Biomedical subjects

D R Danforth

Publications and source records attributed to D R Danforth.

46 records · Page 3Linked to original sources

PubMed1988

125I-luteinizing hormone (LH) binding to soluble receptors from the primate (Macaca mulatta) corpus luteum: effects of ethanol exposure.

In vitro exposure to alcohols unmasks additional binding sites for gonadotropin in cell/membrane preparations of the corpus luteum of rhesus monkeys. In the current study, we compared the effects of ethanol on gonadotropin receptors solubilized from macaque luteal membranes to those on receptors associated with the lipid bilayer. Treatment with 1% Triton X-100 for 30 min at 4C, followed by precipitation with polyethylene glycol, resulted in recovery of 50% more binding sites for 125I-human luteinizing hormone (hLH) than were available in particulate preparations (p less than 0.05). However, the soluble receptors displayed a 3-fold lower affinity for 125I-hLH (p less than 0.05). Conditions which enhanced LH binding to particulates, i.e., 1-8% ethanol at 25C, decreased specific 125I-hLH binding to soluble receptors. Steady-state LH binding to soluble receptors during incubation at 4C was half of that observed at 25C. The presence of 8% ethanol at 4C restored LH binding to levels observed in the absence of ethanol at 25C. Thus, LH binding sites in the primate corpus luteum can be effectively solubilized with Triton X-100. The different binding characteristics of particulate and soluble receptors, including the response to ethanol exposure, suggest that the lipid environment in the luteal membrane modulates the availability and affinity of gonadotropin receptors.

Animals↗

PubMed1988

Prolonged duration of gonadotropin inhibition by a third generation GnRH antagonist.

The dose-response effects of a single administration of Nal-Lys-GnRHant (antagonist) on serum LH and FSH concentrations were compared to the effects of Nal-Glu-GnRHant in monkeys. Twenty ovariectomized monkeys were divided into four sc treatment groups: a) 1.0 mg/kg Nal-Glu-GnRHant; or Nal-Lys-GnRHant at b) 0.3; c) 1.0; d) 3.0 mg/kg. Each monkey received vehicle (propylene glycol/water, 1:1) on day 0, followed by an antagonist preparation on day 11. Serum LH and FSH were measured by RIA; serum LH was also measured by in vitro bioassay. The short-term effects were similar among the four treatment groups. Typically, serum LH declined (p less than 0.05) within 4 to 8 h, achieving maximal reduction by 24 h. Serum FSH levels declined more slowly, but were significantly reduced by 24 h (p less than 0.05). Recovery during the study interval to pretreatment control values occurred in only two groups: a) Nal-Glu-GnRHant (1.0 mg/kg) by day 4 post-treatment and b) Nal-Lys-GnRHant (0.3 mg/kg) by day 2 post-treatment. Monkeys receiving 1.0 or 3.0 mg/kg Nal-Lys-GnRHant had a prolonged inhibition of serum LH and FSH levels. In all animals, serum FSH and LH returned to control levels within 2 months. The duration of gonadotropin inhibition was also prolonged when the Nal-Lys-GnRHant was administered iv. In contrast, Nal-Glu-GnRHant reduced serum LH and FSH for 3 days or less in all monkeys. The serum bioassayable LH levels paralleled those of immunoassayable LH. The prolonged inhibition of gonadotropin secretion following Nal-Lys-GnRHant distinguishes its action from those of previous GnRH antagonists and make this compound of great interest for clinical investigations.

Animals↗

PubMed1988

Subcutaneous implantable infusion device for chronic intravenous pulsatile gonadotropin-releasing hormone treatment.

Preliminary data indicate the potential utility of an implantable subcutaneous device that facilitates chronic intravenous infusion of pulsatile gonadotropin-releasing hormone (GnRH) for ovulation induction. GnRH distribution curves were congruent in control monkeys and those with implanted devices. Tissue tolerance was good in this brief trial. These findings suggest that use of this or a similar implantable device be considered for chronic GnRH administration in human pulse therapy.

Animals↗

PubMed1987

Characterizing pituitary response to a gonadotropin-releasing hormone (GnRH) antagonist in monkeys: tonic follicle-stimulating hormone/luteinizing hormone secretion versus acute GnRH challenge tests before, during, and after treatment.

Pituitary sensitivity to a gonadotropin-releasing hormone (GnRH) challenge test before, during, and after GnRH antagonist administration was compared in four ovariectomized female monkeys receiving GnRH antagonist intramuscularly (IM) at increasing doses of 0.3, 1.0, and 3.0 mg/kg/day over 9 days. Three days before and 3 days after treatment, monkeys received vehicle alone. On experiment days 4, 7, 10, 13, and 16, 100 micrograms of GnRH was administered intravenously (IV) and blood drawn at 0 and 30 minutes. Before treatment, tonic follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were 248 +/- 105 and 178 +/- 31 ng/ml, respectively; after 0.3 mg/kg/day of GnRH antagonist, FSH and LH decreased to 30 +/- 6 and 41 +/- 4 ng/ml, respectively. After treatment with either 1 mg/kg/day or 3 mg/kg/day of GnRH antagonist, both gonadotropins were undetectable in serum. Monkeys with lower initial levels of gonadotropins were suppressed by 48 hours after GnRH antagonist, while those with higher tonic gonadotropins were suppressed 6 days later (FSH: r = 0.992; LH: r = 0.833). The data show that initial physiologic status is predictive of the rapidity of the suppression response induced by a GnRH antagonist and that, after achieving pituitary suppression, responsivity to an IV GnRH challenge test may be restored before normal tonic FSH/LH secretion is regained.

Animals↗

PubMed1987

Identification of gonadotropin surge-inhibiting factor (GnSIF) in follicular fluid and its differentiation from inhibin.

Exposure of rat pituitary cell cultures to charcoal-extracted porcine follicular fluid (pFF) inhibits gonadotropin-releasing hormone (GnRH) stimulation of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release in a dose-dependent manner. The inhibition of GnRH-stimulated gonadotropin secretion (gonadotropin surge-inhibiting factor [GnSIF] activity) by pituitary cells in vitro requires up to 24-48 h preexposure to pFF. One microliter of pFF inhibits approximately 50% of the GnRH-stimulated LH release and is defined as 1 unit of GnSIF activity. Basal LH secretion is unaltered under these conditions. GnSIF activity is distinct from that of inhibin, which selectively suppresses basal release of FSH but not LH. A partially purified preparation of inhibin contains less than 1% as much GnSIF activity as inhibin activity. GnSIF activity is resistant to moderate heat treatment (60 degrees C for 60 min) and is fully recovered after acetone precipitation. Chromatography of pFF on heparin/Sepharose affinity matrix effectively separates inhibin from GnSIF. Whereas inhibin has a high affinity for heparin, GnSIF activity does not associate with this affinity matrix and is recovered in the column void volume. In summary, we have developed an in vitro bioassay for the detection of GnSIF activity in pFF. Moreover GnSIF activity seemingly derives from a molecular entity distinct from inhibin, having different physicochemical characteristics and differential effects on pituitary gonadotropin secretion.

Animals↗

PubMed1986

Characterization of corpora lutea in monkeys after superovulation with human menopausal gonadotropin or follicle-stimulating hormone.

The objective of this study was to characterize the corpora lutea (CL) of superovulatory follicles, which form in nonhuman primates after treatment with exogenous gonadotropins. Adult female rhesus monkeys (n = 15) with amenorrhea or irregular menstrual cycles received im injections of either human menopausal gonadotropin [hMG; equivalent amounts (37.5 IU) of hFSH and hLH] or human FSH (37.5 IU) twice daily for 6 or 9 days. One day later, hCG (1000 IU) was administered to induce ovulation. Serum estradiol levels rose rapidly in hMG-treated monkeys. In contrast, estradiol levels did not rise in FSH-treated animals for 3-4 days, but ultimately reached concentrations comparable to or greater than those in hMG-treated monkeys. Serum progesterone levels were low in all groups before hCG injection, but rose thereafter. Peak progesterone levels were greater (P less than 0.05) in 9- vs. 6-day treatment groups. Serum concentrations of hCG peaked within 24 h of injection and declined to undetectable levels 6-7 days later. The mass of luteinized tissue removed 7 days after hCG injection was markedly (P less than 0.01) increased in hMG- and FSH-treated monkeys compared to that of the active CL of the natural menstrual cycle (n = 6). However, the protein content of luteal tissue from FSH-treated monkeys was less (P less than 0.05) than that in hMG-treated groups or in the CL of the natural cycle. Luteal particulate fractions from all treatment groups had [125I]human LH binding sites, with the Kd for LH interaction comparable to that in the CL of the natural cycle. However, the LH-binding capacity in hMG-treated groups was less (P less than 0.05) than that in the CL of the cycle, when normalized per mg tissue wt or protein. Notably, the binding capacity in FSH-treated groups was comparable to that in the CL cycle when expressed per mg protein. Nevertheless, only after 6 days (not 9 days) of FSH treatment or 9 days (not 6 days) of hMG treatment did tissues have a LH-sensitive (activation constant) or LH-responsive adenylate cyclase comparable to that in the CL of the cycle. Thus, properties of the primate CL after superovulation varied markedly with the type and length of gonadotropin treatment employed for follicular stimulation. The findings support the concept that gonadotropin-regulated events in the developing follicle(s) are important determinants of the subsequent character of the primate CL.

Adenylyl Cyclases↗

PubMed1985

Laboratory instrument interface system (LIIS): a unit for the acquisition, temporary storage and transfer of data to a microcomputer.

A simple method of interfacing clinical and research laboratory equipment with a microcomputer is described. A four-channel buffer system has been constructed which stores data generated from laboratory instruments and then transmits the data directly to a microcomputer. The system is highly flexible with respect to the type of laboratory equipment and model of computer that can be interfaced, and it allows for virtually automatic data acquisition and analysis.

Computers↗

PubMed1985

Evidence for two populations of masked gonadotropin-binding sites in the corpus luteum of the rhesus monkey (Macaca mulatta).

To evaluate the possible existence of masked gonadotropin receptors in the corpus luteum, we characterized the effects of alcohols and neuraminidase on [125I]iodohuman LH binding to in vitro preparations of luteal tissue from the rhesus monkey and pseudopregnant rat. The presence of 1-8% (vol/vol) ethanol enhanced specific LH binding to macaque luteal particulates under steady state conditions (25 C, 20-h incubation), with a maximal effect at 8% ethanol (166% of control uptake; P less than 0.05). However, 1-8% ethanol had no effect on LH binding to rat luteal tissue. Higher concentrations of ethanol (20%) decreased LH binding relative to control values in both species. Ethanol modulation of LH binding to macaque luteal particulates and dispersed cells was a time- and temperature-dependent process. At 4 and 25 C, ethanol increased LH uptake at all times during a 32-h incubation. However, at 37 C, ethanol increased LH uptake at 30 min; binding peaked at 2 h and then returned to control levels within 20 h. The optimal concentration of ethanol for enhancing LH uptake was inversely related to the incubation temperature. The increase in LH binding to macaque luteal particulates in the presence of ethanol was reversible; binding returned to control levels if ethanol was removed before the addition of labeled LH. Longer straightchain alcohols (butanol, pentanol, and octanol) were progressively more potent than ethanol in enhancing LH binding to macaque luteal particulates and dispersed luteal cells. Pretreatment of luteal particulates from either the rat or monkey with neuraminidase increased LH uptake, with a maximal effect (160% of control) at 1 mg/ml enzyme. Scatchard analyses revealed that both ethanol and neuraminidase increased (P less than 0.05) the number of LH-binding sites without altering the affinity for gonadotropin. Moreover, the effects of ethanol and neuraminidase were additive, i.e. increased LH binding during combination of the two treatments approximated the sum of the individual effects. The data suggest that two distinct populations of LH-binding sites are masked within the membranes of the monkey corpus luteum. The ability of two markedly different agents, alcohol and neuraminidase, to increase LH binding indicates that diverse mechanisms may modulate the masking/unmasking of gonadotropin receptors in target cell membranes. Finally, the inability of ethanol to enhance LH binding in the rat suggests species differences in the receptor population or milieu of luteal membranes.

Animals↗

PubMed1985

Modulation of membrane fluidity in the primate (Macaca mulatta) corpus luteum: correlation with changes in gonadotropin binding.

Addition of alcohols to particulate or cellular preparations of the monkey corpus luteum unmasks gonadotropin-binding sites via a temperature-sensitive process. Since alcohols and temperature are known modulators of membrane fluidity, we measured the fluidity of luteal membranes and determined whether the effects of ethanol and temperature on gonadotropin binding correlated with changes in the fluid state of the membrane. The fluidity of membranes from the macaque and rat corpus luteum was estimated from the fluorescence polarization of the lipophilic membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The absorption and emission spectra of DPH incorporated into luteal membranes were typical of those in other systems. Fluorescence intensity increased rapidly during the first 60 min of incubation and reached steady state conditions within 3 h. In contrast, polarization was constant within minutes and was insensitive to pH, ionic strength, tissue concentration, or DPH levels over the ranges tested. Fluorescence polarization was acutely sensitive to the temperature of the assay medium; polarization decreased as temperature increased from 4-50 C, and no phase transitions were observed. Addition of 4-20% and 8-20% ethanol to monkey and rat membranes, respectively, decreased (P less than 0.05) polarization relative to control values. However, ethanol was less effective on rat membranes, such that 20% ethanol was required to elicit a similar change in polarization as 8% ethanol in macaque membranes. The decrease in polarization was reversed to control levels when ethanol was removed from the incubation medium. Changes in fluorescence polarization of DPH-labeled macaque membranes elicited by ethanol and temperature correlated significantly (r = -0.97) with changes in specific [125I]iodohuman LH binding. In contrast, pretreatment of luteal membranes from the monkey and rat with neuraminidase, which unmasks another population of LH-binding sites in both species, did not alter polarization. We conclude that the fluorescence polarization of DPH is a useful tool for estimating membrane fluidity in the corpus luteum. Furthermore, changes in membrane fluidity may play an important role in the masking/unmasking of alcohol-sensitive (but not neuraminidase-sensitive) gonadotropin-binding sites in the macaque corpus luteum. Finally, the lesser effects of ethanol in the rat suggest important species differences in the receptor milieu and composition of luteal membranes.

Animals↗

PubMedDate not supplied

In vivo effects of a potent GnRH antagonist ORG 30850: physiologic evidence that down-regulation of GnRH receptors does not occur.

OBJECTIVE: Our purpose was to determine the pituitary responsiveness to exogenous GnRH in GnRH antagonist-suppressed ovariectomized monkeys. METHODS: This was a prospective experimental non-human primate study performed at the research laboratories of The Jones Institute for Reproductive Medicine. Seventeen long-term ovariectomized cynomolgus monkeys were studied. INTERVENTIONS: The GnRH antagonist ORG 30850 was administered to long-term ovariectomized monkeys assigned to one of six groups: single subcutaneous injections in group A (n = 4), 0.3 mg/kg; group B (n = 4), 1.0 mg/kg; and group C (n = 3), 3.0 mg/kg; and six consecutive daily subcutaneous injections in group D (n = 2), 0.3 mg/kg; group E (n = 2), 1.0 mg/kg; and group F (n = 2), 3.0 mg/kg. Blood samples were collected daily from 10 days before treatment until 22 days after treatment, then weekly for 6 additional weeks. Intravenous GnRH stimulation tests (10 micrograms/kg) were performed on the day after vehicle injection (control) and the day after completion of treatment(s), and then at weekly intervals. The main outcome measures were serum levels of LH, FSH, and ORG 30850. RESULTS: Administration of ORG 30850 resulted in suppression (P < .05) of LH and FSH in all treatment groups. Long-term suppression (greater than 2 weeks) was evident in all primates receiving a cumulative dose of at least 1 mg/kg. Paradoxically, the responsiveness of the pituitary to exogenous GnRH was accentuated during the time of maximal tonic LH/FSH suppression. CONCLUSIONS: ORG 30850 is a potent long-acting GnRH antagonist. Furthermore, the present in vivo demonstration of heightened pituitary responsiveness to exogenous GnRH emphasizes the divergent mechanisms of action of GnRH antagonists and GnRH agonists.

Animals↗