Expression, genetic localization and phylogenic analysis of NAPlr in piscine Streptococcus dysgalactiae subspecies dysgalactiae isolates and their patterns of adherence.

Streptococcus dysgalactiae, the long recognized mammalian pathogen, has currently received a major concern regarding fish bacterial infection. Adhesion to host epithelial cells and the presence of wall-associated plasminogen binding proteins are prerequisites to Streptococcus infection. This is the first study of the occurrence of nephritis-associated plasminogen-binding receptor (NAPlr) and α-enolase genes in piscine S. dysgalactiae subspecies dysgalactiae (SDSD) isolates. Further characterization of surface localized NAPlr of fish SDSD revealed a similar immune-reactive band of 43 KDa as that from porcine S. dysgalactiae subsp. equisimilis (SDSE). The phylogenetic analysis revealed that NAPlr of fish SDSD is more associated with those of mammalian SDSE and Streptococcus pyogenes rather than of other streptococci. Our findings warrant public attention to the possible implication of these virulence genes in dissemination of SDSD to different tissues of infected hosts and to get advantage to new niches. The SDSD adherence patterns were also studied to better understand their pathogenicity. The patterns of adherence of SDSD on two different cell lines showed a different pattern of adherence. Such difference gives an insight about the variance in host susceptibility to infection.

5-aminolevulinic acid combined with sodium ferrous citrate ameliorates H2O2-induced cardiomyocyte hypertrophy via activation of the MAPK/Nrf2/HO-1 pathway.

Hydrogen peroxide (H2O2) causes cell damage via oxidative stress. Heme oxygenase-1 (HO-1) is an antioxidant enzyme that can protect cardiomyocytes against oxidative stress. In this study, we investigated whether the heme precursor 5-aminolevulinic acid (5-ALA) with sodium ferrous citrate (SFC) could protect cardiomyocytes from H2O2-induced hypertrophy via modulation of HO-1 expression. HL-1 cells pretreated with/without 5-ALA and SFC were exposed to H2O2 to induce a cardiomyocyte hypertrophy model. Hypertrophy was evaluated by planar morphometry, (3)H-leucine incorporation, and RT-PCR analysis of hypertrophy-related gene expressions. Reactive oxygen species (ROS) production was assessed by 5/6-chloromethyl-2',7'-ichlorodihydrofluorescein diacetate acetylester. HO-1 and nuclear factor erythroid 2-related factor 2 (Nrf2) protein expressions were analyzed by Western blot. In our experiments, HL-1 cells were transfected with Nrf2 siRNA or treated with a signal pathway inhibitor. We found several results. 1) ROS production, cell surface area, protein synthesis, and expressions of hypertrophic marker genes, including atrial natriuretic peptide, brain natriuretic peptide, atrial natriuretic factor, and ß-myosin heavy chain, were decreased in HL-1 cells pretreated with 5-ALA and SFC. 2) 5-ALA and SFC increased HO-1 expression in a dose- and time-dependent manner, associated with upregulation of Nrf2. Notably, Nrf2 siRNA dramatically reduced HO-1 expression in HL-1 cells. 3) ERK1/2, p38, and SAPK/JNK signaling pathways were activated and modulate 5-ALA- and SFC-enhanced HO-1 expression. SB203580 (p38 kinase), PD98059 (ERK), or SP600125 (JNK) inhibitors significantly reduced this effect. In conclusion, our data suggest that 5-ALA and SFC protect HL-1 cells from H2O2-induced cardiac hypertrophy via activation of the MAPK/Nrf2/HO-1 signaling pathway.

Permanent acceptance of mouse cardiac allografts with CD40 siRNA to induce regulatory myeloid cells by use of a novel polysaccharide siRNA delivery system.

The CD40/CD154 co-stimulatory pathway is crucial in alloimmune response. We developed a novel small interfering RNA (siRNA) delivery system with a poly-dA extension at the 5'-end of the siRNA sense strand that was stably incorporated into 1,3-ß-glucan (schizophyllan, SPG). This was captured and incorporated into dendritic cells (DCs) through its receptor, Dectin-1, specifically silencing CD40 genes (siCD40) to exert immunoregulatory activity. siCD40/SPG-treated CBA mice permanently accepted B10 fully mismatched cardiac allografts. Consistent with graft survival, the infiltration of CD4(+), CD8(+) T cells into the graft was lower, and that the numbers of CD40(low)CD11c(+) DCs cells and CD4(+)Foxp3(+)cells were increased in both the graft and in the recipient spleen. In addition, naive CBA recipients given an adoptive transfer of splenocytes from the primary recipients with siCD40/SPG accepted a heart graft from donor-type B10, but not third-party Balb/c mice. In conclusion, the treatment with siCD40/SPG targeting DCs could generate antigen-specific Tregs, resulting in the permanent acceptance of mouse cardiac allografts. These findings have important implications for clarifying the mechanism underlying the induction of tolerance in DCs, and also highlight the potential of immunomodulation and the feasibility of siRNA-based clinical therapy in the transplantation field.

Development of a novel, nearly insoluble antiadhesive membrane.

BACKGROUND: Sodium hyaluronate/carboxymethylcellulose (HA/CMC) is difficult to use in a moist environment because of its susceptibility to moisture. METHODS: We developed the three-layered nDM-14R membrane. The surface layers are composed of 1-lactide, glycolide and e-caprolactone copolymers. HA/CMC and nDM-14R were used in all these studies. (1) The central region of 1 × 10 cm specimens (n = 5) was moistened for 0, 5, 10, 20, 30 or 60 s, after which the tensile strength was determined; (2) one side of specimens of 1 × 10 cm (n = 5) was moistened with agar gel for 5, 10, 15 or 30 s, after which the adhesion strength was determined, and (3) Rat cecum (n = 10) was scratched, 3 × 3 cm specimens were placed on the scratched area, and adhesions were evaluated on postoperative day 14. RESULTS AND CONCLUSION: (1) The tensile strength of nDM-14R after contact for 10-30 s was greater than that of HA/CMC. (2) The adhesive strength of HA/CMC after contact for 5-10 s was greater than that of nDM-14R. (3) Adhesion scores in treatment groups were significantly lower than in the control group. The results suggest that nDM-14R has the same antiadhesive effect and allows easier placement under moist conditions than HA/CMC.

PD-1/B7-H1 interaction contribute to the spontaneous acceptance of mouse liver allograft.

The programmed death-1 (PD-1)/B7-H1 pathway acts as an important negative regulator of immune responses. We herein investigated the role of the PD-1/B7-H1 pathway in establishing an immunological spontaneous tolerance status in mouse liver allografting. B7-H1 is highly expressed on the donor-derived tissue cells and it is also associated with the apoptosis of infiltrating T cells in the allografts. Strikingly, a blockade of the PD-1/B7-H1 pathway via anti-B7-H1mAb or using B7-H1 knockout mice as a donor led to severe cell infiltration as well as hemorrhaging and necrosis, thus resulting in mortality within 12 days. Furthermore, the expression of the FasL, perforin, granzyme B, iNOS and OPN mRNA in the liver allografts increased in the antibody-treated group in comparison to the controls. Taken together, these data revealed that the B7-H1 upregulation on the tissue cells of liver allografts thus plays an important role in the apoptosis of infiltrating cells, which might play a critical role of the induction of the spontaneous tolerance after hepatic transplantation in mice.

Clinicopathological manifestations and treatment of intestinal transplant-associated microangiopathy.

Intestinal transplant-associated microangiopathy (i-TAM) is an important complication after allogeneic hematopoietic SCT. From 1997 to 2006, 87 of 886 patients with diarrhea after transplantation received colonoscopic biopsy. i-TAM, GVHD and CMV colitis were diagnosed histopathologically. The median duration from transplantation to the onset of diarrhea was 32 days (range: 9-130 days) and that from the onset of diarrhea to biopsy was 12 days (range: 0-74 days). The median maximal amount of diarrhea was 2 l/day (range: 130-5600 ml/day). Histopathological diagnosis included i-TAM (n=80), GVHD (n=26), CMV colitis (n=17) and nonspecific findings (n=2) with overlapping. Among 80 patients with i-TAM, abdominal pain was a major symptom, and only 11 patients fulfilled the proposed criteria for systemic TAM. Non-relapse mortality (NRM) among patients without resolution of diarrhea was 72% and i-TAM comprised 57% of NRM. NRM was 25% among patients without intensified immunosuppression, but was 52, 79 and 100% among those with intensified immunosuppression before diarrhea, after diarrhea, and before and after diarrhea, respectively. In conclusion, i-TAM is a major complication presenting massive refractory diarrhea and abdominal pain, which causes NRM. Avoiding intensified immunosuppression that damages vascular endothelium until the resolution of i-TAM may improve transplant outcome.

New highly potent and specific E6 and E7 siRNAs for treatment of HPV16 positive cervical cancer.

Persistent infection by high-risk types of human papillomaviruses (HPV) is a necessary cause of cervical cancer, with HPV16 the most prevalent, accounting for more than 50% of reported cases. The virus encodes the E6 and E7 oncoproteins, whose expression is essential for maintenance of the malignant phenotype. To select efficacious siRNAs applicable to RNAi therapy for patients with HPV16+ cervical cancer, E6 and E7 siRNAs were designed using siDirect computer software, after which 10 compatible with all HPV16 variants were selected, and then extensively examined for RNAi activity and specificity using HPV16+ and HPV16-cells. Three siRNAs with the highest RNAi activities toward E6 and E7 expression, as well as specific and potent growth suppression of HPV16+ cancer cells as low as 1 nM were chosen. Growth suppression was accompanied by accumulation of p53 and p21(WAF1/CIP1), as well as morphological and cytochemical changes characteristic of cellular senescence. Antitumor activity of one of the selected siRNAs was confirmed by retarded tumor growth of HPV16+ cells in NOD/SCID mice when locally injected in a complex with atelocollagen. Our results demonstrate that these E6 and E7 siRNAs are promising therapeutic agents for treatment of virus-related cancer.

Intestinal thrombotic microangiopathy induced by FK506 in rats.

Thrombotic microangiopathy (TMA) is one of the severe complications after stem cell transplantation (SCT) and is associated with graft-versus-host disease (GVHD) prophylaxis including FK506. In this study, we experimented on rats using FK506 to demonstrate the occurrence of intestinal TMA. FK506 was administrated into Wistar/ST rats intraperitoneally for 7 days. Rats were examined histopathologically after FK506 injection using light and electron microscopy and immunohistochemistry. FK506 concentrations in whole blood were measured by enzyme immunoassay. In the acute phase, hemorrhagic lesions with multifocal erosions and crypt loss were found in the small intestines of all treated rats. Capillary vessels were dilated, and a few platelet thrombi were found. Electron microscopy demonstrated degenerative swelling of endothelial cells and platelet aggregates adhering to the vessel walls. In the later phase, epithelial regenerative failure, characterized by crypt ghosts, was found in the affected mucosa. Apoptotic epithelial cells were increased in number. The extent of intestinal injury was proportional to the whole blood levels of FK506. The intestinal lesions in rats were consistent with TMA and induced by the injection of FK506 alone. Apoptotic enteropathy was also observed and similar to intestinal GVHD. In this study, we established an intestinal TMA model induced by immunosuppressant (Tacrolimus) only without irradiation.

Differential-display analysis of gene expression in livers from normal and partially hepatectomized mice.

Partial hepatectomy, resulting in the removal of approximately 70% of the liver, is widely utilized for studies of liver growth in experimental animals. The regenerative response is proportional to the amount of liver removed. Knowing when and where genes are expressed provides a strong clue as to its biological role. The RNA differential-display (DD) technique facilitates monitoring the differential expression of a large number of activated or suppressed genes under various biological conditions. To reveal mechanisms of liver regeneration, we performed a comparative analysis of gene expression during liver regeneration using DD. We sacrificed male Balb/c mice, aged 10 to 12 weeks, at 0, 24, 48, and 72 hours, and 1 and 2 weeks after PHx. The livers were weighed, and the amount of glutamic-oxaloacetate transaminase in serum measured. We extracted the total RNA from frozen liver tissue and confirmed the RNA quality using a lab-chip system. DD analysis was performed essentially as described by Liang and Pardee. Semiquantitative reverse-transcription polymerase chain reaction was performed to confirm the results of DD analysis. Of the 56 fragments that exhibited changed expression levels during PHx, 39 were cloned and sequenced. There were 31 known genes, 13 unknown genes, and 9 expressed-sequence tags. These results indicated that DD is a powerful approach for monitoring molecular events in the regenerating liver.

Exogenous expression of Fas-ligand or CrmA prolongs the survival in rat liver transplantation.

Modulation of donor organs by transfection of a gene encoding immmunosuppresive molecules has been recognized as a less toxic approach to prevent allograft rejection. Fas-ligand (FasL) plays a critical role in activation-induced cell death of activated cytotoxic lymphocytes. This may provide a potential for induction of "immune privileged sites" to escape the host immune surveillance system. Cytokine response modifier A (CrmA), a gene product of cowpox virus, blocks caspase as well as perforin/granzyme-mediated apoptotic pathways. Therefore, it may suppress intragraft apoptosis. The aim of the present study was to investigate whether transfection of FasL or CrmA genes prolonged the survival of rat liver allografts. Using the high responder rat combination of DA (RT-1(a)) donor to LEW (RT-1(1)) recipient, we performed orthotopic liver transplantation with subsequent delivery of adenoviral vectors containing FasL, CrmA, or LacZ, at a dose of 1 x 10(9) pfu via a recipient tail vein using a Cre-mediated gene expression system. Recipient survival was assessed as well as immunohistochemical examination of the grafts for anti-CD2, TUNEL, and H&E staining. Statistical analysis was performed with the Mann-Whitney U test. The therapeutic groups showed significantly prolonged recipient survival compared with the LacZ-treated control group. Histologic analysis revealed reduced hepatocyte apoptosis in the CrmA-treated group and increased apoptosis of infiltrating mononuclear cells in the FasL-treated group. These data suggested that FasL and CrmA may be potent genes to prolong rat liver allograft survival.

The reduction of hypoxia-induced and reoxygenation-induced apoptosis in rat islets by epigallocatechin gallate.

The survival of transplanted tissue is affected by the detrimental consequences of hypoxia followed by reoxygenation. The majority of transplanted cells undergo apoptosis due to hypoxia and reoxygenation (H/R) injury, but protection from H/R has been less examined. In this study, we examined whether epigallocatechin gallate (EGCG) protected rat islets from H/R injury. Rat islets, freshly prepared from F344 rat strain by collagenase digestion and density centrifugation, were seeded in triplicate at concentrations of 100 per well in 24-well plates for culture under normoxia. The cells were then exposed to hypoxia for 14 hours with or without EGCG, after which they were reoxygenated for 72 hours in a humidified oxygenated CO(2) incubator at 37 degrees C. Apoptosis, lactate dehydrogenase (LDH), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) were evaluated according to the manufacturer's instructions. The H/R induced apoptosis in the islets that was reduced in dose-dependent manner by EGCG treatment. The viability of islets exposed to H/R was assessed by LDH release. H/R reduced viability compared with the controls, while the viability of the islets improved upon EGCG treatment. The secretion of insulin was also decreased by H/R, as well as the dose dependent EGCG protective ability on insulin secretion. The content of 8-OHdG in islets from H/R was also reduced by EGCG. Our results indicated that apoptosis and the decline in insulin secretion by H/R were inhibited by EGCG treatment. EGCG may be considered useful for protection of islets from oxidative injury associated with the transplantation procedure.

Influence of the numbers of islets on the models of rat syngeneic-islet and allogeneic-islet transplantations.

One of the main barriers to widespread application of islet transplantation is the limited availability of human pancreatic islets. The reduction of graft islet mass for transplantation to a recipient is one of the strategies in islet transplantation. However, transplantation of only a small number of islets may result in primary nonfunction. To optimize the sites and numbers of islets for transplantation, we analyzed these factors using pancreatic islets from Lewis or F344 rats transplanted into rats rendered diabetic by streptozotocin (50 mg/kg IV) and confirmed as such prior to transplantation (>300 mg/dL blood glucose). Approximately 500 to 1500 islets were injected via the portal vein or under the renal capsule into the diabetic F344 rats. The blood glucose level of all animals bearing 1500 syngeneic or allogeneic islets transplanted to the liver or under the kidney capsule exhibited restored normoglycemia (<200 mg/dL) at 1 day after transplantation. Graft function deteriorated after only 3 days in three animals (5.8%). The loss of graft function after 3 days occurred in 10 of 28 rats transplanted with 1000 to 1200 syngeneic islets, 4 of 19 rats transplanted with 800 to 900 syngeneic islets, and 7 of 17 rats transplanted with 500 to 600 syngeneic islets. There was no significant difference in the loss of graft function between the sites of transplantation via portal vein or under the kidney capsule. In conclusion, higher frequencies of primary nonfunction occurred with less than 1500 islets transplanted. They were independent of the sites in the rat-islet transplantation model.

CrmA gene expression protects mice against concanavalin-A-induced hepatitis by inhibiting IL-18 secretion and hepatocyte apoptosis.

Activated cytotoxic T-cell-mediated hepatocyte apoptosis via Fas/Fas-ligand and perforin/granzyme pathways are believed to involve the model of concanavalin A (ConA)-induced hepatitis. The purpose of the present study is to investigate whether the cytokine response modifier A (crmA) gene effectively inhibits the hepatocyte apoptosis of ConA-induced hepatitis. We examined survival rates, liver pathology, immune histological changes, and cytokine profiles from mice receiving the recombinant adenovirus vectors containing cre and/or crmA genes, transferred to the liver 3 days before ConA injection, and a crmA gene nonexpression control group. Injection of ConA into mice rapidly led to massive hepatocyte apoptosis, and infiltration of leukocytes, especially CD11b(+) inflammatory cells. In contrast, liver damage was dramatically reduced in the mice that expressed the crmA gene. However, infiltration by CD4(+) cells was not affected. The survival of the mice increased significantly to 100% in the treated group versus the control group. Furthermore, we demonstrated that interleukin (IL)-18 plays an important role in ConA-induced hepatitis, and that crmA expression significantly inhibited IL-18 secretion. Our results showed that the crmA gene effectively inhibits apoptosis induced by ConA hepatitis. This indicates a potential therapeutic usage of crmA for protection from cellular damage due to hepatitis.

Antagonism of ghrelin receptor reduces food intake and body weight gain in mice.

BACKGROUND AND AIMS: Ghrelin, an endogenous ligand for growth hormone secretagogue receptor (GHS-R), is an appetite stimulatory signal from the stomach with structural resemblance to motilin. We examined the effects of the gastric peptide ghrelin and GHS-R antagonists on energy balance and glycaemic control in mice. MATERIALS AND METHODS: Body weight, fat mass, glucose, insulin, and gene expression of leptin, adiponectin, and resistin in white adipose tissue (WAT) were measured after repeated administrations of ghrelin under a high fat diet. Gastric ghrelin gene expression was assessed by northern blot analysis. Energy intake and gastric emptying were measured after administration of GHS-R antagonists. Repeated administration of GHS-R antagonist was continued for six days in ob/ob obese mice. RESULTS: Ghrelin induced remarkable adiposity and worsened glycaemic control under a high fat diet. Pair feeding inhibited this effect. Ghrelin elevated leptin mRNA expression and reduced resistin mRNA expression. Gastric ghrelin mRNA expression during fasting was increased by a high fat diet. GHS-R antagonists decreased energy intake in lean mice, in mice with diet induced obesity, and in ob/ob obese mice; it also reduced the rate of gastric emptying. Repeated administration of GHS-R antagonist decreased body weight gain and improved glycaemic control in ob/ob obese mice. CONCLUSIONS: Ghrelin appears to be closely related to excess weight gain, adiposity, and insulin resistance, particularly under a high fat diet and in the dynamic stage. Gastric peptide ghrelin and GHS-R may be promising therapeutic targets not only for anorexia-cachexia but also for obesity and type 2 diabetes, which are becoming increasingly prevalent worldwide.

Orexin reverses cholecystokinin-induced reduction in feeding.

AIM: This study was designed to investigate the effect of orexin on anorexia induced by cholecystokinin (CCK),a peripheral satiety signal. METHODS: We administered orexin A (0.01-1 nmol/mouse) and CCK-8 (3 nmol/mouse) to mice. Food intake was measured at different time-points: 20 min, 1, 2 and 4 h post-intracerebroventricular (i.c.v.) or intraperitoneal (i.p.) administrations. RESULTS: Intracerebroventricular-administered orexin significantly increased food intake in a dose-dependent manner. The inhibitory effect of i.p.-administered CCK-8 on food intake was significantly negated by the simultaneous i.c.v. injection of orexin in a dose-dependent manner. CONCLUSIONS: Orexin reversed the CCK-induced loss of appetite. Our results indicate that orexin might be a promising target for pharmacological intervention in the treatment of anorexia and cachexia induced by various diseases.

Significance of fetal hemoglobin-containing erythroblasts (F blasts) and the F blast/F cell ratio in myelodysplastic syndromes.

To investigate the relationship between the fetal hemoglobin-containing erythroblasts (F blasts) and apoptosis in myelodysplastic syndromes (MDS), we immunohistochemically assessed F blasts, F cells, and apoptosis in 137 patients with MDS. A marked increase in the number of F blasts in the bone marrow was identified in 116 of 137 patients (84.7%), and the number of F cells was elevated in 54 patients (39.4%). Among the erythroblasts stained by anti-glycophorin C antibody, the mean percentage of F blasts was 14.63 +/- 9.17% in MDS, which was significantly higher than that in non-MDS patients with stress erythropoiesis (4.82 +/- 3.35%, P < 0.01), although there were no significant differences in the number of F cells between these groups. In particular, 62 of the 137 MDS patients (45.3%) had an apparent increase in F blasts but no elevation of F cells. The apoptotic rate was significantly higher in the patients with a F blast/F cell (Fb/Fc) ratio >or=5.0 than in those with a Fb/Fc ratio <1.0 (P < 0.01). The results indicate that F cell precursors are incapable of maturing into functioning end-stage F cells, presumably owing to apoptotic cell death. The measurement of F blasts in the bone marrow is needed for the precise evaluation of fetal-type erythropoiesis in MDS.

Novel human and mouse genes encoding an acid phosphatase family member and its downregulation in W/W(V) mouse jejunum.

BACKGROUND AND AIMS: Interstitial cells of Cajal (ICC) are pacemakers and mediators of motor neurotransmission in gastrointestinal smooth muscles. ICC require cellular signalling via Kit, a receptor tyrosine kinase, for development and maintenance of phenotype. Much of the evidence demonstrating the functions of ICC comes from studies of W/W(V) mice, which have reduced Kit function and reductions in specific populations of ICC. The aim of the present study was to differentially examine gene expression in the small intestines of wild-type and W/W(V) mutant mice. METHODS AND RESULTS: RNA from the jejunums of wild-type and W/W(V) mutants was analysed using a differential gene display method. Eighteen queries were identified as novel genes that were differentially displayed in wild-type and W/W(V) mice. One candidate gene, encoding a novel acid phosphatase-like protein, was significantly suppressed in fed and starved W/W(V) mice. The full length clone of the murine gene and its human counterpart were designated acid phosphatase-like protein 1 (ACPL1). Human ACPL1 cDNA encodes a protein of 428 amino acids with homology to human prostatic acid phosphatase protein. This gene is located at 1q21. ACPL1 was abundantly expressed in the human small intestine and colon. Gene products were found to be cytoplasmic in transfected COS-7 cells. Reverse transcription-polymerase chain reaction analysis revealed expression of ACPL1 mRNA within single isolated ICCs. CONCLUSIONS: Gene analysis showed that ACPL1 was differentially expressed in the small intestines of normal and W/W(V) mice. ICC within the small intestine expressed mRNA for ACPL1. Specific downregulation of ACPL1 in the jejunums of W/W(V) mice and high expression in human intestinal tissue suggest that the ACPL1 gene could be associated with ICC function in mice and humans.

Insulin-like growth factor I enhances gonadotropin-releasing hormone-stimulated luteinizing hormone release from bovine anterior pituitary cells.

The role of insulin-like growth factor I (IGF-I) in the release of luteinizing hormone (LH) is unclear in ruminants. In the present study, the effects of IGF-I on the release of LH stimulated by gonadotropin-releasing hormone (GnRH) were examined in primary cultures of bovine anterior pituitary (AP) cells, and the interaction between estradiol-17beta (E(2)) and IGF-I was characterized. GnRH(100nM)-stimulated LH release from the cultured cells was increased (P<0.05) 12, 24 and 36h after addition of IGF-I (250ng/ml), with a maximum at 12h (48.4ng/ml media versus 35.4ng/ml media in controls). IGF-I at concentrations of 25, 250 and 500ng/ml increased the release by 18.7, 24.2 and 28.9%, respectively (P<0.05), when compared with controls (37.2ng/ml media). E(2) (10nM), IGF-I (250ng/ml) and combined treatment of E(2) plus IGF-I also induced significant increases in LH release (P<0.05). The amounts of LH release after treatment with E(2) alone was 37.3% greater than with IGF-I alone (39.0ng/ml media versus 28.4ng/ml media) (P<0.05). When E(2) and IGF-I were added together (45.6ng/ml media), the release of LH was significantly greater than with either E(2) alone or IGF-I alone (P<0.05). E(2) (10nM) significantly (P<0.05) increased the amount of GnRH bound to the cells by 51.6% when compared with controls, however, IGF-I (250ng/ml) failed to increase GnRH binding. These results show that IGF-I enhances GnRH-stimulated LH release without changing the number of GnRH receptors in cattle, and IGF-I interacts with E(2) to increase the response to GnRH.