The Role of Lhx9 in the Male Reproductive System

Abstract

Leydig cells (LCs) are found within the interstitium of the testes. LCs are crucial to male reproductive function due to their role in testosterone synthesis, a process vital for both organogenesis and secondary sex characteristic development. The generation of LCs differs between foetal and adult testes. During embryonic development, LCs differentiate from progenitors, their numbers peaking at embryonic day 15.5 (E15.5), then degenerating at birth. Postnatally, the stem LC (SLC) population is activated at puberty, providing the precursor pool where the adult LCs (ALCs) will later differentiate from. The exact mechanisms of this process are not well understood. The LIM homeobox 9 gene, Lhx9, is essential for the formation of the gonad, it is also expressed later in development in the testes interstitium in both foetal and adult LCs. The functional role of Lhx9 associated with this expression has been poorly characterised. We hypothesise that the Lhx9 is vital for forming the progenitor population from which LCs later differentiate. The effect of Lhx9 loss on the foetal or adult LC pools has not been studied.

Gene expression was compared between Lhx9+/+ (WT) and Lhx9+/- (HET) mouse testes at two stages: E15.5 (using RT-qPCR), and adult (RNA-Sequencing). In the adult testes, 51 genes were found to be differentially expressed between the WT and HET mice. Additionally, gene ontology analysis revealed enrichment for genes involved in lipid metabolism. This pathway is utilised by LCs for testosterone production. At E15.5, expression of marker genes for progenitor stem cell populations, such as Nestin and Notch, was significantly reduced in the HET testes compared to the WT littermates. Conversely, steroidogenesis genes showed a significant increase in expression; supporting the hypothesis that Lhx9 acts to maintain the LC progenitor population in the testes.

Treatment of testes with ethane dimethanesulfonate (EDS) in vivo causes the death of Leydig cells, allowing analysis of their regenerative capability. Preliminary results of qPCR analysis following EDS treatment in adult HET and WT testes shows altered expression of a few genes, such as a decrease in Nestin in EDS treated HET mice. Together, these results will not only provide further understanding of the role of Lhx9 in foetal and adult testis, but additionally help illustrate delicate balance of progenitor populations and their impact on secondary sex determination and fertility in the adult.