Biomedical subjects

R C Simmen

Publications and source records attributed to R C Simmen.

At least 55 records · Page 3Linked to original sources

PubMed1994

Expression and characterization of recombinant rat placental prolactin-like protein C.

Prolactin-like protein C (PLP-C) is a member of the rat placental family of proteins which are structurally related to pituitary prolactin (PRL). In an effort to characterize the receptor specificity and biological activity of PLP-C, we used a PLP cDNA to express the recombinant protein in a bacterial system. The PLP-C cDNA was modified by oligonucleotide mutagenesis and ligated into a human carbonic anhydrase II (hCAII) expression vector. Following a single step affinity purification, the hCAII-PLP-C fusion protein was digested with enterokinase to release a 25 kDa protein. N-Terminal sequence analysis of the 25 kDa band demonstrated identity with PLP-C. A polyclonal antiserum to the fusion protein cross reacted with seven major proteins in rat placental culture media of which two were the native forms of PLP-C. Recombinant PLP-C was not mitogenic in the Nb2 lymphoma bioassay and did not exhibit high affinity binding to rat PRL receptor. The choice of hCA-II fusion allows for rapid purification of rPLP-C which will aid in further investigation of the biological role of PLP-C.

Amino Acid Sequence↗

PubMed1994

A regulatory element within the uteroferrin gene 5'-flanking region binds a pregnancy-associated uterine endometrial protein.

DNA-protein interactions within two putative regulatory regions distal from the transcription initiation site of the porcine uteroferrin (UF) gene were characterized. These regions, termed XB (-1,600 to -1,129 bp) and AB (-1,128 to -893 bp) exhibited transcriptional enhancer activities within the context of the heterologous SV40 promoter, that were specific to endometrial cells. DNase I and gel-shift assays demonstrated that both fragments contain a heptamer motif TGCTAGA that binds a nuclear protein present in crude and DEAE-fractionated nuclear extracts from porcine endometrium of pregnancy. This heptad sequence, designated as endometrial-associated sequence (EAS), is different from previously described nuclear protein-binding consensus sequences. Mutations in the heptamer motif abolished binding to the nuclear factor, as detected by gel-shift assays. The endometrial nuclear protein that interacts with the heptamer was characterized by Southwestern and UV cross-linking analysis. The protein has an approximate M(r) of 80 kD, is basic (pI 7.7-8.6) and is present in pig endometrium throughout pregnancy. The functional relevance of this DNA-binding protein in the control of UF gene transcription in the endometrium is discussed.

Acid Phosphatase↗

PubMed1994

Retinol-binding protein: immunolocalization of protein and abundance of messenger ribonucleic acid in conceptus and maternal tissues during pregnancy in pigs.

Retinol-binding protein (RBP) is a major secretory product of liver as well as uterine endometrium and periimplantation conceptuses of pigs. The present study examined distribution of RBP protein and abundance of RBP mRNA in pig maternal and conceptus tissues throughout pregnancy. A porcine liver RBP cDNA clone was isolated and used as probe in these studies. Northern blot analysis detected a 1.0-1.1-kb transcript in conceptus, trophoblast, yolk sac, placenta, endometrium, myometrium, oviduct, and numerous fetal tissues. Slot blot analyses of RBP mRNA abundance in endometrium, myometrium, conceptus, trophoblast, chorioallantoic placenta, and embryo/fetus indicated differential RBP gene expression in each tissue. Immunocytochemical localization studies indicated the presence of immunoreactive RBP in endometrial surface and glandular epithelium, circular and longitudinal muscle of myometrium, parenchymal cells of fetal liver, and proximal convoluted tubules of fetal kidney. Results suggest an integrated system of RBP gene expression and protein secretion that allows for transport of retinol from the uterine endometrium to the periimplantation conceptus during early pregnancy and to the fetal-placental unit throughout pregnancy in pigs.

Allantois↗

PubMed1993

Chromosomal organization of the gene encoding porcine antileukoproteinase and functional analysis of the promoter region in endometrial and placental cells.

The apparent preferential expression of the elastase/cathepsin G protease inhibitor antileukoproteinase (ALP) in endometrium of species with epitheliochorial placenta suggests mechanisms of transcriptional regulation unique to these mammalian species. To begin to define the cis-acting regulatory elements involved in the endometrial transcription of the ALP gene, the porcine ALP gene was isolated and characterized. The porcine gene spans at least 13 kb and consists of 5 exons and 4 introns. This genomic structure, except for an additional exon, is similar to that of the human gene where the first three exons encode the signal peptide, trypsin/cathepsin G binding region, and elastase binding region, respectively. The positions of the 16 cysteine residues in exons 2 and 3 of the human gene are conserved in the porcine gene. The porcine gene contains a TATA box at -29 nucleotide (nt), and sequences with limited homology to those which might bind the transcription factors AP-1, AP-2, Sp-1 and Oct-1. The functional promoter activity of the ALP-5' flanking DNA was examined using chimeric ALP-chloramphenicol acetyl transferase (CAT) DNA constructs, after transient transfection in human (ECC-1, Ishikawa) and rabbit (HRE-H9) endometrial and human trophoblastic (JEG-3) cell lines. A 887 nt fragment of the ALP-5'-flanking region (-887ALP-pCAT-E) was active in these cell lines, with the highest promoter activity observed in the ECC-1. Progressive 5' deletion of the 887 nt fragment up to -243 nt had no effect on CAT gene expression in all cell lines, relative to the longest construct.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Non-consensus progesterone response elements mediate the progesterone-regulated endometrial expression of the uteroferrin gene.

A 2.0 kilobase-pair (kb) fragment encompassing the promoter and 5' flanking region of the uteroferrin (UF) gene was previously demonstrated to confer progesterone (P) responsiveness to chimeric UF gene promoter-reporter gene constructs when transfected in endometrial cells. In the present study, transient transfection experiments with the chloramphenicol acetyltransferase reporter gene linked to the sequentially deleted UF gene 5' flanking region and to genomic fragments within this region subcloned into the heterologous SV40 promoter were used to define the progesterone-responsive elements (PRE). The identified PREs are located distal to the promoter in the region between -1754 to -1601 bp and -893 to -678 bp of the UF gene and exhibit only limited similarities to the half-sites of the consensus palindromic PRE. The non-consensus PREs bind the progesterone receptor (PR) and independently exhibit P-dependent enhancer activities within the context of homologous and heterologous promoters in endometrial and placental cell lines. The unique features of these functional PREs suggest that formation of the P-PR complex with its cognate sequences upstream of the UF gene may be less dependent on the sequence per se but may require the binding of nuclear factors proximal to the PRE to stabilize PRE-PRE interactions.

Acid Phosphatase↗

PubMed1993

Expression of c-fos in porcine endometrium during the estrous cycle and early pregnancy.

This study determined the pattern of expression of the c-fos oncogene in endometrium from pregnant and cyclic gilts. A transcript of the expected 2.2-kb size for the c-fos mRNA was detected in porcine endometrium from cyclic and pregnant gilts. Expression of endometrial c-fos mRNA was not different for Days 0, 5, 10, 15, or 18 cyclic or pregnant gilts. However, expression of c-fos mRNA was greater on Day 12 of pregnancy compared to Day 12 of the estrous cycle. Furthermore, expression of c-fos mRNA was related to developmental stages when pig conceptuses secrete estradiol, the primary determinant for maternal recognition of pregnancy. Throughout the estrous cycle and early pregnancy, immunoreactive fos protein was localized to the glandular and surface epithelial cells as well as myometrial cells. Results of this study indicate that endometrial expression of c-fos mRNA increases on Day 12 of pregnancy, coinciding with the establishment of maternal recognition of pregnancy in pigs.

Animals↗

PubMed1992

Characterization and developmental expression of binding sites for the transplacental iron transport protein, uteroferrin, in fetal hematopoietic tissues.

In the pig, iron transport to the developing fetus during pregnancy involves, in part, uteroferrin (UF), a secreted progesterone-induced protein of the uterus. Neonatal pigs suffer from anemia, and the decrease in the synthesis of UF protein in late pregnancy was suggested to be partly responsible for this condition. To examine whether diminished capacity for UF uptake by pig fetuses may also contribute to neonatal anemia, binding sites for 125I-UF were examined in plasma membrane-enriched fractions of fetal liver and spleen, which are sites of fetal hematopoiesis. In addition, changes in the number of these binding sites as a function of fetal development were evaluated. Binding of 125I-UF to liver membrane fractions was displaced by intact UF greater than deglycosylated (aglyco) UF greater than ovalbumin, but not by yeast mannan. Scatchard analysis of radioligand binding showed the presence of a single class of binding sites with a dissociation constant of 10(-7) M. During fetal development and at postpartum (day 5), liver binding sites for UF remained invariant and displayed the same affinity. In contrast, the number of binding sites for UF in fetal spleen increased from midpregnancy to parturition and remained elevated in day 5 neonatal spleen. Affinity cross-linking of 125I-UF to liver membrane-associated binding sites and subsequent analysis by gel electrophoresis and autoradiography demonstrated a single labeled protein complex of Mr 58,000 and 87,000 under denaturing and nondenaturing conditions, respectively. The appearance of these bands was inhibited by intact UF, but not ovalbumin. The characteristics of the membrane-associated binding sites for UF differed from those of the mannose-related receptor previously described in reticuloendothelial cells of fetal liver. The invariant presence of UF binding components in sites of hematopoiesis during fetal development suggests that mechanism(s) unrelated to specific uptake of UF are responsible for neonatal anemia.

Acid Phosphatase↗

PubMed1992

Differential expression, during the estrous cycle and pre- and postimplantation conceptus development, of messenger ribonucleic acids encoding components of the pig uterine insulin-like growth factor system.

The temporal patterns of endometrial expression for mRNAs encoding insulin-like growth factor-I (IGF-I), IGF-II, IGF-binding protein-2 (IGFBP-2), and the type I IGF receptor (IGF-IR) were elucidated in cyclic and pregnant pigs. Peak levels of IGF-I mRNAs occurred on day 12 in cyclic and early pregnant gilts, while IGFBP-2 mRNA levels were lowest on day 10. Pregnant gilt endometrium had higher levels of both RNA classes than the corresponding cyclic endometrium. IGF-II and IGF-IR mRNAs remained low during this period. In pregnant pig endometrium and rat uterus, levels of IGF-I mRNA decreased, while those of IGF-II and IGFBP-2 mRNAs increased with stage of pregnancy. Decreased endometrial production of IGF-I mRNA during pregnancy paralleled that in the myometrium. IGF-II mRNA tissue abundance was placenta greater than endometrium greater than myometrium. In contrast, IGFBP-2 mRNA levels were higher in endometrium than in placenta and myometrium. Endometrial expression of IGF-II mRNAs was limited to surface and glandular epithelial cells; epithelial and stromal cells expressed IGFBP-2 mRNAs at comparable levels. Expression of IGF-IR mRNAs was low and did not change with pregnancy. The endometria of two breeds of pigs that exhibit different levels of prolificacy were also examined for IGF mRNAs. On day 12, endometrium from the Large White breed with high conceptus mortality had higher levels of IGF-II and IGFBP-2 mRNAs than did endometrium from the Meishan breed with low conceptus mortality. Expression of IGF-I mRNAs was higher in endometria of Meishan than Large White gilts on day 12. The differential expression of IGF mRNAs with stage of gestation and the correlation of relative ratios of IGF mRNAs with prolificacy during the critical period of maternal recognition of pregnancy suggest an important role(s) for IGFs in conceptus and fetal development.

Animals↗

PubMed1992

Ontogeny, immunocytochemical localization, and biochemical properties of the pregnancy-associated uterine elastase/cathepsin-G protease inhibitor, antileukoproteinase (ALP): monospecific antibodies to a synthetic peptide recognize native ALP.

Expression of the mRNA encoding the elastase/cathepsin-G protease inhibitor, antileukoproteinase (ALP), is highest in pig uterus during mid- and late pregnancy, suggesting a stage of pregnancy-dependent role for ALP in feto-maternal interactions. To elucidate a function for ALP in these events, immunogenic probes were developed to localize sites of ALP expression in the environment of the developing fetus. Monospecific antibodies raised against a 16-mer synthetic peptide corresponding to residues 21-36 (ALP 16P) of the deduced amino acid sequence of pig uterine ALP were generated by active immunization of sheep. ALP 16P conjugated to keyhole limpet hemocyanin elicited high titer antibodies that were specific to ALP. The antipeptide antibodies were used to characterize pig uterine ALP from allantoic fluids. Uterine ALP has an approximate mol wt of 14,000 and a pI of 8.2 and exhibits elastase inhibitor activity. Amino-terminal amino acid sequencing of uterine ALP indicated the sequence AENALKGGACPPRKIVQC, which has 44% identity with the corresponding region in human bronchial ALP. RIA for ALP, developed using ALP 16P as standard and iodinated tracer, demonstrated the presence of immunoreactive ALP in early, mid-, and late pregnant endometrium and myometrium, placenta, allantoic fluids, fetal cord blood, and fetal liver. ALP was undetectable in the maternal circulation. The ALP levels in endometrium, allantoic fluids, and fetal cord blood changed with the stage of pregnancy; however, ALP content in placenta, myometrium, and fetal liver, although different among tissues, remained invariant during gestation. By immunocytochemical analyses, ALP was localized in the glandular epithelium of the uterus, in placenta, and in fetal liver, consistent with the presence of immunoreactive ALP as measured by RIA. The localization of uterine ALP in placenta and its corresponding transport to fetal circulation provide strong evidence to support a physiological function for the protease inhibitor in the biological mechanisms controlling fetal development in utero.

Amino Acid Sequence↗

PubMed1991

Peptide growth factors and proto-oncogenes in mammalian conceptus development.

Development of mammalian conceptuses (embryo and associated membranes) in utero is an extremely complex, programmed orchestration of cellular activities that include proliferation, differentiation, migration, and death. Failure of conceptuses to develop is a significant, though not well-understood, problem in human and animal reproduction. Increased understanding of molecular and cellular processes in these events is ultimately required to determine the mechanisms responsible for developmental failures. Peptide growth factors, their associated proteins (carrier proteins and membrane receptors), and proto-oncogenes (the normal, cellular counterparts of retroviral oncogenic proteins) are intimately involved in normal and abnormal cellular growth regulation. This paper highlights recent work which suggests the involvement of these proteins in critical aspects of conceptus growth and uterine-conceptus interactions during the peri-implantation period.

Animals↗

PubMed1991

Regulation of synthesis of uterine secretory proteins: evidence for differential induction of porcine uteroferrin and antileukoproteinase gene expression.

Mechanisms regulating the expression of two pregnancy-associated proteins of the porcine uterus, namely the iron-transport protein uteroferrin (UF) and the lysosomal serine proteinase-inhibitor antileukoproteinase (ALP), were investigated by comparing the effects of estrogen (E), progesterone (P4), and conceptuses on the steady-state levels of their mRNAs. For UF, the expression of mRNA with production and secretion of the corresponding protein was also investigated. Progesterone increased the levels of endometrial UF mRNA and of secreted UF, but did not affect the levels of ALP mRNA in ovariectomized gilts that had received P4 treatment for 8 days. Estrogen inhibited the accumulation of endometrial UF mRNA, increased UF secretion in these gilts, but had no effect on levels of ALP mRNA. Administration of E to gilts on Day 11 of the cycle slightly diminished UF mRNA levels at 1 h post-E; had no effect at 6, 12, and 24 h post-E; and increased levels of secreted UF in uterine luminal fluids 24 h post-E. The presence of conceptuses increased levels of endometrial ALP mRNA and decreased UF protein in uterine luminal fluids, but did not affect levels of endometrial UF mRNA. Myometrium, endometrium, and placenta from Day 75 and Day 105 pregnant gilts were also evaluated for ALP and UF mRNA expression to determine regional expression of these steroid-regulated genes. Myometrium and endometrium expressed comparable levels of UF and ALP mRNAs within Days 75 or 105, but placenta did not express detectable levels of mRNA for either protein. Within the myometrium, UF protein is immunolocalized mostly to the inner circular and to a lesser extent to the outer longitudinal layer of smooth muscle. These results indicate that E, P4, and presence of conceptuses differentially affect endometrial expression of ALP and UF mRNAs and secretion of UF.

Acid Phosphatase↗

PubMed1991

Insulin-like growth factors in sheep uterine fluids: concentrations and relationship to ovine trophoblast protein-1 production during early pregnancy.

Uterine luminal fluids (ULFs) from Days 10, 12, 14, and 16 cyclic (C) and pregnant (Px) ewes were analyzed for presence of insulin-like growth factors (IGF-I, IGF-II) and other mitogenic factor(s). IGF content and non-IGF mitogenic activity were measured by IGF RIAs after removal of IGF binding proteins and by stimulatory effects on DNA synthesis of density arrested AKR-2B cells, respectively. ULF IGF-I content was not different between days, but differences in IGF-I between C and Px groups at Day 16 (C greater than Px) were found (p less than 0.05). ULF IGF-II content was not different between C and Px ewes; however, differences among days (p less than 0.01) were apparent. In both C and Px ewes, Day 14 ULF had highest IGF-II content (C: 4.60 +/- 0.98 ng/ml, Px: 5.39 +/- 1.38 ng/ml). In Day 12 and Day 14 (C and Px) ULF, IGF-II concentration was about 10-fold greater than that of IGF-I. AKR-2B mitogenic activity in ULF differed among days (p less than 0.01), but not between C and Px ewes. Highest activity was observed for Day 14 and Px ULF, whereas lowest activity was for Day 10 C and Day 16 Px ULF. Sephadex G-200 gel-filtration chromatography of ULF from Day 14 Px ewes demonstrated mitogenic activity in the column void volume fractions and in the 30-kDa size range of eluted proteins. Day 13 conceptuses were cultured in serum-free medium to define the effect of exogenous IGFs on ovine trophoblast protein-1 (oTP-1) secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Constitutive expression of uterine receptors for insulin-like growth factor-I during the peri-implantation period in the pig.

Insulin-like growth factor-I (IGF-I), synthesized by the uterine endometrium of cyclic and early pregnant gilts, accumulates in the uterine luminal fluid, where it comes in contact with the developing conceptus and the rapidly growing uterus. The uterus and the conceptus thus represent potential target sites for the biological effects of IGF-I, provided high-affinity Type I receptors are present. This study was undertaken to evaluate the expression of functional IGF-I receptors in the endometrium and myometrium of pregnant (Day 10, 12, and 15) gilts and in the endometrium of cyclic (Day 15) and pseudopregnant (Day 15) gilts and to correlate levels of these receptors with temporally regulated uterine production of IGF-I. Specific binding of 125I-IGF-I to endometrial membranes pretreated with MgCl2 (4 M) at 4 degrees C for 16 h, was saturable and membrane concentration-dependent. Competition of 125I-IGF-I binding to endometrial membranes was highest with unlabeled IGF-I greater than IGF-II much greater than insulin, whereas porcine relaxin was noncompetitive. Affinity cross-linking of endometrial membranes with 125I-IGF-I followed by SDS-PAGE and autoradiography revealed two labeled bands of Mr greater than 200,000 and Mr 135,000, with the major band being the Mr 135,000 species. Scatchard analysis of 125I-IGF-I binding to endometrial membranes from Day 12 pregnant gilts revealed a single class of binding sites with a dissociation constant (Kd) = 4.08 +/- 0.09 nM. Membranes prepared from endometrium of Day 10, 12, and 15 pregnant gilts exhibited comparable 125I-IGF-I binding (p greater than 0.05) that was higher (p less than 0.001) than that for the corresponding myometrial membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗

PubMed1991

Expression of messenger RNAs encoding insulin-like growth factor-I, -II, and insulin-like growth factor binding protein-2 in bovine endometrium during the estrous cycle and early pregnancy.

The present study characterized the changes in concentrations of insulin-like growth factors-I and -II (IGF-I and IGF-II) in uterine luminal flushings, and the endometrial mRNA levels of IGF-I, IGF-II, and IGF binding protein-2 (IGFBP-2) obtained from Days 0 through 18 of the bovine estrous cycle and early pregnancy. Concentrations of IGF-I and IGF-II in uterine flushings were greater on Days 0 and 5 than on other days of the estrous cycle or pregnancy. Northern blot analysis of endometrial poly(A)+ RNA revealed two major transcripts of 7.5 and 1.0 Kb for IGF-I, one major transcript of 4.0 Kb for IGF-II, and one major transcript of 1.3 Kb for IGFBP-2. RNA dot-blot analyses indicated that endometrial expression of IGF-I mRNA was unaffected by day of the estrous cycle or status (cyclic vs. pregnancy). However, endometrial expression of IGF-II mRNA was greater in pregnant than in cyclic endometrium on Days 15 and 18. Levels of endometrial IGFBP-2 mRNA increased (p less than 0.05) between Days 10 and 18 of the estrous cycle and early pregnancy. Results suggest that the presence of the bovine conceptus has a stimulatory effect on endometrial expression levels of IGF-II, whereas progesterone appears to be involved with enhancement of IGFBP-2.

Animals↗

PubMed1991

Comparative aspects of conceptus signals for maternal recognition of pregnancy.

Maintenance of corpus luteum (CL) function is essential for establishment of pregnancy in mammals. Estrogens from pig conceptuses (embryo and associated membranes) initiate events that, with prolactin, redirect secretion of the uterine luteolytic hormone prostaglandin F2 alpha (PGF) from an endocrine (to uterine veins) to an exocrine (to uterine lumen) direction to prevent luteolysis. Ovine conceptuses secrete ovine trophoblast protein-1 (oTP-1), which exhibits high amino acid sequence relatedness with alpha II interferons (IFN alpha II) and inhibits synthesis of endometrial receptors for oxytocin and uterine production of luteolytic pulses of PGF. Estrogens and oTP-1 are local antiluteolytic signals to endometrium, whereas human chorionic gonadotrophin (hCG) appears to have a direct luteotrophic effect on CL. A progestational endometrium secretes proteins that serve as growth factors, transport proteins, regulatory proteins and enzymes, as well as transporting nutrients into the uterine lumen to support conceptus development.

Animals↗

PubMed1991

Regulation of the uteroferrin gene promoter in endometrial cells: interactions among estrogen, progesterone, and prolactin.

Expression of the gene for the porcine transplacental iron transport protein uteroferrin (UF) is largely restricted to the uterus, where it is differentially regulated by estrogen (E) and progesterone (P). To study the regulatory mechanisms subserving these effects, a 2-kilobase genomic fragment corresponding to -2005 to 48 nucleotides of the UF gene was ligated up-stream to the reporter gene chloramphenicol acetyltransferase (CAT). This construct (UF-CAT) was transiently transfected into rabbit endometrial (HRE-H9), mouse fibroblastic (AKR-2B), and human choriocarcinoma (JEG-3) cells. The basal gene promoter activity of UF-CAT was exhibited in H9 cells, but not in AKR-2B or JEG-3 cells. In contrast, a simian virus-40 early promoter (SV2) was functional in all three cell lines. The H9 cells were used to examine steroid regulation of the UF gene promoter. The CAT expression in H9 cells primed with E and PRL, but not with E or PRL alone, was stimulated by P. In contrast, basal activity of SV2 in these cells was unaffected by hormones, singly or in combination. To examine the basis for the E/PRL-dependent response to P, levels of P and E receptors in H9 cells were quantified. PRL and E plus PRL increased the number of high affinity sites for P, but had little effect on levels of high affinity sites for E in treated vs. untreated H9 cells. In vivo administration of PRL to cyclic gilts had no effect on levels of endometrial UF mRNA and secreted UF protein; however, E- plus PRL-treated gilts had higher (P less than 0.05) levels of endometrial UF mRNA and luminal UF than PRL-treated gilts. These results demonstrate in vitro functional activity of the UF gene promoter and associated 5' flanking region and suggest that sequences within this region may mediate tissue-specific and steroid hormone-regulated expression of the UF gene. Moreover, interactions among E, PRL, and P modulate UF gene expression in vivo and in vitro.

Acid Phosphatase↗

PubMed1991

Effects of insulin-like growth factor-I on aromatase cytochrome P450 activity and oestradiol biosynthesis in preimplantation porcine conceptuses in vitro.

The effects of insulin-like growth factor-I (IGF-I) on aromatase P450 activity and steroid production in preimplantation pig conceptuses were evaluated in vitro. Conceptuses recovered from gilts on days 10 and 12 of pregnancy were incubated for 6 h in modified Eagle's Minimum Essential Medium (MEM) plus IGF-I (0.1 microgram/ml) or insulin (8.5 micrograms/ml), and conceptuses were monitored for their ability to convert [1,2-3H]beta-testosterone into oestrogens. Aromatase activity of day-10 conceptuses was low and unaffected by IGF-I or insulin. In contrast, basal aromatase activity in day-12 conceptuses was about threefold higher and was further increased by IGF-I (P less than 0.02), but was unaffected by insulin. To determine whether higher aromatase P450 activity was associated with increased oestradiol production, concentrations of oestradiol were determined by radioimmunoassay in culture medium of day-11 and -12 conceptuses, after incubation in MEM alone or in the presence of dehydroepiandrosterone (DHA, 1 microgram/ml) with or without IGF-I (0.1 microgram/ml) or insulin (0.1 or 8.5 micrograms/ml) for 24 h. Conceptuses in MEM plus DHA produced more oestradiol (P less than 0.01) than those in MEM alone. Addition of IGF-I or insulin did not increase the effect of DHA. Basal oestradiol production was dependent on conceptuses size; however, IGF-I or insulin did not affect basal or DHA-stimulated oestradiol production regardless of conceptus size. These findings demonstrate that IGF-I can modulate aromatase activity in vitro, without affecting overall de-novo steroidogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Somatic cell mapping and restriction fragment length polymorphism analysis of bovine insulin-like growth factor I.

The DNA isolated from cow-hamster hybrid somatic cells segregating bovine chromosomes was analyzed by Southern blotting and hybridization with a heterologous [32P]-labeled porcine cDNA probe encoding insulin-like growth factor I (IGF-I). Thirteen of 25 cow-hamster hybrid cell lines exhibited the bovine-specific IGF-I fragment. Analysis for the retention or loss of bovine IGF-I with markers previously screened against the same panel of hybrid cells revealed a 100% concordance with lactate dehydrogenase B of bovine syntenic group U3 located on bovine chromosome 5. Restriction fragment length analyses of genomic DNA from animals representing five breeds (Angus, Polled Hereford, Simmental, Gelbvieh, and Belgian Blue) and from seven half-sib Angus calves indicated that polymorphisms for the genomic composition of the bovine IGF-I gene may exist in cattle populations.

Animals↗