Loss of function mutations in essential genes cause embryonic lethality in pigs.

Lethal recessive alleles cause pre- or postnatal death in homozygous affected individuals, reducing fertility. Especially in small size domestic and wild populations, those alleles might be exposed by inbreeding, caused by matings between related parents that inherited the same recessive lethal allele from a common ancestor. In this study we report five relatively common (up to 13.4% carrier frequency) recessive lethal haplotypes in two commercial pig populations. The lethal haplotypes have a large effect on carrier-by-carrier matings, decreasing litter sizes by 15.1 to 21.6%. The causal mutations are of different type including two splice-site variants (affecting POLR1B and TADA2A genes), one frameshift (URB1), and one missense (PNKP) variant, resulting in a complete loss-of-function of these essential genes. The recessive lethal alleles affect up to 2.9% of the litters within a single population and are responsible for the death of 0.52% of the total population of embryos. Moreover, we provide compelling evidence that the identified embryonic lethal alleles contribute to the observed heterosis effect for fertility (i.e. larger litters in crossbred offspring). Together, this work marks specific recessive lethal variation describing its functional consequences at the molecular, phenotypic, and population level, providing a unique model to better understand fertility and heterosis in livestock.

Conceptus interferon gamma is essential for establishment of pregnancy in the pig†.

Establishment and maintenance of pregnancy in the pig is a complex process that relies on conceptus regulation of the maternal proinflammatory response to endometrial attachment. Following elongation, pig conceptuses secrete interferon gamma (IFNG) during attachment to the endometrial luminal epithelium. The objective here was to determine if conceptus production of IFNG is important for early development and establishment of pregnancy. CRISPR/Cas9 gene editing and somatic cell nuclear transfer technologies were used to create an IFNG loss-of-function study in pigs. Wild-type (IFNG+/+) and null (IFNG-/-) fibroblast cells were used to create embryos through somatic cell nuclear transfer. IFNG expression was not detected in IFNG-/- conceptuses on either day 15 or day 17 of pregnancy. Ablation of conceptus IFNG production resulted in the reduction of stromal CD3+ and mast cells, which localized to the site of conceptus attachment on day 15. The uteri of recipients with IFNG-/- conceptuses were inflamed, hyperemic and there was an abundance of erythrocytes in the uterine lumen associated with the degenerating conceptuses. The endometrial stromal extracellular matrix was altered in the IFNG-/- embryo pregnancies and there was an increased endometrial mRNA levels for collagen XVII (COL17A1), matrilin 1 (MATN1), secreted phosphoprotein 1 (SPP1), and cysteine-rich secretory protein 3 (CRISP3), which are involved with repair and remodeling of the extracellular matrix. These results indicate conceptus IFNG production is essential in modulating the endometrial proinflammatory response for conceptus attachment and survival in pigs.

Temporal and spatial expression of adrenomedullin and its receptors in the porcine uterus and peri-implantation conceptuses.

Adrenomedullin (ADM) is an evolutionarily conserved multifunctional peptide hormone that regulates implantation, embryo spacing, and placentation in humans and rodents. However, the potential roles of ADM in implantation and placentation in pigs, as a litter-bearing species, are not known. This study determined abundances of ADM in uterine luminal fluid, and the patterns of expression of ADM and its receptor components (CALCRL, RAMP2, RAMP3, and ACKR3) in uteri from cyclic and pregnant gilts, as well as conceptuses (embryonic/fetus and its extra-embryonic membranes) during the peri-implantation period of pregnancy. Total recoverable ADM was greater in the uterine fluid of pregnant compared with cyclic gilts between Days 10 and 16 post-estrus and was from uterine luminal epithelial (LE) and conceptus trophectoderm (Tr) cells. Uterine expression of CALCRL, RAMP2, and ACKR3 were affected by day (P < 0.05), pregnant status (P < 0.01) and/or day x status (P < 0.05). Within porcine conceptuses, the expression of CALCRL, RAMP2, and ACKR3 increased between Days 10 and 16 of pregnancy. Using an established porcine trophectoderm (pTr1) cell line, it was determined that 10-7 M ADM stimulated proliferation of pTr1 cells (P < 0.05) at 48 h, and increased phosphorylated mechanistic target of rapamycin (p-MTOR) and 4E binding protein 1 (p-4EBP1) by 6.1- and 4.9-fold (P < 0.0001), respectively. These novel results indicate a significant role for ADM in uterine receptivity for implantation and conceptus growth and development in pigs. They also provide a framework for future studies of ADM signaling to affect proliferation and migration of Tr cells, spacing of blastocysts, implantation, and placentation in pigs.

Disrupting porcine glutaminase does not block preimplantation development and elongation nor decrease mTORC1 activation in conceptuses†.

Elongation of pig conceptuses is a dynamic process, requiring adequate nutrient provisions. Glutamine is used as an energy substrate and is involved in the activation of mechanistic target of rapamycin complex 1 (mTORC1) during porcine preimplantation development. However, the roles of glutamine have not been extensively studied past the blastocyst stage. Therefore, the objective of the current study was to determine if glutaminase (GLS), which is the rate-limiting enzyme in glutamine metabolism, was necessary for conceptus elongation to proceed and was involved in mTORC1 activation. The CRISPR/Cas9 system was used to induce loss-of-function mutations in the GLS gene of porcine fetal fibroblasts. Wild type (GLS+/+) and knockout (GLS-/-) fibroblasts were used as donor cells for somatic cell nuclear transfer, and GLS+/+ and GLS-/- blastocyst-stage embryos were transferred into surrogates. On day 14 of gestation, GLS+/+ conceptuses primarily demonstrated filamentous morphologies, and GLS-/- conceptuses exhibited spherical, ovoid, tubular, and filamentous morphologies. Thus, GLS-/- embryos were able to elongate despite the absence of GLS protein and minimal enzyme activity. Furthermore, spherical GLS-/- conceptuses had increased abundance of transcripts related to glutamine and glutamate metabolism and transport compared to filamentous conceptuses of either genotype. Differences in phosphorylation of mTORC1 components and targets were not detected regarding conceptus genotype or morphology, but abundance of two transcriptional targets of mTORC1, cyclin D1, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha was increased in spherical conceptuses. Therefore, porcine GLS is not essential for conceptus elongation and is not required for mTORC1 activation at this developmental timepoint.

Tauroursodeoxycholic acid/TGR5 signaling promotes survival and early development of glucose-stressed porcine embryos†.

Conditions of impaired energy and nutrient homeostasis, such as diabetes and obesity, are associated with infertility. Hyperglycemia increases endoplasmic reticulum stress as well as oxidative stress and reduces embryo development and quality. Oxidative stress also causes deoxyribonucleic acid damage, which impairs embryo quality and development. The natural bile acid tauroursodeoxycholic acid reduces endoplasmic reticulum stress and rescues developmentally incompetent late-cleaving embryos, as well as embryos subjected to nuclear stress, suggesting the endoplasmic reticulum stress response, or unfolded protein response, and the genome damage response are linked. Tauroursodeoxycholic acid acts via the Takeda-G-protein-receptor-5 to alleviate nuclear stress in embryos. To evaluate the role of tauroursodeoxycholic acid/Takeda-G-protein-receptor-5 signaling in embryo unfolded protein response, we used a model of glucose-induced endoplasmic reticulum stress. Embryo development was impaired by direct injection of tauroursodeoxycholic acid into parthenogenetically activated oocytes, whereas it was improved when tauroursodeoxycholic acid was added to the culture medium. Attenuation of the Takeda-G-protein-receptor-5 precluded the positive effect of tauroursodeoxycholic acid supplementation on development of parthenogenetically activated and fertilized embryos cultured under standard conditions and parthenogenetically activated embryos cultured with excess glucose. Moreover, attenuation of tauroursodeoxycholic acid/Takeda-G-protein-receptor-5 signaling induced endoplasmic reticulum stress, oxidative stress and cell survival genes, but decreased expression of pluripotency genes in parthenogenetically activated embryos cultured under excess glucose conditions. These data suggest that Takeda-G-protein-receptor-5 signaling pathways link the unfolded protein response and genome damage response. Furthermore, this study identifies Takeda-G-protein-receptor-5 signaling as a potential target for mitigating fertility issues caused by nutrient excess-associated blastomere stress and embryo death.

Inhibition of Suv39h1/2 expression improves the early development of Debao porcine somatic cell nuclear transfer embryos.

Suppressor of variegation 3-9 homolog (Suv39h)1 and 2, Histone H3 lysine 9 trimethylation (H3K9me3)-specific methyltransferases, are mainly involved in regulating the dynamic changes of H3K9me3. Regulating Suv39h expression influences the early development of mice somatic cell nuclear transfer (SCNT) embryos, there are few reports concerning their features in domestic animals. The aim of the present study was to characterize the Suv39h function in early development of Debao porcine SCNT embryos. The global level of H3K9me3 and the expression profiles of Suv39h1/2 in porcine early embryos were analysed by immunohistochemistry and qRT-PCR methods, respectively. Their roles in cell proliferation and histone modification of Debao porcine foetal fibroblast cells (PFFs), and developmental competence of porcine SCNT embryos were investigated by shRNA technology. The methylation levels of H3K9me3 and the expression patterns of Suv39h1 and Suv39h2 were similar (p < .05), and both of them displayed higher levels in Debao porcine SCNT embryos compared with that in PA embryos. The global levels of H3K9me3 and the expressions of G9a, HDAC1 and DNMT1 were decreased by combined inhibition of Suv39h1 and Suv39h2 (p < .05), while the expression of HAT1 was increased (p < .05). Downregulation of Suv39h1/2 also promoted cell proliferation and resulted in a significant increase in the expression of CyclinA2, CyclinB and PCNA in PFFs (p < .05). Furthermore, the use of donor somatic nuclei which depleted H3K9me3 by inhibiting Suv39h1/2 expression markedly increased the cleavage rate, the blastocyst rate and the total cell number of blastocysts of Debao porcine SCNT embryos (p < .05). Altogether, the above results indicate that H3K9me3 levels and Suv39h1/2 expressions display similar patterns in porcine early embryo, and low levels of them are critical to cell proliferation of PFFs and early development of SCNT embryos.

Transcriptomic analysis of the porcine anterior pituitary gland during the peri-implantation period.

The peri-implantation period is controlled by signals originating from hypothalamic-pituitary-ovarian axis, uterus and developing embryos. The transcriptomic activity of the anterior pituitary gland may be important for the control of the peri-implantation period. The aim of this study was to determine the alternations in the transcriptomic profile of porcine anterior pituitary gland during the peri-implantation period (days 15-16 of pregnancy) in comparison with established for the respective days of the oestrous cycle. Analysis using a microarray approach indicated that the 651 genes (fold-change ˂1.2; p ≤ .05) were differentially expressed (DEGs) in the anterior pituitary of pigs during the peri-implantation period when compared to cyclic females. Of these DEGs, 404 were upregulated and 247 downregulated. Analysis of occurred relationships among DEGs revealed that some of them are involved in steroid-response and oestrogen synthesis, FSH secretion, immune response, PPAR signalling pathway and the potential for DNA methylation. In conclusion, the altered transcriptomic profile of the porcine pituitary gland in pigs during the peri-implantation period indicates the role of embryos presence in the creation of transcriptomic activity of the pituitary gland in pigs.

Butylparaben Is Toxic to Porcine Oocyte Maturation and Subsequent Embryonic Development Following In Vitro Fertilization.

Parabens are widely used in personal care products due to their antimicrobial effects. Although the toxicity of parabens has been reported, little information is available on the toxicity of butylparaben (BP) on oocyte maturation. Therefore, we investigated the effects of various concentrations of BP (0 µM, 100 µM, 200 µM, 300 µM, 400 µM, and 500 µM) on the in vitro maturation of porcine oocytes. BP supplementation at a concentration greater than 300 µM significantly reduced the proportion of complete cumulus cell expansion and metaphase II oocytes compared to the control. The 300 µM BP significantly decreased fertilization, cleavage, and blastocyst formation rates with lower total cell numbers and a higher rate of apoptosis in blastocysts compared to the control. The BP-treated oocytes showed significantly higher reactive oxygen species (ROS) levels, and lower glutathione (GSH) levels than the control. BP significantly increased the aberrant mitochondrial distribution and decreased mitochondrial function compared to the control. BP-treated oocytes exhibited significantly higher percentage of γ-H2AX, annexin V-positive oocytes and expression of LC3 than the control. In conclusion, we demonstrated that BP impaired oocyte maturation and subsequent embryonic development, by inducing ROS generation and reducing GSH levels. Furthermore, BP disrupted mitochondrial function and triggered DNA damage, early apoptosis, and autophagy in oocytes.

Gene ontology analysis of expanded porcine blastocysts from gilts fed organic or inorganic selenium combined with pyridoxine.

BACKGROUND: Gene ontology analysis using the microarray database generated in a previous study by this laboratory was used to further evaluate how maternal dietary supplementation with pyridoxine combined with different sources of selenium (Se) affected global gene expression of expanded porcine blastocysts. Data were generated from 18 gilts randomly assigned to one of three experimental diets (n = 6 per treatment): i) basal diet without supplemental Se or pyridoxine (CONT); ii) CONT + 0.3 mg/kg of Na-selenite and 10 mg/kg of HCl-pyridoxine (MSeB610); and iii) CONT + 0.3 mg/kg of Se-enriched yeast and 10 mg/kg of HCl-pyridoxine (OSeB610). All gilts were inseminated at their fifth post-pubertal estrus and euthanized 5 days later for embryo harvesting. Differential gene expression between MSeB610 vs CONT, OSeB610 vs CONT and OSeB610 vs MSeB610 was performed using a porcine embryo-specific microarray. RESULTS: There were 559, 2458, and 1547 differentially expressed genes for MSeB610 vs CONT, OSeB610 vs CONT and OSeB610 vs MSeB610, respectively. MSeB610 vs CONT stimulated 13 biological processes with a strict effect on RNA binding and translation initiation. OSeB610 vs CONT and OSeB610 vs MSeB610 impacted 188 and 66 biological processes, respectively, with very similar effects on genome stability, ceramide biosynthesis, protein trafficking and epigenetic events. The stimulation of genes related with these processes was confirmed by quantitative real-time RT-PCR. CONCLUSIONS: Gene expression of embryos from OSeB610 supplemented gilts was more impacted than those from MSeB610 supplemented gilts. Whereas maternal OSeB610 supplementation influenced crucial aspects of embryo development, maternal MSeB610 supplementation was restricted to binding activity.

Iloprost supports early development of in vitro-produced porcine embryos through activation of the phosphatidylinositol 3-kinase/AKT signalling pathway.

Despite evidence of the presence of prostaglandin (PG) I2 in mammalian oviducts, its role in early development of in vitro-produced (IVP) embryos is largely unknown. Thus, in the present study we examined the effects of iloprost, a PGI2 analogue, on the in vitro developmental competence of early porcine embryos and the underlying mechanism(s). To examine the effects of iloprost on the development rate of IVF embryos, iloprost was added to the in vitro culture (IVC) medium and cultured for 6 days. Supplementation of the IVC medium with iloprost significantly improved developmental parameters, such as blastocyst formation rate, the trophectoderm:inner cell mass ratio and cell survival in IVF and parthenogenetically activated (PA) embryos. In addition, post-blastulation development into the expanded blastocyst stage was improved in iloprost-treated groups compared with controls. Interestingly, the phosphatidylinositol 3-kinase (PI3K)/AKT signalling pathway was significantly activated by iloprost supplementation in a concentration-dependent manner (10-1000nM), and the beneficial effects of iloprost on the early development of porcine IVF and PA embryos was completely ablated by treatment with 2.5µM wortmannin, a PI3K/AKT signalling inhibitor. Importantly, expression of the PI3K/AKT signalling pathway was significantly reduced in somatic cell nuclear transfer (SCNT) compared with IVF embryos, and iloprost supported the early development of SCNT embryos, as was the case for IVF and PA embryos, suggesting a consistent effect of iloprost on the IVC of IVP porcine embryos. Together, these results indicate that iloprost can be a useful IVC supplement for production of IVP early porcine embryos with high developmental competence.

Supplement of autologous ooplasm into porcine somatic cell nuclear transfer embryos does not alter embryo development.

Somatic cell nuclear transfer (SCNT) is considered as the technique in which a somatic cell is introduced into an enucleated oocyte to make a cloned animal. However, it is unavoidable to lose a small amount of the ooplasm during enucleation step during SCNT procedure. The present study was aimed to uncover whether the supplement of autologous ooplasm could ameliorate the oocyte competence so as to improve low efficiency of embryo development in porcine SCNT. Autologous ooplasm-transferred (AOT) embryos were generated by the supplementation with autologous ooplasm into SCNT embryos. They were comparatively evaluated with respect to embryo developmental potential, the number of apoptotic body formation and gene expression including embryonic lineage differentiation, apoptosis, epigenetics and mitochondrial activity in comparison with parthenogenetic, in vitro-fertilized (IVF) and SCNT embryos. Although AOT embryos showed perfect fusion of autologous donor ooplasm with recipient SCNT embryos, the supplement of autologous ooplasm could not ameliorate embryo developmental potential in regard to the rate of blastocyst formation, total cell number and the number of apoptotic body. Furthermore, overall gene expression of AOT embryos was presented with no significant alterations in comparison with that of SCNT embryos. Taken together, the results of AOT demonstrated inability to make relevant values improved from the level of SCNT embryos to their IVF counterparts.

Peri-conceptional under-nutrition alters the expression of TRIM28 and ZFP57 in the endometrium and embryos during peri-implantation period in domestic pigs.

DNA methylation is maintained by the main elements of methylation complex-tripartite motif containing 28 (TRIM28) and zinc finger protein 57 (ZFP57). Previously, it was found that the activity of TRIM28 and ZFP57 determines the process of DNA methylation and preserves over-expression of genes. We hypothesized that restricted diet applied during peri-conceptional period may induce changes in the expression of methylation complex in porcine endometrium and embryos during the peri-implantation period. The aim of this study was to detect and determine the expression of TRIM28 and ZFP57 in the endometrium and embryos harvested from gilts during the peri-implantation period (days 15-16 of pregnancy) fed restricted (n = 5) or normal (n = 5) diet during peri-conceptional period. In restricted-diet-fed gilts, endometrial expression of TRIM28 and ZFP57 mRNAs was decreased in comparison with normal-diet-fed gilts (p ≤ .01), while the embryonic expression of TRIM28 and ZFP57 mRNAs was increased in restricted-diet-fed gilts (p ≤ .05). The immunofluorescence showed the presence of TRIM28 and ZFP57 in luminal epithelial (LE), glandular epithelial (GE) and stromal cells (ST) of the endometrium as well as in the embryos. Total endometrial and embryonic abundance of TRIM28 and ZFP57 proteins was significantly higher (p ≤ .05) in restricted-diet-fed gilts than in normal-diet-fed gilts. Female under-nutrition during peri-conceptional period affects the expression of two main elements of methylation complex in the endometrium and in embryos during the peri-implantation period and may have the impact on DNA methylation in these tissues.

Expressed alleles of imprinted IGF2, DLK1 and MEG3 colocalize in 3D-preserved nuclei of porcine fetal cells.

BACKGROUND: To explore the relationship between spatial genome organization and gene expression in the interphase nucleus, we used a genomic imprinting model, which offers parental-specific gene expression. Using 3D FISH in porcine fetal liver cells, we compared the nuclear organization of the two parental alleles (expressed or not) of insulin-like growth factor 2 (IGF2), a paternally imprinted gene located on chromosome 2. We investigated whether its nuclear positioning favors specific locus associations. We also tested whether IGF2 is implicated in long-range chromatin trans-associations as previously shown in the mouse model species for its reciprocal imprinted gene H19. RESULTS: We focused on the 3D position of IGF2 alleles, with respect to their individual chromosome 2 territories. The paternally expressed allele was tagged with nascent RNA. There were no significant differences in the position of the two alleles (p = 0.06). To determine long-range chromatin trans-interactions, we chose 12 genes, some of which are known to be imprinted in mammalian model species and belong to a network of imprinted genes (i.e. SLC38A4, DLK1, MEG3, and ZAC1). We screened them and ABCG2, OSBP2, OSBPL1, RPL32, NF1, ZAR1, SEP15, GPC3 for associations with IGF2 in liver cells. All imprinted genes tested showed an association with IGF2. The DLK1/MEG3 locus showed the highest rate of colocalization. This gene association was confirmed by 3D FISH (in 20 % of the nuclei analyzed), revealing also the close proximity of chromosomes 2 and 7 (in 60 % of nuclei). Furthermore, our observations showed that the expressed paternal IGF2 allele is involved in this association. This IGF2-(DLK1/MEG3) association also occurred in a high percentage of fetal muscle cells (36 % of nuclei). Finally, we showed that nascent IGF2, DLK1 and MEG3 RNAs can associate in pairs or in a three-way combination. CONCLUSION: Our results show that trans-associations occur between three imprinted genes IGF2, DLK1 and MEG3 both in fetal liver and muscle cells. All three expressed alleles associated in muscle cells. Our findings suggest that the 3D nuclear organization is linked to the transcriptional state of these genes.

Left-right asymmetry: lessons from Cancún.

The satellite symposium on 'Making and breaking the left-right axis: implications of laterality in development and disease' was held in June 2013 in conjunction with the 17th International Society for Developmental Biology meeting in Cancún, Mexico. As we summarize here, leaders in the field gathered at the symposium to discuss recent advances in understanding how left-right asymmetry is generated and utilized across the animal kingdom.

Altered expression of chemokines and their receptors at porcine maternal-fetal interface during early and mid-gestational fetal loss.

Chemokines play a significant role in pregnancy, especially during embryonic attachment and placental development. During early pregnancy, immune cells are recruited extensively to the endometrium in several species including pigs. However, this recruitment is solely mediated by the presence of the conceptus in pigs making it a unique feature compared with other species (humans, primates and mice). To understand the biological significance of chemokine expression and immune cell recruitment in the context of fetal loss, we investigate a well-characterized porcine fetal loss model during the window of early pregnancy at gestational day (gd) 20 and mid-pregnancy (gd50). These periods coincide with 25-40 % of conceptus loss. Using targeted quantitative polymerase chain reaction and Western blot approaches, we screened a specific set of chemokines. Comparisons were made with endometrial lymphocytes (ENDO LY), endometrium and chorioallantoic membranes (CAM) associated with spontaneously arresting and healthy conceptus attachment sites (CAS). mRNA expression studies revealed an increased expression of CXCR3 and CCR5 in ENDO LY and of CXCL10, CXCR3, CCL5 and CCR5 in the endometrium associated with arresting CAS at gd20. DARC was decreased in the endometrium at gd50. CCL1 was increased in CAM associated with arresting CAS at gd50. Some of these differences were also noted at the protein level (CXCL10, CXCR3, CCL5 and CCR5) in the endometrium and CAM. CD45+ immunohistochemistry demonstrated a significantly higher localization in ENDO LY in the endometrium associated with healthy versus arresting counterparts. Most of these differences were observed in early pregnancy and might contribute towards a shift in immune cell functions.

Histone H3 lysine 27 trimethylation acts as an epigenetic barrier in porcine nuclear reprogramming.

Aberrant epigenetic reprogramming is the main obstacle to the development of somatic cell nuclear transfer (SCNT) embryos and the generation of induced pluripotent stem (iPS) cells, which results in the low reprogramming efficiencies of SCNT and iPS. Histone H3 lysine 27 trimethylation (H3K27me3), as a repressive epigenetic mark, plays important roles in mammalian development and iPS induction. However, the reprogramming of H3K27me3 in pig remains elusive. In this study, we showed that H3K27me3 levels in porcine early cloned embryos were higher than that in IVF embryos. Then GSK126 and GSK-J4, two small molecule inhibitors of H3K27me3 methylase (EZH2) and demethylases (UTX/JMJD3), were used to regulate the H3K27me3 level. The results showed that H3K27me3 level was reduced in cloned embryos after treatment of PEF with 0.75 µM GSK126 for 48 h, incubation of one-cell reconstructed oocytes with 0.1 µM GSK126 and injection of antibody for EZH2 into oocyte. Meanwhile, the development of the cloned embryos was significantly improved after these treatments. On the contrary, GSK-J4 treatment increased the H3K27me3 level in cloned embryos and decreased the cloned embryonic development. Furthermore, iPS efficiency was both increased after reducing the H3K27me3 level in donor cells and in early reprogramming phase. In summary, our results suggest that H3K27me3 acts as an epigenetic barrier in SCNT and iPS reprogramming, and reduction of H3K27me3 level in donor cells and in early reprogramming phase can enhance both porcine SCNT and iPS efficiency.

The relationship between mitochondrial DNA haplotype and the reproductive capacity of domestic pigs (Sus scrofa domesticus).

BACKGROUND: The maternally inherited mitochondrial genome encodes key proteins of the electron transfer chain, which produces the vast majority of cellular ATP. Mitochondrial DNA (mtDNA) present in the mature oocyte acts as a template for all mtDNA that is replicated during development to meet the specific energy requirements of each tissue. Individuals that share a maternal lineage cluster into groupings known as mtDNA haplotypes. MtDNA haplotypes confer advantages and disadvantages to an organism and this affects its phenotype. In livestock, certain mtDNA haplotypes are associated with improved milk and meat quality, whilst, other species, mtDNA haplotypes have shown increased longevity, growth and susceptibility to diseases. In this work, we have set out to determine whether mtDNA haplotypes influence reproductive capacity. This has been undertaken using a pig model. RESULTS: To determine the genetic diversity of domestic pigs in Australia, we have sequenced the D-loop region of 368 pigs, and identified five mtDNA haplotypes (A to E). To assess reproductive capacity, we compared oocyte maturation, fertilization and development to blastocyst, and found that there were significant differences for maturation and fertilization amongst the haplotypes. We then determined that haplotypes C, D and E produced significantly larger litters. When we assessed the conversion of developmentally competent oocytes and their subsequent developmental stages to offspring, we found that haplotypes A and B had the lowest reproductive efficiencies. Amongst the mtDNA haplotypes, the number of mtDNA variants harbored at >25 % correlated with oocyte quality. MtDNA copy number for developmentally competent oocytes positively correlated with the level of the 16383delC variant. This variant is located in the conserved sequence box II, which is a regulatory region for mtDNA transcription and replication. CONCLUSIONS: We have identified five mtDNA haplotypes in Australian domestic pigs indicating that genetic diversity is restricted. We have also shown that there are differences in reproductive capacity amongst the mtDNA haplotypes. We conclude that mtDNA haplotypes affect pig reproductive capacity and can be used as a marker to complement current selection methods to identify productive pigs.

SMAD7, an antagonist of TGF-beta signaling, is a candidate of prenatal skeletal muscle development and weaning weight in pigs.

SMAD7 promotes and enhances skeletal muscle differentiation by inhibiting transforming growth factor beta (TGF-ß)/activin signaling and bone morphogenetic protein (BMP) pathways. However, its function, the mechanism regulating its translation, and its association with production meat traits remain unclear in pigs. In this study, we explored SMAD7 gene spatio-temporal and tissue distribution, conducted a single nucleotide polymorphism association analysis, and examined regulation of its expression during skeletal muscle development. We found that SMAD7 was positively related to TGF-ß pathway genes and mainly expressed in prenatal developing muscle, and dual luciferase and western blot assays demonstrated that SMAD7 expression was regulated by miRNA-21 at the protein level via inhibition of mRNA translation. Finally, the association analysis showed that a single nucleotide mutation (Exon 4_28816;C/A) was significantly associated with the weaning weight of piglets among Yorkshire pigs. These data indicate that SMAD7 plays a potentially important role in mammalian prenatal skeletal muscle development and is a candidate gene for promoting greater weaning weight in pig breeding.

Islet xenotransplantation: what is the optimal age of the islet-source pig?

BACKGROUND: The need for pig islet xenotransplantation in patients with type 1 diabetes is compelling; however, the ideal age at which islets should be isolated from the donor pig remains uncertain. Pig islet transplantation in primates, as a valuable pre-clinical model, has been explored using adult, neonatal, fetal pig islets, and also pancreatic primordia from pig embryos as beta cell donors. Neonatal pig islets have some advantages over adult and fetal islets, but the optimal age within the first month of life at which neonatal islets should be isolated and transplanted is as yet unclear. METHODS: In an attempt to answer this question, we carried out a literature search, but limited the search primarily to evidence in the clinically-relevant pig-to-non-human primate model. RESULTS: We identified surprisingly few studies in this model directed to this topic. Even in pig-to-rodent models, there were few definitive data. CONCLUSION: From the few data available to us, we conclude that pancreatectomy and islet isolation from neonatal pigs may have advantages over adult pigs and that isolation during the first week of life may have minor advantages over later weeks.

Dynamic changes in nuclear import of a nuclear localisation signal-bearing substrate in 8-cell stage porcine embryos.

Coordinated intracellular trafficking is critically important for proper timing of major cellular events during embryogenesis. Nuclear import mediated by the karyopherin α/ß (importin α/ß) heterodimer is perhaps the best characterised nuclear trafficking system in eukaryotic cells. Seven karyopherin α subtypes have been identified in the domestic pig, and although each karyopherin α subtype transports proteins bearing classical nuclear localisation signals (NLSs), individual karyopherin α subtypes have been shown to preferentially transport specific cargoes. The aim of the present study was to determine the mechanism by which BRN2, a transcription factor previously reported to be transported by the karyopherin α/ß heterodimer, gains access to the nucleus in porcine oocytes and embryos. Using a combination of in vivo and in vitro assays, we tested the hypothesis that discrete karyopherin α subtypes transport BRN2 into the nuclei of porcine oocytes and cleavage stage embryos. Our results show that ectopically expressed BRN2 adopts a nuclear localisation in all nuclei through the 4-cell stage of development, whereas only a subset of blastomeres in 8-cell stage embryos possess nuclear BRN2. This pattern is unique to BRN2 because another ectopically expressed NLS-containing protein is able to adopt a nuclear localisation in all blastomeres of 8-cell stage embryos.