Abstract: PD08-02
Sources of Funding: ASH is a K12 scholar supported by a Male Reproductive Health Research (MRHR) Career Development Physician-Scientist Award (Grant # HD073917-01 to DJL) from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) Program

Introduction

We previously identified a homozygous, 6 nucleotide deletion in exon 22 of the intraflagellar transport 140 (IFT140) gene in a consanguineous family of non-obstructive azoospermic brothers using whole-exome sequencing. Data in Drosophila and mice implicate IFT family proteins in the modulation of cell signaling pathways, which are involved in the differentiation and proliferation of germ cells. The objective of this study is to determine the mechanism by which loss of IFT140 function may disrupt spermatogenesis.

Methods

Ift140 function was silenced in C18-4, a mouse type A spermatogonial cell line. Ift140 mRNA levels were evaluated using quantitative real-time PCR (qPCR). Gene expression studies were performed in triplicate on an oligonucleotide array (Qiagen) of 84 cell signaling genes of various known signaling pathways using Ift140 siRNA and a scrambled control (GE Dharmacon). Genes whose expression was more than 1.5x up or down relative to control were considered dysregulated and were validated and analyzed for statistical significance using REST software (Qiagen). The down-regulation of one target, Fas, was validated at the protein level by immunofluorescent staining. Knockdown and control cultures were treated with Sunitinib (2μM, 8 hours) to induce apoptosis, and a cell survival assay was performed. _x000D_

Results

Ift140 knockdown was 75% efficient in C18-4 cells at 72 hours. Forty-one genes were down-regulated and 25 genes up-regulated. Eighteen genes&[prime] expression changes were indeterminate due to low expression in both groups. The expression of seven genes (Fas, Slc27a4, Serpine1, Bcl2l1, Cebpd, Sqstm1, Socs3) was more than 2x down-regulated while 5 genes (Slc2a1, Stat1, Hes1, Bmp4, Wnt6) were 1.5x to 2x down-regulated. Expression of five genes (Jag1, Csf1, Wisp1, Gata3, Bcl2) was more than 1.5x up-regulated. Fas, Bcl2, Bcl2l1, Slc2a1, Csf1, andBmp4 were significantly dysregulated (p<0.05). Co-immunofluorescent staining showed concordant down-regulation of Fas protein in Ift140-silenced cells. Upon Sunitinib treatment to induce apoptosis, 63.4% of Ift140-silenced cells survived 8 hours compared to 25.7% of scramble-control cells (p<0.05)._x000D_

Conclusions

We observed statistically significant changes in the expression of key genes in the NFkB and TGFβ/BMP/Hedgehog pathways as well as major dysregulation in additional genes of the Notch, Wnt, Jak/Stat, TGFβ, PPAR, p53, and oxidative stress signaling pathways upon Ift140 knockdown in mouse male germ cells. The data suggests that silencing of Ift140 inhibits apoptosis, which may in turn cause spermatogenic failure._x000D_

Funding

ASH is a K12 scholar supported by a Male Reproductive Health Research (MRHR) Career Development Physician-Scientist Award (Grant # HD073917-01 to DJL) from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) Program