Ashley I. Yudin

Diffusion of Macromolecules and Virus-Like Particles in Human Cervical Mucus

To determine whether or not large macromolecules and viruses can diffuse through mucus, we observed the motion of proteins, microspheres, and viruses in fresh samples of human cervical mucus using fluorescent recovery after photobleaching and multiple image photography. Two capsid virus-like particles, human papilloma virus (55 nm, approximately 20,000 kDa) and Norwalk virus (38 nm, approximately 10,000 kDa), as well as most of the globular proteins tested (15-650 kDa) diffused as rapidly in mucus as in saline. Electron microscopy of cervical mucus confirmed that the mesh spacing between mucin fibers is large enough (20-200 nm) for small viruses to diffuse essentially unhindered through mucus. In contrast, herpes simplex virus (180 nm) colocalized with strands of thick mucus, suggesting that herpes simplex virus, unlike the capsid virus particles, makes low-affinity bonds with mucins. Polystyrene microspheres (59-1000 nm) bound more tightly to mucins, bundling them into thick cables. Although immunoglobulins are too small to be slowed by the mesh spacing between mucins, diffusion by IgM was slowed by mucus. Diffusion by IgM-Fc(5 mu), the Fc pentamer core of an IgM with all 10 Fab moieties removed, was comparably slowed by mucus. This suggests that the Fc moieties of antibodies make low-affinity bonds with mucins.

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.

Hyaluronic acid enhances induction of the acrosome reaction of human sperm through interaction with the PH-20 protein.

When capacitated human sperm were treated with hyaluronic acid (HA) for 30 min prior to the addition of progesterone or solubilised human zonae pellucidae, there was a significant increase in the percentage of acrosome reactions. Progesterone treatment alone increased acrosome reactions from 10.5% to 21.8% and pretreatment with 100 micrograms/ml HA resulted in 33.0% acrosome reactions. With zonae pellucidae treatment alone the increase was from 9.0% to 23.5% and with HA pretreatment it was 48.8%. HA treatment alone had no direct effect on acrosome reactions, and the enhancing effect of HA was not removed when sperm were washed prior to the addition of either acrosome reaction agonist. Experiments with sperm 5 min after HA treatment demonstrated that enhancement of acrosome reactions was apparent as early as 1 min after addition of zonae and within 5 min after addition of progesterone. When sperm were pretreated with Fab fragments of anti-PH-20 IgG, then with HA and then with progesterone or zonae pellucidae, there was no enhancement of the acrosome reaction. Fab treatment did not induce acrosome reactions and did not interfere with the action of either agonist in the absence of HA. Sperm that were treated with HA had significantly higher intracellular calcium levels, and pretreatment with Fab reduced this increase to 42.7%. Addition of progesterone to HA-treated sperm was followed by another large increase in intracellular calcium, which was lower when sperm were pretreated with Fab. These results suggest that HA interacts with the PH-20 protein to increase basal levels of intracellular calcium and thereby potentiates the acrosome reaction. The data support the hypothesis that HA in the cumulus matrix may act to prime the fertilising sperm for induction of the acrosome reaction by constituents of the cumulus and/or zona pellucida.

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.

Structure of the cumulus matrix and zona pellucida in the golden hamster: A new view of sperm interaction with oocyte-associated extracellular matrices

SummaryHamster oocyte-cumulus complexes (OCC), with and without sperm, were structurally analyzed by light- and electron microscopy using freeze substitution. This method has yielded a clear picture of the extracellular oocyte investments, the cumulus cell matrix and the zona pellucida. The cumulus matrix has an overall homogeneous fibrillar structure which appears to attach to cumulus cells at their filopodial extensions. The matrix also extends into the outer regions of the zona pellucida. The zona pellucida has a distinct porous configuration throughout its entire structure. During gamete interaction experiments, capacitated hamster sperm with ultrastructurally intact acrosomes were found throughout the matrix. Sperm had dramatic effects on the matrix, resulting in compression and stretching. Sperm found on the zona pellucida had initiated or completed the acrosome reaction. During the initial stages of the acrosome reaction, the matrix was in contact with the sperm. At later stages of the acrosome reaction, there was a complete loss of matrix material in regions near the sperm.

Hamster sperm penetration of the zona pellucida: Kinematic analysis and mechanical implications

There have been few direct observations of penetration of the zona pellucida by spermatozoa, and no detailed description of the kinematics of this process. Such information is important in evaluating the contribution of mechanical thrust by the sperm flagellum to the mechanism of zona penetration by the sperm head. To make such observations, small numbers of hamster spermatozoa were inseminated to cumulus masses slightly compressed (150 micron) between a slide and coverglass. Observations were made with interference contrast optics and videorecorded at 60 fields/sec. A total of 63 penetrating spermatozoa were recorded, of which 21 were penetrating completely cumulus-intact zonae. Direct comparison of penetration angles for cumulus-intact and cumulus-dispersed zonae suggested that the cumulus may be important in reorientation of penetrating spermatozoa, which initially lie flat on the zona surface. The beat shape during zona penetration was more complex than the simple sinusoidal waves used previously in modeling the mechanics of sperm-zona interaction. Motility during zona penetration was bimodal, having high-amplitude, low-frequency lever strokes, alternating with low-amplitude, high-frequency propagated sinusoidal waves. The completely asymmetric lever mode and the oscillatory motions of the curved leading edge of the sperm head within the zona may afford significant mechanical advantages to spermatozoa in forcing their way through that matrix. Initial calculations of the maximum force exerted by the sperm head against the zona material during lever strokes predicted values as high as 2700 mu dyn. This result is two orders of magnitude higher than that previously estimated assuming more simple flagellar motility. Although not conclusive, our observations and analysis support the concept that zona penetration is more efficient when the cumulus is present, and that this may be due, in part, to a mechanical advantage conferred upon the sperm by the cumulus material.

Fine structure of the unistellate sperm of the shrimp, Sicyonia ingentis (Natantia).

Sperm of the prawn Sicyonia ingentis were studied cytochemically and ultrastructurally. Striking cytological differences were noted between these natantian sperm and previously studied reptantian sperm. In general, the S. ingentis sperm are composed of a spherical main body that is partially encompassed by a morphologically diverse cap region, from which extends a single appendage or spike. The main body houses an uncondensed, Feulgen-positive nuclear region that is partially surrounded by a cytoplasmic band. A single layer of small, 600 A, vesicles lines the periphery of the cytoplasmic band. Large membranous vesicles extend from the inner surface of the cytoplasmic band into the nuclear region. The nucleus is separated from the cap or acrosomal complex by a dense plate and a highly organized crystalline lattice, which is composed of geometric squares that are approximately 350 A in dimension. The cap region also contains convoluted membrane pouches; a central granular core; spherical bodies; an electron-dense, saucer-shaped plate; and a large anterior granule. The convoluted membrane pouches and anterior granule are periodic acid-Schiff (PAS) positive. The anterior granule also demonstrates RNAase-stable red fluorescence with acridine orange staining. A spiralled spike, approximately 6 micron long, extends from the anterior end of the cap. The cap and spike are bound by a double membrane, which results from the fusion of the plasma membrane and the convoluted pouch membrane. The sperms acrosome is thought to be composed of the two PAS-positive cap components and the spike.