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.

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.

BackgroundMetabolic stress associated with negative energy balance in high producing dairy cattle and obesity in women is a risk factor for decreased fertility. Non-esterified fatty acids (NEFA) are involved in this pathogenesis as they jeopardize oocyte and embryo development. Growing evidence indicates that maternal metabolic disorders can disturb epigenetic programming, such as DNA methylation, in the offspring. Oocyte maturation and early embryo development coincide with methylation changes and both are sensitive to adverse environments. Therefore, we investigated whether elevated NEFA concentrations affect establishment and maintenance of DNA methylation in oocytes and embryos, subsequently altering transcriptomic profiles and developmental competence of resultant blastocysts.ResultsBovine oocytes and embryos were exposed to different NEFA concentrations in separate experiments. In the first experiment, oocytes were matured in vitro for 24 h in medium containing: 1) physiological (“BASAL”) concentrations of oleic (OA), palmitic (PA) and stearic (SA) acid or 2) pathophysiological (“HIGH COMBI”) concentrations of OA, PA and SA. In the second experiment, zygotes were cultivated in vitro for 6.5 days under BASAL or HIGH COMBI conditions. Developmental competence was evaluated by assessing cleavage and blastocyst rate. Overall gene expression and DNA methylation of resultant blastocysts were analyzed using microarray. DNA methylation data were re-evaluated by pyrosequencing. HIGH COMBI-exposed oocytes and embryos displayed a lower competence to develop into blastocysts compared to BASAL-exposed counterparts (19.3% compared to 23.2% and 18.2% compared to 25.3%, respectively) (P < 0.05). HIGH COMBI-exposed oocytes and embryos resulted in blastocysts with altered DNA methylation and transcriptomic fingerprints, compared to BASAL-exposed counterparts. Differences in gene expression and methylation were more pronounced after exposure during culture compared to maturation suggesting that zygotes are more susceptible to adverse environments. Main gene networks affected were related to lipid and carbohydrate metabolism, cell death, immune response and metabolic disorders.ConclusionsOverall, high variation in methylation between blastocysts made it difficult to draw conclusions concerning methylation of individual genes, although a clear overview of affected pathways was obtained. This may offer clues regarding the high rate of embryonic loss and metabolic diseases during later life observed in offspring from mothers displaying lipolytic disorders.

BACKGROUND: Dnmt3L, a member of the DNA methyltransferase 3 family, lacks enzymatic activity but is required for de-novo methylation of imprinted genes in oocytes and for transposon repression in male germ cells. METHODS: We used northern blots, RT-PCR, 5¢ rapid amplification of complementary DNA (cDNA) ends (RACE), RNase H mapping, real-time/quantitative RT-PCR and in situ hybridization to identify and characterize Dnmt3L transcripts produced during germ cell development. RESULTS: Mouse Dnmt3L uses three sex-specific promoters, not the single promoter previously thought. A promoter active in prospermatogonia drives transcription of an mRNA encoding the full-length protein in perinatal testis, where de-novo methylation occurs. Late pachytene spermatocytes activate a second promoter in intron 9 of the Dnmt3L gene. After this stage, the predominant transcripts are three truncated mRNAs, which appear to be non-coding. We could also detect similar adult testis transcripts in humans. In the mouse ovary, an oocyte-specific promoter located in an intron of the neighbouring autoimmune regulator (Aire) gene produces a transcript with the full open reading frame (ORF). This is the only Dnmt3L transcript found in growing oocytes and is absent in the oocytes of Dnmt3L-/-females. CONCLUSIONS: Sex-specific promoters control Dnmt3L expression in the mouse germ line, mirroring the situation at the Dnmt1 and Dnmt3A loci.

Infertility is an often devastating diagnosis encountered by around one in six couples who are trying to conceive. Moving away from the long-held belief that infertility is primarily a female issue, it is now recognised that half, if not more, of these cases may be due to male factors. Recent evidence has suggested that epigenetic abnormalities in chromatin dynamics, DNA methylation or sperm-borne RNAs may contribute to male infertility. In light of advances in deep sequencing technologies, researchers have been able to increase the coverage and depth of sequencing results, which in turn has allowed more comprehensive analyses of spermatozoa chromatin dynamics and methylomes and enabled the discovery of new subsets of sperm RNAs. This review examines the most current literature related to epigenetic processes in the male germline and the associations of aberrant modifications with fertility and development.