Intragenic sequences in the trophectoderm harbour the greatest proportion of methylation errors in day 17 bovine conceptuses generated using assisted reproductive technologies.

BACKGROUND: Assisted reproductive technologies (ART) are widely used to treat fertility issues in humans and for the production of embryos in mammalian livestock. The use of these techniques, however, is not without consequence as they are often associated with inauspicious pre- and postnatal outcomes including premature birth, intrauterine growth restriction and increased incidence of epigenetic disorders in human and large offspring syndrome in cattle. Here, global DNA methylation profiles in the trophectoderm and embryonic discs of in vitro produced (IVP), superovulation-derived (SOV) and unstimulated, synchronised control day 17 bovine conceptuses (herein referred to as AI) were interrogated using the EmbryoGENE DNA Methylation Array (EDMA). Pyrosequencing was used to validate four loci identified as differentially methylated on the array and to assess the differentially methylated regions (DMRs) of six imprinted genes in these conceptuses. The impact of embryo-production induced DNA methylation aberrations was determined using Ingenuity Pathway Analysis, shedding light on the potential functional consequences of these differences. RESULTS: Of the total number of differentially methylated loci identified (3140) 77.3 and 22.7% were attributable to SOV and IVP, respectively. Differential methylation was most prominent at intragenic sequences within the trophectoderm of IVP and SOV-derived conceptuses, almost a third (30.8%) of the differentially methylated loci mapped to intragenic regions. Very few differentially methylated loci were detected in embryonic discs (ED); 0.16 and 4.9% of the differentially methylated loci were located in the ED of SOV-derived and IVP conceptuses, respectively. The overall effects of SOV and IVP on the direction of methylation changes were associated with increased methylation; 70.6% of the differentially methylated loci in SOV-derived conceptuses and 57.9% of the loci in IVP-derived conceptuses were more methylated compared to AI-conceptuses. Ontology analysis of probes associated with intragenic sequences suggests enrichment for terms associated with cancer, cell morphology and growth. CONCLUSION: By examining (1) the effects of superovulation and (2) the effects of an in vitro system (oocyte maturation, fertilisation and embryo culture) we have identified that the assisted reproduction process of superovulation alone has the largest impact on the DNA methylome of subsequent embryos.

Expression of granulosa cell microRNAs, AVEN and ATRX are associated with human blastocyst development.

A greater understanding of the key molecules associated with embryo development during human-assisted reproduction is imperative for the development of advanced diagnostics. Previous studies have shown that follicular microRNAs (miRNAs) are reliable markers of the polycystic ovarian syndrome (PCOS). Leveraging the utility of miRNAs in PCOS, the aim of this study was to identify miRNAs in human granulosa cells that may be indicative of blastocyst development. Granulosa cells and oocytes were collected from the first follicle aspirated from patients undergoing oocyte retrieval for in vitro fertilization or intracytoplasmic sperm injection. The development of isolated oocytes was recorded, and granulosa cell samples in this study were separated as follows. Group 1-BLAST: granulosa cells from follicles containing an oocyte that fertilized and developed into a blastocyst, and Group 2-FERT: granulosa cells from oocytes that fertilized but failed to reach blastocyst. A panel of 84 miRNAs, related to development and cellular differentiation, was assessed between the two groups using a miScript PCR array. Fourteen miRNAs and one snoRNA were differentially expressed between the groups. In addition, two downstream candidate protein biomarkers, ATRX and AVEN, were also found to be differentially expressed between the groups. The findings of this pilot study reveal follicular abnormalities on a molecular level, which may affect oocyte competence and its potential to develop successfully as an embryo. We encourage additional studies to confirm and expand on our findings and to determine the usefulness of granulosa-borne miRNAs, ATRX, and AVEN as biomarkers.

Subfertility in bulls carrying a nonsense mutation in transmembrane protein 95 is due to failure to interact with the oocyte vestments.

In a recent genome-wide association study, 40 Fleckvieh bulls with exceptionally poor fertility were found to be homozygous for a nonsense mutation in the transmembrane protein 95 (TMEM95) encoding gene. Ejaculates from these individuals exhibited normal sperm concentration, morphology, viability, and motility. However, only 1.7% of inseminations resulted in pregnancies. The aim of this study was to examine the effect of this mutation in TMEM95 on bovine sperm function in vitro. Sperm from homozygous (mt/mt) males had lower in vitro fertility than sperm from wild-type (wt/wt) or heterozygous (wt/mt) bulls (P < 0.01). In addition, early embryo division was affected in the mt/mt group (P < 0.01). This translated into a lower (P < 0.01) blastocyst rate at day 8. Fluorescent staining revealed that TMEM95 is lost after the acrosome reaction. This led us to hypothesize that TMEM95 might be involved in events that lead to sperm-oocyte interaction. After fertilization, a lower number (P < 0.01) of sperm from mt/mt bulls bound to the zona pellucida (ZP). Sperm from mt/mt bulls were also less able to penetrate oocytes with no ZP (P< 0.01). However, when sperm from these animals were injected into mouse oocytes, they could decondense as successfully as sperm from wt/wt bulls. No differences between genotypes were observed in the ability of sperm to retain motility in an ex vivo oviduct, or in the percentage of sperm exhibiting markers for capacitation and acrosomal reaction. These results suggest that fertilization failure in mt/mt bulls is due to the inability of their sperm to interact with the oocyte vestments.

DNA methylation dynamics at imprinted genes during bovine pre-implantation embryo development.

BACKGROUND: In mammals, maternal differentially methylated regions (DMRs) acquire DNA methylation during the postnatal growth stage of oogenesis, with paternal DMRs acquiring DNA methylation in the perinatal prospermatagonia. Following fusion of the male and female gametes, it is widely accepted that murine DNA methylation marks at the DMRs of imprinted genes are stable through embryogenesis and early development, until they are reprogrammed in primordial germ cells. However, the DNA methylation dynamics at DMRs of bovine imprinted genes during early stages of development remains largely unknown. The objective of this investigation was to analyse the methylation dynamics at imprinted gene DMRs during bovine embryo development, from blastocyst stage until implantation. RESULTS: To this end, pyrosequencing technology was used to quantify DNA methylation at DMR-associated CpG dinucleotides of six imprinted bovine genes (SNRPN, MEST, IGF2R, PLAGL1, PEG10 and H19) using bisulfite-modified genomic DNA isolated from individual blastocysts (Day 7); ovoid embryos (Day 14); filamentous embryos (Day 17) and implanting conceptuses (Day 25). For all genes, the degree of DNA methylation was most variable in Day 7 blastocysts compared to later developmental stages (P < 0.05). Furthermore, mining of RNA-seq transcriptomic data and western blot analysis revealed a specific window of expression of DNA methylation machinery genes (including DNMT3A, DNMT3B, TRIM28/KAP1 and DNMT1) and proteins (DNMT3A, DNMT3A2 and DNMT3B) by bovine embryos coincident with imprint stabilization. CONCLUSION: The findings of this study suggest that the DNA methylation status of bovine DMRs might be variable during the early stages of embryonic development, possibly requiring an active period of imprint stabilization.

Epigenetic processes in the male germline.

Sperm undergo some of the most extensive chromatin modifications seen in mammalian biology. During male germline development, paternal DNA methylation marks are erased and established on a global scale through waves of demethylation and de novo methylation. As spermatogenesis progresses, the majority of the histones are removed and replaced by protamines, enabling a tighter packaging of the DNA and transcriptional shutdown. Following fertilisation, the paternal genome is rapidly reactivated, actively demethylated, the protamines are replaced with histones and the embryonic genome is activated. The development of new assays, made possible by high-throughput sequencing technology, has resulted in the revisiting of what was considered settled science regarding the state of DNA packaging in mammalian spermatozoa. Researchers have discovered that not all histones are replaced by protamines and, in certain experiments, various species of RNA have been detected in what was previously considered transcriptionally quiescent spermatozoa. Most controversially, several groups have suggested that environmental modifications of the epigenetic state of spermatozoa may operate as a non-DNA-based form of inheritance, a process known as 'transgenerational epigenetic inheritance'. Other developments in the field include the increased focus on the involvement of short RNAs, such as microRNAs, long non-coding RNAs and piwi-interacting RNAs. There has also been an accumulation of evidence illustrating associations between defects in sperm DNA packaging and disease and fertility. In this paper we review the literature, recent findings and areas of controversy associated with epigenetic processes in the male germline, focusing on DNA methylation dynamics, non-coding RNAs, the biology of sperm chromatin packaging and transgenerational inheritance.

Negative energy balance affects imprint stability in oocytes recovered from postpartum dairy cows.

Ovarian follicle development in post-partum, high-producing dairy cows, occurs in a compromised endogenous metabolic environment (referred to as negative energy balance, NEB). Key events that occur during oocyte/follicle growth, such as the vital process of genomic imprinting, may be detrimentally affected by this altered ovarian environment. Imprinting is crucial for placental function and regulation of fetal growth, therefore failure to establish and maintain imprints during oocyte growth may contribute to early embryonic loss. Using ovum pick-up (OPU), oocytes and follicular fluid samples were recovered from cows between days 20 and 115 post-calving, encompassing the NEB period. In a complimentary study, cumulus oocyte complexes were in vitro matured under high non-esterified fatty acid (NEFA) concentrations and in the presence of the methyl-donor S-adenosylmethionine (SAM). Pyrosequencing revealed the loss of methylation at several imprinted loci in the OPU derived oocytes. The loss of DNA methylation was observed at the PLAGL1 locus in oocytes, following in vitro maturation (IVM) in the presence of elevated NEFAs and SAM. Finally, metabolomic analysis of postpartum follicular fluid samples revealed significant differences in several branched chain amino acids, with fatty acid profiles bearing similarities to those characteristic of lactating dairy cows. These results provide the first evidence that (1) the postpartum ovarian environment may affect maternal imprint acquisition and (2) elevated NEFAs during IVM can lead to the loss of imprinted gene methylation in bovine oocytes.

An integrated platform for bovine DNA methylome analysis suitable for small samples.

BACKGROUND: Oocytes and early embryos contain minute amounts of DNA, RNA and proteins, making the study of early mammalian development highly challenging. The study of the embryo epigenome, in particular the DNA methylome, has been made accessible thanks to the possibility of amplifying specific sequences according to their initial methylation status. This paper describes a novel platform dedicated to the genome-wide study of bovine DNA methylation, including a complete pipeline for data analysis and visualization. The platform allows processing and integrating of DNA methylome and transcriptome data from the same sample. Procedures were optimized for genome-wide analysis of 10 ng of DNA (10 bovine blastocysts). Bovine sperm and blastocysts were compared as a test of platform capability. RESULTS: The hypermethylation of bovine sperm DNA compared to the embryo genome was confirmed. Differentially methylated regions were distributed across various classes of bovine sperm genomic feature including primarily promoter, intronic and exonic regions, non-CpG-island regions (shore, shelf and open-sea) and CpG islands with low-to-intermediate CpG density. The blastocyst genome bore more methylation marks than sperm DNA only in CpG islands with high CpG density. Long-terminal-repeat retrotransposons (LTR), LINE and SINE were more methylated in sperm DNA, as were low-complexity repetitive elements in blastocysts. CONCLUSIONS: This is the first early embryo compatible genome-wide epigenetics platform for bovine. Such platforms should improve the study of the potential epigenetic risks of assisted reproductive technologies (ART), the establishment sequence of embryonic cell lines and potential deviations in both gene expression and DNA methylation capable of having long-term impact.

Designing infographics in health research with patients and the public: A scoping review protocol.

Information graphics or infographics combine visual representations of information or data with text. They have been used in health research to disseminate research findings, translate knowledge and address challenges in health communication to lay audiences. There is emerging evidence of the design of infographics with the involvement of patients and the public in health research. Approaches to involvement include public and patient involvement, patient engagement and participatory research approaches. To date, there has been no comprehensive review of the literature on the design of infographics with patients and the public in health research. This paper presents a protocol and methodological framework for a scoping review to identify and map the available evidence for the involvement of patients and the public in infographics design in health research. It has been informed by preliminary searches and discussions and will guide the conduct and reporting of this review.

DNA methylation plays an important role in promoter choice and protein production at the mouse Dnmt3L locus.

The DNA methyltransferase 3-like (Dnmt3L) protein is a crucial cofactor in the germ line for the de novo methyltransferase Dnmt3a, which establishes imprints and represses transposable elements. We have previously shown that Dnmt3L transcription is regulated via three different promoters in mice, producing transcripts we term Dnmt3L(s) (stem cell), Dnmt3L(o) (oocyte) and Dnmt3L(at) (adult testis). Here we show that both Dnmt3L(s) and Dnmt3L(o) produce full-length proteins but that the Dnmt3L(at) transcripts are not translated. Although not a canonical CpG island, the Dnmt3L(s) promoter is silenced by methylation during somatic differentiation in parallel with germ-cell-specific genes. During oocyte growth, Dnmt3L(s) also becomes heavily methylated and silenced and this requires its own gene product, since there is complete loss of methylation and derepression of transcription from this promoter in oocytes derived from Dnmt3L(-/-) mice. Methylation of the Dnmt3L(s) promoter is established prior to the completion of imprinting and explains the requirement in mouse oocytes for the Dnmt3L(o) promoter, located in an intron of the neighboring unmethylated Aire gene. Overall these results give insight into how and why promoter switching at the mouse Dnmt3L locus occurs and provide one of the first examples of a non-imprinted locus where methylation plays a role in promoter choice. The derepression of the Dnmt3L(s) promoter in the knockout oocytes also suggests that other non-imprinted loci may be dysregulated in these cells and contribute to the phenotype of the resultant mice.

Bovine DNA methylation imprints are established in an oocyte size-specific manner, which are coordinated with the expression of the DNMT3 family proteins.

A subset of genes, known as imprinted genes, is present in the mammalian genome. Genomic imprinting governs the monoallelic expression of these genes, depending on whether the gene was inherited from the sperm or the egg. This parent-of-origin specific gene expression is generally dependent on the epigenetic modification, DNA methylation, and the DNA methylation status of CpG dinucleotides residing in loci known as differentially methylated regions (DMRs). The enzymatic machinery responsible for the addition of methyl (-CH(3)) groups to the cytosine residue in the CpG dinucleotides are known as DNA methyltransferases (DNMTs). Correct establishment and maintenance of methylation patterns at imprinted genes has been associated with placental function and regulation of embryonic/fetal development. Much work has been carried out on imprinted genes in mouse and human; however, little is known about the methylation dynamics in the bovine oocyte. The primary objective of the present study was to characterize the establishment of methylation at maternally imprinted genes in bovine growing oocytes and to determine if the expression of the bovine DNMTs-DNMT3A, DNMT3B, and DNMT3L-was coordinated with DNA methylation during oocyte development. To this end, a panel of maternally imprinted genes was selected (SNRPN, MEST, IGF2R, PEG10, and PLAGL1) and putative DMRs for MEST, IGF2R, PEG10, and PLAGL1 were identified within the 5' regions for each gene; the SNRPN DMR has been reported previously. Conventional bisulfite sequencing revealed that methylation marks were acquired at all five DMRs investigated in an oocyte size-dependent fashion. This was confirmed for a selection of genes using pyrosequencing analysis. Furthermore, mRNA expression and protein analysis revealed that DNMT3A, DNMT3B, and DNMT3L are also present in the bovine oocyte during its growth phase. This study demonstrates for the first time that an increase in bovine imprinted gene DMR methylation occurs during oocyte growth, as is observed in mouse.

Application of multi-omics data integration and machine learning approaches to identify epigenetic and transcriptomic differences between in vitro and in vivo produced bovine embryos.

Pregnancy rates for in vitro produced (IVP) embryos are usually lower than for embryos produced in vivo after ovarian superovulation (MOET). This is potentially due to alterations in their trophectoderm (TE), the outermost layer in physical contact with the maternal endometrium. The main objective was to apply a multi-omics data integration approach to identify both temporally differentially expressed and differentially methylated genes (DEG and DMG), between IVP and MOET embryos, that could impact TE function. To start, four and five published transcriptomic and epigenomic datasets, respectively, were processed for data integration. Second, DEG from day 7 to days 13 and 16 and DMG from day 7 to day 17 were determined in the TE from IVP vs. MOET embryos. Third, genes that were both DE and DM were subjected to hierarchical clustering and functional enrichment analysis. Finally, findings were validated through a machine learning approach with two additional datasets from day 15 embryos. There were 1535 DEG and 6360 DMG, with 490 overlapped genes, whose expression profiles at days 13 and 16 resulted in three main clusters. Cluster 1 (188) and Cluster 2 (191) genes were down-regulated at day 13 or day 16, respectively, while Cluster 3 genes (111) were up-regulated at both days, in IVP embryos compared to MOET embryos. The top enriched terms were the KEGG pathway "focal adhesion" in Cluster 1 (FDR = 0.003), and the cellular component: "extracellular exosome" in Cluster 2 (FDR<0.0001), also enriched in Cluster 1 (FDR = 0.04). According to the machine learning approach, genes in Cluster 1 showed a similar expression pattern between IVP and less developed (short) MOET conceptuses; and between MOET and DKK1-treated (advanced) IVP conceptuses. In conclusion, these results suggest that early conceptuses derived from IVP embryos exhibit epigenomic and transcriptomic changes that later affect its elongation and focal adhesion, impairing post-transfer survival.

Regulation of a bovine nonclassical major histocompatibility complex class I gene promoter.

Studies have shown in humans and other species that the major histocompatibility complex class I (MHC-I) region is involved at a number of levels in the establishment and maintenance of pregnancy. The aim of this study was to characterize how a bovine nonclassical MHC-I gene (NC1) is regulated. Initial serial deletion experiments of a 2-kb fragment of the NC1 promoter identified regions with positive regulatory elements in the proximal promoter and evidence for a silencer module(s) further upstream that cooperatively contributed to constitutive NC1 expression. The cytokines interferon tau (IFNT), interferon gamma (IFNG), and interleukin 4 (IL4) significantly increased luciferase expression in NC1 promoter reporter constructs and endogenous NC1 mRNA levels in a bovine endometrial cell line. In addition, IFNG, IL3, IL4, and progesterone significantly increased Day 7 bovine blastocyst NC1 mRNA expression when supplemented during in vitro embryo culture. Site-directed mutagenesis analysis identified a STAT6 binding site that conferred IL4 responsiveness in the NC1 proximal promoter. Furthermore, methylation treatment of the proximal promoter, which contains a CpG island, completely abrogated constitutive NC1 expression. Overall, the findings presented here suggest that constitutive NC1 expression is regulated positively by elements in the proximal promoter, which are further controlled by upstream silencer modules. The promoter is responsive to IFNT, IFNG, and IL4, suggesting possible roles for these cytokines in bovine preimplantation embryo survival and/or maternal-fetal tolerance. Our studies also suggest that methylation of the proximal promoter, in particular, could play a significant role in regulating NC1 expression.

Epigenetic homogeneity in histone methylation underlies sperm programming for embryonic transcription.

Sperm contributes genetic and epigenetic information to the embryo to efficiently support development. However, the mechanism underlying such developmental competence remains elusive. Here, we investigated whether all sperm cells have a common epigenetic configuration that primes transcriptional program for embryonic development. Using calibrated ChIP-seq, we show that remodelling of histones during spermiogenesis results in the retention of methylated histone H3 at the same genomic location in most sperm cell. This homogeneously methylated fraction of histone H3 in the sperm genome is maintained during early embryonic replication. Such methylated histone fraction resisting post-fertilisation reprogramming marks developmental genes whose expression is perturbed upon experimental reduction of histone methylation. A similar homogeneously methylated histone H3 fraction is detected in human sperm. Altogether, we uncover a conserved mechanism of paternal epigenetic information transmission to the embryo through the homogeneous retention of methylated histone in a sperm cells population.

Alveolar Macrophage Chromatin Is Modified to Orchestrate Host Response to Mycobacterium bovis Infection.

Bovine tuberculosis is caused by infection with Mycobacterium bovis, which can also cause disease in a range of other mammals, including humans. Alveolar macrophages are the key immune effector cells that first encounter M. bovis and how the macrophage epigenome responds to mycobacterial pathogens is currently not well understood. Here, we have used chromatin immunoprecipitation sequencing (ChIP-seq), RNA-seq and miRNA-seq to examine the effect of M. bovis infection on the bovine alveolar macrophage (bAM) epigenome. We show that H3K4me3 is more prevalent, at a genome-wide level, in chromatin from M. bovis-infected bAM compared to control non-infected bAM; this was particularly evident at the transcriptional start sites of genes that determine programmed macrophage responses to mycobacterial infection (e.g. M1/M2 macrophage polarisation). This pattern was also supported by the distribution of RNA Polymerase II (Pol II) ChIP-seq results, which highlighted significantly increased transcriptional activity at genes demarcated by permissive chromatin. Identification of these genes enabled integration of high-density genome-wide association study (GWAS) data, which revealed genomic regions associated with resilience to infection with M. bovis in cattle. Through integration of these data, we show that bAM transcriptional reprogramming occurs through differential distribution of H3K4me3 and Pol II at key immune genes. Furthermore, this subset of genes can be used to prioritise genomic variants from a relevant GWAS data set.

The bovine alveolar macrophage DNA methylome is resilient to infection with Mycobacterium bovis.

DNA methylation is pivotal in orchestrating gene expression patterns in various mammalian biological processes. Perturbation of the bovine alveolar macrophage (bAM) transcriptome, due to Mycobacterium bovis (M. bovis) infection, has been well documented; however, the impact of this intracellular pathogen on the bAM epigenome has not been determined. Here, whole genome bisulfite sequencing (WGBS) was used to assess the effect of M. bovis infection on the bAM DNA methylome. The methylomes of bAM infected with M. bovis were compared to those of non-infected bAM 24 hours post-infection (hpi). No differences in DNA methylation (CpG or non-CpG) were observed. Analysis of DNA methylation at proximal promoter regions uncovered >250 genes harbouring intermediately methylated (IM) promoters (average methylation of 33-66%). Gene ontology analysis, focusing on genes with low, intermediate or highly methylated promoters, revealed that genes with IM promoters were enriched for immune-related GO categories; this enrichment was not observed for genes in the high or low methylation groups. Targeted analysis of genes in the IM category confirmed the WGBS observation. This study is the first in cattle examining genome-wide DNA methylation at single nucleotide resolution in an important bovine cellular host-pathogen interaction model, providing evidence for IM promoter methylation in bAM.

Bi-directional effects of vitamin B12 and methotrexate on Daphnia magna fitness and genomic methylation.

Here we interrogated, using three separate but complementary experimental approaches, the impact of vitamin B12 availability and methotrexate exposure on Daphnia magna, which we hypothesised should have an opposite effect on One carbon metabolism (OCM). OCM is a vital biological process supporting a variety of physiological processes, including DNA methylation. Contrary to mammalian models, this process remains largely unexplored in invertebrates. The purpose of this study was to elucidate the impact of OCM short-term alteration on the fitness and epigenome of the keystone species, Daphnia. We used maternal age at reproduction, brood size and survival rates in combination with DNA methylation sensitive comet assay to determine the effects of vitamin B12 or MTX on fitness and the epigenome. Vitamin B12 had a positive influence on Daphnia fitness and we provide evidence demonstrating that this may be associated with an increased level of genome-wide DNA methylation. Conversely, exposing D. magna to MTX negatively influenced the fitness of the animals and was associated with loss of global DNA methylation, translating in decreased fitness. These results highlight the potential importance of OCM in invertebrates, providing novel evidence supporting a potential role for epigenetic modifications to the genome in D. magna environmental adaptability.

Genomic imprinting effects on complex traits in domesticated animal species.

Monoallelically expressed genes that exert their phenotypic effect in a parent-of-origin specific manner are considered to be subject to genomic imprinting, the most well understood form of epigenetic regulation of gene expression in mammals. The observed differences in allele specific gene expression for imprinted genes are not attributable to differences in DNA sequence information, but to specific chemical modifications of DNA and chromatin proteins. Since the discovery of genomic imprinting some three decades ago, over 100 imprinted mammalian genes have been identified and considerable advances have been made in uncovering the molecular mechanisms regulating imprinted gene expression. While most genomic imprinting studies have focused on mouse models and human biomedical disorders, recent work has highlighted the contributions of imprinted genes to complex trait variation in domestic livestock species. Consequently, greater understanding of genomic imprinting and its effect on agriculturally important traits is predicted to have major implications for the future of animal breeding and husbandry. In this review, we discuss genomic imprinting in mammals with particular emphasis on domestic livestock species and consider how this information can be used in animal breeding research and genetic improvement programs.

Chito-oligosaccharide inhibits the de-methylation of a 'CpG' island within the leptin (LEP) promoter during adipogenesis of 3T3-L1 cells.

Chito-oligosaccharide (COS) is a natural bioactive compound, which has been shown to suppress lipid metabolic genes and lipid accumulation in differentiating adipocytes. Leptin has been identified as a key regulator of energy homeostasis and is known to be under epigenetic regulation during adipogenesis. Hence, the first objective of this experiment was to compare leptin gene (LEP) expression and leptin secretion during the different stages of adipogenesis and to investigate the effect of COS on these processes. As COS inhibited LEP expression during adipogenesis, the second aim was to investigate the methylation dynamics of a 'CpG' island in the proximal region of the LEP promoter during adipogenesis and to determine the effect of COS on this process. Mouse 3T3-L1 cells were stimulated to differentiate in the absence or presence of COS and the levels of leptin mRNA and protein were evaluated on days 0, 2, 4 and 6 post-induction of differentiation (PID). The extent of de-methylation of six CpG sites was evaluated. LEP mRNA transcript and protein could not be detected on either day 0PID or 2PID. In contrast, both were detected on day 4PID (P<0.05) and 6PID (P<0.001) and both were inhibited by COS (P<0.001). Of the six CpG sites analyzed, CpG_52, CpG_62 and CpG_95 became 11.5, 5.0 and 5.0% de-methylated between day 2PID and 6PID, respectively. COS blocked this de-methylation event at CpG_52 (P<0.001), CpG_62 (P<0.01) and CpG_95 (P<0.01) on day 6PID. These data suggest that COS can have an epigenetic effect on differentiating adipocytes, a novel biological function of COS which has potential applications for the manipulation of leptin gene expression, adipogenesis, and conditions within the metabolic syndrome spectrum.

Catsper3 and catsper4 encode two cation channel-like proteins exclusively expressed in the testis.

CATSPER1 and CATSPER2 are two cation channel-like proteins exclusively expressed in the testis and essential for normal sperm motility and male fertility. Using in silico subtraction and database mining, we identified expressed sequence tags encoding two previously uncharacterized cation channel-like proteins structurally homologous to CATSPER1 and CATSPER2. Similar to CATSPER1 and CATSPER2, these two proteins contain a single-ion transport domain comprised of six transmembrane spanning regions, in which the fourth transmembrane region resembles a voltage sensor and a pore-forming region lies between transmembrane regions 5 and 6. The pore contains the consensus sequence T x D x W, which is indicative of a potential calcium-selective channel. The mRNAs for Catsper3 and Catsper4 were detected exclusively in the testis using multitissue Northern blot and RT-PCR analyses. The onsets of both genes coincide with the first appearance of spermatids during testicular development. In situ hybridization analyses revealed that Catsper3 and Catsper4 mRNAs displayed identical localization patterns and were confined to spermatids of steps 1-8. Immunofluorescence and immunohistochemistry analyses demonstrated that these two proteins were expressed within the acrosome of late spermatids and spermatozoa. Our data suggest that CATSPER3 and CATSPER4 are two cation-channel proteins and have roles in acrosome reaction and male fertility.