesus monkey, rabbit, dog, and hamster gamma globulins which had been purified by agar-block electrophoresis. Immunoelectrophoretic analysis demonstrated that these antisera were specific for gamma globulin components in whole serum. The use of these antisera as secondary serological reagents was examined.     

Binding of progesterone to the proteins of the uterine luminal fluid. Identification of uteroglobin as the binding protein.

1. The uterine luminal fluid of rabbits treated with estradiol and progesterone contains a protein factor with high affinity for [3-H] progesterone which is not present in the uterine secretion of control rabbits treated with estradiol. 2. This progesterone dependent factor is shown by gel filtration and polyacrylamide gel electrophoresis to be identical with the uterus specific protein uteroglobin, which seems to be required during the preimplantation phase. Uteroglobin specific antiserum, prepared in guinea pigs, completely inhibits the progesterone binding activity of the proteins of the uterine fluid. 3. Progesterone binding to uteroglobin is dependent upon millimolar concentrations of dithioerythritol. At saturation, one molecule of progesterone binds per uteroglobin molecule and the apparent association constant is 2 x 10-6 M-1 at 0 degrees C. 4. The progesterone binding species of uteroglobin exhibits a molecular weight of around 12 000 on polyacrylamide gels containing dodecylsulfate, and of 15 000 upon gel filtration, indicating a non-globular shape. This molecule is compased of two subunits of similar molecular size which are held together by a disulfide bridge among other forces.     

Immunocytochemical localization of uteroglobin in the genital tract of male rabbits

Summary Using the immunoperoxidase technique and specific goat antiserum to uteroglobin, selective immunochemical staining products are localized in the epididymal epithelium of the caput and proximal corpus region, at the adluminal border of the cauda epididymidis and, as well known, in epithelial cells of the endometrium of pregnant and progesterone-treated rabbits. Specific staining is also seen on spermatozoa. A uteroglobin-like antigen has been similarly localized in alveolar and bronchial epithelial cells of the lung. Testis, prostate, seminal vesicle and ductus deferens do not seem to contain in their tissues immunoreactive uteroglobin-like antigens. Similarly, the uterus and ductus epididymidis of immature rabbits are devoid of immunoreactivity. The presence of uteroglobin-like antigens in tissues other than the endometrium, particularly the ductus epididymidis, stimulates new discussions on the function of this protein in reproductive physiology and fertility research.