Qualitative differences in sperm antibody responses in mice of different inbred strains and sexes

To better understand the immunogenetic basis and potential pathological consequences of anti-sperm humoral immunity, age-matched female mice of 9 different inbred strains were immunized with syngeneic sperm and were tested for qualitative (specificity) and quantitative (titer) antibody differences by radioimmunoassay, immunofluorescence and immunoblot techniques. All mice developed antisperm antibodies, although titers varied considerably between inbred strains. Antisperm antibodies produced in this study did not cross-react with membrane antigens on thymocytes, brain or immature testicular germ cells. Immunoblot tests identified 17 major sperm antigen bands; this approach also revealed considerable inter- and intra-strain variation in antisperm antibody specificities among female mice. In a parallel study C57BL/6 male mice demonstrated significantly lower antisperm antibody titers and an absence of response to certain sperm antigens in immunoblot tests when compared to age-matched females of the same inbred strain. These findings provide evidence that genetic factors (including sex) interact with environmental (nongenetic) factors in the control of immune responses to sperm antigens.     

Reconstruction of humoral response to sperm antigens in scid mice

OBJECTIVE AND DESIGN: Production of specific human antisperm antibodies by using human-SCID mice model with deposited peripheral blood lymphocytes. MATERIALS AND METHODS: Human peripheral blood lymphocytes (PBL's; CD8(+)-negative cell fraction) were grafted to the peritoneal cavity of severely-combined immunodeficient (SCID) mice at concentration of 20-35 x 10(6) cells per mouse. Lymphocytes were obtained from non-sensitized individual (to sperm antigen) and from in vivo primed males (vasectomized). Two sets of experiments were carried out, with 'native' (glycosylated) and enzymatically deglycosylated sperm antigenic extracts. In all applied variants, sperm antigens were administered with Complete and then with Incomplete Freund adjuvant to improve an immune response. RESULTS AND CONCLUSION: This approach allowed us to obtain better pronounced humoral antisperm response, specific to sperm deglycosylated antigens when PBL's were obtained from individuals in vivo sensitized to sperm (after vasectomy).     

Modulation of the immune response to teratocarcinoma in mice sensitized by sperm antigens

Mouse primitive teratocarcinoma cells share a common surface antigen with morulae, preimplantation embryo cells and murine and human spermatozoa. 129/Sv mice were immunized with either spermatozoa and subsequently inoculated with various doses of teratocarcinoma 6050. A significant inhibition or acceleration of tumor growth was observed when compared with controls immunized with compatible fibroblasts. These effects were sex-dependent, both the incidence and tumor growth being suppressed in sperm-immunized males. The opposite effects were observed in sperm-presensitized females. Immune sera obtained from both male and female 129/Sv mice exhibited a high binding activity to human spermatozoa when tested in a cellular radioimmunoassay. Thus, immunization with sperm antigens provides immunotherapeutic and/or enhancing effects in male and female 129/Sv mice, respectively.     

Testicular origin of immunobead-reacting antigens in human sperm.

OBJECTIVE: To investigate, by means of immunobinding technique, the antigenic surface expression of human vasa efferentia sperm as compared with that of ejaculated sperm. DESIGN: Briefly, vasa efferentia sperm, retrieved microsurgically, and donors' ejaculated sperm (controls) were used. PATIENTS, PARTICIPANTS: Men with congenital absence of the vas deferens, undergoing sperm aspiration as part of their infertility treatment, and controls who were donors of our sperm bank were first assayed by direct immunobead test and, if found negative, were exposed to sera containing known high titers of antisperm antibodies and then retested by indirect immunobead test. RESULTS: The data showed no difference in binding titers and class of immunoglobulins between vasa efferentia and ejaculated sperm. CONCLUSIONS: These findings suggest that, in humans, sperm acquire those surface antigens commonly detected by immunobinding test within the testes before their transit through the epididymis.     

Cryoprotective effects of various saccharides on cryopreserved mouse sperm from various strains

Aim  Cryopreservation of mouse sperm commonly uses raffinose, which is a trisaccharide, plus 3% skim milk. Because of the present lack of knowledge of the effectiveness of any other saccharides, we examined the cryoprotective effects of various saccharides on the viability of mouse sperm from various strains to determine which saccharides are the best cryoprotectants for mouse sperm. Methods  Sperm from the caudae epididymides of mature C57BL/6J mice were frozen with monosaccharides (fructose, glucose, rhamnose, xylose), disaccharides (lactose, maltose, sucrose, trehalose) or trisaccharides (melezitose, raffinose) in a range of concentrations (4–33%). After thawing, the optimal concentration was determined to be the concentration in which there was the highest proportion of motile sperm. In addition, sperm of inbred and hybrid mice were frozen with the saccharides at the optimal concentrations and used for in vitro fertilization. Results  The optimal concentration was 12% for the disaccharides and 18% for the trisaccharides. The fertility of all strains, except C57BL/6J, showed the best cryoprotective effects with maltose, melezitose and raffinose when compared with fresh sperm. Conclusion  Maltose, melezitose and raffinose have the best effects when used as a protectant for cryopreservation of mouse sperm.     

免疫性不育相关精子抗原研究进展

抗精子抗体是导致不育的一个重要原因,它们结合于精子上不同部位的不同抗原,从而以不同的机理影响着生育。本文就存在于精子不同部位上和不育相关的几个精子抗原的分子生物学特性及功能作一综述。这些抗原包括P18、P36、TSA-1、SPRASA、YLP12、SAMP32、rSMP-B、BS-63、CABYR、E-3和CV。对这些精子抗原的了解将会为免疫性不育的诊断和治疗以及避孕疫苗的开发提供帮助。     

Consequences of autoimmunity to sperm antigens in vasectomized men.

The development by a large percentage of vasectomized men of sperm autoantibodies is discussed in this monograph chapter. The production of anti-sperm antibodies is attributed to: 1) granuloma formation; 2) increased permeability of epithelial barriers in the rete testis and epididymis; and 3) transport of phagocytic cells to regional lymph nodes. Individual variation in type of antibodies and response to antibody production is documented and is thought to depend on such factors as rate of sperm production, the structure of the blood-testis barrier, surgical technique, and expression of immune response genes. Morphological changes in spermatozoa and testes occurring after vasectomy may be induced by immunological mechanisms. Tests of cell-mediated immunity to sperm antigens are described, and more accurate tests are needed. Animal studies provide evidence that chronic immune stimulation can result in formation of circulating immune complexes, resulting in deleterious systemic effects, including damage to kidneys, blood vessels, and cells of the immune system. Further studies of the autoimmune orchitis phenomenon may aid in avoidance of immunologically mediated side effects of vasectomy.     

Two-dimensional electrophoretic analysis of sperm antigens recognized by sperm immobilizing antibodies detected in infertile women.

In this study, high resolution two-dimensional (2-D) gel electrophoresis was used to identify human sperm antigens recognized by the sera from infertile women having sperm immobilizing (SI) antibodies. Two-D gel electrophoresis was employed to separate Percoll purified human sperm proteins using isoelectric focusing (IEF), followed by polyacrylamide gel electrophoresis (PAGE). Sperm proteins were transferred to the nitrocellulose membranes and immunoblotted with seven sera from infertile women with high titers of SI antibodies and 6 sera from those without SI antibodies. The blots were compared to the 2-D composite image of human sperm proteins [Sperm Protein Encyclopedia] and sperm surface index and the sperm surface proteins recognized by infertile sera were identified. Fifty-two human sperm surface proteins reacted with sera containing SI antibodies, while 35 of these were reactive with the SI-negative control sera. The average numbers of protein spots reacted with test and control sera were 24.6 and 15.0 respectively. A subset of sperm surface proteins which were unique to the SI antibodies were identified by the following criteria; the sperm protein spots which were highly reactive with the infertile sera containing SI antibodies but not reactive with any of the SI-negative infertile sera. The coordinates of 4 prominent immunoreactive sperm proteins were considered as possibly relevant to antibody mediated female infertility.     

Specificity of human sperm antigens solubilized by N-cetylpyridinium chloride

To test the specificity of sperm fractions solubilized by N-acetylpyridinium chloride, human spermatozoa were washed 3 times with phosphate-buffered saline and resuspended in 5 ml of the solubilizing solution. The mixture was ultrasonated for 5 minutes in ice water and then centrifuged for 20 minutes. The supernatant was used for specificity tests. Details of the techniques used are given. Using N-acetylpyridinium chloride, a medium polar cationic detergent, 8 antigenic sperm fractions were extracted from ultrasonated spermatozoa run against human sperm-agglutinating sera in 2-dimensional immuno-electrophoresis. In runs against sperm-immobilizing sera, 9 sperm fractions were visualized. In runs against rabbit antihuman spermatozoa serum 11 fractions were found. Most of the antigenic fractions that were separated showed cross-reactivity with allogenic or xenogenic tissues. Cross-reactivity with human and animal organs was observed in many instances. Sperm as well as species specificities were found in some fractions. None of the fractions found could be detected on the surface membranes of intact human spermatozoa. Specific reactivity against a sperm-immobilizing serum was detected but no specific activity against a sperm-agglutinating serum was found. The fraction designated Gi is considered to be the "immobilizing" antigen. The origin of the antigens detectable on the surface membranes of intact spermatozoa was not determined.     

Vasectomy: consequences of autoimmunity to sperm antigens.

Data from studies examining the effects of vasectomy in a large number of nonhuman primates vasectomized for periods ranging up to 14 years are summarized, and these findings and speculations are used as a framework with which to review the subject of autoimmunity and vasectomy. Attention is directed to autoimmunity to sperm antigens following vasectomy (factors affecting antisperm antibody levels, characteristics of circulating antisperm antibodies, antisperm antibodies in seminal plasma, and cellular immunity following vasectomy), and immunopathology of antisperm autoimmunity (local effects on the male reproductive tract and systemic effects on the male reproductive tract). The 6 hypotheses that have been advanced to explain individual variations in dynamics and types of antisperm antibodies produced following vasectomy are reviewed. 3 tests are commonly used to detect free antisperm antibodies after vasectomy: 1) the spermagglutination test; 2) the sperm immobilization test; and 3) the immunofluorescence test. Spermagglutinating (SA) antibodies, the most common type of antibody produced after vasectomy, occur in approximately 2/3 of vasectomized men and in a majority of vasectomized rhesus monkeys. Sperm-immobilizing (SI) antibodies are also produced in a large percentage (40%) of vasectomized men and rhesus monkeys. About 30% of vasectomized men also have antiprotamine antibodies.     

Effects of different doses of ethanol on sperm parameters,chromatin structure and apoptosis in adult mice

Objective

Chronic ethanol abuse causes reproductive organ failure and infertility in both humans and laboratory animals. Since sperm has a critical role in reproductive function, the objective of this unique study was to evaluate the effects of different doses of ethanol on sperm parameters, chromatin structure and apoptosis in adult mice.

Study design

A total of 36 adult male mice were equally divided into four groups. Group 1 received ethanol (10%, v/v) containing saccharin (0.2%, w/v), group 2 received ethanol (5%, v/v) containing saccharin (0.1%, w/v), group 3 was treated with saccharin (0.2%, w/v) and group 4 served as control and fed on basal diet for 35 days. Finally, the left cauda epididymis of each animal was cut and placed in Ham's F10 medium. Retrieved spermatozoa were used to analyze count, motility, morphology and viability. Sperm chromatin condensation and DNA integrity were assessed by five different tests including chromomycin A3 (CMA3), toluidine blue (TB), sodium dodecyl sulfate (SDS), and SCD (sperm chromatin dispersion) and sperm apoptosis was assessed by TUNEL.

Results

Following ethanol consumption, the sperm count diminished in the ethanol-treated groups. A decrease in sperm motility and an increase in the rate of morphological abnormalities (coiled and broken tails) were seen in the experimental and saccharin groups in comparison with controls. We showed that ethanol consumption can disturb sperm DNA integrity and chromatin remodeling and it may also induce sperm apoptosis. The rates of sperm apoptosis were 51.57 ± 7.45 and 42.85 ± 6.76 in the high ethanol dose and low ethanol dose groups, respectively.

Conclusion

The results showed that alcohol has negative effects on sperm parameters, chromatin/DNA integrity and apoptosis in mice. These alcohol-induced sperm anomalies may be dose-dependent.     

Low fertility in vivo resulting from female factors causes small litter size in 129 inbred mice

Purpose  

129 inbred mice show poor reproductive ability, as evidenced by small litters; however, the exact cause of this is unknown. In the present in vivo study we examined fertility and subsequent post-implantation development in an attempt to clarify the cause of small litter size in 129 mice.     

Developmental studies of sperm surface antigens using sperm-specific monoclonal antibodies

Sperm-specific monoclonal antibodies generated against mouse sperm isoantigens were used to analyze the developmental expressions of sperm surface antigens during different stages of spermatogenesis. Indirect immunofluorescent assays using the freshly isolated testicular cells from mature and immature mice revealed that a number of monoclonal antibodies did not stain the surface of spermatogenic cells in testis. Instead these antibodies reacted with the sperm surface antigens in testis and/or epididymis. Further analysis was performed using frozen testicular sections from mice of day 14 to day 30 after birth. It was generally observed that a significant number of these antibodies reacted with the cytoplasmic components of spermatogenic cells in testis at the postmeiotic stages (e.g. day 22 after birth). After meiosis, the percentage of seminiferous tubules that were stained by immunofluorescence was found to increase with the ages of the mice. The results of this study suggest that some cytoplasmic components (especially acrosomal) of spermatogenic cells are expressed postmeiotically in testis, but later translocated to the sperm surface during very late stages of spermatogenesis.     

不同来源精子行ICSI助孕1662个周期治疗结局分析

目的:探讨不同来源精子行卵胞浆内单精子显微注射(ICSI)助孕的妊娠结局。方法:回顾分析我中心2006年1月~2010年6月1662个ICSI治疗周期,按精子来源分为射出精子来源(重度少、弱精子)组1208周期,附睾穿刺取精(PESA)组324周期,睾丸穿刺取精(TESA)组130周期,比较3组胚胎发育情况和妊娠结局等指标。结果:射出精子组及PESA组受精率、卵裂率及2PN率较TESA组高(79.1%,77.9%vs 73.9%;98.7%,98.8%vs 96.6%;74.6%,73.0%vs 69.5%),TESA组1PN率较射出精子组及PESA组高(3.8%vs 2.2%,2.6%),差异均有统计学意义(P<0.05);3组优质胚胎率、胚胎种植率、临床妊娠率、异位妊娠率、流产率、单胎出生率、双胎出生率、畸形率无统计学差异。结论:PESA及TESA来源精子行ICSI助孕可获得与射出精子相似的妊娠结局。     

Molecular and immunological characteristics of sperm antigens involved in egg binding.

Development of a vaccine(s) based on sperm antigens represents a promising approach to contraception. The utility of an antigen in immunocontraception is contingent upon sperm/testis-specificity and its involvement in the fertilization process. Since, the sperm-zona pellucida (ZP) recognition and binding constitutes the most important event in the fertilization process, molecules involved at this site are attractive candidates for immunocontraception. Using hybridoma technology, subtractive hybridization, and differential display technology, our laboratory has delineated several sperm antigens. These antigens have testis-specific expression and have a role in the fertilization process. The cDNAs encoding for the antigens have been cloned and sequenced. Among these, the fertilization antigen (FA-1) is particularly interesting, because it is involved in immuno-infertility in humans. Using the phage peptide display technique, a novel dodecamer sequence of a approximately 72+/-5 kD antigen, designated as YLP(12), that is testis-specific and involved in human sperm-ZP recognition/binding, was identified. A synthetic 12-mer peptide was generated based on this sequence. In the hemizona assay, YLP(12) peptide and its monovalent Fab' antibodies specifically and significantly inhibited human sperm-ZP binding. Furthermore, the presence of specific antibodies reactive with YLP(12) peptide, were identified in the serum and seminal plasma of immuno-infertile men. Thus, FA-1 and YLP(12) are promising target antigens for the development of contraceptive vaccines as well as for specific diagnosis and treatment of male infertility.     

Characterization of human sperm antigens and antisperm antibodies in infertile patients.

OBJECTIVE: To identify which sperm antigens may elicit the production of functionally important antisperm antibodies. DESIGN: Immunoblot analysis was performed on 69 serum and 9 seminal plasma samples from infertile patients, using detergent extracts of pooled donor sperm as the antigen source. Serum and seminal plasma had been previously tested by an indirect immunobead binding test (IBT); 61 IBT-positive and 17 IBT-negative samples were included in the study. Proteins recognized by IBT-positive but not IBT-negative samples were most likely to be cell surface antigens, whereas proteins recognized by both IBT-positive and IBT-negative samples were probably intracellular. Antibodies directed toward surface antigens would be most likely to affect fertilization. Characterization of sperm surface proteins on both acrosome-intact and -reacted sperm used labeling of cell surface proteins with an N-hydroxysuccinimide ester of biotin, fractionation of sperm heads and tails, and lectin binding to determine glycosylation. RESULTS: Specific immunoreactivity (with respect to IBT results) was observed to 35K, 40 to 45K, 57K, 66K, and 88 to 90K MW proteins. Characterization studies identified an 88K MW glycosylated plasma membrane protein, a 66K MW inner acrosomal membrane protein, a 34K MW inner acrosomal membrane protein, and a 35K MW prominent tail protein. CONCLUSION: Immunological infertility may involve several antigens characterized in this study. Further studies are necessary to determine if antibodies to these specific proteins interfere with sperm function.     

Identification of human sperm antigens reacting with antisperm antibodies from sera and genital tract secretions.

OBJECTIVE: To identify sperm antigens reacting with antisperm antibodies relevant in human infertility. DESIGN: The reactions of separated sperm antigens with antibodies present in sera and genital tract secretions from infertile and fertile females and males were examined by immunoblotting techniques. SETTING: The patients were followed in an outpatient setting of a hospital clinic. PATIENTS: One hundred consecutive infertile males and females, referred for determinations of antisperm antibodies, comprised the study group. Fifty hospital and faculty employees with proven fertility served as a control group. RESULTS: A high proportion of sera from fertile and infertile humans contained antibodies reacting with at least one sperm antigen. However, two discrete bands of antigenic proteins with molecular weights of 44 and 72 kd reacted significantly more frequently with serum antibodies from infertile females than from fertile females. No apparent correlation could be demonstrated between any particular antigen and serum antibodies from infertile males. Nevertheless, antigenic proteins of 62 kd were identified as the major sperm antigens reacting with antibodies present in seminal plasmas from infertile males. CONCLUSIONS: The major sperm antigens reacting with systemic antibodies differ from the antigens recognized by local antisperm antibodies. Sperm antigens exhibiting relative molecular weights of 62 kd are major antigens reactive with local antisperm antibodies from infertile humans.     

Antisperm antibodies to sperm surface antigens in women with genital tract infection

Antisperm antibodies to sperm surface antigens in nulligravid women with primary upper genital tract infections were measured by the sperm mixed agglutination reaction assay. As many as 56% of women with a primary episode of pelvic inflammatory disease had antisperm antibodies. In addition, 69% of those women with no history of genital tract infection but with laparoscopic evidence of past pelvic infection had significant levels of circulating antisperm antibodies. Electroimmunoblots of sperm preparations probed with the sera of women who had either known or presumed upper genital tract infection revealed a uniformly recognized 69 kd antigen. In contrast, women with circulating antisperm antibodies before primary upper genital tract infection recognized up to five distinct sperm antigen determinants of 27, 54, 131, 146, and 174 kd. It is a distinct possibility that genital tract infections may lead to immunopotentiation of antisperm antibodies that could affect fertility.