Arrest of human spermatogenesis at the pachytene stage is frequently accompanied by disruption of the piRNA pathway

Abstract

Recent genetic studies have identified defects in the PIWI-interacting RNA (piRNA) pathway to cause nonobstructive azoospermia. However, limited knowledge of piRNA function in human spermatogenesis makes it difficult to quantify the number of cases affected. Central to the piRNA biogenesis are the transcription factor MYB proto-oncogene like 1 (MYBL1), which transcribes most piRNA clusters, and PIWI-like 1 (PIWIL1), which is critical for the processing of piRNAs. Here, we show that testicular biopsies with complete spermatogenesis ( n  = 55) and biopsies with spermatogenic arrest at the round spermatid stage ( n  = 32) consistently exhibit a robust expression of MYBL1 and PIWIL1 proteins in pachytene spermatocytes and until round spermatids. In contrast, MYBL1 and/or PIWIL1 expression was lost in 59% (19/32) and 42% (15/36) of the biopsies with homogeneous and heterogeneous arrest at the pachytene stage, respectively. Biopsies showing arrest at the earlier leptotene stage ( n  = 17) did not express MYBL1 or PIWIL1 because pachytene spermatocytes were absent. In biopsies without MYBL1 and PIWIL1 protein expression, RNA in situ hybridization revealed a concurrent decrease in PIWIL1 , but not MYBL1 , transcripts. Small RNA sequencing further demonstrated a significant and near-complete loss of pachytene piRNAs, but not pre-pachytene piRNAs, in biopsies without MYBL1 and PIWIL1 protein expression. Although we cannot establish direct causality, our data suggest that defective piRNA processing can be linked to arrest of spermatogenesis at the pachytene stage in half (34/68) of the cases. This establishes the piRNA pathway as a frequent and central determinant of human spermatogenic arrest with a distinctive molecular signature of impaired germ‑cell differentiation.