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Midkine (MK), a heparin-binding growth factor, is associated with the poor prognosis of the pediatric tumor, neuroblastoma. MK would be a druggable target as many studies showed inhibition of its function in various cancers suppressed tumor developments. To establish the therapy targeting MK, identification of its binding partners, and elucidation of its intracellular signaling are needed. It was reported that exogenous MK induced phosphorylation of ribosomal protein S6 (RPS6) downstream of mTOR signaling. Using RPS6 phosphorylation as a marker of MK response, we searched for MK reactive cell lines. We found that MK cell lines expressing less MK tended to respond better to MK. Next, using an MK reactive neuroblastoma cell line, MK-knocked down SH-SY5Y cells, we employed a proximity-dependent biotin identification method, which was invented to evaluate protein-protein interactions by biotinylation. We confirmed that secreted MK fused to the biotin ligase BioID2 (MK-BioID2) was able to biotinylate proteins from the cells. Biotinylated proteins were identified by liquid chromatography-mass spectrometry analyses. Twenty five proteins were found to be overlapped after three independent experiments, among which insulin-like growth binding protein 2 (IGFBP2) was further analyzed. IGFBP2 was indeed detected with immunoblotting after streptavidin pull down of MK-BioID2 labeled cell extract of MK-knocked down SH-SY5Y cells. Our study suggests that the BioID2 method is useful to identify binding partners of growth factors., Competing Interests: The authors have no conflict of interest.

The versatility of IGFBP2, as a secreted protein in cancer cells or a cytoplasmic signaling effector, has been extensively investigated in many malignant cancers. Over the last few decades, IGFBP2, a key member of the IGFBP family, has been identified as an important oncogene in multiple human cancers. In addition, a growing number of studies have shown that IGFBP2 is greatly elevated in serum or tissue in patients with malignant tumors and plays an essential role in several key oncogenic processes, such as tumor cellular proliferation, migration, invasion, angiogenesis, epithelial‑to‑mesenchymal transition, and immunoregulation, which are involved in a variety of signal pathways, usually via an IGF‑independent means. Moreover, growing evidence indicates that aberrant overexpression of IGFBP2 may serve as a useful biomarker for the diagnosis and prognosis of patients, as well as act as a potential therapeutic target for the management of clinical treatment in patients with malignant disease. In the present review, we summarize the current points of view that IGFBP2 performs a role in the initiation and progression of various types of cancer by interacting with several key molecules involved in cancer signaling pathways. We also discuss its potential clinical application value as a diagnostic/prognostic biomarker for patients with malignant tumors.

Purpose: Expression of IGFBP-2 in mice is regulated by leptin. Over-expression of IGFBP-2 is associated with reduced caloric intake and resistance to weight gain. Hormonal variations contributing to weight loss occur very early after bariatric surgery but have not been fully elucidated. We evaluated IGFBP-2 serum changes after bariatric surgery and their relationship with leptin variations to test the hypothesis that an increase of leptin sensitivity may explain some of the effects of gastric bypass., Methods: This is a historical prospective study. Fifty-one obese patients (41 women e 10 men), 9 non-obese surgical controls and 41 lean matched controls were studied. Serum IGFBP-2 and leptin were measured after bariatric bypass surgery at various time points up to 18 months, after non-bariatric laparoscopic surgery in a control group, and in lean matched controls., Results: Compared to lean controls, serum IGFBP-2 levels were lower in obese patients. After gastric bypass, IGFBP-2 significantly increased at 3 days and became normal before the occurrence of relevant changes in body weight, remaining stable up to 18 months after surgery. IGFBP-2/leptin ratio increased early after surgery and became normal after one year., Conclusions: After gastric bypass, serum IGFBP-2 increases in a window of time when variations of hormones mediating the effects of bariatric surgery occur. Our results suggest that IGFBP-2, a leptin-regulated protein, may be an in-vivo marker of leptin action. If this is the case, an early improvement of leptin sensitivity might contribute to the anorectic effect of gastric bypass.

Objective: Sublobar resection may be insufficient for early-stage lung adenocarcinoma with micropapillary or solid components because of the associated higher incidence of locoregional recurrence. This study sought to establish a novel method for rapidly identifying their presence to facilitate decision making for sublobar resection., Methods: Antibody arrays of adhesion and apoptosis molecules were applied for adenocarcinomas with or without micropapillary/solid components to identify differentially expressed proteins. A semi-dry dot-blot system that visualizes the presence of target proteins was used to determine the presence of micropapillary or solid components in a prospective cohort of patients with clinical stage I who underwent operation. Sensitivity and specificity were calculated by comparing semi-dry dot-blot results with pathologic examinations., Results: Insulin-like growth factor-binding protein 2 and P-cadherin were found more frequently in the micropapillary or solid positive group, and these were used as the target proteins in the semi-dry dot-blot system for detection of micropapillary or solid components. A total of 68 nodules with a mean size of 2.3 ± 0.7 cm, including 13 (19.1%) with a micropapillary and 20 (29.4%) with a solid pattern, were recruited. Micropapillary or solid (+) lesions were more likely to have lymph node upstaging, greater diameter, and higher maximum standardized uptake value. The specificity and sensitivity for detecting the minor presence of micropapillary or solid component using the semi-dry dot-blot method were 94.4% (95% confidence interval, 81.3-99.3) and 65.6% (95% confidence interval, 46.8-81.4), respectively. The average test duration was 26.9 ± 2.5 minutes., Conclusions: Detecting insulin-like growth factor-binding protein 2 and P-cadherin via the semi-dry dot-blot method could identify micropapillary or solid components in early-stage lung adenocarcinoma in a short processing time., (Copyright © 2018 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

We aimed to verify the levels of IGFBP2 and SOCS3 in cartilage and chondrocytes of Kashin-Beck disease (KBD) patients and the effects of different selenium concentrations on the protein expression levels. Chondrocytes were cultured with sodium selenite in vitro. Immunohistochemistry and western blotting were used to verify the protein expressions. IGFBP2 and SOCS3 were up-regulated in KBD chondrocytes and decreased with increasing selenium concentrations. IGFBP2 expressed highest in the middle zone of KBD cartilage, SOCS3 expressed higher in the middle and deep zone. IGFBP2 and SOCS3 may be the biomarkers for KBD diagnosis and evaluating the effect of selenium supplement.

MIR-491 is commonly co-deleted with its adjacent CDKN2A on chromosome 9p21.3 in glioblastoma multiforme (GBM). However, it is not known whether deletion of MIR-491 is only a passenger event or has an important role. Small-RNA sequencing of samples from GBM patients demonstrated that both mature products of MIR-491 (miR-491-5p and -3p) are downregulated in tumors compared with the normal brain. The integration of GBM data from The Cancer Genome Atlas (TCGA), miRNA target prediction and reporter assays showed that miR-491-5p directly targets EGFR, CDK6 and Bcl-xL, whereas miR-491-3p targets IGFBP2 and CDK6. Functionally, miR-491-3p inhibited glioma cell invasion; overexpression of both miR-491-5p and -3p inhibited proliferation of glioma cell lines and impaired the propagation of glioma stem cells (GSCs), thereby prolonging survival of xenograft mice. Moreover, knockdown of miR-491-5p in primary Ink4a-Arf-null mouse glial progenitor cells exacerbated cell proliferation and invasion. Therefore, MIR-491 is a tumor suppressor gene that, by utilizing both mature forms, coordinately controls the key cancer hallmarks: proliferation, invasion and stem cell propagation.

Biomarker analysis of blood samples by liquid chromatography (LC) mass spectrometry (MS) is extremely challenging due to the high protein concentration range, characterised by abundant proteins that suppress and mask other proteins of lower abundance. This situation is further aggravated when using fast high-throughput methods, which are necessary for analysis of hundreds and thousands of samples in clinical laboratories. The blood proteins IGF1, IGF2, IBP2, IBP3 and A2GL have been proposed as indirect biomarkers for detection of GH administration and as putative biomarkers for breast cancer diagnosis. We describe a sensitive and scalable method to quantify these 5 proteins of medium and low abundance by selected reaction monitoring (SRM) LC-MS/MS analysis in blood samples. Our method requires 7μL of plasma and reaches a throughput of up to ca. 80 analyses per day. It includes an initial protein precipitation protocol optimised for extraction of low mass proteins from blood samples for reduced signal suppression and increased sensitivity in LC-MS/MS. We benchmarked this method for the analysis of 40 individual blood samples including 20 patients diagnosed with breast cancer., Biological Significance: The interest for MS-based biomarker analysis in body fluids is steadily increasing as proteomics methodology translates into clinical laboratories. We describe a method for detection of 5 distinct proteins of low mass and medium to low abundance, which are of interest in anti-doping and clinical analysis. The analytical setup is simple and robust and is suitable for high-throughput instrument configurations., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Cows with different Insulin-like Growth Factor-I (IGF-I) concentrations showed comparable expression levels of hepatic growth hormone receptor (GHR). Suppressor of cytokine signaling 2 (SOCS2), could be responsible for additional inhibition of the GHR signal cascade. The aims were to monitor cows with high or low antepartal IGF-I concentrations (IGF-I(high) or IGF-I(low)), evaluate the interrelationships of endocrine endpoints, and measure hepatic SOCS2 expression. Dairy cows (n = 20) were selected (240 to 254 days after artificial insemination (AI)). Blood samples were drawn daily (day -17 until calving) and IGF-I, GH, insulin, thyroid hormones, estradiol, and progesterone concentrations were measured. Liver biopsies were taken (day 264 ± 1 after AI and postpartum) to measure mRNA expression (IGF-I, IGFBP-2, IGFBP-3, IGFBP-4, acid labile subunit (ALS), SOCS2, deiodinase1, GHR1A). IGF-I concentrations in the two groups were different (p < 0.0001). However, GH concentrations and GHR1A mRNA expression were comparable (p > 0.05). Thyroxine levels and ALS expression were higher in the IGF-I(high) cows compared to IGF-I(low) cows. Estradiol concentration tended to be greater in the IGF-I(low) group (p = 0.06). It was hypothesized that low IGF-I levels are associated with enhanced SOCS2 expression although this could not be decisively confirmed by the present study.

Objective: The differential diagnosis between preterm and false labour remains one of the most challenging issues in perinatal medicine., Aim: To assess the prognostic importance of the selected biochemical markers in predicting preterm labour., Material and Methods: 74 patients hospitalized due to threatening preterm labour. 51 women gave birth prematurely; the remaining 23 were diagnosed with false labour. We used ELISA arrays to study 13 proteins: IGFBP-1, IGFBP-2, BDNF, L-Selectin, E-Selectin, ICAM-1, PECAM, VCAM-1, MIP-1 delta (MIP-1d) MIP-3β (MIP-3b), Eotaxin-1, Eotaxin-2, BLC., Results: An increased risk of preterm labour should be expected when the serum concentration for: IGFBP-1 > 158.83 pg/ml (sens. 0.608, sp. 0.609, p < 0.0001); MIP-1d < 27.66 pg/ml (sens. 0.627, sp. 0.627, p = 0.021); BDNF >36.54 pg/ml (sens. 0.630, sp. 0.647, p = 0.002); BLC >25.46 pg/ml (sens. 0.588, sp. 0.609, p < 0.001); Eotaxin-1 >1.16 pg/ml (sens. 0.633, sp. 0.652)., Conclusion: There have been reported statistically significant differences in serum concentrations of selected proteins in women with preterm labour and false labour.

Purpose: Our objective was to develop a system to simultaneously and quantitatively measure the expression levels of the insulin-like growth factor (IGF) family proteins in numerous samples and to apply this approach to profile the IGF family proteins levels in cancer and adjacent tissues from patients with hepatocellular carcinoma (HCC)., Experimental Design: Antibodies against ten IGF family proteins (IGF-1, IGF-1R, IGF-2, IGF-2R, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, and Insulin) were immobilized on the surface of a glass slide in an array format to create an IGF signaling antibody array. Tissue lysates prepared from patient's liver cancer tissues and adjacent tissues were then applied to the arrays. The proteins captured by antibodies on the arrays were then incubated with a cocktail of biotinylated detection antibodies and visualized with a fluorescence detection system. By comparison with standard protein amount, the exact protein concentrations in the samples can be determined. The expression levels of the ten IGF family proteins in 25 pairs of HCC and adjacent tissues were quantitatively measured using this novel antibody array technology. The differential expression levels between cancer tissues and adjacent tissues were statistically analyzed., Results: A novel IGF signaling antibody array was developed which allows the researcher to simultaneously detect ten proteins involved in IGF signal pathway with high sensitivity and specificity. Using this approach, we found that the levels of IGF-2R and IGFBP-2 in HCC tissues were higher than those in adjacent tissues., Conclusion: Our IGF signaling antibody array which can detect the expression of ten IGF family members with high sensitivity and specificity will undoubtedly prove a powerful tool for drug and biomarker discovery.

Background: Nutritional factors during a sensitive period can influence child development in a sex-related manner., Objective: Our aim was to investigate whether sex modulates the responses of relevant biochemical parameters and growth to different protein intakes early in life., Design: In a randomized controlled trial, formula-fed infants were assigned to receive formula with higher protein (HP) or lower protein (LP) content. The main outcome measures were insulin-like growth factor (IGF)-1 axis parameters, weight, length, BMI, leptin, and C-peptide/creatinine ratio at 6 mo of age. Dietary intake during the first 6 mo of life was also assessed., Results: The IGF-1 axis response to HP feeding was modulated by sex. Total and free IGF-1 and IGF binding protein 3 concentrations were higher in girls than in boys. Compared with the LP diet, the HP diet was associated with higher IGF-1 and lower IGF binding protein 2 secretion. The response to this HP content formula tended to be stronger in girls than in boys. The HP diet was associated with a higher C-peptide/creatinine ratio. The leptin concentration was higher in girls than in boys and was correlated to the IGF-1 axis parameters. No interaction between sex and nutritional intervention was shown on growth., Conclusions: Our findings show that the endocrine response to a high protein diet early in life may be modulated by sex. The IGF-1 axis of female infants shows a stronger response to the nutritional intervention than does that of male infants, but there is no enhanced effect on growth. This trial was registered at clinicaltrials.gov as NCT00338689.

Purpose: Previous studies from this laboratory revealed that vitreous insulin-like growth factor (IGF) biological activity increases in proliferative diabetic retinopathy and that this activity is normally attenuated by IGFBPs. The goal of this study was to identify and characterize the species involved., Methods: Human and porcine vitreous, plasma, recombinant IGFBP-2, and IGFBP-3 were separated by gel electrophoresis. Functional IGFBPs were detected in Western ligand blots with biotinylated IGF-II. IGFBPs were identified using IGFBP-specific antibodies., Results: Western ligand blots of normal vitreous and plasma detected two major proteins at ∼35 kDa and ∼29 kDa. Western blot analysis of human and porcine vitreous and plasma confirmed the identity of the ∼35-kDa band as IGFBP-2 and the ∼29-kDa band as a fragment of IGFBP-3. Western blot and Western ligand blot analyses of vitreous and plasma proteins separated by two-dimensional gel electrophoresis revealed that the IGFBP-3 fragments in vitreous and plasma have virtually identical profiles. Lyase digestion revealed that the ∼29-kDa IGFBP-3 fragment is a glycoprotein with a peptide core of ∼25 kDa. N-terminal sequence data obtained from vitreous IGFBP-3 revealed that the protein is proteolytically truncated at the C terminus. CONCLUSIONS. Normal human and porcine vitreous contain two major IGFBPs, IGFBP-2 and an ∼29-kDa fragment of IGFBP-3. Both IGFBPs retain biological activity, and IGFBP-3 has one or more glycosylation sites with a protein core of ∼25 kDa. Systematic comparisons indicate that the vitreous IGFBP-3 is similar to and perhaps identical with a previously described IGFBP-3 fragment in plasma with reduced growth factor affinities.

Background and Objective: Insulin-like growth factor-binding proteins (IGFBPs) are crucial regulators of insulin-like growth factor (IGF). They enhance or inhibit IGF functions, but also exhibit IGF-independent effects. In a previous study, we detected, qualitatively, IGFBP-2 and -3 in gingival crevicular fluid using a cytokine antibody array. Here we extended these results using an ELISA to determine the concentrations of IGFBP-2 and -3 in gingival crevicular fluid. In addition, we explored whether the expression of IGFBP-2 and IGFBP-3 correlates with periodontal disease severity., Material and Methods: Gingival crevicular fluid samples from 92 sites of 12 patients affected with periodontal disease and from 100 sites of 19 healthy volunteers, were collected, divided into two groups and analyzed by ELISA for IGFBP-2 and -3 expression. The potential correlation among probing depth, gingival index and the concentrations of IGFBP-2 and -3 was analyzed., Results: Positive correlations were observed between the concentration of IGFBP-2 and probing depth and gingival index, but not for IGFBP-3. The IGFBP-2 concentrations at bleeding on probing-positive sites and at sites with a probing depth of ≥ 4 mm were higher than at bleeding on probing-negative sites and at sites with a probing depth of ≤ 3 mm., Conclusion: These results indicate that IGFBP-2 is a potential novel marker for periodontal disease progression. As IGFBP-2 modulates bone metabolism and cell migration, IGFBP-2 in the gingival crevicular fluid may reflect periodontal disease activity., (© 2010 John Wiley & Sons A/S.)

Myxoid liposarcoma displays variably aggressive behavior and responds poorly to available systemic therapies. Expression profiling followed by tissue microarray validation linked to patient outcome is a powerful approach for validating biological mechanisms and identifying prognostic biomarkers. We applied these techniques to independent series of primary myxoid liposarcomas in an effort to assess markers of adipose differentiation in myxoid liposarcoma and to identify prognostic markers that can be efficiently assessed by immunohistochemistry. Candidate genes were selected based on analysis of expression profiles from 9 primary myxoid/round liposarcomas and 45 other soft tissue tumors, and by reference to publicly available data sets. Protein products were validated on an adipose neoplasm tissue microarray, including 32 myxoid liposarcomas linked to patient outcome. Results were scored visually and correlated with clinical outcome by Kaplan-Meier and Cox regression analyses. In the study, by examining expression patterns of several lipogenic regulatory gene products, an immature adipogenic status was verified in myxoid liposarcomas. We also found that expression levels of the ret proto-oncogene, insulin-like growth factor 1 receptor, and insulin-like growth factor 2 correlate with poor metastasis-free survival, supporting a role for ERK/MAPK and PI3K/AKT pathways in clinically aggressive myxoid liposarcomas.

Preterm infants can inoculate virulent cytomegalovirus (CMV) through their mothers' raw breast milk. Complete virus inactivation is achieved only by heat treatment, but the effect on growth factors has never been assessed systematically. Insulin-like-growth-factor-1-, IGF-2-, insulin-like-growth-factor-binding-protein-2-, and IGFBP-3-concentrations were measured, before and after heating, in 51 breast-milk-samples from 28 mothers, and epidermal-growth-factor-concentrations in a subgroup of 35 samples from 22 mothers. Two heating methods were applied: Short-term (5 s) pasteurisation at 62, 65, and 72 degrees C, and long-term Holder-Pasteurisation (30 min) at 63 degrees C. IGF-1, IGF-2, IGFBP-2, and IGFBP-3 were measured by RIA, and EGF by ELISA. Heating for 30 min decreased significantly IGF-1 by 39.4%, IGF-2 by 9.9%, IGFBP-2 by 19.1%, and IGFBP-3 by 7.0%. In contrast, IGF-1, IGF-2, IGFBP-2, and IGFBP-3 were not altered significantly when using a short heating duration of 5 s, irrespective of the level of temperature, except for IGF-2 at 62 degrees C for 5 s (p = 0.041) and IGFBP-2 at 72 degrees C for 5 s (p = 0.025). Neither long- nor short-time heating methods changed the concentration of EGF. Only short heating methods (5 s, 62-72 degrees C) can preserve, almost completely, the concentrations of IGFs in human milk, whereas Holder-Pasteurization does not.

The growth hormone/insulin-like growth factor (IGF) system plays a critical endocrine role controlling nutrient metabolism in dairy cattle. In liver, growth hormone receptor (GHR) and IGF-1 are dynamically regulated by lactation and energy balance. Less is known about the regulation of GHR, IGF-1, and IGF-binding protein mRNA in reproductive tissues (uterus, ovarian follicle, and corpus luteum). The objective was to determine expression patterns for GHR, IGF-1, and IGF-binding protein (IGFBP)-2 mRNA in the liver, uterus, dominant follicle, and corpus luteum in Holstein cows (n = 21) sampled at 3 times during early lactation. The first postpartum ovulation was induced with an injection of GnRH within 15 d of calving. Nine days after ovulation [23 +/- 1 d postpartum; 20 d in milk (DIM)], the liver, uterus, dominant follicle, and corpus luteum were biopsied. Prostaglandin F(2alpha) and GnRH were injected 7 and 9 d after each biopsy to synchronize the second (41 +/- 1 d postpartum; 40 DIM) and third (60 +/- 1 d postpartum; 60 DIM) tissue collections. Total RNA was isolated and used for mRNA analysis by real-time quantitative reverse transcription PCR. Liver had more GHR, IGF-1, and IGFBP-2 mRNA than the reproductive tissues that were tested. Gene expression for GHR, IGF-1, and IGFPB-2 within tissues did not change across the sampling interval (20 to 60 DIM). The only detected change in gene expression across days was for cyclophilin in uterus (increased after 20 DIM). Parity had an effect on gene expression for GHR in corpus luteum. Neither level of milk production nor body condition score affected the amount of GHR, IGF-1, or IGFBP-2 mRNA in the respective tissues. The repeatability of gene expression within a tissue was 0.25 to 0.5 for most genes. In most instances, expression of a single gene within a tissue was correlated with other genes in the same tissue but was not correlated with the same gene in a different tissue. We did not find evidence for major changes in gene expression within reproductive tissues in postpartum cows. Differences between cows (independent of their BCS and milk production) accounted for a major portion of the variation that we observed.

We have applied an in-depth quantitative proteomic approach, combining isotopic labeling extensive intact protein separation and mass spectrometry, for high confidence identification of protein changes in plasmas from a mouse model of breast cancer. We hypothesized that a wide spectrum of proteins may be up-regulated in plasma with tumor development and that comparisons with proteins expressed in human breast cancer cell lines may identify a subset of up-regulated proteins in common with proteins expressed in breast cancer cell lines that may represent candidate biomarkers for breast cancer. Plasma from PyMT transgenic tumor-bearing mice and matched controls were obtained at two time points during tumor growth. A total of 133 proteins were found to be increased by 1.5-fold or greater at one or both time points. A comparison of this set of proteins with published findings from proteomic analysis of human breast cancer cell lines yielded 49 proteins with increased levels in mouse plasma that were identified in breast cancer cell lines. Pathway analysis comparing the subset of up-regulated proteins known to be expressed in breast cancer cell lines with other up-regulated proteins indicated a cancer related function for the former and a host-response function for the latter. We conclude that integration of proteomic findings from mouse models of breast cancer and from human breast cancer cell lines may help identify a subset of proteins released by breast cancer cells into the circulation and that occur at increased levels in breast cancer.

Extracts of bovine corneal stroma have been shown to activate keratocytes in culture to proliferate. We fractionated stromal extract on a column of Sephacryl S-300 and tested the fractions for mitogenic activity using cell culture and for the presence of IGF-II and its binding protein IGFBP-2 by Western blot. We found that the mitogenic activity in the extract separated into major and minor peaks and that immunologically detectable IGF-II and IGFBP-2 co-eluted with the minor peak. We also compared the effects of 10 ng IGF-II/ml on keratocytes in culture to that of 2 ng TGF-beta/ml over a 7-day culture period. We found that IGF-II and TGF-beta, alone or combined, increased both (3)H-thymidine incorporation and DNA content of the cultures. The phenotype of the cells was determined by using antibodies to alpha-SM (smooth muscle) actin, fibronectin, SPARC, lumican and keratocan in Western blots of cell layers of media. Keratocytes cultured in IGF-II expressed no alpha-SM actin or fibronectin, low levels of SPARC and high levels of lumican and keratocan, indicating a native phenotype. Keratocytes in TGF-beta expressed alpha-SM actin, fibronectin, SPARC and lumican, and expressed no or low levels of keratocan, indicating a myofibroblast phenotype. Keratocytes cultured in IGF-II plus TGF-beta, however, expressed alpha-SM actin, fibronectin, SPARC, lumican, and keratocan by day 7 of culture. The results of this study show that IGF-II to be present in the corneal stroma, to stimulate keratocyte proliferation while maintaining native phenotype and to override the TGF-beta mediated down regulation of keratocan production. The IGF-II in the stroma may serve as a mechanism to immediately activate keratocytes upon wounding and to ameliorate the scarring effects of TGF-beta.

Negative energy balance (NEB) during early lactation in dairy cows leads to an altered metabolic state that has major effects on the production of IGF family members. Low IGF-I concentrations are associated with poor fertility and therefore we aimed to determine whether NEB exerts a direct effect on IGF expression in the postpartum oviduct. Multiparous Holstein cows were allocated to two treatments (each n=6) designed using differential feeding and milking regimes to produce either mild NEB (MNEB) or severe NEB (SNEB). Animals were slaughtered in week 2 of lactation when divergent metabolic profiles were evident. Oviducts were collected for RNA analysis by real-time RT-PCR and in situ hybridisation. Quantitative measures in oviduct gene expression were obtained for all members of the IGF family (IGF-I/II, IGF-binding proteins (IGFBP) 1-6 and receptors for IGF types 1 and 2), insulin A/B, GH, glucocorticoid and oestrogen alpha/beta. Expression of IGFBP-2 and IGFBP-6 (both of which have a high affinity for IGF-II) was decreased in SNEB relative to MNEB (P<0.05). No other gene was altered by NEB, but IGF-II, IGFBP-3, IGFBP-5 and IGFBP-6 all showed differential expression in different regions of the oviduct. These results indicate that, in addition to low circulating IGF-I after calving, NEB may also influence IGF availability in the oviduct indirectly through changes in specific IGFBP expression. It is possible that the predicted increased signalling by IGF-II may perturb embryo development, contributing to the high rates of embryonic mortality in dairy cows.

Aim: To evaluate the postpartum time course of changes in insulin-like growth factors (IGFs) and their binding proteins (IGFBPs)., Methods: Breast milk IGF-I and IGF-II and IGFBP-1, IGFBP-2, and IGFBP-3 levels were determined in 23 women with babies born at term, from day 4 until up to 9 months after birth., Results: The IGFBP-3 levels were highest from day 4 to day 6 and then decreased by days 10-12. In contrast, IGF-I and IGF-II and IGFBP-1 and IGFBP-2 showed little change over the first 2 weeks after birth. Subsequently, all the IGF components showed a moderate decline over approximately the first 1-3 months and then stable levels up to 9 months after birth., Conclusion: Although the possibility cannot be excluded that these changes in levels of IGFs and their binding proteins in human milk represent passive loss from the mammary gland, we speculate that higher early levels of the human milk IGF system contribute to maturation of the infant gut., ((c) 2008 S. Karger AG, Basel)

The aim of this study was to determine the effect of regulation of IGF-I bioavailability on preantral follicle development in vitro. Bovine preantral follicles were cultured for 6 days in serum-free medium with increasing doses of Long R3 (LR3) IGF-I (an analog with low affinity for IGF-binding proteins (IGFBPs)), or human recombinant IGF-I (hrIGF-I). Follicle diameter and estradiol production were measured every second day. On day 6, ratios of oocyte/follicle diameter and oocyte morphology were assessed by histological examination, and IGFBP-2 and -3 were detected by immunocytochemistry and in situ hybridization respectively. Both types of IGF-I increased follicle diameter in a dose-dependent manner (P < 0.05) and increased estradiol production over control levels (P < 0.05). However, follicles treated with LR3 IGF-I and the highest concentration of hrIGF-I (1000 ng/ml) had smaller oocyte/follicle ratios, and increased oocyte degeneration, compared with controls or follicles treated with physiological concentrations of hrIGF-I (P < 0.05). IGFBPs were detected in cultured preantral follicles, indicating a requirement for regulation of IGF bioavailability during the early stages of follicular development. Specifically, IGFBP-3 mRNA was found to be expressed in oocytes, and IGFBP-2 immunoreactivity was detected in oocytes and granulosa cells of cultured follicles. In summary, the regulation of IGF-I bioavailability by IGFBPs is necessary for the co-ordination of oocyte and follicle development in vitro.

Objective: Primary pigmented nodular adrenocortical disease (PPNAD) can occur as an isolated trait or part of Carney complex, a familial lentiginosis-multiple endocrine neoplasia syndrome frequently caused by mutations in PRKAR1A, which encodes the 1alpha regulatory subunit of protein kinase A (PKA). Because alterations in the insulin-like growth factor (IGF) axis, particularly IGF-II and IGF binding protein (IGFBP)-2 overexpression, have been implicated in sporadic adrenocortical tumors, we sought to examine the IGF axis in PPNAD., Design: RNA samples and paraffin-embedded sections were procured from adrenalectomy specimens of patients with PPNAD. Changes in expression of IGF axis components were evaluated by real-time quantitative RT-PCR and immunohistochemistry. NCI-H295R cells were used to study PKA and IGF axis signaling in adrenocortical cells in vitro., Results: IGFBP-2 mRNA level distinguished between the two genetic subtypes of this disease; increased IGFBP-2 expression in PRKAR1A mutation-positive PPNAD tissues was also confirmed by immunohistochemistry. Moreover, PKA inhibitors increased IGFBP-2 expression in NCI-H295R adrenocortical cells, and anti-IGFBP-2 antibody reduced their proliferation., Conclusions: IGFBP-2 expression is increased in PPNAD caused by PRKAR1A mutations, and in adrenocortical cancer cells. This is the first evidence for PKA-dependent regulation of IGFBP-2 expression in adrenocortical cells.

Breast-fed preterm infants often show a better outcome, partly ascribed to the benefit of insulin-like growth factors (IGFs) and their binding proteins (IGFBP). We compared IGF-I, IGF-II, IGFBP-2 and IGFBP-3 levels, measured by radioimmunoassays in milk samples from 30 mothers of preterm (<31 weeks) and from 19 mothers of term (>37 weeks) infants at days 7 and 21 postpartum. Proteolysis of IGFBP-2 within mother's milk and digestion of (125)I-IGF-II and (125)I-IGFBP-2 by gastric juice from neonates were assessed by electrophoretic techniques. Mean concentrations did not differ between preterm and term milk: IGF-I (2.8 +/- 0.2 vs. 2.3 +/- 0.1 ng/ml), IGF-II (12.0 +/- 0.4 vs. 12.2 +/- 0.5 ng/ml), IGFBP-3 (100.0 +/- 5.1 vs. 80.0 +/- 5.8 ng/ml), but did so for IGFBP-2 (3,144 +/- 172 vs. 2,428 +/- 188 ng/ml, p < 0.02). Immunoblots revealed 42% (p < 0.05) more IGFBP-2 fragments of 14 and 25 kDa in preterm milk. Incubation with gastric juice caused cleavage of (125)I-IGFBP-2 and partial cleavage of (125)I-IGF-II. Mutual complexation protected IGF-II and IGFBP-2 from cleavage, suggesting that both are likely to arrive in the bowel in an intact form to exert promotive effects. The results provide further evidence that IGFBP-2 and IGF-II in breast milk are relevant factors for the early development of preterm infants., (Copyright 2007 S. Karger AG, Basel.)

There is evidence that growth factors, such as the insulin-like growth factors (IGFs), are involved in biological and pathological processes in oro-dento-facial tissues. To investigate their roles in tooth movement, root resorption, and repair, the occurrence of components of the IGF system, including the ligands IGF-I and -II, the IGF receptor 1 (IGF1R) and six IGF-binding proteins (IGFBP-1 to -6), was investigated by immunohistochemistry on sections from rat maxillae where the first molar had been moved mesially by means of an orthodontic appliance for 9 d to induce root resorption. After force deactivation on day 0, early repair was studied after a further 5, 7, 10, 12, 14, and 17 d. The immunostaining pattern in the periodontal ligament, cementum, and bone of control animals showed similarities known from studies in human teeth. Increased immunostaining for nearly all components in pressure sides and resorption lacunae indicated an involvement in resorption processes and clastic activities. During early stages of repair, the occurrence of several components (e.g. IGF-II, IGFBP-5 or -6) within lacunae and in cementoblasts showed an involvement in the resorption-repair sequence, which is considered to be a coupling process as known from bone.

The objective was to determine the pattern of IGFBP-2, -3 and -4 gene expression and follicular fluid concentrations of IGFBP-2, -3, -4 and -5 during emergence, selection and dominance of the first follicle wave of the estrous cycle in cattle and during exogenous steroid treatment. Heifers (n = 35) were ovariectomized at 36 (n = 7), 66 (n = 8), 84 (n = 12) and 108 (n = 8) h after the onset of estrus. Heifers in the 84 h ovariectomy group were sub-divided to receive either no treatment (n = 6) or were treated with a progesterone-releasing intravaginal device (n = 6, PRID) and 0.75 mg estradiol benzoate i.m. at the approximate time of ovulation, 30 h post estrus until ovariectomy. Within heifers the four largest follicles recovered following ovariectomy were ranked on size (F1, F2, F3 and F4). At 36 h IGFBP gene expression and follicular fluid IGFBP concentrations were similar in all follicles (F1-F4). Mean diameter of the F1 follicle increased (P < 0.05) between 36 and 84 h with no difference between 84 and 108 h. The F1 follicle had the highest (P < 0.05) concentration of estradiol compared with the F2, F3 and F4 at 84 and 108 h. There was no granulosa cell IGFBP-2 mRNA in F1 follicles at 84 or 108 h. Intrafolliclar IGFBP-2 concentrations were lower (P < 0.05) in the F1 compared with F3 and F4 follicles at 108 h. There was no difference in theca cell IGFBP-4 mRNA expression at 108h, but amounts of follicular fluid IGFBP-4 were lower (P < 0.05) in F1 follicles compared with F3 and F4 follicles at 108 h. IGFBP-3 mRNA was localized in the theca layer of all follicles examined with no difference in expression or follicular fluid concentrations during emergence, selection and dominance of the first follicle wave. IGFBP-5 concentrations were higher (P < 0.05) in follicular fluid of F3 follicles at 108 h compared with the F3 at 36 h. In conclusion follicular dominance was associated with low or decreased follicular fluid concentrations of IGFBP-4 and -5, increased estradiol and differential regulation of IGFBP production.

Elucidating how mitogens facilitate epithelial/stromal interactions is critical given that mitogens regulate mammary gland development and function. IGF-I is a potent mammary cell mitogen that is locally produced in the mammary gland. Since IGF-binding proteins (IGFBPs) regulate IGF-I bioavailability, we characterized the cell-type-specific production of IGFBP in primary bovine mammary epithelial (BME) and fibroblast (BMF) cells. Cells were treated with IGF-I and mRNA levels were analyzed via quantitative real-time (qRT)-PCR and Northern blot analysis. Media conditioned by cells treated with IGF-I for 48 h were analyzed via ligand blotting with 125I-labeled IGF-I and -II and immunoblotting with specific IGFBP antibodies. A reciprocal regulation of IGFBP-3 and -5 by IGF-I was observed between the two cell types. IGF-I induced large dose-dependent increases in IGFBP-3 mRNA and protein levels in BME cells, while IGFBP-5 protein was barely detectable and mRNA levels were detectable only by qRT-PCR. In BMFs, IGF-I induced large increases in IGFBP-5 mRNA and protein while IGFBP-3 mRNA was only slightly increased by IGF-I treatment and the protein was difficult to detect. IGFBP-6 mRNA was detected by Northern blot analysis in both cell types but was not regulated by IGF-I. In BME cells, IGFBP-6 protein levels were readily detectable under basal conditions and were increased by IGF-I. Interestingly, IGFBP-6 protein could not be detected in media conditioned by BMFs. IGFBP-4 mRNA was readily seen by Northern blot analysis in BMFs, however qRT-PCR was required to detect IGFBP-4 mRNA in BME cells. IGF-I increased IGFBP-4 mRNA levels by 2-fold in both cell types. IGFBP-4 protein was only detectable in media conditioned by BME cells when stimulated by IGF-I. In contrast, IGFBP-4 was present in media conditioned by untreated BMFs but was not consistently increased by IGF-I treatment. This was explained by the finding that IGF-I stimulated proteolysis of IGFBP-4, as evidenced by the appearance of two immuno-responsive fragments of 18 and 14 kDa. This proteolysis was specific to IGFBP-4, and was not observed in BME cells. We confirmed the protease to be pregnancy-associated plasma protein A (PAPP-A) by immunoblotting with an antibody against human PAPP-A/proMBP (pro form of eosinophil major basic protein) complex. In vitro immuno-neutralization experiments showed that blocking PAPP-A prevented the ability of IGF-I to stimulate IGFBP-4 proteolysis. IGFBP-2 mRNA and protein levels were observed under basal conditions in both cell types, with no significant regulation by IGF-I. The analysis of cell-type-specific regulation of the IGF system in both primary mammary epithelial cells and stromal cells will assist in the characterization of the mechanisms behind the role of the IGF system in normal mammary physiology and ultimately breast cancer.

Background/aim: Insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) play a role in the normal development of breast tissue, and possibly in breast cancer aetiology. IGFBP2, one of six members of the IGFBP superfamily, acts as regulator of the IGFs and has pleiotropic effects in normal and neoplastic tissues. Because IGFs have mitogenic effects on mammary epithelia, this study investigated IGFBP2 expression in mammary tissues of different benign and malignant entities., Methods: Immunohistochemistry was used to study correlations between the presence and intensity of IGFBP2 staining and tumour type and grade, in addition to steroid hormone receptor status, in 120 breast specimens. Expression was measured by quantitative colour video image analysis and semiquantitative evaluation, and the measurements correlated well (r = 0.92; p<0.05)., Results: Both methods found no significant expression of IGFBP2 in normal glandular cells and hyperplasia (group I). Atypical hyperplasia showed a slightly increased cytoplasmic expression of IGFBP2, and carcinoma in situ showed a distinctive, membrane associated and cytoplasmic expression (group II). Infiltrating carcinomas strongly expressed cytoplasmic IGFBP2 (group III). There were significant differences between group I and II, and between group II and III. There were no significant differences between invasive lobular and invasive ductal carcinoma, or between grades I, II, and III within these entities. There was no significant correlation between IGFBP2 immunostaining and oestrogen or progesterone receptor positivity within the malignant group., Conclusions: IGFBP2 mitogenic signals of autocrine/paracrine regulatory mechanisms may be responsible for the growth of breast carcinomas and IGFBP2 may be an independent indicator of malignancy.

Postnatal development of the ovine uterus primarily involves uterine gland morphogenesis or adenogenesis. Adenogenesis involves the budding differentiation of the glandular epithelium (GE) from the luminal epithelium (LE) and then GE proliferation and coiling/branching morphogenetic development within the stroma between birth (postnatal day or PND 0) and PND 56. Insulin-like growth factor (IGF)-I and IGF-II mRNAs were previously found to be expressed only in the endometrial stroma, whereas the IGF receptor (IGF-1R) mRNA was most abundant in epithelia and in stroma, suggesting that an intrinsic IGF system regulates postnatal development of the uterus. Given that the biological activities of IGFs are modulated by a family of six IGF binding proteins (IGFBPs) and specific proteases, the objective was to determine the effects of age and estrogen disruption on expression of IGFs, IGFBPs and pregnancy-associated plasma protein A (PAPP-A or IGFBP-4 protease) in the ovine uterus. In Study One, circulating levels of IGF-I and IGF-II in the serum of neonatal ewes did not change between PND 0 and PND 56. Levels of immunoreactive IGF-I, IGF-II and IGF-1R protein were most abundant on the apical surface of the endometrial LE and GE. RT-PCR analyses detected expression of IGFBPs (3, 4, 5 and 6) as well as PAPP-A mRNAs in the uterus, but not IGFBP-1 and IGFBP-2 mRNAs. IGFBP-3 and IGFBP-4 mRNAs were expressed specifically in the endometrial stroma and myometrium and increased after birth. PAPP-A mRNA was expressed specifically in the endometrial stroma and increased after birth. In Study Two, ewes were treated from birth with estradiol-17beta valerate (EV), which reduces uterine growth and inhibits endometrial adenogenesis. On PNDs 14 and 56, IGFBP-3 mRNA was decreased in the uterus of EV-treated ewes, but IGF-1R and IGFBP-4 mRNAs were not affected. PAPP-A mRNA was increased by EV treatment on PND 14, but decreased on PND 56. These results support the hypothesis that an intrinsic IGF system in the uterus regulates epithelial-stromal interactions important for postnatal uterine growth and endometrial gland morphogenesis in the sheep.

The period between seasonal anoestrus and cyclicity is characterized in many mares by cyclical growth and regression of large dominant follicles. The insulin-like growth factor (IGF) system plays a key role in follicular growth and regression; therefore, we hypothesized that changes in the IGF system and its binding proteins would modulate onset of cyclicity in mares. Ovaries were obtained from pony mares on the day after detection of an actively growing 30 mm transitional anovulatory follicle, and also at the second or third oestrus of the breeding season on the day after the preovulatory follicle reached 30 mm in diameter. Size of dominant follicles at the time of removal was similar in transition (32 +/- 0.8 mm) and at oestrus (34 +/- 0.6 mm). IGF-I mRNA was present in granulosa cells, with low thecal expression, whereas IGF-II mRNA was confined to the theca layer. Expression of IGF-I and -II mRNAs, and intrafollicular concentrations of oestradiol, were lower (P < 0.01; paired t test) in transitional anovulatory follicles than in preovulatory follicles. Messenger RNA encoding IGFBP-2 was present in both theca and granulosa layers. Steady-state concentrations of mRNA encoding IGFBP-2 mRNA increased (P < 0.001) in theca in preovulatory follicles. Intrafollicular concentrations of IGFBP-2 were higher (P < 0.001) in transitional than in preovulatory follicles. The similarity in circulating concentrations of IGF-I in transitional and cyclic mares, suggested that the somatotrophic axis is not involved in transition from anovulatory to ovulatory cycles. The results suggest that the increased expression of IGF-I and -II mRNAs in preovulatory follicles, along with the decrease in IGFBP-2 concentrations, could increase the bioavailability of intrafollicular IGF in large follicles during the breeding season, and support our hypothesis that intrafollicular IGF bioavailability must exceed a threshold level before ovulation can occur.

Objective: Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) is characterized by high androgen levels, ambiguous genitalia or premature pubarche, increased height velocity and skeletal maturation. Considering the possibility of changes in the IGF system components depending on the state of clinical control, the objective of the present study was to analyse serum IGF-I, IGF-II and IGFBP levels in children with 21-OHD under two states of clinical control., Patients and Design: We studied 12 prepubertal children with 21-OHD CAH aged 4.0 +/- 0.7 years. They were classified as good (GC) or poor control (PC) based on growth rate, signs of adrenal insufficiency or Cushing syndrome, progression of sexual characteristics and serum androgens levels. Blood samples were obtained from each patient in two different states of clinical control (GC and PC) for biochemical measurements., Measurements: IGF-I, IGF-II, IGFBP-1, IGFBP-2 and IGFBP-3 were determined by immunoassays. IGFBPs were also analysed by Western ligand blotting (WLB)., Results: Levels of IGF-I (P = 0.03) and IGFBP-3 (P = 0.01) were higher in GC than in PC while IGFBP-1 (P = 0.004) concentrations were lower in GC patients. A trend towards higher levels of IGF-II (P = 0.08) and lower levels of IGFBP-2 (P = 0.08) was observed in GC children. Increased IGFBP-4 band intensity was observed in GC children (P = 0.03)., Conclusion: Higher levels of IGF-I, IGFBP-3 and IGFBP-4, but lower levels of IGFBP-1, were associated with better control in children with 21-OHD CAH. These findings are different from those observed in children with other causes of increasing androgens levels and are likely to be related to the insufficient glucocorticoid status.

In vitro studies suggest that insulin-like growth factor (IGF)-I is a mitogen for microglia/macrophages. The actions of IGF-I are mediated by IGF-I receptors and modulated by IGF binding proteins (IGFBPs). The aim of this study was to investigate IGF-I receptors and IGFBPs in human microglia in normal brain white matter and active lesions of multiple sclerosis, which contain activated microglia/macrophages. Methods used were immunohistochemistry and confocal laser microscopy. IGF-I receptors were demonstrated in both resting and activated microglia. In resting conditions, microglia displayed no immunoreactivity for any of the six IGFBPs, whereas activated microglia/macrophages were immunoreactive for IGFBP-2 only. Our data suggest an important function for IGFBP-2 in IGF-I actions in activated microglia/macrophages in human brain.

IGF-I and IGF-II have known roles in postnatal development of the mammary gland. In contrast, the function of the high-affinity IGF binding proteins (IGFBPs) in mammary growth and differentiation is largely unknown. The goal of these studies was to determine the patterns and levels of IGFBP expression during postnatal growth of the murine mammary gland. IGFBP-1 to -5 proteins were detected in mammary tissue by immunoblotting during both pubertal and pregnancy-induced growth; however, the regulation of each IGFBP was distinct through these developmental periods. IGFBP-2 to -5 mRNAs were readily detectable in the developing gland by in situ hybridization analyses but were expressed in distinct cellular sites. IGFBP-3 and -5 mRNAs were expressed in the developing epithelial structures and in isolated stromal cells during ductal growth and alveolar differentiation. In the terminal end buds (TEBs), IGFBP-3 mRNA expression was consistent with its localization in the cap cells, whereas IGFBP-5 was highly expressed in the body cells of the TEB. In contrast, IGFBP-2 and -4 mRNAs were expressed predominantly in stromal cells. IGFBP-2 mRNA was localized to restricted sites in the neck of the TEB and along the ductal structures, whereas IGFBP-4 mRNA was widely expressed in the stroma surrounding the epithelial structures. Protein and mRNA expression for most of the IGFBPs decreased during lactational ages. Levels of IGFBP-2 and -5 protein increased after pup removal during forced involution. Taken together, these data suggest important functions for the family of IGFBPs during postnatal growth and differentiation of the mammary epithelium.

Transgenic and knockout models have been used successfully in order to attribute specific functions to distinct growth factors. However, it is not clear which from the different IGF-components are actually altered when growth is affected. Furthermore it is not clear if unique or redundant patterns of IGF-component expression are present under conditions of elevated or reduced growth. To address these questions we have used a unique set of mouse models generated by divergent selection for high and low body growth. The set of mouse models consisted of eight mouse lines established in different laboratories. We have studied systemic and local expression of growth relevant genes in these mouse lines highly diverging for body and carcass weights but also for nose-rump lengths. As a strictly conserved pattern, serum IGF-I levels were dramatically increased in all H-lines if compared with the respective L-lines. By contrast serum IGFBP concentrations did not reveal clear patterns of expression in response to growth selection: IGFBP-3 was elevated in some H-lines, IGFBP-2 was increased in H- or L-lines and IGFBP-4 was similar in H- and L-lines. The fact that IGFBP-2 was the only IGFBP elevated in part of the L-lines, identifies IGFBP-2 as an exclusive although facultative negative effector for growth in the circulation among all other IGFBPs. In muscle tissue from selected breeding groups characterized by specific increases of the carcass weights we found redundant patterns of gene expression indicating the absence of tissue-specific or uniquely fixed expression patterns during growth selection within muscle tissue. The finding that serum but not tissue IGF-I levels were strictly positively correlated with growth during growth selection argues for an important role of endocrine IGF-I for postnatal growth in mice.

Objective: The purpose of this study was to determine the effects of long-term estrogen replacement therapy (ERT) on insulin-like growth factor binding protein (IGFBP)-2, IGFBP-3, collagen and proteoglycan levels in the articular cartilage of the knee joint in a well-characterized monkey model of naturally occurring osteoarthritis (OA). A secondary aim was to evaluate the effect of soy phytoestrogen treatment on these articular cartilage components., Design: Monkeys were ovariectomized and given ERT, soy phytoestrogen treatment or no treatment (control) for 3 years. Ten animals were randomly selected from each of the three groups and the cartilage was dissected from the proximal tibia and distal femur of the knee. Levels of IGFBP-2, IGFBP-3, and total protein were measured in cartilage desorptions, and proteoglycan levels and collagen levels were measured in the cartilage tissue. Sections from the tibial plateau of the opposite knee were immunostained using antibodies directed against IGFBPs and evaluated subjectively., Results: IGFBP-3 levels were significantly higher, and total protein levels were significantly lower in the cartilage desorption samples from the estrogen-treated animals compared to the control animals. There were no significant differences in IGFBP-2, collagen or proteoglycan levels between the estrogen-treated and control groups. Soy phytoestrogen treatment had no significant effect on the levels of any of the cartilage components that were measured. The staining patterns observed by immunohistochemistry suggested local production of IGFBP-2 and IGFBP-3 by articular cartilage chondrocytes., Conclusions: Long-term estrogen treatment results in increased IGFBP-3 levels in articular cartilage without a significant change in IGFBP-2, collagen or proteoglycan content, and IGFBP-3 appears to be synthesized by articular cartilage chondrocytes. Long-term soy phytoestrogen treatment did not have a statistically significant effect on the levels of IGFBP-2, IGFBP-3, collagen or proteoglycan.

We investigated the effect of an isocaloric maternal low-protein diet during pregnancy in rats on the proliferative capacity of cultured fetal hepatocytes. The potential roles of these changes on the IGF-IGF-binding protein (IGFBP) axis, and the role of insulin and glucocorticoids in liver growth retardation, were also evaluated. Pregnant Wistar rats were fed a control (C) diet (20% protein) or a low-protein (LP) diet (8%) throughout gestation. In primary culture, the DNA synthesis of hepatocytes derived from LP fetuses was decreased by approximately 30% compared with control hepatocytes (P < 0.05). In parallel, in vivo moderate protein restriction in the dam reduced the fetal liver weight and IGF-I level in fetal plasma (P < 0.01) and augmented the abundance of 29- to 32-kDa IGFBPs in fetal plasma (P < 0.01) and fetal liver (P < 0.01). By contrast, the abundance of IGF-II mRNA in liver of LP fetuses was unaffected by the LP diet. In vitro, the LP-derived hepatocytes produced less IGF-I (P < 0.01) and more 29- to 32-kDa IGFBPs (P < 0.01) than hepatocytes derived from control fetuses. These alterations still appeared after 3-4 days of culture, indicating some persistence in programming. Dexamethasone treatment of control-derived hepatocytes decreased cell proliferation (54 +/- 2.3%, P < 0.01) and stimulated 29- to 32-kDa IGFBPs, whereas insulin promoted fetal hepatocyte growth (127 +/- 5.5%, P < 0.01) and inhibited 29- to 32-kDa IGFBPs. These results show that liver growth and cell proliferation in association with IGF-I and IGFBP levels are affected in utero by fetal undernutrition. It also suggests that glucocorticoids and insulin may modulate these effects.

The objectives of the present study were to determine how varying frequency of LH pulses as controlled by varying treatments with progesterone (P4) in cattle would affect: (1) concentration of steroid hormones and activity of insulin-like growth factor binding proteins (IGFBPs) in the ovarian follicular fluid and blood plasma, and (2) duration of persistence of largest ovarian follicles. There were four treatment groups (n=7 per group) and a control group (n=5) of mature, non-lactating beef cows. Treatments were: (1) two progesterone releasing intravaginal devices (PRIDs) for 16 days (2PRID); (2) a half PRID for 16 days (0.5PRID); (3) two PRIDs for 8 days, then a half PRID for 8 days (2-0.5PRID); or (4) a half PRID for 8 days, then two PRIDs for 8 days (0.5-2PRID). Treatment was initiated on the fifth day of the estrous cycle, which was designated as Day 0, and continued for 16 days. All P4-treated females were administered prostaglandin F2alpha on Day 0 and 1 to regress their corpora lutea. Frequency of LH pulses was greater during treatment with the smaller dose of P4 compared with treatment with the larger dose of P4 and the control group. Ovarian follicles were classified into five categories based on ultrasonographic observations: growing (G); atretic (A); growing dominant (GD); growing persistent (GP); or atretic persistent (AP). At ovariectomy on Day 16, the largest and second largest follicles collected were re-classified into five categories based on follicular concentration of steroids. Classification of the largest follicle collected on Day 16 was influenced by treatment (P<0.005), with the 2PRID group having A follicles, the 2-0.5PRID group GP follicles, the 0.5-2PRID group AP follicles, and the 0.5PRID group GD and GP follicles. Concentrations of 17beta-estradiol (E2) were greatest in GD and GP follicles (P<0.05). There was less (P<0.05) activity of IGFBP-2 in GD follicles and less (P<0.05) activity of IGFBP-3 in GD and GP follicles than other follicles. Activity of IGFBP-4 and -5 was greater (P<0.05) in A and AP follicles than G, GD, and GP follicles. Maintenance of a frequent release of LH pulses over a 16-day period did not result in maintenance of persistent follicles throughout this period indicating that duration of dominance of these follicles is finite even when there is frequent release of LH pulses. Follicular atresia is associated with greater activity of IGFBP-2, -4, -5, and greater concentrations of P4 in follicles, whereas growing dominant and persistent follicles contained greater concentrations of E2, androstenedione (A4), and less IGFBP-2 activity than follicles of other classes. Follicle classifications based on ultrasonography or follicular concentration of steroids did differ (P<0.05) for the largest follicles from the 2PRID group. Two follicles in this group appeared as GD follicles by ultrasonography, but these were atretic based on follicular steroid contents. Objective 1 of the present study yielded the conclusion that concentrations of steroid hormones in follicular fluid and blood plasma could be predictably controlled by regulating the frequency of LH pulses with varying doses of P4. Objective 2 yielded the conclusion that maintain frequent release of LH pulses over a 16-day period could not maintain persistent follicles throughout this period, indicating that duration of dominance of these follicles is finite even when there is frequent release of LH pulses. Follicular atresia in the present study was associated with increased follicular fluid activity of IGFBP-2, -4, -5, and P4, whereas growing dominant and persistent follicles contained greater concentrations of E2, A4, and less IGFBP-2 activity than follicles of other classes.

Objective: Recently, GnRH antagonists (GnRHants) have been introduced for the prevention of premature LH surges during controlled ovarian hyperstimulation (COH). Here we investigated whether the GnRHants cetrorelix and ganirelix exert effects on the human ovarian IGF system. Since controversy exists on the action of GnRH agonists in the human ovary, we also tested the effect of triptorelin on IGF-II, IGF-binding protein-2 (IGFBP-2) and pregnancy-associated plasma protein-A (PAPP-A) in cultured human granulosa-lutein cells., Design: In vitro cell culture study in a research laboratory of a university hospital., Patients: Cells were obtained from patients treated with different protocols of COH. In addition to gonadotropins they received triptorelin or cetrorelix. Cells were treated with triptorelin, cetrorelix or ganirelix, 1 nmol/l each, for 48 h. IGF-II, IGFBP-2 and PAPP-A were measured by RIA and enzyme immunoassay respectively., Results: GnRHants and triptorelin did not affect IGF-II, IGFBP-2 or PAPP-A., Conclusions: We conclude that GnRHants do not exert any significant effects on the IGF system of granulosa-lutein cells and therefore their introduction into protocols of COH is unlikely to impair ovarian function.

The insulin-like growth factor (IGF) system is an important mediator of postnatal longitudinal growth, and the growth inhibiting effects of glucocorticoid (GC) treatment are suggested to be due to impaired action of the IGF system. However, the precise changes of the IGFs and the IGF-binding proteins (IGFBPs) in the growth plate, occurring upon short-term GC treatment have not been characterized. Prepubertal mice treated daily with dexamethasone (DXM) for 7 days, showed significant growth inhibition of total body length and weight and weight of the liver, thymus and spleen, whereas the weight of the kidneys was not affected. Analysis of the tibial growth plate showed that the total growth plate width significantly decreased to 84.5% of control values, caused by a significant decrease in the proliferative zone. The number of proliferating cell nuclear antigen (PCNA)-positive chondrocytes in the proliferative zone decreased significantly (to 40%) and TUNEL staining showed a significant 1.6-fold increase in apoptotic hypertrophic chondrocytes. In the growth plates, both IGF-I and IGF-II, as well as IGFBP-2 mRNAs were detected, mainly in the proliferative and prehypertrophic zones. DXM treatment significantly decreased the number of chondrocytes expressing IGF-I, whereas the number of chondrocytes expressing IGF-II and IGFBP-2 were not affected. The decrease in IGF-I expression in the growth plate indicates that GC treatment affects IGF-I at the local level of the growth plate, which could contribute to the GC-induced growth retardation.

Wound problems are common in the elderly. We hypothesized that age-related decrements in blood levels of components of the insulin-like growth factor (IGF) system are reflected in the wound environment. In this prospective, observational study IGF-I, IGF-II, IGF-binding protein-2, IGF-binding protein-3, and acid labile subunit were measured by immunoassays in the wound fluid and plasma of young (23.5 +/- 3.3 years) and elderly (78.9 +/- 6.2 years) patients before and daily for 4 days after elective surgery. IGFs, IGFBP-3, and acid labile subunit in plasma were significantly lower in the elderly group (p < 0.0001). The decrements of these proteins in plasma were reflected in corresponding decrements of 25-70% in the wound fluid of elderly patients (p < 0.0001). Additionally, bioavailability of IGF-I was less in the aged. The IGF parameters in the wound displayed a constant ratio with those of blood, suggesting that blood contributes a major share of the IGF that enters the wound during the initial phase of healing. The current data adds to accumulating evidence that a decline in the IGF system in aged patients contributes to the healing deficits observed in the elderly.

The effect of an acute energy deficit on the hormone balance of dominant follicles was studied in six normally-cycling, high-yielding Italian Friesian cows at 60 and 90 days after calving. At 60 days after calving, the cows, which had been fed according to their maintenance and production requirements, were synchronized and follicular fluid was collected from the dominant follicles under ultrasound guidance. At 90 days after calving, the same protocol was used on the same cows, which had been subjected to an acute dietary restriction since the day of the second prostaglandin treatment for synchronization. At the follicular level, the dietary restriction caused a significant reduction (P < 0.05) in the concentration of estradiol-17beta and a significant increase (P < 0.05) in NEFA. There were no significant differences in follicular diameter, follicular concentrations of progesterone, and Insulin-like Growth Factor-I (IGF-I). The amount of IGFBP2 and IGFBP3 in follicular fluid increased. The results suggest that an acute dietary restriction induces substantial changes at the dominant follicle level, despite the fact that the recruitment and selection phase occurred before the cows' diet was restricted.

In this study, we have cloned insulin-like growth factor binding protein (IGFBP)-2 from a marine hermaphroditic fish species, the gilthead sea bream (Sparus aurata), and determined its structure, ontogeny, tissue distribution, and hormonal regulation. The sea bream IGFBP-2 precursor consists of 286 amino acids, including a putative signal peptide of 22 residues and a mature protein of 264 residues. The overall sequence of sea bream preIGFBP-2 is 52-39% identical to that of zebrafish, chick, mouse, rat, pig, sheep, bovine, and human preIGFBP-2. The cysteine-rich N- and C-terminal domains, which are believed to be important for IGF binding, show a greater degree of conservation with a sequence identity of 73-56% and 53-50%, respectively. Sea bream IGFBP-2 contains 18 cysteine residues and their alignment is identical to those of other vertebrate IGFBP-2s. The Arg-Gly-Asp (RGD) sequence, present in the C-terminal domain of all known IGFBP-2, is also present in the sea bream. Northern blot analysis of RNA samples extracted from adult liver and developing larvae, revealed two transcripts of about 1.3 and 2.5 kb. RT-PCR analysis showed that IGFBP-2 was expressed in all adult tissues studied, with the highest levels found in liver and skin. IGFBP-2 transcripts were detected in gonad during reproductive cycle of S. aurata. Highest levels of IGFBP-2 mRNA were found in bisexual young gonads, and the levels decreased with gonad development and relatively high levels of IGFBP-2 mRNA were found in the ovary during spawning. By contrast, testicular IGFBP-2 mRNA levels were very low. RT-PCR detected IGFBP-2 mRNA throughout development in unfertilized eggs, embryos, and larvae, with highest levels observed after day 3 post-hatching, suggesting that this mRNA is the product of both the maternal and embryonic genomes. High variability in steady-state levels of hepatic IGFBP-2 mRNA was noted in adult fish, resulting in a statistically insignificant response to growth hormone treatment. These results suggest that the structure of IGFBP-2 is conserved in sea bream and that IGFBP-2 mRNA is expressed during early development and in gonad during the reproductive cycle, suggesting that it may play a role in gilthead sea bream development and reproduction.

Purpose: Although the illegal use of recombinant human growth hormone (rhGH) to enhance performance is increasing among athletes, no official test for its detection has yet been implemented. The aim of this work was to study how prolonged rhGH administration in trained subjects influences the insulin-like growth factor (IGF) system, in order to evaluate new methods in antidoping tests., Methods: Morning serum growth hormone (GH), IGF-I, IGF binding protein (BP)-2, IGFBP-3, IGF-I/IGFBP-2, and IGFBP-3/IGFBP-2 ratios were evaluated before, during (8th and 15th days), and at the end and after cessation (+3, +6, +9, +12, and +15 d) of a 3-wk treatment with different doses of rhGH (0.09 IU.kg BW(-1).d(-1) for 6 or 3 d a week, i.e., the A and B trials, respectively) in seven well-trained subjects not involved in competitive sports. The blood collections pre- and during treatment were performed immediately before the daily rhGH dose., Results: In both trials, significant increases of IGF-I (higher in the A trial) and IGFBP-3 serum concentrations during rhGH administration were observed. Serum IGFBP-3 remained significantly increased in the A trial 3 d after treatment cessation. In the A trial only, two subjects had IFG-I concentrations exceeding the upper limit of the reference range. No modifications of serum GH, IGFBP-2 and IGF-I/IGFBP-2, and IGFBP-3/IGFBP-2 ratios were observed. The z-score evaluation for IGFBP-3 detected GH exposure in 100% of subjects only at end treatment in A trial., Conclusion: Although IGF-I and IGFBP-3 seem potentially the most specific markers of rhGH assumption, our data suggest that for antidoping purposes a single evaluation of their absolute serum concentration is not a sufficiently secure method to detect rhGH abuse in all subjects, especially in the case of low rhGH doses.

The temporal relationships in the changes in concentrations of follicular fluid factors during follicle selection were characterized in mares. All follicles > or =5 mm were ablated 10 days after ovulation, followed by follicular fluid collection from the three largest follicles (F1, F2, and F3) when F1 of the new wave reached a diameter of 8.0-11.9, 12.0-15.9, 16.0-19.9, 20.0-23.9, 24.0-27.9, or 28.0-31.9 mm (n = 4-8 mares/range). Diameter deviation between F1 and F2 began during the 20.0- to 23.9-mm range, as indicated by a greater difference in diameter between the two follicles at the 24.0- to 27.9-mm range than at the 20.0- to 23.9-mm range. Androstenedione concentrations increased in F1, F2, and F3 between the 16.0- to 19.9- and 20.0- to 23.9-mm ranges. In contrast, estradiol, free insulin-like growth factor (IGF)-1, activin-A, and inhibin-A concentrations increased only in F1 beginning at the 16.0- to 19.9-mm range. As a result, the concentrations of all four factors were higher in F1 than in F2 and F3 at all the later ranges, including the 20.0- to 23.9-mm range (beginning of diameter deviation). Concentrations of progesterone differentially increased in F1, concentrations of androstenedione and IGF-binding protein (IGFBP)-2 increased only in F2 and F3, and concentrations of inhibin-B differentially decreased in F2 and F3 simultaneous with the beginning of deviation. Concentrations of FSH, LH, pro-alphaC inhibin, and total inhibin did not change differentially among follicles. Results indicated that, on a temporal basis, estradiol, free IGF-1, activin-A, and inhibin-A may have played a role in the initiation of follicle deviation. In addition, these four factors as well as progesterone, androstenedione, IGFBP-2, and inhibin-B may have been involved in the subsequent differential development of the follicles.

Cultured human umbilical vein endothelial cells (HUVEC) were used as a model to study transendothelial IGF-I transport, and its deposition into the extracellular matrix (ECM). Specific binding of (125)I-IGF-I to HUVEC monolayers was demonstrated, which was inhibited by aIR-3, a specific antibody directed against the IGF-I receptor. ECM-associated (125)I-IGF-I was approximately 10% of cell-bound IGF-I at 22 degrees C, and increased 4.5-fold at 37 degrees C, indicating that endothelial metabolism is required for the transport. However, neither monensin and cytochalasin B, both of which block endocytosis, nor aIR-3 did inhibit transport of (125)I-IGF-I into the ECM. In order to characterize IGF-I binding to the subendothelial ECM, HUVEC were removed nonenzymatically by treatment with Triton X-100 and ammonia. Specific, saturable binding of (125)I-IGF-I to the isolated ECM was observed, which was protease-sensitive. Antibodies directed against vitronectin inhibited IGF-I binding to the matrix by 35%, while antibodies directed against other ECM proteins had no significant influence on IGF-I binding. Using radioimmunoassays the IGF binding protein-2 was detected in the ECM, while IGFBP-1 and IGFBP-3 were below the detection limits. In order to evaluate functional aspects of IGF-I binding to the matrix, HUVEC were incubated under serum-free conditions in the absence and presence of IGF-I. Under serum-free conditions 48% of cells rounded up and started to detach after 2 hours incubation, while only 23% of the cells started to detach in the presence of IGF-I. These data indicate that IGF-I is transported via a paracellular route across endothelial cells, and becomes bound to the subendothelial ECM. Vitronection seems to be involved in binding of IGF-I to the ECM. ECM-associated IGF-I might play a role in endothelial cell survival and stability.

The presence of insulin-like growth factors (IGF), IGF binding proteins (IGFBP) and IGF receptor type 1 (IGF-IR) in the human corpus luteum was investigated by examining the expression and production of related proteins throughout the lifespan of the corpus luteum and the action of nitric oxide upon their production. The expression of proteins in corpora lutea from the early, mid-and late luteal phases was assessed by immunohisto-chemistry, evaluated by a semi-quantitative analysis and the functional study was performed in corpus luteum explants incubated with nitric oxide donors. IGF-I and -II and IGFBP-1 and -3 were measured in the culture media by specific immunoassays. The results showed that IGF-I and -II, IGFBP-1 to -6 and IGF-IR were detected in the human corpus luteum throughout the luteal phase. Moreover, the expression and production of IGF-I and IGFBP-1 increased progressively from corpora lutea from the early to late luteal phases (P < 0.05), whereas the expression and production of IGFBP-2, -4 and -5 were significantly higher in corpora lutea from the mid-luteal phase (P < 0.05). No differences were observed in the expression of IGF-II, IGFBP-3 and -6 and IGF-IR throughout the lifespan of the corpus luteum. However, functional studies showed that nitric oxide donors elicited a stimulatory action on production of IGF-I in corpora lutea from the early luteal phase (80%) and on production of IGFBP-1 in corpora lutea from the late luteal phase (50%) (P < 0.05), whereas production of IGF-II and IGFBP-3 was not affected by nitric oxide. In conclusion, the components of the IGF-IGFBP system are expressed in the human corpus luteum throughout its lifespan. Nitric oxide regulates IGF-I and IGFBP-1 production, indicating that the growth factors may serve, at least in part, as mediators of the action of nitric oxide in the human corpus luteum.

In contrast to the adult gut, the immature intestine is refractory to subcutaneously infused insulin-like growth factor I (IGF-I). IGF binding protein (IGFBP) mRNA expression was characterized in intestinal tissues from 6-, 19-, and 90-day-old rats to determine if changes in local expression could account for this age-related change in IGF-I potency. For all age groups, IGFBP-3 to -6, but not IGFBP-1 or -2, were detected by Northern blot analysis. IGFBP-3, -4, and -5 were more intensely expressed in the 6-day-old rat intestine compared with weanling or adult tissue. In contrast, IGFBP-6 expression peaked at the time of weaning. In situ hybridization showed IGFBP-3 to -6 expression was confined to cells of the lamina propria and submucosa and also in the muscularis layer for IGFBP-5. Furthermore, the pattern of IGFBP-5 localization in the intestine changed with development. The findings indicate that the expression of IGFBP-3 to -6 is higher in the immature intestine compared with the adult intestine, suggesting locally produced IGFBPs may inhibit systemically derived IGF-I action in the intestine. Therefore, changes to local IGFBP expression may contribute to the varying response of the rat intestine to IGF-I peptides during postnatal development.

Progesterone was used to reduce LH concentrations starting at the time when the largest follicle was > or = 5.7 mm in diameter or well before the expected start of follicle deviation (largest follicle > or = 8.5 mm in diameter). Plasma concentrations of LH, FSH and oestradiol were determined at 4 h intervals in control and progesterone-treated heifers (n = 8 per group). Concentrations of LH were lower (P < 0.05) in the progesterone-treated group, reflecting an absence of the transient increase in LH concentrations that encompasses follicle deviation. An increase in oestradiol and a continued decrease in FSH occurred at the start of follicle deviation in the control cows but not in the treated heifers. In a second experiment, follicular fluid of the two largest follicles of control and progesterone-treated heifers was sampled at the expected start of deviation (n = 8--10 per group). The concentrations of oestradiol, but not androstenedione and free insulin-like growth factor I (IGF-I), in follicular fluid were higher (P < 0.001) in the largest follicle than in the second largest follicle. Progesterone treatment reduced (P < or = 0.02) the concentrations of all three factors in follicular fluid and increased (P < 0.05) the concentrations of insulin-like growth factor binding protein 2 (IGFBP-2). These results confirm that oestradiol contributes to the continued decrease in FSH concentrations after the start of follicle deviation. Prevention of the transient LH increase, the oestradiol increase and the continued FSH decrease did not significantly alter the mean time or follicle diameters characteristic of expected follicle deviation. However, in some treated individuals (three of eight), the observed follicle deviation was delayed. In addition, these results indicate that the secretion of oestradiol into the circulation and the increase in oestradiol and IGF-I and decrease in IGFBP-2 concentrations in the follicular fluid at the start of deviation are functions of the transient increase in LH concentrations that encompasses follicle deviation.

To clarify the role of insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) in postnatal growth regulation, we crossed hemizygous CMV-IGFBP-2 transgenic mice with hemizygous PEPCK-bGH transgenic mice, which are characterized by serum GH levels in the range of 2 microgram/ml. Four genetic groups were obtained: animals carrying both transgenes (GB), the GH (G) or the IGFBP-2 transgene (B), and nontransgenic controls (C). Male offspring were analyzed for serum levels of IGF-I, for serum and tissue levels of IGFBP-2, and for body and organ growth. Serum IGF-I levels were 2- to 3-fold increased (P < 0.001) in the GH-overexpressing groups, with no difference between G and GB mice. Serum IGFBP-2 levels were 4- to 9-fold (P < 0.001) increased both in B and GB vs. C and G mice. Western immunoblot analysis did not reveal differences in tissue IGFBP-2 levels between B and GB mice. IGFBP-2 levels were highest in pancreas, followed by skeletal muscle, heart, kidney, brain, skin, and spleen. No elevation of IGFBP-2 was found in the liver. Body weight gain of G and GB mice was significantly increased vs. C and B mice, resulting in almost 2-fold increased body weights at the age of 15 weeks. However, there was a significant reduction in body weight of GB vs. G mice (17%; P < 0.001) and of B vs. C mice (13%; P < 0.05). This was primarily caused by a marked reduction of carcass weight (GB vs. G, 27%; B vs. C, 21%; P < 0.001). Measurements of nose-rump-length, organ (brain, heart, spleen, liver, pancreas, kidney), and tissue weights (skin, carcass, abdominal fat) in 5- and 15-week-old mice revealed several indications that the growth-inhibiting effect of IGFBP-2 overexpression was more marked in high-GH/IGF-I mice: 1) At 5 weeks of age, GB mice displayed a significant reduction of all growth parameters except for the weight of abdominal fat, when compared with G mice, whereas only brain weight was significantly reduced in B vs. C mice. 2) In 15-week-old animals, a significant reduction in all growth parameters, except for spleen and abdominal fat weights, was seen in GB vs. G mice, whereas only nose-rump-length and the weights of carcass and brain were significantly reduced in B vs. C mice. Our study demonstrates, for the first time, the potential of IGFBP-2 to inhibit GH-stimulated growth in giant transgenic mice, providing further evidence for an inhibitory effect of this IGFBP in vivo.

The objective of this study was to examine the quality of successive dominant follicles (DFs) after induced heat stress. Non-lactating dairy cows expressing estrus at normal intervals were allocated randomly to heat stress (HS; n=8) and control (C; n=8) groups. Cows received GnRH (100 microg, i.m.) on Day 0, a progesterone CIDR-B device on Day 4 and prostaglandin (PGF(2alpha); 25mg, i.m.) on Day 7 upon removal of the CIDR device. The DF and follicles >5mm were aspirated on Day 8, and GnRH (100 microg) injected following aspiration, to initiate a new follicular wave. In this manner, a DF was aspirated every 8 days (one "follicular cycle") for 10 cycles. After the first follicular cycle, HS cows were placed in environmental chambers for 7 days during the second follicular cycle (8h per day at 43.3 degrees C set point and 16h per day at 24 degrees C for 4 days, and 8h per day at 43.3 degrees C set point and 16h per day at 32.2 degrees C set point for 3 days; relative humidity, 40%) and thereafter maintained outdoors with control cows at a mean ambient temperature (18.5 degrees C; range 12.7-26 degrees C). Rectal temperature increased (P<0.001) in HS as compared with C cows (39.28+/-0.01 degrees C versus 38.78+/-0.01 degrees C). Concentrations of estradiol (E(2); 1662+/-189 versus 1493+/-188ng/ml) and progesterone (P(4); 44.7+/-5 versus 54.1+/-5.1ng/ml) in follicular fluid (FF) of DF did not differ between C and HS treatments, respectively. Total FF protein concentration was greater (P<0.05) in HS (99.7+/-2.3mg/ml) than in C (92.7+/-2.3mg/ml). Heat shock protein 90 (Hsp 90) in FF was not altered by heat stress. IGF-II ligand blots were conducted with FF samples (n=79) from four HS and four C cows. There was a predominance of IGFBP-3 in 76 of 79 FF samples, indicating healthy follicular status, and only three FF samples had the lower molecular weight IGFBP-2 indicative of a poor quality follicle. Plasma P(4) and E(2) concentrations did not differ between C and HS groups. The number of class 1 and 3 follicles increased during and just after heat stress, but the number of class 2 follicles did not differ between C and HS cows. Heat stress appeared to induce a decrease in follicular dominance, but GnRH-induced follicular cycles resulted in development of healthy preovulatory follicles in both groups.