Protective effect of lycopene on testicular toxicity induced by Benzo[a]pyrene intake in rats

Journal Pre-proof Protective effect of lycopene on testicular toxicity induced by Benzo[a]pyrene intake in rats Anran Xu, Jianye Wang, Haiyu Wang, Yanqing Sun, Tianyu Hao

PII:

S0300-483X(19)30258-6

DOI:

https://doi.org/10.1016/j.tox.2019.152301

Reference:

TOX 152301

To appear in:

Toxicology

Received Date:

19 March 2019

Revised Date:

13 August 2019

Accepted Date:

26 September 2019

Please cite this article as: Xu A, Wang J, Wang H, Sun Y, Hao T, Protective effect of lycopene on testicular toxicity induced by Benzo[a]pyrene intake in rats, Toxicology (2019), doi: https://doi.org/10.1016/j.tox.2019.152301

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Protective effect of lycopene on testicular toxicity induced by Benzo[a]pyrene intake in rats

Anran Xu1, Jianye Wang2, Haiyu Wang1, Yanqing Sun1, Tianyu Hao1*

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Reproductive Medicine Center, the 960th Hospital of the PLA Joint Logistic Support

Force, Jinan 250031, Shandong, China Reproductive Medicine Center, the Seventh Medical Center of PLA General Hospital,

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Beijing 100700, China

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Corresponding author

Tianyu Hao

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Email: [email protected]

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Force, Jinan 250031, Shandong, China

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Reproductive Medicine Center, the 960th Hospital of the PLA Joint Logistic Support

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Running title: Protective effect of lycopene on testicular toxicity

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Abstract Benzo[a]pyrene (BaP) stimulates male reproductive toxicity. In this study, we want to examine the ameliorative potential of Lycopene (LYC) on BaP-induced testicular toxicity. Adult male Wistar rats were segregated into 5 groups: Control, LYC, BaP, BaP+LYC and BaP+PQ7. Sperm parameters, testosterone level, oxidant and antioxidant parameters were determined. MRNA and protein abundances of key genes were analyzed. Cell death and apoptosis were assessed by trypan blue exclusion and

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Annexin V-FITC staining assay, respectively. LYC inhibited BaP-caused decrease in sperm motility and epididymal sperm concentration, and increase in head, tail and total

abnormal sperm rate. LYC inhibited BaP-caused decrease in testosterone level in serum

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and intratesticular fluids. LYC protected germ cells from BaP-caused oxidative stress. LYC also prevented BaP-caused germ cell death and apoptosis by inhibiting apoptotic

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pathway. Besides, LYC ameliorated BaP-mediated gap-junction dysfunction of sertoli

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cells, as shown by the inhibited sertoli cell death and apoptosis, the upregulation of Bcl2 and Cx43, the downregulation of Cleaved Caspase 3, Bax and CaM, and the decrease in Ca2+ level. LYC ameliorated BaP-caused testicular damage via inhibiting

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oxidative stress and apoptosis, and relieving the gap-junction dysfunction of sertoli cells.

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Keywords: Benzo[a]pyrene, Lycopene, testicular toxicity, sertoli cells, gap junctions

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Introduction Benzo[a]pyrene (BaP) belongs to polycyclic aromatic hydrocarbons family and is a ubiquitous environmental pollutant which may be found in coal tar, automobile exhaust fumes, cigarette smoke and charbroiled food (Chung et al. 2011; Li et al. 2007). Men are usually exposed to BaP because of smoking and BaP has deleterious activities against male reproductive health (Chung et al. 2011; Jeng and Bocca 2013). Hence, the mechanisms underlying BaP-induced testicular toxicity need to be further investigated.

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Lycopene (LYC, C40H56) is a natural non-provitamin A carotenoid present in most red-colored fruits and vegetables, especially in tomatoes (Durairajanayagam et al. 2014; Krishnamoorthy et al. 2013). LYC is an unsaturated linear carotenoid with 11

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conjugated double bonds and possesses anti-oxidative activity (Tvrda et al. 2016). LYC has protective activities against testicular toxicity (Atessahin et al. 2006; Turk et al.

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2007), which makes it a probable therapy option for male reproductive dysfunction.

dysfunction are not clear.

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However, the mechanisms by which LYC protects against male reproductive

Aberrant spermatogenesis and decreased sperm counts will result in male infertility.

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Sertoli cells are the most important cells for offering physical and nutritional support to germ cells during spermatogenesis (Guerrero-Bosagna et al. 2013). Sertoli cells can be

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injured by various environmental toxicants, which subsequently cause defects in

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spermatogenesis (Krishnamoorthy et al. 2013). Moreover, recent evidence shows that sertoli cells is damaged by BaP treatment through breaking down gap junctions (Ji et al. 2018). Gap junctions are involved in various biological processes, such as cell development and differentiation, metabolic support, cell synchronization and so on (Gilleron et al. 2006; Ji et al. 2018). Moreover, gap junctions between sertoli cells in testes play important roles in spermatogenesis (Aravindakshan and Cyr 2005; Li et al. 3

2016; Pointis et al. 2011). On the other hand, LYC is helpful for gap junctional communication (Durairajanayagam et al. 2014; Watanabe et al. 2010). We hypothesized that LYC protected rats against BaP-induced testicular toxicity. To test the above hypothesis, BaP-exposed rats were established through sixty-day continuous oral gavage of BaP. In addition, the relationship between LYC, BaP-caused testicular toxicity and gap junction dysfunction of sertoli cells is also investigated.

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Methods & materials Animals and drug administration

All animal experiments in our study were reviewed by Animal Ethical Committee of

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the 960th Hospital of the PLA Joint Logistic Support Force. Sixty adult male Wistar

rats (180-200 g, from Nanjing Model Animal Institute, Nanjing, China) were equally

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segregated into 5 groups:

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Control group: Corn oil 0.2 ml, oral gavage, once a day

LYC group: LYC (4 mg/kg of body weight), oral gavage, once a day BaP group: BaP (5 mg/kg of body weight), oral gavage, once a day

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BaP+LYC group: BaP (5 mg/kg of body weight) plus LYC (4 mg/kg of body weight), oral gavage, once a day

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BaP+PQ7 group: BaP (5 mg/kg of body weight, oral gavage) along with PQ7 (10 mg/kg

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of body weight, IP), once a day After 60 days, rats were killed and blood and testes were collected. Sertoli cell isolation Rats of different group were killed and testes were harvested. Tissues from decapsulated testes were kept in HBSS solution containing 0.002% soybean trypsin inhibitor and 0.1% collagenase (Sigma, St. Louis, MO) for 8 min. Seminiferous tubules 4

were collected and digested by collagenase for ten min at 37°C. Isolated sertoli cell clusters were forced through 20 G needles and then subjected to 20 mM Tris (pH 7.4) for 4 min to lyse germ cells. Isolation of germ cells Testes were incubated in HBSS supplemented with 5 mM glucose. Seminiferous tubules were subjected to 15-minute-incubation with collagenase at 34 °C. After being washed tubules were digested with 1 mg/ml trypsin and 0.04 mg/ml DNaseI. The cell

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suspension was forced through a 65 μm Nitex mesh and isolated germ cells were washed and resuspended in PBS. Epididymal sperm concentration

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Freshly isolated right epididymis was minced, squashed and incubated in 2 ml isotonic

saline for 3 h. The diluted sperm suspension (10 μl) was transferred to the Improved

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Neubauer hemocytometer and the settled sperms were counted under a light microscope

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(× 400). Measurement of testosterone content

Serum and intratesticular testosterone levels were measured by ELISA kits (R&D

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System, Minneapolis, MN, USA) according to manufacturer's instructions. Sperm motility and morphology

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Sperm motility was analyzed through examining 5 μl of sperm suspensions placed on

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a clean microscope slide at 37°C. The sample was covered with a coverslip, and immediately, we analyzed 100 sperms per sample, with × 400 light microscope magnification. Each spermatozoa was classified into 4 groups: rapid linear progressive motility, slow or sluggish linear or non-linear motility, nonprogressive motility or immotile sperm. Sperms in the first 3 groups were further classified as motile sperm. The number of motile sperm was counted and then divided by 100. 5

The sperm suspension (1 µl) was incubated with eosin-nigrosin. Sperm morphology analysis was performed by examining the smear under light microscope (× 400) and the abnormal sperm was classified according to the previous report (Wyrobek and Bruce 1975) Biochemical assay Reactive oxygen species (ROS) levels were evaluated according to the method previously reported (Banerjee et al. 2016a). Reduced (GSH) and oxidized glutathione

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(GSSG) were analyzed at 350 nm and 412 nm on the spectrophotometer through ophthalaldehyde assay (Sa-Nakanishi et al. 2018). Glutathione peroxidase (GPx) activity was assayed using Kits from Cayman (MI, USA). The levels of thiobarbituric acid

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reactive substances (TBARS) and malondialdehyde (MDA) and the activities of

BioLabs (San Diego, USA).

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superoxide dismutase (SOD) and catalase (CAT) were assayed using kits from Cell

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The measurement of intracellular Ca2+ level

Sertoli cells of different groups were subjected to one-hour-incubation with 10 μM Fluo4/AM (Sigma, St. Louis, MO) at 37°C and washed by PBS. Subsequently, intracellular

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Ca2+ levels were analyzed by FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

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Western blot

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Primary antibodies against Caspase 3, Cleaved Caspase 3, Bax, Bcl2 and Cytosolic Cytochrome c, Cx43 and CaM were bought from Abcam (Cambridge, England). Protein lysates were prepared extracted from germ cells or sertoli cells from different groups. After being quantified, equal protein samples were separated on SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk

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in TBST at 4℃ overnight, and then probed with the above primary antibodies and subjected to one-hour-incubation with secondary antibodies. Reverse Transcriptase PCR (RT-PCR) Total cellular RNA was extracted by Trizol (Invitrogen, Waltham, MA USA) and transcribed into cDNA by Omniscript RT Kit (Qiagen, Valencia, CA, USA). Primers used in this study were listed below: Bcl2-fwd: 5'-CATGCGACCTCTGTTTGATTC-3'

Bax-fwd: 5'-GGCAGACAGTGACCATCTTT-3' Bax-rev: 5'-CCAAAGTGGACCTGAGGTTTAT-3'

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β-Actin-fwd: 5'-GGAGATTACTGCCCTGGCTCCTA-3'

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Bcl2-rev: 5'-GAATGTGTGTGTGTGTGTGTG-3'

β-Actin-rev: 5'-GACTCATCGTACTCCTGCTTGCTG-3'

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Histological evaluation

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Testicular tissues were fixed in formalin, blocked in paraffin and sliced into 5-micron sections. Subsequently, sections were stained with Hematoxylin-eosin (H/E) and examined under light microscope to analyze histological changes.

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Trypan blue exclusion assay

Germ cells of different groups were dyed mixed with trypan blue (Beyotime

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Biotechnology, Beijing, China) for 3 min. Unstained cells were considered as viable

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cells. Dead cells were counted through cell counting chamber. Cell apoptosis assay Annexin V-FITC Apoptosis Detection Kits were bought from Sangon Biotech. Cells were dyed with 5 μl Annexin V-FITC for 15 min and 10 μl PI for 10 min in dark at room temperature. Subsequently, the staining was analyzed detected by flow cytometer (BD Biosciences). 7

Statistics analysis Data were expressed as means ± SEM. One-way ANOVA was followed by Dunnett multiple comparison test and differences with P<0.05 were regarded statistically significant.

Results LYC reversed BaP-induced changes of epididymal sperm characteristics

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Table 1 showed that LYC did not affect sperm motility and epididymal sperm

concentration compared with control, whereas LYC treatment caused the decrease in head (P<0.05), tail (P<0.01) and total abnormal sperm rate (P<0.01). BaP treatment

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caused the decrease in sperm motility and epididymal sperm concentration, and the

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increase in head, tail and total abnormal sperm rate compared with control (Table 1, P<0.001). In contrast, LYC reversed BaP-induced changes of epididymal sperm

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characteristics, as shown by the increase in sperm motility and epididymal sperm concentration, and decrease in head, tail and total abnormal sperm rate (Table 1,

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P<0.001).

LYC inhibited BaP-caused decrease in testosterone level in serum and

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intratesticular fluids

LYC alone had little effect on testosterone level, whereas BaP inhibited testosterone

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production in serum and intratesticular fluid compared to control (Fig. 1A and 1B, P<0.001). However, LYC co-treatment inhibited BaP-caused decrease in testosterone level (Fig. 1A and 1B, P<0.001). LYC protected germ cells from BaP-caused oxidative stress As shown in Fig 2, LYC did not significantly affect the oxidant and antioxidant parameters in testes compared to control. BaP caused the increase in the levels of ROS 8

(Fig. 2A, P<0.001), MDA (Fig. 2B, P<0.001) and TBARS (Fig. 2D, P<0.01), and the decrease in GSH/GSSG ratio (Fig. 2C, P<0.001), GPx activity (Fig. 2E, P<0.001), CAT activity (Fig. 2F, P<0.001) and SOD activity (Fig. 2G, P<0.001) in testes compared to control or LYC. However, LYC administration to BaP-treated rats abolished the above phenomena, as shown by the decrease in the levels of ROS (Fig. 2A, P<0.001), MDA (Fig. 2B, P<0.001) and TBARS (Fig. 2D, P<0.001), and increase in GSH/GSSG ratio (Fig. 2C, P<0.001), GPx activity (Fig. 2E, P<0.001), CAT activity (Fig. 2F, P<0.001)

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and SOD activity (Fig. 2G, P<0.001). LYC prevented BaP-caused germ cell death and apoptosis

Isolated testicular germ cells were used to test whether LYC had protective effect on

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BaP-caused cell death and apoptosis. Fig. 3A showed that germ cell death percentage in BaP group increased to almost 600% of that in control or LYC groups (P<0.001).

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However, LYC co-administration abolished BaP-induced above phenomena (Fig. 3A,

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P<0.01). Apoptosis assay found that LYC inhibited BaP-induced germ cell apoptosis (Fig. 3B). Besides, BaP caused the decrease in tubular sperm cell load, the shedding of germ cells and tubular disorder, whereas LYC co-administration alleviated the above

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symptoms (Fig. 3C).

LYC prevented BaP-caused apoptotic pathway activation in germ cells

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BaP stimulated the expression of Cleaved Caspase 3, Bax and Cytosolic Cytochrome c,

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inhibited Bcl2 expression and nearly had no effect on Caspase 3 expression compared to control or LYC (Fig. 4A). In contrast, BaP+LYC group had lower levels of Cleaved Caspase 3, Bax and Cytosolic Cytochrome c and higher level of Bcl2 than BaP group (Fig. 4A). In addition, RT-PCR assay proved that LYC inhibited BaP-caused Bax upregulation and Bcl2 downregulation (Fig. 4B). Besides, Fig. 4C showed that Bax/Bcl2 ratio in BaP group was increased compared to that in control group (P<0.001), 9

whereas LYC suppressed the above phenomenon (P<0.01). LYC ameliorated BaP-mediated gap-junction dysfunction of sertoli cells BaP stimulated sertoli cell apoptosis compared to control or LYC, whereas LYC inhibited BaP-induced cell apoptosis (Fig. 5A). Moreover, BaP stimulated Cleaved Caspase 3 and Bax upregulation and inhibited Bcl2 expression in sertoli cells compared to control or LYC, whereas LYC treatment along with BaP inhibited Cleaved Caspase 3 and Bax expression and stimulated Bcl2 expression compared to BaP alone (Fig. 5B).

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Next, the relationship between LYC, BaP and gap junctions of sertoli cells was examined. Fig. 5C showed that LYC inhibited BaP-induced the increase in intracellular

Ca2+ level. Moreover, Fig. 5D showed that BaP inhibited Cx43 expression and

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stimulated CaM expression in sertoli cells, whereas LYC or PQ7 co-treatment inhibited the above phenomena. Besides, Fig. 5E demonstrated that germ cell death percentage

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in BaP group was increased compared to that in control or LYC groups (P<0.001),

Discussion

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and P<0.01, respectively).

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whereas it in BaP+LYC or BaP+PQ7 groups was lower than in BaP group (P<0.001

BaP exposure is a common public health problem and identifying potential treatments

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for BaP-induced reproductive toxicity is very important. Previous studies found that

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LYC had protective effects on testes exposed to pollutants, such as polychlorinated biphenyl (Ateşşahin A et al. 2006) and bisphenol A (Tamilselvan et al. 2013). In current study, we found that LYC reversed BaP-induced changes of epididymal sperm characteristics, as shown by the significant increase in sperm motility and epididymal sperm concentration, and decrease in the production of abnormal sperms. Testosterone, the most important male sex hormone, is secreted by testes in males and plays important 10

roles in the development of testes as well as spermatogenesis (Zhao et al. 2018). Hence, testosterone levels were examined in serum and intratesticular fluids of different groups and the results demonstrated that BaP caused the significant decrease in testosterone levels in serum and intratesticular fluids, suggesting that BaP resulted in testicular damage. However, LYC inhibited BaP-induced decrease in testosterone levels. BaP-induced reproductive dysfunctions are highly associated with the oxidative stress (Adedara et al. 2015). Oxidative stress is a state of imbalance between ROS generation

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and the ability to degrade those radicals (Tvrda et al. 2016). Moreover, elevated ROS abundance is proved to be linked with male reproductive dysfunction (Desai et al. 2009). On the other hand, LYC has been proved to have strong antioxidant properties

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(Durairajanayagam et al. 2014; Krishnamoorthy et al. 2013). Moreover, previous studies suggested that LYC supplementation may be used as a powerful therapy for

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male infertility because of its antioxidant properties. For example, LYC inhibited toxic

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effects of polychlorinated biphenyls on sertoli cells via the antioxidant mechanism (Krishnamoorthy et al. 2013). LYC was capable of protecting testes against di-(2ethylhexyl) phthalate-induced oxidative injury (Bahrami et al. 2018). LYC

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administration prevented ferrous ascorbate-caused oxidative injury in bovine spermatozoa (Tvrda et al. 2016). Hence, oxidant and antioxidant parameters in testes of

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different groups were evaluated. The results showed that LYC abolished BaP-caused

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oxidative stress, as shown by the decreased abundances of ROS, MDA and TBARS, increased GSH/GSSG ratio and the enhancement of GPx, CAT and SOD activities, which suggested that LYC may prevent BaP-caused testicular toxicity via an antioxidant mechanism. BaP exposure may induce cell apoptosis in testes (Banerjee et al. 2016b). Spermatogenesis involves germ cell proliferation and differentiation (Ji et al. 2018; 11

Lizama et al. 2009). To test whether the protective effects of LYC is linked with the inhibition of cell death and apoptosis in germ cells, trypan blue exclusion assay and Annexin V-FITC staining assay were conducted. The result showed that LYC cotreatment protected germ cells against BaP-mediated cell death and apoptosis. Moreover, we found that LYC inhibited BaP-mediated the activation of apoptotic pathway, as shown by the downregulation of Cleaved Caspase 3, Bax and Cytosolic Cytochrome c and the upregulation of anti-apoptotic gene Bcl-2 in germ cells.

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In our study, H&E staining assay of paraffin-embedded testes sections was also conducted. Our results showed that LYC alleviated BaP-mediated the decline in tubular sperm cell load, the shedding of germ cells and tubular disorder. Sertoli cells can offer

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physical and nutritional support to germ cells and form blood-testis barriers by tight

junctions (Guerrero-Bosagna et al. 2013; Liu et al. 2018). Structural alterations of

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sertoli cells may result in germ cell apoptosis (Liu et al. 2018). Hence, we hypothesized

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that BaP may also impair sertoli cells’ ability to support the development of germ cells, which subsequently result in injured spermatogenesis. To test this hypothesis, sertoli cell apoptosis in different groups was examined by flow

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cytometry analysis. The results showed LYC co-treatment abolished BaP caused sertoli cell apoptosis. Moreover, LYC administration inhibited BaP-induced the upregulation

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of Cleaved Caspase 3 and Bax and the downregulation of Bcl2. On the other hand, gap

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junction functions of sertoli cells have significant role in spermatogenesis (Aravindakshan and Cyr 2005; Li et al. 2016; Pointis et al. 2011). Cx43, also known as GJA1, is a predominant testicular connexin and Cx43-based gap junction functions in sertoli cells can not be substituted by other connexins (Giese et al. 2012; Li et al. 2016). Besides, studies demonstrate that gap junctions may be modulated by various factors, including intracellular Ca2+ level, CaM, etc (Chipman et al. 2003; Zou et al. 2014). In 12

this study, we found that LYC co-treatment partially alleviated BaP-caused the increase in intracellular Ca2+ level, CaM upregulation and Cx43 downregulation. PQ7, a gap junction enhancer (Shishido et al. 2013), was used in our study and we found that PQ7 co-treatment recovered gap-junction dysfunction in sertoli cells, as shown by the increase in Cx43 protein level and the decrease in CaM protein level. Additionally, trypan blue exclusion assay showed that LYC or PQ7 administration partially suppressed BaP-mediated the elevation in germ cell death percentage. Hence, the above

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results demonstrated that LYC treatment partially ameliorated BaP-induced the gapjunction dysfunction of sertoli cells.

In summary, the current study demonstrated that LYC partially ameliorated BaP-

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induced testicular toxicity via inhibiting oxidative stress, suppressing the apoptosis of germ cells and sertoli cells, and relieving the gap-junction dysfunction of sertoli cells.

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This study provided a further understanding of the mechanisms underlying BaP-

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induced testicular toxicity and supported the use of LYC as a powerful therapeutic compound for the treatment of BaP-induced male reproductive dysfunction. In addition, our finding also suggested that gap junctions may be potential therapeutic targets for

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the treatment of BaP-induced testicular toxicity.

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Conflict of interest

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The authors declare that they have no conflict of interest.

Acknowledgements None.

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efficiency of lycopene on oxidative stress - induced damage in bovine spermatozoa. Journal of animal science and biotechnology 7, 50. Watanabe, H., Okawara, S., Bhuiyan, M. and Fukui, Y. 2010. Effect of lycopene on cytoplasmic maturation

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of porcine oocytes in vitro. Reproduction in domestic animals = Zuchthygiene 45, 838-845. Wyrobek, A.J. and Bruce, W.R. 1975. Chemical induction of sperm abnormalities in mice. Proceedings of the National Academy of Sciences of the United States of America 72, 4425-4429. Zhao, J., Ren, S., C., L., L., H., Z., L. and L., Z. 2018. Di-(2-Ethylhexyl) Phthalate Increases Obesity-Induced Damage to the Male Reproductive System in Mice. Oxid Med Cell Longev. 2018, 1861984. Zou, J., Salarian, M., Chen, Y., Veenstra, R., Louis, C.F. and Yang, J.J. 2014. Gap junction regulation by calmodulin. FEBS letters 588, 1430-1438.

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Figure legends

Fig. 1. Effect of treatment with LYC on testosterone level in serum (A) and intratesticular fluids (B). n = 12. *** P<0.001. One-way ANOVA followed by Dunnett multiple comparison test.

Fig 2. LYC protected germ cells from BaP-mediated oxidative stress. (A-D)

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Graphical representation of ROS level (A), MDA level (B), GSH/GSSG ratio (C) and TBARS level (D). (E-G) Graphical representation of testicular GPx activity (E), CAT

activity (F) and SOD activity (G). n = 12. **P<0.01, ***P<0.001, One-way ANOVA

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followed by Dunnett multiple comparison test.

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Fig 3. LYC prevented BaP-caused germ cell death and apoptosis. (A) Trypan blue

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exclusion assay of germ cell death percentage. (B) Annexin V-FITC staining assay of germ cell apoptosis. (C) H&E staining of paraffin embedded testis sections were visualized at 200× magnification. n = 12. **P<0.01, ***P<0.001. One-way ANOVA

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followed by Dunnett multiple comparison test.

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Fig 4. LYC prevented BaP-caused apoptotic pathway activation in germ cells. (A)

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Western blot of Caspase 3, Cleaved Caspase 3, Bax, Bcl2 and Cytosolic Cytochrome c. (B) RT-PCR of Bax and Bcl2. (C) Expression level of Bax/Bcl2 ratio, n = 3. **P<0.01, ***P<0.001. One-way ANOVA was followed by Dunnett multiple comparison test.

Fig 5. LYC ameliorated BaP-mediated gap-junction dysfunction of sertoli cells. (A) Flow cytometry analysis of sertoli cell apoptosis. Data for apoptotic cells were from Q2 16

+ Q3 (Annexin V-FITC positive). (B) Western blot of apoptosis-related genes. (C) Ca2+ levels in sertoli cells were detected by flow cytometry. (D) Western blot of Cx43 and CaM in sertoli cells. (E) Trypan blue exclusion assay of germ cell death percentage. n = 12. **P<0.01, ***P<0.001. One-way ANOVA followed by Dunnett multiple

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Table 1. Effects of LYC on BaP-mediated changes of epididymal sperm characteristics. parameters Groups

Sperm motility (%)

Epididymal sperm concentration (million/g tissue)

Abnormal sperm rate (%) Head

Tail

Total

75.61±3.01

316.52±6.82

2.41±0.41

3.83±0.69

6.24±1.98

LYC

84.33±1.69

323.63±9.7

1.92±0.56*

1.73±0.62**

3.65±0.95**

BaP

22.72±4.96***

46.75±7.1***

37.89±0.63***

46.52±0.89***

84.45±0.78***

BaP+LYC

81.41±4.51###

320.34±8.6###

3.93±0.36###

4.58±0.93###

8.51±0.95###

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Control

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