Combining single-cell profiling and functional analysis explores the role of pseudogenes in human early embryonic development.

More and more studies have demonstrated that pseudogenes possess coding ability, and the functions of their transcripts in the development of diseases have been partially revealed. However, the role of pseudogenes in maintenance of normal physiological states and life activities has long been neglected. Here we identify pseudogenes that are dynamically expressed during human early embryogenesis, showing different expression pattern from that of adult tissues. We explore the expression correlation between pseudogenes and the parent genes, part due to their shared gene regulatory elements or the potential regulation network between them. The essential role of three pseudogenes, PI4KAP1, TMED10P1, and FBXW4P1, in maintaining self-renewal of human embryonic stem cells is demonstrated. We further find that the three pseudogenes might perform their regulatory functions by binding to proteins or microRNAs. The pseudogene-related single-nucleotide polymorphisms are significantly associated with human congenital disease, further illustrating their importance during early embryonic development. Overall, this study is an excavation and exploration of functional pseudogenes during early human embryonic development, suggesting that pseudogenes are not only capable of being specifically activated in pathological states, but also play crucial functions in the maintenance of normal physiological states.

Characterization of bovine long non-coding RNAs, OOSNCR1, OOSNCR2 and OOSNCR3, and their roles in oocyte maturation and early embryonic development.

In mammals, early embryogenesis relies heavily on the regulation of maternal transcripts including protein-coding and non-coding RNAs stored in oocytes. In this study, the expression of three bovine oocyte expressed long non-coding RNAs (lncRNAs), OOSNCR1, OOSNCR2, and OOSNCR3, was characterized in somatic tissues, the ovarian follicle, and throughout early embryonic development. Moreover, the functional requirement of each transcript during oocyte maturation and early embryonic development was investigated using a siRNA-mediated knockdown approach. Tissue distribution analysis revealed that OOSNCR1, OOSNCR2 and OOSNCR3 are predominantly expressed in fetal ovaries. Follicular cell expression analysis revealed that these lncRNAs are highly expressed in the oocytes, with minor expression detected in the cumulus cells (CCs) and mural granulosa cells (mGCs). The expression for all three genes was highest during oocyte maturation, decreased at fertilization, and ceased altogether by the 16-cell stage. Knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 in immature oocytes was achieved by microinjection of the cumulus-enclosed germinal vesicle (GV) oocytes with siRNAs targeting these lncRNAs. Knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 did not affect cumulus expansion, but oocyte survival at 12 h post-insemination was significantly reduced. In addition, knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 in immature oocytes resulted in a decreased rate of blastocyst development, and reduced expression of genes associated with oocyte competency such as nucleoplasmin 2 (NPM2), growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), and JY-1 in MII oocytes. The data herein suggest a functional requirement of OOSNCR1, OOSNCR2, and OOSNCR3 during bovine oocyte maturation and early embryogenesis.

miRNAs in Follicular and Oviductal Fluids Support Global DNA Demethylation in Early-Stage Embryos.

Global methylation levels differ in in vitro- and in vivo-developed embryos. Follicular fluid (FF) contains extracellular vesicles (EVs) containing miRNAs that affect embryonic development. Here, we examined our hypothesis that components in FF affect global DNA methylation and embryonic development. Oocytes and FF were collected from bovine ovaries. Treatment of zygotes with a low concentration of FF induced global DNA demethylation, improved embryonic development, and reduced DNMT1/3A levels. We show that embryos take up EVs containing labeled miRNA secreted from granulosa cells and the treatment of zygotes with EVs derived from FF reduces global DNA methylation in embryos. Furthermore, the methylation levels of in vitro-developed blastocysts were higher than those of in their vivo counterparts. Based on small RNA-sequencing and in silico analysis, we predicted miR-29b, -199a-3p, and -148a to target DNMTs and to induce DNA demethylation, thereby improving embryonic development. Moreover, among FF from 30 cows, FF with a high content of these miRNAs demethylated more DNA in the embryos than FF with a lower miRNA content. Thus, miRNAs in FF play a role in early embryonic development.

Inhibition of neddylation disturbs zygotic genome activation through histone modification change and leads to early development arrest in mouse embryos.

Post-translational modification and fine-tuned protein turnover are of great importance in mammalian early embryo development. Apart from the classic protein degradation promoting ubiquitination, new forms of ubiquitination-like modification are yet to be fully understood. Here, we demonstrate the function and potential mechanisms of one ubiquitination-like modification, neddylation, in mouse preimplantation embryo development. Treated with specific inhibitors, zygotes showed a dramatically decreased cleavage rate and almost all failed to enter the 4-cell stage. Transcriptional profiling showed genes were differentially expressed in pathways involving cell fate determination and cell differentiation, including several down-regulated zygotic genome activation (ZGA) marker genes. A decreased level of phosphorylated RNA polymerase II was detected, indicating impaired gene transcription inside the embryo cell nucleus. Proteomic data showed that differentially expressed proteins were enriched in histone modifications. We confirmed the lowered in methyltransferase (KMT2D) expression and a decrease in histone H3K4me3. At the same time, acetyltransferase (CBP/p300) reduced, while deacetylase (HDAC6) increased, resulting in an attenuation in histone H3K27ac. Additionally, we observed the up-regulation in YAP1 and RPL13 activities, indicating potential abnormalities in the downstream response of Hippo signaling pathway. In summary, we found that inhibition of neddylation induced epigenetic changes in early embryos and led to abnormalities in related downstream signaling pathways. This study sheds light upon new forms of ubiquitination regulating mammalian embryonic development and may contribute to further investigation of female infertility pathology.

Characterization of agouti-signaling protein (ASIP) in the bovine ovary and throughout early embryogenesis.

The oocyte expresses certain genes during folliculogenesis to regulate the acquisition of oocyte competence. Oocyte competence, or oocyte quality, is directly related to the ability of the oocyte to result in a successful pregnancy following fertilization. Presently, approximately 40 % of bovine embryos will develop to the blastocyst stage in vitro. Characterization of factors regulating these processes is crucial to improve the efficiency of bovine in vitro embryo production. We demonstrated that the secreted protein, agouti-signaling protein (ASIP) is highly abundant in the bovine oocyte and aimed to characterize its spatiotemporal expression profile in the ovary and throughout early embryonic development. In addition to oocyte expression, ASIP was detected in granulosa, cumulus, and theca cells isolated from antral follicles. Both gene expression data and immunofluorescent staining indicated ASIP declines with oocyte maturation which may indicate a potential role for ASIP in the attainment of oocyte competence. Microinjection of zygotes using small interfering RNA targeting ASIP led to a 16 % reduction in the rate of development to the blastocyst stage. Additionally, we examined potential ASIP signaling mechanisms through which ASIP may function to establish oocyte developmental competence. The expression of melanocortin receptor 3 and 4 and the coreceptor attractin was detected in the oocyte and follicular cells. The addition of cortisol during in vitro maturation was found to increase significantly oocyte ASIP levels. In conclusion, these results suggest a functional role for ASIP in promoting oocyte maturation and subsequent embryonic development, potentially through signaling mechanisms involving cortisol.

Lysine-specific demethylase 1 (LSD1) participate in porcine early embryonic development by regulating cell autophagy and apoptosis through the mTOR signaling pathway.

Lysine-specific demethylase 1 (LSD1) stands as the pioneering histone demethylase uncovered, proficient in demethylating H3K4me1/2 and H3K9me1/2, thereby governing transcription and participating in cell apoptosis, proliferation, or differentiation. Nevertheless, the complete understanding of LSD1 during porcine early embryonic development and the underlying molecular mechanism remains unclear. Thus, we investigated the mechanism by which LSD1 plays a regulatory role in porcine early embryos. This study revealed that LSD1 inhibition resulted in parthenogenetic activation (PA) and in vitro fertilization (IVF) embryo arrested the development, and decreased blastocyst quality. Meanwhile, H3K4me1/2 and H3K9me1/2 methylase activity was increased at the 4-cell embryo stage. RNA-seq results revealed that autophagy related biological processes were highly enriched through GO and KEGG pathway analyses when LSD1 inhibition. Further studies showed that LSD1 depletion in porcine early embryos resulted in low mTOR and p-mTOR levels and high autophagy and apoptosis levels. The LSD1 deletion-induced increases in autophagy and apoptosis could be reversed by addition of mTOR activators. We further demonstrated that LSD1 inhibition induced mitochondrial dysfunction and mitophagy. In summary, our research results indicate that LSD1 may regulate autophagy and apoptosis through the mTOR pathway and affect early embryonic development of pigs.

Early embryonic polarity establishment and implications for lineage differentiation.

Polarity establishment is one of the key factors affecting early embryonic development. Polarity establishment begins with myosin phosphorylation in the 8-cell embryo, and phosphorylation activates actin leading to its initiation of contractility. Subsequently, actin undergoes reorganization to form an apical domain rich in microvilli on the non-contacting surface of each blastomere, and form the actomyosin ring that marks the maturation of the apical domain in conjunction with polar protein complexes and others. From the process of polarity establishment, it can be seen that the formation of the apical domain is influenced by actin-related proteins and polar protein complexes. Some zygote genome activation (ZGA) and lineage-specific genes also regulate polarity establishment. Polarity establishment underlies the first cell lineage differentiation during early embryonic development. It regulates lineage segregation and morphogenesis by affecting asymmetric cell division, asymmetric localization of lineage differentiation factors, and activity of the Hippo signaling pathway. In this review, we systematically summarize the mechanisms of early embryonic polarity establishment and its impact on lineage differentiation in mammals, and discuss the shortcomings of the currently available studies in terms of regulatory mechanisms and species, thereby providing clues and systematic perspectives for elucidating early embryonic polarity establishment.

A Mettl16/m6A/mybl2b/Igf2bp1 axis ensures cell cycle progression of embryonic hematopoietic stem and progenitor cells.

Prenatal lethality associated with mouse knockout of Mettl16, a recently identified RNA N6-methyladenosine (m6A) methyltransferase, has hampered characterization of the essential role of METTL16-mediated RNA m6A modification in early embryonic development. Here, using cross-species single-cell RNA sequencing analysis, we found that during early embryonic development, METTL16 is more highly expressed in vertebrate hematopoietic stem and progenitor cells (HSPCs) than other methyltransferases. In Mettl16-deficient zebrafish, proliferation capacity of embryonic HSPCs is compromised due to G1/S cell cycle arrest, an effect whose rescue requires Mettl16 with intact methyltransferase activity. We further identify the cell-cycle transcription factor mybl2b as a directly regulated by Mettl16-mediated m6A modification. Mettl16 deficiency resulted in the destabilization of mybl2b mRNA, likely due to lost binding by the m6A reader Igf2bp1 in vivo. Moreover, we found that the METTL16-m6A-MYBL2-IGF2BP1 axis controlling G1/S progression is conserved in humans. Collectively, our findings elucidate the critical function of METTL16-mediated m6A modification in HSPC cell cycle progression during early embryonic development.

Unveiling Gene Expression Dynamics during Early Embryogenesis in Cynoglossus semilaevis: A Transcriptomic Perspective.

Commencing with sperm-egg fusion, the early stages of metazoan development include the cleavage and formation of blastula and gastrula. These early embryonic events play a crucial role in ontogeny and are accompanied by a dramatic remodeling of the gene network, particularly encompassing the maternal-to-zygotic transition. Nonetheless, the gene expression dynamics governing early embryogenesis remain unclear in most metazoan lineages. We conducted transcriptomic profiling on two types of gametes (oocytes and sperms) and early embryos (ranging from the four-cell to the gastrula stage) of an economically valuable flatfish-the Chinese tongue sole Cynoglossus semilaevis (Pleuronectiformes: Cynoglossidae). Comparative transcriptome analysis revealed that large-scale zygotic genome activation (ZGA) occurs in the blastula stage, aligning with previous findings in zebrafish. Through the comparison of the most abundant transcripts identified in each sample and the functional analysis of co-expression modules, we unveiled distinct functional enrichments across different gametes/developmental stages: actin- and immune-related functions in sperms; mitosis, transcription inhibition, and mitochondrial function in oocytes and in pre-ZGA embryos (four- to 1000-cell stage); and organ development in post-ZGA embryos (blastula and gastrula). These results provide insights into the intricate transcriptional regulation of early embryonic development in Cynoglossidae fish and expand our knowledge of developmental constraints in vertebrates.

Bta-miR-301a targets ACVR1 to influence cleavage time and blastocyst formation rate of early embryos in cattle.

Accumulating evidence indicates that paternally-derived miRNAs play a crucial role in the development of early embryos and are regarded as the key factor in the successful development of somatic cell cloned embryos. In our previous study, bta-miR-301a was found to be highly expressed in bovine sperm, and was delivered into oocytes during fertilization. In this study, bioinformatics, dual luciferase reporter assays, rescue experiments and gain- and loss-of-function experiments indicated that ACVR1 is the target gene of bta-miR-301a in early bovine embryos. By microinjecting bta-miR-301a mimic into embryos of parthenogenetic or somatic cell nuclear transfer, we observed that bta-miR-301a prolonged the first cleavage time of the embryos and increased the blastocyst formation rate. Thus, this study provides preliminary evidence that bta-miR-301a influences remodeling of the microfilament skeleton, prolongs the first cleavage time, and improves the developmental competence of embryos by negatively regulating ACVR1 translation.

Maternal KLF17 controls zygotic genome activation by acting as a messenger for RNA Pol II recruitment in mouse embryos.

Initiation of timely and sufficient zygotic genome activation (ZGA) is crucial for the beginning of life, yet our knowledge of transcription factors (TFs) contributing to ZGA remains limited. Here, we screened the proteome of early mouse embryos after cycloheximide (CHX) treatment and identified maternally derived KLF17 as a potential TF for ZGA genes. Using a conditional knockout (cKO) mouse model, we further investigated the role of maternal KLF17 and found that it promotes embryonic development and full fertility. Mechanistically, KLF17 preferentially binds to promoters and recruits RNA polymerase II (RNA Pol II) in early 2-cell embryos, facilitating the expression of major ZGA genes. Maternal Klf17 knockout resulted in a downregulation of 9% of ZGA genes and aberrant RNA Pol II pre-configuration, which could be partially rescued by introducing exogenous KLF17. Overall, our study provides a strategy for screening essential ZGA factors and identifies KLF17 as a crucial TF in this process.

Transition from totipotency to pluripotency in mice: insights into molecular mechanisms.

Totipotency is the ability of a single cell to develop into a full organism and, in mammals, is strictly associated with the early stages of development following fertilization. This unlimited developmental potential becomes quickly restricted as embryonic cells transition into a pluripotent state. The loss of totipotency seems a consequence of the zygotic genome activation (ZGA), a process that determines the switch from maternal to embryonic transcription, which in mice takes place following the first cleavage. ZGA confers to the totipotent cell a transient transcriptional profile characterized by the expression of stage-specific genes and a set of transposable elements that prepares the embryo for subsequent development. The timely silencing of this transcriptional program during the exit from totipotency is required to ensure proper development. Importantly, the molecular mechanisms regulating the transition from totipotency to pluripotency have remained elusive due to the scarcity of embryonic material. However, the development of new in vitro totipotent-like models together with advances in low-input genome-wide technologies, are providing a better mechanistic understanding of how this important transition is achieved. This review summarizes the current knowledge on the molecular determinants that regulate the exit from totipotency.

High-throughput sequencing reveals hub genes for human early embryonic development arrest in vitro fertilization: a pilot study.

Many clinical studies have shown that embryos of in vitro fertilization (IVF) are often prone to developmental arrest, which leads to recurrent failure of IVF treatment. Early embryonic arrest has always been an urgent clinical problem in assisted reproduction centers. However, the molecular mechanisms underlying early embryonic development arrest remain largely unknown. The objective of this study is to investigate potential candidate hub genes and key signaling pathways involved in early stages of embryonic development. RNA-seq analysis was performed on normal and arrest embryos to study the changes of gene expression during early embryonic development. A total of 520 genes exhibiting differential expression were identified, with 174 genes being upregulated and 346 genes being downregulated. Upregulated genes show enrichment in biosynthesis, cellular proliferation and differentiation, and epigenetic regulation. While downregulated genes exhibit enrichment in transcriptional activity, epigenetic regulation, cell cycle progression, cellular proliferation and ubiquitination. The STRING (search tool for the retravel of interacting genes/proteins) database was utilized to analyze protein-protein interactions among these genes, aiming to enhance comprehension of the potential role of these differentially expressed genes (DEGs). A total of 22 hub genes (highly connected genes) were identified among the DEGs using Cytoscape software. Of these, ERBB2 and VEGFA were upregulated, while the remaining 20 genes (CCNB1, CCNA2, DICER1, NOTCH1, UBE2B, UBE2N, PRMT5, UBE2D1, MAPK3, SOX9, UBE2C, UB2D2, EGF, ACTB, UBA52, SHH, KRAS, UBE2E1, ADAM17 and BRCA2) were downregulated. These hub genes are associated with crucial biological processes such as ubiquitination, cellular senescence, cell proliferation and differentiation, and cell cycle. Among these hub genes, CCNA2 and CCNB1 may be involved in controlling cell cycle, which are critical process in early embryonic development.

NLRP14 Safeguards Calcium Homeostasis via Regulating the K27 Ubiquitination of Nclx in Oocyte-to-Embryo Transition.

Sperm-induced Ca2+ rise is critical for driving oocyte activation and subsequent embryonic development, but little is known about how lasting Ca2+ oscillations are regulated. Here it is shown that NLRP14, a maternal effect factor, is essential for keeping Ca2+ oscillations and early embryonic development. Few embryos lacking maternal NLRP14 can develop beyond the 2-cell stage. The impaired developmental potential of Nlrp14-deficient oocytes is mainly caused by disrupted cytoplasmic function and calcium homeostasis due to altered mitochondrial distribution, morphology, and activity since the calcium oscillations and development of Nlrp14-deficient oocytes can be rescued by substitution of whole cytoplasm by spindle transfer. Proteomics analysis reveal that cytoplasmic UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is significantly decreased in Nlrp14-deficient oocytes, and Uhrf1-deficient oocytes also show disrupted calcium homeostasis and developmental arrest. Strikingly, it is found that the mitochondrial Na+ /Ca2+ exchanger (NCLX) encoded by Slc8b1 is significantly decreased in the Nlrp14mNull oocyte. Mechanistically, NLRP14 interacts with the NCLX intrinsically disordered regions (IDRs) domain and maintain its stability by regulating the K27-linked ubiquitination. Thus, the study reveals NLRP14 as a crucial player in calcium homeostasis that is important for early embryonic development.

Tris(1,3-dichloro-2-propyl) Phosphate Inhibits Early Embryonic Development by Binding to Gsk-3ß Protein in Zebrafish.

Recently, several studies have reported that exposure to tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) results in abnormal development of zebrafish embryos in blastocyst and gastrula stages, but molecular mechanisms are still not clear. This lacking strongly affects the interspecific extrapolation of embryonic toxicity induced by TDCIPP and hazard evaluation. In this study, zebrafish embryos were exposed to 100, 500 or 1000 µg/L TDCIPP, and 6-bromoindirubin-3'-oxime (BIO, 35.62 µg/L) was used as a positive control. Results demonstrated that treatment with TDCIPP or BIO caused an abnormal stacking of blastomere cells in mid blastula transition (MBT) stage, and subsequently resulted in epiboly delay of zebrafish embryos. TDCIPP and BIO up-regulated the expression of ß-catenin protein and increased its accumulation in nuclei of embryonic cells. This accumulation was considered as a driver for early embryonic developmental toxicity of TDCIPP. Furthermore, TDCIPP and BIO partly shared the same modes of action, and both of them could bind to Gsk-3ß protein, and then decreased the phosphorylation level of Gsk-3ß in TYR·216 site and lastly inhibited the activity of Gsk-3ß kinase, which was responsible for the increased concentrations of ß-catenin protein in embryonic cells and accumulation in nuclei. Our findings provide new mechanisms for clarifying the early embryonic developmental toxicity of TDCIPP in zebrafish.

Effect of bovine viral diarrhea virus biotypes exposure on bovine gametes in early embryonic development in vitro.

Bovine viral diarrhea virus (BVDV) is an important viral agent causing reproductive failure in cattle. The objectives of the current study were to investigate the interaction between two BVDV biotypes, cytopathic (CP) and Non-cytopathic (NCP) and bovine gametes during in vitro fertilization (IVF) processing, the existence of the virus within embryonic cells and early embryonic development rates. Sperm and ova were exposed separately to CP and NCP BVDV at two concentrations of 104.5 and 105.5 tissue culture infectious dose 50.00% (TCID50) mL-1 prior to IVF, respectively. After five days post-IVF, early embryonic development rates of infected groups were assessed. Several embryos of each group, normal and degenerated, were selected for a viral assay using reverse transcription polymerase chain reaction technique. The result showed that the early embryonic development rates were decreased in treatment groups. The rates in the CP groups were lower than the NCP groups. In the CP groups, the proportions were, respectively, 10.00, 6.00 and 11.00, and 6.00% in the infected sperm and oocyte groups (104.5 and 105.5 TCID50 mL-1) that were higher than 50.00% in the control group. In NCP groups, the rates were, respectively, 25.00, 18.00 and 24.00, and 21.00% in the infected groups compared to 48.00% in the control group. In the CP groups, no BVDV was detected in normal embryos, whereas, all degenerated embryos were completely virus-positive. In the NCP groups, the virus was detected in both normal and degenerated embryos. In conclusion, this study supported detrimental impacts of CP and NCP BVDV on early embryonic development and the role of sperm and the zona pellucida layer as carriers of the virus.

A paternal protein facilitates sperm RNA delivery to regulate zygotic development.

Sperm contributes essential paternal factors, including the paternal genome, centrosome, and oocyte-activation signals, to sexual reproduction. However, it remains unresolved how sperm contributes its RNA molecules to regulate early embryonic development. Here, we show that the Caenorhabditis elegans paternal protein SPE-11 assembles into granules during meiotic divisions of spermatogenesis and later matures into a perinuclear structure where sperm RNAs localize. We reconstitute an SPE-11 liquid-phase scaffold in vitro and find that SPE-11 condensates incorporate the nematode RNA, which, in turn, promotes SPE-11 phase separation. Loss of SPE-11 does not affect sperm motility or fertilization but causes pleiotropic development defects in early embryos, and spe-11 mutant males reduce mRNA levels of genes crucial for an oocyte-to-embryo transition or embryonic development. These results reveal that SPE-11 undergoes phase separation and associates with sperm RNAs that are delivered to oocytes during fertilization, providing insights into how a paternal protein regulates early embryonic development.

Neurulation of the cynomolgus monkey embryo achieved from 3D blastocyst culture.

Neural tube (NT) defects arise from abnormal neurulation and result in the most common birth defects worldwide. Yet, mechanisms of primate neurulation remain largely unknown due to prohibitions on human embryo research and limitations of available model systems. Here, we establish a three-dimensional (3D) prolonged in vitro culture (pIVC) system supporting cynomolgus monkey embryo development from 7 to 25 days post-fertilization. Through single-cell multi-omics analyses, we demonstrate that pIVC embryos form three germ layers, including primordial germ cells, and establish proper DNA methylation and chromatin accessibility through advanced gastrulation stages. In addition, pIVC embryo immunofluorescence confirms neural crest formation, NT closure, and neural progenitor regionalization. Finally, we demonstrate that the transcriptional profiles and morphogenetics of pIVC embryos resemble key features of similarly staged in vivo cynomolgus and human embryos. This work therefore describes a system to study non-human primate embryogenesis through advanced gastrulation and early neurulation.

Assessment of Escherichia coli-derived Recombinant Human Bone Morphogenic Protein-2 on Fertilization and Early Embryonic Development in Rats.

OBJECTIVE: Bone morphogenetic protein-2 (BMP-2) impacts fertility in women by affecting the menstrual cycle and embryonic development. We aimed to determine the reproductive toxicity of Escherichia coli (E. coli)-derived recombinant human BMP-2 (rhBMP-2) by measuring changes in the reproductive performance and organs in rhBMP-2-treated rats. METHODS: Overall, 88 male and female rats each were categorized into one control and three experimental groups. rhBMP-2 was intravenously administered to the experimental groups at 0.05, 0.15, and 0.50 mg/kg/day, respectively. The male rats were administered rhBMP-2 daily, starting from 28 days before mating until the day of necropsy (48 days), after which they were euthanized and necropsied. The female rats were administered rhBMP-2 daily, starting from 14 days before mating until 7 days after fertilization (22-36 days), after which they were necropsied 13 days after fertilization. RESULTS: No rhBMP-2-related death occurred throughout the study period. All rhBMP-2-treated groups showed swelling in the tail at the site of rhBMP-2 administration. In the high-dose rhBMP-2 group, the male rats showed a slight reduction in body weight and food consumption, whereas the female rats showed a reduction in the weights of the ovary and oviduct. Examining the fertilization status and necropsy showed no effect of rhBMP-2 on fertility and early embryonic development. The no-observed-adverse-effect level of rhBMP-2 was 0.50 mg/kg/day in all rats. CONCLUSION: rhBMP-2 had no reproductive toxicity on the reproductive performance and organs in female and male rats. Therefore, these results provide new toxicology information on E. coli-derived rhBMP-2 as a therapeutic protein.