Analysis of female reproductive traits in Angus beef cattle divergently selected for blood serum insulin-like growth factor I concentration.

Insulin-like growth factor-I (IGF-I) is an anabolic polypeptide involved in reproductive performance in several species. The objectives of this study were to determine relationships of pregnancy rate, and age of heifers at puberty and at first calving with serum IGF-I concentration in Angus beef cattle. Data were obtained from an ongoing divergent selection experiment for IGF-I concentration involving purebred Angus cows. The IGF-I concentrations measured at Days 28, 42, and 56 of the 140-day postweaning test are abbreviated as IGF28, IGF42, and IGF56, respectively. Pregnancy rate did not differ between high and low IGF-I line females (P=0.95; n=2618), but high line heifers tended to be 4.02+/-2.18 days younger (P=0.07; n=281) at first calving. Residual correlations of age of heifers at first calving (AFC) with IGF-I measurements were not significant. The linear and quadratic terms for regression of AFC on IGF-I concentrations were also non-significant. Contrast analysis showed no difference in age at puberty between the high and low IGF-I line heifers (5.3+/-6.4 days earlier in the high line; P=0.43; n=51). Residual correlations of age of heifers at puberty with IGF28, IGF42, IGF56, and mean IGF-I were -0.30 (P=0.03), -0. 22 (P=0.12), -0.35 (P=0.01), and -0.34 (P=0.01), respectively. The observed relationships between female reproductive traits and IGF-I concentration in Angus beef cattle suggest complex and multiple roles for IGF-I in reproduction.

The secretory leukocyte protease inhibitor gene is a target of epidermal growth factor receptor action in endometrial epithelial cells.

The over-expression of epidermal growth factor receptor (EGFR) and its ligands, epidermal growth factor (EGF) and transforming growth factor-alpha, is a common feature of epithelial carcinomas and correlates with neoplastic progression. Secretory leukocyte protease inhibitor (SLPI), a member of the Kazal superfamily of serine anti-proteases, induces proliferation and promotes malignancy of epithelial cells and is expressed at high levels in multiple tumor types. In the present study, we have demonstrated that EGF increases SLPI expression in the human endometrial epithelial cell line Ishikawa in a dose- and time-dependent manner. We have shown that this effect of EGF occurs, in part, at the level of the SLPI promoter and involves the MAP kinase signaling pathway. We have further shown that EGF promotion of cell proliferation, but not induction of cyclin D1 gene expression, involves SLPI. Our results suggest that the regulation of SLPI expression by EGFR ligand(s) may represent a 'feed-forward' mechanism by which the enhanced proliferative and migratory properties of EGFR over-expressing cancer cells are sustained. Increased SLPI expression is likely an important component of altered EGFR signaling in human tumors and may have significant therapeutic implications in cancer progression.

Evaluation of serum insulin-like growth factor binding proteins (IGFBP) in Angus cattle divergently selected for serum IGF-I concentration.

Postweaning serum insulin-like growth factor-I (IGF-I) concentrations and serum IGF binding proteins (IGFBP) were investigated in 68 (1992 Fall-born) and 84 (1999 Fall-born) Angus cattle selected for either high or low serum IGF-I concentrations since 1989. Relative serum levels of IGFBP were determined by [125I]IGF-I Western ligand blotting. IGFBP species of 38-42, 34, 30, and 24 kDa were identified. The 34 kDa species was identified as IGFBP-2 by immunoblot analysis. No significant line effects were observed for any of the IGFBP. In both 1992 and 1999, heifers had higher IGFBP-2 levels than bulls (P<0.0005). In 1992 calves, relative levels of the 38-42 and 24 kDa species were significantly correlated with serum IGF-I concentration. In 1999 calves, none of the IGFBP were correlated with serum IGF-I, although IGFBP-2 was negatively correlated with several measures of body weight. No significant line effects were observed for growth or serum IGF-I traits in 1992 calves. However, 1999 high line calves had higher serum IGF-I concentrations and body weights than low line calves (P<0.05). In both 1992 and 1999 calves, bulls had higher serum IGF-I concentrations and body weights than heifers (P<0.05). Thus, while selection for high versus low serum IGF-I concentrations has resulted in divergence between the selection lines and also in changes in body weights, it has not resulted in changes in serum IGFBP levels. Furthermore, circulating IGFBP-2 appears to be higher in heifers than in bulls, and also appears to be negatively correlated with body weights.

Estimation of (co)variance components for reproductive traits in Angus beef cattle divergently selected for blood serum IGF-I concentration.

The objective of this study was to obtain estimates of (co)variance components for reproductive traits and insulin-like growth factor-I (IGF-I) concentration. Data were from a divergent selection experiment for blood serum IGF-I concentration in Angus beef cattle. Numbers of observations for mean IGF-I concentration of three blood samples taken at d 28, 42, and 56 of the 140-d postweaning test, scrotal circumference (SC), percentage of motile sperm cells (PMSC), percentage of morphologically normal sperm cells (PNSC), age of heifers at first calving (AFC), and calving rate (CR) were 1,848, 825, 596, 765, 294, and 2,092, respectively. Total number of animals in the numerator relationship matrix, including base animals, was 2,864, of which 1,861 were inbred. Estimates of direct heritability for IGF-I concentration of three blood samples collected at d 28, 42, and 56 of the postweaning test and for mean IGF-I concentration were 0.43+/-0.08, 0.51+/-0.09, 0.41+/-0.08, and 0.50+/-0.08, respectively. Estimates of direct heritability for SC, PMSC, PNSC, AFC, and CR were 0.51+/-0.13, 0.08+/-0.12, 0.47+/-0.07, 0.26+/-0.28, and 0.11+/-0.05, respectively. With the exception of age at first calving, estimates of maternal heritability and proportion of phenotypic variance that were due to permanent environmental effects of the dams were smaller than 0.21. Observations for calving rate were entered as either 1 (if calved) or 100 (if not calved). Estimates of additive genetic correlations of mean IGF-I concentration with SC, PMSC, PNSC, AFC, and CR were 0.35+/-0.11, 0.43+/-0.32, 0.00+/-0.03, -0.14+/-0.33, and -0.41+/-0.16, respectively. Environmental and phenotypic correlations for all of the traits with IGF-I measurements were smaller than 0.23. These results suggest that selection for increased serum IGF-I concentration should result in increased scrotal circumference, percent motile sperm cells, and calving rate.

Genetic parameter estimates for serum insulin-like growth factor-I concentration and ultrasound measurements of backfat thickness and longissimus muscle area in Angus beef cattle.

A divergent selection experiment for serum IGF-I concentration began at the Eastern Ohio Resource Development Center in 1989 using 100 spring-calving (50 high line and 50 low line) and 100 fall-calving (50 high line and 50 low line) purebred Angus cows. Following weaning, bull and heifer calves were fed in drylot for a 140-d period. Real-time ultrasound measurements of backfat thickness and longissimus muscle area were taken on d 56 and 140 of the postweaning test. Only ultrasound data from calves born from fall 1995 through spring 1999 were included in the analysis. At the time of this study, IGF-I measurements were available for 1,521 bull and heifer calves, and ultrasound data were available for 636 bull and heifer calves. Data were analyzed by multiple-trait, derivative-free, restricted maximum likelihood methods. Estimates of direct heritability for IGF-I concentration at d 28, 42, and 56 of the postweaning period, and for mean IGF-I concentration were 0.26 +/- 0.07, 0.32 +/- 0.08, 0.26 +/- 0.07, and 0.32 +/- 0.08, respectively. Direct heritabilities for ultrasound estimates of backfat thickness ranged from 0.17 +/- 0.11 to 0.28 +/- 0.12, whereas direct heritabilities for longissimus muscle area ranged from 0.20 +/- 0.10 to 0.36 +/- 0.12, depending on the time of measurement and the covariate used for adjustment (age vs. weight). Direct genetic correlations of IGF-I concentrations with backfat thickness at d 56 and 140 and with longissiumus muscle area at d 56 and 140 averaged 0.02, 0.20, -0.08, and 0.23, respectively, when age was used as the covariate for both IGF-I and ultrasound measurements. Corresponding genetic correlations when age was used as the covariate for IGF-I and weight was used as the covariate for ultrasound measurements were 0.05, -0.07, -0.22, and -0.04, respectively. Therefore, the positive associations of serum IGF-I concentration with backfat thickness and longissimus muscle area at d 140 seem to have been partially mediated by weight. Results of this study do not indicate strong associations of serum IGF-I concentration with fat thickness or muscling of bulls and heifers during the postweaning feedlot period.

Association of single nucleotide polymorphisms in the growth hormone and growth hormone receptor genes with blood serum insulin-like growth factor I concentration and growth traits in Angus cattle.

This study was conducted to identify polymorphisms in the promoter and coding regions of the bovine growth hormone and growth hormone receptor genes and to study association of polymorphisms identified in these genes with growth traits and serum insulin-like growth factor-I (IGF-I) concentration. The denaturing gradient gel electrophoresis method and sequencing were utilized to identify three new single nucleotide polymorphisms in the promoter region of the growth hormone gene in Angus cattle. Polymerase chain reaction-based restriction fragment length polymorphism procedures were developed for rapid determination of the single nucleotide polymorphism genotypes in the growth hormone and the growth hormone receptor genes among Angus calves from lines divergently selected for high or low blood serum IGF-I concentration. The IGF-I concentration and growth traits were analyzed using animal models. The single nucleotide polymorphism in the promoter region of the growth hormone receptor gene was associated with serum IGF-I concentration on d 42 of the postweaning test and with mean IGF-I concentration. The associated effects of the markers need to be verified in other populations.

Estimates of inbreeding depression for serum insulin-like growth factor I concentrations, body weights, and body weight gains in Angus beef cattle divergently selected for serum insulin-like growth factor I concentration.

Data for the current study were obtained from a divergent selection experiment in which the selection criterion was the average serum IGF-I concentration of 3 postweaning blood samples collected from purebred Angus calves. Multiple trait derivative-free REML procedures were used to obtain estimates of inbreeding depression for IGF-I concentration and for BW and BW gains measured from birth to the conclusion of a 140-d postweaning performance test. Included in the analysis were 3,243 animals in the A(-1) matrix, 2,182 of which had valid records for IGF-I concentration. Over the course of the entire selection experiment, inbreeding of the calf averaged 3.3% (SD = 3.1%) and inbreeding of the dam averaged 1.8% (SD = 2.7%). Mean inbreeding levels at the end of the study were 6.82 +/- 0.38% and 4.20 +/- 0.36% for calves and dams, respectively. Annual rates of increase in inbreeding of calves and dams were 0.36 +/- 0.01 (P < 0.0001) and 0.25 +/- 0.01%/yr (P < 0.0001), respectively. Insulin-like growth factor I concentration at d 28 (IGF28), 42 (IGF42), and 56 (IGF56) of the 140-d postweaning test and mean IGF-I concentration decreased by 0.62 +/- 0.88, 1.86 +/- 0.96, 1.92 +/- 0.89, and 1.48 +/- 0.76 ng/mL per 1% increase in inbreeding of calf. Only the regression coefficient for IGF56 differed significantly from zero, although the regression coefficients for IGF42 and mean IGF-I approached significance (P < 0.10). Increases in inbreeding levels of the dams also tended to result in reduced IGF-I concentrations, although the regression coefficients were not significantly different from zero. Inbreeding of calf had highly significant negative effects on all BW and BW gain traits examined, except for birth weight, with regression coefficients ranging from -0.74 +/- 0.20 kg/% increase in calf inbreeding for postweaning BW gain to -1.68 +/- 0.33 kg/% increase in calf inbreeding for off-test BW. Inbreeding of dam had a significant negative effect on birth weight of progeny and tended to have a negative effect on postweaning BW gain (P < 0.10). Preweaning gain of the progeny and BW other than birth weight were not influenced by increases in dam inbreeding. Results indicate that reductions in serum IGF-I concentration due to inbreeding may contribute to the decline in BW and BW gains that is typically associated with increases in inbreeding within populations.

The emerging role of Krüppel-like factors in endocrine-responsive cancers of female reproductive tissues.

Krüppel-like factors (KLFs), of which there are currently 17 known protein members, belong to the specificity protein (Sp) family of transcription factors and are characterized by the presence of Cys(2)/His(2) zinc finger motifs in their carboxy-terminal domains that confer preferential binding to GC/GT-rich sequences in gene promoter and enhancer regions. While previously regarded to simply function as silencers of Sp1 transactivity, many KLFs are now shown to be relevant to human cancers by their newly identified abilities to mediate crosstalk with signaling pathways involved in the control of cell proliferation, apoptosis, migration, and differentiation. Several KLFs act as tumor suppressors and/or oncogenes under distinct cellular contexts, underscoring their prognostic potential for cancer survival and outcome. Recent studies suggest that a number of KLFs can influence steroid hormone signaling through transcriptional networks involving steroid hormone receptors and members of the nuclear receptor family of transcription factors. Since inappropriate sensitivity or resistance to steroid hormone actions underlies endocrine-related malignancies, we consider the intriguing possibility that dysregulation of expression and/or activity of KLF members is linked to the pathogenesis of endometrial and breast cancers. In this review, we focus on recently described mechanisms of actions of several KLFs (KLF4, KLF5, KLF6, and KLF9) in cancers of the mammary gland and uterus. We suggest that understanding the mode of actions of KLFs and their functional networks may lead to the development of novel therapeutics to improve current prospects for cancer prevention and cure.

Response of adult mouse uterus to early disruption of estrogen receptor-alpha signaling is influenced by Krüppel-like factor 9.

Inappropriate early exposure of the hormone-responsive uterus to estrogenic compounds is associated with increased risk for adult reproductive diseases including endometrial cancers. While the dysregulation of estrogen receptor-alpha (ESR1) signaling is well acknowledged to mediate early events in tumor initiation, mechanisms contributing to sustained ESR1 activity later in life and leading to induction of oncogenic pathways remain poorly understood. We had shown previously that the transcription factor Krüppel-like factor 9 (KLF9) represses ESR1 expression and activity in Ishikawa endometrial glandular epithelial cells. We hypothesized that KLF9 functions as a tumor suppressor, and that loss of its expression enhances ESR1 signaling. Here, we evaluated the contribution of KLF9 to early perturbations in uterine ESR1 signaling pathways elicited by the administration of synthetic estrogen diethylstilbestrol (DES) to wild-type (WT) and Klf9 null (KO) mice on postnatal days (PNDs) 1-5. Uterine tissues collected at PND84 were subjected to histological, immunological, and molecular analyses. Compared with WT mice, KO mice demonstrated larger endometrial glands and lower endometrial gland numbers; DES exposure exacerbated these differences. Loss of KLF9 expression resulted in increased glandular ESR1 immunoreactivity with DES, without effects on serum estradiol levels. Quantitative RT-PCR analyses indicated altered expression of uterine genes commonly dysregulated in endometrial cancers (Akt1, Mmp9, Slpi, and Tgfbeta1) and of those involved in growth regulation (Fos, Myc, Tert, and Syk), with loss of Klf9, alone or in concert with DES. Our data support a molecular network between KLF9 and ESR1 in the uterus, and suggest that silencing of KLF9 may contribute to endometrial dysfunctions initiated by aberrant estrogen action.

Nuclear receptor co-regulator Krüppel-like factor 9 and prohibitin 2 expression in estrogen-induced epithelial cell proliferation in the mouse uterus.

Estrogen, acting through its cognate receptor estrogen receptor-alpha (ESR1), is a critical regulator of uterine endometrial epithelial proliferation. Although the dynamic communication between endometrial stromal (ST) and epithelial cells is considered to be an important component in this process, key molecular players in particular compartments remain poorly defined. Here, we used mice null for Krüppel-like factor 9 (KLF9) to evaluate the contribution of this nuclear protein in ST-epithelial interactions underlying proliferative effects of estrogen. We found that in ovariectomized mice administered estradiol-17beta (E(2)) for 24 h, Klf9 null mutation resulted in lack of E(2)-induced proliferative response in all endometrial compartments. We demonstrated a negative association between Klf9 expression and nuclear levels of ESR1 transcriptional corepressor prohibitin (PHB) 2 in uterine ST and epithelial cells of E(2)-treated wild-type (WT) and Klf9 null mice. In early pregnancy uteri of WT mice, the temporal pattern of Klf9 transcript levels was inversely associated with that of Phb2. Deletion of Klf9 up-regulated uterine Phb2 expression and increased PHB2 nuclear localization in endometrial ST and epithelial cells, with no effects on the expression of the related Phb1. In the human endometrial ST cell line treated with E(2) for 24 h, Klf9 siRNA targeting augmented PHB2 transcript and increased nuclear PHB2 protein levels, albeit this effect was not to the extent seen in vivo with Klf9 null mutants. Our findings suggest a novel mechanism for control of estrogen-induced luminal epithelial proliferation involving ST KLF9 regulation of paracrine factor(s) to repress epithelial expression of corepressor PHB2.

Genetic parameter estimates for serum insulin-like growth factor I concentrations, and body weight and weight gains in Angus beef cattle divergently selected for serum insulin-like growth factor I concentration.

Data for the current study were obtained from a divergent selection experiment in which the selection criterion was the average serum IGF-I concentrations of 3 postweaning blood samples collected from purebred Angus calves. Multiple-trait derivative-free REML procedures were used to obtain genetic parameter estimates for IGF-I concentrations and for BW and BW gains measured from birth to the conclusion of a 140-d postweaning performance test. Included in the analysis were 2,674 animals in the A(-1) matrix, 1,761 of which had valid records for IGF-I concentrations. Direct heritability estimates +/- SE for IGF-I concentration at d 28, 42, and 56 of the postweaning period and for mean IGF-I concentrations were 0.44 +/- 0.07, 0.51 +/- 0.08, 0.42 +/- 0.07, and 0.52 +/- 0.08, respectively. Heritability estimates for maternal genetic effects ranged from 0.10 +/- 0.05 to 0.20 +/- 0.06. The proportion of total phenotypic variance due to the maternal permanent environmental effect was essentially zero for all measures of IGF-I concentrations. Genetic correlations of IGF-I concentrations with weaning and post-weaning BW ranged from 0.07 +/- 0.12 to 0.32 +/- 0.11 and generally demonstrated an increasing trend during the postweaning period. Averaged across the various measures of IGF-I, the genetic correlation of IGF-I with preweaning gain was 0.14, whereas the genetic correlation with postweaning gain was 0.29. Genetic correlations between IGF-I and BW gain were positive during all time intervals, except between weaning and the beginning of the postweaning test and from d 84 to 112 of the postweaning period. Environmental and phenotypic correlations of IGF-I with BW and BW gains were generally positive, but small. These results indicate that postweaning serum IGF-I concentration is moderately to highly heritable and has small positive genetic, environmental, and phenotypic correlations with BW other than birth weight and with pre- and postweaning gain. Therefore, if IGF-I proves to be a biological indicator of an economically important trait (e.g., efficiency of feed use for growth) in beef cattle, it should be possible to rapidly change IGF-I concentrations via selection without significantly altering live weight or rate of gain.

Insulin-like growth factor-I and insulin-like growth factor binding protein-2 and -5 in equine seminal plasma: association with sperm characteristics and fertility.

The objectives of this study were 1) to determine whether insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding proteins (IGFBPs) were present in seminal plasma of stallions; 2) to compare semen parameters (IGF proteins, sperm numbers, morphology, and motility) from stallions at sexual rest (SR) and when sexually active (SA); 3) to compare semen parameters between stallions with high and low seminal plasma IGF-I concentrations; and 4) to examine the relationship between seminal plasma IGF-I concentrations and fertility parameters of stallions. Ejaculates were collected from stallions at SR (n = 51) and SA (n = 46). Concentrations of IGF-I and IGFBP-2 in seminal plasma samples were determined by radioimmunoassay. Presence of IGFBPs in equine seminal plasma was verified using immunoprecipitation and Western ligand blot procedures. IGF-I, IGFBP-2, and IGFBP-5 were present in equine seminal plasma. Concentrations of IGF-I, IGF-I/protein, total IGF-I, IGFBP-2, IGFBP-2/protein, and total IGFBP-2 were not significantly different (P > or = 0.13) in seminal plasma between stallions at either SR or SA. At SR, stallions with higher seminal plasma IGF-I had more total IGFBP-2 per ejaculate (P < 0.01), more morphologically normal sperm (P = 0.05), and higher first-cycle pregnancy rates (P = 0.02). At SA, stallions with higher seminal plasma IGF-I had fewer cycles per pregnancy (P = 0.02). An association of seminal plasma IGF-I concentration with sperm motility, sperm morphology, and pregnancy rates in bred mares suggests that IGF-I may play a role in sperm function.