Biomedical subjects

N Ling

Publications and source records attributed to N Ling.

At least 91 records · Page 5Linked to original sources

PubMed1993

Stimulation of progesterone secretion by recombinant follistatin-288 in human granulosa cells.

The effects of recombinant human follistatin (follistatin-288) on basal and hCG-stimulated progesterone secretion were examined in cultured human granulosa cells. Follistatin increased progesterone secretion in a dose-dependent manner. However, follistatin did not augment hCG- or cAMP-stimulated progesterone secretion. Time-course analysis revealed that follistatin increased progesterone secretion after 24 h of incubation. Follistatin also enhanced basal, but not hCG-stimulated, 20 alpha-hydroxyprogesterone accumulation, indicating that the increase in progesterone accumulation was not due to a blockade of the 20 alpha-hydroxylase metabolic pathway. In the presence of the phosphodiesterase inhibitor isobutylmethylxanthine, follistatin significantly increased intracellular cAMP accumulation to levels comparable to those induced by 1 IU/ml hCG. These results provide the first evidence of a stimulatory action of follistatin on progestin secretion in the human ovary, which is accompanied by an increased accumulation of intracellular cAMP levels. Follistatin may well be another potential regulator of steroid hormone production in human granulosa cells during the periovulatory period.

20-alpha-Dihydroprogesterone↗

PubMed1993

Effects of estrous cycle stage and pregnancy on follistatin gene expression and immunoreactivity in rat reproductive tissues: progesterone is implicated in regulating uterine gene expression.

Follistatin, a monomeric protein originally isolated from ovarian follicular fluid, is now believed to be a major local regulator of the multifaceted actions of activin by virtue of its activin-binding properties. In view of the ability of follistatin to stimulate progesterone production from granulosa cells and its presence in newly formed corpora lutea, the following study was conducted to determine the effects of cycle stage and pregnancy on follistatin gene expression and immunoreactivity in the rat ovary and uterus with the intent of gaining additional insights into the regulation of follistatin in these tissues. Decidua and placentas were also examined on days 15, 18, and 21 of pregnancy. Follistatin messenger RNA (mRNA) levels were quantified using a sensitive solution hybridization-RNase protection assay and values normalized to the amount of cyclophilin mRNA present in each sample. Levels of follistatin-like immunoreactivity (FLI) in serum and tissues were estimated using a homologous porcine follistatin RIA. Follistatin message levels in the ovary increased between proestrus and estrus with a return to proestrous values on both days of diestrus. In the nonpregnant uterus, mRNA levels on proestrus were similar to levels measured in uteri taken from hypophysectomized or ovariectomized rats. Interestingly, follistatin gene expression increased almost 3-fold between proestrus and estrus. An additional experiment demonstrated that this increase could be abated by treatment of proestrous rats with pentobarbital which blocks preovulatory rises in serum progesterone levels and could be restored by administration of progesterone to pentobarbital-treated proestrous rats. In pregnant rats, ovarian follistatin message levels on days 3 and 6 of pregnancy were identical to levels observed on day 2 of diestrus. However, an abrupt 4-fold increase in ovarian mRNA levels occurred between days 6 and 9 with a further 58% increase occurring by day 12. This marked increase in message levels was unaccompanied by changes in ovarian FLI levels. A precipitous decrease in transcript levels accompanied by a decline in FLI levels then followed with ovarian gene expression on days 15 through 21 being slightly higher than expression during the initial stages of gestation. Expression of the gene in the decidua and placenta did not vary between days 15 and 21 of pregnancy. Levels of FLI in serum also were invariant during the cycle and pregnancy.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Isomerases↗

PubMed1993

Expression and regulation of insulin-like growth factor-binding protein-1, -2, -3, and -4 messenger ribonucleic acids in purified rat Leydig cells and their biological effects.

The actions of insulin-like growth factor-I (IGF-I) are modified by binding proteins [IGF-binding proteins (IGFBPs)]. Previously, we reported that IGF-I enhances Leydig cell steroidogenesis and that IGF-I mRNA is expressed in Leydig cells. In the present study, we evaluated the expression and regulation of IGFBP-1, -2, -3, and -4 in purified rat Leydig cells and their biological effects. We found that none of the testicular crude interstitial cells, purified Leydig cells, or seminiferous tubules expressed IGFBP-1 mRNA. This indicated that IGFBP-1 mRNA is not expressed in the testis in detectable amounts. In contrast, large amounts of IGFBP-2 with a size of 1.8 kilobases (kb) were expressed in purified Leydig cells, and lesser amounts in crude interstitial cells. Small amounts of IGFBP-2 mRNA were expressed in seminiferous tubules, but none could be detected in liver. IGFBP-3 mRNA was predominantly expressed in purified Leydig cells, crude interstitial cells, and liver, while appreciable amounts were not found in seminiferous tubules. Liver had the highest amounts of IGFBP-4 mRNA, whereas purified Leydig cells and crude interstitial cells had lesser amounts. We next evaluated the pituitary dependency of IGFBP mRNAs in Leydig cells. Purified Leydig cells were isolated from 50-day-old rats 5 days after hypophysectomy. IGFBP-2, -3, and -4 mRNA levels in Leydig cells decreased 22%, 80%, and more than 90%, respectively, after hypophysectomy. In the liver, however, IGFBP-2 mRNA levels increased, and IGFBP-3 mRNA levels decreased, while IGFBP-4 remained unchanged. As expected, hypophysectomy caused decrements in large (7.0- to 7.5-kb; a 75% reduction) and small (0.8- to 1.2-kb; a 90% reduction) IGF-I mRNA levels in Leydig cells. Hypophysectomy also reduced IGF-I mRNA expression in liver. Finally, the effects of IGFBP-2, -3, and -4 on Leydig cell testosterone formation were investigated. hCG in a concentration of 10 ng/ml increased testosterone formation from 0.6 +/- 0.01 to 27.4 +/- 1.01 ng/10(6) cells.h. In the presence of IGF-I (10 ng/ml), testosterone formation was further increased to 58.6 +/- 1.6 ng/10(6) cells.h (P < 0.01). IGFBP-3 (0.1, 1, and 2.5 pmol/ml) caused a dose-dependent inhibition of IGF-I- plus hCG-induced testosterone formation. IGFBP-3 in a concentration of 2.5 pmol/ml completely neutralized the effects of IGF-I on Leydig cell steroidogenesis. IGFBP-4 had a lesser effect, while IGFBP-2 had no effect on IGF-I- plus hCG-induced testosterone formation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Increased follistatin (activin-binding protein) gene expression in rat anterior pituitary tissue after ovariectomy may be mediated by pituitary activin.

For lack of evidence to the contrary, it is now believed that the FSH-suppressing actions of follistatin are due to its ability to bind endogenous pituitary activin. Recent data have demonstrated a role for pituitary activin-B in mediating FSH hypersecretion after ovariectomy (OVX) and during the secondary FSH surge on estrus. Therefore, given that follistatin is produced within anterior pituitary tissue, and considering the potentially important function of follistatin to modulate activin bioactivity, we sought to gain insights into the regulation of follistatin gene expression in the anterior pituitary gland of adult female rats. At the termination of all in vivo investigations, rats were killed, trunk blood was collected for determination of serum LH and FSH levels by RIA, and pituitary tissue was collected, pooled (two or three glands per pool), and processed for determination of follistatin messenger RNA (mRNA) levels by a solution-hybridization RNase protection assay. In the first experiment, pituitary follistatin mRNA levels were significantly (P < 0.01) increased 3 weeks after OVX. Treatment of long-term ovariectomized rats with a Nal-Glu LHRH antagonist restored serum LH levels to precastration levels and suppressed serum FSH concentrations by 70%, but follistatin message levels were not altered. In contrast, treatment of castrated rats with recombinant human follistatin-288 selectively suppressed serum FSH levels (50%) and completely abolished OVX-induced increases in follistatin mRNA levels. Subsequent experiments revealed that OVX-induced increases in follistatin gene expression could be observed in pituitary tissue grafted underneath the kidney capsule of hypophysectomized rats. Furthermore, follistatin mRNA levels were significantly (P < 0.05) higher in pituitary glands taken from estrous rats during the secondary FSH surge (0200 h) than in glands obtained from rats on proestrous morning when serum FSH levels were basal. Because increased steady state follistatin mRNA levels in the latter two instances were associated with selective FSH hypersecretion, and such hypersecretion was previously shown to be dependent to a significant degree on pituitary activin, we next tested the hypothesis that increased pituitary follistatin gene expression is mediated by activin. Using cultures of dispersed pituitary cells, addition of recombinant human activin-A for 72 h increased follistatin mRNA levels 3-fold while enhancing only FSH secretion. Collectively, the present results demonstrate a coupling of follistatin gene expression in the anterior pituitary gland with changes in pituitary FSH secretion under conditions where LH secretion is unaltered. Viewed in the context of previous work, the data also suggest that changes in follistatin mRNA levels may be linked to activin signaling.

Activins↗

PubMed1993

Insulin-like growth factor binding protein-3 gene expression is restricted to involuting corpora lutea in rat ovaries.

There is evidence that insulin-like growth factor binding protein-3 (IGFBP-3) is a part of the intrinsic IGF system in rat CL. Here, we examined when during luteogenesis the IGFBP-3 gene is expressed. IGFBP-3 messenger RNA (mRNA) was characterized by Northern analysis and in situ hybridization techniques. Animals were selected at proestrus (P 1000 h and P 2000 h), estrus (E 0200 h and E 1000 h), diestrus I (DI 1100 h), and diestrus II (DII 1100 h), and in pregnancy (day 12, before luteolysis and day 21, during luteolysis). A single 2.6 kilobase IGFBP-3 transcript was identified at each stage of the estrous cycle; however, the amounts of message varied markedly, being most abundant at P 1000 h, least abundant at P 2000 h, E 0200 h, E 1000 h, and DI, and then more abundant at DII. Corroborating our earlier report, IGFBP-3 mRNA was limited solely to corpora lutea (CL). Newly-formed CL-I at E 0200 h and E 1000 h revealed no IGFBP-3 hybridization. This is the period of early luteinization when cells undergo hypertrophy and capillaries and lymphatics penetrate the granulosa lutein layer. At DI 1100 h, a few cells (12.2 +/- 3.4%) near the central cavity of the CL-I showed a positive hybridization signal for IGFBP-3; this period is commensurate with active luteinization when the vascular tissue develops a distinctly sinusoidal character and progesterone secretion by CL-I increases. At DII 1100 h, more cells in the central area were positive for IGFBP-3 (55.2 +/- 6.4%); this is the period of active luteolysis when P4 secretion has fallen to basal levels. At P 1000 h, a positive IGFBP-3 hybridization signal was detected in most CL-I cells (85.3 +/- 2.8%), and the signal was particularly intense in subtypes of endothelial cells lining venous sinusoids and/or lymphatics and some perivascular cells; this is the period when patches of pyknotic cells appear in the central area of CL-I. At P 2000 h, 45.9 +/- 1.6% of the CL-I cells showed a positive signal; however, the intensity of the signal was much weaker when compared to P 1000 h. During the next cycle, the CL-I become the CL of the second generation (CL-II), which show increased necrosis. Between estrus and diestrus II of the next cycle, a large number of the CL-II cells (approximately 75%) were positive for IGFBP-3 and the signal was very strong in some groups of cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Human plasma free activin and inhibin levels during the menstrual cycle.

The role of inhibin in gonadal function and reproduction has been confirmed by the measurement of plasma inhibin levels, but there has been no clinical data available on activin because of the lack of a good assay method. We measured plasma free activin levels during the normal menstrual cycle using a newly developed competitive protein binding assay with follistatin as the binding protein. Plasma inhibin levels were measured simultaneously using an alpha-subunit N-terminal fragment RIA with recombinant inhibin as the reference standard. Four normal women, aged 23-29 years, were investigated by obtaining plasma at 3-day intervals. Plasma inhibin levels showed some variation during the follicular phase, but a parallel rise in inhibin and progesterone was observed during the luteal phase. These findings confirmed those of previous studies. In contrast, plasma free activin levels did not show any substantial changes during the menstrual cycle. This study suggests that activin has no endocrine role in modulating the pituitary-gonadal axis during the normal menstrual cycle, while changes of inhibin reflect cyclic gonadal function and indicate an endocrine role for this protein in modulating gonadal activity.

Activins↗

PubMed1993

A 12-kilodalton N-glycosylated growth hormone-related peptide is present in human pituitary extracts.

This study was designed to investigate whether N-linked glycosylation could account for the presence of glycosylated GH forms (G-GH) in human pituitary extracts. The study was carried out in commercially available pituitary GH preparations (pitGH). Recombinant GHs obtained from eu- or prokaryotic cells were used as controls. Radioiodinated GHs were incubated in tubes containing Concanavalin-A (Con-A) attached to a Sepharose 4B matrix. Pituitary G-GH forms were selectively displaced from Con-A by adding N-acetyl-D-glucosamine or methyl-alpha-D-mannopyranoside and electrophoresed. Autoradiographies of these gels identified a 12-kilodalton (12K) band as the glycosylated form. Due to the fact that this peptide was partially immunoprecipitated with an anti-GH serum and the absence of detectable PRL in the pitGH extracts, it would indicate that such a glycopeptide was GH related. Endoglycosidase-F treatment of pitGH extracts induced a decrease in the mol wt of that 12K peptide, as indicated by the changes observed in its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This demonstrated that the sugar moieties were N-linked. When recombinant GHs were assayed by a similar method, no specific binding to Con-A was detected. NH2-terminal amino acid sequence analysis from the 12K band demonstrated that this band was composed by three peptides. Peptide 1 corresponds to the GH-N 102-119 sequence. Interestingly, peptide 2 exhibits GH-V 1-18 sequence, while peptide 3 seems to be a novel GH-related peptide. Taken together, these data suggest that the pituitary G-GH form found in human pituitary extracts is derived not from the "normal" GH-N gene, but, rather, from the GH-V gene or another unidentified gene. The potentially important pathophysiological implications of this finding need to be investigated.

Amino Acid Sequence↗

PubMed1993

Novel anti-inflammatory undecapeptides that contain anisolyated glutamic acid derivatives.

In various animal models of tissue injury, corticotropin-releasing factor (CRF) and related peptides inhibit swelling, edema and loss of protein from the vascular compartment. To search for smaller peptide segments of CRF that might retain anti-inflammatory activity, the authors tested peptides similar to the carboxy terminals of ovine (o) and human/rat (h/r) CRF. Also, because h/rCRF(35-39), -Arg-Lys-Leu-Met-Glu-, resembles Arg-Lys-Leu-Leu-Glu-, a sequence found in many intermediate filament proteins, analogous peptides were evaluated. Ovine CRF(21-41), -Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys- Leu-Asp-Ile-Ala-NH2, its carboxy terminal carboxyl derivative, oCRF(21-41)-OH, and the fragments, oCRF(26-41) and oCRF(30-41), were inactive when assayed at 5 mg/kg i.v. on edema induced in the pentobarbital-anesthetized rat's hindpaw after immersion in 58 degrees C water for 1 min. Crude peptides, D-Leu-Ala-Thr-D-Tyr-Arg-Lys-Leu-Leu-Glu-Ile-D-Leu-NH2 and D-Ala-His-Ser-D-Asn-Arg-Lys-Leu-Leu-Glu-Ile-D-Leu-NH2, were found to have activity in this bioassay. Characterization of the structures within the crude mixture revealed that substitution of the glutamic acid residue with an anisolyated glutamic acid (2-amino-5-(methoxyphenyl)-5-oxopentanoic acid) derivative, designated as (A*), increased the overall potency. The glutamyl-anisole derivative was a by-product of the temperature-dependent Friedel-Crafts acylation reaction that occurs during hydrogen fluoride cleavage of glutamyl-containing peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

Decrease in beta-subunit phosphotyrosine correlates with internalization and activation of the endosomal insulin receptor kinase.

In a previous study, we showed that the rat hepatic insulin receptor (IR) kinase of endosomes (ENs) was transiently activated to levels exceeding those of plasma membrane (PM) receptors following insulin injection. Phosphatase treatment of EN receptors abolished IR kinase activation implicating beta-subunit autophosphorylation as a mediator of the activation process (Khan, M. N., Baquiran, G., Brule, C., Burgess, J., Foster, B., Bergeron, J. J. M., and Posner, B. I. (1989) J. Biol. Chem. 264, 12931-12940). In the present study, the phosphotyrosine (PY) content of the IR beta-subunit in PM and ENs was estimated by two different methods. In one method, direct in vivo labeling with 32Pi followed by receptor immunoprecipitation was carried out. In the second method, immunoblotting with antibodies against the submembrane domain of the IR beta-subunit, encompassing residue 960 (alpha 960), and with antibodies against PY (alpha PY) was used to determine the content of PY/beta-subunit in PM and ENs following injection of insulin. By both methods, it was found that the PY content of PM IR was significantly greater than that of IR in ENs. With doses of 1.5 micrograms of insulin/100 g body weight (50% receptor occupancy) or 15 micrograms/100 g body weight (receptor saturation), the PY/beta-subunit of PM IR attained a level 2.0 to 2.5-fold of that observed for the IR of ENs. Surprisingly, the IR of ENs incorporated 3 to 5 times more PY/beta-subunit than those of PM consequent to autophosphorylation. Exogenous IR kinase activity (poly(Glu:Tyr)) in PM changed only slightly with insulin dose. In contrast, EN receptors exhibited a dose-dependent increase in kinase activity coincident with the decrease in PY/beta-subunit levels. A comparison of the proportion of receptor and kinase activity immunoprecipitated by alpha PY both before and after autophosphorylation indicated that ENs but not PM contained a small population of lightly phosphorylated but highly activated receptors. Since Thr12-Lys (IR kinase residues 1142-1153) efficiently inhibited IR autophosphorylation of both PM and EN receptors, Tris phosphorylation of beta-subunit regulatory tyrosines was unlikely. These results may be explicable by a dephosphorylation-dependent activation of IR kinase, as seen with the src family of tyrosine kinases.

Amino Acid Sequence↗

PubMed1992

Melanotropic activity of gamma MSH peptides in melanoma cells.

We studied the pigmentary activity of the peptides gamma 1, gamma 2 and gamma 3 melanocyte stimulating hormone (MSH), which differ in the structure of their C-termini, using hamster and mouse melanoma cell lines responsive to beta-MSH by increasing tyrosinase activity. Gamma 1-MSH alone or in combination with beta-MSH had no effect on either cell line. Gamma 2-MSH alone was biologically inactive but potentiated beta-MSH stimulation of tyrosinase activity. Gamma 3-MSH at high concentration (10 microM) induced tyrosinase activity and dendrite formation in the hamster melanoma line. When added together with beta-MSH, gamma 3-MSH partially inhibited the tyrosinase activity response to beta-MSH. Thus, gamma-MSH peptides have low intrinsic melanotropic activity in mammalian melanoma cells; the specific pigmentary responses appear to be affected by the structure of the C-terminal portion.

Amino Acid Sequence↗

PubMed1992

Localization of the heparin binding site of follistatin.

To define the heparin-binding site of follistatin, the reduced and S-carboxymethylated recombinant human follistatin containing 288 amino acids was digested by Staphylococcus aureus V8. The digested product was subjected to sulfate cellufine column chromatography and the adsorbed peptide fragments eluted with a stepwise gradient of sodium chloride. The recovered column fractions were further purified by reversed-phase high-performance liquid chromatography (HPLC) and the HPLC peaks subjected to amino-terminal sequence analysis. All of the sulfate cellufine-retarded peptide fragments gave the same N-terminal amino acid sequence, which started at residue-68 of human follistatin, suggested that those fragments starting from residue-68 contain the heparin binding site. The multiple fragments might represent the oxidized, non-glycosylated or glycosylated forms of follistatin(68-113) resulting from the V8 digestion. A synthetic peptide corresponding to the region having the amino acid sequence 72-86 of follistatin was able to bind both heparin and sulfate cellufine, as well as compete with recombinant follistatin for binding to heparin. These findings further define the location of the heparin and heparan sulfate-binding site of follistatin at the basic amino acid-rich region comprising the amino acid sequence Lys75-Lys-Cys-Arg-Met-Asn-Lys-Lys-Asn-Lys-Pro-Arg86.

Amino Acid Sequence↗

PubMed1992

Regulation of pulsatile gonadotropin secretion by estrogen, inhibin, and follistatin (activin-binding protein) in ovariectomized rats.

The following study was conducted to examine the effects of estrogen and polypeptides, given either alone or in combination, on pulsatile gonadotropin secretion. One week after ovariectomy, rats received s.c. injections of oil or various doses (0.5, 5, 20 micrograms) of estradiol benzoate (EB) followed 1 day later by i.v. administration of 60 micrograms purified porcine follistatin, 10 micrograms recombinant inhibin, or the appropriate vehicle. Four hours after injection of the nonsteroids, blood was collected at 10-min intervals for 2 h, and the effects on pulsatile hormone release were assessed. Administration of EB alone dose-dependently suppressed mean and trough (lowest point between two pulses) FSH levels and all parameters of pulsatile LH release. Both follistatin and inhibin at the doses employed suppressed mean FSH levels to an equivalent extent (40%). Follistatin, but not inhibin, suppressed FSH pulse amplitude, while neither polypeptide alone influenced FSH pulse frequency or any parameter of pulsatile LH release. The effects of follistatin and EB on mean FSH levels were additive at all EB doses, whereas the effects of inhibin and EB were additive only at the middle EB dose. Follistatin in combination with the lowest EB dose significantly suppressed mean LH levels. These studies are the first to demonstrate that combined treatment with estrogen and the nonsteroids follistatin and inhibin is more efficacious in suppressing FSH release than treatment with either agent alone, thereby indicating that both steroids and nonsteroids are probably important in the physiological regulation of FSH secretion in rats. The additive effects of these compounds on FSH secretion could form the basis for exploring novel contraceptive interventions.

Animals↗

PubMed1992

Gonadotropin and cAMP modulation of IGE binding protein production in ovarian granulosa cells.

Porcine granulosa cells (GC) produce insulin-like growth factor (IGF) binding protein (BP)-3 and IGFBP-2 in culture. A gonadotropin, follicle-stimulating hormone (FSH), dramatically inhibited GC production of these IGFBPs in control cultures and in cultures stimulated by insulin plus epidermal growth factor (EGF) or IGF-I plus EGF. Stimulators of adenylate cyclase (forskolin, cholera toxin) and a derivative of adenosine 3',5'-cyclic monophosphate (cAMP), 8-bromoadenosine 3',5'-cyclic monophosphate, inhibited IGFBP synthesis in a manner similar to FSH. In contrast, the antagonist of cAMP action, (R)-p-adenosine 3',5'-cyclic phosphorothioate [(R)-p-cAMPS], significantly stimulated production of IGFBP-3 and IGFBP-2 compared with controls. This stimulatory effect of (R)-p-cAMPS was counteracted by cotreatment with FSH in a dose-dependent manner. Finally, treatment of GC cultures with FSH plus 3-isobutyl-1-methylxanthine resulted in a significant reduction in cellular content of mRNA coding for IGFBP-3 with no change in IGFBP-2 mRNA. In summary, agents that elevate intracellular cAMP were found to mimic the effects of FSH on IGFBP production.

Animals↗

PubMed1992

Cyclic changes in insulin-like growth factor-binding protein-4 messenger ribonucleic acid in the rat ovary.

We have previously shown that the mRNA for insulin-like growth factor-binding protein-4 (IGFBP-4) is present in adult rat ovaries, being localized predominantly to granulosa cells of atretic follicles. Now we have considered the following questions. What class of atretic follicles expresses IGFBP-4 mRNA? How does IGFBP-4 mRNA expression change during the estrous cycle? In keeping with our earlier work, a strong hybridization signal for IGFBP-4 mRNA was present in subpopulations of follicles throughout the estrous cycle. In all cases, the hybridization signal was localized to granulosa cells. Among the various types of follicles, IGFBP-4 mRNA was present almost exclusively in atretic graafian (antral) follicles. Morphologically, the outer layer of granulosa cells was positive, while cells in the cumulus oophorous were negative. By Northern analysis and in situ hybridization, the levels of IGFBP-4 mRNA were found to change over the estrous cycle. At 1000 h on proestrus (before the LH/FSH surge), the hybridization signal was relatively weak, being restricted in some (but not all) atretic Graafian follicles. At 2000 h on proestrus, (after the LH/FSH surge), essentially all atretic Graafian follicles were strongly positive for the message. The pattern of hybridization was similar at 0200 h on estrus, but the signal was less intense. At 1000 h on estrus, the hybridization signal was variable, ranging from very strong to weak or undetectable in atretic follicles. At this stage, however, the highest levels of IGFBP-4 mRNA were measured by Northern analysis; interestingly, a strong signal became apparent in the stromal cells. On diestrous day 1, the message levels decreased, and the signal was restricted to some atretic follicles. On diestrous day 2, the hybridization signal was very weak. There was virtually no detectable IGFBP-4 mRNA in any healthy follicle. In summary, we found that IGFBP-4 mRNA is 1) not detected in healthy dominant follicles; 2) localized almost exclusively to atretic Graafian follicles, except on estrus when it also appears in stromal cells; 3) localized predominantly to the mural granulosa cells in atretic follicles; and 4) undergoes changes during the cycle, being most prominent around estrous morning. The possibility that IGFBP-4 plays a role in the cyclic destruction of cohort Graafian follicles at estrus, perhaps by mechanisms involving hormones, is discussed.

Animals↗

PubMed1992

Expression of messenger RNA for insulin-like growth factors and insulin-like growth factor binding proteins by experimental breast cancer and normal breast tissue in vivo.

We report the expression of insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) by breast cancer cells and normal breast tissue in vivo. N-nitrosomethyl-urea (NMU)-induced rat mammary tumors synthesize mRNAs for IGF-II and IGFBP-2, -3, and -4. In contrast, normal lactating breast contains only IGFBP-2 and IGF-II messages; IGFBP-3 and -4 mRNAs are absent in this tissue. IGF-I and IGFBP-1 mRNAs are not expressed in either NMU tumors or in normal breast. This is the first report of in vivo expression of IGFBPs and IGF-II messages in breast tumors.

Animals↗

PubMed1992

Localization of insulin-like growth factor-binding protein-5 messenger ribonucleic acid in rat ovaries during the estrous cycle.

To investigate the potential role of insulin-like growth factor-binding protein-5 (IGFBP-5) in ovarian physiology, we employed in situ hybridization and Northern analysis to localize IGFBP-5 mRNA in rat ovaries during the estrous cycle. By Northern analysis, the mRNA was abundant at all stages of the cycle. Two species of mRNAs were detected, with sizes of 6.0 and 1.8 kilobases, respectively. The relative amounts of the two transcripts changed throughout the cycle. By in situ hybridization, IGFBP-5 mRNA was expressed in only a few cell types: 1) granulosa cells of some atretic follicles, 2) some secondary interstitial cells, 3) some corpora lutea, and 4) the surface epithelium. The levels of message in both the granulosa and secondary interstitial cells changed over the cycle. At 1000 h on proestrus (before the LH/FSH surge), the message was expressed in only a few follicles. Interestingly, all were small atretic preantral (200-250 microns) follicles. At 2000 h on proestrus (after the LH/FSH surge), the IGFBP-5 mRNA was more abundant; now almost every atretic preantral follicle showed a strong hybridization signal. At 0200 and 1000 h on estrus, the mRNA appeared for the first time in granulosa cells of some atretic antral follicles and in secondary interstitial cells. Hence, virtually all atretic follicles, preantral and antral, now showed IGFBP-5 gene expression. In contrast to that on proestrus and estrus, the hybridization signal on diestrous days 1 and 2 was much less prominent and was found in only a few atretic preantral follicles. Throughout the cycle, IGFBP-5 mRNA was evident in some corpora lutea, but it was not particularly prominent. Abundant IGFBP-5 mRNA was evident in the surface epithelium, and no change was detected over the cycle. Dominant follicles were devoid of IGFBP-5 mRNA. In conclusion, this paper presents the first evidence that the IGFBP-5 gene is expressed in the adult rat ovary. The IGFBP gene is expressed in a cell-specific manner, e.g. in atretic granulosa, secondary interstitial cells, corpora lutea, and the surface epithelium, and the stage of the cycle significantly affected message levels, especially in atretic granulosa and secondary interstitial cells around estrous morning. These findings suggest that IGBP-5 may be an autocrine/paracrine regulator of ovarian physiology, particularly in relation to preantral follicle atresia.

Animals↗

PubMed1992

Sensitive colorimetric bioassays for insulin-like growth factor (IGF) stimulation of cell proliferation and glucose consumption: use in studies of IGF analogs.

We have established two in vitro bioassay systems for quantification of insulin-like growth factor (IGF) bioactivity. The first assay was used to quantitate mitogenic activity and the second was used to quantitate metabolic activity. Both assays use BALB/c 3T3 fibroblasts grown under serum-free conditions; detection of bioactivity in assays was performed colorimetrically and did not require the use of radioisotopes. The mitogenic bioassay, which requires 48 h for detection, quantitates changes in cell number and provides an index for determining the mitogenic activity of growth factors. Changes in cell number were measured by the enzymatic reduction of exogenously added MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide] to MTT-formazan by mitochondrial enzymes, which was directly correlated to cell number. The metabolic bioassay, which requires 22 h for detection, measures glucose consumption by detecting changes from the initial glucose concentration of conditioned medium after addition of various growth factors. When appropriate standards were established for these bioassays, they demonstrated a high level of reproducibility (coefficients of variation were 0.085-0.096 for the mitogenic bioassay and 0.120-0.191 for the metabolic bioassay). Both assays can be performed in 96-well microtiter plates, without the use of radioisotopes, or the limitations of conventional glucose, amino acid, or thymidine incorporation studies. In initial experiments for assay specificity, epidermal growth factor had no measurable effect in either assay. However, IGF-I, IGF-II, and insulin demonstrated effects on both metabolism and mitogenesis. In the case of the mitogenic bioassay, the maximum mitogenic activation by these growth factors was approximately 180% of control, and these factors demonstrated parallel sigmoidal dose-response curves, ranging from 0.02-2 ng/ml for IGF-I and from 2-200 ng/ml for both IGF-II and insulin. In the metabolic bioassay, in contrast to the mitogenic bioassay, insulin showed a dose-response curve whose shape was different from those of IGF-I and IGF-II. IGF-I and IGF-II stimulated glucose consumption in dose-dependent ranges of 0.02-3 ng/ml and 0.4-40 ng/ml, respectively. However, the dose-response effect of insulin was wider, ranging from 0.1-2000 ng/ml. When these assays were used to measure the bioactivity of IGF analogs, a des(1-3)-IGF-I, which has decreased affinity for IGF binding proteins, demonstrated activity equivalent to IGF-I, a [Leu27]IGF-II, which has markedly diminished affinity for the type 1 IGF receptor, exhibited approximately 0.07% of the potency of IGF-I and 1% of the potency of IGF-II.

3T3 Cells↗

PubMed1992

Expression of insulin-like growth factor-binding protein-2 and -3 messenger ribonucleic acid in the porcine ovary: localization and physiological changes.

Insulin-like growth factor-binding protein (IGFBP)-2 and -3 are the most prevalent IGFBPs in porcine follicular fluid, as determined on ligand blots, but little is known about the localization and regulation of their synthesis in vivo. This study was designed to investigate the localization and cyclic regulation of the mRNA for these two IGFBPs in the porcine ovary, RNA was extracted from whole ovaries morphologically classified as immature, preovulatory, and luteal. Northern hybridization analysis of this RNA showed no significant difference in the expression of IGFBP-2 mRNA in these ovaries (OD for preovulatory, luteal, and immature ovaries, 0.076 +/- 0.01, 0.071 +/- 0.01, and 0.10 +/- 0.008/micrograms RNA, respectively). IGFBP-3 mRNA was not different in immature and preovulatory ovaries, but was 10-fold greater (P less than 0.025) in luteal ovaries. Northern analysis of RNA extracted from ovaries also showed no significant change in IGFBP-2 mRNA on days (d) 11, 16, and 21 of the estrous cycle. IGFBP-3 mRNA tended to decrease between d11-16 with the onset of luteal regression and was significantly decreased in d21 preovulatory ovaries to 22% of the values in d11 ovaries. Granulosa, thecal, and luteal cells were also analyzed for IGFBP mRNA. IGFBP-2 mRNA was most abundant in granulosa cells, lower in thecal cells, and lowest in luteal cells. No IGFBP-3 mRNA could be detected in granulosa cells, and luteal cells expressed 15- to 63-fold greater levels than thecal cells. These results show that IGFBP-2 and -3 mRNAs are expressed in specific ovarian cell types and that their expression appears to be independently regulated during the reproductive cycle. This provides further evidence for the importance of these proteins as paracrine/autocrine regulators of ovarian function.

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