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R C Simmen

Publications and source records attributed to R C Simmen.

At least 73 records · Page 4Linked to original sources
PubMed1991

Characterization of endocrine cell lines immortalized by a temperature-sensitive mutant SV40.

An ideal in vitro model for the study of endocrine functions would be one in which cells could propagate in culture and express their specialized functions. Most endocrine studies to date have relied on primary cell culture or on the use of tumor cell lines. This report describes the characterization of three endocrine cell lines immortalized by transfecting endocrine cells with a temperature-sensitive mutant SV40 virus. Rabbit endometrium (HRE-H9), human placenta (SPA209-10) and rat pituitary (RP) cells were immortalized by SV40 virus, a temperature-sensitive (ts) mutant in the A gene, which encodes the large tumor antigen that is required for the maintenance of transformation. The transformed phenotype of the SV40 tsA mutant-immortalized cell line can be reversed simply by a shift in temperature. At the permissive temperature (34 degrees C), all three types of cells exhibited a transformed phenotype, which is characterized by high cell density growth and by the overgrowth of nontransformed cell layers. However, at the non-permissive temperature (40 degrees C) these cells reverted to a non-transformed phenotype as demonstrated by a marked decrease in the overgrowth of nontransformed layers and by the expression of differentiated functions. At the non-permissive temperature (40 degrees C), the endometrial cell line was capable of synthesizing beta-endorphin, and it exhibited hormonally regulated expression of the transfected hybrid uteroferrin gene construct. The human placenta cell line was capable of secreting GnRH upon stimulation by cAMP, forskolin, theophyllin, PGE, catecholamine and Ca++ channel stimulators. Moreover, the rat pituitary cell line was capable of synthesizing and secreting growth hormone (GH) which was stimulated by GHRH and cAMP. The advantage of the temperature-sensitive cell lines is that a single cell line is the source of both the normal and transformed states; thus, studies are internally controlled. These results demonstrate that tsA mutants of SV40 virus are the best available agents for immortalizing mammalian endocrine cells that retain differentiated functions.

Animals↗
PubMed1990

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)

Animals↗
PubMed1990

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.

Journal Article↗
PubMed1990

Hormonal regulation of insulin-like growth factor gene expression in pig uterus.

The present studies were undertaken to define potential regulation of endometrial insulin-like growth factor-I (IGF-I) synthesis and secretion by steroid hormones and to correlate conceptus- and serum-derived estrogens with these events. Four experimental groups of gilts were studied: 1) prepubertal gilts administered estradiol (E) and/or progesterone (P4) daily for up to 15 days; 2) mature gilts ovariectomized (ovx) on day 2 of the estrous cycle, and then administered E, P4, or E plus P4 from days 4-11 after estrus; 3) unilaterally pregnant gilts on day 12 or 16 after the onset of estrus; and 4) cyclic gilts administered E on day 11 and hysterectomized 1, 6, 12, or 24 h after E. IGF-I mRNA was measured by blot hybridization of total cellular RNAs, while a specific RIA was used to quantitate levels of tissue and uterine luminal fluid (ULF) IGF-I. In prepubertal gilts, E or P4 increased uterine IGF-I mRNA levels and IGF-I protein. Mature, ovx gilts treated with E, P4, or E plus P4 had increased levels of endometrial IGF-I mRNAs and protein and luminal IGF-I. The magnitude of E or P4 induction was comparable and was not additive. Gravid horns on day 12 had higher amounts of IGF-I in ULF compared to day 12 nongravid horns or day 16 gravid and nongravid horns. However, no significant differences in the amounts of endometrial IGF-I mRNA and tissue IGF-I content between horns on each day were detected. Acute treatment of cyclic gilts (day 11) with E increased levels of endometrial IGF-I mRNAs and luminal IGF-I 1 h after treatment. The effect of E was transient and was reversed within 24 h of E injection. IGF-II and IGF-binding protein-2 mRNAs were regulated by E and P4 distinct from IGF-I mRNAs. These results indicate that E and P4 are involved in the normal regulation of uterine IGF-I synthesis and/or secretion. However, the relative contributions of serum and conceptus-derived estrogens in these processes during the preimplantation period in the pig remain to be defined.

Aging↗
PubMed1990

Molecular cloning of the uteroferrin-associated protein, a major progesterone-induced serpin secreted by the porcine uterus, and the expression of its mRNA during pregnancy.

The uteroferrin(Uf)-associated basic proteins (UfAP) are a group of three (Mr = 42K, 48K, and 50K) antigenically related, basic glycoproteins secreted by the porcine uterus under the influence of progesterone (P4) which exist as heterodimers (Mr = 80,000) with the iron-binding acid phosphatase, Uf. Several UfAP cDNA clones from a day-60 pregnant pig uterine endometrial cDNA library have been cloned and sequenced. The UfAP mRNA is approximately 1400 bases long and has a single open reading frame of 1251 bases with two start codons at positions 64 and 79 from the 5'-end. UfAP mRNA content of the endometrium increases as pregnancy proceeds, reaching maximum levels around day 70 and then remaining relatively constant in late gestation (days 70 to 110). The pro-form of the UfAP minus signal sequence appears to be 392 amino acids in length and has four potential N-linked glycosylation sites Asn107, Asn197, Asn243, and Asn315. Comparison of the NH2-terminal sequences of the individual UfAP poly-peptides with the amino acid sequence deduced from the cDNA has indicated a series of at least four posttranslational proteolytic processing steps which generate the various molecular forms of the UfAP. The deduced amino acid sequence of UfAP shares considerable identity with several protease inhibitors and hormone-binding proteins that are members of the serpin superfamily of proteins. The UfAP amino acid sequence also exhibits about 55% sequence identity with the P4-induced uterine milk proteins (UTMP) of the sheep. Since the UfAP and UTMP share many biosynthetic and structural features that include site of biosynthesis in the endometrium, P4-responsiveness, the presence of the mannose 6-phosphate lysosomal recognition marker, and considerable sequence similarity, the UfAP and the UTMP may have homologous function which for both still remains obscure.

Acid Phosphatase↗
PubMed1990

Complementary DNA cloning and regulation of expression of the messenger RNA encoding a pregnancy-associated porcine uterine protein related to human antileukoproteinase.

Antileukoproteinase (ALP) is a low mol wt mucosal secretory protein which, in human tissues, inhibits the activities of the neutral serine lysosomal proteinases elastase and cathepsin-G. In this study a number of recombinant cDNA clones corresponding to porcine ALP (pALP) were isolated from a cDNA library prepared from porcine endometrial poly(A)+ RNAs. The combined nucleotide sequences of the cDNA clones, representing the entire pALP mRNA sequence, are approximately 600 nucleotides long and encode a protein of 114 amino acids. The deduced amino acid sequence of pALP is 68% similar in primary structure to that of human ALP, is cysteine and proline rich, and exhibits a two-domain structure which, in the human protein, is involved in binding trypsin/cathepsin-G and elastase, respectively. However, pALP appears to lack the internal signal sequence of the corresponding human protein. Northern blot analysis of uterine RNAs using pALP cDNAs as probe demonstrated a single mRNA species approximately 0.8 kilobase in length. Uterine expression of pALP mRNA was highest in mid- and late pregnancy and very low or undetectable in early pregnancy. Estrogen and progesterone increased the levels of uterine pALP mRNA in prepubertal gilts, but not to the levels obtained at mid- and late gestation. pALP mRNA was also abundant in adult pig lung, where its expression was constitutive. Lower levels of pALP were found in fetal and neonatal lung and small intestine and in maternal cervix, spleen, and small intestine. Our study on the molecular cloning and analysis of pALP mRNA represents the first report on the porcine proteinase inhibitor and extends the identification of pregnancy-associated uterine proteins, which may play important functions in embryo or fetal development. The control of expression of pALP mRNA, which is distinct from those of other porcine uterine proteins studied to date, should provide additional insights into the mechanisms of regulation of uterine secretory activity.

Amino Acid Sequence↗
PubMed1990

Regulation of uterine and conceptus secretory activity in the pig.

Evidence is presented for the involvement of a number of specific uterine- and conceptus-derived proteins in endometrial differentiation and conceptus or fetal development. These secretory proteins include mitogens (insulin-like growth factor-I and -II, epidermal growth factor, uterine luminal fluid mitogen), binding and transport proteins (uteroferrin, insulin-like growth factor and retinol binding proteins, respectively), protease inhibitors (antileukoproteinase, plasmin/trypsin inhibitor), and trophoblastic specific proteins. Using immunological reagents and specific complementary DNA (cDNA) probes, the tissue origins of several of these proteins have now been identified. In addition, the temporal regulation of messenger RNA (mRNA) production for a number of these proteins has been elucidated. The results suggest that although circulating and locally produced steroid hormones may be involved in regulating the synthetic abilities of these tissues during pregnancy, other, as yet undefined, factors may also mediate these activities. In this paper we present a review of the current knowledge pertaining to the identity, physiological regulation and potential functions of pig maternal and conceptus secretory proteins during pregnancy.

Animals↗
PubMed1989

Molecular cloning of the type 5, iron-containing, tartrate-resistant acid phosphatase from human placenta.

The type 5, iron-containing, tartrate-resistant acid phosphatase (TR-AP) constitutes a relatively minor intracellular isozyme of acid phosphatase in the human that is immunologically related to uteroferrin, a secreted progesterone-induced protein of the porcine uterus. Here, the purification of small amounts of TR-AP from human placenta is described. When a placental lambda gt11 cDNA library was screened with two short 32P-labeled cDNA clones from within the coding region of uteroferrin, a 1412-base pair cDNA was identified that encodes the entire human TR-AP isozyme. This cDNA contains an open reading frame of 969 base pairs, corresponding to a protein of 323 amino acids. A putative signal sequence of 19 amino acids and two potential glycosylation sites are present. The deduced amino acid sequence of the human TR-AP is 85% identical to that of porcine uteroferrin (whose sequence is also reported here in complete form for the first time) and 82% identical to the corresponding regions of a partial amino acid sequence of a bovine spleen phosphoprotein phosphatase. Northern blotting techniques employing a labeled TR-AP cDNA probe revealed the presence of a 1.5-kilobase transcript in white cells from a patient with hairy cell leukemia, in human K562 erythroleukemic cells, and in Epstein-Barr virus-transformed human B-cells, but not in a human T-cell line. Culture of K562 cells in presence of 10(-8) M phorbol 12-myristate 13-acetate ester for 48-72 h enhanced TR-AP activity per cell about 30-fold and led to a corresponding increase in TR-AP mRNA levels.

Acid Phosphatase↗
PubMed1989

cDNA sequence, gene organization, and progesterone induction of mRNA for uteroferrin, a porcine uterine iron transport protein.

The complete nucleotide sequence of porcine uteroferrin mRNA was determined by analysis of overlapping cDNA and genomic clones. The uteroferrin mRNA is 1,424 nucleotides in length and encodes a precursor protein of 338 amino acids, of which 20 residues subsequently are cleaved to form the mature peptide. The uteroferrin gene spans 3.5 kb and consists of three exons and two introns. The first intron separates the 5' untranslated sequences from the translation initiation codon ATG while the other intron interrupts the coding region of the mature protein. Primer extension analysis localized the presumptive transcription initiation site of the mRNA 94 nucleotides 5' of the ATG. No canonical TATA or CAAT sequences were apparent upstream from the mRNA cap site. However, sequences within the 5'-flanking region of the gene exhibit similarities to defined regulatory sequences for iron- and steroid hormone-responsive genes. The steady-state level of uteroferrin mRNA is enhanced by progesterone but not by estrogen alone, although the extent of progesterone induction is lower than at midgestation. The simple organization of the uteroferrin gene, which contrasts with those of the transferrin gene family, and the progesterone induction of uteroferrin mRNA expression suggest that, although this protein may have evolved in a manner distinct from other iron binding proteins, its regulation by steroid hormones may be similar.

Acid Phosphatase↗
PubMed1989

Insulin-like growth factor-I expression during early conceptus development in the pig.

Porcine conceptuses (embryo and associated membranes) in utero undergo developmental morphological transformations coincident with structural and biochemical changes in the uterine endometrium during early gestation. To elucidate a possible role for insulin-like growth factor-I (IGF-I) in these events, porcine endometrial (Days 8, 10, 11, 12, 14, and 30) and conceptus (Days 12, 14, and 16) tissues were characterized for the presence of IGF-I peptide and mRNAs. The corresponding uterine luminal fluids (ULF) at these stages of pregnancy were also analyzed for immunoreactive IGF-I concentration. ULF IGF-I was lowest on Day 8, highest on Day 12, and declined by Day 14. In contrast, endometrial tissue IGF-I content remained constant during this period. Conceptus tissues contained less IGF-I than endometrial tissues; however, conceptus IGF-I values were maximum on Day 12 coincident with peak values for ULF IGF-I. Dot-blot hybridization analyses revealed temporal variation in steady-state levels of IGF-I mRNAs in endometrium. Highest levels of endometrial IGF-I mRNA were detected on Day 12 and were about 4-fold greater than on Day 30 of pregnancy. IGF-I mRNA expression in conceptus tissues on Days 12, 14, and 16 was the same and was significantly less than that in endometrium on Day 12. These results demonstrate the temporal variation of IGF-I mRNA abundance in uterine endometrium and of immunoreactive IGF-I in ULF and in conceptus tissues, with the developmental processes occurring in the conceptuses at early pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

The relationship of insulin-like growth factor-I with postweaning performance in Angus beef cattle.

This study was designed to evaluate the ontogeny of serum IGF-I (SI) concentrations and its relationship to animal performance in a 140-d postweaning feeding trial. Ninety-eight progeny representing six sires (three high and three low feed conversion) and two sexes (43 bulls and 55 heifers) with ad libitum access to feed were allocated by sire and sex to monitor individual weights and pen feed consumption. Blood serum samples were obtained at the beginning of test (average age of 230 d) and every 28 d thereafter until each animal reached a fat thickness (estimated by sonoray) of 8.9 mm. Individual serum samples were acid-ethanol extracted and measured for IGF-I peptide by heterologous RIA. Serum IGF-I concentrations differed (P less than .10) between high (H) and low (L) feed conversion progeny groups at the end of the first 28-d period (125.12 vs 89.52 ng/ml) and tended to differ at the conclusion of the second 28-d period (P less than .15). Weight gains of H and L groups tended to differ in the second and third 28-d periods (P = .11 and .10, respectively). Serum IGF-I concentrations differed (P less than .05) between bulls and heifers for the first through fourth 28-d periods (P less than .01, P less than .05, P less than .10 and P less than .01, respectively). Phenotypic correlations indicated that pens with higher mean SI concentrations at the beginning of the test consumed less feed and had lower cumulative feed:gain ratios.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Differential growth factor content of uterine luminal fluids from large white and prolific Meishan pigs during the estrous cycle and early pregnancy.

Uterine luminal fluids (ULF) from Large White (LW) and prolific Chinese Meishan (MS) gilts were compared with respect to their peptide growth factor content during an estrous cycle and early pregnancy. Insulin-like growth factor-I (IGF-I) was quantitated by RIA; in vitro growth promoting properties of uterine luminal fluid mitogen (ULFM) were measured by [3H]thymidine incorporation into DNA of quiescent AKR-2B fibroblastic cells in culture. Peak concentrations (pg/microgram ULF protein) of IGF-I in ULF of Large White and Meishan gilts, respectively, were: estrous cycle, 9.8 +/- 1.4 (on d 10) and 39.7 +/- 7.8 (on d 12); gestation, 13.1 +/- 3.2 (on d 8 and 10) and 11.9 +/- 2.1 (on d 12), with differences among days (except d 10, P greater than .5) being affected by breed (P less than .10). For both breeds, there was a rapid decline in IGF-I concentrations by d 14 of the cycle and of pregnancy. Uterine luminal fluid mitogen activity was greater (P less than .01) for LW than for MS gilts on d 10 to 14 of an estrous cycle and gestation and diminished in a time-dependent manner in both breeds. No correlation was observed between IGF-I concentrations and uterine weights for either breed. In contrast, a negative correlation between uterine weight and ULFM activity was detected for cyclic (MS: r = -.855, P less than .10; LW: r = -.834, P less than .05) and pregnant (MS: r = -.806, P less than .10; LW: r = -.928, P less than .05) gilts.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

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 (Mr's 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.

Animals↗
PubMed1988

A uterine cell mitogen distinct from epidermal growth factor in porcine uterine luminal fluids: characterization and partial purification.

Uterine luminal fluids (ULF) from early (Days 10 and 12)-pregnant sows contain factors that stimulate DNA synthesis in a variety of cell lines. The major growth factor component in these fluids has been partially purified 200-fold by heat treatment, anion-exchange chromatography, and gel filtration using mouse embryo-derived AKR-2B fibroblasts as an indicator cell line. The ULF mitogen (ULFM) is a polypeptide with an apparent molecular weight of 4800; it is extremely heat stable and resistant to treatment with urea. This mitogen is also present in ULF from cycling sows but is not detectable in uterine cytosolic extracts or in serum isolated from pigs at Day 12 of pregnancy. The addition of this factor to medium containing 0.5% calf serum results in a 50% increase in final cell density of AKR-2B cells. ULFM appears biologically distinct from mouse and human epidermal growth factor (EGF), since its activity is not inhibited by antibody to mouse EGF and it does not compete for binding to human (A431) EGF receptors. In addition, the ULF factor stimulates DNA synthesis in human A431 epidermoid carcinoma cells, whereas EGF is inhibitory. Partially purified ULFM also stimulates DNA synthesis in primary cultures of pig uterine stromal cells. This mitogen activity is dose-dependent and is not inhibited by antibody to mouse EGF. Thus ULFM may act in concert with other peptide growth factors in regulating uterine growth and/or differentiation.

Animals↗
PubMed1988

Molecular cloning and temporal expression during pregnancy of the messenger ribonucleic acid encoding uteroferrin, a progesterone-induced uterine secretory protein.

A lambda gt11 expression library containing cDNA inserts prepared from porcine endometrial mRNA was immunologically screened by using an antiserum developed against porcine uteroferrin (Uf), a glycoprotein that has been strongly implicated in transplacental iron transport in the pregnant pig. Antibody reactive clones (lambda 4a3, 13.1, and 2.2) were isolated after screening 1.5 x 10(5) recombinant phages. Clones 4a3 and 13.1 expressed Uf antigenic determinants in beta-galactosidase fusion proteins and specifically selected antibody which reacted with Uf in immunoblots prepared from uterine cytosolic extracts. In addition, all three cDNA clones collectively contained DNA sequences that encoded an 85-amino acid peptide which corresponded to a region within the carboxyterminal portion of the Uf protein. Northern blot hybridization of these cDNAs to RNAs extracted from whole uterine tissue of pregnant pigs revealed a single uterine poly(A)+ RNA of approximately 1.7 kilobases in length, which was not found in liver and mammary tissue RNAs. The concentration of the Uf mRNA changed in a temporal fashion during pregnancy in a manner that was distinct from that of the progesterone receptor mRNAs. Highest levels of Uf mRNA were found at mid and late pregnancy (days 45-110) and were about 50-fold greater than at day 30 of pregnancy. By contrast, RIA analysis of the uterine tissue extracts showed that maximum amounts of Uf were present at day 60 and then declined sharply. Thus the pattern of Uf mRNA present in the uterus did not parallel the amount of Uf polypeptide that could be recovered from the tissue. The tissue specific and temporal regulation of Uf gene expression emphasizes that the protein plays an important role in uterine activity and/or fetal development.

Acid Phosphatase↗
PubMed1988

Porcine insulin-like growth factor-I (pIGF-I): complementary deoxyribonucleic acid cloning and uterine expression of messenger ribonucleic acid encoding evolutionarily conserved IGF-I peptides.

In order to facilitate studies of insulin-like growth factor-I (IGF-I) expression during the pregnancy-associated development of uterus and mammary gland in the pig model, we have isolated several cDNA clones corresponding to porcine IGF-I (pIGF-I) mRNA. Sequence analysis of two cDNA fragments (sigf. 2 and sigf. 3) revealed an open reading frame encoding in order a putative 25 amino acid (aa) hydrophobic leader peptide, the mature (processed) 70 aa pIGF-I peptide and a 35 aa carboxy-terminal extension (E) peptide. The deduced aa sequence of the pIGF-I peptide is identical to human and bovine IGF-I but differs from that of rat and mouse at three and four residues, respectively. The sequences of the amino- and carboxy-terminal IGF extension peptides are also highly conserved among these species. Northern analysis using sigf. 3 as a probe revealed multiple IGF-I mRNAs (including species of 8000, 2300, and 1200 nucleotides in length) in uteri of pregnant pigs. Highest levels of the uterine IGF-I mRNAs were found at early pregnancy, when increased levels of immunoreactive tissue IGF-I were also observed. Mammary levels of IGF-I mRNAs and protein were considerably lower than that observed for uterus at the same time period. Thus, uterine production of IGF-I appears to be especially significant during early pregnancy in the pig when uterine growth, elevated IGF-I in uterine fluids, and rapid embryonic development are observed.

Amino Acid Sequence↗