Herbert H. Wotiz

An in vitro system for the binding of estradiol to rat uterine nuclei.

Abstract A in vitro system has been developed which is capable of specifically binding estradiol to rat uterine nuclei. Evidence is presented for the enzymatic nature of the process and the need for the presence of the supernatant during the incubation.

Separation and detection of nanogram amounts of steroids

Abstract The use of heptafluorobutyrates as derivatives for the ultra-sensitive measurement of hydroxyl containing steroids and using an electron affinity detector has been described. Following gas chromatography quantities of as little as 10−10 gram of steroid have been detected.

Estrogens and Progesterone in the Sea Urchin (Strongylocentrotus franciscanus) and Pecten (Pecten hericius).

Summary The ovaries of the sea urchin, Strongylocentrotus franciscanus, and the pecten, Pecten hericius, were extracted for steroids. Estrogenic activity equivalent to 10 μg or less per kilo of wet tissue was found in each species by bioassay. The most active estrogenic substance was identified as estradiol-17β. Separations were made by counter-current distribution (29 transfers: upper phase—70% methanol; lower phase—50% chloroformic, 50% carbon tetrachloride), paper chromatography (3 systems: toluene-propylene glycol; ligroin-propylene glycol; benzene-formamide), and bioassay (Astwood method). Progesterone was also identified tentatively by column chromatography and the Hooker-Forbes assay. Other progestational substances were present, one of which may be Δ4-3-keto-pregnen-20β-ol. The occurrence of these steroids in 2 distinctly different phyla indicates a wide distribution and may be of some significance in the evolution of sex hormones.

Dysfunctional Penile Cholinergic Nerves in Diabetic Impotent Men

Impotence in the diabetic man may be secondary to a neuropathic condition of the autonomic penile nerves. The relationship between autonomic neuropathy and impotence in diabetes was studied in human corporeal tissue obtained during implantation of a penile prosthesis in 19 impotent diabetic and 15 nondiabetic patients. The functional status of penile cholinergic nerves was assessed by determining their ability to accumulate tritiated choline (34), and synthesize (34) and release (19) tritiated-acetylcholine after incubation of corporeal tissue with tritiated-choline (34). Tritiated-choline accumulation, and tritiated-acetylcholine synthesis and release were significantly reduced in the corporeal tissue from diabetic patients compared to that from nondiabetic patients (p less than 0.05). The impairment in acetylcholine synthesis worsened with the duration of diabetes (p less than 0.025). No differences in the parameters measured were found between insulin-dependent (11) and noninsulin-dependent (8) diabetic patients. The ability of the cholinergic nerves to synthesize acetylcholine could not be predicted clinically with sensory vibration perception threshold testing. It is concluded that there is a functional penile neuropathic condition of the cholinergic nerves in the corpus cavernosum of diabetic impotent patients that may be responsible for the erectile dysfunction.

Gas Chromatographic Measurement of Plasma Estrogens Using an Electron Capture Detector

Abstract A new method for the measurement of plasma free or total estrone, estradiol and estriol using heptafluorobutyrates and electron capture detection has been described. Normal mean values for non-pregnant females of 0.241, 0.166 and 0.229 μg/100 ml for total E1, E2 and E3 respectively were found. The results are more nearly relatable to urinary excretion patterns than other procedures have indicated. This investigation has been carried out with the assistance of a Research Career Development award (GM K3 15,369) and a Research Grant (CA 03135) from the National Institutes of Health, United States Public Health Service, Bethesda, Maryland, and from Aid for Cancer Research, Brookline, Massachusetts. The authors are grateful to Dr. J. Dingman, Robert B. Brigham Hospital, Boston, Mass, for his advice and assistance in the setting up of the glass fiber paper Chromatographic systems.

Studies in steroid metabolism. XI. Gas chromatographic determination of estrogens in human pregnancy urine.

Abstract A rapid, chemically specific, and reproducible method for the determination of estrogens in pregnancy urine by means of gas-liquid partition chromatography is described. The use of estrogen acetates to facilitate separation on short silicone grease columns has been further established. Further evidence for the slight destruction of urinary estrogen conjugates by mineral acid is demonstrated.

Development and characterization of monoclonal antibodies to a specific domain of human estrogen receptor

We have synthesized three peptides with amino acid sequences identical to those spanning amino acids 201-215, 231-245, and 247-261 of the human estrogen receptor (hER). These peptides were conjugated to keyhole limpet hemocyanin and used as immunogens to develop monoclonal antibodies (MoAbs) to hER. Antibody responses were only elicited by the peptide with amino acid sequence 247-261. Splenocytes from immunized mice were used for hybridoma production. Of the seven MoAbs that recognized the native (functional) form of the ER, four (MoAbs 16, 33, 114, and 213) recognized the ER with high affinity, as demonstrated by the increased sedimentation coefficient of the antibody-complexed ER in sucrose density gradients. Antibodies 318, 35, and 36 bound to ER with low affinity since they immunoprecipitated ER, but the ER-antibody complex appeared to dissociate on sucrose density gradients. The high-affinity MoAbs appear to be site-specific since the peptide competed effectively for binding of the receptor by the antibody. The fact that they reacted with ER from human breast cancer and calf, rat, and mouse uterine tissues suggests that this epitope of the receptor is conserved in these species. Although the DNA-binding region appears to be conserved among the various steroid receptors, these MoAbs did not recognize the native forms of progesterone, androgen, or glucocorticoid receptors. These MoAbs bound to the KCl-activated 4S ER and heat-transformed 5S ER, suggesting that the antibody-binding site is accessible in the monomeric and dimeric forms of ER. The antibodies did not recognize the untransformed 8S ER in the presence of molybdate and without KCl, suggesting that the antibody-binding site in the oligomeric form of ER is inaccessible. The fact that the antibodies did bind to the unoccupied 4S ER was demonstrated by the data obtained with sucrose density gradient analysis followed by postlabeling of ER with [3H]estradiol. The antibodies bound to ERs with high affinity (KD = 0.4 to 1.8 nM). At a fixed concentration of antibody, ERs ranging from 20 to 1,000 fmol were detectable. These MoAbs did not inhibit nuclear or DNA binding of ER in vitro. This can be attributed to the dissociation of the antibodies from ER when the latter interacts with its acceptor site. These results demonstrate the development of site-specific MoAbs to the native form of the hER using synthetic peptides as immunogens.

Competition between estradiol and estriol for end organ receptor proteins

Abstract Evidence is presented that estriol is a potent inhibitor of estradiol binding to specific end organ receptor proteins, suggesting a more than passive role of this material in metabolic processes. The results tend to imply a regulatory function to estriol postulated previously by other investigators. The kinetics of incorporation of radioactive estradiol in the presence of varying amounts of estriol-and vice versa-indicate competitive binding. A similar binding phenomenon has also been demonstrated in a modified in vitro system. Differences in the nature of the binding of these two steroids are discussed.

Immuno‐electron microscopic localization of estradiol receptor in cells of male and female reproductive and non‐reproductive organs

Summary— The localization of estradiol receptor (ER) in various tissues and their distribution in sub‐cellular compartments were studied by means of immunogold‐electron microscopic methods using a site‐directed polyclonal antibody developed against a peptide from the DNA binding site of ER. This method was used to determine the presence and localization of ER in tissues and cells of male and female reproductive and non‐reproductive organs. In the female reproductive tract, endometrial cells and the cells of the corpus luteum were found to contain ER. In non‐reproductive organs of both sexes the following cell types showed significant labeling: hepatocytes, epithelial duodenal cells, striated muscle fibers, cells of the proximal convoluted tubules of the kidney, lymphocytes, neurons, and adipose cells. Alveolar epithelial cells were studied only in female specimens and were labeled by the anti‐ER. Prostatic and epididymal epithelial cells were found to be labeled in the male reproductive organs. In all these cells a higher density of label was found in the nucleus, especially in the space between the clumps of compact chromatin, as was previously found in epithelial endometrial cells. These results suggest that estradiol exerts its effects through a common nuclear mechanism in cells of male and female reproductive and non‐reproductive organs.

Factors involved in the production of specific antibodies to estriol and estradiol

Abstract To determine what effect the site of attachment of the hapten would have on specificity, 6-oxoestrogens were synthesized and conjugated to several carrier proteins, using the mixed anhydride formed between isobutyl chloroformate and the steroid 6-oxime. Estriol was also treated with phosgene to produce a mixture of chloroformates followed by conjugation to bovine serum albumin. Rabbits were immunized, and the specificity of the anti-sera produced was established by cross-reaction measurements, using both direct binding and binding inhibitor assays. Comparison of the immune response showed that antibodies to the randomly linked estriol chloroformate had a high degree of cross-reactivity with estrone and estradiol (30–100%), while those produced to 6-oxo linked estriol showed only a 1–12% cross-reaction. Similar results were obtained for estradiol antibodies. Comparison of antibody formation and specificity of 6-oxoestriol linked to a polylysine copolymer, bovine serum albumin or rabbit serum albumin was made. Also, the antigen was given with complete Freunds adjuvant, adsorbed onto charcoal, entrapped within polyaery lamide gel or in vivo titrated with estrogen implants. Only estrogen specifically linked to bovine serum albumin in Freunds adjuvant gave satisfactory results, while the others yielded antibodies of decreasing titers or low specificity. It is concluded that specificity and titers seem to depend not only on the site of conjugation but also on the carrier, immunization procedure and certain other factors.