Daniel M. Hardy

A Sperm Membrane Protein That Binds in a Species-specific Manner to the Egg Extracellular Matrix Is Homologous to von Willebrand Factor

We have purified a sperm membrane protein, designated zonadhesin, that binds in a species-specific manner to the extracellular matrix (zona pellucida) of the egg, and cloned its cDNA. The cDNA encodes a novel protein with a single transmembrane segment separating a 36 amino acid, highly basic intracellular C terminus from a 2418-amino acid extracellular region. The extracellular sequence specifies a mosaic protein comprising a unique N-terminal domain, a mucin-like domain, and five tandem domains proximal to the membrane that are homologous to prepro von Willebrand factor. The N-terminal and mucin-like domains were absent from zonadhesin that bound to the egg extracellular matrix, suggesting that processing occurs during sperm maturation and/or capacitation. By Northern blotting and in situ hybridization, zonadhesin mRNA was detected only within the testis, where it was expressed primarily in haploid spermatids. The unique domain structure of zonadhesin suggests multiple functions, one of which is to mediate sperm adhesion to the zona pellucida.

Processing, localization and binding activity of zonadhesin suggest a function in sperm adhesion to the zona pellucida during exocytosis of the acrosome.

Zonadhesin is a sperm protein that binds in a species-specific manner to the extracellular matrix ZP (zona pellucida) of the mammalian oocyte. The pig zonadhesin precursor is a 267000-Da mosaic protein with a Type I membrane topology and a large extracellular region comprising meprin/A5 antigen/mu receptor tyrosine phosphatase, mucin and five tandem von Willebrand D (VWD) domains. Multiple mature forms of zonadhesin in the sperm head differ in their avidities for the ZP. To determine the potential functions of zonadhesin forms in gamete adhesion, we characterized the processing, activation and localization of protein in pig spermatozoa. The predominant polypeptides of processed zonadhesin were M(r) 300000 (p300), 105000 (p105) and 45000 (p45). p45 and p105, comprised primarily the D1, D2-D3 domains respectively, and were N-glycosylated. p300 was heavily O-glycosylated, and spanned the meprin/A5 antigen/mu receptor tyrosine phosphatase, mucin and D0 domains. Hydrolysis of the precursor polypeptide occurred in the testis, and N-terminal sequencing of p45 and p105 identified Asp806-Pro and Asp1191-Pro in the D1 and D2 domains respectively as bonds cleaved in the proteins functional maturation. Testicular zonadhesin was extractable with non-ionic detergents, and localized to the developing outer acrosomal membrane of round and elongating spermatids. As spermatozoa transited the epididymis, most of the protein became incorporated into an extraction-resistant fraction, and the proportions of active and of multimeric zonadhesins in the cells increased. Zonadhesin localized to the perimeter of the acrosome in intact ejaculated spermatozoa and to the leading edge of acrosomal matrix overlying cells with disrupted acrosomal membranes. We conclude that the zonadhesin precursor is specifically proteolysed, glycosylated and assembled into particulate structures in the distal parts of the acrosome where it may mediate specific adhesion to the ZP during the initial stages of acrosomal exocytosis.

Hip extension, knee flexion paradox: A new mechanism for non-contact ACL injury

Considering that an athlete performs at-risk sports activities countless times throughout the course of his or her career prior to the instance of anterior cruciate ligament (ACL) injury, one may conclude that non-contact ACL injury is a rare event. Nevertheless, the overall number of non-contact ACL injuries, both in the US and worldwide, remains alarming due to the growing number of recreational and professional athletes participating in high-risk activities. To date, numerous non-contact ACL injury mechanisms have been proposed, but none provides a detailed picture of sequence of events leading to injury and the exact cause of this injury remains elusive. In this perspective article, we propose a new conception of non-contact ACL injury mechanism that comprehensively integrates risk factors inside and outside the knee joint. The proposed mechanism is robust in the sense that it is biomechanically justifiable and addresses a number of confounding issues related to ACL injury.

Zonadhesin Is Essential for Species Specificity of Sperm Adhesion to the Egg Zona Pellucida

Interaction of rapidly evolving molecules imparts species specificity to sperm-egg recognition in marine invertebrates, but it is unclear whether comparable interactions occur during fertilization in any vertebrate species. In mammals, the sperm acrosomal protein zonadhesin is a rapidly evolving molecule with species-specific binding activity for the egg zona pellucida (ZP). Here we show using null mice produced by targeted disruption of Zan that zonadhesin confers species specificity to sperm-ZP adhesion. Sperm capacitation selectively exposed a partial von Willebrand D domain of mouse zonadhesin on the surface of living, motile cells. Antibodies to the exposed domain inhibited adhesion of wild-type spermatozoa to the mouse ZP but did not inhibit adhesion of spermatozoa lacking zonadhesin. Zan−/− males were fertile, and their spermatozoa readily fertilized mouse eggs in vitro. Remarkably, however, loss of zonadhesin increased adhesion of mouse spermatozoa to pig, cow, and rabbit ZP but not mouse ZP. We conclude that zonadhesin mediates species-specific ZP adhesion, and Zan−/− males are fertile because their spermatozoa retain adhesion capability that is not species-specific. Mammalian sperm-ZP adhesion is therefore molecularly robust, and species-specific egg recognition by a protein in the sperm acrosome is conserved between invertebrates and vertebrates, even though the adhesion molecules themselves are unrelated.

Regulatory T cells control tolerogenic versus autoimmune response to sperm in vasectomy

Vasectomy is a well accepted global contraceptive approach frequently associated with epididymal granuloma and sperm autoantibody formation. To understand the long-term sequelae of vasectomy, we investigated the early immune response in vasectomized mice. Vasectomy leads to rapid epithelial cell apoptosis and necrosis, persistent inflammation, and sperm granuloma formation in the epididymis. Vasectomized B6AF1 mice did not mount autoimmune response but instead developed sperm antigen-specific tolerance, documented as resistance to immunization-induced experimental autoimmune orchitis (EAO) but not experimental autoimmune encephalomyelitis. Strikingly, tolerance switches over to pathologic autoimmune state following concomitant CD4+CD25+Foxp3+ regulatory T cell (Treg) depletion: unilaterally vasectomized mice produce dominant autoantibodies to an orchitogenic antigen (zonadhesin), and develop CD4 T-cell– and antibody-dependent bilateral autoimmune orchitis. Therefore, (i) Treg normally prevents spontaneous organ-specific autoimmunity induction by persistent endogenous danger signal, and (ii) autoantigenic stimulation with sterile autoinflammation can lead to tolerance. Finally, postvasectomy tolerance occurs in B6AF1, C57BL/6, and A/J strains. However, C57BL/6 mice resisted EAO after 60% Treg depletion, but developed EAO after 97% Treg reduction. Therefore, variance in intrinsic Treg function—a possible genetic trait—can influence the divergent tolerogenic versus autoimmune response to vasectomy.

Expression of matrix metalloprotease and tissue inhibitor of metalloprotease genes in human anterior cruciate ligament.

Women are more susceptible to anterior cruciate ligament (ACL) injuries than men performing similar athletic activities. Because tissue remodeling may affect ligament strength, we assessed expression of tissue remodeling effector genes in the human ACL. Specifically, we surveyed ACL for RNAs encoding all known matrix metalloproteases (MMPs) and tissue inhibitors of metalloproteases (TIMPs) by reverse transcription/polymerase chain reaction (RT‐PCR). These experiments revealed that mRNAs encoding nine of sixteen MMPs and all four TIMPs are present in the normal ACL. The nine expressed proteases were MMPs 1—3, 7, 9, 11, 14, and 17 (collagenase 1, gelatinase A, stromelysin 1, matrilysin, gelatinase B, stromelysin 3, and membrane types 1 and 4, respectively), and MMP‐18. Genes for MMPs 8, 10, 12, 13, 15, and 16 appeared not to be expressed in ACL, as their mRNAs were not detected using RT‐PCR conditions that did yield positive signals from other tissues (testis or bone). We conclude that numerous genes encoding tissue remodeling effector proteins are expressed in the human ACL.

Age, sex, body anthropometry, and ACL size predict the structural properties of the human anterior cruciate ligament.

Anterior cruciate ligament (ACL) injury continues to be at the forefront of sports injury concerns because of its impact on quality of life and joint health prognosis. One strategy is to reduce the occurrence of this injury by identifying at‐risk subjects based on key putative risk factors. The purpose of our study was to develop models that predict the structural properties of a subjects ACL based on the combination of known risk factors. We hypothesized that the structural properties of the ACL can be predicted using a multi‐linear regression model based on significant covariates that are associated with increased risk of injury, including age, sex, body size, and ACL size. We also hypothesized that ACL size is a significant contributor to the model. The developed models had predictive capabilities for the structural properties of the ACL: load at failure (R2 = 0.914), elongation at failure (R2 = 0.872), energy at failure (R2 = 0.913), and linear stiffness (R2 = 0.756). Furthermore, sex, age, body mass, BMI, and height were contributors (p < 0.05) to all predicted structural properties. ACL minimal area was a contributor to elongation, energy at failure, and linear stiffness (p < 0.05), but not to load at failure. ACL volume was also a contributor to elongation and energy at failure (p < 0.05), but not to linear stiffness and load at failure models. ACL length was not a significant contributor to any structural property. The clinical significance of this research is its potential, after continued development and refinement of the model, for application to prognostic studies that are designed to identify individuals at increased risk for injury to the ligament.

The human anterior cruciate ligament: Sex differences in ultrastructure and correlation with biomechanical properties

The purpose of this study was to investigate the existence of sex‐based differences in the ultrastructural characteristics of the human anterior cruciate ligament (ACL) as the underlying cause of differences in the structural and mechanical properties between sexes. The ACL of six male and six female cadaveric donors were randomly chosen from a pool of 10 male and 10 female ACLs that had previously been tested for their structural and mechanical properties. Eighteen tissue samples from the distal, proximal, and middle sections of the anteromedial and posterolateral bundles were analyzed by transmission electron microscopy. Female ACLs exhibited both lower fibril concentration and lower percent area occupied by collagen fibrils (p < 0.05) compared to males. There was also a difference in the fibril diameters (p < 0.05); donor age, height, body mass, and body mass index contributed significantly to this difference. In females, ACL stiffness and modulus of elasticity were highly correlated to fibril concentration (r = 0.96 and 0.97, respectively); in males ACL failure load and strength were highly correlated to percent area occupied by collagen (r = 0.96 and 0.96, respectively). These differences in ultrastructure may underlie differences in ACL properties between sexes.

Zonadhesin Assembly into the Hamster Sperm Acrosomal Matrix Occurs by Distinct Targeting Strategies During Spermiogenesis and Maturation in the Epididymis

Abstract Zonadhesin is the only sperm protein known to bind in a species-specific manner to the zona pellucida. The zonadhesin precursor is a mosaic protein with a predicted transmembrane segment and large extracellular region composed of cell adhesion, mucin, and tandem von Willebrand D domains. Because the precursor possesses a predicted transmembrane segment and localizes to the anterior head, the mature protein was presumed to be a sperm surface zona pellucida-binding protein. In this study of hamster spermatozoa, we demonstrate that zonadhesin does not localize to the sperm surface but is instead a constituent of the acrosomal matrix. Immunoelectron microscopy revealed that distinct targeting pathways during spermiogenesis and sperm maturation in the epididymis result in trafficking of zonadhesin to the acrosomal matrix. In round spermatids, zonadhesin localized specifically to the acrosomal membrane, where it appeared to be evenly distributed between the outer and inner membrane domains. Subsequent redistribution of zonadhesin resulted in its elimination from the inner acrosomal membrane and restriction to the outer acrosomal membrane of the apical and principal segments and the contents of the posterior acrosome. During sperm maturation in the epididymis, zonadhesin dissociated from the outer acrosomal membrane and became incorporated into the forming acrosomal matrix. These data suggest an important structural role for zonadhesin in assembly of the acrosomal matrix and further support the view that the species specificity of zona pellucida adhesion is mediated by egg-binding proteins contained within the acrosome rather than on the periacrosomal plasma membrane.

Chapter 12 Isolation of Extracellular Matrix Structures from Xenopus laevis Oocytes, Eggs, and Embryos

Publisher Summary The extracellular matrix (ECM) surrounding amphibian eggs is composed of jelly coat layers, the egg envelope, and the fibrous elements of the perivitelline space. The oviposited egg envelope provides an initially penetrable structure to the sperm which is subsequently modified by products released in the cortical reaction to a sperm-impenetrable structure in the block to polyspermy reaction. At fertilization the egg envelope is chemically modified and its macromolecular permeability properties changed. This permeability change (hardening) results in an osmotically driven envelope elevation, owing to the influx of water into the perivitelline space. The jelly after fertilization functions as a “sticky substrate” for the adherence of the zygote to objects in its surroundings, protects the zygote against physical damage, and also provides a microbiological barrier (bacteria are rarely found within the jelly coat layers). Thus, after fertilization the ECM protects the developing embryo and functions as a barrier for the chemical and biological regulation of the embryo environment. This protective function of the ECM persists until the embryo develops into a free swimming tadpole and hatches from the ECM. Thus, the chapter briefly discusses isolation of ECM structures from Xenopus laevis oocytes, eggs, and embryos.