Conserved functional characteristics of the PIWI family members in chicken germ cell lineage

The P-element-induced wimpy testis (PIWI) protein family, one of two subfamily members of Argonautes that regulate gene expression by associating with small noncoding RNAs, plays a role in maintaining the integrity of germ cells and stem cells. PIWI proteins have been identified and characterized in many species, including humans, mice, and zebrafish, but not as yet in avian species. In this study, chicken PIWI-like protein 1 (CIWI) and 2 (CILI) were identified from a 25-wk-old adult male testis cDNA library. Similar to mammals, CIWI and CILI have two conserved functional domains (PIWI/Argonaute/Zwille [PAZ] and PIWI), which bind the 3' end of single-stranded RNAs and the RNase H domain. The similarities between the PAZ and PIWI domain in CIWI were 65% and 80% to those of humans, and 67% and 80% to those of mice, respectively. In addition, the similarities between PAZ and PIWI in CILI were 60% and 61% to humans, and 60% and 63% to mice, respectively. Notably, the PIWI domain in the PIWIL1 protein showed high similarity between mammals and birds (80% between chickens and humans vs. 80% between chickens and mice), but the PIWI domain in PIWIL2 was less similar compared with PIWIL1 (61% between chickens and humans vs. 63% between chickens and mice). Reverse transcription polymerase chain reaction, quantitative real-time polymerase chain reaction, and in situ hybridization were conducted to analyze the expression patterns of PIWI family genes during gametogenesis in adult chickens during gonad development in the embryonic stage. CIWI and CILI showed germ cell-specific expression in both males and females, and CIWI and CILI mRNAs were distinctively expressed in different cell stages of spermatogenesis in testis. Using RNA interference, knockdowns of CIWI and CILI resulted in the increased expression of Chicken Repetitive 1 (CR1) Elements in primordial germ cells. Thus, the CIWI and CILI proteins of the PIWI family are conserved in avian species and play a critical role in germ line development and gametogenesis.