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

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

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

Immunocastration is a considerable alternative to a surgical castration method especially in male animal species for alleviating unwanted male behaviors and characteristics. Induction of high titer of antibody specific for gonadotropin-releasing hormone (GnRH) correlates with the regression of testes. Fusion proteins composed of canine GnRH and T helper (Th) cell epitope p35 originated from canine distemper virus (CDV) F protein and goat rotavirus VP6 protein were produced in E. coli. When these fusion proteins were injected to male dogs which were previously immunized with CDV vaccine, the fusion protein of GnRH-CDV Th cell epitope p35 induced much higher antibody than that of GnRH-rotavirus VP6 protein or GnRH alone. The degeneration of spermatogenesis was also verified in the male dogs immunized with the fusion protein of GnRH-CDV Th cell epitope p35. These results indicate that canine GnRH conjugated to CDV Th cell epitope p35 acted as a strong immunogen and the antibody to GnRH specifically neutralized GnRH in the testes. This study also implies a potential application of GnRH-based vaccines for immunocastration of male pets.
Amino Acid Sequence ; Animals ; Antibodies/blood ; Base Sequence ; Contraception, Immunologic/methods/*veterinary ; Distemper Virus, Canine/*immunology ; Dogs/immunology/*physiology ; Epitopes, T-Lymphocyte/*immunology ; Fertility/immunology ; Gonadotropin-Releasing Hormone/chemistry/*immunology ; Male ; Molecular Sequence Data ; Organ Size ; Recombinant Proteins/immunology ; Spermatogenesis/immunology ; T-Lymphocytes, Helper-Inducer/immunology ; Testis/immunology ; Vaccines, Contraceptive/immunology

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 investigate the expression of FasL in rat cryptorchidism and its significance.

METHODSTwenty-four male SD rats (22-day old) were randomly divided into two groups: unilateral cryptorchid group (n = 12) and pseudo-operation group (n = 12). When the rats were 110-day old, blood samples were taken and the rats were killed for analysis. Immunohistochemical method (SP) was used to detect FasL expression in testes and ELISA method to detect serum antisperm antibody (AsAb).

RESULTSThe positive FasL expression rates in cryptorchid and contralateral testes were significantly higher than those in pseudo-operation group (P < 0.001). The serum AsAb positive rates in the cryptorchid group and the pseudo-operation group were 41.7% and 0, respectively, with significant difference(P < 0.01).

CONCLUSIONSFasL expression upregulating in both testes of the unilateral cryptorchid rat may be a protective response of the testis to autoimmunity.

Animals ; Autoantibodies ; blood ; Cryptorchidism ; immunology ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Fas Ligand Protein ; biosynthesis ; Immunohistochemistry ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spermatozoa ; immunology ; Testis ; metabolism ; Up-Regulation

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

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

OBJECTIVETo simplify the method for separation and cultivation of rat testicular Sertoli cells with high viability, quantity and expression efficiency.

METHODSTesticular Sertoli cells from 2 to 3-week-old male Wistar rats were prepared by digestion with collagenase, trypsin and DNase and cultured together with active lymphocytes to observe their killing effect against lymphocytes. After cell culture for 72 h, the Sertoli cells were morphologically observed by different means and identified with transmission electron microscope. Fas ligand and follicle-stimulating hormone receptor (FSHR) were examined immunohistochemically to identify testicular Sertoli cells. SABC method was used for labeling the Fas ligand on the testicular Sertoli cells.

RESULTSThe viability of the isolated and cultured Sertoli cells was more than 90%, and in in vitro culture, Sertoli cells, which expressed the Fas ligand, could kill the active lymphocytes.

CONCLUSIONThis method improves the efficiency in acquisition of rat testicular Sertoli cells expressing Fas ligand, which are believed to be a potential donor for co-transplantation with parathyroid cells to offer immune privilege.

Animals ; Cell Communication ; immunology ; Cell Separation ; methods ; Cell Survival ; immunology ; Cells, Cultured ; Fas Ligand Protein ; metabolism ; Immunohistochemistry ; Lymphocytes ; cytology ; immunology ; Male ; Microscopy, Electron, Transmission ; Rats ; Rats, Wistar ; Receptors, FSH ; metabolism ; Sertoli Cells ; cytology ; metabolism ; ultrastructure ; Testis ; cytology

OBJECTIVESIn order to identify whether the hens immunized with recombinant human testis prostaglandin D synthase (rhtL-PGDS) DNA can produce anti-L-PGDS antibody.

METHODSThe serum were got from the hens immunized with recombinant plasmid pGEX-2T/htL-PGDS DNA (100 micrograms) every 2 weeks for 2 times. The exist of anti-L-PGDS antibody and its titer were tested with agarose dual immunodiffusion and ELISA with rhtL-PGDS as antigen.

RESULTSThe serum anti-L-PGDS antibody in hen immunized with pGEX-2T/htL-PGDS DNA were confirmed and its titer tested by ELISA was 1:2,048.

CONCLUSIONSIt is feasible to produce anti-L-PGDS antibody by immunizing hens with recombinant pGEX-2T/htL-PGES DNA.

Animals ; Antibodies ; analysis ; Chickens ; DNA, Recombinant ; administration & dosage ; immunology ; Enzyme-Linked Immunosorbent Assay ; Female ; Genetic Vectors ; Humans ; Immunization ; Intramolecular Oxidoreductases ; genetics ; immunology ; Lipocalins ; Male ; Models, Animal ; Plasmids ; genetics ; Testis ; enzymology ; Vaccines, DNA ; administration & dosage ; immunology