Vernon C. Stevens

Effectiveness of prostaglandin F2α in restoration of HMG-HCG induced ovulation in indomethacin-treated rhesus monkeys

Abstract Six mature female rhesus monkeys were treated with HMG-HCG in control cycles at doses adjusted to induce ovulation while avoiding superovulation. Occurrence of ovulation was determined by observation of fresh ovulation points at laparotomy 48 to 120 hours following HCG. In subsequent cycles animals were treated with indomethacin (treatment days 4 through 10) together with the established dose of HMG-HCG. In 8 cycles indomethacin 5 mg/kg was given i.m. once daily; in 9 cycles 10 mg/kg i.m. was administered in 2 divided doses. Following this, PGF 2 α (3 mg t.i.d. s.c.) was administered for 3 days together with indomethacin 10 mg/kg and HMG-HCG, beginning on the day prior to HCG. Determinations of progesterone were performed by RIA on treatment days 4, 7, 10, and 11. Eleven of the 13 control cycles were ovulatory. With indomethacin 5 mg/kg/day, 5 of 8 cycles were ovulatory but ovulation was delayed in 2 instances. Of 9 cycles using indomethacin 10 mg/kg/day only 1 was ovulatory. When PGF 2 α was administered in subsequent cycles along with indomethacin (10 mg/kg) and HMG-HCG, ovulation occurred in 13 of 19 cycles. These data suggest that local ovarian PGF 2 α may be essential in the mechanics of follicle rupture in gonadotropin-treated rhesus monkeys.

The effect of indomethacin on HMG-HCG induced ovulation in the rhesus monkey

The investigation was designed to study the influence of indomethacin on gonadotropin induced ovulation in the rhesus monkey. Six mature female monkeys were treated with HMG-HCG for at least 2 control ovulatory cycles at dosage levels adjusted to induced ovulation while avoiding superovulation. Ovulation was confirmed by observation of the ovaries for fresh ovulation points at laparotomy. Following establishment of an appropriate dosage schedule, treatment was begun with indomethacin (5 mg/kg/day) starting 5 days prior to HCG and continuing to the time of laparotomy. In a second treatment cycle, indomethacin was administered at a dose of 5 mg/kg b.i.d. together with the established dose of HMG-HCG. Ovarian inspection was carried out as in the control cycles. Venous blood was obtained on treatment days 4, 7, 10 and 11 for determination of serum estrone, estradiol and progesterone. Indomethacin administration resulted in ovulation inhibition at a dose of 10 mg/kg/day when ovulation inducing doses of gonadotropins were administered. Peripheral blood steroid levels suggest that follicle maturation and estrogen production was unimparied by indomethacin. These findings indicate that the ovarian synthesis of prostaglandin may be essential in the process of ovulation.

Effects of MSH/ACTH 4–10 on memory, attention and endogenous hormone levels in women☆

Abstract Young women, tested during their menstrual phase or at midcycle, received either 30 mg MSH/ACTH 4–10 or the saline diluent subcutaneously in a doubleblind procedure. Behavioral testing indicated that the peptide significantly facilitated verbal memory and impaired reversal learning ability. Visual memory, field independence, basal heart rate and state anxiety were not influenced by the treatment. Radioimmunoassays of plasma samples collected across the testing period indicated that levels of LH, FSH, 17-β-estradiol, progesterone and cortisol were not significantly altered by the peptide. It was speculated that human sex differences in response to MSH/ACTH 4–10 exist with females exhibiting an enhancement of verbal modalities.

Preparation and Formulation of a Human Chorionic Gonadotropin Antifertility Vaccine: Selection of Adjuvant and Vehicle*

ABSTRACT: Peptides representing sequences of the beta subunit of HCG (Cys‐(Pro)6‐111–145 and 109–145) were coupled to tetanus toxoid (TT) and the resultant conjugates used to test the ability of eight experimental adjuvants and six different vehicles for enhancing antibody responses to the peptides in inbred strains of mice (C3H/He, C57BL/6, DBA/1, and SJL) and outbred rabbits. Antibody levels reactive to both 125I‐peptides and 125I‐HCG were used as criteria of efficacy of these adjuvants and vehicles. In most experiments, groups of animals were immunized with conjugates in complete Freunds adjuvant (CFA) for comparison. Two hydrophilic adjuvants‐NAc‐nor‐Mur‐L.ala‐D.isoGln, CGP 11,637 and NGlycol‐nor‐Mur‐L.α‐abu‐D.isoGln, DT‐1‐were consistently the most effective in stimulating antibody production to both peptides and HCG. An emulsion containing squalene‐water stabilized with arlacel A proved to be superior to other vehicles tested for administering conjugates and adjuvants to animals. Antibody levels produced by animals with different genetic backgrounds were shown to be polymorphic with respect to experimental adjuvants, but responses among groups using the same adjuvant and different vehicles were more consistent. Data were obtained suggesting that the coupling of a peptide antigen and a peptide adjuvant to the same carrier holds promise as a model for vaccine development.

The identification of peptide sequences of human chorionic gonadotropin containing a conformational epitope

A series of overlapping peptides were synthesized representing the entire amino acid sequence of the beta-subunit of human chorionic gonadotropin (hCG) and these were reacted with a monoclonal antibody shown to be specific for hCG. One linear peptide (residues 40-52 of the sequence) reacted significantly with the monoclonal antibody but a conjugate of this peptide to diphtheria toxoid (DT) failed to elicit significant levels of antibodies reactive to hCG in rabbits. The subsequent preparation of an extended peptide (residues 38-57) in which the two cysteines were oxidized to form a loop peptide yielded a highly immunogenic antigen when conjugated to DT. Antibody levels reactive with hCG from loop peptide immunizations of rabbits exceeded those found after immunization with a 37 residue peptide representing the carboxyl terminus of the beta-hCG subunit. The antisera did not react with pituitary glycoprotein hormones with similar sequences.

Observations on the Mechanism of Action of Clomiphene (MRL-41)

Serial follicle stimulaint hormone (FSH) and luteinizing hormone (LH) determinations (by the methods of Steelman and Pohley and Parlow respectively) in 4 23-53 year old anovulatory patients given short-term clomiphene treatment (100-400 mg orally/day for 6 days) were employed in studying the mechanism of clomiphene action. Serial determinations of 3 estrogens (estrone estradiol and estriol) were also determined using thin-layer absorption chromatography before Kober color development. FSH levels rose in all cases during or immediately following clomiphene therapy. LH did not rise prior to a rise in estrogens. The estradiol fraction was found to contain at least 3 different components. During clomiphene therapy the estradiol fraction fell. Since the estradiol fraction was composed of 2 compounds in addition to estradiol and since it fell during treatment it was postulated that it was the unidentified estrogens whose excretion had decreased. It was suggested that this fall in unidentified estrogens may be responsible for the increase in FSH release observed during treatment.

Preparation and Formulation of a Human Chorionic Gonadotropin Antifertility Vaccine: Selection of a Peptide Immunogen*

ABSTRACT: Peptides representing the amino acid sequence of the carboxy‐terminal of the human chorionic gonadotropin (HCG) beta subunit were used in studies to determine whether an immunogen could be prepared that would be suitable for an HCG antifertility vaccine. Peptides of varying length were conjugated to several macro‐molecular carriers, in varying peptide‐carrier ratios, via an N‐ or C‐terminal group, with and without amino acid spacers. Antibody levels to peptides and intact HCG were used as the criteria for conjugate utility. Immunizations with a peptide of 37 amino acid residues elicited the highest antibody levels to HCG. No differences were found between N‐ or C‐terminal conjugates, but peptides with amino acid spacers elicited higher responses than peptides without spacer. Conjugates with a peptide:carrier ratio greater than 20 peptides/105 daltons carrier were more immunogenic than those with a lower ratio. Diphtheria and tetanus toxoids were consistently the most effective carriers for enhancing antibody responses to peptides. It was concluded that conjugates of β‐HCG peptide 109–145 coupled to a toxoid carrier via its N‐terminus in a ratio of 20–30 peptides per 105 daltons carrier was a suitable immunogen for further studies for the development of an anti‐fertility vaccine.

Differentiation and secretory activities of cultured human placental cytotrophoblast

Ultrastructural, autoradiographic, immunofluorescent and biochemical techniques were used to characterize primary cultures of term placental cytotrophoblast in order to gain insight into the differentiation and secretory capacities of the cellular component of human trophoblast. Trypsin treatment of placental villi allowed isolation of a predominantly cytotrophoblast cell population that maintained viability up to 13 weeks in monolayer culture. Autoradiographic studies of tritiated thymidine incorporation identified a smaller diameter mononucleated cell population that was mitotically active and developed into larger diameter mononucleated cells and into multinucleated cells during culture. Ultrastructurally, cultured cells formed desmosomes, had an extensive network of cytoplasmic microfilaments and contained the organelles for hormone synthesis and secretion. These cells secreted steroid hormones, secreted Schwangerschafts protein I, actively incorporated tritiated glycoprotein precursors and expressed surface immunoreactivity for the beta-subunit of human chorionic gonadotrophin (hCG). However, medium concentrations of hCG and human placental lactogen dropped rapidly to undetectable levels after 14 days in primary culture. Cells grown beyond confluence differentiated into 1 to 2 mm structures with a villus-like histology. Our studies indicate that cytotrophoblast can secrete steroids, cytotrophoblast differentiation occurs in vitro in the absence of maternal tissues, hCG synthesis occurs in cultured cytotrophoblast and medium concentrations of placental protein hormones are not the best indicators of cell viability for cultures of cytotrophoblast.

THE EFFECTS OF A NEW ORAL CONTRACEPTIVE ON GONADOTROPIN EXCRETION.

Abstract Six subjects were administered varying doses of ethynodiol diacctate (SC-11800) in combination with 0.1 mg. mestranol and the effects of these drugs on gonadotropin excretion observed. Control cycles were studied on all subjects. The contraceptive action of this drug combination appears to be the inhibition of an ovulatory peak in luteinizing hormone (LH) secretion in midcycle. Doses of 2.0 mg. SC-11800 with 0.1 mg. mestranol appeared to stimulate follicle stimulating hormone (FSH) excretion while LH excretion was prematurely stimulated in one subject during the first treatment cycle. A similar LH response was observed with 0.5 mg. SC-11800 and 0.1 mg. mestranol in another patient, however, FSH levels were uniformly suppressed at this dose. There were no peaks of LH in the second treatment cycles of either the 2.0 mg. or the 0.5 mg. dose of the drug mixture. No effect was seen on FSH excretion at the 1.0 mg. dose of SC-11800 in combination with 0.1 mg. mestranol in either the first or second treatment cycle. The LH excretion patterns of subjects on 1.0 mg. dose showed no ovulatory peaks of this hormone at any time while on treatment.

Progress in the development of human chorionic gonadotropin antifertility vaccines.

Prototype human chorionic gonadotropin (hCG) vaccines have demonstrated the feasibility of effectively eliciting antibodies in women and inhibiting fertility in both humans and nonhuman primates. Also, no serious side‐effects due to immunization against self antigens have been revealed to date. However, the formulations so far tested in clinical trials are not suitable for widespread applications due to problems associated with complexities in production, burdensome application procedures, the need for frequent booster immunizations or cost of manufacture. Current research efforts involve the development of delivery systems to permit annual or biannual intervals between immunizations for protection from pregnancy, procedures for mucosal immunizations, methods to reduce hypersensitivity and local reactions, and procedures for reducing the cost of production. Recent progress in understanding the crystalline structure of the hCG molecule has stimulated further studies to define immunological epitope sequences that might constitute immunogens in future vaccines.