Theodore L. Tollner

A Common Mutation in the Defensin DEFB126 Causes Impaired Sperm Function and Subfertility

A frequent frameshift mutation in defensin DEFB126, a protein that adheres to the surface of human sperm, weakens its ability to penetrate cervical mucus-like gels and causes low fertility. Defensin-Deficient Sperm Get Stuck Like Robert Burns’ best laid schemes of mice and men, the joining of egg and sperm “gang aft agley” (transl., often go awry)—and it’s no wonder, considering the many molecular events that must be correctly executed for successful fertilization. The current clinical tests still fail to explain infertility in almost one-fifth of infertile couples. Now, Tollner et al. pinpoint one more critical cog in this vital process: Men who carry a genetic variant of a certain sperm surface protein are less fertile than normal. This common but life-altering deviation likely accounts for some of the currently unexplained cases of infertility. β-Defensin is a protein made in the paired coils of the epididymis, which carries sperm from testes. This defensin is secreted as the sperm travels by and is integrated into the glycocalyx, a protein-sugar coating on the sperm surface. Surface-hugging β-defensins protect sperm from immune attack and help them to penetrate the cervical mucus in the female. While cloning the human version of this defensin, the authors found a mutated variant that was surprisingly prevalent; about 20% of the European, Chinese, and Japanese men that the authors examined carried the variation on both chromosomes (del/del). Although they did not uniformly display deficiencies usually associated with infertility (such as inadequate semen volume and low sperm motility), sperm from del/del men did show lower lectin binding relative to controls; this measure was shown to be a marker for sperm-associated O-linked oligosaccharides that cannot attach to the mutated defensin. The del/del sperm were poor penetrators of hyaluronic acid, an in vitro surrogate for cervical mucus. But did the presence of the defensin variant actually cause lower fertility? In a group of 509 newly married Chinese couples, the authors showed that it did. Wives of men with the del/del genotype were only 60% as likely to get pregnant as were women who mated with men who carried wild-type or wt/del genotypes, and the time from enrollment in the study to the live birth of a child was 2 months longer in the former group. The impaired fertility among carriers of this deletion might imply that these individuals are headed for extinction, but their prevalence in the population indicates otherwise. How can this be? The authors speculate that carriers of a single copy of the mutated defensin may have an as yet undefined survival advantage over wild-type carriers, an evolutionary situation known as balancing selection. Whatever the reason for variation persistence, our new understanding of β-defensin will enable better appreciation of human fertility and help to keep our reproductive plans on track. A glycosylated polypeptide, β-defensin 126 (DEFB126), derived from the epididymis and adsorbed onto the sperm surface, has been implicated in immunoprotection and efficient movement of sperm in mucosal fluids of the female reproductive tract. Here, we report a sequence variant in DEFB126 that has a two-nucleotide deletion in the open reading frame, which generates an abnormal mRNA. The allele frequency of this variant sequence was high in both a European (0.47) and a Chinese (0.45) population cohort. Binding of the Agaricus bisporus lectin to the sperm surface glycocalyx was significantly lower in men with the homozygous variant (del/del) genotype than in those with either a del/wt or a wt/wt genotype, suggesting an altered sperm glycocalyx with fewer O-linked oligosaccharides in del/del men. Moreover, sperm from del/del carriers exhibited an 84% reduction in the rate of penetration of a hyaluronic acid gel, a surrogate for cervical mucus, compared to the other genotypes. This reduction in sperm performance in hyaluronic acid gels was not a result of decreased progressive motility (average curvilinear velocity) or morphological deficits. Nevertheless, DEFB126 genotype and lectin binding were correlated with sperm performance in the penetration assays. In a prospective cohort study of newly married couples who were trying to conceive by natural means, couples were less likely to become pregnant and took longer to achieve a live birth if the male partner was homozygous for the variant sequence. This common sequence variation in DEFB126, and its apparent effect of impaired reproductive function, will allow a better understanding, clinical evaluation, and possibly treatment of human infertility.

Beta-Defensin 126 on the Cell Surface Protects Sperm from Immunorecognition and Binding of Anti-Sperm Antibodies

Abstract Beta-defensin 126 (DEFB126), formerly known as epididymal secretory protein 13.2 (ESP13.2), coats the entire primate sperm surface until completion of capacitation, and it is a candidate for providing immune protection in the female reproductive tract. To further examine the potential role of DEFB126 as a means of protection from immune recognition, cynomolgus macaque sperm were exposed to a number of treatments that are known to alter sperm surface coats, including capacitation. We used a novel in vivo assay to determine immune recognition: aldehyde-fixed whole sperm injections into rabbits. Following booster injections, immunoblot analyses of whole sperm prepared in various manners was conducted. On Days 60 and 80 post-initial immunization, the antisera showed a remarkably strong reaction to a single 34–36 kDa protein, which was shown to be DEFB126. Sera from rabbits that were immunized with sperm washed more rigorously using Percoll gradients showed an increase in the number and intensity of proteins recognized on whole sperm Western blots, although DEFB126 was still the major immune response. When capacitated sperm, from which most DEFB126 had been released, were used as the immunogen, there was a dramatic increase in the immune recognition to a variety of protein bands. Sperm treated with neuraminidase to remove sialic acid on DEFB126 before fixation were shown to still possess DEFB126, but lacked the sialic acid component of the glycoprotein. These sperm were as immunogenic as capacitated sperm even though the desialylated DEFB126 still covered the entire cell surface. These sperm lost their highly negative charge (the isoelectric point of DEFB126 shifted from pI 3.0 to pI 6.4). Experiments using different sperm plasma membrane protein-specific Igs showed that recognition did not occur when DEFB126 was present, but following capacitation these Igs readily recognized the exposed sperm membrane. Our data suggest that DEFB126 protects the entire primate sperm surface from immune recognition and that the sialic acid moieties are responsible for the cloaking characteristic of this unique glycoprotein.

Macaque sperm coating protein DEFB126 facilitates sperm penetration of cervical mucus.

BACKGROUND Sperm coating protein beta-defensin 126 (DEFB126) is adsorbed onto the entire surface of macaque sperm in the caudal epididymis and is retained on viable sperm collected from the cervix and the uterine lumen of mated female macaques. We investigated the role of sperm coating protein DEFB126 in cervical mucus penetration (CMP). METHODS Cervical mucus (CM) was collected from peri-ovulatory female macaques and loaded into CMP chambers. Sperm were introduced to CMP chambers following treatment with either polyclonal antibodies raised to DEFB126 or seminal plasma proteins (SPPs), 1 mM caffeine+1 mM dibutyryl cyclic adenosine monophosphate (dbcAMP) (induces release of DEFB126 from sperm surface), neuraminidase (NMase) or poly-L-lysine (PLP). Following removal of DEFB126 or SPPs from the sperm surface, sperm were treated with concentrated DEFB126 or concentrated SPPs prior to being introduced to CMP chambers. The numbers of sperm that penetrated and traversed CM were scored over 6 min. RESULTS Treatment of sperm with anti-DEFB126 antibodies, 1 mM caffeine+1 mM dbcAMP, NMase, and PLP resulted in similar and significant levels of inhibition of sperm CMP, whereas addition of anti-SPPs antibodies had no effect. In experiments where DEFB126 and SPPs were removed, CMP capability of sperm was restored by addition of DEFB126 back to the sperm surface, whereas treatment of sperm with concentrated SPPs slightly inhibited sperm penetration. CONCLUSIONS DEFB126 and its high negative charge appears to be critical for the movement of sperm through CM in the macaque, while SPPs adhered to the sperm surface offer no advantage in CMP.

Beta-Defensin 126 on the Surface of Macaque Sperm Mediates Attachment of Sperm to Oviductal Epithelia

Abstract Beta-defensin 126 (DEFB126) coats the entire surface of macaque sperm until sperm become capacitated, and the removal of DEFB126 from over the head of sperm is required for sperm-zona recognition. Viable sperm collected from cervix and the uterine lumen of mated female macaques had DEFB126 coating the entire surface, suggesting that DEFB126 is retained on sperm en route to the oviduct. DEFB126 plays a major role in attachment of sperm to oviductal epithelial cells (OECs). Following treatment to either remove or alter DEFB126, sperm were coincubated with explants of OECs, which were assessed for sperm binding following rinsing to remove superficially attached sperm. Sperm treated with either 1 mM caffeine + 1 mM dibutyryl cyclic adenosine monophosphate (dbcAMP) (induces capacitation and complete release of DEFB126 from sperm), 2 mM caffeine (removes DEFB126 from over the head and midpiece but does not induce capacitation), anti-DEFB126 immunoglobulin, or neuraminidase (cleaves sialic acid from terminal positions on glycosylation sites of DEFB126) resulted in similar and significant levels of inhibition of sperm-OEC binding. Preincubation of OECs with soluble DEFB126 also resulted in significantly reduced sperm-OEC binding. Furthermore, reduced OEC binding capability of sperm lacking DEFB126 could be restored by addition of soluble DEFB126 to the sperm surface prior to incubation with OECs. Finally, purified DEFB126, infused into oviducts in situ, associated primarily with the apical membranes of secretory-type epithelial cells. In summary, treatments of macaque sperm that result in either removal, masking, or alteration of DEFB126 result in loss of sperm-OEC binding that is independent of changes in sperm motility. DEFB126 may be directly involved in the formation of a reservoir of sperm in the oviduct of macaques.

ESP13.2, a Member of the β-Defensin Family, Is a Macaque Sperm Surface-Coating Protein Involved in the Capacitation Process

Abstract Female macaques produced isoantibodies to a limited number of sperm surface proteins following immunization with sperm components released by phosphatidylinositol-specific phospholipase C (PI-PLC). Washed, acrosome-intact, fixed sperm injected into rabbits elicited a major immune response to one of the same PI-PLC-released proteins, which was shown to be a sperm surface-coating protein. After purification and digestion of the glycoprotein, four peptides were analyzed for amino acid sequence, and all had 100% homology with an epididymal secretory protein, ESP13.2, reported previously to be a small, cationic-rich peptide and a member of the β-defensin family. Antibodies to purified ESP13.2 recognized a number of protein bands on Western blots of nonreduced PI-PLC-released sperm components and nonreduced whole-sperm extracts. After chemical disulfide reduction, only a single, broad band from 31 to 35 kDa was recognized by anti-ESP13.2 antibodies. Indirect immunofluorescence showed ESP13.2 over the entire surface of ejaculated macaque sperm. Fluorescence was only slightly reduced after sperm were washed through 80% Percoll. A 24-h incubation in capacitating medium significantly reduced the amount of ESP13.2 over the head and midpiece, whereas exposure of the incubated sperm to dbcAMP and caffeine (capacitation activators) resulted in almost complete loss of ESP13.2 from the sperm surface. After activation, ESP13.2 was the primary component released into the medium as judged electrophoretically. Lignosulfonic acid, a potent inhibitor of macaque fertilization in vitro, completely blocked release of ESP13.2 from the sperm surface, even following treatment with activators. These findings suggest that the β-defensin, ESP13.2, has a function in the capacitation of macaque spermatozoa and may modulate sperm surface-receptor presentation at the time of fertilization.

The importance of mitochondrial metabolic activity and mitochondrial DNA replication during oocyte maturation in vitro on oocyte quality and subsequent embryo developmental competence

Mitochondrial metabolic capacity and DNA replication have both been shown to affect oocyte quality, but it is unclear which one is more critical. In this study, immature oocytes were treated with FCCP or ddC to independently inhibit the respective mitochondrial metabolic capacity or DNA replication of oocytes during in vitro maturation. To differentiate their roles, we evaluated various parameters related to oocyte maturation (germinal vesicle break down and nuclear maturation), quality (spindle formation, chromosome alignment, and mitochondrial distribution pattern), fertilization capability, and subsequent embryo developmental competence (blastocyst formation and cell number of blastocyst). Inhibition of mitochondrial metabolic capacity with FCCP resulted in a reduced percent of oocytes with nuclear maturation; normal spindle formation and chromosome alignment; evenly distributed mitochondria; and an ability to form blastocysts. Inhibition of mtDNA replication with ddC has no detectable effect on oocyte maturation and mitochondrial distribution, although high‐dose ddC increased the percent of oocytes showing abnormal spindle formation and chromosome alignment. ddC did, however, reduce blastocyst formation significantly. Neither FCCP nor ddC exposure had an effect on the rate of fertilization. These findings suggest that the effects associated with lower mitochondrial DNA copy number do not coincide with the effects seen with reduced mitochondrial metabolic activity in oocytes. Inhibiting mitochondrial metabolic activity during oocyte maturation has a negative impact on oocyte maturation and subsequent embryo developmental competence. A reduction in mitochondrial DNA copy number, on the other hand, mainly affects embryonic development potential, but has little effect on oocyte maturation and in vitro fertilization. Mol. Reprod. Dev. 79:392–401, 2012.

Multifunctional glycoprotein DEFB126--a curious story of defensin-clad spermatozoa.

During maturation, the surface of mammalian spermatozoa undergoes dramatic changes leading to the acquisition of properties vital for survival and performance in the female reproductive tract. A prominent change is the addition to the sperm surface of an atypical β-defensin polypeptide. In primates, the β-defensin DEFB126 becomes adsorbed to the entire sperm surface as spermatozoa move through the epididymal duct. DEFB126 has a conserved β-defensin core and a unique long glycosylated peptide tail. The carbohydrates of this domain contribute substantially to the sperm glycocalyx. DEFB126 is critical for efficient transport of sperm in the female reproductive tract, preventing their recognition by the female immune system, and might facilitate the delivery of capacitated sperm to the site of fertilization. A newly discovered dinucleotide deletion in the human DEFB126 gene is unusually common in diverse populations and results in a null allele. Predictably, men who are homozygous for the deletion produce sperm with an altered glycocalyx and impaired function, and have reduced fertility. Insights into the biology of DEFB126 are contributing to a better understanding of reproductive fitness in humans, as well as the development of diagnostics and therapeutics for male infertility.

The carbohydrate structure of DEFB126, the major component of the cynomolgus Macaque sperm plasma membrane glycocalyx.

Based on the amino-acid sequence of the macaque epididymal secretory protein, ESP 13.2 (Q9BEE3/AJ236909), it has now been classified as β-defensin DEFB126. DEFB126 is one of the five β-defensins with genes that are clustered along chromosome 20pl3, and all five proteins have an extended carboxy terminus that continues beyond the 6-cysteine β-defensin core region. This 60-amino acid carboxyl tail extension of the DEFB126 molecule is extraordinarily rich in threonine and serine (40%), many of which appear to be likely candidates for having O-glycosylation. DEFB126 has been shown to coat the entire surface of cynomolgus macaque sperm as they move through the corpus/caudal region of the epididymis. It is a major glycocalyx barrier to the external environment and is retained until the completion of capacitation. Sperm exposed to fluorescein-conjugated poly-L-lysine or Alexa488-histone showed a very uniform fluorescent labeling pattern over the entire sperm surface, almost identical to that observed with anti-DEFB126 Ig label. Sperm surface components that were released following treatment with caffeine/cAMP (in vitro capacitation) were blotted and probed with three different lectins which are known to recognize terminal sialic acid residues, and all three recognized the 35 kDa DEFB126 band. Neuraminidase treatment of sperm shifted the molecular weight of DEFB126 from 34–36 kDa to approximately 38–40 kDa and removed or greatly inhibited sialic acid-specific lectin recognition. O-Glycanase treatment alone was ineffective at removal of the oligosaccharides, but prior treatment with neuraminidase was sufficient to enable the O-glycanase treatment to effectively change the apparent molecular weight to 10 kDa, confirming that a major portion of the molecular mass is associated with the carbohydrate portion. Western blots of neuraminidase-treated DEFB126 showed strong recognition with a number of lectins that identify β-galactose and also lectins that recognize the N-acetylgalactosamine-serine/threonine, the proposed connection site of O-glycosylation. All of the lectins that recognized DEFB126 on Western blots were used to fluorescently probe sperm. The fluorescent patterns that were observed with poly-L-lysine, Alexa488-histone, sialic acid-specific lectins, and galactose-specific lectins showed even distributions over the entire sperm surface and the patterns were identical to sperm labeled with anti-DEFB126 Ig, and all but the antibody did not recognize neuraminidase-treated sperm.

Real-Time Observations of Individual Macaque Sperm Undergoing Tight Binding and the Acrosome Reaction on the Zona Pellucida

Abstract Changes in binding affinity, acrosomal status, and motility of living sperm on the zona pellucida were for the first time in any mammalian species directly observed and analyzed with video microscopy. A single zona was air-dried and rehydrated on a microscope slide, and a coverslip supported by glass beads was added. Capacitated sperm were added together with Alexa-SBTI, a probe for acrosin that can detect the acrosome reaction. The heads of loosely attached sperm oscillated on the zona and the flagella beat symmetrically with a sigmoid-shaped waveform. Tight binding was observed after 16 sec as the sperm head became fixed in place on the zona. The shape of the flagellar beat simultaneously shifted to a more rigid, C-shaped waveform. The first signs of the acrosome reaction were detected within 11 sec of tight binding. Rapid flushing removed approximately 65% of sperm that were loosely attached but only 2% of those that were tightly bound. In the 2 min following the onset of tight binding, the lateral displacement of the flagellum increased by approximately 30% and the beat frequency decreased by 25%. Lignosulfonic acid (LSA) inhibited loose sperm attachment and the development of tight binding. LSA had no effect on the time of the acrosome reaction following tight binding or on changes in motility that followed tight binding. These data suggest that LSA affects the initial attachment or docking of sperm to the zona, a step that may align or recruit one or more specific zona receptors to be responsible for mediating the acrosome reaction.

β-Defensin 22 is a major component of the mouse sperm glycocalyx

Surface components of sperm isolated from the cauda epididymides were stabilized by whole sperm fixation for immunization of rabbits. The resulting immunoglobulins (Igs) recognized a single protein of 130 kDa (non-reduced) or 54-57 kDa (reduced) on western blots of cauda sperm. Igs recognized the same 54-57 kDa protein band on whole tissue blots of the corpus and cauda epididymidis and vas deferens. No immunoreactive bands were detected on blots of the prostate, seminal vesicles, testes, caput epididymis, or any of various non-reproductive tissues. Removal of sperm from the vas deferens prior to blotting eliminated the detection of the sperm antigen. Antibodies raised to synthetic peptides, identical in amino acid sequence to two unique spans of DEFB22, recognized the same 130/54-57 kDa antigen on western blots of both caudal sperm and the purified antigen isolated with the anti-sperm Ig. From indirect immunofluorescence, both the anti-sperm and anti-peptide Igs appeared to localize to the entire sperm surface, a pattern confirmed at the ultrastructural level. Real-time PCR identified the corpus epididymides as the major site of expression of DEFB22, with negligible expression in the testes, caput epididymides, and vas deferens. Immunostaining of epididymal sections showed DEFB22 being released into the lumen at the distal caput/proximal corpus, with sperm becoming intensely coated with DEFB22 as they reached the distal corpus. Most uterine sperm recovered from mice 4 h following copulation exhibited DEFB22 coating the entire sperm surface. By contrast, some sperm recovered from the oviduct and cumulus extracellular matrix showed loss of DEFB22 from the sperm head.