Peculiarities of the microscopic structure of rat testis under the influence of vipera berus berus venom

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Introduction
To date, it has been established that the venom of snakes and vipers includes about 50-200 different components, which usually belong to four main families of toxins: phospholipases A2 (PLA2), metalloproteinases (SVMPs), serine proteases (SVSPs), three-finger toxins (3FTXs) [13,15,16,17].The composition of the venom can vary at the interspecies and intraspecies levels and depends on such factors as features of ontogenesis, nutrition, sex, adaptation to living conditions [14,19,20].
Among the many venoms of other animals, the composition of the venom of the scorpion Leiurus macroctenus, which mainly consists of inorganic salts, nucleotides, amino acids, lipids, enzymes and peptides, attracts attention [19].The most common in European countries are vipers of the Viperidae family, the bites of which are associated with numerous fatal consequences or the development of hemotoxic, cytotoxic, neurotoxic disorders.Among all species of this family, Vipera berus berus and Vipera berus nikolskii are also common in Ukraine [11,21].
The spectrum of clinical manifestations of snake and viper bites is extremely wide.The analysis of scientific literature shows that the most common are local tissue damage, pain, lymphadenitis, hemorrhagic disorders, damage of the nervous, urinary systems, etc [4,5,10].
Enzymes present in the venom enhance the process of poisoning and the spread of the venom by destroying the extracellular matrix.An increase in enzymatic activity during poisoning leads to increased tissue permeability and provides a systemic inflammatory response since poison toxins can quickly spread between all organs and tissues [8,9].
Morpho-functional changes in the organs of the male reproductive system are usually associated with injuries, infectious diseases, age, lifestyle, the presence of bad habits (abuse of alcohol, drugs), the influence of environmental factors, etc.However, the action of natural toxins, in particular those that are components of animal venom, including the venom of snakes and vipers, is not less important.This problem is currently a subject of interest of scientists.The facts of the development of pronounced violations in the structural organization of the male reproductive system under the influence of snake venom, namely Crotalus durissus ssp.and Daboia russelli are proven.In the scientific literature, no data were found regarding the effect of Vipera berus berus venom on organs of the male reproductive system, which determines the relevance of the chosen direction of research.
The aim of the research is to study the features of the microscopic structure of the testis of rats under the influence of Vipera berus berus venom.

Materials and methods
Experimental studies were carried out on white nonlinear male rats.For preliminary acclimatization, the animals were kept for 7 days in the animal facility of Taras Shevchenko National University of Kyiv, and then kept in laboratory conditions at constant temperature (22±3°C), humidity (60±5 %) and light (12 h light/12 h dark cycle), being fed standard rodent food and water ad libitum [6].All experiments were conducted in accordance with the National Institutes of Health Guidelines for the care and use of laboratory animals and the European Council Directive of 24 November 1986 for the Care and Use of Laboratory Animals (86/609/EEC).The research was approved and confirmed by the Bioethics Commission of the NSC "Institute of Biology and Medicine" of the Taras Shevchenko National University of Kyiv (protocol No. 2 dated August 19, 2021).
Vipera berus berus venom was obtained from the V. N. Karazin Kharkiv National University.The lyophilized crude venom was stored at -20°C and then dissolved in saline solution immediately before the experiment.
The animals were conditionally divided into two groups -a control and an experimental group of 10 individuals in each.Experimental rats were injected intraperitoneally with a semi-lethal dose (LD 50 ) (1.576 mg/g -1 ) of Vipera berus berus venom in saline solution.Animals of the control group were injected intraperitoneally with only saline solution.Rats were removed from the experiment 24 hours after exposure to the venom, anesthetized by cervical dislocation.
Testis samples from animals of all groups were taken for microscopic examination.The pieces were fixed in a 10 % formalin solution for 1 day.Further the pieces were dehydrated in alcohols of increasing concentration and embedded in paraffin blocks.Histological preparations of testis were stained with hematoxylin and eosin [12].Histological specimens were studied using a SEO SСAN light microscope.

Results
A morphological study of the testis of animals from the group injected with Vipera berus berus venom showed a lower reactivity of stromal elements damage compared to parenchymatous elements.The dense connective tissue of the capsule and trabeculae does not undergo noticeable changes: the septa do not thicken and do not undergo delamination.Small and medium-diameter blood vessels in the interstitium are characterized by full blood and aggregation of red blood cells in the lumen (Fig. 1 A, Fig. 2  D).In general, the shape of the seminiferous tubules does not change and remains quite regular (see Fig. 1 B).
Sometimes the interstitium was characterized by an increased number of Leydig cells, which may indicate a violation in the hormonal regulation of the organ (see Fig. 2 А, 2 D).
Unlike the stroma, the parenchymal components of the testis are characterized by various microscopic signs of damage.In particular, a general examination of the contents of the seminiferous tubules demonstrates detachment and disorganization of spermatogenic cells at all stages of their development.The lumens of some tubules are empty, which indicates a radical disruption of spermatogenesis processes in them (see Fig. 1 B, 1 C).Such emptiness is most likely determined by the incomplete stop of spermatocytes in the spermatogenesis pathway in such tubules.In the case of spermatogenic hypoplasia, the tubules have a reduced population of germ cells and a poor order of spermatogenesis.
In other tubules, desquamation and disorganization indicate that spermatogenesis is disordered and the tubule lumen is filled with desquamated immature cells (see Fig. 1 D).Spermatogenic cells in seminiferous tubules of animals are characterized by changes in morphology and location.In contrast to animals from the control group, in rats, after the injection of Vipera berus berus venom, spermatogonia lose the regularity of their location and decrease in number (see Fig. 2 А, 2 B).Cells are separated from the basement membrane and do not form a single layer.It is likely that their location below the blood-testicular barrier makes them most vulnerable to venom.In some tubules, spermatogonia are practically not visible (see Fig. 2 B), which indicates the loss of the ability to renew the population of male germ cells, because only spermatogonia can divide by mitosis and perform this function.
Primary spermatocytes are slightly less exposed to snake venom, their general presence in the lumen of the tubules practically does not change, but their location becomes disorganized, they are largely shifted to the center of the seminiferous tubules, where they "mix" with more mature forms of spermatogenic cells (see Fig. 2 C, 2 B).Inside the first line spermatocytes, vacuolization is often seen around the nucleus (see Fig. 2 A).Presumably, this can be explained by the origin of these vacuoles -they are expanded cisterns of the rough endoplasmic reticulum, which try to ensure detoxification processes in the cell.
Spermatids in their location shift to the center of the seminiferous tubules, presumably losing their connection with Sertoli cells.Closer to the center of the tubule lumen, they form an eosinophilic amorphous mass together with mature spermatozoa.An interesting feature is the presence of erythrocytes clusters in the lumen of the tubules, which probably indicates hemorrhages under the influence of snake venom (see Fig. 2 B, 2 C).
In conclusion, it should be noted that the content of seminiferous tubules in research group of animals is characterized by a partial inhibition of proliferation and differentiation of spermatocytes, spermatogenic hypoplasia.

Discussion
The study of the effect of snake venom on the testis is related to their important functions, which have complex mechanisms of regulation.Factors such as active mitosis, the presence of a barrier between the blood and testicles, the spermatogenic cycle and hormonal control complicate the response of this organ to external toxic factors.
However, the general mechanisms of the types of testicular toxicity can be classified according to certain developmental mechanisms.It is necessary to analyze the primary toxicity -the direct effect of the venom on the spermatogenic epithelium.In addition, there are several indirect factors of the effect of the venom on this organ, related to the regulation of its functions and blood supply.In particular, indirect hormonal influence on spermatogenesis and impaired blood circulation in the testicle can also lead to pathological changes.In the histological assessment of testicular toxicity, histological models of toxic lesions caused by various model chemicals, classified on the basis of their mechanisms of action, are very useful [18].Studies of the effects of toxic compounds on the structure and function of the testis strongly suggest that the early changes, rather than those that occur later are particularly useful for understanding their mechanisms of action.This observation correlates with our research, because we study the morphology of the testes 24 hours after the injection of snake venom.B. S. Ajisebiola et al. established that the venom of Naja nigricollis causes the development of morphological and biochemical changes in testicular tissue of experimental animals.In rats, under these conditions, atrophy of the tubular epithelium of the testis, a significant decrease in the number of spermatogenic cells, their necrosis, and distortion of the structure of the germinal epithelium were detected.In the tissue of the organ, a decrease in the activity of catalase, glutathione peroxidase and an increase in the level of malondialdehyde, IL-1β, TNF-α were noted [1].
According to scientists, toxins from the venom of Echis ocellatus snakes cause the development of an inflammatory process in the tissue of the testis of rats, degenerative Fig. 2. The stroma and seminiferous tubules of the testis of rats in the experimental group with the introduction of Vipera berus berus venom.A: vacuolization around the nucleus of spermatocytes (blue arrows); A, B: a small number of irregularly placed spermatogonia (yellow arrows); A, D: Leydig cells in the interstitium (red arrows); B, C: erythrocytes within the amorphous mass in the lumen of seminiferous tubules (green ovals); C, D: cell mass from spermatogenic cells of various degrees of maturity with blurred edges (yellow ovals).Staining with hematoxylin and eosin; x 1000.changes in the germinal epithelium and the epithelium of seminiferous tubules, and tubular atrophy [2].
C. Alberto-Silva and co-authors [3] in experiments on rats injected with Bothrops jararaca snake venom observed a violation of the integrity of the epithelial lining of the seminiferous tubules, the absence of spermatids in them.In some places, degenerative changes of the seminiferous tubules, an increase in their diameter, and an expansion of the lumens were detected.The number of pachytene spermatocytes, rounded spermatids and pluripotent spermatocytes also decreased.At the same time, the general resistance capacity of Sertoli cells also underwent changes.Research of other scientists regarding the effect of the toxic venom components of these snakes demonstrated the presence of microscopic changes in the structure of the germinal epithelium in the adlumenal part of testis, atypical multinucleated cells were present in the lumen of the seminiferous tubules [7].

Conclusions
Injection of Vipera berus berus venom to rats leads to the development of pathogistological changes in the parenchymal elements of the testis of animals, among which desquamation and disorganization of spermatogenic cells of all stages of development, disruption of spermatogenesis processes were the most pronounced.Spermatogenic cells of the seminiferous tubules of the testis were distinguished by changes in morphology and location, lost the regularity of their placement, and their number decreased.The cells detached from the basement membrane and did not form a continuous layer.A characteristic feature was the presence of clusters of erythrocytes in the lumen of the seminiferous tubules, which may indicate the development of hemorrhagic complications.In addition, an increase in the number of Leydig cells was detected, which is probably the cause of changes in the hormonal regulation of the organ.

Fig. 1 .
Fig. 1.The stroma and seminiferous tubules of the testis of rats in the experimental group with the introduction of Vipera berus berus venom.A: full blood vessels with aggregated blood elements in the lumen (red arrow); B: seminiferous tubules of regular shape (green ovals); B, C: gaping space of seminiferous tubules without spermatogenic cells (yellow arrows); D: desquamated immature cells in the lumen of tubules (yellow oval).Staining with hematoxylin and eosin; A, B x200, C x100, D х400.