Sperm specific proteins-potential candidate molecules for fertility control

The increase in population growth rate warrants the development of additional contraceptive methods that are widely acceptable, free from side effects and less expensive. Immunocontraception, and in particular the targeting of antibodies to gamete-specific antigens implicated in sperm egg binding and fertilization, offers an attractive approach to control fertility. The development of a contraceptive vaccine based on sperm antigen represents a promising approach to contraception. In mammals, fertilization is completed by the direct interaction of sperm and egg, a process mediated primarily by sperm surface proteins. Sperm have proteins that are unique, cell specific, immunogenic and accessible to antibodies. A few of the sperm specific proteins have been isolated and characterized. The antibodies raised against the sperm specific antigens have proved to be extremely effective at reducing sperm-egg interaction in vitro; fertility trials in sub-human primates would eventually prove the effectiveness of the sperm antigens in terms of contraceptive efficacy.     

SNARE proteins and caveolin-1 in stallion spermatozoa: possible implications for fertility

Proteins implicated in the "SNARE hypothesis" for membrane fusion have been characterized in the acrosome of several mammalian species, and a functional role for these proteins during the acrosome reaction has been proposed. We have investigated the presence of SNAREs in equine sperm, using semen samples obtained from stallions with varying fertility. Immunocytochemical analysis revealed that members of different SNARE families can be detected on the acrosome of equine sperm, notably in the acrosomal cap and equatorial segment. These proteins include the t-SNARE syntaxin, the v-SNARE synaptobrevin/VAMP, the calcium sensor synaptotagmin, and the ATPase NSF. Also present is caveolin-1, a component of lipid rafts. Stallions with fertility problems presented the worst quality of sperm and acrosomal membrane, and had less sperm cells stained positively for SNAREs and caveolin-1, than sperm from fertile donors (p < 0.001). Ubiquitin surface staining was also performed and it seemed to inversely correlate with stallion fertility, supporting data obtained with the negative staining technique. A male-related problem was confirmed when mares that had failed to impregnate with samples from an infertile stallion were successfully inseminated with sperm from a fertile donor. Furthermore NSF, synaptotagmin and caveolin-1 staining seemed to be useful in predicting stallion fertility, i.e. significantly more sperm cells stained positively for these proteins in samples from fertile males. Although these results need to be expanded on a larger scale, they suggest that acrosomal and surface staining of equine sperm with novel probes may constitute useful tools in predicting stallion fertility.     

Immunocontraceptive effect of Izumo and enhancement by combination vaccination

Sperm proteins are being investigated for their applications in the development of contraceptive vaccines (CV) in several laboratories. In the present study, various synthetic peptides based upon four sperm proteins, namely Izumo, fertilization antigen-1 (FA-1), YLP(12), and SP56 that are involved in various steps of the fertilization cascade were examined for their immunocontraceptive effect. The synthetic peptides were conjugated to four carrier proteins namely keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), chicken gamma-globulin, and chicken ovalbumin. Female mice were immunized with various peptide vaccines and each booster injection was given with the peptide conjugated to a different carrier protein. Two different fertility trials with different doses of the peptide vaccines were conducted to examine the contraceptive effect. Injection of 150 microg of the peptides (Trial II) caused a significantly higher immune response in serum as well as in the vaginal tract causing enhanced contraceptive effect than those observed after injection with 75 microg of the peptides (Trial I). Immunization with the peptide vaccines based upon Izumo, which is involved in sperm-egg plasma membrane fusion, caused a significant reduction in fertility. The contraceptive effect was enhanced by immunizing with the peptides based upon other antigens (FA-1, YLP(12), and SP56), resulting in an overall 73.33% reduction in fertility. When the antibodies against the peptides disappeared after >9-10 months from circulation and genital tract, all the animals regained fertility. These findings indicate for the first time that the immunization with Izumo and other sperm peptides namely FA-1, YLP(12), and SP56 induces antibodies in serum and genital tract that cause a reversible long-term contraceptive effect in female mice. The data further indicate that the proteins involved in sperm-egg fusion can also be used for contraceptive vaccine development. The contraceptive effects are enhanced by immunizing with the multipeptide vaccines.     

Comprehensive profiling of accessible surface glycans of mammalian sperm using a lectin microarray

It is well known that cell surface glycans or glycocalyx play important roles in sperm motility, maturation and fertilization. A comprehensive profile of the sperm surface glycans will greatly facilitate both basic research (sperm glycobiology) and clinical studies, such as diagnostics of infertility. As a group of natural glycan binders, lectin is an ideal tool for cell surface glycan profiling. However, because of the lack of effective technology, only a few lectins have been tested for lectin-sperm binding profiles. To address this challenge, we have developed a procedure for high-throughput probing of mammalian sperm with 91 lectins on lectin microarrays. Normal sperm from human, boar, bull, goat and rabbit were collected and analyzed on the lectin microarrays. Positive bindings of a set of ~50 lectins were observed for all the sperm of 5 species, which indicated a wide range of glycans are on the surface of mammalian sperm. Species specific lectin bindings were also observed. Clustering analysis revealed that the distances of the five species according to the lectin binding profiles are consistent with that of the genome sequence based phylogenetic tree except for rabbit. The procedure that we established in this study could be generally applicable for sperm from other species or defect sperm from the same species. We believe the lectin binding profiles of the mammalian sperm that we established in this study are valuable for both basic research and clinical studies.     

免疫不育疫苗的研究进展

免疫不育疫苗主要以哺乳动物的精子或卵子蛋白以及在受精和胚胎早期发育过程中发挥重要作用的激素为靶抗原。以激素为抗原的不育疫苗产生的不育效果多为不可逆的,且对机体损伤较大。以精子表面抗原制备的疫苗能够诱导产生精子抗体和不育效果,目前已成为避孕研究的一个热点。哺乳动物卵透明带（zona pellucida,ZP）是覆盖于卵母细胞及着床前受精卵外的一层基质,其在调节精卵特异性结合、诱导获能精子发生顶体反应和阻止多精受精等方面发挥着重要作用。ZP相对分子质量较小且免疫原性强,是免疫不育疫苗理想的靶抗原,抗ZP抗体可阻断精卵结合,故可被用作人类避孕和免疫不育控制有害动物种群数量的靶抗原,但人用ZP疫苗免疫机体后造成的卵巢功能损伤和免疫抑制等问题尚有待明确。     

Inhibiting sperm motility for male contraception: will the sperm tail be its "Achilles heel"?

Blocking sperm motility has great appeal as a male contraceptive. A drug that targets sperm motility might have a very rapid onset of action, possibly allowing for administration immediately prior to intercourse and minimizing concerns about compliance. Promising sperm motility targets include transmembrane calcium channels, a unique adenylyl cyclase, and novel flagellar proteins. Future efforts directed towards effectively antagonizing the activities of these or other such targets will be required to completely impair sperm production, function, or both and create a usable male "pill."     

Bull subfertility is associated with low levels of a sperm membrane antigen

During epididymal transit, mammalian spermatozoa acquire new surface antigens that may participate in gamete interaction. We have previously described a 26 kDa (P26h) epididymal hamster sperm protein that is proposed to be involved in fertilization. We have also identified its human homolog, P34H. Variability in the amount of P34H on spermatozoa from fertile and idiopathic infertile men provides strong evidence that this protein is a potential marker of male fertility. Since these sperm antigens constitute a family of proteins with common antigenicity, we have investigated the presence of a related protein in bovine sperm. In the present study, a P26h antiserum recognized two bull sperm proteins of 21 kDa and 25 kDa (MW) on SDS‐PAGE. We showed that P25b could be extracted with detergent as a surface protein, whereas the P21b was associated with non‐soluble intracellular structures. Sonication of whole sperm cell suspensions and subsequent Percoll gradient centrifugation revealed that P21b may be a flagellar protein whereas the P25b may be located in the head region. Western blot analysis was used to determine the amount of P25b and P21b proteins present on spermatozoa obtained from fertile and subfertile bulls. P21b protein levels were similar in fertile and subfertile bulls, but P25b protein levels were variable. Thus, all bulls with high Non‐Return Rates (fertile bulls) demonstrated high amounts of P25b, whereas P25b levels were decreased in semen from subfertile bulls. We conclude that the protein P25b is a potential fertility marker in the bull and consequently may provide an invaluation tool for the evaluation of bull fertility. Mol. Reprod. Dev. 52:57–65, 1999. © 1999 Wiley‐Liss, Inc.     

Comprehensive mapping of the bull sperm surface proteome

While the mechanisms that underpin maturation, capacitation, and sperm–egg interactions remain elusive it is known that these essential fertilisation events are driven by the protein complement of the sperm surface. Understanding these processes is critical to the regulation of animal reproduction, but few studies have attempted to define the full repertoire of sperm surface proteins in animals of agricultural importance. Recent developments in proteomics technologies, subcellular fractionation, and optimised solubilisation strategies have enhanced the potential for the comprehensive characterisation of the sperm surface proteome. Here we report the identification of 419 proteins from a mature bull sperm plasma membrane fraction. Protein domain enrichment analyses indicate that 67% of all the proteins identified may be membrane associated. A large number of the proteins identified are conserved between mammalian species and are reported to play key roles in sperm–egg communication, capacitation and fertility. The major functional pathways identified were related to protein catabolism (26S proteasome complex), chaperonin‐containing TCP‐1 (CCT) complex and fundamental metabolic processes such as glycolysis and energy production. We have also identified 118 predicted transmembrane proteins, some of which are implicated in cell adhesion, acrosomal exocytosis, vesicle transport and immunity and fertilisation events, while others have not been reported in mammalian LC‐MS‐derived sperm proteomes to date. Comparative proteomics and functional network analyses of these proteins expand our system's level of understanding of the bull sperm proteome and provide important clues toward finding the essential conserved function of these proteins.     

Nutlin-3a Decreases Male Fertility via UQCRC2

Ubiquinol-cytochrome-c reductase core protein 2 (UQCRC2) is a component of ubiquinol-cytochrome c reductase complex that is known to correlate with male fertility via spermatogenesis. Simultaneously, nutlin-3a is a small molecule antagonist of mouse double minute 2 repressor (MDM2), activate p53 and induce apoptosis responsible for spermatogenesis. To date, however there are no known effects of nutlin-3a on reproduction. Therefore, present study was designed to investigate the effect of nutlin-3a on male fertility via UQCRC2. In this in vitro trial with mice spermatozoa, we utilized CASA, CTC staining, ATP assay, western blotting, and IVF to measure the main study outcome. The short-term exposure of spermatozoa in nutlin-3a decreases sperm motion kinematics, intracellular ATP production, capacitation, the acrosome reaction, UQCRC2, and tyrosine phosphorylation (TYP) of sperm proteins in a dose-dependent manner. Notably, the decreased UQCRC2 and TYP were associated with reduced sperm kinematics, ATP production, and capacitation, which ultimately led to adverse effects on male fertility such as poor fertilization rates and embryo development. Thus, nutlin-3a may be considered as a potential male contraceptive agent due to its ability to decrease fertility secondary to changes in overall sperm physiology and embryonic development. However, the results of this preliminary study have to be confirmed by additional independent trial.     

Purification and characterization of a Ca2+-binding protein in Lumbricus terrestris.

A Ca2+-binding protein which is capable of activating mammalian Ca2+-activatable cyclic nucleotide phosphodiesterase has been purified from Lumbricus terrestris and characterized. This protein and the Ca2+-dependent protein modulator from bovine tissues have many similar properties. Both proteins have molecular weights of approximately 18,000, isoelectric points of about pH 4, similar and characteristic ultraviolet spectra, and similar amino acid compositions. Both proteins bind calcium ions with high affinity. However, the protein from Lumbricus terrestris binds 2 mol of calcium ions with equal affinity, Kdiss = 6 X 10(-6) M, whereas the Ca2+-dependent protein modulator from bovine tissues binds 4 mol of calcium ions with differing affinities. Although the Ca2+-binding protein of Lumbricus terrestris activates the Ca2+-activatable cyclic nucleotide phosphodiesterase from mammalian tissues, we have failed to detect the existence of a Ca2+-activatable phosphodiesterase activity in Lumbricus terrestris. The activation of phosphodiesterase by the Ca2+-binding protein from Lumbricus terrestris is inhibited by the recently discovered bovine brain modulator binding protein (Wang, J. H., and Desai, R. (1977) J. Biol. Chem. 252, 4175-4184). Since the modulator binding protein has been shown to associate with the mammalian protein modulator to result in phosphodiesterase inhibition, it can be concluded that the Lumbricus terrestris Ca2+-binding protein also associates with the bovine brain modulator binding protein. Attempts to demonstrate the existence of a similar modulator binding protein in Lumbricus terrestris have been unsuccessful.     

Flow cytometric sexing of mammalian sperm

This review reexamines parameters needed for optimization of flow cytometric sexing mammalian sperm and updates the current status of sperm sexing for various species where this technology is currently being applied. Differences in DNA content have provided both a method to differentiate between these sex-determining gametes and a method to sort them that can be used for predetermining sex in mammals. Although the DNA content of all cells for each mammalian species is highly conserved, slight but measurable DNA content differences of sperm occur within species even among cattle breeds due to different sizes of Y-chromosomes. Most mammals produce flattened, oval-headed sperm that can be oriented within a sorter using hydrodynamic forces. Multiplying the percentage the difference in DNA content of the X- or Y-chromosome bearing sperm times the area of the flat profile of the sperm head gives a simple sorting index that suggests that bull and boar sperm are well suited for separation in a flow sorter. Successful sperm sexing of various species must take into account the relative susceptibilities of gametes to the stresses that occur during sexing. Sorting conditions must be optimized for each species to achieve acceptable sperm sexing efficiency, usually at 90% accuracy. In the commercial application of sperm sexing to cattle, fertility of sex-sorted bull sperm at 2 x 10(6)/dose remains at 70-80% of unsexed sperm at normal doses of 10 to 20 x 10(6) sperm. DNA content measurements have been used to identify the sex-chromosome bearing sperm populations with good accuracy in semen from at least 23 mammalian species, and normal-appearing offspring have been produced from sexed sperm of at least seven species.     

Relationships between the dynamics of iatrogenic DNA damage and genomic design in mammalian spermatozoa from eleven species

The dynamic onset of DNA fragmentation in mammalian sperm populations varies widely in different species when the spermatozoa are incubated in vitro at body temperature for several hours, and recent studies have shown that the dynamic rate of DNA fragmentation within a species has considerable predictive value in terms of fertility. The reasons for such variation are unclear, but here we show that differences in protamine sequence and identity could be partially responsible. Sets of 10 normal semen samples from 11 species (ram, goat, boar, white-tailed deer, rabbit, human, domestic and Spanish fighting bull, horse, donkey, rhinoceros, and koala) were cryopreserved, thawed, diluted in an appropriate extender for each species, and then incubated for 4 hr at 37 °C. Semen samples from human infertility patients were also included for comparison with the donors. DNA fragmentation analysis was undertaken immediately after thawing (t(0)) and after 4 hr (t(4)) using the Halomax/Halosperm procedure, and the differences in DNA fragmentation between t(0) and t(4) were examined in the context of the respective protamine genomes. The expression of protamine 2 in a species significantly enhanced the likelihood of sperm DNA fragmentation; greater numbers of cysteine residues in protamine 1 tended to confer increased sperm DNA stability, and there were logical evolutionary relationships between species in terms of their sperm DNA stability. Human spermatozoa from infertility patients exhibited considerably higher DNA instability than the normal semen donors, a difference that could be indirectly attributed to unbalanced protamine 1-to-protamine 2 ratios.     

Evidence of amino acid diversity-enhancing selection within humans and among primates at the candidate sperm-receptor gene PKDREJ

Sperm-egg interaction is a crucial step in fertilization, yet the identity of most interacting sperm-egg proteins that mediate this process remains elusive. Rapid evolution of some fertilization proteins has been observed in a number of species, including evidence of positive selection in the evolution of components of the mammalian egg coat. The rapid evolution of the egg-coat proteins could strongly select for changes on the sperm receptor, to maintain the interaction. Here, we present evidence that positive selection has driven the evolution of PKDREJ, a candidate sperm receptor of mammalian egg-coat proteins. We sequenced PKDREJ from a panel of 14 primates, including humans, and conducted a comparative maximum-likelihood analysis of nucleotide changes and found evidence of positive selection. An additional panel of 48 humans was surveyed for nucleotide polymorphisms at the PKDREJ locus. The regions predicted to have been subject to adaptive evolution among primates show several amino acid polymorphisms within humans. The distribution of polymorphisms suggests that balancing selection may maintain diverse PKDREJ alleles in some populations. It remains unknown whether there are functional differences associated with these diverse alleles, but their existence could have consequences for human fertility.     

Sperm development and motility are regulated by PP1 phosphatases in Caenorhabditis elegans

Sperm from different species have evolved distinctive motility structures, including tubulin-based flagella in mammals and major sperm protein (MSP)-based pseudopods in nematodes. Despite such divergence, we show that sperm-specific PP1 phosphatases, which are required for male fertility in mouse, function in multiple processes in the development and motility of Caenorhabditis elegans amoeboid sperm. We used live-imaging analysis to show the PP1 phosphatases GSP-3 and GSP-4 (GSP-3/4) are required to partition chromosomes during sperm meiosis. Postmeiosis, tracking fluorescently labeled sperm revealed that both male and hermaphrodite sperm lacking GSP-3/4 are immotile. Genetic and in vitro activation assays show lack of GSP-3/4 causes defects in pseudopod development and the rate of pseudopodial treadmilling. Further, GSP-3/4 are required for the localization dynamics of MSP. GSP-3/4 shift localization in concert with MSP from fibrous bodies that sequester MSP at the base of the pseudopod, where directed MSP disassembly facilitates pseudopod contraction. Consistent with a role for GSP-3/4 as a spatial regulator of MSP disassembly, MSP is mislocalized in sperm lacking GSP-3/4. Although a requirement for PP1 phosphatases in nematode and mammalian sperm suggests evolutionary conservation, we show PP1s have independently evolved sperm-specific paralogs in separate lineages. Thus PP1 phosphatases are highly adaptable and employed across a broad range of sexually reproducing species to regulate male fertility.     

Selection of peptides targeting the human sperm surface using random peptide phage display identify ligands homologous to ZP3

Analysis of the surface architecture of human spermatozoa is a necessary step in the development of new approaches to contraception and resolving the causes of human infertility. In this study we have utilized phage display technology to identify peptides that bind with high affinity to the surface of human spermatozoa. Fifteen- and twelve-mer random peptide phage display libraries were screened against paraformaldehyde-fixed spermatozoa and a number of sperm-binding peptides were identified. One peptide, M6, displayed a high level of affinity for the sperm surface and showed sequence homology with a dominant human ZP3 epitope (hZP 25-33). This peptide bound preferentially to the equatorial and post acrosomal domains of the sperm head and exhibited contraceptive activity by virtue of its capacity to impair the fusion of acrosome-reacted spermatozoa with the vitelline membrane of the oocyte. A similar form of contraceptive activity was also observed within an unrelated peptide, K6, derived from screening the 12-mer library. These results indicate that phage display technology is a powerful tool for developing reagents capable of targeting the human sperm surface, providing insights into the composition of this structure and the identity of targets susceptible to contraceptive attack and pathological disruption.     

Glutathione S-transferase Mu 3 is associated to in vivo fertility,but not sperm quality,in bovine

In the dairy breeding industry, pregnancy of dairy cows is essential to initiate milk production, so that high fertility rates are required to increase their productivity. In this regard, sperm proteins that are indicative of sperm quality and/or fertility have become an important target of study. Glutathione S-transferase Mu 3 (GSTM3) has been established as a fertility and sperm quality parameter in humans and pigs and, consequently, it might be a potential biomarker in cattle. For this reason, the present work aimed to determine if GSTM3 could predict sperm quality and in vivo fertility in this species. Sperm quality was assessed with flow cytometry and computer-assisted sperm analysis. Immunoblotting and immunofluorescence analysis were performed to determine the presence and localisation pattern of sperm GSTM3. This enzyme was found to be present in bovine sperm and to be localised along the sperm tail and the equatorial segment of the head. No significant associations between sperm GSTM3 and sperm quality parameters were observed, except a negative association with morphologically abnormal sperm having a coiled tail. In addition, and more relevant, higher levels of GSTM3 in sperm were seen in bulls showing lower in vivo fertility rates. In conclusion, our data evidenced the presence of GSTM3 in bovine sperm. Moreover, we suggest that, despite not being associated with sperm quality, GSTM3 might be an in vivo subfertility biomarker in cattle sperm, and that high levels of this protein could be an indicative of defective spermatogenesis and/or epididymal maturation.     

Male contraception: Another holy grail

The idea that men should participate in family planning by playing an active role in contraception has become more acceptable in recent years. Up to the present the condom and vasectomy have been the main methods of male contraception. There have been and continue to be efforts to develop an acceptable hormonal contraceptive involving testosterone (T) suppression. However the off target affects, delivery of the analogs and the need for T replacement have proven difficult obstacles to this technology. Research into the development of non-hormonal contraception for men is progressing in several laboratories and this will be the subject of the present review. A number of promising targets for the male pill are being investigated. These involve disruption of spermatogenesis by compromising the integrity of the germinal epithelium, interfering with sperm production at the level of meiosis, attacking specific sperm proteins to disrupt fertilizing ability, or interfering with the assembly of seminal fluid components required by ejaculated sperm for acquisition of motility. Blocking contractility of the vas deferens smooth muscle vasculature to prevent ejaculation is a unique approach that prevents sperm from reaching the egg. We shall note the lack of interest by big pharma with most of the support for male contraception provided by the NIH.     

Phosphoproteome analysis of capacitated human sperm. Evidence of tyrosine phosphorylation of a kinase-anchoring protein 3 and valosin-containing protein/p97 during capacitation

Before fertilization can occur, mammalian sperm must undergo capacitation, a process that requires a cyclic AMP-dependent increase in tyrosine phosphorylation. To identify proteins phosphorylated during capacitation, two-dimensional gel analysis coupled to anti-phosphotyrosine immunoblots and tandem mass spectrometry (MS/MS) was performed. Among the protein targets, valosin-containing protein (VCP), a homolog of the SNARE-interacting protein NSF, and two members of the A kinase-anchoring protein (AKAP) family were found to be tyrosine phosphorylated during capacitation. In addition, immobilized metal affinity chromatography was used to investigate phosphorylation sites in whole protein digests from capacitated human sperm. To increase this chromatographic selectivity for phosphopeptides, acidic residues in peptide digests were converted to their respective methyl esters before affinity chromatography. More than 60 phosphorylated sequences were then mapped by MS/MS, including precise sites of tyrosine and serine phosphorylation of the sperm tail proteins AKAP-3 and AKAP-4. Moreover, differential isotopic labeling was developed to quantify phosphorylation changes occurring during capacitation. The phosphopeptide enrichment and quantification methodology coupled to MS/MS, described here for the first time, can be employed to map and compare phosphorylation sites involved in multiple cellular processes. Although we were unable to determine the exact site of phosphorylation of VCP, we did confirm, using a cross-immunoprecipitation approach, that this protein is tyrosine phosphorylated during capacitation. Immunolocalization of VCP showed fluorescent staining in the neck of noncapacitated sperm. However, after capacitation, staining in the neck decreased, and most of the sperm showed fluorescent staining in the anterior head.     

Purification and Characterization of a Sperm Motility Inhibiting Factor from Caprine Epididymal Plasma

Several studies have been reported on the occurrence of sperm motility inhibiting factors in the male reproductive fluids of different mammalian species, but these proteins have not been adequately purified and characterized. A novel sperm motility inhibiting factor (MIF-II) has been purified from caprine epididymal plasma (EP) by Hydroxylapatite gel adsorption chromatography, DEAE-Cellulose ion-exchange chromatography and chromatofocusing. The MIF-II has been purified to apparent homogeneity and the molecular weight estimated by Sephacryl S-300 gel filtration is 160 kDa. MIF-II is a dimeric protein, made up of two subunits each having a molecular mass of 80 kDa as shown by SDS-PAGE. The isoelectric point of MIF-II is 5.1 as determined by chromatofocusing and isoelectric focusing. It is a heat labile protein and maximal active at the pH 6.9 to 7.5. The sperm motility inhibiting protein factor at 2 µg/ml (12.5 nM) level showed maximal motility-inhibiting activity. The observation that the epididymal plasma factor lowered the intracellular cAMP level of spermatozoa in a concentration-dependent manner suggests that it may block the motility of caprine cauda spermatozoa by interfering the cAMP dependent motility function. The results revealed that the purified protein factor has the potential of sperm motility inhibition and may serve as a vaginal contraceptive. The antibody raised against the MIF-II has the potential for enhancement of forward motility of cauda-spermatozoa. This antibody may thus be useful for solving some of the problems of male infertility due to low sperm motility.