Conceptus estrogen and prostaglandins provide the maternal recognition of pregnancy signal to prevent luteolysis during early pregnancy in the pig†.

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.

Oocyte recovery after overnight ovary transport provides an alternative source of cumulus oocyte complexes that are competent to produce live piglets.

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.

FGF2, LIF, and IGF1 (FLI) supplementation during human in vitro maturation enhances markers of gamete competence.

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.

Inactivation of growth differentiation factor 9 blocks folliculogenesis in pigs†.

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.

Profiling development of abdominal organs in the pig.

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.

Disruption of anthrax toxin receptor 1 in pigs leads to a rare disease phenotype and protection from senecavirus A infection.

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.

Challenges and Considerations during In Vitro Production of Porcine Embryos.

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.

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.

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.

Cardiovascular Development and Congenital Heart Disease Modeling in the Pig.

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.

Glutaminolysis is involved in the activation of mTORC1 in in vitro-produced porcine embryos.

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.

Neither gonadotropin nor cumulus cell expansion is needed for the maturation of competent porcine oocytes in vitro†.

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.

Improved cryopreservation of in vitro produced bovine embryos using FGF2, LIF, and IGF1.

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.

A porcine model of phenylketonuria generated by CRISPR/Cas9 genome editing.

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.

Lack of airway submucosal glands impairs respiratory host defenses.

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 (), which initiates SMG development.

Chemical simulation of hypoxia in donor cells improves development of somatic cell nuclear transfer-derived embryos and increases abundance of transcripts related to glycolysis.

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.

Removal of hypotaurine from porcine embryo culture medium does not impair development of in vitro-fertilized or somatic cell nuclear transfer-derived embryos at low oxygen tension.

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% O instead of atmospheric (20%) O . Our objective was to determine the effects of removing HT from the culture medium on porcine preimplantation embryo development. Embryos cultured in 20% O without HT had decreased blastocyst development compared to culture with HT or in 5% O with or without HT.

Ablation of conceptus PTGS2 expression does not alter early conceptus development and establishment of pregnancy in the pig†.

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.

In Vitro Maturation, Fertilization, and Culture of Pig Oocytes and Embryos.

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.

Improvement of in vitro and early in utero porcine clone development after somatic donor cells are cultured under hypoxia.

Genetically engineered pigs serve as excellent biomedical and agricultural models. To date, the most reliable way to generate genetically engineered pigs is via somatic cell nuclear transfer (SCNT), however, the efficiency of cloning in pigs is low (1-3%). Somatic cells such as fibroblasts frequently used in nuclear transfer utilize the tricarboxylic acid cycle and mitochondrial oxidative phosphorylation for efficient energy production.

Glutamine supplementation enhances development of in vitro-produced porcine embryos and increases leucine consumption from the medium.

Improper composition of culture medium contributes to reduced viability of in vitro-produced embryos. Glutamine (Gln) is a crucial amino acid for preimplantation embryos as it supports proliferation and is involved in many different biosynthetic pathways. Previous transcriptional profiling revealed several upregulated genes related to Gln transport and metabolism in in vitro-produced porcine blastocysts compared to in vivo-produced counterparts, indicating a potential deficiency in the culture medium.

Single step production of Cas9 mRNA for zygote injection.

Production of Cas9 mRNA in vitro typically requires the addition of a 5´ cap and 3´ polyadenylation. A plasmid was constructed that harbored the T7 promoter followed by the EMCV IRES and a Cas9 coding region. We hypothesized that the use of the metastasis associated lung adenocarcinoma transcript 1 (Malat1) triplex structure downstream of an IRES/Cas9 expression cassette would make polyadenylation of in vitro produced mRNA unnecessary.