benzo[a]pyrene ( b[a]p ) , a polycyclic aromatic hydrocarbon compound , is produced by incomplete combustion of organic compounds and high - pressure processes .
this compound is commonly present in motor vehicle exhaust , tobacco smoke , grilled , smoked and broiled foods , and emissions from residential and industrial heating sources .
biotransformation of b[a]p occurs as it undergoes initial oxidation by cytochrome p450 enzymes and consequently forms reactive metabolites , for example , b[a]p-9 , bap-7,8-dihydrodiol 9,10-epoxide and quinones .
the active intermediates are capable of covalently bonding to dna   and form dna adducts .
also , they can undergo redox cycling and generate excessive reactive oxygen species , which may alter cell signaling and damage cellular membranes resulting in apoptosis .
previous studies in animal models have demonstrated that direct exposure to b[a]p could cause toxic effects on male reproduction and has been implicated in the pathobiology of adverse reproductive health .
b[a]p has been related to decreased spermatozoa quality [ 3 , 4 ] and germ cell apoptosis after chronic exposure . to date
, limited data has appeared available to depict the impact of exposure to b[a]p on spermatozoa quality in relation to testicular germ cells during spermatogenesis .
spermatogenesis represents a complex and dynamic process of proliferation and differentiation of the transformation of spermatogonia into mature spermatozoa in three major stages , the mitotic stage , the meiotic stage , and the maturation stage .
alterations occurring in any of them could lead to the production of abnormal spermatozoa and reduce proliferation of spermatozoa .
thus , the understanding of processes related to spermatogenesis is critical for assessment of male reproductive health .
the objective of this study was to investigate the toxicological impact of b[a]p exposure on testicular germ cells during spermatogenesis by assessing concentrations of and apoptosis in testicular germ cells and mature spermatozoa .
hsd : icr ( cd1 ) 10-week - old male mice , weighing 3040  g , were used ( harlan laboratories , inc . ) .
mice were caged individually with 12  hr light / dark cycles , given rodent chow ( global rodent diet number 2018 ) , water ad libitum , and enrichment items .
all procedures were performed in accordance with protocols approved by the old dominion university 's institutional animal care and use committee .
each mouse was weighed every other day and was examined daily for behavioral and clinical symptoms . upon arrival
mice ( n = 8 per group ) were randomly assigned to the exposure group and control group .
the exposed mice were gavaged with 1 , 10 , 50 , and 100  mg / kg / day of b[a]p dissolved in 100% corn oil for 30 days .
control animals were only dosed with 20  l of 100% corn oil . on day 30 , mice were anesthetized using intraperitoneal injection of sodium pentobarbital ( 50  mg / ml ) to the surgical plane , confirmed by the toe - pinch method .
reproductive tissues , including testes , seminal vesicles , prostate , epididymis , and vase deferens , were removed and placed on a petri dish and weighed .
the left testes were used for collection of germ cells and stored in the freezing media - rpmi 1640 and 5% glycerol at 80c until germ cell collection .
the testes were fixed with 10% formalin in a 1% phosphate buffer at room temperature for 2448 hours .
, serial sections ( 8  m ) were cut from the middle of each testis using a microtome .
these sections were placed in a water bath at 47c and transferred to charged microscope slides .
sections were dried overnight on glass slides at 35c and deposited in a storage box at room temperature until processed for histology . for staining and morphometric evaluation , sections were deparaffinized and rehydrated using 100% , 95% , and 70% graded ethanol .
images were acquired using an olympus cx41 microscope with a dp72 camera and cellsens standard software .
testes were digested in a 50  ml conical tube containing 10  ml d - hanks solution with 0.5  mg / ml collagenase and 25  g / ml dnase , then incubated at room temperature for 10 minutes with gentle oscillation .
next , the suspension was filtered through a 150 mesh ( 104  m ) filter to collect the seminiferous tubules .
the filtrate was centrifuged at 300  g for 10 minutes to concentrate the interstitial cells .
cells were pelleted by centrifugation ( 300  g for 10 minutes ) , and the supernatant was removed .
the cells were then suspended in 0.2% bovine serum albumin ( bsa ) in rpmi+ by gentle pipetting .
sedimentation at unit gravity was used to separate each type of germ cell , including spermatogonia , pachytene spermatocytes , round spermatids , and elongated spermatids .
the procedures were performed according to those developed for murine spermatogenic cells [ 7 , 8 ] with some modifications .
a small sta - put chamber and sample loading chamber ( pro science glass co. , ontario , canada ) were prepared by sterilization .
both chambers were primed by applying 25  ml rpmi+ through the sample loading chamber using the pump to advance the volume into the sta - put chamber . a 10  ml cell suspension
was loaded onto a 5  ml cushion of 0.2% bsa in rpmi+ in the sample loading chamber , and then 0.2% bsa in rpmi+ was added at a flow rate of 20  ml / min .
the tubing was reversed to allow 10  ml fractions to be drawn from the chamber at a flow rate of 10  ml / min .
each fraction was centrifuged at 900  g for 10  min , and then supernatant was removed .
each fraction was next assessed for cell types , which were identified based on morphology .
elongated spermatids were identified based on shape , placement of cytoplasm , condensed nucleus , and the tail .
each type of cell was counted using a hemocytometer and stored at 80c until apoptosis analysis .
a consistent length of each vas deferens , 1.5  cm , was flushed with 0.5% bsa in rpmi+ to recover spermatozoa .
spermatozoa quality , including concentration , viability , motility , and morphology , was assessed .
the percentage of motility was determined by counting both motile and immotile spermatozoa . for viability analysis ,
at least 100 spermatozoa per sample were assessed from an eosin stained preparation . for morphology assessment ,
at least 100 spermatozoa were classified as either normal , abnormal head , or curly tail .
apoptotic cells were detected by terminal deoxynucleotidyl transferase mediated deoxy - utp nick end labeling ( tunel ) , using an in situ detection kit ( roche diagnostic , manheim , germany ) . to detect apoptotic germ cells in seminiferous tubules ,
testes were fixed in formalin for 24 hours and then embedded in paraffin , and testicular sections were sliced in six  m ribbons on a leica microtome .
the tissue sections were washed with 1% hsa in a 1x phosphorous buffer solution ( pbs ) , permeabilized using 0.1% ( v / v ) triton x-100 , 0.1% ( w / v ) sodium citrate in 1x pbs , and incubated at room temperature for 10 minutes .
the presence of germ cell apoptosis related to dna strand breaks was evaluated by terminal deoxynucleotidyl transferase mediated d'utp nick - end labeling by means of the in situ cell detection kit with fitc - labeled d'utp . a tunel mixture ( roche )
slides were incubated for 60 minutes at 32c , in the dark , with humidity , and were monitored for wetness by adding 1% hsa in 1x pbs as needed .
slides were analyzed immediately using a fluorescent microscope ( nikon eclipse 80i ) with a cool snap ez camera and nis elements br3.2 software .
a minimum of 20 fields of view at 1000x magnification were randomly selected for analysis of cells . to detect apoptotic spermatozoa
, 200  l of spermatozoa at 30  10/ml collected from the previous step were washed twice with 1x pbs/1% hsa .
spermatozoa were fixed to the slides , permeabilized with 0.1% triton x-100 in 0.1% sodium citrate at 4c for two minutes .
each test included both positive and negative controls to ensure the performance of the assay .
cells in the positive control were treated with 50  l of dnase solution , while cells in the negative control did not get treated with the tunel mixture .
after the cells were incubated one hour at 37c , the cells were washed twice with 1% has in pbs .
a minimum of 100 spermatozoa were counted as either positive ( green ) or negative ( absence ) at 1000x under oil immersion , using a nikon eclipse 80i fluorescent / brightfield microscope with the x - cite series 120 and fitc filter .
all data were tested for normality and variance before statistical analysis took place and presented as mean  standard deviation ( sd ) .
multivariate analysis of variances ( manova ) and lsd post hoc tests were utilized to assess statistically significant differences in spermatozoa quality , reproductive organ weight , and apoptotic cells between the exposure group and the control group .
the weight of testes and epididymis did gradually decrease as b[a]p doses increased ( figures 1(c ) and 1(d ) ) .
the cross sections of the seminiferous tubules of mice that showed that exposure to b[a]p at 100  mg / kg / day resulted in the atrophy of seminiferous tubules , reduced width of adluminal compartments of the seminiferous tubules , and altered morphology of spermatogonia and spermatocytes ( figure 2 ) . 

figure 3 shows spermatogenic cells recovered from the testes of mice exposed to b[a]p and their concentrations are shown in figure 4 the b[a]p - treated mice yielded lower concentrations of germ cells , including spermatogonia , pachytene spermatocytes , spermatids , and elongated spermatids in the tubules , as b[a]p doses increased .
mg / kg / day and 100  mg / kg / day induced a significant decrease in the concentrations of germ cells as compared to the control ( p < 0.05 ) .
mice exposed to 50 and 100  mg / kg / day of b[a]p yielded significantly lower concentrations of spermatozoa than the control group ( figure 5(a ) ) . for motility and viability , only b[a]p at 100  mg
/ kg / day induced a significant decrease in percentages of motility and viability as compared with the control ( p = 0.04 and 0.04 , resp . )
percentages of morphology of spermatozoa with normal head decreased but did not reach the significant level .
the percentages of spermatozoa with abnormal head and curly tail remained stable with no significant changes ( figure 5(c ) ) . a cross section of a seminiferous tubule of mice exposed to b[a]p at 100  mg / kg / day showed apoptosis in germ cells as detected using the tunel assay as compared with the control ( figure 6 ) .
mg / kg / day had less than 0.6% of cells with apoptosis ( figure 7 ) .
the apoptosis percentages of those cells started increasing as b[a]p reached 50  mg / kg / day and 100  mg / kg / day . at 100  mg / kg / day , spermatogonia , pachytene spermatocytes , and spermatocytes had significant percentages of apoptosis ( 3.0% , 4.1% , and 3.0% , resp . ) as compared with the control groups ( 0.05% , 0% , and 0% , resp . )
elongated spermatids were the type of germ cells with the highest percentages of cells with apoptosis .
the percentages of elongated spermatids from mice exposed to 1 , 10 , and 50  mg / kg / day were not significantly higher than those in spermatogonia , pachytene spermatocytes , and round spermatids . at 100  mg / kg / day of b[a]p , elongated spermatids ,
however , had a significant increase in the percentage of apoptosis ( 19.2% ) as compared with the control ( 2.1% ) ( p = 0.01 ) .
mature spermatozoa with apoptosis had a similar pattern as elongated spermatids , whose apoptotic cells increased at 10  mg / kg / day of b[a]p and reached a significant level of increase at 50  mg / kg / day and 100  mg / kg / day ( 8.7% and 27.6% , resp . )
as compared with the control groups ( 1.4% ) ( p = 0.04 and 0.009 , resp . )
the current study used rodents for the experimental paradigm to provide knowledge about the influence of exposure to b[a]p on germ cells during spermatogenesis .
each type of germ cell represents steps of the spermatogenic process involving mitotic cell division , meiosis , and the process of spermiogenesis .
exposure to b[a]p , particularly at 50  mg / kg / day and 100  mg / kg / day for 30 days , induced a significant decrease in the levels of germ cells , including spermatogonia , pachytene spermatocytes , round spermatids , and elongated spermatids .
b[a]p could have affected the proliferation of spermatogonia and pachytene spermatocytes in the early spermatogenesis .
such effect led to the lower concentrations of round spermatids and elongated spermatids in the later spermatogenesis .
our results suggest that exposure to b[a]p did not significantly affect remodeling of spermatids during spermiogenesis , which produces the mature streamlined spermatid form .
histological assessment revealed that b[a]p exposure at 50  mg / kg / day and 100  mg / kg / day for 30 days had a significant impact on the seminiferous tubules : reduced width of the adluminal of the seminiferous tubules and the size of the sertoli cell basal membrane , lost integrity of cellular membranes and the atrophy of seminiferous tubules .
that ran parallel with a reduction in the weight and size of testes of mice exposed to b[a]p at 100  mg / kg / day .
the basal compartment of seminiferous tubules is occupied by spermatogonia and early spermatocytes and forms a niche for the developing spermatogonia .
the proliferation and differentiation of spermatogonia up to meiosis occurred within the basal compartment of the seminiferous tubules .
a reduction in the basal membrane of sertoli cells may impact spermatogonial proliferation and survival .
also , bap could cause cellular changes and affect cell signaling pathways of sertoli cells , which involve regulation of growth and differentiation of spermatogenic cells .
another possible mechanism related to the reduction of germ cells could be associated with the destruction of testosterone production in leygid cells . in mammalian male reproduction ,
leygid cells are the major source of testosterone , which is a critical hormone for the initiation and maintenance of spermatogenesis .
b[a]p metabolites have been detected upon accumulation in the male reproductive system , especially in the testis and epididymis of exposed animals .
accumulation of the metabolites of b[a]p could reduce the number of leydig cells via apoptosis . that may lead to altering intratesticular testosterone production in leydig cells , which causes testicular atrophy and an accompanying decrease in the number of germ cells [ 5 , 14 ] .
based on the histological assessment , leydig cells appeared to have increased dna strand breaks ( data not shown ) .
testosterone levels from mice exposed to b[a]p at 50  mg / kg / day and 100  mg / kg / day decreased , which was in line with increased percentages of apoptosis in spermatogonia and pachytene spermatocytes and decreased levels of the germ cells .
b[a]p promotes apoptotic cell death of four types of germ cells . with doses of b[a]p at 1  mg / kg / day and 10  mg / kg / day ,
the apoptotic responses of spermatogonia , pachytene spermatocytes , and round spermatids remained stable , while elongated spermatids with apoptotic responses increased .
although the percentages of apoptotic elongated spermatids were not significant higher than those in other germ cells , the elongated spermatids from mice exposed to 100  mg / kg / day had a more significant increase in apoptosis than other germ cells . during the normal spermatogenesis cycle , elongated spermatids have been known to rarely undergo apoptosis .
our results suggest that at the b[a]p doses for 30-day exposure , spermatogonia and spermatocytes with b[a]p - induced dna damage underwent apoptotic cell death rather than repair . then , during the course of exposure , dna damage was passed on and accumulated in elongated spermatids .
similar results were observed in van loon 's study , showing increased dna breaks in the elongated spermatids after radiation exposure .
the increased dna damage might be due to a cumulative effect in the absence of repair .
the repair mechanisms , for example , nucleotide excision repair [ 16 , 17 ] , of spermatogonia seemingly failed to overcome the insults from b[a]p exposure and led to a reduction in the proliferation of pachytene spermatocytes .
in addition , the high dose of 100  mg / kg / day could damage the blood - testis barrier ( btb ) and directly insult the elongated spermatids .
germ cells during spermatogenesis were susceptible to being insulted by 100  mg / kg / day of b[a]p exposure for 30 days . in the testis ,
junctional proteins of the sertoli cells participated in forming btb , which divides the seminiferous epithelium into basal and adluminal compartments . increased levels of b[a]p - dna adducts were detected in germ cells of mice exposed to 50  mg / kg / day and 100  mg / kg / day ( data not shown ) .
our results suggest that b[a]p at 50  mg / kg / day and 100  mg / kg / day could compromise the function of btb for entry into germ cells , while directly reaching germ cells in the spermatogonia and early spermatocytes .
b[a]p can undergo a futile redox cycle in the presence of nadph to generate ros , which could directly react with germ cells and cause damage to their dna .
spermatozoa production in the testis is a regulated balance between germ cell proliferation and germ cell loss .
mice exposed to b[a]p experienced an increase in germ cell loss and a decrease in their proliferation .
the reduction in germ cell levels appeared to be correlated with depletion of spermatozoa levels in the epididymis and vas deferens .
also , the percentage of apoptosis in germ cells was positively correlated with dna damage in spermatozoa .
low concentrations of spermatozoa with poor morphology were more likely to show high levels of tunel positivity .
our data simply reflect that b[a]p could decrease the proliferation of germ cells that eventually leads to alteration in spermatozoa production and motility in the later stages of spermatogenesis ( figure 5 ) .
we observed that the apoptosis of germ cells appeared to be associated with the depletion of spermatozoa concentrations in the epididymis and vas deferens .
our present study demonstrated that mice exposed to b[a]p at or greater than 50  mg / kg / day for 30 days result in pathological changes such as decreasing spermatogonial cell proliferation and modulating germ cell apoptosis during spermatocytogenesis , the early stage of spermatogenesis .
the results suggest that b[a]p affects the apoptotic process during the early stage of spermatogenesis and leads to decreased spermatozoa quality during sperm maturation .
exposure to b[a]p at higher than 50  mg / kg / day for 30 days influenced germ cell quality by decreasing their concentrations and increasing apoptotic response .
b[a]p can impact germ cells during spermatogenesis , which could lead to decreased proliferation of spermatids and mature spermatozoa .
 the objective of this study was to assess the toxicological effect of exposure to benzo(a)pyrene , b[a]p , on germ cells during spermatogenesis . 
 mice were exposed to b[a]p at 1 , 10 , 50 , and 100  mg / kg / day for 30 days via oral ingestion . 
 germ cells , including spermatogonia , spermatocytes , pachytene spermatocytes , and round spermatids , were recovered from testes of mice exposed to b[a]p , while mature spermatozoa were isolated from vas deferens . 
 reproductive organs were collected and weighed . 
 apoptotic response of germ cells and mature spermatozoa were qualified using the terminal deoxynucleotidyl transferase mediated deoxy - utp nick end labeling ( tunel ) assay . 
 b[a]p exposure at 10  mg / kg / day for 30 days did not significantly alter concentrations of germ cells and mature spermatozoa and apoptotic response in germ cells and mature spermatozoa . 
 exposure to b[a]p at 50 and 100  mg / kg / day induced testicular atrophy and yielded a significant reduction in the concentrations of spermatogonia , spermatocytes , pachytene spermatocytes , and round spermatid cells as compared with the control . also , mature spermatozoa experienced decreased concentrations and viability . 
 b[a]p - exposed mice experienced a significant increase in apoptotic germ cells as compared to the control mice . 
 however , the mice dose concentrations were not relevant for comparison to human exposure . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion