Downregulation of Reproductive Homeobox Gene 6 (Rhox6) Interferes with Male Germ Cell Differentiation.

Abstract

Homeodomain proteins contain a 60 amino acid DNA binding motif called homeobox that regulates a variety of developmental events such as determination of body segment identity, limb development and organogenesis. Recently, a novel cluster of homeobox genes has been discovered on the X chromosome of the mouse. Because of their selective expression in reproductive organs, they are named Reproductive homeobox genes on the X chromosome (Rhox). Rhox6, a member of the Rhox6 family, is expressed in primordial germ cells (PGCs) in the developing gonad and the placenta, although the biological function of Rhox6 in determination of PGCs and placental cells remains unknown. To investigate the Rhox6 function, we generated male chimeric mice by injection of the gene-trapped embryonic stem cells (ESCs) harboring a hypomorphic mutation in Rhox6 into wild-type blastocysts. Almost all of the male chimeric mice exhibited infertility associated with smaller testes, reduced sperm number and motility and abnormal morphology of the epididymal sperm. Next, to obtain a better understanding of the Rhox6 function in germ cell differentiation, we took advantage of in vitro ESC culture. ESCs can differentiate into PGCs when ESCs are cultured as aggregates called embryoid bodies (EBs) in the medium containing Ascorbic acid, 1-Thioglycerol and Transferrin without the leukemia inhibitory factor. By using a mouse ESC line that expresses the enhanced green fluorescent protein (EGFP) under the promoter of Oct3/4, a central regulator of cellular pluripotency, the presence of PGCs within EBs was monitored in real time by EGFP expression. While this ESC line was induced to differentiate into PGCs, expression of Rhox6 was downregulated by stable expression of the short-hairpin (sh) RNA against the Rhox6 transcripts. The level of Rhox6 knockdown was validated by RT-PCR. As a negative control, a scrambled nucleotide sequence that does not show any match with the mouse genome was used to express shRNA molecules. EBs were generated from multiple independent clones that express either one of the shRNAs, which were isolated by electroporation of the ESC line with each vector, followed by drug selection. We found that 0% of the EBs having the shRNA against the Rhox6 transcripts expressed (n=29; the number of clones used to generate EBs is 17) had EGFP-positive cells, whereas 43% of the EBs having the control shRNA expressed (n=35; the number of clones used to generate EBs is 25) had EGFP-positive cells. To validate differentiation of PGCs, these EBs were pooled according to EGFP expression and used to examine expression of PGC markers by semi-quantitative RT-PCR. Because expression of Rex1, a marker for undifferentiated ESCs, was significantly downregulated in these EBs than in undifferentiated ESCs, EGFP expression detected in the EBs indicates the presence of PGCs, but not the presence of undifferentiated ESCs. Rhox6 as well as other PGC markers including Oct3/4, Rnh2, Piwil2, and Fgls were downregulated in the EBs expressing the shRNA against the Rhox6 transcripts, whereas the EBs expressing the control shRNA with EGFP-positive cells expressed Rhox6 and these PGC markers. Collectively, these in vivo and in vitro studies strongly suggest that downregulation of Rhox6 impairs differentiation of PGCs. This study is supported by the Departmental Startup fund including the USDA Cooperative State Research, Education and Extension Service, Hatch project (ILLU-538-323). (poster)