Oral delivery of mouse [d-Leu-4]-OB3, a synthetic peptide amide with leptin-like activity, in male C57BL/6J wild-type and ob/ob mice: effects on energy balance, glycaemic control and serum osteocalcin levels.

BACKGROUND: We have recently shown that intranasal administration of mouse [d-Leu-4]-OB3 reconstituted in Intravail to male Swiss Webster mice resulted in significantly higher bioavailability than commonly used injections methods of delivery. The absorption profile associated with intranasal delivery of mouse [d-Leu-4]-OB3 showed an early peak representing absorption across the nasal mucosa, and a later peak suggesting a gastrointestinal site of uptake. AIM AND METHODS: In the present study, we examined the effects of orally administered (by gavage) mouse [d-Leu-4]-OB3 on energy balance, glycaemic control and serum osteocalcin levels in male C57BL/6J wild-type and ob/ob mice allowed food and water ad libitum or calorie restricted by 40% of normal intake. RESULTS: In wild-type mice fed ad libitum, oral delivery of mouse [d-Leu-4]-OB3 reduced body weight gain, food intake and serum glucose, by 4.4, 6.8 and 28.2% respectively. Serum osteocalcin levels and water intake were essentially the same in control and treated wild-type mice. In ob/ob mice fed ad libitum, mouse [d-Leu-4]-OB3 reduced body weight gain, food intake, water intake and serum glucose by 11.6, 16.5, 22.4 and 24.4% respectively. Serum osteocalcin in ob/ob mice treated with mouse [d-Leu-4]-OB3 was elevated by 62% over controls. Calorie restriction alone caused significant weight loss in both wild-type (9.0%) and ob/ob (4.8%) mice, and mouse [d-Leu-4]-OB3 did not further enhance this weight loss. As expected, serum glucose levels in wild-type and ob/ob mice were significantly reduced by calorie restriction alone. Mouse [d-Leu-4]-OB3 further reduced serum glucose in wild-type mice and normalized levels in ob/ob mice. Calorie restriction alone reduced serum osteocalcin levels by 44.2% in wild-type mice and by 19.1% in ob/ob mice. Mouse [d-Leu-4]-OB3 prevented this decrease in groups of mice. CONCLUSIONS: The results of this study suggest that oral delivery of mouse [d-Leu-4]-OB3 in Intravail is possible and may have potential not only as an alternative therapy in the treatment of human obesity and some of its associated metabolic dysfunctions, but also may help to prevent and/or reverse at least some of the bone loss which accompanies osteoporosis, anorexia nervosa and other wasting diseases.

Intranasal delivery of mouse [D-Leu-4]-OB3, a synthetic peptide amide with leptin-like activity, improves energy balance, glycaemic control, insulin sensitivity and bone formation in leptin-resistant C57BLK/6-m db/db mice.

BACKGROUND: We have recently shown that intranasal administration of mouse [D-Leu-4]-OB3 reconstituted in Intravail(®) to male Swiss Webster mice resulted in significantly higher uptake and bioavailability when compared with commonly used injection methods of delivery. AIM AND METHODS: In this study, we examined the effects of intranasal delivery of mouse [D-Leu-4]-OB3 in Intravail(®) on energy balance, glucose regulation, insulin secretion and serum levels of osteocalcin, a specific and sensitive marker of bone formation. Genetically obese C57BLK/6-m db/db mice were allowed food and water ad libitum and given either Intravail(®) alone or mouse [D-Leu-4]-OB3 in Intravail(®) for 14 days by intranasal instillation. RESULTS: Mouse [D-Leu-4]-OB3 reduced body weight gain, daily food intake, daily water intake and serum glucose by 11.5, 2.2, 4.0 and 61.9%, respectively. Serum insulin levels in db/db mice given mouse [D-Leu-4]-OB3 were approximately threefold lower than those in mice receiving Intravail(®) alone. Mouse [D-Leu-4]-OB3 elevated serum osteocalcin in db/db mice by 28.7% over Intravail(®) treated control mice. CONCLUSIONS: The results of our study indicate that intranasal delivery of biologically active mouse [D-Leu-4]-OB3 in Intravail(®) is feasible and has significant effects on regulating body weight gain, food and water intake, serum glucose, insulin sensitivity and bone formation in leptin-resistant C57BLK/6-m db/db mice.

Nephroblastoma in the rat: histology of a spontaneous tumor, identity with respect to renal mesenchymal neoplasms, and a review of previously recorded cases.

The histology of a spontaneously occurring neoplasm of the rat kidney conforming to a classification of nephroblastoma is described and compared with that of N-nitrosodimethylamine-induced renal mesenchymal tumors. This rat nephroblastoma was an encapsulated epitheloid neoplasm with a uniform histologic pattern. Clumps of densely crowded, hyperchromatic cells frequently associated with central, well-differentiated ducts were supported by a less cellular, interconnecting stroma of loose areolar or mature fibrous connective tissue. Neoplastic cells were organized into primitive, ill-defined tubular formations. The neoplastic cell component strongly resembled metanephrogenic blastema. In contrast, the renal mesenchymal tumor was nonencapsulated and consisted of a heterogeneous mixture of connective tissue elements including fibroblast-like spindle cells, smooth muscle, and embryonic mesenchyme that engulfed and sequestered preexisting renal tubules and glomeruli. The separate morphologic identities and apparently unrelated existence of rat nephroblastoma and renal mesenchymal tumor were stressed. The rat nephroblastoma morphologically resembled the malignant epithelial component of human Wilms' tumor, whereas rat renal mesenchymal tumor appeared to have counter-parts in the mesenchymal component of Wilms' tumor and in congenital mesoblastic nephroma (leiomyomatous hamartoma) of infancy. The histologic descriptions of previously recorded occurrences of spontaneous and experimentally induced rat neoplasma classified as nephroblastoma or its synonyms were reevaluated in comparison to the present case. In all but four instances, in which sufficient histologic detail was provided in previous reports, a consistent histologic pattern emerged for this neoplasm in the rat.

Effects of pyrazole and 3-amino-1,2,4-triazole on the metabolism and toxicity of dimethylnitrosamine in the rat.

Pretreatment of rats with pyrazole or 3-amino-1,2,4-triazole (3-AT) known inhibitors of alcohol metabolism, profoundly inhibited the metabolism of dimethylnitrosamine (DMN), both in terms of [14C]CO2 excretion and of the decline in the blood concentration. Additionally, 4-methylpyrazole, tetraethylthiuram disulfide (disulfiram), methanol, and ethanol inhibited the metabolism of DMN in the whole animal. In parallel experiments with [14C]aminopyrine, no substantial inhibitory effect was found with pyrazole, 3-AT, or disulfiram pretreatment. Investigations into the effects of pyrazole and 3-AT pretreatment on the acute toxicity and hepatotoxicity of DMN showed that pyrazole significantly increased the median lethal dose (LD50) of DMN and provided substantial protection against the hepatotoxicity of DMN, in that centriblobular necrosis was not seen at dose levels of DMN up to 25 mg/kg and early histochemical changes indicative of liver injury were not observed at a dose level of 15 mg DMN/kg. In contrast, 3-AT pretreatment did not affect the LD50 of DMN or provide any protection against the hepatotoxicity of DMN. Further, although both inhibitors delayed the incorporation of radioactivity from [14C]DMN into hepatic subcellular organelles, pyrazole was significantly more effective than was 3-AT.

Effects on 4080 rats of chronic ingestion of N-nitrosodiethylamine or N-nitrosodimethylamine: a detailed dose-response study.

Four thousand eighty inbred rats were maintained from weaning on various different concentrations of N-nitrosodiethylamine (NDEA) or N-nitrosodimethylamine (NDMA). The principal aim was to characterize the dose-response relationship for the effects of these agents on esophageal cancer (NDEA) or on various types of liver cancer (NDEA and NDMA), although NDEA also caused a few tumors of the nasopharynx and NDMA also caused a few tumors of the lung. The numbers of tumors of mesenchymal and Kupffer cells in the liver were too few to allow easy characterization of the dose-response relationships, and although NDMA induced large numbers of bile duct neoplasms, NDEA did not. Thus, the four principal dose-response relationships studied were of NDEA on esophageal or liver cells and of NDMA on bile duct or liver cells. At doses sufficiently high for the median time to death from the disease of interest to be estimated, relationships were observed of the general form (Dose rate) x (median)n = constant where n was about 2.3 for the first three relationships and about 1 for the last one (NDMA on liver cell tumors). By contrast, at doses sufficiently low for longevity to be nearly normal (median survival about 2.5 years), there remained no material dependence on the dose rate of the age distribution of the induced neoplasms. At these low dose rates, the number of liver (but not of esophageal) neoplasms induced by treatment was simply proportional to the dose rate. This finding is not surprising, since the background incidence of liver (but not of esophageal) neoplasms was appreciable. The linear relationship observed at low dose rates (below 1 ppm) suggests that under these experimental conditions, among rats allowed to liver their natural life span, a dose of 1 ppm of NDEA or NDMA in the drinking water will cause about 25% to develop a liver neoplasm, a dose of 0.1 ppm will cause about 2.5% to do so, and a dose of 0.01 ppm will cause about 0.25% to do so, etc., with no indication of any "threshold." (At these low dose rates, the incidence of liver neoplasms appears likely to exceed greatly that of esophageal neoplasms.) In addition, even quite low dose rates of the test agents caused a variety of nonneoplastic liver abnormalities (e.g., hyperplastic nodules, or shrinkage of hepatocytes) at a frequency roughly proportional to the dose rate.

Dose and time relationships for tumor induction in the liver and esophagus of 4080 inbred rats by chronic ingestion of N-nitrosodiethylamine or N-nitrosodimethylamine.

A Weibull analysis is presented of the dose and time relationships for the effects on 4080 inbred rats of chronic ingestion in the drinking water of 16 different doses of N-nitrosodimethylamine (NDMA) or N-nitrosodiethylamine (NDEA). The sites chiefly affected were the liver (by both agents) and the esophagus (by NDEA only). Since the experiment continued on into extreme old age, effects became measurable at doses of only 0.01 to 0.02 mg/kg/day, which is an order of magnitude lower than previously achieved. (After only 2 years of treatment, however, the TD50 doses needed to halve the proportion of tumorless survivors would have been about 0.06 mg/kg/day of NDEA, or about 0.12 mg/kg/day of NDMA.) The general pattern of response was that the natural logarithm of the probability of remaining tumorless was given by the product of two terms, the first (the "Weibull b value") depending on the dose rate but not on the duration of exposure and the second depending not on dose at all but only on duration. For all types of tumor the dependence on duration was fairly similar (and for each the second term was taken to be -t7, where t = years of treatment), but for different types of tumor the dependence on dose rate was quite different. For esophageal tumors, the "Weibull b value" was approximately proportional to the cube of the dose rate of NDEA (males 21 d3, females 11 d3, where d = dose rate in mg/kg adult body weight/day), and the background incidence was unmeasurably low. For liver tumors induced by NDEA, the b value was approximately proportional to the fourth power of dose rate + 0.04 mg/kg/day [males, 19 (d + 0.04)4; females, 32 (d + 0.04)4], although the relationships were somewhat different for the different cell types of liver tumor. This one formula implies both approximate linearity at low doses and an approximately cubic relationship within the higher range of doses that was studied. For liver tumors induced by NDMA, the Weibull b value was approximately proportional to the sixth power of dose rate + 0.1 mg/kg/day [males, 37 (d + 0.1)6; females, 51 (d + 0.1)6], again with some variation between liver cell types, and again implying approximate linearity at low doses. These algebraic formulae should, of course, be trusted only in the range of doses where they were derived, and particularly not above it.(ABSTRACT TRUNCATED AT 400 WORDS)

Chronic nitrosamine ingestion in 1040 rodents: the effect of the choice of nitrosamine, the species studied, and the age of starting exposure.

In parallel with a larger experiment on 4080 rats fed 16 different concentrations of N-nitrosodiethylamine (NDEA) or N-nitrosodimethylamine (NDMA) from 6 weeks of age, a variety of smaller experiments on a total of 1040 rodents were undertaken and are the subject of the present report. Three separate subjects were addressed. Studies of 16 different concentrations of N-nitrosopyrrolidine and N-nitrosopiperidine given from age 6 weeks onwards to small groups of rats yielded dose-response relationships for the effects of N-nitrosopyrrolidine on liver tumors and for those of N-nitrosopiperidine on tumors of the liver and upper gastrointestinal tract that resembled those seen for NDMA and NDEA, respectively, except that N-nitrosopyrrolidine and N-nitrosopiperidine were less potent [the respective dose rates needed to halve the proportion of tumorless survivors after 2 years of treatment being approximately 0.4 (males) and 0.6 (females) mg/kg adult body weight/day for each agent]. Alternatively, it was estimated that the risks to rats from lifelong exposure to 1 microgram/kg adult body weight/day of each agent might be about 0.1%, and that the risks to rats from lower doses would be proportionately less. Studies of 16 different concentrations of NDEA on small groups of female mice and female hamsters yielded the types of dose response that would be expected for upper gastrointestinal tumors, liver cell tumors, and Kupffer cell tumors in mice (no other types of liver tumor being produced, in contrast with previous reports) and for tracheal and liver cell tumors in hamsters (no clear effect on upper gastrointestinal tumors being apparent in hamsters). The dose rates needed to halve the proportion of tumorless survivors after 2 years of treatment were approximately 0.3 mg/kg adult body weight/day, i.e., 5 times that for the same agent in rats. In part, however, this may be because treatment started at an older age in these species. Studies were undertaken of the effects on esophageal and liver tumorigenesis of starting the treatment of rats with NDEA at 3 or at 20 weeks of age instead of at 6 weeks of age (as in the main experiment). Earlier treatment resulted in slightly greater dosage rates, if dosage was measured in mg/kg/day, and hence in a correspondingly more rapid yield of esophageal tumors, but the effect was not large. By contrast, an earlier start to treatment resulted, after a fixed duration of treatment, in animals having a 3-fold higher incidence rate of liver tumors, while a later start resulted in a 2-fold decrease.(ABSTRACT TRUNCATED AT 400 WORDS)

Structure--function relationships of the glycoprotein hormones and their receptors.

The primary structures of the glycoprotein hormones follitropin (FSH), lutropin (LH), human choriogonadotropin (hCG) and thyrotropin (TSH) have been determined, hCG has been crystallized and initial diffraction data obtained. Studies with synthetic peptides have provided information on regions involved in receptor interaction and signal transduction. The receptors for the glycoprotein hormones have been prepared by gene cloning methods and their primary structures deduced. As Leo Reichert and colleagues discuss here, although cAMP is involved in glycoprotein hormone signal transduction, recent evidence also implicates other second messengers, especially Ca2+ and may include both the phosphatidylinositol pathway and activation of Ca2+ channels.

Inhibitory effects of leptin-related synthetic peptide 116-130 on food intake and body weight gain in female C57BL/6J ob/ob mice may not be mediated by peptide activation of the long isoform of the leptin receptor.

We recently reported that intraperitoneal administration of leptin-related synthetic peptide 116-130 [LEP-(116-130)] resulted in reduced food intake and significant weight loss in homozygous female C57BL/6J ob/ob mice. In this study, we used two in vitro bioassays to show that the interaction of LEP-(116-130) with the long form of the leptin receptor (OB-Rb), the receptor isoform that is predominantly expressed in the hypothalamus, is not required for the observed in vivo effects of the peptide on energy balance. LEP-(116-130) was unable to compete the binding of alkaline phosphatase-leptin fusion protein to OB-R. Moreover, LEP-(116-130) was unable to activate signal transduction by OB-Rb in vitro. In homozygous female C57BLKS/J-m db/db mice that do not express OB-Rb, intraperitoneal administration of LEP-(116-130) reduced body weight gain and blood glucose levels but not food intake, which further supports a mechanism of action that does not require peptide interaction with OB-Rb.

Reconstitution of hormone-responsive detergent-solubilized follicle stimulating hormone receptors into liposomes.

An FSH receptor-enriched fraction that responds to exogenous FSH by activation of adenylate cyclase was prepared by ultrafiltration of sucrose density gradient-purified light membranes derived from bovine calf testes homogenates and solubilized with Triton X-100. To further confirm the functional nature of the detergent-solubilized FSH receptor, the extract was incorporated by lipid hydration into large multilamellar vesicles composed of dioleoyl phosphatidylcholine and cholesterol, 2:1 molar ratio. Receptor incorporation was determined by measurement of specific binding of [125I] human FSH ([125I] hFSH). Substitution of dioleoyl phosphatidylcholine with dipalmitoyl phosphatidylcholine or increasing the cholesterol concentration of the vesicles reduced specific binding of [125I]hFSH. Under conditions favoring optimal incorporation of the receptor, specific binding of [125I]hFSH was time and temperature dependent and saturable when increasing concentrations of radioligand were added to a constant amount of proteoliposomes. Reconstituted proteoliposomes bound 1600 fmol FSH/mg protein with an affinity of 3.54 x 10(9) M-1. Inhibition of [125I] hFSH binding by hFSH was comparable to that seen with the membrane-bound receptor (ED50 = 10 ng). Equilibrium binding studies with [3H]Gpp(NH)p indicated that a single class of high affinity GTP binding sites with an association constant (Ka) of 3.33 x 10(7) m-1 which bound 2.19 fmol [3H]Gpp(NH)p/mg protein had also been incorporated into the proteoliposomes. Addition of FSH induced a 2-fold stimulation of [3H]Gpp(NH)p binding, supporting our earlier studies suggesting that the detergent-solubilized FSH receptor is complexed to the G protein. Of particular significance in the present study was the observation that both NaF and FSH stimulated cAMP production in the reconstituted system. In addition to belonging to a class of membrane receptors functionally and physically associated with G protein, this observation suggests that FSH receptors in bovine calf testicular membranes may be associated, at least in part, with adenylate cyclase as well.

The effects of aprotinin on follicle-stimulating hormone binding and signal transduction in bovine calf testis.

Aprotinin (bovine pancreatic trypsin inhibitor), a serine protease inhibitor, caused a dose-dependent inhibition of [125I]human FSH ([ 125I]hFSH) binding to 1) an FSH receptor-enriched light membrane fraction prepared from bovine calf testes homogenates, 2) Triton X-100-solubilized FSH receptor, and 3) proteoliposomes containing incorporated FSH receptor-G-protein-adenylate cyclase (AC) complexes. Equilibrium binding studies with the solubilized receptor indicated that the effect of aprotinin on [125I]hFSH binding was due to a decrease in the Ka of the receptor, with no change in FSH-binding capacity. The rate of association of [125I]hFSH with its receptor was reduced by 50% in the presence of aprotinin, but no effect on dissociation of FSH-receptor complexes was evident. Aprotinin, at a concentration (250 microM) that inhibited binding of [125I]hFSH to the membrane receptor by 25%, completely inhibited basal, fluoride-stimulated and FSH-stimulated AC activity. However, aprotinin, at a concentration (50 microM) that had little effect on [125I]hFSH binding, markedly enhanced basal AC activity (3.4-fold) to the level of fluoride and FSH stimulation. Aprotinin did not inhibit [3H]5'-guanylylimidodiphosphate binding to FSH receptor-enriched membranes, suggesting that its effects on the affinity of the receptor for FSH and on AC activation were not mediated through an interaction with FSH receptor-associated G-protein. No serine protease activity could be detected in any of the receptor or hormone preparations used in this study. The ability of aprotinin to inhibit binding of [125I]hFSH to the Triton X-100-solubilized receptor and to the soluble receptor incorporated into proteoliposomes as well as to the FSH receptor-enriched membrane fraction, all of which are free of serine protease activity, is consistent with the notion that aprotinin may directly interact with the FSH receptor to sterically hinder binding of FSH. Furthermore, the apparent direct effect of aprotinin on basal as well as FSH-stimulated AC activity suggests its general usefulness in studies on the mechanism of signal transduction for ligands thought to operate via the cAMP second messenger system.

Follicle-stimulating hormone receptor-mediated uptake of 45Ca2+ by proteoliposomes and cultured rat sertoli cells: evidence for involvement of voltage-activated and voltage-independent calcium channels.

We have previously reported incorporation into liposomes of Triton X-100-solubilized FSH receptor-G-protein complexes derived from purified bovine calf testis membranes. In the present study we have used this model system to show that FSH induces flux of 45Ca2+ into such proteoliposomes in a hormone-specific concentration-dependent manner. FSH, inactivated by boiling, had no stimulatory effect on 45Ca2+ flux, nor did isolated alpha- or beta-subunits of FSH. Addition of GTP (or its analogs 5'-guanylylimidodiphosphate and guanosine-5'-O-[3-thiotriphosphate]) or sodium fluoride (in the presence or absence of GTP or its analogs) failed to induce 45Ca2+ flux into proteoliposomes, suggesting that the uptake of 45Ca2+ was receptor, and not G-protein, related. Voltage-independent (ruthenium red and gadolinium chloride) and voltage-activated (methyoxyverapamil and nifedipine) calcium channel-blocking agents reduced FSH-stimulated 45Ca2+ flux into proteoliposomes to control levels. FSH also induced uptake of 45Ca2+ by cultured rat Sertoli cells. Ruthenium red and gadolinium chloride had no effect on basal levels of 45Ca2+ uptake or estradiol secretion by cultured rat Sertoli cells, nor did methoxyverapamil or nifedipine. All four calcium channel blockers, however, were able to reduce FSH-induced 45Ca2+ uptake to basal levels and FSH-stimulated conversion of androstenedione to estradiol by up to 50%, indicating an involvement of Ca2+ in FSH-stimulated steroidogenesis. Our results suggest that the well documented changes in intracellular calcium levels consequent to FSH binding may be due, at least in part, to an influx of calcium through FSH receptor-regulated calcium channels.

In vivo effects of follicle-stimulating hormone-related synthetic peptides on the mouse estrous cycle.

We have previously shown that a synthetic peptide amide corresponding to residues 34-37 (TRDL) of the human (h) FSH beta-subunit inhibited binding of [125I]hFSH to bovine calf testis membrane receptors and antagonized FSH-stimulated estradiol biosynthesis in primary cultures of rat Sertoli cells. These in vitro effects would have additional significance if they could be confirmed in an in vivo model system. We have obtained several lines of evidence supporting in vivo effects of TRDL on the mouse estrous cycle. 1) A single i.p. injection of 200 microg/g BW TRDL induced persistent vaginal estrus, characterized by the complete absence of epithelial casts in 87% of the mice treated, as determined by vaginal cytology. 2) A synthetic peptide representing a larger receptor-binding domain of the hFSH beta-subunit, hFSHbeta-(33-53), that contains the TRDL sequence had a similar effect, but hFSHbeta-(38-53) lacking the TRDL sequence, did not. 3) A series of unrelated synthetic peptides, tested at a comparable dose (200 microg/g BW), were also without effect, as was a D-amino acid analog of TRDL, TR(D)DL. 4) Serum estradiol levels at proestrus in TRDL-treated mice were significantly lower than those in vehicle-injected control mice. 5) The effect of estrogen on uterine ballooning and weight gain, seen in all vehicle-injected control mice at proestrus, did not occur in 97% of the mice treated with TRDL. 6) The ovaries of TRDL-treated mice taken during persistent vaginal estrus contained a greater number of large hemorrhagic preovulatory follicles and fewer corpora lutea than those in ovaries taken at estrus from vehicle-injected control mice. Taken together, these results indicate disruption of the normal mouse estrous cycle by the TRDL peptide and represent the first demonstration of in vivo effects of gonadotropin-related synthetic peptides on reproductive processes.

Follicle-stimulating hormone receptor-mediated uptake of 45Ca2+ by cultured rat Sertoli cells does not require activation of cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding proteins or adenylate cyclase.

We have previously reported that FSH stimulates flux of 45Ca2+ into cultured Sertoli cells from immature rats via voltage-sensitive and voltage-independent calcium channels. In the present study, we show that this effect of FSH does not require cholera toxin (CT)- or pertussis toxin (PT)-sensitive guanine nucleotide binding (G) protein or activation of adenylate cyclase (AC). Significant stimulation of 45Ca2+ influx was observed within 1 min, and maximal response (3.2-fold over basal levels) was achieved within 2 min after exposure to FSH. FSH-stimulated elevations in cellular cAMP paralleled increases in 45Ca2+ uptake, suggesting a possible coupling of AC activation to 45Ca2+ influx. (Bu)2cAMP, however, was not able to enhance 45Ca2+ uptake over basal levels at a final concentration of 1000 microM, although a concentration-related increase in androstenedione conversion to estradiol was evident. Exposure of Sertoli cells to CT (10 ng/ml) consistently stimulated basal levels of androstenedione conversion to estradiol but had no effect on basal levels of 45Ca2+ uptake. Similarly, CT had no effect on FSH-induced 45Ca2+ uptake, but potentiated FSH-stimulated estradiol synthesis. PT (10 ng/ml) augmented basal and FSH-stimulated estradiol secretion without affecting 45Ca2+ influx. The adenosine analog N6-phenylisopropyladenosine, which binds to Gi-coupled adenosine receptors on Sertoli cells, inhibited FSH-stimulated androgen conversion to estradiol in a dose-related (1-1000 nM) manner, but FSH-stimulated 45Ca2+ influx remained unchanged. Our results show that in contrast to FSH-stimulated estradiol synthesis, the flux of 45Ca2+ into Sertoli cells in response to FSH is not mediated either directly or indirectly by CT- or PT-sensitive G protein, nor does it require activation of AC. Our data further suggest that the FSH receptor itself may function as a calcium channel.

Prolactin-mediated progesterone secretion by cultured rat granulosa cells: regulation by purified human glycoprotein hormones and their subunits.

The effects of purified alpha- and beta-subunits of human glycoprotein hormones on initial luteinization and subsequent prolactin-mediated progesterone responses of cultured rat granulosa cells were studied. Granulosa cells, obtained from immature female rats 50 h after PMSG treatment, were incubated for 24 h in control medium lacking added hormones or in medium containing hCG or the alpha- or beta-subunit of human (h) FSH, LH, CG, or TSH at 0.1, 0.5, and 1.0 microgram/ml. Cultures were maintained subsequently for 6 days in medium containing 1.0 microgram/ml bovine PRL (bPRL), with medium changes every 48 h. Indices of luteotropic stimulation in response to bPRL were provided by 1) elevated progesterone concentrations determined by RIA of spent media samples, and 2) cytoplasmic lipid accumulation assessed by osmium tetroxide staining following fixation of monolayers after 7 days of culture. Progesterone concentrations in media from cultures incubated in 0.5 or 1.0 microgram/ml hCG were 6-fold higher than in cultures incubated in control medium, while those in media from cultures incubated in 0.5 or 1.0 microgram/ml hFSH alpha, hLH alpha, hCG alpha, hTSH alpha, hLH beta, or hCG beta (but not in hFSH beta or hTSH beta) were from 2- to 4-fold higher than those in control cultures. This enhancement was not evident when subunits were added to the incubation media at the lowest concentration. Progesterone secretion corresponded directly with the degree of cytoplasmic osmiophilia. These results suggest that the alpha-subunit of each of the glycoprotein hormones as well as the beta-subunit of hLH and hCG have the ability to promote progesterone secretion during initial luteinization and to regulate subsequent PRL-mediated steroidogenesis by rat granulosa cells in vitro. Furthermore, these effects are greater than can be accounted for by potential contamination of subunit preparations with undissociated hormones, as demonstrated by dose-response curves.

A new role for follicle-stimulating hormone in the regulation of calcium flux in Sertoli cells: inhibition of Na+/Ca++ exchange.

Elucidation of mechanisms regulating intracellular calcium levels in steroidogenic tissues is important for understanding control of cellular function. We have previously described FSH receptor-mediated flux of 45Ca++ into cultured rat Sertoli cells and receptor-enriched proteoliposomes via voltage-sensitive and voltage-independent calcium channels. In the present study, we report heretofore unrecognized inhibitory effects of FSH on Na+/Ca++ exchange in these two systems. An outwardly directed Na+ gradient, developed by preincubating Sertoli cell monolayers in buffer made hypertonic with NaCl, resulted in uptake of 45Ca++ that was unaffected by calcium channel blocking agents, ruthenium red or methoxyverapamil, but was enhanced by ouabain, a specific inhibitor of Na+/K(+)-ATPase. Sodium-dependent 45Ca++ flux into Sertoli cells was inhibited in a concentration-related manner by increased extracellular Na+ (up to 135 mM). FSH consistently and reproducibly (28.9 +/- 3.8%, 10 separate assays) reduced sodium-dependent 45Ca++ influx in the absence or presence of ouabain. A lesser effect on Na+/Ca++ exchange was seen when Li+ replaced Na+ in the preincubation buffer, and a marked reduction occurred when Sertoli cells were incubated in buffer containing KCl, presumably due to membrane depolarization. FSH-sensitive Na+/45Ca++ exchange was also observed when using FSH receptor-enriched proteoliposomes. Our earlier calcium channel studies indicated that FSH affects Ca++ entry into Sertoli cells via a receptor-mediated process. The results reported here demonstrate that the interaction of FSH with its receptor is associated with changes in Na+/Ca++ exchange as well, and suggest that this activity may also be involved in regulating intracellular free Ca++ levels in the Sertoli cell.

A synthetic peptide corresponding to amino acid residues 34 to 37 of human follicle-stimulating hormone beta-subunit accelerates the onset of puberty in male and female mice.

We have previously reported that a synthetic peptide amide corresponding to residues 34-37 (TRDL, threonine, arginine-aspartic acid-leucine) of the beta-subunit of human FSH induced prolonged vaginal estrus in normally cycling female mice (see Ref. 15). These results represented the first demonstration of an in vivo effect of a gonadotropin-related synthetic peptide on reproductive processes. We have extended these studies to examine possible effects of TRDL on the onset of puberty in female mice. In two replicated experiments, vehicle-injected control mice attained first vaginal estrus by day 39. An ip injection of 200 ug TRDL/g BW to 28-day-old prepubertal female mice, however, accelerated the onset of first vaginal estrus by 7 days in 11 of 12 (11/12) (Exp 1) and 7/9 (Exp 2) mice. Serum estradiol levels were significantly (P = 0.017) elevated in TRDL-treated mice, whereas progesterone was unchanged. Uteri of TRDL-treated mice were significantly (P = 0.003) heavier than uteri of vehicle-injected control animals of the same age and body weight. Intraluminal fluid accumulation (ballooning) at proestrus was absent in 20/21 TRDL-treated females, as were oviductal ova and ovarian corpora lutea. These phenomena are characteristic of the first estrous cycles of female mice isolated from males. To obtain further evidence for in vivo effects of TRDL, we assessed the ability of TRDL to accelerate the onset of puberty in male mice. When given as five consecutive daily ip injections of 200 ug/g BW to 28-day-old prepubertal male mice, TRDL significantly increased testis weight, when compared with vehicle-injected control mice of the same age and BW (171.3 +/- 3.8 mg vs. 151.6 +/- 4.3 mg, P = 0.001) and induced a 6.5-fold increase in serum testosterone levels. These studies confirm the previously reported in vivo activity of a synthetic peptide corresponding to human FSH-beta subunit 34-37 (TRDL) in adult female mice and extend its effects to the acceleration of the onset of puberty in immature male and female mice.

Transglutaminase activity in bovine calf testicular membranes: evidence for a possible role in the interaction of follicle-stimulating hormone with its receptor.

Transglutaminase (TGase) activity, determined by incorporation of [1,4-14C]putrescine into casein, was demonstrated in a light membrane fraction derived from bovine calf testicular homogenates. In common with other TGases thus far identified, testicular TGase is calcium dependent. A concentration-dependent relationship was observed between decreasing TGase activity and increasing concentrations of N-ethylmaleimide and bacitracin, inhibitors of TGase, and the TGase substrate monodansylcadaverine. Although NEM at all concentrations tested had no effect on [125I]human (h) FSH-receptor binding, dissociation of [125I]hFSH from its receptor was enhanced when [125I]hFSH-receptor complexes were formed in the presence of NEM. Dissociation of [125I]hFSH from its receptor also increased in the presence of bacitracin or monodansylcadaverine. Reduced TGase activity always paralleled increases in hormone-receptor dissociation. The inverse relationship between TGase activity and [125I]hFSH-receptor dissociation observed in this study suggests that TGase activity may be involved in the interaction of FSH with its receptor and that protein cross-linking (via peptide bond formation) may be a mechanism whereby some FSH-receptor complexes are stabilized in the bovine testis.

Correlation of follicle-stimulating hormone (FSH)-receptor complex internalization with the sustained phase of FSH-induced calcium uptake by cultured rat Sertoli cells.

We have previously reported that synthetic peptide amides corresponding to regions of the beta-subunit of human FSH [hFSH-beta-(1-15) and hFSH-beta-(51-65)] have the ability to bind calcium and to facilitate its entry into liposomes. In the present study, we have examined the ability of synthetic peptides corresponding to the entire primary structure of hFSH-beta-subunit, to induce calcium influx in cultured rat Sertoli cells. Calcium (as 45Ca2+) uptake in response to 50 microM hFSH-beta-(1-15), hFSH-beta-(21-35), or hFSH-beta-(51-65) peptide amides was 2.5-, 2.4-, and 2.0-fold higher, respectively, than basal uptake. Pretreatment of Sertoli cells for 5 min with phenylarsine oxide (PAO, 80 microM), an inhibitor of receptor-mediated endocytosis, significantly (P < 0.05) reduced 45Ca2+ influx in response to hFSH-beta-(1-15), hFSH-beta-(21-35), and hFSH-beta-(51-65). A delay of 20 min was required, however, before the inhibitory effect of PAO on 45Ca2+ uptake was observed. Specific binding of [125I] hFSH to receptor at 4 C was unaffected by PAO. After 2 h at 37 C, however, approximately 1.6-fold more [125I]hFSH specifically bound at 4 C could be dissociated from the cell surfaces of PAO-pretreated Sertoli cell monolayers, compared to untreated monolayers. This result is consistent with an inhibitory effect of PAO on FSH receptor internalization. Chloroquine (at 100 microM), a lysosomotropic agent known to block FSH degradation, also significantly (P < 0.05) inhibited FSH-induced 45Ca2+ uptake. Extending our earlier studies, these results suggest that the sustained (> 20 min) phase of FSH-induced calcium uptake, also seen in response to synthetic hFSH-beta-(1-15), hFSH-beta-(21-35), and hFSH-beta-(51-65) peptide amides, may occur as a consequence of FSH-receptor complex internalization and FSH degradation. Vesicular uptake of extracellular calcium, which accompanies internalization of FSH-receptor complexes, and release of channel-forming peptides by lysosomal hydrolysis of FSH suggests a novel mechanism whereby FSH increases intracellular calcium levels in Sertoli cells.

Synthetic human follicle-stimulating hormone-beta-(1-15) peptide-amide binds Ca2+ and possesses sequence similarity to calcium binding sites of calmodulin.

To determine the basis for the previously demonstrated calcium requirement for specific binding of FSH to receptor, 11 overlapping peptide amides representing the entire primary structure of human FSH (hFSH)-beta-subunit were tested for their ability to bind 45Ca2+ as an approach to identifying possible calcium binding regions of the hormone. hFSH-beta-(1-15)-peptide amide bound significant amounts of 45Ca2+. This peptide contains an amino acid sequence similar to that found in the loop structures of the calcium-binding domains of calmodulin. The affinity of hFSH-beta-(1-15)-peptide amide for calcium (Kd = 1.2 +/- 0.3 mM) was similar to that previously reported for a synthetic peptide corresponding to calmodulin binding site III. No such sequence is predicted in the recently deduced primary structure of the FSH receptor. FSH-beta-(1-15) may, therefore, be associated with the calcium requirement for specific binding of FSH to receptor. The calcium binding property of this calmodulin-like peptide also correlates well with its ability to induce uptake of calcium into liposomes via transmembrane channel formation, as previously reported by this laboratory.