Testicular Histopathology and Spermatogenesis in Mice with Scrotal Heat Stress

2.1 Mouse model for testicular heat stress

Swiss mature male mice have 8–10 weeks old (20-23 g) were kept in an animal facility at a controlled temperature (25 ± 1°c) and illuminated (12 h light-12 h dark), and with free access to food and water.

The mouse model was established to study the effect of scrotal heat stress on spermatogenesis and male fertility. The lower body (including the scrotum) of the mouse was soaked in a thermally controlled water bath (heat exposure at 37°C, 40°C, or 43°C) 2 times a day 10 minutes apart, every 10 minutes, lasting for 5 consecutive weeks, 6 days a week.

Control mice were treated in the same way, but in water bath maintained at room temperature. After having the bath, mice were dried and examined for any injury or redness to the scrotal skin before being returned to their cages. Mice were kept in different cages and had free access to water and food. All animals are cared for under identical environmental conditions and monitored their general health.

2.2 Tissue processing and hematoxylin–eosin staining

After completing the heat exposure experiment after 5 weeks, all mice was sacrificed under anesthesia. Their testis tissue were harvested for histological morphometric analysis. The testicular specimens were individually immersed into 4% buffered formaldehyde and dehydrated with graded concentrations of ethyl alcohols at room temperature. The testicular specimens were then embedded into paraffin.

The paraffin blocks were cut thinly with a thickness of 5 μm and transferred into gelatinized slides. The sections were deparaffinized with xylene and then rehydrated through a descending series of ethanol and water. Slides were stained with hematoxylin and eosin (H&E) and observed under a light microscope for histopathological analysis.

2.3 Johnsen’s mean testicular biopsy score count

Testicular histological damage and spermatogenesis were assessed using Johnsen’s mean testicular biopsy score under light microscopy [25]. Thirty tubules for each testis were graded and each tubule was given a score from 1 to 10 based on the presence or absence of germ cell types in the testicular seminiferous tubules such as spermatozoa, spermatids, spermatocyte, spermatogonia, germ cells, and Sertoli cells to evaluate histology.

A higher Johnsen’s score indicates a better status of spermatogenesis, while a lower score refers to more severe dysfunction. A score of 1 means no epithelial maturation is considered for the tubules with complete inactivity while a score of 10 means full epithelial maturation is considered for the tubules with maximum activity (Table 1).

3.1 An overview of the spermatogenesis

Mammalian spermatogenesis is a complex system within structurally well-designed seminiferous tubules of the testes (Figure 1a). Germ cells develop in a spatially organized fashion from the basal membrane to the lumen. During the process of spermatogenesis, primary spermatocytes were differentiated from diploid spermatogonia (Spermatogonial stem cells).

Primary spermatocytes undergo to the first meiotic division to generate two secondary spermatocytes. Each secondary spermatocytes go through the second meiosis division to produce haploid daughter spermatids. Spermatid transforms into spermatozoa by the process of metamorphosis (spermiogenesis). Finally, mature sperm were released from the seminiferous epithelium into the tubular lumen. In humans, the spermatogenesis process takes approximately 70 days [26, 28].

3.2 Spermatogenesis complications in mice with heat stress-induced

Globally, animal models have commonly been used to address a variety of biomedical research including, from basic science to immunology and infectious disease, oncology, and behavior or therapies. Laboratory mice are genetically heterogeneous and have been developed to be the powerhouse for biomedical research [29]. Animal models, especially the mouse model was investigated for the study of human infertility or spermatogenesis [30], effects of testes hyperthermia [10], cisplatin-induced testicular injury [31].

Healthy mice without heat stress show the complete process of spermatogenesis in which progenitor spermatogonia develop into mature spermatozoa in the seminiferous tubules (Figure 1b). The germinal epithelium including of cells at different stages of spermatogenic development (spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, and spermatozoa) located in invaginations or in dilations between of Sertoli cells (Figure 1c).

Meanwhile, the heat-stress exposed mice exhibited degenerated and disorganized features of spermatogenic epithelium and reduced spermatogenic cell numbers (Figure 1d and e).

3.3 Evaluation on changes of testicular histology and spermatogenesis in mice exposed to heat stress

Johnsen’s score evaluation system was used in several studies to assess testicular histology, a relatively complete scoring system with a full level of testicular histological scores given a score of 10 to 1 with a decrease in the number of cells in the lumen of the spermatogenesis [25]. This quantitative histological grading system is reliable, easily obtained and simple to prognosis for reproductive capacity in men [32, 33].

Johnsen’s score is also a histopathological predictor of semen quality after a number of treatment indications in male infertility patients to analyze and determine positive prognostic values that help improve fertility [33].

Tung et al. applied the criteria formulated by Johnsen to assess murine testicular histopathology affected by scrotal heat stress [27]. Results showed that Johnsen’s score was significantly reduced in the heat stress groups compared with those in the control group, increasing the possibility of infertility in male mice. The seminiferous tubules were graded (1 to 10) according to a reduction in the number and density of germ cells from the lumen of seminiferous tubules.

Standardization of Johnsen scores in the seminiferous tubule cross-sections of control and heat exposed group mice stained with hematoxylin–eosin was shown in Figure 2. Johnsen scores 8, 9, and 10 indicate spermatogenesis with a few to many spermatozoa present in a section of seminiferous tubular (Figure 2a–c). Scores 6 and 7 indicate seminiferous tubular with no spermatozoa, but spermatids present (Figure 2d and e). Seminiferous tubular with no spermatozoa, no spermatids, but spermatocyte present is evaluated as scores 4 and 5 (Figure 2g and f).

Seminiferous tubular with only spermatogonia as germ cells presence is evaluated as score 3 (Figure 2h), meanwhile tubular with no germ cells but Sertoli cells present is given a score 2 (Figure 2i). Seminiferous tubular with complete absence of cells in the seminiferous tubules is evaluated as score 1 (Figure 2j) [27].

3.4 The effect of heat stress on male reproduction

Genital heat stress has been known as a risk factor for male infertility. However, the exact mechanism causing impaired spermatogenesis is still unclear. Testicular histology after thermal exposure had changed such as decreased epithelial thickness, the appearance of cellular debris, fragmented cells, and absence of sperm and spermatocytes [34, 35]. The impact of prolonged heat stress negatively affects sperm quality and quantity, testicular structure with disruption of the surrounding epithelium [34].

In addition, scrotal heat stress reduces testicular weight, mitochondrial degeneration, dilatation of the smooth endoplasmic reticulum and Leydig cells lose the function of supporting stem cells [13]. Findings from this study support that the testicular structure was disorganized germinal epithelium with marked sloughing or obliteration of lumen; spermatogenesis was interrupted with the absence of many types of sperm cells in male mice after chronic scrotal heat stress exposed for 5 weeks.

The elevated testicular temperature in mammals leads to impair spermatogenesis, germ cells incur damage, decreased sperm motility, and increases the percentage of sperm with an abnormal morphology [3, 36, 37]. Paul et al. also showed the effect of heat stress on testicular function and decreased fertility in mice [38].

In addition, heat stress increases free radicals and oxidative stress resulting in apoptosis of germ cells and increased sperm DNA fracture, loss of sperm integrity [39, 40].

There are many factors that can increase testicular temperature. These factors that affect male fertility can be grouped according to habits, lifestyles [18, 19], occupational factors that must be exposed to high temperatures for long periods [20, 21], and climate change [22]. Depending on the situation, the testicles may experience transient or prolonged heat stress with varying intensity. The effects of high ambient temperature on male fertility tend to accumulate with repeated exposure over a while [7, 18].

Heat-stressed has a direct impact on work performance by increasing the risk of illness and work-related injuries. When workers are exposed to high temperatures, their bodies are unable to activate compensation regimes or recover from stressful working days that put their health in danger [41].

Boni et al. demonstrated high ambient temperature as a serious threat to reproductive function in animals and humans. Comparing parameters between the control group and the group workers exposed to heat were within the limits of normozoo sperm range, there was a serious decline in semen parameters [21]. The bakers expose to high ambient temperature have high infertility rates as shown in Al-Otaibi’s study [20].

There was another large-scale epidemiological study at the Danish infertility clinic in which subjects underwent sperm examinations or infertility treatments, and obtained information on occupational exposure. It was found that the groups of workers exposed to textile dyes and lead, noise, cadmium, and mercury were all potentially infertile [42]. Similarly, Hamerezaee et al. have drawn the same results about the effects of temperature stress on semen quality when studied on workers of the steel industry in Iran. The semen quality of the workers is lower than the normal limit and significantly lower than the control group [43].

Another three-year study conducted in a fertility clinic in New Orleans found that men working in buildings without air conditioning during summertime would reduce their sperm quality. Their semen samples had a significantly lower sperm concentration, number of sperms per ejaculation, and a lower percentage of sperm motility when compared those figures in summer to other seasons of the year [44]. In contrast, a study in the ceramic industry has reported that workers exposed to high temperatures had no significant difference on semen analyses except for the sperm velocity [45].

In conclusion, this chapter shows the evidence for negative effects on histopathological alterations and spermatogenesis arrest following chronic scrotal heat stress.

This unit also characterizes an animal model for studying the male reproductive system and standardized Johnsen scores system to assess murine testicular histopathology in the seminiferous tubule cross-sections.