219 EXPRESSION PATTERN OF THE SUB-CORTICAL MATERNAL COMPLEX IN OVINE OOCYTES AND PRE-IMPLANTATION EMBRYOS

The sub-cortical maternal complex (SCMC) is a multi-protein complex located in the sub-cortex of the oocyte. In mouse, it assembles during oocyte growth and is essential for zygotes to progress beyond the first embryonic cell divisions (Li et al., 2008). At least 4 proteins contribute to the complex: oocyte expressed protein (OOEP), maternal antigen that embryo requires (MATER), transducin-like enhancer of split 6 (TLE6), and ES cell associated transcript 1 (ECAT1), all encoded by maternal effect genes. In mouse, the relative transcripts are degraded during meiotic maturation and ovulation, whereas the SCMC proteins persist in the early embryo. Whereas MATER expression has been studied in several species, the existence of the genes encoding the other components has been assessed in few mammalian species and their pattern of expression during pre-implantation development has been analysed only in mouse (Li et al. 2008 Dev. Cell 15, 416–425). In a previous work (Bebbere et al. 2008 Reprod. Fertil. Dev. 20, 908–915), we assessed MATER existence and pattern of expression in the ovine species. The aim of the present work was to assess the existence of OOEP, TLE6, and FILIA in the ovine species and to analyse the expression pattern of the 4 genes in the oocytes and during pre-implantation embryo development. Total RNA was isolated and reverse transcribed from pools of immature (GV) and in vitro matured (IVM) metaphase II (MII) oocytes, from in vitro fertilized and cultured (IVFC) embryos at the 2-, 4-, 8-, and 16-cell stage and from blastocysts. Three pools of 10 oocyte/embryos were analysed for each class. Primers were designed on the basis of the sequences conserved among orthologs and amplify intron-spanning regions. The PCR products were sequenced, and the alignment, performed with BLASTn, confirmed the homology with the orthologous genes present in public databases. Real-time PCR analysis revealed that all 4 transcripts are present at its highest level in the GV oocyte but decrease during embryo pre-implantation development with a gene-specific pattern. Conversely to the pattern of expression observed in mouse, all 4 transcripts persisted until the 8-cell stage embryo, disappearing only at the 16-cell stage. No transcripts were detected at the blastocyst stage. This study confirms the existence of transcripts related to SCMC also in the ovine species, but highlights species-specific patterns of expression in the 2 species, possibly related to the different time of activation of the embryo genome in mouse and in sheep. The observed expression patterns suggest an involvement of the protein complex in oocyte maturation and in the very first phases of life, possibly in the transition from the maternal to embryonic program of development.

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Signalling pathways and mechanistic cues highlighted by transcriptomic analysis of primordial, primary, and secondary ovarian follicles in domestic cat

Scientific Reports ◽

10.1038/s41598-021-82051-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Shauna Kehoe ◽

Katarina Jewgenow ◽

Paul R. Johnston ◽

Susan Mbedi ◽

Beate C. Braun

Keyword(s):

Gene Expression ◽

Transforming Growth Factor ◽

Ovarian Follicle ◽

Mammalian Species ◽

Expression Patterns ◽

Ovarian Follicles ◽

Domestic Cat ◽

Transcriptional Analysis ◽

Ovarian Folliculogenesis

AbstractIn vitro growth (IVG) of dormant primordial ovarian follicles aims to produce mature competent oocytes for assisted reproduction. Success is dependent on optimal in vitro conditions complemented with an understanding of oocyte and ovarian follicle development in vivo. Complete IVG has not been achieved in any other mammalian species besides mice. Furthermore, ovarian folliculogenesis remains sparsely understood overall. Here, gene expression patterns were characterised by RNA-sequencing in primordial (PrF), primary (PF), and secondary (SF) ovarian follicles from Felis catus (domestic cat) ovaries. Two major transitions were investigated: PrF-PF and PF-SF. Transcriptional analysis revealed a higher proportion in gene expression changes during the PrF-PF transition. Key influencing factors during this transition included the interaction between the extracellular matrix (ECM) and matrix metalloproteinase (MMPs) along with nuclear components such as, histone HIST1H1T (H1.6). Conserved signalling factors and expression patterns previously described during mammalian ovarian folliculogenesis were observed. Species-specific features during domestic cat ovarian folliculogenesis were also found. The signalling pathway terms “PI3K-Akt”, “transforming growth factor-β receptor”, “ErbB”, and “HIF-1” from the functional annotation analysis were studied. Some results highlighted mechanistic cues potentially involved in PrF development in the domestic cat. Overall, this study provides an insight into regulatory factors and pathways during preantral ovarian folliculogenesis in domestic cat.

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Identification, Expression, and Functions of the Somatostatin Gene Family in Spotted Scat (Scatophagus argus)

Genes ◽

10.3390/genes11020194 ◽

2020 ◽

Vol 11 (2) ◽

pp. 194

Author(s):

Peizhe Feng ◽

Changxu Tian ◽

Xinghua Lin ◽

Dongneng Jiang ◽

Hongjuan Shi ◽

...

Keyword(s):

Gene Family ◽

Growth Regulation ◽

Expression Patterns ◽

Open Reading Frames ◽

Pcr Analysis ◽

Sequence Alignments ◽

Scatophagus Argus ◽

Gene Structure And Expression ◽

Liver Fragments

Somatostatins (SSTs) are a family of proteins consisting of structurally diverse polypeptides that play important roles in the growth regulation in vertebrates. In the present study, four somatostatin genes (SST1, SST3, SST5, and SST6) were identified and characterized in the spotted scat (Scatophagus argus). The open reading frames (ORFs) of SST1, SST3, SST5, and SST6 cDNA consist of 372, 384, 321, and 333 bp, respectively, and encode proteins of 123, 127, 106, and 110 amino acids, respectively. Amino acid sequence alignments indicated that all SST genes contained conserved somatostatin signature motifs. Real-time PCR analysis showed that the SST genes were expressed in a tissue specific manner. When liver fragments were cultured in vitro with synthetic peptides (SST1, SST2, or SST6 at 1 μM or 10 μM) for 3 h or 6 h, the expression of insulin-like growth factor 1 and 2 (Igf-1 and Igf-2) in the liver decreased significantly. Treatment with SST5 had no significant effect on Igf-1 and Igf-2 gene expression. This study provides an enhanced understanding of the gene structure and expression patterns of the SST gene family in S. argus. Furthermore, this study provides a foundation for future exploration into the role of SST genes in growth and development.

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Transcription Factor Lbx1 Expression in Mouse Embryonic Stem Cell-Derived Phenotypes

Stem Cells International ◽

10.4061/2011/130970 ◽

2011 ◽

Vol 2011 ◽

pp. 1-7 ◽

Cited By ~ 5

Author(s):

Stefanie Schmitteckert ◽

Cornelia Ziegler ◽

Liane Kartes ◽

Alexandra Rolletschek

Keyword(s):

Transcription Factor ◽

Developmental Stages ◽

Troponin T ◽

Es Cells ◽

Expression Patterns ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cell ◽

Cardiac Cells ◽

Cell Stage

Transcription factor Lbx1 is known to play a role in the migration of muscle progenitor cells in limb buds and also in neuronal determination processes. In addition, involvement of Lbx1 in cardiac neural crest-related cardiogenesis was postulated. Here, we used mouse embryonic stem (ES) cells which have the capacity to develop into cells of all three primary germ layers. Duringin vitrodifferentiation, ES cells recapitulate cellular developmental processes and gene expression patterns of early embryogenesis. Transcript analysis revealed a significant upregulation ofLbx1at the progenitor cell stage. Immunofluorescence staining confirmed the expression of Lbx1 in skeletal muscle cell progenitors and GABAergic neurons. To verify the presence of Lbx1 in cardiac cells, triple immunocytochemistry of ES cell-derived cardiomyocytes and a quantification assay were performed at different developmental stages. Colabeling of Lbx1 and cardiac specific markers troponin T, α-actinin, GATA4, and Nkx2.5 suggested a potential role in early myocardial development.

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171 THE EFFECT OF TRICHOSTATIN A ON THE DEVELOPMENT AND THE GLOBAL GENE EXPRESSION PATTERNS IN MOUSE EMBRYOS DEVELOPING IN VITRO

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab171 ◽

2008 ◽

Vol 20 (1) ◽

pp. 165

Author(s):

X. S. Cui ◽

X. Y. Li ◽

T. Kim ◽

N.-H. Kim

Keyword(s):

Gene Expression ◽

Trichostatin A ◽

Expression Profiles ◽

Expression Patterns ◽

Gene Expression Profiles ◽

Mouse Embryos ◽

Differentially Expressed ◽

Gene Expression Patterns ◽

Cell Stage

Trichostatin A (TSA) is an inhibitor of histone deacetylase and is able to alter gene expression patterns by interfering with the removal of acetyl groups from histones. The aim of this study was to determine the effect of TSA treatment on the development and gene expression patterns of mouse zygotes developing in vitro. The addition of 100 nm TSA to the culture medium did not affect the cleavage of mouse embryos (TSA treatment, 148/150 (99%) v. control, 107/107 (100%)); however, embryos that were treated with TSA arrested at the 2-cell stage (145/148, 98%). We estimated the number of nuclei in control and TSA-treated embryos by propidium iodide staining, taking into account the presence of any cells with two or more nuclei. At 62–63 h post-hCG stimulation, control zygotes had developed to the 4-cell stage and exhibited one nucleus in each blastomere, indicative of normal development. In contrast, we observed tetraploid nuclei in at least one blastomere in 20.8% (11/53) of the embryos that had been treated with TSA. At 28–29 h post-hCG stimulation (metaphase of the 1-cell stage), there was no difference in the mitotic index (as determined by analyzing the microtubule configuration) in the TSA group compared to the control group. At the 2-cell stage, however, we did not observe mitotic spindles and metaphase chromatin in embryos in the TSA treatment group compared to the controls. Interestingly, when embryos were cultured in TSA-free medium from 35 h post-hCG stimulation (S- or early G2-phase of the 2-cell stage) onward, almost all of them (47/50) developed to the blastocyst stage. In contrast, when embryos were cultured in TSA-free medium from 42 h post-hCG stimulation (middle G2-phase of the 2-cell stage) onward, they did not develop to the 4-cell stage. We used Illumina microarray technology to analyze the gene expression profiles in control and TSA-treated late 2-cell-stage embryos. Applied Biosystems Expression System software was used to extract assay signals and assay signal-to-noise ratio values from the microarray images. Our data showed that 897 genes were significantly (P < 0.05; 2-sample t-test) up- or down-regulated by TSA treatment compared to controls. Analysis using the PANTHER classification system (https://panther.appliedbiosystems.com) revealed that the 575 genes that were differentially expressed in the TSA group compared to the control were classified as being associated with putative biological processes or molecular function. Overall, in terms of putative biological processes, more nucleoside, nucleotide, and nucleic acid metabolism, protein metabolism and modification, signal transduction, developmental process, and cell cycle genes were differentially expressed between the TSA and control groups. In terms of putative molecular function, more nucleic acid-binding transcription factor and transferase genes were differentially expressed between the groups. The results collectively suggest that inhibition of histone acetylation in mouse embryos affects gene expression profiles at the time of zygotic genome activation, and this subsequently affects further development.

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214 PRE-IMPLANTATION EMBRYO SEX DETERMINATION BY AMPLIFICATION OF AMELOGENIN GENE IN WATER BUFFALOES (BUBALUS BUBALIS)

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab214 ◽

2016 ◽

Vol 28 (2) ◽

pp. 238

Author(s):

J.-H. Shang ◽

C.-Y. Yang ◽

H.-Y. Zheng ◽

J. Qin ◽

Y.-P. Gu ◽

...

Keyword(s):

Y Chromosome ◽

Natural Science ◽

Water Buffalo ◽

Tooth Enamel ◽

Mammalian Species ◽

Early Embryo ◽

Specific Pattern ◽

Specific Sequence ◽

Embryo Sex

Sex-specific sequence variability of the amelogenin (AMEL) gene has been observed in a variety of mammalian species. AMEL is a multi-copy gene expressed in abundance in the development of mammalian tooth enamel matrix. Its homologous genes are located on the X- or Y-chromosome, respectively. In our study, a pair of primers was designed to allow amplification of a single fragment of 312 bp from buffalo cows. Because the gene is slightly differently encoded on the Y-chromosome, the same primers were expected to produce 2 fragments of 312 and 249 bp from bulls. The primers were successfully applied to single, in vitro-produced Day-6 to Day-8 water buffalo blastocysts by direct lysis of the whole blastocysts, followed by PCR. The amplification on presumed female and male embryos consistently displayed the same sex-specific pattern. Of 100 blastocysts, 43 were determined to be females, 51 males, and 6 unknown (samples missed). We concluded that PCR amplification of the AMEL gene can be used for early embryo sex identification of water buffalo. This work was supported by the National Natural Science Foundation of China under Grant No. 31160456 and the Guangxi Natural Science Foundation of China under Grants No. 2013GXNSFAA019075 and 2014GXNSFAA118116.

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Mesenchymal Stem Cell Differentiation Markers Shared with Soft Tissue Sarcomas

Blood ◽

10.1182/blood.v112.11.4755.4755 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4755-4755

Author(s):

Stefan Wirths ◽

Hans-Joerg Buehring ◽

Lothar Kanz ◽

Joerg T Hartmann ◽

Hans-Georg Kopp

Keyword(s):

Stem Cells ◽

Soft Tissue ◽

Expression Pattern ◽

Cell Lines ◽

Expression Profiles ◽

Expression Patterns ◽

Soft Tissue Sarcomas ◽

Stem Cell Differentiation ◽

Antibody Panel

Abstract Malignant tumors are hypothesized to harbor small populations of self-renewing cancer stem cells. Targeting these cells may be the decisive step to overcome treatment resistance and achieve tumor eradication in cancer patients. Advanced soft tissue sarcomas (STS) are rare tumors with a dismal prognosis and a small number of systemic treatment options. STS may originate from mesenchymal stem cells (MSC); the latter have mainly been isolated from adult bone marrow (BM) as non-hematopoietic, self-renewing cells whose in vitro progeny comprises osteoblasts, chondroblasts, myocytes, and adipocytes. While in vitro expression profiles of MSC have been investigated extensively, the in vivo counterparts of MSC are still hypothetical. To target rare human cell BM populations including MSC, an exclusive antibody panel was developed. The target antigens include platelet-derived growth factor receptor-β (CD140b), HER-2/erbB2 (CD340), the recently described W8B2 antigen as well as several surface antigens identified by novel antibodies. To define the expression pattern of MSC-markers in STS, three STS cell lines were tested for expression of these antigens. In addition, snap-frozen primary STS sections were analyzed by immunohistochemistry using the same antibody panel. All cell lines revealed expression of selected markers including CD340, W8B2, and CD140b. Several MSC markers were restricted to a subpopulation of cells. In addition, leiomyosarcoma cells displayed a different expression pattern as compared to liposarcoma and Ewing’s sarcoma cells. Results of immunohistochemistry analysis of primary leiomyosarcoma tumor samples correlated strongly with expression patterns established by FACS analysis. However, important cytoarchitectural features regarding selected markers were revealed by immunohistochemistry: while primary leiomyosarcomas displayed uniform expression of W7C6, HEK3D6, CD10, and CD318, other markers such as CD34, W5C5, and 57D2 were expressed by tumor endothelia only. Moreover, a population of perivascular tumor cells was found to express the MSC-markers W4A5, W8B2, CD140b, W3D5, and W5C4. Novel MSC-markers are expressed by subpopulations in STS cell lines as well as in primary sarcoma tissue. Further studies on the functional significance of these phenotypical studies are underway and may help to identify novel specific targets recognizing the self-renewing STS-compartment.

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Identification of protein IT of the intestinal cytoskeleton as a novel type I cytokeratin with unusual properties and expression patterns.

The Journal of Cell Biology ◽

10.1083/jcb.111.2.567 ◽

1990 ◽

Vol 111 (2) ◽

pp. 567-580 ◽

Cited By ~ 249

Author(s):

R Moll ◽

D L Schiller ◽

W W Franke

Keyword(s):

Mammalian Species ◽

Expression Patterns ◽

Prostate Gland ◽

Coiled Coil ◽

Immunological Methods ◽

Type I ◽

Peptide Fragments ◽

Binding Assays ◽

Human Intestinal Epithelium

A major cytoskeletal polypeptide (Mr approximately 46,000; protein IT) of human intestinal epithelium was characterized by biochemical and immunological methods. The polypeptide, which was identified as a specific and genuine mRNA product by translation in vitro, reacted, in immunoblotting after SDS-PAGE, only with one of numerous cytokeratin (CK) antisera tested but with none of many monoclonal CK antibodies. In vitro, it formed heterotypic complexes with the type II CK 8, as shown by blot binding assays and gel electrophoresis in 4 M urea, and these complexes assembled into intermediate filaments (IFs) under appropriate conditions. A chymotrypsin-resistant Mr approximately 38,000 core fragment of protein IT could be obtained from cytoskeletal IFs, indicating its inclusion in a coiled coil. Antibodies raised against protein IT decorated typical CK fibril arrays in normal and transformed intestinal cells. Four proteolytic peptide fragments obtained from purified polypeptide IT exhibited significant amino acid sequence homology with corresponding regions of coils I and II of the rod domain of several other type I CKs. Immunocytochemically, the protein was specifically detected as a prominent component of intestinal and gastric foveolar epithelium, urothelial umbrella cells, and Merkel cells of epidermis. Sparse positive epithelial cells were noted in the thymus, bronchus, gall bladder, and prostate gland. The expression of protein IT was generally maintained in primary and metastatic colorectal carcinomas as well as in cell cultures derived therefrom. A corresponding protein was also found in several other mammalian species. We conclude that polypeptide IT is an integral IF component which is related, though somewhat distantly, to type I CKs, and, therefore, we propose to add it to the human CK catalogue as CK 20.

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Transcription of paternal Y-linked genes in the human zygote as early as the pronucleate stage

Zygote ◽

10.1017/s0967199400002100 ◽

1994 ◽

Vol 2 (4) ◽

pp. 281-287 ◽

Cited By ~ 76

Author(s):

Asangla Ao ◽

Robert P. Erickson ◽

Robert M.L. Winston ◽

Alan H Handysude

Keyword(s):

Mammalian Species ◽

Blastocyst Stage ◽

Preimplantation Embryos ◽

Human Embryos ◽

Cell Stage ◽

Gonad Differentiation ◽

Embryonic Genome ◽

Human Preimplantation Embryos

SummaryGlobal activation of the embryonic genome occurs at the 4– to 8–cell stage in human embryos and is marked by continuation of early cleavage divisions in the presence of transcriptional inhibitors. Here we demonstrate, using recerse transcripase–polymerase chin reaction (Rt–PCR), the presence of transcripts for wo paternal Y chromosomal genes, ZFY and SRY in human preimplantation embryos. ZFY transcripts were detected as early as the pronucleate stage, 20–24 h post-insemination In vitro and at intermediate stages up to the blastocyst stage. SRY Transcripts were also detected at 2–cell to blastocyos observed in many mammalian species focuses attention on the role of events in six determination prior to gonad differentiation.

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Effect of medium conditioned with rat hepatoma BRL cells on ‘2-cell block’ of random-bred mouse embryos cultured in vitro

Zygote ◽

10.1017/s0967199408005121 ◽

2009 ◽

Vol 17 (2) ◽

pp. 169-174 ◽

Cited By ~ 3

Author(s):

Masayuki Kobayashi ◽

Yoshinori Terawaki ◽

Koichi Saito ◽

Kano Kasuga ◽

Ikuo Kojima

Keyword(s):

Specific Inhibitor ◽

Reversed Phase ◽

Mouse Embryos ◽

Mouse Strains ◽

Pcr Analysis ◽

Cell Block ◽

Cell Stage ◽

B Subunit ◽

Rat Hepatoma

SummaryThe phenomenon of developmental arrest at the 2-cell stage of zygotes obtained from certain mouse strains during in vitro culture is known as the 2-cell block. The effect of conditioned medium (CM) with rat hepatoma BRL cells on the 2-cell block of CD-1 mouse zygotes was investigated in comparison with that of CM with rat hepatoma Reuber H-35 cells. In control medium with EDTA, 75.4% of 2-cell embryos developed to the 4- to 8-cell stages. In the same conditions, the BRL Mr <10000 fraction inhibited the development of 2-cell embryos to the 4- to 8-cell stages (57.7%), although the inhibition by this fraction was weaker than by the Reuber Mr <10000 fraction (19.8%). As a result of reversed-phase column chromatography, a 2-cell stage specific inhibitor of the cleavage of mouse embryos (Fr.B-25), which separated into the Mr <10000 fraction of the Reuber CM, was detected at a low level in the BRL Mr <10000 fraction. On the other hand, the Mr >10000 fraction of BRL CM accelerated the development of the embryos (90.3%). This beneficial effect was also evident even in the absence of EDTA. RT-PCR analysis revealed that mRNAs encoding the β-A or β-B subunit of activins (Mr ~29000), which are well characterized cytokines that act as releasers of the 2-cell block, were expressed in BRL cells. These results indicate that BRL cells synthesize Fr.B-25 at low levels, and that activins contained in the BRL CM probably contributed to overcoming the 2-cell block of CD-1 zygotes cultured in vitro.

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144 Oct-4 EXPRESSION PATTERN IN THE EQUINE EMBRYO

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab144 ◽

2007 ◽

Vol 19 (1) ◽

pp. 189

Author(s):

Y. H. Choi ◽

H. D. Harding ◽

A. D. Obermiller ◽

K. Hinrichs

Keyword(s):

Embryonic Development ◽

Expression Pattern ◽

Embryo Development ◽

Inner Cell Mass ◽

Ex Vivo ◽

Expression Patterns ◽

Cell Mass ◽

Population Variation ◽

Early Embryonic Development

Oct-4 is a key transcription factor in the control of early embryonic development and maintenance of a pluripotent cell population. Variation in Oct-4 expression patterns during embryo development have been reported among species, and have been related to the time of placental development in those species. This study was conducted to investigate Oct-4 expression pattern during early embryonic development in the horse, a species with relatively delayed placentation. In vitro-produced embryos were obtained from in vitro-matured oocytes via fertilization by intracytoplasmic sperm injection. Ex vivo blastocysts were recovered from mares that had been artificially inseminated. Oct-4 status was determined by immunocytochemistry; photomicrographs were taken at 4 standardized settings to aid in qualitative comparison of the amount of fluorescence. A total of 106 oocytes and embryos were evaluated. Immature oocytes showed Oct-4 expression in the nucleus and cytoplasm, as did early-cleaved embryos (2 to 5 cells, 1 to 2 days). Oct-4 expression in embryos at 3 to 4 days (6 to 12 cells) decreased and was restricted to the cytoplasm. From 5 to 6 days (15 cells to morulae), Oct-4 intensity increased and was exclusively found in the nuclei. In vitro-produced blastocysts (7 to 8 days) expressed Oct-4 equivalently in the trophectoderm and inner cell mass nuclei; culture for 2 to 3 more days (10 to 11 days) did not alter Oct-4 expression. However, when in vitro-produced blastocysts were transferred to the uteri of mares and recovered after 2 to 3 days (IVP-ET), the embryos showed strong expression of Oct-4 within the inner cell mass and limited expression in the trophectoderm, and a similar pattern was seen for ex vivo-recovered embryos. In bigger embryos (such as a 1779-�m ex vivo embryo and a 1121-�m IVP-ET embryo), the trophectoderm lost staining completely. These results suggest that Oct-4 expression is present in both nucleus and cytoplasm in equine oocytes and early-cleaved embryos as a result of maternal mRNA accumulation. Oct-4 protein decreases over the first few days of embryonic development as these stores are used. The shift to greater expression, in the nucleus only, during further embryo development suggests embryonic genome activation. Oct-4 expression in the trophectoderm of in vitro-produced blastocysts was different from that in blastocysts that had been exposed to the uterus (both ex vivo and IVP-ET); this indicates that differentiation of the trophectoderm is dependent upon factors present in the uterine environment. The Oct-4 expression in the trophectoderm of in vitro-produced equine blastocysts thus appears to be an artifact due to in vitro culture; this finding may be applicable to the reported patterns of Oct-4 expression in embryos of other species. This work was supported by the Link Equine Research Endowment Fund, Texas A&M University.

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