Embryonic Development Research Articles

Epigenetic landscape reorganisation and reactivation of embryonic development genes are associated with malignancy in IDH-mutant astrocytoma

Accurate grading of IDH-mutant gliomas defines patient prognosis and guides the treatment path. Histological grading is challenging, and aside from CDKN2A/B homozygous deletions in IDH-mutant astrocytomas, there are no other objective molecular markers used for grading. RNA-sequencing was conducted on primary IDH-mutant astrocytomas (n = 138) included in the prospective CATNON trial, which was performed to assess the prognostic effect of adjuvant and concurrent temozolomide. We integrated the RNA-sequencing data with matched DNA-methylation and NGS data. We also used multi-omics data from IDH-mutant astrocytomas included in the TCGA dataset and validated results on matched primary and recurrent samples from the GLASS-NL study. Since discrete classes do not adequately capture grading of these tumours, we utilised DNA-methylation profiles to generate a Continuous Grading Coefficient (CGC) based on classification scores from a CNS-tumour classifier. CGC was an independent predictor of survival outperforming current WHO-CNS5 and methylation-based classification. Our RNA-sequencing analysis revealed four distinct transcription clusters that were associated with (i) upregulation of cell cycling genes; (ii) downregulation of glial differentiation genes; (iii) upregulation of embryonic development genes (e.g. HOX, PAX, and TBX) and (iv) upregulation of extracellular matrix genes. The upregulation of embryonic development genes was associated with a specific increase of CpG island methylation near these genes. Higher grade IDH-mutant astrocytomas have DNA-methylation signatures that, on the RNA level, are associated with increased cell cycling, tumour cell de-differentiation and extracellular matrix remodelling. These combined molecular signatures can serve as an objective marker for grading of IDH-mutant astrocytomas.

Development of a TBXT-EGFP iPS cell model for screening the early developmental toxicity of typical environmental pollutants

In our daily lives, we are inevitably exposed to a variety of environmental pollutants in numerous ways. Fortunately, recent years have witnessed significant advancements in the field of stem cell toxicology, which have provided new opportunities for research in environmental toxicology. Applying stem cell technology to environmental toxicology, overcomes some of the limitations of traditional screening methods and we can more accurately predict the toxicity of environmental pollutants. However, there are still several aspects of stem cell toxicology models that require improvement, such as increasing the throughput of detection and simplifying detection methods. Consequently, we developed an environmental pollutant toxicity detection model based on TBXT-EGFP iPS cells and screened the developmental toxicity of 38 typical environmental pollutants. Our results indicate that TBBPA-BDBPE, TBBPA-BHEE, DG, and AO2246 may interfere with the expression of TBXT, a critical marker gene for early human embryo development, implying that these environmental pollutants could lead to developmental abnormalities.

Elucidation of the pluripotent potential of bovine embryonic lineages facilitates the establishment of formative stem cell lines.

The establishment of epiblast-derived pluripotent stem cells (PSCs) from cattle, which are important domestic animals that provide humans with milk and meat while also serving as bioreactors for producing valuable proteins, poses a challenge due to the unclear molecular signaling required for embryonic epiblast development and maintenance of PSC self-renewal. Here, we selected six key stages of bovine embryo development (E5, E6, E7, E10, E12, and E14) to track changes in pluripotency and the dependence on signaling pathways via modified single-cell transcription sequencing technology. The remarkable similarity of the gene expression patterns between cattle and pigs during embryonic lineage development contributed to the successful establishment of bovine epiblast stem cells (bEpiSCs) using 3i/LAF (WNTi, GSK3βi, SRCi, LIF, Activin A, and FGF2) culture system. The generated bEpiSCs exhibited consistent expression patterns of formative epiblast pluripotency genes and maintained clonal morphology, normal karyotypes, and proliferative capacity for more than 112 passages. Moreover, these cells exhibited high-efficiency teratoma formation as well as the ability to differentiate into various cell lineages. The potential of bEpiSCs for myogenic differentiation, primordial germ cell like cells (PGCLCs) induction, and as donor cells for cellnuclear transfer was also assessed, indicating their promise in advancing cell-cultured meat production, gene editing, and animal breeding.

Comparison of Galaxy and Unix tools for analyzing the exome sequencing data from syndactyly abnormalities

Syndactyly is a congenital limb abnormality, which manifests as the fusion of digits due to incomplete separation during embryonic development, and its pathogenesis involves intricate genetic and molecular processes. Since Exome sequencing has gained widespread utilization as an invaluable tool for exploring genetic disorders during prenatal development, the Bioinformatic platforms, such as GALAXY and UNIX, play a central role in the analysis process of exome sequencing data, facilitating precise identification and interpretation of genetic variations linked to congenital abnormalities. In this study, we conducted a comparative analysis of exome sequencing data from a 1.5-year-old syndactyly patient using two platforms: GALAXY and UNIX. The UNIX platform identified a total of 275,572 variants, and the GALAXY platform identified 140,291 variants when compared with the Grch38/hg38 reference genome. A comparative analysis identified 126,848 common variants between the platforms. After filtration with the 200 syndactyly-related genes, 1,345 variants were remained. The distribution of these 1,345 variants spans the entirety of the patient's genome, with focal concentrations observed on specific chromosomes including chromosomes 2, 4, and 11. Concurrently, within the top 200 genes implicated in syndactyly, the genes FRAS1, CACNA1C, GLI2, and NOTCH1 exhibit the highest frequency of variants. These data emphasized the impact of the chosen analytical platform on genetic variation detection in congenital limb abnormalities, provided critical insights into the selection of bioinformatic tools for optimizing exome sequencing workflows in the context of limb malformations, contributed to advancements in genetic research and diagnostic methodologies.

Genome-wide analysis of anterior-posterior mRNA regionalization in Stentor coeruleus reveals a role for the microtubule cytoskeleton.

Cells have complex and beautiful structures that are important for their function. However, understanding the molecular mechanisms that produce these structures is a challenging problem due to the gap in size scales between molecular interactions and cellular structures. The giant ciliate Stentor coeruleus is a unicellular model organism whose large size, reproducible structure, and ability to heal wounds and regenerate have historically allowed the formation of structure in a single cell to be addressed using methods of experimental embryology. Such studies have shown that specific cellular structures, such as the membranellar band, always form in particular regions of the cell, which raises the question: what is the source of positional information within this organism? By analogy with embryonic development, in which regionalized mRNA is often used to mark position, we asked whether specific regionalized mRNAs might mark position along the anterior-posterior axis of Stentor . By physically bisecting cells and conducting bulk RNA sequencing, we were able to identify sets of messages enriched in either the anterior or posterior half. We then conducted half-cell RNA-sequencing in paired anteriors and posteriors of cells in which the microtubule cytoskeleton was disrupted by RNAi of β-tubulin or dynein intermediate chains. We found that many messages either lost their regionalized distribution or switched to an opposite distribution, such that anterior-enriched messages in control became posterior-enriched in the RNAi cells, or vice versa. This study indicates that mRNA can be regionalized within a single giant cell and that microtubules may play a role, possibly by serving as tracks for the movement of the messages.

The Influence of Oviductal and Uterine Fluid Supplementation on the In Vitro Development and Quality of Cloned Sheep Embryos.

Somatic cell nuclear transfer (SCNT) has great potential for the replication of high-commercial-value animals, threatened wild species for conservation purposes, and transgenic animals for biomedical purposes. However, SCNT has a low success rate due to intrinsic factors of the technique itself, which leads to low rates of embryonic development and epigenetic alterations in cloned embryos. The objective of this study was to evaluate the effect of OF-UF on the intracellular concentrations of ROS and GSH and the development of cloned and parthenogenetic Ovis aries embryos. The results do not show a beneficial effect on the development of parthenogenetic and cloned embryos at concentrations of 0.5% OF-UF. Furthermore, at 1% OF-UF, an adverse effect was observed in cloned embryos at the blastocyst stage and 2% OF and UF in parthenogenetic embryos during the first divisions. Decreases in ROS and GSH levels were observed in the parthenogenetic blastocysts treated with 1% OF-UF, but not in the clones, in which a higher concentration of GSH and a similar concentration of ROS were observed. No effect of OF-UF was observed on embryonic development and redox balance in sheep embryos cloned via handmade cloning.

Reproductive and developmental toxicity of α-terpineol in Wistar rats

Alpha-terpineol (α-T) is a type of monoterpenoid alcohol commonly present in essential oils, it contributes to a pleasant floral fragrance similar to that of lilacs. This research aimed to evaluate the impact of α-terpineol on the reproductive functions of both male and female rats, including gonadal activity, mating behavior, conception, conceptus development and parturition. Six male and female Wistar rats per group received α-terpineol through gavage at doses of 0, 75, 150 and 300 mg/kg/day. The study revealed changes in body weight gain inhibition, food consumption, azoospermia, decreased testosterone levels (0.7 ± 1.61 ng/mL, 0.7 ± 2.30 ng/mL) as well as histopathological variations in testis and epididymis among males exposed to doses of 150 and 300 mg/kg/day. Moreover, this exposure led to significantly decreased serum T4 levels in both adult males (21.85 ± 12.68 ng/mL, 16.20 ± 9.15 ng/mL) and dams (11.24 ± 12.37 ng/mL, 9.48 ± 11.74 ng/mL) at the dose range of 150 and 300 mg/kg/day without affecting TSH concentrations. In summary, the present study showed that α-terpineol induced reproductive toxicities in male rats. Therefore, a detailed toxicological assessment is highly recommended.

An efficient and easy-to-use protocol for induction of haploids in cucumber through parthenogenic embryo development

BackgroundCucumber (Cucumis sativus L.) is a model crop to study cell biology, including the development of haploids and doubled haploids in vegetable crops. In plant breeding, haploid and doubled haploids are valuable tools for developing pure homozygous inbred lines and accelerating genetic progress by reducing the time required for breeding cycles. Besides, the haploids are also valuable in genomic studies. We are reporting the induction of haploids in cucumber involving gynoecious and parthenocarpic genotypes for the first time. This study aimed to assess the efficient induction of haploids through pollination with gamma-irradiated pollen in cucumber. The effect of gamma irradiation dose on pollen viability and germination, fruit setting percentage, seed development, and haploid embryo development in cucumber hybrid genotypes were studied in detail. The goal was to utilize this information to produce haploid plants for genomics and transformation works in this model vegetable crop.ResultsPollination was done on six cucumber genotypes using varying doses of gamma rays (100, 200, 300, 400, and 500 Gy). Genotypes, doses of irradiation, and embryo developmental stage influenced the successful generation of in-vitro haploid plants. The optimal timeframe for embryo rescue was found to be 25 to 30 days after pollination. Haploid embryos were effectively induced using irradiated pollen at 400 to 500 Gy doses. Parthenogenetic plantlets were analyzed, and their ploidy level was confirmed through stomatal physiology, cytology (mitosis), and flow cytometry methods.ConclusionThrough parthenogenic embryo development, it is possible to induce a large number of haploids in cucumber. This technique’s power lies in its ability to streamline the breeding process, enhance genetic gain, and produce superior cultivars that contribute to sustainable agriculture and food security.

Poly(A)-Specific Ribonuclease Deficiency Leads to Deregulated Expression of Genes Involved in Sex Determination and Gonadal Maturation in Zebrafish

Abstract Background Deadenylation, the process of removal of poly (A) tail of messenger ribonucleic acids (mRNAs), is a rate-limiting step in mRNA stability, and poly(A)-specific ribonuclease (PARN) is the most important exonuclease involved in this process. Besides mRNA stability, PARN is also involved in several other processes including telomere maintenance, noncoding RNA maturation, ribosome biogenesis, and TP53 function. Previously, we have shown that zebrafish PARN null mutants are viable and fertile but turn out to only develop into males, indicating a role in oogenesis. The present study was focused on analyzing the expression of genes involved in sex determination and gonadal development in PARN mutant zebrafish. Materials and Methods Total RNA was extracted and quantitative real-time polymerase chain reaction was performed to determine the expression level of genes involved in gonad development in PARN mutant embryos (4 days postfertilization [dpf]) and adults (120 dpf) in comparison to their wild-type siblings. The expression levels were estimated by the ΔΔCT relative quantification method. Results At 4 dpf, the expression of germ cell-specific genes did not show any significant difference in the null mutants compared to the heterozygous and their wild-type siblings, suggesting no effect on germ cell differentiation due to the loss of PARN. However, the majority of the ovary-associated genes analyzed showed an increased expression in PARN null and heterozygous mutants compared to the wild-type siblings. Intriguingly, the expression of testis-associated genes showed decreased expression in the mutants compared to their wild-type siblings at 4 dpf. In adult stages, as expected, the expression of genes that jointly regulate the proper formation and function of ovaries and testes showed decreased expression in PARN null mutants. Interestingly, the expression of genes involved in the differentiation of testes, despite showing a decreased expression in the mutants, was comparable between the null and heterozygous mutants. Conclusion Taken together, these results suggest that the loss of PARN does not affect germ cell differentiation but affects the sexual differentiation that happens at later stages of development, particularly the process of oogenesis, in zebrafish.

Cryo-EM structure of the human subcortical maternal complex and the associated discovery of infertility-associated variants.

The functionally conserved subcortical maternal complex (SCMC) is essential for early embryonic development in mammals. Reproductive disorders caused by pathogenic variants in NLRP5, TLE6 and OOEP, three core components of the SCMC, have attracted much attention over the past several years. Evaluating the pathogenicity of a missense variant in the SCMC is limited by the lack of information on its structure, although we recently solved the structure of the mouse SCMC and proposed that reproductive disorders caused by pathogenic variants are related to the destabilization of the SCMC core complex. Here we report the cryogenic electron microscopy structure of the human SCMC and uncover that the pyrin domain of NLRP5 is essential for the stability of SCMC. By combining prediction of SCMC stability and in vitro reconstitution, we provide a method for identifying deleterious variants, and we successfully identify a new pathogenic variant of TLE6 (p.A396T). Thus, on the basis of the structure of the human SCMC, we offer a strategy for the diagnosis of reproductive disorders and the discovery of new infertility-associated variants.

The effect of male factors on embryo morphokinetics: a retrospective analysis of 2726 blastocysts.

Male infertility may influence fertilization rates, embryo morphology, and implantation rates in in vitro fertilization (IVF) cycles. Oocyte competence plays a major role in embryo development, but there is a limited understanding of the connection between sperm quality, embryo development, and morphokinetic parameters using donor oocytes. The study evaluated if sperm quality may influence the morphokinetic parameters in IVF cycles. A retrospective multicentric observational cohort study included 747 ICSI cycles using donor oocytes with fresh or frozen sperm. Embryos were cultured in time-lapse incubators until the blastocyst stage. The population was divided into three groups according to sperm concentration, as control group (> 16 mill/mL), severe oligospermia (0-5 mill/mL), and moderate oligospermia group (5-16 mill/mL). Morphokinetic analysis showed no difference in the time from the 2-cell to 6-cell stage of embryo development. A significant difference was observed on day 3 of embryo development, specifically at the 7-cell stage (t7), severe oligospermia 53.37 ± 9.81, moderate oligospermia 56.95 ± 9.78, and control 55.1 ± 8.85h post-insemination (hpi) (p = 0.024), and 8-cell stage (t8), severe oligospermia 55.41 ± 10.83, moderate oligospermia 61.86 ± 12.38 hpi (p < 0.001), and control 58.61 ± 11.33. Accordingly, the synchrony of the four cleavages going from 4 to 8 cells (s3) was found statistically different among the groups in the severe oligospermia 8.05 ± 9.99, moderate oligospermia 11.66 ± 11.04 hpi, and control 8.55 ± 8.58 (p = 0.009). Morphokinetic time ranges were obtained for t6, t7, t8, and s3 in order to identify the good-quality blastocysts. Poor sperm quality is associated with alterations in morphokinetic parameters on day 3 in IVF cycles with donor oocytes, underlining the important role of spermatozoa during embryo development.

Mitochondrial non-energetic function and embryonic cardiac development.

The initial contraction of the heart during the embryonic stage necessitates a substantial energy supply, predominantly derived from mitochondrial function. However, during embryonic heart development, mitochondria influence beyond energy supplementation. Increasing evidence suggests that mitochondrial permeability transition pore opening and closing, mitochondrial fusion and fission, mitophagy, reactive oxygen species production, apoptosis regulation, Ca2+ homeostasis, and cellular redox state also play critical roles in early cardiac development. Therefore, this review aims to describe the essential roles of mitochondrial non-energetic function embryonic cardiac development.

Melatonin decreases excessive polyspermy for single oocyte in pigs through the MT2 receptor

Melatonin supplementation during in vitro maturation (IVM) improves porcine oocyte maturation and embryonic development by exerting antioxidative effects. Nevertheless, the mechanism by which melatonin prevents polyspermy after in vitro fertilization (IVF) remains unclear. Here, we examined the effects of melatonin on cytoplasmic maturation and the incidence of polyspermic penetration in porcine oocytes. No statistically significant difference was observed in the rate of first polar body formation between the groups (Control, Melatonin, Melatonin + Luzindole, and Melatonin + 4-P-PDOT). Interestingly, melatonin supplementation significantly improved the cytoplasmic maturation of porcine oocytes by enhancing the normal distribution of organelles (Golgi apparatus, endoplasmic reticulum and mitochondria) and upregulating organelle-related gene expressions (P < 0.05). However, these promotional effects were counteracted by melatonin antagonists, suggesting that melatonin enhances cytoplasmic maturation through its receptors in porcine oocytes. Melatonin supplementation also significantly improved the rate of diploid and blastocyst formation after IVF by promoting the normal distribution of cortical granules (P < 0.05). In conclusion, melatonin supplementation during in vitro maturation of porcine oocyte improves fertilization efficiency and embryonic developmental competence by enhancing cytoplasmic maturation.

8795 Impact of Retinoic Acid Receptor Alpha Conditional Knockout on Ovarian Follicle Development in the Mouse

Abstract Disclosure: Z. Bogin: None. A. Keisker: None. A. Urbanowski: None. C. White: None. J.L. Kipp: None. The ovaries are the primary endocrine and reproductive organs in females. The ovarian follicle is the basic structural and functional unit of the ovary. When there is abnormal development in ovarian follicles, infertility and reproductive diseases such as polycystic ovarian syndrome (PCOS), premature ovarian failure (POF), and ovarian cancers may occur. Retinoic acid (RA), a biologically active derivative of vitamin A, is a signaling molecule that plays a vital role in various physiological processes, including embryonic development, tissue differentiation, and reproduction. We have shown that RA stimulates granulosa cell proliferation through a cell signaling cascade involving Retinoic Acid Receptors (RARs), which have three isoforms alpha, beta, and gamma. In this study, we characterized a new retinoic acid receptor alpha conditional knockout (RARA cKO) mouse model with RARA deleted in granulosa cells during embryonic development. The body weight and uterus weight of the RARA cKO mice were decreased at week 7 but returned to normal at week 15 and 12 months. The animals were either sub-fertile or infertile with phenotypic evidence of POF. In addition, a variety of ovarian pathologies were observed, including the presence of hemosiderin, increased cyst formation, and abnormal follicle counts. Overall, this study provides evidence of the involvement of retinoic acid signaling, mediated by RARA, in ovarian development and fertility. This study will help to better understand the prevention and treatment of ovarian diseases. Presentation: 6/2/2024

9271 Cholinergic Signaling in the Mouse Embryonic Pancreas Targets Early Endocrine Cells

Abstract Disclosure: C. Duarte: None. J.K. Panzer: None. N. Borowsky: None. A. Caicedo: None. The parasympathetic vagus nerve affects pancreas function by activating local intrapancreatic neurons that release acetylcholine (ACh). These neurons are derived from progenitors of the vagal neural crest and colonize the pancreas during embryonic development. The specific mechanisms by which ACh is released from these neurons influence embryonic development of the islet and, specifically, beta cells remain largely unknown. The purpose of this study was to identify cholinergic signaling components and assess early physiological responses to ACh during murine development of endocrine lineage cells. We extracted pancreatic rudiments from mice at embryonic stages E15 to E18 and stained them for neuronal and endocrine markers, along with components associated with cholinergic signaling. We further performed Ca2+ imaging of the pancreatic rudiments from NGN3CRE-GCaMP3 mice to record responses to ACh in cells of the endocrine lineage. At E15, we observed a significantly higher concentration of glucagon+ cells compared to insulin+ cells, consistent with the earlier differentiation of alpha cells in the developing pancreas. Neuronal body clusters were identified in close proximity to endocrine lineage cells from E15 to E18, with the neuronal network becoming more dispersed and interconnected. At E15, wide distribution of the presynaptic vesicular acetylcholine transporter (VAChT) was observed later becoming localized around insulin+ and glucagon+ cells at E18. Postsynaptic acetylcholinesterase (AChE) colocalized with glucagon+ cells from E15 to E18 and with insulin+ cells by E18. ACh elicited Ca2+ responses in individual endocrine cells as early as E15. Our results show that cholinergic nerve terminals target early endocrine cells and elicit responses to ACh, indicating that local cholinergic neurons innervate and activate endocrine progenitors in the embryonic pancreas. Our current studies are aimed at determining how cholinergic signaling contributes to beta cell differentiation and function. We expect this knowledge to guide future protocols for deriving beta cells from stem cells. Presentation: 6/2/2024

8715 Cholinergic Signaling in the Mouse Embryonic Pancreas Targets Early Endocrine Cells

Abstract Disclosure: C. Duarte: None. J.K. Panzer: None. N. Borowsky: None. A. Caicedo: None. The parasympathetic vagus nerve affects pancreas function by activating local intrapancreatic neurons that release acetylcholine (ACh). These neurons are derived from progenitors of the vagal neural crest and colonize the pancreas during embryonic development. The specific mechanisms by which ACh is released from these neurons influence embryonic development of the islet and, specifically, beta cells remain largely unknown. The purpose of this study was to identify cholinergic signaling components and assess early physiological responses to ACh during murine development of endocrine lineage cells. We extracted pancreatic rudiments from mice at embryonic stages E15 to E18 and stained them for neuronal and endocrine markers, along with components associated with cholinergic signaling. We further performed Ca2+ imaging of the pancreatic rudiments from NGN3CRE-GCaMP3 mice to record responses to ACh in cells of the endocrine lineage. At E15, we observed a significantly higher concentration of glucagon+ cells compared to insulin+ cells, consistent with the earlier differentiation of alpha cells in the developing pancreas. Neuronal body clusters were identified in close proximity to endocrine lineage cells from E15 to E18, with the neuronal network becoming more dispersed and interconnected. At E15, wide distribution of the presynaptic vesicular acetylcholine transporter (VAChT) was observed later becoming localized around insulin+ and glucagon+ cells at E18. Postsynaptic acetylcholinesterase (AChE) colocalized with glucagon+ cells from E15 to E18 and with insulin+ cells by E18. ACh elicited Ca2+ responses in individual endocrine cells as early as E15. Our results show that cholinergic nerve terminals target early endocrine cells and elicit responses to ACh, indicating that local cholinergic neurons innervate and activate endocrine progenitors in the embryonic pancreas. Our current studies are aimed at determining how cholinergic signaling contributes to beta cell differentiation and function. We expect this knowledge to guide future protocols for deriving beta cells from stem cells. Presentation: 6/2/2024

8795 Impact of Retinoic Acid Receptor Alpha Conditional Knockout on Ovarian Follicle Development in the Mouse

Abstract Disclosure: Z. Bogin: None. A. Keisker: None. A. Urbanowski: None. C. White: None. J.L. Kipp: None. The ovaries are the primary endocrine and reproductive organs in females. The ovarian follicle is the basic structural and functional unit of the ovary. When there is abnormal development in ovarian follicles, infertility and reproductive diseases such as polycystic ovarian syndrome (PCOS), premature ovarian failure (POF), and ovarian cancers may occur. Retinoic acid (RA), a biologically active derivative of vitamin A, is a signaling molecule that plays a vital role in various physiological processes, including embryonic development, tissue differentiation, and reproduction. We have shown that RA stimulates granulosa cell proliferation through a cell signaling cascade involving Retinoic Acid Receptors (RARs), which have three isoforms alpha, beta, and gamma. In this study, we characterized a new retinoic acid receptor alpha conditional knockout (RARA cKO) mouse model with RARA deleted in granulosa cells during embryonic development. The body weight and uterus weight of the RARA cKO mice were decreased at week 7 but returned to normal at week 15 and 12 months. The animals were either sub-fertile or infertile with phenotypic evidence of POF. In addition, a variety of ovarian pathologies were observed, including the presence of hemosiderin, increased cyst formation, and abnormal follicle counts. Overall, this study provides evidence of the involvement of retinoic acid signaling, mediated by RARA, in ovarian development and fertility. This study will help to better understand the prevention and treatment of ovarian diseases. Presentation: 6/2/2024

9378 Knockdown Of The Cdh2 Gene In Zebrafish Determines Its Role In Terminal Hormone Differentiation

Abstract Disclosure: B.V. Fernandes: None. C. MacRae: None. L.R. Carvalho: None. M. Junqueira: None. Introduction: Congenital hypopituitarism (CH) is characterized by the deficiency of one or more pituitary hormones. Whole exome sequencing (WES) has allowed the identification of novel/rare variants in genetic diseases. Among CH patients, 85% lack a molecular diagnosis. WES approach in a patients born to consanguineous parents identified the homozygous variant (c.865G&amp;gt;A, p. Val289Ile) in the CDH2 gene that produce N-Cadherin protein responsible for cell-cell adhesion. This variant was predicted as likely pathogenic allelic variant by ACMG criteria was found in a patient with GH, TSH, ACTH, LF/FSH deficiencies associated with an ectopic neurohypophysis and pituitary hypoplasia. CDH2 analysis in a cohort of 219 patients with CH, followed in a single Brazilian center, identified 3 unrelated families with heterozygous variants and incomplete penetrance where 2 were novel, in addition to the p. Val289Ile. The deleterious effect of the homozygous variant on cell adhesion properties was confirmed in permanent transfection assays in L1 fibroblast cell lines. To determine the role of the cdh2 gene in pituitary development and hormone production and secretion, we selected zebrafish as an animal model due to its 75% genetic homology with humans. Knockout of cdh2 using Crispr/cas9 genomic edition resulted in 85% lethality of animals at 48 hours post-fertilization (hpf), precluding analysis of its role during pituitary development. Objective: To produce a cdh2 knockdown in zebrafish for phenotypic and molecular analysis. Materials and Methods: The pET28B-RfxCas13d-His plasmid was transformed, linearized, and followed by in vitro transcription to produce Cas13 mRNA. Three guide RNAs targeting the cdh2 gene were designed to guide Cas13 to the target. The Cas13/RNA guide complex was injected into single-cell embryos, and phenotypic analyses were performed under a stereomicroscope until 96 hpf. To evaluate cdh2, prop1, pit1 and gh gene expression by RT-PCR, embryos were collected at 24, 48, 72, and 96 hpf. Results: Knockdown animals exhibited microcephaly, cardiac edema, and microphthalmia during embryonic development. CDH2 expression was decreased by 75% in the first 24 hours of life and began to recover by 48 hours, gradually increasing until 96 hpf when it equaled to WT. gh expression was statistically decreased at 48h compared to WT, with an increased gh expression similar to cdh2 increasing reaching similar WT levels by 96 hpf. There was no statistically significant difference in prop1 and pit1 expression compared to WT in the analyzed periods. Conclusion: The cdh2 knockdown in zebrafish using Cas13 enzyme was effective with cdh2 decreased expression by 75% presenting a phenotype similar to the knockout model. Decreased gh expression at 48h suggests an important role of cdh2 in hormonal terminal differentiation, which should be corroborated by analysis of other hormone expressions. Presentation: 6/2/2024

9233 Sperm-carried IGF2: a Spark Contributing To Embryo Growth And Development

Abstract Disclosure: R. Cannarella: None. O.J. Rando: None. R.A. Condorelli: None. C. Sandrine: None. S. Romano: None. A. Guglielmino: None. Q. Yin: None. H. Gustafsson: None. F. Mancuso: None. C. Bellucci: None. G. Luca: None. S. La Vignera: None. A.E. Calogero: None. Background: Sperm RNAs can regulate early embryo development. A potential key regulator of growth is insulin-like growth factor 2 (IGF2), a highly conserved paternally-imprinted gene involved in cell growth and proliferation that is expressed in human spermatozoa. Animal research supports the positive impact of IGF2-supplemented cultures on embryo quality. However, the role of sperm-carried IGF2 in the early development of the human embryo is unknown. Objective: To study whether sperm-carried IGF2 mRNA influences the morphokinetics of the human embryo. Design: Prospective, uncontrolled, observational pilot study. Setting: University-affiliated fertility center. Subjects: We enrolled 16 patients undergoing assisted reproductive techniques (ART) for idiopathic or male factor infertility and 10 healthy fertile controls with proven paternity. Couples with female factor infertility due to reduced oocyte reserve were excluded, while those with tubal factor or anovulation were included. Main Outcome Measure(s): After clinical use, an aliquot of the sperm samples employed for ART was used to evaluate IGF2 mRNA levels. Embryo kinetics were recorded and correlated with the amount of IGF2 mRNA, correcting for female age, body mass index (BMI), anti-Müllerian hormone (AMH) levels, and oocyte quality. To provide mechanistic insights into the human data, a transcriptome analysis was performed of parthenotes obtained from twelve-week-old superovulated mice injected with Igf2 mRNA plus Gfp mRNA (experimental group, n=20) or Gfp mRNAs (control group, n=20). Pathway enrichment analysis was then performed using g:Profiler g:GOSt and EnrichR. Results: Sperm IGF2 mRNA negatively correlated with time to 2-cell stage (t2), t3, t4, t5, and time to expanded blastocyst (tEB), independently of maternal age, BMI, AMH, and oocyte quality. IGF2 mRNA index &amp;gt;4.9 predicted the ability of the embryo to reach the blastocyst stage at day 5, with a sensitivity of 100% and specificity of 71.6% (AUC 0.845; p&amp;lt;0.001). The distribution of sperm IGF2 levels differs significantly between morphologically good or poor embryos, with the lowest levels more frequently associated with morphologically poor embryos (61.9%) than with good ones (38.1%) (p=0.0091). Furthermore, sperm-derived IGF2 levels were significantly lower in subfertile men compared to fertile controls with proven paternity. Transcriptome analysis of Igf2-injected embryos demonstrated differential expression of 101 genes activating pathways regulating early embryo development. Conclusion: Sperm-carried IGF2 mRNA can support early embryo development, independently of female factors. Larger studies are needed to confirm this finding and to investigate its role as a clinical marker to predict the chances of obtaining blastocysts that can be implanted in infertile couples undergoing ART. Presentation: 6/1/2024

7110 Sperm-carried IGF2: a Spark Contributing To Embryo Growth And Development

Abstract Disclosure: R. Cannarella: None. O.J. Rando: None. R.A. Condorelli: None. C. Sandrine: None. S. Romano: None. A. Guglielmino: None. Q. Yin: None. H. Gustafsson: None. F. Mancuso: None. C. Bellucci: None. G. Luca: None. S. La Vignera: None. A.E. Calogero: None. Background: Sperm RNAs can regulate early embryo development. A potential key regulator of growth is insulin-like growth factor 2 (IGF2), a highly conserved paternally-imprinted gene involved in cell growth and proliferation that is expressed in human spermatozoa. Animal research supports the positive impact of IGF2-supplemented cultures on embryo quality. However, the role of sperm-carried IGF2 in the early development of the human embryo is unknown. Objective: To study whether sperm-carried IGF2 mRNA influences the morphokinetics of the human embryo. Design: Prospective, uncontrolled, observational pilot study. Setting: University-affiliated fertility center. Subjects: We enrolled 16 patients undergoing assisted reproductive techniques (ART) for idiopathic or male factor infertility and 10 healthy fertile controls with proven paternity. Couples with female factor infertility due to reduced oocyte reserve were excluded, while those with tubal factor or anovulation were included. Main Outcome Measure(s): After clinical use, an aliquot of the sperm samples employed for ART was used to evaluate IGF2 mRNA levels. Embryo kinetics were recorded and correlated with the amount of IGF2 mRNA, correcting for female age, body mass index (BMI), anti-Müllerian hormone (AMH) levels, and oocyte quality. To provide mechanistic insights into the human data, a transcriptome analysis was performed of parthenotes obtained from twelve-week-old superovulated mice injected with Igf2 mRNA plus Gfp mRNA (experimental group, n=20) or Gfp mRNAs (control group, n=20). Pathway enrichment analysis was then performed using g:Profiler g:GOSt and EnrichR. Results: Sperm IGF2 mRNA negatively correlated with time to 2-cell stage (t2), t3, t4, t5, and time to expanded blastocyst (tEB), independently of maternal age, BMI, AMH, and oocyte quality. IGF2 mRNA index &amp;gt;4.9 predicted the ability of the embryo to reach the blastocyst stage at day 5, with a sensitivity of 100% and specificity of 71.6% (AUC 0.845; p&amp;lt;0.001). The distribution of sperm IGF2 levels differs significantly between morphologically good or poor embryos, with the lowest levels more frequently associated with morphologically poor embryos (61.9%) than with good ones (38.1%) (p=0.0091). Furthermore, sperm-derived IGF2 levels were significantly lower in subfertile men compared to fertile controls with proven paternity. Transcriptome analysis of Igf2-injected embryos demonstrated differential expression of 101 genes activating pathways regulating early embryo development. Conclusion: Sperm-carried IGF2 mRNA can support early embryo development, independently of female factors. Larger studies are needed to confirm this finding and to investigate its role as a clinical marker to predict the chances of obtaining blastocysts that can be implanted in infertile couples undergoing ART. Presentation: 6/1/2024