Testis Homogenates Research Articles

Granulocyte Colony-Stimulating Factor Restored Impaired Spermatogenesis and Fertility in an AML-Chemotherapy Mice Model.

Leukemia and treatment of male patients with anticancer therapy (aggressive chemotherapy and/or radiotherapy) may lead to infertility or even permanent male sterility. Their mechanisms of spermatogenesis impairment and the decrease in male fertility are not yet clear. We showed that under acute myeloid leukemia (AML) conditions, alone and in combination with cytarabine (CYT), there was significant damage in the histology of seminiferous tubules, a significant increase in apoptotic cells of the seminiferous tubules, and a reduction in spermatogonial cells (SALL and PLZF) and in meiotic (CREM) and post-meiotic (ACROSIN) cells. In addition, we showed a significant impairment in sperm parameters and fertilization rates and offspring compared to control. Our results showed a significant decrease in the expression of glial cell line-derived neurotrophic factor (GDNF), macrophage colony-stimulating factor (MCSF) and stem cell factor (SCF) under AML conditions, but not under cytarabine treatment compared to control. In addition, our results showed a significant increase in the pro-inflammatory cytokine interleukin-1 (IL-1) alpha in whole testis homogenates in all treatment groups compared to the control. Increase in IL-1 beta level was shown under AML conditions. We identified for the first time the expression of GCSF receptor (GCSFR) in sperm cells. We showed that GCSF injection in combination with AML and cytarabine (AML + CYT + GCSF) extended the survival of mice for a week (from 6.5 weeks to 7.5 weeks) compared to (AML + CYT). Injection of GCSF to all treated groups (post hoc), showed a significant impact on mice testis weight, improved testis histology, decreased apoptosis and increased expression of pre-meiotic, meiotic and post- meiotic markers, improved sperm parameters, fertility capacity and number of offspring compared to the controls (without GCSF). GCSF significantly improved the spermatogonial niche expressed by increased the expression levels of testicular GDNF, SCF and MCSF growth factors in AML-treated mice and (AML + CYT)-treated mice compared to those groups without GCSF. Furthermore, GCSF decreased the expression levels of the pro-inflammatory cytokine IL-12, but increased the expression of IL-10 in the interstitial compartment compared to the relevant groups without GCSF. Our results show for the first time the capacity of post injection of GCSF into AML- and CYT-treated mice to improve the cellular and biomolecular mechanisms that lead to improve/restore spermatogenesis and male fertility. Thus, post injection of GCSF may assist in the development of future therapeutic strategies to preserve/restore male fertility in cancer patients, specifically in AML patients under chemotherapy treatments.

A Chemoproteomics Approach to Determine the Mechanism of Testicular Toxicity for the Bruton’s Tyrosine Kinase Inhibitor CC-292

During drug development, potential safety issues can occur at any time. Understanding the cause of a toxicity can help with deciding on how to advance the drug development program. Chemoproteomics provides a way to help understand the cause of a toxicity wherein the affected tissue is accessible and can be probed with a covalently binding compound that is analogous to the offending drug. In this case, N-(3-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide (CC-292), a covalently binding Bruton's tyrosine kinase inhibitor, had produced testicular toxicity in rodents. Experiments were conducted using a CC-292 analog that could be chemically modified with biotin to probe rodent testes homogenates for potential binding sites that were subsequently recovered with avidin beads. These biotin-tagged proteins undergo trypsin digest on the avidin beads to yield peptides that are identified using mass spectrometry. Two proteins were identified from the testicular homogenates of both rats and mice, namely retinal dehydrogenase 1 (ALDH1A1) and retinal dehydrogenase 2 (ALDH1A2). Literature confirmed a link between inhibition of these enzymes and testicular toxicity. Subsequently, molecular modeling was used to demonstrate that CC-292 can be docked into both the nicotinamide adenine dinucleotide and retinal binding pockets of the analogous human ALDH1A2 enzyme. These data suggest that the off-target binding site for CC-292 on retinal dehydrogenase enzymes may provide a mechanistic explanation to the testicular toxicity observed in rodents and that there may be a potential concern for human male fertility. SIGNIFICANCE STATEMENT: Biotinylated covalently binding drug analogues are used to enrich bound proteins from tissue homogenates wherein toxicity was observed in rodents. Bound proteins were subsequently identified by mass spectroscopy. Competition of the analog binding with the parent inhibitor itself and three-dimensional molecular modeling were used to establish a likely link between the off-targets of CC-292, ALDH1A1, and ALDH1A2 with potential testicular toxicity.

Protective effects of caffeic acid phenethyl ester on cadmium-induced testicular injury: A crucial role of antioxidant enzymes in male mice infertility

Infertility in men is referred to inability to achieve pregnancy in fertile females after at least one year of regular intercourse. The lack of oxygen in the environment may lead to an imbalance of testes production. Swiss mice were alienated into four groups 10 mice/each. This included one negative normal control group I. The induction of infertility was achieved with injection of cadmium chloride at dose 3 mg/kg body weight for four consecutive days for the rest groups. Group III received vehicle (saline) from the second day of induction for the similar period during the experiment. Infertile mice determined depending on alterations in morphology, motility, and reduced sperm count. Group IV was treated with 3 mg/kg of caffeic acid phenethyl ester (CAPE) per day, for 6 days from the second day of cadmium intoxication. Data showed effectiveness of CAPE significantly through improving the antioxidant enzymes SOD, GST and GSH in testes homogenate and GSH-Px in mice serum that were treated compared to those in the untreated group II (P˂0.001). The histopathology, DNA analysis showed marked improvement in spermatogenesis and DNA intact pattern in treated mice testis. Overall, the results demonstrated the ability of CAPE to improve spermatogenic cells. The data analysis indicated the possibility for the future use of CAPE as an inhibitory agent of infertility. Clinical trials and further studies are required to evaluate the definite medical effects of CAPE based on abundant experimental studies, with predictive future applications in human clinical trials.

Quantification of KRAS inhibitor sotorasib in mouse plasma and tissue homogenates using liquid chromatography-tandem mass spectrometry

Sotorasib is a KRAS inhibitor with promising anticancer activity in phase I clinical studies. This compound is currently under further clinical evaluation as monotherapy and combination therapy against solid tumors. In this study, a liquid chromatography-tandem mass spectrometric method to quantify sotorasib in mouse plasma and eight tissue-related matrices (brain, liver, spleen, kidney, small intestine, small intestine content, lung, and testis homogenates) was developed and validated. Protein precipitation using acetonitrile was utilized in 96-well format to extract sotorasib and erlotinib (internal standard) from mouse plasma and tissue homogenates. Separation of the analytes was performed on an Acquity UPLC® BEH C18 column by gradient elution of methanol and 0.1% formic acid in water at a flow rate of 0.6 ml/min. Sotorasib was detected by a triple quadrupole mass spectrometer with positive electrospray ionization in selected reaction monitoring mode. A linear calibration range of 2–2,000 ng/ml of sotorasib was achieved during the validation. Accuracy values were in the range of 90.7–111.4%, and precision values (intra- and interday) were between 1.7% and 9.2% for all tested levels in all investigated matrices. The method was successfully applied to investigate the plasma pharmacokinetics and tissue accumulation of sotorasib in female wild-type mice.

Potential therapeutic effects of Ulva lactuca water fraction on monosodium glutamate-induced testicular and prostatic tissue damage in rats.

Male infertility is considered one of the most critical health problems that are expected to expand worldwide. Ulva lactuca is a species of green seaweeds which is known to be a rich source of many important nutrients. Accordingly, this study is designated to investigate the therapeutic role of Ulva lactuca water fraction (UL) against monosodium glutamate (MSG)-induced male reproductive system disorders in male rats. Ulva lactuca methanolic crude extract was prepared firstly, and then water-dissolved compounds of this crude methanolic extract were separated. Ulva lactuca water fraction active phenolic compounds were determined using high-performance liquid chromatography (HPLC). Thirty-two male rats were divided equally into four groups; male infertility was induced in sixteen experimental animals by MSG at dose of 15mg/Kg for 45days. Eight infertile animals were treated with 100mg/Kg of Ulva lactuca water fraction for 30days. The rest of the animals were divided into two control groups; one control group (eight animals) was used to study the effect of UL on healthy rats at dose of 100mg/Kg for 30days and healthy control group (eight animals). Semen quality parameters (concentration and motility ratio), serum testosterone, prostate-specific antigen (PSA), and phosphatases were estimated by using standard protocols. Moreover, prooxidants and endogenous antioxidant enzymes were measured in prostate and testis homogenates. In addition, relative expression of pro-inflammatory genes (inducible nitric oxide synthase (i-NOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α alpha (TNF-α), and tumor protein (P53)) were assessed in testicular and prostatic tissues. Finally, histological alterations were measured by hematoxylin and eosin (H&E) stain. Results revealed that Ulva lactuca water fraction contains active phenolic constituents responsible for its antioxidant bioactivity. Oral administration of MSG significantly induced histological alterations. Oxidative stress was observed with elevated levels of nitric oxide (NO), thiobarbituric acid reactive substances (TBARS), and xanthine oxidase (XO) activity in both testis and prostate tissues. MSG adversely affected prostate function via elevation of PSA, prostatic acid phosphatases (PAPs), and total acid phosphatases (TAPs). In addition, it upregulated pro-inflammatory genes in testis and prostate tissues. Meanwhile, MSG reduced serum testosterone, semen quality, and antioxidant enzyme activities (glutathione peroxidase (GPx), glutathione-s-transferase (GST), and superoxide dismutase (SOD)). Treatment with UL notably ameliorated the state of oxidative stress and downregulated the expression of pro-inflammatory gene markers. This study highlighted the potential efficacy of Ulva lactuca water fraction on MSG-induced male infertility in rats. Therapeutic effect of UL on oxidative stress and inflammation induced by MSG in testicular and prostatic tissues.

Effect of simultaneous coffee/caffeine and ethanol administration on sperm quality and reproductive hormones: an experimental study in Sprague Dawley rats

Chronic alcohol or coffee/caffeine consumption has been associated with negative effects on male reproductive system. The present study evaluated the possible interaction (combination treatment) of coffee/caffeine and alcohol and its resultant consequence on sperm parameters in rats. Ten groups of adult Sprague Dawley rats (n = 5 per group) were orally gavaged with different quantities of coffee (≈5, 10, and 20 mg/kg caffeine); 30% v/v ethanol (0.5, 1, and 2 g/kg); coffee + ethanol or vehicle (distilled water) daily for 60 days. Sperm parameters, serum sex hormone levels, together with antioxidants and lipid peroxidation status of testis homogenate were evaluated. Single and combined administration of coffee/caffeine and ethanol caused a reduction in sperm count dose-dependently (p = 0.0002), but this effect was highest in the combined treatment. Sperm viability, motility, and morphology were unaffected by coffee or ethanol administration, but the combination produced detrimental effects on them. While changes in follicle stimulating hormone were not significant, testosterone and luteinizing hormone levels were decreased significantly and dose-dependently in all experimental groups (p < 0.05–0.0001). This effect was most pronounced in the groups that received the combination. Testicular superoxide dismutase and catalase activities, and reduced glutathione and malondiadehye (MDA) levels were not affected by coffee/caffeine, but ethanol decreased (p < 0.05) the antioxidants and elevated MDA levels mostly at 2 g/kg. The combination reduced (p < 0.0001) the antioxidants and increased MDA levels in testis homogenates at all doses. Combination of coffee/caffeine and ethanol potentiates their detrimental effects on sperm quality and reproductive hormones via reduction in testicular antioxidants activities and elevation of lipid peroxidation in rats.

Modulatory effect of Jatropha gossypifolia leaf on testicular lipidomics and plasma lipoproteins of rats exposed to cypermethrin

The use of pesticides such as cypermethrin (Cyp) has become hazardous resulting into public and environmental health issues in the world with several studies pointing to a decline in reproductive health coupled with increased risk of infertility. Thus, pesticide safety and effects on human exposure in relation to general and reproductive health are of paramount concern. This study was aimed at investigating the biochemical alterations in the lipidomics of plasma components and reproductive organ of male rats exposed to cypermethrin. The animals were grouped into eight comprising seven animals each and orally treated for 28 days: control, 20 mg/kg body weight of Cyp, Cyp+JG(50 mg/kg), Cyp+JG(100 mg/kg), Cyp+ALA(50 mg/kg), JG(50 mg/kg), JG(100 mg/kg) and ALA(50 mg/kg). Lipid profile analyses were carried out on the plasma, HDL, vLDL+LDL, and testis homogenate of the rats as described by the diagnostic kit manual. Results obtained from this study showed that cypermethrin administration significantly elevated triglyceride (105.22%) and not significant cholesterol level (15.28%) in the plasma; significantly increased testicular triglyceride (96.46%) and cholesterol (64.99%) concentrations; significantly decreased HDL triglyceride (46.57%) and cholesterol (38.37%); significantly increased the concentration of vLDL+LDL triglyceride (126.28 %) and cholesterol (76.09%) in comparison with the control. Co-administration with both doses of J. gossypifolia (JG) leaf extract and alpha-lipoic acid (ALA) significantly reversed these alterations. Thus, the phytoconstituents of Jatropha gossypifolia plant extract possesses modulatory potentials that can mitigate the effect of cypermethrin on the reproductive organ and plasma lipidomics especially in the treatment of heart related disorders caused by hyperlipidemia.

Hot-humid season restraint stress duration on sperm indices and oxidative biomarkers on rats

Restraint stress is usually occasioned by excessive production of reactive oxygen species (ROS) thereby causing potential damage to the normal spermatozoa by inducing lipid peroxidation. This study investigated the possible time in which restraint stress induces maximum effects on sperm motility, concentration, and oxidative stress markers in male Wistar rats. Animals were randomly divided into three groups of three animals each (n=3). Group I- normal control (undisturbed), Group II- 3 h stress group, Group III- 6 h stress group. Restraint stress was induced by placing rats in specially constructed restraint meshes for both 3 and 6 hours (between 9.00-15.00 h) for 21 days. Testes homogenate were evaluated for Malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD) and reduced glutathione (GSH). The 3 h stress group showed a significant decrease in sperm motility and concentration (5.4 ± 0.24) when compared to the normal control group (10.0 ± 0.45) and 6 h stress group (7.3 ± 0.89) (P

Methyl parathion causes genetic damage in sperm and disrupts the permeability of the blood-testis barrier by an oxidant mechanism in mice

Methyl parathion (Me-Pa) is an extremely toxic organophosphorus pesticide still used in developing countries. It has been associated with decreased sperm function and fertility and with oxidative and DNA damage. The blood-testis barrier (BTB) is a structure formed by tight junction (TJ) proteins in Sertoli cells and has a critical role in spermatogenesis. We assessed the effect of repeated doses of Me-Pa (3−12 mg/kg/day for 5 days, i.p.) on sperm quality, lipid oxidation, DNA integrity, and BTB permeability in adult male mice and explored oxidation as a mechanism of toxicity. Me-Pa caused dose-dependent effects on sperm quality, lipoperoxidation, and DNA integrity. Testis histology results showed the disruption of spermatogenesis progression and atrophy of seminiferous tubules. The pesticide opened the BTB, as evidenced by the presence of a biotin tracer in the adluminal compartment of the seminiferous tubules. This effect was not observed after 45 days of exposure when a spermatogenic cycle had completed. The coadministration of the antioxidant α-tocopherol (50 mg/kg/day for 5 days, oral) prevented the effects of Me-Pa on sperm quality, DNA and the BTB, indicating the importance of oxidative stress in the damage generated by Me-Pa. As evidenced by immunochemistry, no changes were found in the localization of the TJ proteins of the BTB, although oxidation (carbonylation) of total proteins in testis homogenates was detected. Our results show that Me-Pa disturbs the BTB and that oxidation is involved in the observed toxic effects on sperm cells.

INFLUENCE OF NANOSIZED SILICA OXIDE SUSPENSION ON NITRIC OXIDE PRODUCTION IN RATS TESTES DURING CHRONIC NITRATE-FLUORIDE INTOXICATION

In the Poltava region of Ukraine there is a high possibility of combined excessive intake of nitrates and fluorides, since the waters of the Buchaksʹkii aquifer are rich in fluorine ions, moreover, the Poltava region is a region with a developed agriculture.&#x0D; The purpose of this work is to determine the effect of nanosized silicon oxide suspension on the changes in the activity of nitric oxide cycle components, components, and, namely the total activity of NO-synthases (NOS), the activity of constitutive forms of NOS (cNOS), inducible form of NOS (iNOS), nitrate reductase, nitrite reductase, nitrite reductase, nitrite reductase in the testes of rats under the conditions of chronic combined nitrate-fluorodide intoxication.&#x0D; Materials and methods. The study was conducted on 15 mature male Wistar rats weighing 200-253 g. The animals were randomly divided into 3 groups of 5 animals in each. The first group received daily saline solution through a gastric tube. The second group (chronic combined intoxication group) received sodium nitrate and fluoride in a dosage of 500 mg / kg of sodium nitrate and 10 mg / kg of sodium fluoride. The third group against the background of chronic combined intoxication modelling additionally received a suspension of nanosized silicon oxide is a dosage of 100 mg / kg of active substance.&#x0D; Results. Chronic combined intoxication increases total NOS activity by 65.9% compared to the control group. The increase in NO production by NOS is the result of a 4-fold increase in iNOS activity, since the activity of cNOS under these conditions does not change significantly. Nitrite reductase-dependent NO production increases by 66.7%, nitrate reductase activity increases by 77.8%. Nitrite content in the testes homogenate grows in 2.17 times. Hydrolytic cleavage of L-arginine by arginase increases in 2.88 times. The introduction of nanosized silicon oxide suspension under the conditions of chronic combined intoxication did not statistically significantly change the total NOS activity and cNOS activity compared to the group exposed to chronic intoxication. However, iNOS activity reduces in 3.69 times. Nitrate reductase activity decreases in 1.6 times, while nitrite reductase activity decreases in 45%. The activity of arginases lowered in 2.38 times. The nitrite content of the testes cut down by 23.6%.&#x0D; Conclusions. Chronic combined nitrate-fluoride intoxication leads to the development of hyperproduction of nitric oxide in the testes of rats by activating the inducible isoform of NO synthase and nitrite reductases. The use of nanosized silica suspension during chronic combined intoxication is an effective method to correct nitric oxide overproduction in the testes of rats.

Bioanalytical assay for the new-generation ROS1/TRK/ALK inhibitor repotrectinib in mouse plasma and tissue homogenate using liquid chromatography-tandem mass spectrometry

Repotrectinib, a next-generation ROS1/TRK/ALK tyrosine kinase inhibitor, overcomes resistance due to acquired solvent-front mutations involving ROS1, NTRK1-3, and ALK. A bioanalytical assay for quantification of repotrectinib in mouse plasma and seven tissue-related matrices (brain, liver, spleen, kidney, small intestinal tissue, small intestinal content, and testis homogenates) was developed and validated using liquid chromatography with tandem mass spectrometric detection in a high-throughput 96-well format. Protein precipitation was performed by adding acetonitrile, also containing the internal standard axitinib, to 10-µl samples for all matrices. Chromatographic separation of analytes was done on an ACQUITY UPLC® BEH C18 column by gradient elution using ammonium hydroxide in water and methanol. Compounds were monitored with positive electrospray ionization using a triple quadruple mass spectrometer in selected reaction monitoring mode. The method was successfully validated in the 1–1000 ng/ml calibration range. Precisions (intra- and interday) were in the range of 1.3–8.7% and accuracies were in between 90.5% and 107.3% for all levels in all matrices. The developed method was successfully applied to investigate the plasma pharmacokinetics and tissue accumulation of repotrectinib in wild-type mice.

Heat induced differential pattern of DNA fragmentation in male germ cells of rats.

Spermatogenesis being a highly dynamic processes is highly vulnerable to various stresses including heat stress. Though, the relationship between physiological temperature and male germ cells is certainly immense, the magnitude of spermatozoal damage after exposure to heat is evidently degree and dose dependent. Further, there are contradictory reports related to germ cells apoptosis in relation to temperatures. Thus, currently the dynamics of temperature and time dependence on germ cell apoptosis were studied by modulating the heat treatment strategies. It was observed that the rate of apoptosis increased initially then decreased with time. The DNA fragmentation in the 10,000×g supernatant of testis homogenate of rats that received heat treatment for 15-min, 30-min as well as 45-min treatment with 15-min intermittent period was found to be almost equal. In various heat treated animals, the apoptosis was found to be maximum after day-1 of treatments, which then followed a decreased pattern. These results indicate that there may be an initial induction of apoptosis in the germ cells, which later primed or programmed the other germ cells to activate protective mechanisms against heat induced DNA damage and thus protecting germ cell population to undergo apoptosis at later durations.

Bioanalytical assay for the novel TRK inhibitor selitrectinib in mouse plasma and tissue homogenates using liquid chromatography-tandem mass spectrometry

Selitrectinib is a next generation tropomyosin receptor kinase (TRK) inhibitor developed to overcome acquired resistance to first generation TRK inhibitors. The drug is a cyclic analogue of larotrectinib. An existing bioanalytical assay for larotrectinib was therefore redesigned for selitrectinib. The assay used liquid chromatography-electrospray tandem mass spectrometry in positive selected reaction monitoring mode. Mouse plasma and tissue homogenates of brain, heart, kidney, liver, lung, small intestine, spleen, and testis were pretreated using acetonitrile protein precipitation with larotrectinib added as internal standard. Successful validation using current guidelines was obtained in the range 0.5–1000 ng/ml. Precision was within 5–12% and accuracy within 91–108% for all matrices investigated. The drug was stable in all matrices under the relevant storage conditions. Pharmacokinetics and tissue distribution of selitrectinib were monitored in a pilot study in mice demonstrating the applicability of the presented assay.

Effects of soya milk on reproductive hormones during puberty in male Wistar rats.

Puberty is considered a critical period on development that involved sexual maturation and morphological changes. Isoflavones have been described as endocrine disruptors in male rats. Therefore, the present study attempt to evaluate the effect that daily intake of low and high doses of isoflavones exert into the hormonal regulation that take place during puberty by analyzing the levels of serum and testes steroid and pituitary hormones. 108 male pre-puberal Wistar rats (30days old) were randomly divided into three groups; control, low and high doses of isoflavones. Experimental animals were daily dosed orally with low and high doses of a mixture of two soy isoflavones (genistein and daidzein) during 6weeks. An EIA was performed in serum and testes homogenates for analyzing FSH, LH, P5, P4, DHEA, A4, T, DHT, SO4E1 and E2 hormone concentrations. Results revealed a decrease of an oestrogen environment in testes stimulates the secretion of FSH and LH leading to the production of androgens in the testes at the onset of puberty. Low doses of isoflavones resulted in a significant increase of testes oestrogens that consequently produced a delay on the onset of puberty; however at high doses of isoflavones the maintained oestrogenic environment in the testes prevent the stimulation of the secretion of pituitary hormones and the production of T abolishing the onset of puberty. These results clarify the hormonal mechanisms that take place on puberty and determine the effect of high and low doses of isoflavones at the onset of puberty.

Antioxidant Role of Beta Carotene: Protection against Cadmium Induced Testicular Toxicity

system. The purpose of present study was to investigate the protective role of Beta carotene on cadmium induced testicular damage. Materials and Methods: The present study was conducted following approval from Institutional Bioethical Committee and strict internationally accepted guidelines, for the usage of animals in experimental study were. Rats were divided into four groups with 8 rats in each.The Gr. I rats were administered with the single dose of normal saline intraperitoneally. Group II received Beta carotene (10 mg/kg bw) orally for 30 days. Group III received a single dose of 1 mg/kg bw cadmium chloride and Group IV received Beta carotene for 30 days prior to cadmium administration. After the desired protocol, rats were sacrificed and both the testes were removed for biochemical and histopathological evaluation. One testis was fixed in Bouvins fluid and processed or histopathological studies. The levels of lipid peroxides (LPO) and glutathione (GSH) and superoxide dismutase (SOD) were detected in the tissue homogenates of other testis. Results: In the present study, the level of lipid peroxidation (LPO) was significantly high and GSH and SOD (P<0.001) were low in cadmium treated rats compared to normal control. Pre-treatment with beta carotene showed a protective effect by decreasing LPO and increasing GS Hand SOD level (P<0.001). The morphological changes like atrophy of tubules, edema and decreased spermatogenesis in the testis of rats exposed to cadmium chloride. But, antioxidant showed the normal architecture of the testis. Conclusion: Results of the present study showed the antioxidative role of beta carotene in protecting the testis from cadmium induced toxicity.

Melatonin protects methotrexate-induced testicular injury in rats.

Melatonin possesses anti-inflammation and anti-oxidant potentials. However, whether NF-E2 related factor 2 (Nrf2) pathway and nuclear factor-kappaB (NF-κB) pathway are involved in the protective effect of melatonin are unknown. We aim to explore the regulatory effect of melatonin on methotrexate-induced testicular injury. Sprague Dawley (SD) rats were randomly assigned in sham group, methotrexate group and melatonin group, with 8 rats in each group. Testis tissues were collected 10 days after animal procedures. Pathological lesions and cell apoptosis in testis tissues were evaluated using HE (hematoxylin and eosin) staining and TUNEL assay, respectively. Oxidative stress in rat testis was accessed using relative commercial kits. Western blot was performed to detect protein expressions of relative genes in Nrf2 pathway and NF-κB pathway in rat testis tissues. Activities of SOD, GSH, CAT and T-AOC in testis homogenate in melatonin group were remarkably higher than those of methotrexate group (p < 0.05). On the contrary, levels of MDA, ROS and inflammatory factors (TNF-α, IL-1β, IL-6 and KC-GRO) were markedly decreased after melatonin treatment. Besides, melatonin group showed alleviated pathological lesions and cell apoptosis in testis. Western blot results demonstrated that melatonin treatment upregulated expressions of Nrf2, GSR, GCLm, HO-1 and NQO-1 in testis. However, protein expressions of NF-κB, TNF-α, VCAM-1, ICAM-1 and MCP-1 were downregulated. Melatonin protects methotrexate-induced testicular damage in rats by improving antioxidant capacity and inhibiting inflammatory response via Nrf2 and NF-κB pathways.

Pleurotus tuber-regium mushrooms in the diet of rats ameliorates reproductive and testicular injury caused by carbon tetrachloride

BackgroundThe incidence of male infertility arising from male sexual dysfunction is high especially in the sub saharan Africa. African foods may hold promise to reverse this trend. The aim of this study therefore is to evaluate the improvement of the reproductive and testicular injuries mediated by CCl4 by the use of a wild edible mushroom, P. tuber-regium.MethodsSixty rats were divide into six groups. Group I received 3 mL/kg olive oil by intraperitoneal injections twice weekly. Group II received 3 mL/kg (30% in olive oil) injected twice weekly i.p, Groups III, IV and V received 100 mg, 200 mg and 500 mg wild edible P. tuber-regium (33.3% in feed) daily in addition to 3 mL/kg CCl4 in oil injected twice weekly i.p. Group VI received 500 mg P. tuber-regium (33.3% in feed) daily. After 13 weeks, the animals were sacrificed and testes weighed.Testicular counts and viability were evaluated. Serum levels of FSH, LH, testosterone, estrogen and prolactin were assayed. Malondialdehyde (MDA), ascorbic acid, α-tocopherol, superoxide dismutase (SOD), catalase, total glutathione and peroxidase were determined in testis homogenate. Also, histopathological examinations of the testes were performed.ResultsAdministration of CCl4 to rats significantly (p < 0.01) increased the relative testicular mass in treated group when compared to control group. Also, CCl4 administration decreased significantly (p < 0.01) the levels of sperm motility, epidydymal and testicular sperm count and viability ratio in the CCl4 group when compared to the control group. Exposure to CCl4 decreased significantly the levels of FSH, LH and testosterone when compared to the control while increasing the levels of estrogen, prolactin and MDA when compared to the control. The levels of ascorbic acid, α-tocopherol, SOD, catalase, total glutathione and peroxidase decreased significantly (p < 0.01) in treated groups when compared to control group. These changes were reversed by diets containing Pleurotus tuber-regium mushrooms in a dose-dependent manner. Photomicrographs also showed that P. tuber-regium prevented the edema, spermatogenic distortions and maturation arrest observed in the CCl4 only group.ConclusionP. tuber-regium is effective in protecting the testes from the free radical injuries mediated by CCl4.

Protective role of Limonium bonduelli extract against non-enzymatic peroxidation in brain and testes induced by iron in vitro.

&lt;p&gt;Infertility and Neurodegerative diseases have been linked to oxidative stress arising from peroxidation of membrane biomolecules and high levels of iron have been reported to play an important role. The present study sought to determine the antioxidant activity and protective ability of &lt;em&gt;n&lt;/em&gt;-butanol extract of &lt;em&gt;Limonium bonduelli&lt;/em&gt; on lipid peroxidation induced by FeSO&lt;sub&gt;4&lt;/sub&gt; in rat brain and testes homogenates &lt;em&gt;in vitro&lt;/em&gt;. &lt;em&gt;n&lt;/em&gt;-butanol extract of the aerial parts (leaves and flowers)&lt;em&gt; &lt;/em&gt;was prepared, and the ability of the extract to inhibit FeSO&lt;sub&gt;4&lt;/sub&gt; induced lipid peroxidation in isolated rat brain and testes was determined using spectrophotometric method. The study revealed that the extract inhibited malondialdehyde (MDA) production in FeSO&lt;sub&gt;4 &lt;/sub&gt;induced lipid peroxidation in the brain and testes in a dose-dependent manner and the highest percentage of the inhibition was 89.80% similar to vitamin C in the same concentration (100 µg/mL) in brain and 82.33% in testes (200 µg/mL). &lt;em&gt;Limonium bonduelli&lt;/em&gt; extract&lt;strong&gt; &lt;/strong&gt;demonstrated important anti-lipid peroxidative effect, which may be useful in preventing the progress of various oxidative stress related diseases. The higher inhibitory effect of the extract could be attributed to its phytochemical content.&lt;/p&gt;

Protective role of hesperidin against \u03b3-radiation-induced oxidative stress and apoptosis in rat testis

BackgroundGamma (γ) ray, an electromagnetic radiation, is occasionally accompanying the emission of an alpha or beta particle. Exposure to such radiation can cause cellular changes such as mutations, chromosome aberration and cellular damage which depend upon the total amount of energy, duration of exposure and the dose. Ionizing radiation can impair spermatogenesis and can cause mutations in germ cells. In general, type B spermatogonia are sensitive to this type of radiation. The current study was carried out to evaluate the protective role of hesperidin (H), as a polyphenolic compound, on rat testis injury induced by γ-radiation.MethodsRats were divided into groups including C group (control rats), R (irradiated) group (rats irradiated with γ-radiation), Vehicle (V) group (rats administered with dimethylsulfoxide “DMSO”), H group (rats administered with H only), HR and RH groups (rats treated with H before and after exposure to γ-radiation, respectively). Malondialdehyde (MDA: the end product of lipid peroxidation “LPO”) and xanthine oxidase (XO: it generates reactive oxygen species “ROS”) in testes homogenate as well as nitric oxide (NO: as ROS) in mitochondrial matrix were determined. The apoptotic markers including DNA-fragmentation (DNAF) in testes homogenate and calcium ions (Ca2+) in mitochondrial matrix were determined. Superoxide dismutase (SOD) and catalase (CAT) activities in testes homogenate, while reduced glutathione “GSH” in nuclear matrix were determined. Also histopathological examination for testes tissues through electron microscope was studied.ResultsExposure of rats to γ-radiation (R group) increased the levels of MDA, NO, DNAF, Ca2+ and XO activity, while it decreased GSH level, SOD and CAT activities as compared to the C groups; γ-radiation increased oxidative stress (OS), LPO, apoptosis and induced testes injuries. These results are in agreement with the histopathological examination. In contrast, treatment with H before or after exposure to γ-radiation (HR and RH groups, respectively) decreased the levels of MDA, NO, DNAF and Ca2+ but increased GSH level and the activities of SOD, CAT and XO as compared to R group and this indicates that H decreased OS, LPO and apoptosis. Also, the histopathological results showed that H improved testis architecture and this is related to the antioxidant and anti-apoptotic activities of H contents. Protection is more effective when H is given before rather than after exposure. Finally, administration of H to healthy rats for a short period had no adverse affect on testes cells.ConclusionHesperidin showed antioxidant and anti-apoptotic activities. It has a protective role against OS, injury and apoptosis induced by γ-radiation in testes. Protection is more effective when H is given before rather than after exposure.Graphical .

Connexin 43 expression in the testes during postnatal development of finasteride-treated male rat offspring.

IntroductionHormone-dependent events that occur throughout the first wave of spermatogenesis, such as cellular communication within seminiferous epithelium during early postnatal testis maturation, are important for adult male fertility. Any changes in the T/DHT ratio in male progeny born from females fertilized by finasteride-treated male rats can result in impairment of testicular physiology. The aim of the study was to verify whether finasteride has a transgenerational effect on the expression of connexin 43 (Cx43), a gap junction protein in testes of the F1 generation.Material and methodsThe subjects of the study were 7, 14, 21/22, 28, and 90-day-old Wistar male rats born by females fertilized by finasteride-treated rats (F1:Fin). The offspring born by untreated rats were used as controls (F1:Control). Connexin 43 was evaluated in the seminiferous epithelium by immunohistochemistry, and in the testis homogenates by Western blot and qRT-PCR. The Cx43 mRNA and protein expression was correlated with intratesticular levels of T and DHT by Spearman’s rank correlation coefficient.ResultsWe observed a difference in the Cx43 expression in the testis of male rats born by female rats fertilized by finasteride-treated male rats, as compared to the control on following PND (7, 22 and 28 PND, p < 0.001; 14 PND, p < 0.01); and a strong, positive correlation between Cx43 with DHT was only in the F1:Fin group (mRNA: rs = +0.51, p = 0.004; protein: rs = +0.54, p = 0.002).ConclusionsFinasteride treatment of male adult rats may cause changes in the communication between the testicular cells of their offspring, leading to a defective course of spermatogenesis.