UDC: 611.6+59.084:615.37
Kashchenko S.A., Zakharov A.A.
Department of histology, cytology and embryology of State Institution of Lugansk People’s Republic «St. Luke Lugansk State Medical University». Lugansk, 50–years of Lugansk Defense bl., 1g.

Influence of immunostimulation on the testes morphostructure of reproductive period animals

Abstract. Background. In recent years, the influence of various exogenous factors on the human body has significantly increased, which is due to the deterioration of the ecological situation, the expansion of the spectrum of drugs used in practical medicine, etc. Particularly relevant is the issue of environmental immunosuppression, characteristic of large industrial regions, including the Donbass. Changes in the functioning of regulatory systems naturally affect the work of the whole organism. It has been clinically proven that the inhibition of the functioning of the immune system causes certain shifts in the work of the male reproductive system, however, there is no morphological confirmation of this, or the available data are contradictory and do not give a complete picture of changes in the morphogenesis of organs. Aim. To study the influence of immunostimulation on the structure of testes of experimental animals of the reproductive period.

Key words: immunostimulation; imunofan; testes; rats.

Contact person:

Zakharov Aleksey Aleksandrovich
Candidate of Medical Sciences, Associate Professor of the Department of Histology, Cytology and Embryology,
SI LPR «St. Luke Lugansk State Medical University»; 91045, Lugansk,
50th anniversary of the Defense of Lugansk bl., 1g. Phone: +380509678705, e–mail: Этот адрес электронной почты защищен от спам-ботов. У вас должен быть включен JavaScript для просмотра.

Introduction. In recent years, the influence of various exogenous factors on the human body has significantly increased, which is due to the deterioration of the ecological situation, the expansion of the spectrum of drugs used in practical medicine, etc. Particularly relevant is the issue of environmental immunosuppression, characteristic of large industrial regions, including the Donbass [3, 9]. The regulation of the organism's activity is carried out by cooperating the activities of the nervous, endocrine and immune systems, which necessarily react to changes in the environment [8]. Changes in the functioning of regulatory systems naturally affect the work of the whole organism.
Significant interest in their study in recent years is due to the growing number of immunopathological conditions among the population, among which the leading role is played by immunodeficiencies. It has been clinically proven that the systemic suppression of the functioning of the immune system causes certain shifts in the work of the male reproductive system, but there is no morphological confirmation, or the available data are contradictory and do not give a complete picture of organ morphogenesis changes [6]. Recently, preparations of the immunomodulating series have been widely used to correct immunosuppressive states. One of the new representatives of this group is imunofan, created using nanotechnology. This drug is approved in clinical practice; questions concerning its influence on the structure of the immune system are covered only in single publications, whereas structural changes in the organs of the male reproductive system at immunostimulation with imunofan have not been studied at all.

Aim. To study the influence of immunostimulation on the structure of testes of experimental animals of the reproductive period.
Material and methods. The study was carried out on 60 mature white male rats, obtained from the laboratory vivarium of the SI LPR «St. Luke Lugansk State Medical University». When working with animals guided by the Directive 2010/63 / EU of the European Parliament and the Council of the European Union for the protection of animals used for scientific purposes [4]. Imunofan is the representative of the IV generation of thymic hormone derivatives, created with the help of nanotechnology. The drug was administered according to the scheme on the 1st, 3rd, 5th, 7th, 9th day of the experiment at a dosage of 0.7 μg/kg of body weight of the animal [7]. The controls were rats that received 0.9% sodium chloride solution in equivalent volumes and according to the same scheme. The animals were taken out from the experiment at 1, 7, 15, 30 and 60 days after the drug was discontinued. The organs were weighed on a torsion balance, the relative mass was calculated, and linear dimensions were determined by means of a caliper: length, width and thickness. According to the formula for the volume of the ellipsoid of revolution, the volume of the organ was calculated:

where A is the length, B is the width, and C is the thickness. Testes were subjected to standard histological wiring, the resulting sections 4–6 μm thickness were stained with hematoxylin–eosin and photographed using the Olympus CX–41 automated morphometric complex. Micromorphometric measurements of objects were carried out by analyzing the obtained digital images in the computer program ASCON "Compass–3D 17.0", using a calibration file created with the help of photographs of the object–micrometer in similar
modes of shooting. The study used only those tubules along which the cut was perpendicular to its central axis. The following parameters were calculated: the larger and smaller diameters, the area of the tubule, the height and area of the epitheliospermatogenous layer. The index of spermatogenesis (I) was calculated by the formula:

where A is the number of rows of spermatogenic cells found in each tubule (1 row – spermatogonia, 2 – spermatocytes, 3 – spermatids, 4 – spermatozoa); n is the number of studied tubules [5]. The obtained data was processed using the licensed program "StatSoftStatistica v6.0". The methods of parametric statistics were applied, since the use of the Shapiro–Wilk criterion permitted the establishment of a normal distribution of the indicators in the sample. The reliability of the differences between the indices of the experimental and control groups was determined with the help of the Student–Fisher criterion with an error probability p<0.05 permissible for biomedical research.

Results and discussion. Testes of the rat are located in the scrotum, directly connected with the abdominal cavity, have an oval shape, each has the cranial and caudal poles, the medial and lateral surfaces, the ventral and dorsal margins. Outside the organ is covered
with a connective tissue capsule that extends into the body by the septum, dividing it into lobules containing convoluted seminiferous tubules, which are located in different planes. Epitheliospermatogenous layer densely adjoins the wall of the tubule and is represented by sustantocytes, as well as by rows of sperm progenitors located at different stages of spermatogenesis (Fig. 1).

The organometric parameters of the examined organs of the rats of the control groups underwent progressive changes in connection with the regular processes of their morphogenesis (Table).

After the end of the imunofan administration, the principle of the structure of the testes did not change, but changes in the organometric parameters of the organs were revealed. Thus, a statistically significant difference in parameters was established on the 30th and 60th days after the end of the drug administration: the absolute mass increased by 7.12%, and 10.84%, respectively. The values of the relative mass changed unidirectionally and synchronously: the increase in the indicators was 5.77%, and 6.59%, respectively, in the same observation period. Linear and volume parameters of organs after application of immunosuppressor underwent similar changes. So, significant deviations were established on 30 and 60 days (Fig. 2).

After the end of the drug administration, the changes in the micromorphometric parameters of the testes were observed at the same time, with a tendency to increase with respect to the control groups of the animals. After the application of imunofan, the morphometric parameters of the convoluted seminiferous tubules significantly exceeded the control data.

Thus, the larger and smaller diameters increased by 5.67%, 9.17%, and 5.12%, 8.35%, respectively, on 30 and 60 days of observation. The areas of the tubule and epitheliospermatogenous layer increased synchronously, by 8.05%, 10.36%, and 7.31%, 10.04%
(Figure 3).

The height of the latter, as well as the index of spermatogenesis, statistically significantly exceeded the control group of animals. Thus, on the 30th and 60th days after the immunostimulant was discontinued, these parameters increased by 7.91%, 13.31% and 10.08%, 9.64%, respectively. At the 1st, 7th and 15th days of the observation, not all the examined organ parameters differed significantly from the control data.
It has been proved that immunosuppressive influences change the morphogenesis of testes, which is accompanied by their dysfunction in the form of disturbances of spermatogenesis up to azoospermia [1, 10]. The obtained results testify to the development of the re–adaptation processes in the testes after the application of the immunomodulator against the background of the ecological immunosuppression, which is reflected in the prevalence of the morphometric parameters of the organ under study over the control group of the animals. So, J. Gold and V. Vardhani (2017) in studies in mice have demonstrated an increase in the level of testicular DNA after the use of an immunostimulant [11]. This can be explained by the properties of imunofan to increase the stability of the genetic material of cells to adverse exogenous effects.

The ability of the drug to stimulate the production of cytokines normalizes the function of immunocompetent cells, which is manifested in the stabilization of the immune homeostasis of the testes. At the same time, the long–acting phase of imunofan is accompanied by an intense antioxidant and detoxifying effect, which prevents damage to actively dividing gonad cells. Also I.V. Bobrysheva (2013) established facts confirming the mutual regulation of the immune and endocrine systems both by direct influence on effector cells and by hormonal regulation of the hypothalamic–pituitary system [2]. Consequently, the use of imunofan can modulate the activity of tropic cells of the adenohypophysis, which, in turn, affects the morphogenesis of the testes.

Conclusions.
1. In response to the use of imunofan, there is an active reaction from the testes of animals of reproductive age.
2. Statistically significant changes in the organ's parameters of the organ are noted in the late periods of the experiment (30 and 60 days of observation), which is explained by the pharmacodynamic features of the drug, which has the most intensive stabilizing, antioxidant and detoxifying effect during the long phase of action.
3. Micromorphometric data of testes confirm the development of readaptation processes at late observation periods after application of imunofan against the background of environmental immunosuppression.
4. The data obtained cause interest in the study of the morphological features of the organ of immature animals and rats of pronounced senile changes.

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