Sperm Adhesion Molecule Research Articles

Sperm Adhesion Molecule 1 (SPAM1) Distribution in Selected Human Sperm by Hyaluronic Acid Test.

The failures of binding to the oocyte zona pellucida are commonly attributed to defects in the sperm recognition, adhesion, and fusion molecules. SPAM1 (sperm adhesion molecule 1) is a hyaluronidase implicated in the dispersion of the cumulus-oocyte matrix. Therefore, the aim of this study was to characterize the SPAM1 distribution in the different physiological conditions of human sperm. Specifically, we evaluated the location of the SPAM1 protein in human sperm before capacitation, at one and four hours of capacitation and after hyaluronic acid (HA) selection test by fluorescence microscopy. Sperm bound to HA were considered mature and those that crossed it immature. Our results detected three SPAM1 fluorescent patterns: label throughout the head (P1), equatorial segment with acrosomal faith label (P2), and postacrosomal label (P3). The data obtained after recovering the mature sperm by the HA selection significantly (p < 0.05) highlighted the P1 in both capacitation times, being 79.74 and 81.48% after one hour and four hours, respectively. Thus, the HA test identified that human sperm require the presence of SPAM1 throughout the sperm head (P1) to properly contact the cumulus-oocyte matrix. Overall, our results provide novel insights into the physiological basis of sperm capacitation and could contribute to the improvement of selection techniques.

P–026 Seminal plasma exosomes: a promising source of biomarkers for fertility evaluation

Abstract Study question Do exosomes from seminal plasma have a role in male fertility? Summary answer Exosomes isolated from seminal plasma have a pivotal role during spermatogenesis and sperm maturation and may represent eligible biomarkers for male fertility/infertility. What is known already During their journey along the male reproductive tract, exosomes contained in seminal fluid are involved in the transfer of several molecules to the maturing sperm. Exosomes are extracellular vesicles (EVs) released by all the cells; they carry a cargo of nucleic acids, proteins and lipids. In the male genital tract, they are released at various levels and their composition differs between men of proven fertility and infertile male patients. Recent studies reported the proteomic profile of exosomes, revealing the presence of several proteins with a well know role in sperm maturation and fertilizing ability acquiring. Study design, size, duration This prospective study consisted of 36 Caucasian men; according to seminal parameters (WHO 2010) they were divided in normozoospermic (N; n = 12), oligoasthenoteratozoospermic (OAT: n = 12) and azoospermic (A; n = 12). Semen samples were collected between October 2020 and January 2021 at the Assisted Reproductive Unit, Siena University Hospital (Italy) after institutional ethical approval and signed written consent from all the participants. Participants/materials, setting, methods Ejaculated sperm were analyzed according to WHO–2010 criteria and divided into the three groups: N, OAT and A. Exosomes were isolated by an in-house modified ExoGAG®-polymer precipitation-based protocol and characterized for size and ultrastructure by Nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). The exosomal proteins were extracted and analyzed by 2D-electrophoresis and the identified profiles were examined by applying bioinformatic tools. The expression of selected genes was evaluated by digital droplets PCR (ddPCR). Main results and the role of chance The present work is readily providing an improvement of the standard ExoGAG® protocol and underlines its advantages over more conventional EVs isolation protocols used to date for recovery from seminal fluid: the number of recovered EVs and their size were finely included in the range of exosomes. This isolation protocol provides samples suitable for proteomic analyses, representing the first 2D-electrophoresis reference map of exosome-pay loaded proteins in N respect to OAT/A groups and providing an innovative and comprehensive functional overview of its proteins. Moreover, the STRING protein-protein interaction analysis revealed the deregulation of specific pathways (e.g. signaling proteins, chromatin packaging and/or remodeling, protein folding and apoptosis) in A and OAT in comparison with N group. Gene expression by ddPCR analysis highlighted that most of the analyzed genes are modulated in according to seminal parameters, in particular: GAPDHS (Glyceraldehyde–3-Phosphate Dehydrogenase, Spermatogenic); SPAM1 (Sperm Adhesion Molecule–1) encoding a members of hyaluronidase family; ADAM2 (ADAM Metallopeptidase Domain–2) that plays an important role in sperm-egg interactions; CRISP1,2,3 (Cysteine Rich Secretory Protein 1,2,3) expressed in the epididymis and secreted into the epididymal lumen; CLGN (Calmegin) encoding a testis-specific chaperone protein and PGK2 (Phosphoglycerate Kinase–2) expressed in the later stages of spermatogenesis. Limitations, reasons for caution This study represents a preliminary experiment. We suggest further comparative studies in larger study cohorts. Wider implications of the findings: This pilot study, demonstrating the unique proteomic and transcriptomic pattern of exosomes in N/OAT/A groups, supports the importance of exosomes in sperm production and maturation. This methodological set-up is expected to open new ways for advancement in the use of exosomes as fertility biomarkers, making possible personalized approaches in ART. Trial registration number Not applicable

Arylsulfatase A Remodeling during Human Sperm In Vitro Capacitation Using Field Emission Scanning Electron Microscopy (FE-SEM).

Capacitation drives sperm biophysical and biochemical changes for sperm-oocyte interactions. It is a well-known fact that the molecular complex arylsulfatase A (ARSA), hyaluronidase sperm adhesion molecule 1 (SPAM1), and heat shock protein 2 (HSPA2) plays a significant role in sperm–zona pellucida (ZP) binding. However, the time-dependent capacitation effects on the sperm surface ARSA presence and specific topographic distributions remain to be elucidated. Here, we quantified the ARSA density and specific membrane domain locations before (US) and after in vitro capacitation (one and four hours; CS1–CS4) in human sperm using high-resolution field emission scanning electron microscopy (FE-SEM) and immunogold labeling. Our results showed a significant and progressive capacitation-mediated increase of labeled spermatozoa from the US (37%) to CS4 (100%) physiological conditions. In addition, surface mapping revealed a close relationship between the ARSA residues and their acrosomal repositioning. Compared with the ARSA surface heterogeneous distribution found in US, the CS1–4 conditions exhibited clustering on the peri-acrosomal region, showing that time-dependent capacitation also induced a ARSA residue dramatic translocation on sperm surfaces. Our findings provide novel insights into the molecular remodeling events preceding sperm-oocyte interactions.

Fermented Maillard reaction products attenuate stress-induced testicular dysfunction in mice

Chronic stress can cause psychological diseases and affect male fertility and the reproductive system. Maillard reaction of milk proteins improves their functional and nutritional properties through modification of proteins. Previously, we determined that Maillard reaction product (MRP) from milk casein and MRP fermented (FMRP) with Lactobacillus rhamnosus 4B15 (4B15) had anti-anxiolytic effects in mice under chronic stress. Therefore, we aimed to investigate the effects of MRP and FMRP on chronic stress-induced testicular dysfunction in mice through quantitative real-time PCR (qRT-PCR) and in situ hybridization analysis. Mice were pretreated with MRP and FMRP for 10 wk; simultaneously, from the third week of the experimental period, they were exposed to unpredictable chronic mild stress (UCMS) for 7 wk. The expression levels of the luteinizing hormone subunit β (Lhb) and follicle-stimulating hormone subunit β (Fshb) were remarkably reduced after exposure to UCMS. However, treatment with MRP and FMRP inhibited the UCMS-induced reduction, with FMRP showing especially significant inhibition. Moreover, the expression of steroidogenesis-related genes [luteinizing hormone receptor (Lhr), follicle-stimulating hormone (Fshr), 3-β hydroxysteroid dehydrogenase 2 (Hsd3b2), and steroidogenic acute regulatory protein (StAR)] were significantly reduced in response to UCMS. In contrast, the transcript levels of these genes were highest in the MRP-treated mice. Mice pretreated with FMRP also exhibited higher levels of gene expression compared with the nonstressed mice. Moreover, UCMS significantly downregulated the expression of genes associated with testicular function [i.e., a disintegrin and metallopeptidase domain 5 (Adam5), Adam29, bone morphogenetic protein 2 (Bmp2), tektin 3 (Tekt3), and sperm adhesion molecule 1 (Spam1)]. However, the administration of MRP and FMRP prevented the UCMS-induced reduction in the expressions of above genes. The localization of Lhr, Srd5a2, Adam29, and Spam1 was confirmed by in situ hybridization analysis and the results were consistent with those of qRT-PCR. Consequently, these results indicated that MRP and FMRP, manufactured by the heat treatment of milk casein and fermentation with probiotic 4B15, have the potential to prevent chronic stress-induced testicular dysfunction.

How does secondary hypogonadism affect the spermatozoa proteome? Lessons from a porcine animal model

Secondary hypogonadism is a consequence of congenital or acquired diseases that affect the hypothalamus and/or pituitary gland, impairing secretion of gonadotrophin-releasing hormone (GnRH). Androgen deficiency resulting from reduced GnRH secretion is likely to have disrupting effects in epididymal epithelial cells, impairing the sperm maturation process. The aim of this study was to describe changes in the proteome of epididymal spermatozoa in a porcine model of secondary hypogonadism. Cauda epididymal spermatozoa were obtained from 10 boars previously immunised against GnRH (Vivax; Pfizer) and from 10 healthy boars. Protein extracts were analysed by multidimensional protein identification technology. In all, 1322 unique proteins were identified in the protein extracts of cauda epididymal spermatozoa, with significant changes in the abundance of key proteins involved in sperm metabolism (enolase, pyruvate dehydrogenase), acrosome reaction and capacitation (oxoprolinase, acrosomal protein SP-10, dihydrolipoyl dehydrogenase) and sperm–oocyte interactions (zona pellucida-binding protein, zonadhesin, sperm adhesion molecule 1). In addition, the abundance of mitochondrial proteins was severely affected, with significant changes in proteins of Complex I and II, as well as ATPase of the oxidative phosphorylation chain. The proteins identified in this study are potential sperm biomarkers of testicular and epididymal dysfunction related to disruption of the hypothalamus–pituitary–testis axis.

SPAM1/HYAL5 double deficiency in male mice leads to severe male subfertility caused by a cumulus-oocyte complex penetration defect.

The glycosylphosphatidylinositol-anchored sperm hyaluronidases (Hyals), sperm adhesion molecule 1 (SPAM1) and HYAL5, have long been believed to assist in sperm penetration through the cumulus-oocyte complex (COC), but their role in mammalian fertilization remains unclear. Previously, we have shown that mouse sperm devoid of either Spam1 or Hyal5 are still capable of penetrating the COC and that the loss of either Spam1 or Hyal5 alone does not cause male infertility in mice. In the present study, we found that Spam1/Hyal5 double knockout (dKO) mice produced significantly fewer offspring compared with wild-type (WT) mice, and this was due to defective COC dispersal. A comparative analysis between WT and Spam1/Hyal5 dKO epididymal sperm revealed that the absence of these 2 sperm Hyals resulted in a marked accumulation of sperm on the outside of the COC. This impaired sperm activity is likely due to the deficiency in the sperm Hyals, even though other somatic Hyals are expressed normally in the dKO mice. The fertilization ability of the Spam1/Hyal5 dKO sperm was restored by adding purified human sperm Hyal to the in vitro fertilization medium. Our results suggest that Hyal deficiency in sperm may be a significant risk factor for male sterility.-Park, S., Kim, Y.-H., Jeong, P.-S., Park, C., Lee, J.-W., Kim, J.-S., Wee, G., Song, B.-S., Park, B.-J., Kim, S.-H., Sim, B.-W., Kim, S.-U., Triggs-Raine, B., Baba, T., Lee, S.-R., Kim, E. SPAM1/HYAL5 double deficiency in male mice leads to severe male subfertility caused by a cumulus-oocyte complex penetration defect.

Detection of extracellular vesicles in the mouse vaginal fluid: Their delivery of sperm proteins that stimulate capacitation and modulate fertility.

Extracellular vesicles (EVs) were isolated by ultracentrifugation of vaginal luminal fluid (VLF) from superovulated mice and identified for the first time using transmission electron microscopy. Characterized by size and biochemical markers (CD9 and HSC70), EVs were shown to be both microvesicular and exosomal and were dubbed as "Vaginosomes" (VGS). Vaginal cross-sections were analyzed to visualize EVs in situ: EVs were present in the lumen and also embedded between squamous epithelial and keratinized cells, consistent with their endogenous origin. Western blots detected Plasma membrane Ca2+ -ATPase 1 (PMCA1) and tyrosine-phosphorylated proteins in the VGS cargo and also in uterosomes. Flow cytometry revealed that following coincubation of caudal sperm and VLF for 30 min, the frequencies of cells with the highest Sperm adhesion molecule 1 (SPAM1), PMCA1/4, and PMCA1 levels increased 16.4-, 8.2-, and 27-fold, respectively; compared with control coincubated in phosphate buffered saline (PBS). Under identical conditions, sperm tyrosine-phosphorylated proteins were elevated ~3.3-fold, after VLF coincubation. Progesterone-induced acrosome reaction (AR) rates were significantly (p < 0.001) elevated in sperm coincubated with VGS for 10-30 min, compared with PBS. Sperm artificially deposited in the vaginas of superovulated females for these periods also showed significant (p < 0.01) increases in AR rates, compared with PBS. Thus in vitro and in vivo, sperm acquire from the vaginal environment factors that induce capacitation, explaining recent findings for their acrosomal status in the isthmus. Overall, VGS appear to deliver higher levels of proteins involved in preventing premature capacitation and AR than those promoting them. Our findings which have implications for humans open the possibility of new approaches to infertility treatment with exosome therapeutics.

Genes Encoding Mammalian Oviductal Proteins Involved in Fertilization are Subjected to Gene Death and Positive Selection

Oviductal proteins play an important role in mammalian fertilization, as proteins from seminal fluid. However, in contrast with the latter, their phylogenetic evolution has been poorly studied. Our objective was to study in 16 mammals the evolution of 16 genes that encode oviductal proteins involved in at least one of the following steps: (1) sperm–oviduct interaction, (2) acrosome reaction, and/or (3) sperm–zona pellucida interaction. Most genes were present in all studied mammals. However, some genes were lost along the evolution of mammals and found as pseudogenes: annexin A5 (ANXA5) and deleted in malignant brain tumor 1 (DMBT1) in tarsier; oviductin (OVGP1) in megabat; and probably progestagen-associated endometrial protein (PAEP) in tarsier, mouse, rat, rabbit, dolphin, and megabat; prostaglandin D2 synthase (PTGDS) in microbat; and plasminogen (PLG) in megabat. Four genes [ANXA1, ANXA4, ANXA5, and heat shock 70 kDa protein 5 (HSPA5)] showed branch-site positive selection, whereas for seven genes [ANXA2, lactotransferrin (LTF), OVGP1, PLG, S100 calcium-binding protein A11 (S100A11), Sperm adhesion molecule 1 (SPAM1), and osteopontin (SPP1)] branch-site model and model-site positive selection were observed. These results strongly suggest that genes encoding oviductal proteins that are known to be important for gamete fertilization are subjected to positive selection during evolution, as numerous genes encoding proteins from mammalian seminal fluid. This suggests that such a rapid evolution may have as a consequence that two isolated populations become separate species more rapidly.

SPAM1 and PH-20 are two gene products expressed in bovine testis and present in sperm.

Sperm adhesion molecule 1 (SPAM1) is a sperm protein possessing a hyaluronidase domain in its N-terminus and a zona pellucida-binding domain in its C-terminus. Our previous studies showed that bovine spermatozoa potentially have 2 SPAM1 isoforms that present different C-terminal domains, different origins (testis and epididymis) and different locations in spermatozoa. In this study, two approaches were taken to characterize the different SPAM1 isoforms. First, 3'-RACE experiments were done to determine the sequence of the 3' regions of the potential transcripts. Second, by in silico analyses, we aimed to determine whether our antibody that recognizes the N-terminal domain of SPAM1 detects two SPAM1 isoforms or two highly similar, although different, proteins. We found that the 3' regions of SPAM1 transcripts from bovine testis and caput epididymis were identical. Nevertheless, two transcript variants that differ by 90 nucleotides, encoded by an entire exon, are expressed in both tissues. Only the protein encoded by the longest SPAM1 transcript variant was confirmed in ejaculated bull spermatozoa by mass spectrometry. In silico analyses revealed a highly similar protein to SPAM1, PH-20, that could potentially be recognized by our N-terminal antibody. The presence of PH-20 transcripts was confirmed in bovine testis and the protein is present in ejaculated spermatozoa. Our N-terminal antibody possibly recognizes both SPAM1 and the highly homologous protein PH-20 instead of two SPAM1 isoforms. Identifying the proteins implicated in the fertilization process is crucial in order to elucidate their roles and to better understand the complex process of fertilization.

Constitutive expression of recombinant human hyaluronidase PH20 by Pichia pastoris

PH20 is known as sperm adhesion molecule 1 (SPAM1) and also has hyaluronidase function to preferentially hydrolyze the glycosidic linkage of hyaluronic acid (HA). A DNA fragment containing core domain of human PH20 gene was cloned into a constitutive expression plasmid (pGAPZαC) of Pichia pastoris to produce a fusion protein with α factor signal in the N-terminus and 6×His as well as c-Myc tags in the C-terminus. The resulting plasmid pGAPZαC-PH20 was integrated into the genome of P.pastoris strain GS115. Functional recombinant human PH20 (rHuPH20) was successfully expressed and secreted by the recombinant P.pastoris transformant. Highest hyaluronidase activity of 2mU/mL could be obtained at 3 day in an YPD culture. After purified by phenylboronic acid resin adsorption, rHuPH20 with a specific activity of 230mU/mg was obtained. Via periodic acid-Schiff staining and zymogram analysis, the partially purified rHuPH20 was determined to be highly glycosylated to various extents with molecular mass in the range of 100-300kDa. The enzyme showed a maximal activity at pH 5.0 but no appreciable activity at pH ≤3 and pH ≥8. The hyaluronidase activity could be stably maintained at 4°C but lost 40% after incubating at 30°C for 4h. Both N-acetyl cysteine and glutathione showed a half maximal inhibitory concentration (IC50) of 8mM against rHuPH20.

The impact of oxidative stress on chaperone-mediated human sperm-egg interaction.

How does oxidative stress impact upon human sperm-egg interaction and in particular the formation of zona pellucida-receptor complexes on the sperm surface? Oxidative stress during human sperm capacitation resulted in the chemical alkylation of the molecular chaperone heat shock protein A2 (HSPA2), a concomitant reduction in surface expression of the zona pellucida-receptor arylsulphatase A (ARSA) and a severe loss of zona pellucida binding ability. An inability to bind to the zona pellucida is commonly encountered in the defective spermatozoa generated by male infertility patients; however, the underlying mechanisms remain unresolved. Recent studies have revealed that zona pellucida binding is mediated by molecular chaperones, particularly HSPA2, that facilitate the formation of multimeric zona pellucida-receptor complexes on the surface of mammalian spermatozoa during capacitation. Spermatozoa were collected from healthy normozoospermic donors (n = 15). Low levels of oxidative stress were induced in populations of non-capacitated spermatozoa by a 1 h treatment with 4-hydroxynonenal (4HNE) or hydrogen peroxide (H2O2) and then these insults were removed and cells were capacitated for 3 h. Motility, membrane fluidity, protein tyrosine phosphorylation and lipid raft distribution were evaluated after sperm capacitation to determine the impact of oxidative stress on this process. The surface expression of ARSA and sperm adhesion molecule 1 (SPAM1) was observed using fluorescence microscopy, and the ability of treated cells to interact with homologous human zonae pellucidae was assessed through gamete co-incubation. Proximity ligation was used to evaluate the state of the HSPA2-laden zona pellucida-receptor complex and an immunoprecipitation approach was taken to establish the chemical alkylation of HSPA2 by the cytotoxic lipid aldehyde 4HNE. The validity of these findings was then tested through treatment of oxidatively stressed cells with the nucleophile penicillamine in order to scavenge lipid aldehydes and limit their ability to interact with HSPA2. All experiments were performed on samples pooled from two or more donors per replicate, with a minimum of three replicates. The oxidative treatments employed in this study did not influence sperm motility or capacitation-associated changes in membrane fluidity, tyrosine phosphorylation and lipid raft redistribution. However, they did significantly impair zona pellucida binding compared with the capacitated control (P < 0.01). The reduction in zona pellucida binding was associated with the impaired surface expression (P < 0.02) of a zona pellucida-receptor complex comprising HSPA2, SPAM1 and ARSA. Proximity ligation and immunoprecipitation assays demonstrated that impaired zona pellucida binding was, in turn, associated with the chemical alkylation of HSPA2 with 4HNE and the concomitant disruption of this zona pellucida-receptor complex. The use of penicillamine enabled a partial recovery of ARSA surface expression and zona pellucida adherence in H2O2-treated cells. These data suggest that the ability of low levels of oxidative stress to disrupt sperm function is mediated by the production of lipid aldehydes as a consequence of lipid peroxidation and their adduction to the molecular chaperone HSPA2 that is responsible for co-ordinating the assembly of functional zona pellucida-receptor complexes during sperm capacitation. While these results extend only to one particular zona pellucida-receptor complex, we postulate that oxidative stress may more broadly impact upon sperm surface architecture. In this light, further study is required to assess the impact of oxidative stress on additional HSPA2-laden protein complexes. These findings link low levels of oxidative stress to a severe loss of sperm function. In doing so, this work suggests a potential cause of male infertility pertaining to a loss of zona pellucida recognition ability and will contribute to the more accurate diagnosis and treatment of such conditions.

Hyaluronidase 2: a novel germ cell hyaluronidase with epididymal expression and functional roles in mammalian sperm.

To initiate the crucial cell adhesion events necessary for fertilization, sperm must penetrate extracellular matrix barriers containing hyaluronic acid (HA), a task thought to be accomplished by neutral-active hyaluronidases. Here we report that the ~57 kDa hyaluronidase 2 (HYAL2) that in somatic tissues has been highly characterized to be acid-active is present in mouse and human sperm, as detected by Western blot, flow cytometric, and immunoprecipitation assays. Immunofluorescence revealed its presence on the plasma membrane over the acrosome, the midpiece, and proximal principal piece in mice where protein fractionation demonstrated a differential distribution in subcellular compartments. It is significantly more abundant in the acrosome-reacted (P = 0.04) and soluble acrosomal fractions (P = 0.006) (microenvironments where acid-active hyaluronidases function) compared to that of the plasma membrane where neutral hyaluronidases mediate cumulus penetration. Using HA substrate gel electrophoresis, immunoprecipitated HYAL 2 was shown to have catalytic activity at pH 4.0. Colocalization and coimmunoprecipitation assays reveal that HYAL2 is associated with its cofactor, CD44, consistent with CD44-dependent HYAL2 activity. HYAL2 is also present throughout the epididymis, where Hyal2 transcripts were detected, and in the epididymal luminal fluids. In vitro assays demonstrated that HYAL2 can be acquired on the sperm membrane from epididymal luminal fluids, suggesting that it plays a role in epididymal maturation. Because similar biphasic kinetics are seen for HYAL2 and SPAM1 (Sperm adhesion molecule 1), it is likely that HYAL2 plays a redundant role in the catalysis of megadalton HA to its 20 kDa intermediate during fertilization.

Nanoparticle incorporation of melittin reduces sperm and vaginal epithelium cytotoxicity.

Melittin is a cytolytic peptide component of bee venom which rapidly integrates into lipid bilayers and forms pores resulting in osmotic lysis. While the therapeutic utility of free melittin is limited by its cytotoxicity, incorporation of melittin into the lipid shell of a perfluorocarbon nanoparticle has been shown to reduce its toxicity in vivo. Our group has previously demonstrated that perfluorocarbon nanoparticles containing melittin at concentrations <10 µM inhibit HIV infectivity in vitro. In the current study, we assessed the impact of blank and melittin-containing perfluorocarbon nanoparticles on sperm motility and the viability of both sperm and vaginal epithelial cells. We found that free melittin was toxic to sperm and vaginal epithelium at concentrations greater than 2 µM (p<0.001). However, melittin nanoparticles were not cytotoxic to sperm (p = 0.42) or vaginal epithelium (p = 0.48) at an equivalent melittin concentration of 10 µM. Thus, nanoparticle formulation of melittin reduced melittin cytotoxicity fivefold and prevented melittin toxicity at concentrations previously shown to inhibit HIV infectivity. Melittin nanoparticles were toxic to vaginal epithelium at equivalent melittin concentrations ≥20 µM (p<0.001) and were toxic to sperm at equivalent melittin concentrations ≥40 µM (p<0.001). Sperm cytotoxicity was enhanced by targeting of the nanoparticles to the sperm surface antigen sperm adhesion molecule 1. While further testing is needed to determine the extent of cytotoxicity in a more physiologically relevant model system, these results suggest that melittin-containing nanoparticles could form the basis of a virucide that is not toxic to sperm and vaginal epithelium. This virucide would be beneficial for HIV serodiscordant couples seeking to achieve natural pregnancy.

Investigation of the mechanisms by which the molecular chaperone HSPA2 regulates the expression of sperm surface receptors involved in human sperm-oocyte recognition

A unique characteristic of mammalian spermatozoa is that, upon ejaculation, they are unable to recognize and bind to an ovulated oocyte. These functional attributes are only realized following the cells' ascent of the female reproductive tract whereupon they undergo a myriad of biochemical and biophysical changes collectively referred to as 'capacitation'. We have previously shown that this functional transformation is, in part, engineered by the modification of the sperm surface architecture leading to the assembly and/or presentation of multimeric sperm-oocyte receptor complexes. In this study, we have extended our findings through the characterization of one such complex containing arylsulfatase A (ARSA), sperm adhesion molecule 1 (SPAM1) and the molecular chaperone, heat shock 70kDa protein 2 (HSPA2). Through the application of flow cytometry we revealed that this complex undergoes a capacitation-associated translocation to facilitate the repositioning of ARSA to the apical region of the human sperm head, a location compatible with a role in the mediation of sperm-zona pellucida (ZP) interactions. Conversely, SPAM1 appears to reorient away from the sperm surface, possibly reflecting its primary role in cumulus matrix dispersal preceding sperm-ZP recognition. The dramatic relocation of the complex was completely abolished by incubation of capacitating spermatozoa in exogenous cholesterol or broad spectrum protein kinase A (PKA) and tyrosine kinase inhibitors suggesting that it may be driven by alterations in membrane fluidity characteristics and concurrently by the activation of a capacitation-associated signal transduction pathway. Collectively these data afford novel insights into the sub-cellular localization and potential functions of multimeric protein complexes in human spermatozoa.

The Molecular Chaperone HSPA2 Plays a Key Role in Regulating the Expression of Sperm Surface Receptors That Mediate Sperm-Egg Recognition

A common defect encountered in the spermatozoa of male infertility patients is an idiopathic failure of sperm–egg recognition. In order to resolve the molecular basis of this condition we have compared the proteomic profiles of spermatozoa exhibiting an impaired capacity for sperm-egg recognition with normal cells using label free mass spectrometry (MS)-based quantification. This analysis indicated that impaired sperm–zona binding was associated with reduced expression of the molecular chaperone, heat shock 70 kDa protein 2 (HSPA2), from the sperm proteome. Western blot analysis confirmed this observation in independent patients and demonstrated that the defect did not extend to other members of the HSP70 family. HSPA2 was present in the acrosomal domain of human spermatozoa as a major component of 5 large molecular mass complexes, the most dominant of which was found to contain HSPA2 in close association with just two other proteins, sperm adhesion molecule 1 (SPAM1) and arylsulfatase A (ARSA), both of which that have previously been implicated in sperm-egg interaction. The interaction between SPAM1, ARSA and HSPA2 in a multimeric complex mediating sperm-egg interaction, coupled with the complete failure of this process when HSPA2 is depleted in infertile patients, provides new insights into the mechanisms by which sperm function is impaired in cases of male infertility.

Sexual size dimorphism predicts rates of sequence evolution of SPerm Adhesion Molecule 1 (SPAM1, also PH-20) in monkeys, but not in hominoids (apes including humans)

Based on a dataset comprising coding DNA sequences of 23 anthropoid primates, we herein investigate if rates of sequence evolution of SPerm Adhesion Molecule1 (SPAM1, also PH-20), which participates in sperm–egg interaction, is lower in more sexually dimorphic species. For comparison, we analyze sequence evolution of apolipoproteinA-IV (APOA4) and apolipoprotein A-V (APOA5), which should evolve under less or even no sexual selection given their expression in blood, digestive tract, liver, and lungs. Regression analyses provides significant support for a negative dependence of SPAM1 derived branch-specific ratios of non-synonymous to synonymous substitution rates (dN/dS) on sexual size dimorphism (SSD) in a subsample comprising New World and Old World monkeys. We moreover observed a tendency for a positive correlation of substitution rates of SPAM1 with relative testes weight (RTW) and significantly lowered dN/dS estimates in uni-male and uni-male/multi-male breeding monkeys. Importantly, the pattern was not reproduced when analyzing partial APOA4 and APOA5 sequences. These findings illustrate that different levels of sperm competition, probably fueled by female cryptic choice, account for species-specific sequence evolution of SPAM1 in monkeys. Remarkably, present data do not support a correlation of species-specific sequence evolution of SPAM1 with sexual selection levels in hominoids (apes including humans). This can partly be ascribed to a relaxation of functional constraint of SPAM1 in some hominoid species. Additional factors confounding regression analyses specifically in hominoids might be higher levels of sperm competition than reflected by SSD and RTW in some species, a rather strong effect of female mate choice on paternity rates in others, and – in particular in humans – socio-cultural factors not measurable by SSD and RTW.

SPAM1 (sperm adhesion molecule 1 (PH-20 hyaluronidase, zona pellucida binding))

Review on SPAM1 (sperm adhesion molecule 1 (PH-20 hyaluronidase, zona pellucida binding)), with data on DNA, on the protein encoded, and where the gene is implicated.

Germ-cell hyaluronidases: their roles in sperm function

Hyaluronidases (hyases) are a family of enzymes that catalyse the breakdown of hyaluronic acid (HA), which is abundant in the extracellular matrix. Two unlinked gene clusters encode these six proteins: three each in the somatic (or ubiquitous) acid-active subgroup and the neutral-active germ-cell subgroup. This review analyses the data on the expression and role of hyases in gamete biology and fertilization, using electronic databases until October 2010. Evidence indicates that hyases are membrane proteins with multifunctional essential, enzymatic and non-enzymatic, roles (cumulus penetration, zona binding and HA receptor) in fertilization. While sperm adhesion molecule-1 (SPAM1), which has neutral and acidic (bimodal) activity, is the widely conserved mammalian sperm hyase, it co-exists with an acidic hyase in murine and human spermatozoa. Thus, sperm function depends on the concerted activity of both germ cell and 'somatic' hyases. Some hyases are in low abundance in the ovary, somatic testicular cells, the male accessory organs and the male and female genital tracts where they are secreted and acquired by spermatozoa. The latter opens up the possibility of treating hyase-deficient spermatozoa via assisted reproductive technology. The findings challenge the existing classification of hyases, and support the notion that hyase activities are polygenic traits controlled by as many as five hyase genes in mice. Multiple sperm hyases may function cooperatively in a quantitative system and/or serve redundant roles. Unsolved problems include functional redundancy, which can be addressed by double gene-knockouts, and identifying the murine hyase(s) involved in zona binding or whether this role shows species specificity.

Acidic hyaluronidase activity is present in mouse sperm and is reduced in the absence of SPAM1: Evidence for a role for hyaluronidase 3 in mouse and human sperm

The molecular mechanisms underlying sperm penetration of the physical barriers surrounding the oocyte have not been completely delineated. Although neutral-active or "reproductive" hyaluronidases (hyases), exemplified by Sperm Adhesion Molecule 1 (SPAM1), are thought to be responsible for hyaluronan digestion in the egg vestments and for sperm-zona binding, their roles in mouse sperm have been recently questioned. Here we report that acidic "somatic" Hyaluronidase 3 (HYAL3), a homolog of SPAM1 with 74.6% structural similarity, exists in two isoforms in human ( approximately 47 and approximately 55 kDa) and mouse ( approximately 44 and approximately 47 kDa) sperm, where it resides on the plasma membrane over the head and midpiece. Mouse isoforms are differentially distributed in the soluble (SAP), membrane (MBP), and acrosome-reacted (AR) fraction where they are most abundant. Comparisons of zymography of Hyal3 null and wild-type (WT) AR and MBP fractions show significant HYAL3 activity at pH 3 and 4, and less at pH 7. At pH 4, a second acid-active hyase band at approximately 57 kDa is present in the AR fraction. HYAL3 activity was confirmed using immunoprecipitated HYAL3 and spectrophotometry. In total proteins, hyase activity was higher at pH 6 than at 4, where Spam1 nulls had significantly (P < 0.01) diminished activity implicating an acidic optima for murine SPAM1. Although fully fertile, Hyal3 null sperm showed delayed cumulus penetration and reduced acrosomal exocytosis. HYAL3 is expressed in epididymal tissue/fluid, from where it is acquired by caudal mouse sperm in vitro. Our results reveal concerted activity of both neutral- and acid-active hyaluronidases in sperm.

Evidence for the Participation of Sperm Proteasomes in Zona Pellucida Degradation and Zona-Induced Acrosomal Exocytosis During Fertilization in the Domestic Pig.

Realization of importance of the ubiquitin-proteasome pathway (UPP) in mammalian fertilization is continuing to grow with every new publication. The UPP is known to participate in multiple steps of mammalian fertilization, including sperm capacitation, acrosomal exocytosis (AE), and sperm-zona pellucida (ZP) penetration. In the UPP, a chaperone protein ubiquitin binds in tandem fashion to proteins that need to be recycled, and targets them for proteolysis by a multi-subunit protease, the 26S proteasome. We have been studying the participation of the 26S proteasome in the ZP-induced AE and sperm-ZP penetration during porcine fertilization. The 26S proteasome is a ~2 MDa, complex consisting of a 19S regulatory complex (role: ubiquitinated substrate recognition, deubiquitination and unfolding) and the 20S core (role: substrate degradation). Three distinct proteolytic activities reside in the 20S core: trypsin-like, chymotrypsin-like and caspase-like. In the presence of proteasomal inhibitors, porcine sperm are unable to fertilize oocytes, which may be due either to inhibition of acrosomal exocytosis and/or degradation of the ZP matrix. To understand this, fresh, capacitated boar spermatozoa were incubated with solubilized porcine ZP proteins with or without proteasomal inhibitors MG 132 (inhibits chymotrypsin-like core activity), clasto-lactacystin β-lactone (targets trypsin-like activity) and epoxomicin (targets chymotrypsin-like activity). After two-hour incubation, soluble ZP proteins and exocytosed acrosomal ghosts were separated from sperm pellets by centrifugation. They were analyzed for evidence of ZP protein degradation by the sperm proteasomes and for the accumulation of undegraded sperm acrosomal proteins. Results show that solubilized ZP proteins readily bind to acrosomal caps of the capacitated boar spermatozoa and induce AE. Presence of proteasomal inhibitors during sperm-ZP coincubation protected the zona glycoprotein ZPB from being degraded, without affecting its deglycosylation. Additionally, release of the 26S proteasomes with the acrosomal ghosts was reduced in the presence of proteasomal inhibitors. Acrosomal ghost-associated proteins such as Sperm Adhesion Molecule 1 (SPAM1), Lactadherin/SP47, Beta-Acrosin, Zona Pellucida-Binding Protein 2 (ZPBP2/IAM38) and Proacrosin-binding Protein (SP32), were protected from proteasomal degradation and accumulated in the ghost fraction. Thus, it appears that proteasomes are released from the sperm acrosome during ZP-induced acrosomal exocytosis. However, since acrosomal ghost remains zona bound and surrounds the sperm head during early stages of sperm-ZP penetration, the resident acrosomal proteasomes could assist the spermatozoon during the formation of the fertilization slit. A second pool of proteasomes remains associated with the inner acrosomal membrane (IAM) of the exocytosed spermatozoa; it may provide the fertilizing spermatozoon with a mean to digest the zona, as it advances through the fertilization slit. Collectively these results explain why the inhibition of sperm proteasomal activity prevents porcine fertilization. This model provides tools to modulate fertilization in vitro and points to a new direction in which to contemplate non-hormonal contraceptive design. Supported by USDA-NRI grant #2007-01319 and by seed funding from the F21C Program of UM (PS). (platform)