DISORDER OF SPERMATOGENESIS UNDER CONDITIONS OF EXPERIMENTAL CHRONIC KIDNEY DISEASE

Purpose - to estimate the disorder of spermatogenesis under conditions of experimentalchronic kidney disease (EChD).Material and methods. The study was carried out in two series of experiments on malemice with EChD, the model of which was created by immunizing animals with a kidneyhomogenate. The first series of experiments was devoted to the study of: sperm count(sperm concentration (mln / ml)) and the number of abnormal sperm forms; the ratio ofcells of different generations of spermatogenic epithelium (%) in the testes; pathways ofcell death of cells of the testes and epididymis (spermatocytes (primary) and spermatozoa).The fertile qualities of males were assessed in the second series of experiments, afterreplanting them to intact females. Pre- and post-implantation embryonic mortality andthe number of living fetuses per female mouse have been investigated. The research resultswere compared with the performance of animals in the control groups for each series.Results. No significant changes in the number of spermatozoa were found under EChDconditions (p> 0.05). An increase in the number of abnormal spermatozoa (22%) andthose with primary abnormalities (p <0.05) was found. Among the generations of testescells, a decrease in the number of spermatids and living spermatocytes (primary) (15%)was established, with an increase in the number of cells with apoptosis and necrosisamong them (p <0.05). The number of living cells of the epididymis (spermatozoa) alsodecreased (17.8%), with the growth of cells with apoptosis and necrosis among them (p<0.05). There was an increase in the pre- and post-implantation mortality of embryos (p<0.05); decrease in the number of living fetuses (p <0.05).Conclusions. Under conditions of four-time treatment with renal homogenate (EChD)there is a disorder of spermatogenesis in male mice. Experimental model of kidneydamage, proposed by us, can be useful for studying other aspects and consequences ofkidney pathology, and both for establishment of the features and detection of possiblepathogenetic links in the development of spermatogenesis disorder under conditions ofchronic kidney disease and search of the effective ways to correct it in future.

Efficiency of Combined Use of Fullerene C60 and Bovine Serum Albumin for Rehabilitation of Vitrified Fragments of Rat Immature Seminiferous Tubules

Background. Today, cryopreserved reproductive tissues are used to treat some forms of male infertility. However, after long-term preservation of fragments of seminiferous tubules of testes (FSTT) in a low-temperature bank (-196 °С) their morphological and functional characteristics decrease reversibly. To solve this problem after freezing-thawing, the use of rehabilitation media with special additives is promising. Due to the fact that serum albumin and fullerene C60 have powerful protective and antioxidant properties, their use allows to stabilize the plasma membrane, osmotic pressure, and reduce free radicals that make them promising candidates to use in the development of rehabilitation media for biological objects after cryopreservation. Objective. The efficacy of fullerene C60, bovine serum albumin (BSA), and their combination as components of rehabilitation medium of vitrified FSTT of immature rats was studied. Methods. Vitrified-thawed samples of FSTT were incubated (22 °C) for 30 minutes in Leibovitz's medium with addition of 15 mg/mL C60, 5 g/L BSA or their combination. Control samples were incubated in the medium without C60 or BSA addition. Metabolic activity (MTT test), histomorphological data, total antioxidant status (TAS), reactive oxygen species (ROS) production, activity of g-glutamyltransferase (gGGT), and glucose-6-phosphate dehydrogenase were determined in the samples after rehabilitation in the investigated media. Results. The use of C60 led to the increase of metabolic (by 1.26 times) and TAS (by 1.74 times) activities, to the decrease in the number of ROS+ cells (by 1.35 times) and to the improvement of the spermatogenic epithelium binding to the basement membrane versus control sample. Application of BSA did not significantly affect the studied biochemical indices but decreased the number of tubules with desquamation of spermatogenic epithelium in histological sections. The combined use of BSA and C60 had the best effect among investigated rehabilitation media that led to the increase of metabolic activity (by 1.51 times), TAS activity (by 1.78 times), gGGT activity (by 1.59 times), histostructure restoration and the decrease in the number of ROS+ cells (by 1.45 times) compared to the control samples. Conclusions. The use of C60 and BSA combination increases the metabolic and antioxidant activity of vitrified FSTT and also has a positive effect on their histostructural characteristics compared to control samples. It should be noted that the effect of С60 and BSA addition to rehabilitation medium exceeds the results of using the investigated additives separately (by the metabolic and gGGT activity as well as architectonics of vitrified FSTT). These data relate to reproductive medicine and can be used to develop an effective rehabilitation protocol for vitrified FSTT.

Morphological testicular changes following the experimental inguinal hernia repair modeling

Purpose of the study. To evaluate morphological changes in the testes in experimental animals after tension-free inguinal hernia repair modeling.Materials and methods. The study included male rabbits, aged 120 days, weighing 3.8 ± 0.9 kg. All rabbits were divided into two groups depending on the type of operation: in the first group (n = 10) of animals, we made a model of tension-free inguinal hernia repair and used a polypropylene mesh; in the second group (n = 10) of animals, we left the structures of the inguinal canal intact. Morphological assessment of spermatogenesis was performed after 40 days. All morphometric measurements were carried out on strictly cross-sections of the convoluted seminal tubules.Results. In rabbits of group 1, the volume of the testicle was significantly three times less than in animals of group 2 (p < 0.05). In the animals of the first group, a significant deterioration in spermatogenesis was observed (p < 0.05). Histological examination of sections of the testes of these animals showed that hypoplasia of the spermatogenic epithelium was present in the convoluted seminal tubules, in 1/8 of the tubules there was subtotal aplasia of the spermatogenic epithelium, Sertoli-Cell-Only Syndrome was detected only in 2 %. Atrophy of the convoluted seminal tubules was not recorded at this period of observation. In the animals of the control group, almost 90 % of cases of spermatogenesis disorders were not detected.Conclusions. This experimental study on rabbits showed that after using a polypropylene mesh for inguinal canal plastic, inhibition of germ cell maturation occurs after 1.5 months. Therefore, in men of reproductive age, it is necessary to use polypropylene mesh implants with caution in terms of performing inguinal hernia repair.

Expression of the NSE, SP, NFH and DβH in Normal and Cryptorchid Testes of Bactrian Camel

Background: Neuroendocrine substances play important roles in regulating the normal physiological functions of testicles.The purpose of this study is to explore the localization and effects of four neuroendocrine markers(NSE, SP, NFH and DβH) in normal and cryptorchid testes of Bactrian camels.Methods: The cryptorchid testes were located in the abdominal cavity and were collected by orchiectomy. Fresh testes tissues were processed into small pieces and then divided into three samples. One sample was frozen in liquid nitrogen for western blotting hybridization reaction, while the other sample was fixed with 4% paraformaldehyde solution for histochemical analysis, and the third fixed in glutaraldehyde for transmission electron microscopy observation.Results: The results showed that cryptorchidism caused a reduction in layers of spermatogenic epithelium and decreased glycogen positivity in the basement membrane.The ultrastructure revealed that macrophages were always found around the Leydig cells, which were crowded with swelling mitochondria in cryptorchidism. Expression of NSE in the Leydig cells of cryptorchidism was significantly weakened compared to that in the normal group(p<0.01). We found that SP was always distributed along the nerve fibers in normal testes and was expressed in the Leydig cells of cryptorchidism. However, expression of NFH in the cryptorchidic tissue was strongly positive in spermatogenic epithelium, with limited expression in Leydig cells and no expression in peritubular myoid cells. Therefore, expression of DβH in the Sertoli cells was comparatively strong in both the normal and cryptorchidism groups.NFH and DβH expression was significantly increased in the cryptorchidism group compared with the nomal group(p<0.01).Conclusions: These findings indicated that the underdeveloped seminiferous epithelium and pathological changes in cryptorchid tissue in Bactrian camels were potentially related to a disorder in glycoprotein metabolism.Our results suggest that NSE and SP could helpful to judge the pathological changes of cryptorchidism. The present study provides the first evidence at the protein level for the existence of NFH and DβH in Sertoli and Leydig cells in Bactrian camel cryptorchidism and provides a more in-depth understanding of neuroendocrine regulation is crucial for animal cryptorchidism.

The Use of Fullerene C60 to Preserve Testicular Tissue after Cryopreservation

Autologous transplantation of cryopreserved fragments of an immature testis is an actively developing approach to save fertility in patients facing a gonadotoxic therapy. The use of bioavailable fullerene C60 as a powerful antioxidant opens up a new potential for the prevention and correction of ischemic-reperfusion pathological processes in tissues including those associated with freezing-thawing procedure. In this work, we aimed to study the antioxidant status, apoptotic/necrotic processes, and morphological characteristics of cryopreserved fragments of the seminiferous tubules of testis (CrFSTT) of immature rats after incubation in media with different concentrations of fullerene C60 (10, 15, and 20 μg/mL). Our results indicated that the addition of C60 in a concentration of 15 μg/mL decreased ROS production, cytochrom C release, and degree of histological damage of spermatogenic epithelium as well as increased the activity of the mitochondria, antioxidant defense system, and cell density in histological sections of CrFSTT compared to the control. Fullerene C60 at investigated concentrations did not impact significantly on apoptosis in cells of CrFSTT but, after incubation with 15 μg/mL C60, a percentage of living cells was 1.2-fold higher and a value of necrotic ones in this group was 1.6-fold lower than the control samples ( p < 0.05 ). Relative amount of cells of the spermatogonia germ layer did not differ between the studied concentrations. The general analysis of obtained data showed that the C60 addition in the concentration of 15 μg/mL was the most optimal for the rehabilitation of CrFSTT. The results can be used for the development of an effective rehabilitation medium for the cryopreserved testicular tissue.

MORPHOLOGYCAL CHANGES IN THE TESTES OF EXPERIMENTAL ANIMALS IN CHRONIC ALCOHOL POISONING

The experiment with the chronic effects of alcohol on experimental animals reflects the picture of long-term use of alcoholic beverages by a person with alcoholism. It is impossible to fully study the ultrastructural changes in internal organs, including the reproductive system, in humans, both in acute and chronic alcohol consumption, but the results obtained in the experiment can be extrapolated to humans. The results obtained showed that changes in the parenchymal-stromal elements of the testicles during chronic alcoholization are reduced to dystrophic transformation of the spermatogenic epithelium. The testicular stroma is characterized by pronounced circulatory disorders with plethora and stasis in the postcapillaries and small veins, edema, phenomena of perivascular and interstitial sclerosis, with foci of pronounced hyalinosis, most expressed by 2-3 months of alcoholism. By 6 months of the model experiment, there was no dynamics of the growth of morphological changes, partial adaptation to constant intoxication with ethyl alcohol.

Morphological features of spermatogenesis in rats after targeted irradiation with electrons of variable doses

Introduction. Today, a primary concern of humanity is male infertility. Recent figures suggest that the disease affects 186 million people worldwide, and its prevalence increases as men get older. In literature the radiation therapy effect on the male reproductive system is debated. Over the past decade, scientific and technological progress has definitely accelerated the development of the radiation therapy methods, and as new devices have emerged, there is a need to carefully select new dosing regimens. The aim of the experimental study was to evaluate morphological spermatogenesis indicators after targeted irradiation with electrons of variable doses. Materials and methods. Wistar rats (n=70) were once subjected to targeted ionizing irradiation on a pulsed electron accelerator NOVAC 11 with doses of 2 Gy, 4 Gy, 6 Gy, 8 Gy, 10 Gy, and 12 Gy, and a week later spermatogenesis was assessed with light microscopy. Results. In the experimental groups, we revealed a decrease in the number of germ cells; signs of degenera-tive changes and lysis of spermatids and spermatozoa; the appearance of intense pyknotic nuclei in primary spermatocytes; and a sharp decrease in the number of spermatogonia and Sertoli and Leydig cells without change in their structure. In the irradiation groups of 8–12 Gy, there was the deepest damage to the tubules, up to spermatogenic epithelium aplasia, and extensive vacuolization. Conclusion. In the testes, we observed dose-dependent progressive pathological changes in tissue structure as well as a decrease in the number of spermatogonia and other germ cells on day 7 after irradiation with a pulsed electron accelerator NOVAC 11. Keywords: electron irradiation, male infertility, spermatogenesis, seminal globs

MORPHOMETRIC EVALUATION OF SPERMATOGENIC EPITHELIOCYTES STRUCTURE UNDER THE RUBOMYCIN INFLUENCE IN RATS

Background. Over the last 20 years, the quality of men’s sperm around the world has deteriorated twice. The causes are poor environment, stress, sedentary lifestyle, prevalence of sexually transmitted infections, obesity, alcohol abuse and various stimulants. The structure and function of the testes, when exposed to various drugs and chemicals, attracts the attention of present researchers. Objectives. The aim of the research was to study the structural changes of the spermatogenic epithelium in testicular damage caused by rubomycin hydrochloride. Methods. The spermatogenic epithelium of the testes of 30 white adult white male rats, which were divided into 2 groups, was morphometrically examined. The 1st group comprised 15 experimental intact animals, the 2nd – 15 rats, in which testicular damage with rubomycin hydrochloride was simulated. Euthanasia of rats was performed by bloodletting under thiopental anesthesia one month after the experiment beginning. Quantitative morphological parameters were statistically processed. In spermatocytes of the 1st P-order, spermatogonia and spermatids of testes, their height, diameter of nuclei, nuclear-cytoplasmic ratio and relative volumes of damaged cells of spermatogenic epithelium were evaluated. Results. It was established that spermatogenic epithelial cells were damaged in the simulated experimental conditions, which was morphometrically confirmed by changes in nuclear-cytoplasmic relations in the 1st, 2nd order spermatocytes, spermatogonia and spermatids. The relative volumes of damaged spermatogenic epithelial cells also increased significantly. The most significant morphometric parameters were altered in spermatids. Thus, the nuclear-cytoplasmic ratio in the studied cells was statistically significantly (p <0.001) increased by 7.4% compared with the similar control morphometric parameter. The relative volume of damaged spermatids in these experimental conditions reached (32.50±0.18) %, which with a high statistically significant difference (p<0.001) exceeded the same control value in almost 15.8 times. Optically, histological preparations of the testes showed severe vascular disorders, characterized by dilation and plethora mostly of venous vessels, which pointed to violation of venous drainage of the studied organs. Conclusions. According to the attained research results it has been established that nuclear-cytoplasmic relations are an objective and valuable informative indicator of a functional condition of cells and their structural changes in pathological conditions. When rubomycin hydrochloride is exposed to experimental animals, the most significant changes in nuclear-cytoplasmic relations are found in spermatic spermatozoa.

Evaluation of Rats’ Spermatogenesis State under Irradiation Exposure and Influence of Natural Origin Drugs

Male infertility is probably to be affected by intense heat exposure and extreme exposure to pesticides, irradiation, electromagnetic radiation and other hazardous substances. The authors of most of the works agree that the cells of the basement membrane of the tubules and spermatogonia, which are quickly exhausted after irradiation, are the most vulnerable in the testes. The type and extent of these changes depends on the dose, duration and frequency of radiation. The participation of connective tissue in almost all typical pathological conditions has been shown: inflammation, allergy, carcinogenesis, hypoxia, free radical oxidation processes, etc. The purpose of the work was to study evaluation the state of rats' spermatogenesis under conditions of the total irradiation exposure and to correct the pathological process with drugs of natural origin Chondroitin sulfate and "Bioglobin-U". Material and methods. A model of total single irradiation with a dose of 3 Gy was used to obtain the pathology of the testes, after which the substance Chondroitin sulfate was administered at a dose of 60 mg / kg; comparison drug Tribestan at a dose of 60 mg/kg; the drug "Bioglobin-U" at a dose of 200 μl / kg and Chondroitin sulfate + Tribestan in the above doses. Results and discussion. 30 days of exposure of single total irradiation with a dose of 3 Gy led to significant disruption of the morphological state of the testes and spermatogenic epithelium of rats. Thus, in rats with irradiation, the number of normal spermatogonia in the convoluted tubules was reduced by 5.7 times, there were practically no convoluted tubules with spermatocytes in the metaphase of the 2nd maturation division (with the 12th stage of meiosis) and tubules with desquamated spermatogenic epithelium, the index of spermatogenesis decreased by almost 6.5 times. The introduction of natural origin drugs led to an improvement in the quantitative and qualitative indicators of spermatogenesis. Conclusion. In the conditions of irradiation exposure, Chondroitin sulfate has more significant regenerative properties, the effect of which is almost 1.9 times better than the effect of the reference drug Tribestan. The effectiveness of "Bioglobin-U" in these experimental conditions was somewhat inferior to Chondroitin sulfate, but this drug was almost 1.4 times more effective than the reference drug Tribestan. The combination of Tribestan and the substance Chondroitin sulfate was more than 1.4 times more effective than the reference drug Tribestan itself