Mechanical damage to the testicles is a known trigger for impaired spermatogenesis. This damage can result from sports, domestic, or workplace injuries and typically presents as compression or bruising. Although these incidents are common, they have not been sufficiently studied to date. The initial damage from mechanical impact initiates primary damage, which in turn triggers secondary processes that are usually autoimmune or infectious, along with impaired blood supply [1,2].

According to Tullington and Blecker [3], traumatic lesions of the genitourinary tract, ranging from minor wounds to complex injuries, can lead to shock and multiple organ failure. Trauma is a leading cause of death among individuals aged 15 to 24 years and accounts for 30% of annual intensive care hospitalisations. One particularly challenging scenario involves blunt trauma to the scrotum, which can result in testicular dislocation. This condition is difficult to diagnose due to various accompanying disorders. The most common causes of dislocation include riding injuries and road traffic accidents, especially motorcycle accidents where the scrotum impacts the fuel tank during a sudden stop. The most severe complication is the loss of blood supply, leading to necrosis. In such instances, surgical intervention is crucial to prevent infertility [4]. One risk following dislocation is impaired spermatogenesis. The initial histological changes in the organ become apparent after 4 months. These changes include disrupted spermatid generation, absence of sperm, hyalinisation, and atrophy of the seminiferous tubules, and the emergence of alternative germ cells. Post-surgery, the complete restoration of spermatogenesis can take up to 15 years [5,6].

As noted by Chouhan et al. [7], mechanical pelvic trauma accounts for 6% to 15% of disorders affecting the bladder, urethra, and testicular structure and function. Another adverse outcome of trauma to the scrotum and groin area is varicocele, which involves varicose veins of the spermatic cord. The severe stage of this disease is characterised by visibly swollen veins and scrotum, along with a reduction in testicle size. Fluid stagnation in the tissues, caused by circulatory disorders, can provoke the development of atrophy, potentially leading to infertility in 10% of cases. Another factor contributing to infertility is a sustained local increase in temperature, which reduces oxygen concentration in the scrotum and, consequently, impedes sperm maturation. If the spermogram is normal, treatment involves conservative methods such as stabilising blood pressure, therapeutic exercises, and massage. However, if there are abnormal spermogram results, testicular atrophy, or severe pain, surgical intervention is recommended. This procedure involves freeing the arteries from the pathologically transformed veins [8-10]. Blunt trauma to the testicle, often overlooked, can ultimately impair spermatogenesis and result in a loss of fertility.

The purpose of this study was to perform cytological and histological examinations of testicular tissue in a line of laboratory rats following mechanical trauma. Investigating this aspect in model organisms can help build a database that may be predictive for medical practice.

In the postnatal period, assuming no pathologies develop, the gonads of white rats are typically elliptical, featuring elastic contents and a smooth surface. The organ weighs approximately 600 mg. The testicle is encased in a compacted protein shell that is about 35 µm thick. Within the haematoxylin-eosin staining system, the testicular tissue appears white-purple. Cross-sectional analysis reveals sinuous seminiferous tubules of spherical or ellipsoid shape, interstitial connective tissue that separates these tubules, spermatogenic epithelium at various stages of development inside the basement membrane, and the tubular lumen where spermatozoa are released (Figure 1).

Spermatogenesis occurs in various stages within the seminiferous tubules. These tubules are encased by a tubular membrane composed of connective tissue fibres. A layer of myoid cells, uniformly distributed within loose connective tissue, adheres to the exterior of the basement membrane. The ratio of interstitial connective tissue to seminiferous tubule content is 1:30 [11,12]. The spermatogenic epithelium's first (outer) layer in the seminiferous tubule consists of spermatogonia, which possess a dark, optically dense nucleus and are situated on the basement membrane (Figure 2). The second layer, moving towards the central canal, comprises spermatocytes. These are large cells featuring a prominent nucleus and an extensive cytoplasmic rim. The third layer is made up of early spermatids, while late elongated spermatids form a layer encircling the tubular lumen, some of which develop flagella. Spermatozoa, characterised by a tail pointing into the tubular lumen and a dark elongated head facing the tubular periphery, are found in groups of 6 to 8 within the lumen of the tubule [13,14].

Blood vessels are identified in the interstitial tissue, which is composed of loose connective cells. Within this interstitial layer, Leydig cells, responsible for producing testosterone, are situated. These cells are large, oval or polygonal in shape, and contain a prominent round nucleus. They are found either individually or in clusters of five to seven cells [15]. Puberty in rats is a complex, multi-stage process. A critical marker of this process is the composition of the spermatogenic layer, which varies in the ratio of spermatogonia, spermatocytes, spermatids, and spermatozoa at different stages of puberty. In sexually mature animals at 2 months of age, in the absence of pathologies, the spermatogenic cells comprise 70% spermatozoa, 12% spermatogonia, 9% spermatocytes, and 8% spermatids. This distribution suggests that the stage of premeiotic spermatogenesis is complete by 2 months, leading to a stabilisation of cell abundance [16]. The predominance of spermatozoa in the cell population of sexually mature animals may suggest that inhibition of spermatocyte apoptosis is occurring in mature individuals. During the neonatal period, the spermatogenic cells consist of gonocytes; spermatogonia emerge 3 to 6 days post-birth, but they are only clearly quantifiable by day 60. The surface area of the seminiferous tubules expands as the animal grows; however, once puberty is reached, it remains constant thereafter. This parameter serves as an indicator of gonadal puberty [17,18]. This data is valuable for documenting and elucidating the cytological and histological changes that occur following trauma.

This study was approved by the Institutional Review Board of Vasyl Stefanyk Precarpathian National University (approval number: E-1285). The study was conducted without human participation. Informed consent is not required.

The histological studies focused on sexually mature male rats. For the experiment, 32 animals weighing between 300 and 350 g and aged 3 to 4 months were selected. The animals were divided into two groups: group 1 (control) consisted of healthy males kept under standard vivarium conditions without any physical damage (six individuals). Group 2 (experimental) comprised males with testicular tissue damage caused by blunt mechanical trauma without rupture of the protein membrane (26 individuals). The same method of blunt mechanical trauma, which did not rupture the protein membrane, was uniformly applied to all subjects in the experimental group. One method to achieve consistent injury is the use of a controlled impact device that delivers a constant force to the testicular area. This device is designed to ensure uniform injury across subjects, thereby minimising variability in injury severity. Screening was conducted at 7, 14, 30, and 90 days post-injury.

Group 2 was divided into four subgroups, depending on the time of the experiment:

 • 2.1: 7 days after injury (six individuals)

 • 2.2: 14 days after injury (six individuals)

 • 2.3: 30 days after injury (six individuals)

 • 2.4: 90 days after injury (eight individuals).

To visualize morphological changes in blood vessels, a 10% solution of Paris blue pigment was administered to both experimental and control animals via the abdominal aorta.

Ethical standards are observed in accordance with several regulatory documents, namely: Law of Ukraine No. 3447-IV “On the protection of animals from cruelty” [19]; European convention for the protection of vertebrate animals used for experimental and other scientific purposes [20]; Universal declaration on bioethics and human rights [21]; and Declaration of principles on tolerance [22].

Histological samples of the testis were fixed in Buena’s solution at approximately 25 °C for 2 days. Subsequently, the samples underwent three dehydration cycles in 100% diethylenedioxide, each lasting 30 minutes at 30 °C. Following dehydration, the samples were incubated in a 3% colloidal nitrocellulose solution, which also contained ethanol and diethyl ether in a 50/50 volume ratio, at the same temperature and duration. The treated samples were then immersed in orthoxylol for 20 minutes at 30 °C and embedded in a liquid mixture of 90% paraffin and 10% lanolin (two cycles, 25 minutes each, at 60 °C) to prepare them for sectioning using freezing and angle microtomes. Staining was performed using solutions of haematoxylin/eosin, and the periodic acid-Schiff test, after which the final preparations were mounted in Canadian balsam.

An eyepiece micrometre was utilised for general morphometry. Microscopy facilitated the assessment of various parameters, including the nature of vascular filling, the extent of damage to the spermatogenic epithelium, the number of cells in phase VII of the cycle, the nuclear volume in Leydig cells, the diameter of vessels, and the diameter and number of convoluted seminiferous tubules, as well as the thickness of the tubule wall. Micrographs were captured using the Leitz DIAPLAN microscopic system, equipped with a Nikon D-60 camera featuring a trinocular objective (×200; ×400; ×900) and a Linvatec halogen illuminator. For enhanced contrast, gaussian pyramid module 2.5 and fine gaussian pyramid module 3.5 filters were employed. To analyse the structural conformations of the tissue, thin spatially oriented sections measuring 5 to 7 µm in thickness were prepared, followed by imaging on a Philips CM200F transmission electron microscope at magnifications of ×5,000 and ×10,000. Image adjustments were conducted using the FS Viewer. Statistical analysis of the numerical data was carried out using Excel (Microsoft) and GraphPad Prism ver. 9.0 (GraphPad Software Inc.) software suites, with the threshold for statistical significance set at p<0.05.

As a result of blunt mechanical trauma, several morphological, morphometric, and histological abnormalities are observed in rat testicular tissue. Specifically, 1 week after the injury, alterations in the structure of the basal membrane of the spermatogenic epithelium were noted; it became expanded and twisted. The interstitium shows infiltration and swelling. In supporting epithelial cells, nuclear deformation occurs, the morphology of the tight junctions between cells is disrupted, the cytoplasm becomes lucent, and mitochondrial cristae are diminished. In the cells lining the walls of testicular capillaries, there is nuclear deformation and uneven chromatin condensation. On the surface, protrusions of various sizes and shapes, which are not characteristic of unaffected cells, were observed. Mitochondrial cristae are partially homogenised, and the organelle matrix appears lighter. In myoid cells, myofilaments are diminished, mitochondrial cristae are homogenised, and the perinuclear space is reduced; similar to the capillary endothelial cells, a change in nuclear shape is observed. In damaged interstitial cells, signs similar to those described above were found: reduced mitochondrial cristae, depleted cytoplasm, and unevenly condensed nuclear chromatin (Figure 3).

A month after the experiment began, several deeper destructive changes were detected in the tissue. The number of spermatogenic epithelial cells was rapidly decreasing. The basement membrane of the seminiferous tubules became delaminated, and their diameter narrowed. Structural damage to the endoplasmic reticulum and Golgi apparatus occurred alongside mitochondrial reduction and deformation of the myoid cell nucleus. The structure of the basement membrane of the spermatogenic epithelium remained twisted. In the cells lining the walls of capillaries, the nucleus became deformed, the cytoplasm vacuolised, the shape of organelles was disturbed, and the capillaries themselves narrowed [23]. In endocrine cells, the cytosolic volume decreased, organelles were completely reduced, and the nuclei deformed. In supporting cells, chromatin remained unevenly condensed, and mitochondrial cristae were reduced. A large number of vacuoles appeared in the cytoplasm and matrix of mitochondria. In the structure of the junction of these cells, the volume of the cisternae of the Golgi apparatus increased, filaments disappeared, and the space between the cytolemmas narrowed. Testicular tissue was depleted by the capillary bed; capillaries were narrowed (Figure 4).

By day 90 of the study, the content of spermatogenic epithelial cells continues to decline rapidly. A similar pattern of destruction is observed in the haemato-testicular barrier, akin to that detected after the first month. Some capillaries are diminished. In endocrine cells, the compartments of the reticulum become narrower, mitochondrial cristae degrade, and both an irregular nucleus and hyperchromatisation are observed. In the nuclei of myoid cells, chromatin condensation is noted along the periphery, and their irregular shape is maintained. The spatial structure and integrity of the cytolemmas within the connections of supporting cells are disrupted, and their organelles become irregularly shaped. Capillary endothelial cells exhibit fragmentation of the basal layer, altered nuclei, organelle reduction, and hypervacuolarisation. In the structure of the seminiferous tubules' wall, there is an increase in collagen fibres, and the wall develops a folded structure. Some seminiferous tubules are empty, while others contain few spermatozoa. The capillary network remains sparse, and the lumens of the vessels continue to be narrowed (Figure 5).

Images obtained from a transmission electron microscope on day 30 of the experiment reveal capillary microclasmatosis, characterised by the detachment of structural elements from the surface of vascular wall cells, which then enter the intercellular space (Figure 6).

Figure 6 illustrates nuclear swelling and irregularities in the basement membrane. Sertoli cells are characterised by a brightened cytoplasm and a thickened basement membrane that forms protrusions. Myoid cells display a dark nucleus, vacuolated cytoplasm, and a thickened cytoplasmic membrane with an uneven surface (Figure 7).

In Leydig cells, there was a marked reduction in mitochondrial cristae, accompanied by a stiffening of the nuclear envelope, accumulation of chromatin at the periphery of the nucleus, and an increased number of vacuoles in the cytoplasm (Figure 8).

In addition to cytological and histological changes, physical trauma also leads to changes in the morphometric parameters of the affected organ (Figure 9). Specifically, on the first day of screening (7 days post-trauma), the weight of the injured testicle decreased by 202 mg compared to the control (p≤0.05). This weight loss may be attributed to parenchymal atrophy, as evidenced by a 50 µm narrowing in the diameter of the seminiferous tubules (compared to the control sample, p≤0.05). The protein membrane of the affected organ thickens due to oedema and the accumulation of connective tissue. The capillary network is partially destroyed, resulting from a reduction in blood vessels whose diameters expand unevenly. Hemosiderin accumulates in the membrane and parenchyma due to the breakdown of haemoglobin and the denaturation of ferritin. The membrane of the seminiferous tubules thickens, and the volume of Leydig cell nuclei decreases to 80 µm³. Significant damage to the spermatogenic epithelium is observed in about 20% of the tubules; in 10% of these, only spermatogonia and supporting cells are present in the vicinity of the tubule membrane.

Two weeks after the experiment began, the weight of the damaged organ decreased by 227 mg (p≤0.05) compared to that of healthy animals. In some areas, the blood vessel walls were deformed and remained unevenly expanded. The concentration of microvessels increased, while the diameter of the seminiferous tubules narrowed by 58 µm (p≤0.05) relative to the control. Additionally, the number of empty and severely damaged tubules increased, and the volume of the Leydig cell nucleus averaged 77 µm³. One month post-trauma, the testicular weight had decreased by 267 mg, and the tubule diameter had reduced by 63 µm compared to the healthy sample (p≤0.05). Sclerotisation of the seminiferous tubule membrane accompanied the thickening of the connective tissue. The number of spermatids and spermatocytes decreased by 200 and 87 units, respectively, compared to the control (p≤0.05) (Figure 10). The size of Leydig cell nuclei was measured at 75 µm. Only 33% of the tubules remained intact; severe cell damage was observed in 21% of the tubules, and 16% were found to be empty.

As shown in Figure 8, from day 7 of the experiment onward, there was a significant decrease in the number of spermatocytes and spermatids compared to the control group, while the number of spermatogonia remains unchanged. By day 90, only 67% of spermatocytes and 77% of spermatids are retained relative to the baseline measurement. Three months post-injury, the organ's weight has decreased by 307 mg, and the volume of Leydig cell nuclei has reduced to 71 µm (p≤0.05). The parenchyma undergoes atrophy, leading to deformation of the capillary network surrounding the seminiferous tubules. Additionally, over 50% of the tubules exhibit severe damage, accompanied by a significant reduction in the number of spermatogenic cells (p≤0.05).

The focal deformity of the vascular walls in the capillary network of severely affected tubules is likely caused by microhaematomas and infiltration occurring shortly after injury. Disruption of the hematotesticular barrier—which includes the integrity of supporting cells, blood vessels, and tubule walls—may lead to the subsequent development of an autoimmune reaction post-trauma. One month following the trauma, the negative progression of structural changes becomes more pronounced: 16% of the tubules are empty, and there is a significant reduction in the content of spermatogenic cells. Similar phenomena are observed in men following inguinal canal surgery and in the diagnosis of an oblique inguinal hernia [24,25]. Testicular injury can result in the post-traumatic accumulation of exudate, the development of oedema, and consequently, disruptions in thermoregulation and spermatogenesis [26]. The results of the study conducted at the model facility may have predictive value for medical practice.

Mechanical trauma disrupts blood circulation in the affected organ. The present study demonstrated this disruption through the detection of local rearrangement of blood vessels. Tanriverdi et al. [27] have also demonstrated impaired blood circulation in rats experiencing testicular torsion. In severe cases, this disruption of blood flow can lead to ischemia and tissue atrophy due to an inadequate supply of oxygen and nutrients. Ischemia subsequently triggers an inflammatory response, during which neutrophils release oxygen free radicals. These radicals initiate oxidative stress, characterised by the destruction of cell membranes and the initiation of an apoptosis cascade in germ cells. For accurate biochemical analysis post-trauma, several markers of oxidative stress were identified. The extent of cell apoptosis and the characteristics of spermatogenesis were also documented in the tissues. Oxidative stress was mitigated by famotidine, a histamine receptor blocker. As anticipated, the group subjected to trauma exhibited increased nitric oxide (NO) content, a higher germ cell apoptosis index, and reduced activity of antioxidant enzymes. The Johnsen index, a measure of spermatogenesis, also showed a significant decline in the study sample. Additionally, we observed inhibition of the spermatogenesis process, which was reflected in a decrease in the levels of spermatogonia and spermatocytes.

Another study by Wei et al. [28] also demonstrated the adverse effects of testicular torsion in laboratory rats. The torsion was maintained for 2 hours. Four hours later, the development of ischemia was assessed by monitoring the expression of antioxidant defence enzymes, such as catalase and superoxide dismutase, and by measuring the levels of malondialdehyde (MDA). As anticipated, histological samples from the experimental group showed decreased enzyme expression and increased MDA concentration, indicative of ischemia. Three months post-trauma, spermatogenesis was evaluated by measuring testicular weight, the diameter of the seminiferous tubules, the number of germ cells, and the Johnson score. The experimental group exhibited a 40% reduction in testicular weight, a 30% reduction in tubular diameter, and a 70% reduction in germ cell count. These findings align with those of the current study, further validating the long-term negative impact of trauma on germ cell maturation. However, the adverse effects can be mitigated with biologically active plant compounds, specifically salidroside. In samples treated with this compound, there was a restoration of antioxidant enzyme synthesis and a reduction in tissue MDA levels [29,30].

Results from a proteomic analysis conducted by Ouh et al. [31] demonstrate the destructive effects of mechanical damage at the cellular, tissue, molecular, and biochemical levels. The experimental group of rats underwent mechanical trauma for 1 hour through twisting. Protein expression levels were analysed using electrophoretic separation and Western blotting. In the experimental group, protein expression related to germ cell division and maturation, gene expression regulation, antioxidant protection, and apoptosis protection was found to be nearly three times lower than in healthy animals. Additionally, histopathological alterations in germ cells within the seminiferous tubules were observed. These tubules contained only a few primary and secondary spermatocytes or germ cells with pyknotic nuclei and disorganised cytoplasm; markers of apoptosis were detected in spermatogonia, spermatocytes, and several cells within the seminiferous tubules. This study yielded similar cytological findings. The number of germ cells decreased post-injury due to the activation of caspase cascades leading to cell death.

It should be noted that not only ischemia but also the moment of reperfusion is hazardous for testicular tissue. A gradual increase in pressure within the damaged organ poses one of the initial risks to spermatogenesis. During reperfusion, epithelial growth factor (EGF) plays a pivotal role. It is primarily synthesised by the submandibular salivary glands in rats and transported via the bloodstream. This protein is expressed locally, allowing for paracrine, autocrine, and endogenous regulation. It influences the division of endothelial and epithelial cells, keratinocytes, and fibroblasts, and facilitates communication between immature germ cells and Sertoli cells. In the early stages of reperfusion following testicular ischemia, the endogenous levels of this regulator increase. As a consequence of torsion and reperfusion, reductions in testicular weight and volume, tubule diameter, and seminal epithelial thickness are observed in Wistar rats. Following treatment with exogenous EGF and fasciotomy in affected animals, the volume of the damaged organ is restored to normal, and the rate of apoptosis decreases. The effects of EGF last for 2 days, providing sufficient time to normalise blood flow and pressure. In addition to growth factors, several antioxidants can mitigate the adverse effects of reperfusion, including resveratrol, trapidil, N-acetylcysteine, and methylprednisolone. Typically, these agents are administered to animals in a single dose immediately before reperfusion [32].

Severe damage to rat testes after trauma can be prevented at the molecular level by reducing the accumulation of nitrogen (II) oxide and by inhibiting NO synthase. This enzyme uses L-arginine and nicotinamide adenine dinucleotide as substrates, oxidising them to citrulline and NO. Research by Kapucu and Akgun-Dar [33] demonstrated that L-arginine methyl ester inhibits this enzyme, likely by competing with L-arginine for the binding centre. Additionally, leptin suppresses the expression level of inducible NO synthase. Rats treated with leptin and L-arginine methyl ester exhibited fewer destructive histopathological changes in testicular tissue compared to those in the post-injury samples without these inhibitors. Animals with inhibited synthase likely do not produce significant oxidative stress, thus preserving cell integrity. One marker of initial tissue damage after mechanical injury to rat testicles is the protein aquaporin 1 (AQP1), primarily found in the interstitial connective tissue and vascular endothelium of the testes. Its role is to regulate cell volume by facilitating water intake, which leads to tissue oedema. Following the onset of oedema, the weight of the interstitial tissue increases, spermatogenic tubules atrophy, and tubular cells are shed. In injured animals, the level of mRNA transcription and expression of this protein rises and is detectable within 48 hours. Preventing its accumulation can avert tissue oedema and subsequent damage [34]. Other indicators of potential testicular dysfunction after injury include decreased overall antioxidant activity and testosterone levels, increased quantitative levels of caspase-3 (involved in the apoptosis cascade), pro-inflammatory interleukin-1-beta, tumour necrosis factor-alpha, strong positive expression of nuclear factor erythroid 2-related factor 2 (Nrf2; a nuclear factor that protects against oxidative stress), and negative expression of proliferating cell nuclear antigen (PCNA; nuclear proliferative antigen) [35].

As can be seen from the data in the literature, a comprehensive study of molecular genetics and biochemistry in damaged tissues can enhance our understanding and prevention of their destruction mechanisms. Therefore, future research should focus on examining alterations at the molecular level in these tissues, including changes in the expression of marker proteins and genes.

In conclusion, the experimental findings suggest that blunt mechanical trauma to the ovarian tissue is associated with significant morphological, morphometric, and histological alterations in rat tissues. Within the first week following the injury, the basal membrane of the spermatogenic epithelium expanded and twisted, with observed alterations in the structure of intercellular junctions and a reduction in the number of cells in the VII cycle. One month after the experiment began, there was a further decrease in the number of epithelial cells and a deterioration in the structure of the seminiferous tubules, accompanied by a reduction in the number of spermatids and sperm cells. By the 90th day, the decline in epithelial cell numbers continued, along with a disruption of the barrier between blood and ovarian tissue and deformation of blood vessels. Microscopic examination also revealed changes in the structure and shape of the nuclei of various cell types. By day 90, additional changes included a reduction of the Golgi complex and endoplasmic reticulum in endocrine cells, vacuolisation in endothelial cells, Leydig cells, and supporting cells. Capillaries reorganised and narrowed, with uneven thickening of their walls. The basement membrane swelled and twisted, the interstitium became infiltrated, and the structure of the Sertoli cell junctions was compromised. Half of the seminiferous tubules were completely or partially reduced; the walls of intact tubules were enriched with collagen fibres. The diameter of the seminiferous tubules decreased by 50 microns (1 week after injury) and by 63 microns (1 month after the experiment started). By day 90, the diameter of the tubules had decreased by 30% compared to the control. The ratio and quantity of spermatogenic cells also changed: the content of spermatids and spermatocytes decreased, while the number of spermatogonia remained stable. By day 90, 77% of spermatids and 67% of spermatocytes remained intact. Damage to blood vessels, the walls of seminiferous tubules, and the integrity of Sertoli cells led to the destruction of the haemato-testicular barrier.

To better assess the extent of destruction and understand its mechanisms on a larger scale, it is advisable to measure the expression levels of marker genes and proteins, which could be a potential focus for further experiments.