DYNAMICS OF GLYCOSAMINOGLYCANS CONTENT IN THE TESTES OF RATS AFTER ADMINISTRATION OF NATURAL HERBAL PRODUCTS *

It is possible that testicular dysfunctions, including idiopathic infertility in men, may be caused by changes in connective tissue, which are characterized by disorders of its metabolism, the content of collagen and glycosaminoglycans (GAGs). Such patients quite often have the anomalies of the genital system. It is the anomalies in the development of connective tissue structures of the venous wall and not compensated state of the valve apparatus that lead to varicocele. The prevalence of varicocele among infertile patients reaches 40 %, whereas male population in general has this disease only in 15 % of cases. Changes in GAG content may reflect the functional status of the semen, which ensures normal spermatogenesis. Therefore, the study of this indicator can be used to explain the mechanisms of infertility and to find ways to correct both hypo- and infertile conditions in men. of in the samples of testes and correction of this condition with such drugs as Tribestan, Chondroitin in alcohols of increasing strength and poured into paraffin. Detection of GAGs on sections was a metachromatic reaction by staining with toluidine blue at pH 2.5: there occurred a change in color from blue to purple color of various degrees of saturation due to the combination of a high degree of dissociation of the acid groups of the substrate. Results. It was shown the histochemical methods and metachromatic reaction with toluidine blue showed that intact sexually mature male rats’ testes had a sufficient number of glycosaminoglycans, which was visualized in the tape of the spermatogenic epithelium and small areas of metachromaticemia. The results of histochemical study of glycosaminoglycans content in animals with induced gonadopathy by administration of serotonin hydrochloride indicated glycosaminoglycans deficiency. Administration of Chondroitin sulfate, «Bioglobin-U» and Tribestan during serotonin lesions of the testes significantly increased glycosaminoglycans in the tubules and intercranial stroma.

According to the results of a modern study of the frequency of action of factors that can affect spermatogenesis (Androbase ® database), the cause of male infertility is often unknown. In this case varicocele, hypogonadism, infectious diseases of the male genital organs and cryptorchidism are the most common diseases in pathozoospermia (14.8 %; 10.1 %; 9.3 %, and 8.4 %, respectively) [1].
It is possible that testicular dysfunctions, including idiopathic infertility in men, may be caused by changes in connective tissue [2,3], which are characterized by disorders of its metabolism, the content of collagen and glycosaminoglycans (GAGs) [4,5]. Such patients quite often have the anomalies of the genital system. It is the anomalies in the development of connective tissue structures of the venous wall and not compensated state of the valve apparatus that lead to varicocele. The prevalence of varicocele among infertile patients reaches 40 %, whereas male population in general has this disease only in 15 % of cases [4,5].
It is known that human spermoplasm has a molecular composition that is not inferior in complexity to blood serum (Petrunin D.D. et al., 1996; Loko F., 2001). One of its features is a high concentration of proteoglycans which are high molecular weight carbohydrate-protein compounds. They are among the most fully studied biochemical components of human tissues with the exception of the reproductive Проблеми ендокринної патології № 2, 2020 Експериментальні дослідження system (Nikolaev A. A., 2000;Gainullina L. H. et al., 2003). According to the research of R. V. Vetoshkin [6,7], the concentration of proteoglycans in the rats' spermoplasma averages 0.77-0.82 mg/ml. These compounds in the spermoplasma are composed of approximately 70 % of Chondroitin sulfate proteoglycans and only 30 % of Heparan sulfate proteoglycans.
Today it has been proven that spermiogenic epithelium and Sertoli cells also synthesize glycosaminoglycans, which are then secreted into ejaculates. Chondroitin-4-sulfate, Chondroitin-6-sulfate, Keratansulfate and hyaluronic acid are known to be components of the tissue of epididymis and testes of rats [2]. Song X. X. et al. in their research showed the effect of Chondroitin sulfate C on the capacity and parameters of fertilization of sperm in animals in vitro [8]. The study showed that chondroitin sulfate C was an effective factor in the acti vity of sperm in pigs and could transfer sperm from the condensed state and consequently prevent the germ cells from spontaneous acrosome reaction, which facilitates their penetration into the oocyte. According to the research conducted by Makolinets K.V. et al., chondroitin sulfates can be used as biochemical markers of GAG metabolism [9]. Changes in GAG content may reflect the functional status of the semen, which ensures normal spermatogenesis. Therefore, the study of this indicator can be used to explain the mechanisms of infertility and to find ways to correct both hypo-and infertile conditions in men.
Therefore, the aim of our work was to study the content of glycosaminoglycans in the samples of the testes affected with serotonin hydrochloride and correction of this condition with such drugs as Tribestan, Chondroitin sulfate and Bioglobin-U.

MATERIALS AND METHODS
Animals were kept in the vivarium of the State Institution «V. Danilevsky Institute for Endocrine Pathology Problems of NAMS of Ukraine» in natural light and standard diet recommended for this species of animals and with water access ad libitum. The study was performed on sexually mature male rats weighing 280-350 g of the Wistar population. A serotonin lesion model was used to obtain the pathology of the testes; rats were given Serotonin hydrochloride (Alfa Aesar ® ) for 14 days, subcutaneously at a dose of 5 mg/kg body weight).
In the mechanism of the testes pathology model development, a leading role is played by the vasoconstrictive effect of serotonin hydrochloride which causes the disturbed trophic and pathological changes observed in varicocele in men, traumas and congestion in the gonads, etc. [10]. As noted in previous studies, Serotonin hydrochloride at a dose of 5 mg/kg body weight disrupts the metabolism of Sertoli and Ladig cells in rats. During the reproduction of this pathology, the synthetic and reparative acti vity of the testes decreased, as a consequence there was fragmentation of membranes of the endoplasmic reticulum, reduction of the number of ribosomes, polys and secretory granules, increase in the number of secondary ly-sosomes, as well as reduction of lamellar cytoplasmic degeneration, and decrease in the level of testosterone [11].
The reference drug was Tribestan, which is prescribed for the treatment of hypogonadism, disorders of spermatogenesis, namely oligo-and asthenospermia, which includes an extract of anchors (Tribulus terrestris) containing steroid saponins of furastanol type. The drug restores Leydig cells, stimulates spermatogenesis, increases sperm count, increases sperm motility [12].
Chondroitin sulfate, «Bioglobin-U» and Tribestan were given three days before the start of Serotonin hydrochloride administration, during Serotonin administration (14 days) and for three days after the last Serotonin injection once daily.
The animals were divided into six groups: the 1 st group was intact control; the 2 nd was control pathology, i.e. a pathology of the semen, in which animals were administered serotonin hydrochloride (Alfa Aesar ® ) for 14 days subcutaneously at a dose of 5 mg/kg body weight; the 3 rd group was control pathology + Tribestan (manufactured by Sopharma, Bulgaria) at a dose of 60 mg/kg body weight; the 4 th group was control pathology + substance Chondroitin sulfate (manufactured by «Sigma», USA), at a dose of 60 mg/kg body weight; the 5 th group was control pathology + «Bioglobin-U» (manufactured by CJSC «Biolik», Kharkiv), at a dose of 200 μl/kg body weight; and the 6 th group was control pathology + substance Chondroitin sulfate + Tribestan in the above specified dose. The euthanasia of rats was performed on the 21 st day of the experiment, the testes were isolated. There are 8 male rats in each group.
In this work we performed histochemical studies of rat testes. For the histochemical study of GAGs content in the samples of testes, they were fixed in 10 % formalin solution, dehyd rated in alcohols of increasing strength and poured into paraffin. Detection of GAGs on sections was a metachromatic reaction by staining with toluidine blue at pH 2.5: there occurred a change in color from blue to purple color of various degrees of saturation due to the combination of a high degree of dissociation of the acid groups of the substrate [13]. The degree of color saturation was evaluated accor ding to the principle of cell counting in histological samp les using a semi-quantitative method for visual assessment of the color intensity of microstructures during histochemical reactions [14]. A quantitative assessment of the degree of color intensity was expressed in points: 1) orthochromatic reaction -0 point; 2) low metachromatic reaction -1 point; 3) average metachromatic reaction -2 points; 4) significant metachromatic reaction -3 po ints; 5) extremely significant metachromatic reaction -4 points. The investigation has been carried out according to the National «General Principles for Animal Research Ethics» (Ukraine, 2001) which corresponds to the «European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes», (Strasburg, 1985), and «Guide for Research Ethic Committee», 2012.
The data was processed using the software package «Statistica v. 10.0». If the data were not consistent with the normal distribution, the comparison of indicators was performed according to the non-parametric Wilcoxon criterion with calculation of the median (Me) and percentiles (25 % and 75 %). Critical level of significance was assumed to be equal to or less than 0.05 (p < 0.05).

RESULTS AND THEIR DISCUSSION
In the gonads of intact rats, violet staining in the ribbon of the spermatogenic epithelium was detected in the tubules by staining with toluidine blue. Small areas of metachromasia (the same purple color) were visualized in the inter-canal stroma, indicating the presence of GAGs. The degree of color saturation was 4.00 ± 0.53 (3.30 ÷ 0.00) points. The intact connective tissue in young intact adult rats (body weight 280-350 g) was combined with the normal morphological condition of the testes, which tracked all stages of spermatogenesis and their functional activity (namely, the normal level of sex hormones, quantitative and qualitative indicators of spermatogenesis).
Reproduction of Serotonin pathology of gonads in the seminiferous tubules and intercranial stroma shows an orthochromatic reaction (almost complete absence of purple color) when staining with toluidine blue, which advocates a deficiency of GAGs compared with intact control (Fig. 1). The degree of color saturation was 0.00 ± 0.20 (0.00 ÷ 0.08) points. Such a histochemical picture in serotonin lesions of the testes may explain the fact that the level of Tc decreases in the animals with experimental (control) pathology, because the decrease in the amount of loose connective tissue providing «nutrition» of Leydig cells in the intact animals, probably, led to the reduction of their functional activity level. Violation of the functional state of the testes in case of serotonin loading was reported by other authors [10] and by us in earlier research [11].
In addition, as it was expected, serotonin causes significant submicroscopic changes of the organisms of glondulocytes and dystrophicdestructive disorders in the ultrastructure of their mitochondria accompanied by hypotestosteronemia. Moreover, animals with serotonin pathology had an intensive decrease in the number of ribosomes and polysomes in the cytoplasm of Sertoli cells [11], the so-called supporting cells, which also ensure the normal Проблеми ендокринної патології № 2, 2020 Експериментальні дослідження state of spermatogonia and other spermatogenesis cells. The decrease of ribosomes and polysomes in the cytoplasm of Sertoli cells together with a very small number of GAGs, may cause abnormality of spermogram.
Chondroitin sulfate, administered to rats with serotonin pathology of the testes, signifi-cantly increased the content of GAGs in the intercranial stroma and in the tubules, as evidenced by the presence of purple staining zones in the intercranial stroma and areas of location of the germ cells (Fig. 1). The degree of color saturation in this group of animals was 3.00 ± 0.33 (3.00 ÷ 3.00) points. Metachromatic reaction with toluidine blue. Administration of Bioglobin-U, together with serotonin was also accompanied by a rather pronounced metachromatic reaction visualized in the tubules and inter-canal stroma, which indicated an increase of GAGs under such experimental conditions (Fig. 2). The degree of color saturation was 3.00 ± 0.33 (3.00 ÷ 3.00) points.
Tribestan administration to animals with experimental gonadopathy, contributed to the appearance of metachromatic zones in the ribbon of the spermatogenic epithelium and intercranial stroma, but the severity of the reaction was moderate, especially 2.00 ± 0.00 (2.00 ÷ 2.00) points. However, the combined administration of Chondroitin sulfate and Tribestan to rats with serotonin pathology of the testes contributed to the increase of metachromasia in the inter-canal stroma compared with the separate administration of these substances (Fig. 2). The degree of color saturation in this group of animals was 3.50 ± 0.33 (3.00 ÷ 4.00) points. The restoration of the quantitative content of chondroitin sulfates in the testes of rats coincided with the revealed increase in the degree of metachromasia according to the histological examination, which indicated the interrelation of the GAGs content in the spermiogenic epithelium, and may be the basis for a more complete restoration of the functional state of testes and potentiate Tribestan action for some forms of infertility. Morphological and histological examinations of gonadopathy animals treated with Chondroitin sulfate, Bioglobin-U, Tribestan, and Tribbestan + Chon droitin sulfate correlate with spermatogenesis and male sex hormone level previously determined in these animals [14,15].
Thus, the results of histochemical examination of rats' testicular tissues by chondroitin sulfate content may indicate a positive effect of GAG-containing drugs, in particular, Chondroitin sulfate and «Bioglobin-U», on the morpho-functional state of gonads in rats.
К л ю ч е в ы е с л о в а : Хондроитина сульфат, серотонина гидрохлорид, варикоцеле, гликозаминогликаны, метахромазия, сперматозоиды. It is possible that testicular dysfunctions, including idiopathic infertility in men, may be caused by changes in connective tissue, which are characterized by disorders of its metabolism, the content of collagen and glycosaminoglycans (GAGs). Such patients quite often have the anomalies of the genital system. It is the anomalies in the development of connective tissue structures of the venous wall and not compensated state of the valve apparatus that lead to varicocele. The prevalence of varicocele among infertile patients reaches 40 %, whereas male population in general has this disease only in 15 % of cases. Changes in GAG content may reflect the functional status of the semen, which ensures normal spermatogenesis. Therefore, the study of this indicator can be used to explain the mechanisms of infertility and to find ways to correct both hypo-and infertile conditions in men.

DYNAMICS OF GLYCOSAMINOGLYCANS CONTENT
Therefore, the aim of our work was to study the content of glycosaminoglycans in the samples of the testes affected with serotonin hydrochloride and correction of this condition with such drugs as Tribestan, Chondroitin sulfate and Bioglobin-U.
Materials. A serotonin lesion model was used to obtain the pathology of the testes; rats were given Serotonin hydrochloride (Alfa Aesar®) for 14 days, subcutaneously at a dose of 5 mg/kg of body weight) and on the background of it was administered Chondroitin sulfate at a dose of 60 mg/kg of body weight, Bioglobin-U at a dose of 200 μl/kg of body weight and Tribestan -60 mg/kg of body weight. In this work we performed histochemical studies of rat testes. For the histochemical study of GAGs content in the samples of testes, they were fixed in 10 % formalin solution, dehydrated in alcohols of increasing strength and poured into paraffin. Detection of GAGs on sections was a metachromatic reaction by staining with toluidine blue at pH 2.5: there occurred a change in color from blue to purple color of various degrees of saturation due to the combination of a high degree of dissociation of the acid groups of the substrate.
Results. It was shown the histochemical methods and metachromatic reaction with toluidine blue showed that intact sexually mature male rats' testes had a sufficient number of glycosaminoglycans, which was visualized in the tape of the spermatogenic epithelium and small areas of metachromaticemia. The results of histochemical study of glycosaminoglycans content in animals with induced gonadopathy by administration of serotonin hydrochloride indicated glycosaminoglycans deficiency. Administration of Chondroitin sulfate, «Bioglobin-U» and Tribestan during serotonin lesions of the testes significantly increased glycosaminoglycans in the tubules and intercranial stroma.
K e y w o r d s : Chondroitin sulfate, Serotonin hydrochloride, varicocele, glycosaminoglycans, metachromatic reaction, sperm.