Discovery of tsRNAs in mature sperm

In year 2012, by analyzing mature sperm small RNAs by RNA-seq in mice, we serendipitously found that the mature sperm contain a unique subset of tRNA–derived small RNAs (tsRNAs), mainly from 5′ transfer RNA halves and ranging in size from 29-34 nucleotides. tsRNAs show a drastic increase at late- spermatogenesis and post- spermatogenesis during epididymis maturation, suggesting regulated tRNA cleavage or/and selective concentrating mechanisms at these stages. tsRNAs are enriched in the sperm head and thus could be delivered into oocytes during fertilization. Like tRNAs, tsRNAs are highly conserved, leading us to propose that tsRNAs might serve as an ancient paternal element with evolutionarily conserved functions. (Cell Res 2012; Highlighted in Cell Res 2013)

Hidden information in sperm small RNAs: tRNA-derived small RNA (tsRNA) & rRNA-derived small RNA (rsRNA)

Discovery of tsRNAs in serum, across vertebrate species

After identifying tsRNAs as a new type of sperm small RNAs, we further discovered that tsRNAs are also abundantly and conservatively present in the serum of a wide range of vertebrates (from fish to human), and that serum tsRNAs are sensitive to pathological conditions (e.g. active infection) in mice, monkey and human beings. Importantly, we found that tsRNAs extracted from serum are more stable than chemically synthetic tsRNAs of the same sequence, suggesting RNA modifications in tsRNAs would contribute to their stabilization in the serum, thus represent another layer of information. (J Mol Cell Biol 2014)

Sperm tsRNAs/rsRNAs, and RNA modifications, contribute to epigenetic inheritance of paternally acquired traits

Parallel with the discovery of tsRNAs in sperm and serum, we are fascinated about the increasing evidence that certain acquired traits during paternal exposure can be “memorized” in the sperm and inherited by the offspring, leading to a resurrection of the ideas of 'Lamarckian inheritance' and 'Darwin's Pangenesis' (Nat Rev Genet 2016Nat Rev Mol Cell Biol 2018). Recently, we found changes in expression profiles in sperm tsRNAs of mice with a paternal high-fat diet (HFD) (Science 2016), and later on also found another type of small RNAs, rRNA-derived small RNAs (rsRNAs), co-exist with tsRNAs in the sperm and they are sensitively regulated by HFD (Nat Cell Biol 2018). By injecting sperm total RNAs or different RNA fractions into normal zygotes, we found that the tsRNA/rsRNA-enriched 30-40nt sperm RNA fraction, along with associated RNA modifications, represent a carrier of paternal epigenetic information that contributes to intergenerational inheritance of diet-induced metabolic disorder (Science 2016; Nat Cell Biol 2018). We also demonstrated that a RNA methyltransferase, Dnmt2, can shape the sperm RNA ‘coding signature’ by regulating RNA modifications and tsRNA/rsRNA biogenesis, and affect RNAs’ secondary structures that may together contribute to the effect of epigenetic inheritance (Nat Cell Biol 2018). 

Mammalian pre-implantation embryos provide a unique opportunity to study how a single cell diverges from a totipotent state into different fates within limited rounds of cell division. There are two longstanding questions about this process: When does the first blastomere-to-blastomere asymmetry emerge and how does this relate to the development of distinct cell fates? It remains debatable whether the first bifurcation of cell fate emerges randomly at the morula stage, or if it has been initiated at earlier stages before morphological differences arise. Combining single-cell RNA-seq analysis and mathematical modeling, we recently showed that the very first symmetry-breaking process involves both chance separation and defined transcriptional circuits (Development 2015; Highlighted in Development 2015), a new framework for future  investigations.

Single-cell analysis: bridging randomness and determinism

From our single-embryo transcriptome analysis, small biases at molecular level will inevitably emerge at the 2-cell embryo stage, following a binomial distribution due to the cleavage division. At this stage, the blastomere-to-blastomere distribution seems random but during subsequent zygotic transcriptional activation, a “bistable pattern” emerges in some genes. Several lineage specifiers show a strong bias between different blastomeres thus providing potential for further increased asymmetry subsequently (Development 2015). These observations suggest a scenario of how order is created from a seemingly random process through the differential triggering of existing master regulators by the emergence of their small bias. As a triumph of our hypothesis, we further showed symmetry breaking driven by heterogeneous LincGET expression since 2-cell mouse embryo (Cell 2018).

Symmetry-breaking in mammalian early embryo: when and how?

With the increasing understandings of the information capacity mediated by sperm RNAs and RNA modifications and their involvement as novel layers of paternal hereditary information beyond DNA, we proposed the concept of 'sperm RNA code' (Nat Rev Endocrionl 2019). We are interested in exploring how the sperm RNA code intereact with epigenetic reprogramming, and embryonic translational programming such as ribosome heterogeneity that may influence offspring phenotypes (Nat Rev Endocrionl 2019)
To decode the sperm RNA code, we developed a small RNA analyzing software SPORTS1.0 with Tong Zhou lab (Genomics Proteomics Bioinformatics 2018) to facilitate the analyses of tsRNAs and rsRNAs. More recently, we develop PANDORA-seq to overcome RNA modifications that prevent small RNA detection in traditional RNA-seq (Nat Cell Biol 2021). PANDORA-seq uncovers a new and surprising small RNA landscape that is in fact, dominated by tsRNA/rsRNA, rather than miRNA in many tissues/cells. In mouse mature sperm, PANDORA-seq reveals that miRNAs take a tiny potion and tsRNAs are more abundant, while rsRNAs are dominant. Interestingly, tsRNAs are much more enriched in the sperm heads compared to that of whole sperm, suggesting yet to be discovered function in the nuclei (Nat Cell Biol 2021)

The 'sperm RNA Code' and how to decode it

Old dog, new tricks? novel function of Aquaporins beyond simple permeability

Discovery of Aquaporin-3 for rapid sperm osmoadaptation

In the journey from the male to female reproductive tract, mammalian sperm experiences a natural osmotic decrease (e.g., in mouse, from ~415 mOsm in the cauda epididymis to ~310 mOsm in the uterine cavity). On one hand, the hypotonic stress upon ejaculation is beneficial for the onset of mouse sperm motility (an evolutionary trait from fish sperm). However, this is a double-edged sword because the hypotonic stress could also cause potential harm to sperm function by inducing un-wanted swelling. To counteract this negative impact, mammalian sperm have acquired mechanisms for rapid transmembrane water movement to efficiently regulate cell volume. However, the specific sperm proteins responsible for this rapid osmoadaptation remain elusive. Using a knockout model, we discovered that Aquaporin-3 (AQP3) is an essential membrane protein for sperm regulatory volume decrease (RVD) upon physiological hypotonicity, balancing the “trade-off” between hypotonic induced sperm motility and cell swelling, thereby optimizing postcopulatory sperm behavior (Cell Res 2011).
However, we found that AQP3's role in sperm osmoadaptation cannot be fully explained by considering AQPs as inert pores simply for water permeability, the conventional view. We have discussed alternative possibilities for AQP3’s role in osmosensing or mechanosensing to regulate the subsequent RVD process (Acta Pharmacol Sin 2011).
Actually, in addition to our discovery, emerging evidence of other AQPs’ (such as AQP4, AQP5) having a role in cell volume regulation, can also not be fully explained by considering AQPs as inert pores simply for water permeability. We are now looking at the potential roles of AQPs as mechanosensors, especially in tissues with very low water permeability such as bladder, urethra etc.

AQP3's role as mechanosensor?

Compartmentalized intracellular reactions: an alternative sourece of heterogeneity

The game of fate: deterministic & stochastic factors

in addition to the potential influence of stochastic events such as gene expression noise and uneven random segregation at cell division, we propose that cell-to-cell heterogeneity may also be initiated by pre-existing molecular inhomogeneity regulated by intracellular compartmentalization (Nat Commun 2018). The evidence for a spatially confined subcellular transcriptome together with the idea of compartmentalized reaction space provides a viable explanation of how mechanical cues, e.g. cell–cell contact during development, can alter transcriptional patterns and cell fate. In general, within a compartmentalized intracellular reaction space, the property of the space itself (volume and shape) has an essential influence on reaction rate. Morphological changes to a cell, either due to intrinsic factors such as physical constraints arising during development or due to external forces, could alter the reaction space. This would then lead to region-specific changes in biochemical reaction rates and products that could alter intracellular properties. It is possible that different cleavage division patterns of the mammalian embryo can result in differential cell–cell contact that affects blastomere geometry, which create differential reaction spaces and thus cell properties that bias cell fate (Nat Commun 2018)
PANDORA-seq (right optic) reads more hidden
'RNA code' than traditional RNA-seq (let optic)
 Inspired by the movie: National Treasure