Sublethal sperm freezing damage: Manifestations and solutions.

Semen cryopreservation is an important tool for artificial breeding, species conservation and human reproductive medicine. However, sublethal freezing damage remains an important limitation of the cryopreservation process, inevitably leading to reduced fertility in vivo. This review explores several facets of sublethal freezing damage, touching on cryocapacitation, the generation of reactive oxygen species and alterations to sperm proteins, lipids and sugars. The effects of sublethal freezing damage on sperm performance in vivo are also discussed, examining fertile lifespan and interaction with female reproductive tract immune cells, mucus and oviductal cells. Finally, the cryoprotective potential of whole seminal plasma and individual proteins are explored.

Paternal priming of maternal tissues to optimise pregnancy success.

The question of 'how does the allogeneic fetus survive gestation in the face of the maternal immune system?' has yet to be definitively answered. Several acceptable mechanisms exist to facilitate survival of the semi-allogeneic fetus in various species; paramount is the immunological separation of maternal and fetal tissues during gestation. However, keen observation of the maternal immune system during pregnancy has noted maternal immune tolerance to paternal-specific antigens. A mechanism by which the maternal immune system tolerates specific paternal antigens expressed on the fetus would be far more beneficial than the previously proposed immune indolence that would leave the mother susceptible to infection. In species like human or rodent, implantation occurs days after fertilisation and, as such, the mechanisms to establish antigen-specific tolerance must be initiated very early during pregnancy. We and others propose that these mechanisms are initiated at the time of insemination when paternal antigens are first introduced to the maternal immune system. Indeed, a new paradigm demonstrating the importance of paternal-maternal communication at the time of insemination is becoming evident as it relates to maternal tolerance to fetal antigen and ultimately pregnancy success.

A panoramic view to relationships between reproductive failure and immunological factors.

PURPOSE: To examine the possible roles of various immunological factors in recurrent miscarriage and unexplained infertility. METHODS: The synthesis and review of the relevant current literature in English language. RESULTS: Substantial evidence suggests that antiphospholipid antibodies, lupus anticoagulant, antisperm antibodies, antithyroid antibodies, anti-endometrial antibodies, antiovarian antibodies, anti-C trachomatis antibodies, cytokines, and immunological events in endometriosis and premature ovarian failure due to immunologic factors may contribute to reproductive failure including unexplained infertility and/or non-chromosomal recurrent miscarriage. CONCLUSIONS: Elimination or suppression of the immunological factors related with reproductive failure might occupy an important place in the treatment of unexplained infertility and non-chromosomal recurrent miscarriage.

DNA immunization against proacrosin impairs fertility in male mice.

PROBLEM: Evaluation of proacrosin/acrosin ability to induce an immune response in male mice after genetic immunization and assessment of animal fertility. METHOD OF STUDY: Mice received 50 µg per animal of a plasmid containing the human proacrosin cDNA (pSF2-Acro) (control: empty plasmid, pSF2). The humoral response was evaluated by ELISA and immunocytochemistry. In vivo fertility was assessed by mating immunized males with control females. The effect of antibodies upon Ca(+2)-ionophore-induced acrosomal exocytosis (AE) and in vitro sperm-zona pellucida (ZP) binding was also studied. RESULTS: pSF2-Acro-immunized mice developed high levels of specific antibodies (P < 0.05) that recognized the sperm acrosomal cap. The number of fertile mice was lower (P = 0.027) in pSF2-Acro-immunized animals than in controls. Litter size was smaller (P < 0.05) in the pSF2-Acro group compared with controls. A negative correlation (P < 0.05) between antibody levels and litter size was found. Antiproacrosin/acrosin antibodies inhibited sperm-ZP binding (P < 0.0001) and Ca(+2)-ionophore-induced AE (P < 0.05). CONCLUSION: DNA immunization against proacrosin elicits an immune response in male mice associated with abnormal sperm functions and reduced fertility.

Follicle-stimulating hormone receptor (FSHR)-derived peptide vaccine induced infertility in mice without pathological effect on reproductive organs.

In a previous study it was found that priming with recombinant human follicle-stimulating hormone receptor (rhFSHR) protein (F140) and boosting with a peptide containing amino acids 32-44 from FSHR showed a specific immune response and fertility inhibition in adult male mice. However, this priming and boosting led to damage of the reproductive organs. Therefore, to eliminate this damage, the peptide prime-boost strategy was explored as a possible means of avoiding the pathological change while maintaining infertility. Immunisation with the peptide prime-boost strategy led to decreased fertility 10 weeks after vaccination, which is consistent with Balb/C mice treated with the protein prime-peptide boost regime. In contrast to the cellular swelling and spotty necrosis in spermatogonia observed in the protein-primed mice, the mice receiving peptide priming did not display pathological damage in seminiferous tubules and interstitial cells. Thus, the prime-boost immune regime with the FSHR-derived peptide potentially provides a much safer candidate for a contraceptive vaccine.

Diagnosis and treatment of immunologically infertile women with sperm-immobilizing antibodies in their sera.

Detection of sperm-immobilizing antibodies in women may have relevance for diagnosis of immunological infertility. Infertile women in whom sperm-immobilizing antibodies are detected can be refractory to conventional treatments such as timed intercourse or intrauterine insemination (IUI) because the antibodies secreted in the female reproductive tract might impair sperm passage, inhibit fertilization, and prevent normal post-fertilization processes. Hence, manipulation of gametes and embryos from patients with sperm-immobilizing antibodies should be carried out with additional care to avoid fertilization failure resulting from the presence of antibodies during in vitro fertilization (IVF). Moreover, the reasons for the why majority of women do not develop sperm-immobilizing antibodies on exposure to sperm is not clear. The production of sperm-immobilizing antibodies is likely to occur in women with particular HLA haplotypes after repeated exposure to sperm. Characterization of sperm-immobilizing antibodies may help in the identification and characterization of sperm specific antigens that can be used as candidate antigens for the development of sperm based contraceptive vaccines.

Immunocontraceptive potential of the Ig-like domain of Izumo.

To investigate whether the Ig-like domain of sperm protein Izumo or the other part of the protein could be used as an immunocontraceptive antigen, three partially overlapping cDNA fragments (PA, PB, and PC), together covering entire mouse Izumo, were cloned, expressed, and purified. PB contains the whole Ig-like domain of mouse Izumo. The anti-PB antibody significantly inhibited the fusion of sperm with zona-free mouse eggs with no effect on sperm motility, while anti-PA and anti-PC antibodies virtually had no effect on sperm-egg fusion at the same concentration. Furthermore, in the presence of anti-PB antibody, the anti-sperm reactivity could be competitively inhibited by recombinant PB protein. The PB-specific antibody staining was restricted to the acrosome region in acrosome-reacted mouse spermatozoa by indirect immunofluorescence. Active immunization with the PB antigen sharply raised the antibody titers in mouse that were enough to cause a significant reduction in fertility compared to the PA and PC immunized groups. In conclusion, our data indicate that the Ig-like domain of Izumo plays an important role in the fertilization process, as verified by the dose-dependent reduction in fertilization rates in mouse IVF trials and mouse mating assay. These results indicate that the Ig-like domain of Izumo might be a new candidate for the development of a contraceptive vaccine.

The antibody against a nuclear autoantigenic sperm protein can result in reproductive failure.

To study whether the antibody against the testis form of the nuclear autoantigenic sperm protein (tNASP) could result in reproductive failure, we successfully cloned and expressed a 339-bp cDNA fragment of mouse tNASP (mtNASP). Using mouse as a model, recombinant mtNASP (rmtNASP) and a synthetic peptide, human tNASP(393-408) (htNASP(393-408)), were investigated for their antifertility effect. Active immunization with rmtNASP or the synthesized peptide raised high antibody titers in the immunized mice. Sperm-egg binding and fusion assay were carried out in 8-10-week-old BALB/c mice. Sperm-egg binding and in vitro fertilization of mouse oocytes were inhibited by co-incubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of varying concentrations of the antisera against rmtNASP. There was a significant antifertility effect in animals immunized with rmtNASP or the synthesized peptide. The effect on fertility in the mice immunized with the synthesized peptide was reversible. Our data indicate that active immunization with rmtNASP antigen may induce a strong antibody response that causes an inhibition of fertility.

Immunoglobulin superfamily member IgSF8 (EWI-2) and CD9 in fertilisation: evidence of distinct functions for CD9 and a CD9-associated protein in mammalian sperm-egg interaction.

On the mouse egg, the tetraspanin CD9 is nearly essential for sperm-egg fusion, with another tetraspanin, CD81, playing a complementary role. Based on what is known about these proteins, egg tetraspanins are likely to be involved in regulation of membrane order through associations with other egg membrane proteins. Here, we identify a first-level interaction (stable in 1% Triton X-100) between CD9 and the immunoglobulin superfamily member IgSF8 (also known as EWI-2), the first evidence in eggs of such an interaction of CD9 with another protein. We also compared the effects of antibody-mediated perturbation of IgSF8 and CD9, evaluating the robustness of these perturbations in IVF conditions that heavily favour fertilisation and those in which fertilisation occurs less frequently. These studies demonstrate that IgSF8 participates in mouse gamete interactions and identify discrete effects of antibody-mediated perturbation of CD9 and IgSF8. An anti-IgSF8 antibody had moderate inhibitory effects on sperm-egg binding, whereas an anti-CD9 antibody significantly inhibited sperm-egg fusion and, in certain assays, had an inhibitory effect on binding as well. The present study highlights the critical importance of design of IVF experiments for the detection of different effects of experimental manipulations on gamete interactions.

In situ reconstruction of humoral immune response against sperm: comparison of SCID and NOD/SCID mouse models.

PROBLEM: Comparison of two types of immunocompromised mouse strains (SCID and NOD/SCID) for production of human antisperm antibodies (AsA). METHOD OF STUDY: Human peripheral blood lymphocytes (PBL) were grafted to mouse peritoneal cavity and sensitized with natively glycosylated and N-deglycosylated sperm extracts. RESULTS AND CONCLUSION: NOD/SCID mice inoculated with hu-PBLs exhibited higher AsA titres with a tendency for greater sperm agglutination than human AsA raised in SCID mouse model. A comparison between 'native' and deglycosylated sperm extracts revealed higher agglutination titres by sera induced with the latter ones. Inhibitory effect of human polyclonal AsA in sperm penetration assay, however, produced opposite results to that for agglutination. Western immunoblotting was used to evaluate reactive sperm antigens prior to and after in situ sensitization showing multiple bands different from positive reactions brought by original sera of in vivo primed AsA-positive males. It seems that in situ generated AsA recognized sperm entities different from those revealed by blood donor's sera samples.

The involvement of beta-1,4-Galactosyltransferase and N-Acetylglucosamine residues in fertilization has been lost in the horse.

BACKGROUND: In human and rodents, sperm-zona pellucida binding is mediated by a sperm surface Galactosyltransferase that recognizes N-Acetylglucosamine residues on a glycoprotein ZPC. In large domestic mammals, the role of these molecules remains unclear: in bovine, they are involved in sperm-zona pellucida binding, whereas in porcine, they are not necessary. Our aim was to clarify the role of Galactosyltransferase and N-Acetylglucosamine residues in sperm-zona pellucida binding in ungulates. For this purpose, we analyzed the mechanism of sperm-zona pellucida interaction in a third ungulate: the horse, since the Galactosyltransferase and N-Acetylglucosamine residues have been localized on equine gametes. METHODS: We masked the Galactosyltransferase and N-Acetylglucosamine residues before the co-incubation of gametes. Galactosyltransferase was masked either with an anti-Galactosyltransferase antibody or with the enzyme substrate, UDP Galactose. N-Acetylglucosamine residues were masked either with a purified Galactosyltransferase or with an anti-ZPC antibody. RESULTS AND DISCUSSION: The number of spermatozoa bound to the zona pellucida did not decrease after the masking of Galactosyltransferase or N-Acetylglucosamine. So, these two molecules may not be necessary in the mechanism of in vitro sperm-zona pellucida interaction in the horse. CONCLUSION: The involvement of Galactosyltransferase and N-Acetylglucosamine residues in sperm-zona pellucida binding may have been lost during evolution in some ungulates, such as porcine and equine species.

The molecular basis of allorecognition in ascidians.

The process of allorecognition consists of an ability to discriminate self from non-self. This discrimination is used either to identify non-self cells and reject them ("non-self histocompatibility") or to identify self cells and reject them (as in the avoidance of self-fertilization by hermaphrodites ("self incompatibility"). The molecular basis governing these two distinct systems has been studied recently in hermaphroditic ascidian urochordates. Harada et al. postulated two highly polymorphic self-incompatibility loci, Themis (A and B), that are transcribed from both strands, forward to yield sperm (s-) trans-membrane antigen, and reverse to yield the egg vitelline coat (v-) receptor. De Tomaso et al. characterized a candidate histocompatibility locus, encoding a highly variable immunoglobulin. Nyholm et al. isolated its candidate allorecognition receptor, fester. Only a minute similarity was found in the structure of the genes involved. It appears that ascidian harbor two very separate types of labeling and recognition genetic systems: one for self and the other for non-self.

Immunogenicity of a multi-component recombinant human acrosomal protein vaccine in female Macaca fascicularis.

A vaccine formula comprised of five recombinant human intra-acrosomal sperm proteins was inoculated into female monkeys to test whether specific antibodies to each component immunogen could be elicited in sera and whether antibodies elicited by the vaccine affected in vitro fertilization. Acrosomal proteins, ESP, SLLP-1, SAMP 32, SP-10 and SAMP 14, were expressed with his-tags, purified by nickel affinity chromatography and adsorbed to aluminum hydroxide. Five female cynomolgus monkeys were inoculated intramuscularly three times at monthly intervals. All five monkeys developed both IgG and IgA serum responses to each recombinant immunogen on Western blots. Each serum stained the acrosome of human sperm and bound to the cognate native protein on Western blots of human sperm extracts. By ELISA, all monkeys developed IgG to each immunogen, with the highest average absorbance values to ESP, SAMP 32 and SP-10, followed by lower values for SLLP-1 and SAMP 14. IgA was also generated to each component immunogen with the highest average absorbance values to SLLP-1 and SP-10. For antigens that induced an IgA response, the duration of the IgA response was longer than the IgG response to the same antigens. This study supports the concept that a multivalent contraceptive vaccine may be administered to female primates evoking both peripheral (IgG) and mucosal (IgA) responses to each component immunogen following an intramuscular route of inoculation with a mild adjuvant, aluminum hydroxide, approved for human use.

Family of sperm associated antigens: relevance in sperm-egg interaction and immunocontraception.

Overpopulation is a global problem of significant magnitude, with grave implications for the future. Development of new contraceptives is necessary, since current forms of birth control are unavailable, impractical and/or too expensive to many individuals due to sociological, financial, or educational limitations. A novel contraceptive strategy that is receiving considerable attention is that of immunocontraception. The targeting of antibodies to gamete-specific antigens implicated in sperm function, sperm-egg binding and fertilization offers an attractive approach to the growing global problem of over population. The sermatozoon has proteins that are unique, cell specific, immunogenic and accessible to antibodies. Immunological interaction with such molecules can cause block of sperm binding to the oocyte and thus fertilization. Modern biotechnologies (such as sperm proteomics, the determination of molecular and structural details of sperm proteins, and the modelling of protein-ligand interaction using X-ray and/or NMR structures to name a few) are trying to make intervention into the domain of human reproduction possible through the development of a variety of new methods and products to control fertility. The present article highlights the various sperm associated antigens involved in various aspects of sperm-egg interaction.

Detection of antizona pellucida antibodies in the sera from premature ovarian failure patients by a highly specific test.

OBJECTIVE: To develop a highly specific test for the detection of antizona pellucida (ZP) antibodies in the sera from premature ovarian failure (POF) patients. DESIGN: Laboratory study. SETTING: University hospital. PATIENT(S): Twenty-seven idiopathic POF patients, 30 control women, and 30 healthy males. INTERVENTION(S): Anti-ZP antibodies were detected by the microdot assay using a very small amount of human ZP or porcine ZP. The effect of anti-ZP antibodies on sperm-ZP binding was examined by hemizona assay. MAIN OUTCOME MEASURE(S): Results from the microdot assay and hemizona assay. RESULT(S): By the microdot assay using human ZP, the sera from POF patients reacted significantly stronger than those of control women and healthy males. However, no obvious difference could be found by the same assay using porcine ZP among these three groups. Anti-ZP antibodies against sera from some POF patients showed significant blocking effects on sperm-ZP binding assessed by hemizona assay. Anti-ZP antibodies were detected in 7 of 27 POF patients, while none were detected in control women and healthy males. CONCLUSION(S): Some idiopathic POF patients have anti-ZP antibodies in their sera, which were detected with high specificity by a newly developed microdot assay using a very small amount of human ZP.

Glycosylation related actions of glycodelin: gamete, cumulus cell, immune cell and clinical associations.

Glycodelin is an example of a glycoprotein whose complex-type glycans mediate biological actions in human reproduction and immune reactions. Being attached to an identical protein backbone, glycodelin oligosaccharides vary significantly from one reproductive tissue to another and have an effect on its own secretion and role in cell communication. For instance, uterine glycodelin-A inhibits sperm-oocyte interaction by binding on the sperm head. This is a glycosylation-dependent phenomenon, in which fucosyltransferase-5 plays a key role. Glycodelin-S from seminal plasma binds evenly around the sperm head and maintains an uncapacitated state in the spermatozoa, until the isoform is detached during sperm passage through the cervix. Glycodelin-F from follicular fluid and Fallopian tube binds to the acrosomal region of the sperm head, thereby inhibiting both the sperm-oocyte binding and premature progesterone-induced acrosome reaction. The cumulus cells surrounding the oocyte can capture glycodelin-A and -F from the surrounding environment and convert these isoforms to a cumulus cell isoform, glycodelin-C. It differs by glycosylation from the other isoforms, and it too attaches on the sperm head, with the highest density in the equatorial region. Glycodelin-C is capable of detaching the sperm-bound inhibitory isoforms so that the sperm-oocyte binding is enhanced. Glycodelin-A also has immunosuppressive actions directed to cellular, humoral and innate immunity. Although these actions depend mainly on the protein backbone, glycosylation also plays a part. Glycosylated glycodelin may be involved in the protection of spermatozoa against maternal immune reactions, and glycodelin also has apoptogenic activity. Some glycosylation patterns of glycodelin may mask its apoptogenic domain. This review updates the recent research and clinical associations of glycodelin, highlighting the role of glycosylation.

Immunization with autologous CD46 generates a strong autoantibody response in rats that targets spermatozoa.

CD46, a membrane complement regulator, has been implicated as pathogen receptor, T cell activator and contributor to spermatozoa-egg interactions. In man, a role in the fertilization process was suggested by its localization on the acrosome. In rodents, CD46 is expressed only on the spermatozoal acrosome, suggesting an essential role at this site. This restricted expression led us to ask whether immunization with CD46 would generate anti-CD46 antibody responses that might target spermatozoa and influence fertility. We immunized male and female rats with rat CD46. Strong immune responses were generated in all rats and immune sera stained CD46 in testis extracts and in situ in testis and sperm. Incubation of spermatozoa with immune sera caused deposition of immunoglobulin and C3b in an acrosome pattern and reduced motility. We mated immune male rats with naïve females and female immune rats with naïve males. The incidence of pregnancy and number of fetuses were not different in matings involving immune male or female rats compared to controls. Testis sections from immune rats revealed no immunoglobulin deposition on CD46-positive sperm precursors, suggesting that acrosomal CD46 was inaccessible in this location. A minority of spermatozoa harvested from epididymis of immune rats had immunoglobulin and C3b bound to the acrosome, suggesting that anti-CD46, present in genital tract fluids, bound after acrosome reaction. These data demonstrate that the restricted expression of CD46 allows strong anti-CD46 responses in rats that target spermatozoa in vitro and in vivo. The anti-CD46 response did not influence fertility, perhaps reflecting the considerable redundancy for fertilization in rodents.

[The immunocontraceptive effects of Lagurus lagurus and Mus musculus zona pellucida 3 DNA vaccine].

To investigate the immunocontraception effects of different kinds of mice and seek the relatively effective species-specific DNA vaccines, we constructed four corresponding immunocontraceptive recombinant plasmids: pcDNA3-mzp3 (pcD-M), pcDNA3-Izp3 (pcD-L), pcDNA3-aat-comp-mzp3 (pcD-ACM) and pcDNA3-aat-comp-lzp3 (pcD-ACL) using zona pellucida 3 gene of Lagurus lagurus (Lzp3) and Mus musculus (Mzp3) respectively to immunize NIH mice. With the introduction of hydrnamic transfection instead of traditional Hela cell transfection to study the genes expression in vivo, the results indicated that all four recombinants could be expressed in livers of mice; Histogram pattern of ELISA showed that all of the recombinants in mice could elicit high quantity and lasting specific anti-ZP3 antibody; Antifertility experiment showed that Mzp3 and Lzp3 groups both enhanced sterile effects (P < 0.05), especially the group of pcD-ACM had a significant difference compared with control group (P < 0.01). Histology of ovarian sections demonstrated that pcD-M and pcD-L groups had no disruption of follicular development while pcD-ACL and pcD-ACM did the opposite. The present studies proved that L. lagurus zona pellucida 3 gene (Lzp3) and M. musculus zona pellucida 3 gene (Mzp3) had immunocontraception effects and primarily presumed that they didn't possess species specificity.