In vitro fertilization program in white rhinoceros.

In brief: To save endangered rhinoceros species, assisted reproductive technologies are warranted. We here report in vitro blastocyst generation of the Near-Threatened Southern white rhinoceros and, for the first time, also of the technically Extinct Northern white rhinoceros. Abstract: The Anthropocene is marked by a dramatic biodiversity decline, particularly affecting the family Rhinocerotidae. Three of five extant species are listed as Critically Endangered (Sumatran, Javan, black rhinoceros), one as Vulnerable (Indian rhinoceros), and only one white rhino (WR) subspecies, the Southern white rhinoceros (SWR), after more than a century of successful protection is currently classified as Near Threatened by the IUCN, while numbers again are declining. Conversely, in 2008, the SWR's northern counterpart and second WR subspecies, the Northern white rhinoceros (NWR), was declared extinct in the wild. Safeguarding these vanishing keystone species urgently requires new reproductive strategies. We here assess one such strategy, the novel in vitro fertilization program in SWR and - for the first-time NWR - regarding health effects, donor-related, and procedural factors. Over the past 8 years, we performed 65 procedures in 22 white rhinoceros females (20 SWR and 2 NWR) comprising hormonal ovarian stimulation, ovum pick-up (OPU), in vitro oocyte maturation, fertilization, embryo culture, and blastocyst cryopreservation, at an efficiency of 1.0 ± 1.3 blastocysts per OPU, generating 22 NWR, 19 SWR and 10 SWR/NWR hybrid blastocysts for the future generation of live offspring.

In Vitro-Produced Equine Blastocysts Exhibit Greater Dispersal and Intermingling of Inner Cell Mass Cells than In Vivo Embryos.

In vitro production (IVP) of equine embryos is increasingly popular in clinical practice but suffers from higher incidences of early embryonic loss and monozygotic twin development than transfer of in vivo derived (IVD) embryos. Early embryo development is classically characterized by two cell fate decisions: (1) first, trophectoderm (TE) cells differentiate from inner cell mass (ICM); (2) second, the ICM segregates into epiblast (EPI) and primitive endoderm (PE). This study examined the influence of embryo type (IVD versus IVP), developmental stage or speed, and culture environment (in vitro versus in vivo) on the expression of the cell lineage markers, CDX-2 (TE), SOX-2 (EPI) and GATA-6 (PE). The numbers and distribution of cells expressing the three lineage markers were evaluated in day 7 IVD early blastocysts (n = 3) and blastocysts (n = 3), and in IVP embryos first identified as blastocysts after 7 (fast development, n = 5) or 9 (slow development, n = 9) days. Furthermore, day 7 IVP blastocysts were examined after additional culture for 2 days either in vitro (n = 5) or in vivo (after transfer into recipient mares, n = 3). In IVD early blastocysts, SOX-2 positive cells were encircled by GATA-6 positive cells in the ICM, with SOX-2 co-expression in some presumed PE cells. In IVD blastocysts, SOX-2 expression was exclusive to the compacted presumptive EPI, while GATA-6 and CDX-2 expression were consistent with PE and TE specification, respectively. In IVP blastocysts, SOX-2 and GATA-6 positive cells were intermingled and relatively dispersed, and co-expression of SOX-2 or GATA-6 was evident in some CDX-2 positive TE cells. IVP blastocysts had lower TE and total cell numbers than IVD blastocysts and displayed larger mean inter-EPI cell distances; these features were more pronounced in slower-developing IVP blastocysts. Transferring IVP blastocysts into recipient mares led to the compaction of SOX-2 positive cells into a presumptive EPI, whereas extended in vitro culture did not. In conclusion, IVP equine embryos have a poorly compacted ICM with intermingled EPI and PE cells; features accentuated in slowly developing embryos but remedied by transfer to a recipient mare.

Induced pluripotent stem cells and cerebral organoids from the critically endangered Sumatran rhinoceros.

Less than 80 Sumatran rhinos (SR, Dicerorhinus sumatrensis) are left on earth. Habitat loss and limited breeding possibilities are the greatest threats to the species and lead to a continuous population decline. To stop the erosion of genetic diversity, reintroduction of genetic material is indispensable. However, as the propagation rate of captive breeding is far too low, innovative technologies have to be developed. Induced pluripotent stem cells (iPSCs) are a powerful tool to fight extinction. They give rise to each cell within the body including gametes and provide a unique modality to preserve genetic material across time. Additionally, they enable studying species-specific developmental processes. Here, we generate iPSCs from the last male Malaysian SR Kertam, who died in 2019, and characterize them comprehensively. Differentiation in cells of the three germ layers and cerebral organoids demonstrate their high quality and great potential for supporting the rescue of this critically endangered species.

Ethical Analysis of the Application of Assisted Reproduction Technologies in Biodiversity Conservation and the Case of White Rhinoceros (Ceratotherium simum) Ovum Pick-Up Procedures.

Originally applied on domestic and lab animals, assisted reproduction technologies (ARTs) have also found application in conservation breeding programs, where they can make the genetic management of populations more efficient, and increase the number of individuals per generation. However, their application in wildlife conservation opens up new ethical scenarios that have not yet been fully explored. This study presents a frame for the ethical analysis of the application of ART procedures in conservation based on the Ethical Matrix (EM), and discusses a specific case study-ovum pick-up (OPU) procedures performed in the current conservation efforts for the northern white rhinoceros (Ceratotherium simum cottoni)-providing a template for the assessment of ART procedures in projects involving other endangered species.

Naïve-like pluripotency to pave the way for saving the northern white rhinoceros from extinction.

The northern white rhinoceros (NWR) is probably the earth's most endangered mammal. To rescue the functionally extinct species, we aim to employ induced pluripotent stem cells (iPSCs) to generate gametes and subsequently embryos in vitro. To elucidate the regulation of pluripotency and differentiation of NWR PSCs, we generated iPSCs from a deceased NWR female using episomal reprogramming, and observed surprising similarities to human PSCs. NWR iPSCs exhibit a broad differentiation potency into the three germ layers and trophoblast, and acquire a naïve-like state of pluripotency, which is pivotal to differentiate PSCs into primordial germ cells (PGCs). Naïve culturing conditions induced a similar expression profile of pluripotency related genes in NWR iPSCs and human ESCs. Furthermore, naïve-like NWR iPSCs displayed increased expression of naïve and PGC marker genes, and a higher integration propensity into developing mouse embryos. As the conversion process was aided by ectopic BCL2 expression, and we observed integration of reprogramming factors, the NWR iPSCs presented here are unsuitable for gamete production. However, the gained insights into the developmental potential of both primed and naïve-like NWR iPSCs are fundamental for in future PGC-specification in order to rescue the species from extinction using cryopreserved somatic cells.

The ART of bringing extinction to a freeze - History and future of species conservation, exemplified by rhinos.

The ongoing mass extinction of animal species at an unprecedented rate is largely caused by human activities. Progressive habitat destruction and fragmentation is resulting in accelerated loss of biodiversity on a global scale. Over decades, captive breeding programs of non-domestic species were characterized by efforts to optimize species-specific husbandry, to increase studbook-based animal exchange, and to improve enclosure designs. To counter the ongoing dramatic loss of biodiversity, new approaches are warranted. Recently, new ideas, particularly the application of assisted reproduction technologies (ART), have been incorporated into classical zoo breeding programs. These technologies include semen and oocyte collection, artificial insemination, and in-vitro embryo generation. More futuristic ideas of advanced ART (aART) implement recent advances in biotechnology and stem-cell related approaches such as cloning, inner cell mass transfer (ICM), and the stem-cell-associated techniques (SCAT) for the generation of gametes and ultimately embryos of highly endangered species, such as the northern white rhinoceros (Ceratotherium simum cottoni) of which only two female individuals are left. Both, ART and aART greatly depend on and benefit from the rapidly evolving cryopreservation techniques and biobanking not only of genetic, but also of viable cellular materials suitable for the generation of induced pluripotent stem cells (iPSC). The availability of cryopreserved materials bridges gaps in time and space, thereby optimizing the available genetic variability and enhancing the chance to restore viable populations.

An Ethical Assessment Tool (ETHAS) to Evaluate the Application of Assisted Reproductive Technologies in Mammals' Conservation: The Case of the Northern White Rhinoceros (Ceratotherium simum cottoni).

Assisted reproductive technologies (ARTs) can make a difference in biodiversity conservation. Their application, however, can create risks and raise ethical issues that need addressing. Unfortunately, there is a lack of attention to the topic in the scientific literature and, to our knowledge, there is no tool for the ethical assessment of ARTs in the context of conservation that has been described. This paper reports the first applications of the Ethical Assessment Tool (ETHAS) to trans-rectal ovum pick-up (OPU) and in vitro fertilization (IVF) procedures used in a northern white rhinoceros (Ceratotherium simum cottoni) conservation project. The ETHAS consists of two checklists, the Ethical Evaluation Sheet and the Ethical Risk Assessment, and is specifically customized for each ART procedure. It provides an integrated, multilevel and standardized self-assessment of the procedure under scrutiny, generating an ethical acceptability ranking (totally, partially, not acceptable) and a risk rank (low, medium, high), and, hence, allows for implementing measures to address or manage issues beforehand. The application of the ETHAS to the procedures performed on the northern white rhinoceros was effective in ensuring a high standard of procedures, contributing to the acceptability and improved communication among the project's partners. In turn, the tool itself was also refined through an iterative consultation process between experts and stakeholders.

Laboratory Production of Equine Embryos.

Assisted reproduction technologies (ART) are well developed in humans and cattle and are gaining momentum also in the equine industry because of the fact that the mare does not respond to superovulation but can donate large numbers of oocytes through ovum pick up (OPU). After collection, the oocytes can be fertilized by intracytoplasmic sperm injection (ICSI) using a variety of stallion semen samples, even of poor quality, and the resulting embryos can establish high pregnancy rates after cryopreservation and transfer. The discoveries that equine oocytes can be held at room temperature without loss of viability and that an increase in vitro maturation time can double the number of embryos produced are fueling the uptake of the OPU technique by several clinics that are shipping oocytes of their client's mares to specialized ICSI laboratories for embryo production and freezing. In this article, we present a retrospective analysis of 10 years of work at Avantea with a special focus on the last 3 years. Based on our data, an average production of 1.7 to 2 embryos per OPU-ICSI procedure can be obtained from warmblood donor mares with a pregnancy rate of 70% and a foaling rate in excess of 50%. OPU-ICSI offers the added value of freezing embryos that allows the development of embryo commercialization worldwide to the benefit of top horse breeders who are endorsing this technology as never before.

A prematuration approach to equine IVM: considering cumulus morphology, seasonality, follicle of origin, gap junction coupling and large-scale chromatin configuration in the germinal vesicle.

Several studies report that a two-step culture where mammalian oocytes are first kept under meiosis-arresting conditions (prematuration) followed by IVM is beneficial to embryo development. The most promising results were obtained by stratifying the oocyte population using morphological criteria and allocating them to different culture conditions to best meet their metabolic needs. In this study, horse oocytes were characterised to identify subpopulations that may benefit from prematuration. We investigated gap-junction (GJ) coupling, large-scale chromatin configuration and meiotic competence in compact and expanded cumulus-oocyte complexes (COCs) according to follicle size (<1, 1-2, >2cm) and season. Then we tested the effect of cilostamide-based prematuration in compact COCs collected from follicles <1 and 1-2cm in diameter on embryo development. Meiotic competence was not affected by prematuration, whereas COCs from follicles 1-2cm in diameter yielded embryos with a higher number of cells per blastocyst than oocytes that underwent direct IVM (P<0.01, unpaired Mann-Whitney test), suggesting improved developmental competence. Oocytes collected from follicles <1cm in diameter were not affected by prematuration. This study represents an extensive characterisation of the functional properties of immature horse oocytes and is the first report of the effects of cilostamide-based prematuration in horse oocyte IVM on embryo development.

Mitochondrial DNA replication is initiated at blastocyst formation in equine embryos.

Intracytoplasmic sperm injection is the technique of choice for equine IVF and, in a research setting, 18-36% of injected oocytes develop to blastocysts. However, blastocyst development in clinical programs is lower, presumably due to a combination of variable oocyte quality (e.g. from old mares), suboptimal culture conditions and marginal fertility of some stallions. Furthermore, mitochondrial constitution appears to be critical to developmental competence, and both maternal aging and invitro embryo production (IVEP) negatively affect mitochondrial number and function in murine and bovine embryos. The present study examined the onset of mitochondrial (mt) DNA replication in equine embryos and investigated whether IVEP affects the timing of this important event, or the expression of genes required for mtDNA replication (i.e. mitochondrial transcription factor (TFAM), mtDNA polymerase γ subunit B (mtPOLB) and single-stranded DNA binding protein (SSB)). We also investigated whether developmental arrest was associated with low mtDNA copy number. mtDNA copy number increased (P<0.01) between the early and expanded blastocyst stages both invivo and invitro, whereas the mtDNA:total DNA ratio was higher in invitro-produced embryos (P=0.041). Mitochondrial replication was preceded by an increase in TFAM but, unexpectedly, not mtPOLB or SSB expression. There was no association between embryonic arrest and lower mtDNA copy numbers.

Embryos and embryonic stem cells from the white rhinoceros.

The northern white rhinoceros (NWR, Ceratotherium simum cottoni) is the most endangered mammal in the world with only two females surviving. Here we adapt existing assisted reproduction techniques (ART) to fertilize Southern White Rhinoceros (SWR) oocytes with NWR spermatozoa. We show that rhinoceros oocytes can be repeatedly recovered from live SWR females by transrectal ovum pick-up, matured, fertilized by intracytoplasmic sperm injection and developed to the blastocyst stage in vitro. Next, we generate hybrid rhinoceros embryos in vitro using gametes of NWR and SWR. We also establish embryonic stem cell lines from the SWR blastocysts. Blastocysts are cryopreserved for later embryo transfer. Our results indicate that ART could be a viable strategy to rescue genes from the iconic, almost extinct, northern white rhinoceros and may also have broader impact if applied with similar success to other endangered large mammalian species.

Comparative Analysis of AGE and RAGE Levels in Human Somatic and Embryonic Stem Cells under H2O2-Induced Noncytotoxic Oxidative Stress Conditions.

The accumulation of advanced glycation end products (AGEs) occurs in ageing and in many degenerative diseases as a final outcome of persistent oxidative stress on cells and organs. Environmental alterations taking place during early embryonic development can also lead to oxidative damage, reactive oxygen species (ROS) production, and AGE accumulation. Whether similar mechanisms act on somatic and embryonic stem cells (ESC) exposed to oxidative stress is not known; and therefore, the modelling of oxidative stress in vitro on human ESC has been the focus of this study. We compared changes in N ε -carboxymethyl-lysine (CML) advanced glycation end products and RAGE levels in hESC versus differentiated somatic cells exposed to H2O2 within the noncytotoxic range. Our data revealed that hESC accumulates CML and RAGE under oxidative stress conditions in different ways than somatic cells, being the accumulation of CML statistically significant only in somatic cells and, conversely, the RAGE increase exclusively appreciated in hESC. Then, following cardiac and neural differentiation, we observed a progressive removal of AGEs and at the same time an elevated activity of the 20S proteasome. We conclude that human ESCs constitute a unique model to study the consequence of an oxidative environment in the pluripotent cells of the embryo during the human preimplantation period.

Global gene expression profiling and senescence biomarker analysis of hESC exposed to H2O2 induced non-cytotoxic oxidative stress.

BACKGROUND: Human embryonic stem cells (hESCs) potentially offer new routes to study, on the basis of the Developmental Origins of Health and Disease (DOHaD) concept, how the maternal environment during pregnancy influences the offspring's health and can predispose to chronic disease in later life. Reactive oxygen species (ROS), antioxidant defences and cellular redox status play a key function in gene expression regulation and are involved in diabetes and metabolic syndromes as in ageing. METHODS: We have, therefore, designed an in vitro cell model of oxidative stress by exposing hESCs to hydrogen peroxide (H2O2) during 72 h, in order to resemble the period of preimplantation embryonic development. RESULTS: We have analysed the global gene expression profiles of hESCs (HUES3) exposed to non-cytotoxic H2O2 concentrations, using Illumina microarray HT-12 v4, and we found the differential expression of 569 upregulated and 485 downregulated genes. The most affected gene ontology categories were those related with RNA processing and splicing, oxidation reduction and sterol metabolic processes. We compared our findings with a published RNA-seq profiling dataset of human embryos developed in vitro, thereupon exposed to oxidative stress, and we observed that one of the common downregulated genes between this publication and our data, NEDD1, is involved in centrosome structure and function. CONCLUSIONS: Therefore, we assessed the presence of supernumerary centrosomes and showed that the percentage of cells with more than two centrosomes increased acutely with H2O2 treatment in hESCs (HUES3 and 7) and in a control somatic cell line (Hs27), inducing a premature entry into senescence.

Pyruvate antioxidant roles in human fibroblasts and embryonic stem cells.

Oxidative stress has been related to multiple diseases, especially during early embryonic development, when environmental alterations can lead to long-term deleterious effects. In vitro studies of oxidative stress have been mainly focused on somatic cells, but embryonic stem cells (ESCs) represent a promising model of early embryonic development as they are the in vitro equivalent to pluripotent cells in the embryo. Human fibroblasts and ESCs were exposed to different pro-oxidant agents (hydrogen peroxide, tert-butyl hydroperoxide (TBHP), and rotenone) and antioxidants (sodium pyruvate, N-acetylcysteine, Trolox, and sodium selenite) during a 72 h oxidative stress treatment. Then, cell viability, oxidative stress, mitochondrial activity, and gene expression were analyzed, focusing on the antioxidant effect of pyruvate. Pyruvate protected both somatic and pluripotent cells against different pro-oxidant agents, showing strong ROS scavenging capacity, protecting mitochondrial membrane potential, and regulating gene expression and cell metabolism through different mechanisms in fibroblasts and ESCs. In fibroblasts, pyruvate avoided NFKß nuclear translocation and the upregulation of genes related to the oxidative stress response, while in ESCs pyruvate stimulated the expression of genes involved in anaerobic glycolysis. Fibroblasts and ESCs reacted in different ways to oxidative stress and antioxidant treatment, and pyruvate was the most complete antioxidant, protecting both cell types at different levels.

Maternal age and in vitro culture affect mitochondrial number and function in equine oocytes and embryos.

Advanced maternal age and in vitro embryo production (IVP) predispose to pregnancy loss in horses. We investigated whether mare age and IVP were associated with alterations in mitochondrial (mt) DNA copy number or function that could compromise oocyte and embryo development. Effects of mare age (<12 vs ≥12 years) on mtDNA copy number, ATP content and expression of genes involved in mitochondrial replication (mitochondrial transcription factor (TFAM), mtDNA polymerase γ subunit B (mtPOLB) and mitochondrial single-stranded DNA-binding protein (SSB)), energy production (ATP synthase-coupling factor 6, mitochondrial-like (ATP-synth_F6)) and oxygen free radical scavenging (glutathione peroxidase 3 (GPX3)) were investigated in oocytes before and after in vitro maturation (IVM), and in early embryos. Expression of TFAM, mtPOLB and ATP-synth-F6 declined after IVM (P<0.05). However, maternal age did not affect oocyte ATP content or expression of genes involved in mitochondrial replication or function. Day 7 embryos from mares ≥12 years had fewer mtDNA copies (P=0.01) and lower mtDNA:total DNA ratios (P<0.01) than embryos from younger mares, indicating an effect not simply due to lower cell number. Day 8 IVP embryos had similar mtDNA copy numbers to Day 7 in vivo embryos, but higher mtPOLB (P=0.013) and a tendency to reduced GPX3 expression (P=0.09). The lower mtDNA number in embryos from older mares may compromise development, but could be an effect rather than cause of developmental retardation. The general down-regulation of genes involved in mitochondrial replication and function after IVM may compromise resulting embryos.

Transferability and inter-laboratory variability assessment of the in vitro bovine oocyte fertilization test.

The dramatic increase in the number of animals required for reproductive toxicity testing imposes the validation of alternative methods to reduce the use of laboratory animals. As we previously demonstrated for in vitro maturation test of bovine oocytes, the present study describes the transferability assessment and the inter-laboratory variability of an in vitro test able to identify chemical effects during the process of bovine oocyte fertilization. Eight chemicals with well-known toxic properties (benzo[a]pyrene, busulfan, cadmium chloride, cycloheximide, diethylstilbestrol, ketoconazole, methylacetoacetate, mifepristone/RU-486) were tested in two well-trained laboratories. The statistical analysis demonstrated no differences in the EC50 values for each chemical in within (inter-runs) and in between-laboratory variability of the proposed test. We therefore conclude that the bovine in vitro fertilization test could advance toward the validation process as alternative in vitro method and become part of an integrated testing strategy in order to predict chemical hazards on mammalian fertility.

A comparative transcriptomic study on the effects of valproic acid on two different hESCs lines in a neural teratogenicity test system.

A number of in vitro toxicity assays based on human embryonic stem cells (hESCs) are under development in order to provide alternative methods for the screening of chemicals and drugs and to reduce the number of animals needed for developmental toxicity assessment. The major challenge is to demonstrate the reliability of these in vitro methods by correlating the in vitro produced results to the available in vivo data. In this context transcriptomic approaches associated to toxicogenomic database analysis give the possibility to screen, annotate and cluster high numbers of genes and to identify the molecular changes that univocally mark the toxicity induced processes or are indicative of the early initiating events that lead to cellular toxicity. In this retrospective study we compare microarray transcriptomic data derived from two different hESCs lines (HUES1 and H9) exposed to valproic acid (VA) while applying the same differentiation protocol. We present the results of this comparative analysis in light of the known teratogenic effects of VA. The results show molecular changes in the processes of neural development, neural crest migration, apoptosis and regulation of transcription, indicating a good correspondence with the available in vivo data. We also describe common toxicological signatures and provide an interpretation of the observed qualitative differences referring to known biological features of the two hESCs lines.

Ovum pick up, intracytoplasmic sperm injection and somatic cell nuclear transfer in cattle, buffalo and horses: from the research laboratory to clinical practice.

Assisted reproductive techniques developed for cattle in the last 25 years, like ovum pick up (OPU), intracytoplasmic sperm injection (ICSI), and somatic cell nuclear transfer, have been transferred and adapted to buffalo and horses. The successful clinical applications of these techniques require both the clinical skills specific to each animal species and an experienced laboratory team to support the in vitro phase of the work. In cattle, OPU can be considered a consolidated technology that is rapidly outpacing conventional superovulation for embryo transfer. In buffalo, OPU represents the only possibility for embryo production to advance the implementation of embryo-based biotechnologies in that industry, although it is still mainly in the developmental phase. In the horse, OPU is now an established procedure for breeding from infertile and sporting mares throughout the year. It requires ICSI that in the horse, contrary to what happens in cattle and buffalo, is very efficient and the only option because conventional IVF does not work. Somatic cell nuclear transfer is destined to fill a very small niche for generating animals of extremely high commercial value. The efficiency is low, but because normal animals can be generated it is likely that advancing our knowledge in that field might improve the technology and reduce its cost.

Derivation of neural precursors from bovine preimplantation embryos.

Induction of neural differentiation from embryonic pluripotent stem cells (ES/EpiSc), both of mouse and primates, has been extensively published by several research teams. However, direct derivation of organized neuroectoderm in vitro from blastocyst stage embryos of rodents or primates has not been reported so far. Here we describe a method of direct neural differentiation from the inner cell mass cells of preimplantation bovine embryos, without the intermediate step of ES/EpiSc cells derivation (Lazzari et al., Stem cells 24:2514-2521, 2006). Proliferating neural precursors cells lines, and both central and peripheral nervous system derivatives, can be obtained providing a unique in vitro model of early neurulation events in mammals.

Development of a neural teratogenicity test based on human embryonic stem cells: response to retinoic acid exposure.

The aim of this study was the development of an alternative testing method based on human embryonic stem cells for prenatal developmental toxicity with particular emphasis on early neural development. To this purpose, we designed an in vitro protocol based on the generation of neural rosettes, representing the in vitro counterpart of the developing neural plate and neural tube, and we challenged this complex cell model with retinoic acid (RA), a well-known teratogenic agent. The cells were exposed to different concentrations of RA during the process of rosettes formation. Morphological and molecular parameters were evaluated in treated as compared with untreated cells to detect both cytotoxicity and specific neural toxicity. Transcriptomic analysis was performed with microarray Affymetrix platform and validated by quantitative real-time PCR for genes relevant to early neural development such as HoxA1, HoxA3, HoxB1, HoxB4, FoxA2, FoxC1, Otx2, and Pax7. The results obtained demonstrated that neural rosette forming cells respond to RA with clear concentration-dependent morphological, and gene expression changes remarkably similar to those induced in vivo, in the developing neural tube, by RA exposure. This strict correspondence indicates that the neural rosette protocol described is capable of detecting specific teratogenic mechanisms causing perturbations of early neural development and therefore represents a promising alternative test for human prenatal developmental toxicity.