Male gamete copies to characterize genome inheritance and generate progenies

Male gamete copies to characterize genome inheritance and generate progenies | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Biological Sciences - Article Male gamete copies to characterize genome inheritance and generate progenies Gianpiero Palermo, Philip Xie, Takumi Takeuchi, Stephanie Cheung, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4682261/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Male factor infertility accounts for approximately 30% of infertile couples. When spermatozoa are extremely scarce, replicating the male gamete to fertilize a large cohort of oocytes would be ideal. Additionally, patients with inherited disorders currently rely on pre-implantation genetic diagnosis (PGD) to select healthy embryos, which raises ethical concerns due to the generation of multiple embryos to select one healthy conceptus. Therefore, it would be beneficial to decode the genetics of a single sperm cell before conceptus generation. In this study, we demonstrated the feasibility of replicating the sperm genome via androgenesis and selecting the desired gamete before fertilization to preserve a specific paternal genotype, confirmed by phenotypic observation and genetic testing, in a murine model. We achieved satisfactory pre-implantation developmental rates with replicated male gametes and were able to generate healthy offspring. Specifically, using 8-cell stage androgenetic embryos, a single spermatozoon can yield up to three conceptuses carrying the identical paternal haplotype. Biological sciences/Developmental biology/Embryology Health sciences/Medical research/Preclinical research Biological sciences/Stem cells/Embryonic stem cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Male infertility affects about 30% of the infertile population and about 9–15% of overall population ( 1 ) Intracytoplasmic sperm injection (ICSI) offers a promising solution by allowing a single selected spermatozoon to fertilize an oocyte regardless of the quantity and type of motility of the male gamete ( 2 ) Even in cases where the spermatozoa are functionally impaired such as globozoospermia, ICSI can still grant fertilization when used in conjunction with assisted oocyte activation ( 3 ). However, in cases when the spermatozoa are extremely scarce, it would be ideal to be able to replicate the male gamete to be able to fertilize a large cohort of oocytes. In addition, there are patients who carry inherited disorders, and the current treatment plan is solely dependent the selection of embryos available through pre-implantation genetic diagnosis (PGD). This can be ethically debatable due to the fact that several embryos are generated in order to select one normal conceptus ( 4 ). Therefore, it would be ideal to decipher the genetic of a single sperm cell prior the generation of the conceptus. There are situations where inherited disorders manifest due to the heterogeneity of germ line. This gamete heterogeneity is relevant in cases of structural chromosomal abnormality occurring in 5–15% of infertile men ( 5 ), including balanced and unbalanced translocations, where the affected individual generates a heterogenous sperm population whether healthy or abnormal ( 6 – 8 ). On the other hand, gamete heterogeneity can also be characterized in term of monogenic diseases caused by gene mutations, estimated to affect about 6% the human population ( 9 ). In cases of autosomal recessive disorders, phenotypically healthy carriers produce a heterogenous population of germ cells due to the heterozygosity ( 10 ). Nonetheless, even in individuals who are not carriers of diseases, de novo germline mutation may occur and accumulate over years with frequency of 45–60 de novo mutation per genome in a generation ( 11 ). For example, unaffected individuals may pass on pathological de novo variants of alleles, such as X-linked TEX11 mutation and autosomal dominant PTPN11 mutation responsible Noonan Syndrome ( 12 – 14 ). This awareness has stimulated attempts to replicate sperm genome to identify the healthy gametes. Indeed, androgenetic embryos can be generated by injection of a single spermatozoon into an enucleated oocyte ( 15 ), these embryos can yield haploid embryonic stem cells (haESCs) to study a recessive phenotype, or to be utilized in reproductive semi-cloning ( 16 , 17 ). Generation of haESCs has been successful, however, as expected, the utilization of haESC line is hindered by low blastocyst development averaging at 12.8% and an even lower efficiency in haESC derivation up to 40% ( 18 ). Therefore, in order to obtain one haESC line, at least 20 haploid embryos must be sacrificed even in optimal conditions. In addition, haploidy in mammalian somatic cells is an unstable situation and tend to self-diploidized due to endo-cycling and failed cytokinesis that can, and become more manifested when uncoupling of DNA replication and centrosome duplication cycles are more common ( 19 – 22 ). Once haESCs are achieved and purified, the utilization of such cells as a source of male gamete were able to yield offspring of desired genotype, albeit at a very low rate ( 23 , 24 ). In addition, concerns include the epigenetic profile of the male gamete with consequential genetic imprinting. In this study, we plan to replicate the mammalian male gamete by injecting a single spermatozoon into an enucleated oocyte and subsequently use these replicated cells to fertilize oocytes to generate conceptuses. We closely monitored the pre-implantation development of conceptuses by time-lapse microscopy, and post-implantation development by assessing the health and reproductive ability of the offspring. To confirm the provenance of the paternal genome, we sequenced replicated pseudo-gametes and compared it to the genomes of resulting offspring. We also assessed the uniformity of the genotype among the siblings originated from one single spermatozoon. METHODS Study Design In this work, we carried out a preliminary study where we assessed the ability of haploid androgenetic and gynogenetic constructs to develop into blastocysts in comparison to a mouse ICSI control. In the following step, we assessed the ability to generate conceptuses by utilizing haploid androgenetic pseudo-blastomere at different cleavage stage as a source of male gamete. Haploid pseudo-blastomeres were isolated from embryos at 2-cell, 4-cell and 8-cell. Resulting constructs were assessed up to full pre-implantation development. In some embryos, chromosomal analyses were carried out to confirm ploidy. In order to prove the embryo developmental competence of these biparental zygotes in some pseudo-pregnancy mice, hatching blastocysts were transferred. This allowed us to assess the development up to adulthood and related reproductive potential. In a later series of experiment, to confirm the origin of the male genome, we generated haploid androgenetic embryos as a source of male gamete using GFP transgenic male mice (Fig. 1). This project was approved through the IACUC of Weill Cornell Medical College, protocol number 0605-493A. Ovarian Stimulation, Oocyte Collection, and Oocyte Preparation To harvest mouse metaphase II oocytes, 8–12 weeks old B6D2F1 mice (Jackson Laboratory) were stimulated by intra-peritoneal (IP) injection of 0.1-0.2cc of ready-to-use pregnant mare serum gonadotropin / inhibin superovulation reagent (CARD HyperOva®, Cosmo Bio, Japan). Forty-eight hours after stimulation, eventual oocyte maturation and ovulation was triggered by IP injection of 7.5 IU of human chorionic gonadotropin (hCG, Sigma-Aldrich, St. Louis, MO, USA). Up to 16 hours after hCG trigger, mice were euthanized by cervical dislocation and dissected to retrieve oviducts containing cumulus-oocyte-complexes (COCs). COCs were then isolated from the ampullae of the oviducts and treated with 80 IU/ml hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA) followed by mechanical denudation using microcapillary. Oocytes were serially washed thrice in potassium simplex optimized media (KSOM, Cosmo Bio, Japan) and placed in an incubator at 37°C and 5% CO2 in equilibrated KSOM for use in subsequent steps. Spermatozoa Collection and Preparation Euthanasia was performed on male B6D2F1 or heterozygous B6-EGFP mice aged 12–16 weeks by cervical dislocation. The lower abdomen was sanitized with betadine where incision was then made surgically, and bilateral cauda epididymis identified and excised. They were placed in modified human tubal fluid (HTF) media (Cosmo Bio, Japan) on heat block for transport. Spermatozoa were extracted from cauda epididymis and diluted to 3 million/ml for insemination. Preparation of ooplasts for haploid androgenetic embryos To generate haploid androgenetic embryos, mature oocytes from B6D2F1 mice were enucleated using techniques based on previous literatures ( 25 – 27 ). Briefly, denuded oocytes were transferred into HEPES-buffered M2 media (Sigma-Aldrich, St. Louis, MO, USA) droplets supplemented with 5 µg/ml cytochalasin B (Sigma-Aldrich, St. Louis, MO, USA) on specialized glass dish (FluoroDish™, WPI.inc, Sarasota, FL, USA). Instead of nuclear staining commonly used in cloning, oocyte metaphase II spindle were visualized under stain-free polarized microscopy (Oosight™, Hamilton-Thorne, Beverly, MA, USA). The metaphase II spindle were then held by a holding pipette and rotated to have spindle located at 3 o'clock. A laser (LYKOS, Hamilton-Thorne, Beverly, MA, USA) or a piezo pulse (PMM-150, PrimeTech, Japan) was then applied to breach the zona. Next, a micropipette was advanced into the perivitelline space through the breach and a gentle suction was applied to remove the spindle. Enucleated oocytes were then placed in KSOM at least 1 hour until next procedure. Generation of haploid androgenetic embryos Piezo-actuated ICSI were performed to generate haploid androgenetic embryos using enucleated oocytes. The piezo-ICSI procedure is performed similarly to standard protocol ( 28 ). A single mouse spermatozoon from B6D2F1 or heterozygous B6-EGFP were decapitated mechanically, and the isolated head was suctioned into the micropipette in 7% polyvinylpyrrolidone (Fujifilm Irvine Scientific, Santa Ana, CA, USA). Individual ooplasts, placed in HEPES-buffered M2 media, was then positioned by a holding pipette, and rotated so the breach created previously was placed at 3 o'clock. The injection pipette containing the spermatozoa were the advanced into the perivitelline space of the ooplasts creating an invagination. Finally, a weak piezo pulse was applied to penetrate the oolemma with the sperm deposited into the cytoplasm. Post-ICSI oocytes inseminated by B6D2F1 spermatozoa were the cultured in KSOM for 24h, 36h, or 48h to generate 2-cell, 4-cell, or 8-cell haploid androgenetic embryos, respectively. In our modified experiment using B6-EGFP gametes, haploid androgenetic embryos were cultured in G-TL media (Vitrolife, Gothenburg, Sweden) designed for time-lapse microscopy. Each haploid androgenetic embryos were transferred into the microwell of EmbryoSlide (Vitrolife, Gothenburg, Sweden) covered with mineral oil, and the EmbryoSlide was then loaded into EmbryoScope set to take picture every 10 minutes for time-lapse microscopy in order to observe embryo morphokinesis (Video 1). Haploid androgenetic embryos displaying extensive fragmentation, abnormal morphokinetics or arrested development were excluded. Recipient Oocyte Preparation and Generation of Biparental Zygotes To prepare recipient oocytes for the replicated male gamete, mature oocytes from B6D2F1 mice were chemically activated by 2.5h incubation in Calcium-free CZB medium supplied with 10 mM SrCl 2 and cultured in KSOM for 2–3 more hours until a female pronucleus is present and a secondary polar body is extruded ( 29 ). In our early experiment using B6D2F1 androgenotes, a single pseudo-blastomere were isolated from 2-cell, 4-cell or 8-cell haploid androgenetic embryos with each pseudo-blastomere sized 15 µm in diameter and transferred into the perivitelline space of an activated parthenote. Each grafted oocyte was then aligned between 2 microelectrodes (ECF-100, BTX, Cambridge, UK) connected to an electro cell manipulator (BTX 200, BTX, Cambridge, UK). The grafted construct was positioned so the axes of the oocyte and pseudo-blastomere were aligned parallel to the electrodes. Electrofusion was conducted by single or double 1.0 kV/cm DC pulse with 50–99 µs duration within electrolytic M2 media. After washing and cultured for 3 minutes, reconstructed zygotes were monitored for fusion and survival. In our modified experiment protocol using B6-EGFP androgenotes, resulting 8-cell haploid embryos were examined for the presence of GFP signal (Fig. 2 ). GFP-positive embryos were selected and isolated by micromanipulation similar as described above. Instead of electrofusion, a modified virus-mediated method was employed. Each haploid androgenetic pseudo-blastomere was first coated with inactivated Sendai virus (HVJ-E, Cosmo Bio, Japan) and subsequently transferred into the perivitelline space of an activated parthenote ( 30 , 31 ) (Video 2). Reconstructed zygotes were monitored for fusion 15 minutes after micromanipulation. All reconstructed zygotes were cultured in G-TL media in EmbryoSlide. Embryo morphokinesis was recorded and evaluated (Video 3). Embryos with extensive fragmentation, direct uneven cleavage, or delayed morphokinetics were excluded. Blastocysts were collected and transferred into uterine cavity into 2.5 day-post-coitus CD1 mice previously mated with vasectomized CD-1 male. Karyotyping and Genetic Analysis In early set of experiments, karyotyping with Giemsa staining was used to assess ploidy. Haploid pseudo-blastomere or blastomere from reconstructed embryos were mechanically isolated by micromanipulation and transferred in media containing 0.06 µl/ml Colcemid (Sigma-Aldrich, St. Louis, MO, USA) to pause mitosis. After overnight culture, the blastomeres was transferred to a hypotonic media containing 40% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) for 15 minutes on a glass slide, subsequently fixed by methanol:acetic acid (3:1) and stained by 2% Giemsa solution ( 32 ). Chromosomal spread was then observed in microscope at 100x magnification. To confirm inheritance of paternal genome, Whole Exome Sequencing (WES) were employed to identify genetic similarity within a triad consisting of DNA extracted from one haploid blastomere and DNA from two offspring generated from the sibling haploid blastomere originated from the same spermatozoon. DNA extraction and amplification were performed using a commercial kit (Repli-G Single Cell; Qiagen, Hilden, Germany). Purified DNA was normalized at 20 ng/µl and sent to an external laboratory (Genewiz; Azenta Life Sciences; South Plainfield, NJ) where a 150-bp paired-end exome sequencing on an Illumina HiSeq 2500 platform was performed, as previously described ( 33 ). Quality controls of each sample were performed by quantitative polymerase chain reaction, and poor-quality nucleotides were removed (error rate < 0.01). Variant detection was performed using CLC Genomics Server 9.0, and the detected variants were annotated to identify unique gene mutations. All genomic coordinates were based on reference mouse genome assembly GRCm38 (mm10). Statistical Analysis and Bioinformatics The χ 2 test were used for comparison of fertilization, embryo development and pregnancy outcome. Statistical analysis was completed using IBM SPSS statistical software. P -value of < 0.05 was considered significant. Copy number variant determination and annotation of gene mutation were performed using CLC Genomics Server 9.0 modules with Next Generation Sequencing (NGS) core tools. Integrative Genomics Viewer (IGV, version 2.17.0) was used to visualize CNV comparing to GRCm38 (mm10) reference genome ( 34 ). Results Generation of Haploid Androgenetic Embryos to Replicate Sperm Genome In a series of 17 experiment using 85 mice, a total number of 535 oocytes were enucleated with a survival rate at 97.0% (519/535). Sperm injections were performed on all enucleated oocytes resulted in 471 mononucleated constructs (90.8%). In female counterpart, a total of 895 intact Metaphase II oocytes were activated and 96.4% (863/895) developed a single pronucleus and extruded a second polar body. Control oocytes fertilized at a rate of 97.8%. Cleavage rate of haploid B6D2F1 androgenetic embryos and gynogenetic counterparts into 2-cell and 4-cell were comparable to control zygotes. However, decrease in development from 8-cell onward was apparent for both haploid embryo types and become dramatic in blastocysts stage particularly in androgenotes. The same experiment was repeated in heterozygous transgenic strain (B6-EGFP) and followed the same trend observed in B6D2F1 androgenotes (Table 1). Haploid androgenetic embryos generated using B6-GFP spermatozoa expressing GFP were used (Figure 2). To verify the haploidy status of the embryos generated with sole male genome, karyotyping was performed on blastomeres from 2-cell, 4-cell and 8-cell embryos and evidenced a haploidy rate of 88.2% (Table 2, Figure 3). Haploid Androgenetic Blastomeres as Gametes Subsequently we used 2-cell, 4-cell, 8-cell stage androgenetic pseudo-blastomeres as male gamete to generate biparental constructs and additional sets were carried out using 8-cell blastomeres from B6-GFP mice (Table 3). Interestingly, the reconstruction (survived grafting) and fusion (presence of 2PN in the biparental constructs) rate was extremely successful in all categories of constructs reaching over 95%, similar to respective ICSI survival and fertilization rate of control zygotes. Biparental zygotes generated from all types of blastomeres were capable to support full pre-implantation development. While overall experimental constructs have development rate similar to control zygotes, in a sub-analysis comparing each type of conceptuses, lower compaction ( P<0.001 ) and blastulation ( P<0.0001 ) rates were observed in constructs created from 8-cell blastomeres. In experiment on B6-EGFP gametes, resulting biparental embryos expressed GFP (Figure 4). To confirm ploidy status of these biparental zygotes, chromosome spreads were prepared from the biparental constructs and preliminary result revealed diploidy at a rate of 83.3%. Haploid Androgenetic Blastomeres as Gametes Can Support Livebirth and Maintaining Paternal Haplotype To prove the post-implantation developmental ability. Overall, a total of 240 blastocysts generated from different stages of haploid blastomeres were transferred in a total of 25 surrogates resulting in 17 pregnancies. A total of 54 experimental embryos implanted (22.5%) and yielded 37 full-term development (15.4%) with 33 live birth (13.8%). Interestingly, the sex of the offspring trended to female, where only 13 (39.4%) were male and 20 (60.1%) were female. When comparing pregnancy outcome between experiments using different type of haploid blastomeres, we observed that live birth rate decreases as the haploid blastomere stage were more advanced (Table 4). In experiment with GFP mice, all pups (10/10) were confirmed to express green fluorescence signal (Figure 5). To undoubtfully confirm the inheritance of paternal genome and conservation of the identical haplotype between conceptuses that originated from the same gamete, WES revealed identical nucleotide sequence on Ahr, and Nnt genes, which all demonstrated unique mutations comparing to GRCm38 mm10 reference genome (Figure 6). Discussion Our study demonstrated the feasibility of replicating the sperm genome via androgenesis and selecting the desired gamete before fertilization to preserve a specific paternal genotype confirmed by phenotypical observation and corroborated by genetic testing. We achieved a satisfactory pre-implantation developmental rate of the conceptuses generated using those replicated male gametes and were able to generate healthy offspring. Specifically, in our experiment using 8-cell stage androgenetic embryos, a single spermatozoon can yield up to 3 conceptuses that carries the identical paternal haplotype. Interestingly, the embryo development of haploid pseudo-embryos reported in the first part of the study, whether androgenetic or parthenogenetic (Table 1 ), demonstrated a similar trend observed in previous studies, particularly the work done on digyneic and dispermic embryos, which underscores the importance of interdependence and complementation of both paternal and maternal genome in mammalian embryogenesis ( 35 ). Once the haploid androgenetic pseudo-blastomeres were used as gametes, the conceptuses generated demonstrated comparable embryo development comparing to control (Table 2 ) when 2- or 4-cell stage blastomeres were used. The utilization of 8-cell stage blastomere is less successful possibly caused by an intrinsic imprinting or epigenomic heterogeneity between sibling blastomeres since mouse embryo polarization occurs at 8-cell stage ( 36 – 39 ). However, more recent studies have suggested that the loss of totipotency can occur even in earlier stage at 2-cell in ~ 30% of the blastomeres ( 40 ) and further amplified in 4-cell stage embryos ( 41 ). Therefore, when an 8-cell stage pseudo-blastomere that is already differentiated was used as a male gamete, embryos generated by such cell may result in embryo development arrest, implantation failure and post-implantation development arrest. Another explanation for why 8-cell androgenetic pseudo-blastomeres yielded less blastocyst per embryo reconstruction could be attributed to a higher level of asynchrony of epigenome between the native maternal genome and replicated paternal genome. During murine preimplantation development, zygotic genome activation occurs at 2-cell stage ( 42 ) and the embryo genome expression is heavily regulated by the paternal chromatin ( 43 ). Therefore, when a pseudo-gamete was used to generate a zygote, the intricate regulation of zygote genome activation and epigenome modification may be disrupted, therefore causing a lower pre-implantation development rate and low implantation rate. In contrary, when the nucleus of 2-cell or 4-cell were used as the source of paternal genome, the blastocysts rates were reported at 80.2% and 81.1% respectively, which is comparative to control (80.8%) and higher than experimental embryos generated from 8-cell stage pseudo-blastomeres (60.7%). This observation maybe supported by the fact that the haploid genome of earlier stage pseudo-blastomere did not underwent extensive demethylation comparing to later stage embryos ( 44 ), and therefore, the 2-cell and 4-cell stage pseudo-blastomere retains a more gamete-like epigenome. Nonetheless, despite a higher blastocyst rate per embryo reconstruction experiment, the eventual success rate per spermatozoon is still comparable. The use of 8-cell stage pseudo-blastomere is still preferred since more male gamete copies can be used to fertilize a larger cohort of oocytes. When considering the use of this approach in higher mammal and even in human reproduction, this method of gamete replication is preferred over the utilization of haploid embryonic stem cells. Conventionally, haploid androgenetic embryonic stem cells (haESCs) are generated from haploid androgenetic embryos which has limitations including a low blastulation rate at 10–20%, supported by the data reported in our study at 11.2% ( 23 , 45 ), a low cell line derivation rate about 20% ( 23 ), and prone to a rapid loss of haploidy unable to be blocked by FACS purification ( 46 , 47 ). Comparing to haESC with low haploidy rate at 2–13% ( 17 ), as a precursor to haESCs, androgenetic blastomeres retain their haploidy at a higher rate (88.2%, Table 4). Additionally, Y-chromosome bearing haploid androgenetic blastocyst are unable to yield haESC lines, which further inhibits the application of haESCs in reproductive semi-cloning ( 48 , 49 ). However, our technique using haploid androgenetic blastomeres as gametes were able to generate male offspring. We do, however, observed a trend toward more viable female offspring (60.6%) comparing to male (39.4%), which could be explained by that an X-chromosome-bearing haploid blastomere has higher embryo competence comparing to the Y-chromosome-bearing counterpart. This trend was indeed true in early studies done on androgenetic and parthenogenetic embryos, where androgenones (two male pronuclei) arrest earlier while parthenogenetic embryos arrest post-implantation, signifying the role of X chromosome in these peculiar embryonic constructs ( 50 ). During the generation of haploid androgenetic embryos, the sperm DNA decondenses in the ooplasm during pronuclear stage. The availability of an unraveled sperm genome creates a conducive environment for heritable genome editing experiments favoring the fore-coming progeny. This strategy can ensure the uniformity of the eventual gamete genotype, and the subsequent use of edited gamete can therefore prevent genetic mosaicism sometimes observed following CRISPR-Cas9-mediated genome editing on diploid embryo ( 51 , 52 ). One major concern of this technique is the species specificity. This tailored technique is currently optimized for murine gametes with limited generalizability if applied in human gametes due to the different physiology between rodent and primate reproduction. One major uncertainty is the role of sperm centrosome. In murine reproduction, the zygotic spindle is formed from the oocyte’s own microtubule organization center (MTOC), while the mitotic spindle of human zygote is contributed by the sperm proximal centriole ( 53 , 54 ). Therefore, whether the human haploid blastomere will carry a functional MTOC still needs to be investigated. In term of epigenetic modification, we hypothesize that our technique may perform better on human gametes. Mouse zygotic genome activation occurs as early as 2-cell stage and this explains why the development of embryos generated from 8-cell stage blastomere is lower. Contrarily, higher mammal, including primates, has a later zygotic genome activation occur at 8-cell stage ( 55 ). Therefore, the maintenance of a more gamete-like epigenome may compensate for the development of conceptuses generated using our technique. When considering clinical application of male gamete replication, our technique represents an alternative to traditional PGD of embryos. Screening gametes before fertilization may be more ethically palatable than embryo selection by PGD. The ability to identify healthy gametes prior to insemination can prevent the generation of excessive embryos with pathological genetic condition, therefore reducing embryo wastage. Moreover, this technique can also benefit men with severe oligozoospermia or even cryptozoospermia. 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Maemura et al. , Totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage. Scientific Reports 11 , 11167 (2021). J. Y. Nothias, S. Majumder, K. J. Kaneko, M. L. DePamphilis, Regulation of gene expression at the beginning of mammalian development. J Biol Chem 270 , 22077-22080 (1995). H.-T. Bui et al. , Essential role of paternal chromatin in the regulation of transcriptional activity during mouse preimplantation development. REPRODUCTION 141 , 67-77 (2011). K. Iqbal, S.-G. Jin, G. P. Pfeifer, P. E. Szabó, Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine. Proceedings of the National Academy of Sciences 108 , 3642-3647 (2011). T. Kono, Y. Sotomaru, Y. Sato, T. Nakahara, Development of androgenetic mouse embryos produced by in vitro fertilization of enucleated oocytes. Mol Reprod Dev 34 , 43-46 (1993). T. Cui, Z. Li, Q. Zhou, W. Li, Current advances in haploid stem cells. Protein & Cell 11 , 23-33 (2020). U. Elling et al. , Derivation and maintenance of mouse haploid embryonic stem cells. Nature Protocols 14 , 1991-2014 (2019). K. E. Latham, H. Akutsu, B. Patel, R. Yanagimachi, Comparison of gene expression during preimplantation development between diploid and haploid mouse embryos. Biol Reprod 67 , 386-392 (2002). K. E. Latham, B. Patel, F. D. M. Bautista, S. M. Hawes, Effects of X Chromosome Number and Parental Origin on X-Linked Gene Expression in Preimplantation Mouse Embryos1. Biology of Reproduction 63 , 64-73 (2000). J. A. Thomson, D. Solter, The developmental fate of androgenetic, parthenogenetic, and gynogenetic cells in chimeric gastrulating mouse embryos. Genes Dev 2 , 1344-1351 (1988). M. Mehravar, A. Shirazi, M. Nazari, M. Banan, Mosaicism in CRISPR/Cas9-mediated genome editing. Developmental Biology 445 , 156-162 (2019). I. Lamas-Toranzo et al. , Strategies to reduce genetic mosaicism following CRISPR-mediated genome edition in bovine embryos. Scientific Reports 9 , 14900 (2019). P. Xie et al. , Sperm centriolar factors and genetic defects that can predict pregnancy. Fertil Steril 120 , 720-728 (2023). G. Palermo, S. Munné, J. Cohen, The human zygote inherits its mitotic potential from the male gamete. Human Reproduction 9 , 1220-1225 (1994). J. Taubenschmid-Stowers et al. , 8C-like cells capture the human zygotic genome activation program in vitro. Cell Stem Cell 29 , 449-459.e446 (2022). Tables Tables 1 to 4 are available in the Supplementary Files section Additional Declarations There is NO Competing Interest. Supplementary Files Video1.avi Embryo development of haploid androgenetic embryo Video2.mov Grafting a haploid androgenetic pseudo-blastomere onto an intact oocyte Video3.avi Embryo development of embryo generated from fertilization of oocyte using androgenetic blastomere as male gamete Table1.docx Table 1 Table2.docx Table 2 Table3.docx Table 3 Table4.docx Table 4 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4682261","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":328156994,"identity":"a8456a21-5b6e-4fab-88c5-ab6bf3c7bedd","order_by":0,"name":"Gianpiero Palermo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYFCCBAaGB0CKH4glICLMjQeABH4tQMQg2QDXwthAnBaDA8RqkXfPPfghoeJetPG1wwdvMNTY5JuzNwK1VFgnNuDQYnjmXbJEwpni3G2305ItGI6lWe7sOQjUciYdt5YZOQYSiW0JQC05ZhKMDYcNDG4kNhxgbDuMT4vxj8R/CbmbZ+d/g2i5/xCo5R9uLfISQMMTGxJyN0jnsEFtAXofyMCpxYDnXZpFwrGE3Bm304yBjDQDgzNAhyUcSzfGaUt77uEbH2oScvtnJz8EMmwMDI4fPvjgQ421LE5bDvAg8RIwGNhsaeDBIzsKRsEoGAWjAAQAyq5lwlJlaoQAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-8440-4560","institution":"Weill Cornell Medicine","correspondingAuthor":true,"prefix":"","firstName":"Gianpiero","middleName":"","lastName":"Palermo","suffix":""},{"id":328156995,"identity":"b3f12b4d-f6ce-4eb9-8678-551ed9e41dd9","order_by":1,"name":"Philip Xie","email":"","orcid":"","institution":"Weill Cornell 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18:40:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4682261/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4682261/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62115920,"identity":"a979dae1-14d0-4396-b5e3-8361190fbf69","added_by":"auto","created_at":"2024-08-09 12:50:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":172935,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTimeline – 8-cell stage haploid male pseudo-blastomere NT\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4682261/v1/3861944204d08b985794df66.png"},{"id":62116597,"identity":"f4d6d05b-5d4e-4c62-902a-3d3a342187dc","added_by":"auto","created_at":"2024-08-09 12:58:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":249524,"visible":true,"origin":"","legend":"\u003cp\u003eEight-cell Haploid androgenetic embryos generated from B6-GFP sperm a) phase contrast, and b) fluorescent\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4682261/v1/e3d6c8c1aea35e913e7a1468.png"},{"id":62115926,"identity":"488f676b-4f54-4ea2-b788-d72e18d86e77","added_by":"auto","created_at":"2024-08-09 12:50:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":267904,"visible":true,"origin":"","legend":"\u003cp\u003eChromosome spread of haploid androgenetic blastomere\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4682261/v1/826a8b02419f68e2d66a80b1.png"},{"id":62116601,"identity":"1e5dcc4e-5d24-4915-9e03-fca1d9191f40","added_by":"auto","created_at":"2024-08-09 12:58:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":453030,"visible":true,"origin":"","legend":"\u003cp\u003eHatching blastocyst generated from embryo reconstruction using haploid pseudo-blastomere generated using B6-GFP sperm.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4682261/v1/93b5285e303ec9a541d83ed7.png"},{"id":62115924,"identity":"eaf90735-b05a-429f-95d7-72a6a3da209b","added_by":"auto","created_at":"2024-08-09 12:50:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":700508,"visible":true,"origin":"","legend":"\u003cp\u003ePups generated from reconstructed embryos using a). B6D2F1 2-cell androgenotes, b). B6D2F1 4-cell androgenotes, c). B6-GFP 8-cell androgenotes and d). dead pup with abnormality.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4682261/v1/1db8e0cdfc71e6af6c493939.png"},{"id":62115930,"identity":"76761467-81aa-40d5-9ded-d5d6b07a24b2","added_by":"auto","created_at":"2024-08-09 12:50:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":145563,"visible":true,"origin":"","legend":"\u003cp\u003eWES results identified exact nucleotide sequence between 3 offspring derived from the exact original spermatozoon\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4682261/v1/0ec13b6614aeac75e389bb69.png"},{"id":62117090,"identity":"cd7c53be-2b1e-4bec-b7ff-a799501a75d5","added_by":"auto","created_at":"2024-08-09 13:06:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2729844,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4682261/v1/889e8fdc-9853-4362-ba44-8aef33d60e33.pdf"},{"id":62116598,"identity":"0d7f6f52-b245-43d6-9444-b362896c9ad3","added_by":"auto","created_at":"2024-08-09 12:58:10","extension":"avi","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3948092,"visible":true,"origin":"","legend":"\u003cp\u003eEmbryo development of haploid androgenetic embryo\u003c/p\u003e","description":"","filename":"Video1.avi","url":"https://assets-eu.researchsquare.com/files/rs-4682261/v1/8ab3e7ab1ef7eedea6410cfc.avi"},{"id":62115922,"identity":"2df09ce5-4a13-423d-888b-33cbabd50457","added_by":"auto","created_at":"2024-08-09 12:50:10","extension":"mov","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2183644,"visible":true,"origin":"","legend":"\u003cp\u003eGrafting a haploid androgenetic pseudo-blastomere onto an intact oocyte\u003c/p\u003e","description":"","filename":"Video2.mov","url":"https://assets-eu.researchsquare.com/files/rs-4682261/v1/73ad29737bfde6b9ed88ac99.mov"},{"id":62115929,"identity":"80f2a9e8-587b-4ea9-bf1c-638cd317b40e","added_by":"auto","created_at":"2024-08-09 12:50:10","extension":"avi","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":10161422,"visible":true,"origin":"","legend":"Embryo development of embryo generated from fertilization of oocyte using androgenetic blastomere as male gamete","description":"","filename":"Video3.avi","url":"https://assets-eu.researchsquare.com/files/rs-4682261/v1/6dd4759f1749dec238fb672f.avi"},{"id":62116600,"identity":"73b21d4f-6654-4196-b3d9-02fc8508bbb6","added_by":"auto","created_at":"2024-08-09 12:58:10","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":16258,"visible":true,"origin":"","legend":"\u003cp\u003eTable 1\u003c/p\u003e","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4682261/v1/38011c548a5c74d14eeb4888.docx"},{"id":62115923,"identity":"f6385033-135d-45b9-bdb1-a086cb4e2097","added_by":"auto","created_at":"2024-08-09 12:50:10","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":15341,"visible":true,"origin":"","legend":"Table 2","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4682261/v1/fced5bc12120620e8615dcfb.docx"},{"id":62115931,"identity":"ce96f3a7-abe0-4ce9-a396-b7260f13835d","added_by":"auto","created_at":"2024-08-09 12:50:10","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":16493,"visible":true,"origin":"","legend":"\u003cp\u003eTable 3\u003c/p\u003e","description":"","filename":"Table3.docx","url":"https://assets-eu.researchsquare.com/files/rs-4682261/v1/80a18e6c34d5e82eb7f75759.docx"},{"id":62115928,"identity":"e3d9dad8-964e-4f71-99b5-5b95056be6c0","added_by":"auto","created_at":"2024-08-09 12:50:10","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":16597,"visible":true,"origin":"","legend":"\u003cp\u003eTable 4\u003c/p\u003e","description":"","filename":"Table4.docx","url":"https://assets-eu.researchsquare.com/files/rs-4682261/v1/65bd34dd7a105c52ab063165.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Male gamete copies to characterize genome inheritance and generate progenies","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eMale infertility affects about 30% of the infertile population and about 9\u0026ndash;15% of overall population (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Intracytoplasmic sperm injection (ICSI) offers a promising solution by allowing a single selected spermatozoon to fertilize an oocyte regardless of the quantity and type of motility of the male gamete (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) Even in cases where the spermatozoa are functionally impaired such as globozoospermia, ICSI can still grant fertilization when used in conjunction with assisted oocyte activation (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). However, in cases when the spermatozoa are extremely scarce, it would be ideal to be able to replicate the male gamete to be able to fertilize a large cohort of oocytes. In addition, there are patients who carry inherited disorders, and the current treatment plan is solely dependent the selection of embryos available through pre-implantation genetic diagnosis (PGD). This can be ethically debatable due to the fact that several embryos are generated in order to select one normal conceptus (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Therefore, it would be ideal to decipher the genetic of a single sperm cell prior the generation of the conceptus.\u003c/p\u003e \u003cp\u003eThere are situations where inherited disorders manifest due to the heterogeneity of germ line. This gamete heterogeneity is relevant in cases of structural chromosomal abnormality occurring in 5\u0026ndash;15% of infertile men (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), including balanced and unbalanced translocations, where the affected individual generates a heterogenous sperm population whether healthy or abnormal (\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). On the other hand, gamete heterogeneity can also be characterized in term of monogenic diseases caused by gene mutations, estimated to affect about 6% the human population (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). In cases of autosomal recessive disorders, phenotypically healthy carriers produce a heterogenous population of germ cells due to the heterozygosity (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Nonetheless, even in individuals who are not carriers of diseases, \u003cem\u003ede novo\u003c/em\u003e germline mutation may occur and accumulate over years with frequency of 45\u0026ndash;60 \u003cem\u003ede novo\u003c/em\u003e mutation per genome in a generation (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). For example, unaffected individuals may pass on pathological \u003cem\u003ede novo\u003c/em\u003e variants of alleles, such as X-linked \u003cem\u003eTEX11\u003c/em\u003e mutation and autosomal dominant \u003cem\u003ePTPN11\u003c/em\u003e mutation responsible Noonan Syndrome (\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis awareness has stimulated attempts to replicate sperm genome to identify the healthy gametes. Indeed, androgenetic embryos can be generated by injection of a single spermatozoon into an enucleated oocyte (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), these embryos can yield haploid embryonic stem cells (haESCs) to study a recessive phenotype, or to be utilized in reproductive semi-cloning (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Generation of haESCs has been successful, however, as expected, the utilization of haESC line is hindered by low blastocyst development averaging at 12.8% and an even lower efficiency in haESC derivation up to 40% (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Therefore, in order to obtain one haESC line, at least 20 haploid embryos must be sacrificed even in optimal conditions. In addition, haploidy in mammalian somatic cells is an unstable situation and tend to self-diploidized due to endo-cycling and failed cytokinesis that can, and become more manifested when uncoupling of DNA replication and centrosome duplication cycles are more common (\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Once haESCs are achieved and purified, the utilization of such cells as a source of male gamete were able to yield offspring of desired genotype, albeit at a very low rate (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). In addition, concerns include the epigenetic profile of the male gamete with consequential genetic imprinting.\u003c/p\u003e \u003cp\u003eIn this study, we plan to replicate the mammalian male gamete by injecting a single spermatozoon into an enucleated oocyte and subsequently use these replicated cells to fertilize oocytes to generate conceptuses. We closely monitored the pre-implantation development of conceptuses by time-lapse microscopy, and post-implantation development by assessing the health and reproductive ability of the offspring. To confirm the provenance of the paternal genome, we sequenced replicated pseudo-gametes and compared it to the genomes of resulting offspring. We also assessed the uniformity of the genotype among the siblings originated from one single spermatozoon.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Design\u003c/h2\u003e \u003cp\u003eIn this work, we carried out a preliminary study where we assessed the ability of haploid androgenetic and gynogenetic constructs to develop into blastocysts in comparison to a mouse ICSI control. In the following step, we assessed the ability to generate conceptuses by utilizing haploid androgenetic pseudo-blastomere at different cleavage stage as a source of male gamete. Haploid pseudo-blastomeres were isolated from embryos at 2-cell, 4-cell and 8-cell. Resulting constructs were assessed up to full pre-implantation development. In some embryos, chromosomal analyses were carried out to confirm ploidy. In order to prove the embryo developmental competence of these biparental zygotes in some pseudo-pregnancy mice, hatching blastocysts were transferred. This allowed us to assess the development up to adulthood and related reproductive potential. In a later series of experiment, to confirm the origin of the male genome, we generated haploid androgenetic embryos as a source of male gamete using GFP transgenic male mice (Fig.\u0026nbsp;1). This project was approved through the IACUC of Weill Cornell Medical College, protocol number 0605-493A.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eOvarian Stimulation, Oocyte Collection, and Oocyte Preparation\u003c/h2\u003e \u003cp\u003eTo harvest mouse metaphase II oocytes, 8\u0026ndash;12 weeks old B6D2F1 mice (Jackson Laboratory) were stimulated by intra-peritoneal (IP) injection of 0.1-0.2cc of ready-to-use pregnant mare serum gonadotropin / inhibin superovulation reagent (CARD HyperOva\u0026reg;, Cosmo Bio, Japan). Forty-eight hours after stimulation, eventual oocyte maturation and ovulation was triggered by IP injection of 7.5 IU of human chorionic gonadotropin (hCG, Sigma-Aldrich, St. Louis, MO, USA). Up to 16 hours after hCG trigger, mice were euthanized by cervical dislocation and dissected to retrieve oviducts containing cumulus-oocyte-complexes (COCs).\u003c/p\u003e \u003cp\u003eCOCs were then isolated from the ampullae of the oviducts and treated with 80 IU/ml hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA) followed by mechanical denudation using microcapillary. Oocytes were serially washed thrice in potassium simplex optimized media (KSOM, Cosmo Bio, Japan) and placed in an incubator at 37\u0026deg;C and 5% CO2 in equilibrated KSOM for use in subsequent steps.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSpermatozoa Collection and Preparation\u003c/h2\u003e \u003cp\u003eEuthanasia was performed on male B6D2F1 or heterozygous B6-EGFP mice aged 12\u0026ndash;16 weeks by cervical dislocation. The lower abdomen was sanitized with betadine where incision was then made surgically, and bilateral cauda epididymis identified and excised. They were placed in modified human tubal fluid (HTF) media (Cosmo Bio, Japan) on heat block for transport. Spermatozoa were extracted from cauda epididymis and diluted to 3\u0026nbsp;million/ml for insemination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of ooplasts for haploid androgenetic embryos\u003c/h2\u003e \u003cp\u003eTo generate haploid androgenetic embryos, mature oocytes from B6D2F1 mice were enucleated using techniques based on previous literatures (\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Briefly, denuded oocytes were transferred into HEPES-buffered M2 media (Sigma-Aldrich, St. Louis, MO, USA) droplets supplemented with 5 \u0026micro;g/ml cytochalasin B (Sigma-Aldrich, St. Louis, MO, USA) on specialized glass dish (FluoroDish\u0026trade;, WPI.inc, Sarasota, FL, USA). Instead of nuclear staining commonly used in cloning, oocyte metaphase II spindle were visualized under stain-free polarized microscopy (Oosight\u0026trade;, Hamilton-Thorne, Beverly, MA, USA). The metaphase II spindle were then held by a holding pipette and rotated to have spindle located at 3 o'clock. A laser (LYKOS, Hamilton-Thorne, Beverly, MA, USA) or a piezo pulse (PMM-150, PrimeTech, Japan) was then applied to breach the zona. Next, a micropipette was advanced into the perivitelline space through the breach and a gentle suction was applied to remove the spindle. Enucleated oocytes were then placed in KSOM at least 1 hour until next procedure.\u003c/p\u003e \u003cp\u003eGeneration of haploid androgenetic embryos\u003c/p\u003e \u003cp\u003ePiezo-actuated ICSI were performed to generate haploid androgenetic embryos using enucleated oocytes. The piezo-ICSI procedure is performed similarly to standard protocol (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). A single mouse spermatozoon from B6D2F1 or heterozygous B6-EGFP were decapitated mechanically, and the isolated head was suctioned into the micropipette in 7% polyvinylpyrrolidone (Fujifilm Irvine Scientific, Santa Ana, CA, USA). Individual ooplasts, placed in HEPES-buffered M2 media, was then positioned by a holding pipette, and rotated so the breach created previously was placed at 3 o'clock. The injection pipette containing the spermatozoa were the advanced into the perivitelline space of the ooplasts creating an invagination. Finally, a weak piezo pulse was applied to penetrate the oolemma with the sperm deposited into the cytoplasm.\u003c/p\u003e \u003cp\u003ePost-ICSI oocytes inseminated by B6D2F1 spermatozoa were the cultured in KSOM for 24h, 36h, or 48h to generate 2-cell, 4-cell, or 8-cell haploid androgenetic embryos, respectively. In our modified experiment using B6-EGFP gametes, haploid androgenetic embryos were cultured in G-TL media (Vitrolife, Gothenburg, Sweden) designed for time-lapse microscopy. Each haploid androgenetic embryos were transferred into the microwell of EmbryoSlide (Vitrolife, Gothenburg, Sweden) covered with mineral oil, and the EmbryoSlide was then loaded into EmbryoScope set to take picture every 10 minutes for time-lapse microscopy in order to observe embryo morphokinesis (Video 1). Haploid androgenetic embryos displaying extensive fragmentation, abnormal morphokinetics or arrested development were excluded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eRecipient Oocyte Preparation and Generation of Biparental Zygotes\u003c/h2\u003e \u003cp\u003eTo prepare recipient oocytes for the replicated male gamete, mature oocytes from B6D2F1 mice were chemically activated by 2.5h incubation in Calcium-free CZB medium supplied with 10 mM SrCl\u003csub\u003e2\u003c/sub\u003e and cultured in KSOM for 2\u0026ndash;3 more hours until a female pronucleus is present and a secondary polar body is extruded (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our early experiment using B6D2F1 androgenotes, a single pseudo-blastomere were isolated from 2-cell, 4-cell or 8-cell haploid androgenetic embryos with each pseudo-blastomere sized 15 \u0026micro;m in diameter and transferred into the perivitelline space of an activated parthenote. Each grafted oocyte was then aligned between 2 microelectrodes (ECF-100, BTX, Cambridge, UK) connected to an electro cell manipulator (BTX 200, BTX, Cambridge, UK). The grafted construct was positioned so the axes of the oocyte and pseudo-blastomere were aligned parallel to the electrodes. Electrofusion was conducted by single or double 1.0 kV/cm DC pulse with 50\u0026ndash;99 \u0026micro;s duration within electrolytic M2 media. After washing and cultured for 3 minutes, reconstructed zygotes were monitored for fusion and survival.\u003c/p\u003e \u003cp\u003eIn our modified experiment protocol using B6-EGFP androgenotes, resulting 8-cell haploid embryos were examined for the presence of GFP signal (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). GFP-positive embryos were selected and isolated by micromanipulation similar as described above. Instead of electrofusion, a modified virus-mediated method was employed. Each haploid androgenetic pseudo-blastomere was first coated with inactivated Sendai virus (HVJ-E, Cosmo Bio, Japan) and subsequently transferred into the perivitelline space of an activated parthenote (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) (Video 2). Reconstructed zygotes were monitored for fusion 15 minutes after micromanipulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAll reconstructed zygotes were cultured in G-TL media in EmbryoSlide. Embryo morphokinesis was recorded and evaluated (Video 3). Embryos with extensive fragmentation, direct uneven cleavage, or delayed morphokinetics were excluded. Blastocysts were collected and transferred into uterine cavity into 2.5 day-post-coitus CD1 mice previously mated with vasectomized CD-1 male.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eKaryotyping and Genetic Analysis\u003c/h2\u003e \u003cp\u003eIn early set of experiments, karyotyping with Giemsa staining was used to assess ploidy. Haploid pseudo-blastomere or blastomere from reconstructed embryos were mechanically isolated by micromanipulation and transferred in media containing 0.06 \u0026micro;l/ml Colcemid (Sigma-Aldrich, St. Louis, MO, USA) to pause mitosis. After overnight culture, the blastomeres was transferred to a hypotonic media containing 40% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) for 15 minutes on a glass slide, subsequently fixed by methanol:acetic acid (3:1) and stained by 2% Giemsa solution (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Chromosomal spread was then observed in microscope at 100x magnification.\u003c/p\u003e \u003cp\u003eTo confirm inheritance of paternal genome, Whole Exome Sequencing (WES) were employed to identify genetic similarity within a triad consisting of DNA extracted from one haploid blastomere and DNA from two offspring generated from the sibling haploid blastomere originated from the same spermatozoon. DNA extraction and amplification were performed using a commercial kit (Repli-G Single Cell; Qiagen, Hilden, Germany). Purified DNA was normalized at 20 ng/\u0026micro;l and sent to an external laboratory (Genewiz; Azenta Life Sciences; South Plainfield, NJ) where a 150-bp paired-end exome sequencing on an Illumina HiSeq 2500 platform was performed, as previously described (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Quality controls of each sample were performed by quantitative polymerase chain reaction, and poor-quality nucleotides were removed (error rate\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Variant detection was performed using CLC Genomics Server 9.0, and the detected variants were annotated to identify unique gene mutations. All genomic coordinates were based on reference mouse genome assembly GRCm38 (mm10).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis and Bioinformatics\u003c/h2\u003e \u003cp\u003eThe χ\u003csup\u003e2\u003c/sup\u003e test were used for comparison of fertilization, embryo development and pregnancy outcome. Statistical analysis was completed using IBM SPSS statistical software. \u003cem\u003eP\u003c/em\u003e-value of \u0026lt;\u0026thinsp;0.05 was considered significant. Copy number variant determination and annotation of gene mutation were performed using CLC Genomics Server 9.0 modules with Next Generation Sequencing (NGS) core tools. Integrative Genomics Viewer (IGV, version 2.17.0) was used to visualize CNV comparing to GRCm38 (mm10) reference genome (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eGeneration of Haploid Androgenetic Embryos to Replicate Sperm Genome\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;In a series of 17 experiment using 85 mice, a total number of 535 oocytes were enucleated with a survival rate at 97.0% (519/535). Sperm injections were performed on all enucleated oocytes resulted in 471 mononucleated constructs (90.8%). In female counterpart, a total of 895 intact Metaphase II oocytes were activated and 96.4% (863/895) developed a single pronucleus and extruded a second polar body. Control oocytes fertilized at a rate of 97.8%. Cleavage rate of haploid B6D2F1 androgenetic embryos and gynogenetic counterparts into 2-cell and 4-cell were comparable to control zygotes. However, decrease in development from 8-cell onward was apparent for both haploid embryo types and become dramatic in blastocysts stage particularly in androgenotes. The same experiment was repeated in heterozygous transgenic strain (B6-EGFP) and followed the same trend observed in B6D2F1 androgenotes (Table 1). Haploid androgenetic embryos generated using B6-GFP spermatozoa expressing GFP were used (Figure 2). To verify the haploidy status of the embryos generated with sole male genome, karyotyping was performed on blastomeres from 2-cell, 4-cell and 8-cell embryos and evidenced a haploidy rate of 88.2% (Table 2, Figure 3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHaploid Androgenetic Blastomeres as Gametes\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Subsequently we used 2-cell, 4-cell, 8-cell stage androgenetic pseudo-blastomeres as male gamete to generate biparental constructs and additional sets were carried out using 8-cell blastomeres from B6-GFP mice (Table 3). Interestingly, the reconstruction (survived grafting) and fusion (presence of 2PN in the biparental constructs) rate was extremely successful in all categories of constructs reaching over 95%, similar to respective ICSI survival and fertilization rate of control zygotes. Biparental zygotes generated from all types of blastomeres were capable to support full pre-implantation development. While overall experimental constructs have development rate similar to control zygotes, in a sub-analysis comparing each type of conceptuses, lower compaction (\u003cem\u003eP\u0026lt;0.001\u003c/em\u003e) and blastulation (\u003cem\u003eP\u0026lt;0.0001\u003c/em\u003e) rates were observed in constructs created from 8-cell blastomeres. In experiment on B6-EGFP gametes, resulting biparental embryos expressed GFP (Figure 4). To confirm ploidy status of these biparental zygotes, chromosome spreads were prepared from the biparental constructs and preliminary result revealed diploidy at a rate of 83.3%.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHaploid Androgenetic Blastomeres as Gametes Can Support Livebirth and Maintaining Paternal Haplotype\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;To prove the post-implantation developmental ability. Overall, a total of 240 blastocysts generated from different stages of haploid blastomeres were transferred in a total of 25 surrogates resulting in 17 pregnancies. A total of 54 experimental embryos implanted (22.5%) and yielded 37 full-term development (15.4%) with 33 live birth (13.8%). Interestingly, the sex of the offspring trended to female, where only 13 (39.4%) were male and 20 (60.1%) were female. When comparing pregnancy outcome between experiments using different type of haploid blastomeres, we observed that live birth rate decreases as the haploid blastomere stage were more advanced (Table 4). In experiment with GFP mice, all pups (10/10) were confirmed to express green fluorescence signal (Figure 5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;To undoubtfully confirm the inheritance of paternal genome and conservation of the identical haplotype between conceptuses that originated from the same gamete, WES revealed identical nucleotide sequence on \u003cem\u003eAhr,\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Nnt\u003c/em\u003e\u0026nbsp; genes, which all demonstrated unique mutations comparing to GRCm38 mm10 reference genome (Figure 6).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study demonstrated the feasibility of replicating the sperm genome via androgenesis and selecting the desired gamete before fertilization to preserve a specific paternal genotype confirmed by phenotypical observation and corroborated by genetic testing. We achieved a satisfactory pre-implantation developmental rate of the conceptuses generated using those replicated male gametes and were able to generate healthy offspring. Specifically, in our experiment using 8-cell stage androgenetic embryos, a single spermatozoon can yield up to 3 conceptuses that carries the identical paternal haplotype.\u003c/p\u003e \u003cp\u003eInterestingly, the embryo development of haploid pseudo-embryos reported in the first part of the study, whether androgenetic or parthenogenetic (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), demonstrated a similar trend observed in previous studies, particularly the work done on digyneic and dispermic embryos, which underscores the importance of interdependence and complementation of both paternal and maternal genome in mammalian embryogenesis (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Once the haploid androgenetic pseudo-blastomeres were used as gametes, the conceptuses generated demonstrated comparable embryo development comparing to control (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) when 2- or 4-cell stage blastomeres were used. The utilization of 8-cell stage blastomere is less successful possibly caused by an intrinsic imprinting or epigenomic heterogeneity between sibling blastomeres since mouse embryo polarization occurs at 8-cell stage (\u003cspan additionalcitationids=\"CR37 CR38\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). However, more recent studies have suggested that the loss of totipotency can occur even in earlier stage at 2-cell in ~\u0026thinsp;30% of the blastomeres (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e) and further amplified in 4-cell stage embryos (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Therefore, when an 8-cell stage pseudo-blastomere that is already differentiated was used as a male gamete, embryos generated by such cell may result in embryo development arrest, implantation failure and post-implantation development arrest.\u003c/p\u003e \u003cp\u003eAnother explanation for why 8-cell androgenetic pseudo-blastomeres yielded less blastocyst per embryo reconstruction could be attributed to a higher level of asynchrony of epigenome between the native maternal genome and replicated paternal genome. During murine preimplantation development, zygotic genome activation occurs at 2-cell stage (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e) and the embryo genome expression is heavily regulated by the paternal chromatin (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Therefore, when a pseudo-gamete was used to generate a zygote, the intricate regulation of zygote genome activation and epigenome modification may be disrupted, therefore causing a lower pre-implantation development rate and low implantation rate. In contrary, when the nucleus of 2-cell or 4-cell were used as the source of paternal genome, the blastocysts rates were reported at 80.2% and 81.1% respectively, which is comparative to control (80.8%) and higher than experimental embryos generated from 8-cell stage pseudo-blastomeres (60.7%). This observation maybe supported by the fact that the haploid genome of earlier stage pseudo-blastomere did not underwent extensive demethylation comparing to later stage embryos (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e), and therefore, the 2-cell and 4-cell stage pseudo-blastomere retains a more gamete-like epigenome. Nonetheless, despite a higher blastocyst rate per embryo reconstruction experiment, the eventual success rate per spermatozoon is still comparable. The use of 8-cell stage pseudo-blastomere is still preferred since more male gamete copies can be used to fertilize a larger cohort of oocytes.\u003c/p\u003e \u003cp\u003eWhen considering the use of this approach in higher mammal and even in human reproduction, this method of gamete replication is preferred over the utilization of haploid embryonic stem cells. Conventionally, haploid androgenetic embryonic stem cells (haESCs) are generated from haploid androgenetic embryos which has limitations including a low blastulation rate at 10\u0026ndash;20%, supported by the data reported in our study at 11.2% (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), a low cell line derivation rate about 20% (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), and prone to a rapid loss of haploidy unable to be blocked by FACS purification (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Comparing to haESC with low haploidy rate at 2\u0026ndash;13% (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), as a precursor to haESCs, androgenetic blastomeres retain their haploidy at a higher rate (88.2%, Table\u0026nbsp;4).\u003c/p\u003e \u003cp\u003eAdditionally, Y-chromosome bearing haploid androgenetic blastocyst are unable to yield haESC lines, which further inhibits the application of haESCs in reproductive semi-cloning (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). However, our technique using haploid androgenetic blastomeres as gametes were able to generate male offspring. We do, however, observed a trend toward more viable female offspring (60.6%) comparing to male (39.4%), which could be explained by that an X-chromosome-bearing haploid blastomere has higher embryo competence comparing to the Y-chromosome-bearing counterpart. This trend was indeed true in early studies done on androgenetic and parthenogenetic embryos, where androgenones (two male pronuclei) arrest earlier while parthenogenetic embryos arrest post-implantation, signifying the role of X chromosome in these peculiar embryonic constructs (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDuring the generation of haploid androgenetic embryos, the sperm DNA decondenses in the ooplasm during pronuclear stage. The availability of an unraveled sperm genome creates a conducive environment for heritable genome editing experiments favoring the fore-coming progeny. This strategy can ensure the uniformity of the eventual gamete genotype, and the subsequent use of edited gamete can therefore prevent genetic mosaicism sometimes observed following CRISPR-Cas9-mediated genome editing on diploid embryo (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOne major concern of this technique is the species specificity. This tailored technique is currently optimized for murine gametes with limited generalizability if applied in human gametes due to the different physiology between rodent and primate reproduction. One major uncertainty is the role of sperm centrosome. In murine reproduction, the zygotic spindle is formed from the oocyte\u0026rsquo;s own microtubule organization center (MTOC), while the mitotic spindle of human zygote is contributed by the sperm proximal centriole (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). Therefore, whether the human haploid blastomere will carry a functional MTOC still needs to be investigated. In term of epigenetic modification, we hypothesize that our technique may perform better on human gametes. Mouse zygotic genome activation occurs as early as 2-cell stage and this explains why the development of embryos generated from 8-cell stage blastomere is lower. Contrarily, higher mammal, including primates, has a later zygotic genome activation occur at 8-cell stage (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). Therefore, the maintenance of a more gamete-like epigenome may compensate for the development of conceptuses generated using our technique.\u003c/p\u003e \u003cp\u003eWhen considering clinical application of male gamete replication, our technique represents an alternative to traditional PGD of embryos. Screening gametes before fertilization may be more ethically palatable than embryo selection by PGD. The ability to identify healthy gametes prior to insemination can prevent the generation of excessive embryos with pathological genetic condition, therefore reducing embryo wastage. Moreover, this technique can also benefit men with severe oligozoospermia or even cryptozoospermia. By replicating scarce spermatozoa, the abundance of sperm copies permits the insemination of entire oocyte cohort using only few available male gametes.\u003c/p\u003e \u003cp\u003eIn conclusion, haploid androgenetic blastomeres can function as replicated spermatozoa, demonstrated satisfactory blastocyst development, and yielded healthy offspring. This replication of sperm genome allowed us to select gametes of desired genotype prior to the generation of an embryo.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eC. L. R. Barratt\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e, The diagnosis of male infertility: an analysis of the evidence to support the development of global WHO guidance-challenges and future research opportunities. \u003cem\u003eHum Reprod Update\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 660-680 (2017).\u003c/li\u003e\n \u003cli\u003eG. D. 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When spermatozoa are extremely scarce, replicating the male gamete to fertilize a large cohort of oocytes would be ideal. Additionally, patients with inherited disorders currently rely on pre-implantation genetic diagnosis (PGD) to select healthy embryos, which raises ethical concerns due to the generation of multiple embryos to select one healthy conceptus. Therefore, it would be beneficial to decode the genetics of a single sperm cell before conceptus generation. In this study, we demonstrated the feasibility of replicating the sperm genome via androgenesis and selecting the desired gamete before fertilization to preserve a specific paternal genotype, confirmed by phenotypic observation and genetic testing, in a murine model. We achieved satisfactory pre-implantation developmental rates with replicated male gametes and were able to generate healthy offspring. Specifically, using 8-cell stage androgenetic embryos, a single spermatozoon can yield up to three conceptuses carrying the identical paternal haplotype.","manuscriptTitle":"Male gamete copies to characterize genome inheritance and generate progenies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-09 12:50:05","doi":"10.21203/rs.3.rs-4682261/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"44ff0f7f-71de-4f68-84d6-354a8a979df1","owner":[],"postedDate":"August 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":34731573,"name":"Biological sciences/Developmental biology/Embryology"},{"id":34731574,"name":"Health sciences/Medical research/Preclinical research"},{"id":34731575,"name":"Biological sciences/Stem cells/Embryonic stem cells"}],"tags":[],"updatedAt":"2024-11-20T17:53:24+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-09 12:50:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4682261","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4682261","identity":"rs-4682261","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}