(369) Generation and characterization of Rhox homeobox transcription factor mutants in primordial germ cells of mice

James A. MacLean^1,2; Jenna Douglas¹; Cynthia M. R. Francis¹; Lisette A. Maddison^1,2; Miranda L. Bernhardt^1,2; Kanako Hayashi^1,2

1. School of Molecular Biosciences, Washington State University, Pullman, WA USA

2. Center for Reproductive Biology, Washington State University, Pullman, WA USA

Abstract Text:

Homeobox genes encode transcription factors that have well-known roles in tissue development.  Our work has centered on the discovery and characterization of the Reproductive Homeobox X-linked (Rhox) gene cluster.  The Rhox family represents a distinct class of homeobox genes, which are selectively expressed in the developing embryo, postnatal and adult gonads, and accessory tissues associated with mammalian reproduction.  The largest and best-known Rhox cluster is found in mice.  However, all mammalian genomes examined to-date possess genes encoding RHOX factors in the syntenic region of the X chromosome.  The restriction to normal reproductive tissues is conserved between species, especially in the gonads.  The disruption of human RHOX gene expression and naturally occurring mutations has been linked to oligospermia.

Our prior research has focused on the role of Rhox5 and Rhox8, the only members of the cluster that are expressed in somatic Sertoli cells of the gonad and we have demonstrated that ablation of either one results in male subfertility.  The goal of this study was to generate mice lacking both Rhox6 and Rhox9 using CRISPR/Cas9 and characterize the impact on germ cell colonization of the testis and ultimately male fertility.  Previously, standard targeting strategies have been untenable as the two genes reside 74.7 kb apart on the X chromosome and breeding single knockouts would rarely produce a double.  Ablation of both genes is likely necessary as Rhox6 is 95% identical to Rhox9 and is expressed at the same place and time.  In support of this, Rhox9-null mice, generated by homologous recombination before Rhox6 was known to exist, had no phenotype.  This is most likely due to redundancy with RHOX6, which we will test by dual ablation (DKO). 

To date, we have generated Rhox6-null mice and are screening putative DKO founder lines.  We expect that loss of both RHOX factors will lead to improper differentiation of PGC and severely compromised spermatogenesis.  Eight additional members of the Rhox cluster have germ-cell restricted expression in the testis.  However, these other RHOX factors are not likely to functionally compensate for RHOX6/9 in PGC as they are all expressed later in testis development and predominantly postnatally in the seminiferous tubules. While a postnatal role for RHOX10 in supporting the germ stem cell niche that maintains adult spermatogenesis is known.  The actions of Rhox6 and Rhox9 in prenatal gonads, specifically their role in primordial germ cells (PGC), represents a new window for RHOX factor action which has not previously been investigated systematically using transgenic animals by any group.  However, in vitro spermatogenesis systems have suggested a potential role for Rhox6 in embryonic stem cell renewal and PGC differentiation, giving strength to the premise the knockout will be interesting.