Phylogenomic Analyses Reveal the Evolutionary Origin of the Inhibin α-Subunit, a Unique TGFβ Superfamily Antagonist

Transforming growth factor-beta (TGFβ) homologues form a diverse superfamily that arose early in animal evolution and control cellular function through membrane-spanning, conserved serine-threonine kinases (RII and RI receptors). Activin and inhibin are related dimers within the TGFβ superfamily that share a common β-subunit. The evolution of the inhibin α-subunit created the only antagonist within the TGFβ superfamily and the only member known to act as an endocrine hormone. This hormone introduced a new level of complexity and control to vertebrate reproductive function. The novel functions of the inhibin α-subunit appear to reflect specific insertion-deletion changes within the inhibin β-subunit that occurred during evolution. Using phylogenomic analysis, we correlated specific insertions with the acquisition of distinct functions that underlie the phenotypic complexity of vertebrate reproductive processes. This phylogenomic approach presents a new way of understanding the structure-function relationships between inhibin, activin, and the larger TGFβ superfamily

Role of PCSK5 Expression in Mouse Ovarian Follicle Development: Identification of the Inhibin α- and β-Subunits as Candidate Substrates

Inhibin and activin are essential dimeric glycoproteins belonging to the transforming growth factor-beta (TGFβ) superfamily. Inhibin is a heterodimer of α- and β-subunits, whereas activin is a homodimer of β-subunits. Production of inhibin is regulated during the reproductive cycle and requires the processing of pro-ligands to produce mature hormone. Furin is a subtilisin-like proprotein convertase (proconvertase) that activates precursor proteins by cleavage at basic sites during their transit through the secretory pathway and/or at the cell surface. We hypothesized that furin-like proconvertases are central regulators of inhibin α- and β-subunit processing within the ovary. We analyzed the expression of the proconvertases furin, PCSK5, PCSK6, and PCSK7 in the developing mouse ovary by real-time quantitative RT-PCR. The data showed that proconvertase enzymes are temporally expressed in ovarian cells. With the transition from two-layer secondary to pre-antral follicle, only PCSK5 mRNA was significantly elevated. Activin A selectively enhanced expression of PCSK5 mRNA and decreased expression of furin and PCSK6 in cultured two-layer secondary follicles. Inhibition of proconvertase enzyme activity by dec-RVKR-chloromethylketone (CMK), a highly specific and potent competitive inhibitor of subtilisin-like proconvertases, significantly impeded both inhibin α- and β-subunit maturation in murine granulosa cells. Overexpression of PC5/6 in furin-deficient cells led to increased inhibin α- and βB-subunit maturation. Our data support the role of proconvertase PCSK5 in the processing of ovarian inhibin subunits during folliculogenesis and suggest that this enzyme may be an important regulator of inhibin and activin bioavailability

Detection of allelic expression utilizing RNA fluorescent in situ hybridization in mouse embryonic cells

grantor: University of TorontoGenomic imprinting in mammals is defined as a reversible epigenetic process which renders the two parental genomes functionally nonequivalent in the developing embryo. Monoallelic expression is observed exclusively in seventeen imprinted genes identified to date, however the time at which transcriptional silencing of one allele occurs is variable throughout development and dependent on the gene in question. RNA fluorescent in situ hybridization (FISH) is utilised in this study as a method of detecting when one allele of an imprinted gene undergoes transcriptional silencing in the embryonic development of the mouse. The aim of this work is to utilize this sensitive FISH technique, which targets the nascent transcripts of genes, in order to visualise transcriptionally active alleles. Examination of the levels of active transcription in each individual cell in a spectrum of tissues will reveal if all nuclei act synonymously and determine at what embryonic stage an imprinted locus is silenced. This thesis attempts to optimize the conditions for RNA FISH in two models--cultured cell lines and embryonic tissues of the mouse.M.Sc

A Comparative Analysis of Hippo Signaling Pathway Components during Murine and Bovine Early Mammalian Embryogenesis

The time required for successful blastocyst formation varies among multiple species. The formation of a blastocyst is governed by numerous molecular cell signaling pathways, such as the Hippo signaling pathway. The Hippo signaling pathway is initiated by increased cell–cell contact and via apical polarity proteins (AMOT, PARD6, and NF2) during the period of preimplantation embryogenesis. Cell–cell contact and cell polarity activate (phosphorylates) the core cascade components of the pathway (mammalian sterile twenty like 1 and 2 (MST1/2) and large tumor suppressor 1 and 2 (LATS1/2)), which in turn phosphorylate the downstream effectors of the pathway (YAP1/TAZ). The Hippo pathway remains inactive with YAP1 (Yes Associated protein 1) present inside the nucleus in the trophectoderm (TE) cells (polar blastomeres) of the mouse blastocyst. In the inner cell mass (ICM) cells (apolar blastomeres), the pathway is activated with p-YAP1 present in the cytoplasm. On the contrary, during bovine embryogenesis, p-YAP1 is exclusively present in the nucleus in both TE and ICM cells. Contrary to mouse embryos, transcription co activator with PDZ-binding motif (TAZ) (also known as WWTR1) is also predominantly present in the cytoplasm in all the blastomeres during bovine embryogenesis. This review outlines the major differences in the localization and function of Hippo signaling pathway components of murine and bovine preimplantation embryos, suggesting significant differences in the regulation of this pathway in between the two species. The variance observed in the Hippo signaling pathway between murine and bovine embryos confirms that both of these early embryonic models are quite distinct. Moreover, based on the similarity of the Hippo signaling pathway between bovine and human early embryo development, bovine embryos could be an alternate model for understanding the regulation of the Hippo signaling pathway in human embryos

Modulation of GSTA1 levels mediate changes in Caco-2 cell growth.

<p>Effect of (A) GSTA1 down-regulation and (B) GSTA1-V5 overexpression on Caco-2 cell viability evaluated by MTS assay over three days. Asterisks depict significant differences between controls and the cells with GSTA1 modulated levels (*, <i>p</i>≤0.05; and **, <i>p</i>≤0.01). (C) Effect of GSTA1-V5 over-expression on cellular proliferation at 72 h as determined by BrdU incorporation in Caco-2 cells. Bars indicated by different letters differ significantly from one another (p≤0.001). Values represent the mean ± S.E. of four independent experiments with three replicates each.</p

NaB (10 mM) causes GSTA1-JNK complex dissociation without activating JNK in Caco-2 cells.

<p>(A) Representative western blot of GSTA1 (∼25 KDa) and GST Pi (∼26 KDa) protein levels in GSTA1-JNK complexes. Cells were transiently transfected with GSTA1 siRNA and non-specific (NS) siRNA for 72 h and treated with 10 mM NaB. GSTA1-JNK complexes were then pulled-down from cell lysates using c-Jun fusion beads. (B) Representative western blot of phosphorylated JNK (∼54 KDa and 46 KDa) and phosphorylated p38 (∼43 KDa) protein expression in preconfluent Caco-2 cells with the treatment of 10 mM NaB. β-actin (∼42 KDa) was used as a protein loading control.</p

GSTA1 down-regulation increases the percentage of Caco-2 cells in the S phase.

<p>(A) Changes of cell cycle phase distribution in GSTA1 down-regulated Caco-2 cells as compared to controls. (B) Graphic representation of percent of cells in G1, S and G2 phase of cell cycle in non-transfected control, GSTA1 siRNA and NS siRNA transfected Caco-2 cells. Asterisks depict significant differences between control and GSTA1 down-regulated cells (*, p≤0.05; and **, p≤0.01).</p

Modulation of GSTA1 does not affect NaB-induced differentiation.

<p>Preconfluent Caco-2 cells were transiently transfected with 40 nM of GSTA1 siRNA or non-specific (NS) siRNA and after 72 h, cells were treated with 1 mM NaB for 72 h; (A) GSTA1 activity (nmol/mg/min) was determined in GSTA1 down-regulated cells. (B) AlkP activity (µmol/mg/min) was measured to determine the effect of GSTA1 down-regulation on NaB-induced differentiation. (C) Preconfluent Caco-2 cells were transiently transfected with one µg of either GSTA1-V5 or empty vector (EV) for 48 h and were treated with 1 mM NaB for 48 h. AlkP activity (µmol/mg/min) was measured to determine the effect of GSTA1 over-expression on NaB-induced differentiation. Values represent the mean ± S.E. of three independent experiments with six replicates each. Bars indicated by different letters differ significantly from one another (p≤0.001).</p