Abstract
Infertility caused by male factors is routinely diagnosed by assessing traditional semen parameters. Growing evidence has indicated that the tsRNAs carried in sperm act as epigenetic factors and potential biomarkers for the assessment of sperm quality. We recently demonstrated that tRNAGln-TTG derived small RNAs played notable roles in the first cleavage of a porcine embryo. However, the function of human sperm tRNAGln-TTG derived small RNAs as a diagnostic biomarker and its role in early embryo development remains unclear. In this study, we found that human sperm tRNAGln-TTG derived small RNAs were highly associated with sperm quality. By microinjecting the antisense sequence into human tripronuclear (3PN) zygotes followed by single-cell RNA-sequencing, we found that human sperm tRNAGln-TTG derived small RNAs participated in the development of a human embryo. Furthermore, Gln-TTGs might influence embryonic genome activation by modulating noncoding RNA processing. These findings demonstrated that human sperm tRNAGln-TTG derived small RNAs could be potential diagnostic biomarkers and could be used as a clinical target for male infertility.
Introduction
Infertility is a common problem for couples with approximately one out of eight failing to conceive after 1 year of trying (Kelley et al. 2019). The diagnostic tests indicate that 1/3 male factors and 1/3 female factors caused the sterility, however, the other 1/3 are regarded as unexplained or idiopathic infertility (Turner et al. 2020). Sperm concentration and motility are classic diagnostic tests for male problems, however, the sperm carried contents could serve as the potential diagnostic criteria as well (Olds-Clarke 2003, Mortimer 2018). Growing evidence suggests that the epigenetic factors that are carried in sperm, including histone methylation, DNA methylation, and RNAs, do have an impact on embryo development (Jenkins et al. 2017). RNAs are distributed in the head and residual cytoplasm of the sperm (Godia et al. 2018). Sperm RNAs not only participate in the regulation of embryo development, but also reflect if the male is healthy (Platts et al. 2007, Jodar et al. 2015). Hence, the role of human sperm RNAs as potential diagnostic biomarkers for infertility needs to be explored.
The total RNAs that contain long noncoding RNAs and small noncoding RNAs in human spermatozoa amount to 10–400 fg per sperm (Goodrich et al. 2007). Specifically, small noncoding RNAs play roles in governing genome integrity, post-transcriptional regulation, early embryo development and intergenerational inheritance (Chen et al. 2016b). In human sperm, 56% of small noncoding RNAs are from tRNA derived small RNAs (tsRNAs), 18% from rRNA derived small RNAs (rsRNAs), 6% from micro RNAs (miRNAs), and 4% from piRNAs (Hua et al. 2019). Among them, tsRNAs, rsRNAs, and miRNAs are significantly associated with sperm quality (Hua et al. 2019). Notably, tsRNAs dynamically present in germ cells during spermatogenesis and sperm maturation, and are highly abundant in sperm (Peng et al. 2012, Sharma et al. 2018, Chen et al. 2020). However, it is still unclear whether tsRNAs can be used as the biomarker for male fertility.
The known tsRNAs so far are classified into two groups. One group known as tRNA halves, which is around 35 nt in length (Fu et al. 2009), is predominantly stress-induced and generated by cleavages at the anticodon loop. The second group generated by cleavages at the D or T loop in a size of around 20 nt is similar to small interfering RNAs (siRNAs) and miRNAs (Sobala & Hutvagner 2011). Several novel functions of tsRNAs have recently been identified, including regulation of RNA stability, translation, stress responses, and cell proliferation (Yamasaki et al. 2009, Emara et al. 2010, Ivanov et al. 2011, Goodarzi et al. 2015). Sperm tsRNAs delivered into the embryos act as the acquired epigenetic factors reflecting the environment stress (Chen et al. 2016a). In addition, tRNAGly-GCC derived small RNAs mediated the expression of mouse endogenous retrovirus type L (MERVL)-linked genes in early embryo development (Sharma et al. 2016). However, the function of tsRNAs in human sperm remain largely unknown. Notably, we recently demonstrated that porcine sperm tRNAGln-TTG derived small RNAs regulated the first cleavage of embryo through mediating cell cycle-associated genes and retrotransposon elements (Chen et al. 2020). Because pig as a species are a medical model, it triggers the question whether tRNAGln-TTG derived small RNAs have similar roles in human embryo development.
Here, we analyzed previously published data on human sperm small RNA to uncover the dynamics of tRNAGln-TTG derived small RNAs. Moreover, by using tripronuclear (3PN) human zygotes, we explored the function of tRNAGln-TTG derived small RNAs in human embryo development. Our study revealed that human sperm tRNAGln-TTG derived small RNAs were significantly associated with embryo quality. In addition, tRNAGln-TTG derived small RNAs played a role in the early development of human embryos. Knocking down tRNAGln-TTG derived small RNAs could disorder the embryonic genome activation (EGA) by disturbing noncoding RNA processing. Thus, we uncovered a novel role of tsRNAs in human embryos, and provided evidence that tRNAGln-TTG derived small RNAs could serve as potential diagnostic biomarkers for human fertility with the potential as a clinical target of human sterility.
Materials and methods
Study design
Given the reported association between sperm tsRNAs and intergenerational inheritance of the acquired metabolic disorder, and that tsRNAs could be used as potential biomarkers of the embryo quality, we aimed to uncover the regulatory roles of tsRNAs in early embryo development. Pigs (Sus scrofa) are increasingly utilized as a large animal model in biomedical research due to their high similarity to humans in terms of anatomy and physiology. Previously, we used pigs as model animals to study the potential function of tsRNAs in spermatozoa. After the sense and antisense sequences of tsRNAs were microinjected into porcine embryos, we found that tRNAGln-TTG derived small RNAs could regulate the early embryo development by mediating the expression of cell cycle associated genes. Hence, we proposed that human sperm tRNAGln-TTG derived small RNAs also functions in human embryo. The sense and antisense of tsRNAs were microinjected into human 3PN embryos followed by single-cell RNA-sequencing.
Ethical guidelines
The experiments of 3PN human zygotes followed the ethical standards of Helsinki Declaration and national legislation and were approved by the Chengdu Xinan Women’s Hospital. The patients who donated their 3PN zygotes for research purposes signed informed consent forms.
Tripronuclear human embryo collection, culture and RNA microinjection
Mature human oocytes were inseminated in fertilization medium for 4 h. The fertilization state was examined 16–20 h after insemination. Human embryos were assessed and those with three pronuclei were selected for cryopreservation by using the vitrification kit (KITAZATO, Japan). For recovery, the embryos were warmed with the thawing solution at 37°C for 1 min, and then transferred to the dilution solution and warmed at 35.5°C for 3 min. Later, the embryos were transferred to the washing solution I at 35.5°C for 5 min and then transferred to washing solution II at 37°C for another 5 min. Then, the recovered embryos were microinjected with the 20 μM synthesized sense, antisense of Gln-TTGs, and the scrambled sequences, respectively. Once the cytoplasm of embryo was expand, the microinjected embryo was kept for the future experiment. The microinjected embryos were cultured in vitrolife G1 cleavage (Vitrolife) medium till the eight-cell stage and transferred to vitrolife G2 blastocyst medium (Vitrolife) for 72 h. The microinjection sequences are listed below:
To avoid any influence on embryo development, videos of the different embryo stages were captured by microscope. The developmental rates of embryos in each stage were analyzed according to the method previously reported (Balaban et al. 2011). Blastocyst grading schemes based on the degree of expansion and hatching status (2–6), inner cell mass grading (A–C), and trophectoderm grading (A–C). We divided blastocysts into three quality groups: good (3AA, 4AA, 5AA, 6AA, 3AB, 4AB, 5AB, 6AB, 3BA, 4BA, 5BA, and 6BA), average (2BB, 3BB, 4BB, 5BB, and 6BB), and poor (2BC, 3BC, 4BC, 5BC, 6BC, 2CB, 3CB, 4CB, 5CB, 6CB, 2CC, 3CC, 4CC, 5CC, and 6CC).
Embryo collection and single-cell RNA-sequencing
To conduct single-cell RNA sequencing (Chen et al. 2016a), five embryos per group were pooled. Embryos developed to four-cell, eight-cell, and blastocyst embryos were collected in the lysis buffer and stored at −80°C for the use of single-cell RNA-sequencing. Embryos were lysed to release all RNAs, and the external RNA control consortium (ERCC) was added into the lysate. The RNAs were then reversely transcribed into the first-strand cDNA by using the Smart-Seq2 method (Picelli et al. 2014). Briefly, the first-strand cDNA was synthesized in the mixture of RT enzyme, buffer, oligo-dT primers with common sequence and TSO primers. PCR amplification was used to synthesize the second-strand cDNA. These cDNAs were recovered by the Beckman Ampure XP magnetic beads. 20 ng amplified cDNA was used to construct the single-cell library. The library was sonic to the 200 bp long fragments, which were further end-repaired and ligated with sequencing adaptor for PCR amplification. The 300−350 bp DNA fragments were purified to generate the final sequencing library. The library was sequenced on an Illumina Hiseq 2500 platform to generate 125 bp paired-end reads. The total reads acquired from samples were used for further analysis.
Single-cell RNA-sequencing analysis
For single-cell RNA-sequencing analysis, the Perl script was used to filter the raw data. Briefly, the public primer sequences were trimmed from the reads, then the adaptors were removed from reads, and the low-quality sequencing output was removed to obtained clean data. Acquired clean data were mapped to the Ensemble reference genome (Homo_sapiens.GRCh38.94.chr) with HISAT2 v2.1.0. Reads for each gene was counted by HTSeq vp.6.0. The fragments per kilobase million mapped reads (FPKM) were calculated to estimate the gene expression level. DESeq2 was used to analyze DEGs. Genes that satisfied the criteria of fold change ≥2, P-value ≤ 0.05, and q-value ≤ 0.05 were considered to be DEGs. The enrichment of GO terms was calculated by hypergeometric test.
Statistical analysis
Experiments were repeated at least three times (n = 3). Statistical analyses were performed with either Student’s t-test or one-way ANOVA followed by Turkey’s test using SPSS V23.0 statistical software (IBM). Data were presented as the mean ± s.e.m. and differences were considered statistically significant at *P ≤ 0.05 and highly significant at **P ≤ 0.01.
Results
Gln-TTGs in sperm were associated with human embryo quality
To probe tsRNA function in human embryo development, we first analyzed the published RNA-sequencing data (Hua et al. 2019) and identified the expression patterns of tRNAGln-TTGs derived small RNAs in two groups of human sperm samples. One group is the high-quality sperms that produce a high ratio of superior quality embryos, and the second group is the low-quality sperms that produce a high ratio of inferior quality embryos. An analysis of the results showed that the tRNAGln-TTGs derived small RNAs were grouped into 11 families (Supplementary Table 1, see section on supplementary materials given at the end of this article). These tsRNA families showed identical 5’ sequences and differences at 3’ sequences. We found that tsRNA family 2 of Gln-TTGs dominated in both high-quality sperms (98.74%) and low-quality sperms (98.52%) (Fig. 1A). Therefore, the tsRNA family 2 that was termed as Gln-TTGs was chosen for further study. Our investigation showed that the length of Gln-TTGs ranged from 17 to 45 nt and peaked in between 30 and 36 nt in both high-quality and low-quality sperms (Fig. 1B). Notably, over 50% of Gln-TTGs with 30, 31, 32, 33, and 36 nt were the top 5 abundant tsRNAs of Gln-TTGs (Fig. 1C). In the high-quality sperms, the proportion of Gln-TTGs with 30, 31, 32, 33, and 36 nt were 10.41%, 9.63%, 12.91%, 24.43%, and 9.45%, respectively (Fig. 1C). On the other hand, the proportion of Gln-TTGs with 30, 31, 32, 33, and 36 nt in the low-quality sperms were 10.33%, 8.96%, 11.95%, 21.88%, and 8.54%, respectively (Fig. 1C).
Expression pattern of Gln-TTGs in human sperm. (A) The composition of tRNAGln-TTG derived small RNA family in human sperm. (B) The length distribution of Gln-TTGs in human sperm. (C) Composition of top five abundant tsRNA in human sperm Gln-TTGs.
Citation: Reproduction 161, 2; 10.1530/REP-20-0415
Expression pattern of Gln-TTGs in human sperm. (A) The composition of tRNAGln-TTG derived small RNA family in human sperm. (B) The length distribution of Gln-TTGs in human sperm. (C) Composition of top five abundant tsRNA in human sperm Gln-TTGs.
Citation: Reproduction 161, 2; 10.1530/REP-20-0415
Expression pattern of Gln-TTGs in human sperm. (A) The composition of tRNAGln-TTG derived small RNA family in human sperm. (B) The length distribution of Gln-TTGs in human sperm. (C) Composition of top five abundant tsRNA in human sperm Gln-TTGs.
Citation: Reproduction 161, 2; 10.1530/REP-20-0415
To verify whether the Gln-TTGs in sperm could be used to predict the embryo quality, the difference in quantity of total Gln-TTGs between high-quality sperms and low-quality sperms were compared. Results revealed that Gln-TTGs were differentially enriched in these two groups (Fig. 2A). To further verify the relationship between the quantity of Gln-TTGs in sperms and the quality of human embryos, we compared the top 5 abundant tsRNAs of Gln-TTGs in these two groups. Results indicated that tsRNAs with 30, 31, 32, 33, and 36 nt were differentially presented in the high-quality and low-quality sperms (Fig. 2B, C, D and E). Together, Gln-TTGs in human sperm were associated with human embryo quality and could be used as potential biomarkers.
The differential expression of Gln-TTGs in high and low quality of human sperm. (A) Total Gln-TTGs in two groups of human sperms. (B, C, D, E and F) The differential expression patterns of 30 nt (B), 31 nt (C), 32 nt (D), 33 nt (E), and 36 nt (F) in two groups. Each dot represents an individual sperm.
Citation: Reproduction 161, 2; 10.1530/REP-20-0415
The differential expression of Gln-TTGs in high and low quality of human sperm. (A) Total Gln-TTGs in two groups of human sperms. (B, C, D, E and F) The differential expression patterns of 30 nt (B), 31 nt (C), 32 nt (D), 33 nt (E), and 36 nt (F) in two groups. Each dot represents an individual sperm.
Citation: Reproduction 161, 2; 10.1530/REP-20-0415
The differential expression of Gln-TTGs in high and low quality of human sperm. (A) Total Gln-TTGs in two groups of human sperms. (B, C, D, E and F) The differential expression patterns of 30 nt (B), 31 nt (C), 32 nt (D), 33 nt (E), and 36 nt (F) in two groups. Each dot represents an individual sperm.
Citation: Reproduction 161, 2; 10.1530/REP-20-0415
Sperm Gln-TTGs influence the cleavage of 3PN human embryos
To determine whether Gln-TTGs participated in human embryo development, the synthesized sense (the OE group), the antisense oligonucleotides of human Gln-TTGs (the Anti group), and the scrambled sequence (the NC group) were microinjected into 3PN human embryos, respectively (Fig. 3A). The rates of four-cell embryos and morula in Anti group exhibited the lowest value compared to the NC and OE groups. The Anti group also showed the lowest rate of eight-cell embryos, while the OE group exhibited the highest rate of eight-cell embryos (Fig. 3B). Meanwhile, the blastocyst rate in the Anti group was lowest on days 5 and 6 of incubation, while it was highest in the OE group on day 6 although it was lower than that of the NC group on day 5 (Fig. 3B, C and Supplementary Table 3). Together, these results indicated that sperm Gln-TTGs could influence the cleavage of human embryos.
Gln-TTGs influence the cleavage of 3PN human embryos. (A) Images of the collected 3PN human embryos. (B) The developmental rate of 3PN human embryos (n > 30 per test) with different RNA microinjection. Data are presented as the mean ± s.e.m. *P < 0.05, **P < 0.01, n = 3. (C) Representative graphs of 3PN human embryos in the three groups on day 5. Microinjection RNA of scrambled sequence (NC), sense sequence (OE), and antisense sequence (Anti). Bar = 100 µm.
Citation: Reproduction 161, 2; 10.1530/REP-20-0415
Gln-TTGs influence the cleavage of 3PN human embryos. (A) Images of the collected 3PN human embryos. (B) The developmental rate of 3PN human embryos (n > 30 per test) with different RNA microinjection. Data are presented as the mean ± s.e.m. *P < 0.05, **P < 0.01, n = 3. (C) Representative graphs of 3PN human embryos in the three groups on day 5. Microinjection RNA of scrambled sequence (NC), sense sequence (OE), and antisense sequence (Anti). Bar = 100 µm.
Citation: Reproduction 161, 2; 10.1530/REP-20-0415
Gln-TTGs influence the cleavage of 3PN human embryos. (A) Images of the collected 3PN human embryos. (B) The developmental rate of 3PN human embryos (n > 30 per test) with different RNA microinjection. Data are presented as the mean ± s.e.m. *P < 0.05, **P < 0.01, n = 3. (C) Representative graphs of 3PN human embryos in the three groups on day 5. Microinjection RNA of scrambled sequence (NC), sense sequence (OE), and antisense sequence (Anti). Bar = 100 µm.
Citation: Reproduction 161, 2; 10.1530/REP-20-0415
Gln-TTGs regulate developmental and signal transduction-associated genes
To probe the underlying mechanisms of sperm Gln-TTGs in early human embryo development, single-cell RNA sequencing was conducted to analyze the transcriptomes of four-cell, eight-cell, and blastocyst embryos, which were treated by Gln-TTGs. At the four-cell stage, there were 1655 and 1647 differentially expressed genes (DEGs) in the Anti and OE groups vs the NC group (Fig. 4A, B and Supplementary Table 2). GO–-BP annotation analysis showed that the expression of developmental and signal transduction-associated genes was altered by the depletion or overexpression of Gln-TTGs (Fig. 4C and D). At the eight-cell stage, 1381 DEGs in Anti group and 1400 DEGs in OE group were identified (Fig. 5A, B and Supplementary Table 2). These genes also participated in the development and signal transduction (Fig. 5C and D). At the blastocyst stage, Anti group has 2015 DEGs, and OE group has 1512 DEGs (Fig. 6A, B and Supplementary Table 2). Most of these genes were annotated to regulate signal transduction and developmental process (Fig. 6C and D). Overall, the sequencing data revealed that Gln-TTGs regulated the human embryo development at the early stages by orchestrating developmental and signal transduction-associated genes.
Single-cell sequencing of four-cell embryos. (A) Heatmap of DEGs between Gln-TTG-Anti four-cell and Gln-TTG-NC four-cell embryos. (B) Heatmap of DEGs between Gln-TTG-OE four-cell and Gln-TTG-NC four-cell embryos. (C) The enriched biological processes in the Gln-TTG-Anti four-cell embryos revealed by GO analysis. (D) The enriched biological processes in the Gln-TTG-OE four-cell embryos revealed by GO analysis.
Citation: Reproduction 161, 2; 10.1530/REP-20-0415
Single-cell sequencing of four-cell embryos. (A) Heatmap of DEGs between Gln-TTG-Anti four-cell and Gln-TTG-NC four-cell embryos. (B) Heatmap of DEGs between Gln-TTG-OE four-cell and Gln-TTG-NC four-cell embryos. (C) The enriched biological processes in the Gln-TTG-Anti four-cell embryos revealed by GO analysis. (D) The enriched biological processes in the Gln-TTG-OE four-cell embryos revealed by GO analysis.
Citation: Reproduction 161, 2; 10.1530/REP-20-0415
Single-cell sequencing of four-cell embryos. (A) Heatmap of DEGs between Gln-TTG-Anti four-cell and Gln-TTG-NC four-cell embryos. (B) Heatmap of DEGs between Gln-TTG-OE four-cell and Gln-TTG-NC four-cell embryos. (C) The enriched biological processes in the Gln-TTG-Anti four-cell embryos revealed by GO analysis. (D) The enriched biological processes in the Gln-TTG-OE four-cell embryos revealed by GO analysis.
Citation: Reproduction 161, 2; 10.1530/REP-20-0415
Single-cell sequencing of eight-cell embryos. (A) Heatmap of DEGs between Gln-TTG-Anti eight-cell and Gln-TTG-NC eight-cell embryos. (B) Heatmap of DEGs between Gln-TTG-OE eight-cell and Gln-TTG-NC eight-cell embryos. (C) The enriched biological processes in the Gln-TTG-Anti eight-cell embryos revealed by GO analysis. (D) The enriched biological processes in the Gln-TTG-OE eight-cell embryos revealed by GO analysis.
Citation: Reproduction 161, 2; 10.1530/REP-20-0415
Single-cell sequencing of eight-cell embryos. (A) Heatmap of DEGs between Gln-TTG-Anti eight-cell and Gln-TTG-NC eight-cell embryos. (B) Heatmap of DEGs between Gln-TTG-OE eight-cell and Gln-TTG-NC eight-cell embryos. (C) The enriched biological processes in the Gln-TTG-Anti eight-cell embryos revealed by GO analysis. (D) The enriched biological processes in the Gln-TTG-OE eight-cell embryos revealed by GO analysis.
Citation: Reproduction 161, 2; 10.1530/REP-20-0415
Single-cell sequencing of eight-cell embryos. (A) Heatmap of DEGs between Gln-TTG-Anti eight-cell and Gln-TTG-NC eight-cell embryos. (B) Heatmap of DEGs between Gln-TTG-OE eight-cell and Gln-TTG-NC eight-cell embryos. (C) The enriched biological processes in the Gln-TTG-Anti eight-cell embryos revealed by GO analysis. (D) The enriched biological processes in the Gln-TTG-OE eight-cell embryos revealed by GO analysis.
Citation: Reproduction 161, 2; 10.1530/REP-20-0415
Single-cell sequencing of blastocyst embryo. (A) Heatmap of DEGs between Gln-TTG-Anti blastocyst and Gln-TTG-NC blastocyst embryos. (B) Heatmap of DEGs between Gln-TTG-OE blastocyst and Gln-TTG-NC blastocyst embryos. (C) The enriched biological processes in the Gln-TTG-Anti blastocyst embryos revealed by GO analysis. (D) The enriched biological processes in the Gln-TTG-OE blastocyst embryos revealed by GO analysis.
Citation: Reproduction 161, 2; 10.1530/REP-20-0415
Single-cell sequencing of blastocyst embryo. (A) Heatmap of DEGs between Gln-TTG-Anti blastocyst and Gln-TTG-NC blastocyst embryos. (B) Heatmap of DEGs between Gln-TTG-OE blastocyst and Gln-TTG-NC blastocyst embryos. (C) The enriched biological processes in the Gln-TTG-Anti blastocyst embryos revealed by GO analysis. (D) The enriched biological processes in the Gln-TTG-OE blastocyst embryos revealed by GO analysis.
Citation: Reproduction 161, 2; 10.1530/REP-20-0415
Single-cell sequencing of blastocyst embryo. (A) Heatmap of DEGs between Gln-TTG-Anti blastocyst and Gln-TTG-NC blastocyst embryos. (B) Heatmap of DEGs between Gln-TTG-OE blastocyst and Gln-TTG-NC blastocyst embryos. (C) The enriched biological processes in the Gln-TTG-Anti blastocyst embryos revealed by GO analysis. (D) The enriched biological processes in the Gln-TTG-OE blastocyst embryos revealed by GO analysis.
Citation: Reproduction 161, 2; 10.1530/REP-20-0415
Gln-TTGs regulate the embryonic genome activation
We found that depletion of Gln-TTGs delayed the embryo development from four-cell stage to eight-cell stage. Coincidentally, this is exactly when human embryonic genome activation (EGA) occurs with increasing gene expression (Yan et al. 2013). Therefore, it is possible that Gln-TTGs are involved in the EGA. At this point, we analyzed the gene expression pattern of embryos from four-cell to eight-cell stage. Compared to the four-cell stage, there were 1950 genes that were upregulated at eight-cell stage in both NC and Anti groups, in which 1901 genes were uniquely upregulated in Anti group (Fig. 7A), indicating the disordered EGA from four-cell to eight-cell stage. In addition, 1813 genes were upregulated at eight-cell stage vs four-cell stage in NC and OE groups, in which 1629 genes were uniquely upregulated in OE group (Fig. 7B). Interestingly, GO–-BP annotation analysis uncovered that the uniquely up-regulated genes at eight-cell stage in Anti and OE groups were related to noncoding RNA processing (Fig. 7C and D).
Gln-TTGs influence the EGA of 3PN human embryos. (A) Upregulated genes in Gln-TTG-NC and Gln-TTG-Anti eight-cell groups comparing with four-cell counterparts. (B) Upregulated genes in Gln-TTG-NC and Gln-TTG-OE eight-cell groups comparing with four-cell counterparts. (C) GO analysis of the uniquely upregulated genes in the Gln-TTG-Anti eight-cell embryos. (D) GO analysis of the uniquely upregulated genes in the Gln-TTG-OE eight-cell embryos.
Citation: Reproduction 161, 2; 10.1530/REP-20-0415
Gln-TTGs influence the EGA of 3PN human embryos. (A) Upregulated genes in Gln-TTG-NC and Gln-TTG-Anti eight-cell groups comparing with four-cell counterparts. (B) Upregulated genes in Gln-TTG-NC and Gln-TTG-OE eight-cell groups comparing with four-cell counterparts. (C) GO analysis of the uniquely upregulated genes in the Gln-TTG-Anti eight-cell embryos. (D) GO analysis of the uniquely upregulated genes in the Gln-TTG-OE eight-cell embryos.
Citation: Reproduction 161, 2; 10.1530/REP-20-0415
Gln-TTGs influence the EGA of 3PN human embryos. (A) Upregulated genes in Gln-TTG-NC and Gln-TTG-Anti eight-cell groups comparing with four-cell counterparts. (B) Upregulated genes in Gln-TTG-NC and Gln-TTG-OE eight-cell groups comparing with four-cell counterparts. (C) GO analysis of the uniquely upregulated genes in the Gln-TTG-Anti eight-cell embryos. (D) GO analysis of the uniquely upregulated genes in the Gln-TTG-OE eight-cell embryos.
Citation: Reproduction 161, 2; 10.1530/REP-20-0415
To gain more insight of Gln–TTGs regulation, we then compared our data with the sequencing data from normal human embryos reported previously (Yan et al. 2013). We found 2513 and 2319 uniquely upregulated genes at eight-cell stage in Anti and OE group, respectively (Fig. 8A and B), and these uniquely upregulated genes were also enriched in noncoding RNA processing (Fig. 8C and D), supporting the notion that Gln–TTGs are involved in the EGA of human embryos and indicating that Gln–TTGs influence human embryo EGA by regulating noncoding RNA processing. These results indicated that Gln–TTGs could mediate human embryonic genome activation.
Sperm Gln-TTGs influence the EGA of 3PN human embryos vs normal embryo. (A) Upregulated genes in Gln-TTG-Anti and normal eight-cell groups in comparing with four-cell counterparts. (B) Upregulated genes in Gln-TTG-OE and normal eight-cell groups comparing with four-cell counterparts. (C) GO analysis of the uniquely upregulated genes in the Gln-TTG-Anti eight-cell embryos. (D) GO analysis of the uniquely upregulated genes in the Gln-TTG-OE eight-cell embryos.
Citation: Reproduction 161, 2; 10.1530/REP-20-0415
Sperm Gln-TTGs influence the EGA of 3PN human embryos vs normal embryo. (A) Upregulated genes in Gln-TTG-Anti and normal eight-cell groups in comparing with four-cell counterparts. (B) Upregulated genes in Gln-TTG-OE and normal eight-cell groups comparing with four-cell counterparts. (C) GO analysis of the uniquely upregulated genes in the Gln-TTG-Anti eight-cell embryos. (D) GO analysis of the uniquely upregulated genes in the Gln-TTG-OE eight-cell embryos.
Citation: Reproduction 161, 2; 10.1530/REP-20-0415
Sperm Gln-TTGs influence the EGA of 3PN human embryos vs normal embryo. (A) Upregulated genes in Gln-TTG-Anti and normal eight-cell groups in comparing with four-cell counterparts. (B) Upregulated genes in Gln-TTG-OE and normal eight-cell groups comparing with four-cell counterparts. (C) GO analysis of the uniquely upregulated genes in the Gln-TTG-Anti eight-cell embryos. (D) GO analysis of the uniquely upregulated genes in the Gln-TTG-OE eight-cell embryos.
Citation: Reproduction 161, 2; 10.1530/REP-20-0415
Discussion
Sperm epigenetic signatures have been efficiently applied in various fertility-related diseases and/or predictors for success in assisted reproductive techniques (ART) (Jodar et al. 2015, Jenkins et al. 2016). Recently, researchers reported the small RNA expression pattern of human sperms from groups of a high rate of good quality embryos and low rate of good quality embryos, and found that ten tsRNAs were differentially presented in these two groups, suggesting that tsRNAs might be the useful biomarkers for the clinical evaluation of sperm quality (Hua et al. 2019). Intriguingly, by further analyzing their sequencing data, we found that the total level of Gln-TTGs in two groups of human sperms was significantly different. In addition, the top 5 abundant Gln-TTGs of different lengths including 30, 31, 32, 33, and 36 nt were all differentially presented in these two groups. Furthermore, up to 40% of men from couples with recurrent pregnancy loss showed normal sperm density and motility (Boostanfar et al. 2015), suggesting that routine assessment of semen parameters may miss some information. Hence, developing novel diagnostic tests based on sperm RNA contents would help the treatment of male factor-induced sterility. The tsRNAs in human sperm such as Gln-TTGs may serve as potential biomarkers.
The 3PN zygotes invariably fail to develop in vivo, but can develop to blastocysts in vitro (Balakier 1993, Munne & Cohen 1998), therefore, the 3PN zygotes become the ideal alternative resource used for human embryo development in vitro. Injection of the antisense Gln-TTGs into 3PN human zygotes could reduce the developmental rate of four-cell, eight-cell, and blastocyst embryos, suggesting that the antisense sequence blocks the nascent Gln-TTGs that are derived from either sperm or oocyte presenting in the embryo. These results corroborated a similar role for human Gln-TTGs and their potential as a biomarker. Additionally, we found that overexpression of Gln-TTGs significantly promoted human embryo development, raising an issue whether tsRNAs could exert their roles without modifications. A recent study showed that in vitro transcribed tRNAThr-derived small RNAs stimulated translation in cells from Trypanosoma brucei, excluding the important roles of modifications in this tsRNA molecule (Fricker et al. 2019). Consistently, the outcome of the analysis revealed that overexpression of Gln-TTGs influenced a wide array of biological processes such as development, signal transduction and noncoding RNA processing, similar to the results from the Gln-TTG-Anti group. Thus, we provide further evidence that specific tsRNA molecules could, at least during early embryo development, exert their roles without modifications.
After fertilization, zygotes with both paternal and maternal genomes must be reprogrammed into the totipotent state with transcriptional silence (Tadros & Lipshitz 2009). The resumption of embryo development requires the switch from maternal-to-embryo-derived transcripts and proteins, termed as maternal-to-embryonic transition (Graf et al. 2014). This crucial process contains EGA and the degradation of maternal molecules (Graf et al. 2014). The mechanism underlying the initiation of EGA is generally conserved, though the time points when EGA occurs differs in species, for example, at the two-cell stage in mice, and the four- to eight-cell stage in pigs and humans (Ostrup et al. 2013, Eckersley-Maslin et al. 2018). It has been reported that transient inhibition of EGA results in developmental arrest mostly at the two-cell stage in mice (Abe et al. 2018). Here, we found that depletion of Gln-TTGs in 3PN human embryos delayed embryo development, along with disordered EGA, while overexpression of Gln-TTGs accelerated the human embryo development, also with disordered EGA, therefore for the first time demonstrating that tsRNAs could influence the embryo cleavage by regulating EGA.
Histone modification, chromatin remodeling and maternal effects are associated with EGA (Li et al. 2013, Schulz & Harrison 2019). In mice, the active marker H3K4 is established in the transcription start sites (TSSs) of a group of genes at the two-cell stage, exactly when EGA occurs (Dahl et al. 2016, Liu et al. 2016, Zhang et al. 2016). The accessible chromatin profiling in early embryos has also revealed that the sharp open chromatin peaks in promoter regions become increasingly visible during development from the two-cell stage to blastocysts with inner cell mass, due to the initiation of gene expression by EGA (Wu et al. 2016). In addition, ablation of genes encoding the maternal transcription factors for pluripotency, for example, OCT4 and SOX2, results in embryonic arrest at the cleavage stages, along with the abnormal initiation of EGA (Li et al. 2013). It has been reported that tRNALeu-CAG-derived small RNAs bind to two ribosomal protein mRNAs (RPS28 and RPS15) to enhance their translation and further regulate ribosome biogenesis (Kim et al. 2017). In this study, we found that depletion or overexpression of Gln-TTGs induced the disordered EGA, and that the perturbed genes were mainly involved in tRNA and rRNA processing as well as ribosome biogenesis. Therefore, it would be interesting to validate whether Gln-TTGs acts as regulatory factors for ribosome biogenesis during early human embryo development in the future study.
Generally, ART, including intrauterine insemination, in vitro fertilization or intracytoplasmic sperm injection, is the available treatment for sterility. However, infertile couples using artificial insemination only achieve a pregnancy rate of 44%, and the pregnancy rate of intracytoplasmic sperm injection per cycle reaches just 12% (Hennebicq et al. 2018). Although ART has been significantly improved, the complicated process can never guarantee a healthy pregnancy (Hu et al. 2018). Indeed, the live birth rate after ART is about 30% and decreases with increasing maternal age (Adamson et al. 2018). In this case, an improvement to this current treatment might be supplemented with human sperm RNA contents. The finding that overexpression of Gln-TTGs improved the developmental rate of human embryo suggests that Gln-TTGs may potentially act as a clinical target for the treatment of male infertility.
In summary, the bioinformatic analysis revealed that human sperm Gln-TTGs were associated with sperm quality. By using 3PN human zygotes, we demonstrate that sperm Gln-TTGs were involved in the cleavage of human embryos by regulating developmental processes and signal transduction and in EGA, probably by modulating noncoding RNA processing, a mechanism illustrated in Fig. 9. Many couples suffering from male infertility are treated by in vitro fertilization or intracytoplasmic sperm injection, which are complicated and with some limitations as well. Our findings provide references for improving future diagnostic tests and clinical treatments with paternally derived factors and for facilitating safe ART in human infertility.
Schematic diagram of tsRNA functions in human embryo development. Microinjection of sense/antisense RNAs changed the embryo development. Gln-TTGs are involved in the cleavage of human embryos by regulating developmental processes and signal transduction and in EGA of embryos probably by modulating noncoding RNA processing.
Citation: Reproduction 161, 2; 10.1530/REP-20-0415
Schematic diagram of tsRNA functions in human embryo development. Microinjection of sense/antisense RNAs changed the embryo development. Gln-TTGs are involved in the cleavage of human embryos by regulating developmental processes and signal transduction and in EGA of embryos probably by modulating noncoding RNA processing.
Citation: Reproduction 161, 2; 10.1530/REP-20-0415
Schematic diagram of tsRNA functions in human embryo development. Microinjection of sense/antisense RNAs changed the embryo development. Gln-TTGs are involved in the cleavage of human embryos by regulating developmental processes and signal transduction and in EGA of embryos probably by modulating noncoding RNA processing.
Citation: Reproduction 161, 2; 10.1530/REP-20-0415
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/REP-20-0415.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This study was supported in part by the National Key R&D Program of China (2018YFD0501001) to W Z; the Natural Science Foundation of Shaanxi Province, China (Grant No. 2019JQ-430) to Y Z.
Author contribution statement
X C and W Z conceived the study. X C, X M, Q S and H L performed the experiments. X C and Z L performed the bioinformatic analyses. X C and Y Z drafted the original manuscript. H L and W Z revised the manuscript. W Z supervised the whole study. All authors read and approved the final manuscript.
Acknowledgements
The authors thank Dr Enkui Duan for critical suggestions and comments. Dr Huayan Wang for English language editing.
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