Genome-wide profiling of DNA 5-hydroxymethylcytosine during rat Sertoli cell maturation.

Sertoli cells have dual roles during the cells' lifetime. In the juvenile mammal, Sertoli cells proliferate and create the structure of the testis, and during puberty they cease to proliferate and take on the adult role of supporting germ cells through spermatogenesis. Accordingly, many genes expressed in Sertoli cells during testis formation are repressed during spermatogenesis. 5-Hydroxymethylcytosine (5hmC) is a DNA modification enzymatically generated from 5mC and present in all investigated mammalian tissues at varying levels. Using mass spectrometry and immunofluorescence staining we identified a substantial Sertoli cell-specific global 5hmC increase during rat puberty. Chemical labeling, pull-down and sequencing of 5hmC-containing genomic DNA from juvenile and adult rat Sertoli cells revealed that genes that lose or gain 5hmC belong to different functional pathways and mirror the functions of the cells in the two different states. Loss of 5hmC is associated with genes involved in development and cell structure, whereas gain of 5hmC is associated with genes involved in cellular pathways pertaining to the function of the adult Sertoli cells. This redistribution during maturation shows that 5hmC is a dynamic nucleotide modification, correlated to gene expression.

Sequencing of FTO and ALKBH5 in men undergoing infertility work-up identifies an infertility-associated variant and two missense mutations.

OBJECTIVE: To assess whether men with reduced semen quality exhibit genetic variants in the genes coding for the messenger RNA methylation erasers FTO and ALKBH5. DESIGN: DNA of men undergoing infertility work-up was extracted and the FTO and ALKBH5 genes were sequenced. Statistical analysis was used to study the correlation between the identified ALKBH5 and FTO variants and sperm quality. SETTING: University hospital infertility clinic. PATIENT(S): Semen samples from 77 unselected men that had been referred to Oslo University Hospital for routine semen analysis as part of infertility work-up. INTERVENTION(S): Not applicable. MAIN OUTCOME MEASURE(S): Immunohistochemistry and Western blot were used to confirm the presence of ALKBH5 and FTO in human testis. DNA extraction from samples was followed by Illumina MiSeq amplicon high throughput sequencing and sequence alignment. Variant calling was carried out using GATK's UnifiedGenotyper. Standard semen parameter analysis was performed according to World Health Organization guidelines. RESULT(S): We found an FTO genetic variant to be associated with reduced semen quality. We also identified two FTO missense variants, one mutation (p.Cys326Ser) was located in the important linker between the two protein domains; the other mutation (p.Ser256Asn) was situated in a flexible loop able to interact with other molecules. CONCLUSION(S): The discovery of two missense mutations with potentially detrimental effect on the functionality of the methylation eraser protein FTO, as well as a genetic variant of the same protein that is associated with altered semen quality could suggest that aberrant demethylation of messenger RNA is a factor involved in reduced male fertility.