Immunoassay of androgen binding protein in blood: a new approach for study of the seminiferous tubule.

Androgen binding protein, a secretory product of seminiferous tubules, was isolated by means of affinity chromatography. A radioimmunoassay was developed and used to identify androgen binding protein in rat plasma. The ability to measure a testicular protein in blood provides a new method for investigation of seminiferous tubular physiology.

Hormonal regulation of rat androgen-binding protein (ABP) messenger ribonucleic acid and homology of human testosterone-estradiol-binding globulin and ABP complementary deoxyribonucleic acids.

Complementary DNA clones coding for rat androgen-binding protein (rABP) were isolated from a rat testis cDNA library constructed in the bacteriophage lambda gt11. The library was screened immunochemically, using two different antibodies against rABP. The identity of the isolated clones was confirmed by epitope selection and DNA sequence analysis. The mRNA encoding rABP could be detected in the testes of 20- and 46-day-old-rats, but not in the 10-day-old rats by hybridization with 32P-labeled rABP cDNA in a Northern blot of poly(A)+-RNA fractioned by agarose gel electrophoresis. No hybridization signal was seen with poly(A)+-RNA isolated from kidney and liver. The rABP mRNA appeared as a single species with a size of 1.65 kilobase, sufficient to encode a protein of 42,000 daltons. The concentration of rABP mRNA in the testes of 37-day-old hypophysectomized rats increased after treatment with testosterone and FSH, given alone or in combination. Sequence and hybridization analysis of cDNAs for rABP, human testosterone-estradiol-binding globulin, and human ABP demonstrates that the cDNAs for human testosterone-estradiol binding globulin and human ABP have greater sequence similarity with each other than either has with rABP.

Androgen receptor distribution in rat testis: new implications for androgen regulation of spermatogenesis.

The distribution of the androgen receptor (AR) in the adult rat testis was determined by biotin-streptavidin immunoperoxidase, employing tissue embedded in polyester wax which preserves antigenicity without compromising tissue preservation. The antibody probe used, which has been characterized previously, was an affinity purified, rabbit polyclonal antibody raised to the amino terminus peptide of the rat AR. Within the interstitial compartment, AR immunostaining was detected in some Leydig cells and all smooth muscle cells forming the walls of blood vessels, but endothelial cells of blood vessels were negative. Furthermore, in those Leydig cells that were clearly identified as exhibiting AR immunostaining, the intensity of the reaction varied. In the seminiferous tubules AR immunostaining was observed in all peritubular myoid cell nuclei, but not in the distal layer of lymphatic endothelial cells. In Sertoli cells, nuclear AR immunostaining was stage specific. Moderate AR immunostaining first became evident at late stage IV or early stage V 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, the spermatids in which nuclear elongation is apparent but chromatin condensation has not yet begun. Next, with onset of chromatin condensation, nuclear AR immunostaining in elongated spermatids was not discerned concomitant with its detection in the cytoplasm of the germ cells. These results are interpreted in the following manner: 1) The presence of AR in Leydig cells is consistent with the hypothesis that androgens modify Leydig cell activity in an autocrine fashion. Further, that not all Leydig cells exhibited AR immunostaining at steady state suggests a differential, functional activity of these cells within the population. 2) The intense AR immunostaining of smooth muscle cells present in the interstitium indicates that these cells are targets for androgens. 3) AR immunoreactivity in both Sertoli and peritubular myoid cells suggests their involvement in the androgenic control of spermatogenesis. The stage specific AR immunoreactivity in Sertoli cells, however, may be more indicative of a specific androgen response during these stages, whereas peritubular cells may participate in the tonal maintenance of spermatogenesis. 4) The specific presence of AR in step 11 elongated spermatids may suggest that androgens can act directly on germ cells to regulate spermatogenesis.

The epididymis contributes minimally to serum androgen-binding protein in the rat: a whole body kinetic study.

The half-times, MCRs, and secretion rates of androgen-binding protein (rABP) were determined in male rats under a variety of conditions. After orchiectomy, the disappearance of endogenous immunoassayable rABP from serum was described by a single exponential term with half-lives of 21 +/- 0.2 and 20 +/- 0.8 h at 25 and 90 days, respectively. The MCR (milliliters per g/day) was not affected by age or hormonal status of the animals. The secretion rate of rABP into the blood was higher in the immature animals than in adults. The decrease in serum rABP concentrations after 20-25 days of age was due to a decrease in the rate of secretion into blood rather than an increase in MCR, a finding consistent with the observation that after formation of the blood-testis barrier, most of the rABP is secreted into the seminiferous tubular lumen. The disappearance curve after injection of purified epididymal rABP was best described by two exponential terms. The first component disappeared very rapidly and the second more slowly, with a half-time corresponding to that of endogenous rABP. The MCR calculated from the latter component was the same as that for endogenous rABP. Having established the kinetic parameters for rABP in serum, a series of experiments was conducted to determine whether it was possible for the epididymis to release this protein into the blood. The apparent half-time of rABP measured in rats in which the testes had been removed and the epididymides left intact was found to be 65 +/- 3 to 70 +/- 5 h in three separate experiments. This increase over the actual half-life of rABP (20 h) was due to the release of rABP from the epididymis into the blood. A similar experiment was performed in an identical group of animals (testes removed, epididymides intact) that had been treated with testosterone via Silastic implants. In these animals the apparent half-time (24 +/- 4 to 28 +/- 2 h; three experiments) closely approximated the actual half-life (20 h). These findings indicate that androgens retarded degeneration of the epididymides, thus minimizing their release of rABP into blood. Our experimental findings suggest the following conclusions. The dramatic rise and subsequent decline of serum rABP concentrations that occur before puberty are due to changes in the secretion rate rather than in the MCR, which is unaffected by age or hormonal states.(ABSTRACT TRUNCATED AT 400 WORDS)

The heterogeneity of rat androgen-binding protein in serum differs from that in testis and epididymis.

Fractionation of testicular, epididymal, and serum extracts containing rat androgen-binding protein (rABP) on a Concanavalin A-Sepharose (Con A) column resolved two peaks of immunoreactive protein. The first peak was present in the void volume, and the other was bound by the column and specifically eluted by alpha-methyl-D-glucoside. These two peaks of immunoreactive rABP have been designated form I and form II for the portions of rABP that do not and do bind, respectively, to Concanavalin A. In the course of studying this heterogeneity, we observed that the distribution of the two forms of rABP was the same in the blood and cytosols prepared from testis and epididymis of young rats before the formation of the blood-testis barrier; that is, the ratio of form I to form II ranged from 1:1 to 1:2. Similar heterogeneity was observed in extracts of the reproductive tract from mature animals. However, the blood of adult rats contained reduced amounts of form I relative to form II, so that their ratio was about 1:5. Subsequent studies of infertile rats heterozygous for the Hre gene (Hre/ +), in which total rABP secretion was decreased, and of their normal littermates, indicated that the reduced amount of form I ABP in the sera of mature rats is typical of adult animals regardless of strain or genetic abnormality. The reduced amount of form I relative to form II observed in the blood of adult rats could result from either reduced secretion or increased metabolic clearance of form I in the blood compartment. To distinguish between these possibilities, the blood clearance of the two forms was estimated after orchiectomy. The disappearance rate of form I was not significantly different from that of either form II or unfractionated serum. These results are consistent with reduced release into blood of form I relative to form II rABP rather than increased clearance of form I in adult animals.

Receptor-mediated endocytosis of an extracellular steroid-binding protein (TeBG) in MCF-7 human breast cancer cells.

Previous studies suggested that an extracellular steroid-binding protein, testosterone-estradiol-binding globulin (TeBG), can enter a variety of cells. Experiments were conducted to determine whether uptake of TeBG occurs by nonspecific fluid phase endocytosis or via a specific receptor-mediated process. In human breast carcinoma cells (MCF-7) maintained on serum-free medium, exposure to radiolabeled TeBG resulted in cellular uptake, which reached a plateau by 6 h and could be inhibited 80% by competition with unlabeled TeBG. Uptake was temperature dependent with cell-associated radioactivity at 37 C being 1.6-fold higher than at 4 C. Subsequent exposure of cells to pronase resulted in release of the cell-associated TeBG by 88% and 44% at 4 C and 37 C, respectively. After transfer to media devoid of TeBG, approximately 35% of cell-associated radioactivity was release into the medium at 37 C; it was not possible to distinguish whether this was released from the cell surface or from inside the cell. Investigation of the localization of TeBG-gold complexes by electron microscopy revealed that TeBG first binds to the plasmalemma. Within 15 min label appears in receptosomes, which fuse to form multivesicular endosomes. By 1 h all label is observed in multivesicular endosomes and lysosomes, most of which are in the Golgi zone. Localization of the internalized radioactivity using classical cell fractionation techniques showed it appears in a symmetrical band exhibiting the same buoyant density as the lysosomal marker acid phosphatase. The observations reported here show that: 1) TeBG binds to MCF-7 cells; 2) some of the bound TeBG is taken up via a mechanism with all the characteristics of receptor-mediated endocytosis; and 3) within these cells TeBG is localized in endosomes and lysosomes.

Measurement of a follicle-stimulating hormone-responsive protein of Sertoli cell origin using an enzyme-linked immunoblot assay.

Many proteins secreted by Sertoli cell-enriched cultures are maximally stimulated by a combination of FSH and testosterone. Since very few are stimulated primarily by FSH, we thought it pertinent to identify such proteins. Sertoli cell-enriched cultures were prepared from testes of 20-day-old rats and grown in serum-free medium containing insulin, transferrin, and epidermal growth factor and in such medium supplemented with FSH, testosterone, or FSH plus testosterone. Media were fractionated using HPLC, and proteins were identified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. A protein designated CMB-2, with an apparent mol wt of 22,000, was shown to increase in response to FSH. Antiserum was raised using denatured protein eluted from SDS-polyacrylamide gels as the antigen, and a specific immunoassay using a combination of SDS-polyacrylamide gel electrophoresis and Western blotting was developed. The production of CMB-2 by primary Sertoli cell-enriched cultures was found to increase in a dose-dependent manner in response to FSH (30-1000 ng/ml); secretion was not significantly affected by testosterone (2 X 10(-13) M). An investigation of the tissue distribution of CMB-2 showed that the puberty, CMB-2 is secreted into the rete testis and accumulates in the epididymis in high concentration. We conclude that CMB-2 will be a useful marker to study the action of FSH on the rat testis.

Human testicular androgen-binding protein shares immunodeterminants with serum testosterone-estradiol-binding globulin.

In the human, there are two glycoproteins, testosterone-estradiol-binding globulin (hTeBG) and androgen-binding protein (hABP), which bind testosterone. Although these two proteins have similar physicochemical properties, they can be distinguished on the basis of origin and lectin binding. hTeBG is made in the liver and exhibits high affinity for Concanavalin A (Con A), while hABP from the testes is only partially bound to this lectin. That is, when testicular extracts were applied to Con A-Sepharose columns, a portion of the testosterone-binding material showed no interaction with the lectin and eluted in the void volume (peak I), while the remainder interacted strongly and could be eluted with alpha-methyl-D-glucoside (peak II). These observations are consistent with the proposal that peak I contains only hABP, whereas peak II contains hTeBG and/or hABP with carbohydrate units that permit binding to Con A. To further study the properties of these binding proteins, a hTeBG RIA using a monospecific antiserum was employed to compare the proteins in testes and serum. The results indicated that the testosterone-binding activities in peaks I and II of testicular extracts could not be distinguished immunologically from hTeBG in sera of normal women. These findings suggested that hTeBG and hABP share common epitopes. We next determined whether hABP was secreted into the blood or amniotic fluid by fractionating these fluids in Con A-Sepharose columns. Unlike testicular extracts, male serum and amniotic fluid contained single immunoreactive and steroid-binding species which bound specifically to Con A. We conclude from these observations that hABP (peak I), peak II activity, and hTeBG have common immunodeterminants and that if hABP is secreted into the blood of men, then its carbohydrate chains bind to Con A, making it indistinguishable from hTeBG under these conditions.

Binding of an extracellular steroid-binding globulin to membranes and soluble receptors from human breast cancer cells (MCF-7 cells).

Studies of MCF-7 breast cancer cells demonstrated that sex hormone-binding globulin (SHBG) is internalized by receptor-mediated endocytosis. The present study demonstrated specific binding of SHBG to receptor on membranes isolated from MCF-7 cells. Scatchard analysis of these binding studies suggested that SHBG binds to a single class of sites on membranes. The analysis yielded a dissociation constant (Kd) at 37 C of 3 x 10(-8) M and a binding capacity of 48 +2- 0.12 pmol/mg protein. A procedure for solubilizing the SHBG receptor from MCF-7 membranes used buffers containing protease inhibitors, 10% glycerol, and 10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Solubilization of the receptor resulted in a 5-fold increase in its binding capacity (246 +/- 14 pmol/mg protein) and a 10-fold decrease in binding affinity (Kd at 37 C = 2 x 10(-7) M).

Sertoli cell maturation is impaired by neonatal passive immunization with antiserum to luteinizing hormone-releasing hormone.

Male rats treated with a single injection of antiserum to LHRH (LHRH-AS) at 5 days of age have small testes as adults. In the present investigation, the serial maturation of the hypothalamic-pituitary-gonadal axis was studied in young male rats passively immunized with LHRH-AS. Testicular and epididymal weights, serum androgen and gonadotropin levels, testicular receptors for human CG (hCG), and androgen binding protein (ABP) concentrations in serum, testis, and epididymis were compared in developing animals treated with a single ip injection of LHRH-AS or normal rabbit serum. Rats treated with LHRH-AS had lower serum concentrations of ABP at all ages; the highest levels were on days 22-24, which were several days later than controls. Testicular weight was about 60% that of the control at all ages from 10-90 days. A reduction in epididymal weight to 80% that of the control was seen only in adults at days 60 and 90. Testicular ABP content increased steadily with age, but its concentration peaked at day 17 for controls and day 22 for LHRH-AS treated animals. Both testicular and epididymal ABP content were commensurate with testicular weight in controls and treated rats through day 45. Similarly, hCG-receptor content and concentration increased steadily with age, but differences between control and treated groups paralleled testicular weight. These results suggest an effect of LHRH blockade at a critical period which impairs early testicular growth and causes a permanent reduction in growth. Sertoli cell function and hCG-receptor appearance are impaired in proportion to this reduction.

Immunocytochemical localization of androgen-binding protein in the male rat reproductive tract.

The localization of androgen-binding protein (ABP) in the reproductive tract of young adult male rats was studied with the peroxidase-antiperoxidase technique using frozen sections and light microscopy. Within the seminiferous tubules, a positive reaction was noted in the apical portion of the epithelium, apparently in spermatids and/or Sertoli cells. ABP was localized in granules in the apical cytoplasm of the principal epithelial cells of the proximal part of the caput epididymis and in the epithelial cells of the ductuli efferentes. The cells in the distal part of the caput as well as the corpus and cauda of the epididymis did not contain ABP. Numerous coated vesicles and multivesicular bodies were present in the supranuclear cytoplasm of the epididymal epithelium where ABP was taken up. The results indicate that ABP is taken up from the lumen by epithelial cells of the ductuli efferentes and proximal part of the caput epididymis.

Differential regulation of testicular transferrin and androgen-binding protein secretion in primary cultures of rat Sertoli cells.

Specific RIAs for rat transferrin (rTF) and androgen-binding protein (rABP) were used to determine whether the secretion of these proteins was coordinately regulated in the Sertoli cell under a variety of conditions. Sertoli cell-enriched primary cultures were prepared from the testes of 20-day-old rats, and rTF and rABP were assayed in medium from the same culture. There was a strong effect of cell density on both rABP and rTF secretion per cell, with increased secretion per cell at high densities. Human TF (hTF), FeSO4, and desferrioxamine had little or no effect on rTF secretion. The age of the animal at the time of preparation of cells for culture had a strong effect on the pattern of rTF and rABP secretion in vitro; however, the effects of animal age, time in culture, and medium supplementation differed for the two proteins. In cultures prepared from 20-day-old animals, insulin, epidermal growth factor, and testosterone stimulated both rTF and rABP secretion, although to different extents. Retinoic acid was required for the stimulation and maintenance of rTF secretion, but had no effect on rABP secretion in the presence of insulin, hTF, and epidermal growth factor. Conversely, FSH and isoproterenol stimulated rABP, but not rTF, secretion. These data suggest that the secretion of rABP and rTF by Sertoli cells is under differential control.

Spermatogenesis in vitro: completion of meiosis and early spermiogenesis.

In vitro formation of haploid spermatids has not been convincingly demonstrated in mammals. To investigate this problem we selected defined segments of rat seminiferous tubules containing late pachytene and diakinetic primary spermatocytes (Stages XII and XIII of the cycle) for culture in a chemically defined medium. After 2 days, most spermatocytes completed both meiotic divisions, and by 6 days the tubular epithelium developed morphologic characteristics of Stage V in which the newly formed spermatids had acrosomic systems characteristic of step 5 spermiogenesis. The seminiferous tubules also differentiated biochemically as evidenced by increased production of proteins characteristically secreted by Stage V. Since this in vitro differentiation of the germinal epithelium occurred in the absence of testosterone and FSH, we conclude that late pachytene spermatocytes and their associated Sertoli cells have all the information required for both meiotic divisions and early spermiogenesis.

Influx of testosterone-binding globulin (TeBG) and TeBG-bound sex steroid hormones into rat testis and prostate.

The availability of testosterone and estradiol to Sertoli and prostate cells is dependent upon 1) the permeability properties of the blood-tubular barrier (BTB) of the testis or prostate cell membrane, and 2) sex steroid binding to plasma proteins, such as albumin or testosterone-binding globulin (TeBG). Sex steroid influx into these tissues was studied after in vivo arterial bolus injections of [3H]testosterone or [3H]estradiol in anesthetized rats. Both testosterone and estradiol were readily cleared across the BTB or prostate cell membrane in the absence of plasma proteins and in the presence of human pregnancy serum, in which testosterone or estradiol are 80-95% distributed to TeBG. The extravascular extraction of [3H]TeBG across the BTB or prostate plasma membrane [73 +/- 2% (+/- SE) and 92 +/- 9%, respectively] was significantly greater than extraction of [3H]albumin or other plasma space markers and indicative of a rapid first pass clearance of TeBG by Sertoli or prostate cells. In summary, these studies indicate that 1) testosterone and estradiol are readily cleared by Sertoli and prostate cells; 2) albumin- and TeBG-bound sex steroids represent the major circulating pool of bioavailable hormone for testis or prostate; and 3) the TeBG-sex steroid complex may be nearly completely available for influx through the BTB or prostate plasma membrane.

Radioimmunoassay of testosterone-estradiol-binding globulin in humans: a reassessment of normal values.

Antiserum against human testosterone-estradiol-binding globulin (hTeBG) was prepared in rabbits, and its specificity was demonstrated by crossed immunoelectrophoresis. A RIA for the measurement of hTeBG in serum was developed. With this assay, hTeBG was readily measured in 1-2 microliters serum. Interassay coefficients of variation for pools of serum from men, women, and women in late pregnancy were 7.7%, 5.5%, and 6.1%, respectively. Interassay coefficients of variation for the same pools were 10%, 12%, and 12%, respectively. The TeBG levels in a number of nonselected subjected determined by the present method show good correlations with those obtained by steady state polyacrylamide gel electrophoresis and dextran-coated charcoal assay. The concentrations of TeBG determined by the RIA in sera from men, women, and women in late pregnancy were 18 +/- 9 (n = 12), 54 +/- 13 (n = 8), and 374 +/- 55 (n = 6) pmol/ml (mean +/- SD), respectively.

The cDNA-deduced primary structure of human sex hormone-binding globulin and location of its steroid-binding domain.

We have sequenced a cDNA for sex hormone-binding globulin (SHBG) isolated from a phage lambda gt11 human liver cDNA library. The library was screened with a radiolabeled rat androgen-binding protein (ABP) cDNA, and the abundance of SHBG cDNAs was 1 in 750,000 plaques examined. The largest human SHBG cDNA (1194 base-pairs) contained a reading frame for 381 amino acids. This comprised 8 amino acids of a signal peptide followed by 373 residues starting with the known NH2-terminal sequence of human SHBG, and ending with a termination codon. The predicted polypeptide Mr of SHBG is 40,509, and sites of attachment of one O-linked (residue 7) and two N-linked oligosaccharide (residues 351 and 367) chains were identified. Purified SHBG was photoaffinity-labeled with delta 6-[3H]testosterone and cleaved with trypsin. The labeled tryptic fragment was isolated by reverse-phase HPLC, and its NH2-terminal sequence was determined. The results suggest that a portion of the steroid-binding domain of SHBG is located between residue 296 and the 35 predominantly hydrophilic residues at the C-terminus of the protein.

Genetic variations in human testosterone-estradiol binding globulin.

The human testosterone-estradiol-binding globulin (hTeBG) is a plasma heterogeneous glycoprotein with high affinity for a number of circulating steroid hormones. The heterogeneity originates from differential glycosylation of a common protein precursor. Analysis of desialylated hTeBG by isoelectric focusing (IEF) has revealed that microheterogeneity could be partly attributed to variability in sialic acid content or rearrangement of amino acid composition. We have studied this possibility by the analysis of desialylated serum hTeBG by Western blotting of proteins previously separated on IEF-gels. Two distinct well-defined IEF patterns were identified. The most frequent consisted of two major IEF-bands of equal color intensity. The other pattern consisting of four IEF-bands was present in only 5.55% of the total serum samples analyzed. Family studies showed that these phenotypes were autosomally inherited with a simple Mendelian transmission and allele frequencies had an excellent agreement between the observed and expected phenotypes. Androgen affinity constants and serum concentrations of hTeBG variant were similar to those of normal hTeBG. Molecular analyses of each of the exons of hTeBG gene by denaturing gradient gel electrophoresis revealed the presence of a point mutation in exon 8. The studies presented herein confirm and extend previous reports on the existence of structural variants of hTeBG. In addition, the mutation reported in this study is probably the same as that recently identified within numerous ethnic groups throughout the world, thus further supporting the concept of a two allele gene worldwide concoding hTeBG.

The extracellular sex steroid-binding proteins of testis and liver. Structure-function studies.

Though the existence of extracellular sex steroid-binding proteins has been known for a number of years, we are still only on the threshold of understanding their biological role. Through efforts such as those described above, we are beginning to examine the structure of these macromolecules and correlating them with present known functions. As our understanding of the function of these proteins evolves, we will be further able to ascribe structural domains.

Developmental changes in brain and serum binding of testosterone and in brain capillary uptake of testosterone-binding serum proteins in the rabbit.

Developmental changes in the brain uptake of circulating testosterone and of testosterone-binding proteins, such as testosterone-binding globulin (TeBG) or albumin, may play a role in the sexually dimorphic changes in brain structure that are mediated by circulating testosterone. The present studies examine developmental changes in binding of testosterone in both the serum and brain compartments in postnatal rabbits in vivo and developmental changes in the uptake of [3H]TeBG or [3H]albumin by capillaries isolated from developing rabbit brain. The results show that between 10 and 15 days postnatally both the brain sequestration of testosterone and rabbit serum binding of the hormone are markedly increased relative to the newborn period. In addition, both [3H]TeBG and [3H]albumin were taken up by microvessels isolated from 28-day-old rabbit brain, and this process for [3H]TeBG was more active in capillaries obtained from neonatal rats as opposed to adult rats. In summary, these studies show that the binding systems for testosterone are modulated in a parallel fashion in both the serum and brain compartments. In addition, uptake mechanisms for serum testosterone-binding proteins such as TeBG and, to a lesser extent, albumin exist in the capillaries of developing rabbits. These brain capillary plasma protein uptake systems may allow for the distribution into brain of circulating serum proteins such as TeBG and, to a lesser extent, albumin, in developing rabbits.

Culture of ciliated and nonciliated cells from rat ductuli efferentes.

The isolation and culture of ciliated and nonciliated cells from rat ductuli efferentes is described. Fragments of epithelium obtained after two collagenase digestions attached to plastic and to extracellular matrix and could be maintained in culture for at least 2 weeks. Ciliary beating in cells grown on epididymal extracellular matrix-coated plastic could be observed for up to 7 days in culture. Although cells maintained on this substrate retained organelles characteristic of cells in vivo, they assumed a flattened, squamous appearance. In contrast, cells growing on the surface of permeable supports impregnated with extracellular matrix were polarized and exhibited a cuboidal/columnar appearance. Androgen binding protein conjugated to colloidal gold was taken up by these cells via coated pits and was found sequentially in uncoated endosomes, multivesicular bodies and lysosomes.