Mammalian Embryonic Development Research Articles

Homeostatic regulation of lipid droplet content in mammalian oocytes and embryos.

Lipid droplets (LDs) consist of a core of neutral lipids such as triacylglycerols and cholesteryl esters covered by a phospholipid monolayer. Recent studies have shown that LDs not only store neutral lipids but are also associated with various physiological functions. LDs are found in most eukaryotic cells and vary in size and quantity. It has long been known that mammalian oocytes contain LDs. Porcine and bovine oocytes contain substantial amounts of LDs, which cause their cytoplasm to darken, whereas mouse and human oocytes are translucent due to their low LD content. A sufficient amount of LDs in mammalian oocytes has been thought to be associated with oocyte maturation and early embryonic development, but the necessity of LDs has been questioned because embryonic development proceeds normally even when LDs are removed. However, recent studies have revealed that LDs play a crucial role during implantation and that maintaining an appropriate amount of LDs is important for early embryonic development, even in mammalian species with low amounts of LDs in their oocytes. This suggests that a fine-tuned balance of LD content is essential for successful mammalian embryonic development. In this review, we discuss the physiological importance of LDs in mammalian oocytes and preimplantation embryos based on recent findings on LD biology.

Insights into the Evolution of Spermatogenesis-Related Ubiquitin-Proteasome System Genes in Abdominal Testicular Laurasiatherians.

During embryonic development in mammals, the testicles generally descend into the scrotum, making the testicular temperature 2–4 °C lower than the core temperature via heat exchange and clearance, and thus more beneficial for normal spermatogenesis. Failure to descend, known as cryptorchidism, carries a series of risks such as infertility and testicular cancer. However, some mammals have evolved abdominal testes while maintaining healthy reproduction. To explore the underlying molecular mechanism, we conducted comparative genomic analyses and functional assays on the spermatogenesis-related ubiquitin–proteasome system (UPS) genes essential to sperm formation in representative laurasiatherians. Here, positive selection and rapid evolution of spermatogenesis-related UPS genes were identified in the abdominal testicular laurasiatherians. Moreover, potential convergent amino acids were found between distantly related species with similar abdominal testicles and functional analyses showed RNF8 (V437I) in abdominal testicular species (437I) has a stronger ubiquitination ability, which suggests that the mammals with abdominal testes might exhibit enhanced sperm cell histone clearance to maintain sperm formation. This evidence implies that, in response to “cryptorchidism injury”, spermatogenesis-related UPS genes in the abdominal testicular species might have undergone adaptive evolution to stabilize sperm formation. Thus, our study could provide some novel insights into the reproductive adaptation in abdominal testicular mammals.

Cep55 regulation of PI3K/Akt signaling is required for neocortical development and ciliogenesis.

Homozygous nonsense mutations in CEP55 are associated with several congenital malformations that lead to perinatal lethality suggesting that it plays a critical role in regulation of embryonic development. CEP55 has previously been studied as a crucial regulator of cytokinesis, predominantly in transformed cells, and its dysregulation is linked to carcinogenesis. However, its molecular functions during embryonic development in mammals require further investigation. We have generated a Cep55 knockout (Cep55-/-) mouse model which demonstrated preweaning lethality associated with a wide range of neural defects. Focusing our analysis on the neocortex, we show that Cep55-/- embryos exhibited depleted neural stem/progenitor cells in the ventricular zone as a result of significantly increased cellular apoptosis. Mechanistically, we demonstrated that Cep55-loss downregulates the pGsk3β/β-Catenin/Myc axis in an Akt-dependent manner. The elevated apoptosis of neural stem/progenitors was recapitulated using Cep55-deficient human cerebral organoids and we could rescue the phenotype by inhibiting active Gsk3β. Additionally, we show that Cep55-loss leads to a significant reduction of ciliated cells, highlighting a novel role in regulating ciliogenesis. Collectively, our findings demonstrate a critical role of Cep55 during brain development and provide mechanistic insights that may have important implications for genetic syndromes associated with Cep55-loss.

The evolutionary acquisition and mode of functions of promoter-associated non-coding RNAs (pancRNAs) for mammalian development.

Increasing evidence has shown that many long non-coding RNAs (lncRNAs) are involved in gene regulation in a variety of ways such as transcriptional, post-transcriptional and epigenetic regulation. Promoter-associated non-coding RNAs (pancRNAs), which are categorized into the most abundant single-copy lncRNA biotype, play vital regulatory roles in finely tuning cellular specification at the epigenomic level. In short, pancRNAs can directly or indirectly regulate downstream genes to participate in the development of organisms in a cell-specific manner. In this review, we will introduce the evolutionarily acquired characteristics of pancRNAs as determined by comparative epigenomics and elaborate on the research progress on pancRNA-involving processes in mammalian embryonic development, including neural differentiation.

Asymmetric expression of maternal mRNA governs first cell-fate decision.

The goal of preimplantation development is to establish the fates of the embryonic and extra-embryonic cells. However, when and how cell fates are determined during early mammalian embryonic development remains unclear. We report that the high mobility group (HMG) protein family member HMGA1 was distributed differentially in mouse two-cell blastomeres. Knockdown of Hmga1 expression in one of the two cells reduced the number of cells contributing to the inner cell mass (ICM), suggesting that differential distribution of HMGA1 in the blastomeres in two-cell mouse embryos affected the selection of embryonic cell lineages. Mechanistically, HMGA1 promotes the expression of the ICM-specific gene Sox2. The results of this study show that mouse embryos demonstrate heterogeneity as early as the two-cell stage, and that these differences are related to cell-fate differentiation in early mouse embryos.

Challenges on the effect of cell phone radiation on mammalian embryos and fetuses: a review of the literature.

Cell phones operate with a wide range of frequency bands and emit radiofrequency-electromagnetic radiation (RF-EMR). Concern on the possible health hazards of RF-EMR has been growing in many countries because these RF-EMR pulses may be absorbed into the body cells, directly affecting them. There are some in vitro and in vivo animal studies related to the consequences of RF-EMR exposure from cell phones on embryo development and offspring. In addition, some studies have revealed that RF-EMR from cellular phone may lead to decrease in the rates of fertilization and embryo development, as well as the risk of the developmental anomalies, other studies have reported that it does not interfere with in vitro fertilization or intracytoplasmic sperm injection success rates, or the chromosomal aberration rate. Of course, it is unethical to study the effect of waves generated from cell phones on the forming human embryos. Conversely, other mammals have many similarities to humans in terms of anatomy, physiology and genetics. Therefore, in this review we focused on the existing literature evaluating the potential effects of RF-EMR on mammalian embryonic and fetal development.

Generation of a homozygous CRISPR/Cas9-mediated knockout human iPSC line for PTCH1 gene

PTCH1 is the receptor protein of Hedgehog signaling pathway, and Hedgehog pathway plays a vital role in mammalian embryonic development. However, the specific biological role of PTCH1 is incompletely understood for embryonic development. Here, we used a CRISPR/Cas9 genome editing approach to generate a homozygous PTCH1 knock-out iPSC line (SCMCi001-A-1) from a healthy donor, which will be a valuable in vitro model to study the pathogenic mechanism of PTCH1 dysfunction in congenital disease.

P–803 Novel culture conditions for the improvement of the in vitro expansion of human male fetal germ cells

Abstract Study question Do different ECMs/substrates and growth media culture conditions improve in vitro male human primordial germ cell (hPGC) expansion? Summary answer We achieved in vitro expansion improvement of male hPGCs with specific growth factors such as LIF, EGF, FGF2 and GDNF on gelatin- and vitronectin-coating cultures. What is known already PGCs are the precursors of male and female gametes, which are specified during early mammalian post-implantation embryonic development. PGCs undergo sequential cell divisions to differentiate into pro-spermatogonia (pSPG). In vitro propagation of pSPG could be important to understand the transition to spermatogonial stem cells (SSCs), important for fertility preservation in patients with infertility. Here, we aimed at performing a comparative analysis on in vitro feeder-free culture systems, based on different extracellular matrix (ECM) and growth media culture conditions, to support the expansion of the male germline stem cell populations using second trimester human male gonads as primary material. Study design, size, duration We collected human 2nd trimester male fetal gonads from elective abortions. Male gonads were dissected in saline solution (0.9% NaCl) and were either fixed overnight in 4% paraformaldehyde (PFA) for immunohistochemistry or disaggregated by enzymatic digestion for in vitro culture. Participants/materials, setting, methods After differential plating, fetal cells were cultured for 6 days. Disaggregated gonads were cultured with two different growing media (Medium 1 supplemented with LIF, EGF, FGF–2 and GDNF and Medium 2 supplemented with RA, BMP 4 and Activin A) on gelatin, laminin, vitronectin or matrigel coated plates. Cultured cells were immunostained, quantified for the expression of DDX4 and POU5F1 after 3 days (D3) and 6 days (D6) of culture. Main results and the role of chance We pursued to evaluate whether germ cells dissociated from a pool of male fetal gonads could propagate in vitro when cultured for D6 in different conditions. We observed that expansion of POU5F1-positive early PGCs and DDX4-positive late PGCs was only observed when cells were plated on gelatin or vitronectin and cultured with Medium 1, containing the growth factors LIF, EGF, FGF2 and GDNF. However, a reduced percentage of PGCs was observed in all four different coatings when grown with Medium 2, containing RA, BMP4 and Activin A. We analyzed the relative expression of the PGC markers POU5F1, DDX4 and MAGEA4 in histological sections of gonads from embryos at 18.5 weeks of gestation. Two populations of hPGCs were observed: ∼10–30% of the gonadal cells expressed solely DDX4 (late PGCs), whereas less than 10% of gonadal cells expressed POU5F1 (early PGCs). SOX9 and STARD1 expression was evaluated, confirming the presence of Sertoli cells and Leydig cells, respectively. Limitations, reasons for caution Due to the limited and difficulty to obtain human fetal tissue, a limited number of samples were used. Wider implications of the findings: We expanded human male fetal germ cells in vitro for D6 on gelatin and vitronectin coated plates with Medium 1, containing growth factors LIF, EGF, FGF2 and GDNF. Our findings provide a 2D culture system to expand hPGCs that could be useful to study propagation to pSPGs and eventually SSCs. Trial registration number Not applicable

Structural and biophysical characterization of the selenoprotein SelW1 from Chlamydomonas reinhardtii

Selenoprotein W is widespread among pro- and eukaryotic organisms. It possesses antioxidant activity and plays pivotal roles in mammalian embryonic development and cellular functions. A very simple, prototypical selenoprotein W is SelW1 from Chlamydomonas.The U14C mutant of SelW1 was isolated and biophysically characterized. It contains an intramolecular disulfide bond and is thermally stable up to 70 °C. NMR resonance assignment of reduced and oxidized SelW1 showed that SelW1 adopts a thioredoxin fold. Interestingly, both forms show two additional sets of resonance for amino acid residues near the termini and have basically identical dynamic behavior.Since SelW1 from Chlamydomonas resembles the ancestor of mammalian selenoproteins in certain aspects, this study lays the basis for future characterization of SelW1 function and possible interaction partners.

Development in a Dish-In Vitro Models of Mammalian Embryonic Development.

Despite decades of research, the complex processes of embryonic development are not fully understood. The study of mammalian development poses particular challenges such as low numbers of embryos, difficulties in culturing embryos in vitro, and the time to generate mutant lines. With new approaches we can now address questions that had to remain unanswered in the past. One big contribution to studying the molecular mechanisms of development are two- and three-dimensional in vitro model systems derived from pluripotent stem cells. These models, such as blastoids, gastruloids, and organoids, enable high-throughput screens and straightforward gene editing for functional testing without the need to generate mutant model organisms. Furthermore, their use reduces the number of animals needed for research and allows the study of human development. Here, we outline and discuss recent advances in such in vitro model systems to investigate pre-implantation and post-implantation development.

TRIM71 Deficiency Causes Germ Cell Loss During Mouse Embryogenesis and Is Associated With Human Male Infertility.

Mutations affecting the germline can result in infertility or the generation of germ cell tumors (GCT), highlighting the need to identify and characterize the genes controlling germ cell development. The RNA-binding protein and E3 ubiquitin ligase TRIM71 is essential for embryogenesis, and its expression has been reported in GCT and adult mouse testes. To investigate the role of TRIM71 in mammalian germ cell embryonic development, we generated a germline-specific conditional Trim71 knockout mouse (cKO) using the early primordial germ cell (PGC) marker Nanos3 as a Cre-recombinase driver. cKO mice are infertile, with male mice displaying a Sertoli cell-only (SCO) phenotype which in humans is defined as a specific subtype of non-obstructive azoospermia characterized by the absence of germ cells in the seminiferous tubules. Infertility in male Trim71 cKO mice originates during embryogenesis, as the SCO phenotype was already apparent in neonatal mice. The in vitro differentiation of mouse embryonic stem cells (ESCs) into PGC-like cells (PGCLCs) revealed reduced numbers of PGCLCs in Trim71-deficient cells. Furthermore, TCam-2 cells, a human GCT-derived seminoma cell line which was used as an in vitro model for PGCs, showed proliferation defects upon TRIM71 knockdown. Additionally, in vitro growth competition assays, as well as proliferation assays with wild type and CRISPR/Cas9-generated TRIM71 mutant NCCIT cells showed that TRIM71 also promotes proliferation in this malignant GCT-derived non-seminoma cell line. Importantly, the PGC-specific markers BLIMP1 and NANOS3 were consistently downregulated in Trim71 KO PGCLCs, TRIM71 knockdown TCam-2 cells and TRIM71 mutant NCCIT cells. These data collectively support a role for TRIM71 in PGC development. Last, via exome sequencing analysis, we identified several TRIM71 variants in a cohort of infertile men, including a loss-of-function variant in a patient with an SCO phenotype. Altogether, our work reveals for the first time an association of TRIM71 deficiency with human male infertility, and uncovers further developmental roles for TRIM71 in the germline during mouse embryogenesis.

A case for revisiting Nodal signaling in human pluripotent stem cells.

Nodal is a transforming growth factor-β (TGF-β) superfamily member that plays a number of critical roles in mammalian embryonic development. Nodal is essential for the support of the peri-implantation epiblast in the mouse embryo and subsequently acts to specify mesendodermal fate at the time of gastrulation and, later, left-right asymmetry. Maintenance of human pluripotent stem cells (hPSCs) in vitro is dependent on Nodal signaling. Because it has proven difficult to prepare a biologically active form of recombinant Nodal protein, Activin or TGFB1 are widely used as surrogates for NODAL in hPSC culture. Nonetheless, the expression of the components of an endogenous Nodal signaling pathway in hPSC provides a potential autocrine pathway for the regulation of self-renewal in this system. Here we review recent studies that have clarified the role of Nodal signaling in pluripotent stem cell populations, highlighted spatial restrictions on Nodal signaling, and shown that Nodal functions in vivo as a heterodimer with GDF3, another TGF-β superfamily member expressed by hPSC. We discuss the role of this pathway in the maintenance of the epiblast and hPSC in light of these new advances.

Pluripotency and Growth Factors in Early Embryonic Development of Mammals: A Comparative Approach.

The regulation of early events in mammalian embryonic development is a complex process. In the early stages, pluripotency, cellular differentiation, and growth should occur at specific times and these events are regulated by different genes that are expressed at specific times and locations. The genes related to pluripotency and cellular differentiation, and growth factors that determine successful embryonic development are different (or differentially expressed) among mammalian species. Some genes are fundamental for controlling pluripotency in some species but less fundamental in others, for example, Oct4 is particularly relevant in bovine early embryonic development, whereas Oct4 inhibition does not affect ovine early embryonic development. In addition, some mechanisms that regulate cellular differentiation do not seem to be clear or evolutionarily conserved. After cellular differentiation, growth factors are relevant in early development, and their effects also differ among species, for example, insulin-like growth factor improves the blastocyst development rate in some species but does not have the same effect in mice. Some growth factors influence genes related to pluripotency, and therefore, their role in early embryo development is not limited to cell growth but could also involve the earliest stages of development. In this review, we summarize the differences among mammalian species regarding the regulation of pluripotency, cellular differentiation, and growth factors in the early stages of embryonic development.

Self-organized signaling in stem cell models of embryos.

Mammalian embryonic development is a complex process driven by self-organization. Understanding how a fertilized egg develops into an embryo composed of more than 200 cell types in precise spatial patterns remains one of the fundamental challenges in biology. Pluripotent stem cells have been used as in vitro models for investigating mammalian development, and represent promising building blocks for regenerative therapies. Recently, sophisticated stem cell-based models that recapitulate early embryonic fate patterning and morphogenesis have been developed. In this article, we review recent advances in stem cell models of embryos in particular focusing on signaling activities underpinning cell fate decisions in space and time.

Limited proteolysis by acrosin affects sperm-binding and mechanical resilience of the mouse zona pellucida

The encounter of oocyte and sperm is the key event initiating embryonic development in mammals. Crucial functions of this existential interaction are determined by proteolytic enzymes, such as acrosin, carried in the sperm head acrosome, and ovastacin, stored in the oocyte cortical granules. Ovastacin is released upon fertilisation to cleave the zona pellucida, a glycoprotein matrix surrounding the oocyte. This limited proteolysis hardens the oocyte envelope, and thereby provides a definitive block against polyspermy and protects the developing embryo. On the other hand, acrosin, the renowned and most abundant acrosomal protease, has been thought to enable sperm to penetrate the oocyte envelope. Depending on the species, proteolytic cleavage of the zona pellucida by acrosin is either essential or conducive for fertilisation. However, the specific target cleavage sites and the resulting physiological consequences of this proteolysis remained obscure. Here, we treated native mouse zonae pellucidae with active acrosin and identified two cleavage sites in zona pellucida protein 1 (ZP1), five in ZP2 and one in ZP3 by mass spectrometry. Several of these sites are highly conserved in mammals. Remarkably, limited proteolysis by acrosin leads to zona pellucida remodelling rather than degradation. Thus, acrosin affects both sperm binding and mechanical resilience of the zona pellucida, as assessed by microscopy and nanoindentation measurements, respectively. Furthermore, we ascertained potential regulatory effects of acrosin, via activation of latent pro-ovastacin and inactivation of fetuin-B, a tight binding inhibitor of ovastacin. These results offer novel insights into the complex proteolytic network modifying the extracellular matrix of the mouse oocyte, which might apply also to other species.

Centrosomal protein FOR20 knockout mice display embryonic lethality and left-right patterning defects.

Centrosomal protein FOR20 has been reported to be crucial for essential cellular processes, including ciliogenesis, cell migration, and cell cycle in vertebrates. However, the function of FOR20 during mammalian embryonic development remains unknown. To investigate the invivo function of the For20 gene in mammals, we generated For20 homozygous knockout mice by gene targeting. Our data reveal that homozygous knockout of For20 results in significant embryonic growth arrest and lethality during gestation, while the heterozygotes show no obvious defects. The absence of For20 leads to impaired left-right patterning of embryos and reduced cilia in the embryonic node. Deletion of For20 also disrupts angiogenesis in yolk sacs and embryos. These results highlight a critical role of For20 in early mammalian embryogenesis.

Humanization of a tandem repeat in IG-DMR causes stochastic restoration of paternal imprinting at mouse Dlk1-Dio3 domain.

The Dlk1-Dio3 imprinted domain, regulated by an intergenic differentially methylated region (IG-DMR), is important for mammalian embryonic development. Although previous studies have reported that DNA methylation of a tandem repeated array sequence in paternal IG-DMR (IG-DMR-Rep) plays an essential role in the maintenance of DNA methylation in mice, the function of a tandem repeated array sequence in human IG-DMR (hRep) is unknown. Here, we generated mice with a human tandem repeated sequence, which replaced the mouse IG-DMR-Rep. Mice that transmitted the humanized allele paternally exhibited variable methylation status at the IG-DMR and were stochastically rescued from the lethality of IG-DMR-Rep deficiency, suggesting that hRep plays a role in human IG-DMR for the regulation of imprinted expression. Moreover, chromatin immunoprecipitation analysis showed that TRIM28 was enriched in hypermethylated paternal hRep without ZFP57. Our results suggest that hRep contributes to the maintenance of human IG-DMR methylation imprints via the recruitment of TRIM28.

FoxL1+ mesenchymal cells are a critical source of Wnt5a for midgut elongation during mouse embryonic intestinal development.

Wnt5a is a non-canonical Wnt ligand that is essential for normal embryonic development in mammals. The role of Wnt5a in early intestinal development has been examined in gene ablation models, where Wnt5a-/- mice exhibit strikingly shortened intestines. However, the exact cellular source of Wnt5a has remained elusive, until a recent study found that FoxL1-expressing mesenchymal cells (FoxL1+ cells), which are localized directly beneath the intestinal epithelium, express Wnt5a. To determine whether FoxL1+ cells are a required source of Wnt5a during intestinal development, we derived FoxL1-Cre; Wnt5af/f mice, which is the first mouse model to ablate Wnt5a in a cell type-specific manner in the intestine in vivo. Our results show that Wnt5a deletion in FoxL1+ cells during fetal life causes a shortened gut phenotype in neonatal mice, and that our model is sufficient to increase rate of apoptosis in the elongating epithelium, thus explaining the shortened gut phenotype. However, in contrast to previous studies using Wnt5a null mice, we did not observe dysregulation of epithelial structure or apical-basal protein localization. Altogether, our findings establish a developmental role for FoxL1+ mesenchymal cells in controlling non-canonical Wnt signaling during midgut elongation.

Heightened activation of embryonic megakaryocytes causes aneurysms in the developing brain of mice lacking podoplanin

AbstractDuring early embryonic development in mammals, including humans and mice, megakaryocytes (Mks) first originate from primitive hematopoiesis in the yolk sac. These embryonic Mks (eMks) circulate in the vasculature with unclear function. Herein, we report that podoplanin (PDPN), the ligand of C-type lectin-like receptor (CLEC-2) on Mks/platelets, is temporarily expressed in neural tissue during midgestation in mice. Loss of PDPN or CLEC-2 resulted in aneurysms and spontaneous hemorrhage, specifically in the lower diencephalon during midgestation. Surprisingly, more eMks/platelets had enhanced granule release and localized to the lower diencephalon in mutant mouse embryos than in wild-type littermates before hemorrhage. We found that PDPN counteracted the collagen-1–induced secretion of angiopoietin-1 from fetal Mks, which coincided with enhanced TIE-2 activation in aneurysm-like sprouts of PDPN-deficient embryos. Blocking platelet activation prevented the PDPN-deficient embryo from developing vascular defects. Our data reveal a new role for PDPN in regulating eMk function during midgestation.

The Role of Dexamethasone in the Era of “Dex-CSDH” Randomized Controlled Trial. A Multicenter, Prospective Study on Specific Subset of Patients with Chronic Subdural Hematoma (CSDH) Treated with Dexamethasone Alone or Surgery

Accessory or ectopic breast tissue is residual breast tissue that persists after normal embryonic mammary development. It can occur anywhere along the embryonic mammary streak, but is most commonly located in the axilla. Accessory breast tissue can consist of any component of the breast and may be functional or non-functional and its development is hormone dependent like normal breasts. Overall, the prevalence of accessory breast tissue in women ranges from 0.4% to 6%, and in men, from 1% to 3% [1]. Usually they are asymptomatic and do not need any intervention unless they start causing discomfort. Diagnosis of accessory breast tissue is important because they are subject to all diseases of the breast including carcinomas [2]. Here we report a case of asymptomatic accessory breast tissue in bilateral chest wall of a 32-year Nepalese multi Para female patient