Expression of proto-oncogene c-kit receptor in rats (Rattus norvegicus) and identification of a mutant mRNA transcript implicated in spermatogenic failure

Abstract

The pleiotropic proto-oncogene c-kit receptor, implicated in hematopoiesis and melanogenesis, is also known to play an important role in germ cell proliferation and differentiation although the mechanisms for the latter remain unknown. We studied c-kit expression by RT-PCR in various tissues of both fertile and infertile Brown Norway rats. Using different sets of primers, several regions from within the extracellular domain were amplified, cloned, and sequenced. One set of primers, in addition to the expected 352-bp amplicon, revealed a 276-bp transcript, although its biological functions remain unknown. These two transcripts showed varying levels of expression in different tissues of infertile rats against nearly uniform expression in the fertile animals. Significantly, the 352 bp testis transcript showed mutational hotspots from nucleotide 84-266 in the infertile rats. Analysis of testis and brain genomic DNA from these infertile rats showed mutations only in the testis suggesting this to be a postzygotic event. In contrast, no mutation was detected in the genomic DNA of testis and brain of the fertile rats. Protein expression studies showed complete absence of the cytoplasmic kinase domain and soluble c-kit protein in one of the infertile rats. Histological examination of testis of these infertile animals showed stem cell depletion resulting in fewer germ cells. Based on these results, we infer that 352-bp mutant mRNA transcript is implicated in the spermatogenic failure.