Pseudopregnant Females Research Articles

Ovarian support cell in vitro maturation (OSC-IVM) results in healthy murine live births with no evidence of reprotoxicology in a multigenerational study

Research questionDoes application of human stem cell-derived ovarian support cells (OSCs) for in vitro maturation (IVM) have a safe reproductive toxicity profile? DesignA total of 70 B6/CBA 6–8-week-old stimulated female mice were used to collect immature oocytes (n=2,025) at the germinal vesicle stage. Oocytes were retrieved denuded (n=930) or with cumulus cells (COCs, n=1,095). These were randomly assigned to traditional IVM media (MediCult-IVMTM, Origio) or the same IVM media (MediCult-IVMTM, Origio) further supplemented with human OSCs (FertiloTM, Gameto Inc.). Metaphase II (M2) oocytes were inseminated by intracytoplasmic sperm injection and cultured to blastocyst stage. Vitrified blastocysts were transferred to pseudopregnant females, and F1 offspring were evaluated for various health and developmental metrics. Select F1 mice were outbred to produce the F2 generation, assessing live births, sex ratio, morphology, behavior, and health. ResultsIn mice OSC-IVM generally led to a decreased maturation rate compared to Traditional-IVM (p=0.0101). Subsequent embryo culture yielded similar cleavage rates between the four groups (p=0.7547). However, there was a significant distinction in the blastocyst formation with OSC-IVM showing higher blastocyst formation rates for both denuded oocytes (p=0.0408) and COCs (p=0.0063). Live births, sex ratio, development, and behavior in F1 and F2 generations revealed no adverse effects or significant differences, confirming the absence of long-term toxicity. ConclusionsThis study demonstrates the safety of the OSC-IVM system, with no adverse effects on embryo development or offspring health and fertility across generations.

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.

Pronuclear transfer rescues poor embryo development of in vitro-grown secondary mouse follicles.

Pronuclear transfer rescues poor embryo development of in vitro-grown secondary mouse follicles.

Knockdown of Placental Major Facilitator Superfamily Domain Containing 2a in Pregnant Mice Reduces Fetal Brain Growth and Phospholipid Docosahexaenoic Acid Content.

Docosahexaenoic acid (DHA) is an n-3 long chain polyunsaturated fatty acid critical for fetal brain development that is transported to the fetus from the mother by the placenta. The lysophosphatidylcholine (LPC) transporter, Major Facilitator Superfamily Domain Containing 2a (MFSD2a), is localized in the basal plasma membrane of the syncytiotrophoblast of the human placenta, and MFSD2a expression correlates with umbilical cord blood LPC-DHA levels in human pregnancy. We hypothesized that placenta-specific knockdown of MFSD2a in pregnant mice reduces phospholipid DHA accumulation in the fetal brain. Mouse blastocysts (E3.5) were transduced with an EGFP-expressing lentivirus containing either an shRNA targeting MFSD2a or a non-coding sequence (SCR), then transferred to pseudopregnant females. At E18.5, fetuses were weighed and their placenta, brain, liver and plasma were collected. MFSD2a mRNA expression was determined by qPCR in the brain, liver and placenta and phospholipid DHA was quantified by LC-MS/MS. MFSD2a-targeting shRNA reduced placental mRNA MFSD2a expression by 38% at E18.5 (n = 45, p < 0.008) compared with SCR controls. MFSD2a expression in the fetal brain and liver were unchanged. Fetal brain weight was reduced by 13% (p = 0.006). Body weight, placenta and liver weights were unaffected. Fetal brain phosphatidyl choline and phosphatidyl ethanolamine DHA content was lower in fetuses with placenta-specific MFSD2a knockdown. Placenta-specific reduction in expression of the LPC-DHA transporter MFSD2a resulted in reduced fetal brain weight and lower phospholipid DHA content in the fetal brain. These data provide mechanistic evidence that placental MFSD2a mediates maternal-fetal transfer of LPC-DHA, which is critical for brain growth.

Porcine-specific expression ofthe three functional CYP19paralogs in early conceptus, placenta, and gonads.

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 expression was detected in conceptuses and gonads, respectively. Cyp19a2 transcripts were identified on both conceptuses and placenta samples. Transcripts for Cyp17a1 were detected predominantly in conceptus and gonads. In the endometrium of Day 21 pregnant, as well as Days 12 and 17 pseudopregnant females, we did not detect 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 secretion of estrogen from the conceptus and the placenta.

The effect of calcitriol on the development and implantation capacity of embryos from hyper-stimulated mice.

The evidence concerning the role of vitamin D (VD) in reproduction is still inconclusive. Calcitriol was given to superovulated female mice at the time of FSH injection (Group A), or at day 0.5 of pregnancy (Group B). The retrieved and cultured embryos were transferred to the uteri of pseudopregnant females. Ten animals from each group conceived naturally, and at day 7.5 of pregnancy, the implantation sites were counted. Serum hormone concentrations were determined by ELISA. The expression of CD70, PD-L1, OX-40L, and PIBF on extracellular vesicles (EVs) was tested by flow cytometry. Calcitriol treatment did not alter serum oestradiol concentrations, while 25(OH) D levels significantly decreased in both treated groups. Progesterone concentrations were significantly higher in group A and lower in group B than in the controls. On EVs produced by group B embryos PIBF, CD70, and OX-40L expression were significantly lower, while that of PD-L1 was significantly higher than that of controls. Calcitriol treatment decreased the fertilization rate in group A, and the blastulation rate of cultured embryos in group B, while the implantation capacity of the embryos was not affected, suggesting that depending on the time of administration, VD has an adverse effect on oocyte maturation and embryo development, but not on the implantation rates.

Inducing Pseudopregnancy in Female Mice Without the Need for Vasectomized Males Prior to Non-Surgical Embryo Transfer or Artificial Insemination.

For successfully maintaining pregnancy with embryo transfer or artificial insemination,female recipient mice must be induced into a pseudopregnant state. Female mice are traditionally paired overnight with vasectomized males, and the following morning, the presence of a copulation plug is assessed. To increase the efficiency of producing pseudopregnant females, a cervical manipulation technique has been standardized to be used in combination with non-surgical embryo transfer or artificial insemination techniques in mice. The blunt end of a small plastic rod is inserted vaginally to contact the cervix and is vibrated for 30 s by contact with a trimmer. The procedure is quick and does not require anesthesia or analgesia. This technique increases the reliability and predictability of producing pseudopregnant females and entirely eliminates the requirement for vasectomized males. For CD1 mice, the efficiency of pseudopregnancy induction using cervical manipulation was 83% for females in estrus (N = 76) but only 38% of females in estrus were plugged by vasectomized males (N = 24). Artificial insemination in CD1 mice was performed by estrus synchronization with hormones, cervical manipulation, and the uterine transfer of sperm. Artificial insemination recipients receiving cervical manipulation (N = 76) had a pregnancy rate of 72% and an average litter size of 8.3 pups. This method can also be used to produce pseudopregnant females for non-surgical embryo transfer. Therefore, inducing pseudopregnancy by cervical manipulation is a convenient and efficient alternative to mating with a vasectomized male when performing non-surgical assisted reproduction techniques. Using cervical manipulation provides 3Rs (replacement, reduction, and refinement) benefits for assisted reproduction techniques by reducing the number of animals required and eliminating the necessity for surgically altered males.

Expression of Toll-Like Receptors in the Lung Tissue of Mouse Fetuses Generated by in vitro Embryo Culture and Embryo Transfer

Mouse fetuses generated by in vitro embryo culture and embryo transfer exhibit impaired lung development, altered composition of pulmonary epithelial cells associated with downregulation of several genes involved in lung development and toll-like receptor (TLR) signaling pathway. The aims of the present study were to determine the expression of all TLRs and to examine if the expression of TLRs, along with genes involved in TLR signaling pathway, is altered in the lung tissue of mouse fetuses generated through embryo culture and embryo transfer. Two experimental (EGs) and one control (CG) group were included in the study. Embryos cultured at 5% CO2-95% air for 95 h or less than 24 h were transferred to pseudo-pregnant females to obtain fetuses comprising EGin vitro (n = 18) and EGin vivo (n = 18), respectively. Fetuses obtained from naturally ovulating females on day 18 of pregnancy served as the CG (n = 18). Western blot and immunohistochemistry were used to determine the expression of TLR proteins. The expression of transcripts encoding TLRs, and the genes involved in TLR signaling pathway (Lbp, Pik3r1, Pik3cb, Nfkbia, and Fos), was determined using qRT-PCR. While all TLRs were expressed by cells lining the bronchial/bronchiolar epithelium of lung tissues in all groups, some of the TLRs were expressed in a specific pattern. When compared to CG, the expression of transcripts encoding TLR-2, -3, -4, -5, -7, -8, -9, -12, -13, Lbp, Pik3r1, Pik3cb, Nfkbia, and Fos was significantly downregulated in both EGs. It appears that stress imposed on embryos at preimplantation stages of development is associated with downregulation of TLRs, along with some of the genes involved in TLR signaling pathway, in the lung tissue during the perinatal period. It remains to be determined if downregulation of TLRs, along with the genes involved in TLR signaling pathway, has any functional consequences in the adult lung tissue.

Scheduled simple production method of pseudopregnant female mice for embryo transfer using the luteinizing hormone-releasing hormone agonist

The use of mice as experimental animal models has been a practice since the development of genetically engineered mouse models (GEMMs) in the early 1980s. New technologies, including genome editing, have helped in the time- and cost-efficient generation of GEMMs. However, methods for preparing pseudopregnant females, essential for the generation of GEMMs, remain less advanced. This study proposes a new method to achieve simple production of pseudopregnant female mice using a luteinizing hormone-releasing hormone agonist (LHRHa). A 20 µg LHRHa/mouse was identified as the best dose for inducing estrus synchronization. However, the frequency of copulation was 40% on a single injection. With sequential injections of 20 µg LHRHa/mouse on Days-1 and -2, followed by pairing on Day-5, 74% of LHRHa-treated females copulated with male mice. Moreover, LHRHa treatment did not affect fetal and postnatal development. Eventually, successful generation of offspring via embryo transfer was attained using LHRHa-treated pseudopregnant females. LHRHa administration method is efficient in producing pseudopregnant female mice for the generation of GEMMs, and we expect that it will contribute towards advancing the clinical research.

Embryological Aspects of Creating a New Humanized Transgenic Mouse Line with an Integrated Human Gene &lt;i&gt;HLA A*02:01:01:01&lt;/i&gt;

The accumulation of scientific data in the field of pharmacogenetics requires the creation of adequate biomodels to reflect the immunogenetic characteristics of different population groups. We have obtained the ancestors of a new humanized transgenic mouse line carrying the human HLA-A*02:01:01:01 gene, which is characteristic of the Russian population. The new biomodels was created using the pronuclei microinjection method of a linearized fragment of genetically engineered DNA construct into zygotes, followed by overnight cultivation in CO2 incubator and transfer of potentially modified embryos at the stage of two 4.0 blastomere to pseudopregnant foster females. A total of 91 living offspring were obtained and analyzed, with 18 pups carrying the target genome modification. The resulting transgenic animals were used to create a new line of mouse biomodels carrying the human HLA-A*02:01:01:01 gene.

OR25-4 Active Testosterone Treatment Impairs In Vitro Fertilization (IVF) Outcomes in a Female Mouse Model for Gender-Affirming Testosterone with Improvement in Outcomes Following Cessation of Testosterone Treatment

Abstract Introduction The impact of testosterone (T) on reproductive potential is poorly understood. Masculinizing hormones have demonstrated mixed effects on ovarian histology and data on assisted reproductive outcomes are limited to small case series. There is consensus among major medical societies to encourage fertility preservation counseling prior to initiation of gender-affirming hormone care, however, it is uncertain whether a break in T treatment prior to undergoing oocyte cryopreservation is beneficial. We hypothesized that T treatment would not have an impact on IVF outcomes. Methods C57BL/6N female mice were implanted with silastic tubing with either 10 mg testosterone enthanate in ethanol (n = 20) or ethanol alone (n = 18) at 10 weeks of age. Four groups of C57BL/6N female mice were used in this study: 1) current T implant 2) current sham implant 3) T cessation 4) control cessation. Biweekly blood samples and vaginal cytology were undertaken to monitor T levels and estrous cycles. For groups 1 and 2, mice were stimulated twelve weeks following implantation. For groups 3 and 4, implants were removed after twelve weeks and mice were stimulated two weeks after explantation. All mice were stimulated with 0.2 mL intraperitoneal CARD HyperOva followed 48 hours later by 7.5 IU intraperitoneal human chorionic gonadotropin (hCG). Oocytes were collected 14 hours after hCG injection and fertilized in vitro with sperm from 12-week-old B6D2F1/J male mice. 2-cell embryos were transferred into the oviducts of pseudopregnant recipient females. The study was powered to detect a 25% difference in 2-cell embryos. Data were analyzed using Chi-squared and unpaired t-tests with Prism 9.0. Results All T treated mice ceased cycling within one week of implantation and developed clitoromegaly. The current T implant group had fewer total oocytes retrieved (17.00 vs 36.00; p &amp;lt; 0.0001), mature oocytes (13.00 vs 28.10; p = 0.0002) and 2-cell embryos (12.78 vs 26.90; p = 0.0007) as compared to the current sham implant group. There was no significant difference in maturity (76.47% vs 78.06%; p = 0.6937) or fertilization rate (75.16% vs 74.72%; p = 0.9161). Pseudopregnant females who had 2-cell embryos transferred from current T implant mice were less likely to have a live birth than those with transfers from current sham implant mice (25.00% vs 80.00%; p = 0.0196). In contrast, comparison of IVF outcomes in the T cessation group with corresponding control cessation group revealed no significant differences between total oocytes (22.30 vs 30.75; p = 0.0708), mature oocytes (20.80 vs 27.75; p = 0.1288), or 2-cell embryos (20.40 vs 27.38; p = 0.104). Conclusion In a mouse model of gender-affirming testosterone, active T treatment negatively impacted IVF outcomes. Improved outcomes following cessation of T treatment support reversibility of T impact on reproductive potential. Presentation: Monday, June 13, 2022 11:45 a.m. - 12:00 p.m.

Altered Expression of Pulmonary Epithelial Cell Markers in Fetal and Adult Mice Generated by in vitro Embryo Culture and Embryo Transfer

Lung development is impaired in mice generated through transfer of in vitro-derived blastocysts. The main objective of the current study was to determine if the composition of epithelial cells in the fetal and adult lung tissue is altered in mice generated through transfer of in vitro-derived blastocysts. The study comprised two experimental (EGs) and two control (CGs) groups. Fetuses (18.5 d.p.c.) and adult mice (8 weeks old) of the EGs (EG_fetus, n = 18; EG_adult, n = 15) were produced by the transfer of day 5 F2 blastocysts to pseudo-pregnant females. F2 fetuses and adult mice derived from naturally ovulating females served as the CGs (CG_fetus, n = 18; CG_adult, n = 15). The expression of Tuba-1a (a marker of ciliated cells), Foxj-1 (a marker of motile ciliated cells), Uch-L1 (a marker of neuroendocrine cells), Cldn-10 (a marker of club cells), Aqp-5 (a marker of type I alveolar cells), and Sp-C (a marker of type II alveolar cells) was determined using Western blot, immunohistochemistry/immunofluorescence, and quantitative RT-PCR analyses. Weight of fetuses as well as adult mice is decreased in mice comprising the EGs. Impaired lung development observed in EG_fetus was associated with altered expression of Tuba-1a, Foxj-1, Cldn-10, Uch-L1, Sp-C, and Aqp-5. Morphology of the adult lung tissue was similar between the groups except for a significant increase in the thickness of the epithelia in EG_adult. The expression of Cldn-10 and Sp-C was also altered in EG_adult. It remains to be determined whether altered expression of these genes has any long-term impact on epithelial cell functions in the adult lung tissue.

Short-Term Exposure with Long-Term Effects: The Ketone Body, Beta-Hydroxybutyrate, Alters Mouse Embryo Developmental Programming and Reduces Viability

Background: Diet-induced nutritional changes in the maternal reproductive tract stimulate embryonic adaptations to maintain growth and survival, directly impacting child and adult health via developmental programming. This is plausibly facilitated by an interrelationship between metabolism and epigenetic control of gene expression, termed ‘metaboloepigenetics’, resulting in heritable modifications that affect long-term offspring health and viability. The ketogenic diet (KD) is increasingly popular amongst sub-fertile women trying to conceive, and results in elevated maternal ketone levels, including beta-hydroxybutyrate (bOHB). The impact of high bOHB levels on embryonic development, physiology, and developmental programming remains unknown. Aim: To assess how bOHB affects preimplantation mouse embryo development, metaboloepigenetic regulation, and post-transfer viability. Method: Preimplantation mouse embryos were cultured in vitro with or without 2 mM bOHB, representing serum concentrations with KD consumption. Day 5 blastocyst cell number and lineage allocation was assessed via differential nuclear stain, metabolism of bOHB and carbohydrates was assessed by ultramicrofluorescence, and acetylation of histone 3 lysine 9 (H3K9ac) and lysine 27 (H3K27ac) were assessed by immunofluorescence. Day 4 blastocysts were transferred to pseudo-pregnant females for analysis of embryonic day 14.5 placental and fetal development. Results: A reduction in total and trophectoderm cell number (P&lt;0.05) was observed, indicating reduced viability. Blastocysts were shown to consume bOHB in vitro, with uptake increasing with bOHB concentration. Further, bOHB increased glycolytic rate (P&lt;0.01), while epigenetic analyses revealed H3K9ac and H3K27ac were unaffected by bOHB. Of significance, post-transfer implantation rates were reduced (P&lt;0.05), concurrent with smaller placental diameter (P&lt;0.01) and fetal crown-rump length (P&lt;0.05), indicating developmental programming alterations. Conclusion: These data infer that bOHB-stimulated metabolic alterations are a contributing factor to ketone-induced developmental programming, which may incorporate epigenetic components other than H3K9ac or H3K27ac. Therefore, consumption of a KD during conception and pregnancy may have negative impacts on the long-term viability and health of offspring.

Development of humanized ACE2 mouse and rat models for COVID‐19 research

To date (17 November 2021), there has been more than 254 million confirmed cases of COVID‐19, and more than 5 million death globally (World Health Organization. https://covid19.who.int/). The virus causing COVID‐19 is called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2). SARS‐CoV‐2 infects host cells by the binding of its spike protein to the cellular surface protein angiotensin‐converting enzyme 2 (ACE2). The predicted 29 amino acid residues of ACE2 that interact with SARS‐CoV‐2 spike protein receptor binding domain (RBD) vary between human ACE2 and mouse or rat ACE2. Therefore, wildtype mice and rats show lower SARS‐CoV‐2 infection rate and mild symptoms compared to what is seen in humans. Small animal models that recapitulate human COVID‐19 disease are urgently needed for better understanding the transmission and therapeutic measurement. Currently, scientists use either mouse‐adapted SAS‐CoV‐2 (SAS‐CoV‐2 MA) models or random transgenic mouse models that artificially express human ACE2 under the control of cytokeratin 18 promoter or a constitutive promoter. SAS‐CoV‐2 MA may not completely reflect all aspects of the original human‐tropic SAS‐CoV‐2 and the current transgenic human ACE2 mouse models typically have high mortality rate caused by neuroinvasion and encephalitis due to very high human ACE2 expression. To overcome these limitations, we have developed humanized ACE2 mouse and rat models using CRISPR‐Cas9. Specifically, we inserted a ~3kb human ACE2 cDNA cassette into the mouse and rat Ace2 gene loci to ensure that human ACE2 expression is under the control of rodent Ace2 promoter and regulatory elements, while simultaneously disabling the rodent Ace2 gene. To accomplish this, CRISPR gRNAs targeting close to the translation initiation site of Ace2 were screened in cultured mouse and rat cells. Then CRISPR/Cas9 complex and donor DNA were subsequently microinjected into one‐cell stage embryos which were subsequently implanted into pseudo pregnant females. Resulting pups were screened for correct knockin by junction PCR and insert PCR, and the PCR products were Sanger sequenced. Targeted Locus Amplification (TLA) further confirmed the integration sites and transgene sequence. RT‐qPCR and Western blot analysis data showed that, in our models, human ACE2 is expressed in tissues expressing endogenous Ace2 (such as lung, kidney, and GI tract), while rodent endogenous Ace2 is absent from these tissues. Further breeding data indicated that both hemizygous and homozygous humanized ACE2 animals appear to be normal and fertile. Most importantly, animals displayed symptoms after infection with SARS‐CoV‐2. In summary, these data suggest that our humanized ACE2 models can be valuable for COVID‐19 research.

In vitro production of viable eggs from isolated mouse primary follicles by successive culture.

To produce viable eggs from single primary follicles in vitro, primary follicles containing oocytes (average 39.0 ± 0.2 µm in diameter) were isolated from the ovaries of 1-week-old mice, and cultured in combination with culture membranes for the first 8 days up to the secondary follicle stage, followed by the next 12 days to the later stages. After culture with a combination of first and second culture membranes using high and low adhesion characteristics, the average oocyte diameters of the surviving follicles increased by almost two-fold in all four groups. Further, the oocyte maturation rate was the highest (74.1%) in the culture group with low adhesion with collagenase and high adhesion. In this culture group, when the O2 concentration was changed from 20% in the first culture to 5% in the second culture, the cleavage rate increased to 47.5%, which was comparable to the level of the in vivo control (34.6%). Finally, 39 embryos at the 2- to 8-cell stages were transferred into the oviducts of three pseudopregnant females, and eight live pups (20.5%) were obtained. Of the eight pups, six survived for at least six months and were fertile. The present study shows successive in vitro cultures of single isolated primary follicles for the production of viable eggs. We believe that this culture system, with a combination of culture membranes under controlled O2 conditions, is applicable to other mammalian species, including humans.

Comparison of methods of surgical transplantation of mice embryos into pseudopregnant female recipients

There are a lot of factors that are able to influence the result of embryo transfer to pseudopregnant females, including the transplantation procedure itself. To determine the possible role of the surgical transplantation method on the embryo transfer result, we analyzed the data of transplantation of 6715 microinjected embryos into the oviduct of 471 pseudopregnant female by three ways — into the infundibulum, into the ampullae by the oviduct muscular wall puncture or incision. A significant increase in the pregnancy rate of recipients, as well as the birth rate in total recipients in oviduct puncture/incision groups, has been shown, however, the birth rate in pregnant recipients does not differ between all experimental groups. Survival of pups is also significantly higher in oviduct puncture/incision groups.

Fixing Pitx3 gene Mutation with CRISPR-Cas9 Intervention in Zygotic Aphakia-Prone Mice – A Research Protocol

Introduction: Despite the drawbacks of gene-editing, CRISPR-Cas9 remains a versatile tool for editing mutated genes. The mutated Pitx3 gene may cause aphakia in mice and Parkinsonism in humans. Pitx3 gene codes for the Pitx3 transcription factor involved in many tasks including the differentiation of dopaminergic neurons in the substantia nigra, tyrosine hydroxylase expression, dopamine transporter and lens development. We aim to edit mutated Pitx3 genes using CRISPR-Cas9 in embryonic-dopaminergic progenitor-neurons of mice and compare to wildtypes (WT) that do not undergo CRISPR-Cas9 intervention. Methods: We will use 5 female mice, 2 homozygous for mice aphakia/Pitx3 mutation (Ak) and 3 standard pseudo-pregnant females. 2-male homozygous (+/+ Ak) mice with Ak will mate with the aphakic females (+/+ Ak). Dopamine levels in both (aphakic/non-aphakic) mice will be determined. We will extract embryonic stem cells (ESC) from aphakic female’s uterus and treat with CRISPR-Cas9 and homology-directed repair (HDR). Both treated and untreated ESC will be divided between 3 pseudo-pregnant females. Post-parturition, intervention success will be assessed using presence of optical lens, dopamine levels and restriction sequence expression in treated/untreated progeny. Results: We expect to observe that untreated ESC zygotes (no CRISPR-Cas9 and HDR treatment) will not express normal Pitx3. This will result in low dopamine levels (inappropriate dopamine levels for their age group) and an aphakic mice progeny. Likewise, treated ESC zygotes will express normal Pitx3, hence have normal dopamine levels and non-aphakic phenotype. Discussion: Progeny of both groups (treated/non-treated) will be examined for aphakia, dopamine levels and restriction sequence. Dopamine levels of treated mice will be compared to dopamine levels of WT mice. Should none of the treated progeny develop lenses or have normal dopamine levels, these mice will be sacrificed to target the restriction sequence in the inserted gene — helping to evaluate why the inserted gene is unexpressed. Conclusion: This research examines CRISPR-Cas9 and HDR use on embryos with mutated genes that impair lens development and dopaminergic-neuron differentiation in humans and mice. It has potential of qualifying for clinical trials as the technique may be used to fix heritable disease-inducing genes that decrease longevity and quality of life in humans.

GONAD: A new method for germline genome editing in mice and rats.

Recent advances in the CRISPR/Cas9 system have demonstrated it to be an efficient gene-editing technology for various organisms. Laboratory mice and rats are widely used as common models of human diseases; however, the current standard method to create genome-engineered animals is laborious and involves three major steps: isolation of zygotes from females, ex vivo micromanipulation of zygotes, and implantation into pseudopregnant females. To circumvent this, we recently developed a novel method named Genome-editing via Oviductal Nucleic Acids Delivery (GONAD). This method does not require the ex vivo handling of embryos; instead, it can execute gene editing with just one step, via the delivery of a genome-editing mixture into embryos in the oviduct, by electroporation. Here, we present a further improvement of GONAD that is easily applicable to both mice and rats. It is a rapid, low-cost, and ethical approach fulfilling the 3R principles of animal experimentation: Reduction, Replacement, and Refinement. This method has been reconstructed and renamed as "improved GONAD (i-GONAD)" for mice, and "rat improved GONAD (rGONAD)" for rats.

EXTENDED EXPOSURE TIME TO EMBRYOGLUE™ Results IN IMPROVED MOUSE FETAL DEVELOPMENT

Human data point towards positive effects of extended incubation times in EmbryoGlue. In the mouse, Gardner and collaborators demonstrated a beneficial effect on implantation after 5 minutes incubation in medium with high levels of hyaluronan. Clinical practice using EmbryoGlue has confirmed its beneficial effect (Heymann et al., 2020). The aim of this study was to evaluate the effect of extended incubation times of mouse embryos in EmbryoGlue on implantation and fetal development. Mouse embryos from hybrid B6CBAF1 females were cultured from the one-cell stage up to day-4 in G-TL before incubation in the transfer medium (Control: G-TL or Test: EmbryoGlue). Transfer dishes with 0.5 mL of medium under oil were prepared and equilibrated overnight. Day-4 blastocysts were randomly allocated to the control and test group. Embryos were incubated for 10 minutes, 1 hour or 4 hours prior to transfer. Transfer procedures were performed using synchronized CD1 pseudopregnant females. For each transfer, the uterine horns were randomized and 4-5 embryos from the control and test group were replaced in each recipient. In total, 90 to 95 blastocysts were transferred from each group in 18 to 19 embryo transfer procedures. Outcome was assessed 10 days after embryo transfer. Implantation sites and fetal development were determined and crown-rump length, fetal and placental weight measured. Results of implantation, loss rate and fetal development are summarized in the table. No significant differences in implantation rates between the control and the test were observed at the different incubation times. A significantly lower (p<0.05) rate of implantation loss after 4 h exposure to EmbryoGlue resulted in increased fetal development in this group (p<0.05) compared to the corresponding control. No statistical differences were found in the mean values of crown-rump length, fetal and placental weight between the different groups. Compared to a medium with low levels of hyaluronan, exposure of mouse embryos to a medium containing high levels of hyaluronan for 4 h before transfer reduces loss rates after implantation and increases fetal development with no effect on fetal outcome parameters on day 14 of gestation.

Ovarian stimulation reduces fetal growth by dysregulating uterine natural killer cells in mice.

Ovarian stimulation is associated with a higher risk of low birth weight. However, the precise mechanisms by which ovarian stimulation increases the chances of low birth weight remain unclear. In this mouse model study, in vivo developed blastocysts that were not exposed to gonadotropins were transferred into pseudopregnant females that had mated naturally (the control group), pseudopregnant females that had been administered a low dose of ovulation-stimulating hormone (the L-SO group) and pseudopregnant females that had been administered a high dose of ovulation-stimulating hormone (the H-SO group). The embryo implantation rate and fetal weight were significantly lower in the L-SO and H-SO groups than in the control group. The density of Dolichos biflorus agglutinin (DBA)+ uterine natural killer (uNK) cells in the decidua basalis was significantly lower in the L-SO and H-SO groups than in the control group. Ovarian stimulation also downregulated a variety of cytokines related to uNK cells that are involved in placental angiogenesis and trophoblast invasion. Collectively, our findings indicate that ovarian stimulation impairs DBA+ uNK cell density in the decidua basalis, which may downregulate uNK-related cytokine secretion and influence placental angiogenesis and restrict fetal growth in mice.