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Human spermatogenesis requires an orchestrated expression of numerous genes in various germ cell subtypes. Therefore, the genetic landscape of male infertility is highly complex. Known genetic factors alone account for at least 15% of male infertility. However, ~40% of infertile men remain undiagnosed and are classified as idiopathic infertile men. We performed exome sequencing in 47 idiopathic infertile men (discovery cohort), followed by replication study (40 variants in 33 genes) in 844 infertile men and 709 controls using Sequenom MassARRAY® based genotyping. We report 17 variants in twelve genes that comprise both previously reported (DNAH8, DNAH17, FISP2 and SPEF2) and novel candidate genes (BRDT, CETN1, CATSPERD, GMCL1, SPATA6, TSSK4, TSKS and ZNF318) for male infertility. The latter have a strong biological nexus to human spermatogenesis and their respective mouse knockouts are concordant with human phenotypes. One candidate gene CETN1, identified in this study, was sequenced in another independent cohort of 840 infertile and 689 fertile men. Further, CETN1 variants were functionally characterized using biophysical and cell biology approaches. We demonstrate that CETN1 variant- p.Met72Thr leads to multipolar cells, fragmented nuclei during mitosis leading to cell death and show significantly perturbed ciliary disassembly dynamics. Whereas CETN1–5′ UTR variant; rs367716858 leads to loss of a methylation site and increased reporter gene expression in vitro. We report a total of eight novel candidate genes identified by exome sequencing, which may have diagnostic relevance and can contribute to improved diagnostic workup and clinical management of male infertility.

A multibranched hierarchy of regulatory genes controls all aspects of somatic sexual development in Drosophila melanogaster. One branch of this hierarchy is headed by the fruitless (fru) gene and functions in the central nervous system, where it is necessary for male courtship behavior as well as the differentiation of a male-specific abdominal structure, the muscle of Lawrence (MOL). A preliminary investigation of several of the mutations described here showed that the fru gene also has a sex-nonspecific vital function. The fru gene produces a complex set of transcripts through the use of four promoters and alternative splicing. Only the primary transcripts produced from the most distal (P1) promoter are sex-specifically spliced under direction of the sex-determination hierarchy. We have analyzed eight new fru mutations, created by X-ray mutagenesis and P-element excision, to try to gain insight into the relationship of specific transcript classes to specific fru functions. Males that lack the P1-derived fru transcripts show a complete absence of sexual behavior, but no other defects besides the loss of the MOL. Both males and females that have reduced levels of transcripts from the P3 promoter develop into adults but frequently die after failing to eclose. Analysis of the morphology and behavior of adult escapers showed that P3-encoded functions are required for the proper differentiation and eversion of imaginal discs. Furthermore, the reduction in the size of the neuromuscular junctions on abdominal muscles in these animals suggests that one of fru's sex-nonspecific functions involves general aspects of neuronal differentiation. In mutants that lack all fru transcripts as well as a small number of adjacent genes, animals die at an early pupal stage, indicating that fru's function is required only during late development. Thus, fru functions both in the sex-determination regulatory hierarchy to control male sexual behavior through sex-specific transcripts and sex-nonspecifically to control the development of imaginal discs and motorneuronal synapses during adult development through sex-nonspecific transcript classes.