RESUMO
The Simpson-Golabi-Behmel syndrome (SGBS) cell strain is widely considered to be a representative in vitro model of human subcutaneous white pre-adipocytes. These cells achieve a transient expression of classical brown markers, such as uncoupling protein 1, peaking at day 14 of differentiation and decreasing thereafter. Adipocyte browning process involves dynamic changes in lipid droplet (LD) dimension, in mitochondria morphology, and in the expression of brown-specific marker genes. This study analyzes SGBS transient phenotypic transformation by quantifying the heterogeneity of LDs, mitochondrial dynamics, and a panel of genes involved in adipocyte differentiation and browning. LDs at 21 days of differentiation were larger than in the previous stages, without any change in the number per cell. The expression of genes such as peroxisome peroxisome proliferator-activated receptor γ, leptin, and lipase E significantly raised from 0 to 21 days. Adiponectin was significantly upregulated at 14 days of differentiation. Brown-specific marker PR domain containing 16 was highly expressed at D0. The variability of mitochondrial shape and interconnectivity reflects differences in the relative rates of fusion and fission, resulting in a significant shift from a networked shape at D7 to a fragmented and swollen one at D14 and D21. The transient phenotype experienced by this cellular model should be considered whether used in studies involving the stimulation of adipocyte browning and could be an interesting human model to further elucidate the browning process in the absence of any stimulation.
Assuntos
Adipócitos/patologia , Arritmias Cardíacas/patologia , Diferenciação Celular , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Gigantismo/patologia , Cardiopatias Congênitas/patologia , Deficiência Intelectual/patologia , Adipócitos/metabolismo , Arritmias Cardíacas/metabolismo , Células Cultivadas , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Gigantismo/metabolismo , Cardiopatias Congênitas/metabolismo , Humanos , Deficiência Intelectual/metabolismo , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , FenótipoRESUMO
High-throughput RNA sequencing of human Simpson-Golabi-Behmel syndrome cells (SGBS) was performed during the time-course of adipogenic differentiation at day 4 (D04), 6 (D06), 8 (D08), and 10 (D10) to characterize transcriptomic changes and to identify key patterns involved in adipogenesis and browning. In the comparisons, 932 and 384 overlapping transcripts were consistently up- and down-regulated, respectively. Combining the results of protein-protein interaction network analysis MCODE and CytoHubba, 55 up-regulated hub genes from four clusters and 9 down-regulated genes were identified. The up-regulated hub genes were mainly enriched in brown adipocyte differentiation, extracellular matrix organization, and valine, leucine, and isoleucine degradation. The enrichment of downregulated hub genes was related to NRF2 signalling and glutathione metabolism, indicating that oxidative stress also plays a role. Analysis of overlapping down-regulated genes, targets of transcription factors, revealed enrichment in the IL-18 signalling pathway, which is involved in browning process and extracellular matrix organization via actomyosin mechanics and integrin-extracellular matrix interactions. Finally, the comparison transcriptomic analysis with the gene signature reported by BATLAS and PROFAT web-based tools showed an increased percentage of the brown phenotype, confirming that differentiated SGBS cells at D06, D08, and D10 gradually acquire BAT-like function.
Assuntos
Doenças Genéticas Ligadas ao Cromossomo X , Gigantismo , Deficiência Intelectual , Arritmias Cardíacas , Cardiopatias Congênitas , Humanos , Transcriptoma/genéticaRESUMO
Obesity is a growing threat. In recent years, the finding of functional brown adipose tissue (BAT) in adult humans implemented the studies of anti-obesity therapies based on triggering energy expenditure. The activation of BAT thermogenesis and the recruitment of brite (brown-in-white) adipocytes are under noradrenergic control. Brain-derived neurotrophic factor (BDNF), if centrally administered, enhances thermogenesis through sympathetic activation, but its direct effect on adipocytes is still unclear. The phenotypic change from fat storing to thermogenic adipocytes is recognized by the presence of multilocular lipid droplets (LDs) and fissed mitochondria that tend to surround LDs, maximizing the efficiency of fatty acid release for thermogenesis. BDNF treatment on differentiated 3T3-L1 adipocytes was compared to negative (CTRL) and positive (norepinephrine, NE) controls. BDNF significantly increased small globular mitochondria percentage (>150% CTRL), while the area surface and elongation index of branched tubules were respectively 55% and 10% lower than NE. Canonical discriminant analysis of mitochondria morphological data clearly separated differentially treated cells with 85% of the total variance. The expression of brown markers and mitochondrial dynamic genes was significantly affected by BDNF. Investigating the pathways involved in adipocyte BDNF stimulation could clarify its role in thermogenesis and its possible local regulation.
Assuntos
Adipócitos/metabolismo , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Dinâmica Mitocondrial/efeitos dos fármacos , Termogênese/efeitos dos fármacos , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Animais , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Análise Discriminante , Perfilação da Expressão Gênica , Humanos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fenótipo , Transcrição Gênica/efeitos dos fármacosRESUMO
Stress activates the hypothalamo-pituitary-adrenal axis leading to enhanced glucocorticoid secretion and concurrently disrupts ovarian cycle. Plant polyphenols are known to posses antioxidant and anti-inflammatory proprieties. This could be of interest for ovarian cycle when stressing conditions lead to progesterone enhancement and hamper normal reproduction activity. The present study examined whether ovarian follicular development and progesterone secretory pattern are affected by exogenous ACTH administration in heifers. Moreover, the effect of grape polyphenols in endometrium of heifers, under adrenocorticotropic hormone challenge, is evaluated in terms of transcriptional patterns of genes related to inflammation, oxidative stress and endometrial functions. At day 14 of synchronized estrous cycle, Holstein Friesian heifers received injections of either saline (CTR group) or adrenocorticotropic hormone (ACT group) agonist every 12 h for 7 days. Another group (POL group) of animals received the same treatment plus an oral supplementation of 15 g/day of grape skin extract. Cortisol and progesterone were analysed in the blood samples collected at days 0, 3, 6, 9, 12, 14, 17, 21, 24 of the estrous cycle. Endometrial biopsies were collected at diestrus (day 18) and at estrus and a panel of gene expressions were quantified by real-time PCR. ACTH administration increased both cortisol (P<0.001) and progesterone concentrations (P<0.01) compared to CTR group. PGHS-2 was significantly (P<0.01) up-regulated in the POL group compared to ACT and CTR groups at diestrus and at estrus. FOXO3 and TIS11b were down-regulated in the CTR group compared to ACT and POL groups. The PGHS-2, SOD2 (P<0.05), FOXO3 and TIS11b (P<0.10) genes were down-regulated at estrus in all groups compared to diestrus. An interesting role of polyphenols in modulating the expression levels of PGHS-2 in endometrial tissue and on the activation of TIS11b and SOD2 through c-AMP-dependent signalling was suggested.
Assuntos
Hormônio Adrenocorticotrópico/farmacologia , Endométrio/efeitos dos fármacos , Flavonoides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fenóis/farmacologia , Proteínas/genética , Estresse Fisiológico/veterinária , Vitis/química , Animais , Bovinos , Ciclo-Oxigenase 2/genética , Feminino , Fatores de Transcrição Forkhead/genética , Hormônios/farmacologia , Hidrocortisona/sangue , Folículo Ovariano/diagnóstico por imagem , Folículo Ovariano/efeitos dos fármacos , Extratos Vegetais/farmacologia , Polifenóis , Progesterona/sangue , Proteínas de Ligação a RNA/genética , Estresse Fisiológico/induzido quimicamente , Fatores de Tempo , UltrassonografiaRESUMO
Antlers are the cranial appendages of deer that regenerate each year. This renewal provides a model to explore molecules involved in mammalian organ regeneration. The cellular distributions of the brain-derived neurotrophic factor (BDNF) and the isoforms of its cognate receptor Trk tyrosine kinase receptor (TrkB) were localized by immunohistochemistry in sections of growing red deer antler. BDNF and TrkB full length were widely expressed in the integument, perichondrium, periosteum and bone. The truncated isoform receptor was particularly evidenced in integument and vascular inner dermis, but very light reaction was observed in cartilage and bone, both at the site of endochondral and intramembranous ossification. These observations were also assessed at transcriptional level by RT-PCR analyses. The highest expression of all genes significantly occurred in chondroprogenitor cells; however the full-length TrkB receptor was down regulated in osteocartilaginous compartments, in which the truncated isoform was up regulated. The truncated isoform is a dominant-negative receptor that inhibits the full length receptor signalling, even if the truncated isoform not only has this function. This study establishes the presence of BDNF and its receptor in the different cellular compartments of growing antler. Their transcripts assessed by RT-PCR indicate a local synthesis of these molecules that may contribute to the modulation of antler growth, acting as autocrine and/or paracrine factors independently of nerve supply. Among the plethora of other molecular signals and growth factors affecting the antler growth, the local production of BDNF and its cognate receptor could be of interest in understanding their role in antler renewal and to delineate the different involvement of the receptor isoforms.
Assuntos
Chifres de Veado/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Cervos/metabolismo , Receptor trkB/metabolismo , Animais , Chifres de Veado/citologia , Osso e Ossos/metabolismo , Cartilagem/metabolismo , Condrócitos/fisiologia , Derme/metabolismo , Humanos , Imuno-Histoquímica , Isomerismo , Masculino , Osteogênese , Transdução de Sinais , Células-Tronco/metabolismoRESUMO
Obesity is the result of energy intake chronically exceeding energy expenditure. Classical treatments against obesity do not provide a satisfactory long-term outcome for the majority of patients. After the demonstration of functional brown adipose tissue in human adults, great effort is being devoted to develop therapies based on the adipose tissue itself, through the conversion of fat-accumulating white adipose tissue into energy-dissipating brown adipose tissue. Anti-obesity treatments that exploit endogenous, pharmacological and nutritional factors to drive such conversion are especially in demand. In the present review, we summarize the current knowledge about the various molecules that can be applied in promoting white-to-brown adipose tissue conversion and energy expenditure and the cellular mechanisms involved.
Assuntos
Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Termogênese , Tecido Adiposo Marrom/efeitos dos fármacos , Tecido Adiposo Branco/efeitos dos fármacos , Animais , Fármacos Antiobesidade/farmacologia , Dieta , Modelos Animais de Doenças , Metabolismo Energético , Humanos , Obesidade/tratamento farmacológicoRESUMO
During the peripartum period, high-yielding dairy cows experience metabolic stress, which alters their homeostasis and exposes the cows to illness. The aim of this study was to quantify the expression levels of genes involved in antioxidant defences during the transition period in the blood of dairy cows and to evaluate the regulative activity on these genes of natural antioxidants in the diet. Three groups of 7 heifers each, at the 7th month of pregnancy, were used. Starting from 3 weeks before the expected calving date (-22 days), the three groups were allotted to the following experimental treatments: control (CTR, basal diet); lycopene (LYC, basal diet + lycopene 540 mg/day) and grape polyphenols (POL, basal diet + grape polyphenols 10 g/day). Blood was sampled at 22 and 8 days before and 8, 15 and 22 after calving and analysed for the expression level of glutathione peroxidase (GPx) and superoxide dismutase (Cu/ZnSOD) using the real-time PCR technique with LUX (Light Upon eXtension) fluorogenic primers. During the peripartum period (-22 days until + 22 days from calving), Cu/ ZnSOD mRNA expression decreased (p<0.05) in the CTR and LYC groups, but increased at 15 days after calving in the POL group. No significant differences were found in GPx mRNA expression. The results suggest that grape polyphenols may have a controlling effect on peripartum metabolic stress through modulation of superoxide dismutase expression.
Assuntos
Antioxidantes/farmacologia , Bovinos/metabolismo , Glutationa Peroxidase/biossíntese , Leucócitos Mononucleares/enzimologia , Prenhez/metabolismo , RNA Mensageiro/biossíntese , Superóxido Dismutase/biossíntese , Animais , Carotenoides/farmacologia , Feminino , Flavonoides/farmacologia , Glutationa Peroxidase/sangue , Glutationa Peroxidase/genética , Licopeno , Estresse Oxidativo/efeitos dos fármacos , Fenóis/farmacologia , Polifenóis , Período Pós-Parto/efeitos dos fármacos , Período Pós-Parto/metabolismo , Gravidez , Prenhez/efeitos dos fármacos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Superóxido Dismutase/sangue , Superóxido Dismutase/genéticaRESUMO
The study of cell turnover of mammary gland helps to gain understanding of the subtle modulation that largely determines milk production. To evaluate the effect of diet composition on cell turnover of mammary gland, pluriparous sheep were allotted to three experimental groups and fed from day 33 after lambing a control diet (BD), a high starch (HS) diet or a high fat (HF) diet. Biopsies of mammary gland tissue were collected at 50 days after lambing for in situ detection of cell death and RT-PCR analysis of bax, bcl-2, caspase-3 and GST expressions. Both apoptotic and proliferation indexes were significantly higher (P<0.05) in the BD group compared to HS and HF groups. The relative expressions of GST were significantly greater (P<0.05) in HF group compared to the BD and HS groups. There was a significant increase in the ratio of bcl-2 to bax mRNA in the HS and HF groups. Availability of energy substrates for mammary gland can interfere with the cell fate, modulating genes involved in the control of oxidative stress which, in turn, can indirectly regulate cell apoptosis and proliferation.
Assuntos
Metabolismo Energético/fisiologia , Lactação/fisiologia , Glândulas Mamárias Animais/fisiologia , Ovinos/fisiologia , Fenômenos Fisiológicos da Nutrição Animal , Animais , Apoptose , Proliferação de Células , Carboidratos da Dieta/administração & dosagem , Gorduras na Dieta/administração & dosagem , Feminino , Lactação/metabolismo , Glândulas Mamárias Animais/citologia , Estresse Oxidativo/fisiologia , Ovinos/metabolismoRESUMO
Immunoelectron microscopy was used to study the subcellular localisation of apoptosis-inducing factor (AIF) in involuting bovine mammary tissue. AIF was detected using a polyclonal antibody and secondary anti-rabbit antibody conjugated to colloidal gold particles under optimised conditions. The polyclonal antibody appeared specific for the bovine antigen and immunocytological examination identified specific localisation of AIF in mitochondria, cytoplasm and nucleus of mammary epithelial cells in involuting mammary tissue, suggesting a subcellular translocation in cells undergoing apoptosis.
Assuntos
Flavoproteínas/metabolismo , Glândulas Mamárias Animais/metabolismo , Proteínas de Membrana/metabolismo , Animais , Apoptose , Fator de Indução de Apoptose , Bovinos , Feminino , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/ultraestrutura , Microscopia Imunoeletrônica , Frações Subcelulares/metabolismo , Frações Subcelulares/ultraestruturaRESUMO
Immunopresence of estrogen receptor α (ERα), ß (ERß) and progesterone receptor (PR) were examined in the ewe mammary gland from prepubertal stage to involution. Immunolocalization of ERα revealed a strong positive staining in nuclei of cells composing terminal ductular units (TDUs) in prepubertal ewes. A mild immunoreactivity was identified in early lactating gland. During late lactation immunoreactive product to ERα was observed in the cytoplasm of glandular epithelial cells in all alveoli. In mammary glands at involution ERα positivity was clearly nuclear, with weak to moderate cytoplasmic staining. Cytoplasmic strong immunostaining for ERß was detected in cells of TDUs, whereas some stromal cells exhibited nuclear staining. A nuclear ERß immunostaining was observed at early lactation, instead during late lactation, the positivity for ERß showed only a moderate cytoplasmic distribution. At involution, ERß positivity was very moderate and detected just in the cytoplasm of shrunken alveoli. Scattered nuclear staining of PR was observed just in mammary glands at early lactation. These results showed that in the mammary glands of sheep both estrogen receptor isoforms were displayed during lactation cycle and that PR appeared just at early lactation, reflecting their regulatory role in alveolar cells.
Assuntos
Glândulas Mamárias Animais/química , Receptores de Estrogênio/análise , Receptores de Progesterona/análise , Animais , Feminino , Imuno-Histoquímica , Lactação , Microscopia , OvinosRESUMO
This review focuses on the pro-apoptotic and anti-apoptotic Bcl-2 family members involved in apoptosis, which is the predominant process controlling cell remodelling during post-lactational mammary gland involution. The members of the Bcl-2 protein family, whose expression levels are under the control of lactogenic hormones, internally control this mechanism also during lactation. They can physically interact with each other, sometimes in an antagonistic manner. Mammary glands undergo repeated cycles of structural development, functional differentiation and regression, therefore provide a unique model for investigating this family of proteins that regulate the fate of the secretory cells and consequently milk yield. The involvement of Bcl-2 family members is reviewed in mammary tissue during morphogenesis, at different stages of lactation cycle and in comparison with dairy and laboratory animals.
Assuntos
Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Apoptose , Feminino , Janus Quinases/metabolismo , Lactação , Glândulas Mamárias Animais/fisiologia , Camundongos , Gravidez , Transdução de SinaisRESUMO
The aim of the present research was to investigate the regulation of gene expression in ovine blood leukocytes during ACTH-induced cortisol release and the effect of dietary administration of botanicals to counteract the evoked response in polymorphonucleate cells (PMNCs). Thirty-six homogeneous Sarda sheep (age, 18±4.1 mo; BW, 38.7±1.3 kg) were allotted to six groups of six sheep each. One group was used as a negative control (Saline) and five groups were treated, every 12 h for 48 h, with 0.5 mL of ACTH agonist (250 µg/mL of tetracosactrin). Before ACTH treatment, four of the five ACTH-treated groups were separated and fed for 22 d with a basal diet supplemented with extracts from Echinacea angustifolia roots (PO+ACTH), Echinacea angustifolia flowers (EA+ACTH), Andrographis paniculata (AP+ACTH), and the bark of Larix decidua milled (LB+ACTH). Control groups (Saline and ACTH) were fed with the same basal diet without botanicals. Total RNA was extracted from blood samples collected before (T0) and after 3 h (T3) and 51 h (T51) from the first ACTH injection, and transcriptome analysis was performed using a custom oligoarray, designed from 12,194 Ovis aries UniGenes on a CombiMatrix platform. At T3, treatment with ACTH caused down-regulation of transcripts (P<0.001) involved in "response to stress" (GADD45A, GADD45B, WRNIP1, and XRCC6) and in "innate immune response" (MAPK3 and NFkBIB). At T51, treatment with ACTH caused down-regulation (P<0.001) of genes involved in "immune response" (IFNG and IL2) and up-regulation (P<0.001) of NF-κB1 and TP53. Each botanical produced a different (P<0.001) molecular signature for these genes at T3 and T51. The most active botanical in modulating transcriptome modifications in PMNCs after ACTH-induced cortisol release was Larix decidua Mill bark followed by Polinacea roots. These botanicals can be viewed as promising feed supplements in ruminants to cope with conditions associated with increased concentrations of plasma cortisol.
Assuntos
Hormônio Adrenocorticotrópico/farmacologia , Ração Animal/análise , Dieta/veterinária , Regulação da Expressão Gênica/efeitos dos fármacos , Extratos Vegetais/farmacologia , Ovinos/sangue , Fenômenos Fisiológicos da Nutrição Animal , Animais , Flores/química , Globinas/genética , Globinas/metabolismo , Imunidade Inata , Casca de Planta/química , Extratos Vegetais/administração & dosagem , Extratos Vegetais/química , Raízes de Plantas/química , Reação em Cadeia da Polimerase , RNARESUMO
The dietary effect of non-steroidal anti-inflammatory drug (NSAID) or curcumin on the gene expression of peripheral white blood cells in osteoarthritis (OA) affected dogs was investigated using a 44K oligo microarray. Two groups of OA dogs and one group of healthy dogs (6 dogs each) were clinically evaluated and blood was sampled before (T0) and after 20days (T20) of dietary administration of NSAID (NSAID group) or curcumin (CURCUMIN group). Differentially expressed genes (P<0.05) in comparison to the control group were identified with MeV software and were functional annotated and monitored for signaling pathways and candidate biomarkers using the Ingenuity Pathways Analysis (IPA). After 20days of treatment, the differentially expressed transcripts significantly (P<0.05) decreased from 475 to 173 in NSAID group and from 498 to 141 in CURCUMIN group. Genes involved in "inflammatory response" and in "connective tissue development and function" dramatically decreased at T20. Other genes, included in "cellular movement", "cellular compromise" and "immune cell trafficking", were differentially expressed at T0 but not at T20 in both groups. Specific molecular targets of CURCUMIN, not observed for NSAID, were the IkB up regulation in the "TNRF1 signaling pathway" and IL18 down regulation in the "role of cytokines in mediating communication between immune cells". The activity of CURCUMIN was also evidenced from the inhibition of macrophages proliferation (HBEGF), related to a strong down regulation of TNFα and to activation of fibrinolysis (SERPINE1). The results would suggest that curcumin offers a complementary antinflammatory support for OA treatment in dogs.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Curcumina/uso terapêutico , Doenças do Cão/tratamento farmacológico , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Osteoartrite/veterinária , Administração Oral , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Sequência de Bases , Curcumina/administração & dosagem , Suplementos Nutricionais , Doenças do Cão/sangue , Doenças do Cão/genética , Cães , Feminino , Expressão Gênica/efeitos dos fármacos , Marcadores Genéticos , Masculino , Osteoartrite/sangue , Osteoartrite/tratamento farmacológico , Osteoartrite/genética , RNA/sangue , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Transcriptoma/efeitos dos fármacosRESUMO
Mammary gland remodelling is strictly related to intracellular signals and stem cell biology. Among the best candidates to identify the nature and development of mammary cells are cytokeratin 19 (CK19), the Na-K-Cl cotransporter (NKCC1) and receptor of estrogen alpha (ERα). In this study, we analyzed the expression of these genes in ewe mammary glands from prepubertal stage to involution. Using Real time PCR we showed that NKCC1 transcription was significantly down regulated during lactation and at involution in comparison to the expression measured in the prepubertal group. No significant differences were found in CK19 expression, whereas ERα transcription was significantly down regulated before lambing, during lactation and at involution. In situ hybridization analysis confirmed quantitative data and localized the CK 19 transcript at basal and luminal compartment of terminal ductal unit (TDU) of prepubertal mammary glands. NKCC1 expression was also present in lactating glands and ERα in connective tissue surrounding TDU. The characterization and identification of mammary developmental markers in the tissue of dairy animals is necessary to gain knowledge in mammary gland biology.
Assuntos
Envelhecimento/genética , Receptor alfa de Estrogênio/genética , Queratina-19/genética , Glândulas Mamárias Animais/metabolismo , Simportadores de Cloreto de Sódio-Potássio/genética , Fatores Etários , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Lactação/genética , Glândulas Mamárias Animais/crescimento & desenvolvimento , Parto/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ovinos , Membro 2 da Família 12 de Carreador de Soluto , Transcrição GênicaRESUMO
It is thought that the regenerative capacity of the mammary gland following post-lactation involution resides in multipotent stem cells within the luminal tissue. Adult stem cells make up a small percentage of the cells found in mature organ systems, however to define useful markers has long been a challenge. c-Kit (KIT) and its ligand stem cell factor (KITLG), ATP-binding cassette sub-family G member 2 (ABCG2) and Musashi 1 (MSI1) are good candidate to identify progenitor cells in their niche. Using real-time PCR we showed that KIT, KITLG and MSI1 expressions were up regulated before lambing and at involution relatively to prepubertal stage. The in situ hybridization analysis for KIT gene confirmed and localized the expression in luminal epithelial cells. The changes in the expression profile of putative stem cell markers in mammary glands of sheep suggest that they modify with the progression of lactation cycle, being up regulated during differentiation and down regulated during lactation.
Assuntos
Células-Tronco Hematopoéticas/citologia , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/fisiologia , Células-Tronco Multipotentes/citologia , Ovinos/fisiologia , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Células Epiteliais/metabolismo , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Hibridização In Situ , Lactação , Glândulas Mamárias Animais/metabolismo , Células-Tronco Multipotentes/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , RNA Ribossômico 18S/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fator de Células-Tronco/genética , Fator de Células-Tronco/metabolismoAssuntos
Lactação/fisiologia , Glândulas Mamárias Animais/fisiologia , Animais , Apoptose , Bovinos , Feminino , Regulação da Expressão Gênica , Glândulas Mamárias Animais/citologia , Antígeno Nuclear de Célula em Proliferação/análise , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína X Associada a bcl-2RESUMO
Mammary glands are special tissue characterized by proliferation of the epithelium, during puberty and pregnancy and by programmed cell death, during involution. In this study, apoptosis was identified by TUNEL staining and then related to cell proliferation, as determined by Ki-67 staining. The apoptotic index was at its highest at 8 days of involution, whereas the proliferation index was at its highest during lactation. Caspase-3 was immunolocalised only in mast cells and along the basal membrane in the mammary tissue at -10 days from lambing, 150 days of lactation and at 8 days of involution. This finding could indicate that caspase-3 is not involved in sheep mammary gland apoptosis, but that other proteins - such as apoptosis inducing factor (AIF) - can trigger apoptosis, through the mitochondrial pathway, in a caspase-independent manner. The expression of genes involved in the regulation of lactation and apoptosis was also investigated and determined relatively to -10 days from lambing. The relative expression level of LALBA, reached its maximum during lactation, whereas the expressions of BCL2, BCL2L1, BAX, STAT5A, STAT3, IGFBP5 and FOXO3A, increased significantly during involution in correlation with apoptotic index. This work shows for the first time the turnover of mammary cells and the interaction of their signals during the complete lactation cycle in sheep. The data on gene expression can contribute to elucidate the mechanisms controlling milk production and cell turnover in this species.
Assuntos
Glândulas Mamárias Animais/citologia , Ovinos/genética , Animais , Apoptose/fisiologia , Caspase 3/metabolismo , Proliferação de Células , Feminino , Perfilação da Expressão Gênica , Marcação In Situ das Extremidades Cortadas , Lactação/genética , Glândulas Mamárias Animais/metabolismo , Parto , GravidezRESUMO
Several recent studies demonstrated that development, function and remodelling of mammary glands involved multipotent cells, but no specific molecular markers for mammary epithelial stem cells were revealed. These studies principally concerned human and mouse mammary tissue, but mammary stem cells could be a valuable tool in agricultural production and bioengineering in farm animals. The Musashi-1 (Msi 1) gene encodes an RNA binding protein, which is likely to be associated with self-renewal of neural, intestinal and mammary progenitor cells and is believed to influence the Notch signalling pathway. In this study Musashi-1 expression was detected using immunohistochemistry and in situ hybridisation analysis on mammary glands of ewes at different developmental stages. The protein expression was observed in the epithelial cells at all stages examined. In situ hybridization analysis showed that Msi 1 mRNA has an expression pattern similar to the encoded protein, with positive staining in both nuclei and cytoplasm of ductal, secretory and stromal cells. Ultrastructural in situ analysis confirmed the nuclear and cytoplasmatic expression of Msi. Quantitative analysis of Msi 1 gene expression showed a strong correlation with that of Ki-67, that is a marker of cell proliferation. This is the first report outlining expression of Msi 1 in ovine mammary glands during a complete cycle of lactation.
Assuntos
Glândulas Mamárias Animais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ovinos/metabolismo , Células-Tronco/metabolismo , Animais , Biomarcadores/metabolismo , Feminino , Imuno-Histoquímica , Hibridização In Situ , Proteínas do Tecido Nervoso/genética , Proteínas de Ligação a RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Antlers are the only mammalian appendages capable of epimorphic regeneration and thus provide a unique model for investigating the mechanisms that underlie mammalian regeneration. Antlers elongate by a modified endochondral ossification process while intramembranous ossification takes place concurrently around the antler shaft. In this study, sites of apoptosis in the growing antler tip were identified by TUNEL staining and related to cell proliferation, as determined by PCNA staining. Bcl-2 and bax were identified by RT-PCR and bax was also immunolocalized in tissue sections. The apoptotic index was high in perichondrium, undifferentiated mesenchymal cells and cellular periosteum but was low in skin. The proliferation index was high in mesenchyme, skin (specifically in hair follicles) and cellular periosteum; it was low in fibrous perichondrium and periosteum, and barely detectable in cartilage. Both bcl-2 and bax were found to be more highly expressed in the perichondrium/mesenchyme and non-mineralized cartilage than in skin and mineralized cartilage. Bax was immunolocalized in mesenchyme cells, chondroprogenitors, chondrocytes, osteoblasts, osteocytes and osteoclasts. In conclusion, this study shows that programmed cell death plays a necessary role in regenerating antlers, as it does during skeletal development, bone growth and bone remodelling. The high level of apoptosis and proliferation in mesenchymal progenitor cells confirms that this represents the antler 'growth zone'. In fact, the percentage of TUNEL-positive cells in the mesenchymal growth zone (up to 64%) is higher than that recorded in any other adult tissue. This extensive cell death probably reflects the phenomenal rate of morphogenesis and tissue remodelling that takes place in a growing antler. The local and/or systemic factors that control the balance between cell growth and apoptosis in antler tissues now need to be determined.
Assuntos
Chifres de Veado/fisiologia , Cervos/fisiologia , Regeneração , Animais , Chifres de Veado/citologia , Apoptose , Biomarcadores/análise , Proliferação de Células , Genes bcl-2 , Imuno-Histoquímica/métodos , Marcação In Situ das Extremidades Cortadas , Masculino , Antígeno Nuclear de Célula em Proliferação/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína X Associada a bcl-2/genéticaRESUMO
The scleral bony ring (s.b.r.) of Coturnix coturnix japonica was examined and evaluated morphometrically in its characteristic formations (scleral ossicles, overlapping sectors). The modal class 14-14, the most representative, was compared to others having minor or major numbers of ossicles with complex or simple overlaps in the left and right eyes. It was observed that the ossicles, numbered as in the literature, are ordered in a characteristic manner to form the s.b.r. The ossicle dimensions are different and numbers 4 and 7 are always smaller and larger. The s.b.r. with different numbers of ossicles compared to the model class 14-14 (0,0) showed an evident compensatory behaviour in all classes. In fact, when the ossicle number decreased, single ossicle area increased, retaining a constant s.b.r. area.