Assessment of Testis Histopathological Changes and Spermatogenesis in Male Mice Exposed to Chronic Scrotal Heat Stress

J Anim Behav Biometeorol (2020) 8:174-180 ISSN 2318-1265 ORIGINAL ARTICLE Assessment of testis histopathological changes and spermatogenesis in male mice exposed to chronic scrotal heat stress Tung Nguyen Thanh ▪ Phuoc Dang Van ▪ Thuan Dang Cong ▪ Tam Le Minh ▪ Quoc Huy Nguyen Vu T Nguyen Thanh (Corresponding author) P Dang Van Institute of Biomedical Research, Hue University of Medicine Institute of Biomedical Research, Hue University of and Pharmacy, Hue University, 6 Ngo Quyen Street, Hue, Medicine and Pharmacy, Hue University, 6 Ngo Quyen Vietnam. Street, Hue, Vietnam. Department of Histology, Embryology, Pathology and Forensic Medicine Center, Thua Thien Hue Province, Forensic, Hue University of Medicine and Pharmacy, Hue Vietnam. University, 6 Ngo Quyen Street, Hue, Vietnam. email: [email protected]; T Le Minh ▪ QH Nguyen Vu [email protected] Department of Obstetrics and Gynecology, Hue University of Medicine and Pharmacy, Hue University, 6 Ngo Quyen T Dang Cong Street, Hue, Vietnam. Department of Histology, Embryology, Pathology and Forensic, Hue University of Medicine and Pharmacy, Hue University, 6 Ngo Quyen Street, Hue, Vietnam. Received: April 04, 2020 ▪ Accepted: April 20, 2020 ▪ Published Online: April 22, 2020 Abstract Elevation of scrotal temperature may be injurious to system. Johnsen scores system was standardized to assess spermatogenesis and leading cause male infertility. Scrotal murine testicular histopathology in the seminiferous tubule heat stress reduces the number and motility of spermatozoa, cross-section. Collectively, these results indicated a negative fertilization ability of the surviving sperm and poor impact on histopathological alterations and spermatogenesis fertilization-embryo. This study was designed to investigate arrest following chronic scrotal heat stress. the effect of heat stress (at 37 ºC, 40 ºC and 43 ºC) on Keywords: Johnson scoring system, male reproductive histopathological features of testicular tissue in scrotal heat system, mouse model, testicular tissue exposed male mice. Experimental and control groups were sacrificed after completion of five weeks heat exposure Introduction period. The testes were fixed and stained hematoxylin-eosin (H&E) for qualitative and quantitative analysis of The scrotal temperature in most mammals is normally histopathological alterations and spermatogenesis according below the core body temperature by 2 – 8 °C, playing an to Johnson scoring system. The results indicated that mice important role in spermatogenesis that occurs in the testes exposed to heat-stress mice exhibited degenerated and normally. Increases in testicular temperature may be disorganized features of spermatogenic epithelium and detrimental to spermatogenesis and ultimately cause problems reduced spermatogenic cells. Heat stress exposure (40 ºC and in fertility (Shadmehr et al 2018; Lin et al 2015). Elevation of 43 ºC) shows the significantly reduced Johnson score scrotal temperature interrupts spermatogenesis, resulting in compared to the control condition (P < 0.05 and P < 0.001, reduced the number and motility of spermatozoa, fertilization respectively). Meanwhile, scrotal heat exposure at 37 ºC did ability of the surviving sperm and poor fertilization-embryo not reach significantly changes in Johnsen’s testicular (Shadmehr et al 2018; Rasooli et al 2010). High scrotum histopathological score. The seminiferous tubule structure and temperature increases oxidation, resulting in disrupted spermatogenesis were completely disrupted in mice exposed thermoregulation of the testicle such as reduction of to 43 °C. Percentage of high Johnsen score point was antioxidant enzymes production and heat shock protein decreased in mice in heat-stress exposure group, while the production, and an increase in apoptosis and cell death (Kumar ratio of low Johnsen score points was gradually increase. Roy et al 2016). Heat stress reduces the ability to regulate the Spermatogenesis in male mice exposed to chronic scrotal heat temperature of the scrotum, causes oxidative stress, thus stress is disrupted at 43 °C. In conclusion, this study attempted causing cellular reactions including mitochondrial to develop an animal model for studying the male reproductive dysfunction, increase the ROS and reduces the production of doi.org/10.31893/jabb.20023 J Anim Behav Biometeorol (2020) 8:174-180 175 anti-ROS enzymes, reduces the increased apoptotic rate. After completing the heat exposure experiment after 5 weeks, Besides, heat stress acts inhibitory on the structure and all mice were sacrificed under anesthesia. Their testis tissue function of the testes, leading to decrease male fertility was harvested for histological morphometric analysis. (Rasooli et al 2010). Tissue processing and hematoxylin-eosin staining Scrotal heat stress causes a variety of mechanisms that occur in the testes including oxidative stress response, heat At the end of the surgical procedure, the testicular shock response, cell cycle checkpoints, DNA repair and specimens were individually immersed into 4% buffered apoptosis and cell death (Lin et al 2015; Zhang et al 2015). formaldehyde, dehydrated with graded concentrations of ethyl Scrotal heat stress interrupts spermatogenesis in male mice alcohols and embedded tissues into paraffin blocks. Paraffin (Lin et al 2015). Spermatogonial germ cells are also sections of 5 μm thick were obtained transferred onto gelatin- influenced by heat stress, resulting in the elimination and coated slides, deparaffinized with xylene and then rehydrated absence of them in the seminiferous tubules, Sertoli and through a descending series of ethanol and water. Slides were Leydig cells are degenerated (Rasooli et al 2010). stained with hematoxylin and eosin (H&E) and observation Mice are animal models commonly used in the study under a light microscope for histopathological analysis. of the effects of heat stress because they are readily available, easy to find, cost-effective, easy to handle and have Histological morphometric analysis similarities in thermostats such as a human. Several studies The testicular tissue was evaluated in random order by have used experimental mouse models to assess a pathologist, who was blinded to the study group allocations environmental stress and exercise stress (Rakesh et al 2014). with Zeiss Axioplan-2 Imaging light microscopy (KS-300 Previous research has evaluated the testicular response after Imaging System). Testicular damage and spermatogenesis transient scrotal temperature exposure leading to germ cell were assessed histopathologically using Johnsen’s mean death and complex stress response (Paul et al 2009). However, testicular biopsy score (MTBS) (Johnsen 1970). By using a animal model for heat stress study is not yet well established. 40x magnification, thỉty tubules for each animal were graded This study aims to establish a mouse model for scrotal heat and each tubule was given a score from 1 to 10 based on the stress and investigate the histomorphometric change of presence or absence of germ cell types in the testicular testicular tissue in scrotal heat-stress exposed male mice based seminiferous tubules such as spermatozoa, spermatids, on Johnsen score of testicular histopathology. spermatocyte, spermatogonia, germ cells and Sertoli cells to evaluate histology. A higher Johnsen’s score indicates a better Materials and Methods status of spermatogenesis, while a lower score refers to more Animals severe dysfunction. Score 1 means no epithelial maturation considered for the tubules with complete inactivity while a Adult male Swiss mice with 8-10 weeks old (20-23g) score 10 means full epithelial maturation considered for the were purchased from Pasteur Institute of Nha Trang, Viet tubules with maximum activity (Table 1). Nam. Mice were kept in animal facility in a controlled temperature (25±1 °C) and photoperiod (12h light: 12h dark), Statistical analysis with free access to food and water. Animals were randomly Results were analyzed using SPSS version 18. All data divided into 4 groups of 5 mice each: Control group, 37 °C were reported as mean ± standard deviation (SD). To heat exposure group, 40 °C heat exposure group, 43 °C heat evaluation of significant differences, the comparison of means exposure group. between every two experimental groups was done by a Experimental heat stress computer program for Student’s t-test. Differences were considered to be statistically significant if P < 0.05. The lower half of the mouse's body, including the scrotum, was submerged in a temperature-controlled water Results bath (37 °C, 40 °C or 43 °C) for 10 minutes, twice per day, at 10 minutes intervals, 6 days per week for 5 weeks. Control Heat stress-induced spermatogenesis complications mice were treated within the same way, but in water bath Mice without heat stress (control group: bathing in 25 maintained at 25 °C. After having the bath, mice were dried °C water) showed the complete process of spermatogenesis in and examined for any injury or redness to the scrotal skin which progenitor spermatogonia develop into mature before returned to their cages. The four groups were kept in spermatozoa in the seminiferous tubules (Figure 1a). The different cages and had free access to water and food. All germinal epithelium including of cells at different stages of animals were cared for under the identical environmental spermatogenic development (spermatogonia, primary conditions. The mice were monitored their general health. spermatocytes, secondary spermatocytes,

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