R. C. M. Simmen

Maternal and neonatal somatomedin C/insulin-like growth factor-I (IGF-I) and IGF binding proteins during early lactation in the pig☆

RIA for insulin-like growth factor-I (IGF-I) was performed on Tris-neutralized, acid-ethanol extracts of porcine, bovine, ovine and human mammary secretions, and porcine maternal and neonatal sera. High levels (50-500 ng/ml) of immunoreactive IGF-I were present in the colostrum of all three animal species. IGF-I was also identified in porcine milk, though at levels 10- to 100-fold reduced relative to that in colostrum. Maternal (pig) sera was characterized by IGF-I concentrations intermediate between that in colostrum and that in milk. IGF-I levels were relatively low in serum of newborn pigs and exhibited an approximately 1.4-fold increase between Days 3 and 7 of postnatal life. Fractionation of pig colostrum in nondenaturing, gel-filtration columns demonstrated association of endogenous IGF-I with two prominent binding proteins (Mrs of 150,000 and 50,000 for the complexes). A third immunoreactive component was also observed to elute in the column void volume fractions (Mr greater than 158,000). The 150,000 and 50,000 Mr complexes were also present in serum obtained from sows at term. In contrast, IGF-I immunoreactivity in porcine milk was localized exclusively in the 150,000 Mr complex. Incubation of porcine colostrum and milk with 125I-IGF-I revealed a prominent, unoccupied IGF binding protein corresponding to that of the 150,000 Mr complex, whereas serum obtained from sows at term displayed both the 150,000 and 50,000 Mr unoccupied forms. Fractionation of (pooled) serum obtained from porcine neonates immediately at birth revealed a heterogeneous pattern of IGF-I immunoreactivity which included both the 150,000 and 50,000 Mr forms. Qualitative differences in this chromatographic pattern were apparent in serum at 6 hr postnatal and after ingestion of colostrum had occurred. The unoccupied IGF binding proteins in newborn pig serum were solely of the small size class. These results demonstrate that mammary secretion of IGF-I and its binding proteins are temporally regulated during the period immediately surrounding parturition. Physiologic alterations in the serum IGF-I profile during early postnatal life may reflect in part the uptake and/or response of the neonate to maternal IGF-I.

Lactational variation and relationship to postnatal growth of insulin-like growth factor-I in mammary secretions from genetically diverse sows.

Mammary secretions obtained from four groups of sows at parturition and on days 7, 14 and 21 of lactation were defatted and assayed for total protein and insulin-like growth factor-I (IGF-I). Sows (n = 57) represented two breeds (Landrace and Duroc) and two genetic lines (selected for differences in sow productivity index, SPI) within each breed. Colostrum of Duroc sows was 4-6 fold and 30-60 fold greater in protein (P less than .001) and IGF-I (P less than .001) concentrations, respectively, than the corresponding day 7 milk from these sows. In contrast, the colostrum of Landrace sows was 2-3 fold and 30-50 fold greater in protein (P less than .001) and IGF-I (P less than .001) concentrations, respectively, than the corresponding day 7 milk. The IGF-I content in milk from Duroc sows did not differ among days 7, 14 and 21 of lactation, whereas the IGF-I content of day 7 milk from Landrace sows exceeded those for the corresponding 14 day and 21 day secretion (P less than .05). IGF-I concentration in days 14 and 21 milk was higher in Duroc (P less than .001 respectively) than Landrace sows. No significant differences in total protein or IGF-I content of mammary secretions were observed between the selected and control lines within each breed.(ABSTRACT TRUNCATED AT 250 WORDS)

Nuclear receptor co-regulator Kruppel-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.

Response of adult mouse uterus to early disruption of estrogen receptor-α 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.

Isolation, characterization, and developmental expression of pig intestinal fatty acid-binding proteins

The goal of this study was to characterize and quantify intestinal fatty acid-binding proteins of the pig. Small intestinal mucosa from 13-19 kg pigs was homogenized and centrifuged to obtain cytosol. Isolation of fatty acid-binding proteins from delipidated cytosol was achieved using molecular sieve, oleic acid affinity, and ion exchange chromatography. Fatty acid-binding protein isolation was monitored using a fatty-acid binding assay in conjunction with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Antisera to rat liver-fatty acid-binding protein cross reacted with an isolated intestinal fatty acid-binding protein of Mr = 13,000, whereas antisera to rat intestine-fatty acid-binding protein was not cross reactive with isolated pig intestinal proteins. These experiments identify a pig intestinal fatty acid-binding protein that exhibits strong immunochemical similarity to rat liver-fatty acid-binding protein. Cytosol prepared from intestinal mucosa of pigs at -4, 2, 4, 7, 15, 22, 28, and 35 d of age was assayed for fatty acid-binding protein activity. Preweaning fatty acid-binding protein activity in cytosol was maximal at 7 days of age when expressed as total jejunal fatty acid binding per kilogram bodyweight, intestinal or mucosal weight or milligram total protein. After weaning (21 d), fatty acid-binding protein activities declined to 28 days, but increased again by 35 days. Total soluble fatty acid-binding protein activity in pig intestine is regulated during postnatal development and this may account in part for the altered intestinal absorption of lipids observed in young pigs at weaning.