Contribution of CBX4 to cumulus oophorus cell phenotype in mice and attendant effects in cumulus cell cloned embryos

Abstract

Cumulus oophorus cells play an essential role in oocyte development. They are also widely employed as donor cells for cloning by somatic cell nuclear transfer. Our previous studies revealed that Cbx4 mRNA was overexpressed in cloned two-cell embryos. These data indicated that CBX4 may regulate normal cumulus cell differentiation and that its overexpression in clones could contribute to aberrant gene regulation. We used siRNA-mediated knockdown of Cbx4 to assess its role in determining cumulus cell phenotype and compared the effects of this knockdown to published data for aberrant gene regulation in cloned embryos. We observed widespread effects on the expression of genes related to diverse processes in cultured cumulus cells, including cell assembly/proliferation and DNA replication/repair, endocrine function, carbohydrate and lipid metabolism, inflammation, and cell morphology, with apparent effects of CBX4 in promoting cumulus cell proliferation and survival and inhibiting differentiation. Overall, the data implicate CBX4 as a key component in the pathway integrating endocrine signals, intraovarian paracrine factors, and oocyte-derived factors in the control of cumulus cell functions. We also observed altered expression of 25 cumulus cell markers of oocyte quality, indicating an important role of CBX4 in production of high quality oocytes. Finally, we found that about one-quarter of the genes showing aberrant transcription in cloned embryos are sensitive to Cbx4 knockdown in cumulus cells, consistent with a role for aberrant Cbx4 regulation in elaborating abnormal cloned embryo characteristics.

Keywords: chromatin; cloning; differentiation; follicle; nuclear reprogramming; ovary; polycomb; somatic cell nuclear transfer.

Fig. 1.

Confirmation of Cbx4 gene expression knockdown after siRNA treatment. Cells were lysed at 72 h of siRNA treatment and processed either for RNA or protein isolation. A: qRT-PCR analysis revealing reduced expression of Cbx4 mRNA. B: Western blot revealing reduction at the level of CBX4 protein.

Fig. 2.

Ingenuity Pathway Analysis (IPA) network 1 (see Supplemental Table S6). Names of molecules that are affected by Cbx4 siRNA treatment are preceded by ↑ (increased) and ↓ (decreased by treatment), and their symbols filled gray; darker shades of gray correspond to higher fold-change. Upstream regulators are marked with a thicker border. IPA employs the following symbols in its networks: A, activation; CP, chemical-protein interaction; E, expression; I, inhibition; LO, localization; M, biochemical modification; MB, group/complex membership; nTRR, nontargeting RNA-RNA interaction; P, phosphorylation/dephosphorylation; PD, protein-DNA interaction; PP, protein-protein interaction; PR, Protein-RNA binding; RB, regulation of binding; RE, reaction; T, transcription; TR, translocation; UB, ubiquitination; line, binding only; line with filled arrowhead, downstream effect; line with bar and arrowhead, inhibition and downstream effect; line with outlined arrowhead, translocation; solid line, direct interaction; broken line, indirect interaction; vertical diamond, enzyme; horizontal diamond, peptidase; horizontal oval, transcription regulator; vertical oval, transmembrane receptor; trapezoid, transporter; down-pointing triangle, kinase; square, cytokine; vertical rectangle, G-protein coupled receptor, horizontal rectangle, ligand-dependent nuclear receptor; dashed square, growth factor; dashed rectangle, ion channel; double circle, complex/group; circle, other.

Fig. 3.

IPA network 2 (see Supplemental Table S6). Symbols are as described in Fig. 2.

Fig. 4.

IPA network 8 (see Supplemental Table S6). Symbols are as described in Fig. 2.

Fig. 5.

IPA network 18 (see Supplemental Table S6). Symbols are as described in Fig. 2.

Fig. 6.

Summary of connections between CBX4 expression and cumulus cell characteristics. CBX4 activates or inhibits the expression of a variety of upstream regulators, which in turn regulate downstream gene networks that control the indicated cellular processes. *The observed effects of Cbx4 siRNA on FOXO1 and NUPR1 are opposite to the effects predicted by IPA on the basis of changes in the expression of downstream network members.