Interaction between poly(A)-binding protein PABPC4 and nuclear receptor corepressor NCoR1 modulates a metabolic stress response.

Mitochondria are organelles known primarily for generating ATP via the oxidative phosphorylation process. Environmental signals are sensed by whole organisms or cells and markedly affect this process, leading to alterations in gene transcription and, consequently, changes in mitochondrial function and biogenesis. The expression of mitochondrial genes is finely regulated by nuclear transcription factors, including nuclear receptors and their coregulators. Among the best-known coregulators is the nuclear receptor corepressor 1 (NCoR1). Muscle-specific knockout of NCoR1 in mice induces an oxidative phenotype, improving glucose and fatty acid metabolism. However, the mechanism by which NCoR1 is regulated remains elusive. In this work, we identified the poly(A)-binding protein 4 (PABPC4) as a new NCoR1 interactor. Unexpectedly, we found that silencing of PABPC4 induced an oxidative phenotype in both C2C12 and MEF cells, as indicated by increased oxygen consumption, mitochondria content, and reduced lactate production. Mechanistically, we demonstrated that PABPC4 silencing increased the ubiquitination and consequent degradation of NCoR1, leading to the derepression of PPAR-regulated genes. As a consequence, cells with PABPC4 silencing had a greater capacity to metabolize lipids, reduced intracellular lipid droplets, and reduced cell death. Interestingly, in conditions known to induce mitochondrial function and biogenesis, both mRNA expression and PABPC4 protein content were markedly reduced. Our study, therefore, suggests that the lowering of PABPC4 expression may represent an adaptive event required to induce mitochondrial activity in response to metabolic stress in skeletal muscle cells. As such, the NCoR1-PABPC4 interface might be a new road to the treatment of metabolic diseases.

Multimodal and non-linear optical microscopy applications in reproductive biology.

A plethora of optical techniques is currently available to obtain non-destructive, contactless, real time information with subcellular spatial resolution to observe cell processes. Each technique has its own unique features for imaging and for obtaining certain biological information. However none of the available techniques can be of universal use. For a comprehensive investigation of biological specimens and events, one needs to use a combination of bioimaging methods, often at the same time. Some modern confocal/multiphoton microscopes provide simultaneous fluorescence, fluorescence lifetime imaging, and four-dimensional imaging. Some of them can also easily be adapted for harmonic generation imaging, and to permit cell manipulation technique. In this work we present a multimodal optical workstation that extends a commercially available confocal microscope to include nonlinear/multiphoton microscopy and optical manipulation/stimulation tools. The nonlinear microscopy capabilities were added to the commercial confocal microscope by exploiting all the flexibility offered by the manufacturer. The various capabilities of this workstation as applied directly to reproductive biology are discussed. Microsc. Res. Tech. 79:567-582, 2016. © 2016 Wiley Periodicals, Inc.

Measurement of the hydrodynamic radius of quantum dots by fluorescence correlation spectroscopy excluding blinking.

One of the most important properties of quantum dots (QDs) is their size. Their size will determine optical properties and in a colloidal medium their range of interaction. The most common techniques used to measure QD size are transmission electron microscopy (TEM) and X-ray diffraction. However, these techniques demand the sample to be dried and under a vacuum. This way any hydrodynamic information is excluded and the preparation process may alter even the size of the QDs. Fluorescence correlation spectroscopy (FCS) is an optical technique with single molecule sensitivity capable of extracting the hydrodynamic radius (HR) of the QDs. The main drawback of FCS is the blinking phenomenon that alters the correlation function implicating in a QD apparent size smaller than it really is. In this work, we developed a method to exclude blinking of the FCS and measured the HR of colloidal QDs. We compared our results with TEM images, and the HR obtained by FCS is higher than the radius measured by TEM. We attribute this difference to the cap layer of the QD that cannot be seen in the TEM images.

Aspects related to the technique and the utilization of sexed semen in vivo and in vitro

The development of methods capable of selecting the sex of animals has always been a great challenge for humankind. Separating X chromosome- bearing and Y chromosome-bearing sperm based on DNA difference using flow cytometry is the only technique that has achieved a useful level of progress, especially in cattle. This allowed the technique to be commercially applicable in this species. Thorough this review, aspects related to the sexing technique (flow cytometry) and to the sperm will be discussed along with the in vitro use of sexed semen. Besides, number of sperm used and semen deposition location, the influence of reproductive status (heifers vs. cows), time of insemination and production of embryos in superovulated donors will be reviewed.(AU)

Aspects related to the technique and the utilization of sexed semen in vivo and in vitro

The development of methods capable of selecting the sex of animals has always been a great challenge for humankind. Separating X chromosome- bearing and Y chromosome-bearing sperm based on DNA difference using flow cytometry is the only technique that has achieved a useful level of progress, especially in cattle. This allowed the technique to be commercially applicable in this species. Thorough this review, aspects related to the sexing technique (flow cytometry) and to the sperm will be discussed along with the in vitro use of sexed semen. Besides, number of sperm used and semen deposition location, the influence of reproductive status (heifers vs. cows), time of insemination and production of embryos in superovulated donors will be reviewed.

The anti-oestrogen fulvestrant (ICI 182,780) reduces the androgen receptor expression, ERK1/2 phosphorylation and cell proliferation in the rat ventral prostate.

This study proposed to investigate further the role of oestrogens during pubertal growth of rat ventral prostate, by analysing the effect of anti-oestrogen fulvestrant (ICI 182,780) on the expression of androgen (AR) and oestrogen receptors (ESR1 and ESR2), mitogen-activated protein kinase (ERK1/2) phosphorylation, and expression of Ki-67, a biomarker for cell proliferation. Ventral prostates were obtained from 90-day-old rats treated once a week for 2 months with vehicle (control) or ICI 182,780 (10 mg/rat, s.c.). Transcripts for AR, ESR1 and ESR2 were evaluated by quantitative real-time polymerase chain reaction. Expression of AR, ESR1, ESR2, total and phospho-ERK1/2 was analysed by Western blot or immunofluorescence. Ki-67-positive cells and myosin heavy chain were detected by immunohistochemistry. Cylindrical epithelial cells slightly taller, epithelial dysplasia and an increase in smooth muscle layer were observed in the ventral prostate from ICI 182,780-treated rats. ICI 182,780 did not change the mRNA, but decreased the protein levels for AR in the ventral prostate. The expression of ESR1 (mRNA and protein) was upregulated by ICI 182,780, but no changes were observed on ESR2 expression (mRNA and protein). ICI 182,780 decreased the phosphorylation state of ERK1/2, with no changes in total ERK1/2 levels. Ki-67-positive cells in the ventral prostate were also decreased by ICI 182,780. In conclusion, ICI 182,780 induces downregulation of AR expression and may block the translocation of ESR1 and ESR2 from the nucleus to the plasma membrane, decreasing ERK1/2 phosphorylation and prostatic epithelial cell proliferation. These findings provide a basis for physiological roles of oestrogen in the ventral prostate. Further studies with fulvestrant are necessary in benign prostate hyperplasia and prostatic cancer models.

Biomechanical responses of different rat tendons to nandrolone decanoate and load exercise.

Androgenic-anabolic steroids (AAS) have been associated with an increased incidence of tendon rupture. The aim of this study was to compare the biomechanical properties of the rat calcaneal tendon (CT), superficial flexor tendon (SFT), and deep flexor tendon (DFT), and to determine the effect of jump training in association with AAS. Animals were separated into four groups: sedentary, trained, AAS-treated sedentary rats (AAS), and AAS-treated and trained animals. Mechanical testing showed that the CT differed from the DFT and SFT, which showed similar mechanical properties. Jump caused the CT to exhibit an extended toe region, an increased resistance to tensional load, and a decreased elastic modulus, characteristics of an elastic tendon capable of storing energy. AAS caused the tendons to be less compliant, and the effects were reinforced by simultaneous training. The DFT was the most affected by training, AAS, and the interaction of both, likely because of its involvement in the toe-off step of jumping, which we suggest is related to the rapid transmission of force as opposed to energy storage. In conclusion, tendons are differently adapted to exercise, but responded equally to AAS, showing reduced flexibility, which is suggested to increase the risk of tendon rupture in AAS consumers.

Métodos de avaliação da morfologia e função espermática: momento atual e desafios futuros / Methods of the assessment of morphology and function of sperm: actual moment and future challenges

Com o propósito de obter resultados que demonstrem maior repetibilidade na avaliação da morfologia tanto quanto na função espermática, diversas técnicas laboratoriais tem sido desenvolvidas. Surgiram sistemas que utilizam a análise computadorizada de imagens, métodos de “coloração” empregando corantes fluorescentes (sondas fluorescentes) em microscopia de epifluorescência ou citometria de fluxo, que aumentaram a possibilidade de uma análise mais criteriosa da integridade estrutural dos espermatozóides. Os autores descrevem experiências, por mais de uma década, com a utilização destas biotécnicas, fazendo uma análise crítica, bem como apontando os desafios futuros, inclusive na área de biologia molecular espermática.(AU)

In order to obtain results showing greater repeatability in morphology and sperm function assessment, several laboratorial techniques have been developed continuously. Systems that utilize computerized image analysis, staining methods using fluorescent dies (fluorescent probes) in epifluorescence microscopy or flow cytometry, that allowed a more judicious evaluation of sperm structure integrity arose. Authors describe experiences, over a decade, with the use of these biotechiques, critically analyzing and pointing out future challenges, including the field of sperm molecular biology.(AU)

Métodos de avaliação da morfologia e função espermática: momento atual e desafios futuros / Methods of the assessment of morphology and function of sperm: actual moment and future challenges

Com o propósito de obter resultados que demonstrem maior repetibilidade na avaliação da morfologia tanto quanto na função espermática, diversas técnicas laboratoriais tem sido desenvolvidas. Surgiram sistemas que utilizam a análise computadorizada de imagens, métodos de “coloração” empregando corantes fluorescentes (sondas fluorescentes) em microscopia de epifluorescência ou citometria de fluxo, que aumentaram a possibilidade de uma análise mais criteriosa da integridade estrutural dos espermatozóides. Os autores descrevem experiências, por mais de uma década, com a utilização destas biotécnicas, fazendo uma análise crítica, bem como apontando os desafios futuros, inclusive na área de biologia molecular espermática.

In order to obtain results showing greater repeatability in morphology and sperm function assessment, several laboratorial techniques have been developed continuously. Systems that utilize computerized image analysis, staining methods using fluorescent dies (fluorescent probes) in epifluorescence microscopy or flow cytometry, that allowed a more judicious evaluation of sperm structure integrity arose. Authors describe experiences, over a decade, with the use of these biotechiques, critically analyzing and pointing out future challenges, including the field of sperm molecular biology.

Death switch for gene therapy: application to erythropoietin transgene expression

The effectiveness of the caspase-9-based artificial "death switch" as a safety measure for gene therapy based on the erythropoietin (Epo) hormone was tested in vitro and in vivo using the chemical inducer of dimerization, AP20187. Plasmids encoding the dimeric murine Epo, the tetracycline-controlled transactivator and inducible caspase 9 (ptet-mEpoD, ptet-tTAk and pSH1/Sn-E-Fv’-Fvls-casp9-E, respectively) were used in this study. AP20187 induced apoptosis of iCasp9-modified C2C12 myoblasts. In vivo, two groups of male C57BI/6 mice, 8-12 weeks old, were injected intramuscularly with 5 µg/50 g ptet-mEpoD and 0.5 µg/50 g ptet-tTAk. There were 20 animals in group 1 and 36 animals in group 2. Animals from group 2 were also injected with the 6 µg/50 g iCasp9 plasmid. Seventy percent of the animals showed an increase in hematocrit of more than 65 percent for more than 15 weeks. AP20187 administration significantly reduced hematocrit and plasma Epo levels in 30 percent of the animals belonging to group 2. TUNEL-positive cells were detected in the muscle of at least 50 percent of the animals treated with AP20187. Doxycycline administration was efficient in controlling Epo secretion in both groups. We conclude that inducible caspase 9 did not interfere with gene transfer, gene expression or tetracycline control and may be used as a safety mechanism for gene therapy. However, more studies are necessary to improve the efficacy of this technique, for example, the use of lentivirus vector.

Death switch for gene therapy: application to erythropoietin transgene expression.

The effectiveness of the caspase-9-based artificial "death switch" as a safety measure for gene therapy based on the erythropoietin (Epo) hormone was tested in vitro and in vivo using the chemical inducer of dimerization, AP20187. Plasmids encoding the dimeric murine Epo, the tetracycline-controlled transactivator and inducible caspase 9 (ptet-mEpoD, ptet-tTAk and pSH1/Sn-E-Fv'-Fvls-casp9-E, respectively) were used in this study. AP20187 induced apoptosis of iCasp9-modified C2C12 myoblasts. In vivo, two groups of male C57BI/6 mice, 8-12 weeks old, were injected intramuscularly with 5 microg/50 g ptet-mEpoD and 0.5 microg/50 g ptet-tTAk. There were 20 animals in group 1 and 36 animals in group 2. Animals from group 2 were also injected with the 6 microg/50 g iCasp9 plasmid. Seventy percent of the animals showed an increase in hematocrit of more than 65% for more than 15 weeks. AP20187 administration significantly reduced hematocrit and plasma Epo levels in 30% of the animals belonging to group 2. TUNEL-positive cells were detected in the muscle of at least 50% of the animals treated with AP20187. Doxycycline administration was efficient in controlling Epo secretion in both groups. We conclude that inducible caspase 9 did not interfere with gene transfer, gene expression or tetracycline control and may be used as a safety mechanism for gene therapy. However, more studies are necessary to improve the efficacy of this technique, for example, the use of lentivirus vector.

Stromal remodelling is required for progressive involution of the rat ventral prostate after castration: identification of a matrix metalloproteinase-dependent apoptotic wave.

Prostate epithelial-cell apoptosis occurs in response to androgen deprivation. We have hypothesized that continued regression would require stromal changes. Studying apoptosis kinetics up to the 14th day after castration, we identified successive waves of apoptosis, with a prominent peak on day 11. This peak was associated with caspase-3 activity, nuclear translocation of apoptosis-inducing factor and clusterin expression. The apoptosis peak on day 11 was preceded by increased MMP-2 and MMP-7 activation, and MMP-9 expression on days 9 and 10. Treatment with the matrix metalloproteinases inhibitors doxycyclin, hydrocortisone, or GM6001 caused significant reduction in the apoptosis rate on day 11. The present data demonstrate that prostatic epithelial-cell deletion at the 11th day after castration was induced by focal degradation of the extracellular matrix associated with stromal remodelling.

Oestrogen imprinting causes nuclear changes in epithelial cells and overall inhibition of gene transcription and protein synthesis in rat ventral prostate.

Oestrogen exposure during the early post-natal period affects male growth, physiology, and susceptibility to disease in adult life. The prostate gland is susceptible to this oestrogen imprinting, showing a reduced expression of the androgen receptor and inability to respond to androgen stimulus. In this context, we decided to study key signalling regulators of ventral prostate (VP) functioning after early postnatal exposure to high-dose oestrogen. Our results showed a decrease of mTOR phosphorylation and its direct downstream target 4EBP. It is known that mTOR-induced signalling is a pivotal pathway of cell metabolism, which is able to control gene transcription and protein synthesis. We then decided to investigate other indicators of a reduced metabolism in the oestrogenized prostate, and found that the luminal epithelial cells were shorter, less polarized and had smaller nuclei containing more compacted chromatin, suggesting that a general mechanism of regulating gene expression and protein synthesis could be installed in the epithelium of the oestrogenized VP. To evaluate this idea, we analysed nucleolar morphology, and measured the amount of ribosomes and the level of methylation of the 45S ribosomal RNA promoter region. These data indicated that the nucleolus was dismantled and that the methylation at the 45S promoter was increased ( approximately five-fold). Taken together, the results support the idea that the oestrogenized prostate maintains a very low transcriptional level and protein turnover by affecting canonical signalling pathways and promoting nuclear and nucleolar changes.

A new paradigm in the periodontal disease progression: gingival connective tissue remodeling with simultaneous collagen degradation and fibers thickening.

Bacterial dental plaque is considered to be the main cause of periodontal diseases, but progression of the disease is also related to the host inflammatory response. The earliest affected tissue is the gingiva, but the specific mechanisms involved in the onset of this condition remain unclear. Frequently, collagen degradation is pointed as the main marker of periodontal disease progression, but the organization of the fibers in the gingival tissue is still unknown. The aim of the present study was to investigate the gingival extracellular matrix in a model of ligature-induced periodontal disease. Analysis of the microbiota indicated a progressive increase in the ratio of Gram-negative/Gram-positive microorganisms. There was no difference in the organization of reticulin fibers next to the epithelial basement membrane, whereas the arrangement of collagen fibers in the gingival connective tissue was significantly affected. Animals with inflammation presented a reduction of 35% in the total area occupied by collagen fibers. However, these fibers were thicker and more densely packed. These alterations involve type I, type III and type VI collagens as determined by immunohistochemistry. The results demonstrated the occurrence of marked reorganization of the gingival extracellular matrix in response to the inflammatory process, indicating a new paradigm in the periodontal disease progression: collagen degradation and fibers thickening, simultaneously.

The influence of type I diabetes mellitus in periodontal disease induced changes of the gingival epithelium and connective tissue.

Periodontal disease constitutes the most frequent chronic diseases in human dentition. Bacterial plaque is the main etiologic agent, although it is the host immune response that causes periodontal tissue destruction. Diabetes is considered an important risk factor, not only for the onset but also for progression of the disease. The aim of this study was to analyze structural changes in the rat gingival epithelium and connective tissue in response to the experimental periodontal disease induced by the ligature technique, under the influence of diabetes. The results showed that experimental periodontal disease is characterized by marked inflammation, affecting both the epithelial and connective tissues, causing degeneration of the dermal papilla, increase in the number of inflammatory cells, destruction of reticulin fibers, and accumulation of dense collagen fibers (fibrosis). These changes were worsened by diabetes, apparently by hampering the inflammatory response and affecting tissue repair of the affected tissues.

The influence of type I diabetes mellitus on the expression and activity of gelatinases (matrix metalloproteinases-2 and -9) in induced periodontal disease.

BACKGROUND AND OBJECTIVE: Periodontal disease corresponds to a group of lesions that affect the tooth-supporting tissues present in the dental follicle. Although bacterial plaque is important, the immune response also contributes to the destruction of periodontal tissues. Diabetes mellitus is closely associated with the development, progression and severity of periodontal disease because it not only affects extracellular matrix organization but also the tissue response to inflammation. The objective of the present investigation was to study the influence of diabetes on experimental periodontal disease by evaluating the degradation of extracellular matrix through the analysis of matrix metalloproteinase (MMP)-2 and MMP-9 expression and activity, using immunofluorescence, zymography and real-time reverse transcription-polymerase chain reaction. MATERIAL AND METHODS: Wistar rats were divided into normal and diabetic groups and evaluated 0, 15 and 30 d after the induction of periodontal disease by ligature. RESULTS: MMP-2 and -9 were detected in epithelial cells, in the blood vessel endothelium and in connective tissue cells. The same profile of enzymatic expression of MMP-2 and -9 was observed in normal and diabetic animals, with a peak in activity at day 15 of inflammation. However, in diabetic animals, MMP-2 gelatinolytic activity was reduced after the inflammatory stimulus, whereas that of MMP-9 was increased. MMP-2 gene expression decreased with inflammation in both normal groups and groups with diabetes. In contrast, MMP-9 expression increased in normal animals and decreased in diabetic animals after inflammation. CONCLUSION: The results suggest the involvement of MMP-2 and -9 in the dynamics of periodontal disease and that variation in their expression levels results in differences in tissue organization and wound healing in normal and diabetic animals.

The prima donna of epigenetics: the regulation of gene expression by DNA methylation

This review focuses on the mechanisms of DNA methylation, DNA methylation pattern formation and their involvement in gene regulation. Association of DNA methylation with imprinting, embryonic development and human diseases is discussed. Furthermore, besides considering changes in DNA methylation as mechanisms of disease, the role of epigenetics in general and DNA methylation in particular in transgenerational carcinogenesis, in memory formation and behavior establishment are brought about as mechanisms based on the cellular memory of gene expression patterns.

The prima donna of epigenetics: the regulation of gene expression by DNA methylation.

This review focuses on the mechanisms of DNA methylation, DNA methylation pattern formation and their involvement in gene regulation. Association of DNA methylation with imprinting, embryonic development and human diseases is discussed. Furthermore, besides considering changes in DNA methylation as mechanisms of disease, the role of epigenetics in general and DNA methylation in particular in transgenerational carcinogenesis, in memory formation and behavior establishment are brought about as mechanisms based on the cellular memory of gene expression patterns.

Early effects of estrogen on the rat ventral prostate.

Complex interactions between androgen and estrogen (E2) regulate prostatic development and physiology. We analyzed the early effects of a high single dose of E2 (25 mg/kg body weight) and castration (separately or combined) on the adult 90-day-old male Wistar rat ventral prostate. Androgen levels, prostate weight, and the variation in the relative and absolute volume of tissue compartments and apoptotic indices were determined for 7 days. Castration and exogenous E2 markedly reduced ventral prostate weight (about 50% of the control), with a significant reduction in the epithelial compartment and increased stroma. The final volume of the epithelium was identical at day 7 for all treatments (58.5% of the control). However, E2 had an immediate effect, causing a reduction in epithelial volume as early as day 1. An increase in smooth muscle cell volume resulted from the concentration of these cells around the regressing epithelium. The treatments resulted in differential kinetics in epithelial cell apoptosis. Castration led to a peak in apoptosis at day 3, with 5% of the epithelial cells presenting signs of apoptosis, whereas E2 caused an immediate increase (observed on day 1) and a sustained (up to day 7) effect. E2 administration to castrated rats significantly increased the level of apoptosis by day 3, reaching 9% of the epithelial cells. The divergent kinetics between treatments resulted in the same levels of epithelial regression after 7 days (approximately 30% of control). These results show that E2 has an immediate and possibly direct effect on the prostate, and anticipates epithelial cell death before reducing testosterone to levels as low as those of castrated rats. In addition, E2 and androgen deprivation apparently cause epithelial cell death by distinct and independent pathways.

Early effects of estrogen on the rat ventral prostate

Complex interactions between androgen and estrogen (E2) regulate prostatic development and physiology. We analyzed the early effects of a high single dose of E2 (25 mg/kg body weight) and castration (separately or combined) on the adult 90-day-old male Wistar rat ventral prostate. Androgen levels, prostate weight, and the variation in the relative and absolute volume of tissue compartments and apoptotic indices were determined for 7 days. Castration and exogenous E2 markedly reduced ventral prostate weight (about 50 percent of the control), with a significant reduction in the epithelial compartment and increased stroma. The final volume of the epithelium was identical at day 7 for all treatments (58.5 percent of the control). However, E2 had an immediate effect, causing a reduction in epithelial volume as early as day 1. An increase in smooth muscle cell volume resulted from the concentration of these cells around the regressing epithelium. The treatments resulted in differential kinetics in epithelial cell apoptosis. Castration led to a peak in apoptosis at day 3, with 5 percent of the epithelial cells presenting signs of apoptosis, whereas E2 caused an immediate increase (observed on day 1) and a sustained (up to day 7) effect. E2 administration to castrated rats significantly increased the level of apoptosis by day 3, reaching 9 percent of the epithelial cells. The divergent kinetics between treatments resulted in the same levels of epithelial regression after 7 days (about 30 percent of control). These results show that E2 has an immediate and possibly direct effect on the prostate, and anticipates epithelial cell death before reducing testosterone to levels as low as those of castrated rats. In addition, E2 and androgen deprivation apparently cause epithelial cell death by distinct and independent pathways.