Validation of commonly used reference genes for sleep-related gene expression studies

<p>Abstract</p> <p>Background</p> <p>Sleep is a restorative process and is essential for maintenance of mental and physical health. In an attempt to understand the complexity of sleep, multidisciplinary strategies, including genetic approaches, have been applied to sleep research. Although quantitative real time PCR has been used in previous sleep-related gene expression studies, proper validation of reference genes is currently lacking. Thus, we examined the effect of total or paradoxical sleep deprivation (TSD or PSD) on the expression stability of the following frequently used reference genes in brain and blood: <it>beta-actin (b-actin), beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH)</it>, and <it>hypoxanthine guanine phosphoribosyl transferase (HPRT)</it>.</p> <p>Results</p> <p>Neither TSD nor PSD affected the expression stability of all tested genes in both tissues indicating that <it>b-actin, B2M, GAPDH </it>and <it>HPRT </it>are appropriate reference genes for the sleep-related gene expression studies. In order to further verify these results, the relative expression of <it>brain derived neurotrophic factor (BDNF) </it>and <it>glycerol-3-phosphate dehydrogenase1 (GPD1) </it>was evaluated in brain and blood, respectively. The normalization with each of four reference genes produced similar pattern of expression in control and sleep deprived rats, but subtle differences in the magnitude of expression fold change were observed which might affect the statistical significance.</p> <p>Conclusion</p> <p>This study demonstrated that sleep deprivation does not alter the expression stability of commonly used reference genes in brain and blood. Nonetheless, the use of multiple reference genes in quantitative RT-PCR is required for the accurate results.</p

The influence of sleep deprivation and obesity on DNA damage in female Zucker rats

OBJECTIVE: The aim of this study was to evaluate overall genetic damage induced by total sleep deprivation in obese, female Zucker rats of differing ages. METHOD: Lean and obese Zucker rats at 3, 6, and 15 months old were randomly distributed into two groups for each age group: home-cage control and sleep-deprived (N = 5/group). The sleep-deprived groups were deprived sleep by gentle handling for 6 hours, whereas the home-cage control group was allowed to remain undisturbed in their home-cage. At the end of the sleep deprivation period, or after an equivalent amount of time for the home-cage control groups, the rats were brought to an adjacent room and decapitated. The blood, brain, and liver tissue were collected and stored individually to evaluate DNA damage. RESULTS: Significant genetic damage was observed only in 15-month-old rats. Genetic damage was present in the liver cells from sleep-deprived obese rats compared with lean rats in the same condition. Sleep deprivation was associated with genetic damage in brain cells regardless of obesity status. DNA damage was observed in the peripheral blood cells regardless of sleep condition or obesity status. CONCLUSION: Taken together, these results suggest that obesity was associated with genetic damage in liver cells, whereas sleep deprivation was associated with DNA damage in brain cells. These results also indicate that there is no synergistic effect of these noxious conditions on the overall level of genetic damage. In addition, the level of DNA damage was significantly higher in 15-month-old rats compared to younger rats

Progesterone reduces erectile dysfunction in sleep-deprived spontaneously hypertensive rats

BACKGROUND: Paradoxical sleep deprivation (PSD) associated with cocaine has been shown to enhance genital reflexes (penile erection-PE and ejaculation-EJ) in Wistar rats. Since hypertension predisposes males to erectile dysfunction, the aim of the present study was to investigate the effects of PSD on genital reflexes in the spontaneously hypertensive rat (SHR) compared to the Wistar strain. We also extended our study to examine how PSD affect steroid hormone concentrations involved in genital events in both experimental models. METHODS: The first experiment investigated the effects of PSD on genital reflexes of Wistar and SHR rats challenged by saline and cocaine (n = 10/group). To further examine the impact of the PSD on concentrations of sexual hormones, we performed a hormonal analysis of testosterone and progesterone in the Wistar and in SHR strains. Since after PSD progesterone concentrations decreased in the SHR compared to the Wistar PSD group we extended our study by investigating whether progesterone (25 mg/kg or 50 mg/kg) or testosterone (0.5 mg/kg or 1.0 mg/kg) administration during PSD would have a facilitator effect on the occurrence of genital reflexes in this hypertensive strain. RESULTS: A 4-day period of PSD induced PE in 50% of the Wistar rats against 10% for the SHR. These genital reflexes was potentiated by cocaine in Wistar rats whereas this scenario did not promote significant enhancement in PE and EJ in hypertensive rats, and the percentage of SHR displaying genital reflexes still figured significantly lower than that of the Wistar strain. As for hormone concentrations, both sleep-deprived Wistar and SHR showed lower testosterone concentrations than their respective controls. Sleep deprivation promoted an increase in concentrations of progesterone in Wistar rats, whereas no significant alterations were found after PSD in the SHR strain, which did not present enhancement in erectile responses. In order to explore the role of progesterone in the occurrence of genital reflexes, SHR were treated daily during the sleep deprivation period with progesterone; after the administration of this hormone and challenge with cocaine, we observed a significant increase in erectile events compared with the vehicle PSD SHR+cocaine group. CONCLUSION: Our data showed that the low frequency of genital reflexes found in SHR sleep deprived rats may be attributed to the lower concentrations of progesterone in these rats, based on the observation that progesterone replacement increased genital reflexes in this strain

Impairment of male reproductive function after sleep deprivation

Objective: To evaluate the influence of sleep loss on sexual behavior, hormone levels, sperm parameters, and testis-specific gene expression in male rats.Design: Experimental research.Setting: Animal laboratory.Animal(s): Male adult Wistar-Hannover rats.Intervention(s): Sexually experienced rats were subjected to paradoxic sleep deprivation (PSD) for 96 hours or sleep restriction (SR) for 21 days or kept in their home cage as control (CTRL).Main Outcome Measure(s): Sexual behavior, hormone levels, sperm parameters and expression of stress and nitric oxide-related genes were evaluated.Result(s): PSD significantly decreased sexual behavior compared with the CTRL group, whereas SR had no effect. the PSD group had significantly lower testosterone levels than the CTRL group. Both PSD and SR groups had lower sperm viabilities than the CTRL group. the decrease in the number of live sperm compared with the CTRL group was larger in the PSD group than in the SR group. Regarding testicular gene expression, both PSD and SR led to an increase of iNOS and hydroxysteroid 11 beta-dehydrogenase 1 expressions compared with the CTRL group. These changes were more pronounced in the PSD group. A significant increase in endothelial nitric oxide synthase expression was observed in the PSD groups compared with the CTRL group. No changes were observed in dimethylarginine dimethylaminohydrolase 1 and casein kinase 2 beta-polypeptide expressions.Conclusion(s): Sleep loss can promote marked changes in the male reproductive system of rats, particularly affecting spermatic function in part by interfering in the testicular nitric oxide pathway. (C) 2015 by American Society for Reproductive Medicine.Associacao Fundo de Apoio a Pesquisa (AFIP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)MEC-SeSU (PET) fellowshipUniversidade Federal de São Paulo, Dept Psychobiol, São Paulo, BrazilUniv Fed Goias, Dept Pharmacol, Goias, BrazilUniversidade Federal de São Paulo, Dept Psychobiol, São Paulo, BrazilFAPESP: 2014/15259-2FAPESP: 11/12325-6FAPESP: 12/05396-7Web of Scienc

Sexual dysfunction and epilepsy: the reasons beyond medications

Universidade Federal de São Paulo, Dept Psicobiol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Psicobiol, São Paulo, BrazilWeb of Scienc

Paradoxical Sleep Deprivation Influences Sexual Behavior in Female Rats

Introduction.Sleep disturbances are a frequent complaint in women and are often attributed to hormonal fluctuations during the menstrual cycle. Rodents have been used as models to examine the effects of sleep deprivation on hormonal and behavioral changes. Among the many comorbidities common to sleep disorders, sexual behavior remains the least well studied.Aim.To determine whether paradoxical sleep deprivation (PSD) can affect sexual receptivity (male acceptance) and proceptivity (male solicitation) behaviors in female rats.Methods.Female Wistar rats were subjected to PSD or were maintained as controls. After this period, the estrous cycle (proestrus, estrus, and diestrus) was determined, and all females were placed with a sexually experienced male. in order to investigate the role of hormones in sexual behavior, we included additional groups that were artificially induced to be sexually receptive via administration of a combination of estradiol and progesterone.Main Outcome Measurements.Receptivity and proceptivity behaviors, as well as progesterone and corticosterone concentrations were monitored.Results.Selective sleep loss caused a significant increase in proceptivity and receptivity behaviors in females exclusively during the proestrus phase. the rejection response was increased in PSD rats during the estrus and diestrus phases, as compared with PSD-receptive and proestrus females. PSD reduced progesterone levels during the proestrus phase relative to the respective control group during the same phase of the estrous cycle. the PSD-proestrus females that displayed the most robust sexual response exhibited greater concentrations of corticosterone than PSD-diestrus females, with an absence of sexual solicitation behaviors.Conclusions.PSD produced a distinct response in the hormonal profile that was consistent with the phase of the estrous cycle. These results show that sleep loss can affect sexual motivation and might lead to important clinical implications, including alterations in female physiology and reproductive abnormalities. Andersen ML, Alvarenga TAF, Guindalini C, Perry JC, Silva A, Zager A, and Tufik S. Paradoxical sleep deprivation influences sexual behavior in female rats. J Sex Med 2009;6:2162-2172.Associacao Fundo de Incentivo Psicofarmacologia (AFIP)Fundacao de Amparo Pesquisa do Estado de São PauloConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Psychobiol, BR-04024002 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Psychobiol, BR-04024002 São Paulo, BrazilFundacao de Amparo Pesquisa do Estado de São Paulo: 98/14303-3Fundacao de Amparo Pesquisa do Estado de São Paulo: 06/58274-5Fundacao de Amparo Pesquisa do Estado de São Paulo: 07/56620-6Fundacao de Amparo Pesquisa do Estado de São Paulo: 06/58275-1CNPq: 558924/2008 5Web of Scienc