ObjectiveTo investigate the effects of Qiangjing Tablets (QJT) on sperm quality and the MAPK signaling pathway in the SD rat model of asthenospermia (AS).

METHODSA total of 100 male SD rats were randomly divided into five groups of equal number, blank control, AS model control, high-dose QJT, medium-dose QJT, and low-dose QJT. All the rats were intragastrically administered ORN at 200 mg/kg/d for establishment of the AS model except those in the blank control group, which were given 1% CMC sodium solution at 1 ml/100 g by gavage. Meanwhile the animals of the high-, medium-, and low-dose QJT groups were gavaged with QJT at 6700, 3300 and 1700 mg/kg/d, respectively, qd 6 days a week for 20 days. Then the testis issue and the apoptosis of the testicular cells were observed under the electron microscope, the expression of vimentin in the testis was determined with the immunohistochemical SP method, that of ERK1/2 detected by Western blot, and the concentration of TGF-β1 in the semen measured by ELISA.

RESULTSThe AS model controls showed round nuclei of spermatocytes, homogeneously distributed chromatins, broken or lost mitochondria, and expanded rough endoplasmic reticulum in the testis tissue. In comparison, the rats of the high-, medium-, and low-dose QJT groups exhibited round nuclei of spermatocytes, homogeneously distributed chromatins, and well-structured mitochondria, rough endoplasmic reticulum and ribosome, which were all similar those of the blank controls. Compared with the blank controls, the AS model rats manifested significantly increased expressions of ERK1/2 (1.00 ± 0.00 vs 1.26 ± 0.10, P<0.01) and vimentin (0.16 ± 0.01 vs 0.17 ± 0.01, P<0.01) and apoptosis rate of cells in the testis tissue (［9.20 ± 3.07］ vs ［42.20 ± 9.17］ %, P<0.01), but decreased level of TGF-β1 in the semen (［627.67 ± 26.07］ vs ［566.73 ± 68.44］ ng/ml, P<0.05). In comparison with the model controls, the rats of the high- and medium- -dose QJT groups presented remarkably down-regulated expressions of ERK1/2 (1.26 ± 0.10 vs 1.14 ± 0.08, P<0.01; 1.26 ± 0.10 vs 1.18 ± 0.05, P<0.05) and vimentin (0.17 ± 0.01 vs 0.16 ± 0.01, P<0.01; 0.17 ± 0.01 vs 0.17 ± 0.09, P<0.05) and decreased rate of cell apoptosis (［42.20 ± 9.17］ vs ［21.60 ± 5.94］ %, P<0.01; ［42.20 ± 9.17］ vs ［33.95 ± 6.39］ %, P<0.05). The concentration of TGF-β1 in the semen was markedly lower in the high-dose QJT than in the AS model control group (［621.78 ± 30.80］ vs ［566.73 ± 68.44］ ng/ml, P < 0.05).

CONCLUSIONSQiangjing Tablets could improve semen quality in asthenospermia rats by acting against oxidative stress.

Animals ; Apoptosis ; Asthenozoospermia ; enzymology ; Drugs, Chinese Herbal ; pharmacology ; Male ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Mitogen-Activated Protein Kinases ; drug effects ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Semen ; Semen Analysis ; Signal Transduction ; Spermatozoa ; Testis ; metabolism ; ultrastructure ; Transforming Growth Factor beta1 ; metabolism ; Vimentin ; metabolism

Objective: To elucidate the possible role of human lysozyme-like protein 4 (LYZL4) in fertilization and characterize its enzymatic properties. METHODS: The localization of LYZL4 in human spermatozoa was investigated by immunofluorescence staining, the sources of LYZL4 on the sperm surface examined by RT-PCR, and the role of LYZL4 in fertilization assessed by the zona-free hamster egg penetration test. The recombinant plasmid pPIC9K-LYZL4 was constructed and its expression induced with methanol after transformed into competent Pichia pastoris GS115. The recombinant LYZL4 protein (rLYZL4) was purified from the fermentation supernatant and subsequently identified by Western blot. The hyaluronan binding ability of rLYZL4 was determined by ELISA and the muramidase activity, hyaluronidase activity, and free radical scavenging ability examined by spectrophotometric methods. RESULTS: Immunodetection with a specific antiserum localized LYZL4 on the acrosomal membrane of mature spermatozoa, which was exclusively secreted from the testis and epididymis as shown by RT-PCR. Immunoneutralization of LYZL4 significantly decreased the number of human spermatozoa bound to zona-free hamster eggs in a dose-dependent manner in vitro. The recombinant protein was expressed successfully by the P. pastoris strain GS115. Purified rLYZL4 exhibited a potent hyaluronan binding ability and a strong free radical scavenging ability but no muramidase or hyaluronidase activity. CONCLUSIONS LYZL4 secreted from the testis and epididymis is localized on the acrosomal membrane of mature spermatozoa and plays a role in sperm-egg binding as well as in binding hyaluronan and scavenging free radicals, which suggests that it might be a multi-functional molecule contributive to sperm protection and sperm-egg binding.
Acrosome ; enzymology ; Animals ; Blotting, Western ; Cricetinae ; Enzyme-Linked Immunosorbent Assay ; Epididymis ; Female ; Fertilization ; physiology ; Free Radical Scavengers ; metabolism ; Humans ; Hyaluronic Acid ; metabolism ; Male ; Muramidase ; analysis ; physiology ; Pichia ; Plasmids ; metabolism ; Recombinant Proteins ; analysis ; metabolism ; Sperm-Ovum Interactions ; physiology ; Spermatozoa ; enzymology ; Testis

Angiotensin-converting enzyme functions in the male reproductive system, but the extent of its function in reproduction is not fully understood. The primary objective of this work was to investigate the relationship between the testicular isoform of angiotensin-converting enzyme present in human spermatozoa and semen parameters, human embryo quality, and assisted reproduction success. A total of 81 semen samples and 635 embryos from couples undergoing oocyte donation cycles at the IVI Bilbao Clinic were analyzed. Semen parameters, embryos quality, and blastocyst development were examined according to the World Health Organization standards and the Spanish Association of Reproduction Biology Studies criteria. The percentage of testicular angiotensin-converting enzyme-positive spermatozoa and the number of molecules per spermatozoon were analyzed by flow cytometry. Both parameters were inversely correlated with human sperm motility. Higher percentages of testicular angiotensin-converting enzyme-positive spermatozoa together with fewer enzyme molecules per spermatozoon were positively correlated with better embryo quality and development. Our results suggest that embryos with a higher implantation potential come from semen samples with higher percentages of testicular angiotensin-converting enzyme-positive cells and fewer enzyme molecules per spermatozoon. Based on these findings, we propose that testicular angiotensin-converting enzyme could be used to aid embryologists in selecting better semen samples for obtaining high-quality blastocysts during in vitro fertilization procedures.
Adult ; Embryo Implantation/physiology* ; Embryo Transfer ; Embryonic Development/physiology* ; Fertility/physiology* ; Fertilization in Vitro ; Humans ; Male ; Middle Aged ; Peptidyl-Dipeptidase A/metabolism* ; Sperm Motility/physiology* ; Spermatozoa/enzymology* ; Testis/enzymology*

OBJECTIVEThis study was designed to evaluate the toxic effects of Atrazine (ATZ) on the reproductive system of male rats.

METHODSMale Sprague-Dawley rats were exposed to ATZ by gavage at dosages of 0, 38.5, 77, and 154 mg/kg bw/day for 30 d. The toxic effects of ATZ to rats were assessed through histopathologcal observation, spermatozoa quality evaluation, testicular marker enzyme indicators, antioxidant capacity and reproductive hormone levels.

RESULTSSignificant adverse effects on reproductive system were observed in rats exposed to ATZ at different dosages compared with 0 mg/kg group, including an irregular and disordered arrangement of the seminiferous epithelium in 154 mg/kg group; a decreased spermatozoa number and an increased spermatozoa abnormality rate in 77 and 154 mg/kg groups; decreased levels of acid phosphatase (ACP), alkaline phosphatase (AKP), lactic dehydrogenase (LDH), and succinate dehydrogenase (SDH) with the increasing of ATZ concentration; a decreased level of total antioxidant capacity (TAC) in a dose-dependent manner, and a decreased reduced glutathione (GSH) level and an increased malondialdehyde (MDA) content in 154 mg/kg group; and decreased serum levels of testosterone (T) and inhibin-B (INH-B) and an increased serum level of follicle stimulating hormone (FSH) in 77 and 154 mg/kg groups, and an increased serum level of luteinizing hormone (LH) in 154 mg/kg group.

CONCLUSIONThese results suggested that relatively high doses of ATZ could exert reproductive toxicity of male rats.

Animals ; Antioxidants ; metabolism ; Atrazine ; toxicity ; Body Weight ; drug effects ; Herbicides ; toxicity ; Hormones ; blood ; Male ; Organ Size ; drug effects ; Rats ; Rats, Sprague-Dawley ; Sperm Count ; Spermatozoa ; abnormalities ; drug effects ; Testis ; drug effects ; enzymology ; pathology ; Toxicity Tests, Chronic

OBJECTIVETo explore the effects of Jingui Shenqi Pill (JSP) on the testis telomerase activity in mice of Shen-yang deficiency syndrome (SYDS).

METHODSThe SYDS model was prepared in 30 mice by over-fatigue and sexual overstrain. They were randomly divided into the model group and the JSP group, 15 in each group. Another 15 normal male mice were selected as the normal group. Mice in the normal group were fed routinely, with distilled water administered intragastrically at the daily dose of 0.1 mL/10 g. Mice in the model group were also administered intragastrically with distilled water at the daily dose of 0.1 mL/10 g while modeling establishment. Mice in the treatment group were administered intragastrically with JSP suspension at 0.1 mL/10 g (the concentration was 0.241 g/mL). The intervention lasted for 4 weeks. Four weeks later, the testis telomerase activity was detected in the three groups by ELISA.

RESULTSThe SYDS model was replicated successfully by over-fatigue and sexual overstrain. JSP could improve the signs of mice of SYDS. Compared with the normal group, the activity of testis telomerase decreased in the model group (P < 0.01). Compared with the model group, the testis telomerase activity markedly increased in the treatment group (P < 0.01).

CONCLUSIONSThe testis telomerase activity in mice of SYDS caused by over-fatigue and sexual overstrain obviously decreased, when compared with that in mice of the normal group. JSP could recover its activity.

Animals ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Male ; Mice ; Mice, Inbred Strains ; Telomerase ; metabolism ; Testis ; drug effects ; enzymology ; Yang Deficiency ; drug therapy ; metabolism

OBJECTIVETo observe the effects of metformin on epididymal sperm quality and antioxidant function of the testis in diet-induced obesity rats.

METHODSThirty-two male SD rats were fed on high-fat food for 8 weeks to make obesity models, and another 8 were included as normal controls. Twenty-four of the model rats were equally randomized into a model control group to be fed continuously on high-fat food, a metformin group to be fed on normal food with metformin, and a normal food group. By the end of the 12th week, all the rats were killed for the determination of Lee's index, the organ coefficients of the testis and epididymis, epididymal sperm concentration, sperm motility, grade a + b sperm percentage, and the contents of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) in the testicular homogenate.

RESULTSLee's index was significantly increased in the model control group (P < 0.01) as compared with the other three. Lee's index was markedly higher in the normal control than in the metformin group (P < 0.05). The organ coefficients of the testis and epididymis were significantly decreased in the model control group (P < 0.01) as compared with the other three. Sperm concentration and motility and the percentage of a + b sperm were significantly decreased in the model control in comparison with the other three groups (P < 0.05 or P < 0.01). Sperm concentration was remarkably higher in the normal control than in the metformin and normal food groups (P < 0.05). The content of SOD (U/mg prot) was significantly lower in the model control (90.92 +/- 4.06) than in the normal control and metformin groups (101.69 +/- 8.49 and 102.04 +/- 10.67) (P < 0.05); that of GSH-Px (U) obviously higher in the normal control (28.32 +/- 2.28) than in the model control (23.49 +/- 1.08, P < 0.01), the metformin (25.73 +/- 2.14, P < 0.05) and the normal food group (25.77 +/- 2.19, P < 0.05), but evidently lower in the model control than in the metformin group (P < 0.05); and that of MDA (nmol/mg prot) significantly higher in the model control (2.68 +/- 0.76) than in the normal control (1.84 +/- 0.31, P < 0.01), the metformin (1.88 +/- 0.33, P < 0.01), and the normal food group (2.14 +/- 0.35, P < 0.05).

CONCLUSIONMetformin therapy and improved diet could improve sperm quality and promote the antioxidant ability of the testis in diet-induced obesity rats.

Animals ; Epididymis ; drug effects ; Glutathione Peroxidase ; analysis ; Male ; Malondialdehyde ; analysis ; Metformin ; pharmacology ; Obesity ; metabolism ; Rats ; Rats, Sprague-Dawley ; Sperm Count ; Sperm Motility ; Spermatozoa ; drug effects ; Superoxide Dismutase ; analysis ; Testis ; drug effects ; enzymology

OBJECTIVETo investigate the location of heme oxygenase (HO) enzyme in the human testis, and explore the correlation of the expression of HO enzyme with azoospermia by analyzing its different expression levels in the testes of nonobstructive azoospermia, obstructive azoospermia and normal men.

METHODSWe detected the location of the cells expressing HO enzyme in the human testis tissue using immunohistochemistry, determined the mRNA and protein expression levels of HO-1 and HO-2 in the testes of azoospermia patients and normal healthy men by RT-fluorescence quantitative PCR (RT-FQ-PCR) and Western blot, and explored the correlation of HO expressions with the pathogenesis of azoospermia.

RESULTSHO-1 enzyme was expressed mainly in the Sertoli cells and HO-2 enzyme chiefly in the germ cells of the testis tissue. RT-FQ-PCR showed that the expression of HO-1 in the testis tissue was significantly lower in the nonobstructive azoospermia than in the normal and obstructive azoospermia groups (P < 0.05), with no significant difference between the latter two. Western blot revealed no obvious difference between the expression level of HO-1 protein and that of HO-1 mRNA. There were no differences in the expression level of HO-2 protein among the three groups.

CONCLUSIONThe expression level of HO enzyme is significantly decreased in the testis tissue of nonobstructive azoospermia patients, and the expression of HO-1 protein is consistent with that of HO-1 mRNA. As HO-1 protects the testis tissue against various stress injuries through its antioxidant, anti-inflammatory and anti-apoptotic effects, its decreased expression level may be correlated with spermatogenic dysfunction, and therefore considered as a possible mechanism of nonobstructive azoospermia.

Azoospermia ; enzymology ; metabolism ; Case-Control Studies ; Heme Oxygenase (Decyclizing) ; metabolism ; Heme Oxygenase-1 ; metabolism ; Humans ; Male ; Spermatogenesis ; Testis ; enzymology ; metabolism

To explore the functions of human ribonuclease 9 (RNase 9), we constructed a mammalian fusion expression vector pcDNA-hRNase9, prepared recombinant human RNase 9-His fusion protein from HEK293T cells and determined its N-terminal amino acid sequences. According to the determined mature protein, recombinant human RNase 9 was prepared in E. coli. Ribonucleolytic activity and antibacterial activity of recombinant human RNase 9 were detected, and the distribution of human RNase 9 on tissues and ejaculated spermatozoa and in vitro capacitated spermatozoa were analyzed via indirect immunofluorescence assay. The results showed that recombinant human RNase 9 did not exhibit detectable ribonucleolytic activity against yeast tRNA, but exhibited antibacterial activity, in a concentration/time dependent manner, against E. coli. Immunofluorescent analyses showed that the predicted human RNase 9 was present throughout the epididymis, but not present in other tissues examined, and human RNase 9 was also present on the entire head and neck regions of human ejaculated spermatozoa and in vitro capacitated spermatozoa. These results suggest that human RNase 9 may play roles in host defense of male reproductive tract.
Adult ; Amino Acid Sequence ; Anti-Infective Agents ; metabolism ; Blotting, Western ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Epididymis ; enzymology ; Escherichia coli ; enzymology ; Genetic Vectors ; Humans ; Male ; Molecular Sequence Data ; Recombinant Fusion Proteins ; chemistry ; metabolism ; Ribonuclease, Pancreatic ; metabolism ; Ribonucleases ; chemistry ; metabolism ; Seminal Plasma Proteins ; chemistry ; metabolism ; Spermatozoa ; metabolism ; Testis ; enzymology ; Young Adult

OBJECTIVETo investigate the effects of 3,4-dichloroaniline (3,4-DCA) on activities of testicle enzymes as biological markers in rats.

METHODSFifty male rats were randomly divided into 5 groups (n=10). One group was left untreated and used as a solvent control (administered orally by corn oil), while the other 4 groups were treated with 3, 4-DCA. Corn oil was used as a solvent, and 3,4-DCA was diluted into tested concentrations (39, 81, 170, and 357 mg/kg). All the groups orally administered 3,4-DCA or corn oil, once a day for 4 weeks. The testicle tissue was homogenized in a 0.1 mol/L potassium phosphate buffer (0.1 mol/L, pH 7.2). The crude homogenate was centrifuged at 6000 rpm for 5 min at 4 degrees C. The supernatant obtained was used as an enzyme extract for determination of the enzyme activities.

RESULTSCompared with the control, the activities of ALP, ACP, and SDH were increased significantly at a lower level of 3,4-DCA, and decreased at a higher level of 3, 4-DCA, whreas the activities of LDH, LDH-X, and G6PDH were inhibited significantly with the increased 3,4-DCA concentration. Organ coefficient "organ weight/total body weight x 100" of testis, liver, and spleen increased significantly with the increased 3,4-DCA concentration. These results suggest that 3,4-DCA toxicity to the male reproductive system was associated with the activities of testicular enzymes which are the sensitive biochemical endpoints reflecting 3,4-DCA toxicity to the male reproductive system.

CONCLUSION3,4-DCA has toxicity to the reproductive system in male rats.

Aniline Compounds ; toxicity ; Animals ; Biomarkers ; analysis ; Body Weight ; drug effects ; Male ; Organ Size ; drug effects ; Rats ; Rats, Wistar ; Testis ; drug effects ; enzymology ; Toxicity Tests

AIMTo evaluate the effects of melatonin on antioxidant enzyme levels and histopathologic changes in dizocilpine (MK-801)-induced psychosis model rat testis.

METHODSA total of 24 adult male Wistar-Albino rats were divided into three groups with 8 in each. Group I was used as control. Rats in Group II were injected with MK-801 (0.5 mg/kg body weight i.p. for 5 days). In addition to MK-801, melatonin (50 mg/kg body weight i.p. once a day for 5 days) was injected into the rats in Group III. The testes were harvested bilaterally for biochemical and histopathological examinations. Antioxidant enzyme activities, malondialdehyde, protein carbonyl and nitric oxide (NO) levels in testicular tissues were analyzed using spectrophotometric analysis methods. Histopathological examinations of the testes were also performed.

RESULTSMK-801 induced testicular damage, which resulted in significant oxidative stress (OS) by increasing the levels of antioxidant enzymes. The malondialdehyde, protein carbonyl and NO levels were increased in testicular tissues of rats. Treatment with melatonin led to significant decrease in oxidative injury. Administration of melatonin also reduced the detrimental histopathologic effects caused by MK-801.

CONCLUSIONThe results of the present study showed that MK-801 cause OS in testicular tissues of rats and treatment with melatonin can reduce the harmful effects of MK-801.

Animals ; Antioxidants ; pharmacology ; Disease Models, Animal ; Dizocilpine Maleate ; adverse effects ; pharmacology ; Male ; Malondialdehyde ; Melatonin ; pharmacology ; Mental Disorders ; chemically induced ; Nitric Oxide ; Oxidative Stress ; drug effects ; Protein Carbonylation ; Psychotropic Drugs ; adverse effects ; pharmacology ; Rats ; Rats, Wistar ; Testis ; drug effects ; enzymology ; pathology