BackgroundA valuable tool for both research and industry, in vitro fertilization (IVF) has applications range from gamete selection and preservation of traits to cloning. Although IVF has achieved worldwide use, with approximately 339,685 bovine embryos transferred in 2010 alone, there are still continuing difficulties with efficiency. It is rare to have more than 40% of fertilized in vitro cattle oocytes reach blastocyst stage by day 8 of culture, and pregnancy rates are reported as less than 45% for in vitro produced embryos. To investigate potential influences in-vitro fertilization (IVF) has on embryonic development, this study compares in vivo- and in vitro-derived bovine blastocysts at a similar stage and quality grade (expanded, excellent quality) to determine the degree of transcriptomic variation beyond morphology using RNA-Seq.ResultsA total of 26,906,451 and 38,184,547 fragments were sequenced for in vitro and in vivo embryo pools, respectively. We detected expression for a total of 17,634 genes, with 793 genes showing differential expression between the two embryo populations with false discovery rate (FDR) < 0.05. There were also 395 novel transcribed units found, of which 45 were differentially expressed (FDR < 0.05). In addition, 4,800 genes showed evidence of alternative splicing, with 873 genes displaying differential alternative splicing between the two pools (FDR < 0.05). Using GO enrichment analysis, multiple biological pathways were found to be significantly enriched for differentially expressed genes (FDR < 0.01), including cholesterol and sterol synthesis, system development, and cell differentiation.ConclusionsThus, our results support that IVF may influence at the transcriptomic level and that morphology is limited in full characterization of bovine preimplantation embryos.

Recently, we identified biallelic mutations of SLC25A46 in patients with multiple neuropathies. Functional studies revealed that SLC25A46 may play an important role in mitochondrial dynamics by mediating mitochondrial fission. However, the cellular basis and pathogenic mechanism of the SLC25A46-related neuropathies are not fully understood. Thus, we generated a Slc25a46 knock-out mouse model. Mice lacking SLC25A46 displayed severe ataxia, mainly caused by degeneration of Purkinje cells. Increased numbers of small, unmyelinated and degenerated optic nerves as well as loss of retinal ganglion cells indicated optic atrophy. Compound muscle action potentials in peripheral nerves showed peripheral neuropathy associated with degeneration and demyelination in axons. Mutant cerebellar neurons have large mitochondria, which exhibit abnormal distribution and transport. Biochemically mutant mice showed impaired electron transport chain activity and accumulated autophagy markers. Our results suggest that loss of SLC25A46 causes degeneration in neurons by affecting mitochondrial dynamics and energy production.

Maternal nutrition exclusively during the periconceptional period can induce remarkable effects on both oocyte maturation and early embryo development, which in turn can have lifelong consequences. The objective of this study was to evaluate the effect of maternal methionine supplementation on the transcriptome of bovine preimplantation embryos. Holstein cows were randomly assigned to one of two treatments differing in level of dietary methionine (1.89 Met vs. 2.43 Met % of metabolizable protein) from calving until embryo flushing. High quality preimplantation embryos from individual cows were pooled and then analyzed by RNA sequencing. Remarkably, a subtle difference in methionine supplementation in maternal diet was sufficient to cause significant changes in the transcriptome of the embryos. A total of 276 genes out of 10,662 showed differential expression between treatments (FDR <0.10). Interestingly, several of the most significant genes are related to embryonic development (e.g., VIM, IFI6, BCL2A1, and TBX15) and immune response (e.g., NKG7, TYROBP, SLAMF7, LCP1, and BLA-DQB). Likewise, gene set enrichment analysis revealed that several Gene Ontology terms, InterPro entries, and KEGG pathways were enriched (FDR <0.05) with differentially expressed genes involved in embryo development and immune system. The expression of most genes was decreased by maternal methionine supplementation, consistent with reduced transcription of genes with increased methylation of specific genes by increased methionine. Overall, our findings provide evidence that supplementing methionine to dams prior to conception and during the preimplantation period can modulate gene expression in bovine blastocysts. The ramifications of the observed gene expression changes for subsequent development of the pregnancy and physiology of the offspring warrant further investigation in future studies.

Primary microcephaly is a congenital brain malformation characterized by a head circumference less than three standard deviations below the mean for age and sex and results in moderate to severe mental deficiencies and decreased lifespan. We recently studied two children with primary microcephaly in an otherwise unaffected family. Exome sequencing identified an autosomal recessive mutation leading to an amino acid substitution in a WD40 domain of the highly conserved Coatomer Protein Complex, Subunit Beta 2 (COPB2). To study the role of Copb2 in neural development, we utilized genome-editing technology to generate an allelic series in the mouse. Two independent null alleles revealed that Copb2 is essential for early stages of embryogenesis. Mice homozygous for the patient variant (Copb2R254C/R254C) appear to have a grossly normal phenotype, likely due to differences in corticogenesis between the two species. Strikingly, mice heterozygous for the patient mutation and a null allele (Copb2R254C/Zfn) show a severe perinatal phenotype including low neonatal weight, significantly increased apoptosis in the brain, and death within the first week of life. Immunostaining of the Copb2R254C/Zfnbrain revealed a reduction in layer V (CTIP2+) neurons, while the overall cell density of the cortex is unchanged. Moreover, neurospheres derived from animals with Copb2 variants grew less than control. These results identify a general requirement for COPB2 in embryogenesis and a specific role in corticogenesis. We further demonstrate the utility of CRISPR-Cas9 generated mouse models in the study of potential pathogenicity of variants of potential clinical interest.

The progesterone receptor (PGR) gene is a key factor in the initiation and maintenance of pregnancy and in embryo development. Currently, it is unknown what variants of the PGR gene are related to fertility traits in cattle. Identification of such variants would allow the implementation of marker-assisted selection in breeding schemes. The objective of this study was to investigate the association of single nucleotide polymorphisms (SNP) of PGR with fertility traits in Holstein dairy cattle. An in vitro fertilization system was used to maximize the efficiency of the identification of genetic factors affecting fertility. This in vitro fertilization system would allow the assessment of fertilization and embryonic survival rates independently of influences from the uterine environment. A total of 5,566 fertilization attempts were performed, and a total of 3,679 embryos were produced using oocytes from 324 Holstein cows and semen from 10 Holstein bulls. Sequencing of pooled DNA samples from ovaries revealed an SNP (G/C) in intron 3 of PGR. A generalized linear model was used to analyze the association of this SNP with fertilization and embryonic survival rates for each ovary. Oocytes obtained from CC ovaries showed a 61% fertilization rate, compared with 68 and 69% for GC and GG ovaries, respectively. The survival rate of embryos produced from GG ovaries was 5 and 6% higher than that of GC and CC ovaries . These results indicate that the PGR SNP could be used in marker-assisted selection breeding programs in Holstein dairy cattle.

Heat shock proteins (HSP) are among the first proteins produced during embryonic development and are crucial to cell function; their activities include folding, unfolding, transport, and localization of proteins and differentiation and regulation of the embryonic cell cycle. Although HSP have been extensively studied in humans and mice, limited information exists on the roles of these genes in bovine embryos. As such, the objectives of this study were to profile the expression of HSP and their splice variants in bovine embryos (degenerates vs. blastocysts) and to carry out association analysis with fertility traits. Quantitative real time PCR analysis revealed differences in expression that ranged from 1.5- to 7.6-fold in degenerate embryos compared with blastocysts. Interestingly, all members of the HSP40 family were found to be upregulated in degenerate embryos. Analysis of current bovine Ensembl data showed that 13 HSP genes have 1 transcript each, 2 genes have 2 transcripts each, and 2 have 3 transcripts each. Some splice variants show differential expression between degenerate embryos and blastocysts, whereas others were not expressed at all in embryos, which implies different functions of these transcripts in embryonic development. The most significant differentially expressed genes were further investigated for association with fertility and development traits. Single nucleotide polymorphisms in DNAJC15 and DNAJC27 were found to be associated with blastocyst rate and fertilization rate, respectively. Collectively, the roles of HSP in fertilization and early development of cattle embryos are important.

We previously reported a mutation in the cholesterol biosynthesis gene, hydroxysteroid (17-beta) dehydrogenase 7 (Hsd17b7rudolph), that results in striking embryonic forebrain dysgenesis. Here we describe abnormal patterns of neuroprogenitor proliferation in the mutant forebrain, namely, a decrease in mitotic cells within the ventricular zone (VZ) and an increase through the remainder of the cortex by E11.5. Further evidence suggests mutant cells undergo abnormal interkinetic nuclear migration (IKNM). Furthermore, intermediate progenitors are increased at the expense of apical progenitors by E12.5, and post-mitotic neurons are expanded by E14.5. In vitro primary neuron culture further supports our model of accelerated cortical differentiation in the mutant. Combined administration of a statin and dietary cholesterol in utero achieved partial reversal of multiple developmental abnormalities in the Hsd17b7rudolph embryo, including the forebrain. These results suggest that abnormally increased levels of specific cholesterol precursors in the Hsd17b7rudolph embryo cause cortical dysgenesis by altering patterns of neurogenesis.

Primary microcephaly is a congenital brain malformation characterized by a head circumference less than three standard deviations below the mean for age and sex and results in moderate to severe mental deficiencies and decreased lifespan. We recently studied two children with primary microcephaly in an otherwise unaffected family. Exome sequencing identified an autosomal recessive mutation leading to an amino acid substitution in a WD40 domain of the highly conserved Coatomer Protein Complex, Subunit Beta 2 (COPB2). To study the role of Copb2 in neural development, we utilized genome editing technology to generate an allelic series in the mouse. Two independent null alleles revealed that Copb2 is essential for early stages of embryogenesis. Mice homozygous for the patient variant (Copb2 R254C/R254C) appear to have a grossly normal phenotype, likely due to differences in corticogenesis between the two species. Strikingly, mice heterozygous for the patient mutation and a null allele (Copb2 R254C/Znf ) show a severe perinatal phenotype including low neonatal weight, significantly increased apoptosis in the brain, and death within the first week of life. Immunostaining of the Copb2 R254C/Znf brain revealed a reduction in layer V (CTIP2 + ) neurons, while the overall cell density of the cortex is unchanged. Moreover, disruption of Copb2 in mouse neurospheres resulted in reduced proliferation. These results identify a general requirement for COPB2 in embryogenesis and a specific role in corticogenesis. We further demonstrate the utility of CRISPR-Cas9 generated mouse models in the study of potential pathogenicity of variants of potential clinical interest.peer-reviewed)