Testis Homogenates Research Articles

Subpopulation of mouse testicular germ cells were differently affected following experimental autoimmune orchitis

OBJECTIVE: To investigate the effect of induced experimental autoimmune orchitis (EAO) on the levels of pre-meiotic, meiotic and post-meiotic cells. DESIGN: Control and EAO mice were used. MATERIALSANDMETHODS: To develope murine EAO, testes of adult mice were homogenized, mixed with complete Freund’s adjuvant and injected subcutaneously into tail, neck and foot pad of the mice. Pertussis toxin was used as a co-adjuvant (EAO group; n1⁄410). Adjuvant control groups (n1⁄410) received a similar treatment, by replacing testis homogenate with saline. After 50 days, testes were confirmed for EAO by histology and weight and used to isolate cells from the seminiferous tubule (ST) by enzymatic digestion of the ST. RNAwas extracted from the ST isolated cells and real time PCR analysis was performed to evaluate the expression levels of markers related to spermatogenesis. Cells at different stages of spermatogenesis were also examined in testis from control and EAO mice by immunofluorescence staining (IF) using specific antibody for each marker. RESULTS: The expression levels of markers related to pre-meiotic cells (Oct4 and Vasa) were significantly lower in testis from EAO compared to control mice (P<0.01 and P<0.05 respectively). However, Dazl was significantly higher in EAO compared to control mice (P<0.05). CD-9 and GFRa1 were similar in EAO and control mice. The markers of meiotic (Crem-1 and SRF-1) and post-meiotic cells (acrosin and SP-10) were significantly lower in EAO compared to control mice (P<0.01, P<0.05, P<0.001, P<0.001 respectively). IF of the spermatogenic stages confirmed the results of PCR. CONCLUSION: Our results show for the first time the effect of EAO on testicular germ cell development. Our results suggest that distinct subpopulation of testicular germ cells may differently affected by orchitis. These cells could be used in future therapeutic strategies for male infertility associated with orchitis. Supported by: This study was supported by GIF.

Involvement of soluble Fas Ligand in germ cell apoptosis in testis of rats undergoing autoimmune orchitis

Experimental autoimmune orchitis (EAO) is a model of chronic inflammation and infertility useful for studying immune and germ cell (GC) interactions. EAO is characterized by severe damage of seminiferous tubules (STs) with GCs that undergo apoptosis and sloughing. Based on previous results showing that Fas–Fas Ligand (L) system is one of the main mediators of apoptosis in EAO, in the present work we studied the involvement of Fas and the soluble form of FasL (sFasL) in GC death induction. EAO was induced in rats by immunization with testis homogenate and adjuvants; control (C) rats were injected with adjuvants; a group of non-immunized normal (N) rats was also studied. Activation of Fas employing an anti-Fas antibody decreased viability (trypan blue exclusion test) and induced apoptosis (TUNEL) of GCs from STs of N and EAO rats, an effect more pronounced on GCs from EAO STs. By Western blot we detected an increase in sFasL content in the testicular fluid of rats with severe EAO compared to N and C rats. By intratesticular injection of FasL conjugated to Strep-Tag molecule (FasL-Strep, BioTAGnology) and its immunofluorescent localization, we demonstrated that sFasL is able to enter the adluminal compartment of the STs. Moreover, FasL-Strep induced GC apoptosis in testicular fragments of N rats. By flow cytometry, we detected an increase in the number of membrane FasL-expressing CD4+ and CD8+ T cells in testis during EAO development but no expression of FasL by macrophages. Our results demonstrate that sFasL is locally produced in the chronically inflamed testis and that this molecule is able to enter the adluminal compartment of STs and induce apoptosis of Fas-bearing GCs.

Inhibitory Effect of Aqueous Extract of Moringa oleifera and Newbuoldia laevis Leaves on Ferrous Sulphate and Sodium Nitroprusside Induced Oxidative Stress in Rat’s Testes in Vitro

Oxidative stress has been identified as one of the factors that affects fertility status. Therefore, this study sought to investigate the inhibitory effect of aqueous extract of Moringa oleifera and Newbuoldia laevis leaves on FeSO4 and Sodium Nitroprusside (SNP) induced lipid peroxidation in rat testes in vitro. Incubation of the testes tissue homogenate in the presence of FeSO4 and SNP caused a significant increase in the malondialdehyde (MDA) contents of the testes. The aqueous extract from both Moringa oleifera and Newbuoldia laevis leaves caused a significant decrease in the MDA contents of the testes in a dose-dependent manner. However, aqueous extract from Moringa oleifera leaf (EC50 = 0.29 mg/ml) had a significant (P2+ induced lipid peroxidation in the rat testes homogenate than that of Newbuoldia laevis leaf extract (EC50 = 0.58 mg/ml); while there was no significant (P2+ chelating and reducing power. Therefore, these plants have potential to prevent oxidative stress in testes and improve fertility outcomes.

Evaluation of CAG repeat length of androgen receptor expressing cells in human testes showing different pictures of spermatogenic impairment

The androgen receptor (AR) is a ligand-activated transcriptional factor with crucial importance for spermatogenesis. Its transactivation domain consists of a polymorphic sequence of 9-36 cytosin-adenin-guanin (CAG) repeats. Within the physiological range an increased CAG repeat length is assumed to correlate with the reduced androgen sensitivity resulting in impaired spermatogenesis. In 33 testes of 32 patients showing different histological pictures ranging from normal spermatogenesis, hypospermatogenesis to severe spermatogenetic impairment such as maturation arrest, Sertoli cell only Syndrome (SCO) and mixed atrophy, CAG repeat length was assessed in lymphocyte DNA, DNA/mRNA from testis homogenate and in mRNA of AR expressing Sertoli cells within the seminiferous tubules, and interstitial Leydig cells collected by the laser-assisted cell picking. The latter examination was performed to detect a possible somatic mosaicism of CAG repeat length in different testicular cell populations. CAG repeat lengths varied from 12 to 27 repeats, i.e., within the physiological range. We found deviating CAG repeat numbers in different fractions of AR expressing Sertoli and Leydig cells indicating tissue heterogeneity. We did not find a correlation of CAG repeat length to testicular histology or AR expression, and testosterone or luteinizing hormone levels even in biopsies showing mixed atrophy. Additionally, we evaluated the expression pattern of the AR-dependent gene androgen binding protein (ABP), and did not find a correlation to CAG repeat, but a significant reduction of ABP mRNA related to severe spermatogenic impairment in the monomorphic histologies. These data suggest other factors than CAG repeat to be responsible for severe spermatogenic impairment including mixed atrophy.

Micro-encapsulated secretory leukocyte protease inhibitor decreases cell-mediated immune response in autoimmune orchitis

AimsWe previously reported that recombinant human Secretory Leukocyte Protease Inhibitor (SLPI) inhibits mitogen-induced proliferation of human peripheral blood mononuclear cells. To determine the relevance of this effect in vivo, we investigated the immuno-regulatory role of SLPI in an experimental autoimmune orchitis (EAO) model. Main methodsIn order to increase SLPI half life, poly-ε-caprolactone microspheres containing SLPI were prepared and used for in vitro and in vivo experiments. Multifocal orchitis was induced in Sprague–Dawley adult rats by active immunization with testis homogenate and adjuvants. Microspheres containing SLPI (SLPI group) or vehicle (control group) were administered s.c. to rats during or after the immunization period. Key findingsIn vitro SLPI-release microspheres inhibited rat lymphocyte proliferation and retained trypsin inhibitory activity. A significant decrease in EAO incidence was observed in the SLPI group (37.5%) versus the control group (93%). Also, SLPI treatment significantly reduced severity of the disease (mean EAO score: control, 6.33±0.81; SLPI, 2.72±1.05). In vivo delayed-type hypersensitivity and ex vivo proliferative response to testicular antigens were reduced by SLPI treatment compared to control group (p<0.05). SignificanceOur results highlight the in vivo immunosuppressive effect of released SLPI from microspheres which suggests its feasible therapeutic use.

Effect of selenium on carbimazole-induced testicular damage and oxidative stress in albino rats

Carbimazole is an antithyroid drug used in treatment of hyperthyroidism. The present investigation studied the effect of carbimazole on testicular activity in albino rats and the ameliorative role of selenium. Treating rats with carbimazole (1.35 mg/kg b.w) daily for 8 weeks caused reduction in the body and testes weight. Moreover, the diameters of the seminiferous tubules and heights of their germinal epithelium were significantly reduced. Testes of treated rats showed many histological alterations included congestion of blood vessels, hemorrhage, degeneration of interstitial tissue and degeneration of spermatogenic cells with apoptosis and necrosis. Histochemical results revealed reduction in polysaccharides, total proteins and nucleic acids contents in testicular tissue. In addition, the level of testosterone, luteinizing hormone (LH), T 3, T 4 and thyroid stimulating hormone (TSH) was significantly decreased in sera of treated animals. Moreover, a high lipid peroxidation with a decrease in the enzymatic antioxidant status, superoxide dismutase (SOD) and catalase (CAT) was recorded in testes homogenate. Treating animals with carbimazole and selenium showed an improvement in the histological structure as well as histochemical components of the testis with an increase in the number of spermatogenic cells. There was an increase in testosterone, LH, T 3, T 4 and TSH levels. Moreover, administration of selenium led to decrease in malondialdehyde and increase in catalase and superoxide dismutase activities. It is suggested that the curative effect of selenium against testicular damage induced by carbimazole may be due to its antioxidant properties.

On the different lipolytic capability of diverse organs from young adult guinea pigs. A chromatographic study

In this study we conducted in-vitro incubation of whole-tissue homogenate of young adult guinea pig heart, kidney, testis, and spleen at pH 7.4 and 38°C for 60 min. Subsequent TLC analysis revealed: (a) a noticeably high level of MLCL and a very low level of CL in control testis, indicating possible in-vivo deacylation of CL (b) a high level of CL in control guinea pig heart and kidney samples, and no MLCL detected (c) MLCL was only produced (in both heart and kidney) subsequent to in-vitro incubation (d) a very low level of CL was present in control samples of guinea pig spleen and no MLCL was produced subsequent to in-vitro incubation (e) guinea pig heart contains both ethanolamine (PE) and choline plasmalogens (PC) (f) PE plasmalogen is the only alkenyl species present in guinea pig kidney, testes, and spleen (g) sphingomyelin (SM) is uniquely higher in kidney than testis and spleen and very low in the heart (h) subsequent to in-vitro incubation, ceramide is produced in the kidney and testes only, with...

Effect of vinyl chloride on reproductive and endocrine system of male rats

To explore the effect of vinyl chloride on reproductive and endocrine system of male rats. Male SD rats were administered with vinyl chloride at dose of (0, 10, 100, 1000 mg/kg) for 14 and 28 days, respectively. The levels of testosterone (T), inhibin B, luteinizing hormone (LH), follicle stimulating hormone (FSH) and estradiol (E2) were measured in serum and testis homogenates. Histopathological examinations were performed for testis with electron microscopy. Compared with the control group after 14-day exposure, T and E2 serum levels of 1000, 100, 10 mg/kg groups decreased, InhB and LH levels of three dose groups increased. LH serum levels of 100 mg/kg increased significantly statistically compared with control group (P < 0.05). After 28-day exposure, T serum levels of 100, 1000 mg/kg groups were (10.90 +/- 1.56), (8.52 +/- 2.85) ng/ml respectively (P < 0.05), InhB serum levels of 100, 1000 mg/kg groups were (31.40 +/- 6.21), (28.39 +/- 5.67) pg/ml respectively. Both of T and InhB serum levels of 100, 1000 mg/kg groups decreased significantly (P < 0.05). Serum FSH levels of 10, 100, 1000 mg/kg groups decreased significantly compared with control group (P < 0.05). Compared with groups of 14-day exposure, serum InhB and LH levels of 10, 100, 1000 mg/kg groups decreased significantly statistically after 28 days. T and InhB testis levels of 100, 1000 mg/kg groups were 8.05 +/- 2.19),(6.75 +/- 1.94) ng/mg pro and (39.32 +/- 5.55), (35.53 +/- 8.71) pg/mg pro respectively, which decreased significantly compared with control group (P < 0.05). Leydig cell and Sertoli cell were damaged according to histopathological examinations. Vinyl chloride has adverse effects on reproductive and endocrine system of male rats and may change their serum and testis homogenate levels of hormones.

Natural regulatory T cells (Treg) determine post vasectomy sequlae: autoimmune orchitis (EAO) versus testis-specific tolerance. (83.3)

Abstract Vasectomy (Vx), a popular male contraceptive, causes a persistent epididymal granuloma. Using unilateral Vx (uni-Vx) as an inducer of a localized danger signal, we queried whether Treg control the transition to autoimmunity. When Treg were depleted (CD25 Ab) in uni-Vx mice, frequent and bilateral EAO developed. This was accompanied by T cell activation in the regional lymph node and a robust testis antibody response. CD4+ T cells that produced IFNγ predominated in the diseased testes. CD4+ T cells are sufficient for disease induction as in vivo depletion of CD4+ cells blocked the emergence of EAO and CD4+ cells from uni-Vx mice were able to adoptively transfer disease. We also identified sperm Zonadhesin (Zan) as the major orchitogenic autoantigen. By immunoblot, autoantibodies from uni-Vx mice prominently recognized Zan, and immunization with Zan in adjuvant induced EAO. As EAO induction in Vx men would be devastating, we depleted Treg two weeks following uni-Vx and found they were free of EAO. To test whether uni-Vx in fact induces tolerance to testicular antigens, we compared uni-Vx and Sham-Vx mice in their response to EAO induction by immunization with testis homogenate in CFA. Indeed, uni-Vx mice developed significantly lower EAO scores and autoantibody and T cell proliferative responses. Thus, Treg protect Vx mice from post-Vx EAO by blocking propagation of danger signals to pathogenic autoimmunity while granting systemic tolerance to testis antigens.

Distribution of a novel binding site for angiotensins II and III in mouse tissues

A novel binding site for angiotensins II and III that is unmasked by parachloromercuribenzoate has been reported in rat, mouse and human brains. Initial studies of this binding site indicate that it is not expressed in the adrenal, liver or kidney of the rat and mouse. To determine if this binding site occurs in other mouse tissues, 8 tissues were assayed for expression of this binding site by radioligand binding assay and compared with the expression of this binding site in the forebrain. Particulate fractions of homogenates of testis, epididymis, seminal vesicles, heart, spleen, pancreas, lung, skeletal muscle, and forebrain were incubated with 125I-sarcosine 1, isoleucine 8 angiotensin II in the presence or absence of 0.3 mM parachloromercuribenzoate plus 10 µM losartan and 10 µM PD123319 (to saturate AT 1 and AT 2 receptors). Specific (3 µM angiotensin II displaceable) high affinity binding occurred in the testis > forebrain > epididymis > spleen > pancreas > lung when parachloromercuribenzoate was present. Binding could not be reliably observed in heart, skeletal muscle and seminal vesicles. High affinity binding of 125I-sarcosine 1, isoleucine 8 angiotensin II was observed in the absence of parachloromercuribenzoate in the pancreas on occasion. This suggests that this novel angiotensin binding site may have a functional role in these tissues.

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The Effect of Eurycoma longifolia on Sperm Quality of Male Rats

The present study investigated the effects of a standardized methanol extract of E. longifolia Jack containing the major quassinoid constituents of 13alpha(21)-epoxyeurycomanone (1), eurycomanone (2), 13alpha,21-dihydroeurycomanone (3) and eurycomanol (4) on the epididymal spermatozoa profile of normal and Andrographis paniculata induced infertile rats. The standardized MeOH extract at doses of 50, 100 and 200 mg/kg, the EtOAc fraction (70 mg/kg), and standardized MeOH extract at 200 mg/kg co-administered with the EtOAc fraction of A. paniculata at 70 mg/kg were each given orally to male Sprague-Dawley albino rats for 48 consecutive days. The spermatozoa count, morphology, motility, plasma testosterone level and Leydig cell count of the animals were statistically analyzed by ANOVA with a post-hoc Tukey HSD test. The results showed that the sperm count of rats given the standardized MeOH extract alone at doses of 50, 100 and 200 mg/kg were increased by 78.9, 94.3 and 99.2%, respectively when compared with that of control (p < 0.01). The low count, poor motility and abnormal morphology of the spermatozoa induced by the A. paniculata fraction were significantly reversed by the standardized MeOH extract of E. longifolia (p < 0.001). The plasma testosterone level of the rats treated with the standardized MeOH extract at 200 mg/kg was significantly increased (p < 0.01) when compared with that of the control and infertile animals. The spermatocytes in the seminiferous tubules and the Leydig cells appeared normal. Testosterone level was significantly higher in the testes (p < 0.01) than in the plasma after 30 days of oral treatment with the standardized MeOH extract. Interestingly, eurycomanone (2) alone was detected in the rat testis homogenates by HPLC-UV and confirmed by LC/MS, and may have contributed towards the improvement of sperm quality. Thus, the plant may potentially be suitable for the management of male infertility.

Testicular Steroid Hormone Secretion in the Boar and Expression of Testicular and Epididymal Steroid Sulphatase and Estrogen Sulphotransferase Activity

Spermatogenesis and epididymal function depend on testicular steroids with estrogens being important regulatory factors. However, testicular estrogen secretion shows distinct species specificities, with the boar being characterized by the production of high amounts of estrone [E1] and estronesulphate [E1S]. As the boar testis also expresses estrogen sulphatase [StS] and sulphotransferase [EST] the present paper is based on the hypothesis that local availability of biologically active estrogens results from an interplay between estrogen synthesis and local activities of StS and EST. Blood was collected during castration of 37 boars, aged between 98 (peripubertal) to 2 793 (old sexually mature) days, from the testicular vein and artery and peripheral circulation; E1, E1S, testosterone [T] and progesterone [P] were determined by established RIA-procedures. Similarly seminal plasma from 21 sexually mature boars was assessed. StS- and EST-activity were determined in testicular- and epididymal homogenates of 3 sexually mature boars (200 d) using (3)H-E1S resp. (3)H-E1 as substrate. Immunohistochemistry [IHC] was applied to locate EST- expressing cells in testes in relation to age and in the epididymis of 3 mature boars. Hormone data showed a high variability. A significant age dependent increase was only observed for E1 and E1S in the peripheral circulation with absolute values being highest for E1S (5-60 nmol/l), followed by T (2.6-14 nmol/l), P (0.5-1.48 nmol/l) and E1 (0.24-0.84 nmol/l). Testicular vein concentrations always exceeded those in the testicular artery with the differences being significant for E1 and P, group 1. Concentrations in the testicular artery and peripheral vein plasma were not different but higher (p<0.0001) than those in seminal plasma with the exception of E1. StS activity was higher (p<0.001) in the testis than the epididymis. EST activity was high in epididymal homogenates and at the level of detection in testis homogenates. IHC located EST in virtually all epididymal epithelial cells. In the testis the number of positive staining Leydig cells decreased (p<0.05) from 72% in the premature to 57% in the mature boars. The provision of biologically active estrogens to the testicular and epididymal compartment is controlled by a complex regulatory system, with the sulphatase pathway being an important component. P is a secretory product of the testis, E1 and E1S are not actively enriched in seminal plasma.

Acid phosphatases in the human testis after prolonged estrogen treatment.

The testes of 2 autopsied adult men and 6 subjects, suffering from prostatic carcinoma, were analysed for acid phosphatase activities, Two of the prostatic patients had been receiving estrogen treatment at least for a year and had completely regressed testes. Testes of other subjects contained well-defined tubules with different spermatogenic cells in abundance. The total acid phosphatase activity, assayed in the homogenate, showed a marked reduction in the testes of estrogen-treated subjects. Enzymes were separated by cellulose chromatography or by gel filtration combined with cellulose chromatography. Three activity peaks were resolved by the former and four by the latter technique, when homogenates of the control testes were used. In contrast, two to three strongly reduced activities could be discerned from the testes of estrogen-treated subjects. The specific activity of each enzyme after fractionation was compared between control and regressed testes. No difference was observed in the activities of enzyme I. Enzyme II was markedly reduced in the regressed testes, but was clearly present. Enzymes III and IV were either totally absent or showed a marked reduction in the regressed testes. It is suggested that a correlation exists between the destruction of germ cells, as a consequence of estrogen treatment, and the marked reduction in the activity of enzymes III and IV.

Studies on the male antifertility agent--gossypol acetic acid. V. Effect of gossypol acetic acid on the fertility of male rats.

Gossypol acetic acid was given to male rats at a dose of 7.5 mg/rat/day six days a week for ten weeks. After nine weeks of gossypol treatment no implantation sites were observed in the females mated with gossypol treated males. After ten weeks of gossypol treatment all the spermatozoa in the vas deferens were non-motile. Gossypol treatment did not affect the body weight and the weights of the accessory sex organs. Plasma LH and FSH levels, hCG binding in testis and succinic dehydrogenase (SDH) and adenosine triphosphatase (ATPase) activities in liver, kidney and testis were not affected by gossypol treatment. Histological observations of the testis revealed partial damage to the seminiferous tubules. Single high doses of gossypol did not induce significant changes in the body weight and weights of testis and accessory sex organs. ATPase activity in the testis was reduced significantly after gossypol treatment, the enzyme activity in liver and kidney, was however, affected at high doses only. Gossypol treatment had no effect on the histoarchitecture of the testis. Intratesticular administration of gossypol evoked localized damage in the testis. Gossypol treatment had no effect on I125 FSH binding to the rat testis homogenate in vitro.

Methoxychlor‐induced alteration in the levels of HSP70 and clusterin is accompanied with oxidative stress in adult rat testis

Methoxychlor, an organochlorine pesticide, has been reported to induce abnormalities in male reproductive tract. However, the insight into the mechanisms of gonadal toxicity induced by methoxychlor is not well known. We investigated whether treatment with methoxychlor would alter the levels of stress proteins, heat shock proteins (HSP), and clusterin (CLU), and oxidative stress-related parameters in the testis of adult male rats. Animals were exposed to a single dose of methoxychlor (50 mg/kg body weight) orally and were terminated at various time points (0, 3, 6, 12, 24, and 72 h) using anesthetic ether. The levels of HSP70, CLU, and the activities of superoxide dismutase (SOD), catalase, and lipid peroxidation levels were evaluated in a 10% testis homogenate. A sequential reduction in the activities of catalase and SOD with concomitant increase in the levels of thiobarbituric acid reactive substance (TBARS) was observed. These changes elicited by methoxychlor were very significant between 6-12 h of posttreatment. Immunoblot analysis of HSP revealed the expression of HSP72, an inducible form of HSP, at certain time points (3-24 h) following exposure to methoxychlor. Similarly, the levels of secretory CLU (sCLU) were also found to be elevated between 3-24 h of treatment. The present data demonstrate methoxychlor-elicited increase in the levels of inducible HSP72 and sCLU, which could be a part of protective mechanism mounted to reduce cellular oxidative damage.

Characterization of an Alternative Splice Variant of LKB1

LKB1 is an upstream activating kinase for the AMP-activated protein kinase (AMPK) and at least 12 other AMPK-related kinases. LKB1 therefore acts as a master kinase regulating the activity of a wide range of downstream kinases, which themselves have diverse physiological roles. Here we identify a second form of LKB1 generated by alternative splicing of the LKB1 gene. The two LKB1 proteins have different C-terminal sequences generating a 50-kDa form (termed LKB1L) and a 48-kDa form (LKB1S). LKB1L is widely expressed in mouse tissues, whereas LKB1S has a restricted tissue distribution with predominant expression in the testis. LKB1S, like LKB1L, forms a complex with MO25 and STRAD, and phosphorylates and activates AMPK both in vitro and in intact cells. A phosphorylation site (serine 431 in mouse) and a farnesylation site (cysteine 433 in mouse) within LKB1L are not conserved in LKB1S raising the possibility that these sites might be involved in differential regulation and/or localization of the two forms of LKB1. However, we show that phosphorylation of serine 431 has no effect on LKB1L activity and that both LKB1L and LKB1S have similar patterns of subcellular localization. These results indicate that the physiological significance of the different forms of LKB1 is not related directly to differences in the C-terminal sequences but may be due to their differential patterns of tissue distribution.

ORIGINAL ARTICLE: LPS Increases the Expression Levels of IL‐18, ICE and IL‐18 R in Mouse Testes

Problem This study examined the effect of intraperitoneal administration of lipopolysaccharide (LPS) to adult male mouse on the expression levels of interleukin‐18 (IL‐18), IL‐18 receptor (IL‐18R) and the IL‐1β converting enzyme (ICE) (IL‐18 family) in their testes and spleen (control).Method of study Adult mice were injected (intraperitoneally; i.p.) with saline (control) or LPS (2, 20, 100 μg/mL; 100 μL/mouse). After 3 and 24 hr, testes and spleen were collected. Testicular tissue was examined for IL‐18 (by ELISA, real time PCR, and western blot analysis), IL‐18R and ICE (western blot and real time PCR analysis) and spleen tissue was examined for the IL‐18 family by real time PCR analysis. Student’s t‐test was used for statistical analysis.Results Homogenates of mouse testes contain and express basal levels of IL‐18, ICE and IL‐18 R. The expression levels of IL‐18, ICE and IL‐18R were significantly increased 3 and 24 hr after intraperitoneal injection of LPS to mature mouse, as examined by ELISA, western blot and real time PCR analysis. However, the expression levels of IL‐18, ICE and IL‐18Rα in the spleen increased significantly only after 24 hr of LPS stimulation, as examined by real time PCR.Conclusion Our results demonstrate that LPS increases the expression levels of the IL‐18 family in mouse testis and spleen, but the time of expression differs between the two organs. The presence of IL‐18 in the testes might be involved in the regulation of physiological and infection/inflammatory processes, and may be part of the autocrine/paracrine factors that control spermatogenesis. Further studies should be performed to confirm this possibility.

Protective effects of vitamin E and curcumin on l-thyroxine-induced rat testicular oxidative stress

Present study examines effects of curcumin and vitamin E on oxidative stress parameters, antioxidant defence enzymes and oxidized (GSSG) and reduced glutathione (GSH) levels in testis of l-thyroxine (T 4)-induced hyperthyroid rats. The oxidative stress in T 4-treated rat testis was evident from elevation in oxidative stress parameters such as lipid peroxide and protein carbonyl contents, decrease in superoxide dismutase (SOD) and catalase (CAT) activities and increase in glutathione peroxidase (GPx) activity. This is accompanied with decrease in number and mortality of epididymal sperms. When the T 4-treated rats were fed with vitamin E and/or curcumin, the lipid peroxide and protein carbonyl contents in crude homogenates of testes decreased to normal level. Treatment of curcumin and/or vitamin E to T 4-treated rats resulted in elevation of SOD level in postmitochondrial fraction (PMF) and mitochondrial fraction (MF) and CAT in PMF, with decreased GPx activity in MF. However, curcumin or vitamin E was unable to change GPx activity alone but in together they elevated the GPx in PMF of T 4-treated rat testis. Both the antioxidants are incapable of producing significant changes in GSH:GSSG ratio of PMF of T 4-treated rats. In MF, GSH:GSSG ratio elevated and decreased respectively by curcumin and vitamin E treatments to T 4-treated rats, however, in together these antioxidants caused an elevated GSH:GSSG ratio with a value less than when vitamin E given alone to T 4-treated rats. Vitamin E not the curcumin elevates total sperm count and percentage of live sperm impaired by hyperthyroid state. In summary, both vitamin E and curcumin are efficient in protecting testis from oxidative stress generated by T 4 mainly in restoring antioxidant enzymes to the level of euthyroid animals up to some extent but vitamin E is more efficient than curcumin.

Oxidative alterations induced by d-aspartic acid in prepubertal rat testis in vitro: A mechanistic study

The objective of the present study was to investigate the oxidative induction response following in vitro treatment with D-aspartic acid (DA) in prepubertal rat testis (homogenates, explants, and cell suspensions). In all three preparations, DA enhanced (P<0.001) lipid peroxidation, manifest as increased reactive oxygen species (ROS) and malondialdehyde (MDA). Further, DA-induced oxidative induction was potentiated (P<0.001) in the presence of iron (5 microM) and 3-amino triazole and mercaptosuccinate (P<0.001), known inhibitors of the peroxide metabolizing enzymes, catalase and glutathione peroxidase, respectively. Testis homogenates exposed to L-arginine (LA) per se had reduced (P<0.001) endogenous levels of ROS and MDA; furthermore, pre-incubation with L-arginine markedly suppressed (P<0.001) DA-induced oxidative induction, suggesting an antagonistic action, perhaps due to LA-derived nitric oxide. In conclusion, DA caused significant oxidative induction in prepubertal rat testis, but this action was abrogated by L-arginine. The relevance of this phenomenon in vivo merits further study, as both of these molecules have specific physiological functions in the testis.