Effects of chronic cigarette smoke exposure and alcohol exposure on testicular histopathology, morphometry, and gene expression in adult male rats

Document Type : Research Paper

Authors

1 Department of Human Anatomy and Histology, Medical College, University of Babylon, Iraq.

2 Department of Human Anatomy and Histology, Medical College, University of Babylon, Iraq

Abstract

Objective
Cigarette smoking and alcohol are two important lifestyle factors that affect male reproductive health. However, the effects of individual and combined chronic exposure to cigarette smoke and alcohol on testicular architecture, selected gene expression profiles, and spermatogenesis remain incompletely understood. The aim of this study was to investigate the chronic effects of cigarette smoke and alcohol on histological, morphometrical and molecular status in testicular tissue in adult male rats.
Materials and methods
Forty adult male Sprague-Dawley rats were randomly divided in four groups (n = 10 per group). These groups were control, cigarette smoke-exposed, alcohol-exposed, and combined cigarette smoke- and alcohol-exposed groups. The exposure period was 8 weeks. Testicular histopathological and morphometric changes were evaluated. Moreover, the expression of genes related to oxidative stress (Nrf2 and HO-1), apoptosis (Bax and Bcl-2), steroidogenesis (StAR and CYP11A1), and spermatogenesis (DAZL and SYCP3) was assessed using quantitative real-time PCR (qRT-PCR).
Results
The results showed that exposure to cigarette smoke, alcohol or their combination significantly reduced the expression of Nrf2, HO-1, StAR, CYP11A1, DAZL, Bcl-2 and SYCP3. While, it increased the expression of Bax (P < 0.05). These molecular changes were accompanied by significant histopathological and morphometric changes. For example, it caused a decrease in the diameter of the seminiferous tubules, a decrease in the thickness of the germinal epithelium, a decrease in the density of spermatogenic cells and a decrease in the density of Leydig cells. The most pronounced changes were observed in the combined exposure group.
Conclusions
Chronic cigarette smoking and alcohol drinking negatively impact the structure and function of the testes through mechanisms of oxidative stress, apoptosis, inhibition of steroidogenesis, and disordered spermatogenesis. The combined exposure generates amplifying damage which far exceeds what each factor causes in isolation, suggesting that such lifestyle habits may represent major male infertility risk factors together.

Keywords

References

Agarwal, A., Virk, G., Ong, C., & du Plessis, S. S. (2014). Effect of oxidative stress on male reproduction. The World Journal of Men’s Health, 32(1), 1-17. https://doi.org/10.5534/wjmh.2014.32.1.1
Aitken, R. J. (2020). Oxidative stress and male reproductive health. Reproduction, 159(4), R143-R156. https://doi.org/10.1530/REP-19-0452
Aitken, R. J., & Roman, S. D. (2008). Antioxidant systems and oxidative stress in the testes. Oxidative Medicine and Cellular Longevity, 1(1), 15-24. https://doi.org/10.4161/oxim.1.1.6843
Alavi, M., Mozafari, M. R., Ghaemi, S., Ashengroph, M., Hasanzadeh Davarani, F., & Mohammadabadi, M. (2022). Interaction of epigallocatechin gallate and quercetin with spike glycoprotein (S-glycoprotein) of SARS-CoV-2: In silico study. Biomedicines, 10(12), Article e3074. https://doi.org/10.3390/biomedicines10123074
Arabpour, Z., Mohammadabadi, M., & Khezri, A. (2021). The expression pattern of p32 gene in femur, humeral muscle, back muscle and back fat tissues of Kermani lambs. Agricultural Biotechnology Journal, 13(4), 183-200. https://doi.org/10.22103/jab.2022.18782.1371
Creasy, D. M. (2001). Pathogenesis of male reproductive toxicity. Toxicologic Pathology, 29(1), 64-76. https://doi.org/10.1080/019262301301418865
Emanuele, M. A., & Emanuele, N. V. (2001). Alcohol and the male reproductive system. Alcohol Research & Health, 25(4), 282-287. https://pmc.ncbi.nlm.nih.gov/articles/PMC6705705/
Guo, S., Zhang, Y., Fei, C., Liu, X., Xia, W., Luo, M., Wei, G., Qin, W., Xiong, C., Li, H., Yin, Y., He, X., & Zhou, L.-Q. (2025). Deciphering meiotic chromatin organization by SYCP3. Nucleic Acids Research, 53(11), Article gkaf460. https://doi.org/10.1093/nar/gkaf460
Harlev, A., Agarwal, A., Gunes, S. O., Shetty, A., & du Plessis, S. S. (2015). Smoking and male infertility: An evidence-based review. The World Journal of Men’s Health, 33(3), 143-160. https://doi.org/10.5534/wjmh.2015.33.3.143
Jo, Y., & Stocco, D. M. (2004). Regulation of steroidogenesis and steroidogenic acute regulatory protein in R2C cells by DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene-1). Endocrinology, 145(12), 5629-5637. https://doi.org/10.1210/en.2004-0941
Kazemipour, E., Sasan, H., & Mohammadabadi, M. (2025). The effect of the intrinsic resistance of Shigella flexneri 2a to spectinomycin on the efficiency of the CRISPR/Cas9 system. Agricultural Biotechnology Journal, 17(3), 177-200. https://doi.org/10.22103/jab.2025.25382.1717
Li, H., Liang, Z., Yang, J., Wang, D., Wang, H., Zhu, M., Geng, B., & Xu, E. Y. (2019). DAZL is a master translational regulator of murine spermatogenesis. National Science Review, 6(3), 455-468. https://doi.org/10.1093/nsr/nwy163
Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods, 25(4), 402-408. https://doi.org/10.1006/meth.2001.1262
Oremosu, A. A., & Akang, E. N. (2015). Impact of alcohol on male reproductive hormones, oxidative stress and semen parameters in Sprague-Dawley rats. Middle East Fertility Society Journal, 20(2), 114-118. https://doi.org/10.1016/j.mefs.2014.07.001
Sharma, R., Harlev, A., Agarwal, A., & Esteves, S. C. (2016). Cigarette smoking and semen quality: A new meta-analysis examining the effect of the 2010 World Health Organization laboratory methods for the examination of human semen. European Urology, 70(4), 635-645. https://doi.org/10.1016/j.eururo.2016.04.010
Shin, E. Y., Park, S., Choi, W. Y., & Lee, D. R. (2021). Rapid differentiation of human embryonic stem cells into testosterone-producing Leydig cell-like cells in vitro. Tissue Engineering and Regenerative Medicine, 18(4), 651-662. https://doi.org/10.1007/s13770-021-00359-8
Stocco, D. M. (2007). The role of StAR in Leydig cell steroidogenesis. In A. H. Payne & M. P. Hardy (Eds.), The Leydig cell in health and disease (Contemporary Endocrinology). Humana Press. https://doi.org/10.1007/978-1-59745-453-7_9
Tesarik, J. (2025). Lifestyle and environmental factors affecting male fertility, individual predisposition, prevention, and intervention. International Journal of Molecular Sciences, 26(6), Article 2797. https://doi.org/10.3390/ijms26062797
Xia, K., Luo, P., Yu, J., He, S., Dong, L., Gao, F., Chen, X., Ye, Y., Gao, Y., Ma, Y., Yang, C., Zhang, Y., Yang, Q., Han, D., Feng, X., Wan, Z., Cai, H., Ke, Q., Wang, T., ... & Xiang, A. P. (2024). Single-cell RNA sequencing reveals transcriptomic landscape and potential targets for human testicular ageing. Human Reproduction, 39(10), 2189-2209. https://doi.org/10.1093/humrep/deae199
Zhang, D. D., & Hannink, M. (2003). Distinct cysteine residues in Keap1 are required for Keap1-dependent ubiquitination of Nrf2 and for stabilization of Nrf2 by chemopreventive agents and oxidative stress. Molecular and Cellular Biology, 23(22), 8137-8151. https://doi.org/10.1128/MCB.23.22.8137-8151.2003
Zhao, H., Song, L., Ma, N., Liu, C., Dun, Y., Zhou, Z., Yuan, D., & Zhang, C. (2021). The dynamic changes of Nrf2 mediated oxidative stress, DNA damage and base excision repair in testis of rats during aging. Experimental Gerontology, 152, Article 111460. https://doi.org/10.1016/j.exger.2021.111460
Zhu, W. B., Zhao, H. B., Wang, G. Z., Wang, Z. L., & Wang, S. Q. (2025). The effects of testicular aging on Leydig cells and the application of stem cells in restoring Leydig cells function. Reproductive Biology and Endocrinology, 23(1), Article 147. https://doi.org/10.1186/s12958-025-01487-9
Agricultural Biotechnology Journal
Volume 18, Issue 4
August 2026
Pages 575-592

Files

Share

How to cite

Statistics