ABSTRACT
Background: Presenilin 1 (PSEN1) is one of the genes linked to the prevalence of early onset Alzheimer's disease. In mice, inactivation of Psen1 leads to developmental defects, including vertebral malformation and neural development. However, little is known about the role of PSEN1 during the development in other species. Objective: To investigate the role of PSEN1 in vertebral development and the pathogenic mechanism of neurodegeneration using a pig model. Methods: CRISPR/Cas9 system was used to generate pigs with different mutations flanking exon 9 of PSEN1, including those with a deleted exon 9 (Δexon9). Vertebral malformations in PSEN1 mutant pigs were examined by X-ray, micro-CT and micro-MRI. Neuronal cells from the brains of PSEN1 mutant pigs were analyzed by immunoflourescence, followed by image analysis including morphometric evaluation via image J and 3D reconstruction. Results: Pigs with a PSEN1 null mutation (Δexon9-12) died shortly after birth and had significant axial skeletal defects, whereas pigs carrying at least one Δexon9 allele developed normally and remained healthy. Effects of the null mutation on abnormal skeletal development were also observed in fetuses at day 40 of gestation. Abnormal distribution of astrocytes and microglia in the brain was detected in two PSEN1 mutant pigs examined compared to age-matched control pigs. The founder pigs were bred to establish and age PSEN1ΔE9/+ pigs to study their relevance to clinical Alzheimer's diseases. Conclusions: PSEN1 has a critical role for normal vertebral development and PSEN1 mutant pigs serves as novel resources to study Alzheimer's disease.
ABSTRACT
Conceptus estrogens and prostaglandins have long been considered the primary signals for maternal recognition of pregnancy (MRP) in the pig. However, loss-of-function studies targeting conceptus aromatase genes (CYP19A1 and CYP19A2) and prostaglandin-endoperoxide synthase 2 (PTGS2) indicated that conceptuses can not only signal MRP without estrogens or prostaglandins but can maintain early pregnancy. However, complete loss of estrogen production leads to abortion after day 25 of gestation. Although neither conceptus estrogens nor prostaglandins had a significant effect on early maintenance of CL function alone, the two conceptus factors have a biological relationship. To investigate the role that both conceptus estrogens and prostaglandins have on MRP and maintenance of pregnancy, a triple loss-of function model (TKO) was generated for conceptus CYP19A1, CYP19A2 and PTGS2. In addition, a conceptus CYP19A2-/- model (A2KO) was established to determine the role of placental estrogen during later pregnancy. Estrogen and prostaglandin synthesis were greatly reduced in TKO conceptuses which resulted in a failure to inhibit luteolysis after day 15 of pregnancy despite the presence of conceptuses in the uterine lumen. However, A2KO placentae not only maintained functional CL but were able to maintain pregnancy to day 32 of gestation. Despite the loss of placental CYP19A2 expression, the allantois fluid content of estrogen was not affected as the placenta compensated by expressing CYP19A1 and CYP19A3, which are normally absent in controls. Results suggest conceptuses can signal MRP through production of conceptus PGE or stimulating PGE synthesis from the endometrium through conceptus estrogen. Failure of conceptuses to produce both factors results in failure of MRP and loss of pregnancy.
ABSTRACT
COVID-19 impacted abattoirs worldwide. The processing lines became a hotspot for the spread of COVID-19 resulting in plant restructuring and ultimately a critical loss of pig material for research. Commercial sources of pig oocytes are available but are costly and companies were already operating at a maximum capacity for supplying the oocyte needs around the United States. Here, we provide an alternative source of oocytes that are competent to produce live, healthy piglets.
Subject(s)
COVID-19 , Swine Diseases , Female , Animals , Swine , Ovary , Ovarian Follicle , Oocytes , Oocyte Retrieval/veterinary , COVID-19/veterinary , Cumulus Cells , In Vitro Oocyte Maturation Techniques/veterinary , In Vitro Oocyte Maturation Techniques/methodsABSTRACT
STUDY QUESTION: Does a chemically defined maturation medium supplemented with FGF2, LIF, and IGF1 (FLI) improve in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) obtained from children, adolescents, and young adults undergoing ovarian tissue cryopreservation (OTC)? SUMMARY ANSWER: Although FLI supplementation did not increase the incidence of oocyte meiotic maturation during human IVM, it significantly improved quality outcomes, including increased cumulus cell expansion and mitogen-activated protein kinase (MAPK) expression as well as enhanced transzonal projection retraction. WHAT IS KNOWN ALREADY: During OTC, COCs, and denuded oocytes from small antral follicles are released into the processing media. Recovery and IVM of these COCs is emerging as a complementary technique to maximize the fertility preservation potential of the tissue. However, the success of IVM is low, especially in the pediatric population. Supplementation of IVM medium with FLI quadruples the efficiency of pig production through improved oocyte maturation, but whether a similar benefit occurs in humans has not been investigated. STUDY DESIGN, SIZE, DURATION: This study enrolled 75 participants between January 2018 and December 2021 undergoing clinical fertility preservation through the Fertility & Hormone Preservation & Restoration Program at the Ann & Robert H. Lurie Children's Hospital of Chicago. Participants donated OTC media, accumulated during tissue processing, for research. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants who underwent OTC and include a pediatric population that encompassed children, adolescents, and young adults ≤22 years old. All participant COCs and denuded oocytes were recovered from media following ovarian tissue processing. IVM was then performed in either a standard medium (oocyte maturation medium) or one supplemented with FLI (FGF2; 40 ng/ml, LIF; 20 ng/ml, and IGF1; 20 ng/ml). IVM outcomes included meiotic progression, cumulus cell expansion, transzonal projection retraction, and detection of MAPK protein expression. MAIN RESULTS AND THE ROLE OF CHANCE: The median age of participants was 6.3 years, with 65% of them classified as prepubertal by Tanner staging. Approximately 60% of participants had been exposed to chemotherapy and/or radiation prior to OTC. On average 4.7 ± 1 COCs and/or denuded oocytes per participant were recovered from the OTC media. COCs (N = 41) and denuded oocytes (N = 29) were used for IVM (42 h) in a standard or FLI-supplemented maturation medium. The incidence of meiotic maturation was similar between cohorts (COCs: 25.0% vs 28.6% metaphase II arrested eggs in Control vs FLI; denuded oocytes: 0% vs 5.3% in Control vs FLI). However, cumulus cell expansion was 1.9-fold greater in COCs matured in FLI-containing medium relative to Controls and transzonal projection retraction was more pronounced (2.45 ± 0.50 vs 1.16 ± 0.78 projections in Control vs FLIat 16 h). Additionally, MAPK expression was significantly higher in cumulus cells obtained from COCs matured in FLI medium for 16-18 h (chemiluminescence corrected area 621,678 vs 2,019,575 a.u., P = 0.03). LIMITATIONS, REASONS FOR CAUTION: Our samples are from human participants who exhibited heterogeneity with respect to age, diagnosis, and previous treatment history. Future studies with larger sample sizes, including adult participants, are warranted to determine the mechanism by which FLI induces MAPK expression and activation. Moreover, studies that evaluate the developmental competence of eggs derived from FLI treatment, including assessment of embryos as outcome measures, will be required prior to clinical translation. WIDER IMPLICATIONS OF THE FINDINGS: FLI supplementation may have a conserved beneficial effect on IVM for children, adolescents, and young adults spanning the agricultural setting to clinical fertility preservation. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Department of Obstetrics and Gynecology startup funds (F.E.D.), Department of Surgery Faculty Practice Plan Grant and the Fertility & Hormone Preservation & Restoration Program at the Ann & Robert H. Lurie Children's Hospital of Chicago (M.M.L. and E.E.R.). M.M.L. is a Gesualdo Foundation Research Scholar. Y.Y.'s research is supported by the internal research funds provided by Colorado Center of Reproductive Medicine. Y.Y., L.D.S., R.M.R., and R.S.P. have a patent pending for FLI. The remaining authors have no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.
Subject(s)
Fibroblast Growth Factor 2 , In Vitro Oocyte Maturation Techniques , Pregnancy , Female , Adolescent , Humans , Child , Animals , Swine , Young Adult , Adult , Fibroblast Growth Factor 2/metabolism , Oocytes/metabolism , Hormones , Dietary Supplements , Insulin-Like Growth Factor I/metabolismABSTRACT
Growth differentiation factor 9 (GDF9) is a secreted protein belonging to the transforming growth factor beta superfamily and has been well characterized for its role during folliculogenesis in the ovary. Although previous studies in mice and sheep have shown that mutations in GDF9 disrupt follicular progression, the exact role of GDF9 in pigs has yet to be elucidated. The objective of this study was to understand the role of GDF9 in ovarian function by rapidly generating GDF9 knockout (GDF9-/-) pigs by using the CRISPR/Cas9 system. Three single-guide RNAs designed to disrupt porcine GDF9 were injected with Cas9 mRNA into zygotes, and blastocyst-stage embryos were transferred into surrogates. One pregnancy was sacrificed on day 100 of gestation to investigate the role of GDF9 during oogenesis. Four female fetuses were recovered with one predicted to be GDF9-/- and the others with in-frame mutations. All four had fully formed oocytes within primordial follicles, confirming that knockout of GDF9 does not disrupt oogenesis. Four GDF9 mutant gilts were generated and were grown past puberty. One gilt was predicted to completely lack functional GDF9 (GDF9-/-), and the gilt never demonstrated standing estrus and had a severely underdeveloped reproductive tract with large ovarian cysts. Further examination revealed that the follicles from the GDF9-/- gilt did not progress past preantral stages, and the uterine vasculature was less extensive than the control pigs. By using the CRISPR/Cas9 system, we demonstrated that GDF9 is a critical growth factor for proper ovarian development and function in pigs.
Subject(s)
Growth Differentiation Factor 9 , Ovarian Follicle , Animals , Female , Mice , Bone Morphogenetic Protein 15/genetics , Bone Morphogenetic Protein 15/metabolism , Growth Differentiation Factor 9/genetics , Growth Differentiation Factor 9/metabolism , Oocytes/metabolism , Ovarian Follicle/metabolism , Ovary/metabolism , Sexual Maturation , Sheep , SwineABSTRACT
The pig is an ideal model system for studying human development and disease due to its similarities to human anatomy, physiology, size, and genome. Further, advances in CRISPR gene editing have made genetically engineered pigs viable models for the study of human pathologies and congenital anomalies. However, a detailed atlas illustrating pig development is necessary for identifying and modeling developmental defects. Here we describe normal development of the pig abdominal system and show examples of congenital defects that can arise in CRISPR gene edited SAP130 mutant pigs. Normal pigs at different gestational ages from day 20 (D20) to term were examined and the configuration of the abdominal organs was studied using 3D histological reconstructions with episcopic confocal microscopy, magnetic resonance imaging (MRI) and necropsy. This revealed prominent mesonephros, a transient embryonic organ present only during embryogenesis, at D20, while the developing metanephros that will form the permanent kidney are noted at D26. By D64 the mesonephroi are absent and only the metanephroi remain. The formation of the liver and pancreas was observed by D20 and complete by D30 and D35 respectively. The spleen and adrenal glands are first identified at D26 and completed by D42. The developing bowel and the gonads are identified at D20. The bowel appears completely rotated by D42, and testes in the male were descended at D64. This atlas and the methods used are excellent tools for identifying developmental pathologies of the abdominal organs in the pig at different stages of development.
Subject(s)
Gene Editing , Kidney , Abdomen/diagnostic imaging , Animals , Clustered Regularly Interspaced Short Palindromic Repeats , Gene Editing/methods , Genetic Engineering , Humans , Male , SwineABSTRACT
Senecavirus A (SVA) is a cause of vesicular disease in pigs, and infection rates are rising within the swine industry. Recently, anthrax toxin receptor 1 (ANTXR1) was revealed as the receptor for SVA in human cells. Herein, the role of ANTXR1 as a receptor for SVA in pigs was investigated by CRISPR/Cas9 genome editing. Strikingly, ANTXR1 knockout (KO) pigs exhibited features consistent with the rare disease, GAPO syndrome, in humans. Fibroblasts from wild type (WT) pigs supported replication of SVA; whereas, fibroblasts from KO pigs were resistant to infection. During an SVA challenge, clinical symptoms, including vesicular lesions, and circulating viremia were present in infected WT pigs but were absent in KO pigs. Additional ANTXR1-edited piglets were generated that were homozygous for an in-frame (IF) mutation. While IF pigs presented a GAPO phenotype similar to the KO pigs, fibroblasts showed mild infection, and circulating SVA nucleic acid was decreased in IF compared to WT pigs. Thus, this new ANTXR1 mutation resulted in decreased permissiveness of SVA in pigs. Overall, genetic disruption of ANTXR1 in pigs provides a unique model for GAPO syndrome and prevents circulating SVA infection and clinical symptoms, confirming that ANTXR1 acts as a receptor for the virus.
Subject(s)
Picornaviridae Infections , Picornaviridae , Swine Diseases , Alopecia , Animals , Anodontia , Growth Disorders , Optic Atrophies, Hereditary , Phenotype , Picornaviridae/genetics , Rare Diseases , Receptors, Peptide , SwineABSTRACT
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.
Subject(s)
Embryo, Mammalian/metabolism , Interferon-gamma/metabolism , Pregnancy, Animal/metabolism , Sus scrofa/embryology , Animals , Embryonic Development , Female , PregnancyABSTRACT
Genetically modified pigs have become valuable tools for generating advances in animal agriculture and human medicine. Importantly, in vitro production and manipulation of embryos is an essential step in the process of creating porcine models. As the in vitro environment is still suboptimal, it is imperative to examine the porcine embryo culture system from several angles to identify methods for improvement. Understanding metabolic characteristics of porcine embryos and considering comparisons with other mammalian species is useful for optimizing culture media formulations. Furthermore, stressors arising from the environment and maternal or paternal factors must be taken into consideration to produce healthy embryos in vitro. In this review, we progress stepwise through in vitro oocyte maturation, fertilization, and embryo culture in pigs to assess the status of current culture systems and address points where improvements can be made.
Subject(s)
Embryo, Mammalian/physiology , Research Embryo Creation/methods , Swine/embryology , Animals , Embryo, Mammalian/metabolism , Embryonic Development , Fertilization in Vitro , In Vitro Oocyte Maturation TechniquesABSTRACT
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.
Subject(s)
Blastocyst/metabolism , Embryo, Mammalian/embryology , Embryonic Development/genetics , Glutaminase/genetics , Mechanistic Target of Rapamycin Complex 1/genetics , Sus scrofa/embryology , Animals , Embryo Transfer , Embryo, Mammalian/enzymology , Female , Glutaminase/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolismABSTRACT
Background Modeling cardiovascular diseases in mice has provided invaluable insights into the cause of congenital heart disease. However, the small size of the mouse heart has precluded translational studies. Given current high-efficiency gene editing, congenital heart disease modeling in other species is possible. The pig is advantageous given its cardiac anatomy, physiology, and size are similar to human infants. We profiled pig cardiovascular development and generated genetically edited pigs with congenital heart defects. Methods and Results Pig conceptuses and fetuses were collected spanning 7 stages (day 20 to birth at day 115), with at least 3 embryos analyzed per stage. A combination of magnetic resonance imaging and 3-dimensional histological reconstructions with episcopic confocal microscopy were conducted. Gross dissections were performed in late-stage or term fetuses by using sequential segmental analysis of the atrial, ventricular, and arterial segments. At day 20, the heart has looped, forming a common atria and ventricle and an undivided outflow tract. Cardiac morphogenesis progressed rapidly, with atrial and outflow septation evident by day 26 and ventricular septation completed by day 30. The outflow and atrioventricular cushions seen at day 20 undergo remodeling to form mature valves, a process continuing beyond day 42. Genetically edited pigs generated with mutation in chromatin modifier SAP130 exhibited tricuspid dysplasia, with tricuspid atresia associated with early embryonic lethality. Conclusions The major events in pig cardiac morphogenesis are largely complete by day 30. The developmental profile is similar to human and mouse, indicating gene edited pigs may provide new opportunities for preclinical studies focused on outcome improvements for congenital heart disease.
Subject(s)
Heart Defects, Congenital/embryology , Heart/embryology , Organogenesis/physiology , Animals , Disease Models, Animal , Magnetic Resonance Imaging, Cine/methods , Microscopy, Confocal , SwineABSTRACT
Glutamine supplementation to porcine embryo culture medium improves development, increases leucine consumption, and enhances mitochondrial activity. In cancer cells, glutamine has been implicated in the activation of mechanistic target of rapamycin complex 1 (mTORC1) to support rapid proliferation. The objective of this study was to determine if glutamine metabolism, known as glutaminolysis, was involved in mTORC1 activation in porcine embryos. Culture with 3.75 mM GlutaMAX improved development to the blastocyst stage compared to culture with 1 mM GlutaMAX, and culture with 0 mM GlutaMAX decreased development compared to all groups with GlutaMAX. Ratios of phosphorylated to total MTOR were increased when embryos were cultured with 3.75 or 10 mM GlutaMAX, which was enhanced by the absence of leucine, but ratios for RPS6K were unchanged. As another indicator of mTORC1 activation, colocalization of MTOR and a lysosomal marker was increased in embryos cultured with 3.75 or 10 mM GlutaMAX in the absence of leucine. Culturing embryos with glutaminase inhibitors decreased development and the ratio of phosphorylated to total MTOR, indicating reduced activation of the complex. Therefore, glutaminolysis is involved in the activation of mTORC1 in porcine embryos, but further studies are needed to characterize downstream effects on development.
Subject(s)
Blastocyst/metabolism , Glutamine/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Animals , Blastocyst/cytology , Blastocyst/drug effects , Cells, Cultured , Culture Media/pharmacology , Embryo Culture Techniques/veterinary , Embryonic Development/drug effects , Embryonic Development/physiology , Female , Fertilization in Vitro/methods , Fertilization in Vitro/veterinary , Glutamine/pharmacology , Male , Signal Transduction/drug effects , SwineABSTRACT
In-vitro maturation (IVM) of oocytes from immature females is widely used in assisted reproductive technologies. Here we illustrate that cumulus cell (CC) expansion, once considered a key indicator of oocyte quality, is not needed for oocytes to mature to the metaphase II (MII) stage and to gain nuclear and cytoplasmic competence to produce offspring. Juvenile pig oocytes were matured in four different media: (1) Basal (-gonadotropins (GN) - FLI); (2) -GN + FLI (supplement of FGF2, LIF, and IGF1); (3) +GN - FLI; and (4) +GN + FLI. There was no difference in maturation to MII or progression to the blastocyst stage after fertilization of oocytes that had been matured in -GN + FLI medium and oocytes matured in +GN + FLI medium. Only slight CC expansion occurred in the two media lacking GN compared with the two where GN was present. The cumulus-oocytes-complexes (COC) matured in +GN + FLI exhibited the greatest expansion. We conclude that FLI has a dual role. It is directly responsible for oocyte competence, a process where GN are not required, and, when GN are present, it has a downstream role in enhancing CC expansion. Our study also shows that elevated phosphorylated MAPK may not be a necessary correlate of oocyte maturation and that the greater utilization of glucose by COC observed in +GN + FLI medium probably plays a more significant role to meet the biosynthetic needs of the CC to expand than to attain oocyte developmental competence. Gene expression analyses have not been informative in providing a mechanism to explain how FLI medium enhances oocyte competence without promoting CC expansion.
Subject(s)
Cumulus Cells/metabolism , Fertilization in Vitro/veterinary , Gonadotropins/metabolism , Oocytes/physiology , Sus scrofa/physiology , AnimalsABSTRACT
In vitro embryo production systems are limited by their inability to consistently produce embryos with the competency to develop to the blastocyst stage, survive cryopreservation, and establish a pregnancy. Previous work identified a combination of three cytokines [fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1)], called FLI, that we hypothesize improve preimplantation development of bovine embryos in vitro. To test this hypothesis, FLI was supplemented into oocyte maturation or embryo culture medium. Embryos were produced in vitro using abattoir-derived oocytes and fertilized with sperm from a single bull known to have high fertility. After an 18-20 h fertilization period, putative zygotes were cultured in synthetic oviductal fluid (SOF) for 8 days. The addition of FLI to the oocyte maturation medium increased (P < 0.05) the dissociation of transzonal projections at 12, 18, and 24 h of maturation, as well as, the proportion of oocytes that reached the metaphase II stage of meiosis. Additionally, lipid content was decreased (P < 0.05) in the blastocyst stage embryo. The addition of FLI during the culture period increased development to the blastocyst stage, cytoskeleton integrity, and survival following slow freezing, as well as, decreased post thaw cell apoptosis (P < 0.05). In conclusion, the supplementation of these cytokines in vitro has the potential to alleviate some of the challenges associated with the cryo-survival of in vitro produced bovine embryos through improving embryo development and embryo quality.
Subject(s)
Cattle/embryology , Cryopreservation/veterinary , Embryo, Mammalian/embryology , Fibroblast Growth Factor 2 , Insulin-Like Growth Factor I , Leukemia Inhibitory Factor , Animals , Blastocyst/cytology , Blastocyst/drug effects , Blastocyst/ultrastructure , Cryopreservation/methods , Embryo, Mammalian/drug effects , Embryo, Mammalian/ultrastructure , Female , Fertilization in Vitro/methods , Fertilization in Vitro/veterinary , Fibroblast Growth Factor 2/administration & dosage , Fibroblast Growth Factor 2/pharmacology , In Vitro Oocyte Maturation Techniques/methods , In Vitro Oocyte Maturation Techniques/veterinary , Insulin-Like Growth Factor I/administration & dosage , Insulin-Like Growth Factor I/pharmacology , Leukemia Inhibitory Factor/administration & dosage , Leukemia Inhibitory Factor/pharmacology , PregnancyABSTRACT
Phenylalanine hydroxylase-deficient (PAH-deficient) phenylketonuria (PKU) results in systemic hyperphenylalaninemia, leading to neurotoxicity with severe developmental disabilities. Dietary phenylalanine (Phe) restriction prevents the most deleterious effects of hyperphenylalaninemia, but adherence to diet is poor in adult and adolescent patients, resulting in characteristic neurobehavioral phenotypes. Thus, an urgent need exists for new treatments. Additionally, rodent models of PKU do not adequately reflect neurocognitive phenotypes, and thus there is a need for improved animal models. To this end, we have developed PAH-null pigs. After selection of optimal CRISPR/Cas9 genome-editing reagents by using an in vitro cell model, zygote injection of 2 sgRNAs and Cas9 mRNA demonstrated deletions in preimplantation embryos, with embryo transfer to a surrogate leading to 2 founder animals. One pig was heterozygous for a PAH exon 6 deletion allele, while the other was compound heterozygous for deletions of exon 6 and of exons 6-7. The affected pig exhibited hyperphenylalaninemia (2000-5000 µM) that was treatable by dietary Phe restriction, consistent with classical PKU, along with juvenile growth retardation, hypopigmentation, ventriculomegaly, and decreased brain gray matter volume. In conclusion, we have established a large-animal preclinical model of PKU to investigate pathophysiology and to assess new therapeutic interventions.
Subject(s)
Liver/metabolism , Phenylalanine Hydroxylase/genetics , Phenylalanine/genetics , Phenylketonurias/genetics , Adolescent , Adult , Animals , CRISPR-Cas Systems/genetics , Diet , Disease Models, Animal , Gene Editing , Humans , Liver/drug effects , Phenotype , Phenylalanine/metabolism , Phenylalanine/pharmacology , Phenylketonurias/diet therapy , Phenylketonurias/metabolism , Phenylketonurias/pathology , SwineABSTRACT
Submucosal glands (SMGs) are a prominent structure that lines human cartilaginous airways. Although it has been assumed that SMGs contribute to respiratory defense, that hypothesis has gone without a direct test. Therefore, we studied pigs, which have lungs like humans, and disrupted the gene for ectodysplasin (EDA-KO), which initiates SMG development. EDA-KO pigs lacked SMGs throughout the airways. Their airway surface liquid had a reduced ability to kill bacteria, consistent with SMG production of antimicrobials. In wild-type pigs, SMGs secrete mucus that emerges onto the airway surface as strands. Lack of SMGs and mucus strands disrupted mucociliary transport in EDA-KO pigs. Consequently, EDA-KO pigs failed to eradicate a bacterial challenge in lung regions normally populated by SMGs. These in vivo and ex vivo results indicate that SMGs are required for normal antimicrobial activity and mucociliary transport, two key host defenses that protect the lung.
Subject(s)
Ectodysplasins/genetics , Exocrine Glands/immunology , Respiratory Mucosa/immunology , Staphylococcus aureus/physiology , Sus scrofa/immunology , Animals , Ectodysplasins/immunology , Female , Gene Knockout Techniques , Male , Sus scrofa/geneticsABSTRACT
Hypotaurine (HT) is a routine component of porcine embryo culture medium, functioning as an antioxidant, but its requirement may be diminished as most embryo culture systems now use 5% O2 instead of atmospheric (20%) O2 . Our objective was to determine the effects of removing HT from the culture medium on porcine preimplantation embryo development. Embryos cultured in 20% O2 without HT had decreased blastocyst development compared to culture with HT or in 5% O2 with or without HT. Notably, differences in blastocyst development or total cell number were not detected between embryos cultured in 5% O2 with or without HT. After culture in 5% O2 without HT and embryo transfer, healthy fetuses were retrieved from two pregnancies on Day 42, confirming in vivo developmental competence. Transcript abundance of proapoptotic markers was decreased in embryos cultured without HT regardless of oxygen tension; however, assays for apoptosis did not demonstrate differences between groups. Additionally, no differences were observed in the development or apoptosis of somatic cell nuclear transfer-derived embryos cultured in 5% O2 with or without HT. With decreased utility in 5% O2 , removing HT from porcine embryo culture medium would also have economic advantages because it is undoubtedly the most expensive component.
ABSTRACT
To improve efficiency of somatic cell nuclear transfer (SCNT), it is necessary to modify differentiated donor cells to become more amendable for reprogramming by the oocyte cytoplasm. A key feature that distinguishes somatic/differentiated cells from embryonic/undifferentiated cells is cellular metabolism, with somatic cells using oxidative phosphorylation (OXPHOS) while embryonic cells utilize glycolysis. Inducing metabolic reprogramming in donor cells could improve SCNT efficiency by priming cells to become more embryonic in nature before SCNT hypoxia inducible factor 1-α (HIF1-α), a transcription factor that allows for cell survival in low oxygen, promotes a metabolic switch from OXPHOS to glycolysis. We hypothesized that chemically stabilizing HIF1-α in donor cells by use of the hypoxia mimetic, cobalt chloride (CoCl2 ), would promote this metabolic switch in donor cells and subsequently improve the development of SCNT embryos. Donor cell treatment with 100 µM CoCl2 for 24 hr preceding SCNT upregulated messenfer RNA abundance of glycolytic enzymes, improved SCNT development to the blastocyst stage and quality, and affected gene expression in the blastocysts. After transferring blastocysts created from CoCl2 -treated donor cells to surrogates, healthy cloned piglets were produced. Therefore, shifting metabolism toward glycolysis in donor cells by CoCl2 treatment is a simple, economical way of improving the in vitro efficiency of SCNT and is capable of producing live animals.
ABSTRACT
Pig conceptuses secrete estrogens (E2), interleukin 1 beta 2 (IL1B2), and prostaglandins (PGs) during the period of rapid trophoblast elongation and establishment of pregnancy. Previous studies established that IL1B2 is essential for rapid conceptus elongation, whereas E2 is not essential for conceptus elongation or early maintenance of the corpora lutea. The objective of the present study was to determine if conceptus expression of prostaglandin-endoperoxide synthase 2 (PTGS2) and release of PG are important for early development and establishment of pregnancy. To understand the role of PTGS2 in conceptus elongation and pregnancy establishment, a loss-of-function study was conducted by editing PTGS2 using CRISPR/Cas9 technology. Wild-type (PTGS2+/+) and null (PTGS2-/-) fibroblast cells were used to create embryos through somatic cell nuclear transfer. Immunolocalization of PTGS2 and PG production was absent in cultured PTGS2-/- blastocysts on day 7. PTGS2+/+ and PTGS2-/- blastocysts were transferred into surrogate gilts, and the reproductive tracts were collected on either days 14, 17, or 35 of pregnancy. After flushing the uterus on days 14 and 17, filamentous conceptuses were cultured for 3 h to determine PG production. Conceptus release of total PG, prostaglandin F2⺠(PGF2α), and PGE in culture media was lower with PTGS2-/- conceptuses compared to PTGS2+/+ conceptuses. However, the total PG, PGF2α, and PGE content in the uterine flushings was not different. PTGS2-/- conceptus surrogates allowed to continue pregnancy were maintained beyond 30 days of gestation. These results indicate that pig conceptus PTGS2 is not essential for early development and establishment of pregnancy in the pig.
Subject(s)
Blastocyst/metabolism , Cyclooxygenase 2/metabolism , Embryo Implantation/physiology , Embryonic Development/physiology , Endometrium/metabolism , Animals , Animals, Genetically Modified , CRISPR-Cas Systems , Cyclooxygenase 2/genetics , Dinoprost/metabolism , Dinoprostone/metabolism , Female , Gene Expression Regulation, Developmental , Nuclear Transfer Techniques , Pregnancy , SwineABSTRACT
Assisted reproductive technologies in the pig are critical for the production of genetically modified pigs as models of human disease and to improve production agriculture. Methods of oocyte maturation, fertilization, and culture all play an extremely important role in how the embryo, fetus, and offspring will develop. In this chapter, we discuss the historical methods and recent advances that have been essential in promoting efficient and competent embryo development. Here we describe the procedures that can be used to mature, fertilize, and culture pig embryos to the blastocyst stage.