Paternal contributions: new functional insights for spermatozoal RNA

Abstract

Whereas the presence of RNA in mature ejaculate spermatozoa is now established, its functional significance, if any, is still a matter of debate. This reflects the accepted description that spermatozoa are highly differentiated, specialized cells of minimal cytoplasm and compacted nucleus that are transcriptionally inactive. A significant proportion of the RNA required for the later, haploid stages of terminal spermatogenic differentiation (spermiogenesis) is synthesized prior to transcriptional arrest then stably stored until its translation during spermiogenesis. Spermatozoal RNAs, including messenger RNAs (mRNAs) are therefore considered to be stored remnants. Any role in fertilization and early development has, until recently, seemed unlikely, since the oocyte contains large stores of maternal mRNAs known to be required for early embryonic development prior to zygotic genome activation. Although the spermatozoon can deliver its RNA to the oocyte at fertilization, it has been generally assumed that compared to the oocyte RNA reserve, the spermatozoan payload is too small to be functional in embryo development. However, the debate continues as recent studies suggest that in specific instances sperm RNA is functional. This review presents and discusses the functional significance of spermatozoal RNA in relation to some recent advances in the field.

Figure 1

Functions of sperm RNA. Different outcomes proposed for RNA populations, mRNA, antisense and microRNA, retained in mature spermatozoa. The RNA transferred to the oocyte at fertilization may function in early embryo development. mRNA could be translated, e.g. PLCzeta to sustain Ca²⁺ oscillation. Some mRNAs will be degraded, e.g. protamine. Antisense and microRNAs may epigenetically modify and modulate early embryonic gene expression. Sperm RNA may also have a structural role. For example, nuclear RNA may target chromosome regions remaining histone-bound. RNA located in the midpiece may be translated under certain conditions, e.g. capacitation.

Figure 2

Localization of RNA to the nuclear matrix of human sperm nuclei. The histones and protamines were extracted using a solution containing 10 mM DTT and 2M NaCl. The DNA free of those proteins loops away and forms a halo around the core of DNA that remains attached to the nuclear matrix. The RiboGreen reagent was used to localize RNA to the human sperm halos. The specificity of the signal to RNA was verified by treating the halos with DNAse 1 (signal present) of RNAse (signal absent) prior to in situ hybridization.

Figure 3

Localization of the ropporin 1 transcript in human sperm nucleus. A) Localization in human sperm nucleus partially decondensed in the presence of DTT and Heparin. B) Localization in sperm halo preparations using 10 mM DTT and 2M NaCl to extract the protamines and histones respectively. The specificity of the signal for RNA was verified on decondensed nuclei and halo preparations treated with DNAse 1 (signal present) and RNAse (signal absent) before in situ hybridization.

Figure 4

Comparative Genomic Hybridization of histone and protamine bound spermatozoa DNA. Alignment of CGH profiles for histone (green) and protamine (red) probe signals with Ensembl gene density profiles for chromosomes 12 and 16. Note the tendency for gene density to more closely correspond with histone rather than protamine profiles.