Epigenetic consequences of artificial reproductive technologies to the bovine imprinted genes SNRPN, H19/IGF2, and IGF2R.

Animal breeders have made widespread use of assisted reproductive technologies to accelerate genetic improvement programs aimed at obtaining more, better and cheaper food products. Selection approaches have traditionally focused on Mendel's laws of inheritance using parental phenotypic characteristics and quantitative genetics approaches to choose the best parents for the next generation, regardless of their gender. However, apart from contributing DNA sequence variants, male and female gametes carry parental-specific epigenetic marks that play key roles during pre- and post-natal development and growth of the offspring. We herein review the epigenetic anomalies that are associated with artificial reproductive technologies in current use in animal breeding programs. For instance, we demonstrate that bovine embryos and fetuses derived by in vitro culture and somatic cell nuclear transfer show epigenetic anomalies in the differentially methylated regions controlling the expression of some imprinted genes. Although these genomic imprinting errors are undetected in the somatic tissues after birth, further research is warranted to examine potential germ cell transmission of epimutations and the potential risks of reproducing cattle using artificial reproductive technologies.

In vitro culture and somatic cell nuclear transfer affect imprinting of SNRPN gene in pre- and post-implantation stages of development in cattle.

BACKGROUND: Embryo in vitro manipulations during early development are thought to increase mortality by altering the epigenetic regulation of some imprinted genes. Using a bovine interspecies model with a single nucleotide polymorphism, we assessed the imprinting status of the small nuclear ribonucleoprotein polypeptide N (SNRPN) gene in bovine embryos produced by artificial insemination (AI), in vitro culture (IVF) and somatic cell nuclear transfer (SCNT) and correlated allelic expression with the DNA methylation patterns of a differentially methylated region (DMR) located on the SNRPN promoter. RESULTS: In the AI group, SNRPN maternal expression is silenced at day 17 and 40 of development and a third of the alleles analyzed are methylated in the DMR. In the IVF group, maternal transcripts were identified at day 17 but methylation levels were similar to the AI group. However, day-40 fetuses in the IVF group showed significantly less methylation when compared to the AI group and SNRPN expression was mostly paternal in all fetal tissues studied, except in placenta. Finally, the SCNT group presented severe loss of DMR methylation in both day-17 embryos and 40 fetuses and biallelic expression was observed in all stages and tissues analyzed. CONCLUSION: Together these results suggest that artificial reproductive techniques, such as prolonged in vitro culture and SCNT, lead to abnormal reprogramming of imprinting of SNRPN gene by altering methylation levels at this locus.

Characterization of mitochondrial replication and transcription control during rat early development in vivo and in vitro.

In vitro culture (IVC), used in assisted reproductive technologies, is a major environmental stress on the embryo. To evaluate the effect of IVC on mitochondrial transcription and the control of mtDNA replication, we measured the mtDNA copy number and relative amount of mRNA for mitochondrial-related genes in individual rat oocytes, zygotes and embryos using real-time PCR. The average mtDNA copy number was 147 600 (+/-3000) in metaphase II oocytes. The mtDNA copy number was stable throughout in vivo early development and IVC induced an increase in mtDNA copy number from the 8-cell stage onwards. Gapd mRNA levels vary during early development and IVC did not change the patterns of these housekeeping gene transcripts. Polrmt mRNA levels did not vary during early development up to the morula stage but increased at the blastocyst stage. IVC induced the up-regulation of Polrmt mRNA, one of the key genes regulating mtDNA transcription and replication, at the blastocyst stage. An increase in mt-Nd4 mRNA preceded the blastocyst-related event observed in nuclear-encoded Gapd and Polrmt, suggesting that the expression of mitochondrial encoded genes is controlled differently from nuclear encoded genes. We conclude that the IVC system can perturb mitochondrial transcription and the control of mtDNA replication in rat embryos. This perturbation of mtDNA regulation may be responsible for the abnormal physiology, metabolism and viability of in vitro-derived embryos.

The influence of nuclear content on developmental competence of gaur x cattle hybrid in vitro fertilized and somatic cell nuclear transfer embryos.

In nondomestic and endangered species, the use of domestic animal oocytes as recipients for exotic donor nuclei causes the normal pattern of cytoplasmic inheritance to be disrupted, resulting in the production of nuclear-cytoplasmic hybrids. Evidence suggests that conflict between nuclear and cytoplasmic control elements leads to a disruption of normal cellular processes, including metabolic function and cell division. This study investigated the effects of nuclear-cytoplasmic interactions on the developmental potential of interspecies embryos produced by in vitro fertilization and somatic cell nuclear transfer: cattle x cattle, gaur x cattle, hybrid x cattle. Cattle control and hybrid embryos were examined for development to the blastocyst stage and blastocyst quality, as determined by cell number and allocation, apoptosis incidence, and expression patterns of mitochondria-related genes. These analyses demonstrated that a 100% gaur nucleus within a domestic cattle cytoplasmic environment was not properly capable of directing embryo development in the later preimplantation stages. Poor blastocyst development accompanied by developmental delay, decreased cell numbers, and aberrant apoptotic and related gene expression profiles, all signs of disrupted cellular processes associated with mitochondrial function, were observed. Developmental potential was improved when at least a portion of the nuclear genome corresponded to the inherited cytoplasm, indicating that recognition of cytoplasmic components by the nucleus is crucial for proper cellular function and embryo development. A better understanding of the influence of the cytoplasmic environment on embryonic processes is necessary before interspecies somatic cell nuclear transfer can be considered a viable alternative for endangered species conservation.

Bovine SNRPN methylation imprint in oocytes and day 17 in vitro-produced and somatic cell nuclear transfer embryos.

Findings from recent studies have suggested that the low survival rate of animals derived via somatic cell nuclear transfer (SCNT) may be in part due to epigenetic abnormalities brought about by this procedure. DNA methylation is an epigenetic modification of DNA that is implicated in the regulation of imprinted genes. Genes subject to genomic imprinting are expressed monoallelically in a parent of origin-dependent manner and are important for embryo growth, placental function, and neurobehavioral processes. The vast majority of imprinted genes have been studied in mice and humans. Herein, our objectives were to characterize the bovine SNRPN gene in gametes and to compare its methylation profile in in vivo-produced, in vitro-produced, and SCNT-derived Day 17 elongating embryos. A CpG island within the 5' region of SNRPN was identified and examined using bisulfite sequencing. SNRPN alleles were unmethylated in sperm, methylated in oocytes, and approximately 50% methylated in somatic samples. The examined SNRPN region appeared for the most part to be normally methylated in three in vivo-produced Day 17 embryos and in eight in vitro-produced Day 17 embryos examined, while alleles from Day 17 SCNT embryos were severely hypomethylated in seven of eight embryos. In this study, we showed that the SNRPN methylation profiles previously observed in mouse and human studies are also conserved in cattle. Moreover, SCNT-derived Day 17 elongating embryos were abnormally hypomethylated compared with in vivo-produced and in vitro-produced embryos, which in turn suggests that SCNT may lead to faulty reprogramming or maintenance of methylation imprints at this locus.

Short- and long-term skin graft survival in cattle clones with different mitochondrial haplotypes.

In contrast to nuclear DNA, cytoplasmic genes may differ among cloned animals due to the presence of polymorphic mitochondrial DNA haplotypes in the host oocytes, raising doubts about histocompatibility among clones. Three bovine clones were generated by nuclear transfer; dermal fibroblasts from a fetus were used as donor cells, whereas oocytes from abbatoir-derived ovaries were used as recipient cells. The mitochondrial DNA (sequencing of coding and non-coding regions) and nuclear DNA (13 microsatellite markers) of cloned and control animals were characterized to identify potential polymorphisms. Skin auto- and allografts were transplanted on the adult clones and a non-related animal as a measure of immunological reactivity. Nuclear DNA of cloned animals was genetically identical but differed in all microsatellites of the non-related control. Amounts of donor cell mitochondrial DNA in the skin ranged from 1 to 2.6% among clones. Few differences in heteroplasmy were observed between skin and WBC of the clones, indicating limited mitochondrial DNA segregation in tissues during pre- and post-natal development to adulthood. Sequencing of the remaining oocyte-derived mitochondrial DNA haplotype identified polymorphisms in coding and non-coding regions, confirming their origin from unrelated maternal lineages. Nonetheless, skin transplants between clones were accepted for the 92 d study period, whereas third-party grafts were rejected. In conclusion, the nuclear transfer-generated adult bovine clones used in this study were immunologically compatible with one another despite differences in their mitochondrial DNA haplotypes.

Molecular control of mitochondrial function in preimplantation mouse embryos.

Mitochondria play a key role in a number of physiological events during all stages of life, including the very first stages following fertilization. It is, therefore, important to understand the mechanisms controlling mitochondrial activity during early embryogenesis to determine their role in development outcome. The objective of this study was to investigate the molecular control of mitochondrial transcription and mitochondrial DNA (mtDNA) replication in mouse preimplantation embryos. We estimated the mtDNA copy number and characterized the expression patterns of two mitochondrial genes and several nuclear genes that encode mitochondrial transcription and replication factors throughout preimplantation development. Mitochondrial gene transcripts were present in larger quantities in morula and blastocyst stage embryos relative to other stages. A significant increase in the amount of mRNA for nuclear genes encoding mtDNA transcription factors was observed in eight-cell stage embryos. Although a similar increase in the mRNA levels of nuclear genes encoding mtDNA replication factors was observed in morula and blastocyst stage embryos, the number of mtDNA molecules remained stable during preimplantation stages, suggesting that nuclear-encoded mitochondrial transcription factors are involved in the regulation of mtDNA transcription during early development. Although transcripts of replication factors are abundant at the morula and blastocyst stage, mtDNA replication did not occur until the blastocyst stage, suggesting that the inhibition of mtDNA replication is controlled at the post-transcriptional level during early embryogenesis.

Role of the mitochondrial genome in preimplantation development and assisted reproductive technologies.

Our fascination for mitochondria relates to their origin as symbiotic, semi-independent organisms on which we, as eukaryotic beings, rely nearly exclusively to produce energy for every cell function. Therefore, it is not surprising that these organelles play an essential role in many events during early development and in artificial reproductive technologies (ARTs) applied to humans and domestic animals. However, much needs to be learned about the interactions between the nucleus and the mitochondrial genome (mtDNA), particularly with respect to the control of transcription, replication and segregation during preimplantation. Nuclear-encoded factors that control transcription and replication are expressed during preimplantation development in mice and are followed by mtDNA transcription, but these result in no change in mtDNA copy number. However, in cattle, mtDNA copy number increases during blastocyst expansion and hatching. Nuclear genes influence the mtDNA segregation patterns in heteroplasmic animals. Because many ARTs markedly modify the mtDNA content in embryos, it is essential that their application is preceded by careful experimental scrutiny, using suitable animal models.