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. 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.

Porcine-specific expression of the three functional CYP19 paralogs in early conceptus, placenta, and gonads.

In brief: Aromatase catalyzes the synthesis of estrogens and has been shown to have an important role during the establishment of pregnancy in the pig. This study confirmed the differential expression of the three aromatase isoforms. Abstract: Although three porcine aromatase isoforms have been identified, their gene expression profiles in reproduction are still poorly understood. Here, we identified by Sanger sequencing unique nucleotide signatures for the three paralogous copies of Cyp19 and analyzed by RT-PCR the occurrence of the Cyp19 and Cyp17a1 transcripts at different tissues and stages of conceptus and fetal-placental development. Cyp19a1 and Cyp19a3 expressions were detected in conceptuses and gonads, respectively. Cyp19a2 transcripts were identified on both the conceptuses and the placenta samples. Transcripts for Cyp17a1 were detected predominantly in conceptus and gonads. In the endometrium of day 21 pregnant females, as well as days 12 and 17 pseudopregnant females, we did not detect the expression of Cyp19a1, Cyp19a2, or Cyp19a3. In our study, we have demonstrated distinct transcriptional regulation for the three functional Cyp19 paralogs and a potential role for Cyp17a1 in controlling the secretion of estrogen from the conceptus and the placenta.

Gene editing provides a tool to investigate genes involved in reproduction of pigs.

CRISPR-Cas9 gene editing technology provides a method to generate loss-of-function studies to investigate, in vivo, the specific role of specific genes in regulation of reproduction. With proper design and selection of guide RNAs (gRNA) designed to specifically target genes, CRISPR-Cas9 gene editing allows investigation of factors proposed to regulate biological pathways involved with establishment and maintenance of pregnancy. The advantages and disadvantages of using the current gene editing technology in a large farm species is discussed. CRISPR-Cas9 gene editing of porcine conceptuses has generated new perspectives for the regulation of endometrial function during the establishment of pregnancy. The delicate orchestration of conceptus factors facilitates an endometrial proinflammatory response while regulating maternal immune cell migration and expansion at the implantation site is essential for establishment and maintenance of pregnancy. Recent developments and use of endometrial epithelial "organoids" to study endometrial function in vitro provides a future method to screen and target specific endometrial genes as an alternative to generating a gene edited animal model. With continuing improvements in gene editing technology, future researchers will be able to design studies to enhance our knowledge of mechanisms essential for early development and survival of the conceptus.

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.

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.

Mammalian Placentation: A Tribute to E.C. Amoroso's Contributions to Placenta Development.

Establishment of viviparity in mammals evolved through not only the long-term retainment of the fetus within the maternal uterus but differentiation and expansion of cell layers to form functional membranes to exchange O2/CO2 and nutrients between the placenta and maternal circulations. Development of a fetal placental vascular circulation to interact with the maternal uterus is critical to the survival of all species. However, the fascination with the mammalian placenta is the robust variation in types, form, attachment, invasiveness, structure, cell differentiation, endocrine function, and regulation of the maternal immune system. Despite the obvious role of the placenta to support fetal development, mammals have evolved multiple strategies to give live birth at term. The placenta and the maternal-fetal interface during pregnancy can be quite simple to very complex. Professor E.C. Amoroso contributed greatly to the study of comparative placentation in animals. His paper "Placentation" in Marshall's Physiology of Reproduction published in 1952 remains the standard for comparative placental anatomy today. The present volume on "Mammalian Placentation" brings together current reviews for leading experts to diversity of placentation in a number of mammalian species. Chapters will discuss viviparity, blastocyst formation, and placentation in the cow, pig, horse, mouse, dog, primate, human, elephant, and marsupials.

Implantation and Placentation in Ruminants.

In comparison to many other mammalian species, ruminant ungulates have a unique form of placentation. Ruminants initially display an epitheliochorial type of placentation; however, during the period of placental attachment, trophoblast giant binucleate cells (BNC) develop within the chorion to migrate and fuse with the uterine surface epithelium to form syncytial plaques. Binucleate cell migration and fusion continues throughout pregnancy but never appears to breach the basal lamina, beneath the uterine surface or luminal epithelium. Therefore, the semi-invasive type of placentation in ruminants is classified as synepitheliochorial. The endometrium of ruminant species also contains unique specialized aglandular structures termed "caruncles" in which the chorioallantois (cotyledons) interdigitates and forms highly vascularized fetal-maternal "placentomes." This chapter will discuss the current knowledge of early conceptus development during the peri-attachment period, establishment of pregnancy, conceptus attachment, and placentation in ruminant ungulates. The features of placentomes, BNCs, fetomaternal hybrid cells, and multinucleated syncytial plaques of the cotyledonary placenta of ruminant species will be reviewed to highlight the unique form of placentation compared to the placentae of other artiodactyls.

Gene editing to investigate the role of conceptus factors in the establishment of pregnancy in the pig.

Development of viviparity in mammals requires that the placenta evolves as an intermediate interface between the fetus and maternal uterus. In addition to the retention of the fetus and secretion of nutrients to support growth and development to term, it is essential that viviparous species modify or inhibit the maternal immune system from recognizing the semi-allogeneic fetus. Following blastocyst hatching from its zona pellucida, trophoblast differentiation provides the initial communication to the maternal endometrium to regulate maintenance of progesterone production from the corpus luteum and biological pathways in uterine and conceptus development necessary in the establishment and maintenance of pregnancy. Many conceptus factors have been proposed to serve in the establishment and maintenance of pregnancy. CRISPR-Cas9 gene-editing technology provides a specific and efficient method to generate animal models to perform loss-of-function studies to investigate the role of specific conceptus factors. The utilization of CRISPR-Cas9 gene editing has provided a direct approach to investigate the specific role of conceptus factors in the development and establishment of pregnancy in the pig. This technology has helped address a number of questions concerning peri-implantation development and has altered our understanding of maternal recognition and maintenance of pregnancy in the pig.

Inactivation of porcine interleukin-1ß results in failure of rapid conceptus elongation.

Conceptus expansion throughout the uterus of mammalian species with a noninvasive epitheliochorial type of placentation is critical establishing an adequate uterine surface area for nutrient support during gestation. Pig conceptuses undergo a unique rapid morphological transformation to elongate into filamentous threads within 1 h, which provides the uterine surface to support development and maintain functional corpora lutea through the production of estrogen. Conceptus production of a unique interleukin 1ß, IL1B2, temporally increases during the period of trophoblast remodeling during elongation. CRISPR/Cas9 gene editing was used to knock out pig conceptus IL1B2 expression and the secretion of IL1B2 during the time of conceptus elongation. Trophoblast elongation occurred on day 14 in wild-type (IL1B2+/+) conceptuses but did not occur in ILB2-null (IL1B2-/-) conceptuses. Although the morphological transition of IL1B2-/- conceptuses was inhibited, expression of a number of conceptus developmental genes was not altered. However, conceptus aromatase expression and estrogen secretion were decreased, indicating that IL1B2 may be involved in the spatiotemporal increase in conceptus estrogen synthesis needed for the establishment of pregnancy in the pig and may serve to regulate the proinflammatory response of endometrium to IL1B2 during conceptus elongation and attachment to the uterine surface.

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. 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⍺ (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.

New perspective on conceptus estrogens in maternal recognition and pregnancy establishment in the pig†.

The proposed signal for maternal recognition of pregnancy in pigs is estrogen (E2), produced by the elongating conceptuses between days 11 to 12 of pregnancy with a more sustained increase during conceptus attachment and placental development on days 15 to 30. To understand the role of E2 in porcine conceptus elongation and pregnancy establishment, a loss-of-function study was conducted by editing aromatase (CYP19A1) using CRISPR/Cas9 technology. Wild-type (CYP19A1+/+) and (CYP19A1-/-) fibroblast cells were used to create embryos through somatic cell nuclear transfer, which were transferred into recipient gilts. Elongated and attaching conceptuses were recovered from gilts containing CYP19A1+/+ or CYP19A1-/- embryos on day 14 and 17 of pregnancy. Total E2 in the uterine flushings of gilts with CYP19A1-/- embryos was lower than recipients containing CYP19A1+/+ embryos with no difference in testosterone, PGF2α, or PGE2 on either day 14 or 17. Despite the loss of conceptus E2 production, CYP19A1-/- conceptuses were capable of maintaining the corpora lutea. However, gilts gestating CYP19A1-/- embryos aborted between days 27 and 31 of gestation. Attempts to rescue the pregnancy of CYP19A1-/- gestating gilts with exogenous E2 failed to maintain pregnancy. However, CYP19A1-/- embryos could be rescued when co-transferred with embryos derived by in vitro fertilization. Endometrial transcriptome analysis revealed that ablation of conceptus E2 resulted in disruption of a number biological pathways. Results demonstrate that intrinsic E2 conceptus production is not essential for pre-implantation development, conceptus elongation, and early CL maintenance, but is essential for maintenance of pregnancy beyond 30 days .

Altering rat sexual behavior to teach hormonal regulation of brain imprinting.

In this teaching laboratory, students design and perform an experiment to determine estrogen's role in imprinting the brain of neonatal rats to express either male or female sexual behavior. A discussion question is provided before the laboratory exercise in which each student is asked to search the literature and provide written answers to questions and to formulate an experiment to test the role of estrogen in imprinting the mating behavior of male and female rats. Students discuss their answers to the questions in laboratory with the instructor and design an experiment to test their hypothesis. In male rats, testosterone is converted by aromatase expressed by neurons in the brain to estrogen. Production of estrogen in the brain of neonatal rats imprints mating behavior in males, where a lack of estrogen action in the brain imprints female sexual behavior. The model involves administering exogenous testosterone to imprint male behavior in female pups or administration of an aromatase inhibitor (letrozole) or an estrogen receptor antagonist (ICI 182,780) to imprint female sexual behavior in male pups. In the model, litters of neonatal pups are treated with either carrier (control), testosterone propionate, aromatase inhibitor (letrozole), or an estrogen receptor antagonist (ICI 182,780) postnatally on days 1 and 3. Alteration of mating behavior is evaluated through the numbers of males and females that breed and establish pregnancy. This is a very simple protocol that provides an excellent experiment for student discussion on the effects of hormone action on imprinting brain sexual behavior.

Utilizing a rat delayed implantation model to teach integrative endocrinology and reproductive biology.

In this teaching laboratory, the students are directed in an exercise that involves designing and performing an experiment to determine estrogen's role in regulating delayed implantation (diapause) in female rats. To encourage active participation by the students, a discussion question is provided before the laboratory exercise in which each student is asked to search the literature and provide written answers to questions and to formulate an experiment to test the role of ovarian estrogen in inducing implantation in female rats. One week before the laboratory exercise, students discuss their answers to the questions with the instructor to develop an experiment to test their hypothesis that estrogen is involved with inducing implantation in the rat. A rat delayed implantation model was established that utilizes an estrogen receptor antagonist (ICI 182,780), which inhibits the action of ovarian estrogens. Groups of mated females are treated with either carrier (control) or ICI 182,780 (ICI) every other day, starting on day 2 postcoitus (pc) until day 8 pc. One-half of the females receiving ICI are injected with estradiol-17ß on day 8 pc to induce implantation 4 days after the controls. If the ICI-treated females are not administered estradiol, embryo implantation occurs spontaneously ~4 days after the last ICI injection on day 8. This is a very simple protocol that is very effective and provides an excellent experiment for student discussion on hormone action and the use of agonists and antagonists.

Rapid conceptus elongation in the pig: An interleukin 1 beta 2 and estrogen-regulated phenomenon.

Establishment and maintenance of pregnancy in the pig involves activating many physiological, cellular, and molecular signaling pathways between the developing conceptus and hormonally regulated maternal endometrium. Rapid elongation of the pig trophoblast allows for the establishment of sufficient placental surface area for the transport of nutrients to the fetus throughout pregnancy. Estrogens secreted by the conceptus during elongation act on uterine epithelia to induce secretion of uterine factors required for conceptus development and for preventing endocrine secretion of prostaglandin F2α, which would cause luteolysis. Thus, trophoblast expansion within the uterine lumen during early gestation is an essential process for implantation and maintenance of pregnancy in species with an epitheliochorial form of placentation. In the pig, rapid conceptus elongation involves the unique expression of interleukin-1 beta 2 (IL1B2), which establishes pro-inflammatory effects that may be tempered by the spatiotemporal secretion of estrogen from the conceptuses. The present review provides current information on pig conceptus remodeling and signaling via estrogen and IL1B2 pathways, as well as endometrial responses to those conceptus factors leading to establishment of pregnancy.

Activation of the transcription factor nuclear factor-kappa B in uterine luminal epithelial cells by interleukin 1 Beta 2: a novel interleukin 1 expressed by the elongating pig conceptus.

Conceptus mortality is greatest in mammals during the peri-implantation period, a time when conceptuses appose and attach to the uterine surface epithelium while releasing proinflammatory molecules. Interleukin 1 beta (IL1B), a master proinflammatory cytokine, is released by the primate, rodent, and pig blastocyst during the peri-implantation period and is believed to be essential for establishment of pregnancy. The gene encoding IL1B has duplicated in the pig, resulting in a novel gene. Preliminary observations indicate that the novel IL1B is specifically expressed by pig conceptuses during the peri-implantation period. To verify this, IL1B was cloned from mRNA isolated from Day 12 pig conceptuses and compared with IL1B cloned from mRNA isolated from pig peripheral blood leukocytes (PBLs). The pig conceptuses, but not the PBLs, expressed a novel IL1B, referred to here as interleukin 1 beta 2 (IL1B2). Porcine endometrium was treated with recombinant porcine interleukin 1 beta 1 (IL1B1), the prototypical cytokine, and IL1B2 proteins. Immunohistochemistry and real-time RT-PCR were used to measure activation of nuclear factor-kappa B (NFKB) and NFKB-regulated transcripts, respectively, within the endometrium. Both IL1B1 and IL1B2 activated NFKB in the uterine luminal epithelium within 4 h. The NFKB activation and related gene expression, however, were lower in endometrium treated with IL1B2, suggesting that the conceptus-derived cytokine may have reduced activity within the uterus. In conclusion, the peri-implantation pig conceptus expresses a novel IL1B that can activate NFKB within the uterine surface epithelium, likely creating a proinflammatory microenvironment during establishment of pregnancy in the pig.

Introduction.

Establishment and maintenance of pregnancy in a number of mammalian species depends upon a tightly regulated interaction between the semiallogeneic conceptus and the maternal uterine endometrium.The term "Maternal Recognition of Pregnancy" is attributed to Roger V. Short's paper titled "Implantation and the Maternal Recognition of Pregnancy" which was published in proceedings from the 1969 Symposium on Foetal Autonomy.Professor Short's landmark paper stimulated increased interest in elucidating how the conceptus signals its presence to assure maintenance of the corpus luteum beyond the normal length of the estrous or menstrual cycle to allow pregnancy to be established and maintained.Ten years following publication of Professor Short's paper, a Ciba Foundation Symposium entitled "Maternal Recognition of Pregnancy" brought together leading scientists to discuss the multiple mechanisms and pathways by which different viviparous species establish a successful pregnancy.The present volume on "Regulation of Implantation and Establishment of Pregnancy in Mammals" brings together current reviews from leading experts to address the diversity of mechanisms by which species establish and maintain pregnancy.Implantation in mice, dogs, pigs, cattle, sheep, horses, primates, humans and species in which embryonic diapause occurs are discussed.Reviews will provide current knowledge on the role of endometrial steroid receptors, adhesion factors, cytokines, interferons, steroids, prostaglandins, growth factors and immune cells involved with regulation of conceptus development.

Implantation and Establishment of Pregnancy in the Pig.

Establishment of pregnancy in the pig is initiated through the release of estrogens from the rapidly elongating conceptuses. Release of estrogens from the developing conceptuses alters the movement of endometrial prostaglandin F2α from being released into the vasculature (endocrine secretion) to sequestering in the uterine lumen (exocrine secretion). Rapid trophoblast elongation, which is unique to the pig, may be triggered through production of interleukin 1ß2 (IL1B2) by conceptuses. Trophoblast elongation through the uterine horns provides the mechanism to allow conceptus-endometrial interactions essential for the implantation, placentation, and maintenance of pregnancy in the pig. This chapter provides current information on conceptus signaling pathways and endometrial responses to those conceptus factors leading to establishment of pregnancy.

Maternal recognition of pregnancy in the pig: A servomechanism involving sex steroids, cytokines and prostaglandins.

Maternal recognition of pregnancy (MRP) is a term utilized in mammals to describe pathways in which the conceptus alters the endometrial environment to prevent regression of corpora lutea to ensure continued production of progesterone (P4) required for establishment and maintenance of pregnancy. For nearly 40 years after publication of the endocrine/exocrine theory, conceptus estrogen (E2) was considered the primary maternal recognition signal in the pig. Conceptus production of prostaglandin E2 (PGE2) was also considered to be a major factor in preventing luteolysis. An addition to E2 and PGE2, pig conceptuses produce interleukin 1B2 (IL1B2) and interferons (IFN) delta (IFND) and gamma (IFNG). The present review provides brief history of the discovery of E2, PGs and IFNS which led to research investigating the role of these conceptus secreted factors in establishing and maintaining pregnancy in the pig. The recent utilization of gene editing technology allowed a more direct approach to investigate the in vivo roles of IL1B2, E2, PGE2, AND IFNG for establishment of pregnancy. These studies revealed unknown functions for IFNG and ILB2 in addition to PGE2 and E2. Thus, pregnancy recognition signal is via a servomechanism in requiring sequential effects of P4, E2, IL1B2, PGE2 and IFNG. Results indicate that the original established dogma for the role of conceptus E2 and PGs in MRP is a far too simplified model that involves the interplay of numerous mechanisms for inhibiting luteolysis, inducing critical elongation of the conceptuses and resolution of inflammation in pigs.

Improvements in pig agriculture through gene editing.

Genetic modification of animals via selective breeding is the basis for modern agriculture. The current breeding paradigm however has limitations, chief among them is the requirement for the beneficial trait to exist within the population. Desirable alleles in geographically isolated breeds, or breeds selected for a different conformation and commercial application, and more importantly animals from different genera or species cannot be introgressed into the population via selective breeding. Additionally, linkage disequilibrium results in low heritability and necessitates breeding over successive generations to fix a beneficial trait within a population. Given the need to sustainably improve animal production to feed an anticipated 9 billion global population by 2030 against a backdrop of infectious diseases and a looming threat from climate change, there is a pressing need for responsive, precise, and agile breeding strategies. The availability of genome editing tools that allow for the introduction of precise genetic modification at a single nucleotide resolution, while also facilitating large transgene integration in the target population, offers a solution. Concordant with the developments in genomic sequencing approaches, progress among germline editing efforts is expected to reach feverish pace. The current manuscript reviews past and current developments in germline engineering in pigs, and the many advantages they confer for advancing animal agriculture.

Uterine progesterone receptor expression, conceptus development, and ovarian function in pigs treated with RU 486 during early pregnancy.

Establishment of pregnancy in the pig depends on down-regulation of progesterone receptor (PGR) in the uterine luminal and glandular epithelium during the first week after breeding. The present study evaluated the regulation of endometrial PGR by progesterone and the possible role of endometrial tumor necrosis factor (ligand) superfamily member 11 (TNFSF11) and nuclear factor-kappa B (NFKB) activation in PGR expression. Mature, cycling gilts were inseminated (Day 0) and assigned to either untreated control (n = 9) or one of two treatments that employed RU 486 to block progesterone action either before (treatment 1 [T1]) or after (treatment 2 [T2]) the initiation of PGR down-regulation. The T1 gilts were treated with RU 486 (400 mg/day) on Days 3-5 of pregnancy (n = 9), and T2 gilts were treated with RU 486 on Days 6 and 7 of pregnancy (n = 9). Uteri and ovaries were collected on Day 8 or 12 of gestation. The diameter of the conceptuses in T1 gilts was approximately half that in controls by Day 8, and normal conceptuses were not collected from any T1 gilts on Day 12. Endometrial PGR mRNA was more abundant in T1 and T2 gilts compared with control gilts. The PGR-B protein decreased from Day 8 to Day 12 in the luminal epithelium and, to some extent, in superficial glandular epithelium in control and T2 gilts. In T1 gilts, the PGR-B protein remained elevated (i.e., failed to undergo down-regulation) on Day 12. Blocking PGR action early in the cycle (i.e., on or before Day 5), therefore, prevented normal conceptus development, caused elevated PGR mRNA, and prevented the decrease in PGR protein that typically occurs in pigs. We could not confirm a role for NFKB activation in PGR down-regulation, because pigs with extreme differences in PGR and TNFSF11 expression (T1 and controls) had similar NFKB activation on Day 8. Activated NFKB within the luminal epithelium and glandular epithelium (both superficial and deep) was observed in T2 and control pigs on Day 12 when elongating conceptuses (presumably releasing interleukin 1 beta to activate NFKB) were recovered. Gilts treated with RU 486 had greater ovarian follicular growth and greater plasma estradiol concentrations. We conclude that the mechanisms controlling PGR down-regulation are progesterone-dependent and occur between Day 3 and Day 6 of pregnancy. NFKB activation did not appear to have a role in PGR down-regulation within the period that we studied. Blocking progesterone action after Day 6 did not reverse the process of PGR down-regulation, nor did it appear to affect the development of conceptuses collected on Day 12.