Mature Human Sperm Research Articles

Hv1 Structure and Function: Insights from Evolutionary Information

The voltage gated proton channel, HV1, is the most selective ion channel known, and plays an important role in innate immunity, human sperm maturation, tumorigenesis, and dinoflagellate bioluminescence, to name a few examples. When the gene was identified in 2006, HV1 was found to be homologous to the voltage sensor domains of classic voltage gated cation channels; these homologous domains do not conduct ions. Phylogenetic analysis of VSDs showed that HV1, voltage sensing phosphatases, and c15orf27 are three twigs that occupy a branch distinct from other voltage gated ion channels, and that this branch is part of the larger group comprising voltage gated sodium and calcium channels, which split from potassium channels at least a billion years ago. We used a combination of sequence and phylogenetic analysis, structural modeling, and electrophysiological experimentation to identify the proton selectivity filter of HV1, an important milestone in elucidating the functional components of the protein. We proposed a “signature sequence” that defines all presently known HV1, and used it to identify the first dinoflagellate HV1, despite only 15% identity with hHV1. We combined molecular dynamics and sampling methods with other approaches, to help us choose and experimentally validate a refined model that we are using to help us discover the determinants of proton specific conduction.

Expression pattern of PRM2, HSP90 and WNT5A in male partners of couples experiencing idiopathic recurrent miscarriages

Recurrent pregnancy loss affects approximately 3–5% of couples attempting to have a child. It is defined as the occurrence of two or more consecutive pregnancy failures before 20 weeks of gestation. In about 40% of cases, the cause remains unknown and are defined as idiopathic recurrent miscarriages (iRM) (Stephenson and Kutteh 2007). However, most of these diagnostics tests are performed in the female partner, with the male’s contribution remaining relatively underexplored (only paternal karyotyping is done). Paternal factor may be the underlying aetiology in RM (Lalancette et al. 2008) and molecular abnormalities in sperm may have an adverse effect on embryonic development. Certain portion of sperm genome remains transcriptionally active and codes for transcripts which are of paramount developmental importance (Krawetz 2005; Gil-Villa et al. 2010). Among these, the role of long-lived sperm RNA which are not translated is controversial. Sperm possess a remarkable set of long-lived messenger RNAs (mRNAs) that have been hypothesized to be required for embryogenesis. Some of these spermatozoal mRNAs are also found in zygote, indicating that these transcripts may be functionally critical during embryonic development (Ostermeier et al. 2004). The delivery of certain sperm transcripts to the oocyte, which could translate to proteins, may be critical for the early stages of embryogenesis and/or implantation. In the vast population of mRNA, transcripts of winglesstype MMTV integration site family, member 5A (WNT5A), heat shock protein 90 (HSP90) and protamine 2 (PRM2) are present in human mature spermatozoa as examined by microarray and serial analysis gene expression (SAGE) technology (Wykes et al. 2000; Zhao et al. 2006). These genes

Comparative analysis of human reproductive proteomes identifies candidate proteins of sperm maturation

Male reproductive proteomes provide basis for studying gene products and its involvement or regulation in sperm physiology. Here, a comparative study between these proteomes was performed to find potential proteins and functions associated with human sperm maturation. Seven reproductive proteomes associated with human sperm physiology were integrated. Gene ontology analysis were performed using DAVID and Panther tools to determine enriched functions. Total of 270 proteins overlapped between epididymal, prostatic milieu and sperm proteome were thought to be candidate proteins involved in sperm maturation, and they showed enriched functions of proteasomal protein catabolic process and protein folding. 34 epididymal milieu proteins and 274 prostatic milieu proteins were contributed to the composition of seminal fluids proteome. Literatures have confirmed the involvements in sperm maturation of many of these proteins The spatial expressions of 24 epididymal milieu proteins involved in chaperone and antioxidant activity were authenticated by real-time RT-PCR. These proteins may serve as candidate molecules for future studies of sperm maturation and male infertility.

Potassium channel in human sperm identified by whole cell patch clamping plays a role in normal sperm physiology

To characterise and identify the functional role of potassium channels in motile human sperm by whole cell patch clamping. Whole cell patch clamping of motile human sperm was used to identify a native K+ conductance. Pharmacology and ionic substitution characterised the biophysical properties of the channel. Function assays in the presence and absence of identified channel blockers were used to identify of the role of the channel in normal sperm function. Spermatozoa from healthy donors with normal semen parameters (WHO 2010) were isolated by swim up and incubated under capacitating conditions for 4 hours (25mM NaHCO3, 6% CO2 at 37°C). Currents were recorded under voltage clamp using ramp and leak subtracted step protocols (-80 to 80mV, holding potential -80mV). Standard pipette and bath solutions were designed to mimic quasi-physiological ionic conditions. Motility was assessed using computer aided sperm analysis and Kremer tests were used to assess the cells ability to penetrate viscous media in the presence of the putative potassium channel blocker Quinidine. Chi2, ANOVA and students t-test were used to identify statistical significance where appropriate. Statistical significance P<0.05. Data showed an outwardly rectifying current only active when depolarised past -40mV. Replacing pipette potassium with cesium (n=8) abolished the depolarization-induced current. Putative K+ blocker quinidine [0.3mM] completely inhibited the outward current, depolarised the resting membrane potential, reduced progressive motility (n=9) and penetration into viscous media (n=6) by ∼50% and 70% respectively (P<0.001). Qunidine also reduced straight line velocity and average path velocity by 22 and 31%, respectively (P<0.005). Human sperm possess K+ channels involved in normal sperm function that may play a role in male fertility. This is the first time this has been documented in mature human sperm.

LDH-C4: a target with therapeutic potential for cancer and contraception

The lactate dehydrogenase (LDH) has been studied widely because it exists in various isozymic forms. The association of A and B subunits of LDH can generate five tetrameric isozymes, but the finding of the sixth isozyme in mature human testis and sperm indicated the presence of an additional subunit of LDH, designated as LDH-X (also termed LDH-C4 due to tetrameric nature of C-subunit). LDH-C4 isozyme is an iso-, allo-, and auto-antigen present in mammalian sperm cells. The synthesis of LDH-C4 in the testis takes place during sexual maturation, and it is the predominant fraction in mature spermatozoa. Though, originally considered to be testis specific, LDH-C or Ldh3 in mice was later detected in the murine oocyte and early embryo. Ldh3 in mouse supports its role in energy production in spermatids that favor lactate as substrate and in spermatozoa with a characteristic aerobic glycolytic path to yield ATP. During last two decades, cancer/testis-associated genes (CTAs) which are expressed only in the germinal epithelium of the testis are also expressed in some cancer cells, but not in non-cancerous somatic tissues. The CTAs are considered promising candidates for diagnosis and immunotherapy of cancer. The sperm-specific Ldh-c gene has been shown to express in a broad spectrum of human tumors, with high frequency in lung cancer, melanoma, and breast cancer; the protein being expressed virtually in all tumor types tested. Accordingly, LDH-C4 is the unique target for contraception in both males and females and offers potential future for immunotherapy of different types of cancers. As LDH-C has a preference for lactate as a substrate, LDH-C activation in cancer may depend on lactate for ATP production. The major aim of this article is to review the salient features of LDH-C subunit and the immune responses of LDH-C4 in homologous and heterologous species in relation to its role in acceptance or rejection of the allograft and its application in contraception and immunotherapy of cancer, directly or indirectly through the regulation of its substrate, the lactate.

Cloning and expression of Tssk1 &amp; Tssk2 in mice and the presence &amp; localization of them in mature sperm

We sought to experimentally verify if testis specific serine/threonine kinases (Tssks) play a role in spermatogenesis and/or the regulation of sperm function. Purified Tssk proteins were obtained based on cloning and expression of mouse Tssk1 and Tssk2. Tssk1 and Tssk2 were detected in mature mouse and human sperm by western blotting. Immunofluorescence indicated that Tssk1 is distributed in the acrosome and the entire flagellum of mouse sperm while Tssk2 was mainly distributed in post-acrosomal region. There was no alteration in the distribution pattern of Tssk1 and Tssk2 in non-capacitated and capacitated sperm. Tssk2 distribution remained unchanged after induced acrosome reaction but no signals were detected in the acrosome for Tssk1, which was present before the acrosome reaction, though signals in flagellum were undisturbed. In human sperm, Tssk1 was found in neck and flagellum while Tssk2 was found in the equatorial region. Our results suggest Tssk1 and/or Tssk2 do play an important role(s) in the regulation of sperm function.

Different Isoforms of Hsp90 Present in Human Mature Spermatozoa and Hsp90 Is Associated with Capacitation and Motility Activated by Progesterone.

Heat shock protein 90 (Hsp90) plays an important role in a variety of cells. Since only a few studies on Hsp90 in human mature sperm have been reported, more information of Hsp90 is required. The aim of this study was to provide evidence on location, distribution and expression characteristics of Hsp90 of human mature sperm, and its involvement in sperm capacitation and motility. The location of Hsp90 and its phosphorylation expression was examined by indirect immunofluorescence staining. We found that Hsp90 was mainly located in the neck and midpiece of the tail in sperm. Co-immunolocation of Hsp90 protein and tyrosine phosphorylation was not entirely overlaid during capacitation. Hsp90 protein expressions of different individuals were analyzed by Western blotting and quantified by Image J software. Interestingly, different isoforms of Hsp90 were present in human mature sperm: only single form about 83 kDa or 98 kDa was detected in some individuals; both isoforms of 83 kDa and 98 kDa were detected in other individuals. Moreover, the expression of the 83 kDa isoform could be inhibited by geldanamycin and the other tyrosine phosphorylated proteins except Hsp90 was changed by geldanamycin during capacitation. The acrosome reaction and motility parameters of spermatozoa were evaluated by chlorotetracycline staining and measured by computer assist sperm analyzer, respectively. The results showed that capacitation and some motility parameters activated by progesterone were affected by variant concentrations of geldanamycin. In summary, it is demonstrated that Hsp90 mainly locates in the neck and middle piece of the tail in human sperm, various isoforms of Hsp90 are present in mature sperm of different individuals for the first time, and Hsp90 is involved in the capacitation and motility via tyrosine phosphorylation of other proteins in this study. This research was supported by the Natural Science Foundation of China (Nos. 81000244 and 81170554), the Nature Science Foundation of Zhejiang Province (No. Y2100058).

Calcium Channel CatSper is a Non-Genomic Progesterone Receptor of Human Sperm

Elevation of intracellular Ca2+ regulate sperm motility, chemotaxis, capacitation and the acrosome reaction, and play a vital role in the ability of the sperm cell to reach and fertilize the egg. In mammalian spermatozoa, the flagellar pH-dependent Ca2+ channel CatSper is the main pathway for calcium entry as measured by the whole-cell patch clamp technique. Mouse CatSper channel is activated by alkaline intracellular pH, but human CatSper requires also a ligand. By applying the patch-clamp technique to mature human spermatozoa, we found that nanomolar concentrations of female hormone progesterone activate CatSper and this action explains the mechanism of non-genomic action of progesterone on human sperm cells. Progesterone is released by cumulus cells surrounding the oocyte and serves as a chemoattractant for human spermatozoa.Interestingly, human CatSper can be further potentiated by prostaglandins, but apparently through a binding site other than that of progesterone. Behavior of CatSper channel gradually changes during spermiogenesis with CatSper channel being fully operational only in mature spermatozoa. Physiological regulation of CatSper channel by different components of seminal plasma as well as hormones of female reproductive tract will be discussed.

Rediscovering sperm ion channels with the patch-clamp technique

Upon ejaculation, mammalian spermatozoa have to undergo a sequence of physiological transformations within the female reproductive tract that will allow them to reach and fertilize the egg. These include initiation of motility, hyperactivation of motility and perhaps chemotaxis toward the egg, and culminate in the acrosome reaction that permits sperm to penetrate the protective vestments of the egg. These physiological responses are triggered through the activation of sperm ion channels that cause elevations of sperm intracellular pH and Ca(2+) in response to certain cues within the female reproductive tract. Despite their key role in sperm physiology and their absolute requirement for the process of fertilization, sperm ion channels remain poorly understood due to the extreme difficulty in application of the patch-clamp technique to spermatozoa. This review covers the topic of sperm ion channels in the following order: first, we discuss how the intracellular Ca(2+) and pH signaling mediated by sperm ion channels controls sperm behavior during the process of fertilization. Then, we briefly cover the history of the methodology to study sperm ion channels, which culminated in the recent development of a reproducible whole-cell patch-clamp technique for mouse and human cells. We further discuss the main approaches used to patch-clamp mature mouse and human spermatozoa. Finally, we focus on the newly discovered sperm ion channels CatSper, KSper (Slo3) and HSper (H(v)1), identified by the sperm patch-clamp technique. We conclude that the patch-clamp technique has markedly improved and shifted our understanding of the sperm ion channels, in addition to revealing significant species-specific differences in these channels. This method is critical for identification of the molecular mechanisms that control sperm behavior within the female reproductive tract and make fertilization possible.

Proteomic characterization of the human sperm nucleus

Generating a catalogue of sperm nuclear proteins is an important first step towards the clarification of the function of the paternal chromatin transmitted to the oocyte upon fertilization. With this goal, sperm nuclei were obtained through CTAB treatment and isolated to over 99.9% purity without any tail fragments, acrosome or mitochondria as assessed by optical microscopy and transmission electron microscopy. The nuclear proteins were extracted and separated in 2-D and 1-D gels and the 2-D spots and 1-D bands were excised and analysed to identify the proteins through LC-MS/MS. With this approach, 403 different proteins have been identified from the isolated sperm nuclei. The most abundant family of proteins identified are the histones, for which several novel members had not been reported previously as present in the spermatogenic cell line or in the human mature spermatozoa. More than half (52.6%) of the proteins had not been detected in the previous human whole sperm cell proteome reports. Of relevance, several chromatin-related proteins, such as zinc fingers and transcription factors, so far not known to be associated with the sperm chromatin, have also been detected. This provides additional information about the nuclear proteins that are potentially relevant for epigenetic marking, proper fertilization and embryo development.

Gene Expression in the Epididymis of Normal and Vasectomized Men: What Can We Learn About Human Sperm Maturation?

Anatomically, the human epididymis is unusual when compared with the excurrent duct of other eutherian mammals. Furthermore, clinical observations suggest that it may not be as important for sperm maturation as is the case for laboratory animals. In contrast, hierarchical clustering of microarray data of epididymides from normal men revealed 2274 modulated qualifiers between the epididymal segments, 1184, 713, and 269 of them being highly expressed in the caput, corpus, and cauda, respectively. The organization of qualifiers according to their similarities by gene ontology indicated that caput transcripts are dedicated to cell-cell adhesion, whereas the corpus is characterized by genes involved in response to other organisms (ie, defense mechanisms) and the cauda transcriptome is specialized in muscle contraction and establishment of localization. A region-specific gene expression pattern thus characterizes the human epididymis as in animal models. In humans, vasectomies have consequences on the epididymal transcriptome. Cluster analysis revealed that 1363 genes are expressed in both normal and vasectomized epididymides, whereas 911 and 660 of them are specifically expressed in normal and vasectomized epididymides, respectively. Three of the affected genes are particularly interesting because of their involvement in sperm biochemical remodeling during epididymal transit: dicarbonyl/l-xylulose reductase, Niemann-Pick disease, type C2, and cysteine-rich secretory protein 1. In some vasovasostomized men, these modifications in gene expression induced by vasectomy are irreversible, thus affecting the biochemical parameters, and potentially, the function of their ejaculated sperm. This may explain the discrepancies between a surgically successful vasovasostomy and fertility recovery.

Genes for embryo development are packaged in blocks of multivalent chromatin in zebrafish sperm

In mature human sperm, genes of importance for embryo development (i.e., transcription factors) lack DNA methylation and bear nucleosomes with distinctive histone modifications, suggesting the specialized packaging of these developmental genes in the germline. Here, we explored the tractable zebrafish model and found conceptual conservation as well as several new features. Biochemical and mass spectrometric approaches reveal the zebrafish sperm genome packaged in nucleosomes and histone variants (and not protamine), and we find linker histones high and H4K16ac absent, key factors that may contribute to genome condensation. We examined several activating (H3K4me2/3, H3K14ac, H2AFV) and repressing (H3K27me3, H3K36me3, H3K9me3, hypoacetylation) modifications/compositions genome-wide and find developmental genes packaged in large blocks of chromatin with coincident activating and repressing marks and DNA hypomethylation, revealing complex "multivalent" chromatin. Notably, genes that acquire DNA methylation in the soma (muscle) are enriched in transcription factors for alternative cell fates. Remarkably, whereas H3K36me3 is located in the 3' coding region of heavily transcribed genes in somatic cells, H3K36me3 is present in the promoters of "silent" developmental regulators in sperm, suggesting different rules for H3K36me3 in the germline and soma. We also reveal the chromatin patterns of transposons, rDNA, and tDNAs. Finally, high levels of H3K4me3 and H3K14ac in sperm are correlated with genes activated in embryos prior to the mid-blastula transition (MBT), whereas multivalent genes are correlated with activation at or after MBT. Taken together, gene sets with particular functions in the embryo are packaged by distinctive types of complex and often atypical chromatin in sperm.

Progesterone activates the principal Ca2+ channel of human sperm

Steroid hormone progesterone released by cumulus cells surrounding the egg is a potent stimulator of human spermatozoa. It attracts spermatozoa towards the egg and helps them penetrate the egg's protective vestments. Progesterone induces Ca(2+) influx into spermatozoa and triggers multiple Ca(2+)-dependent physiological responses essential for successful fertilization, such as sperm hyperactivation, acrosome reaction and chemotaxis towards the egg. As an ovarian hormone, progesterone acts by regulating gene expression through a well-characterized progesterone nuclear receptor. However, the effect of progesterone upon transcriptionally silent spermatozoa remains unexplained and is believed to be mediated by a specialized, non-genomic membrane progesterone receptor. The identity of this non-genomic progesterone receptor and the mechanism by which it causes Ca(2+) entry remain fundamental unresolved questions in human reproduction. Here we elucidate the mechanism of the non-genomic action of progesterone on human spermatozoa by identifying the Ca(2+) channel activated by progesterone. By applying the patch-clamp technique to mature human spermatozoa, we found that nanomolar concentrations of progesterone dramatically potentiate CatSper, a pH-dependent Ca(2+) channel of the sperm flagellum. We demonstrate that human CatSper is synergistically activated by elevation of intracellular pH and extracellular progesterone. Interestingly, human CatSper can be further potentiated by prostaglandins, but apparently through a binding site other than that of progesterone. Because our experimental conditions did not support second messenger signalling, CatSper or a directly associated protein serves as the elusive non-genomic progesterone receptor of sperm. Given that the CatSper-associated progesterone receptor is sperm specific and structurally different from the genomic progesterone receptor, it represents a promising target for the development of a new class of non-hormonal contraceptives.

Activity of nitric oxide synthase in mature and immature human spermatozoa

Nitric oxide (NO) is known to be involved in multiple signal transduction pathways of male germ cells, including sperm capacitation. In somatic cells, NO production was found to be part of apoptosis signalling. The aim of our study was to further clarify the role of NO in spermatozoa by investigation of NO synthase activity with regard to sperm maturity and sperm apoptosis signalling. Semen specimens from 19 healthy donors were subjected to density gradient centrifugation to separate the predominantly mature and immature sperm fraction. NO synthase activity was evaluated using diaminofluoresceine-2-diacetate by FACS. Apoptosis signalling was monitored by flowcytometric analyses of caspase-3 (CP3) and integrity of the transmembrane mitochondrial potential (TMP). TUNEL assay was used to detect DNA fragmentations. Maturity of human spermatozoa was associated with increased NO synthase activity and inactivated apoptosis signalling (lower levels of disrupted TMP, active CP3 and DNA fragmentations, P < 0.05). Activation of apoptosis signalling was significantly negatively correlated to NO production, indicating a rather anti-apoptotic effect of NO. This might underline the recently proposed role of NO in physiological sperm signal transduction, e.g. during capacitation.

Kinetics of human male pronuclear development in a heterologous ICSI model

To evaluate human sperm nuclear chromatin decondensation in a heterologous ICSI system using hamster ova injected with human sperm. Frozen hamster oocytes were injected with Triton X-100 treated sperm and fixed at different time points post ICSI. Oocytes injected with non-treated sperm served as controls. Male pronuclear decondensation was evaluated after staining with DAPI. Sperm cells with partially destroyed membranes and depletion of the acrosome decondense more rapidly and to a greater extent than membrane/acrosome intact cells. Marked variability in pronuclear size was observed for any time point post ICSI, which most probably reflects the heterogeneity in the mature human sperm population. Remodeling of male gamete nuclei in this heterologous ICSI mimics events that occur during natural fertilization in humans and therefore this approach may be used for studies of human sperm chromosomes transformations.

H2A.Bbd: an X-chromosome-encoded histone involved in mammalian spermiogenesis

Despite the identification of H2A.Bbd as a new vertebrate-specific replacement histone variant several years ago, and despite the many in vitro structural characterizations using reconstituted chromatin complexes consisting of this variant, the existence of H2A.Bbd in the cell and its location has remained elusive. Here, we report that the native form of this variant is present in highly advanced spermiogenic fractions of mammalian testis at the time when histones are highly acetylated and being replaced by protamines. It is also present in the nucleosomal chromatin fraction of mature human sperm. The ectopically expressed non-tagged version of the protein is associated with micrococcal nuclease-refractory insoluble fractions of chromatin and in mouse (20T1/2) cell line, H2A.Bbd is enriched at the periphery of chromocenters. The exceedingly rapid evolution of this unique X-chromosome-linked histone variant is shared with other reproductive proteins including those associated with chromatin in the mature sperm (protamines) of many vertebrates. This common rate of evolution provides further support for the functional and structural involvement of this protein in male gametogenesis in mammals.

Characterization of a novel cell-surface protein expressed on human sperm

Precise sperm-oocyte interaction is a critical event for successful fertilization. However, the identity of molecules involved in this process in humans remains largely unknown. This report describes the identification and characterization of a novel cell-surface protein and its potential role in human sperm-oocyte interaction. We previously identified an orphan guanylyl cyclase receptor (mouse GC-G) highly enriched in mouse testis and involved in sperm activation. By using a comparative genomic approach, we found the homologue gene in human (hGC-G) composed of 21 exons, spanning a minimum of 48 kb on chromosome 10q25. Real-time RT-PCR analysis revealed hGC-G mRNA selectively expressed in testis but with low or no expression in all other tissues examined. Compared with mGC-G, the hGC-G transcript contains three 1-bp deletions and two in-frame termination codons, which results in a short putative receptor-like polypeptide. Western blot analysis with an anti-hGC-G-specific antibody confirmed the protein expression of hGC-G in human sperm lysate. Flow cytometry and confocal immunofluorescence analysis demonstrated the localization of hGC-G protein on the acrosome cap and equatorial segment of mature human sperm. In addition, an integrin-binding Arg-Gly-Asp (RGD) motif was found in the extracellular domain of hGC-G. Pre-incubation of the hGC-G RGD peptide with zona pellucida-free oocytes greatly decreased the binding of human sperm to hamster oocytes, which suggests a role for hGC-G role in sperm-oocyte interaction. hGC-G is a novel surface protein on human sperm and potentially mediates sperm-oocyte interaction through its RGD-containing motif.

Distinctive chromatin in human sperm packages genes for embryo development.

SummaryAs nucleosomes are widely replaced by protamine in mature human sperm, epigenetic contributions of sperm chromatin to embryo development have been considered highly limited. However, we find the retained nucleosomes significantly enriched at loci of developmental importance including imprinted gene clusters, miRNA clusters, HOX gene clusters, and the promoters of stand-alone developmental transcription and signaling factors. Importantly, histone modifications localize to particular developmental loci. H3K4me2 is enriched at certain developmental promoters, whereas large blocks of H3K4me3 localize to a subset of developmental promoters, regions in HOX clusters, certain non-coding RNAs, and generally to paternally-expressed imprinted loci, but not paternally-repressed loci. Notably, H3K27me3 is significantly enriched at developmental promoters that are repressed in early embryos, including many bivalent (H3K4me3/H3K27me3) promoters in embryonic stem cells. Finally, developmental promoters are generally DNA hypomethylated in sperm, but acquire methylation during differentiation. Taken together, epigenetic marking in sperm is extensive, and correlated with developmental regulators.

Influence of freeze-thawing on hyaluronic acid binding of human spermatozoa

Mature human spermatozoa have at least three specific hyaluronic acid (HA) binding proteins present on their sperm membrane. These receptors play a role in the acrosome reaction, hyaluronidase activity, hyaluronan-mediated motility and sperm-zona and sperm-oolemmal binding. Cryopreservation of spermatozoa can cause ultrastructural and even molecular damage. The aim of this study was to investigate if HA binding receptors of human spermatozoa remain functional after freeze-thawing. Forty patients were enrolled in the study. Semen samples were analysed before and after cryopreservation. Parameters analysed included concentration, motility, morphology and hyaluronan binding. Samples were frozen in CBS straws using a glycerol-glucose-based cryoprotectant. HA binding was studied using the sperm-hyaluronan binding assay. Freeze-thawing resulted in a significant decline in motility: the percentage of motile spermatozoa reduced from 50.6 to 30.3% (P < 0.001). HA binding properties of frozen-thawed spermatozoa remained unchanged after the freeze-thawing process: 68.5 +/- 17.1% spermatozoa of the neat sample were bound to HA, as were 71.3 +/- 20.4 of the frozen-thawed sample. This study indicates that freeze-thawing did not alter the functional hyaluronan binding sites of mature motile spermatozoa, and therefore will not alter their fertilizing potential.

A Bayesian deconvolution strategy for immunoprecipitation-based DNA methylome analysis

DNA methylation is an indispensible epigenetic modification required for regulating the expression of mammalian genomes. Immunoprecipitation-based methods for DNA methylome analysis are rapidly shifting the bottleneck in this field from data generation to data analysis, necessitating the development of better analytical tools. In particular, an inability to estimate absolute methylation levels remains a major analytical difficulty associated with immunoprecipitation-based DNA methylation profiling. To address this issue, we developed a cross-platform algorithm-Bayesian tool for methylation analysis (Batman)-for analyzing methylated DNA immunoprecipitation (MeDIP) profiles generated using oligonucleotide arrays (MeDIP-chip) or next-generation sequencing (MeDIP-seq). We developed the latter approach to provide a high-resolution whole-genome DNA methylation profile (DNA methylome) of a mammalian genome. Strong correlation of our data, obtained using mature human spermatozoa, with those obtained using bisulfite sequencing suggest that combining MeDIP-seq or MeDIP-chip with Batman provides a robust, quantitative and cost-effective functional genomic strategy for elucidating the function of DNA methylation.