Spatial molecular anatomy of germ layers in the gastrulating cynomolgus monkey embryo.

During mammalian embryogenesis, spatial regulation of gene expression and cell signaling are functionally coupled with lineage specification, patterning of tissue progenitors, and germ layer morphogenesis. While the mouse model has been instrumental for understanding mammalian development, comparatively little is known about human and non-human primate gastrulation due to the restriction of both technical and ethical issues. Here, we present a spatial and temporal survey of the molecular dynamics of cell types populating the non-human primate embryos during gastrulation.

3D Genome of macaque fetal brain reveals evolutionary innovations during primate corticogenesis.

Elucidating the regulatory mechanisms of human brain evolution is essential to understanding human cognition and mental disorders. We generated multi-omics profiles and constructed a high-resolution map of 3D genome architecture of rhesus macaque during corticogenesis. By comparing the 3D genomes of human, macaque, and mouse brains, we identified many human-specific chromatin structure changes, including 499 topologically associating domains (TADs) and 1,266 chromatin loops.

Trio deep-sequencing does not reveal unexpected off-target and on-target mutations in Cas9-edited rhesus monkeys.

CRISPR-Cas9 is a widely-used genome editing tool, but its off-target effect and on-target complex mutations remain a concern, especially in view of future clinical applications. Non-human primates (NHPs) share close genetic and physiological similarities with humans, making them an ideal preclinical model for developing Cas9-based therapies. However, to our knowledge no comprehensive in vivo off-target and on-target assessment has been conducted in NHPs.

In vitro culture of cynomolgus monkey embryos beyond early gastrulation.

Gastrulation is a key event in embryonic development when the germ layers are specified and the basic animal body plan is established. The complexities of primate gastrulation remain a mystery because of the difficulties in accessing primate embryos at this stage. Here, we report the establishment of an in vitro culture (IVC) system that supports the continuous development of cynomolgus monkey blastocysts beyond early gastrulation up to 20 days after fertilization.

Single-cell RNA-sequencing reveals the existence of naive and primed pluripotency in pre-implantation rhesus monkey embryos.

Naive pluripotency exists in epiblast cells of mouse pre-implantation embryos. However, whether the naive pluripotency is transient or nonexistent in primate embryos remains unclear. Using RNA-seq in single blastomeres from 16-cell embryos through to hatched blastocysts of rhesus monkey, we constructed the lineage segregation roadmap in which the specification of trophectoderm, epiblast, and primitive endoderm is initiated simultaneously at the early blastocyst stage.

Transcriptome analyses of rhesus monkey preimplantation embryos reveal a reduced capacity for DNA double-strand break repair in primate oocytes and early embryos.

Preimplantation embryogenesis encompasses several critical events including genome reprogramming, zygotic genome activation (ZGA), and cell-fate commitment. The molecular basis of these processes remains obscure in primates in which there is a high rate of embryo wastage. Thus, understanding the factors involved in genome reprogramming and ZGA might help reproductive success during this susceptible period of early development and generate induced pluripotent stem cells with greater efficiency.

Decrease in expression of maternal effect gene Mater is associated with maternal ageing in mice.

Study Hypothesis: What factors in mouse oocytes are involved in the ageing-related decline in oocyte quality?

Study Finding: The maternal effect gene Mater is involved in ageing-related oocyte quality decline in mice.

What Is Known Already: Premature loss of centromere cohesion is a hallmark of ageing-related oocyte quality decline; the maternal effect gene Mater (maternal antigen that embryos require, also known as Nlrp5) is required for preimplantation embryo development beyond the 2-cell stage, and mRNA expression of Mater decreases with maternal ageing.

Study Design, Samples/materials, Methods: Mater protein expression level in mature oocytes from 7 young (5-8 weeks old) to 7 old mice (41-68 weeks old) was compared by immunoblotting analysis.

Maternal-effect Floped gene is essential for the derivation of embryonic stem cells in mice.

Floped (official name Ooep) is specifically and abundantly expressed in mouse oocytes and embryonic stem cells (ESCs). Depletion of Floped from oocytes leads to early embryonic arrest at the 2-cell stage. Although crucial in cleavage stage development, its roles in early embryos as well as in ESCs remain completely unknown.

The available time window for embryo transfer in the rhesus monkey (Macaca mulatta).

Much effort has been focused on improving assisted reproductive technology procedures in humans and nonhuman primates (NHPs). However, the pregnancy rate after embryo transfer (ET) has not been satisfactory, indicating that some barriers still need to be overcome in this important procedure. One of the key factors is embryo–uterine synchronicity, which is little known in NHPs.

[Role of PTEN in mouse pre-implantation development].

The PI3K/Akt signal transduction pathway plays an important role in pre-implantation embryonic development. The tumor suppressor gene PTEN negatively regulates the PI3K/Akt pathway. Although PI3K is constitutively activated during pre-implantation embryonic development, currently no evidence shows the presence and possible involvement of PTEN in early embryo development.

Derivation of rhesus monkey parthenogenetic embryonic stem cells and its microRNA signature.

Parthenogenetic embryonic stem cells are considered as a promising resource for regeneration medicine and powerful tools for developmental biology. A lot of studies have revealed that embryonic stem cells have distinct microRNA expression pattern and these microRNAs play important roles in self-renewal and pluripotency of embryonic stem cells. However, few studies concern about microRNA expression pattern in parthenogenetic embryonic stem cells, especially in non-human primate--the ideal model species for human, largely due to the limited rhesus monkey parthenogenetic embryonic stem cells (rpESCs) available and lack of systematic analysis of the basics of rpESCs.

MicroRNA profiling of rhesus macaque embryonic stem cells.

Background: MicroRNAs (miRNAs) play important roles in embryonic stem cell (ESC) self-renewal and pluripotency. Numerous studies have revealed human and mouse ESC miRNA profiles. As a model for human-related study, the rhesus macaque is ideal for delineating the regulatory mechanisms of miRNAs in ESCs.

Optimization of ethylene glycol concentrations, freezing rates and holding times in liquid nitrogen vapor for cryopreservation of rhesus macaque (Macaca mulatta) sperm.

Ethylene glycol (EG) has been speculated to be the most appropriate penetrating cryoprotectant for cryopreservation of rhesus macaque sperm due to its higher permeability coefficient. The present study aimed to determine the optimal EG concentration, freezing rate and holding time in liquid nitrogen (LN(2)) vapor for rhesus sperm cryopreservation. Among six tested EG concentrations (0, 0.

Transgenic rhesus monkeys produced by gene transfer into early-cleavage-stage embryos using a simian immunodeficiency virus-based vector.

The development of transgenic technologies in monkeys is important for creating valuable animal models of human physiology so that the etiology of diseases can be studied and potential therapies for their amelioration may be developed. However, the efficiency of producing transgenic primate animals is presently very low, and there are few reports of success. We have developed an improved methodology for the production of transgenic rhesus monkeys, making use of a simian immunodeficiency virus (SIV)-based vector that encodes EGFP and a protocol for infection of early-cleavage-stage embryos.

Directional freezing as an alternative method for cryopreserving rhesus macaque (Macaca mulatta) sperm.

The objective was to develop a freezing protocol using a directional freezing (DF) technique for cryopreservation of rhesus macaque sperm and achieve a survival rate comparable to that achieved with a conventional freezing (CF) technique. Rhesus macaque sperm frozen with a DF technique, with cooling rates of 12 or 16 °C/min, had higher post-thaw motility (P < 0.05) than those cooled at 7 °C/min (59.

Impairments in embryonic genome activation in rhesus monkey somatic cell nuclear transfer embryos.

Somatic cell nuclear transfer (SCNT) is a remarkable process in which a somatic cell nucleus is acted upon by the ooplasm via mechanisms that today remain unknown. Here we show the developmental competence (% blastocyst) of embryos derived from SCNT (21%) was markedly (p < 0.05) impaired compared with those derived from in vitro fertilization (IVF) (42.

A comparison of the protective action of added egg yolks from five avian species to the cryopreservation of bull sperm.

Cryopreservation of domestic animal sperm has been widely used for artificial insemination (AI), and egg yolk is one of the most commonly used cryoprotectants during the freezing-thawing process. The objectives of this study were to compare the effectiveness of egg yolk from five avian species (domestic chicken, domestic duck, domestic goose, Japanese quail or domestic pigeon) and to optimize the concentration of egg yolk on the cryopreservation of bull sperm in terms of frozen-thawed sperm progressive motility and viability. The results were two-fold.

Superovulatory response to a low dose single-daily treatment of rhFSH dissolved in polyvinylpyrrolidone in rhesus monkeys.

To simplify the procedure for superovulation in the rhesus monkey, this study was designed using polyvinylpyrrolidone (PVP) solution as a solvent for gonadotropins. Thirty-five cycling females (aged 5-8 years old) were divided into six groups during the breeding season (November- February). The groups were as follows: Group I, animals received twice-daily 35 IU recombinant human follicle-stimulating hormone (rhFSH) dissolved in 0.

Epigenetic marks in cloned rhesus monkey embryos: comparison with counterparts produced in vitro.

Until now, no primate animals have been successfully cloned to birth with somatic cell nuclear transfer (SCNT) procedures, and little is known about the molecular events that occurred in the reconstructed embryos during preimplantation development. In many SCNT cases, epigenetic reprogramming of the donor nuclei after transfer into enucleated oocytes was hypothesized to be crucial to the reestablishment of embryonic totipotency. In the present study, we focused on two major epigenetic marks, DNA methylation and histone H3 lysine 9 (H3K9) acetylation, which we examined by indirect immunofluorescence and confocal laser scanning microscopy.

Cryopreservation of cynomolgus monkey (Macaca fascicularis) spermatozoa in a chemically defined extender.

Aim: To establish a method for cynomolgus monkey sperm cryopreservation in a chemically defined extender.

Methods: Semen samples were collected by electro-ejaculation from four sexually mature male cynomolgus monkeys. The spermatozoa were frozen in straws by liquid nitrogen vapor using egg-yolk-free Tes-Tris mTTE synthetic extender and glycerol as cryoprotectant.