Timothy J. O'Brien

Correlation of semen transferrin concentration and sperm fertilizing capacity.

To determine whether a correlation exists between semen transferrin and sperm fertilizing capacity, transferrin concentration was determined in the semen of 52 male patients referred for the hamster ova penetration test (group 1) and in 17 men participating in the human in vitro fertilization program (group 2). In both groups 1 and 2 seminal transferrin levels were also compared to semen volume, sperm density, and motility. Serum follicle-stimulating hormone and luteinizing hormone concentrations that were determined in 34 individuals (24 in group 1, 10 in group 2) showed no correlation with seminal transferrin levels. In conclusion, low seminal transferrin levels correlated with low sperm density and with poor fertilizing ability of human oocytes in vitro.

Isoelectric focusing in a Sephadex column

Abstract An isoelectric focusing technique on a Sephadex column is described. This technique provides for the fractionation of amphoteric compounds in a pH gradient and allows for better than 95% recovery of the focused material. Focusing in this system over a pH range of 3.5–10 reaches equilibrium in approximately 5 hr and thereby reduces the exposure of more labile proteins to long focusing times.

The detection of estrogen receptors in gynecologic tumors using immunoperoxidase and the dextran-coated charcoal assay

The dextran‐coated charcoal receptor assay for demonstrating functional estrogen and progesterone receptors was used to evaluate the receptor content of gynecological tumors. An immunocytochemical method, the immunoperoxidase antiperoxidase method, that may detect estrogen receptors, was also employed on the same specimens utilizing sections from formalin‐fixed paraffin embedded blocks. The results by the two methods were compared and were correlated with the state of differentiation of the tumors. According to the dextran‐coated charcoal method, two cases of well differentiated endometrial adenocarcinoma were strongly positive for both estradiol and progesterone receptors, and three moderately differentiated cases contained lesser amounts of both types of receptors. Six cases of undifferentiated adenocarcinoma ranged from no detectable receptors to very high values, while of five cases of squamous cell carcinoma of the cervix, only two were positive for estradiol receptors by the dextran‐coated charcoal method. Staining of tissue sections from these same cases using the immunoperoxidase method, demonstrated a positive correlation to the dextran‐coated charcoal assay for estrogen receptors. The Dextran‐coated charcoal method is presently being used clinically as a screening measure for statistical probability of a patients response to hormone therapy. The degree of positive correlation shown here suggests that use of the immunoperoxidase method may have further potential for diagnostic and clinical use, and merits further investigation.

Trophoblastic disease monitoring: Evaluation of pregnancy-specific β1-glycoprotein

Pregnancy-specific beta 1-glycoprotein (SP1) was evaluated as a potential marker protein for monitoring trophoblastic disease. Four patients with post-molar pregnancy accompanied by spontaneous titer remission and three patients with nonmetastatic trophoblastic disease were found to have regression curves for both human chorionic gonadotropin (hCG) and SP, which closely followed each other. Of three patients with metastatic choriocarcinoma, two were shown to have discordant hCG and SP1 patterns, SP1 in both cases was plateauing or rising while hCG continued to fall. Two other patients are described, one with a spontaneous remission, and one who previously had had choriocarcinoma and was found to have low levels of hCG with higher levels of SP1.

Isoelectric heterogeneity of human uterine estrogen binding proteins

Upon Isoelectric Focusing (IEF) of premenopausal uterine myometrial cytosol, specific binding of estradiol (E2) can be shown at elution pHs (EpH) of 4.0-4.4, 5.0-5.2, 5.8-6.2 and 7.5-8.0. Pre-adsorption of premenopausal uterine cytosol by Concanavalin A Sepharose (Con-A) or precipitation with 30% ammonium sulfate results in loss of estradiol binding at EpHs 4.4 and 5.0. The estradiol binding sites that bind to Con-A are present in plasma and have been shown to be Sex Hormone Binding Globulin (EpH = 5.0) and Estrogen Binding Protein (EpH = 4.4). After Con-A adsorption premenopausal cytosol preincubated with 2 nM 3HE2 reveals a single peak on IEF at EpHs congruent to 6.0, while preincubation with 40 nM 3HE2 reveals specific binding peaks at EpHs of congruent to 6.0 and 7.5-8.0. Postmenopausal uterine cytosol preincubated with either 2 or 40 nM 3H-E2 on IEF reveals EpH = 5.8-6.0 binding only. Post-labeling of IEF fractions with 20 nM 3HE2 demonstrates one peak at EpH 5.8-6.0 in postmenopausal tissue and two peaks (5.8-6.2 and 7.5-8.0) in premenopausal tissue. Scatchard analysis of postmenopausal cytosol demonstrates a single population of binding sites with a dissociation constant (Kd) of 10(-10) M. Premenopausal cytosol on Scatchard analysis contains two estradiol binding populations with Kds of 10(-10) and 10(-9) M. The data suggest that the 10(-10) M E2 binding population has a EpH of 5.8-6.2, while the 10(-9) M component has an EpH of 7.5-8.0.

Modulation of protein expression in endometrial adenocarcinoma cells by in vitro exposure to estradiol and progesterone.

Protein maps of in vitro hormone-modulated adenocarcinoma cells from an endometrial tumor are described. It is noted that mapping of tumor tissues by means of the two-dimensional analysis described by OFarrell and associates of 35S-methionine-labeled proteins can provide a characteristic map for each tumor and that the methionine-containing proteins of tumor cells can be independently modulated by the in vitro additions of estradiol and progesterone. Such protein modulation could be indicative of hormonal therapeutic responsiveness of individual endometrial adenocarcinomas and other hormone receptor-positive tumors. Tumor maps may further provide data for the identification and isolation of new tumor markers which might be correlated with the radiotherapeutic and chemotherapeutic sensitivity of the malignancy.

Fractionation of RNA polymerase subspecies from soybean hypocotyls by isoelectric focusing in Sephadex

Abstract RNA polymerase enzymes isolated from chromatin of 6-day-old soybean hypocotyls are resolved into two major and one minor species of activity by DEAE-Sephadex chromatography. A soluble form of the enzyme, isolated from the postchromatin supernatant fraction, shows a broad peak of activity when fractionated by this method. The elution characteristics and α-amanitin sensitivity data indicate the two major chromatin-bound activities to be Class I and III enzymes, while the minor chromatin-bound activity and the soluble enzyme are representative of the Class II enzymes. In contrast to these profiles, fractionation of these enzyme preparations by the new method of isoelectric focusing in Sephadex G-15 yields five distinct chromatin-bound and four soluble subspecies. The relationships of these observed activities to their parent DEAE classes are investigated, showing two subspecies within the Class I and III RNA polymerase enzymes, respectively, and four subspecies within the Class II enzymes.

Developmental restrictions on hormone modulated gene transcription. I. Effects of auxin on template capacity and chromatin-bound RNA polymerase☆

Abstract The auxin induced growth response of the developing soybean hypocotyl has been employed as a model system to investigate the transcriptional mechanisms which may in part determine this tissues hormonal responsiveness during early differentiation. Thus, the molecular components present at the responsive (4 day old) developmental stage, but absent at the unresponsive (8 day) stage, are regarded here as developmental “off-switches” which may commit the older tissue to the synthesis of a limited ensemble of gene products. Such commitments are considered central to the onset of the terminal developmental stages of maturity and senescence. The initial studies have focused on the RNA synthetic capacity of chromatin isolated from control and auxin-induced 4 and 8 day old seedlings. While the young tissue displayed a 160% increase in chromatin bound RNA polymerase activity in vitro , the older plant showed only an 18% increase in this parameter following auxin treatment. Investigations with an exogenous source of RNA polymerase ( Escherichia coli ) ruled out template inaccessibility to RNA polymerase as a limiting component in 8-day unresponsive chromatin. In addition, new hormone induced transcripts as determined by nearest neighbor analysis of the RNA synthesized in vitro were detected if the reaction was templated by 4-day hormone induced chromatin and its bound RNA polymerases. However, auxin specific transcripts were not observed with 8-day induced chromatin unless the reaction was supplemented with an exogenous RNA polymerase. The findings reported herein suggest that limitations on hormone controlled gene transcription may in part be determined at the RNA polymerase level, and suggest a developmentally programmed change in the specificity of the endogenous RNA polymerase enzymes.

Developmental transitions between chromatin-bound and soluble RNA polymerase subspecies in the soybean hypocotyl.

RNA polymerase enzymes isolated from soybean hypocotyl tissue during successive developmental stages (2-8 days old) have been fractionated by Sephadex column isoelectric focusing. Both the enzymes bound to chromatin and those enzymes free in the soluble phase were investigated during development with respect to their distribution within these two pools. All observed activites were classified according to their alpha-amanitin sensitivity and isoelectric points. Two Class I subspecies (Ia, Ib) and two Class III subspecies (IIIa, IIIb) were continually present bound to chromatin throughout the developmental sequences except the IIb form which was absent at the latest stage. However, a great multiplicity (9 total) of Class II activities (totally inhibited by alpha-amanitin) were observed to be bound to chromatin at the 2nd day stage. These forms were first released from the chromatin complex and recovered in a soluble pool (4th day stage). Subsequent hypocotyl development was accompanied by the gradual disappearance of these Class II subspecies from this pool (6th day) until only two soluble species and one chromatin-bound Class II activity remained (8th day). These observations indicate that the early development of this tissue is accompanied by a dramatic alteration in the conplexity of chromatin-bound RNA polymerase subspecies. Such events may in part determine the domain of RNA secies synthesized at successive developmental stages.

Developmental restrictions on hormone modulated gene transcription. II. Hormone induced interactions of RNA polymerase with chromatin

Chromatin-bound and soluble RNA polymerase subspecies have been isolated and fractionated by isoelectric focusing at various times (0, 6, 12 and 18 h) following auxin treatment of 4 day (responsive) and 8 day (unresponsive) soybean hypocotyls. Young 4 day seedlings displayed two well defined phases of auxin induced gene transcription. Phase I (6 h) evidenced the selective dissociation of many RNA polymerase subspecies from the chromatin complex which was accompanied by the retention of three class II enzymes. Phase II occurred after 12 h of treatment when the dissociated enzymes including some species which were soluble in the 0 h controls became re-associated with chromatin. These induced RNA polymerases may be responsible for the synthesis of auxin induced RNAs. In contrast, the unresponsive 8 day hypocotyl did not display two phases of auxin induction. Phase one, the dissociation of the chromatin bound enzymes, occurred at 12 h (compared to 6 h for the 4 day seedling) and was not followed by the later translocation of any soluble enzymes towards the chromatin complex. The results support earlier findings suggesting that the developmental phasing out of RNA polymerase subspecies limits the hormone induced growth response of this tissue and thus is regarded as an off switch for the transcription of such hormone controlled gene sequences.