George M. Butterstein

A Detailed Examination of the in vivo and in vitro Effects of ACTH on Gonadotropin Secretion in the Adult Rat

These studies were designed to: (1) determine the effects of continuous infusion of synthetic ACTH(1-24) on postcastration changes in serum and pituitary LH, FSH and prolactin in the male rat; (2) assess the effects of adrenalectomy on the gonadotropin and prolactin response to ACTH, and (3) test the hypothesis that ACTH may directly (not via adrenal factors) alter gonadotropin secretion at the brain and/or pituitary level. Adult male rats were either orchidectomized (ORX) or orchidectomized-adrenalectomized (ORX-ADX), and were treated continuously for 6 days with ACTH(1-24) (10 micrograms/day) or saline using an osmotic minipump. Animals were killed on day 6 following castration. ACTH treatment reduced serum LH and prolactin levels in ORX rats to mean values +/- SE of 204 +/- 25 and 37 +/- 3 ng/ml respectively, compared to 366 +/- 72 and 62 +/- 7 ng/ml in saline-treated ORX animals. Serum FSH concentrations were not altered by ACTH administration. Pituitary concentrations of LH and FSH, but not prolactin were enhanced by ACTH treatment. Adrenalectomy had no effect on serum and pituitary gonadotropin and prolactin levels, but abolished the effects of ACTH on these parameters. Central (intracerebroventricular) infusion of ACTH(1-24) (6 micrograms/day X 4 days) failed to alter the rise in serum LH in male rats following orchidectomy. Acute treatment with large doses of ACTH of perifused anterior pituitary glands from male rats and chronic treatment with ACTH of enzymatically dispersed anterior pituitary cells from female rats did not influence basal or GnRH-stimulated LH secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Alpha-fetoprotein inhibits frog metamorphosis: implications for protein motif conservation.

Alpha-fetoprotein (AFP) is a tumor-associated fetal protein which has served as a marker for both oncogenic and ontogenetic growth. A growth regulatory segment on human AFP contains amino acid sequence identity and similarity with Rana and Xenopus albumin molecules. This study assessed the ability of both intact mammalian AFP and a derived peptide to influence thyroid induction of tail resorption during Rana catesbeiana metamorphosis. After AFP and other proteins/peptides were pre-incubated with triiodothyronine (T3) for 1 h, they were added to intact tadpoles in 300 ml of water. Human and/or mouse AFP, at a concentration of 70 ng/ml, completely inhibited T3-induced tail loss when measured over a 5 day period. In contrast, albumin and other proteins were without affect. A peptide (P149) with the sequence of human AFP residues # 447-480 also completely blocked the tail response at a concentration of 33 ng/ml, whereas a scrambled version of this peptide was without activity. The present peptide segment derived from mammalian AFP might represent a highly conserved serum protein motif in the vertebrate phyla since it is capable of influencing growth, differentiation and transformation phenomenon in amphibians.

Effect of α-Fetoprotein and Derived Peptides on Insulin- and Estrogen-Induced Fetotoxicity

Both insulin and estrogen are well recognized as growth-promoting substances at physiological concentrations, but they function as teratogens at high doses. Both agents can affect alterations in fetal and maternal serum human α-fetoprotein (HAFP) levels during pregnancy. In the present study, we have employed animal models of both insulin and estrogen fetotoxicity and teratogenicity in order to study the growth-regulatory properties of HAFP and its derived peptides (HAFP/PEP). We report here the effects of HAFP/PEP on fetotoxicity, congenital malformations, and growth retardation in developing chick and murine fetuses. In the insulin model, HAFP/PEP were effective in reducing both fetal mortality and anatomic anomalies, with the result that growth-retarded fetuses were produced. With HAFP/PEP treatment, fetal demise was reduced by as much as 73 and 63% in murine and chick fetuses, respectively, while fetal anomalies were diminished by 50% during chick development. Genebank searches of identity/similarity in a HAFP/PEP fragment identified matches with a number of proteins associated with glucose, pH, ionic, osmotic, and oxidative stresses, and with heat shock, in addition to stress proteins related to protein folding/unfolding processes. It was proposed that the peptide segment on HAFP may represent a topographic ‘hotspot’, sensitive to stress/shock conditions, which exhibits a propensity for conformational alteration in the tertiary structure of the fetal protein.

Review and proposed action of alpha-fetoprotein growth inhibitory peptides as estrogen and cytoskeleton-associated factors.

The (H) human growth‐promoting factor, alpha‐fetoprotein (AFP), has been reported to possess a growth inhibitory motif as an occult epitope in the compactly folded circulating form of the protein. Intermediate unfolded forms of the human HAFP molecule induced by stress, shock, and high ligand concentrations have revealed the presence of an encrypted growth‐suppressive segment on the third domain of HAFP. A purified linear synthetic 34‐mer segment termed the “growth inhibitory peptide” (GIP) exhibits various oligomeric forms with complex aggregation behaviors, in which dominant trimeric forms were found to be suppressive in assays of estrogen‐induced growth. While several amino acid analogs of the cysteines of the GIP retained inhibitory activity, heavy metal binding and pre‐incubation of the peptides with a variety of cations and hormone ligands were found to influence the outcomes of growth bioassays. Smaller segments of the original 34‐mer were each found to display growth activities of their own, with the middle segment (P149b) also showing hydrophobic dye‐binding properties. Studies of amino acid sequence identity further revealed that the GIP sequences displayed identity/similarity matches to both cytoplasmic and nucleus‐cytoskeleton‐associated proteins, and experimental evidence served to support these findings. That is, the peptide was capable of modulating tubulin polymerization, cell shape, and cell‐surface aggregation phenomena reminiscent of a microtubule‐associated protein. Immunofluorescence studies further pinpointed the localization of the GIP to cytoplasmic regions of high cytoskeletal density in the cell. Because of the involvement of the GIP in experimental models of the estrogen receptor/cytoskeleton, a mechanism of action is forwarded in which the linear GIP is proposed to be a G‐coupled receptor binding ligand that is translocated across the plasma membrane via receptor‐mediated endocytosis. Thus, it was predicted that the linear GIP and possibly its peptidic segments serve as decoy ligands to cell‐surface receptors in order to gain access to the cytoplasmic compartment of the cell.

Increase in Serum Levels of Luteinizing Hormone (Lh) in Pine Voles, Microtus pinetorum, after Induction of Estrus and Copulation

and reproductive behavior in microtine rodents. Pp. 197-217, in Mammalian olfaction, reproductive processes, and behavior (R. L. Doty, ed.). Academic Press, New York, 344 pp. RISSMAN, E. F., AND R. E. JOHNSTON. 1985. Female reproductive development is not activated by male California voles exposed to family cues. Biol. Reprod., 32:352-360. SCHADLER, M. H. 1981. Postimplantation abortion in pine voles (Microtus pinetorum) induced by strange males and pheromones of strange males. Biol. Reprod., 25:295-297. SOKAL, R. R., AND F. J. ROHLF. 1969. Biometry. W. H. Freeman and Company, San Francisco, 776 pp. STEHN, R. A., AND F. J. JANNETT. 1981. Male-induced abortion in various microtine rodents. J. Mamm., 62:295-297. STEHN, R. A., AND M. E. RICHMOND. 1975. Maleinduced pregnancy termination in the prairie vole, Microtus ochrogaster. Science, 187:1211-1213.

A pollicle-stimulating hormone receptor ecto-domain epitope that is a target for receptor immunoneutralization yet does not affect ligand contact and activation

The follicle-stimulating hormone receptor (FSHR) large extracellular domain suggests that interaction of ligand with receptor is likely to be complex. Residues 265–296 of the FSHR are part of a sequence primarily nonhomologous with other glycoprotein hormone receptors. A reasonable hypothesis is that this sequence of the FSHR plays a role in binding FSH. Flow cytometry studies of this region revealed that antibody x179 against peptide R265–S296 bins to humans FSHR expressed by CHO cells and can be competed against by preincubating the cells with hFSH. These results suggested that the region corresponding to residues 265–296 in the extracellular domain of the FSHR is involved in binding to hormone. To test this hypothesis 10 scanning alanine mutants of rFSH at the 265–296 epitope were generated, and the binding characteristics of these mutants were studied. Their affinity constants for 125I-hFSH did not deviate greatly from that of wild-type FSHR, in which some mutants exhibited an approximately two- to threefold reduction in Ka compared to wild-type receptor, and no mutation abolished signal transduction. These results lead to rejection of the hypothesis that this region contains residues critical for conveying hormone specificity and receptor-dependent hormone action.

Effect of particle size on the prolonged action of subcutaneous danazol in male and female rats

OBJECTIVE To investigate the effects of two different particle sizes of danazol in male and female rats. DESIGN Prospective, vehicle-controlled study. SETTING Undergraduate college research facility and medical research laboratory. ANIMALS 18 castrated male rats and 18 cycling female rats. INTERVENTION(S) Preparations of danazol with particle sizes of 2.05 microm or 5.2 microm were administered as a single subcutaneous injection of 400 mg/kg to castrated male rats or estrus-cycling females. MAIN OUTCOME MEASURE(S) Serum LH level in males and estrous cycle length in females. RESULT(S) In males, both preparations significantly (P<. 001) suppressed serum LH by day 5 of treatment. Gonadotropin levels remained low throughout the 35-day study in rats that received the larger-particle danazol, whereas LH levels began to increase after 25 days in rats that received smaller-particle danazol. In females, the normal 4- to 5-day estrous cycle interval was prolonged to 38.7 days (P<.001) in those that received the larger-particle danazol and 25.5 days in those that received the smaller-particle danazol. CONCLUSION(S) The results demonstrate the prolonged effectiveness of a single subcutaneous dose of danazol and indicate that one might be able to predict the effective duration of activity by changing the particle size of the danazol administered.

Danazol: Prolonged suppression of gonadotropins after subcutaneous administration in the castrate male rat

OBJECTIVE Our purpose was to compare oral versus subcutaneous administration of danazol for its effect on elevated serum luteinizing hormone and follicle-stimulating hormone levels in castrated male rats. STUDY DESIGN A single dose of danazol, either 100 or 400 mg/kg, was administered by gastric intubation or injected subcutaneously. Jugular venipuncture blood samples were taken at 0, 3, 24, and 48 hours and at 7, 10, 15, and 25 days, and serum levels of luteinizing hormone and follicle-stimulating hormone were determined by radioimmunoassay. RESULTS Gonadotropin levels returned to control values 96 hours after oral administration, whereas 400 mg/kg of danazol administered subcutaneously resulted in suppression of gonadotropins for 25 days. CONCLUSIONS Subcutaneous administration of danazol results in an unexpectedly prolonged suppression of serum gonadotropins compared with the same dose administered orally. A change from oral administration to a prolonged-release subcutaneous preparation of danazol may enhance the use of this drug in clinical situations and may lessen undesirable side effects.

Release of Luteinizing Hormone in Prepubertal and Middle-Aged Ovariectomized Rats

Abstract Concentrations of circulating LH were determined in conscious, free-moving ovariectomized rats. All of the animals had been ovariectomized at 24 days of age. Between 30 and 90 days there was an increase in mean blood LH concentrations; a more vigorous pulsatile release of LH characterized by an increase in amplitude and frequency of LH release; and an elevated responsiveness to LHRH administration. Rats which had been ovariectomized for 1 year still had elevated blood LH levels but had episodic pulses of reduced amplitude and a decrease in responsiveness to LHRH. These data suggest that important alterations occur with age in the neuroendocrine mechanisms responsible for the release of LH.

Effect of Parturition Time on the Response to Neonatal Androgen in Female Rats

Summary Female rats were cesarean delivered on days 22, 23 and 24 of gestation and treated with 100 μg testosterone propionate on various neonatal days. Certain mothers were administered progesterone prior to delivery of the offspring. Prematurely delivered rats had a longer critical period for androgenization than animals delivered on day 24 indicating that the responsiveness of the hypothalamus to androgens is age dependent. Although early testosterone treatment of the prematurely delivered rat resulted in a failure of vaginal opening, androgen treatment on subsequent days (5–10) advanced the timing of vaginal opening, while progesterone administration to the mother prevented this effect.