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Patterning and growth are fundamental features of embryonic development that must be tightly coordinated. To understand how metabolism impacts early mesoderm development, we used mouse embryonic stem-cell-derived gastruloids, that co-expressed glucose transporters with the mesodermal marker T/Bra. We found that the glucose mimic, 2-deoxy-D-glucose (2-DG), blocked T/Bra expression and abolished axial elongation in gastruloids. However, glucose removal did not phenocopy 2-DG treatment despite a decline in glycolytic intermediates. As 2-DG can also act as a competitive inhibitor of mannose in protein glycosylation, we added mannose together with 2-DG and found that it could rescue the mesoderm specification both in vivo and in vitro. We further showed that blocking production and intracellular recycling of mannose abrogated mesoderm specification. Proteomics analysis demonstrated that mannose reversed glycosylation of the Wnt pathway regulator, secreted frizzled receptor Frzb. Our study showed how mannose controls mesoderm specification in mouse gastruloids., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Implantation of the human embryo begins a critical developmental stage that comprises profound events including axis formation, gastrulation and the emergence of haematopoietic system 1,2 . Our mechanistic knowledge of this window of human life remains limited due to restricted access to in vivo samples for both technical and ethical reasons 3-5 . Stem cell models of human embryo have emerged to help unlock the mysteries of this stage 6-16 . Here we present a genetically inducible stem cell-derived embryoid model of early post-implantation human embryogenesis that captures the reciprocal codevelopment of embryonic tissue and the extra-embryonic endoderm and mesoderm niche with early haematopoiesis. This model is produced from induced pluripotent stem cells and shows unanticipated self-organizing cellular programmes similar to those that occur in embryogenesis, including the formation of amniotic cavity and bilaminar disc morphologies as well as the generation of an anterior hypoblast pole and posterior domain. The extra-embryonic layer in these embryoids lacks trophoblast and shows advanced multilineage yolk sac tissue-like morphogenesis that harbours a process similar to distinct waves of haematopoiesis, including the emergence of erythroid-, megakaryocyte-, myeloid- and lymphoid-like cells. This model presents an easy-to-use, high-throughput, reproducible and scalable platform to probe multifaceted aspects of human development and blood formation at the early post-implantation stage. It will provide a tractable human-based model for drug testing and disease modelling., (© 2023. The Author(s).)

Researchers are leveraging what we have learned from model organisms to understand if the same principles arise in human physiology, development, and disease. In this collection of Voices, we asked researchers from different fields to discuss what tools and insights they are using to answer fundamental questions in human biology., Competing Interests: Declaration of interests V.G.S. serves as an advisor to and/or has equity in Branch Biosciences, Ensoma, and Cellarity, all unrelated to the present work. F.M. is a co-founder of and has equity in Harbinger Health, has equity in Zephyr AI, serves as a consultant for Harbinger Health and Zephyr AI, and is on the board of directors of Exscientia Plc., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Investigating human development is a substantial scientific challenge due to the technical and ethical limitations of working with embryonic samples. In the face of these difficulties, stem cells have provided an alternative to experimentally model inaccessible stages of human development in vitro 1-13 . Here we show that human pluripotent stem cells can be triggered to self-organize into three-dimensional structures that recapitulate some key spatiotemporal events of early human post-implantation embryonic development. Our system reproducibly captures spontaneous differentiation and co-development of embryonic epiblast-like and extra-embryonic hypoblast-like lineages, establishes key signalling hubs with secreted modulators and undergoes symmetry breaking-like events. Single-cell transcriptomics confirms differentiation into diverse cell states of the perigastrulating human embryo 14,15 without establishing placental cell types, including signatures of post-implantation epiblast, amniotic ectoderm, primitive streak, mesoderm, early extra-embryonic endoderm, as well as initial yolk sac induction. Collectively, our system captures key features of human embryonic development spanning from Carnegie stage 16 4-7, offering a reproducible, tractable and scalable experimental platform to understand the basic cellular and molecular mechanisms that underlie human development, including new opportunities to dissect congenital pathologies with high throughput., (© 2023. The Author(s).)

Technologies to reproduce specific aspects of early mammalian embryogenesis in vitro using stem cells have skyrocketed over the last several years. With these advances, we have gained new perspectives on how embryonic and extraembryonic cells self-organize to form the embryo. These reductionist approaches hold promise for the future implementation of precise environmental and genetic controls to understand variables affecting embryo development. Our review discusses recent progress in cellular models of early mammalian embryo development and bioengineering advancements that can be leveraged to study the embryo-maternal interface. We summarize current gaps in the field, emphasizing the importance of understanding how intercellular interactions at this interface contribute to reproductive and developmental health., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

How cells build embryos is still a major mystery. Many unresolved questions require the study of the processes that pattern and shape the embryo in live specimens, in toto, across spatial and temporal scales. In mammalian embryogenesis, this remains a major challenge as the embryo develops in utero, precluding easy accessibility. For human embryos, technical, ethical and legal limitations further hamper the in-depth investigation of embryogenesis, especially beyond gastrulation stages. This has resulted in an over-reliance on model organisms, particularly mice, to understand mammalian development. However, recent efforts show critical differences between rodent and primate embryos, including timing, architecture and transcriptional regulation. Thus, a human-centric understanding of embryogenesis is much needed. To empower this, novel in vitro approaches, which coax human pluripotent stem cells to form embryonic organoids that model embryo development, are pivotal. Here, we summarize these emergent technologies that recapitulate aspects of human development "in a dish". We show how these technologies can provide insights into the molecular, cellular and morphogenetic processes that fuel the formation of a fully formed fetus, and discuss the potential of these platforms to revolutionize our understanding of human development in health and disease. Despite their clear promise, we caution against over-interpreting the extent to which these in vitro platforms model the natural embryo. In particular, we discuss how fate, form and function - a tightly coupled trinity in vivo, can be disconnected in vitro. Finally, we propose how careful benchmarking of existing models, in combination with rational protocol design based on an increased understanding of in vivo developmental dynamics and insights from mouse in vitro models of embryo development, will help guide the establishment of better models of human embryo development., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Apico-basal polarization of cells within the embryo is critical for the segregation of distinct lineages during mammalian development. Polarized cells become the trophectoderm (TE), which forms the placenta, and apolar cells become the inner cell mass (ICM), the founding population of the fetus. The cellular and molecular mechanisms leading to polarization of the human embryo and its timing during embryogenesis have remained unknown. Here, we show that human embryo polarization occurs in two steps: it begins with the apical enrichment of F-actin and is followed by the apical accumulation of the PAR complex. This two-step polarization process leads to the formation of an apical domain at the 8-16 cell stage. Using RNA interference, we show that apical domain formation requires Phospholipase C (PLC) signaling, specifically the enzymes PLCB1 and PLCE1, from the eight-cell stage onwards. Finally, we show that although expression of the critical TE differentiation marker GATA3 can be initiated independently of embryo polarization, downregulation of PLCB1 and PLCE1 decreases GATA3 expression through a reduction in the number of polarized cells. Therefore, apical domain formation reinforces a TE fate. The results we present here demonstrate how polarization is triggered to regulate the first lineage segregation in human embryos., Competing Interests: MZ, MS, AM, CZ, BS, MB, RM, RP, MM, ME, ST, RS, AC, SF, VG, HZ, KW, ZC, ES, Md, MZ No competing interests declared, (© 2021, Zhu et al.)

Understanding human development is of fundamental biological and clinical importance. Despite its significance, mechanisms behind human embryogenesis remain largely unknown. Here, we attempt to model human early embryo development with expanded pluripotent stem cells (EPSCs) in 3-dimensions. We define a protocol that allows us to generate self-organizing cystic structures from human EPSCs that display some hallmarks of human early embryogenesis. These structures mimic polarization and cavitation characteristic of pre-implantation development leading to blastocyst morphology formation and the transition to post-implantation-like organization upon extended culture. Single-cell RNA sequencing of these structures reveals subsets of cells bearing some resemblance to epiblast, hypoblast and trophectoderm lineages. Nevertheless, significant divergences from natural blastocysts persist in some key markers, and signalling pathways point towards ways in which morphology and transcriptional-level cell identities may diverge in stem cell models of the embryo. Thus, this stem cell platform provides insights into the design of stem cell models of embryogenesis., (© 2021. The Author(s).)

Diabetes mellitus (DM) has profound effects on the female mammalian reproductive system, and early embryonic development, reducing female reproductive outcomes and inducing developmental programming in utero. However, the underlying cellular and molecular mechanisms remain poorly defined. Accumulating evidence implicates endoplasmic reticulum (ER)-stress with maternal DM associated pathophysiology. Yet the direct pathologies and causal events leading to ovarian dysfunction and altered early embryonic development have not been determined. Here, using an in vivo mouse model of Type 1 DM and in vitro hyperglycaemia-exposure, we demonstrate the activation of ER-stress within adult ovarian tissue and pre-implantation embryos. In diabetic ovaries, we show that the unfolded protein response (UPR) triggers an apoptotic cascade by the co-activation of Caspase 12 and Cleaved Caspase 3 transducers. Whereas DM-exposed early embryos display differential ER-associated responses; by activating Chop in within embryonic precursors and Caspase 12 within placental precursors. Our results offer new insights for understanding the pathological effects of DM on mammalian ovarian function and early embryo development, providing new evidence of its mechanistic link with ER-stress in mice.

Breaking embryonic symmetry is an essential prerequisite to shape the initially symmetric embryo into a highly organized body plan that serves as the blueprint of the adult organism. This critical process is driven by morphogen signaling gradients that instruct anteroposterior axis specification. Despite its fundamental importance, what triggers symmetry breaking and how the signaling gradients are established in time and space in the mammalian embryo remain largely unknown. Stem cell-based in vitro models of embryogenesis offer an unprecedented opportunity to quantitatively dissect the multiple physical and molecular processes that shape the mammalian embryo. Here we review biochemical mechanisms governing early mammalian patterning in vivo and highlight recent advances to recreate this in vitro using stem cells. We discuss how the novel insights from these model systems extend previously proposed concepts to illuminate the extent to which embryonic cells have the intrinsic capability to generate specific, reproducible patterns during embryogenesis., (Published by Elsevier Inc.)

Stem cell-based embryo models by cultured pluripotent and extra-embryonic lineage stem cells are novel platforms to model early postimplantation development. We showed that induced pluripotent stem cells (iPSCs) could form ITS (iPSCs and trophectoderm stem cells) and ITX (iPSCs, trophectoderm stem cells, and XEN cells) embryos, resembling the early gastrula embryo developed in vivo. To facilitate the efficient and unbiased analysis of the stem cell-based embryo model, we set up a machine learning workflow to extract multi-dimensional features and perform quantification of ITS embryos using 3D images collected from a high-content screening system. We found that different PSC lines differ in their ability to form embryo-like structures. Through high-content screening of small molecules and cytokines, we identified that BMP4 best promoted the morphogenesis of the ITS embryo. Our study established an innovative strategy to analyze stem cell-based embryo models and uncovered new roles of BMP4 in stem cell-based embryo models., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The development of mouse embryos can be partially recapitulated by combining embryonic stem cells (ESCs), trophoblast stem cells (TS), and extra-embryonic endoderm (XEN) stem cells to generate embryo-like structures called ETX embryos. Although ETX embryos transcriptionally capture the mouse gastrula, their ability to recapitulate complex morphogenic events such as gastrulation is limited, possibly due to the limited potential of XEN cells. To address this, we generated ESCs transiently expressing transcription factor Gata4, which drives the extra-embryonic endoderm fate, and combined them with ESCs and TS cells to generate induced ETX embryos (iETX embryos). We show that iETX embryos establish a robust anterior signaling center that migrates unilaterally to break embryo symmetry. Furthermore, iETX embryos gastrulate generating embryonic and extra-embryonic mesoderm and definitive endoderm. Our findings reveal that replacement of XEN cells with ESCs transiently expressing Gata4 endows iETX embryos with greater developmental potential, thus enabling the study of the establishment of anterior-posterior patterning and gastrulation in an in vitro system., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

At implantation, the mouse embryo undergoes a critical transformation which requires the precise spatiotemporal control of signalling pathways necessary for morphogenesis and developmental progression. The role played by such signalling pathways during this transition are largely unexplored, due to the inaccessibility of the embryo during the implantation when it becomes engulfed by uterine tissues. Genetic studies demonstrate that mutant embryos for BMPs die around gastrulation. Here we have aimed to dissect the role of BMPs during pre-to post-implantation transition by using a protocol permitting the development of the embryo beyond implantation stages in vitro and using stem cells to mimic post-implantation tissue organisation. By assessing both the canonical and non-canonical mechanisms of BMP, we show that the loss of canonical BMP activity compromises the extra-embryonic ectoderm development. Our analyses demonstrate that BMP signalling maintains stem cell populations within both embryonic/extra-embryonic tissues during pre-to post-implantation development. These results may provide insight into the role played by BMP signalling in controlling early embryogenesis., Competing Interests: Declaration of competing interest The authors declare no competing or financial interests., (Published by Elsevier Inc.)

Mammalian blastocysts comprise three distinct cell lineages essential for development beyond implantation: the pluripotent epiblast, which generates the future embryo, and surrounding it the extra-embryonic primitive endoderm and the trophectoderm tissues. Embryonic stem cells can reintegrate into embryogenesis but contribute primarily to epiblast lineages. Here, we show that mouse embryonic stem cells cultured under extended pluripotent conditions (EPSCs) can be partnered with trophoblast stem cells to self-organize into blastocyst-like structures with all three embryonic and extra-embryonic lineages. Morphogenetic and transcriptome profiling analyses reveal that these blastocyst-like structures show distinct embryonic-abembryonic axes and primitive endoderm differentiation and can initiate the transition from the pre- to post-implantation egg cylinder morphology in vitro., (Published by Elsevier Inc.)

Purpose: Almost every female classic galactosemia patient develops primary ovarian insufficiency (POI). The unique pathophysiology of classic galactosemia, with a severely reduced follicle pool at an early age, requires a new therapeutic approach. This study evaluated the effect of dehydroepiandrosterone (DHEA) on ovarian tissue in a galactose-induced POI rat model., Methods: Pregnant rats were fed with either a normal or a 35% galactose-containing diet from day 3 of conception continuing through weaning of the litters. Galactose-exposed female offspring were further divided into 5 groups on PND21. The first group received no application. Treatment groups were fed orally by gavage once daily with sesame oil (group 2), or DHEA at doses of 0.1 mg/kg (group 3), 1 mg/kg (group 4) or 10 mg/kg (group 5) until PND70. Fertility rates of mothers with galactosemia, body weights (BWs), and ovarian weights of the litters from PND21 to PND70 were recorded. Ovarian follicle count, immunohistochemistry for proliferation and apoptosis marker expressions and TUNEL for cell death assessment were performed in offspring ovaries., Results: Decreased fertility, ovarian/body weights were observed under galactosemic conditions, together with decreased follicle number and increased atresia. Improved postnatal development, primordial follicle recruitment and follicular growth were observed after DHEA treatment. After DHEA treatment, the expression of Ki67 protein was found to be increased; elevated expression of cleaved-caspase-3 under galactosemia was found to be reduced., Conclusions: Our data suggests that DHEA treatment may be a potentially useful clinical therapy to improve ovarian ageing in women with POI-induced by galactosemia.

Purpose: The aim of the present study is to investigate role of FoxO transcription factors in preimplantation embryo development by knocking down FoxO1, FoxO3, and FoxO4 genes and also to assess cell cycle arrest related proteins, p53 and p21, and apoptosis-related proteins, fas ligand (FASL), and cleaved caspase 3., Methods: Knockdown of FoxOs using siRNA was confirmed utilizing RT-PCR and qRT-PCR in gene level and using immunofluorescence in protein level. Following knockdown of FoxO1, FoxO3, and FoxO4 in two-cell mouse embryos with or without resveratrol treatment; developmental competence of embryos and expression patterns of SIRT1, p53, p21, FASL, and CLEAVED CASPASE 3 proteins in embryos by immunofluorescence were assessed after 48 h. ROS levels were measured in knockdown embryos. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to determine resveratrol dose., Results: Successful knockdown of FoxO genes in mouse embryos utilizing a non-invasive siRNA method was achieved. Significantly, knockdown of FoxO genes impaired preimplantation embryo development which cannot be prevented by resveratrol treatment. Immunofluorescence results showed that resveratrol could protect embryos from cell cycle arrest and apoptosis. FOXO proteins regulate apoptosis and cell cycle related proteins in mouse preimplantation embryos. Moreover, there might be an autofeedback mechanism where FOXO1, FOXO3, and FOXO4 regulate SIRT1 protein expression., Conclusions: These results suggest that FOXO transcription factors could contribute to mouse preimplantation embryo development, and it remains to investigate whether they have crucial roles in human preimplantation embryo and infertility.

Embryonic stem cells can be incorporated into the developing embryo and its germ line, but, when cultured alone, their ability to generate embryonic structures is restricted. They can interact with trophoblast stem cells to generate structures that break symmetry and specify mesoderm, but their development is limited as the epithelial-mesenchymal transition of gastrulation cannot occur. Here, we describe a system that allows assembly of mouse embryonic, trophoblast and extra-embryonic endoderm stem cells into structures that acquire the embryo's architecture with all distinct embryonic and extra-embryonic compartments. Strikingly, such embryo-like structures develop to undertake the epithelial-mesenchymal transition, leading to mesoderm and then definitive endoderm specification. Spatial transcriptomic analyses demonstrate that these morphological transformations are underpinned by gene expression patterns characteristic of gastrulating embryos. This demonstrates the remarkable ability of three stem cell types to self-assemble in vitro into gastrulating embryo-like structures undertaking spatio-temporal events of the gastrulating mammalian embryo.

Mammalian embryogenesis requires the coordination of embryonic and extra-embryonic tissues to enable implantation into the uterus and post-implantation development to establish the body plan. Mouse embryonic stem cells (ESCs) are a useful tool for studying pluripotent embryonic tissue in vitro. However, they cannot undertake correct embryogenesis alone. Many attempts to model the early embryo in vitro involve the aggregation of ESCs into spheroids of variable size and cell number that undertake germ-layer specification but fail to recapitulate the characteristic architecture and arrangement of tissues of the early embryo. Here, we describe a protocol to generate the first embryo-like structures by directing the assembly of mouse ESCs and extra-embryonic trophoblast stem cells (TSCs) in a 3D extracellular matrix (ECM) into structures we call 'polarized embryo-like structures'. By establishing the medium and culture conditions needed to support the growth of both stem cell types simultaneously, embryonic architecture is generated within 4 d of co-culture. This protocol can be performed by those proficient in standard ESC culture techniques and can be used in developmental studies to investigate the interactions between embryonic and extra-embryonic tissues during mammalian development.

Mammalian embryogenesis requires intricate interactions between embryonic and extraembryonic tissues to orchestrate and coordinate morphogenesis with changes in developmental potential. Here, we combined mouse embryonic stem cells (ESCs) and extraembryonic trophoblast stem cells (TSCs) in a three-dimensional scaffold to generate structures whose morphogenesis is markedly similar to that of natural embryos. By using genetically modified stem cells and specific inhibitors, we show that embryogenesis of ESC- and TSC-derived embryos-ETS-embryos-depends on cross-talk involving Nodal signaling. When ETS-embryos develop, they spontaneously initiate expression of mesoderm and primordial germ cell markers asymmetrically on the embryonic and extraembryonic border, in response to Wnt and BMP signaling. Our study demonstrates the ability of distinct stem cell types to self-assemble in vitro to generate embryos whose morphogenesis, architecture, and constituent cell types resemble those of natural embryos., (Copyright © 2017, American Association for the Advancement of Science.)

Purpose: The purpose of the present study is to understand Twist-related protein 1 (Twist1) spatiotemporal expression patterns and functions during early embryo development., Methods: We performed whole-mount double immunofluorescence staining and reverse transcription (RT)-PCR analysis of the Twist1 protein and gene throughout the preimplantation development in mice., Results: We determined that after compaction, the expression of Twist1 becomes developmentally differentiated and targeted in the inner cells of embryos. In blastocysts at E4.5, uniform staining of the inner cell mass was apparent, and it had been gradually translocated to the nucleus of hatched embryonic cells at E4.75. Furthermore, the effect of potential regulators of Twist on its expression level during blastocyst development was also sought. Accordingly, Twist1 expression appeared to be upregulated in both mRNA and protein level following culture of embryos in the presence of high glucose., Conclusions: Our study revealed the dynamic Twist localization within the early stage of embryo. The results are discussed in terms of potential roles of Twist1 in the processes of lineage segregation, hatching, and implantation in post-compaction embryos and in blastocysts.

Purpose: Azoospermia is one of the major causes of male infertility and is basically classified into obstructive (OA) and non-obstructive azoospermia (NOA). The molecular background of NOA still largely remains elusive. It has been shown that the poly(A)-binding proteins (PABPs) essentially play critical roles in stabilization and translational control of the mRNAs during spermatogenesis., Methods: In the present study, we aim to evaluate expression levels of the PABP genes, EPAB, PABPC1, and PABPC3, in the testicular biopsy samples and in the isolated spermatocyte (SC) and round spermatid (RS) fractions obtained from men with various types of NOA including hypospermatogenesis (hyposperm), RS arrest, SC arrest, and Sertoli cell-only syndrome (SCO)., Results: In the testicular biopsy samples, both PABPC1 and PABPC3 mRNA expressions were gradually decreased from hyposperm to SCO groups (P < 0.05), whereas there was no remarkable difference for the EPAB expression among groups. The expression levels of cytoplasmically localized PABPC1 and PABPC3 proteins dramatically reduced from hyposperm to SCO groups (P < 0.05). In the isolated SC and RS fractions, the EPAB, PABPC1, and PABPC3 mRNA expressions were gradually decreased from hyposperm to SC arrest groups (P < 0.05). Similarly, both PABPC1 and PABPC3 proteins were expressed at higher levels in the SC and RS fractions from hyposperm group when compared to the SC and RS fractions from either RS arrest or SC arrest group (P < 0.05)., Conclusion: Our findings suggest that observed significant alterations in the PABPs expression may have an implication for development of different NOA forms.

Embryonic poly(A)-binding protein (EPAB) and poly(A)-binding protein, cytoplasmic 1 (PABPC1) play critical roles in translational regulation of stored maternal mRNAs required for proper oocyte maturation and early embryo development in mammals. Superovulation is a commonly used technique to obtain a great number of oocytes in the same developmental stages in assisted reproductive technology (ART) and in clinical or experimental animal studies. Previous studies have convincingly indicated that superovulation alone can cause impaired oocyte maturation, delayed embryo development, decreased implantation rate and increased postimplantation loss. Although how superovulation results in these disturbances has not been clearly addressed yet, putative changes in genes related to oocyte and early embryo development seem to be potential risk factors. Thus, the aim of the present study was to determine the effect of superovulation on Epab and Pabpc1 gene expression. To this end, low- (5IU) and high-dose (10IU) pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotrophin (hCG) were administered to female mice to induce superovulation, with naturally cycling female mice serving as controls. Epab and Pabpc1 gene expression in germinal vesicle (GV) stage oocytes, MII oocytes and 1- and 2-cell embryos collected from each group were quantified using quantitative reverse transcription-polymerase chain reaction. Superovulation with low or high doses of gonadotropins significantly altered Epab and Pabpc1 mRNA levels in GV oocytes, MII oocytes and 1- and 2-cell embryos compared with their respective controls (P<0.05). These changes most likely lead to variations in expression of EPAB- and PABPC1-regulated genes, which may adversely influence the quality of oocytes and early embryos retrieved using superovulation.

CD90 (i.e., THY1) and CD105 (i.e., endoglin) are glycoproteins known as mesenchymal stem cell markers that are expressed in various cell types including male and female gonadal cells. We aimed to determine ovarian and testicular expression of CD90 and CD105 in various cell types during postnatal development in mice. The present study was carried out on male (C57BL/6) and female (Balb/C) mice during critical stages of gonadal development. Immunohistochemical localization of CD90 and CD105 was determined in the ovaries obtained at postnatal days (PND) -1, -7, -21 and -60 and in the testes obtained at PND6, -8, -16, -20, -29, -32 and -88. The relative expression of CD90 and CD105 was evaluated by ImageJ software and data were analyzed by analysis of variance. The relative expression of CD90 and CD105 varied during postnatal development and increased significantly in the adult ovary (PND60) and testis (PND88) compared to the early postnatal gonads. In the ovaries, the expression of CD90 was significantly higher in somatic cells in comparison to germ cell compartments. In the testis, CD90 expression was greater in germ cells and Sertoli cells compared to other cell types. Expression of CD105 was higher in germ cells than somatic cells of both the ovary and testis. In addition to different expression of CD90 and CD105 during various developmental stages, also their altered expression in particular cell types suggests specific roles of these glycoproteins in physiological processes of mouse gonads., (Copyright © 2015 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Preimplantation embryo development is an important and unique period and is strictly controlled. This period includes a series of critical events that are regulated by multiple signal-transduction pathways, all of which are crucial in the establishment of a viable pregnancy. The p38 mitogen-activated protein kinase (MAPK) signalling pathway is one of these pathways, and inhibition of its activity during preimplantation development has a deleterious effect. The molecular mechanisms underlying the deleterious effects of p38 MAPK suppression in early embryo development remain unknown. To investigate of the effect of p38 MAPK inhibition on late preimplantation stages in detail, we cultured 2-cell stage embryos in the presence of SB203580 for 48 h and analysed the 8-cell, morula, and blastocyst stages. We determined that prolonged inhibition of the p38 MAPK altered the expression levels of Glut1 and Glut4, decreased glucose uptake during the 8-cell to blastocyst transition, changed the expression levels of transcripts which will be important to lineage commitment, including Oct4/Pou5f1, Nanog, Sox2, and Gata6, and increased cell death in 8-16 cell stage embryos onwards. Strikingly, while the expression levels of Nanog, Gata6 and Oct4/Pou5f1 mRNAs were significantly decreased, Sox2 mRNA was increased in SB203580-treated blastocysts. Taken together, our results provide important insight into the biological processes controlled by the p38 MAPK pathway and its critical role during preimplantation development., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Purpose: Embryonic poly(A)-binding protein (EPAB) and poly(A)-binding protein, cytoplasmic 1 (PABPC1) bind poly(A) tails of mRNAs and mediate their translational regulation in germ cells and early preimplantation embryos. Although expression patterns and possible functions of the Epab and Pabpc1 genes have been examined in vertebrate germ cells and early embryos, their expression levels and cellular localizations in the postnatal mouse ovaries remained elusive., Methods: In the present study, we first aimed to characterize expression levels of the Epab and Pabpc1 genes in the prepubertal (1-, 2-, and 3-week old), pubertal (4-, 5-, and 6-week old), postpubertal (16-week and 18-week old), and aged (52-, 60-, and 72-week old) mouse ovaries by using quantitative real-time polymerase chain reaction (qRT-PCR)., Results: Epab mRNA was predominantly expressed in the prepubertal ovaries when compared to later developmental periods. However, Pabpc1 transcript was highly generated in the prepubertal and pubertal mouse ovaries except for 1-week old ovary than those of other developmental terms. In the prepubertal mouse ovaries, RNA in situ hybridization localized both Epab and Pabpc1 transcripts in the cytoplasm of oocytes and granulosa cells of all follicular stages. Consistently, Epab and Pabpc1 gene expression were detected in the cumulus cells and MII oocytes obtained from cumulus oocyte complexes (COCs). Ovarian follicle counting in the postnatal ovaries revealed that total number of follicles was higher in the prepubertal ovaries in comparison with later stages of development., Conclusion: As a result, Epab and Pabpc1 expression exhibit differences at postnatal ovary development stages and both genes are transcribed in the granulosa cells and oocytes. These findings suggest that EPAB may predominantly play roles in translational regulation of the mRNAs during early oogenesis and folliculogenesis, but PABPC1 most likely perform these roles in the later terms of ovarian development along with EPAB protein.

Objective: To study the effect of increased endoplasmic reticulum (ER) stress as a major nongenomic mechanism for arrested blastocyst development., Design: Cell and animal study., Setting: The Ohio State University and Yale University., Animal(s): Mice., Intervention(s): Pregnant mare serum gonadotropin and hCG were administered IP; two cell embryos were collected 48 hours after hCG administration., Main Outcome Measure(s): Blastocyst development rate., Result(s): No morphological difference was detected in control versus tunicamycin- (TM) treated embryos until the blastocyst stage. On day 4 of embryonic development, TM treatment reduced blastocyst formation from 79% to 4% and induced nuclear fragmentation. TM treatment caused 2-fold and 2.6-fold increase in binding immunoglobulin protein and spliced-X-box binding protein 1 mRNA expression, respectively. By comparison, the tauroursodeoxycholic acid + TM combination reversed the effect of TM alone on blastocyst formation to near control levels., Conclusion(s): These results indicate that increased ER stress during in vitro embryo development triggers an unfolded protein response (UPR) that negatively affects blastocyst formation and suggests that activation of UPR signaling may account for low rates of blastocyst development., (Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

In the developmental process of the early mammalian embryo, it is crucial to understand how the identical cells in the early embryo later develop different fates. Along with existing models, many recently discovered molecular, cellular and developmental factors play roles in cell position, cell polarity and transcriptional networks in cell fate regulation during preimplantation. A structuring process known as compaction provides the "start signal" for cells to differentiate and orchestrates the developmental cascade. The proper intercellular junctional complexes assembled between blastomeres act as a conducting mechanism governing cellular diversification. Here, we provide an overview of the diversification process during preimplantation development as it relates to intercellular junctional complexes. We also evaluate transcriptional differences between embryonic lineages according to cell- cell adhesion and the contributions of adhesion to lineage commitment. These series of processes indicate that proper cell fate specification in the early mammalian embryo depends on junctional interactions and communication, which play essential roles during early morphogenesis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Telomeres are located at the ends of all eukaryotic chromosomes and protect them from deleterious events such as inappropriate DNA repair, illegitimate recombination or improper segregation of the chromosomes during mitotic or meiotic divisions. However, telomeres gradually shorten primarily due to successive rounds of genomic DNA replication and also as the result of the adverse effects of oxidative stress, genotoxic agents, diseases related to ageing and environmental factors on the nuclear materials of dividing or non-dividing cells. Germline cells, proliferative granulosa cells, early embryos, stem cells, highly proliferative somatic cells and many cancer cells contain the enzyme telomerase so that they are capable of elongating the shortened telomeres. Although numerous studies have revealed the length of telomeres and telomerase activity in oocytes, granulosa cells and early embryos, only a few studies have analyzed and compared the work performed on distinct mammalian species. In this comprehensive review article, we compare and discuss telomere length and telomerase activity in oocytes, granulosa cells and early embryos in different mammalian species including mice, bovines and humans.

Objective: Investigation of the structure of vascular malformations highlights the pathogenic mechanisms underlying their clinical behavior. One of the vascular malformations is called cerebral cavernous malformation (CCM). However, the ultrastructural features of the vascular malformations are not defined in detail., Methods: We aimed to investigate the ultrastructural features of CCMs using transmission (TEM), scanning (SEM) electron microscopy, and also immunohistochemistry methods with antibodies against CCM proteins such as CCM2 and CCM3. CCM tissues (n=6) microsurgically excised from patients for conventional indications., Results: CCM2 and CCM3 were strongly detected in the vascular endothelium. However, there was a very weak immunostaining in stroma. SEM observations revealed that there were ruptures and damages in the luminal endothelium, possibly due to the damage of intercellular junctions. TEM observations also showed a few ruptures and detachments between the endothelium and basal lamina as observed with partially damages and disconnections. The architecture of pericytes showed protrusions and shrinkages. Our results suggest that the thin vessel walls of CCMs were lacking of subendothelial support and intact basal lamina underlying the endothelial cells., Conclusion: This study is so far the first study attempting to show human CCM lesions with SEM. We believe that an understanding of the ultrastructural features of these lesions by light and electron microscopy techniques would help to understand the pathology of these diseases., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We aimed at investigating the effects of sulfite-induced lipid peroxidation and apoptosis mediated by secretory phospholipase A2 (sPLA2) on somatosensory evoked potentials (SEP) alterations in rats. Thirty male albino Wistar rats were randomized into three experimental groups as follows; control (C), sodium metabisulfite treated (S), sodium metabisulfite+quinacrine treated (SQ). Sodium metabisulfite (100 mg/kg/day) was given by gastric gavage for 5 weeks and 10 mg/kg/day quinacrine was applied as a single dose of intraperitoneal injection for the same period. The latencies of SEP components were significantly prolonged in the S group and returned to control levels following quinacrine administration. Plasma-S-sulfonate level was increased in S and SQ groups. TBARS levels in the S group were significantly higher than those detected in controls. Quinacrine significantly decreased brain TBARS levels in the SQ group compared with the S group. Quinacrine treatment did not have an effect on the increased sPLA2 level of the sulfite administered group. Immunohistochemistry showed that sulfite caused an increase in caspase-3 and TUNEL positive cells, restored to control levels via quinacrine administration. This study showed that sPLA2 might play a role in ingested sulfite-induced SEP alterations, oxidative stress, apoptotic cell death and DNA damage in the brain., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Modification of poly(A) tail length constitutes the main posttranscriptional mechanism by which gene expression is regulated during spermatogenesis. Embryonic poly(A)-binding protein (EPAB) and somatic cytoplasmic poly(A)-binding protein (PABPC1) are the 2 key proteins implicated in this pathway. In this study we characterized the temporal and spatial expression of Epab and Pabpc1 in immature (D6-D32) and mature (D88) mouse testis and in isolated spermatogenic cells. Both Epab and Pabpc1 expression increased during early postnatal life and reached their peak at D32 testis. This was due to an increase in both spermatogonia (SG) and spermatocytes. In the mature testis, the highest levels of Epab were detected in SG, followed by round spermatids (RSs), while the most prominent Pabpc1 expression was detected in spermatocytes and RSs. Our findings suggest that PABPC1 may play a role in translational regulation of gene expression by cytoplasmic polyadenylation, which occurs in spermatocytes, while both EPAB and PABPC1 may help stabilize stored polyadenylated messenger RNAs in RSs.

Objective: In this follow-up study, we aimed to assess national and international pharmaceutical companies' Web sites using guidelines of The Association of Research-Based Pharmaceutical Companies (AIFD) and Pharmaceutical Manufacturers Association of Turkey (IEIS) to define whether there has been progress since 2004., Materials and Methods: We used two national guidelines in order to evaluate the Web sites of pharmaceutical companies in our study. The first guideline was from IEIS, and the second was from AIFD, which was issued recently. We collected our data between February 1 and April 30, 2011. Data analyses were performed using SPSS version 15.0. Chi squared test was done for comparing the two assessments in different years (2004 and 2011)., Results: In general, the progress that we were expecting was not satisfactory for both the international and national companies. The percentage of unmet criteria increased for "links" (from 48.0% to 52.0%) and for "mentioning the responsible person/firm for the Web site design" (from 40.6% to 59.4%) among national companies. We observed statistically significant progress only for the "information for the public" criterion (from 52.9% to 100%) among international companies. On the other hand, there was progress and a statistically significant difference in terms of not displaying any "drug ads" on the Web sites of national firms (from 55.9% to 87.5%), availability of "mail address" (from 88.2% to 100%), "telephone number" (from 88.2% to 100%), "indication of the target group" (from 23.5% to 52.1%), and "disclaimer stating the given information cannot replace a physician or pharmacist" (from 29.4% to 53.1%)., Conclusions: Our major recommendation to pharmaceutical companies is to update their Web sites with evidence-based scientific information about themselves and their products using international and national standards. From the companies' perspective, this should be a priority responsibility based on the ethical aspect of individual and community health.

Cerebral cavernous malformation (CCM) is one of the most common types of vascular malformations of the central nervous system, affecting nearly one in 200 people. CCM lesions are characterized by grossly dilated vascular channels lined by a single layer of endothelium. Genetic linkage analyses have mapped three CCM loci to CCM1, CCM2 and CCM3. All three causative genes have now been identified allowing new insights into CCM pathophysiology. We focused on the CCM2 protein that might take place in blood vessel formation; we report here the expression patterns of CCM2 in prenatal development and adult human neocortex by means of immunohistochemistry and Western blot analysis. CCM2 was obviously detected in vascular endothelium and neuroglial precursor cells during development and also observed in arterial endothelium, neurons, some of the glial cells in adult neocortex. The expression patterns suggest that it could be one of the arterial markers whether this is a cause or a consequence of an altered vascular identity. CCM2 might play a role during vasculogenesis and angiogenesis during human brain development. Furthermore, with this study, CCM2 have been described for the first time in developing human neocortex., (Copyright © 2011 ISDN. All rights reserved.)

The objective of this study was to find all the existing Web sites of Turkish community pharmacies and evaluate their "quality" in terms of Health on the Net (HON) Code of conduct principles. Multiple Internet search engines were used (google.com, yahoo.com, altavista.com, msn.com). While searching on the Internet, "eczane (pharmacy)" and "eczanesi (pharmacy of)" key words were used. The Internet search lasted for 2 months starting from March 1, 2007 until May 1, 2007. SPSS ver. 11.5 statistical program (SPSS, Inc., Chicago, IL) was used for data entry and analysis. At the end of the Internet search via all the indicated search engines, a total of 203 (all different from each other) community pharmacy Web sites were determined; of these, 14 were under construction and 6 were not accessible. As a result, 183 community pharmacy Web sites were included in the study. All of the Web sites could be accessed (100%). However, the availability of some characteristics of the pharmacies were quite poor. None of the pharmacies met all of the HON principles. Only 11 Web sites were appropriate in terms of complementarity (6.0%). Confidentiality criteria was met by only 14 pharmacies (7.7%). Nine pharmacies (4.9%) completed the "attribution" criteria. Among 183 pharmacy Web sites, the most met HON principle was the "transparency of authorship" (69 pharmacy Web sites; 37.7%). Because of the results of our study, the Turkish Pharmacists Association can take a pioneer role to apply some principles such as HON code of conduct in order to increase the quality of Turkish community pharmacists' Web sites.