EGFRvIII-mediated transactivation of receptor tyrosine kinases in glioma: mechanism and therapeutic implications.

A truncation mutant of the epidermal growth factor receptor, EGFRvIII, is commonly expressed in glioma, an incurable brain cancer. EGFRvIII is tumorigenic, in part, through its transactivation of other receptor tyrosine kinases (RTKs). Preventing the effects of this transactivation could form part of an effective therapy for glioma; however, the mechanism by which the transactivation occurs is unknown. Focusing on the RTK MET, we show that MET transactivation in U87MG human glioma cells in vitro is proportional to EGFRvIII activity and involves MET heterodimerization associated with a focal adhesion kinase (FAK) scaffold. The transactivation of certain other RTKs was, however, independent of FAK. Simultaneously targeting EGFRvIII (with panitumumab) and the transactivated RTKs themselves (with motesanib) in an intracranial mouse model of glioma resulted in significantly greater survival than with either agent alone, indicating that cotargeting these RTKs has potent antitumor efficacy and providing a strategy for treating EGFRvIII-expressing gliomas, which are usually refractory to treatment.

Glioma-specific Domain IV EGFR cysteine mutations promote ligand-induced covalent receptor dimerization and display enhanced sensitivity to dacomitinib in vivo.

A feature of many gliomas is the amplification of the epidermal growth factor receptor (EGFR), resulting in its overexpression. Missense mutations or deletions within the extracellular domain are associated with this amplification and can lead to constitutive activation of the receptor, with the Domain I/II deletion, EGFRvIII, being the most common. These changes have also been associated with increased sensitivity to EGFR inhibition using small molecule inhibitors. We have expressed, in human glioma cells, EGFR containing four glioma-specific EGFR missense mutations within Domain IV (C620Y, C624F, C628Y and C636Y) to analyze their biological properties and sensitivity to EGFR inhibition. One of these mutants, C620Y, exhibited an enhanced basal phosphorylation, which was partially dependent on an EGFR-ligand autocrine loop. All Domain IV mutants responded equally as well as wildtype EGFR (wtEGFR) to ligand stimulation. Biochemical analysis revealed that a pre-formed, disulfide-bonded dimer associated with these mutations was underglycosylated, inactive and cytoplasmically retained. Ligand stimulation resulted in the formation of a tyrosine-phosphorylated, disulfide-bonded dimer for all Domain IV mutants but not for wtEGFR. Following treatment with the next-generation, irreversible pan-ErbB inhibitor dacomitinib, the C620Y, C624F and EGFRvIII mutants were inactivated, covalently dimerized and were retained in the cytoplasm, resulting in cell-surface receptor loss and, for C620Y and C624F, decreased binding of EGF. Dacomitinib treatment significantly reduced the in vivo growth of human glioma xenografts bearing C620Y, but not wtEGFR. Collectively, these data indicate that the unique biochemical traits of Domain IV EGFR cysteine mutants can be exploited for enhanced sensitivity to EGFR small molecule inhibitors, with potential clinical applications.

The expanding role of recombinant gonadotropins in assisted reproduction.

Using recombinant gonadotropins for assisted reproduction of domestic species is still in its infancy. Yet, the purity, potency and pathogen-free nature of recombinant gonadotropins make them attractive alternatives to tissue-derived gonadotropic agents. In this study, the authors summarize the work to date using recombinant gonadotropins to enhance the - fertility of domestic animals and they discussed their recent studies examining the biopotency of single chain analogues of human gonadotropins. In these studies, single chain analogues of follicle stimulating hormone (Fc alpha), chorionic gonadotropin (CG beta alpha) or a gonadotropin construct with dual activity (FcCG beta alpha) were administered to sheep pre-treated with antisera directed against GnRH. Ovulation was induced 3 days after analogue administration using hCG (1000 IU, iv). Although Fc alpha or CG beta alpha alone induced only modest oestradiol production during the pre-hCG period, serum concentrations of oestradiol were markedly increased (p < 0.05) 3 days after administration of FcCG beta alpha or the Fc alpha + CG beta alpha combination. Final ovarian weight was significantly increased (p < 0.05) in animals receiving Fc alpha, Fc alpha + CG beta alpha or FcCG beta alpha. Collectively, these observations demonstrate that the single chain analogues of the human gonadotropins are active in sheep.

Dietary restriction reduces the rate of estradiol clearance in sheep (Ovis aries).

Three experiments were designed to test the effect of dietary restriction on clearance of 17beta-estradiol (E(2)) in sheep. A preliminary experiment examined the effect of a 4-d fast on the rate of E(2) clearance in wethers. The second experiment tested the hypothesis that either long-term restriction (7 wk) or a 5-d fast would increase steroid-binding capacity of serum by increasing the concentration of sex hormone-binding globulin (SHBG) in the blood of ovariectomized ewes. In Exp. 3, we hypothesized that nutrition-dependent regulation of E(2) clearance by the liver would result in divergence in biliary extraction of E(2) in fed and fasted wethers receiving comparable levels of exogenous E(2). A marked difference in E(2) clearance between fed and fasted wethers was noted in the preliminary study. Relative to ad libitumfed wethers, a 4-d fast decreased E(2) clearance by 52%. Serum concentrations of SHBG were increased in long-term energy-restricted and fasted ewes, relative to the concentration in maintenancefed ewes (P = 0.015). Furthermore, a 5-d fast nearly doubled serum steroid-binding capacity in wethers. The E(2) concentration in bile was 2 times greater in fasted than in fed wethers. This fasting-dependent increase in biliary E(2) may be reflective of the increased serum E(2) in fasted animals, because each 1 pg/mL increase in serum E(2) increased bile E(2) by 0.86 +/- 0.12 pg/mL, independent of nutrition (P = 0.002). Our results demonstrate that the rate of clearance of E(2) is decreased during nutritional restriction. Additionally, these data indicate that altered SHBG expression, enterohepatic recirculation, or both are involved in the decreased E(2) clearance during dietary restriction.

Aggressive behavior is reduced in bulls actively immunized against gonadotropin-releasing hormone.

The purpose of this research was to compare the frequency of aggressive behavior's in beef bulls actively immunized against gonadotropin-releasing hormone relative to contemporary nonimmunized control bulls and surgically castrated steers. Eight males were assigned to each ofthese treatments in each of 4 yr. Immunized males were treated with a GnRH-keyhole-limpet hemocyanin (KLH) conjugate at approximately 4 mo of age. A secondary (booster) immunization was administered at 12 mo. Steers were castrated at 4 mo of age. Animals in each treatment in each year were housed as a single group prior to testing. At approximately 16 mo of age, each group of eight animals was placed in a 10- x 16-m enclosure for 20 min on five occasions at 2 to 3 d intervals. An observer recorded butts initiated by each animal as well as participation in bouts of sparring. Relative to control bulls, immunocastration reduced the frequency of butts initiated (P < 0.05) and participation in sparring bouts (P < 0.05) to levels typically observed in steers (P > 0.05). These observations indicate that active immunization against GnRH reduces the incidence of aggressive behavior in male beef cattle and are consistent with our postulate that immunoneutralization of GnRH is an effective alternative to surgical castration in the management of beef cattle.

Passive immunization of rams (Ovis aries) against GnRH: effects on antibody titer, serum concentrations of testosterone, and sexual behavior.

The effect of immunoneutralization of gonadotropin-releasing hormone (GnRH) on serum concentrations of testosterone and sexual behavior was evaluated in sexually mature male sheep. In Experiment 1, GnRH1 rams (n=16) were passively immunized against GnRH (300 ml antiserum), control rams were either passively immunized against keyhole limpet hemocyanin (KLH, n=15) or surgically castrated (Wethers1, n=4). Sexual performance of the rams was assessed weekly for 3 weeks before and 6 weeks after immunization, using ovarihystertomized ewes actively immunized against GnRH. Experiment 2 evaluated the effects of repeated immunization. Rams were immunized with two aliquots (400 and 300 ml, respectively) of anti-GnRH sera (GnRH, n=5) or normal sheep serum (NSS, n=4), 2 weeks apart. Surgically castrated animals were used as a second control group (Wethers2). Administration of anti-GnRH sera, but neither anti-KLH nor NSS sera, resulted in marked reduction (P<0.05) in serum concentrations of testosterone. Sexual behavior was not consistently affected by administration of one aliquot of anti-GnRH sera, however repeated immunizations resulted in more persistent reduction in serum concentrations of testosterone and more consistent suppression of sexual behavior.

Identification of a determinant of epidermal growth factor receptor ligand-binding specificity using a truncated, high-affinity form of the ectodomain.

Murine and human epidermal growth factor receptors (EGFRs) bind human EGF (hEGF), mouse EGF (mEGF), and human transforming growth factor alpha (hTGF-alpha) with high affinity despite the significant differences in the amino acid sequences of the ligands and the receptors. In contrast, the chicken EGFR can discriminate between mEGF (and hEGF) and hTGF-alpha and binds the EGFs with approximately 100-fold lower affinity. The regions responsible for this poor binding are known to be Arg(45) in hEGF and the L2 domain in the chicken EGFR. In this study we have produced a truncated form of the hEGFR ectodomain comprising residues 1-501 (sEGFR501), which, unlike the full-length hEGFR ectodomain (residues 1-621, sEGFR621), binds hEGF and hTGF-alpha with high affinity (K(D) = 13-21 and 35-40 nM, respectively). sEGFR501 was a competitive inhibitor of EGF-stimulated mitogenesis, being almost 10-fold more effective than the full-length EGFR ectodomain and three times more potent than the neutralizing anti-EGFR monoclonal antibody Mab528. Analytical ultracentrifugation showed that the primary EGF binding sites on sEGFR501 were saturated at an equimolar ratio of ligand and receptor, leading to the formation of a 2:2 EGF:sEGFR501 dimer complex. We have used sEGFR501 to generate three mutants with single position substitutions at Glu(367), Gly(441), or Glu(472) to Lys, the residue found in the corresponding positions in the chicken EGFR. All three mutants bound hTGF-alpha and were recognized by Mab528. However, mutant Gly(441)Lys showed markedly reduced binding to hEGF, implicating Gly(441), in the L2 domain, as part of the binding site that recognizes Arg(45) of hEGF.

Overexpressed growth hormone (GH) synergistically promotes carcinogen-initiated liver tumour growth by promoting cellular proliferation in emerging hepatocellular neoplasms in female and male GH-transgenic mice.

BACKGROUND/AIMS: Growth hormone (GH), when overexpressed in male and female GH-transgenic mice, is known to induce liver tumours within 1 year. This study aimed to gain a clearer understanding of the interaction between GH and tumour cells in vivo. METHODS/RESULTS: The carcinogen diethylnitrosomine (DEN) was administered to neo-natal transgenic and non-transgenic mice maintained in a "hepatocarcinogenesis resistant" genetic background (C57BL/6J). Macroscopic, microscopic and liver weight/body weight ratio analyses revealed that carcinogen-induced hepatocarcinogenesis was dramatically accelerated in young GH-transgenic mice compared to non-transgenic counterparts. Image analysis of microscopic hepatocellular neoplasms showed rapidly increasing tumour burdens, and neoplastic foci size over time in young adult GH-transgenic mice. The magnitude of enhanced tumour growth was equivalent in both male and female transgenic mice, whereas much lower and sexually dimorphic tumour growth rates (males>females) were observed in non-transgenic mice treated with DEN. BrdU labelling experiments demonstrated that rapid tumour growth in carcinogen-treated GH-transgenic mice was due to the promotion of cell proliferation in emerging lesions. Tumour cell proliferation in young GH-transgenic mice was 2.6- and 4-fold higher, respectively, than that observed in similar age male and female non-transgenic mice. Interestingly, both GH-transgenic and non-transgenic mice displayed progressively slower tumour growth rates in older animals. CONCLUSION: Overall, GH synergistically promotes carcinogen-induced hepatocarcinogenesis in both sexes of GH-transgenic mice by stimulating tumour cell proliferation.

Gonadal function, sexual behavior, feedlot performance, and carcass traits of ram lambs actively immunized against GnRH.

The effect of active immunization against GnRH on production, carcass, and behavioral traits was examined in ram lambs fed to a uniform slaughter weight. Lambs (initial BW = 32.6+/-1 kg) were stratified by BW and assigned at random to one of four treatment groups (n = 12 lambs/group). Lambs were untreated, castrated, or actively immunized against GnRH using a GnRH-keyhole limpet hemocyanin conjugate (1 mg) emulsified with either Freund's complete adjuvant (FCA) or another oil-based adjuvant (ISA). Animals were housed individually and slaughtered at 58 kg BW. Immunoneutralization of GnRH reduced (P < .05) testes weight and the concentration of testosterone in serum at slaughter. Suppression of testicular size and function was most clearly evident in animals immunized using FCA. Final anti-GnRH titer was also highest in lambs immunized using FCA. Several measures of sexual behavior (frequency of mounts and ejaculations) were also reduced (P < .05) in animals immunized using FCA. The duration of the feeding period was greater (P < .05) for castrated lambs than for untreated lambs, and intermediate feeding periods were required for FCA and ISA lambs. Average daily gain was greater (P < .05) in untreated than in castrated, FCA, or ISA lambs. Similarly, feed efficiency for untreated lambs was greater (P < .05) than for castrated, FCA, or ISA lambs, but feed efficiency did not differ among castrated, FCA, or ISA lambs. Longissimus muscle area, lean and bone maturity, overall quality, muscling score, flank streaking, and color of fat did not differ among treatments. Intact, FCA, and ISA lambs had more (P < .05) desirable yield grades, less (P < .05) backfat, and less (P < .05) marbling than castrated lambs. In summary, immunization against GnRH decreased testicular weight and reduced (P < .05) feedlot performance and sexual behavior to levels comparable to those of castrated males. Partitioning of nutrients for growth and deposition of fat, however, seems to differ among immunologically castrated and physically castrated lambs. This difference in nutrient partitioning may be due to residual testicular activity in immunized lambs.

Effect of duration of infusion of stress-like concentrations of cortisol on follicular development and the preovulatory surge of LH in sheep.

Stress-like levels of cortisol suppress follicular growth and development and block or delay the preovulatory surge of LH when cortisol is continuously administered during the late luteal and early follicular phases of the ovine oestrous cycle. We postulated that cortisol infusion of shorter duration would have a similar effect. To test this hypothesis the oestrous cycles of mature ewes were synchronized using progestin-treated vaginal pessaries. Ewes were randomly assigned to one of four treatment groups. Animals received cortisol (0.1mg/kg/h; n=8) or vehicle alone (n=8) beginning 5 days before, and continuing for 5 days after, pessary removal (PR). Additional groups received cortisol only during the 5 days period before (n=7), or the 5 days period after (n=8), PR. Continuous delivery of cortisol established stable serum concentrations of cortisol of 72.0+/-2.5ng/ml within 6h of initiation of infusion. Serum concentrations of oestradiol increased progressively during the period after PR in control animals receiving vehicle alone and the preovulatory surge of LH was evident in all control animals (eight of eight) 55.5+/-5.0h after PR. In contrast, follicular development and the preovulatory surge of LH were evident during the period of cortisol infusion in only one of eight animals receiving stress-like levels of cortisol over the entire 10-day infusion period. Similarly, neither follicular development nor surge-like secretion of LH were evident during the infusion period in animals (zero of eight) receiving cortisol during the 5-day period after PR. This cortisol-dependent suppression of ovarian activity in sheep receiving stress-like levels of cortisol during the 5 days after PR was temporary and follicular development, the ovulatory surge of LH, and subsequent luteal function were evident in six of eight ewes after cessation of cortisol delivery. Similarly, follicular development and the preovulatory surge of LH were noted within 5 days after PR in four of seven ewes receiving cortisol only during the 5-day period prior to PR. Collectively, these data indicate that stress-like levels of cortisol reduce fertility of sheep by suppressing follicular development and the preovulatory surge of LH. Additionally, cortisol delivery during the follicular phase has a more profound suppressive effect on follicular development than cortisol administration during the luteal phase.

Structure and function of the type 1 insulin-like growth factor receptor.

The type 1 insulin-like growth factor receptor (IGF-1R), a transmembrane tyrosine kinase, is widely expressed across many cell types in foetal and postnatal tissues. Activation of the receptor following binding of the secreted growth factor ligands IGF-1 and IGF-2 elicits a repertoire of cellular responses including proliferation, and the protection of cells from programmed cell death or apoptosis. As a result, signalling through the IGF-1R is the principal pathway responsible for somatic growth in foetal mammals, whereas somatic growth in postnatal animals is achieved through the synergistic interaction of growth hormone and the IGFs. Forced overexpression of the IGF-1R results in the malignant transformation of cultured cells: conversely, downregulation of IGF-1R levels can reverse the transformed phenotype of tumour cells, and may render them sensitive to apoptosis in vivo. Elevated levels of IGF-IR are observed in a variety of human tumour types, whereas epidemiological studies implicate the IGF-1 axis as a predisposing factor in the pathogenesis of human breast and prostate cancer. The IGF-1R has thus emerged as a therapeutic target for the development of antitumour agents. Recent progress towards the elucidation of the three-dimensional structure of the extracellular domain of the IGF-1R represents an opportunity for the rational assembly of small molecule antagonists of receptor function for clinical use.

Effect of stress-like concentrations of cortisol on the feedback potency of oestradiol in orchidectomized sheep.

The effect of stress-like concentrations of cortisol on oestradiol-induced change in LH secretion and GnRH receptor expression was evaluated in orchidectomized sheep (wethers). Twenty-four wethers were assigned at random to one of the four treatment groups in a 2x2 factorial design (n=6 wethers/group). Wethers received cortisol (90 microg/kg/h; groups 2 and 4) or a comparable volume of cortisol delivery vehicle (groups 1 and 3) by continuous infusion for 48 h. During the final 24 h of infusion, wethers received oestradiol (6 ng/kg/h; groups 3 and 4) or oestradiol delivery vehicle (groups 1 and 2). The pattern of LH secretion was assessed during a 3-h period of intensive blood collection beginning 21 h after initiation of oestradiol infusion. Although neither cortisol nor oestradiol alone affected (P>0.05) mean serum concentration of LH or LH pulse frequency, serum LH and the frequency of secretory episodes of LH were significantly reduced (P<0.05) in wethers receiving cortisol and oestradiol in combination. Anterior pituitary tissue was collected at the end of the infusion period. Oestradiol increased (P<0.05) tissue concentrations of GnRH receptor and GnRH receptor mRNA. Although cortisol alone did not affect (P>0.05) basal concentrations of receptor or receptor mRNA, the magnitude of oestradiol-induced increase in GnRH receptor and GnRH receptor mRNA was significantly reduced in wethers receiving cortisol and oestradiol concurrently. Conversely, steady-state concentrations of mRNA encoding the LHbeta and FSHbeta subunits were increased (P<0.05) in wethers receiving cortisol. These observations demonstrate that stress-like concentrations of cortisol act in concert with oestradiol to suppress LH secretion. In addition, cortisol blocks oestradiol-dependent increase in pituitary tissue concentrations of GnRH receptor and GnRH receptor mRNA.

Properties of an insulin receptor with an IGF-1 receptor loop exchange in the cysteine-rich region.

The insulin receptor (IR) and the insulin-like growth factor-I receptor (IGF-1R) show differential binding of insulin and IGFs. The specificity determinants for IGF-1 binding are known to be located in the cysteine-rich (Cys-rich) region between residues 223 and 274 of human IGF-1R, which includes a loop that protrudes into the putative ligand binding site. In this report we have replaced residues 260-277 of human IR with residues 253-266 of the human IGF-1R to produce an IR-based, cysteine loop exchange chimaera, termed hIR-Cys loop exchange (CLX), in which all 14 amino acid residues in the exchanged loop differ from wild-type insulin receptor. This loop exchange had a detrimental effect on the efficiency of pro-receptor processing and on the binding of the mouse monoclonal antibody 83-7. However, this antibody, which binds hIR but not hIGF-1R, was still capable of immunoprecipitating the mature chimaeric receptor, indicating that the conformational epitope recognised by this antibody is not primarily determined by the loop region exchanged. The loop exchange did not significantly affect the ability of insulin to displace bound radiolabelled insulin, but increased the capacity of IGF-1 to competitively displace labelled insulin by at least 10 fold.

High, persistent hepatocellular proliferation and apoptosis precede hepatocarcinogenesis in growth hormone transgenic mice.

AIMS/BACKGROUND: Growth hormone (GH) transgenic mice are known to develop hepatocellular adenomas and carcinomas. In order to understand more about hepatocarcinogenesis in the GH-transgenic mouse model we quantitated the rates of hepatocellular proliferation and apoptosis in these mice. METHODS: Two lines of GH-transgenic mice and non-transgenic control mice were generated and sacrificed at regular intervals between one and nine months. Hepatocellular replication was measured by in vivo incorporation of bromodeoxyuridine (BrdU) and counting BrdU-positive nuclei in histological liver sections. Serial sections taken from these mouse livers were also assessed for rates of hepatocellular apoptosis using the in situ end-labelling of fragmented DNA (TUNEL) method. RESULTS: High levels of hepatocellular replication were sustained life-long in this model. Increased rates of hepatocellular proliferation preceded the onset of hepatic inflammation, a prominent feature in the liver pathology of GH-transgenic mice. In tumour tissue, cellular proliferation was up to 17-fold greater than in surrounding non-tumour tissue. Apoptosis rates were also elevated in non-tumour regions of GH-transgenic mouse livers compared to controls. Interestingly, large dysplastic hepatocytes were common in the fraction of cells undergoing apoptosis, especially in older mice with inflamed livers. The increase in the rate of hepatocellular apoptosis in GH-transgenic animals largely balanced the augmented levels of proliferation seen in these mice. In tumour tissue, however, the profound increase in the number of proliferating tumour cells outstripped the increase in apoptosis. CONCLUSION: Relatively high and enduring levels of hepatocellular replication and apoptosis precede hepatocarcinogenesis in GH-transgenic mice. Increased cellular proliferation and resistance to apoptosis were evident in tumour growth in older animals.

Effect of stress-like concentrations of cortisol on gonadotroph function in orchidectomized sheep.

The effect of stress-like concentrations of cortisol (C) on the feedback potency of estradiol (E2) was assessed using 32 orchidectomized sheep (wethers) assigned at random to 1 of 4 treatment groups in a 2 x 2 factorial design. Wethers received C (3. 6 mg/50 kg per hour; groups 2 and 4) or a comparable volume of C delivery vehicle (groups 1 and 3) as a continuous infusion for 7 days. During the final 48 h of infusion, wethers received E2 (0.3 microg/50 kg/h; groups 3 and 4) or E2 delivery vehicle (groups 1 and 2). The pattern of LH secretion was assessed during a 4-h period of intensive blood collection beginning 44 h after initiation of E2 infusion. Gonadotroph responsiveness (LH secretion induced by GnRH challenge [500 ng, i.v.]) was determined 48 h after E2 delivery was begun. Although the frequency of secretory episodes of LH was not affected (p > 0.05) by infusion of C or E2 alone, LH pulse frequency was significantly decreased in wethers receiving C and E2 in combination. In contrast, neither the magnitude of basal gonadotroph responsiveness nor the extent of E2-dependent augmentation of responsiveness was significantly affected by stress-like concentrations of C. In a second experiment, the effect of C on the magnitude of E2-induced increase in pituitary concentration of GnRH receptor and GnRH receptor mRNA was assessed using 32 additional wethers. Continuous infusion of E2 for 48 h increased (p < 0.05) tissue concentrations of GnRH receptor and GnRH receptor mRNA. Concurrent delivery of C did not affect (p > 0.05) E2-induced increase in GnRH receptor mRNA but significantly reduced the magnitude of the E2-dependent increase in pituitary concentration of GnRH receptor. Collectively, these data indicate that stress-like concentrations of C enhance the negative feedback potency of E2 and reduce estrogen-dependent augmentation of the concentration of GnRH receptor in pituitary tissue.

Effect of stress-like concentrations of cortisol on estradiol-dependent expression of gonadotropin-releasing hormone receptor in orchidectomized sheep.

The effect of stress-like concentrations of cortisol (C) on estrogen-dependent expression of GnRH receptor was evaluated using orchidectomized sheep (wethers; n = 6 animals per group). C (5.0 mg/50 kg per hour; groups 1-4) or a comparable volume of vehicle (groups 5-8) was delivered by continuous infusion for 48 h. During the final 24 h of infusion, animals received concurrent infusion of estradiol (E2) at rates of 0 (groups 1 and 5), 0.5 (groups 2 and 6), 2.0 (groups 3 and 7), or 8.0 (groups 4 and 8) microg/50 kg per hour. Pituitary tissue was collected at the end of infusion. Although C did not affect (p > 0.05) the basal concentration of GnRH receptor or GnRH receptor mRNA, it reduced (p < 0.05) the increase in receptor and receptor mRNA induced by concurrent administration of 0. 5 microg E2/50 kg per hour. In contrast, the increase in GnRH receptor expression induced by higher levels of estrogen stimulation was not affected (p > 0.05) by concurrent administration of C. The effect of C on the temporal pattern of E2-dependent increase in GnRH receptor expression was assessed using wethers receiving E2 (0.5 microg/50 kg per hour) by continuous infusion for 0 (groups 1 and 5), 24 (groups 2 and 6), 48 (groups 3 and 7), or 72 h (groups 4 and 8). Animals received C (5.0 mg/50 kg per hour; groups 1-4) or vehicle (groups 5-8) beginning 24 h before, and continuing throughout, the E2 delivery period. Stress-like concentrations of C reduced (p < 0. 05) the increase in GnRH receptor and receptor mRNA induced after 24 h of E2 stimulation. However, the suppressive effect of C was transient, and tissue levels of GnRH receptor and receptor mRNA were comparable after 72 h of E2 infusion in animals receiving C or vehicle alone. Collectively these observations demonstrate that C suppresses estrogen-dependent increase in tissue concentrations of GnRH receptor and receptor mRNA. However, this effect of C is transient and not evident in animals receiving moderate to high levels of estrogen stimulation. This transient suppression of GnRH receptor expression may account, at least in part, for the anti-gonadal effect of glucocorticoids.

Effect of stress-like concentrations of cortisol on follicular development and the preovulatory surge of LH in sheep.

Stress-like concentrations of cortisol increase the negative feedback potency of oestradiol in castrated male sheep. A similar cortisol-dependent response in female sheep might be expected to suppress gonadotrophin secretion and impair follicular development and ovulation. The oestrous activity of 21 female sheep was synchronized using progestogen-treated vaginal pessaries to test this hypothesis. Stress-like concentrations of cortisol (60-70 ng ml-1) were established by continuous infusion of cortisol (80 micrograms kg-1 h-1; n = 13) beginning 5 days before, and continuing for 5 days after, pessary removal. Control animals (n = 8) received a comparable volume of vehicle (50% ethanol-saline) over the 10 day infusion period. Serum concentrations of oestradiol increased progressively in control sheep during the 48 h immediately after pessary removal. This increase in serum oestradiol was blocked or significantly attenuated in sheep receiving stress-like concentrations of cortisol. Preovulatory surge-like secretion of LH was apparent in control animals 58.5 +/- 2.1 h after pessary removal. In contrast, surge-like secretion of LH was not observed during the 5 days after pessary removal in 54% (7 of 13) of sheep receiving cortisol. Moreover, the onset of the surge was significantly delayed in the cortisol-treated ewes that showed surge-like secretion of LH during the infusion period. The ability of episodic pulses of exogenous GnRH to override the anti-gonadal effect of cortisol was examined in a second study. Oestrous activity of 12 ewes was synchronized using progestogen-containing pessaries as described above. Ewes were randomly assigned to one of three treatment groups (n = 4 ewes per group). Animals received cortisol (100 micrograms kg-1 h-1; groups 1 and 2) or a comparable volume of vehicle (group 3) beginning 5 days before, and continuing for 2 days after, pessary removal. Pulses of GnRH (4 ng kg-1 h-1, i.v.; group 1) or saline (groups 2 and 3) at 1 h intervals were initiated at pessary removal and continued for 48 h. Serum concentrations of oestradiol were not significantly increased after pessary removal in sheep receiving cortisol alone. Conversely, serum concentrations of oestradiol increased progressively during the 48 h after pessary removal in control ewes and in ewes receiving cortisol and GnRH. At the end of infusion, serum concentrations of oestradiol did not differ (P > 0.05) between control (7.7 +/- 0.8 pg ml-1) ewes and ewes receiving cortisol and episodic GnRH (6.4 +/- 1.3 pg ml-1). Moreover, these values were significantly greater (P < 0.05) than the serum concentrations of oestradiol in animals receiving cortisol (1.0 +/- 0.4 pg ml-1) alone. Collectively, these data indicate stress-like concentrations of cortisol block or delay follicular development and the preovulatory surge of LH in sheep. In addition, episodic GnRH overrides cortisol-induced delay in follicular maturation.

The effect of season and melatonin on GnRH-induced LH secretion in oestradiol-treated orchidectomized sheep.

The biphasic effect of oestradiol (E2) on gonadotrope responsiveness is clearly evident in orchidectomized sheep (wethers) receiving E2 and hourly pulses of GnRH. We hypothesized that the duration of E2-induced reduction in gonadotrope responsiveness differed between the breeding (November) and anoestrous (May) seasons in sheep. To test this hypothesis wethers (n = 6/group) were infused (i.v.) with E2 (2 micrograms/50 kg per h) and received hourly pulses of GnRH (200 ng/50 kg per pulse) or saline in May or November. The pattern of LH secretion during the 72 h infusion period was determined. Serum concentrations of LH did not differ with season in control wethers receiving vehicle alone. Similarly, continuous infusion of E2 resulted in a 3-fold reduction in serum LH, irrespective of season. This E2-induced suppression of serum LH was reversed by concurrent episodic delivery of GnRH. The interval between initiation of infusion and return of pretreatment concentrations of LH was taken as a measure of the duration of E2-induced suppression of gonadotrope responsiveness. The duration of this E2-dependent response varied with season, with suppression of gonadotrope responsiveness more prolonged (P < 0.05) in May (36.7 +/- 2.9 h) than in November (14.3 +/- 1.1 h). In a companion study we examined the effect of melatonin on the duration of E2-induced suppression of gonadotrope responsiveness. Wethers received blank or melatonin-containing implants in March. Sixty days after implant insertion (mid-May) wethers received E2 (2 micrograms/50 kg per h) and hourly pulses of GnRH (200 ng/50 kg per pulse) or saline for 72 h. Continuous delivery of E2 alone resulted in a 3-fold decrease in serum concentrations of LH in both control and melatonin-treated wethers. The duration of E2-induced suppression of gonadotrope responsiveness in animals receiving E2 and GnRH was extended (P < 0.05) in wethers with blank implants (48.0 +/- 0.7 h), relative to the duration of suppression in melatonin-treated wethers (14.5 +/- 1.0 h). Taken together these data indicate that E2-induced suppression of gonadotrope responsiveness is more extended during the anoestrous season. However, this seasonal effect can be reversed by continuous administration of melatonin.

Testis function, carcass traits, and aggressive behavior of beef bulls actively immunized against gonadotropin-releasing hormone.

We assessed testis function, aggressive behavior, and carcass traits in beef bulls actively immunized against GnRH at 1, 4, or 6 mo of age. In addition, we examined the effect of combining immunization with insertion of estrogen-containing implants (Synovex C) at 1 mo of age. Unimmunized bulls and steers were included as control animals. All immunized calves received a secondary immunization at 12 mo of age. Anti-GnRH titer was evident at slaughter in all immunized animals. Neither age at primary immunization nor implant status affected (P > .05) anti-GnRH titer at slaughter. Immunization, but not implant status, reduced (P < .05) serum concentration of testosterone and testis weight at slaughter. The final live weight and feedlot gain of immunized and unimmunized bulls were comparable (P > .05). In contrast, aggressive behavior was reduced (P < .05) and carcass quality was improved (P < .05) by immunization. These data suggest that active immunization against GnRH is a practical, noninvasive alternative to physical castration in the management of bull calves.

Growth hormone preferentially induces the rapid, transient expression of SOCS-3, a novel inhibitor of cytokine receptor signaling.

Four members (SOCS-1, SOCS-2, SOCS-3, and CIS) of a family of cytokine-inducible, negative regulators of cytokine receptor signaling have recently been identified. To address whether any of these genes are induced in response to growth hormone (GH), serum-starved 3T3-F442A fibroblasts were incubated with GH for various time points, and the expression of the SOCS gene family was analyzed by Northern blotting. GH stimulated the rapid, transient induction of SOCS-3 mRNA, peaking 30 min after the initiation of GH exposure and declining to basal levels by 2 h. Expression of the other SOCS genes (SOCS-1, SOCS-2, CIS) was also up-regulated by GH, although to a lesser extent than SOCS-3 and with differing kinetics. SOCS-3 expression was also strongly induced in 3T3-F442A cells treated with leukemia-inhibitory factor (LIF), with weaker induction of SOCS-1 and CIS being observed. The preferential induction of SOCS-3 mRNA was also observed in hepatic RNA isolated from the livers of mice that had received a single supraphysiological dose of GH intraperitoneally. Co-transfection studies revealed that constitutive expression of SOCS-1 and SOCS-3, but not SOCS-2 or CIS, blocked GH-induced transactivation of the GH-responsive serine protease inhibitor 2.1 gene promoter.