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L C Giudice

Publications and source records attributed to L C Giudice.

At least 91 records · Page 5Linked to original sources
PubMed1994

Identification of insulin-like growth factor-binding protein-3 (IGFBP-3) fragments and IGFBP-5 proteolytic activity in human seminal plasma: a comparison of normal and vasectomized patients.

Previous studies have demonstrated that insulin-like growth factor (IGF) peptides, IGF-binding proteins (IGFBPs), and IGFBP-3 proteolytic activity, are present in human seminal plasma (SP). In this study, we have further characterized the IGFBPs in SP using immunoprecipitation and Western ligand blotting, Western immunoblotting, affinity cross-linking and immunoprecipitation, and RIA of IGFBP-3 using two different assays and have identified additional proteolytic activities for IGFBP-4 and IGFBP-5 in SP. Immunoprecipitation with antibodies to IGFBP-2, IGFBP-3, and IGFBP-4, before and after affinity cross-linking, demonstrated that intact IGFBP-2 and IGFBP-4 are present in SP, but intact IGFBP-3 is absent. Low mol wt fragments of IGFBP-3, which did not bind to IGF-I or IGF-II on Western ligand blot and did not cross-link to IGF-II, were demonstrated on Western immunoblot and were measurable by two different RIAs. Proteolytic activities for IGFBP-4 and IGFBP-5 were demonstrated in SP by incubation with the respective iodinated IGFBPs. On comparing the proteolytic activity for IGFBP-4 by purified prostate-specific antigen (PSA; a known IGFBP-3 protease in SP) or by SP with measured equivalent concentrations of PSA, the dose response and fragment patterns were identical. With IGFBP-5, however, proteolysis by purified PSA was different from that by SP with measured equivalent concentrations of PSA: 1) proteolysis by pure PSA was less efficient than matched concentrations of SP; 2) the pattern of fragments after proteolysis by pure PSA was different from that after proteolysis by matched concentrations of SP; and 3) proteolysis by purified PSA was significantly inhibited by phenylmethylsulfonylfluoride and aprotinin, but proteolysis by SP was not. We conclude that human SP contains intact IGFBP-2 and IGFBP-4, but has only IGFBP-3 fragments with low affinity for IGF peptides; that PSA is able to proteolyze IGFBP-4 and IGFBP-5 (as well as IGFBP-3); and that an additional IGFBP-5 protease is probably present in SP. There was no significant difference in any of these findings in SP from normal volunteers, vasectomized patients, or patients with idiopathic azoospermia. The roles of IGFBPs and IGFBP proteases in the male reproductive system and male infertility remain to be further elucidated.

Blotting, Western↗
PubMed1994

Insulin-like growth factor system gene expression in human endometrium during the menstrual cycle.

To study the cellular patterns of gene expression of the insulin-like growth factor (IGF) system in human endometrium during the menstrual cycle, we used in situ hybridization histochemistry to localize messenger ribonucleic acids (mRNAs) encoding IGF-I and -II, their receptors, and their binding proteins (IGFBPs) in fresh-frozen endometrial tissue obtained from cycling women. IGF-I and IGF-II mRNAs are both expressed diffusely throughout endometrial stroma and are not detected in endometrial epithelium. Endometrial IGF-I mRNA is significantly more abundant during the proliferative than the secretory phase of the menstrual cycle, whereas the reverse is true for IGF-II. Type I and type II IGF receptor mRNAs are both present in endometrial stroma, but are relatively more abundant in endometrial epithelium, and neither shows distinctive cyclic changes. IGFBP-2, -4, -5, and -6 mRNAs demonstrate a diffuse stromal pattern of expression, whereas IGFBP-1 and -3 are more focally concentrated in selected subpopulations of endometrial cells. IGFBP-1 mRNA is not detected in proliferative endometrium and demonstrates a very heterogeneous pattern of expression in secretory endometrium, where it is intensely abundant in a patchy distribution of stromal and epithelial cells. IGFBP-3 mRNA is primarily concentrated in endometrial capillaries and is increased in the secretory phase, largely due to the intense vascularization of endometrial glands during this phase. IGFBP-5 mRNA is more abundant in the proliferative phase, but all other IGFBP mRNAs are relatively increased in the secretory phase of the menstrual cycle. These findings support the view that the IGF system plays a fundamental role in endometrial biology, acting via autocrine and/or paracrine mechanisms, with IGF-I and IGFBP-5 being dominant in the proliferative phase, and IGF-II and the other IGFBPs predominant in the secretory phase of the menstrual cycle.

Carrier Proteins↗
PubMed1993

Insulin-like growth factor regulation of human endometrial stromal cell function: coordinate effects on insulin-like growth factor binding protein-1, cell proliferation and prolactin secretion.

The insulin-like growth factor (IGF) autocrine/paracrine system is believed to be involved in endometrial differentiation, but there is limited information on the specific cellular functions regulated by IGFs in uterine tissues and their regulation of IGF-binding proteins (IGFBPs). We have investigated the regulation by insulin, IGF-I, and IGF-II, of IGFBP secretion in human endometrial stromal cells decidualized in vitro, and examined the interrelationship between the induced changes in IGFBP levels and the biological responses of stromal cells to IGFs. IGFBPs in conditioned media were analyzed by Western ligand blotting, and IGFBP-1 was quantified by an immunoenzymometric assay (IEMA). In the absence of peptides, decidualized stromal cells secreted 25.5 +/- 3.2 micrograms/day per 10(6) cells of IGFBP-1. Insulin caused a dose-dependent reduction of IGFBP-1 secretion (half-maximal inhibition at < 1 ng/ml) to a maximum of 1% of control values. Northern analysis using a specific cDNA probe showed the expression in decidualized stromal cells of a single 1.5 kb transcript for IGFBP-1, which was absent in insulin-treated cells. The effects of IGF-I and IGF-II on IGFBP-1 secretion were biphasic, with initial stimulation (200-250%) that peaked at 1 and 10 ng/ml, respectively, followed by inhibition at higher concentrations (half maximal inhibition at 3 ng/ml and 30 ng/ml, respectively). The decrease in IGFBP-1 levels in decidualized stromal cultures was associated with the induction of mitogenesis by IGF-I and IGF-II, while IGF effects on prolactin secretion paralleled those of IGFBP-1 secretion, with stimulation (243-324%) in the low concentration range followed by inhibition at higher concentrations. These data indicate that endometrial stromal cell IGFBP-1 is regulated by insulin, at concentrations that are compatible with insulin acting via its own receptor, while the effects of IGF-I and IGF-II on IGFBP-1 secretion, are suggestive of their acting probably through the type I IGF receptor. The present study describes distinct effects of the IGFs on stromal cell IGFBPs, that correlate with changes in the proliferative and secretory responses of decidualized stromal cells to the IGFs. Our findings suggest that complex IGF-IGFBP interactions may participate in the regulation of endometrial cell function, and support a role for IGF-II in stromal cell mitogenesis during decidualization, and as a local regulator of decidual cell function during the late secretory phase and early pregnancy.

Actins↗
PubMed1993

Expression and localization of matrilysin, a matrix metalloproteinase, in human endometrium during the reproductive cycle.

OBJECTIVE: We studied the expression of a matrix metalloproteinase, matrilysin, in the human endometrium to determine whether metalloproteinase genes are expressed during the reproductive cycle. Matrix metalloproteinases are a tightly regulated family of enzymes that degrade components of the extracellular matrix and basement membrane; they play important roles in growth and development and in invasion and metastasis of tumors and thus are likely enzymes participating in the dynamic structural changes occurring in endometrium during the reproductive cycle. STUDY DESIGN: In situ and Northern nucleic acid hybridization and immunohistochemistry were used to detect and localize matrilysin ribonucleic acid and protein in normal endometrial tissue. RESULTS: Matrilysin protein and matrilysin messenger ribonucleic acid are abundant in proliferative, late secretory, and menstrual endometrial epithelium but are not detected in early or mid secretory endometrium. CONCLUSION: The expression of the matrilysin gene is regulated in endometrium during the reproductive cycle, implying an important role for matrilysin in endometrial physiologic characteristics.

Blotting, Northern↗
PubMed1993

Insulin-like growth factor (IGF), IGF binding protein (IGFBP), and IGF receptor gene expression and IGFBP synthesis in human uterine leiomyomata.

Oestradiol is important in the growth of uterine leiomyomata and may act primarily or secondarily through mediators such as growth factors, including the insulin-like growth factors (IGF-I and IGF-II), mitogenic peptides. IGF binding proteins (IGFBPs) modulate IGF actions at their target cells. The objective of this study was to examine the possible steroid dependence of IGF, IGFBP and IGF receptor gene expression and IGFBP synthesis in uterine leiomyomata, using tissues from women cycling normally and made hypo-oestrogenic by a gonadtrophin-releasing hormone agonist (GnRHa). Using a solution hybridization ribonuclease protection assay, anti-sense RNA probes for IGF-I, IGF-II and beta-actin (control) were hybridized with total RNA isolated from leiomyomata exposed in vivo to a range of serum oestradiol (< 40-240 pg/ml) and progesterone (0-10 ng/ml) concentrations. IGF-I gene expression was most abundant in leiomyomata obtained during the late proliferative phase of the cycle and was undetectable in leiomyomata from hypo-oestrogenic patients. IGF-II gene expression was not dependent on endogenous steroid concentrations or cycle stage. IGFBP gene expression was investigated by Northern blotting. The order of relative abundance of IGFBP mRNAs was IGFBP-4 >>> IGFBP-3 >> IGFBP-5 > IGFBP-2 and was not dependent on the in-vivo oestrogen status. Type I and type II IGF receptor gene expression was investigated by polymerase chain reaction using gene-specific primers. Type I and type II IGF receptor mRNAs were detected in leiomyomata and were not dependent on cycle stage or in-vivo oestrogen status. Explant cultures of leiomyomata and myometrium synthesized IGFBP-3 (mol. wt = 38-43 kDa), IGFBP-4, and binding proteins of mol. wt = 34 and 31 kDa. Identification of IGFBP-2 was inconclusive, and IGFBP-1 was not detected. These data support the hypothesis that IGF-I, but not IGF-II, may be a mediator of oestradiol action in the growth of uterine leiomyomata, and that IGFBPs may further modulate, by an autocrine or paracrine mechanism, IGF-I action in this tissue.

Base Sequence↗
PubMed1993

Comparison of functional response of rat, macaque, and human ovarian cells in hormonally defined medium.

A serum-free medium has been developed which supports in vitro function by ovarian cells derived from rat, monkey, and human tissue. This granulosa cell medium (GCM) consists of Dulbecco's Modified Eagle's Medium: Ham's F-12 medium (1:1, v:v) supplemented with insulin, transferrin, aprotinin, selenium, fibronectin, penicillin, and streptomycin. Ovarian cells from three species were compared: rat, macaque, and human. Four types of ovarian cultures were examined: 1) purified granulosa cell cultures and 2) co-cultures containing granulosa-theca-stroma cells, 3) luteal cells, and 4) granulosa-lutein (harvested from in vitro fertilization cultures) cells. Each cell type was characterized by its response to FSH or hCG when cultured in GCM. Morphologic responses to FSH were observed in GCM in rat granulosa and granulosa-theca-stroma cell cultures, macaque and human granulosa-lutein cells, and human granulosa-theca-stroma cell cultures. The FSH-stimulated cells retracted and became rounded, leaving long intercellular connections. Luteal cells did not retract in response to FSH, and the cells remained firmly attached to the fibronectin matrix. Steroidogenic regulation of the GCM-cultured ovarian cells was monitored following stimulation of the cultures with FSH. The ability of the cells to aromatize testosterone was first examined. Rat granulosa cell cultures and granulosa-theca-stroma cell cultures, macaque granulosa-lutein cell cultures, and human granulosa-theca-stroma cell cultures all accumulated estradiol when given FSH and testosterone for 48 h. Moreover, these cell types as well as human luteal cells were able to metabolize 25-hydroxy [1,2-3H]cholesterol to various steroid metabolites. The data indicate that GCM supports normal granulosa cell morphologic response to FSH.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Insulin-like growth factor binding proteins in sera of pregnant nonhuman primates.

Insulin-like growth factors (IGFs) are mitogenic peptides that are important for fetal and maternal tissue growth during pregnancy. They circulate complexed primarily with a serum binding protein, IGFBP-3, which regulates the availability of the IGFs to their target tissues. We previously reported that in pregnant women, serum IGFBP-3 levels, assessed by Western ligand blotting, decline markedly beginning at 6 weeks gestation due to a circulating protease that cleaves IGFBP-3 into a 29-kilodalton (kDa) protein and lower mol wt (M(r)) fragments. In the current study, we compared IGFBP profiles, IGFBP-3 and IGFBP-1 levels, and IGFBP protease activities in sera from pregnant and nonpregnant women, baboons, and rhesus monkeys, using Western ligand blotting, IGFBP-specific immunoassays, IGFBP-3 protease assay, and zymographic gel electrophoresis. Serum IGFBP profiles in nonpregnant human and nonhuman primates were similar and were not cycle-dependent. IGFBP-3 (37-43 kDa), IGFBP-2 (31 kDa), and IGFBP-1 (28 kDa) were identified in all three species using IGFBP-specific human antisera. A 24-kDa IGFBP was also present and is believed to be IGFBP-4. Serum IGFBP-1 levels increased throughout gestation in human and nonhuman primates. Serum IGFBP-2 and putative IGFBP-4 were barely detectable in all three species from midgestation to term, but increased several days postpartum. In contrast, serum IGFBP-3 profiles differed markedly between species during gestation. Rather than the decrease seen in human pregnancy serum, there was an increase in circulating IGFBP-3 levels in nonhuman primates. Furthermore, for both baboon and rhesus monkey, the M(r) of serum IGFBP-3 was about 2 kDa greater in pregnant than in nonpregnant animals, and deglycosylation studies suggested that the higher M(r) forms may be alternatively glycosylated or may have a unique primary structure. As in nonpregnant women, serum IGFBP-3 protease activity was absent in nonpregnant and pregnant baboons. However, rhesus monkey serum contained a calcium-dependent protease that cleaved recombinant human IGFBP-3 into unique fragments, compared to the human pregnancy enzyme. Unlike human pregnancy serum, which proteolyzes IGFBP-3, in human nonpregnancy serum, rhesus serum incubated under similar conditions did not result in proteolysis of rhesus IGFBP-3, suggesting that the IGFBP-3 protease in human pregnancy serum is not present in the circulation of the rhesus monkey. To assess proteolytic activity in these sera, zymographic polyacrylamide gel analysis, using gelatin as a substrate, was performed. A minor band of proteolytic activity (72 kDa) was observed in all three species throughout gestation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Regulation of insulin-like growth factor binding protein production by human luteinizing granulosa cells cultured in defined medium.

The production of insulin-like growth factor (IGF) binding proteins (IGFBPs) by human luteinizing granulosa cells obtained at oocyte harvest for in vitro fertilization was studied in defined medium. Three species of IGFBP were noted on Western ligand blotting of conditioned medium. A 31-33 kilodalton (kDa) doublet identified as IGFBP-2 by immunoprecipitation was consistently seen and was shown by metabolic labeling to be synthesized de novo. IGFBP-2 production was inhibited by hCG (100 ng/mL) to 32 +/- 8% of levels in control cultures; a similar inhibition was seen with dibutyryl cAMP and forskolin, but not with FSH. IGFBP-2 production was also inhibited by IGF-II and [Leu27]IGF-II (ED50 for both 3 ng/mL) but not by IGF-I at up to 30 ng/mL. In addition to IGFBP-2, a 35-45 kDa IGFBP identified by immunoprecipitation as IGFBP-3 was variably present on ligand blots of control conditioned medium and was consistently found in medium from cells treated with IGF-I or IGF-II (ED50 3 ng/mL) but not with insulin at 2 mg/mL. No de novo synthesis of IGFBP-3 was noted, suggesting that IGFBP-3 may be released from the cell surface by ligand. A 24-kDa IGFBP consistent with IGFBP-4 was also variably noted in basal cultures. Since IGFBPs are believed to sequester IGF peptides and inhibit their action on the granulosa, control of IGFBP production may be an important step in regulating gonadotropin and IGF action in the human granulosa cell.

Autoradiography↗
PubMed1993

Differential expression of messenger ribonucleic acids encoding insulin-like growth factors and their receptors in human uterine endometrium and decidua.

During the menstrual cycle, the endometrium undergoes characteristic changes in response to circulating sex steroids. Intense mitotic activity of glands and stroma occurs in the proliferative (estradiol-dominant) phase, and glandular secretion and stromal differentiation in the secretory (progesterone-dominant) phase. The insulin-like growth factors (IGF-I and IGF-II) promote cellular growth and differentiation and have been proposed to participate in these cyclic endometrial events, acting as mediators of steroid hormones. The objective of this study was to determine whether the messenger RNAs (mRNAs) encoding the IGF peptides and the type I and type II IGF receptors are differentially expressed in human endometrium during the menstrual cycle and in early pregnancy. A solution hybridization ribonuclease protection assay, using 32P-labeled riboprobes for IGF-I, IGF-II, and beta-actin (control), revealed IGF-I gene expression primarily in proliferative and early secretory endometrium and abundant IGF-II gene expression in mid-late secretory endometrium and early pregnancy decidua. Northern analysis, using IGF-I and IGF-II complementary DNA probes, revealed multiple IGF-I mRNAs [2-7.6 kilobase (kb)], expressed primarily in proliferative and early secretory endometrium, and IGF-II mRNAs (1.4-6.0 kb), expressed primarily in secretory endometrium and in early pregnancy decidua. The 7.6-kb IGF-I mRNA and the 6.0-kb IGF-II mRNA were most abundantly expressed. IGF-IEa and IGF-IEb mRNA splicing variants were present in a ratio of about 9:1, respectively. Type I and type II IGF receptor gene expression in endometrium was investigated using specific riboprobes and the ribonuclease protection assay. Messenger RNAs encoding both receptors were more abundantly expressed in the secretory phase and during early pregnancy, compared to the proliferative phase. These results show that mRNAs encoding the IGF peptides and their receptors are differentially expressed in human endometrium, depending on the steroid hormone milieu. The preferential expression of IGF-I mRNA in the proliferative phase supports the hypothesis that IGF-I is an estromedin in human endometrium. The expression of endometrial IGF-II mRNA in the mid to late secretory phase and in early pregnancy supports a role for IGF-II in differentiative functions of the endometrium, perhaps including endometrial tissue shedding in the menstrual cycle or remodeling during early pregnancy.

Base Sequence↗
PubMed1993

The potential roles of intraovarian peptides in normal and abnormal mechanisms of reproductive physiology.

Normal and abnormal follicular growth and steroidogenesis depend on gonadotropins as well as intraovarian peptide and polypeptide growth factors, which may mediate or potentiate gonadotropin action. Epidermal growth factor is mitogenic to ovarian granulosa and is a potent inhibitor of granulosa aromatase. It may be involved in the apparent arrest of follicular development commonly seen in women with polycystic ovarian syndrome as well as in the blunted response to gonadotropins seen in this syndrome. Insulin-like growth factors are also mitogenic to ovarian granulosa, but in contrast to epidermal growth factor, insulin-like growth factor-I, both alone and in synergy with gonadotropins, is a potent stimulus of aromatase and granulosa estradiol production. Insulin-like growth factor binding proteins-2 and -4, known inhibitors of insulin-like growth factor action, are higher in follicular fluid from atretic and polycystic ovarian syndrome follicles compared with estrogenic follicles and may be inhibitors of gonadotropin action in follicle selection and in polycystic ovarian syndrome. Cytokines including interleukins, tumor necrosis factor-alpha and interferon-gamma also appear to play a role in modulating ovarian steroidogenesis. Activins, inhibins, and follistatin (activin-binding protein) also affect follicular development and steroidogenesis and may play a role in dominant follicle selection and follicular atresia.

Activins↗
PubMed1992

Measurement and characterization of insulin-like growth factor binding protein-3 in human biological fluids: discrepancies between radioimmunoassay and ligand blotting.

The inability to detect insulin-like growth factor binding protein-3 (IGFBP-3) in some circumstances by Western ligand blot analysis has emphasized the need to characterize IGFBPs by both ligand binding and immunological techniques. In this study, we have: 1) characterized and quantified IGFBP-3 in nonpregnancy, pregnancy, and fetal cord serum, follicular, peritoneal, and amniotic fluid, seminal plasma, cerebrospinal fluid (CSF), and urine; 2) established a new IGFBP-3 RIA that detects both intact and fragments of IGFBP-3; 3) identified both intact and fragments of IGFBP-3 by Western immunoblot techniques; and 4) addressed the discordance between Western ligand blot analysis and RIA by assessing fluids for IGFBP proteolytic activity. All fluids examined, except pregnancy serum, CSF, and amniotic fluid, displayed a 44-34-kilodalton (kDa) IGFBP-3 doublet by Western ligand blot analysis. Western immunoblot analysis using specific IGFBP-3 antiserum showed a 44-34-kDa IGFBP-3 doublet and a 28-kDa fragment in nonpregnancy serum, fetal cord serum, follicular fluid, and peritoneal fluid. The immunoreactive 42-38-kDa doublet was faint in urine and seminal plasma. IGFBPs in CSF did not cross-react with IGFBP-3 antiserum. Pregnancy serum and amniotic fluid contained only the 28-kDa fragment when compared against equal volumes of nonpregnancy serum. With the development of an IGFBP-3 RIA, IGFBP-3 could be accurately measured; urine, CSF, and seminal plasma contained the lowest levels of IGFBP-3 at 27 +/- 3 ng/ml (mean +/- SEM), 110 +/- 26 ng/ml, and 209 +/- 56 ng/ml, respectively. In increasing concentration: fetal cord serum contained 753 +/- 101 ng/ml; peritoneal fluid, 1124 +/- 130 ng/ml; follicular fluid, 2356 +/- 211 ng/ml; nonpregnancy serum, 3556 +/- 508 ng/ml; pregnancy serum, 3718 +/- 842 ng/ml; and amniotic fluid, 5150 +/- 688 ng/ml. The measurable concentrations of IGFBP-3 in CSF and the high concentrations measured in pregnancy serum and amniotic fluid conflicted with Western blot analysis. Thus, fluids were assessed for IGFBP proteolytic activity by incubation with a source of IGFBP-3, either nonpregnancy serum or purified IGFBP-3. All fluids displayed some proteolytic activity with either assay. Fluids with little protease activity (nonpregnancy serum, follicular fluid, and urine) showed a close relationship between immunoassayable IGFBP-3 by RIA and IGFBP-3 band intensity by Western ligand blot. Fluids with high proteolytic activity (pregnancy serum, CSF, seminal plasma, peritoneal fluid, and amniotic fluid) gave discrepant IGFBP-3 values between RIA and Western ligand blot.(ABSTRACT TRUNCATED AT 400 WORDS)

Amniotic Fluid↗
PubMed1992

Follicular fluid insulin-like growth factor binding protein profiles in polycystic ovary syndrome.

Insulin-like growth factor binding proteins (IGFBPs) are believed to modulate the actions of IGF-I and IGF-II at the cellular level. We have examined, by Western ligand blot analysis, the IGFBP profiles in follicular fluid (FF) from patients with polycystic ovarian syndrome (a disorder of ovarian folliculogenesis), compared to FF from atretic and developing (estrogenic) follicles from normally cycling women. IGFBPs with apparent mol wts (Mr) of 41.5, 38.5, 31, 28, and 24kDa were detected in PCOS FF. The profile of IGFBPs in PCOS FF was indistinguishable from that seen in atretic follicles in cycling women. However, higher levels of the 31, 28, and 24kDa IGFBPs were observed in PCOS FF, compared to healthy, estrogenic follicles. Using specific antisera, the 41.5 and 38.5kDa IGFBPs were identified as IGFBP-3, and the 31kDa IGFBP as IGFBP-2. IGFBP-1, however, was not appreciably detectable in PCOS FF, by Western ligand blotting. Endoglycosidase F treatment of FF decreased the Mr of the 28kDa IGFBP to 24kDa, and neither the 28kDa nor the 24kDa IGFBP was immunoprecipitated by antibodies to IGFBP-1, -2, or -3. Elevated levels of 28kDa and 24kDa IGFBPs in PCOS FF may represent glycosylated and core forms of IGFBP-4. The data presented herein show that in PCOS FF, as well as in FF from atretic follicles from normally cycling women, IGFBP-2 and 28 and 24kDa IGFBPs are present in greater amounts, compared to levels in FF from healthy, developing, estrogenic follicles. One or more of the IGFBP species elevated in atretic and PCOS follicles may bind IGFs in FF, thereby inhibiting IGF action on the granulosa during normal folliculogenesis.

Blotting, Western↗
PubMed1992

Insulin-like growth factor binding protein profiles in human ovarian follicular fluid correlate with follicular functional status.

The insulin-like growth factors (IGF), IGF-I and IGF-II, influence ovarian follicular development. The IGF binding proteins (IGFBPs) comprise at least six distinct species which may modulate IGF action. We examined IGFBP profiles in follicular fluid (FF) from developing human ovarian follicles with aromatase activity as well as from androgen-dominant, atretic follicles. Western ligand blot analysis of FF from both atretic and active follicles revealed the presence of IGFBP-3 and IGFBP-2, confirmed by immunoprecipitation with specific antiserum, as well as 28 kilodalton (kDa) and 24 kDa IGFBPs. In FF specimens obtained during the follicular phase, IGFBP-2 and the 28 kDa and 24 kDa species were present in 3-, 6-, and 19-fold higher amounts in atretic compared to healthy developing follicles, respectively, and were present in greater amounts in atretic FF than in serum. In atretic FF obtained during the luteal phase, lower levels of IGFBP-2 and the 28 and 24 kDa IGFBPs were seen than in atretic follicles in the follicular phase. IGFBP-3 levels were comparable in all types of follicles and in serum. The 28 kDa IGFBP is glycosylated, and its level varied in parallel with the 24 kDa IGFBP, suggesting that these are variants of the recently identified IGFBP-4. IGFBP-1 was not detectable by immunoprecipitation and ligand blotting of FF obtained during the follicular phase. These data suggest that one or more IGFBPs present in higher levels in FF from atretic compared to healthy, developing follicles may play an important role in folliculogenesis and follicular atresia.

Adult↗
PubMed1992

Prostate-specific antigen (PSA) is an insulin-like growth factor binding protein-3 protease found in seminal plasma.

Insulin-like growth factor binding protein-3 (IGFBP-3), the major serum carrier protein for the IGFs, is absent from Western ligand blots of seminal plasma, but is detectable by RIA. IGFBP-3 protease activity has recently been described in pregnancy serum. We investigated the possibility that seminal plasma contains an IGFBP-3 protease, by incubating seminal plasma with 125I-labeled human IGFBP-3. Seminal plasma was found to have potent IGFBP-3 protease activity with a cleavage pattern different from that of pregnancy serum. Prostate-specific antigen (PSA) is a serine protease found in semen. Autoradiographs measuring IGFBP-3 protease activity demonstrated that purified PSA cleaved IGFBP-3, yielding a cleavage pattern identical to that of seminal plasma. IGFBP-2 and -4 in seminal plasma were not degraded by PSA. Cleavage of IGFBP-3 by PSA resulted in a marked reduction in the binding affinity of the fragments to IGF-I, but not IGF-II. We speculate that PSA may serve to modulate IGF function within the reproductive system or in prostate cancer by altering IGF-IGFBP-3 interactions.

Autoradiography↗
PubMed1992

Steroid and peptide regulation of insulin-like growth factor-binding proteins secreted by human endometrial stromal cells is dependent on stromal differentiation.

Endometrial stromal cells undergo decidual transformation, in response to epidermal growth factor (EGF) and progesterone. Since insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) are believed to be involved in endometrial differentiation, and insulin regulates IGFBP production in a variety of cells, we have investigated the modulatory roles of EGF, progesterone, and insulin on IGFBP secretion by long term cultures of human endometrial stromal cells. Without insulin, the principal IGFBP secreted into conditioned medium, detected by Western ligand blotting, was a 28-kilodalton (kDa) IGFBP, identified by immunoprecipitation as IGFBP-1. This was observed only when the stromal cells were decidualized. With increasing insulin, IGFBP-1 decreased to undetectable levels. Concomitantly, IGFBP-2 increased, as did a 24-kDa IGFBP (believed to be IGFBP-4) and a 28-kDa IGFBP, shown to be a glycoprotein by endoglycosidase sensitivity (and believed to be glycosylated IGFBP-4). In the nondecidualized state, insulin increased the secretion of IGFBP-3, IGFBP-2, and the 24-kDa IGFBP, which were slightly inhibited by EGF and relatively unaffected by progesterone alone. In the absence of insulin, progesterone weakly stimulated IGFBP-1 secretion, which increased markedly when the cells were decidualized by combined treatment with EGF and progesterone. These data show that IGFBP-3, IGFBP-2, IGFBP-1, and presumably IGFBP-4 and its glycosylated form are differentially regulated by peptide and steroid hormones in endometrial stromal cells and that their regulation is a function of stromal differentiation.

Carrier Proteins↗
PubMed1992

Identification of insulin-like growth factor binding proteins in human oviduct.

OBJECTIVE: To determine if human oviduct expresses messenger ribonucleic acids (mRNAs) encoding insulin-like growth factor binding proteins (IGFBPs), if oviductal epithelium secretes IGFBPs into conditioned medium (CM), and if IGFBP secretion is regulated by steroid hormones. DESIGN: Northern blots of RNA, isolated from late proliferative phase human fimbria and oviductal isthmus, were probed with complementary deoxyribonucleic acids encoding IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-4. In addition, oviductal ampullary epithelial cells were cultured with and without estrogen and/or progesterone (P), and IGFBPs were examined in CM by Western ligand blot analysis and identified using specific antisera. SETTING: Tissue was obtained from hysterectomy specimens at Stanford University Hospital, a private teaching institution. PATIENTS, PARTICIPANTS: Patients undergoing hysterectomy for benign disease. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Transcripts of IGFBP mRNA and IGFBPs secreted into CM were detected by autoradiography of Northern and Western ligand blots, respectively. RESULTS: Messenger RNA transcripts encoding IGFBP-2, -3, and -4 were detected, whereas IGFBP-1 mRNA was barely detectable in oviductal tissue. In CM, IGFBP-2 and IGFBP-3 were detected, as was a unique 24-kd IGFBP, although IGFBP-1 was not observed. Estrogen and/or P did not regulate the secretion of these IGFBPs by cultured oviductal epithelium. CONCLUSIONS: Human oviduct expresses mRNAs encoding IGFBP-2, IGFBP-3, and IGFBP-4, and in vitro oviductal epithelium secretes IGFBP-2, IGFBP-3, and a unique binding protein of 24 kd, which may be the recently identified IGFBP-4.

Animals↗