Identification of PIWIL1 Isoforms and Their Expression in Bovine Testes, Oocytes, and Early Embryos1

Abstract

PIWI proteins are members of the larger Argonaute family and bind to specific 24-32 nucleotide RNAs called PIWI-interacting RNAs (piRNAs). PIWI-interacting RNAs direct PIWI-mediated suppression of retrotransposon expression in the male germline in humans and mice, but their roles in bovine reproduction and embryogenesis are unknown. Although the majority of research in mammals has focused on the functions of PIWI proteins during spermatogenesis, this family of proteins and their associated piRNAs have recently been identified in early embryos. The goals of this study were to characterize the expression of PIWIL1 in bovine testis, oocytes, and early embryos. A full-lengthPIWIL1transcript and protein was found in the testis, specifically in the germs cells of mature seminiferous tubules. RNA-immunoprecipitation demonstrated the presence of putative piRNAs with a mean length of 30 nucleotides bound to PIWIL1 in testes. 3'-Rapid amplification of cDNA ends analysis ofPIWIL1transcripts in testes and oocytes revealed two shorter isoforms in addition to the full-length transcript that was only present in testes. TruncatedPIWIL1isoforms in oocytes and testes were confirmed through amplification of their unique intronic fragments. Expression profiling ofPIWIL1through early embryogenesis demonstrated peak mRNA expression at the 2-cell stage with decreasing levels through to the blastocyst. PIWIL1-YFP fusion plasmids were produced for each isoform and expressed in HEK 293 cells, demonstrating nuclear exclusion and size-specific banding of the different isoforms. These data represent the first comprehensive characterization of PIWIL1 in bovine, revealing functional similarities with PIWIL1 in other species and suggest tissue-specific expression of several isoforms.

PIWIs, piRNAs and Retrotransposons: Complex battles during reprogramming in gametes and early embryos

“…Following discoveries in Drosophila , one mouse PIWI homolog, MIWI (PIWI‐like 1, or PIWIL1), was shown to be a cytoplasmic protein required for normal mammalian spermatogenesis (Deng et al., ). The expression of PIWIL1 begins at 14 days post‐partum (dpp) in pachytene spermatocytes and persists to the round spermatid stage (Deng et al., ) with similar patterns in the dog (Stalker et al., ) and bull (Russell et al., ). PIWIL1 functions to maintain GSCs through post‐transcriptional gene silencing (PTGS) of transposable elements (TEs).…”

“…One of the most important and widely recognized pathways controlling TE expression during reprogramming is the PIWI (P‐element‐Induced WImpy testes) pathway, named for the phenotype of Drosophila mutants carrying genetic defects in this pathway. Much of the initial mechanistic work has been performed in Drosophila and murine models; however, recent studies (Kowalczykiewicz, Pawlak, Lechniak, & Wrzesinski, ; Russell et al., , ; Stalker, Russell, Co, Foster, & LaMarre, ) have confirmed that these pathways are also present in domestic animals and appear to play similar roles. Table summarizes the PIWI proteins in different species.…”

“…However, the factors that control their expression (transcription factors, epigenetic state) are still not fully understood. Cluster expression occurs in Drosophila nurse cells and in developing spermatocytes of mice and other animals (Aravin et al., ; Brennecke et al., ; Girard et al., ; Lau, Ohsumi, Borowsky, Kingston, & Blower, ; Russell et al., ). They are transcribed from a single transcription start site, either uni‐ or bidirectionally, by RNA Polymerase II (Pol II).…”

“…Subsequent studies have provided strong evidence that the turnover of mRNAs expressed during the pachytene stage of spermatogenesis and is mediated by the piRNA pathway in multiple species (Gebert et al., ; Gou et al., ; Reuter et al., ). Very recently, we have identified piRNA‐like RNAs with sequences that appear to target mRNAs present in bovine testes and oocytes (Russell et al., b), suggesting that they may participate in mRNA turnover during bovine spermatogenesis and the maternal to zygotic transition during embryo development. Some controversy remains in this area, particularly in the early embryo, and it will be important to functionally validate the role of the PIWI pathway in this context before it can be definitively implicated in these processes in mammals.…”

Epigenetic dynamics and interplay during spermatogenesis and embryogenesis: implications for male fertility and offspring health

“…Generally, piRNAs are associated with the PIWI proteins and other adjuncts, to form the piRNA induced silencing complex (piRISC) in germ granules (Figure 3 ). Taking into account the expression patterns of piRNAs and PIWI proteins in male germ cells and early embryos, we believe that piRISC participates in the gene regulation at the transcriptional, post-transcriptional and even translational levels to some extent [ 34 , 35 ]. The piRISC regulates gene silencing and maintains the genome stability through cleaving of the target mRNAs, or directing the DNA methylation dependent silencing towards target loci in the genome [ 36 ].…”