Generation of biologically active retro‐genes upon interaction of mouse spermatozoa with exogenous DNA

Abstract

Mature spermatozoa of most animal species can spontaneously take up foreign DNA molecules which can be delivered to embryos upon fertilization. Following this procedure, transgenic animals of various species have been generated. We recently discovered a reverse transcriptase (RT) activity in mouse spermatozoa that can reverse-transcribe exogenous RNA molecules into cDNA copies. These cDNA copies are transferred to embryos at fertilization, mosaic propagated as non-integrated structures in tissues of founder individuals and further transmitted to F1 progeny. Reverse-transcribed sequences behave as functional genes, being correctly expressed in tissues of F0 and F1 animals. To learn more about this mechanism and further characterize the reverse transcription step, we have now incubated spermatozoa with a plasmid harboring a green fluorescent protein (EGFP) retrotransposition cassette interrupted by an intron in the opposite orientation to the EGFP gene. We found that reverse-transcribed spliced EGFP DNA sequences are generated in sperm cells and transmitted to embryos in IVF assays. After implantation in foster mothers, embryos developed into mice that expressed EGFP in the blood vessel endothelia of a variety of organs. The EGFP-encoding cDNA sequences were detected in positive tissues as extrachromosomal mosaic-propagated structures, maintained in low-copy number (<1 copy/genome), and mosaic transmitted from founders to the F1 progeny. These results indicate that an efficient machinery is present in mature spermatozoa, which can transcribe, splice, and reverse-transcribe exogenous DNA molecules. This mechanism is implicated in the genesis and non-Mendelian propagation of new genetic information besides that contained in chromosomes.

Comparative profiling of small RNAs of pig seminal plasma and ejaculated and epididymal sperm

“…It is believed that the sperm RNA is residual to spermatogenesis, and, according to this hypothesis, sperm RNA is a 'by-product' of the gradual shutdown of the nuclear transcriptional apparatus, and sperm does not support any nuclear gene transcription. However, Pittoggi and coworkers found that the transcription and splicing activities are occasionally initiated in mature sperm (Pittoggi et al 2006). Moreover, a very high abundance of sperm adhesins and most CRISP and Fn-type II mRNAs were found in ejaculated sperm, most of which exhibited weak or no expression in the testes (Song et al 2010(Song et al , 2011.…”

piRNAs derived from ancient viral processed pseudogenes as transgenerational sequence-specific immune memory in mammals

“…Such an effect is feasible and consistent with current models of the mammalian piRNA system if ancient bornaviruses were transmitted vertically via the gamete. Indeed, the ancient bornaviruses giving rise to EBLNs infected germ cells; otherwise a noncanonical mechanism of gene transfer from soma to germline, in violation of Weismann's law, was responsible for EBLN formation (Pittoggi et al 2006). Of note, modern bornaviruses can be transmitted vertically (Okamoto et al 2003;Kerski et al 2012).…”

“…We hypothesize that, lacking RNA-dependent RNA-polymerases used to amplify RNAs for viral silencing in organisms for which this function is well-established (Baulcombe 2004;Rechavi et al 2011), some metazoans could use a multistep process: reverse transcription of viral transcripts followed by RNA polymerization templated on viral complementary DNA (cDNA). Sufficient reverse transcriptase activity acting on viral RNA is present in some mammalian cells to allow laboratory time-scale experiments (Pittoggi et al 2006;Horie et al 2010); LINE-1 ORF2p has recently been suggested to be the responsible enzyme in human cells (Shimizu et al 2014). While no function has been ascribed to cDNA generated by this activity in mammals, millions of years ago).…”

Spermatozoal RNA: why is it there and what does it do?

“…These polymerases were originally reported in human spermatozoa (and seminal plasma) back in the 1970s [43,44]. More intriguing is the more recent observation that a DNA construct containing an intronic sequence can be processed by a spermatozoal machinery such that the intron is correctly spliced out [45].…”