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F A Simmen

Publications and source records attributed to F A Simmen.

At least 37 records · Page 2Linked to original sources
PubMed1998

A distal regulatory region of the insulin-like growth factor binding protein-2 (IGFBP-2) gene interacts with the basic helix-loop-helix transcription factor, AP-4.

Insulin-like growth factor binding protein-2 (IGFBP-2), the predominant IGFBP in the fetal circulation and an induced protein during several types of malignancies, belongs to a family of structurally related proteins that bind the mitogens, IGF-1 and IGF-2. The present study focused on functional analysis of the 5 '-flanking region (approximately 1.3 kb) of the IGFBP-2 gene to identify nuclear factors that mediate hepatic transcription of this gene. Luciferase (LUC) reporter constructs containing progressive deletions of 5'-flanking DNA and the intact promoter of the porcine IGFBP-2 gene were examined for functional activity by transient transfection of human HepG2 liver cells. LUC activity of the transfected reporter gene driven by the IGFBP-2 promoter and flanking sequences to -1397 (numbering relative to initiation codon at +1) was 22-fold higher than that of promoterless parent LUC vector. This activity was decreased by 60% with deletion of sequences to -874 bp, and dropped to basal levels with further truncation to -764 bp. The region between -874 and -765 bp (110 bp) functioned as a potent stimulator of heterologous SV40 promoter activity (110 bp/SV40-LUC construct) and was found to contain two noncontiguous basic helix-loop-helix (bHLH) transcription factor binding motifs (E-boxes [CAN NTG]: CACCTG and CAAATG). In electrophoretic mobility shift assays, nuclear proteins prepared from HepG2 cells formed two complexes (C1, C2) with double-stranded oligonucleotides containing either HLH sequence, mutations of which resulted in loss of complex formation. Southwestern blot analysis identified an HepG2 nuclear protein with molecular mass of 48 kDa, similar to that of the bHLH transcription factor AP-4, which bound the CACCTG motif. Cotransfection of HepG2 cells with the 110-bp/SV40-LUC construct and an expression vector encoding human AP-4 increased IGFBP-2 fragment-dependent SV40 promoter activity by 16-fold. This AP-4-mediated stimulation was lost following block mutation of both bHLH motifs within the IGFBP-2 110-bp fragment. Results demonstrate the functional importance of sequences upstream of the promoter in IGFBP-2 gene transcription and identify a novel mechanism by which bHLH proteins potentially may affect cell proliferation and differentiation via induction of IGFBP-2 synthesis.

DNA-Binding Proteins↗
PubMed1998

Proteolysis of insulin-like growth factor-binding proteins (IGFBPs) within the pig uterine lumen associated with peri-implantation conceptus development.

Pig conceptuses undergo morphological development from spherical to filamentous forms during days 10 to 12 of pregnancy, coincident with a high content of mRNAs encoding insulin-like growth factor (IGF)-I in the uterine endometrium and secretion of IGF-I into the uterine lumen. The potential regulation by developing conceptuses of the bioavailability of IGF-binding proteins (IGFBPs) within the uterine microenvironment was investigated. Uterine luminal flushings (ULFs) were obtained between days 10 and 18 of pregnancy and the presence of specific IGFBPs was detected by ligand blot analysis. ULFs collected at days 10 and 11 of pregnancy contained 46 and 43 kDa IGFBP-3, several IGFBPs of about 30 kDa including IGFBP-2, and an unidentified 26 kDa IGFBP; IGFBP-3 was the most abundant. By day 12, however, IGFBPs were substantially diminished or undetectable. Examination of the morphology of flushed conceptuses revealed that the loss of IGFBPs in ULF was associated with the transition from spherical to filamentous morphology. The abundance of IGFBP-3 mRNA in uterine endometrium, as monitored by blot-hybridization, was not altered in a similar way, suggesting that lack of IGFBP-3 in 'filamentous' ULF resulted from proteolysis rather than from decreased expression of the IGFBP-3 gene. Consistent with this, incubation of 'spherical' ULF with or without added 'filamentous' ULF at 37 degrees C resulted in the disappearance of endogenous IGFBP-3 only in 'spherical + filamentous' ULF. The protease activity in 'filamentous' ULF was inhibited by EDTA, but unlike matrix metalloproteinases, was not zinc ion-dependent or inhibited by 1,10-phenanthroline. Moreover, this activity was partially inhibited by the serine protease inhibitor aprotinin, but not by 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), a known inhibitor of plasmin. The IGFBP protease activity of ULF may therefore comprise a group of enzymes including an unidentified serine protease. The results suggest that elongating pig conceptuses induce IGFBP protease activity which may increase the intrauterine bioavailability of IGF.

Animals↗
PubMed1997

Closely related genes encode developmental and tissue isoforms of porcine cytochrome P450 aromatase.

We examined the chromosomal basis for the synthesis of tissue (ovary, endometrium/placenta, and peri-implantation blastocyst) isoforms of cytochrome P450 aromatase in the pig. DNA fragments derived from three distinct porcine aromatase chromosomal genes were cloned and characterized. The porcine type III aromatase gene encoding the blastocyst aromatase isoform was found to consist of nine coding exons and two mutually exclusive, 5' untranslated exons (designated E1A and E1B), collectively spanning 30 kb or more. The porcine type II aromatase gene, encoding the endometrial/placental aromatase isoform, was identified by cloning of a genomic DNA fragment spanning the corresponding exons 7, 8, and 9. The DNA inserts of two other phage clones encompassed exons 2, 3, and 4 of a third chromosomal gene (type I) encoding the ovarian aromatase isoform. All intron-exon junctions in these genomic fragments were found to be identical in relative positions to those of the single-copy human aromatase gene. Comparisons of cDNA and genomic sequences indicated that nucleotide sequence variation was not uniform across the corresponding exons of these genes and that the corresponding intronic sequences were conserved. The type II and type III aromatase genes were localized to the same regional location (q16-17) on swine chromosome 1, which is homologous to the human chromosome 15 region (q21.1) in which the human aromatase gene resides. Results demonstrate that the three aromatase genes characterized in the present study appear to be similar in their overall structural organization and most likely are clustered, which could have resulted from at least two independent gene duplication events. The presence of multiple aromatase genes constitutes a newly described mechanism by which aromatase enzyme biosynthesis and functional activity can be regulated in a tissue and temporal fashion and serves to highlight further the complexity of aromatase gene expression in mammals. Moreover, the presence of a unique aromatase gene that is highly expressed in pig blastocysts may constitute a paradigm for other mammals (e.g., equids, rabbit, hamster) whose peri-implantation blastocysts are estrogenic.

Animals↗
PubMed1997

A developmental switch in expression from blastocyst to endometrial/placental-type cytochrome P450 aromatase genes in the pig and horse.

Pig blastocysts exhibit a transient period of estrogen production at periimplantation, with a second, more sustained period of estrogen synthesis occurring in endometrium and placenta at later pregnancy. Previously we reported the isolation of cDNA clones encoding a novel isoform of cytochrome P450 aromatase (the terminal enzyme in the estrogen biosynthetic pathway) from porcine periimplantation blastocysts. The present study investigated pregnancy-associated expression, in blastocysts and maternal reproductive tract tissues of this and an additional mRNA transcript encoding a distinct P450 aromatase isoform. Restriction endonuclease and nucleotide sequence analyses of 44 cDNA clones demonstrated that the major aromatase mRNA in periimplantation blastocysts and early-pregnancy endometrium and placenta (blastocyst-type) differed in sequence from the major aromatase mRNA expressed in endometrium and placenta at midpregnancy (endometrial-type). The deduced blastocyst and endometrial aromatase isoform protein sequences had 93% similarity. A third type of aromatase mRNA, deleted in exons 4-6 sequences, also was cloned from blastocysts. This cDNA was identical in nucleotide sequence to the blastocyst full-length aromatase cDNA and specified an open reading frame of 354 amino acids for a putative aromatase-related protein containing the heme-binding domain. Expression of this shorter mRNA in blastocysts was confirmed by reverse transcription polymerase chain reaction. The 5'-untranslated exon sequences in the transcripts encoding the blastocyst-type aromatase isoform were distinct from that of the endometrial type, consistent with differential expression of multiple chromosomal genes. In periimplantation equine embryos, however, embryonic and placental 5'-untranslated exon-containing transcripts were coexpressed. Results identify an aromatase isoform expressed in the endometrium and placenta at midpregnancy, demonstrate a transition in synthesis of aromatase isoform-specific mRNAs during placental development, and suggest the preferential involvement of the blastocyst aromatase isoform in synthesis of estrogenic molecules that may function in embryo-maternal signaling at periimplantation.

Amino Acid Sequence↗
PubMed1997

Cell-type expression, immunolocalization, and deoxyribonucleic acid-binding activity of basic transcription element binding transcription factor, an Sp-related family member, in porcine endometrium of pregnancy.

Basic transcription element binding (BTEB) protein is a newly identified member of the C2H2 zinc finger family that also includes the transcription factors, Sp1, Sp2, Sp3, and Sp4. This family of proteins binds GC-rich motifs widely distributed in gene promoters, resulting in distinct activation or repression of transcriptional activities. Whereas Sp proteins are ubiquitously expressed, expression of BTEB appears more limited and has not been documented in the female reproductive tract of any mammalian species. This study was designed to identify and characterize the cellular distribution of BTEB in the porcine endometrium and placenta at known stages of pregnancy. Northern analysis of uterine endometrium detected BTEB mRNA that corresponds in size (5 kilobases) to that of the major BTEB transcript in rat brain. The steady-state levels of BTEB mRNA were higher (p < 0.05) in endometrium than placenta at corresponding days of pregnancy, although for each tissue, the levels did not change with pregnancy stage (p > 0.05). Luminal epithelial (LE), glandular epithelial (GE), and stromal (ST) cells isolated from pregnancy endometrium expressed the BTEB gene, but mRNA abundance varied with cell type (LE, GE > ST). Western blot analysis using an antiserum generated against the N-terminal region of a porcine BTEB fusion protein produced in Escherichia coli revealed the presence of BTEB protein only in endometrium, not in placenta. Immunohistochemical studies localized BTEB predominantly to the nuclei of endometrial GE and LE cells. Consistent with the presence of functional BTEB protein, binding to a double-stranded oligonucleotide containing multiple GC motifs was demonstrated in nuclear extracts prepared from endometrium and from endometrial LE and GE, but not ST, cells by electrophoretic mobility shift assay. These results demonstrate the preferential endometrial cell-type expression of BTEB and suggest its regulatory role in pregnancy-associated endometrial epithelial gene expression.

Animals↗
PubMed1996

The unique endometrial expression and genomic organization of the porcine IGFBP-2 gene.

The insulin-like growth factor-binding proteins (IGFBPs-1-6) modulate the mitogenic and differentiative actions of the IGFs and may have IGF-independent functions. This study examined the gene expression and pregnancy-regulation of the IGF/IGFBP system in porcine uterine endometrium and myometrium during the periimplantation period and later stages of pregnancy. Endometrial IGFBP-2 mRNA abundance exhibited stage of pregnancy-dependent induction; whereas little or no IGFBP-2 mRNA was found in myometrium. IGFBP-2 protein was immunolocalized to the endometrial glandular and luminal epithelia (staining on day 60 > day 12) with minimal or no immunostaining of uterine stroma observed. IGFBP-3 and IGFBP-4 transcript levels became elevated in endometrium after implantation; whereas, IGFBP-5 and IGFBP-6 mRNAs were in greater abundance in periimplantation than post-implantation endometrium. IGFBP-1 transcripts, in contrast, could not be identified in porcine endometrium or myometrium of pregnancy. As a pre-requisite to understanding the pregnancy-induction and endometrial-specificity of the uterine-expressed IGFBP-2 gene, cosmids encompassing the pig IGFBP-2 chromosomal locus were isolated and characterized. This gene is comprised of four exons that span > 29 kb and encode a 316 amino acid precursor protein. All four exons were found to be G/C rich with exon 1 and immediate 5' flank exhibiting hallmarks of a CpG island. This latter region was devoid of TATA and CAAT motifs. Results identify the preferential endometrial expression of different IGFBP genes at either the periimplantation or post-implantation periods, perhaps reflecting distinct actions of these proteins at the embryo-maternal and feto-maternal interfaces, respectively. Interactions of steroid receptors, endometrial transcription factors and their corresponding cis elements may confer the unique uterine expression of the IGFBP-2 gene.

Amino Acid Sequence↗
PubMed1996

Control and management of ovarian follicles in cattle to optimize fertility.

Experiments were designed to elucidate the control of ovarian follicle turnover and the impact of follicular dynamics on the subsequent fertility of dairy cattle. An experimental model was established to examine the interrelationships of gene expression for steroid enzymes, the insulin-like growth factor system and inhibin production as associated with follicle selection, dominance and atresia. Follicular dynamics during the postpartum period and the oestrous cycle are shown to be altered markedly by the metabolic demands of lactation. The feeding of ruminally-inert fat stimulated follicular development and improved reproductive performance. The development of persistent follicles during oestrus synchronization causes a reduction in fertility that can be corrected by recruitment and selection of a new ovulatory follicle after the injection of a gonadotrophin-releasing hormone agonist. Present systems of oestrus synchronization need to consider both synchronization of follicular development and corpus luteal regression in order to optimize fertility. With current systems manipulating follicle development, the potential to implement a timed insemination programme to improve reproductive management exists. Ovulation of the first-wave dominant follicle with human chorionic gonadotrophin provides a means to markedly enhance concentrations of plasma progesterone in the luteal phase.

Animals↗
PubMed1996

Insulin-like growth factor system in bovine first-wave dominant and subordinate follicles.

Estradiol (E2)-active (Day 5 [D5]), transitional E2-active (D8), atretic (D12 - 1), and E2-sustained dominant follicles (DF, D12 + 1) and associated subordinate follicles (SF) were obtained through use of an experimental model described here. The ovary bearing the DF was surgically removed by colpotomy, and individual follicles were utilized to study changes in concentrations of insulin like growth factor-I (IGF-I) and -II (IGF-II) and changes in amounts and proportions of the different IGF-binding proteins (IGFBP) present in follicular fluid (FF). The ratio of FF E2 to progesterone (EPR) was utilized to classify follicles into E2 active (EPR > 1) and E2 inactive (EPR < 1). The IGF-I and IGF-II concentrations in FF were similar among experimental groups and between DF and SF. Six different molecular mass bands (49, 43, 35, 30, 28, and 22 kDa) were detected by ligand blot in FF of DF and SF. Immunoprecipitation analysis identified four IGFBPs (-2, -3, -4, and -5) in FF. The 35-kDa band corresponded to IGFBP-2, the 49- and 43-kDa bands to IGFBP-3, the 28- and 22-kDa bands to IGFBP-4, and the 30-kDa band to IGFBP-5. No IGFBP-6 was found by immunoprecipitation. Absolute amounts and proportions of low molecular mass IGFBPs (-2, -4, and -5) were increased with atresia of the DF and in SF compared to E2-active DF. Conversely, although absolute amounts of IGFBP-3 remained unchanged, their proportion in FF decreased in SF compared to DF. The ratio of IGF-I to IGFBPs decreased with atresia of the DF, possibly leaving less bioavailable IGF-I to increase FSH action at the level of the follicle.

Animals↗
PubMed1996

Effects of growth hormone and pregnancy on expression of growth hormone receptor, insulin-like growth factor-I, and insulin-like growth factor binding protein-2 and -3 genes in bovine uterus, ovary, and oviduct.

The effects of growth hormone (GH) and pregnancy on insulin-like growth factor (IGF)-I, IGF binding protein (IGFBP)-2, and IGFBP-3 mRNA in reproductive tissues were studied in cattle. Lactating dairy cows were inseminated at estrus and treated with 25 mg/day GH (n = 8) or saline (n = 8) for 16 days. Corpus luteum (CL), ovary (CL removed), oviduct, endometrium, and myometrium were collected at the end of treatment. Messenger RNA for GH receptor, IGF-I, IGFBP-2, IGFBP-3, and actin were measured by nuclease protection assays. The CL contained more GH receptor mRNA than the other reproductive tissues examined. Expression of IGF-I mRNA was highest in myometrium, with lower amounts found in endometrium; the CL expressed the least amount of IGF-I mRNA. The IGFBP-2 mRNA was most abundant in endometrium and least abundant in CL. Expression of IGFBP-3 mRNA was detected in all reproductive tissues examined. However, endometrium, a tissue that expressed the most IGFBP-2 mRNA, had the lowest amount of IGFBP-3 mRNA. The GH receptor mRNA was decreased in cows treated with GH whereas the mRNA for IGF-I, IGFBP-2, or IGFBP-3 was not changed. In the reproductive tissues evaluated, cows that contained a conceptus at tissue collection (pregnant) had higher amounts of IGF-I mRNA than did nonpregnant cows. In summary, the level of mRNA encoding GH receptor, IGF-I, IGFBP-2, and IGFBP-3 varied within the tissues examined, suggesting that these genes may play a variety of roles in the bovine female reproductive tract. Supplemental GH failed to change the expression of IGF-I, IGFBP-2, and IGFBP-3 mRNA, possibly because of low GH receptor mRNA levels in tissues other than CL. A direct action of GH on IGF-I, IGFBP-2, or IGFBP-3 gene expression within cow reproductive tissues was not supported because the amount of IGF-I, IGFBP-2, or IGFBP-3 mRNA was not altered by GH.

Actins↗
PubMed1996

Molecular cloning and characterization of an estrogen-dependent porcine oviductal secretory glycoprotein.

A family of estrogen-dependent porcine oviductal secretory glycoproteins (POSPs) that exhibit structural similarities are synthesized and secreted into the oviductal lumen at proestrus, estrus, and metestrus. The objectives of this study were to clone the POSP cDNA, obtain the full-length cDNA and protein sequence, examine tissue specificity and species distribution, characterize its regulation, and establish its identity by comparison to other known protein, RNA, or DNA sequences. A full-length cDNA of 2022 base pairs was obtained with an open reading frame of 1581 nucleotides, coding for a deduced protein of 527 amino acids (57 970 M(r)). The deduced protein contained three potential N-glycosylation sites, a consensus heparin-binding site, and potential O-glycosylation sites. Amino acid analysis of POSP-E3 confirmed the presence of a 21-amino acid signal sequence. Northern blot analysis revealed an oviduct-specific mRNA species of 2.25 kb in the infundibulum (INF), ampulla (A), and isthmus (I). An mRNA of similar size was detected in the oviduct of the sheep, cow, and rabbit, and one of slightly greater size (2.8 kb) in the mouse and hamster oviduct but not in the horse or alligator oviduct. Dot blot analysis indicated that steady-state levels of POSP mRNA were significantly greater (p = 0.0001) in the A than in the INF or I regardless of day of the estrous cycle and were greater on Day 0 (estrus; p = 0.0001) regardless of location. Further, steady-state mRNA levels were significantly increased (p = 0.02) on Days 0 and 1, declining rapidly to Day 2 through Day 15 of the estrous cycle. Steady-state POSP mRNA levels were significantly greater (p < 0.003) in ovariectomized gilts treated with estradiol valerate than those treated with other steroid regimens, vehicle, or no treatment (Control), consistent with estrogen control of mRNA expression. The POSP protein exhibited significant identity to oviductal glycoproteins from the baboon, cow, hamster, human, mouse, and sheep, to several mammalian nonoviductal glycoproteins; and to several chitinases. POSP joins a growing subfamily of the chitinase gene family that lacks chitinase enzymatic activity.

Alligators and Crocodiles↗
PubMed1996

Regulation of insulin-like growth factor I biosynthesis in porcine granulosa cells.

In the present studies we examined the regulation of insulin-like growth factor I (IGF-I) expression in porcine granulosa cells in vitro. Using Northern analysis and ribonuclease protection assays with exon-specific probes, we identified the IGF-I messenger RNA (mRNA) transcripts present in these cells under basal and hormone-stimulated conditions. We also assessed changes in secreted IGF-I using Western blots and correlated the change in protein secretion after hormone treatment with changes in mRNA levels. By analogy to the human IGF-I gene and its transcription, two major transcripts of approximately 1 and 7.5 kilobases, seen in freshly isolated granulosa cells and follicle wall and in single passaged granulosa (MDGp1) cells, most likely correspond to IGF-IA. Minor transcripts of 3-4 kilobases, which appeared after FSH or forskolin treatments or in control cells after long exposure of the autoradiographs, were attributed to incompletely processed RNA precursors. Ribonuclease protection assay analysis using probes to detect alternative use of exon 5 or exon 6 indicated that most, if not all, of the transcripts contained only exon 6 sequence (IGF-IA). Both class 1 and class 2 transcripts were identified using exon 1- and exon 2-specific probes, respectively. GH increased steady state levels of IGF-I mRNA 3-fold, FSH increased it approximately 10-fold, and forskolin maximally increased it 12- to 15-fold. Estradiol had no effect alone or in combination with the other treatments. All treatments that increased IGF-I mRNA coordinately increased both class 1 and class 2 transcripts, with the increase in class 1 greater than that in class 2. Multiple forms of IGF-I protein were seen under basal conditions and after hormone treatment. These were identified based on mRNA analysis and biochemical methods as both glycosylated and nonglycosylated IGF-IA prohormone, incompletely processed forms of prohormone, and the mature peptide. Changes in the levels of total protein were similar to the changes in mRNA (GH, 3-fold; FSH and forskolin, 10- to 20-fold). All forms of the protein changed coordinately, suggesting that these hormones had no major effect on the intracellular processing mechanism. IGF-binding protein-3 was able to bind to all IGF-I forms. These data conclusively demonstrate FSH and GH induction of ovarian IGF-I. The porcine granulosa cell culture system used in these studies should be an excellent system for studying the hormonal regulation of IGF-I expression.

Animals↗
PubMed1996

Molecular cloning of spermidine/spermine N1-acetyltransferase from the periimplantation porcine uterus by messenger ribonucleic acid differential display: temporal and conceptus-modulated gene expression.

Using messenger RNA (mRNA) differential display, we isolated several putative differentially expressed complementary DNAs (cDNAs) from the periimplantation (days 11-12) endometrium of unilaterally pregnant pigs. Nucleotide sequence analysis revealed that one cDNA clone was 87% homologous to human spermidine/ spermine N1-acetyltransferase (SSAT) over a stretch of 201 bp and represents the porcine homologue of this cDNA. A second differentially expressed cDNA encoded the porcine equivalent of the human fragile X mental retardation gene (FMR1), whereas a third specified an open reading frame with significant homology to the Escherichia coli N-acetylglucosamine transfer protein. Because SSAT is the rate-limiting enzyme in polyamine metabolism and polyamines are required cytosolic components for cell growth and differentiation, we characterized the expression of the porcine SSAT gene as a potential marker for endometrial growth and/or differentiation during early pregnancy. Further, using the consensus sequence from human and mouse cDNAs, PCR primers were designed and used to generate a 568-bp cDNA fragment from gravid endometrium that encompassed the entire open reading frame for porcine SSAT and which was subsequently used for Northern hybridization analysis. Two distinct SSAT transcripts, a major species of 1.3 kilobase pairs (kb) and a minor species of 3.5 kb were detected in endometrium, each with similar temporal patterns of expression. The levels of SSAT mRNA were higher (P = 0.03) in gravid than in nongravid uterine endometrium of unilaterally pregnant pigs on days 11-12. Similarly, SSAT mRNAs were more abundant (P = 0.0004) in day 12 pregnant than in day 12 cyclic, and in days 30, 60, 90, and 105 pregnant pig endometria. Uterine endometrial luminal epithelial (LE), glandular epithelial (GE), and stromal (ST) cells expressed the SSAT gene, but mRNA abundance varied among cell types (LE > GE > ST). Expression of SSAT gene in ovariectomized gilts treated with estrogen (E2, 100 microg/day), progesterone (P4, 200 mg/day) or E2 + P4 for 11 days was highest (P = 0.03) in the endometria of the P4 group. In contrast, E2 (10 nM), P4 (10 nM) and E2 + P4 had no effect on SSAT mRNA abundance in uterine endometrial explants from day 12 pregnant pigs. However, steady-state SSAT mRNA levels were induced in day 12 pregnant uterine explants by conditioned medium from day 12 filamentous but not spherical conceptuses. These data demonstrate that the temporal induction of the endometrial SSAT gene during periimplantation is modulated by a factor(s) secreted by the periimplantation conceptus and suggest that this enzyme may have an important role in uterine endometrial growth, remodeling and/or differentiation during periimplantation.

Acetyltransferases↗
PubMed1996

Molecular cloning of cytochrome P450 aromatase complementary deoxyribonucleic acid from periimplantation porcine and equine blastocysts identifies multiple novel 5'-untranslated exons expressed in embryos, endometrium, and placenta.

To facilitate studies on the molecular mechanisms involved in the unique temporal expression of the P450 aromatase gene in porcine periimplantation embryos, a complementary DNA (cDNA) library was prepared from day 12 porcine embryo messenger RNAs (mRNAs) and screened with a cDNA fragment encoding the amino-terminus of human aromatase. Two size variants of nearly full-length cDNA clones (designated clones 33F and 34B; 2470 and 2588 bp, respectively) were isolated and sequenced. Clones 33F and 34B encoded identical aromatase proteins of 503 amino acids, but differed in size due to alternative polyadenylation signal usage for the corresponding mRNAs. Using the 5'-rapid amplification of cDNA ends procedure, two classes of cDNA clones that contained distinct putative exon 1 sequences (E1A and E1B, respectively), but were otherwise identical in exon 2 and 3 sequences, were isolated. The E1A DNA sequence was identical to the 5'-end specified by clones 33F and 34B, except for an additional 23 nucleotides. The E1A and E1B exons showed no significant homology with each other or with the aromatase cDNA sequences from other species, including human. Expression of E1A-containing aromatase mRNA was higher than that of E1B in day 12 blastocysts, although both mRNAs were expressed at low levels in porcine endometrium and placenta of early pregnancy and in ovary at periestrus. To determine whether the E1A or E1B sequence was common to aromatase mRNAs in other steroidogenic preimplantation embryos, aromatase cDNA clones spanning exons 1-3 were isolated from equine embryos by a combination of RT-PCR and 5'-rapid amplification of cDNA ends. Equine embryonic exon 1 cDNA sequence had no homology with corresponding regions in aromatase transcripts of pigs or other species; in contrast, exon 2 and 3 cDNA sequences predicted an amino acid sequence with significant homology to aromatase. Results from the present study demonstrate alternative splicing of a novel first exon(s) in P450 aromatase gene transcripts in porcine preimplantation embryos and in endometrium and placenta of pregnancy. The lack of homology among equine, porcine, human, and bovine aromatase 5'-untranslated exons may indicate divergence in the corresponding regulatory motifs of these regions among mammalian species. These observations suggest the potential significance of activation of the E1A promoter and/or splicing of novel aromatase 5'-exons in the transient production of estrogens by periimplantation blastocysts.

Alternative Splicing↗
PubMed1995

Effects of somatotropin on the conceptus, uterus, and ovary during maternal recognition of pregnancy in cattle.

Effects of recombinant bovine somatotropin (rbST) on ovarian and uterine function and the production of components of the insulin-like growth factor (IGF) system were examined during the period of maternal recognition of pregnancy in cattle. Lactating dairy cows were treated with 25 mg/d rbST (n = 8) or saline (n = 8) for 16 d after estrus. Ovaries, uteri, and conceptuses were collected on Day 17 after estrus. The length (millimeters) of the conceptus was recorded. The concentration of IGF-I and the content of IGF-binding proteins (BP) in uterine flushings were determined. Corpora lutea (CL) were weighed, and the number of follicles (> or = 2 mm in diameter) were counted. Follicular fluid from the largest and second-largest follicles was assayed for the concentration of IGF-I, IGFBP, progesterone, and estradiol. The length of the conceptus and the total amount of IGF-I in uterine fluid were similar for rbST and control. Recombinant bST increased 1) the weight of the CL, 2) the number of largest follicles (10 to 15 mm in diameter), 3) the concentration of IGF-I in the follicular fluid, 4) the follicular fluid content of IGFBP of the largest estrogenic follicle, and 5) the quantity of IGFBP in uterine flushings. The concentration of progesterone in the follicular fluid tended to be increased in rbST-treated cows, whereas the concentration of estradiol was similar to that of control cows. The concentration of progesterone in plasma was similar for rbST compared with control. In conclusion, the administration of rbST in lactating dairy cows for 16 d after estrus did not alter the growth of the conceptus collected on Day 17. The greatest responses to rbST were found within the ovary, where rbST increased the weight of the CL and altered the amount of IGF-I and IGFBP in the follicular fluid.

Animals↗
PubMed1995

Developmental regulation of steroidogenic enzyme gene expression in the periimplantation porcine conceptus: a paracrine role for insulin-like growth factor-I.

During periimplantation, the estrogen biosynthetic capability of the porcine conceptus is transient, coincident with morphological development (trophoblast elongation), and temporally associated with maximal uterine release of insulin-like growth factor-I (IGF-I) into the luminal fluid. We investigated the possible linkages of conceptus steroidogenesis and IGF-I by characterizing the developmental expression of the pig conceptus cytochrome P450 aromatase (P450arom) and cytochrome P450 17 alpha-hydroxylase (P45017 alpha) genes during periimplantation (days 8, 10, 12, 15, and 18) and the potential regulation by IGF-I of conceptus P450arom messenger RNA (mRNA) content. Primer pairs derived from porcine P450arom and P45017 alpha DNA sequences were used in reverse transcription-polymerase chain reaction analysis of conceptuses of differing morphologies and developmental stages. RNA transcripts for P450arom and P45017 alpha were readily detected only in conceptuses obtained at day 12 of pregnancy, with RNA levels varying according to morphological stage. P45017 alpha gene expression was maximal in spherical conceptuses greater than 5 mm in diameter, whereas P450arom mRNA was expressed in small (3-5 mm) and large (6-10 mm) spherical (large >> small), as well as day 12, filamentous conceptuses. Using primers derived from the human IGF-I receptor (IGF-IR) DNA sequence, constitutive expression of IGF-IR mRNA in porcine conceptuses throughout early pregnancy was demonstrated. Constitutively low expression of conceptus RNA transcripts for IGF-I ligand was similarly observed. However, uterine luminal fluid concentrations of IGF-I on day 12 were highly correlated with conceptus morphology, with maximal IGF-I in uteri possessing filamentous conceptuses. In vitro addition of IGF-I to day 12 conceptuses of filamentous or spherical morphologies resulted in distinct effects on P450arom mRNA abundance. Filamentous conceptuses had increased amounts of P450arom mRNA when treated with IGF-I. In contrast, IGF-I decreased the levels of P450arom mRNA in spherical conceptuses. IGF-I treatment did not alter mRNA expression of the IGF-IR gene in conceptuses of either morphology. These results demonstrate that transient expression of P450arom and P45017 alpha genes in perimplantation porcine conceptuses, document the constitutive expression of the conceptus IGF-IR gene, and support the involvement of endometrial IGF-I in modulation of embryonic P450arom mRNA abundance.

Amino Acid Sequence↗
PubMed1994

Transient expression of the cytochrome P450 aromatase gene in elongating porcine blastocysts is correlated with uterine insulin-like growth factor levels during peri-implantation development.

The insulin-like growth factors (IGFs-I and -II) are mediators of cellular growth and differentiation. The expression of these growth factor genes is temporally and hormonally regulated in the uterus during pregnancy, suggesting potentially important roles in embryonic development, implantation, and successful progression of pregnancy. A known regulator of uterine IGF-I secretion is estrogen, which is produced by pre-implantation mammalian embryos of several species and whose amounts may be influenced by growth factors via their effects on the transcriptional activities of steroidogenic enzyme genes. We have previously proposed that within the uterine microenvironment, a positive feedback loop may link uterine secretion of IGFs with embryonic production of estrogens to maintain and coordinate the timing of biological signals essential for embryo development. The present study examined the temporal relationships between the levels of conceptus cytochrome P450 aromatase mRNA and protein and concentrations of IGF-I and -II in uterine luminal fluids of pigs. A DNA fragment encoding a highly conserved region among mammalian aromatase P450 proteins was isolated by hybridization screening of a porcine genomic DNA library with a human aromatase P450 cDNA fragment as probe. A synthetic oligopeptide DDVIDGYPVKKGTNI within this highly conserved region was used to generate an antiserum in sheep that recognized a protein of M(r) 49,000 in Western blot analysis of porcine ovarian, placental, endometrial, and conceptus extracts. A radioimmunoassay (RIA) for aromatase P450 was established and validated using this antiserum. RIA demonstrated highest levels of aromatase P450 protein in extracts of days 10, 11, and 12 porcine conceptuses with significantly diminished levels in elongated conceptuses at days 15 and 18. In the conceptus, aromatase P450 was localized to the inner cell layer (hypoblast) of the trophectoderm. A major mRNA transcript of approximately 3 kb in length was demonstrated by Northern blot analysis of conceptus RNA with a porcine aromatase P450 antisense RNA probe. The relative levels of aromatase P450 mRNA were higher in conceptuses at day 12 than at days 15 and 18, in parallel with the levels of aromatase P450 protein. RIA of uterine luminal fluids demonstrated maximal concentrations of IGF-I at day 12, which were significantly decreased by day 15, and increased concentrations of IGF-II by day 12, which were maintained until day 18 of pregnancy. These results demonstrate that the transient expression of conceptus aromatase P450 mRNA and protein in elongating pig blastocysts is coincident with their capacity to secrete estrogens and with the rapidly changing concentrations of IGFs within the uterine microenvironment.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Ontogeny of the porcine insulin-like growth factor system.

The developmental expression of the individual components of the insulin-like growth factor (IGF) system in pigs was examined. Serum IGF-I and IGF-binding protein-3 (IGFBP-3) levels were low during fetal life and increased during postnatal development. Levels of mRNAs encoding these proteins were not greater for liver than for nonhepatic tissues (skeletal muscle, lung, kidney) and did not increase during the postnatal period, whereas hepatic growth hormone (GH) receptor mRNA expression was increased postnatally. Serum IGF-II levels exceeded IGF-I levels at all developmental stages examined and both exhibited postnatal increases. IGF-II mRNA abundance, in contrast, was high in the fetal tissues with the exception of lung and declined during the perinatal transition. Hepatic IGFBP-2 mRNA and serum IGFBP-2 levels increased during the latter half of gestation and then declined postnatally. The levels in muscle and liver of type I IGF receptors and the corresponding mRNAs also exhibited postnatal decreases. The discordance of changes in hepatic IGF-I and IGF-II mRNA abundance with serum IGF levels during the postnatal period does not support the concept that liver is the primary endocrine source of IGFs in the young pig. The postnatal increases in serum IGF levels may reflect decreased plasma clearance rates of these peptides which may be related to the transition in IGFBP type from IGFBP-2 to IGFBP-3 in blood and the reduced tissue expression of IGF receptors.

Animals↗