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

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

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

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

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

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

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

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

Secretion of insulin-like growth factor (IGF)-I and -II and IGF binding proteins (IGFBPs) in fetal stromal-vascular (S-V) cell cultures obtained before and after the onset of adipogenesis in vivo.

The present study examined the influence of dexamethasone (DEX) treatment on preadipocyte differentiation and insulin-like growth factor binding protein (IGFBP) secretion in stromal-vascular (S-V) cell cultures established from subcutaneous adipose tissue obtained from nine 75 day and four 50 day pig fetuses. Cultures of S-V cells from four young pigs (5-7 days old) were also studied. Each fetal S-V cell culture represented 1 pool of S-V cells/dam. Cultures were seeded and plated in 10% FBS from day 0-3 and treated with insulin (ITS) + 10 nM DEX from day 3-6 (late DEX treatment). Alternatively, cultures were seeded and plated in 10% FBS + 80 nM DEX from day 0-3 and treated with insulin alone from day 3-6 (early DEX treatment). Conditioned media was collected on day 6 of culture after 3 days of conditioning, and prepared for subsequent 125I-IGF-I ligand blot analysis for IGFBPs and RIA for IGF-I and IGF-II. Early and late DEX increased (P<0.05) preadipocyte (AD-3+) recruitment but only early DEX increased preadipocyte differentiation (lipid + and C/EBP alpha+) by day 6 in S-V cultures from 75 day fetuses. Levels of IGFBP-2, IGFBP-4, IGF-I and IGF-II in media conditioned by 75 day fetal S-V cultures were not influenced by late DEX. However, late DEX reduced levels of 29 kDa IGFBPs and markedly increased (P<0.05) IGFBP-3 levels in 75 day S-V media. Late DEX also markedly increased (P<0.05) IGFBP-3 levels in 50 day S-V media but had little influence on other IGFBPs. Early DEX treatment increased (P<0.05) IGFBP-4 levels in 75 day S-V media but had little to no influence on levels of IGF-I, IGF-II and other IGFBPs. These studies indicate that IGFBP-4 may regulate local metabolism during preadipocyte differentiation, whereas IGFBP-3 may antagonize preadipocyte differentiation by targeting IGF-I away from differentiating cells and towards growing cells.

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

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

Increased expression of the Zn-finger transcription factor BTEB1 in human endometrial cells is correlated with distinct cell phenotype, gene expression patterns, and proliferative responsiveness to serum and TGF-beta1.

Basic transcription element binding (BTEB, also designated BTEB1) protein is a member of the Sp-family of GC-box binding transcription factors that exhibit distinct patterns of expression in many cell types and tissues. A role for BTEB1 in the regulation of cell growth and gene transcription has been invoked, but little is known about the molecular mechanisms underlying these activities. The present study examined the functional consequences of high and low BTEB1 expression in the human endometrial carcinoma cell line Hec-1-A, by deriving stable clonal lines that expressed sense (S) and anti-sense (As) rat BTEB1 constructs. Clonal S lines, with BTEB1 mRNA and protein levels higher than in corresponding parent (N) and As lines, displayed enhanced DNA synthesis upon 3[H]-thymidine incorporation, in serum-containing but not in serum-free medium, and increased cell cycle kinetics, concomitant with the induction in expression of the genes for the cell cycle-associated components cyclin D1, PCNA, cyclin-dependent kinase (Cdk) inhibitor p21, and Cdk2. Compared to N and As lines, S lines also had diminished ability to grow in multi-layers and exhibited increased mRNA levels for plasminogen activator inhibitor-1 (PAI-1), secretory leukocyte protease inhibitor (SLPI), and tissue inhibitor of metalloproteinases (TIMP)-2. In serum-free medium, S, but not N nor As lines, had enhanced DNA synthesis with transforming growth factor (TGF)-beta1, albeit all lines demonstrated similar responses to insulin-like growth factor-I and to epidermal growth factor, respectively. The higher DNA synthesis in S relative to N and As, lines upon exogenous TGF-beta1 addition, was observed in concert with increased expression of cyclins D1 and E and p21, genes. Moreover, S and As lines had increased mRNA levels for TIMP-1, TIMP-2, PAI-1, and beta-catenin, and diminished SLPI, and to a lesser extent, Cdk4 mRNA levels, with TGF-beta1 treatment. These results suggest that BTEB1 may mediate cell growth, in part, by modulating gene expression levels of distinct cell cycle and growth-associated proteins. The correlation between serum- and TGF-beta1 induction of DNA synthesis with increased BTEB1 expression further suggests that BTEB1 may constitute an important downstream regulatory component of various signaling pathways utilized by serum-associated and other growth factors in endometrial epithelial cells.

Polyamine- and insulin-like growth factor-I-mediated proliferation of porcine uterine endometrial cells: a potential role for spermidine/spermine N(1)-acetyltransferase during peri-implantation.

Insulin-like growth factor-I (IGF-I) and the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) are progesterone-regulated genes with maximal expression at peri-implantation in the porcine uterine endometrium. However, while IGF-I stimulates cell proliferation, SSAT, by acetylating the naturally occurring polyamines (PA) spermine (SPM) and spermidine (SPD), typically functions as a cell growth inhibitor. The present study examined the functional relationships of IGF-I, SSAT, and PA in the control of endometrial cell proliferation. Northern blot analysis indicated that SSAT mRNA levels change with distinct pregnancy stages, in contrast to those for the PA biosynthetic enzyme ornithine decarboxylase (ODC). Primary cultures of luminal and glandular epithelial (LE, GE) and stromal (ST) cells isolated from Day 12 pregnant pig endometrium had IGF-I mRNA levels for ST > LE > GE cells. The mRNA levels for SSAT and ODC were transiently diminished by IGF-I treatment, but only in GE cells. By contrast, SPM and SPD increased SSAT mRNA levels in GE and ST cells, but increased ODC mRNA levels only in GE cells. IGF-I, putrescine (PUT), and SPM individually increased cellular DNA synthesis as measured by tritiated thymidine incorporation in GE and ST cells, while SPD had an effect only in ST cells. IGF-I enhanced the proliferative effect of each PA in GE cells, but only of SPD in ST cells. The mitogen-activated protein kinase inhibitor, PD98059, inhibited the induction by SPM of GE cell DNA synthesis but not that of IGF-I. Wortmannin, a phosphatidylinositol-3-kinase inhibitor had no effect on either IGF-I or SPM induction of GE cell DNA synthesis. The relative concentrations of SPM, SPD, and PUT in uterine luminal fluids differed, with the levels for each PA higher at pregnancy Day 12 than at 11.5. These results suggest that IGF-I and PA act through distinct signaling pathways to mediate cell-type-specific growth of early pregnancy pig uterine endometrium. Further, SSAT, through its control of intracellular PA levels, likely plays a modulatory role in the establishment of an optimal uterine environment for successful embryo attachment.

Mapping of expressed sequence tags from a porcine early embryonic cDNA library.

The goal of this study was to identify and map genes expressed during the elongation phase of embryogenesis in swine. Expressed sequence tags were analysed from a previously described porcine cDNA library prepared from elongating swine embryos. Average insert length of randomly selected clones was approximately 600 bp, with a range from < 100 to > 2500 bp. Single-pass, coding strand sequences from 1132 independent clones were compared with the GenBank non-redundant (nr) database via BLASTN analysis to identify potential porcine homologous of known genes. Among these sequences, 781 (69%) showed significant (score > 300) homology to non- mitochondrial sequences previously deposited in GenBank. Sequences matching interleucin 1 beta and thymosin beta 10 were most frequently observed (24 and 18 clones, respectively), in addition to matches with 310 other distinct genes. No significant match in the GenBank nr database was obtained for 303 sequences. Analysis demonstrated that 151 (50%) had open reading frames (ORF) extending at least 50 codons from the first base of the clone insert. Genetic markers were developed and used to map a subset of 17 genes, selected on the basis of function or of the ability to design primers that successfully amplified porcine genomic DNA, to 10 different porcine chromosomes, providing a set of mapped markers corresponding to genes expressed during conceptus elongation.

Molecular cloning of the porcine acid-labile subunit (ALS) of the insulin-like growth factor-binding protein complex and detection of ALS gene expression in hepatic and non-hepatic tissues.

The acid-labile subunit (ALS) is an approximately 85 kDa N-glycoprotein that is known primarily as a component of the systemic insulin-like growth factor-binding protein (IGFBP) complex. We have amplified, using a PCR, three overlapping porcine ALS genomic DNA fragments that together encode the distal region of the signal peptide through to the COOH-terminus. The compiled sequence of 1775 nucleotides of the three overlapping DNAs and the deduced amino acid sequence of the mature porcine ALS (pALS) protein exhibited 84/81%, 79/77%, 79/78% and 84/79% identities with respect to those of the human, the rat, the mouse and the baboon respectively. Four conserved cysteine residues in the NH(2)-terminal domain and 20 leucine-rich repeats in the central domain also were identified at identical positions in the porcine ALS. By using Northern blot analysis, with a genomic DNA fragment as the probe, it was determined that a 2.2 kb ALS mRNA was induced in the liver during the late fetal stage, and hepatic ALS mRNA abundance was increased post-natally. Moreover, hepatic ALS mRNA abundance was increased by daily injection of porcine somatotropin (100 microg/kg body weight) in cross-bred market pigs each weighing approximately 100 kg. The ALS mRNA was not detected by Northern analysis in any non-hepatic tissue examined. However, results of a more sensitive solution hybridization/RNAse protection assay indicated that low levels of ALS mRNA were also present in adult muscle, spleen, ovary and uterus, but not in lung, kidney, oviduct and placenta. Taken together, the present results suggest that although liver is the primary organ that expresses the ALS gene under somatotropin stimulation, some non-hepatic tissues also express the gene at low levels in the pig.

Expression of insulin-like growth factor binding proteins (IGFBPs) before and during the hormone sensitive period of adipose tissue development in the fetal pig.

The present study examined the influence of fetal age and thyroxine (T4) and growth hormone (GH) treatment, on the expression of insulin-like growth factor binding proteins (IGFBPs) in fetal pigs. On day 70 of gestation fetuses were either hypophysectomized (hypox), hypox and implanted with T4 pellets, or left intact, and were recovered 5, 10, 15 and 20 days following hypox and T4 pellet placement. Intact fetuses were also recovered from several dams at 50 days of gestation. In additional dams, hypox fetuses (day 70) were implanted with GH loaded Alzet mini-pumps on day 90, and control, untreated, and GH-treated hypox fetuses were recovered on day 105 of development. Subcutaneous adipose tissue, serum and other fetal tissues were collected at the time of recovery and prepared for subsequent ligand blot analysis with 125I -IGF-1 and immunoblot analysis with IGFBP antibodies. The main effect of IGFBP was significant (P <0.01) for age associated changes in serum IGFBP percentages. Between 50 and 75 days of fetal development the levels of 29 kDa IGFBPs in adipose tissue and serum markedly increased. In contrast, IGFBP-2 levels decreased and IGFBP-4 levels increased in adipose tissue while IGFBP-2 levels increased and levels of IGFBP-4 and -3 decreased in serum. Fetal hypox decreased adipose tissue IGFBP levels in a time and IGFBP-dependent manner. For instance, IGFBP-2 and 29 kDa IGFBP levels decreased much faster after fetal hypox than did IGFBP-3 levels whereas IGFBP-4 levels did not decrease. The main effect of IGFBP was significant (P<0.01) for T4-induced changes in adipose tissue IGFBP levels. T4 treatment increased adipose tissue levels of 29 kDa IGFBPs but did not influence IGFBP-2,-3 and -4 levels. GH treatment had no influence on adipose tissue or serum IGFBP levels. These studies indicate that IGFBP-1 (one of the 29 kDa IGFBPs) may be the major IGFBP mediator of the influence of T4 on fetal development.

Catalytic differences between porcine blastocyst and placental aromatase isozymes.

Two isozymes of porcine aromatase, the placental and the blastocyst forms, were expressed in CHO cells using the mammalian cell transfection method. Using an 'in-cell' assay (a 3H-water release method), catalytic parameters of the porcine placental aromatase were found to be very similar to those of the human enzyme; however, the activity of the blastocyst isozyme was found to be one-thirtieth that of the placental isozyme. Product isolation assay (using testosterone as the substrate) revealed that the major steroid products were 17beta-estradiol and 19-nortestosterone. The product ratio of estradiol/19-nortestosterone was found to be 94 : 6 for the porcine placental form, 6 : 94 for the porcine blastocyst form, and 92 : 8 for the human wild-type aromatase. Therefore, the porcine blastocyst aromatase isozyme catalyzes mainly androgen 19-desmethylation rather than aromatization. In addition, inhibition profile analyses on the placental and blastocyst isozymes were performed using three steroidal inhibitors [4-hydroxyandro-stenedione (4-OHA), 7alpha-(4'-amino)phenylthio-1, 4-androstandiene-3,17-dione (7alpha-APTADD), and bridge (2, 19-methyleneoxy) androstene-3,17-dione (MDL 101,003)], and four nonsteroidal inhibitors [aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole (R83842)]. While the two isozymes of porcine aromatase share 93% amino-acid sequence identity, our results indicate that the two porcine aromatase isozymes have distinct responses to various aromatase inhibitors.

Porcine ovarian cells express messenger ribonucleic acids for the acid-labile subunit and insulin-like growth factor binding protein-3 during follicular and luteal phases of the estrous cycle.

It has recently been shown that, in follicular fluid, as in the circulation, insulin-like growth factors (IGFs)-I and -II exist in a ternary complex with IGF binding protein-3 (IGFBP-3) and the acid-labile subunit (ALS). The current study was designed to determine whether ovarian follicular and luteal cells could synthesize IGFBP-3 and ALS. Ovaries were collected, during the follicular and early luteal phases, from mature pigs whose cycles were synchronized with PGF2alpha. We studied IGFBP-3 and ALS messenger RNA (mRNA) by in situ hybridization. These transcripts were colocalized with aromatase mRNA, a marker of healthy granulosa cells. IGFBP-3 mRNA was equally expressed in granulosa cells of all growing follicles. In contrast, granulosa cell ALS mRNA levels were higher (P < 0.05) in preantral and small antral follicles than in large antral follicles. In thecal cells, expression of mRNA for IGFBP-3, ALS and cyclin D1 (a marker of cell proliferation) was restricted to healthy (aromatase-expressing) follicles. In those follicles, thecal expression of IGFBP-3 mRNA was low or absent in preantral follicles but increased (P < 0.05) in antral follicles. Thecal cell ALS transcripts peaked in small antral follicles (P < 0.05) and then declined. In granulosa cells of atretic follicles, transcripts for aromatase were greatly reduced, whereas IGFBP-3 mRNA levels remained high. In contrast, ALS transcript levels were greatly reduced in both granulosa (P < 0.05) and thecal cells (P < 0.001) of atretic follicles. After ovulation, IGFBP-3 mRNA was moderately expressed in granulosa luteins but strongly detected in a few theca-derived cells and in vascular endothelial cells. This study demonstrates that follicular fluid IGFBP-3 and ALS, like the IGFs, originate (at least in part) from the ovary. The ability of follicular cells to synthesize, assemble, and store all components of the ternary complex may be critical in determining the bioavailability of follicular IGF-I and -II.

Pregnancy-dependent expression of leukaemia inhibitory factor (LIF), LIF receptor-beta and interleukin-6 (IL-6) messenger ribonucleic acids in the porcine female reproductive tract.

Leukaemia inhibitory factor (LIF) and interleukin-6 (IL-6) are candidate embryo-maternal signalling molecules which are present within the uterine luminal micro-environment. We examined the relative expression of the mRNAs encoding LIF and IL-6, as well as the LIF-binding subunit (LIFR-beta) of the LIF receptor and, as a potential downstream cytokine-responsive gene, beta(2)-microglobulin (beta(2)m), in porcine peri-implantation conceptuses, and in placenta and endometrium during early and mid-pregnancy. Peri-implantation spherical and filamentous conceptuses expressed LIFR-beta and beta(2)m mRNAs with no LIF mRNA present. Rapid development in days 11/12 spherical conceptuses to the filamentous stage was accompanied by transiently increased IL-6 gene expression. The corresponding endometrium, in contrast, expressed LIF in addition to these other mRNAs. LIFR-beta, IL-6 and beta(2)m, but not LIF mRNAs, were expressed in the Jag-1 cell line, an in vitro model for porcine day 14 trophoblast. The greatest steady-state amounts of LIF, LIFR-beta and IL-6 mRNAs in both the endometrium and placenta were evident at the post-implantation stages (days 30 and 60>day 18 of pregnancy). Treatment of porcine endometrial explants with human recombinant (hr)LIF or hrIL-6 resulted in no change in, or diminished, the presence of endometrial beta(2)m mRNA, respectively. Addition of LIF to peri-implantation conceptus explant cultures, in contrast, induced beta(2)m mRNA synthesis. These results highlight the potential importance of both the endometrium and placenta as sources, as well as targets, of these cytokines throughout pregnancy. Cytokine modulation of beta(2)m, a known in vitro mitogen, may constitute one mechanism for local control of trophoblast and endometrial proliferation.

Multiple isoforms of porcine aromatase are encoded by three distinct genes.

Cytochrome P450 aromatase, a product of the CYP 19 gene and the terminal enzyme in the estrogen biosynthetic pathway, is synthesized by the ovary, endometrium, placenta, and peri-implantation embryos in the pig and other mammals, albeit to varying levels, implying its functional role(s) in pregnancy events. The aromatase produced by the pig tissues exists as three distinct isoforms (type I - ovary, type II - placenta, and type III - embryo), with presumed differences in substrate specificities, expression levels, activity, and mode of regulation. In order to delineate the molecular mechanisms whereby estrogen synthesis is regulated in these diverse tissues, the present study examined if these aromatase isoforms represent products of multiple genes or of a single gene via complex splicing mechanisms. Porcine genomic DNA from a single animal was used as a template in the polymerase chain reaction (PCR) to amplify isoform-specific sequences corresponding to exons 4 and 7, respectively. Nucleotide sequence analysis of the generated fragments revealed the presence of only clones corresponding to the three known aromatase types. Screening a porcine Bacterial Artificial Chromosome (BAC) library for aromatase gene by PCR yielded a single clone approximately 80 kb in length. Southern blot analysis, using probes specific for exons 1A-1B, 2-3, 4-9, and 10 sequences indicated that the BAC genomic clone contains the entirety of the coding exons as well as the proximal promoter region. Sequence analysis of the fragment generated with exon 4 primers determined that this BAC clone contains only the type II gene. The presence and relative orientation of the untranslated 5'- exons 1A and 1B, previously demonstrated for the type III isoform were evaluated in the BAC clone and genomic DNA by PCR. The 265 bp fragment generated from both PCR reactions was confirmed by sequence analysis to contain exons 1A and 1B that are located contiguous to each other and separated by only three bp. A diagnostic procedure for typing aromatase isoforms was developed, based on the presence of specific restriction sites within isoform-specific exons. The use of this protocol confirmed the existence of only three aromatase isoforms in the porcine genome and indicated changes in aromatase types expressed by the uterine endometrium as a function of pregnancy stage. The presence of distinct genes encoding each of the aromatase isoform predicts important differences in the mechanisms underlying the molecular evolution and regulation of porcine aromatase, unique from those of other mammals, and suggests a critical role for P450 aromatase steroidal products in uterine functions related to pregnancy events.

Expression and regulatory function of the transcription factor Sp1 in the uterine endometrium at early pregnancy: implications for epithelial phenotype.

The uterus during early pregnancy synthesizes a complex array of signaling molecules with specific spatial and temporal modes of expression and which are critical for embryo implantation and subsequent development. The mechanism(s) underlying the differential pattern of synthesis of these pregnancy-associated proteins is not understood very well. The present study evaluated the expression and trans-activation potential of the transcription factor Sp1 in the early pregnancy porcine endometrium to determine its temporal and functional association with the endometrial epithelial-specific genes encoding the transplacental iron-transport protein uteroferrin (UF) and an Sp-family member, basic transcription element-binding (BTEB) protein. Two identical Sp1 clones (717 bp) were isolated from a porcine endometrial cDNA library by polymerase chain reaction (PCR). The nucleotide sequence of these clones encodes a partial protein sequence of 238 amino acids encompassing the Zn-finger region and had significant identities with the corresponding regions in the rat and human proteins. By using a specific antibody raised against human Sp1, porcine endometrial Sp1 was found to exhibit a molecular weight of 110 kDa, was localized predominantly in the nuclei of glandular and luminal epithelial cells, and appeared to exist as a phosphorylated protein. Northern blot analysis demonstrated three distinct size transcripts of approximately 3.5, 5, and 8 kb for endometrial Sp1. The expression of Sp1 mRNA and protein, determined by RT-PCR and by its ability to bind Sp1 consensus motif in gel mobility shift assays, respectively, overlapped with, but did not parallel that of UF mRNA during early pregnancy. The effect of increased Sp1 expression on UF gene promoter activity was examined using a human Sp1 expression vector that was transiently transfected into primary cultures of pig endometrial glandular epithelial cells. Sp1 increased (P < 0.05) the promoter activities of various UF promoter-Luciferase reporter constructs by 2 to 4-fold, over those transfected with empty expression vector. Co-transfection of a BTEB expression vector with the Sp1 expression vector modified the effect of Sp1 on UF promoter activity in the shortest construct. These results suggest that Sp1 mediates the regulation of endometrial epithelial gene expression during pregnancy, and that this function is likely altered in vivo by co-expression of other family members, including BTEB.

Complex mediation of uterine endometrial epithelial cell growth by insulin-like growth factor-II (IGF-II) and IGF-binding protein-2.

The coexpression of IGF (-I and -II) peptides, corresponding receptors, and IGF binding proteins (IGFBPs) in uterine endometrium suggests that a significant component of IGF action in this tissue is via autocrine or paracrine pathways, or both. The present study examined whether IGF-II and a major uterine-expressed IGF-II binding protein, IGFBP-2, modulate endometrial epithelial cell mitogenesis. Serum-deprived porcine endometrial glandular epithelial (GE) cells of early pregnancy were treated with various concentrations of IGFs, recombinant porcine (rp) IGFBP-2, or both, and examined for changes in cellular mitogenesis by incorporation of [(3)H]thymidine into DNA. Recombinant human (rh) IGF-II stimulated DNA synthesis in a dose-dependent manner. Human [Leu(27)]-IGF-II, an analog with selective affinity for the IGF-II (type II) receptor, increased thymidine uptake by twofold compared with untreated GE cells. When added in combination with an equimolar concentration of rhIGF-I, [Leu(27)]-IGF-II or rhIGF-II stimulated thymidine incorporation to a greater extent than did rhIGF-I alone. Ligand blot analysis of GE cell conditioned medium revealed the presence of four IGFBPs with molecular masses of 48, 31, 23, and 15 kDa. Physiological concentrations of rpIGFBP-2 (nM range) increased both basal and IGF-induced DNA synthesis in GE cells. At equimolar concentrations, Des(1-6)IGF-II (an IGF-II analog with much reduced affinity for IGFBPs) and rpIGFBP-2 had additive effects on GE cell mitogenesis, suggesting that the IGFBP-2 modulation of uterine cell growth may involve both IGF-dependent and IGF-independent pathways. Our results demonstrate the complex interplay of IGF system components in uterine endometrial epithelial growth regulation in vitro, identify IGF-II and IGFBP-2 as locally coexpressed uterine epithelial cell mitogens, and suggest the presence of a functional signaling pathway by which IGF-II stimulates epithelial cell proliferation via the type II receptor.

Trans-activation functions of the Sp-related nuclear factor, basic transcription element-binding protein, and progesterone receptor in endometrial epithelial cells.

The present study examined the trans-activation potential of basic transcription element-binding protein (BTEB), a recently identified member of the Sp family of GC box-binding transcription factors, on the expression of the gene encoding the pregnancy-associated, epithelial-specific, and progesterone (P)-induced porcine uterine endometrial secretory protein, uteroferrin (UF). Endometrial expression of BTEB, P receptor (PR), and UF genes was analyzed by RT-PCR as a function of pregnancy stage and cell type and was correlated with the levels of endometrial BTEB that were quantified by Western blot and/or electrophoretic mobility shift assay. PR, BTEB, and UF messenger RNAs (mRNAs) were present in early (day 12) and mid(day 60) pregnancy pig endometrium, although expression levels varied for each mRNA (UF, day 12 << day 60; PR and BTEB, day 12 = day 60). Within the endometrium, glandular epithelial (GE) cells manifested higher amounts of UF mRNA than stromal fibroblastic cells, whereas both cell types had comparable amounts of BTEB and PR mRNAs. Expression of BTEB, however, was limited to endometrial GE cells. A BTEB expression vector (pcDNA-3BTEB) was used to examine the effect of increased BTEB protein on UF gene expression and promoter activity in primary cultures of pig endometrial GE cells. Cells transiently transfected with pcDNA-3BTEB had 2-fold higher UF mRNA levels than those transfected with the empty expression vector (pcDNA-3). Further, cells cotransfected with a UF promoter-luciferase (-1935UF-Luc) reporter gene and the BTEB expression vector had 2-fold higher Luc activity than those cotransfected with reporter gene and pcDNA-3. This effect of BTEB was not observed in transfected endometrial stromal fibroblastic cells, but was apparent in the human endometrial epithelial carcinoma cell lines ECC-1 and Hec-1-A, which exhibit low levels of BTEB protein and low or undetectable PR mRNA levels, respectively. The respective contributions of BTEB and PR to the modulation of UF promoter activity were examined by cotransfection of Hec-1-A and ECC-1 cells with expression plasmids for BTEB and PR and one of two UF promoter constructs (-831UF-Luc or -1935UF-Luc) in the absence or presence of P. The increase in UF promoter activity with BTEB was mimicked by PR in a P-dependent manner in both cell lines. The combined effect of PR/P and BTEB appeared additive in Hec-1-A cells and was synergistic in ECC-1 cells. These results highlight the cell context dependence of the trans-activation potential of BTEB and suggest its unique role, in concert with PR, in directing the temporal expression of endometrial epithelial genes of pregnancy.

Altered IGF-I and IGFBPs in senescent male and female rats.

The effects of senescence on muscle characteristics and the insulin-like growth factor I (IGF-I) pathway were assessed in male and female BN/F344 rats. The mass and total ATPase activity of gastrocnemius and plantaris muscles were reduced with age and to a greater extent in males than in females. The mass and total ATPase activity of soleus muscle were not significantly altered with age. Circulating IGF-I was also significantly reduced with age, 60% in females and 21% in males. Circulating IGF-binding protein 3 (IGFBP-3) was reduced with age. In liver and gastrocnemius muscle, mRNAs for IGF-1, IGFBP-2, and IGFBP-3 were analyzed in young and aged males of two strains, BN/F344 and Sprague-Dawley. In BN/F344 rats, liver mRNAs were unchanged with age. Also in BN/F344 rats, muscle mRNAs for IGFBP-2, and IGFBP-3 displayed nonsignificant trends toward increase with age. In aged Sprague-Dawley males, liver mRNA for IGF-I was increased 15% and muscle mRNA for IGFBP-2 was increased 110%. Thus, different age-related changes in the growth hormone (GH)/IGF pathway occur in males and females between the sexes and strains. These changes may play a role in the muscle atrophy associated with senescence.