ABSTRACT
OBJECTIVE: To analyze steroidogenesis-related gene expression in the rat ovary exposed to melatonin supplementation. METHODS: Thirty-two virgin adult female rats were randomized to two groups as follows: the control group GI received vehicle and the experimental group GII received melatonin supplementation (10 µg/night per animal) for 60 consecutive days. After the treatment, animals were anesthetized and the collected ovaries were immediately placed in liquid nitrogen for complementary deoxyribonucleic acid microarray analyses. A GeneChip(®) Kit Rat Genome 230 2.0 Affymetrix Array was used for gene analysis and the experiment was repeated three times for each group. The results were normalized with the GeneChip(®) Operating Software program and confirmed through analysis with the secondary deoxyribonucleic acid-Chip Analyzer (dChip) software. The data were confirmed by real-time reverse transcription polymerase chain reaction analysis. Genes related to ovarian function were further confirmed by immunohistochemistry. RESULTS: We found the upregulation of the type 9 adenylate cyclase and inhibin beta B genes and the downregulation of the cyclic adenosine monophosphate response element modulator and cytochrome P450 family 17a1 genes in the ovarian tissue of GII compared to those of the control group. CONCLUSION: Our data suggest that melatonin supplementation decreases gene expression of cyclic adenosine monophosphate, which changes ovarian steroidogenesis.
Subject(s)
Adenylyl Cyclases/genetics , Gene Expression/drug effects , Inhibin-beta Subunits/genetics , Melatonin/pharmacology , Ovary/drug effects , Adenylyl Cyclases/metabolism , Animals , Cyclic AMP/metabolism , Cyclic AMP Response Element Modulator/genetics , Cyclic AMP Response Element Modulator/metabolism , Dietary Supplements , Female , Inhibin-beta Subunits/metabolism , Melatonin/metabolism , Models, Animal , Ovary/metabolism , RNA, Complementary/isolation & purification , Random Allocation , Rats, Wistar , Real-Time Polymerase Chain Reaction/methods , Steroid 17-alpha-Hydroxylase/drug effects , Steroid 17-alpha-Hydroxylase/genetics , Steroid 17-alpha-Hydroxylase/metabolism , Tissue Array Analysis/methods , Up-RegulationABSTRACT
Members of the TGF-ß superfamily are involved in numerous cell functions; however, except for myostatin, their roles in the regulation of muscle growth in fish are completely unknown. We measured tgf-ß1, tgf-ß2, tgf-ß3, inhibin ßA (inh) and follistatin (fst) gene expression during muscle growth recovery following a fasting period. We observed that tgf-ß1a and tgf-ß2 expression were quickly down-regulated after refeeding and that tgf-ß3 reached its highest level of expression 7days post-refeeding, mirroring myogenin expression. Inh ßA1 mRNA levels decreased sharply after refeeding, in contrast to fst b2 expression, which peaked at day 2. No significant modification of expression was observed for tgf-ß1a, tgf-ß1b, tgf-ß1c and tgf-ß6 during refeeding. In vitro, tgf-ß2 and inh ßA1 expression decreased during the differentiation of satellite cells, whereas tgf-ß3 expression increased following the same pattern as myogenin. Surprisingly, fst b1 and fst b2 expression decreased during differentiation, whereas no variation was observed in fst a1 and fst a2 expression levels. In vitro analyses also indicated that IGF1 treatment up-regulated tgf-ß3, inh ßA1 and myogenin expression, and that MSTN treatment increased fst b1 and fst b2 expression. In conclusion, we showed that the expression of tgf-ß2, tgf-ß3 and inh ßA1 is dynamically regulated during muscle growth resumption and satellite cell differentiation, strongly suggesting that these genes have a role in the regulation of muscle growth.
Subject(s)
Cell Differentiation/genetics , Inhibin-beta Subunits/genetics , Muscle Development/genetics , Oncorhynchus mykiss , Satellite Cells, Skeletal Muscle/physiology , Transforming Growth Factor beta2/genetics , Transforming Growth Factor beta3/genetics , Animals , Cell Differentiation/drug effects , Cells, Cultured , Gene Expression Regulation, Developmental/drug effects , Growth Hormone/pharmacology , Inhibin-beta Subunits/metabolism , Insulin-Like Growth Factor I/pharmacology , Muscles/drug effects , Muscles/physiology , Myostatin/pharmacology , Oncorhynchus mykiss/genetics , Oncorhynchus mykiss/growth & development , Oncorhynchus mykiss/metabolism , Satellite Cells, Skeletal Muscle/drug effects , Transforming Growth Factor beta2/metabolism , Transforming Growth Factor beta3/metabolismABSTRACT
Since their initial discovery, TGF-ß superfamily members have been considered multifunctional growth and differentiation factors in many cell types. Various studies have clearly demonstrated the key roles of specific TGF-ß members in muscle growth, including myostatin and inhibin as well as genes, such as follistatin. By binding to TGF-ß members, follistatin prevents TGF-ß from binding to its receptors and thus neutralizes its activity. Here, we report the identification of the gene sequences of four TGF-ß isoforms and three paralogs of TGF-ß1, which we called TGF-ß1a, TGF-ß1b and TGF-ß1c, four sequences of inhibin ßA paralogs; and two sequences of follistatin paralogs from rainbow trout. A phylogenetic analysis clearly indicated the existence of four monophyletic clades, corresponding to TGF-ß1, -ß2, -ß3 and -ß6. Based on their sequence identity TGF-ß1a and -ß1c are grouped together, whereas TGF-ß1b appears more divergent even though it is grouped within the TGF-ß1 clade. Alignments and phylogenetic analyses showed that the protein sequences of TGF-ß, inhibin ßA and follistatin are extremely well conserved (>90%) relative to each other; however, their regulation and expression patterns are different. TGF-ß2 and -ß3 showed the most abundant expression in muscle and were the main TGF-ß members expressed in this tissue. Follistatin and inhibin ßA paralogs were expressed in all tissues examined but with different patterns. Our identification of multiple copies of TGF-ß, inhibin ßA and follistatin with different expression patterns suggests non-redundant functions for these paralogs in rainbow trout.
Subject(s)
Follistatin/metabolism , Genome , Inhibin-beta Subunits/metabolism , Oncorhynchus mykiss/genetics , Transforming Growth Factor beta/metabolism , Amino Acid Sequence , Animals , Follistatin/genetics , Inhibin-beta Subunits/genetics , Molecular Sequence Data , Oncorhynchus mykiss/metabolism , Organ Specificity , Phylogeny , RNA, Messenger/metabolism , Transcriptome , Transforming Growth Factor beta/geneticsABSTRACT
Cystic ovarian disease (COD) is an important cause of infertility in dairy cattle. Although many researchers have focused their work on the endocrine changes related to this disease, evidence indicates that intraovarian components play an important role in follicular persistence. Activin, inhibin, and follistatin participate as intraovarian regulatory molecules involved in follicular cell proliferation, differentiation, steroidogenesis, oocyte maturation, and corpus luteum function. Given the importance of these factors in folliculogenesis, we examined the expression and immunolocalization of activin/inhibin ßA-subunit, inhibin α-subunit, and follistatin in the ovaries of healthy estrus-synchronized cows and in those of cows with spontaneous or adrenocorticotropic hormone (ACTH)-induced COD. We also studied inhibin B (α ßB) levels in serum and follicular fluid. We found an increased expression of the ßA-subunit of activin A/inhibin A, the α-subunit of inhibin, and follistatin in granulosa cells of spontaneous follicular cysts by immunohistochemistry, and decreased concentrations of inhibin B (α ßB) in the follicular fluid of spontaneous follicular cysts. These results, together with those previously obtained, indicate that the expression of the components of the activin-inhibin-follistatin system is altered. This could lead to multiple alterations in important functions in the ovary like the balance between pro- and anti-apoptotic factors, follicular proliferation/apoptosis, and steroidogenesis, which may contribute to the follicular persistence and endocrine changes found in cattle with COD.
Subject(s)
Cattle Diseases/etiology , Follistatin/physiology , Inhibin-beta Subunits/physiology , Inhibins/physiology , Ovarian Cysts/etiology , Adrenocorticotropic Hormone , Animals , Blood/metabolism , Case-Control Studies , Cattle , Cattle Diseases/diagnostic imaging , Cattle Diseases/metabolism , Female , Follicular Fluid/metabolism , Follistatin/metabolism , Inhibin-beta Subunits/metabolism , Inhibins/metabolism , Ovarian Cysts/diagnostic imaging , Ovarian Cysts/metabolism , Ovarian Cysts/veterinary , Protein Subunits , UltrasonographyABSTRACT
During ovarian follicular growth and development, only a few follicles actually ovulate. Recently, it was found that follicular atresia is triggered by granulosa cell apoptosis, but the molecular mechanism of follicular atresia was not understood. Using flow cytometry, we found that miR-34a promotes granulosa cell apoptosis in pig ovarian follicles. In addition, inhibin beta B was found to be a miR-34a target gene, based on luciferase reporter assays, quantitative RT-PCR and Western blotting. Taken together, our data indicate that miR-34a plays an important role in granulosa cell apoptosis by targeting the INHBB gene in the porcine ovary.
Subject(s)
Apoptosis/genetics , Inhibin-beta Subunits/metabolism , MicroRNAs/genetics , Ovary/metabolism , Animals , Female , Flow Cytometry , Granulosa Cells/pathology , Inhibin-beta Subunits/genetics , Ovarian Follicle/growth & development , Ovarian Follicle/metabolism , Sus scrofa , SwineABSTRACT
Bovine mammary gland morphogenesis and differentiation are regulated by actions of growth factors including members of the transforming growth factor ß superfamily. Activins A and B, which are members of the transforming growth factor ß superfamily, bind selectively to ActRIB and ActRIIA receptors and their biological effects are antagonized by inhibins and follistatins. In the present paper we evaluated gene and protein expression of the activin and inhibin subunits ßA, ßB, and α-inhibin and follistatin and ActRIB and ActRIIA receptors in the mammary gland of nonpregnant and pregnant heifers. Mammary glands were obtained from nonpregnant Nelore (Bos indicus) heifers (n=9) and from primigravid Nelore heifers during early (n=9), mid (n=6), and late (n=5) pregnancy. Specimens of mammary tissue were analyzed by real-time PCR and immunohistochemistry. The ßA and α-inhibin subunits and ActRIB and ActRIIA mRNA expression was higher in the early-pregnancy group compared with the nonpregnant group. In the mid-pregnancy group, the subunits ßA, ßB, and α-inhibin as much as follistatin mRNA expression was higher compared with the nonpregnant group, whereas ActRIB transcripts were absent in the late-pregnancy group. Immunostaining of these proteins, with the exception of ActRIB, was observed in the mammary tissue sections at all time points analyzed; these findings are in agreement with the observed pattern of mRNA expression. Staining and mRNA expression for ActRIB were undetected in the late-pregnancy group. In summary, the present study demonstrated that the activin-related proteins, ßA, ßB, and α-inhibin subunits, as much as follistatin and ActRIB and ActRIIA receptors display different patterns of expression regarding time of gestation in the bovine mammary gland. The modulation of the expression pattern during gestation suggests that activin-related proteins may play a key role in regulating bovine mammary branching morphogenesis and epithelial differentiation.
Subject(s)
Activins/metabolism , Mammary Glands, Animal/metabolism , Pregnancy/metabolism , Activin Receptors, Type I/metabolism , Activin Receptors, Type II/metabolism , Animals , Cattle , Cell Differentiation , Female , Follistatin/metabolism , Gene Expression , Gestational Age , Inhibin-beta Subunits/metabolism , Inhibins/metabolism , Transforming Growth Factor beta/metabolismABSTRACT
Transforming growth factor-beta 1 (TGF-beta1) and activin A (ActA) induce similar intracellular signaling mediated by the mothers against decapentaplegic homolog (SMAD) proteins. TGF-beta1 is a potent antimitogenic factor for thyroid follicular cells, while the role of ActA is not clear. In our study, the proliferation of TPC-1, the papillary thyroid carcinoma cell line, was reduced by both recombinant ActA and TGF-beta1. Due to the concomitant expression of TGF-beta1 and ActA in thyroid tumors, we investigated the effects of either TGF-beta1 or ActA gene silencing by RNA interference in TPC-1 cells in order to distinguish the specific participation of each in proliferation and intracellular signaling. An increased proliferation and reduced SMAD2, SMAD3, and SMAD4 mRNA expression were observed in both TGF-beta1 and ActA knockdown cells. Recombinant TGF-beta1 and ActA increased the expression of inhibitory SMAD7, whereas they reduced c-MYC. Accordingly, we detected a reduction in SMAD7 expression in knockdown cells while, unexpectedly, c-MYC was reduced. Our data indicate that both TGF-beta1 and ActA generate SMADs signaling with each regulating the expression of their target genes, SMAD7 and c-MYC. Furthermore, TGF-beta1 and ActA have an antiproliferative effect on thyroid papillary carcinoma cell, exerting an important role in the control of thyroid tumorigenesis.
Subject(s)
Activins/metabolism , Inhibin-beta Subunits/metabolism , Signal Transduction/physiology , Smad Proteins/metabolism , Thyroid Neoplasms/pathology , Transforming Growth Factor beta/metabolism , Activins/genetics , Activins/pharmacology , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic , Gene Silencing , Genes, myc , Humans , Immunohistochemistry/methods , Inhibin-beta Subunits/genetics , Inhibin-beta Subunits/pharmacology , Polymerase Chain Reaction/methods , RNA, Small Interfering , Recombinant Proteins/pharmacology , Smad Proteins/genetics , Smad2 Protein/genetics , Smad3 Protein/genetics , Smad4 Protein/genetics , Smad7 Protein/genetics , Thyroid Neoplasms/metabolism , Transfection/methods , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/pharmacologyABSTRACT
In order to test the hypothesis that transforming growth factor beta (TGF-beta) acts by FS regulation on bovine granulosa cells in in vitro differentiation, we analyzed the effect of TGF-beta1 on follistatin mRNA expression in three differentiation states of bovine granulosa cells. We showed a positive regulation of FS mRNA after TGF-beta1 (1 ng/ml) treatment of freshly isolated granulosa cells from small-medium antral follicles (2-8 mm). This effect was abolished by the addition of exogenous follistatin (100 ng/ml), suggesting that this effect could be mediated by activin. Although these cells showed a similar effect on FS mRNA expression after treatment with activin-A, a soluble form of activin receptor type IIA was unable to inactivate the TGF-beta effect. When we tested the TGF-beta effect on FS mRNA in different granulosa cell states, TGF-beta1 regulation was associated with progesterone production only in freshly isolated cells. The amount of total activin-A produced by first passage cells (dedifferentiated cells), was ten times smaller than the one measured in a conditioned medium from freshly isolated cells (mature cells). The TGF-beta1-dependent FS mRNA expression persisted in first passage cells without changes with FS addition. On the other hand, the BGC-1 granulosa cell line (immature cells) produced large amounts of activin-A regulated by TGF-beta1 and an invariable steady state of FS mRNAs. In summary, our results showed that FS mRNA expression is regulated by TGF-beta1 independently of activin effects in differentiated granulosa cells.