RESUMO
The nutritional health of dogs and cats is important to pet owners around the world. Nutrition is inextricably linked to the health of the gastrointestinal system and vice versa. Gastrointestinal signs, such as vomiting, diarrhea, anorexia, or weight loss, are one of the most common reasons that dog and cat owners make non-routine appointments with veterinarians. Those patients are evaluated systematically to identify and/or rule out the causes of the symptoms. Some causes of chronic diarrhea are within the gastrointestinal tract while others are secondary to pathogenic factors outside the digestive system. Some useful biomarkers of chronic intestinal disease (enteropathy) exist in serum and feces. After determination that the clinical signs are due to primary gastrointestinal disease and that there is no parasitism, specific diets are used for at least two weeks. There are several types of diets for pets with chronic enteropathies. There are limited ingredient diets and hydrolyzed protein diets with reduced levels of allergens. There are also highly digestible and fiber-enhanced diets. Some diets contain probiotics and/or prebiotics. If symptoms do not improve and the patient is stable, a diet from a different class may be tried. For chronic enteropathies, the prognosis is generally good for symptom resolution or at least improvement. However, if interventions with novel diets do not ameliorate the symptoms of chronic enteropathy, then antibiotic, anti-inflammatory, or immunosuppressant therapy or further, more invasive diagnostics such as taking an intestinal biopsy, may be indicated. Pancreatitis is a common gastrointestinal disease in dogs and cats and patients may present with mild to severe disease. Many patients with mild to moderate disease can be successfully treated with early supportive care, including feeding a low-fat diet. A novel pharmaceutical, fuzapladib (Panoquell-CA1) looks very promising for treating more severe forms of acute pancreatitis in dogs. Maintenance on a low-fat diet may prevent pancreatitis in at-risk dogs. Future advances in medicine will allow pet owners and veterinarians to use dietary management to maximize the health of their dogs and cats.
Assuntos
Doenças do Gato , Doenças do Cão , Gastroenteropatias , Doenças Inflamatórias Intestinais , Pancreatite , Gatos , Cães , Humanos , Animais , Doenças do Gato/diagnóstico , Doenças do Gato/terapia , Doença Aguda , Doenças do Cão/diagnóstico , Doenças do Cão/terapia , Dieta , Gastroenteropatias/diagnóstico , Gastroenteropatias/terapia , Gastroenteropatias/veterinária , Diarreia/diagnóstico , Diarreia/terapia , Diarreia/veterináriaRESUMO
Males of some species, from horses to humans, require medical help for subfertility problems. There is an urgent need for novel molecular assays that reflect spermatozoal function. In the last 25 years, studies examined RNAs in spermatozoa as a window into gene expression during their development and, more recently, for their functions in early embryo development. In clinics, more dense spermatozoa are isolated by density gradient centrifugation before use in artificial insemination to increase pregnancy rates. The objectives of the current study were to discover and quantify the microRNAs in stallion spermatozoa and identify those with differential expression levels in more dense versus less dense spermatozoa. First, spermatozoa from seven stallions were separated into more dense and less dense populations by density gradient centrifugation. Next, small RNAs were sequenced from each of the 14 RNA samples. We identified 287 different mature microRNAs within the 11,824,720 total mature miRNA reads from stallion spermatozoa. The most prevalent was miR-10a/b-5p. The less dense spermatozoa had fewer mature microRNAs and more microRNA precursor sequences than more dense spermatozoa, perhaps indicating that less dense spermatozoa are less mature. Two of the most prevalent microRNAs in more dense stallion spermatozoa were predicted to target mRNAs that encode proteins that accelerate mRNA decay. Nine microRNAs were more highly expressed in more dense spermatozoa. Three of those microRNAs were predicted to target mRNAs that encode proteins involved in protein decay. Both mRNA and protein decay are very active in late spermiogenesis but not in mature spermatozoa. The identified microRNAs may be part of the mechanism to shut down those processes. The microRNAs with greater expression in more dense spermatozoa may be useful biomarkers for spermatozoa with greater functional capabilities.
Assuntos
Biomarcadores , MicroRNAs , Espermatozoides , Cavalos , Masculino , Animais , Espermatozoides/fisiologia , Espermatozoides/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Biomarcadores/metabolismoRESUMO
Glucocorticoids impair testosterone synthesis by an unknown mechanism. Stallions treated with the synthetic glucocorticoid dexamethasone had testes collected at 6 or 12 hours postinjection. The testicular expression of selected genes encoding nuclear receptors and steroidogenic enzymes was measured. At 6 hours, dexamethasone treatment decreased levels of NR0B2, NR4A1, NR5A1, and NR5A2 messenger RNAs (mRNAs) and NR5A2 mRNA levels remained depressed at 12 hours. In contrast, dexamethasone increased levels of NFKBIA mRNA at both time points. At 6 hours, dexamethasone did not alter levels of NR0B1, NR2F1, NR2F2, NR3C1, CYP11A1, CYP17A1, CYP19A1, DHCR24, GSTA3, HSD3B2, HSD17B3, LHCGR, or STAR mRNAs. In primary cultures of Leydig cells, 10 -9 and 10 -7 M dexamethasone decreased levels of NR4A1 and NR5A1 mRNAs and increased those of NFKBIA mRNA. Our discovery that dexamethasone downregulates NR4A1, NR5A1, and NR5A2 genes, known to be important for testicular functions, may be part of the mechanism by which glucocorticoids acutely decreases testosterone.
Assuntos
Dexametasona/efeitos adversos , Regulação para Baixo/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Receptores Nucleares Órfãos/biossíntese , Testosterona/biossíntese , Animais , Sistema Enzimático do Citocromo P-450/biossíntese , Dexametasona/farmacologia , Cavalos , MasculinoRESUMO
Marinobufagenin (MBG) is a cardiotonic steroid that increases in the circulation in preeclampsia. Preeclampsia and eclampsia are associated with cerebral edema. Therefore, we examined the effects of MBG on human brain microvascular endothelial cells (HBMEC) in vitro. MBG enhanced the permeability of HBMEC monolayers at 1-, 10-, and 100-nM doses, but had no effect at 0.1 nM. Agilent Human Gene Expression microarrays were utilized in these studies. MBG treatment (10 nM for 12 h) downregulated concentrations of the soluble VEGFR transcript sFLT by 59% but did not alter those of FLTv3 mRNA (determined by quantitative PCR). When treated and control HBMEC transcriptomes were interrogated on microarrays, 1,069 genes appeared to be regulated by MBG. Quantitative RT-PCR confirmed that MBG treatment upregulated ENKUR mRNA concentrations by 57%. Its protein product interacts with calmodulin and calcium channel proteins. MBG treatment downregulated several genes whose protein products are involved in cell adhesion (ITGA2B, FERMT1, CLDN16, and TMEM207) and cell signaling (GRIN2C, SLC8A1, and ESR1). The level of downregulation ranged from 22 to 66%. Altogether, MBG actively enhanced the permeability of HBMEC monolayers while downregulating genes involved in adhesion. MBG treatment had variable effects on ENKUR, GRIN2C, and SLC8A1 genes, all associated with calcium transport. These studies provide the basis for future investigations of MBG actions in normal physiology and disease.
Assuntos
Encéfalo/irrigação sanguínea , Bufanolídeos/farmacologia , Cardiotônicos/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Permeabilidade da Membrana Celular/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica/fisiologia , Humanos , Técnicas In Vitro , Receptores de Ácido Caínico/genética , Receptores de Ácido Caínico/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/metabolismo , Análise Serial de Tecidos , Receptor de GluK2 CainatoRESUMO
In vitro fertilization does not occur readily in the horse. This may be related to failure of equine sperm to initiate hyperactivated motility, as treating with procaine to induce hyperactivation increases fertilization rates. In mice, hyperactivated motility requires a sperm-specific pH-gated calcium channel (CatSper); therefore, we investigated this channel in equine sperm. Motility was assessed by computer-assisted sperm motility analysis and changes in intracellular pH and calcium were assessed using fluorescent probes. Increasing intracellular pH induced a rise in intracellular calcium, which was inhibited by the known CatSper blocker mibefradil, supporting the presence of a pH-gated calcium channel, presumably CatSper. Hyperactivation was associated with moderately increased intracellular pH, but appeared inversely related to increases in intracellular calcium. In calcium-deficient medium, high-pH treatment induced motility loss, consistent with influx of sodium through open CatSper channels in the absence of environmental calcium. However, sperm treated with procaine in calcium-deficient medium both maintained motility and underwent hyperactivation, suggesting that procaine did not act via opening of the CatSper channel. CATSPER1 mRNA was identified in equine sperm by PCR, and CATSPER1 protein was localized to the principal piece on immunocytochemistry. Analysis of the predicted equine CATSPER1 protein revealed species-specific differences in structure in the pH-sensor region. We conclude that the CatSper channel is present in equine sperm but that the relationship of hyperactivated motility to calcium influx is weak. Procaine does not appear to act via CatSper in equine sperm, and its initial hyperactivating action is not dependent upon external calcium influx.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/metabolismo , Cavalos/fisiologia , Motilidade dos Espermatozoides/genética , Espermatozoides/fisiologia , Animais , Canais de Cálcio/genética , Sinalização do Cálcio/genética , Cavalos/genética , Concentração de Íons de Hidrogênio , Espaço Intracelular/metabolismo , Cinética , Masculino , RNA Mensageiro/metabolismo , Espermatozoides/metabolismo , Distribuição TecidualRESUMO
The glutathione transferase A3-3 (GST A3-3) homodimeric enzyme is the most efficient enzyme that catalyzes isomerization of the precursors of testosterone, estradiol, and progesterone in the gonads of humans and horses. However, the presence of GST A3-3 orthologs with equally high ketosteroid isomerase activity has not been verified in other mammalian species, even though pig and cattle homologs have been cloned and studied. Identifying GSTA3 genes is a challenge because of multiple GSTA gene duplications (e.g., 12 in the human genome); consequently, the GSTA3 gene is not annotated in most genomes. To improve our understanding of GSTA3 gene products and their functions across diverse mammalian species, we cloned homologs of the horse and human GSTA3 mRNAs from the testes of a dog, goat, and gray short-tailed opossum, the genomes of which all currently lack GSTA3 gene annotations. The resultant novel GSTA3 mRNA and inferred protein sequences had a high level of conservation with human GSTA3 mRNA and protein sequences (≥70% and ≥64% identities, respectively). Sequence conservation was also apparent for the 12 residues of the "H-site" in the 222 amino acid GSTA3 protein that is known to interact with the steroid substrates. Modeling predicted that the dog GSTA3-3 may be a more active ketosteroid isomerase than the corresponding goat or opossum enzymes. However, expression of the GSTA3 gene was higher in liver than in other dog tissue. Our results improve understanding of the active sites of mammalian GST A3-3 enzymes, inhibitors of which might be useful for reducing steroidogenesis for medical purposes, such as fertility control or treatment of steroid-dependent diseases.
Assuntos
Glutationa Transferase , Cabras , Humanos , Cavalos/genética , Cães , Animais , Bovinos , Suínos , RNA Mensageiro/genética , Glutationa Transferase/metabolismo , Cabras/genética , Cabras/metabolismo , Gambás/genética , Gambás/metabolismo , Esteroides/química , Isomerases/genética , Isomerases/metabolismo , CetosteroidesRESUMO
In addition to their well-established role in detoxication, glutathione transferases (GSTs) have other biological functions. We are focusing on the ketosteroid isomerase activity, which appears to contribute to steroid hormone biosynthesis in mammalian tissues. A highly efficient GST A3-3 is present in some, but not all, mammals. The alpha class enzyme GST A3-3 in humans and the horse shows the highest catalytic efficiency with kcat/Km values of approximately 107 M-1s-1, ranking close to the most active enzymes known. The expression of GST A3-3 in steroidogenic tissues suggests that the enzyme has evolved to support the activity of 3ß-hydroxysteroid dehydrogenase, which catalyzes the formation of 5-androsten-3,17-dione and 5-pregnen-3,20-dione that are substrates for the double-bond isomerization catalyzed by GST A3-3. The dehydrogenase also catalyzes the isomerization, but its kcat of approximately 1 s-1 is 200-fold lower than the kcat values of human and equine GST A3-3. Inhibition of GST A3-3 in progesterone-producing human cells suppress the formation of the hormone. Glutathione serves as a coenzyme contributing a thiolate as a base in the isomerase mechanism, which also involves the active-site Tyr9 and Arg15. These conserved residues are necessary but not sufficient for the ketosteroid isomerase activity. A proper assortment of H-site residues is crucial to efficient catalysis by forming the cavity binding the hydrophobic substrate. It remains to elucidate why some mammals, such as rats and mice, lack GSTs with the prominent ketosteroid isomerase activity found in certain other species. Remarkably, the fruit fly Drosophila melanogaster, expresses a GSTE14 with notable steroid isomerase activity, even though Ser14 has evolved as the active-site residue corresponding to Tyr9 in the mammalian alpha class.
RESUMO
Honey bee (Apis mellifera) queens have a remarkable organ, the spermatheca, which successfully stores sperm for years after a virgin queen mates. This study uniquely characterized and quantified the transcriptomes of the spermathecae from mated and virgin honey bee queens via RNA sequencing to identify differences in mRNA levels based on a queen's mating status. The transcriptome of drone semen was analyzed for comparison. Samples from three individual bees were independently analyzed for mated queen spermathecae and virgin queen spermathecae, and three pools of semen from ten drones each were collected from three separate colonies. In total, the expression of 11,233 genes was identified in mated queen spermathecae, 10,521 in virgin queen spermathecae, and 10,407 in drone semen. Using a cutoff log2 fold-change value of 2.0, we identified 212 differentially expressed genes between mated and virgin spermathecal queen tissues: 129 (1.4% of total) were up-regulated and 83 (0.9% of total) were down-regulated in mated queen spermathecae. Three genes in mated queen spermathecae, three genes in virgin queen spermathecae and four genes in drone semen that were more highly expressed in those tissues from the RNA sequencing data were further validated by real time quantitative PCR. Among others, expression of Kielin/chordin-like and Trehalase mRNAs was highest in the spermathecae of mated queens compared to virgin queen spermathecae and drone semen. Expression of the mRNA encoding Alpha glucosidase 2 was higher in the spermathecae of virgin queens. Finally, expression of Facilitated trehalose transporter 1 mRNA was greatest in drone semen. This is the first characterization of gene expression in the spermathecae of honey bee queens revealing the alterations in mRNA levels within them after mating. Future studies will extend to other reproductive tissues with the purpose of relating levels of specific mRNAs to the functional competence of honey bee queens and the colonies they head.
Assuntos
Abelhas/genética , Transcriptoma , Animais , Abelhas/fisiologia , Feminino , Genes de Insetos , Inseminação , Masculino , Reprodução , Sêmen/fisiologia , Comportamento Sexual Animal , Espermatozoides/fisiologiaRESUMO
In the pig, transforming growth factor beta (TGFB), TGFB receptors (TGFBRs), and integrins are present during the peri-implantation period. Latency-associated peptide (LAP), a part of latent TGFB, can bind to integrin heterodimers via its Arg-Gly-Asp (RGD) sequence; therefore, ligand-receptor interactions between TGFB and TGFBRs, along with LAP and integrin heterodimers, may be functional in mediating events supporting conceptus elongation and attachment. With the use of surgically implantable osmotic pumps, we were able to maintain pregnancy with the aim of mechanistically altering in vivo receptor-ligand interactions involving TGFB with TGFBRs and LAP with integrins during porcine pregnancy. Day 9 pregnant gilts received intrauterine infusions of LAP-RGD, a recombinant mutant of LAP (LAP-RGE), or vehicle control and were ovariohysterectomized on Day 13 or 24 of pregnancy. We hypothesized that intrauterine infusion of LAP-RGD would decrease downstream signaling of TGFB while increasing LAP-integrin interactions and that net effect would enhance conceptus survival and attachment early in the peri-implantation period but possibly increase the chance of abnormal placentation later in pregnancy. Additionally, we hypothesized that infusion of LAP-RGE would disrupt TGFB signals but not alter integrin signaling, and thus the net result would be decreased conceptus survival and abnormal development. Unexpectedly, LAP-RGD intrauterine infusions resulted in a reduction of conceptus elongation, whereas infusions of LAP-RGE permitted implantation and placentation but resulted in larger fetal weight, allantois length, and allantoic fluid volume. Results suggest TGFB and integrins are contributing factors in the regulation of conceptus elongation and placental and fetal size.
Assuntos
Blastocisto/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Placenta/efeitos dos fármacos , Prenhez , Fator de Crescimento Transformador beta1/administração & dosagem , Animais , Blastocisto/citologia , Vias de Administração de Medicamentos , Implantação do Embrião/efeitos dos fármacos , Feminino , Peso Fetal/efeitos dos fármacos , Idade Gestacional , Bombas de Infusão/veterinária , Tamanho do Órgão , Osmose , Placenta/citologia , Gravidez , Prenhez/efeitos dos fármacos , Suínos , Fator de Crescimento Transformador beta1/farmacologia , ÚteroRESUMO
The process of implantation is mediated by a complex network of signaling and adhesive factors. In the pig, latent and active transforming growth factor beta (TGFB), TGFB receptors (TGFBR), and integrins (ITGs) are present during the peri-implantation period. TGFB signals via TGFBR and activates downstream effector SMAD proteins 2 and 3 (p-SMAD2/3). Latency-associated peptide (LAP), part of the latent TGFB complex, is known to bind to ITG heterodimers and activate TGFB. We hypothesize that active TGFBs and TGFBRs along with LAP and ITGs functionally interact at the conceptus-maternal interface to mediate events essential for conceptus development and attachment in pigs. Uteri and conceptuses from days 10, 12, 16, 20, and 24 pregnant gilts were immunostained for TGFB, LAP, and ITG subunits (ITGAV, ITGB1, ITGB3, ITGB5, ITGB6, and ITGB8). Activation of TGFBRs was evaluated by the presence of phosphorylated downstream effector SMAD2/3. Binding of LAP to ITGs was also evaluated using porcine trophectoderm cells. Abundant active TGFB was detected at the apical surfaces of epithelia at the conceptus-maternal interface, and p-SMAD2/3 was detected at both conceptus attachment and nonattachment sites during implantation. Separate aggregates of LAP, ITGB1, ITGB5, and later ITGB3 were detected at the porcine conceptus-maternal interface, and binding of LAP to ITGs on apical surfaces was demonstrated. Results suggest that functional LAP-ITG adhesion complexes support conceptus attachment and promote TGFB activation leading to TGFB interaction with TGFBR supporting events of porcine implantation.
Assuntos
Implantação do Embrião , Integrinas/metabolismo , Peptídeos/metabolismo , Precursores de Proteínas/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Útero/metabolismo , Animais , Biotinilação , Linhagem Celular , Feminino , Idade Gestacional , Imuno-Histoquímica , Cadeias beta de Integrinas/metabolismo , Integrina beta1/metabolismo , Integrina beta3/metabolismo , Fosforilação , Gravidez , Ligação Proteica , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Recombinantes/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Suínos , Trofoblastos/metabolismoRESUMO
BACKGROUND: It is not unusual for stallions to have fertility problems. For many, artificial insemination with more dense spermatozoa (isolated by density gradient centrifugation) results in greater pregnancy rates compared with the rates when using unfractionated spermatozoa. RNAs in spermatozoa delivered to the oocyte at conception are required for embryo development. Novel molecular assays of spermatozoa that reflect function are needed to predict the fertility of stallions. OBJECTIVES: To describe and compare the RNA populations in more dense and less dense spermatozoa from stallions. MATERIALS AND METHODS: Spermatozoa from five stallions were separated into more dense and less dense populations by density gradient centrifugation. Complementary DNA libraries were made from each of the ten total RNA samples after ribosomal RNA removal. Next-generation sequencing characterized the RNA populations in more and less dense spermatozoa. Quantitative reverse transcription-PCR was used to confirm differential expression of selected RNAs. RESULTS: Stallion spermatozoa contain 11 215 RNAs, with the most prevalent RNA being a 1492 base long non-coding RNA. The levels of 159 RNAs were greater in more dense spermatozoa, while levels of seven other RNAs were greater in less dense spermatozoa. Quantitative reverse transcription-PCR confirmed the threefold greater levels of solute carrier family 26 member 8 (SLC26A8) mRNA in less dense spermatozoa, and sixfold and threefold greater expression levels of the SCP2 sterol binding domain containing 1 (SCP2D1) and spermatogenesis-associated protein 31D1 (SPATA31D1) mRNAs in more dense spermatozoa, respectively. DISCUSSION AND CONCLUSION: We identified 11 215 RNAs in stallion spermatozoa and 166 with differential expression between more dense and less dense fractions. Many prevalent RNAs were also found in bull, boar, and human spermatozoa. Many differentially expressed RNAs are known to be testis- or spermatozoa-specific. Our results may lead to identification of an RNA population in spermatozoa that is optimal for establishing successful pregnancies.
Assuntos
Fertilidade/genética , RNA Longo não Codificante , RNA Mensageiro , Espermatozoides/metabolismo , Animais , Bovinos , Centrifugação com Gradiente de Concentração , Cavalos , Humanos , Masculino , RNA Longo não Codificante/análise , RNA Longo não Codificante/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , SuínosRESUMO
Glutathione transferases (GSTs) comprise a superfamily of enzymes prominently involved in detoxication by making toxic electrophiles more polar and therefore more easily excretable. However some GSTs have developed alternative functions. Thus, a member of the Alpha class GSTs in pig and human tissues is involved in steroid hormone biosynthesis, catalyzing the obligatory double-bond isomerization of Δ5-androstene-3,17-dione to Δ4-androstene-3,17-dione and of Δ5-pregnene-3,20-dione to Δ4-pregnene-3,20-dione on the biosynthetic pathways to testosterone and progesterone. The human GST A3-3 is the most efficient steroid double-bond isomerase known so far in mammals. The current work extends discoveries of GST enzymes that act in the steroidogenic pathways in large mammals. The mRNA encoding the steroid isomerase GST A3-3 was cloned from testis of the horse (Equus ferus caballus). The concentrations of GSTA3 mRNA were highest in hormone-producing organs such as ovary, testis and adrenal gland. EcaGST A3-3 produced in E. coli has been characterized and shown to have highly efficient steroid double-bond isomerase activity, exceeding its activities with conventional GST substrates. The enzyme now ranks as one of the most efficient steroid isomerases known in mammals and approaches the activity of the bacterial ketosteroid isomerase, one of the most efficient enzymes of all categories known today. The high efficiency and the tissue distribution of EcaGST A3-3 support the view that the enzyme plays a physiologically significant role in the biosynthesis of steroid hormones.
Assuntos
Glutationa Transferase/metabolismo , Isoenzimas/metabolismo , Esteroides/metabolismo , Sequência de Aminoácidos , Animais , Cavalos , Masculino , Homologia de Sequência , Estereoisomerismo , Especificidade por SubstratoRESUMO
In pigs, expression and amounts of biologically active TGFbetas at the conceptus-maternal interface increase significantly as conceptuses elongate and begin the implantation process. Before their activation, secreted TGFbetas are noncovalently associated with their respective, isoform-specific latency-associated peptides (LAPs), which contain the Arg-Gly-Asp (RGD) amino acid sequence that serves as a ligand for numerous integrins. Objectives of this study were to determine whether TGFbeta1 increases production of fibronectin by porcine trophectoderm, whether porcine trophectoderm adheres specifically to fibronectin and LAP, and whether functional interactions between porcine trophectoderm and the two TGFbeta-associated proteins, fibronectin and LAP, are integrin mediated. Porcine trophectoderm cells (pTr2) were cultured in presence of TGFbeta1, LAP, or pan-neutralizing anti-TGFbeta antibody; TGFbeta specifically increased (P < 0.05) fibronectin mRNA levels, as determined by Northern and slot blot analyses. Immunofluorescence microscopy demonstrated a TGFbeta-induced increase in fibronectin in pTr2 cells. In dispersed cell adhesion assays, adhesion of pTr2 cells to fibronectin was inhibited by an RGD-containing peptide (P < 0.05) and pTr2 cells attached to recombinant LAP but not to an LAP mutant, which contained an RGE sequence rather than the RGD site (P < 0.05). Fibronectin- and LAP-coated microbeads induced integrin activation at apical surfaces of both trophectoderm and uterine luminal epithelial cells, as indicated by aggregation and transmembrane accumulation of talin detected with immunofluorescence microscopy. Cell surface biotinylation and immunoprecipitation revealed integrin subunits alphav and beta1 on apical membranes of pTr2 cells. These results suggest multiple effects of TGFbeta at the porcine conceptus-maternal interface, including integrin-mediated conceptus-maternal communication through LAP.
Assuntos
Blastocisto/citologia , Implantação do Embrião/fisiologia , Endométrio/citologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Blastocisto/metabolismo , Adesão Celular/fisiologia , Linhagem Celular Transformada , Endométrio/metabolismo , Feminino , Fibronectinas/genética , Fibronectinas/metabolismo , Integrinas/metabolismo , Oligopeptídeos/metabolismo , RNA Mensageiro/análise , Transdução de Sinais/fisiologia , Suínos , Talina/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta1RESUMO
The preovulatory surge of estrogen up-regulates estrogen receptor-alpha (ER) gene expression in the uterus during the estrous/menstrual cycles of female mammals. Previously, we demonstrated that the 5-fold increase in ER mRNA levels in endometrium of ovariectomized ewes treated with a physiological dose of estradiol (E2) is entirely due to an increase in ER mRNA stability. Our current work confirms that the E2 effect is specific to ER mRNA. The sequence of ER mRNA, cloned from sheep endometrium, shows a high degree of conservation with those of other species, even in the 5'- and the very long 3'-untranslated regions. In a cell-free assay, ER mRNA demonstrates greater stability with endometrial extracts from E2-treated ewes compared with those from untreated ovariectomized ewes. The E2-enhanced stability of ER mRNA was ablated by prior treatment of the extracts with proteinase K, 70 C heat, and oxidizing and alkylating reagents, indicating that a protein is responsible for stabilization of the message. The 3'-untranslated region of ER mRNA contains discrete sequences required for E2-enhanced stability, four of which were identified by extensive deletion mutant analyses. Transfer of two of the four minimal E2-modulated stability sequences conferred E2-enhanced stability to a heterologous RNA. These minimal E2-modulated stability sequences contain a common 10-base, uridine-rich sequence that is predicted to reside in a loop structure. Throughout our studies, estrogen stabilization of ER mRNA in sheep endometrium resembled that of vitellogenin mRNA in frog liver, indicating conservation of this ancient mechanism for enhancing gene expression in response to estrogen.
Assuntos
Regiões 3' não Traduzidas , Proteínas de Transporte , Endométrio/fisiologia , Estradiol/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Receptores de Estrogênio/genética , Animais , Sequência de Bases , Sequência Conservada , Estradiol/farmacologia , Feminino , Magnésio/metabolismo , Magnésio/farmacologia , Dados de Sequência Molecular , Poliadenilação , Proteínas/metabolismo , RNA Mensageiro/efeitos dos fármacos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptores de Estrogênio/efeitos dos fármacos , Sequências Reguladoras de Ácido Ribonucleico , Ovinos , Regulação para Cima , Útero/fisiologiaRESUMO
The purpose of this study was to identify an endometrial cell line that maintained the E2 up-regulation of estrogen receptor (ER) mRNA by enhanced message stability and to assess its dependence on ER protein. Estradiol (E2) effects on gene expression were measured in three cell lines: one immortalized from sheep endometrial stroma (ST) and two from human endometrial adenocarcinomas (Ishikawa and ECC-1). E2 up-regulated ER mRNA levels in ST and Ishikawa cells, but down-regulated ER mRNA levels in ECC-1 cells. E2 up-regulated progesterone receptor (PR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and transforming growth factor-alpha (TGF-alpha) in both Ishikawa and ECC-1 cells. The selective estrogen receptor modulator ICI 182,780 antagonized the E2-induced up-regulation of ER and/or PR mRNA levels in all three cells, while another, GW 5638, antagonized the up-regulation of PR mRNA in Ishikawa and ECC-1 cells. In mechanistic studies, E2 had no effect on ER mRNA stability in ST cells and it destabilized ER mRNA in ECC-1 cells. Thus, Ishikawa cells appear to be the most physiologically relevant cell line in which to study the up-regulation of ER mRNA levels by enhanced mRNA stability. Its antagonism by ICI 182,780 reveals that ER protein is involved in this E2 response.
Assuntos
Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/metabolismo , Neoplasias do Endométrio/tratamento farmacológico , Endométrio/citologia , Estradiol/análogos & derivados , Estradiol/farmacologia , Regulação da Expressão Gênica , RNA Mensageiro/metabolismo , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Células Estromais/efeitos dos fármacos , Animais , Antineoplásicos Hormonais/farmacologia , Northern Blotting , Células Cultivadas , Clonagem Molecular , Regulação para Baixo , Endométrio/efeitos dos fármacos , Endométrio/metabolismo , Antagonistas de Estrogênios/farmacologia , Feminino , Fulvestranto , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores de Progesterona/metabolismo , Ovinos , Fatores de Tempo , Fator de Crescimento Transformador alfa/metabolismo , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Three selective estrogen receptor modulator (SERM) drugs which included 4-OH-tamoxifen (Tam), EM-800 (EM) and GW 5638 (GW) were investigated to determine their ability to inhibit estradiol-responsive gene expression in sheep endometrium. The uteri of ovariectomized ewes (10 ewes per SERM group) were infused with 10(-7)M SERMs for 24h prior to hysterectomy. Five ewes from each group received 50 microg 17beta-estradiol (E2) and the remaining five ewes received vehicle 18 h prior to hysterectomy. Northern blot analyses and in situ hybridization demonstrated that E2 treatment increased estrogen receptor (ER), progesterone receptor (PR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and cyclophilin (CYC) mRNA levels in most endometrial cells examined. Tam and GW exhibited characteristics similar to E2 by increasing ER gene expression, but they antagonized the E2-induced increases in PR and CYC mRNA levels. EM acted as an E2-agonist of GAPDH gene expression, but antagonized the E2 up-regulation of ER, PR and CYC gene expression in most endometrial cells. Immunohistochemistry determined that EM decreased ER protein levels in the glandular epithelium, and the SERMs investigated antagonized increases in PR protein levels in endometrium. In conclusion, GW and EM exhibit fewer agonist effects than Tam on endometrial gene expression. EM demonstrated the greatest antagonism of E2-enhanced levels of ER, PR and CYC, likely due to the inhibition of ER gene expression at both mRNA and protein levels.
Assuntos
Endométrio/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Animais , Benzopiranos/farmacologia , Northern Blotting , Cinamatos/farmacologia , Ciclofilinas/biossíntese , Endométrio/citologia , Endométrio/metabolismo , Endométrio/fisiologia , Estradiol/genética , Estradiol/farmacologia , Feminino , Gliceraldeído-3-Fosfato Desidrogenases/biossíntese , Imuno-Histoquímica , Hibridização In Situ , Propionatos/farmacologia , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Receptores de Estrogênio/biossíntese , Receptores de Progesterona/biossíntese , Moduladores Seletivos de Receptor Estrogênico/química , Ovinos , Estilbenos/farmacologia , Tamoxifeno/química , Tamoxifeno/farmacologiaRESUMO
We examined in vivo effects of selective estrogen receptor modulators (SERMs) 4-OH-tamoxifen (Tam), GW 5638 (GW) and EM-800 (EM) on myometrial gene expression. The uteri of ovariectomized ewes were infused with 10(-7)M of one SERM via indwelling catheters for 24h preceding hysterectomy. Half of the ewes in each SERM group received an intramuscular injection of 50 microg 17beta-estradiol (E2) 18 h prior to hysterectomy. Northern blot analysis and in situ hybridization demonstrated that E2 increased estrogen receptor (ER), progesterone receptor (PR) and cyclophilin (CYC) gene expression in the cells of both inner layer of myometrium (IM) and outer layer of myometrium (OM) as well as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression in OM. Tam also increased ER mRNA levels in OM. EM appeared to increase ER gene expression, but antagonized E2's up-regulation of PR and CYC gene expression in both IM and OM. Tam and GW also antagonized E2 up-regulation of PR gene expression in OM but not IM. No SERM affected GAPDH gene expression with or without E2. Immunohistochemistry indicated that E2 increased nuclear ER and PR protein levels in both IM and OM. EM was unique in up-regulating ER protein levels, opposite to its effects in endometrial cells. All SERMs tested antagonized this increase in PR immunostaining preferentially in OM compared to the IM layer. These results illustrate gene and cell layer-specific effects of SERMs in sheep myometrium.
Assuntos
Estradiol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Miométrio/efeitos dos fármacos , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Animais , Benzopiranos/farmacologia , Northern Blotting , Cinamatos/farmacologia , Ciclofilinas/biossíntese , Estradiol/genética , Feminino , Gliceraldeído-3-Fosfato Desidrogenases/biossíntese , Imuno-Histoquímica , Hibridização In Situ , Miométrio/citologia , Miométrio/metabolismo , Miométrio/fisiologia , Propionatos/farmacologia , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Receptores de Estrogênio/biossíntese , Receptores de Progesterona/biossíntese , Moduladores Seletivos de Receptor Estrogênico/química , Ovinos , Estilbenos/farmacologia , Tamoxifeno/química , Tamoxifeno/farmacologiaRESUMO
In the testis of the 1.5-year-old horse, spermatogenesis initiates locally in grossly light, central areas that contrast with grossly dark, peripheral areas that are as yet inactive in spermatogenesis. Gene expression was compared between "light" and "dark" tissues of 1.5-year-old horse testes to identify mechanisms important to the initiation of spermatogenesis. Microarrays containing human cDNAs were used to assess expression levels of 9132 genes simultaneously in matched pairs of dark and light testis tissues from 3 prepubertal colts. In all 3 analyses, dysferlin (DYS), down-regulated in ovarian cancer 1 (DOC1), and Golgi apparatus protein 1 (GLG1) genes were preferentially expressed in dark tissues, while outer dense fiber of sperm tails (ODF2) and phosphodiesterase 3B (PDE3B) genes were more highly expressed in light testis tissue (>1.7 balanced difference value, Incyte GEM tools software). Expression levels of 88 additional genes appeared to be different between dark and light tissues in 2 of the 3 microarray analyses. The preferential expression of DYS, DOC1, ODF2, and PDE3B genes in dark or light testis tissues was confirmed on Northern blots and localized to cell types by in situ hybridization. Future studies to determine the role of genes regulated during the initiation of spermatogenesis may aid in elucidating molecular mechanisms during this critical time as well as in identifying new therapies for enhancing male fertility.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Cavalos/fisiologia , Maturidade Sexual/fisiologia , Espermatogênese/fisiologia , Testículo/fisiologia , Animais , Escuridão , Luz , Masculino , Testículo/crescimento & desenvolvimentoRESUMO
Estrogens upregulate estrogen receptor (ER) and progesterone receptor (PR) gene expression in endometrium immediately before ovulation to prepare it for nurturing embryos. Most in vitro model systems have lost the ability to upregulate expression of the ER gene in response to estradiol (E2) or the ability to express the ER gene at all. Here, we used explant cultures from control and E2-treated ewes and assessed expression of four genes (ER, PR, glyceraldehyde 3-phosphate dehydrogenase [GAPDH], and cyclophilin [CYC] genes) that are upregulated by E2 in vivo on Northern blots. In cultures from control and E2-treated ewes, ER and PR messenger ribonucleic acid (mRNA) levels dropped significantly during 24 h of culture in the absence of E2. Glyceraldehyde 3-phosphate dehydrogenase mRNA levels increased 300% in explants from control ewes to match the higher levels in the endometrium of the E2-treated ewe (in vivo and in explant culture). The only effect of E2 in the explant cultures was to prevent the decrease in PR mRNA. The new selective ER modulator, EM-800 (EM), decreased ER and PR mRNA levels in explants from control ewes but upregulated GAPDH and CYC mRNA levels. The EM treatment in vitro mimicked that of E2 by increasing the half-life of ER mRNA in endometrial explants. These data illustrate distinct, gene-specific effects of the explant culture process, E2, and EM on the expression of endometrial genes.
Assuntos
Endométrio/citologia , Estradiol/farmacologia , Receptores de Estrogênio/genética , Receptores de Esteroides/genética , Animais , Ciclofilinas/genética , Feminino , Gliceraldeído-3-Fosfato Desidrogenases/genética , Técnicas de Cultura de Órgãos/métodos , Ovariectomia , RNA Mensageiro/genética , RNA Ribossômico 18S/genética , Receptores de Progesterona/genética , Receptores de Esteroides/efeitos dos fármacos , Valores de Referência , Ovinos , Regulação para CimaRESUMO
A single physiological dose of estradiol up-regulates estrogen receptor-alpha(ER), progesterone receptor (PR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), c-fos, cyclophilin, and actin mRNAs in the endometrium of ovariectomized ewes. Therefore, we hypothesized that these genes would be up-regulated by the preovulatory surge of estrogen which occurs on the evening of Day 15 in the estrous cycle of sheep. ER and PR mRNA concentrations increased between Day 15 and Day 1 in cyclic ewes in most endometrial epithelial cells, while GAPDH mRNA increased in epithelial and stromal cells in the deep endometrium. Day 15 pregnant ewes had lower expression of ER, PR, GAPDH, cyclophilin and actin genes. For ER and GAPDH mRNAs, the greatest reduction occurred in the superficial endometrium. Ovariectomized ewes demonstrated concentrations of ER, PR, and GAPDH mRNAs that were similar to those in the cyclic ewes. While concentrations of c-fos mRNA did not differ between groups, those of cyclophilin and actin mRNAs were lower in the pregnant and ovariectomized ewes. In conclusion, ER, PR and GAPDH gene expression rose during estrus in endometrial cells with the highest ER gene expression and were repressed in pregnant ewes in superficial endometrial cells with the greatest PR gene expression.