3.1 Function of sperm miRNA in early embryo development
The function of sperm-borne miRNAs was ignored for a long time as they were thought to simply be remnants or byproducts of spermatogenesis. Recently, promising results were obtained regarding the regulation of sperm-borne miRNAs in early embryonic development [17, 18, 30]. One of the six miRNAs (miR-34c) expressed in sperm and zygotes, but not in oocytes and preimplantation embryos, was required for the first cell division in vitro . However, in vivo research using miR-34b/c and miR-449 double knockout (miR-dKO) mice demonstrated that the two miRNA clusters were indispensable for spermatogenesis and fertility, but not for fertilization or preimplantation development . The conflicting results were mainly due to the off-target effects of the inhibitor of miR-34c used in the former study, indicating that other sperm-borne miRNAs might be involved in early embryonic development. This assumption was further verified by germline-specific Dicer and Drosha conditional knockout in mice, which resulted in failure of zygotic genome activation and embryonic development . One member of this miRNA family, miR-34b, was differentially expressed in the sperm of young bulls; however, the blastocyst rates were not significantly affected , indicating that this miRNA does not have a lethal influence on bovine embryos. Molecular and cellular function analysis using IPA predicts that the differentially expressed sperm-borne miRNAs from young bulls will potentially influence the gene expression, cellular assembly and organization, and cellular function and maintenance at 2-cell stage, while later at blastocysts stage, these miRNAs will be likely to affect RNA damage and repair, protein synthesis, and small molecule biochemistry (Fig. 3). As in other species, it is therefore quite possible that the dominant biofunctions of these miRNAs is to carry paternal epigenetic effects.
3.2 sncRNAs as epigenetic inheritance factors
Epigenetic factors, including DNA methylation, non-coding RNAs, histone modification, and others, are more sensitive to environmental conditions compared to genetic information. Epigenetic information in sperm was proven to translate the environmental experiences of the father who could then transfer these acquired traits to his progeny . In a previous study from our lab, sperm from prepubertal bulls were characterized to bear a different DNA methylation pattern compared to sperm from post-pubertal bulls . Moreover, in the mouse, the global reprogramming during early embryonic development does not erase all methylation marks from the sperm, leaving a few regions for inter or transgenerational inheritance . In our study mentioned above, there were over 2,600 differentially methylated regions between sperm of bulls at 10 and 16 months of age, including one of them located on an imprinted gene . Recently, contradictory evidence cast some doubts on whether sperm DNA methylation is either the primary or the persistent epigenetic carrier of paternally acquired traits [33, 34]. Hence, other epigenetic factors such as sperm-borne sncRNAs are gaining more and more attention as transgenerational inheritance factors from the paternal side. The direct causal relationship between sperm RNAs and paternally inherited characteristics was first reported in a study in which total sperm RNA from traumatized mice was injected into wild type zygotes resulting in offspring with altered behavior and metabolism . Catenin β1 (Ctnnb1), which is involved in stress pathways, was validated as a target of miR-375 which was differentially expressed in mentally stressed mice. The expression of Ctnnb1 was inhibited in F2 offspring . This finding was further supported by various independent studies on the transgenerational inherited paternal acquired traits following mental stress , toxicant exposure , and diet changes . Moreover, paternal age is another factor related to the health of offspring. Advanced paternal age in human increases the probability of genetic disorders such as schizophrenia, bipolar disorder, and autism, as reviewed by Sharma et al. . Given that increased age generally also includes exposure to other complex environmental factors, such as physiological age, nutrition and lifestyle, genetics stability, hormones, senile diseases, oxidative stress, and telomere shortening , paternal effects of advanced age depend on both genetic and epigenetic changes. However, in our study of a relatively short age span in bulls (from 10 to 16 months old), epigenetic changes, especially sncRNAs, seemed to be affected by a young paternal age. In this study, fourteen miRNAs and one tsRNA were differentially expressed in sperm of younger bulls. Considering the vast amount of miRNAs stored in oocytes, sperm miRNAs that are also present in oocytes may have limited purposes, if any, in fertilization or early embryogenesis . Thus, we finally selected ten miRNAs that were exclusively expressed in paternal gametes, based on published transcriptomic data of bovine oocytes [20, 21], to focus on specific sperm effects.
Several of the paternal differentially expressed sncRNAs in our study were associated with environmentally induced epigenetic inheritance of metabolic traits. A high-fat and/or high-sugar diet (Western-like diet) induced obesity and type II diabetes in mice (F0) and these traits were inherited by the resulting progenies . The expression of several miRNAs, including miR-19b was inhibited in sperm of F0 mice . Intriguingly, when miR-19b was injected into wild type one-cell embryos, the offspring (here referred as R1) exhibited similar metabolic disorders as the F0 and F1 mice, and this pathological phenotype was further inherited after mating with healthy partners . In another study focusing on mental stress, offspring of running males had suppressed reinstatement of juvenile fear memory; meanwhile, the expression of sperm-borne miR-19b and miR-133a was altered in these fathers, indicating the potential involvement of these miRNAs in the effects of paternal exercise on the anxiolytic behavioral phenotype of offspring .
3.3 Metabolism related pathways in early embryo development
Normal development of the early embryo promotes further later normal development. For example, in bovine embryos, a faster cleavage of the zygote into a 2-cell embryo is positively correlated to a higher probability of the embryo becoming a viable blastocyst. In the present study, the transcriptome of bovine 2-cell embryos and genes related to developmental capacity were used to assess the value of target mRNA by sperm miRNA . We then used a functional analysis (IPA) of the targets of paternal age-related sperm miRNAs to find pathways that might be affected. Although IPA were not specific to cattle, but the enrichment analysis results were similar to DAVID analysis (data not shown). Moreover, IPA provides an activation z-score which infers to the activation status of biological function (activated or inhibited) . Our results indicated that the pathways targeted were related to metabolism and totipotency which are immediately important for embryo quality and survival.
In the two contrasts, insulin-like growth factor 1 (IGF1) signaling was predicted to be activated by the interaction of sperm miRNA and maternal targets in 2-cell embryos generated from sperm from younger bulls (10 and 12 months), compared to 16-month-old bulls. Insulin-like growth factor 1 levels are higher in younger growing animals and possibly involved in metabolic programming [41-43]. The addition of physiological concentrations of IGF1 (10-100 ng/mL) improved the developmental competence of bovine early embryos in vitro [44-46]. In mice, a study using a high IGF1 concentration, i.e. 130 nM (201 ng/mL), which mimics the pathological condition of polycystic ovary syndrome, induced apoptosis in blastocysts via down regulation of the IGF1 receptor (IGF1R)  and a p53 dependant mechanism . Similar results were also observed in bovine blastocysts supplemented with a supraphysiological concentration of IGF1 (1000 ng/mL), including induced apoptosis and decreased TP53 protein expression, but without inhibition of IGF1R . Hence, the beneficial or adverse effects of IGF1 are concentration dependant and abnormally over activated IGF1 signaling might impair early embryogenesis.
Activation of TGF-β signaling was predicted in sperm from 10-month-old vs 16-month-old bulls. This pathway is involved in the regulation of several aspects of early embryonic development, such as embryonic patterning, cell fate determination, and dynamic movements . The regulatory role of this pathway is exerted beyond the 2-cell stage in mouse embryos , indicating that it could possibly be programmed at the 2-8 cell stage in bovine embryos and impact blastocyst differentiation. The Rho family GTPases, a family of small signaling G proteins, are involved in sperm induced egg activation . In zygotes, inhibition of cell division cycle 42 (Cdc42), a member of the Rho family, significantly decreased in vitro development to the morula or blastocyst stage . The involvement of Rho family GTPases in polarisation of early mouse blastomeres was also demonstrated .
The IPA predicted that several insulin-related pathways were impacted by paternal age. Insulin activates its receptor to phosphorylate and recruit many substrates, including IRS and Shc, to modulate various canonical pathways, such as the PI3K/Akt, AMPK, and Ras/MAPK pathways [55, 56]. These pathways transduce the diverse effects of insulin on energy-associated cellular functions, such as glucose and lipid metabolism, protein synthesis, growth, and survival . Although insulin transcripts are not detectable at the preimplantation stage in bovine embryos, the mRNA encoding insulin receptors, IGFI, and IGFII can be detected starting at the 1-cell stage , indicating that bovine embryos are sensitive to insulin. Similar to other mammals, cattle fed a restricted diet exhibited a reduced basal insulin concentration and improved pancreatic insulin sensitivity . Hence, the insulin-related pathways predicted as activated in response to the different miRNAs in sperm from younger bulls might be associated with the prepubertal condition when energy is primarily devoted to growth, somewhat mimicking a feed restriction context. Transgenerational inheritance of paternal metabolic disorders was observed in various domestic farm animals . As a consequence, embryos from younger bulls might have altered metabolism compared to embryos from adult bulls.
3.4 Pre-implantation embryo developmental competence
Transcriptome analysis of two-cell embryos with different developmental dynamics revealed that DEGs in the earlier or later cleaving embryos were related to the cell cycle, gene expression, RNA processing, and protein degradation functions . Intriguingly, some of these DEGs were targets of the miRNAs related to bull age identified in the present study. The canonical pathway analysis of this dataset demonstrated that the IP3 pathway, PI3K pathway, and pluripotency related pathways could be impacted by paternal age and could therefore affect the developmental competence of two-cell embryos. The IP3 pathway has been identified as a common mechanism to activate the metabolism of unfertilized oocytes by inducing Ca2+ oscillations in various species, from marine invertebrates to mammals . During bovine oocyte fertilization, the IP3 pathway is required for Ca2+ release, and its inhibition impairs the first embryonic interphase . The PI3K pathway is widely studied as a mediator of cell growth, proliferation, differentiation, and survival signals. In mouse preimplantation embryos, the p85 and p110 subunits of PI3K were expressed from the one cell to the blastocyst stage . Moreover, deletion of 3‐phosphoinositide‐dependent protein kinase 1 (PDK1) arrested mouse embryos at the two-cell stage as a result of the inhibition of embryonic genome activation (EGA) . Pluripotency is the capacity of embryonic stem cells to differentiate into all cell types of a body and is controlled by several key regulators (OCT4, NANOG, SOX2, etc.). In bovine embryos, Oct4 and Sox2 transcripts were present at the pre-EGA phase [65, 66]. Meanwhile, during the maternal-to-embryonic transition, transcripts of genes involved in the maintenance of pluripotency of embryos were also observed . Hence, the paternal age-related miRNAs may potentially target pathways associated with two-cell competence to further impact early embryonic development.
In a previous study, we generated blastocysts in vitro with semen from different bulls of the same age as the ones used in the present study . The transcriptome of these blastocysts was influenced by paternal age, and canonical pathways related to the DEGs resulted in altered energy production and protein synthesis. Due to their relatively short half life, miRNAs from sperm might not survive to the blastocyst stage. However, the predicted upstream regulators of DEGs identified in blastocysts could mediate their effects on the transcriptome of blastocysts. In this case, we found four and three upstream regulators that were the targets of differentially expressed miRNAs in the 10 vs 16 months and 12 vs 16 months contrasts. Intriguingly, when we identified the downstream targets of these regulators and redid the IPA analysis, the results of the small fraction were highly consistent with the overall DEGs of the blastocyst transcriptome associated with the age effect, indicating that sperm miRNAs could be directly involved in the intergenerational inheritance of factors associated with paternal age.