High-fructose consumption suppresses insulin signaling pathway accompanied by activation of macrophage and apoptotic markers in rat testis.

Dietary high-fructose may cause metabolic disturbances; however, its effect on the reproductive system is little understood. The insulin signaling pathway is critical in testicular development, maintenance of microcirculation and spermatogenesis. Therefore, in this study, we aimed to investigate the impact of dietary high-fructose on insulin signaling pathway as well as macrophage and apoptotic markers in testicular tissue of rats. Fructose was administered to male Wistar rats as a 20% solution in drinking water for fifteen-week. Gene expression of ir-ß, irs-1, irs-2, pi3k, akt, mtor, and enos in the testicular samples was determined by real-time PCR. Protein expression of IR, IRS-1, IRS-2, PI3K, Akt, phospho-Akt (p-Akt), mTOR, eNOS, phospho-eNOS (p-eNOS), and GLUT5 was established by analysis of Western Blot. Testicular expression of occludin, CD163, CD68, caspase-8, and caspase-3 was analyzed by using immunohistochemical assay. Testicular level of fructose was measured by colorimetric method. Dietary high-fructose decreased mRNA expressions of irs-1, irs-2, pi3k, and mtor in the testicular tissue of rats. Also, this dietary intervention impaired protein expressions of IR, IRS-1, IRS-2, PI3K, p-Akt, mTOR, eNOS, and p-eNOS as well as p-Akt/Akt and p-eNOS/eNOS ratios in the testis of rats. However, a high-fructose diet increased the expression of CD163, CD68, caspase-8 and caspase-3, but decreased that of occludin, in the testicular tissue of rats. The high-fructose consumption in rats suppresses testicular insulin signaling but activates macrophages-related factors and apoptotic markers. These changes induced by dietary fructose could be related to male reproductive dysfunction.

Expression of RSPH6A in the first wave of rat spermatogenesis and oxidative stress conditions: Attenuation by melatonin.

Purpose: Here, we report, for the first time, the temporal expression and localization of axonemal radial spoke head homolog A (RSPH6A) protein during the first wave of rat spermatogenesis and in oxidative stress conditions. Methods: For the developmental study, testes were collected from rats at different developmental stages (7, 14, 21, 28, 35, 42, and 60 postnatal days); for in vivo treatment, 24 rats were treated with cadmium and/or melatonin. From each sample, western blot (WB) and immunofluorescence (IF) analyses for RSPH6A were performed. Results: RSPH6A expression starts at 21 PND alongside the appearance of I spermatocytes (SPC) with a significant increase up to 60 PND. Data were confirmed by IF analysis, showing that RPSH6A expression is restricted to I and II SPC, spermatids, and mature sperm. In vivo experiments showed that the expression and localization of RSPH6A in the testis and epididymal spermatozoa of adult rats treated with cadmium were impaired. Interestingly, melatonin (an antioxidant), given together with Cd, can counteract its damaging effects. Conclusions: All combined data confirm that RSPH6A contributes to the onset of fertility by acting on sperm motility, raising the possibility of using RSPH6A as a marker for normal fertility in the general population.

Differential Expression and Localization of EHBP1L1 during the First Wave of Rat Spermatogenesis Suggest Its Involvement in Acrosome Biogenesis.

The identification and characterization of new proteins involved in spermatogenesis is fundamental, considering that good-quality gametes are basic in ensuring proper reproduction. Here, we further analyzed the temporal and spatial localization during the first spermatogenic wave of rat testis of EHBP1L1, which is involved in vesicular trafficking due to the CH and bMERB domains, which bind to actin and Rab8/10, respectively. Western blot and immunofluorescence analyses showed that EHBP1L1 protein expression started at 21 days post-partum (dpp) concomitantly with the appearance of primary spermatocytes (I SPC). In subsequent stages, EHBP1L1 specifically localized together with actin in the perinuclear cytoplasm close to the acrosomal and Golgian regions of spermatids (SPT) during the different phases of acrosome biogenesis (AB). Moreover, it was completely absent in elongated SPT and in mature spermatozoa, suggesting that its role was completed in previous stages. The combined data, also supported by our previous report demonstrating that EHBP1L1 mRNA was expressed by primary (I) and secondary (II) SPC, lead us to hypothesize its specific role during AB. Although these results are suggestive, further studies are needed to better clarify the underlying molecular mechanisms of AB, with the aim to use EHBP1L1 as a potential new marker for spermatogenesis.

A novel xeno-organoid approach: exploring the crosstalk between human iPSC-derived PGC-like and rat testicular cells.

Specification of germ cell-like cells from induced pluripotent stem cells has become a clinically relevant tool for research. Research on initial embryonic processes is often limited by the access to foetal tissue, and in humans, the molecular events resulting in primordial germ cell (PGC) specification and sex determination remain to be elucidated. A deeper understanding of the underlying processes is crucial to describe pathomechanisms leading to impaired reproductive function. Several protocols have been established for the specification of human pluripotent stem cell towards early PGC-like cells (PGCLC), currently representing the best model to mimic early human germline developmental processes in vitro. Further sex determination towards the male lineage depends on somatic gonadal cells providing the necessary molecular cues. By establishing a culture system characterized by the re-organization of somatic cells from postnatal rat testes into cord-like structures and optimizing efficient PGCLC specification protocols, we facilitated the co-culture of human germ cell-like cells within a surrogate testicular microenvironment. Specified conditions allowed the survival of rat somatic testicular and human PGCLCs for 14 days. Human cells maintained the characteristic expression of octamer-binding transcription factor 4, SRY-box transcription factor 17, and transcription factor AP-2 gamma and were recovered from the xeno-organoids by cell sorting. This novel xeno-organoid approach will allow the in vitro exploration of early sex determination of human PGCLCs.

GABAergic Alterations in the Rat Testis after Methamphetamine Exposure.

Gamma-aminobutyric acid (GABA), GABA-A receptors and GABA transporter 1 (GAT1) were reported to be involved in the proliferation of Leydig cells, testosterone production and spermatogenesis. Since methamphetamine (METH) has been reported to have adverse effects on testis and its functions, the aim of this study was therefore to determine the changes of GABAergic activity in testis after METH exposure. Male Sprague-Dawley rats were divided into control, acute binge (AB-METH), escalating dose (ED METH) and escalating dose-binge (ED-binge METH) groups. After sacrifice, rat testes were removed and used to estimate GABA concentration and the expression of GABA-A receptor, GAD1, GAD2 and GAT1 genes by using HPLC and RT-PCR, respectively. The GABA concentration was significantly increased in all METH-administrated groups. In addition, significant increases of GABA-A α1 receptor and GAD1 genes expression were found in the ED-binge METH group. Gene expressions of GAT1 were numerically decreased in all METH-administrated rats and reached significant in the ED METH group. These results indicated a compensatory upregulation of GABA production and its functions in testis after METH exposure. Thus, these changes might represent a homeostatic response of GABAergic to the adverse effects of METH.

Ameliorative effect of zinc oxide nanoparticles on cyclophosphamide induced testicular injury in adult rat.

Despite its wide range of application, cyclophosphamide (CP) exhibits a wide range of adverse effects including reproductive toxicity. The emerging field of zinc oxide nanoparticles (ZnO NPs) therapy may provide a new hope for prevention of CP induced gonadal toxicity. Herein, we aim to investigate the possible role of ZnO NPs as a new strategy to protect against CP induced testicular injury. Sixty adult male albino rats were divided into 3 groups; control, CP treated and CP + ZnO NPs treated groups. CP group was injected with CP (5 mg/kg/day), whereas CP + ZnO NPs group was concomitantly injected with CP and ZnO NPs (5 mg/kg/day). Testicular specimens were processed for histological, ultrastructural and c-kit immunohistochemical study. Biochemical analysis for tissue malondialdehyde and serum testosterone was done in addition to sperm morphology assay and cytogenetic study. Our results revealed that CP induced deleterious testicular histopathological, biochemical and genetic alterations that were effectively prevented by ZnO NPs.

The effects of melatonin and colchicine on ischemia-reperfusion injury in experimental rat testicular torsion model.

PURPOSE: The aim of the present study is to investigate the efficiency of colchicine and melatonin in an experimental rat testicular torsion model in the light of histological and biochemical data. METHODS: A total of 34 Wistar albino male rats were randomly divided into 5 groups as: Group C (control, n=6), Group S (sham; underwent only left scrotal exploration, n=7), Group TD (torsion and detorsion; 6h of ischemia and 7days of reperfusion, n=7), Group TD/M (TD+Melatonin; 6h of ischemia and 7days of reperfusion and 7days of 17mg/kg intraperitoneal melatonin per day, n=7), group TD/Col (TD+Colchicine; 6h of ischemia and 7days of reperfusion and 7days of 1mg/kg oral colchicine per day, n=7). Histopathologic evaluation of seminiferous tubule deterioration was performed by Johnsen's scoring system. Total antioxidant status (TAS), total oxidant status (TOS), IL-6, TNF alpha levels were analyzed in each group. RESULTS: The histopathologic scores, total antioxidant status (TAS), total oxidant status (TOS), IL-6, TNF alpha levels in groups C and TD/Col were significantly lower than groups TD and TD/M (P<.001). CONCLUSION: Our study results revealed that colchicine reduced testicular ischemia-reperfusion injury in experimental rat testis torsion model. Although detorsion of testis is crucial for the preserving the testicular viability, antioxidant and anti-inflammatory treatment modalities like colchicine might help to reduce ischemia-reperfusion injury in detorsed testis.

Protective role of garlic oil against oxidative damage induced by furan exposure from weaning through adulthood in adult rat testis.

Furan is produced in a wide variety of heat-treated foods via thermal degradation. Furan contamination is found to be relatively high in processed baby foods, cereal products, fruits juices, and canned vegetables. Several studies have demonstrated that furan is a potent hepatotoxin and hepatocarcinogen in rodents. However, few studies have investigated the toxic effects of furan in the testis. In addition, the exact mechanism(s) by which furan exerts toxicity in the testis has not been fully elucidated. In this study, we investigated the potential of furan exposure from weaning through adulthood to induce oxidative stress in adult rat testis, as well as the potential of garlic oil (GO) to ameliorate the induced toxicity. Our results reveal that furan administration significantly reduced serum testosterone levels and increased the levels of malondialdehyde (MDA); furthermore, furan administration decreased significantly the enzymatic activity of testicular antioxidants, including glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) and induced histopathological alterations in the testis. GO co-administration ameliorated the reduction in testosterone levels and dramatically attenuated the furan-induced oxidative and histopathological changes. In addition, Go significantly down-regulated the increased caspase-3 and cytochrome P450 2E1 (CYP2E1) expression in the furan-treated testis. To the best of our knowledge, this study is the first to demonstrate the furan-induced oxidative changes in the adult rat testis and the protective role of GO to ameliorate these changes through its antioxidant effects and its ability to inhibit CYP2E1 production.

Changes of sperm quality and hormone receptors in the rat testis after exposure to methamphetamine.

Methamphetamine (METH) is known to damage neurons and induce psychosis. It can also induce apoptosis in seminiferous tubules and affect sperm quality. The present study was carried out to investigate the effect of a rat model of METH addiction on sperm quality and expression of progesterone receptors (PR) and estrogen receptors (ER) in the testis. Sperm quality parameters including sperm motility, sperm morphology and sperm concentration were examined. Protein and gene expressions PR, ERα and ERß were studied using immunohistochemistry and reverse transcriptase-polymerase chain reaction, respectively. The percentages of normal sperm motility and normal sperm morphology were significantly decreased in animals receiving METH, especially in escalating dose (ED METH) and escalating dose-binge (ED-binge METH) groups when compared with control. In addition, sperm concentrations in ED METH and ED-binge METH groups were numerically decreased. PR, ERα and ERß immunoreactive cells were significantly decreased in spermatogonia, spermatogenic cells and especially in Sertoli cells in all METH-treated groups. Furthermore, messenger RNA expression of PR, ERα and ERß were also significantly decreased in all METH-treated animals. These results indicate that METH can induce abnormal sperm quality. These changes of sperm quality may relate to the reduction of PR, ERα and ERß expressions in male germ cells and Sertoli cells which are essential for spermatogenesis and development of sperm.

Determination of 17α-hydroxylase-C17,20-lyase (P45017α) enzyme activities and their inhibition by selected steroidal picolyl and picolinylidene compounds.

17α-hydroxylase-C17,20-lyase (P45017α) is a key regulator enzyme of the steroid hormone biosynthesis in both the adrenals and the testes. Inhibition of this enzyme can block androgen synthesis in an early step, and may thereby be useful in the treatment of several androgen-dependent diseases. We developed radio-substrate in vitro incubation methods for the determination of the distinct 17α-hydroxylase and C17,20-lyase activities of the enzyme using rat testicular homogenate as enzyme source. With this method we have studied the inhibiting activity of selected steroidal picolyl and picolinylidene compounds. Tests revealed a substantial inhibitory action of the 17-picolinyliden-androst-4-en-3-one compound.

A short-term in vivo screen using fetal testosterone production, a key event in the phthalate adverse outcome pathway, to predict disruption of sexual differentiation.

This study was designed to develop and validate a short-term in vivo protocol termed the Fetal Phthalate Screen (FPS) to detect phthalate esters (PEs) and other chemicals that disrupt fetal testosterone synthesis and testis gene expression in rats. We propose that the FPS can be used to screen chemicals that produce adverse developmental outcomes via disruption of the androgen synthesis pathway more rapidly and efficiently, and with fewer animals than a postnatal one-generation study. Pregnant rats were dosed from gestational day (GD) 14 to 18 at one dose level with one of 27 chemicals including PEs, PE alternatives, pesticides known to inhibit steroidogenesis, an estrogen and a potent PPARα agonist and ex vivo testis testosterone production (T Prod) was measured on GD 18. We also included some chemicals with "unknown" activity including DMEP, DHeP, DHEH, DPHCH, DAP, TOTM, tetrabromo-diethyl hexyl phthalate (BrDEHP), and a relatively potent environmental estrogen BPAF. Dose-response studies also were conducted with this protocol with 11 of the above chemicals to determine their relative potencies. CD-1 mice also were exposed to varying dose levels of DPeP from GD 13 to 17 to determine if DPeP reduced T Prod in this species since there is a discrepancy among the results of in utero studies of PEs in mice. Compared to the known male reproductive effects of the PEs in rats the FPS correctly identified all known "positives" and "negatives" tested. Seven of eight "unknowns" tested were "negatives", they did not reduce T Prod, whereas DAP produced an "equivocal" response. Finally, a dose-response study with DPeP in CD-1 mice revealed that fetal T Prod can be inhibited by exposure to a PE in utero in this species, but at a higher dose level than required in rats.Key words. Phthalate Syndrome, Fetal endocrine biomarkers, Phthalate adverse outcome pathway, testosterone production, fetal rat testis.

Testicular effects of monensin, a golgi interfering agent in male rats.

OBJECTIVE: The present study was undertaken to explore the effects of monensin, a potent Golgi disturbing agent on male fertility. METHODS: Male Wistar rats were administered monensin at the dose levels of 2.5, 5, and 10 mg/kg b wt. Animals were sacrificed after 67 days of the treatment. The activities of lactate dehydrogenase (LDH), ATPase, acid phosphatase and thiamine pyrophosphatase (TPPase) were measured in the testis. Cytochemical assay of Golgi body marker enzyme, thiamine pyrophosphatase was also performed. Ultrastructural changes in testis were studied by Transmission electron microscopy. Sperm number and motility were also examined. RESULTS AND DISCUSSION: The alterations in the activities of above mentioned enzymes indicate the pronounced effect of the drug on the functioning of spermatogenic cells. The findings from electron microscopy such as membrane disruption, swelling and disintegration of Golgi apparatus strongly suggest the interference of monensin with the functioning of Golgi apparatus in the spermatogenic cells. Data from the sperm number and motility as well as the fertility studies and the resulted litter size further points towards the antifertility effects of monensin in male rats. CONCLUSION: The findings from the present study strongly indicated the effects of monensin on the testis, involving alterations in key enzyme activities and changes at the ultrastructural level.

Gibberellic acid acts as an agonist of steroidogenesis in male rats.

Testicular steroidogenesis has significant implication in male reproductive function. Although the effects of various signalling molecules on testicular functions have been studied earlier, the influence of the plant hormone gibberellic acid (GA3 ) on steroidogenesis has not been investigated. Acute (4 h) and subacute (15 days) studies using this compound through oral administration (150 µg day(-1) ) to groups of normal and diabetic Wistar male rats were therefore carried out. Results indicate that (i) enhanced activity of steroidogenic markers 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD), elevated tissue testosterone (T) content, increased steroidogenic acute regulatory protein (StAR) and androgen binding protein (ABP) levels with reduced lipid peroxidation and improved antioxidant defence in this treatment group of normal and diabetic rat testis, and (ii) elevated lipid peroxidation and diminished antioxidant defence, with insignificant change in 3ß-HSD and 17ß-HSD activity and testosterone level in acute treatment group of normal and diabetic rats testis, were noted. The observed increase in the activity of testicular 3ß-HSD and 17ß-HSD along with elevated testosterone content established GA3 as an inducer of steroidogenesis in rat.

Effects of Temperature Change on Heat Shock Protein 70 Expression in Rat Testes / 대한비뇨기과학회지

PURPOSE: It has been reported that exposure of the testes to elevated temperatures results in decreased spermatogenesis. The aim of this study was to investigate the effects of temperature change on the expression of heat shock protein 70 (HSP 70) during spermatogenesis in rat testes. MATERIALS AND METHODS: Twenty-seven Sprague-Dawley rats (200-230g) were randomly divided into control, hot bath and hot bath followed by cold bath, groups. The hot bath consisted of immersion in a 41-43 degrees C water bath for 10 minutes, and the cold bath consisted of immersion in a 18-20 degrees C water bath for 3 minutes. Each bathing was performed twice a day, three times a week, for a total of four weeks. Hematoxylin & Eosin staining was performed to evaluate the degree of spermatogenesis, and Western blot & immunohistochemistry were performed to investigate the expression of HSP 70 in the rat testes. RESULTS: From the histological tests, the spermatogenesis was severely impaired in hot bath group, but preserved in hot bath followed by cold bath group. The expression of HSP 70 in the hot bath group increased 1.5 times compared to that in the control group (p=0.075). However, the hot followed by cold bath group showed similar findings to those in the controls (p=0.934). Immunohistochemical analysis for the expression of HSP 70 demonstrated significant elevations in the hot bath group, and HSP 70 immunoreactivity was found in the Leydig cells and fibroblasts in all three groups, but the levels of expression of the HSP 70 in the control, and the hot followed by cold bath, groups were similar. CONCLUSIONS: The results of this study demonstrate an elevation in the expression of the HSP 70 only occurred in the hot bath group, which suggests the induction of a coping mechanism for exposure to high temperature. As the levels of expression of the HSP 70 in the control, and hot followed by cold bath, groups were comparable, is suggestive of the levels of HSP 70 being consistent with those seen with normal spermatogenesis.

An Experimental Study on the Effects of X-ray Irradiation and Hyperthermia on the Rat Testis / 대한치료방사선과학회지

The effects of both hyperthermia alone and X-ray irradiation combined with hyperthermia on rat testis have been investigated. The histological changes were observed on 15 and 30 days after treatment. There was no histological change of rat testis by hyperthermia alone. The earliest change by x-ray irradiation was the degeneration of the spermatogonia of the seminiferous tubule, which was appeared in 2 gy group. Necrosis of the spermatogonia was severe in 6 gy group and complete atrophy was developed in 8 gy group. With increased dose of radiation, the degrees of changes of tubules was increased. In combined group of X-ray irradiation and hyperthermia, the histological change of the seminiferous tubule was more severe than X-ray alone group. Necrosis and atrophy of the spermatogonia were appeared in 2 gy and complete atrophy of spermatogonia was seen in 6 gy group. Thermal enhancement ratio (calculated at the complete atrophy of the spermatogonia) was 1.3 in this experiment. There was no difference in observation time inverval between 15 and 30 days after each treatment in all groups.

An Effect of Anti-Testicular Rabbit Serum on Rat Testis / 대한비뇨기과학회지

Evidence of the antigenicity of testis and semen was first presented at the end of the last century. Landsteiner (1899), Metchnikoff (1900), and Metalnikoff (1900) demonstrated the induction of a spermotoxic antibody in animals sensitized with testicular homogenate or semen; this antibody was capable of immobilizing sperm cells. The earliest manifestation of homologous type of antisperm sensitization (Kennedy, 1924) was the immobilization of spermatozoa, and in some cases atrophy of germinal epithelium, following repeated injection of testicular homogenate or epidydimal sperm. Ryoo and Kim (1982) reported that spermatogenesis was adversely affected with degeneration and sloughing of germinal cells of the seminiferous tubules in the mice which were immunized with testis homogenate plus complete Freund's adjuvant. The purpose of this study was to observe the effect of antitesticular rabbit serum produced against rat testis on spermatogenesis in rat. The results were as follows: 1.Theseminiferous tubules showed mild to moderate impairment of spermatogenesis such as degeneration and exfoliation of germinal epithelium in all experimental groups. Intraluminal spermatozoa of seminiferous tubules were decreased in number. Interspaces of seminiferous tubules were wider than normal and were infiltrated with mononuclear cells with some hemorrhage. 2. Intraluminal spermatozoa of the epididymides were markedly decreased in number but immature sperm cells were observed much more often than in normal control group.

Effect of Cadmium Chloride on the Mature Rat Testis / 대한비뇨기과학회지

For the study of the effects of cadmium chloride and zinc acetate on the testis and relationship between regeneration of degenerated testis and mast cells, the Sprague-Dawley strain albino rats weighing from 180gm to 250 gm were used. Twenty seven rate were injected with cadmium chloride in the doses of 0.03 mM per kg body weight and twenty seven rats were injected the same amount of cadmium chloride and 3.0 mM per kg body weight of zinc acetate simultaneously. The testes were removed under ether anesthesia at different time intervals (3 hours, 6 hours, 12 hours, 1 day, 2 days, 4 days, 1 week, and 4 weeks) and the sections were stained with hematoxylin and eosin, and toluidine blue, respectively. Through the histological and gross observation the following results were obtained. From 3 hours after the injection of cadmium chloride, the testis began to have degenerative changes in the interstitium and tubules changes began at 2 days after the injection of cadmium chloride. Active regeneration of the damaged interstitium was recognized at 2 weeks after the injection of cadmium chloride while the tubules were still in the state of necrosis. No regenerative changes of the tubular epithelium were observed throughout the experiment. The first appearance of tissue mast cells in the interstitium was observed at 2 days after the injection of cadmium chloride and the cells were increased in number as the time elapsed. The simultaneous injection of zinc acetate prevented the destruction of testes caused by toxic effect of cadmium chloride and no tissue mast cells were found in the interstitium of the testes throughout the experiment. The testes were diminished in the weight and size after the treatment of cadmium chloride but those mgross change were not found in the testes treated with zinc acetate simultaneously.