Objective: To prepare a polyclonal antibody against human lysozyme-like protein 4 (LYZL4) expressed in the prokaryotic system and identify the distribution of LYZL4 in the testis. METHODS: The full-length cDNA of LYZL4 was cloned into the pET32a plasmid and the expression of the recombinant LYZL4 (rLYZL4) was induced by IPTG. The rLYZL4 was purified by Ni-NTA and chitin affinity chromatography respectively and its bactericidal activity was observed by bilayer agar plate diffusion assay. The purified rLYZL4 was used as an immunogen to generate the polyclonal antibody, followed by examination of the antibody titer by ELISA and its specificity by Western blot. The distribution of LYZL4 in human tissue, sperm and seminal plasma was identified and its subcellular localization in the testis was determined by immunohistochemistry. RESULTS: rLYZL4 was expressed efficiently in the prokaryotic system and exhibited no bacteriolytic activity against M. lysodeikticus and E. coli. The anti-rLYZL4 polyclonal antibody could bind the recombinant protein with a high sensitivity and specificity. LYZL4 was identified in the testis, epididymis and sperm protein extracts and localized in the acrosomal region of round and elongating spermatids. CONCLUSIONS An anti-rLYZL4 polyclonal antibody was successfully prepared using the prokaryotic expression system. LYZL4 was detected in the acrosomal region of round and elongating spermatids, suggesting an association with the structure and function of the acrosome.
Acrosome ; immunology ; Animals ; Antibodies ; analysis ; Blotting, Western ; DNA, Complementary ; Enzyme-Linked Immunosorbent Assay ; Epididymis ; immunology ; Escherichia coli ; Humans ; Immunohistochemistry ; Male ; Muramidase ; genetics ; immunology ; Plasmids ; Recombinant Proteins ; genetics ; Semen ; immunology ; Spermatozoa ; immunology ; Testis ; immunology

Objective: To investigate the influence of different procedures of testicular sperm retrieval on the levels of serum inhibin B (INHB), antisperm antibodies (AsAb), follicle-stimulating hormone (FSH), and testosterone (T) in patients with azoospermia. METHODS: We randomly assigned 210 azoospermia patients to receive testicular sperm extraction (TESE, n = 50), testicular sperm aspiration (TESA, n = 56), testicular fine needle aspiration (TEFNA, n = 64), or microscopic TESE (micro-TESE, n = 40). We measured the levels of serum INHB, FSH, and T and the positive rate of AsAb before and at 1 and 3 months after surgery. RESULTS: Compared with the baseline, the levels of serum FSH at 1 and 3 months after surgery showed no statistically significant differences in the TESE (［8.51 ± 4.34］ vs ［8.76 ± 3.07］ and ［7.24 ± 3.32］ IU/L, P >0.05), TESA (［7.70 ± 2.72］ vs ［7.90 ± 4.57］ and ［8.04 ± 3.65］ IU/L, P >0.05), TEFNA (［6.04 ± 3.17］ vs ［6.08 ± 2.70］ and ［6.10 ± 3.32］ IU/L, P >0.05), or micro-TESE group (［6.59 ± 2.74］ vs ［6.89 ± 1.78］ and ［6.75 ± 2.57］ IU/L, P >0.05); the positive rate of AsAb (IgM) was significantly increased at 1 month in the TESE (0.00 vs 14.00%, P <0.05) and micro-TESE groups (2.50% vs 15.00%, P <0.05), while the serum T level markedly decreased in the two groups (［16.52 ± 6.25］ vs ［9.25 ± 5.76］ nmol/L and ［14.16 ± 5.45］ vs ［8.23 ± 4.12］ nmol/L, P <0.05); the levels of serum INHB were remarkably reduced at 1 and 3 months in the TESE (［70.56 ± 23.17］ vs ［42.63 ± 15.34］ and ［44.05 ± 18.47］ pg/ml, P <0.05), TESA (［68.71 ± 14.74］ vs ［40.55 ± 20.51］ and ［42.11 ± 19.34］ pg/ml, P <0.05), TEFNA (［76.81 ± 27.04］ vs ［46.31 ± 19.28］ and ［48.32 ± 20.54］ pg/ml, P <0.05), and micro-TESE groups (［74.74 ± 28.35］ vs ［45.27 ± 18.83］ and ［47.64 ± 28.34］ pg/ml, P <0.05), but with no statistically significant differences among the four groups (P >0.05). CONCLUSIONS Different procedures of testicular sperm retrieval have different impacts on the testicular function and AsAb in patients with azoospermia.
Antibodies ; blood ; Azoospermia ; blood ; physiopathology ; Follicle Stimulating Hormone ; blood ; Humans ; Inhibins ; blood ; Male ; Sperm Retrieval ; Spermatozoa ; immunology ; Testis ; metabolism ; physiopathology ; Testosterone ; blood

Animals ; Humans ; Male ; Mice ; Testis ; immunology ; virology ; Zika Virus ; immunology ; isolation & purification

OBJECTIVETo explore the feasibility of evaluating complete ischemia-reperfusion injury (IRI) of the testis by contrast-enhanced ultrasonography with microbubbles (MB) targeted to P-selectin (MBp) in rabbits.

METHODSWe randomly divided 30 healthy adult rabbits into five groups of equal number (control, 0.5 h IRI, 1 h IRI, 2 h IRI, and 4 h IRI), prepared phospholipid MB and MBp, and performed contrast-enhanced ultrasonography of the bilateral testes with MB or MBp at an interval of 20 min at different times after IRI. When MB or MBp disappeared completely in the healthy testis at 4 to 5 min after intravenous injection, we recorded the power of the first frame (F-P) in the IRI testes followed by immunohistochemical staining of the testis tissue.

RESULTSCEU with MBp achieved a significantly higher F-P than that with MB in all the IRI groups (P < 0.05), which was (8.34 +/- 1.20) versus (1.87 +/- 0.25) 10(-5) AU at 2 hours, but there was no significant difference between MB and MBp in the control rabbits (0 AU, P > 0.05). Immunohistochemistry showed a significantly time-dependent increase in the expression of P-selectin in the vascular endothelial cells of the IRI testes, but not in those of the control.

CONCLUSIONContrast-enhanced ultrasonography with MBp can be used to evaluate the inflammatory reaction of testicular ischemia-reperfusion injury.

Animals ; Antibodies ; Disease Models, Animal ; Male ; Microbubbles ; P-Selectin ; immunology ; Rabbits ; Reperfusion Injury ; diagnostic imaging ; Testis ; blood supply ; Ultrasonography

OBJECTIVETo establish a rat model of anti-sperm antibody (AsAb)-mediated immune infertility, and investigate the effects of serum AsAb positive on the Fas/Fas-L apoptosis pathway in testis tissue and testicular germ cells of pubertal male rats.

METHODSThirty 5-week-old Wistar male rats were included in this study, 10 killed for preparation of sperm suspension, 10 as normal controls, and the other 10 made models of AsAb-positive immune infertility (experimental group). Four weeks after modeling, the testes of the rats were harvested for observation of the changes in the testis tissue under the light microscope and detection of the expressions of Fas, Fas-L and Caspase-3 proteins by immunohistochemistry.

RESULTSCompared with the control group, the experimental group showed obvious apoptotic changes in the testis tissue and remarkably increased expressions (OD value) of Fas (161.87 +/- 5.37 vs 176.97 +/- 4.58), Fas-L (150.27 +/- 8.65 vs 187.52 +/- 7.76) and Caspase-3 (120.37 +/- 6.76 vs 157.65 +/- 7.38) (P < 0.01).

CONCLUSIONSerum AsAb affected the infertility of pubertal male rats, and its mechanisms might be associated with up-regulated expression of Fas, Fas-L and Caspase-3 proteins in the Fas/Fas-L apoptotic pathway.

Animals ; Apoptosis ; Autoantibodies ; immunology ; Caspase 3 ; metabolism ; Fas Ligand Protein ; metabolism ; Germ Cells ; cytology ; immunology ; metabolism ; Infertility, Male ; Male ; Rats ; Rats, Wistar ; Signal Transduction ; Testis ; cytology ; metabolism ; fas Receptor ; metabolism

The testis is an immune privileged organ where germ cells are protected from autoimmune attack to ensure its reproductive function. Immune tolerance is important for the normal development and function of the testis. Notwithstanding its immune-privileged status, the imbalance between the tolerogenic and the efferent limb of the testicular immune response may lead to autoimmune damage in inflammatory or infected circumstances. Testicular immune regulation is a complex system involving multiple factors and the study of the regulation mechanisms of the testis is of great significance for access to new therapeutic targets. Currently, testicular immunoregulation is thought to be related with blood-testis barrier, Sertoli cells, immune cells, cytokines and androgen.
Humans ; Immune Tolerance ; Inflammation ; Male ; Testis ; immunology ; pathology

Zearalenone (ZEA), a nonsteroidal estrogenic mycotoxin, is known to cause testicular toxicity in animals. In the present study, the effects of ZEA on spermatogenesis and possible mechanisms involved in germ cell injury were examined in rats. Ten-week-old Sprague-Dawley rats were treated with 5 mg/kg i.p. of ZEA and euthanized 3, 6, 12, 24 or 48 h after treatment. Histopathologically, spermatogonia and spermatocytes were found to be affected selectively. They were TUNEL-positive and found to be primarily in spermatogenic stages I-VI tubules from 6 h after dosing, increasing gradually until 12 h and then gradually decreasing. Western blot analysis revealed an increase in Fas and Fas ligand (Fas-L) protein levels in the ZEA-treated rats. However, the estrogen receptor (ER)alpha expression was not changed during the study. Collectively, our data suggest that acute exposure of ZEA induces apoptosis in germ cells of male rats and that this toxicity of ZEA is partially mediated through modulation of Fas and Fas-L systems, though ERalpha may not play a significant role.
Animals ; Antigens, CD95/*immunology ; Apoptosis/*drug effects/immunology ; Estrogens, Non-Steroidal/*toxicity ; Fas Ligand Protein/*immunology ; Histocytochemistry ; Immunoblotting ; In Situ Nick-End Labeling ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spermatocytes/cytology/*drug effects/immunology ; Spermatogenesis/drug effects/immunology ; Spermatogonia/drug effects/immunology ; Testis/cytology/*drug effects/immunology ; Zearalenone/*toxicity

Ubiquitin conjugating enzyme functions as the second enzyme required for protein ubiquitination and plays an important role in ubiquitin transferring and substrate specific recognition. UBE2W, a newly described member of E2 family, was formerly reported probably involving in phototransduction or retinal degeneration in Drosophila. In this study, we report that murine UBE2W harbors a typical UBC domain and is highly conserved in different vertebrate homologues. GST-tagged UBE2W was expressed in E. coli BL21 (DE3) and purified with GST affinity chromatography. Using this antigen, we generated and further separated rabbit polyclonal antibody of UBE2W, of which the activity and specificity were confirmed by immunoblotting of transiently expressed myc-UBE2W fusion protein. Wide expression of UBE2W was found in brain, muscle, heart, lung, liver, spleen, kidney and testis of mouse with the generated antibody, indicating the functional importance of this novel protein. Furthermore, the UBE2W highly expression was confined to the adult testis and was developmental stage-specific.
Animals ; Antibodies ; metabolism ; Antibodies, Monoclonal ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Male ; Mice ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Testis ; metabolism ; Tissue Distribution ; Ubiquitin-Conjugating Enzymes ; immunology ; metabolism

OBJECTIVETo establish rat models of FSH autoantibody and to investigate the effect of FSH autoantibody on the spermatogenic capability of rat testis.

METHODSThirsty 21-day old SD rats were randomly divided into an experimental and a control group of equal number. A specific polypeptide corresponding to the rat FSHbeta subunit was synthesized and coupled to (keyhole limpet hemocyanin) KLH. The rats in the experimental group were immunized with polypeptide-KLH and these in the control group with KLH. Further immunization was performed every 2 weeks for 7 times. On the 77th, 91st and 105th day of the immunization, 5 rats from the experimental group and another 5 from the control group were killed. Then the structures of the seminiferous tubule and epididymal sperm were observed by light and electron microscope, respectively. Meanwhile, the counts of sperms and the percentage of swelled sperm were calculated. And the level of serum testosterone was detected by enzyme-linked immunospecific assay (ELISA).

RESULTSThe titer of the anti-polypeptide antibody was 1:200 on the 49th day of the immunization, and reached 1:400 on the 63rd. Compared with the control group, the percentage of swelled sperm significantly decreased on the 91st day (60.4 +/- 6.23 vs 50.60 +/- 3.05, P < 0.05), and the number of spermatogenic cells and sperms in seminiferous tubules reduced on the 105th day in the experimental group, the counts of sperms (46.08 +/- 6.56 vs 32.53 +/- 3.41) and the percentage of swelled sperm (60.60 +/- 5.86 vs 48.60 +/- 3.85) significantly lower (P < 0.05), while the level of serum T significantly higher than that in the control group (P < 0.05).

CONCLUSIONFSH autoantibody might cause testis dyszoospermia.

Animals ; Autoantibodies ; physiology ; Follicle Stimulating Hormone ; immunology ; Hemocyanins ; immunology ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Sperm Count ; Spermatozoa ; physiology ; Testis ; physiology

AIMTo investigate the expression and subcellular localization of chemokine-like factor superfamily 2 (CKLFSF2) in human testis and its potential role in spermatogenesis.

METHODSA specific polyclonal antibody against CKLFSF2 was raised. The expression and cellular localization of CKLFSF2 in the seminiferous tubules was checked by immunohistochemistry method. Also, in situ hybridization was applied to localize the mRNA distribution. The EGFP-CKLFSF2 fusion protein was expressed in COS-7 cells to localize its subcellular location in vitro. In addition, the abnormal expression of CKLFSF2 in testes of patients with male infertility was assayed by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry methods.

RESULTSHaving a close correlation with spermatogenesis defects, CKLFSF2 was specifically expressed in meiotic and post-meiotic germ cells, which were localized to the endoplasmic reticulum (ER) near the Golgi apparatus.

CONCLUSIONCKLFSF2 could play important roles in the process of meiosis and spermiogenesis, and might be involved in the vesicular transport or membrane apposition events in the endoplasmic reticulum.

Animals ; Antibody Specificity ; COS Cells ; Cercopithecus aethiops ; Chemokines ; biosynthesis ; immunology ; Endoplasmic Reticulum ; metabolism ; Germ Cells ; metabolism ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Infertility, Male ; metabolism ; MARVEL Domain-Containing Proteins ; Male ; Meiosis ; Microscopy, Confocal ; Spermatogenesis ; physiology ; Testis ; metabolism