SMA20/PMIS2 Is a Rapidly Evolving Sperm Membrane Alloantigen with Possible Species-Divergent Function in Fertilization.

Immunodominant alloantigens in pig sperm membranes include 15 known gene products and a previously undiscovered Mr 20,000 sperm membrane-specific protein (SMA20). Here we characterize SMA20 and identify it as the unannotated pig ortholog of PMIS2. A composite SMA20 cDNA encoded a 126 amino acid polypeptide comprising two predicted transmembrane segments and an N-terminal alanine- and proline (AP)-rich region with no apparent signal peptide. The Northern blots showed that the composite SMA20 cDNA was derived from a 1.1 kb testis-specific transcript. A BLASTp search retrieved no SMA20 match from the pig genome, but it did retrieve a 99% match to the Pmis2 gene product in warthog. Sequence identity to predicted PMIS2 orthologs from other placental mammals ranged from no more than 80% overall in Cetartiodactyla to less than 60% in Primates, with the AP-rich region showing the highest divergence, including, in the extreme, its absence in most rodents, including the mouse. SMA20 immunoreactivity localized to the acrosome/apical head of methanol-fixed boar spermatozoa but not live, motile cells. Ultrastructurally, the SMA20 AP-rich domain immunolocalized to the inner leaflet of the plasma membrane, the outer acrosomal membrane, and the acrosomal contents of ejaculated spermatozoa. Gene name search failed to retrieve annotated Pmis2 from most mammalian genomes. Nevertheless, individual pairwise interrogation of loci spanning Atp4a-Haus5 identified Pmis2 in all placental mammals, but not in marsupials or monotremes. We conclude that the gene encoding sperm-specific SMA20/PMIS2 arose de novo in Eutheria after divergence from Metatheria, whereupon rapid molecular evolution likely drove the acquisition of a species-divergent function unique to fertilization in placental mammals.

Degraded RNA from Human Anterior Cruciate Ligaments Yields Valid Gene Expression Profiles.

Correlating gene expression patterns with biomechanical properties of connective tissues provides insights into the molecular processes underlying the tissue growth and repair. Cadaveric specimens such as human knees are widely considered suitable for biomechanical studies, but their usefulness for gene expression experiments is potentially limited by the unavoidable, nuclease-mediated degradation of RNA. Here, we tested whether valid gene expression profiles can be obtained using degraded RNA from human anterior cruciate ligaments (ACLs). Human ACL RNA (N = 6) degraded in vitro by limited ribonuclease digestion resemble highly degraded RNA isolated from cadaveric tissue. PCR threshold cycle (Ct) values for 90 transcripts (84 extracellular matrix, 6 housekeeping) in degraded RNAs variably ranged higher than values obtained from their corresponding non-degraded RNAs, reflecting both the expected loss of target templates in the degraded preparations as well as differences in the extent of degradation. Relative Ct values obtained for mRNAs in degraded preparations strongly correlated with the corresponding levels in non-degraded RNA, both for each ACL as well as for the pooled results from all six ACLs. Nuclease-mediated degradation produced similar, strongly correlated losses of housekeeping and non-housekeeping gene mRNAs. RNA degraded in situ yielded comparable results, confirming that in vitro digestion effectively modeled degradation by endogenous ribonucleases in frozen and thawed ACL. We conclude that, contrary to conventional wisdom, PCR-based expression analyses can yield valid mRNA profiles even from RNA preparations that are more than 90% degraded, such as those obtained from connective tissues subjected to biomechanical studies. Furthermore, legitimate quantitative comparisons between variably degraded tissues can be made by normalizing data to appropriate housekeeping transcripts.

Rapid divergence of a gamete recognition gene promoted macroevolution of Eutheria.

BACKGROUND: Speciation genes contribute disproportionately to species divergence, but few examples exist, especially in vertebrates. Here we test whether Zan, which encodes the sperm acrosomal protein zonadhesin that mediates species-specific adhesion to the egg's zona pellucida, is a speciation gene in placental mammals. RESULTS: Genomic ontogeny reveals that Zan arose by repurposing of a stem vertebrate gene that was lost in multiple lineages but retained in Eutheria on acquiring a function in egg recognition. A 112-species Zan sequence phylogeny, representing 17 of 19 placental Orders, resolves all species into monophyletic groups corresponding to recognized Orders and Suborders, with <5% unsupported nodes. Three other rapidly evolving germ cell genes (Adam2, Zp2, and Prm1), a paralogous somatic cell gene (TectA), and a mitochondrial gene commonly used for phylogenetic analyses (Cytb) all yield trees with poorer resolution than the Zan tree and inferior topologies relative to a widely accepted mammalian supertree. Zan divergence by intense positive selection produces dramatic species differences in the protein's properties, with ordinal divergence rates generally reflecting species richness of placental Orders consistent with expectations for a speciation gene that acts across a wide range of taxa. Furthermore, Zan's combined phylogenetic utility and divergence exceeds those of all other genes known to have evolved in Eutheria by positive selection, including the only other mammalian speciation gene, Prdm9. CONCLUSIONS: Species-specific egg recognition conferred by Zan's functional divergence served as a mode of prezygotic reproductive isolation that promoted the extraordinary adaptive radiation and success of Eutheria.

Exposed and Sequestered Antigens in Testes and Their Protection by Regulatory T Cell-Dependent Systemic Tolerance.

Continuous exposure of tissue antigen (Ag) to the autoantigen-specific regulatory T cells (Treg) is required to maintain Treg-dependent systemic tolerance. Thus, testis autoantigens, previously considered as sequestered, may not be protected by systemic tolerance. We now document that the complete testis antigen sequestration is not valid. The haploid sperm Ag lactate dehydrogenase 3 (LDH3) is continuously exposed and not sequestered. It enters the residual body (RB) to egress from the seminiferous tubules and interact with circulating antibody (Ab). Some LDH3 also remains inside the sperm cytoplasmic droplets (CD). Treg-depletion in the DEREG mice that express diphtheria toxin receptor on the Foxp3 promoter results in spontaneous experimental autoimmune orchitis (EAO) and Ab to LDH3. Unlike the wild-type male mice, mice deficient in LDH3 (wild-type female or LDH3 NULL males) respond vigorously to LDH3 immunization. However, partial Treg depletion elevated the wild-type male LDH3 responses to the level of normal females. In contrast to LDH3, zonadhesin (ZAN) in the sperm acrosome displays properties of a sequestered Ag. However, when ZAN and other sperm Ag are exposed by vasectomy, they rapidly induce testis Ag-specific tolerance, which is terminated by partial Treg-depletion, leading to bilateral EAO and ZAN Ab response. We conclude that some testis/sperm Ag are normally exposed because of the unique testicular anatomy and physiology. The exposed Ag: 1) maintain normal Treg-dependent systemic tolerance, and 2) are pathogenic and serve as target Ag to initiate EAO. Unexpectedly, the sequestered Ags, normally non-tolerogenic, can orchestrate de novo Treg-dependent, systemic tolerance when exposed in vasectomy.

Immunocontraceptive target repertoire defined by systematic identification of sperm membrane alloantigens in a single species.

Sperm competence in animal fertilization requires the collective activities of numerous sperm-specific proteins that are typically alloimmunogenic in females. Consequently, sperm membrane alloantigens are potential targets for contraceptives that act by blocking the proteins' functions in gamete interactions. Here we used a targeted proteomics approach to identify the major alloantigens in swine sperm membranes and lipid rafts, and thereby systematically defined the repertoire of these sperm-specific proteins in a single species. Gilts with high alloantibody reactivity to proteins in sperm membranes or lipid rafts produced fewer offspring (73% decrease) than adjuvant-only or nonimmune control animals. Alloantisera recognized more than 20 potentially unique sperm membrane proteins and five sperm lipid raft proteins resolved on two-dimensional immunoblots with or without prior enrichment by anion exchange chromatography. Dominant sperm membrane alloantigens identified by mass spectrometry included the ADAMs fertilin α, fertilin ß, and cyritestin. Less abundant alloantigens included ATP synthase F1 ß subunit, myo-inositol monophosphatase-1, and zymogen granule membrane glycoprotein-2. Immunodominant sperm lipid raft alloantigens included SAMP14, lymphocyte antigen 6K, and the epididymal sperm protein E12. Of the fifteen unique membrane alloantigens identified, eleven were known sperm-specific proteins with uncertain functions in fertilization, and four were not previously suspected to exist as sperm-specific isoforms. De novo sequences of tryptic peptides from sperm membrane alloantigen "M6" displayed no evident homology to known proteins, so is a newly discovered sperm-specific gene product in swine. We conclude that alloimmunizing gilts with sperm membranes or lipid rafts evokes formation of antibodies to a relatively small number of dominant alloantigens that include known and novel sperm-specific proteins with possible functions in fertilization and potential utility as targets for immunocontraception.

Egress of sperm autoantigen from seminiferous tubules maintains systemic tolerance.

Autoimmune responses to meiotic germ cell antigens (MGCA) that are expressed on sperm and testis occur in human infertility and after vasectomy. Many MGCA are also expressed as cancer/testis antigens (CTA) in human cancers, but the tolerance status of MGCA has not been investigated. MGCA are considered to be uniformly immunogenic and nontolerogenic, and the prevailing view posits that MGCA are sequestered behind the Sertoli cell barrier in seminiferous tubules. Here, we have shown that only some murine MGCA are sequestered. Nonsequestered MCGA (NS-MGCA) egressed from normal tubules, as evidenced by their ability to interact with systemically injected antibodies and form localized immune complexes outside the Sertoli cell barrier. NS-MGCA derived from cell fragments that were discarded by spermatids during spermiation. They egressed as cargo in residual bodies and maintained Treg-dependent physiological tolerance. In contrast, sequestered MGCA (S-MGCA) were undetectable in residual bodies and were nontolerogenic. Unlike postvasectomy autoantibodies, which have been shown to mainly target S-MGCA, autoantibodies produced by normal mice with transient Treg depletion that developed autoimmune orchitis exclusively targeted NS-MGCA. We conclude that spermiation, a physiological checkpoint in spermatogenesis, determines the egress and tolerogenicity of MGCA. Our findings will affect target antigen selection in testis and sperm autoimmunity and the immune responses to CTA in male cancer patients.

Evaluation of an Algorithm to Predict Menstrual-Cycle Phase at the Time of Injury.

CONTEXT: Women are 2 to 8 times more likely to sustain an anterior cruciate ligament (ACL) injury than men, and previous studies indicated an increased risk for injury during the preovulatory phase of the menstrual cycle (MC). However, investigations of risk rely on retrospective classification of MC phase, and no tools for this have been validated. OBJECTIVE: To evaluate the accuracy of an algorithm for retrospectively classifying MC phase at the time of a mock injury based on MC history and salivary progesterone (P4) concentration. DESIGN: Descriptive laboratory study. SETTING: Research laboratory. PARTICIPANTS: Thirty-one healthy female collegiate athletes (age range, 18-24 years) provided serum or saliva (or both) samples at 8 visits over 1 complete MC. MAIN OUTCOME MEASURE(S): Self-reported MC information was obtained on a randomized date (1-45 days) after mock injury, which is the typical timeframe in which researchers have access to ACL-injured study participants. The MC phase was classified using the algorithm as applied in a stand-alone computational fashion and also by 4 clinical experts using the algorithm and additional subjective hormonal history information to help inform their decision. To assess algorithm accuracy, phase classifications were compared with the actual MC phase at the time of mock injury (ascertained using urinary luteinizing hormone tests and serial serum P4 samples). Clinical expert and computed classifications were compared using κ statistics. RESULTS: Fourteen participants (45%) experienced anovulatory cycles. The algorithm correctly classified MC phase for 23 participants (74%): 22 (76%) of 29 who were preovulatory/anovulatory and 1 (50%) of 2 who were postovulatory. Agreement between expert and algorithm classifications ranged from 80.6% (κ = 0.50) to 93% (κ = 0.83). Classifications based on same-day saliva sample and optimal P4 threshold were the same as those based on MC history alone (87.1% correct). Algorithm accuracy varied during the MC but at no time were both sensitivity and specificity levels acceptable. CONCLUSIONS: These findings raise concerns about the accuracy of previous retrospective MC-phase classification systems, particularly in a population with a high occurrence of anovulatory cycles.

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 subject's 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.

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.

Infertility with impaired zona pellucida adhesion of spermatozoa from mice lacking TauCstF-64.

Fertilization is a multistep process requiring spermatozoa with unique cellular structures and numerous germ cell-specific molecules that function in the various steps. In the highly coordinated process of male germ cell development, RNA splicing and polyadenylation help regulate gene expression to assure formation of functional spermatozoa. Male germ cells express tauCstF-64 (Cstf2t gene product), a paralog of the X-linked CstF-64 protein that supports polyadenylation in most somatic cells. We previously showed that loss of tauCstF-64 causes male infertility because of major defects in mouse spermatogenesis. Surprisingly, although Cstf2t(-/-) males produce very few recognizable spermatozoa, some of the spermatozoa produced are motile. This led us to ask whether these Cstf2t(-/-) sperm were fertile. A motile cell-enriched population of spermatozoa from Cstf2t-null males dispersed cumulus cells of cumulus-oocyte complexes normally. However, motile spermatozoa from Cstf2t-null males failed to fertilize cumulus-intact mouse eggs in vitro. In addition, sperm adhesion to the zona pellucida (ZP) of cumulus-free eggs was significantly decreased, indicating tauCstF-64 is required for production of spermatozoa capable of ZP interaction. Acrosomal proteins involved in sperm-ZP recognition, including zonadhesin, proacrosin, SPAM1/PH-20, and ZP3R/sp56, were normally distributed in the apical head of Cstf2t(-/-) spermatozoa. We conclude that tauCstF-64 is required not only for expression of genes involved in morphological differentiation of spermatids but also for genes having products that function during interaction of motile spermatozoa with eggs. To our knowledge, this is the first demonstration that a gene involved in polyadenylation has a negative consequence on sperm-ZP adhesion.

Zonadhesin D3-polypeptides vary among species but are similar in Equus species capable of interbreeding.

Zonadhesin is a rapidly evolving protein in the sperm acrosome that confers species specificity to sperm-zona pellucida adhesion. Though structural variation in zonadhesin likely contributes to its species-specific function, the protein has not previously been characterized in organisms capable of interbreeding. Here we compared properties of zonadhesin in several animals, including the horse (Equus caballus), donkey (E. asinus), and Grevy's zebra (E. grevyi) to determine if variation in zonadhesin correlates with ability of gametes to cross-fertilize. Zonadhesin localized to the apical acrosomes of spermatozoa from all three Equus species, similar to its localization in other animals. Likewise, in horse and donkey testis, zonadhesin was detected only in germ cells, first in the acrosomal granule of round spermatids and then in the developing acrosomes of elongating spermatids. Among non-Equus species, D3-domain polypeptides of mature, processed zonadhesin varied markedly in size and detergent solubility. However, zonadhesin D3-domain polypeptides in horse, donkey, and zebra spermatozoa exhibited identical electrophoretic mobility and detergent solubility. Equus zonadhesin D3-polypeptides (p110/p80 doublet) were most similar in size to porcine and bovine zonadhesin D3-polypeptides (p105). Sequence comparisons revealed that the horse zonadhesin precursor's domain content and arrangement are similar to those of zonadhesin from other large animals. Partial sequences of horse and donkey zonadhesin were much more similar to each other (>99% identity) than they were to orthologous sequences of human, pig, rabbit, and mouse zonadhesin (52%-72% identity). We conclude that conservation of zonadhesin D3-polypeptide properties correlates with ability of Equus species to interbreed.

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.

Anterior cruciate ligament biology and its relationship to injury forces.

Anterior cruciate ligament injury is determined by two variables: the ultimate failure load of the ligament and the mechanical load applied to the ligament. All factors that contribute to anterior cruciate ligament injury must do so by affecting one or both of these two basic variables. Some factors, such as sex hormones and tissue remodeling, have a multifaceted effect on the failure load of the anterior cruciate ligament and the magnitude of the load applied to it. The model also illustrates the potentially profound effects that sex hormones and tissue remodeling likely have on female susceptibility to anterior cruciate ligament injuries.

Zonadhesin assembly into the hamster sperm acrosomal matrix occurs by distinct targeting strategies during spermiogenesis and maturation in the epididymis.

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.

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 protein's 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.

The cystatin-related epididymal spermatogenic protein inhibits the serine protease prohormone convertase 2.

The cystatin-related epididymal spermatogenic (CRES) protein is related to the family 2 cystatins of the cystatin superfamily of cysteine protease inhibitors. However, CRES lacks sequences important for cysteine protease inhibitory activity and is specifically expressed in reproductive and neuroendocrine tissues. Thus, CRES is distinct from cystatins and may perform unique tissue-specific functions. The purpose of the present study was to determine whether CRES functions as a protease inhibitor in in vitro assays. In contrast to mouse recombinant cystatin C, recombinant CRES did not inhibit the cysteine proteases papain and cathepsin B, suggesting that it probably does not function as a typical cystatin. CRES, however, inhibited the serine protease prohormone convertase 2 (PC2), a protease involved in prohormone processing in the neuroendocrine system, whereas cystatin C showed no inhibition. CRES did not inhibit subtilisin, trypsin, or the convertase family members, PC1 and furin, indicating that it selectively inhibits PC2. Kinetic analysis showed that CRES is a competitive inhibitor of PC2 with a K(i) of 25 nM. The removal of N-terminal sequences from CRES decreased its affinity for PC2, suggesting that the N terminus may be important for CRES to function as an inhibitor. These studies suggest that CRES is a cross-class inhibitor that may regulate proprotein processing within the reproductive and neuroendocrine systems.

Identification and characterization of cystatin-related epididymal spermatogenic protein in human spermatozoa: localization in the equatorial segment.

Our earlier studies in mouse have shown that the cystatin-related epididymal spermatogenic (CRES) protein is highly expressed in elongating spermatids in the testis and is present in mouse sperm acrosomes, suggesting specific roles in sperm function, fertilization, or both. However, whether the human CRES gene is similar to that of the mouse and is expressed in germ cells has not yet been determined. Therefore, the present study was undertaken to characterize the human ortholog of mouse CRES: Northern blot and in situ hybridization experiments showed that CRES is highly expressed in the human testis, specifically within clusters of round spermatids. Furthermore, reverse transcription-polymerase chain reaction detected CRES mRNA in the epididymis. Western blot analysis of protein lysates prepared from human testis and ejaculated spermatozoa showed a predominant 19-kDa protein and a minor 14-kDa protein. However, in contrast to the acrosomal localization of CRES protein in mouse spermatozoa, indirect immunofluorescence of human spermatozoa treated with methanol/acetic acid using anti-human CRES antibodies revealed that CRES was strictly localized to the equatorial segment. Furthermore, the same staining was observed in both capacitated and acrosome-reacted spermatozoa. To determine whether CRES was associated with the plasma membrane, live spermatozoa were incubated with CRES antibody after capacitation and acrosome reaction. Only acrosome-reacted spermatozoa showed a weak but specific equatorial staining. Taken together, these studies show that CRES protein is present in the sperm equatorial segment and becomes accessible to the extracellular environment during fertilization.

Cubilin, a binding partner for galectin-3 in the murine utero-placental complex.

Galectin-3 is a lectin important in animal development and regulatory processes and is found selectively localized at the implantation site of the mouse embryo. To better understand the role of galectin-3 at the maternal-fetal interface, a binding partner was isolated and characterized. Homogenates of uteroplacental tissue were incubated with immobilized recombinant galectin-3, and specifically bound proteins were eluted using lactose. The principal protein, p400, had an M(r) of 400,000 in SDS-PAGE. Physical properties of p400 and amino acid sequences of seven tryptic peptides were similar to cubilin from rats, humans, and dogs, identifying p400 as the murine ortholog of cubilin. This was further supported by the tissue distribution observed only in yolk sac, kidney, and ileum with monospecific antiserum for p400. Cubilin occurred in yolk sac epithelium throughout pregnancy, but galectin-3 was there only during the last week. Unexpectedly, cubilin was found only in perforin-containing granules of uterine natural killer (uNK) cells, although galectin-3 occurred throughout the cell cytoplasm. In situ hybridization revealed cubilin mRNA in yolk sac epithelium but not uNK cells, implying that yolk sac-derived cubilin is endocytosed by uNK cells via galectin-3. This is consistent with cubilin being an endogenous partner of galectin-3 at the maternal-fetal interface and suggests an important role for cubilin in uNK cell function.

The Menstrual Cycle, Sex Hormones, and Anterior Cruciate Ligament Injury.

OBJECTIVE: To determine if anterior cruciate ligament (ACL) injuries in female athletes occur randomly or correlate with a specific phase of the menstrual cycle. DESIGN AND SETTING: Female athletes who sustained ACL injuries reported the days of their menstrual cycles and provided saliva samples for sex-hormone determination. Salivary sex-hormone profiles were assessed to confirm the self-reported menstrual histories. SUBJECTS: A total of 38 female athletes (20 college, 15 high school, 1 middle school, 2 recreational) with recent ACL injuries participated in the study over a 3-year period. MEASUREMENTS: Athletes with recent ACL injuries completed a questionnaire defining the injury, the last menstrual cycle, prior knee injury, school, and type of birth control used (if any). Each subject provided a 30-cc saliva sample within 72 hours of injury. Saliva samples were placed into sealed containers and frozen at -20 degrees C. We obtained 13 additional control samples from uninjured females to test the correlation between saliva and serum sex-hormone levels. Progesterone and estrogen were assayed by radioimmunoassay. Physical examination, magnetic resonance imaging, or surgery confirmed the injury in all subjects. RESULTS: The correlations between saliva and serum estrogen and progesterone were 0.73 (alpha =.01) and 0.72 (alpha =.01), respectively. Ten of 27 athletes who reported their cycle day at time of injury sustained an ACL injury immediately before or 1 to 2 days after the onset of menses. We rejected the null hypothesis that such high frequency was due to random chance. CONCLUSIONS: A significantly greater number of ACL injuries occurred on days 1 and 2 of the menstrual cycle. Salivary sex-hormone levels correlated with the reported cycle day.