sleep deprivation ( sd ) is a frequent condition in modern life due to the demands of our lifestyles , and sleep loss may trigger several alterations in organisms , including immunological effects [ 25 ] , memory deficits [ 68 ] , and hormonal abnormalities [ 914 ] . shift work , obstructive sleep apnea , and other sleep disturbances may cause metabolic syndrome , which is strongly associated with male sex hormone alterations . for instance , men affected by sd showed accentuated reduction of testosterone levels or hypogonadism . additionally , obstructive sleep apnea syndrome is frequently associated with decreased testosterone concentrations , fatigue , and erectile dysfunction [ 18 , 19 ] . our laboratory group has consistently demonstrated the effects of paradoxical sleep deprivation ( psd ) on overall hormonal profiles in male rats ; specifically we have found lower testosterone and estrone levels , but higher levels of progesterone , prolactin , corticosterone , and adrenocorticotropic hormone ( acth ) [ 11 , 12 ] . interestingly , in contrast to humans , these rats showed increased genital reflexes , reflected in penile erections and ejaculatory frequency [ 912 , 20 , 21 ] . in the male rat 's reproductive system , 4-day psd decreased the weight of the seminal vesicle and ventral prostate , indicating reduced steroidogenesis or diminished expression of androgens in target glands . indeed , psd is inherently a stress condition , and other stress modalities that have been examined , such as immobilization , also lead to lower testosterone concentrations [ 9 , 10 ] . this can impair gonadal function by altering testicular maturation and the production of maturing sperm . moreover , it has been demonstrated that after castration , blood androgen levels as well as blood flow in the prostate ( which is controlled by androgen levels ) decrease abruptly . therefore , our objective was to examine the effect of psd ( 96  h ) and chronic sleep restriction ( 21 days ) on hormonal profiles and prostate histological patterns . wistar male rats , aged 3 months and weighing 300350  g at the beginning of the experiment , were obtained from the instituto nacional de farmacologia ( infar ) colony at unifesp . the rats were housed inside standard polypropylene cages in a temperature - controlled ( 23  1c ) room with a 12:12  h light - dark cycle ( lights on at 06:00 hours ) . all procedures used in the present study complied with the guide for care and use of laboratory animals , and the experimental protocol was approved by the unifesp ethical committee ( 0876/09 ) . nave rats were randomly distributed into three groups : paradoxical sleep deprivation ( psd ) , sleep restriction ( sr ) , and home - cage controls ( ctrl ) . the chronic partial sr group was subjected to sr for 18  h ( beginning at 16:00  h ) per day for 21 consecutive days ( sr21 n = 10 ) . after each 18  h sleep deprivation period , the rats were allowed to sleep for 6  h ( sleep window beginning at 10:00 ) . throughout the sr procedure , rats slept an average of 3040% of the time , corresponding to 8 - 9  h per day . this sleep interval ( 10:0016:00  h ) was chosen because this is when paradoxical sleep attains its highest expression , and slow wave - sleep homeostatic pressure is generated [ 2527 ] .    the sd procedure consisted of placing rats in a container with a narrow cylindrical platform 6.5  cm in diameter surrounded by water about 1  cm below the platform surface . each rat was placed individually in the container , and food and water were available ad libitum throughout the entire experimental period . at the onset of a paradoxical sleep episode , rats experienced loss of muscle tonus and fell into the water , thus being awakened . after the psd / sr period , the rats were brought to an adjacent room and decapitated with minimum discomfort between 09:00  h and noon . the control group rats were euthanized at the same time as the experimental group and the other 50% were euthanized on the first day of the experimental procedure . blood was collected in glass tubes and centrifuged to obtain samples of serum or plasma . each hormone assay of the samples from the five groups was analyzed on the same day . testosterone concentration ( 6.7% ) was measured by chemiluminescent immunometric assay ( immulite automated analyses ; diagnostic products corporation , los angeles , ca , usa ) , with a detection threshold of 10  ng  dl . corticosterone ( 10.3% ) concentrations were assayed by a double - antibody ria method specifically for rats and mice , using a commercial kit ( icn biomedicals , costa mesa , ca , usa ) . the sensitivity of the assay is 0.25  ng  dl .   for light microscopy , the ventral prostates were removed and immediately fixed by a 24  h immersion in karnovsky fixative ( 0.1  m phosphate buffer , ph 7.2 , containing 4% paraformaldehyde and 2.5% glutaraldehyde ) . fixed tissues were dehydrated in a graded ethanol series and embedded in paraffin or glycol methacrylate resin ( historesin embedding kit , leica , heidelberg , germany ) . sections were cut at 3  m and stained with hematoxylin and eosin ( h&e ) for general histological studies . analyses were carried out using either a zeiss jenaval light microscope ( carl zeiss , jena , germany ) or an olympus bx60 ( hamburg , germany ) . random prostatic fields from h&e sections were analyzed using the software image - pro - plus version 4.5 for windows ( media cybernetic ) . stereological analyses were carried out using weibel 's multipurpose graticulate with 130 points and 60 test lines , to compare the relative proportion ( relative volume ) of each prostatic tissue component ( epithelium , lumen , and stroma ) , as described by huttunen and collaborators ( 1981 ) and modified by vilamaior and collaborators ( 2006 ) for prostatic tissue [ 29 , 30 ] . thirty microscopic fields were chosen at random . in summary , the relative values were determined by counting the coincident points of the test grid and dividing them by the total number of points . the absolute volume of each of these compartments was determined by multiplying the relative volume value by the mean prostatic weight , based on the determination that 1  mg of fresh rat ventral tissue had a volume of approximately 1  mm . for the morphometrical analyses , 200 random measurements of epithelium height were also obtained . after 96  h psd , testosterone was 87% lower than in the control group ( 436.0  68.0 versus 58.0  8.2  ng  dl ;  p < 0.01 ) . the 21-day sr group 's testosterone levels fell 65% in regarding control group ( 436.0  68.0 versus 152.7  22.2  ng  dl ; the anova test showed the effect of the psd and sr groups on corticosterone levels [ f(3.49 ) = 13.92 ; p < 0.001 ] . subsequent post hoc analysis revealed that corticosterone concentrations after 96  h of psd ( p < 0.001 ) were higher than in the control 8 group and 21 days of sr . furthermore , corticosterone levels after 21 days of sr were statistically different from the control group ( p < 0.01 ) . structural analysis of the ventral prostate using ordinary light microscopy demonstrated that the ventral prostate tissue was affected in the psd 96-h and sr 21-day protocols . epithelial infoldings of apoptotic figures appeared and interestingly , there was an accumulation of fat cells in prostatic gland stroma , particularly in rats subjected to the sr procedure . morphometric and stereological evaluation of prostatic compartments ( table 1 ) showed regression of epithelium height in psd96 and sr groups ( p < 0.001 ) relative to the control group . the relative epithelial volume did not show any differences relative to the control groups . on the other hand , relative volumes of the stroma and lumen in the sr group however , the absolute volume of the lumen in the sr group declined relative to both the control and psd96 rats ( p < 0.001 ) . the purpose of this study was to evaluate the effects of 96-hour sd and 21-day sr on prostate morphology in rats . the main findings were that prostate tissue responded to 96  h psd with the presence of apoptotic bodies and diminished epithelium height , which indicates regression of the prostatic secretion function ( glandular atrophy ) . our data confirm previous findings about how sleep deprivation affects testosterone and corticosterone concentrations [ 9 , 10 , 31 , 32 ] . the abrupt fall in testosterone levels may be influenced by an elevation of hormones involved in the hypothalamic - pituitary - adrenal ( hpa ) axis , such as corticosterone . therefore , it seems that glucocorticoids have the ability to suppress endocrine signaling in the reproductive axis [ 9 , 10 , 31 , 33 ] . in vitro studies indeed , glucocorticoids directly inhibit transcription of genes responsible for the enzymatic mechanism that activates testosterone synthesis . hales and payne   reported that after treating leydig cells with glucocorticoid in vitro , the gene transcription and subsequent synthesis of the protein responsible for cholesterol cleavage ( which produces testosterone ) was also inhibited . high glucocorticoid concentrations downmodulate the reproductive axis at the hypothalamus and pituitary levels , showing that they have deleterious effects on the expression of gonadotrophin - releasing hormone ( gnrh ) mrna and reduce lh plasma concentrations . as shown by andersen and coworkers , plasma testosterone reduction during sleep deprivation is directly related to higher corticosterone levels [ 11 , 12 ] . prostatic morphogenesis is determined not genetically , but by exposure to androgens produced by the testes in the fetus [ 36 , 37 ] . it has been shown that androgen stimulation at the correct time during embryogenesis can modulate prostate tissue growth in both males and females . several studies have shown that the prostate undergoes more marked alterations in epithelial and stromal compartments after hormonal and surgical ablation . these procedures lead to a decrease in glandular function , with consequent declines in epithelium thickness and stromal volume [ 39 , 40 ] . in the current study , we found diminished epithelium height in rats subjected to psd 96  h and sr . maintenance of prostate physiology depends on circulating androgens and more specifically on dihydrotestosterone levels [ 42 , 43 ] . therefore , this hormone seems to be more potent than testosterone in stimulating epithelium growth and maintenance , although they have the same ability to prevent cell death after castration . androgens play a central role in regulating growth and maintenance of the active functional state of the prostate gland in mammals and can stimulate and inhibit the death of epithelial glandular cells [ 24 , 45 ] . there is strong evidence that the low levels of circulating androgens seen in the psd96 group may have started the apoptotic process in the rat ventral prostate , which led to diminished volume and thus glandular atrophy and decreased function . ribeiro and collaborators ( 2008 ) showed that dexamethasone treatment in male rats induced atrophy and reduced the proliferative capacity of epithelial cells . smooth muscle cells showed an atrophic contractile pattern and high electron density of the mitochondrial matrix . the high concentration of circulating corticosterone may have led to the reduced epithelium height in the experimental groups studied here . in addition , accumulation of fat cells in the stroma was observed in the group of rats subjected to sr21 . we do not know what impact reduced circulating levels of androgens has in this phenomenon , since we found that animals castrated after 21 days did not present this phenomenon ; it is not even known whether this may be related to high levels of corticosterone or some other hormone . taken as a whole , our results suggest that both paradoxical sleep deprivation and sleep restriction strongly influence the ventral prostate alterations observed in this study . these observed morphological alterations could be induced by two mechanisms mediated by sd and/or sr : the drastic reduction in testosterone levels ( important for organ morphology maintenance ) or the increase in corticosterone levels , which can act directly on the cytoplasmic receptors of the prostate cells .
we investigated the effect of 96  h paradoxical sleep deprivation ( psd ) and 21-day sleep restriction ( sr ) on prostate morphology using stereological assays in male rats . 
 after euthanasia , the rat ventral prostate was removed , weighed , and prepared for conventional light microscopy . 
 microscopic analysis of the prostate reveals that morphology of this gland was altered after 96  h of psd and 21 days of sr , with the most important alterations occurring in the epithelium and stroma in the course of both procedures compared with the control group . 
 both 96  h psd and 21-day sr rats showed lower serum testosterone and higher corticosterone levels than control rats . 
 the significance of our result referring to the sleep deprivation was responsible for deep morphological alterations in ventral prostate tissue , like to castration microscopic modifications . 
 this result is due to the marked alterations in hormonal status caused by psd and sr .