Biomedical subjects

R L Stouffer

Publications and source records attributed to R L Stouffer.

At least 109 records · Page 6Linked to original sources

PubMed1988

Chronic exposure of the developing corpus luteum in monkeys to chorionic gonadotropin: persistent progesterone production despite desensitization of adenylate cyclase.

The transient steroidogenic response of the macaque corpus luteum to chronic human CG (hCG) treatment beginning on days 9-10 of the luteal phase (i.e. stimulated early pregnancy) is associated with decreased numbers and affinity of available receptors for gonadotropin and homologous desensitization of adenylate cyclase. This study determined if similar changes in the receptor-adenylate cyclase system accompany the persistent steroidogenic response which occurs when hCG treatment begins earlier in the luteal phase. Female rhesus monkeys received increasing doses of hCG (15 up to 5760 LU) twice daily beginning 5-6 days after the midcycle LH surge. The levels of circulating progesterone increased (P less than 0.05) within 24 h of initial hCG exposure and did not decrease throughout the 10-day regimen. The corpus luteum was removed after 0 (n = 8), 6 (n = 4), or 10 (n = 4) days of hCG treatment. Whereas the numbers of available [125I]hCG binding sites in luteal particulates remained unchanged by 10 days of hCG exposure, the dissociation constant (Kd) for gonadotropin binding was greater than at day 0 (6.17 +/- 1.41 vs. 0.91 +/- 0.06 X 10(-10) M, P less than 0.05). Since the number of binding sites occupied by injected hCG increased with treatment (7.81 +/- 1.55 fmol/mg wet wt at day 10), the total number (available + occupied) of gonadotropin receptors was 3-fold greater (P less than 0.05) at day 10 than at day 0. Adenylate cyclase activity in luteal homogenates, assessed by conversion of [alpha-32P]ATP to [32P]cAMP, was stimulated on day 0 by hCG (2.7 +/- 0.7 X control, at 250 nM hCG), prostaglandin E2 (2.5 + 0.5 X control, at 0.5 mM), and prostaglandin I2 (2.3 +/- 0.5 X control at 0.5 mM) as well as forskolin (100 microM) and 5'-guanylyl-imidodiphosphate (50 microM). In contrast, cAMP production by day 6 of treatment was insensitive to hCG, but remained responsive to prostaglandin E2, prostaglandin I2, and nonhormonal activators. We conclude that CG treatment in the early luteal phase did not prevent the development of gonadotropin receptors to levels typically observed in the functional corpus luteum of the menstrual cycle. Also, many changes in the gonadotropin receptor-adenylate cyclase system in macaque luteal tissue were similar after CG treatment beginning on days 5-6 or days 9-10 of the luteal phase.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenylyl Cyclases↗

PubMed1988

Intraluteal infusion of a prostaglandin synthesis inhibitor, sodium meclofenamate, causes premature luteolysis in rhesus monkeys.

The physiological significance of locally produced prostaglandins (PGs) in the regulation of the functional lifespan of the primate corpus luteum is unknown. In the current study, the PG synthesis inhibitor sodium meclofenamate was administered to adult female rhesus monkeys beginning in the midluteal phase of the menstrual cycle. Meclofenamate was infused continuously for 7 days into the corpus luteum (100 micrograms/h, n = 6) or the jugular vein (100 micrograms/h, n = 3; 1000 micrograms/h, n = 3) via osmotic minipump. As controls, PBS was infused into the corpus luteum (n = 7) or jugular vein (n = 5). In some of the monkeys receiving intraluteal infusions, chronic aortic and utero-ovarian venous catheters were implanted, and blood samples were collected on alternate days for the measurement of PGE and PGF2 alpha by RIA. Saphenous venous blood was collected daily, and progesterone and cortisol levels were determined by RIA. LH levels were determined by the mouse Leydig cell bioassay. Progesterone levels over 5 days preceding treatment were not different among groups. A decline in progesterone levels on day 1 after surgery was observed in all treatment groups and was accompanied by a 1-day elevation in cortisol levels. Thereafter, five of seven monkeys who received intraluteal infusions of PBS displayed normal progesterone patterns during treatment and normal luteal phase lengths of 15.4 +/- 1.2 days (mean +/- SEM). In six monkeys that received intraluteal infusions of meclofenamate, progesterone levels typically fell to less than 1 ng/ml within 72 h after initiation of infusion; progesterone levels during 7 days of intraluteal infusion were significantly lower (P less than 0.01) in meclofenamate- vs. PBS-treated monkeys. Meclofenamate infusion into the corpus luteum significantly shortened (P less than 0.01) the luteal phase to 10.5 +/- 1.0 days. In contrast, progesterone levels during 7 days of meclofenamate infusion into the jugular vein did not differ from those in PBS-treated monkeys, and the length of the luteal phase was unaltered. LH levels, measured daily, did not differ among groups either before or during treatment. Although an venous/arterial gradient in PGE was detected at the time of surgery, we were unable to detect a significant gradient across the ovary in PGE or PGF2 alpha at any time after surgery in monkeys treated with either PBS or meclofenamate. The present data suggest an obligatory luteotropic role for locally produced metabolites of arachidonic acid, but a physiological role for either PGE or PGF2 alpha in regulating the primate corpus luteum remains equivocal.

Animals↗

PubMed1988

Immunocytochemical localization of estradiol and progesterone receptors in the monkey ovary throughout the menstrual cycle.

Both estradiol and progesterone may act locally to modulate ovarian function in various species. This study examined the distribution of estradiol and progesterone receptors (ER and PR, respectively) within the primate ovary throughout the menstrual cycle. Ovaries were collected from rhesus or cynomolgus monkeys during the early, mid-, and late (n = 3-6/stage) follicular and luteal phases of the cycle. The tissues were processed for indirect immunocytochemical localization of receptors with specific monoclonal antibodies against ER (H222 and D75) and PR (JZB39). Specific immunocytochemical staining, as determined by comparing adjacent tissue sections incubated with either receptor antibodies or a nonspecific antibody, was exclusively nuclear. Both ER and PR were localized in the germinal epithelium of ovaries at all stages of the cycle. ER was not detected in any other ovarian structure (i.e. stroma, follicles, interstitial tissue, or corpora lutea) regardless of the stage of development. However, ER was detected in other estrogen-responsive tissues, e.g. the oviduct of the monkey and corpora lutea of the pseudopregnant rabbit. In the monkey ovary, PR was detected in stromal and interstitial tissues as well as theca interna and externa of healthy and atretic follicles at all stages of the cycle. The granulosa cells of some primordial and primary follicles demonstrated staining for PR. However, the granulosa layer of follicles that developed beyond the primary stage were consistently negative for PR. Only the granulosa layer of large preovulatory follicles that showed signs of luteinization after the LH surge showed staining for PR equivalent to that in the theca. Monkey corpora lutea exhibited specific nuclear staining for PR. Moreover, the percentage of receptor-positive nuclei in the corpus luteum varied (P less than 0.05) between the early (28 +/- 3%), mid (48 +/- 1%)-, and late (4 +/- 2%) luteal phase of the cycle. Nonfunctional (serum progesterone less than 0.5 ng/ml) regressing corpora lutea did not exhibit for staining for PR. Luteal cells that were PR positive also contained histochemically detectable 3 beta-hydroxysteroid dehydrogenase. These data are consistent with the concept of a receptor-mediated autocrine or paracrine role for progestins, but not estrogens in the gametogenic and endocrine functions of the primate ovary throughout the menstrual cycle.

3-Hydroxysteroid Dehydrogenases↗

PubMed1987

Stimulatory and inhibitory effects of prostaglandins on the gonadotropin-sensitive adenylate cyclase in the monkey corpus luteum.

Detailed analysis of the action of prostaglandins (PGs) on the corpus luteum in primate species is very limited. In this study we examined the response of the adenylate cyclase system to PGs in homogenates prepared from the corpus luteum of rhesus monkeys at midluteal phase of the menstrual cycle. The conversion of [alpha 32p] ATP to [32p] cyclic AMP (cAMP) was assessed in the absence (control activity; 50 microM GTP) and presence of various concentrations of seven PGs and arachidonic acid, either alone or in combination with 250 nM hCG. Cyclic AMP production increased up to three-fold in the presence of PGD2, PGE2, PGI2 or PGF2 alpha; however PGA2, PGB2, 13, 14-dihydro-15-keto PGE2 and arachidonic acid alone did not alter cAMP levels. In dose-response studies, adenylate cyclase was 10 and 100-fold more sensitive to PGD2 (Vmax at 1 X 10(-5) M) than to PGE2 or to PGI2 and PGF2 alpha, respectively. Activity in the presence of hCG plus either PGD2, PGE2, PGI2 or PGF2 alpha did not differ from that for hCG (or the PG) alone. In contrast, addition of PGA2 or arachidonate inhibited (p less than 0.05) hCG-stimulated cAMP production by 50 and 100 percent. We conclude that the gonadotropin-sensitive adenylate cyclase of the macaque corpus luteum is also modulated by several PGs. These factors may either mimic (e.g., PGD2, PGE2, PGI2) or suppress (PGA2) gonadotropin-stimulated cAMP production and possibly cAMP-mediated events in luteal cells.

Adenylyl Cyclases↗

PubMed1987

Elution and rebinding of gonadotropins to receptors in corpora lutea: comparisons among treatments and species.

Several treatment regimens have been used to dissociate bound gonadotropins from their target tissues to quantify numbers of occupied receptors. To compare the efficacy of these methods, we evaluated the ability of a number of treatments to elute bound gonadotropins from corpora lutea of various species. In addition, we examined the capacity of luteal tissue to rebind gonadotropins after efficacious elution (greater than 90% dissociation of bound gonadotropin). Particulate (20,000 g) preparations of luteal tissue from pseudopregnant rats and from nonpregnant pigs and rhesus monkeys were incubated with 125I-labeled human luteinizing hormone (hLH, NIH-LH-11) or 125I-labelled chorionic gonadotropin (hCG, CR119) for 20 h at 25 degrees C to occupy gonadotropin receptors. Heat treatment (60 degrees C) eluted greater than 80% of bound 125I-hLH from rat tissue within 30 min, but similar treatment dissociated only 35% of 125I-hLH bound to porcine tissue (p less than 0.01). Likewise, heat treatment eluted only 57% of 125I-hLH bound to macaque tissue. At 4 degrees C, the following treatments dissociated greater than 90% of specifically bound 125I-hLH from porcine and rat luteal particulates within 5 min: acetic acid (pH 2.3), formic acid (pH 2.3), propionic acid (pH 2.3), acetic acid: HCl (pH 2.3 and 3.3), and MgCl2 (4 M). After 1-2 h at 4 degrees C, exposure to urea (4 M) or acetic acid:HCl (pH 4.3) also eluted greater than 90% of bound 125I-hLH.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Receptors for chorionic gonadotropin in the corpus luteum of the rhesus monkey during simulated early pregnancy: lack of down-regulation.

Stimulation of the primate corpus luteum (CL) by endogenous CG in early pregnancy or by exogenous hCG in simulated early pregnancy is transient, possibly due to a drop in the number of available gonadotropin receptors in the CL. The objective of the current study was to determine if this reduction in available CG receptors in the CL is due to down-regulation of the total receptor population or occupation of receptors by CG. CL were removed from rhesus monkeys (n = 27) during prolonged CG exposure in simulated early pregnancy. Luteal tissue was homogenized, and a particulate preparation (20,000 g) was formed. Bound gonadotropin was eluted from half of each tissue preparation with 0.05 M Na acetate-HCl in 0.1% gelatin (pH 3.3); the other half was treated with control buffer (0.05 M Tris-HCl, pH7.4). The hCG in the eluate was quantified by RIA and used to estimate the number of occupied receptors. Numbers and affinities of receptors were estimated by Scatchard analyses of specific [125I]hCG binding. Serum concentrations of progesterone increased within 9 h of hCG treatment and declined between 3 and 10 days of treatment. Whereas the number of available receptors declined from 0 h to 10 days of hCG treatment, the number of receptors occupied by hCG increased throughout treatment from 0.19 +/- 0.01 fmol/mg tissue (mean +/- SE) after 2 h of treatment to 7.76 +/- 1.73 fmol/mg after 10 days of treatment (P less than 0.05). The total number of receptors (available plus occupied) did not change throughout the 10-day treatment period (11.93 +/- 2.11 fmol/mg at 0 h vs. 9.64 +/- 1.86 at 10 days; P greater than 0.10). The dissociation constants (Kd) for CG binding were greater (P less than 0.05) after 6 days (4.10 +/- 0.57 X 10(-10) M) and 10 days (5.24 +/- 0.54 X 10(-10) M) of treatment than after 0-3 days (0.95 +/- 0.06 X 10(-10) M) of treatment. Notably, the percentage of total receptors able to rebind hCG after elution declined from 0 h (79.0 +/- 5.4%) to 10 days (23.0 +/- 3.2%; P less than 0.05). Rebindability was highly correlated with the number of available (r = 0.906; P less than 0.05) and occupied (r = -0.999; P less than 0.01) CG receptors. Thus, the reduction in available gonadotropin-binding sites in the CL during simulated early pregnancy is largely due to occupancy rather than down-regulation of receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

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

Adenylate cyclase in human ovarian cancers: sensitivity to gonadotropins and nonhormonal activators.

Adenylate cyclase activity in particulate preparations of ovarian tumors from 47 women was determined by measuring the conversion of phosphorus 32-labeled adenosine triphosphate to phosphorus 32-labeled cyclic adenosine monophosphate. Ovarian cancers typically exhibited an active adenylate cyclase which was stimulated by 50 mumol/L 5'-guanylylimidodiphosphate and 10 mmol/L of sodium fluoride. This activity was comparable to that in particulates of normal postmenopausal ovaries and was independent of the class of tumor. There was no significant increase in adenylate cyclase activity in any epithelial or germinal tumor in the presence of either 250 nmol/L of human chorionic gonadotropin or 333 nmol/L human follicle-stimulating hormone. However, cyclic adenosine monophosphate production by two sex cord stromal tumors was stimulated by follicle-stimulating hormone, but not by human chorionic gonadotropin. Follicle-stimulating hormone stimulated a threefold increase in activity in the granulosa-theca cell tumor, with an activation constant (57 nmol/L) similar to that in follicle-stimulating hormone-responsive rat ovaries. Prostaglandin E1 (50 mumol/L) increased cyclic adenosine monophosphate production by epithelial tumors more than twofold. These data suggest that sex cord stromal tumors, unlike the more common epithelial tumors, can be modulated directly by gonadotropin.

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

Antibody production in rhesus monkeys following prolonged administration of human chorionic gonadotropin.

Recently, a model for early pregnancy in the rhesus monkey was developed which involved administration of a 10-day treatment regimen of human chorionic gonadotropin (hCG). The current studies were designed to determine whether monkeys produced antibodies in response to this hCG regimen and, if so, to characterize the specificity of the antibodies and their effects on subsequent menstrual cycles. Based on Sephadex chromatography and immunoglobulin precipitation with protein A, serum collected from hCG-treated monkeys contained binding components for 125I-hCG. Binding was specific for human gonadotropins (hCG, human luteinizing hormone [LH], human follicle-stimulating hormone [FSH]) and did not cross-react with macaque LH/FSH. Shortening the hCG regimen to less than or equal to 6 days did not result in significant binding. The 10-day hCG regimen did not alter subsequent menstrual cycles, but attenuated 17 beta-estradiol production in response to human menopausal gonadotropin. In conclusion, the 10-day regimen of hCG treatments used to simulate early pregnancy in rhesus monkeys induced the production of serum components with characteristics of hCG antibodies. These antibodies did not alter the regulation of the menstrual cycle, but attenuated the response to exogenous human gonadotropins.

Animals↗

PubMed1985

Adenylate cyclase in the corpus luteum of the rhesus monkey. I. General properties and optimal assay conditions.

To characterize the adenylate cyclase system of the primate corpus luteum, we assayed the conversion of [alpha-32P] ATP to [32P]cAMP in preparations of luteal tissue obtained from rhesus monkeys on days 17-19 of the menstrual cycle. Basal, gonadotropin (hCG; 250 nM)- sensitive, and guanine nucleotide (5'-guanylyl-imidodiphosphate [GMP-P(NH)P]; 10 microM)-sensitive cAMP production were influenced by the pH, osmolality, and ionic strength of the assay buffer. As the concentration of Mg+2 increased from 2 to 10 mM, adenylate cyclase activity was enhanced; however, the relative stimulation by hCG plus GMP-P(NH)P was maximal when the Mg+2 concentration approximated that of ATP plus EDTA. In contrast, the presence of Ca+2 inhibited basal, hCG-stimulated, and GMP-P(NH)P-stimulated cAMP production. Adenylate cyclase activity was substrate dependent at ATP concentrations from 0.7-4.5 mM; however, higher concentrations of ATP did not alter cAMP production. The relative stimulation by hCG and GMP-P(NH)P was independent of ATP levels when the ATP to Mg ratio was constant. The rate of cAMP production was constant during 30 min of incubation at 37 C, with the ATP concentration maintained at greater than 87% of initial levels. Adenylate cyclase activity was 10-fold greater in luteal tissue from the superovulated rat than in that from the cycling rhesus monkey; however, relative stimulation by hCG and GMP-P(NH)P was qualitatively similar in the two species. Thus, we have demonstrated the existence of an adenylate cyclase system in the corpus luteum of the rhesus monkey during the menstrual cycle and have established some general properties and optimal assay conditions for the gonadotropin-sensitive adenylate cyclase.

Adenosine Triphosphate↗

PubMed1985

Adenylate cyclase in the corpus luteum of the rhesus monkey. II. Sensitivity to nucleotides, gonadotropins, catecholamines, and nonhormonal activators.

The sensitivity of the adenylate cyclase of the primate corpus luteum to various nucleotides, gonadotropins, catecholamines, and nonhormonal activators was assessed in homogenates of luteal tissue obtained from rhesus monkeys at the midluteal phase of the menstrual cycle. The conversion of [alpha-32P]ATP to [32P]cAMP was used to monitor adenylate cyclase activity. GTP, the GTP analog 5'-guanylyl-imidodiphosphate, and ITP stimulated adenylate cyclase activity in the presence or absence of exogenous hormone; however CTP, UTP, GMP, and guanosine did not. The gonadotropins, human (h) LH and hCG, stimulated cAMP production in a dose-dependent manner. Maximal stimulation of adenylate cyclase was achieved at 100 nM hLH and hCG, and the activation constant was 20 nM for both hormones. The addition of GTP increased maximal activation of adenylate cyclase by hLH or hCG, but did not alter sensitivity to the hormones. Neither hFSH nor the isolated subunits of hCG stimulated cAMP production. Deglycosylated hCG (native hCG with 70% of the carbohydrate moieties removed) did not stimulate adenylate cyclase activity. However, hLH and intact hCG failed to enhance cAMP production in the presence of an equimolar amount of deglycosylated hCG. The adenylate cyclase of macaque luteal tissue did not respond to the addition of isoproterenol, epinephrine, or phenylephrine. Furthermore, these catecholamines did not affect hCG stimulation of adenylate cyclase. The nonhormonal activators of adenylate cyclase, forskolin and fluoride, stimulated cAMP production in a dose-dependent manner, with maximal stimulation at 100 microM and 10 mM, respectively. Thus, the macaque corpus luteum at the midluteal phase of the menstrual cycle contains a guanine nucleotide-regulated adenylate cyclase which is equally sensitive to the pituitary and placental gonadotropins, hLH and hCG. However, removal of carbohydrate moieties from hCG endows the molecule with gonadotropin-antagonistic properties in the primate. The adenylate cyclase system of the macaque corpus luteum was not responsive to catecholamines; thus, the primate may lack a potential mechanism for control of luteal function that is available to many nonprimate species.

Adenylyl Cyclases↗

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↗

PubMed1985

Adenylate cyclase in the corpus luteum of the rhesus monkey. III. Changes in basal and gonadotropin-sensitive activities during the luteal phase of the menstrual cycle.

The activity of adenylate cyclase was examined in corpora lutea (CL) obtained from rhesus monkeys at specific stages in the luteal phase of the menstrual cycle [3-5, 6-8, 9-12, 13-15, and 16 days (menses) after the midcycle LH surge]. The conversion of [alpha-32P]ATP to [32P]cAMP was used to monitor adenylate cyclase activity. cAMP production in luteal homogenates was assessed in the absence (basal activity) and presence of maximum stimulatory doses of forskolin (100 microM), 5'-guanylylimidodiphosphate [GMP-P(NH)P; 50 microM], GTP (50 microM), and GTP plus increasing doses of hLH and hCG. Basal activity was low in the early luteal phase (days 3-5; mean +/- SE, 1.2 +/- 0.2 pmol cAMP/mg protein X min), increased (P less than 0.05) by the midluteal phase (days 6-8 and 9-12, 2.1 +/- 0.4 and 2.0 +/- 0.3 pmol/mg X min, respectively), and then declined (P less than 0.05) during the late luteal phase (days 13-15 and 16-menses, 1.6 +/- 0.3 and 1.2 +/- 0.5 pmol/mg X min, respectively). Activity stimulated by GTP and GMP-P(NH)P [e.g. GMP-P(NH)P approximately 12 times basal level] followed the same pattern as basal activity during the luteal phase. In contrast, cAMP production in the presence of forskolin did not change significantly throughout the luteal phase. In the midluteal phase (days 6-8 and 9-12; n = 12), hCG and human LH (hLH) stimulated adenylate cyclase in a similar dose-dependent manner. Maximal stimulation of cAMP production by hCG was about 10% greater (P less than 0.05) than that by hLH; the activation constant was 12.3 nM for hCG and 28.3 nM for hLH. The maximal response to hLH and hCG as well as the sensitivity of adenylate cyclase to activation by hLH were greater (P less than 0.05) in the midluteal phase than in the early or late luteal phase. Decreased basal, gonadotropin-stimulated, and guanine nucleotide-stimulated cAMP production and diminished sensitivity of adenylate cyclase to hLH correlated with a decline (P less than 0.05) in circulating progesterone and luteal weight during the late luteal phase. Thus, the adenylate cyclase system of the rhesus monkey CL undergoes significant changes during the luteal phase which are associated with the development and regression of the CL of the menstrual cycle. Mechanisms that modulate gonadotropin and nucleotide activation of adenylate cyclase without interfering directly with the catalytic unit are implicated in the changes that accompany luteolysis.

Adenylyl Cyclases↗

PubMed1984

Comparisons of gonadotropin binding sites and progesterone concentrations among the corpora lutea of individual pigs.

In polyovular species, it is unclear whether the characteristics of each individual corpus luteum (CL), such as mass, progesterone concentration and receptors for luteinizing hormone (LH), are representative of those of its cohorts during the ovarian cycle. The current study was performed 1) to characterize the conditions for estimation of binding parameters for LH receptors in porcine CL, and 2) to compare LH binding sites, luteal progesterone concentrations and luteal masses among CL of ovaries within individual pigs. Gonadotropin binding sites in porcine CL were characterized via specific binding of 125I-human (h) LH to 20,000 X g particulate fractions of luteal tissue. Specific binding was directly proportional to tissue content and was detectable at the lowest content tested (0.5 mg tissue equivalents/tube). Specific uptake of 0.25 ng LH by 5.0 mg tissue equivalents was time- and temperature-dependent; steady-state binding was achieved within 20 h at 37 and 25 degrees C. Binding of LH after 20 h incubation at 37 degrees C (4718 +/- 192 cpm, means +/- SEM) and 25 degrees C (4112 +/- 340 cpm) was greater than that at 4 degrees C (1930 +/- 5 cpm, P less than 0.01). Luteal particulates from individual CL of ovaries collected from four mature nonpregnant pigs (13-23 CL/pig) were incubated with eight concentrations of 125I-hLH. Steady-state binding depended upon hormone concentration until reaching saturation at 2.5 ng 125I-hLH/tube. Scatchard analyses yielded linear plots. Binding capacities for LH ranged among pigs from 0.71 +/- 0.03 to 3.69 +/- 0.13 fmol/mg CL equivalents and receptor affinities (Kd) ranged from 0.92 +/- 0.05 to 4.89 +/- 0.41 X 10(-11) M.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1984

Induction of relaxin secretion in rhesus monkeys by human chorionic gonadotropin: dependence on the age of the corpus luteum of the menstrual cycle.

Peripheral concentrations of immunoreactive relaxin are undetectable in primates during the nonfertile menstrual cycle, but become measurable during the interval when chorionic gonadotropin (CG) rises in early pregnancy. The objectives of the current study were to determine if exogenous CG, administered in a dosage regimen which invoked patterns and concentrations resembling those of early pregnancy, would induce relaxin secretion in nonpregnant rhesus monkeys, and whether the induction was dependent on the age of the corpus luteum (CL) at the onset of treatment. Female rhesus monkeys received twice-daily i.m. injections of increasing doses of human CG (hCG) for 10 days beginning in the early (n = 4), mid (n = 6) or late (n = 4) luteal phase of the menstrual cycle [5.3 +/- 0.3, 8.3 +/- 0.5, and 12.0 +/- 0.4 days after the midcycle luteinizing hormone (LH) surge, respectively; means +/- SEM]. Whereas immunoreactive relaxin was nondetectable in the luteal phase of posttreatment cycles, detectable levels of relaxin were observed in 2 of 4, 5 of 6, and 3 of 4 monkeys during hCG treatment in the early, mid and late luteal phase, respectively. Although CG treatment rapidly enhance progesterone levels, the appearance of relaxin was deferred; relaxin was first detectable 9.0 +/- 1.0 and 4.7 +/- 1.9 days after the onset of CG treatment at early and late luteal phases. Patterns of relaxin concentrations differed among groups (P less than 0.05, ANOVA; split plot design) and relaxin levels were lowest (P less than 0.01) in monkeys treated during the early luteal phase.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1984

Persistent versus transient stimulation of the macaque corpus luteum during prolonged exposure to human chorionic gonadotropin: a function of age of the corpus luteum.

The rescue of the primate corpus luteum (CL) by CG in early pregnancy is transient, despite a continued rise in circulating CG. To investigate the mechanisms resulting in this response, female rhesus monkeys were given im injections of increasing doses of hCG for 10 days during the menstrual cycle to mimic the pattern of circulating macaque CG observed in early pregnancy. Treatment began 5.3 +/- 0.3 (early luteal phase; n = 4; mean +/- SE), 8.3 +/- 0.5 (midluteal phase; n = 6), or 12.0 +/- 0.4 (late luteal phase; n = 4) days after the midcycle LH surge. Femoral venous blood was collected daily from CG-treated animals from day 8 of the menstrual cycle until the onset of menses. Serum hCG, LH, progesterone (P), and 17 beta-estradiol (E) were measured by RIA. When CG treatment began during the early luteal phase, serum P increased within 9 h after the initial injection (P less than 0.05); elevated concentrations of P persisted through treatment (range, 5.7-10.8 ng/ml) and decreased only after termination of CG injections. When treatment began during the midluteal phase, serum P increased within 9 h (P less than 0.05), peaked the third day of CG treatment (10.4 +/- 2.2 ng/ml), and then declined despite a continued rise in serum CG. Initiation of CG treatment late in the luteal phase also elicited a transient but less profound (peak, 6.4 +/- 0.5 ng/ml) increase in P concentrations. Although P patterns differed among treatment groups (P less than 0.01, by analysis of variance, split plot design), E patterns did not (P greater than 0.05). Serum E increased between 9 and 24 h after onset of treatment and remained elevated until 3-4 days after cessation of CG injections. In summary, treatment that elicited pregnancy-like increases in peripheral CG beginning in the midluteal phase of the menstrual cycle resulted in a transient stimulation of luteal P production similar to that observed in macaques during early pregnancy. This scheme provides a useful model for studying the primate CL during simulated early pregnancy. In contrast, CG administration begun early in the luteal phase resulted in persistent, rather than transient, stimulation of luteal P production. We conclude that the mechanisms involved in the transitory response of the primate CL to circulating CG during pregnancy are inoperative during the early luteal phase of the menstrual cycle.

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