Early Human Embryogenesis Research Articles

Expression of MUL, a gene encoding a novel RBCC family ring-finger protein, in human and mouse embryogenesis.

We studied the expression of MUL, a gene encoding a novel member of the RING-B-Box-Coiled Coil family of zinc finger proteins that underlies the human inherited disorder, Mulibrey nanism. In early human and mouse embryogenesis MUL is expressed in dorsal root and trigeminal ganglia, liver and in epithelia of multiple tissues.

Immunohistochemical Expression of S-100 Protein in Human Embryonal Fat Cells

The aim of the present study was to investigate immunocytochemically the presence of S-100 protein in subcutaneous fat cells during early human embryogenesis (6–12 weeks of gestation). We found that preadipocytes in the subcutaneous tissue which were at different stages of differentiation were positive for S-100 protein. The other cells in the embryonal subcutis (mesenchymal cells differentiating into fibroblasts and fibrocytes and endothelial cells) showed a negative reaction for S-100. Our results imply that the S-100 protein is expressed from the beginning of lipidogenesis and possibly acts as a factor regulating lipid storage and body fat formation. It can be used as a reliable biochemical marker of human fat cell differentiation and for distinguishing them from the mesenchymal and the fibroblast cells in the human embryonal subcutis.

Expression of the Sonic hedgehog (SHH ) gene during early human development and phenotypic expression of new mutations causing holoprosencephaly.

Holoprosencephaly (HPE), the most common developmental defect of the forebrain and the face, is genetically heterogeneous. One of the genes involved, Sonic hedgehog ( SHH ), on 7q36, has been identified as the first HPE-causing gene both in mouse and humans. In order to delineate the phenotype of specific SHH mutations, we described the expression of the SHH gene during early human embryogenesis and investigated the phenotype of novel SHH mutations. In situ hybridization studies were performed on paraffin-embedded human embryo sections at three different development stages. These studies show that SHH is expressed in the notochord, the floorplate, the brain, the zone of polarizing activity and the gut. We also report on the phenotype of four novel mutations identified in 40 HPE families (two in isolated HPE and two in familial HPE). Expressivity ranged from alobar HPE to microcephaly and hypoplasia of the pituitary gland in one family, and from HPE to an asymptomatic form in another family. No SHH mutation was found in six polymalformed cases combining HPE with other defects, such as skeletal, limb, cardiac, anal and/or renal anomalies. This study confirms the genetic heterogeneity of HPE, and further demonstrates that SHH mutations are associated with a broad spectrum of cerebral midline defects.

The role of structural integrity of the fertilising spermatozoon in early human embryogenesis.

While the fertilising spermatozoon supplies the active centre directing the human zygote's first mitotic division, the relative contributions of the sperm head and tail (as well as the importance of the sperm's general structural integrity) to subsequent developmental processes remain incompletely studied. The sperm nucleus contains paternal chromatin necessary for restoration of a diploid genome, but the functional role of the sperm tail (either attached or dissected) in early human embryonic growth is not known. In this investigation using oocytes donated by in vitro fertilisation patients, human oocytes were injected with isolated sperm heads (n = 73), isolated sperm flagella (n = 11) or both (dissected sperm heads + free sperm tails, n = 26). The formation of bipronucleate zygotes was recorded for each method. Among oocytes surviving injection with isolated sperm heads, 44 of 66 (67%) formed two pronuclei. Of oocytes receiving only sperm tails, 2 of 11 (18%) displayed two pronuclei, but a single polar body was evident in both cases. When dissected spermatozoa parts (head + tail) were jointly injected, 12 of 26 (46%) developed two pronuclei. From embryos resulting from each of these three fertilisation regimes, blastomere biopsies were obtained and subjected to multiprobe fluorescent in situ hybridisation (FISH) analysis to detect mosaicism or aneuploidy arising from these experimental treatments. Only embryos with growth sufficient to permit sampling of at least two blastomeres were evaluated, and FISH analysis was successful in 25 of 29 (86%) embryos tested. Of 12 embryos derived from injection of an isolated sperm head, only one was normal diploid; the remaining 11 were mosaic. Both embryos resulting from injection of an unattached sperm tail were mosaic. Of 11 embryos generated from oocyte injection with sperm head + tail segments, 10 (91%) were mosaic and only one was normal diploid. Results from this study show that injection of isolated sperm segments can permit oocyte activation and bipronuclear formation. However, a high rate of mosaicism in human embryos originating from disrupted sperm or sperm components suggests that more than a 'sum of parts' is needed for later development. The structural integrity of the intact fertilising spermatozoon appears to contribute to normal human early embryogenesis.

Functional Expressions of fms and M-CSF During Trophoectodermal Differentiation of Human Embryonal Carcinoma Cells

A human embryonal carcinoma (EC) cell line, NCR-G3 (G3), is capable of differentiating into a variety of cell types in vitro, including epithelial, muscle, neural and trophoectodermal cells. The production of human chorionic gonadotropin (hCG), a trophoectoderm-specific hormone, begins 7 days after retinoic acid (RA) treatment and peaks on day 12–13. In this study, we used G3 cells to investigate the biological significance of macrophage colony-stimulating factor (M-CSF), also called colony-stimulating factor-1 (CSF-1), and fms, a receptor tyrosine kinase for M-CSF. The mRNA of c-fms is constitutively expressed in both undifferentiated and differentiated G3 cells. Immunoprecipitation with anti-fms antibodies or flow cytometry revealed that differentiated G3 cells express fms on the cell surface. However, we were unable to demonstrate expression of fms on the surface of undifferentiated G3 cells. Expression of M-CSF mRNA and protein, however, was upregulated by RA treatment prior to hCG production. In order to investigate whether expression of both molecules is biologically functional in G3 cells, we conducted experiments using anti-M-CSF and fms antibodies with neutralizing activity and gene transfer to achieve over-expression of fms in G3 cells. As a result, we observed that hCG production following treatment with both neutralizing antibodies was more than 90 per cent inhibited, and that hCG production increased significantly as a result of over-expression of fms in G3 cells. Our results enabled us to show that M-CSF and fms play important functional roles in the differentiation of G3 cells into trophoectoderm. G3 cells are well suited to serve as an experimental model of human early embryogenesis and of placental differentiation.

Spatial and Temporal Expression of the Cystathionine β-Synthase Gene During Early Human Development

We report the cystathionine-β synthase (CBS) gene expression pattern during early human embryogenesis (3 to 6 weeks post conception) by in situ hybridization and in fetal and adult tissue by Northern Blot analysis. Probes were chosen to recognize either the common sequence to all known CBS mRNAs or the sequences of two different major exons 1 issued of we have previously identified. We demonstrate by in situ hybridization that CBS is continuously expressed from the earliest stages studied (22 days post conception) during embryogenesis in the tissues of developing embryos which will after birth present clinical abnormalities in homocystinuria patients. It is expressed at an especially high level in the neural and cardiac systems until the liver primordium appears. In embryonic central nervous system, the whole neural tube and primary brain vesicles are labeled. Secondary brain vesicles labeling are dependent on the neuroepithelium differentiation. The ventricular layer of the rhombencephalon, cranial nerve nuclei and then after cerebellar cortex derived from rhombencephalon ventricular layer are strongly labeled. Thalamus and other derivatives of the diencephalon plate, the neuroblastic layer of the retina, lens and dorsal root ganglia are labeled. After 35 days post conception, CBS mRNAs was detected in endocardial cells and in cells derived from the neural crest of the heart and in particular developing mesodermic regions such as the primitive hepatocytes of the liver, mesonephros vesicles, various endocrine glands and developing bones. We could not detect tissue specificity of different probes at this embryonic stage. Northern blot analysis consistently detected mRNA species in fetal 25 weeks post conception brain, liver and kidney. The common cDNA probe revealed the 2.5 and 3.7 kb mRNA species from brain, liver and kidney. The exon 1b probe detected only the 2.5 kb mRNA and the exon 1c probe the 3.7 kb mRNA in these three tissues. In adult tissue, the 1b probe detected only the 2.5 kb mRNA and the 1c probe only the 3.7 kb mRNA in the liver.

Blastocyst development from supernumerary embryos after intracytoplasmic sperm injection: a paternal influence?

The success of intracytoplasmic sperm injection (ICSI) warrants further study on the role of paternal factors in early human embryogenesis. To investigate whether poor sperm parameters can influence embryo development, we examined the development of ICSI-fertilized embryos to the blastocyst stage. We present results of blastocyst development from supernumerary ICSI embryos after co-culture on monkey kidney epithelial cells. In addition, we compare the development of supernumerary embryos to the blastocyst stage after ICSI and in-vitro fertilization (IVF). Of 168 supernumerary ICSI embryos, 45 (26.8%) developed to blastocysts. Sperm concentration and morphology did not influence blastocyst development. In contrast, blastocysts arose from spermatozoa that had a significantly higher (P = 0.015) forward progressive motility compared with spermatozoa from those patients who failed to produce blastocysts (42.7% versus 28.2%, respectively). Overall the rate of embryo development to the blastocyst stage after ICSI was lower (26.8%) than that after IVF (47.3%). When the rate of blastocyst development was calculated for patients with three or more supernumerary embryos, it remained significantly higher for the IVF patients than for the ICSI patients (45.6% versus 30.0%). There was no significant difference in the mean cell number and quality of the supernumerary embryos between the IVF and ICSI patients. This study confirms previous reports that have postulated that abnormal spermatozoa may manifest a negative paternal effect on preimplantation embryo development.

Uptake of fluorescent probe DII-acetylated-LDL in microglia of embryonic mouse brain slices are spatially and temporally regulated

We report the cystathionine-β synthase (CBS) gene expression pattern during early human embryogenesis (3 to 6 weeks post conception) by in situ hybridization and in fetal and adult tissue by Northern Blot analysis. Probes were chosen to recognize either the common sequence to all known CBS mRNAs or the sequences of two different major exons 1 issued of we have previously identified. We demonstrate by in situ hybridization that CBS is continuously expressed from the earliest stages studied (22 days post conception) during embryogenesis in the tissues of developing embryos which will after birth present clinical abnormalities in homocystinuria patients. It is expressed at an especially high level in the neural and cardiac systems until the liver primordium appears. In embryonic central nervous system, the whole neural tube and primary brain vesicles are labeled. Secondary brain vesicles labeling are dependent on the neuroepithelium differentiation. The ventricular layer of the rhombencephalon, cranial nerve nuclei and then after cerebellar cortex derived from rhombencephalon ventricular layer are strongly labeled. Thalamus and other derivatives of the diencephalon plate, the neuroblastic layer of the retina, lens and dorsal root ganglia are labeled. After 35 days post conception, CBS mRNAs was detected in endocardial cells and in cells derived from the neural crest of the heart and in particular developing mesodermic regions such as the primitive hepatocytes of the liver, mesonephros vesicles, various endocrine glands and developing bones. We could not detect tissue specificity of different probes at this embryonic stage. Northern blot analysis consistently detected mRNA species in fetal 25 weeks post conception brain, liver and kidney. The common cDNA probe revealed the 2.5 and 3.7 kb mRNA species from brain, liver and kidney. The exon 1b probe detected only the 2.5 kb mRNA and the exon 1c probe the 3.7 kb mRNA in these three tissues. In adult tissue, the 1b probe detected only the 2.5 kb mRNA and the 1c probe only the 3.7 kb mRNA in the liver.

Characterization of the Complete Genomic Structure of the Human WNT-5A Gene, Functional Analysis of its Promoter, Chromosomal Mapping, and Expression in Early Human Embryogenesis

We report the complete genomic organization of the human WNT-5A gene, which encodes a cysteine-rich growth factor involved in cell-cell signaling during growth and differentiation. The gene comprises five exons with the terminal exon coding for a large 3'-untranslated region of approximately 6.5 kilobase pairs and utilizes multiple polyadenylation signals to generate at least four discrete transcripts. We discovered a new leader exon interrupted by a 411-base pair intron that was retained in our original cDNA cloning. The promoter region was located in a GpC-rich island and harbored numerous cis-acting elements including several GC boxes and Sp1, AP1, and AP2 binding motifs. It lacked TATA or CAAT boxes typical of housekeeping and growth factor genes. In support of this, primer extension revealed extension two transcription start sites. Transient cell transfection assays showed functional promoter activity for the 3.9-kilobase pair 5'-flanking region. Interestingly, internal and 5' deletions revealed tha the distal promoter was not required for full transcriptional activity and that the first 631 base pairs of WNT-5A harbored the strongest promoter activity. Using a panel of rodent-human hybrid DNAs carrying portions of chromosome 3p, we mapped the gene to 3p14.2-p21.1, between a constitutional and a familial renal cell carcinoma-associated translocation. In situ hybridization analyses of early human embryos at 28-42 days of gestation revealed that WNT-5A transcripts were not restricted to the developing brain and limbs but were also observed in the mesenchyme bordering the pharyngeal clefts and pouches and in the developing gonads and kidneys. The relatively high expression in the celomic epithelium and in the precursors of follicles and seminiferous tubules suggest a novel role for WNT-5A in germ-cell differentiation. This study provides the molecular basis for discerning the regulation of the WNT-5A gene and offers the opportunity to investigate genetic disorders linked to this important gene.

Cystic fibrosis gene expression in early human embryogenesis

Cystic fibrosis gene expression in early human embryogenesis

The role of genomic imprinting in implantation

To outline the possible role of the imprinted genes in early human embryogenesis and implantation. Literature review. Studies examining the issues of genomic imprinting, implantation, gestational trophoblastic diseases, placental gene expression, and trophoblast invasion. Certain genes have been shown to be expressed either in the embryo or in the uterine decidua before implantation. Some of these have been shown to be parentally imprinted, that is, expressed either from their paternal or maternal origin. The paternally expressed genes are linked to placental proliferation and invasiveness. Clinical and basic data from different disciplines indicate that genomic imprinting may be crucial to the process of implantation.

Aberrant alternative splicing of human zinc finger gene ZNF268 in human hematological malignancy

The human ZNF268 gene was initially described as a gene associated with early human embryogenesis and was later implicated in human leukemia due to the identification of an alternatively splice form in leukemia patients. To systematically evaluate the correlation of ZNF268 with human hematological malignancy, expression of different alternatively spliced forms of ZNF268 mRNA in peripheral blood of 45 patients with hematological malignancies and 17 healthy donors were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR. We demonstrated that presence of ZNF268a, ZNF268c, ZNF268f and ZNF268g were significantly different between the patients and healthy donors (P<0.05). Our study thus suggests that aberrant alternative splicing of ZNF268 is a potential prognostic factor of and may contribute to human hematological malignancies.

Development of human embryos to the hatched blastocyst stage in the presence or absence of a monolayer of Vero cells

In a prospective randomized study, excess embryos from 100 women undergoing in-vitro fertilization were cultured from the 2-cell to the hatched-blastocyst stage in the presence or absence of a confluent monolayer of Vero cells. The frequencies of fragmentation, developmental arrest, multinucleation and blastocyst formation were observed for 254 embryos over 7 days in culture. The number of nucleated cells, and fine structure of trophectoderm and inner cell mass were analysed at the expanded blastocyst stage on day 5.5 post-insemination. The frequency of hatching from the zona pellucida was determined between days 6 and 7 post-insemination. With respect to these developmental parameters, the findings indicate that no overt or statistically significant improvement in early human embryogenesis occurs in the co-culture system.

Factors Regulating Interaction between Trophoblast and Human Endometrium

Implantation is a crucial step in human reproduction. Disturbances of this process are responsible for pregnancy failure after both in vivo and in vitro fertilization. The endometrium provides the implanting embryo with a unique substratum where the embryo communicates with biochemical signals, attaches itself, penetrates and grows without blood circulation. The highly proliferative phase of the cytotrophoblast, during early human embryogenesis, may be due to endogenous production of growth factors that may establish autocrine/short range paracrine stimulator loops which explain the tumor-like properties of these tissues. Endometrial BM penetration and stroma invasion may be due to the proteolytic capability of the human embryo. It is suggested that collagenase and the urokinase-like plasminogen activator are responsible for this activity. To clarify the molecular mechanisms involved in human embryo implantation several models are suggested: culture of blastocysts, culture of endometrial cells, and endometrial explant co-culture. Human blastocysts cultured with whole perfused human uteri make it possible to recognize some aspects of the entire implantation process and give us the possibility of improving the benefits provided by new technologies in reproductive medicine and reducing embryonic loss at an early stage.

Embryogenesis of the human pancreatic islets: a light and electron microscopic study.

The developing pancreases of twenty human embryos and fetuses, 8–23 weeks gestational age, were obtained under conditions optimum for cellular preservation and studied by light and electron microscopy. Primitive pancreatic tubules were the only epithelial structures present at 8 and 8.5 weeks. Alpha cells were first identified at 9 weeks, followed by delta and subsequently beta cells at 10.5 weeks. Endocrine cell progenitors, mixed endocrine cells, or mixed exocrine endocrine cells were not identified. Although potential sampling errors preclude a definitive recitation of the order of islet cell appearance, the alpha and delta cells are numerically the predominant pancreatic endocrine cells during early human embryogenesis. The observation of frequent, well-preserved delta cells and the identification of one undergoing mitotic division support their legitimacy as an independent cell type. Cytoplasmic glycogen was present in primitive tubular cells and differentiated exocrine cells but was excluded from endocrine cells. Other endocrine cells tentatively identified in the developing human pancreas are serotonin, gastrin, epinephrine and norepinephrine cells.

Penicillins

<h3>SUMMARY</h3> X chromosome inactivation (XCI) is a developmental regulatory process that initiates with remarkable diversity in various mammalian species. Here we addressed the contribution of XCI regulators, most of which are lncRNA genes characterized in the mouse, to this mechanistic diversity. By combining analysis of single-cell RNA-seq data from early human embryogenesis with various functional assays in naïve and primed pluripotent stem cells and in differentiated cells, we demonstrate that <i>JPX</i> is a major regulator of <i>XIST</i> expression in human and in mouse. However, the underlying mechanisms differ radically between species and require <i>Jpx</i> RNA in the mouse and the act of transcription of <i>JPX</i> locus in the human. Moreover, biogenesis of <i>XIST</i> is affected at different regulatory steps between these species. This study illustrates how diversification of LRGs modes of action during evolution provide opportunities for innovations within constrained gene regulatory networks. <h3>Graphical abstract</h3>