apoptosis or programmed cell death plays an important role in the homeostasis and normal function 
 of all tissues .
it is morphologically characterized by disruption of the cell skeleton , cell 
 shrinkage , membrane blebbing , nuclear condensation , cell disruption into small membrane - enclosed 
 fragments , and phagocytosis by neighbouring cells .
it is characterized by the activation of endogenous endonucleases with 
 subsequent cleavage of chromatin dna into internucleosomal fragments .
the induction and execution of apoptosis require the cooperation of a series of molecules . among 
 them , the caspase - cascade signaling system is vital in the process of apoptosis .
generally , the caspase family proteases can be activated through three 
 pathways , mediated by either mitochondrion / cytochrome - c , cell surface receptors , or endoplasmic 
 reticulum [ 46 ] . 

caspase-3 , the major effector caspase , is synthesized as an inactive proenzyme and processed during 
 apoptosis into its active form . in this study , caspase-3 was selected because it represents the 
 point of intersection of extrinsic , intrinsic , and mitochondrial pathways .
smoking is one of the most important health issues and the most wide - spread habituation 
 worldwide .
it is well established that smoking is associated with a significant reduction in sperm 
 concentration and quality .
recent studies have also 
 demonstrated that the apoptotic processes may participate in abnormal spermatogenesis in 
 nonobstructive azoospermia , such as maturation arrest and sertoli cell - only syndrome [ 9 , 10 ] .
the rise of tobacco use among pregnant women has promoted concern over the potential adverse 
 effects on their offspring .
maternal smoking during pregnancy is well known to be associated with a 
 variety of adverse neonatal outcomes .
but the effects of intrauterine exposure to cigarette smoke on 
 newborns ' gonads are also intriguing .
unfortunately there are only a few studies related to this 
 subject in the literature .
therefore , in this study , we aimed to investigate the effect of cigarette 
 smoke exposure during intrauterine period on neonatal rat testis .
the 
 research was consistent with world medical association declaration of helsinki and 
 the guide for the care and use of laboratory animals of the institute for 
 laboratory animal research of the national research council . a prospective ,
the rats were randomized to two groups ( n = 13 , n = 12 ) , either 
 to be exposed to room air or cigarette smoke with the walton smoking machine ( process and 
 instruments corp . ,
the rats were exposed to 2r4f kentucky tobacco research and 
 development center reference cigarettes ( 9.7  mg of tar , 0.85  mg of nicotine , and 
 11.7  mg of total particulate matter ) one hour twice a day for a total of 10 cigarettes per 
 day , initiating from proestrous period and during their pregnancies .
each cycle on the machine 
 included one puff of cigarette smoke of 2-second duration , followed by a 28-second hold period , for 
 a smoke exposure time of 30 seconds per cycle .
this was followed by a 30-second purge of fresh air 
 before the second puff is taken to repeat the cycle .
the remaining rats were exposed to room air 
 with the same machine and served as controls .
next morning , the 
 female rats that had spermatozoa on their vaginal smears were accepted pregnant ( n 
 = 16 ) .
the newborn male rats ( n = 21 ) were categorized as group 1 and group 2 .
group 1 
 included 15 newborn male rats that were exposed to cigarette smoke during their intrauterine life . 

group 2 included 6 newborn male rats that were exposed to room air during their intrauterine life . 

after fixation with 10% formalin , the tissues were washed under running tap water for 24 hours 
 and dehydrated with 50 , 60 , 70 , 80 , 90 , 96 , and 100% concentrated ethanol .
the specimens were then 
 laid in a 1  :  1 ratio of immersion oil and absolute alcohol for 1 hour , followed by 
 immersion oil overnight for transparency .
after the application of xylol , the specimens were made 
 into paraffin blocks using a 1  :  1 xylol and paraffin mixture for 1 hour and 
 paraffin for 6 hours in an incubator .
4  m thick sections were 
 dehydrated and dyed with hematoxylin harris ( biostain code : rr sp67-d / biostain ready reagents ltd .
, 
 unit 2a , arrow trading estate , manchester ) . to detect apoptosis , immunohistochemical analyses with anti - caspase-3 primary antibodies were 
 performed .
after embedding in paraffin , 4  m thick sections were 
 taken onto poly - l - lysine coated slides .
after incubating at 37c overnight and 60c 
 for an hour , the sections were dewaxed in xylene for 15 minutes twice and rehydrated with descendent 
 ethanol gradient for 10 minutes and distilled water for 5 minutes .
after dewaxing and rehydration 
 with xylene , alcohol , and distilled water , respectively , the slides were taken into microwave 
 irradiation containing 0.1  m citrate buffer ( catalog number ap-9003 - 500 , lot ct14070 , 
 labvision corporation , fremont , ca , usa ) for 5 minutes at 650 w and for 3  5 minutes at 550 
 w. after 20 minutes at room temperature , the sections were encircled with a pap pen ( super pap pen , 
 pn im3580 , beckman coulter company , france ) . by a new washing with distilled water and then with 
 phosphate - buffered saline ( pbs , lot 0446b002 , labvision corporation , fremont , ca , usa ) , endogenous 
 peroxidase activity was blocked by using 3% hydrogen peroxide ( catalog number ta-125-hp , thermo 
 scientific , fremont , ca , usa ) for 20 minutes . after washing with pbs , ultra v block ( catalog number 
 ta-125-ub , thermo scientific , fremont , ca , usa ) was applied for 5 minutes .
after a 1 hour application of primary antibodies ( caspase-3 ; catalog number rb-1197 , thermo 
 scientific , fremont , ca , usa ) diluted ( 1  :  100 concentration ) with antibody diluents 
 ( catalog number ta-125-ud thermo scientific , fremont , ca , usa ) , the samples were washed with pbs 
 again
. then secondary antibody , biotinylated goat antipolyvalent ( catalog number tp-125-bn , thermo 
 scientific , fremont , ca , usa ) was applied for 20 minutes .
the sections were washed with pbs and 
 treated with streptavidin peroxidase ( tp-125-hr , thermo scientific , fremont , ca , usa ) for 20 
 minutes . by a new washing with pbs ,
finally , the specimens were placed for 10 minutes in dab 
 ( 3,3 diaminobenzidine ) ( thermo scientific , catalog number ta-125-hd , fremont , ca , usa ) and 
 stained with hematoxylin harris ( biostain code : rr sp67-d / biostain ready reagents ltd . , unit 2a , 
 arrow trading estate , manchester ) .
the slides were covered with xylol - based mounting , evaluated under leica dm 4000 b light 
 microscope ( wetzlar , germany ) , and photographed with leica qwin prov 3.4.0 ( calidris and softhard 
 technology ltd . , switzerland ) . in situ
terminal deoxynucleotidyl transferase - mediated dutp nick end - labeling ( tunel ) 
 immunofluorescent staining was used for detection of dna damage .
the formalin fixed , 
 paraffin - embedded tissues were dewaxed in xylene and rehydrated through a graded series of ethanol 
 and double - distilled water .
then they were placed in a plastic jar and incubated for 5 minutes in 
 350  w microwave irradiation containing 200  ml 0.1  m citrate buffer , ph 6.0 . 

after rinsing the slides twice with pbs , 50  l tunel reaction 
 mixture ( catalog number 11 684 795 910 , roche diagnostics , roche diagnostics gmbh , roche applied 
 science , mannheim , germany ) per sample was added
. then the samples were incubated for 60 minutes at 
 37c in a humidified atmosphere in the dark . finally , the slides were rinsed three times 
 with pbs and analyzed under a fluorescence microscope with a wavelength in the range of 
 450500  nm ( e.g. , 488  nm ) and detection in the range of 
 515565  nm ( green ) .
the tunel - positive cells which also showed 
 apoptotic nuclear morphology were defined as apoptotic and the percentage of these cells to total 
 cell number was recorded as apoptotic index . an immunohistochemical score ( hscore )
was calculated as the sum of the percentages of positively 
 stained epithelial cells multiplied by the weighted intensity of staining : hscore = 
 pi(i + 1 ) , where  i
 
 represents staining intensity ( 0 = no expression , 1 = mild , 2 = moderate , and 3 = intense ) and 
  pi  is the percentage of stained cells for each intensity .
the numbers of sertoli cells were quantified in cross sections of seminiferous tubules stained by 
 h and e. the number of sertoli cells per tubule was computed in 30 tubules per animal using a light 
 binocular microscope at 400 . only sertoli cells 
 exhibiting typical morphological nuclear features and evident nucleolus
spss 15.0 for windows was used for the statistical analysis . whether the distributions of 
 variables were normal or not was determined by means of the kolmogorov smirnov test .
student 's 
 t - test was used for the comparison of normal distributed variables .
for the comparison of the groups , the primary outcomes were defined as apoptotic index and 
 hscores and the secondary outcomes were defined as sertoli cell count and birth weights of newborn 
 male rats . when hematoxylin - eosin stain preparation was compared , the cells showing apoptotic morphology 
 with pyknotic nuclei , nuclear condensation , and condensed eosinophilic cytoplasm were more 
 remarkable in group 1 ( figure 2 ) .
the immunofluorescent 
 technique also demonstrated increased dna damage in seminiferous epithelium in group 1 ( figure 2 ) .
it was 49.61 
  9.57 for intrauterine smoke exposure group ( group 1 , n = 15 ) and it was 
 29.98  2.34 for intrauterine room air exposure group ( group 2 , n = 6 ) .
when the slides were evaluated immunohistochemically for the detection of caspase-3 activity , 
 cytoplasmic and nuclear caspase-3 immunostaining was detected both in the germ and sertoli cells in 
 group 1 ( figure 3 ) .
there was a significant increase in the 
 incidence of sertoli cell apoptosis , detected by caspase-3 immunostaining , in group 1 ( hscore = 
 210.6  41.9 ) when compared to group 2 ( hscore = 100.0  17.8 ) ( p = 
 0.001 ) .
there was also a significant increase in the incidence of germ cell apoptosis in group 1 
 ( hscore value for group 1 is 214.0  46.26 and hscore value for group 2 is 93.33  
 10.32 , p < 0.001 ) . when the sertoli cell counts were determined , the median value for group 1 was 9.0 ( 611 ) 
 and 11.5 ( 1013 ) for group 2 .
when the birth weights of newborn male rats were analyzed , the mean birth weight of group 1 was 
 significantly lower than group 2 ( 3.08  0.26 versus 4.47  0.40 , p 
 < 0.001 ) .
spermatogenesis is a dynamic process of cell differentiation characterized by mitotic and meiotic 
 divisions , which transform the stem spermatogonia into final mature spermatozoa . throughout this 
 process of development and differentiation ,
a considerable number of germ cells die due to apoptosis 
 to control the overproduction of male gametes .
it is well known that the paracrine signaling between sertoli cells regulates the process of germ 
 cell death . in the presence of exogenous stimulants , such as cadmium , nicotine , lead , and benzopiren 
 that adversely affect testicular function , nonphysiologic
apoptosis and dna fractures can occur 
 which leads to impaired spermatogenesis and infertility . in a study by lin et al . in 1997
increased apoptosis was detected in testis biopsy specimens of 
 azoospermic and severe oligozoospermic patients and this finding was suggested as one of the causes 
 of male infertility . in the following years numerous 
 investigations were composed for the effects of cigarette smoking on impaired spermatogenesis and 
 increased apoptosis has been found to occur during maturation arrest , hypospermatogenesis , and 
 sertoli cell only syndrome [ 9 , 16 , 17 ] . despite the well - known effects of cigarette smoke on the reproductive performance of the 
 individuals , we have a limited data for its effects on the newborns gonads .
epidemiological studies 
 have demonstrated that sperm production and testicular volumes are dose dependently reduced in the 
 adult sons of mothers who smoked during pregnancy .
other 
 studies showed that sons exposed to maternal smoking in uteri have a reduction in sperm 
 concentration of between 20 and 48% in comparison to unexposed ones [ 19 , 20 ] . in our study
we determined an increased apoptosis and dna damage , in the gonads of the newborns 
 of the study group .
abnormality of the oxidant - antioxidant balance may be one of the mechanisms 
 leading to testicular tissue damage following chronic inhalation of cigarette smoke .
it is suggested that activation of ahr by environmental toxicants and ahr - induced apoptotic 
 pathways may be another mechanism .
activation of 
 coagulation in the intervillous space in addition to vasoconstrictor effects of carbon monoxide and 
 nicotine could reduce the oxygen transport to the fetus which can also cause an increase in 
 apoptosis .
it is well known that tobacco use is associated with fetal growth restriction and low birth 
 weight . in our study
we detected lower birth weights in the in uteri smoke exposed group which 
 concurs well with the literature . in a study by wang et al .
smoking during pregnancy is shown to 
 result in an average of 377 grams of decrease in birth weight . in another study
when the placentas of smoker mothers were analyzed , premature aging 
 and degenerative changes were detected and it was suggested to be the cause of low birth weight 
 newborns . despite remarkable advances in the treatment of severe male factor infertility , significant 
 numbers of men can not be effectively treated because of deficiencies in our understanding of 
 testicular failure pathophysiology .
therefore , the mechanisms responsible for abnormal 
 spermatogenesis should be clarified in order to develop and improve treatment options .
but apart from those studies , this study evaluated the effect of exposure to 
 cigarette smoke in the intrauterine period which , to our knowledge , was not undertaken before .
chronic cigarette smoke is shown to induce apoptosis in the rat testis . and in our previous report , we showed that tobacco smoke exposure during 
 intrauterine period promotes granulosa cell apoptosis in newborn rat ovaries . here in this study , using immunohistochemical and immunofluorescent 
 method , we demonstrated significantly increased apoptosis and dna damage in the newborn male gonads 
 of intrauterine cigarette smoke exposed group and we hope that these data will be an inspiration for 
 the future studies .
 

 objectives . to investigate the effect of cigarette smoke exposure during 
 intrauterine period on neonatal rat testis . methods . 
 twenty - five rats were 
 randomized to be exposed to cigarette smoke with the walton smoking machine or to room air during 
 their pregnancies . the newborn male rats ( n = 21 ) were grouped as group 1 
 ( n = 15 ) which were exposed to cigarette smoke during intrauterine life and group 2 
 ( n = 6 ) which were exposed to room air during intrauterine life . 
 the orchiectomy 
 materials were analyzed with tunel immunofluorescent staining for detection of dna damage . to detect 
 apoptosis , immunohistochemical analyses with caspase-3 were performed . 
 primary outcomes were 
 apoptotic index and immunohistochemical scores ( hscores ) ; secondary outcomes were sertoli - cell count 
 and birth - weight of rats . results . 
 sertoli cell apoptosis was increased in group 1 
 ( hscore = 210.6  41.9 ) when compared to group 2 ( hscore = 100.0  17.8 ) 
 ( p = 0.001 ) . 
 sertoli cell count was decreased in group 1 ( p = 
 0.043 ) . 
 the hscore for the germ cells was calculated as 214.0  46.2 in group 1 and 93.3 
  10.3 in group 2 ( p = 0.001 ) referring to an increased germ cell apoptosis 
 in group 1 . 
 the apoptotic indexes for group 1 were 49.6  9.57 and 29.98  2.34 for 
 group 2 ( p = 0.001 ) . 
 the immunofluorescent technique demonstrated increased dna 
 damage in seminiferous epithelium in group 1 . 
 conclusions . 
 intrauterine exposure to 
 cigarette smoke adversely affects neonatal testicular structuring and diminishes testicular 
 reserve . 
 
1. Introduction
2. Material and Method
3. Results
4. Discussion
5. Conclusion