The Function Study Of Pnldc1 In Mouse Spermatogenesis

Sperm is the transmitter of paternal genetic information.Spermatogenic disorders inevitably lead to male sterility.The integrity of sperm genome is of great significance to the correct transmission of genetic information.The study of spermatogenesis-related regulatory mechanisms is of great significance for the diagnosis and treatment of infertility.A role for small non-coding RNAs in the regulation of spermatogenesis has always been a a research hotspot for reproductive medicine.Small non-coding RNAs expressed in spermatogenic cells include microRNAs(microRNAs),endogenous small interfering RNAs(endo-siRNAs)and piRNAs(PIWI-interacting RNAs).PiRNAs bind with PIWI proteins to form piRNA-induced silencing complexes(piRISC),silencing transposons and maintaining the integrity of germ cell genome.Playing important regulatory roles in many stages of spermatogenesis,piRNA-PIWI complexes participate in De Novo DNA methylation and may regulate the expression of protein coding genes in round spermatozoa.PiRNAs are produced through primary biogenensis and secondary biogenensis.Primary biogenensis begins with transcription of piRNA precursors.PiRNA precursors are cleaved into piRNA intermediates by endonuclease Zuc/MitoPLD.To form mature primary piRNAs,piRNA intermediates binding with PIWI proteins are trimmed at 3' terminals,followed by 2'-O-methylation.The maturation of secondary piRNAs also requires 3' end trimming and methylation.The exonucleases involved in the 3' end trimming of piRNA intermediates have been mysteries.A recent study in silkworms showed that Pnldc1,a member of the Parn exonuclease superfamily,was involved in the 3' end trimming of piRNA intermediates.A study in C.elegans showed that Parn-1,the homolog of Pnldc1,was also involved in the 3' end trimming of piRNA intermediates.However,the function of Pnldc1 in mammals has not been reported yet.In this study,we revealed the molecular function of Pnldc1 in mice and its impact on spermatogenesis.Pnldc1 is predominantly expressed in mouse testes.At the 1st to 3nd weeks after birth,the expression of Pnldc1 increased continuously.However,at the 4th to 8th weeks after birth,the expression of Pnldc1 dropped gradually.Further analysis confirmed that Pnldc1 was expressed in mouse spermatogonia,spermatocytes and round spermatozoa.We generated Pnldc1 knockout mice with CRISPR/Cas9.Male homozygotes were sterile with smaller testes.No sperm was observed in epididymis of male homozygotes.Histological analysis showed mixed spermatogenic abnormalities in homozygous males: meiotic arrest and spermatogenic arrest.Under normal circumstances,transposons are silenced by piRNA pathway.Abnormal activation of transposons indicates defects in piRNA biogenesis and function.Retrotransposon LINE1 was abnormally activated in spermatogenic cells of homozygous males.We then analyzed the expression of piRNA pathway proteins in spermatogenic cells of homozygous males.MILI,MIWI,TDRD1 and TDRKH were abnormally polarized in homozygous males.Mitochondria and intermitochondrial cement(IMC)are closely related to piRNA biogenesis.In many piRNA pathway mutants,formation of IMC and distribution of mitochondria were affected.Electron microscopy revealed that although IMC existed in spermatocytes,the distribution of mitochondria was polarized due to Pnldc1 knockout.We further explored the impact of Pnldc1 knockout on piRNA biogenesis.To analyzed the expression of piRNA precursors after Pnldc1 knockout,RNA-seq was carried out on Pnldc1-knockout testes and widtype controls.Quantitative analysis showed that the expression of piRNA precursors was not affected by Pnldc1 knockout.The influence on piRNA maturation by Pnldc1 knockout was demonstrated through small RNA sequencing.In Pnldc1 knockout males,generation of piRNA intermediates(generation of the 5' end of piRNAs)was unaffected.However,the 3' end trimming of piRNA intermediates was impeded by Pnldc1 knockout.In conclusion,mouse Pnldc1 participates in the 3' end trimming of piRNA intermediate without affecting the generation of piRNA precursors and intermediates.After Pnldc1 knockout,transposon silencing was impaired due to 3'-end extensions of piRNAs.Retrotransposon LINE1 was abnormally activated in Pnldc1-knockout mice,which damaged the genomic integrity of spermatogenic cells.At the meanwhile,regulation on proteion-coding genes of piRNAs with 3'-end extensions was also impacted,which eventually lead to spermatogenic disorders and male infertility.