Pseudopregnant Mice Research Articles

Stimulation of Angiotensin II Receptor Subtype 2 Reduces Preeclampsia-like Symptoms in a Mouse Model of Preeclampsia.

Angiotensin II (AngII) receptor subtype 1 (AT1R) is involved in the pathogenesis of preeclampsia (PE). Angiotensin II receptor subtype 2 (AT2R) can antagonize the effects of AT1R, but its effects during pregnancy are not known. We investigated the effect of AT2R on the pathogenesis of PE using a mouse model and recently developed AT2R agonist (compound 21 [C21]). Blastocysts collected from pregnant imprinting control region (ICR) mice were incubated with adenovirus containing the CD40L gene and transferred into the uterine horns of pseudo-pregnant ICR mice to express PE-like features. Osmotic pumps were placed subcutaneously on the dorsal side with C21 or saline. C21 reduced the plasma soluble fms-like tyrosine kinase 1 (sFlt-1) concentration, ameliorating hypertension. The splenic T and B cell profiles in model mice were analyzed by flow cytometry. The gated percentage of IFN-γ-positive Th cells was significantly increased and the percentage of plasma cells in B cells was significantly decreased; however, the percentages were not altered by C21. sFlt-1 and soluble endoglin concentrations in plasma were measured with an enzyme-linked immunosorbent assay, and sFlt-1 was reduced. C21 could become a candidate PE drug as it ameliorated the pathophysiology of PE as a result of decreased production of sFlt-1.

P-796 A novel method of somatic cell nuclear transfer to generate artificial oocytes

Abstract Study question Can we identify an alternative method to Sendai-Virus fusion for somatic cell nuclear transfer (SCNT) aiming at generating artificial oocyte? Summary answer The utilization lipofectamine proved itself as a valid alternative method to virus-mediated SCNT, allowing successful membrane fusion and supporting proper embryo development. What is known already SCNT involves transferring a diploid cell’s nucleus into an enucleated oocyte. Early attempts of SCNT involves direct injection and electrofusion but both approaches can affect oolemma integrity. To avoid mechanical damage to oolemma, fusogenic agents have been employed for SCNT including polyethylene glycol (PEG) and more efficient Hemagglutinating Virus of Japan Envelope (HVJ-E). However, despite its high efficiency, HVJ-E is difficult to be used in human because it is a virus derivative, and a recent shortage of this compound made it not readily available. On the other hand, cationic phospholipid is fusogenic and is widely utilized in transfection experiments. Study design, size, duration In the past 4 months, SCNT was carried out through PEG-mediated, HVJ-E-mediated, or novel lipofectamine-mediated fusion using cumulus cells as nuclear donor. Post-SCNT oocytes were inseminated and cultured up to the blastocyst stage. Embryo development and morphokinetics were assessed and compared among the three SCNT experimental cohorts. In some cases, blastocysts were transferred into pseudo-pregnant mice, post-natal development were monitored. Fertilization, embryo development, and offspring wellbeing were monitored. Participants/materials, setting, methods Oocytes from B6D2F1 mice were enucleated by micromanipulation. The ooplasts were allocated to 3 cohorts: 1) exposure to 45% PEG, 2) subzonal transfer of donor cell with HVJ-E, or 3) subzonal transfer the donor cell exposed to 1.75% lipofectamine. Reconstructed oocytes were monitored for fusion 30 minutes after exposure to fusogens and inseminated by spermatozoa from B6-EGFP mice to confirm paternal genome provenance. Fertilization and embryo development were monitored in a time-lapse system. Main results and the role of chance A total of 165 oocytes were enucleated and 160 (97.0%) survived and allocated to PEG-fusion (n = 50), Sendai-virus-fusion (n = 41) and lipofecamine-fusion (n = 80) cohorts. Fusion rate was 56.0% (28/50) for the PEG-fusion cohort, while Sendai-virus and lipofectamine-fusion cohorts yielded higher fusion rate at 92.7%(38/41) and 86.3%(69/80), respectively (P<0.0001). Control oocytes yielded 96.7% (145/150) fertilization rate and developed to 2-cell stage at 93.8% (136/145), and yielded an eventual blastocyst rate at 80.7% (117/145). Despite a lower oocyte reconstruction rate, PEG-fusion cohort fertilized at 89.3% (25/28), but cleavage rate to 2-cell stage was lower at 48.0% (12/25, P<0.0001) and did not yield any blastocyst. The HVJ-E cohort yielded a fertilization rate of 94.7% (36/38) and cleavage rate at 97.2% (35/36) similar to control. The blastocyst rate was observed at 22.9% (8/35, P<0.0001) when comparing to control. Lipofectamine-fusion cohort yielded 91.3% (63/69) fertilization rate and subsequent cleavage rate at 95.2% (60/63). Albeit at a lower blastocyst development rate comparing to control, the lipofectamine cohort yielded similar full-preimplantation rate at 19.0% (12/63) when comparing to HVJ-E cohort. All blastocysts (n = 20) were transferred into 2 pseudo-pregnant mice and 3 healthy female offspring were yielded to date. Limitations, reasons for caution HVJ-E mediated membrane fusion has been well established with proven safety record; however, its recent shortage has led to a temporary cessation of its utilization. Our lipofectamine-mediated cell fusion technique yielded satisfactory fusion, fertilization, blastocyst formation and some live offspring, however the safety and efficacy still remain to be validated. Wider implications of the findings This novel lipofectamine-mediated cell fusion has broader implications for advancing cloning technologies. Lipofection is a non-viral approach, overcomes shortage of HVJ-E and therefore propose itself as a more appealing cell fusion method for human SCNT. Trial registration number N/A

P-253 Development of embryos from aged mothers transferred to the uterus of young mice

Abstract Study question Is the development of embryos from advanced maternal age (AMA) mice, similar to those from young maternal age (YMA) mice when transferred to young uteri? Summary answer AMA mouse embryos are characterized by altered organ morphology, particularly of the brain, which may predispose to neurodevelopmental disorders. What is known already Human and animal studies have linked AMA pregnancy to elevated risk of neurodevelopmental disorders and behavioural alterations in offspring, primarily attributed to compromised uterine (maternal) conditions. In human, it was also suggested that postnatal care of older mother may influence offspring behaviour. Animal studies revealed that behavioural alterations of AMA offspring are programmed during pregnancy and so they persist in litters fostered by young mouse. It remains unclear whether an embryo of older mother will undergo normal development after embryo transfer to younger female’s uterus. Study design, size, duration Embryos for Advanced Maternal Age, AMA (n = 105) and control Young Maternal Age, YMA (n = 44) groups were collected from thirty females 8-14 months old and six females 3-5 months old, respectively and transferred to pseudo-pregnant mice for further development. At embryonic day 16 (E16) foetuses were collected, fixed for histology and evaluated using Theiler and Kaufman’s classification of mouse development, where E16 corresponds to Theiler stage 24 (TS24) Participants/materials, setting, methods CBA x C57BL6 hybrid mice were used throughout the experiment. At E16, embryo survival rate (number of foetuses collected / number of transferred embryos) was scored for both groups. Foetuses were assessed for visible abnormalities and underwent histological evaluation of organs accordingly to TS 24. Parameters such as size/diameter, signs of vacuolization, cell density and layers of cerebral cortex in brain of foetuses were analysed. Values were considered different when p < 0.05, Mean±SD; Mann-Whitney test. Main results and the role of chance At E16, the survival of AMA embryos did not differ significantly from YMA control (39/105; 37% vs. 20/44; 45%, respectively) however, 5% of AMA foetuses (2/39) displayed oedema or cerebral haemorrhage. Histological analysis revealed alterations in the head/brain of AMA in comparison to YMA foetuses, such as smaller head size (head area in mid-sagittal section: 25.25±4.87 vs. 31.39±3.79 mm2, p < 0.05), thinner cerebral cortex (435.78±12.95 vs. 515.22±17.78µm, p < 0.005), reduced ratio of cortical plate (CP) to cerebral cortex thickness (0.20±0.03 vs. 0.26±0.009, p < 0.05) and an increased ratio of ventricular zone (VZ) to cerebral cortex thickness (0.29±0.002 vs. 0.24±0.002, p < 0.05). Moreover, decreased cellular density in CP layer (10378.86 vs. 12066.40 cells/mm2, P < 0.05) was noted. AMA brains were characterised by high degree of cellular vacuolisation, particularly in medulla oblongata, suggestive for spongiosis (1550.27 vs. 571.92 vacuoles/mm2, p < 0.05). These brain alterations may cause neurodevelopmental disorders in offspring. AMA foetuses had: smaller bronchial diameter (31.52 vs. 36.62µm, p < 0.05), no ossification centres and reduced skin wrinkling, corresponding to TS23 (that is previous developmental stage). These observations may suggest a delayed development of AMA foetuses. Limitations, reasons for caution The results of controlled animal experiments play a key role in developing conceptual models and formulating hypothesis about target species. However, extrapolation of these results to humans requires caution because species-specific developmental features may differ. Wider implications of the findings Our study demonstrates that advanced maternal age (AMA) impacts oocyte/embryo programming in mice. Morphological changes in AMA-conceived foetuses suggest potential risks for neurodevelopmental and other health issues in offspring, even when carried in the uterus of a young female, emphasizing the far-reaching effects of AMA on progeny health. Trial registration number not applicable

O-185 Far away so close: remote ICSI using an AI-powered robot

Abstract Study question Can intracytoplasmic sperm injection (ICSI) be performed remotely using a personal computer and an intelligent robotic micromanipulation system? Summary answer An AI-enhanced robotic micromanipulation system was developed that allows successful remote execution of ICSI by experts or non-experts. What is known already ICSI has become the method of choice for fertilization of eggs during IVF, but it remains technically demanding, requiring manual control of highly specialized equipment by expert embryologists. Limited automation of the procedure has been achieved but most steps are still performed manually. Remote performance of ICSI has never been attempted. Study design, size, duration To achieve automated ICSI, we explored individual commands for each step, issued from an offsite computer. Steps included AI-assisted sperm selection, laser immobilization, random oocyte positioning, opening a path in the zona pellucida prior to needle entry, and Piezo-mediated oolemma breakage. The modified protocols were optimized using mouse oocytes and mouse sperm and then tested using the automated system to inject hamster oocytes with human sperm. Finally, the system was evaluated in consenting patients. Participants/materials, setting, methods The system consists of an inverted microscope, a motorized stage, motorized micromanipulators, laser, piezo-electric actuator, and microinjectors, all controlled by proprietary digital and AI-driven software. Development of this system was a collaborative effort across four countries in three laboratories, including an engineering and embryo research facility, an experimental embryology laboratory, and an IVF clinic. The pilot clinical procedures were performed after full IRB review and trial registration. Main results and the role of chance To assess the safety of using a laser on sperm tails for immobilization, manual ICSI was performed on mouse oocytes with mouse sperm immobilized using different laser settings. Once the method was optimized, fertilization (n = 176, 98.8%) and blastocyst formation (80.7%) in the experimental group were similar to those in the controls without laser (n = 173, 99.4% and 80.6%, respectively) (p > 0.05). Subsequently, 90 blastocysts from the experimental group were transferred to pseudopregnant mice resulting in 40.4% live pups. This was comparable to the results with control embryos (n = 90, 47.8%) (p = 0.098). The remotely controlled robotic system was then tested in hamster oocytes, demonstrating survival (n = 102, 94.1%) similar to the manually injected control group (n = 102, 97.1%) (p = 0.3). In the clinical pilot trial in four patients, 22 oocytes were assigned to remote ICSI, achieving survival (95%), fertilization (81%), and blastocyst formation (65%) comparable to manually performed ICSI controls on sibling oocytes. These results are within the benchmark values established in the Vienna consensus. Throughout all experiments, the system allowed users to successfully perform ICSI remotely issuing commands by pressing single buttons on a computer screen from various locations, ranging in distance from a few meters to over 9,000 kilometers away from the laboratory. Limitations, reasons for caution This experimental ICSI system requires operators in the laboratory to insert and position the microtools and handle the eggs and sperm before and after ICSI. The system also requires a broadband internet connection with at least 50MBPs speed in upload and download. Latency or delayed reaction is a rare possibility. Wider implications of the findings The functionality of a remotely controlled automated ICSI system was validated. The system demonstrates efficiency gains, even with remote operation. The procedures can be programmed to achieve full microscopic ICSI using a single command. This system represents a significant advancement in automation of IVF. Trial registration number Clinicaltrials.gov: NCT06074835 IRB approval: RA-2023-01 IRB ID: CONBIOÉTICA-09-CEI-001-20170131

Establishment of a humanized ST6GAL1 mouse model for influenza research.

This study aimed to construct and characterize a humanized influenza mouse model expressing hST6GAL1. Humanized fragments, consisting of the endothelial cell-specific K18 promoter, human ST6GAL1-encoding gene, and luciferase gene, were microinjected into the fertilized eggs of mice. The manipulated embryos were transferred into the oviducts of pseudopregnant female mice. The offspring were identified using PCR. Mice exhibiting elevated expression of the hST6GAL1 gene were selectively bred for propagation, and invivo analysis was performed for screening. Expression of the humanized gene was tested by performing immunohistochemical (IHC) analysis. Hematologic and biochemical analyses using the whole blood and serum of humanized hST6GAL1 mice were performed. Successful integration of the human ST6GAL1 gene into the mouse genome led to the overexpression of human SiaT ST6GAL1. Seven mice were identified as carrying copies of the humanized gene, and the invivo analysis indicated that hST6GAL1 gene expression in positive mice mirrored influenza virus infection characteristics. The IHC results revealed that hST6GAL1 was expressed in the lungs of humanized mice. Moreover, the hematologic and biochemical parameters of the positive mice were within the normal range. A humanized influenza mouse model expressing the hST6GAL1 gene was successfully established and characterized.

Disruption of early embryonic development in mice by polymethylmethacrylate nanoplastics in an oxidative stress mechanism

Polymethylmethacrylate (PMMA) has been used in many products, such as acrylic glass, and is estimated to reach 5.7 million tons of production per year by 2028. Thus, nano-sized PMMA particles in the environment are highly likely due to the weathering process. However, information on the hazards of nanoplastics, including PMMA in mammals, especially reproductive toxicity and action mechanism, is scarce. Herein, we investigated the effect of PMMA nanoplastics on the female reproductive system of mice embryos during pre-implantation. The treated plastic particles in embryos (10, 100, and 1000 μg/mL) were endocytosed into the cytoplasm within 30 min, and the blastocyst development and indices of embryo quality were significantly decreased from at 100 μg/mL. Likewise, the transfer of nanoplastic-treated embryos at 100 μg/mL decreased the morula implantation rate on the oviduct of pseudopregnant mice by 70%, calculated by the pregnant individual, and 31.8% by the number of implanted embryos. The PMMA nanoplastics at 100 μg/mL significantly increased the cellular levels of reactive oxygen species in embryos, which was not related to the intrinsic oxidative potential of nanoplastics. This study highlights that the nanoplastics that enter systemic circulation can affect the early stage of embryos. Thus, suitable action mechanisms can be designed to address nanoplastic occurrence.

Pseudopregnant mice generated from Piwil1 deficiency sterile mice.

Vasectomized mice play a key role in the production of transgenic mice. However, vasectomy can cause great physical and psychological suffering to mice. Therefore, there is an urgent need to find a suitable replacement for vasectomized mice in the production of transgenic mice. In this study, we generated C57BL/6J mice (Piwil1 D633A-INS99, Piwil1mt/mt) with a 99-base insertion in the Miwi (Piwil1) gene using CRISPR/Cas9 technology and showed that Piwil1mt/+ heterozygous mice were normally fertile and that homozygous Piwil1mt/mt males were sterile and females were fertile. Transplantation of normal fertilized eggs into wild pseudopregnant females following mating with Piwil1mt/mt males produced no Piwil1mt/mt genotype offspring, and the number of offspring did not differ significantly from that of pseudopregnant mice following mating and breeding with ligated males. The CRISPR‒Cas9 system is available for generating Miwi-modified mice, and provides a powerful resource to replace ligated males in assisted reproduction research.

P124 Elucidating the role of risk variants in the BIM gene enhancer in T-Cell dysregulation and rheumatoid arthritis pathogenesis

Abstract Background/Aims The balance governing T-cell responses is pivotal for immune homeostasis. Disruptions to this balance may lead to aberrant behaviours, potentially resulting in autoimmunity. Rheumatoid Arthritis (RA), an autoimmune disorder primarily manifesting in the joints, is characterised by a defective immune response, resulting in chronic inflammation. Enhanced T-cell apoptosis resistance in this context can contribute to sustained activation and abnormal proliferation in a pro-inflammatory environment. Understanding these disease mechanisms could lead to improvements in clinical care. Genome-wide association studies (GWAS) have identified hundreds of single nucleotide polymorphisms (SNPs) associated with RA, preferentially enriched in non-coding, T-cell-specific putative enhancers. Enhancers are DNA elements that can regulate the transcription of specific genes, often from many kilobases away. Our previous work identified BCL2L11, or BIM, as the probable causal gene within the RA locus rs13396472, despite the GWAS variants' intronic positioning relative to ACOXL. The RA risk variant disrupts a distant enhancer regulating the BIM gene, pivotal for apoptosis. Given the role of apoptosis in immune tolerance, disruption of this pathway driven by RA risk variants could pave the way for autoimmune responses. Our aim was to investigate the effect of this enhancer on T-cell proliferation and function in an in vivo model. Methods We mapped the human RA-locus to the mouse genome and utilised CRISPR-Cas9 to delete this putative RA enhancer in mice. Two sgRNAs, combined with Cas9, were microinjected into C57BL6/J zygote pronuclei, followed by embryo transfer to pseudo-pregnant mice. PCR and Sanger sequencing confirmed enhancer deletion in pups, establishing an RA-enhancer deletion colony. To gauge cell proportion differences, murine splenocytes or thymocytes were stained with specific fluorochrome markers and analysed via a flow cytometer. For the antigen-induced arthritis (AIA) model, wildtype and RA-enhancer deletion mice were subjected to arthritis induction via intra-articular injection of methylated bovine serum albumin, following prior immunisation, and monitored for arthritis signs. Results We observed an increased CD4+CD8+ double positive (DP) T-cell population in aged mice homozygous and heterozygous for the RA-enhancer deletion, but not in younger and wild-type controls. Knockout mice exhibited visibly enlarged spleens in response to the immunisation step prior to AIA induction. Further work will establish the mechanisms underlying this and disease progression after arthritis induction. These observations underscore a potential impact this enhancer might have on immune responses and survival upon immune system alteration. Conclusion Our research indicates that perturbing mouse non-coding sequences syntenic to human GWAS loci can provide a physiological model to elucidate novel mechanisms underlying RA, potentially suggesting therapeutic targets. Future research will explore the mechanism by which the enhancer deletion leads to an increase in DP T-cells and the altered phenotype, and the implications of such a change for T- and B-cell biology and RA. Disclosure S. Rossi: None. E. Richards: None. J. Ding: None. C. Shi: None. M. Menon: None. J. Gibbs: None. R. Grencis: None. A. Adamson: None. G. Orozco: None.

A Potential Role of CD82/KAI1 during Uterine Decidualization in Mice.

The tumor metastasis suppressor gene CD82/KAI1 has been demonstrated to impact human trophoblast invasion and migration. Communication between trophoblasts and decidual stromal cells plays a crucial role in controlling the normal invasiveness of trophoblasts. However, whether CD82/KAI1 is involved in decidualization and what role it plays remain unclear. CD82/KAI1 demonstrates specific spatiotemporal expression patterns in stromal cells undergoing decidualization during pregnancy. This is observed in both naturally pregnant females post-implantation and pseudopregnant mice undergoing induced decidualization, as detected through in situ hybridization and immunofluorescence. CD82/KAI1 expression showed a significant time-dependent increase in cultured stromal cells after 24 and 48 h of progesterone (P4) and estrogen (E2) treatment. This was accompanied by a notable upregulation of decidualization markers, including cyclin D3 and PR. After transducing stromal cells with the adenovirus-overexpressing CD82/KAI1 for 48 h, the expression of cyclin D3 protein increased. Meanwhile, there was an attenuated expression of CD82/KAI1 due to an adenovirus siRNA knockdown, whereas cyclin D3 and PR expressions were not affected. Our findings suggest a potential role of CD82/KAI1 in regulating the process of decidualization, providing insights into stromal cell differentiation.

Blastocyst-Derived Lactic Acid May Regulate S100A6 Expression and Function in Mouse Decidualization via Stimulation of Uterine Epithelial Arachidonic Acid Secretion.

(1) Background: Inflammatory responses are implicated in embryo implantation, decidualization, pregnancy maintenance and labor. Both embryo implantation and decidualization are essential to successful pregnancy in rodents and primates. S100A6 is involved in inflammation, tumor development, apoptosis and calcium homeostasis. S100A6 is strongly expressed in mouse decidua, but the underlying mechanisms of how S100A6 regulates implantation and decidualization are poorly defined. (2) Methods: Mouse endometrial stromal and epithelial cells are isolated from day 4 pseudopregnant mouse uteri. Both immunofluorescence and Western blotting are used to analyze the expression and localization of proteins. The molecular mechanism is verified in vitro by Western blotting and the quantitative polymerase chain reaction. (3) Results: From days 4 to 8 of pregnancy, S100A6 is specifically expressed in mouse subluminal stromal cells. Blastocyst-derived lactic acid induces AA secretion by activating the luminal epithelial p-cPLA2. The epithelial AA induces stromal S100A6 expression through the COX2/PGI2/PPAR δ pathway. Progesterone regulates S100A6 expression through the progesterone receptor (PR). S100A6/RAGE signaling can regulate decidualization via EGFR/ERK1/2 in vitro. (4) Conclusions: S100A6, as an inflammatory mediator, is important for mouse implantation and decidualization.

Attenuated retinoic acid signaling is among the early responses in mouse uterus approaching embryo attachment.

The uterus is transiently receptive for embryo implantation. It remains to be understood why the uterus does not reject a semi-allogeneic embryo (to the biological mother) or an allogeneic embryo (to a surrogate) for implantation. To gain insights, we examined uterine early response genes approaching embryo attachment on day 3 post coitum (D3) at 22 hours when blue dye reaction, an indication of embryo attachment, had not manifested in mice. C57BL/6 pseudo-pregnant (control) and pregnant mouse uteri were collected on D3 at 22 hours for microarray analysis. The self-assembling-manifold (SAM) algorithm identified 21,858 unique probesets. Principal component analysis indicated a clear separation between the pseudo-pregnant and pregnant groups. There were 106 upregulated and five downregulated protein-coding genes in the pregnant uterus with fold change (fc) >1.5 and q value <5%. Gene ontology (GO) analysis of the 106 upregulated genes revealed 38 significant GO biological process (GOBP) terms (P <0.05), and 32 (84%) of them were associated with immune responses, with a dominant natural killer (NK) cell activation signature. Among the top eight upregulated protein-coding genes, Cyp26a1 inactivates retinoic acid (RA) while Lrat promotes vitamin A storage, both of which are expected to attenuate RA bioavailability; Atp6v0d2 and Gjb2 play roles in ion transport and transmembrane transport; Gzmb, Gzmc, and Il2rb are involved in immune responses; and Tdo2 is important for kynurenine pathway. Most of these genes or their related pathways have functions in immune regulations. RA signaling has been implicated in immune tolerance and immune homeostasis, and uterine NK cells have been implicated in immunotolerance at the maternal-fetal interface in the placenta. The mechanisms of immune responses approaching embryo attachment remain to be elucidated. The coordinated effects of the early response genes may hold the keys to the question of why the uterus does not reject an implanting embryo.

Intraperitoneal administration of mouse kisspeptin-10 to mice during estrus stage induces pseudopregnancy.

A simple method for producing pseudopregnant mice supports pup production. In this study, pregnant ICR were obtained mice without mating with vasectomized mice via administration of mouse Kisspeptin-10 (mKp-10) and transferring blastocysts to the uterus. Blastocyst transfer after mKp-10 administration to mice with gapping and reddish pink vagina resulted in 65.2% (15/23) pregnancies, and 39.1% (34/87) of the transferred blastocysts showed full-term growth. Vaginal smears were observed for accurate estrus cycle determination, and subsequent administration of mKp10 to mice during the estrus stage and blastocyst transfer resulted in 95.2% (20/21) pregnancies and 50.7% (104/205) birth rates. Regarding 2-cell transfer after administration of mKp-10, 100% (8/8) of the mice became pregnant, and 45.0% (36/80) of the embryos were born. Administration of mKp-10 to mice during the estrus stage is a convenient way to generate pseudopregnant mice.

Generation of Mouse Model of Hemophilia A by Introducing Novel Mutations, Using CRISPR/Nickase Gene Targeting System.

Developing mouse models of hemophilia A has been shown to facilitate in vivo studies to explore the probable mechanism(s) underlying the disease and to examine the efficiency of the relevant potential therapeutics. This study aimed to knockout (KO) the coagulation factor viii (fviii) gene in NMRI mice, using CRISPR/Cas9 (D10A/nickase) system, to generate a mouse model of hemophilia A. Two single guide RNAs (sgRNAs), designed from two distinct regions on NMRI mouse FVIII (mFVIII) exon 3, were designed and inserted in the pX335 vector, expressing both sgRNAs and nickase. The recombinant construct was delivered into mouse zygotes and implanted into the pseudopregnant female mice's uterus. Mutant mice were identified by genotyping, genomic sequencing, and mFVIII activity assessment. Two separate lines of hemophilia A were obtained through interbreeding the offspring of the female mice receiving potential CRISPR-Cas9-edited zygotes. Genomic DNA analysis revealed disruptions of the mfviii gene reading frame through a 22-bp deletion and a 23-bp insertion in two separate founder mice. The founder mice showed all the clinical signs of hemophilia A including; excessive bleeding after injuries, and spontaneous bleeding in joints and other organs. Coagulation test data showed that mFVIII coagulation activity was significantly diminished in the mFVIII knockout (FVIIIKO) mice compared to normal mice. The CRISPR/nickase system was successfully applied to generate mouse lines with the knockout fviii gene. The two novel FVIIIKO mice demonstrated all clinical symptoms of hemophilia A, which could be successfully inherited. Therefore, both of the developed FVIIIKO mouse lines are eligible for being considered as proper mouse models of hemophilia A for in vivo therapeutic studies.

The presence of blastocyst within the uteri facilitates lumenal epithelium transformation for implantation via upregulating lysosome proteostasis activity

ABSTRACT The mammalian endometrium is covered by the lumenal epithelium (Le), which directly interacts with the blastocyst and plays an important role in the establishment of reciprocal crosstalk between the embryo and receptive uterus during implantation. However, the effect of the blastocyst on uterine differentiation during the window of receptivity is far from well understood. Through transcriptomic profiling of the uterine Le isolated by laser capture microdissection (LCM), it was demonstrated that global gene expression changes occurred in Le between pseudopregnant mice without embryos and pregnant mice with embryos. Some differentially expressed genes, including upregulated Areg (amphiregulin), Ihh (Indian hedgehog), Lifr (leukemia inhibitory factor receptor) and downregulated Msx1 (msh homeobox 1), Pgr (progesterone receptor), and Gata2 (GATA binding protein 2) in pregnant mice, have been reported to regulate the establishment of uterine receptivity. Besides, we found that blastocysts induced an increase in both the number and acidification of lysosome, consistent with enhanced lysosomal hydrolase activity in uterine Le. Further exploration uncovered that blastocyst-derived IGF2 was involved into the activation of epithelial STAT3 to induce lysosomal hydrolase expression, and inhibition of lysosomal function derails both uterine receptive maker gene expressions and embryo implantation. Finally, based on the proteomic data of both epithelia and the separated lysosome, it was revealed that CLDN1 (claudin 1) and MUC1 (mucin 1, transmembrane), two well-known downregulated molecules for successful implantation, are degraded by epithelial lysosome. In brief, our data demonstrated that blastocysts induced normal epithelium differentiation with lysosome activation to promote the uterine epithelial differentiation for embryo implantation. Abbreviations ACTB: actin beta; AREG: amphiregulin; ATP6V0A4: ATPase, H+ transporting, lysosomal V0 subunit A4; Baf A1: bafilomycin A1; BSA: bovine serum albumin; CLDN1: claudin 1; CTSB: cathepsin B; DEGs: differentially expressed genes; E2: 17β-estradiol; ESR: estrogen receptor; GATA2: GATA binding protein 2; GLA: galactosidase, alpha; GO: gene ontology; HBEGF: heparin-binding EGF-like growth factor; IGF1R: insulin-like growth factor 1 receptor; Ihh: Indian hedgehog; ISH: in situ hybridization; LAMP1: lysosomal-associated membrane protein 1; LCM: laser capture microdissection; Le: lumenal epithelium; LGMN: legumain; LIF: leukemia inhibitory factor; LIFR: LIF receptor alpha; MSX1: msh homeobox 1; MUC1: mucin 1, transmembrane; P4: progesterone; PBS: phosphate-buffered saline; PCA: principal component analysis; PPT1: palmitoyl-protein thioesterase 1; PGR: progesterone receptor; PSP: pseudopregnancy; PTGS2/COX2: prostaglandin-endoperoxide synthase 2; qPCR: quantitative real-time polymerase chain reaction; SP: pregnancy; TFEB: transcription factor EB.

Placental deficiency of the (pro)renin receptor ((P)RR) reduces placental development and functional capacity.

The (pro)renin receptor ((P)RR; also known as ATP6AP2) is a multifunctional receptor. The (P)RR activates the tissue renin-angiotensin system (RAS) and is also involved in regulating integral intracellular pathways such as V-ATPase and Wnt/β-catenin signalling. Given this, the (P)RR may be associated with essential pathways in placentation, however its role within the context of pregnancy remains poorly characterised. The first trimester/extravillous trophoblast cell line, HTR-8/SVneo, underwent an siRNA knockdown where they were incubated for 24h with a negative control siRNA or siRNA targeting ATP6AP2 mRNA. xCELLigence real-time cell analysis was performed to assess the effect of ATP6AP2 mRNA knockdown on HTR-8/SVneo cell proliferation, migration, and invasion. In subsequent experiments, GFP-encoding lentiviral packaged gene-constructs were used to knockdown (P)RR expression in the trophectoderm of C57/BL6/CBA-F1 mouse blastocysts. Blastocysts were incubated for 6h with vehicle (no-virus), control virus (non-targeting shRNA and GFP), or (P)RR-knockdown virus ((P)RR shRNA and GFP) before transfer into recipient pseudo-pregnant Swiss CD1 female mice. Fetal and placental tissues were collected and assessed at embryonic age (EA) 10 and 18. (P)RR levels were measured in the labyrinth zone of day 18 placentae and stereological Merz grid analysis was performed to determine the volumetric distribution of trophoblasts, fetal capillaries, and the maternal blood space. We showed that a reduction of ATP6AP2 expression in HTR-8/SVneo cells in vitro, impaired trophoblast proliferation, migration, and invasion. In vivo, decreasing placental labyrinth (P)RR expression adversely effected placental physiology, decreasing placental trophoblast number and total surface area available for exchange, while also increasing maternal blood space. Additionally, decreased (P)RR affected placental efficacy evident by the reduced fetal-placental weight ratio. Our study shows that the (P)RR is necessary for appropriate placental development and function.

Embryo-derive TNF promotes decidualization via fibroblast activation.

Decidualization is a process in which endometrial stromal fibroblasts differentiate into specialized secretory decidual cells and essential for the successful establishment of pregnancy. The underlying mechanism during decidualization still remains poorly defined. Because decidualization and fibroblast activation share similar characteristics, this study was to examine whether fibroblast activation is involved in decidualization. In our study, fibroblast activation-related markers are obviously detected in pregnant decidua and under in vitro decidualization. ACTIVIN A secreted under fibroblast activation promotes in vitro decidualization. We showed that arachidonic acid released from uterine luminal epithelium can induce fibroblast activation and decidualization through PGI2 and its nuclear receptor PPARδ. Based on the significant difference of fibroblast activation-related markers between pregnant and pseudopregnant mice, we found that embryo-derived TNF promotes CPLA2α phosphorylation and arachidonic acid release from luminal epithelium. Fibroblast activation is also detected under human in vitro decidualization. Similar arachidonic acid-PGI2-PPARδ-ACTIVIN A pathway is conserved in human endometrium. Collectively, our data indicate that embryo-derived TNF promotes CPLA2α phosphorylation and arachidonic acid release from luminal epithelium to induce fibroblast activation and decidualization.

Plastic used in in vitro fertilization procedures induces massive placental gene expression alterations

The exposure to plastic derivatives during human life is deleterious. Infants conceived using ART (IVF or ICSI) have twice as many risks of major birth defects compared to naturally conceived infants. Could plastic ware used during ART trigger defects in the fetal development? Three groups of blastocysts were transferred to pseudopregnant mice. One was obtained after IVF and embryo development in plastic ware, the second in glass ware. The third, was obtained invivo by natural mating. On day 16.5 of pregnancy, females were sacrificed and fetal organs collected for gene expression analysis. Fetal sex was determined by RT-PCR. RNA was extracted from a pool of five placental or brain samples coming from at least two litters from the same group and analyzed by hybridisation onto the mouse Affymetrix 430.2.0 GeneChips, confirmed by RT-qPCR for 22 genes. This study highlights a major impact of plastic ware on placental gene expression (1121 significantly deregulated genes), while glassware was much closer to invivo offspring (only 200 significantly deregulated genes). Gene Ontology indicated that the modified placental genes were mostly involved in stress, inflammation and detoxification. A sex specific analysis revealed in addition a more drastic effect on female than male placentas. In the brains, whatever the comparison, less than 50 genes were found deregulated. Embryos incubated in plastic ware resulted in pregnancy with massive alterations of placental gene expression profile in concerted biological functions. There were no obvious effects on the brains. Besides other effects, this suggests that plastic ware in ART could be a cause of the increased level of pregnancy disorders observed recurrently in ART pregnancies. This study was funded by two grants from the Agence de la Biomedecine in 2017 and 2019.

MP01-08 THE TESTIS AND EPIDIDYMIS-SPECIFIC WFDC LOCUS GENES ARE REQUIRED FOR MALE FERTILITY

You have accessJournal of UrologyCME1 Apr 2023MP01-08 THE TESTIS AND EPIDIDYMIS-SPECIFIC WFDC LOCUS GENES ARE REQUIRED FOR MALE FERTILITY Katarzyna Kent, Kimia Bakhshandeh, Kanchan Yadav, Martin Matzuk, Larry Lipshultz, and Thomas Garcia Katarzyna KentKatarzyna Kent More articles by this author , Kimia BakhshandehKimia Bakhshandeh More articles by this author , Kanchan YadavKanchan Yadav More articles by this author , Martin MatzukMartin Matzuk More articles by this author , Larry LipshultzLarry Lipshultz More articles by this author , and Thomas GarciaThomas Garcia More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003212.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Despite 96 million years of evolution separating humans and rodents, 11 closely-related reproductive tract-specific genes in human—SPINT3, WFDC6, EPPIN, WFDC8, WFDC9, WFDC10A, WFDC11, WFDC10B, WFDC13, SPINT4, and WFDC3—and the 13 reproductive tract-specific orthologs in mouse (listed below), form highly conserved syntenic gene clusters indicative of a conserved, combined critical functions. Further, despite significant progress towards a non-hormonal male contraceptive targeting the protein encoded by one of these genes, EPPIN, and associations found between gene mutations in EPPIN and an increased risk of idiopathic male infertility, neither EPPIN nor any of the closely-related genes within the WFDC locus have been explored functionally through genetically manipulated mouse models. To clarify the involvement of these genes in male fertility, we generated and studied mice lacking a combined total of 13, 10, 5, or 4 genes within the conserved gene cluster. METHODS: Custom gRNAs were assembled into ribonucleoprotein complexes with Cas9 nuclease and electroporated into zygotes harvested from superovulated B6D2F1 females. Embryos were cultured overnight to the 2-cell stage before being transferred into the oviducts of pseudopregnant ICR mice. Founder mutations in pups born were identified by PCR and Sanger sequencing. RESULTS: Founder mouse containing a -173.2 kb deletion, corresponding to deletion of all 13 genes (Spint3, Wfdc6a, Eppin, Wfdc8, Wfdc6b, Wfdc16, Wfdc9, Wfdc10, Wfdc11, Wfdc13, Spint4, Spint5, Wfdc3) were expanded for further study. Likewise, founder mice containing either a -115.4 kb deletion (10 genes deleted from Spint3 to Wfdc13), a -46.7 kb deletion (5 genes deleted from Wfdc16 to Wfdc13), and a -28.8 kb deletion (4 genes deleted from Wfdc6a to Wfdc6b) were expanded for further study. We also generated and expanded a single knockout of Wfdc3. The 13 gene, 10 gene, and 4 gene knockouts were all infertile due to a block of spermatogenesis and azoospermia. The 5 gene knockouts were infertile due to asthenozoospermia, while the single knockout of Wfdc3 was fertile with no obvious reproductive phenotype. CONCLUSIONS: Our knockout mouse studies, the first of its kind to study an entire locus of closely related genes, have at last clarified the functional requirement of WFDC locus genes in male fertility, which has implications towards improved clinical diagnosis of men with infertility. Source of Funding: This study has been supported by National Institutes of Health R01 HD106056 and R01 HD095341 (to T.X.G.) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e4 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Katarzyna Kent More articles by this author Kimia Bakhshandeh More articles by this author Kanchan Yadav More articles by this author Martin Matzuk More articles by this author Larry Lipshultz More articles by this author Thomas Garcia More articles by this author Expand All Advertisement PDF downloadLoading ...

CRISPR/Cas9-mediated knockin of IRES-tdTomato at Ins2 locus reveals no RFP-positive cells in mouse islets

Using the CRISPR/Cas9 genomic editing technology, we constructed a transgenic mouse model to express specific fluorescent protein in pancreatic β cells, which harbor tdTomato exogenous gene downstream of the Ins2 promoter in C57BL/6 J mice. The Ins2-specific single-guide RNA-targeted exon2 was designed for the CRISPR/Cas9 system and Donor vector was constructed at the same time. Then Cas9, sgRNA, and Donor vector were microinjected in vitro into the mouse zygotes that were implanted into pseudo-pregnant mice. We obtained homozygotes through mating heterozygotes, and verified the knockin effect through genotype identification, in vivo imaging, and frozen section. Six F0 mice and stable inherited Ins2-IRES-tdTomato F1 were obtained. Genome sequencing results showed that the knockin group had no change in the Ins2 exon compared with the control group, while only the base sequence of tdTomato was added and no base mutation occurred. However, in vivo imaging and frozen section did not observe the expression of red fluorescent protein (RFP), and the protein expression of knockin gene tdTomato was negative. As a result, the expressions of tdTomato protein and fluorescence intensity were low and the detection threshold was not reached. In the CRISP/Cas9 technique, the exogenous fragment of IRES connection would affect the transcription level of the preceding gene, which in turn would lead to low-level expression of the downstream gene and affect the effect of gene insertion.

Improved Genome Editing via Oviductal Nucleic Acids Delivery (i-GONAD): Protocol Steps and Additional Notes.

The clustered regularly interspaced short palindromic repeats (CRISPR) technology has made it possible to produce genome-edited (GE) animals more easily and rapidly than before. In most cases, GE mice are produced by microinjection (MI) or by in vitro electroporation (EP) of CRISPR reagents into fertilized eggs (zygotes). Both of these approaches require ex vivo handling of isolated embryos and their subsequent transfer into another set of mice (called recipient or pseudopregnant mice). Such experiments are performed by highly skilled technicians (especially for MI). We recently developed a novelgenome editing method, called "GONAD (Genome-editing via Oviductal Nucleic Acids Delivery)," which can completely eliminate the ex vivo handling of embryos. We also made improvements to the GONAD method, termed "improved-GONAD (i-GONAD)." The i-GONAD method involves injection of CRISPR reagents into the oviduct of an anesthetized pregnant female using a mouthpiece-controlled glass micropipette under a dissecting microscope, followed by EP of the entire oviduct allowing the CRISPR reagents to enter into the zygotes present inside the oviduct, in situ. After the i-GONAD procedure, the mouse recovered from anesthesia is allowed to continue the pregnancy to full term to deliver its pups. The i-GONAD method does not require pseudopregnant female animals for embryo transfer, unlike the methods relying on ex vivo handling of zygotes. Therefore, the i-GONAD method can reduce the number of animals used, compared to the traditional methods. In this chapter, we describe some newer technical tips about the i-GONAD method. Additionally, even though the detailed protocols of GONAD and i-GONAD have been published elsewhere (Gurumurthy et al., Curr Protoc Hum Genet 88:15.8.1-15.8.12, 2016 Nat Protoc 14:2452-2482, 2019), we provide all the protocol steps of i-GONAD in this chapter so that the reader can find most of the information, needed for performing i-GONAD experiments, in one place.