Soma-to-germline transmission of RNA in mice xenografted with human tumour cells: possible transport by exosomes

Abstract

Mendelian laws provide the universal founding paradigm for the mechanism of genetic inheritance through which characters are segregated and assorted. In recent years, however, parallel with the rapid growth of epigenetic studies, cases of inheritance deviating from Mendelian patterns have emerged. Growing studies underscore phenotypic variations and increased risk of pathologies that are transgenerationally inherited in a non-Mendelian fashion in the absence of any classically identifiable mutation or predisposing genetic lesion in the genome of individuals who develop the disease. Non-Mendelian inheritance is most often transmitted through the germline in consequence of primary events occurring in somatic cells, implying soma-to-germline transmission of information. While studies of sperm cells suggest that epigenetic variations can potentially underlie phenotypic alterations across generations, no instance of transmission of DNA- or RNA-mediated information from somatic to germ cells has been reported as yet. To address these issues, we have now generated a mouse model xenografted with human melanoma cells stably expressing EGFP-encoding plasmid. We find that EGFP RNA is released from the xenografted human cells into the bloodstream and eventually in spermatozoa of the mice. Tumor-released EGFP RNA is associated with an extracellular fraction processed for exosome purification and expressing exosomal markers, in all steps of the process, from the xenografted cancer cells to the spermatozoa of the recipient animals, strongly suggesting that exosomes are the carriers of a flow of information from somatic cells to gametes. Together, these results indicate that somatic RNA is transferred to sperm cells, which can therefore act as the final recipients of somatic cell-derived information.

Figure 1. Outline of the general procedure used for the stepwise detection of EGFP expression from tumor to sperm cells.

An A-375 melanoma derivative cell line stably expressing the EGFP reporter gene was obtained by infecting with an engineered letiviral vector. EGFP RNA, DNA and proteins were detected both in whole A-375 cells and in A-375-released exosomes. Cells were then xenografted in nude mice, 45 days after inoculation the animals were sacrificed and both blood-released exosomes and epidydimal spermatozoa were analyzed for EGFP-containing RNA.

Figure 2. Characterization of EGFP-expressing A-375 cells.

A: EGFP-specific RT-PCR amplification from RNA extracted from whole A-375 cells, either EGFP-infected (lane 1) or non-infected (lane 3), and from the extracellular exosome-containing fraction of infected (lane 5) and non-infected (lane 4) A-375 cells. In lane 2 no RNA was added to the amplification mix. GAPDH was used as standard control. B: PCR amplification of DNA extracted from whole A-375 cells, EGFP-infected (lane 1) and non-infected (lane 4), and from the extracellular exosomal fraction from infected (lane 6) or non-infected (lane 5) A-375; no DNA- and no primer-reactions were loaded for control in lanes 2 and 3, respectively. C: Western immunoblotting analysis of protein extracts from: non-infected A-375 cells (lane 1) and exosomal fraction (lane 2), and from infected A-375 cells (lane 3) and their exosomal fraction (lane 4). CD81 was used as a marker of exosomes.

Figure 3. EGFP-specific RNA in circulating blood from A-375/EGFP-inoculated mice.

A: Ethidium bromide staining of specific RT-PCR products from RNA extracted from EGFP-infected A-375 cells (lane 1) and blood-purified extracellular exosomal fraction from inoculated (lane 4) and non-inoculated (lane 5) mice. No RNA and no primers were added to the amplification mix in lanes 2 and 3, respectively. B: EGFP hybridization pattern. The gel in A was blotted on filter, hybridized with ³²P-end labelled EGFP-specific probe, washed and autoradiographed. C: GAPDH-specific amplification products from the same samples.

Figure 4. EGFP RNA is present in spermatozoa of mice inoculated with EGFP-infected A-375-cells.

A: Murine protamine 2 gene (Prm2) amplification products used to select DNA-free RNA samples. Exemplifying gel of Prm2-specific PCR amplification products of intron-containing DNA from the mouse sperm genome (lane 1) and RT-PCR products from RNA extracted from spermatozoa of non-inoculated (lane 2) and EGFP-expressing A-375⁺ inoculated (lane 3) mice, both showing the spliced Prm2 form. B: Southern blot hybridization of RT-PCR amplified RNA from: spermatozoa of non-inoculated control (lane 2) and A-375⁺ inoculated (lane 3) mice, and from non-infected (lane 4) and EGFP-infected (lane 5) A-375 whole cells. Hybridization was carried out with an EGFP-specific internal probe. Lane 1 is a no-RNA control. The bottom panel shows RT-PCR amplification products from the same samples using GAPDH-specific primers as a loading control. C: Southern blot hybridization of RT-PCR amplified RNA from spermatozoa from a control mouse (lane 2) and from a single EGFP-expressing A375⁺ inoculated mouse (lane 3); lane 1 shows a no RNA reaction. As in B, the bottom panel shows GAPDH amplification from the same samples. D: RT-PCR amplification with (+RT) or without (-RT) reverse transcription step of RNA extracted from sperm-depleted epididymis from two inoculated EGFP mice. No EGFP-specific amplification products were visible by ethidium bromide staining (EtBr) nor by Southern blot hybridization (Hyb) using an EGFP radioactive probe. The bottom panel shows GAPDH amplification from the same samples.