The role of protein arginine methyltransferase 7 in human developmentally arrested embryos cultured in vitro

Abstract

Human embryos of in vitro fertilization (IVF) are often susceptible to developmental arrest, which greatly reduces the efficiency of IVF treatment. In recent years, it has been found that protein arginine methyltransferase 7 (PRMT7) plays an important role in the process of early embryonic development. However, not much is known about the relationship between PRMT7 and developmentally arrested embryos. The role of PRMT7 in developmentally arrested embryos was thus investigated in this study. Discarded human embryos from IVF were collected for experimental materials. Quantitative real-time polymerase chain reaction (qRT-PCR) and confocal analyses were used to identify PRMT7 mRNA and protein levels in early embryos at different developmental stages, as well as changes in the methylation levels of H4R3me2s. Additionally, PRMT7 was knocked down in the developmentally arrested embryos to observe the further development of these embryos. Our results demonstrated that PRMT7 mRNA and protein levels in arrested embryos were significantly increased compared with those in control embryos; meanwhile, the methylation levels of H4R3me2s in arrested embryos were also increased significantly. Knockdown of PRMT7 could rescue partially developmentally arrested embryos, and even individual developmentally arrested embryos could develop into blastocysts. In conclusion, over-expression of PRMT7 disrupts the early embryo development process, leading to early embryos developmental arrest, but these developmentally arrested defects could be partially rescued by knockdown of the PRMT7 protein.

Introduction

In vitro fertilization and embryo transfer (IVF-ET) is currently a conventional treatment for infertility. Although significant progress has been made in reproductive medicine and embryo culture conditions in vitro since the birth of the first IVF baby in 1978, the efficiency of IVF-ET treatment is still very low [1]. As the women’s age increases, the pregnancy rate will be lower [2–4]. Studies have shown that ∼10% of human IVF embryos will be permanently arrested at the early stages of cleavage in culture, and 40% of patients will have at least one arrested embryo during each treatment cycle [5]. Less than 50% of IVF embryos can develop to the stage of blastocyst, and many of them cannot maintain further development after embryo transfer [6,7]. Nevertheless, to date, we know little about the molecular mechanism underlying early embryonic developmental defects.

Previous studies have shown that embryos cultured in vitro are often prone to developmental arrest; cattle and sheep embryos both occur at 8–16 cell stages, and human embryos usually occur at 4–8 cell stages [8–10]. Early embryonic development is the fastest stage of embryonic cell proliferation and differentiation, accompanied by large-scale zygotic genes transcription initiation. Recent studies have revealed that epigenetic regulation plays an important role in these biological processes. Histone methylated modification is one of the important regulatory mechanisms of epigenetics. In the process of early embryonic development, histone methylation modification has a dynamic variation and may have very important functions [11–14].

Some studies have shown that protein arginine methyltransferase (PRMT) plays a key role in early embryonic development [15,16]. Some researchers indicated that inhibiting the expression of PRMT1/PRMT8 genes in zebrafish one-cell embryos leads to developmental defects in the gastrulation of early embryos and retards embryonic development in later stages [17–19]. In the study of mouse embryonic stem cells, it was found that PRMT6 is essential for maintaining the pluripotency of embryonic stem cells. If PRMT6 expression is inhibited, it will lead to overexpression of germ layer differentiation factors and accelerate embryonic stem cell differentiation [20]. PRMT3^-/--deficient mice showed early embryonic developmentally arrested defects [21]. As a type II arginine methylase, PRMT7 usually catalyzes mono-methylation or symmetric di-methylation of histones to induce gene silencing of downstream substrates. H4R3me2s (histone symmetric di-methylation at H4R3), a repressive mark, is positively regulated by the PRMT7 [22–24]. Our previous studies also showed that PRMT7 can regulate the gastrulation movement of early zebrafish embryos by regulating the signaling pathways of its downstream molecules 2-OST and syntenin. When the PRMT7 gene expression was suppressed in zebrafish one-cell embryos, the embryos delayed or even arrested the epiboly process of early embryonic development [25,26]. Based on these findings, we hypothesized that PRMT7 may play a crucial role in the early embryonic development process.

In this study, we found that PRMT7 mRNA and protein have significantly high expression in developmentally arrested embryos. Meanwhile, the methylation level of H4R3me2s in arrested embryos was also increased significantly. Knockdown of PRMT7 in arrested embryos could partially rescue those developmentally arrested embryos.

Materials and Methods

Human embryo collection and culture

Women with male factor or fallopian tube etiology infertility were enrolled from 2018 to 2019 at the Reproductive Center of Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine. All patients who donated their discarded embryos received counseling prior to signing an informed consent form.

To collect early developmental embryos, in vitro fertilized eggs were cultured until the eight-cell stage using a G-1™ PLUS human embryos culture medium (Vitrolife, Göteborg, Sweden). The G-2™ PLUS human embryos culture medium (Vitrolife) was used to culture the eight-cell to the blastocyst stage. The control embryos (at six to eight-cell stage) and blastocyst were from polyspermic embryos and collected at ‘Day 3’ and ‘Day 5’ post-fertilization. Embryos that were arrested at the two or less than five-cell stage were collected at ‘Day 3’ post-fertilization, and these arrested embryos had no signs of degeneration at ‘Day 5’ post-fertilization.

A total of 245 developmentally arrested embryos were collected in this study. This study was approved and guided by the Ethics Committee of Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine.

Quantitative real-time polymerase chain reaction

Discarded embryos (∼10) were collected and lysed in 50 μl of lysis buffer (0.2% Triton X-100 and 4 IU RNase inhibitor), and total RNA was isolated by using the Arcturus PicoPure RNA isolation kit (Applied Biosystems, Carlsbad, USA) according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed with the ABI7500 system (ABI, Foster City, USA) using the AceQ qPCR SYBR Green Master Mix Kit (Vazyme, Nanjing, China). Standard reactions (10 μl) were as follows: 5 μl of qPCR master mix, 0.25 μl of forward primer (10 μM), 0.25 μl of reverse primer (10 μM), 1 μl of template, and 3.5 μl of diethypyrocarbonate (DEPC)-treated water. The reaction conditions were as follows: 95ºC denaturation for 5 min, followed by 40 cycles of 95°C for 15 s, 60°C for 30 s, and followed by one cycle of 95°C for 15 s, 60°C for 30 s, and 95°C for 15 s. GAPDH was used as an internal reference. The qPCR experiments were repeated three times. The gene expression levels were calculated using the 2^−ΔΔCt method. The primer sequences were as follows: PRMT7 forward 5ʹ-GCAGGTCATCGTCCCTCCC-3ʹ, reverse 5ʹ-CACCTGAGCTCGGCCAGA-3ʹ; GAPDH forward 5ʹ-AGAAGGCTGGGGCTCATTTG-3ʹ, reverse 5ʹ-AGGGGCCATCCACAGTCTTC-3ʹ.

Immunofluorescence analysis

Embryos were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 30 min at room temperature, and permeabilized in PBS containing 0.1% Triton X-100 for 30 min. Subsequently, embryos were incubated for 1 h at room temperature in blocking solution (3% bovine serum albumin in PBS), followed by incubation with anti-PRMT7 antibody (1:100, ab181214; Abcam) or anti-H4R3me2s antibody (1:100, ab5823; Abcam) overnight at 4°C. Embryos were then washed three times with PBS with Tween-20 (PBST) and incubated with Alexa Fluor-conjugated secondary antibody (SA00006-4; Proteintech, Rosemont, USA) for 1 h, followed by staining for 5 min in 10 μl 4,6-diamino-2-phenyl indole (DAPI). Then, embryos were washed twice with PBST, and immunofluorescence images were obtained using an LSM8780 laser-scanning confocal microscope (ZEISS, Jena, Germany).

PRMT7 lentivirus construction and embryo microinjection

The PRMT7 cDNA fragment was amplified as mentioned above, cloned into the pLKO.1-EGFP plasmid (HarO, Shanghai, China), and sequenced to confirm that the correct pLKO.1-EGFP-shhPRMT7 recombinant plasmid was constructed. The primer sequences were as follows: pLKO.1-EGFP-shhPRMT7-F 5ʹ-AUUUUUCUCCACAAUCUUCAC-3ʹ, pLKO.1-EGFP-shhPRMT7-R: 5ʹ-GAAGAUUGUGGAGAAAAAUGG-3ʹ. Lipofactamine 2000 (Invitrogen, CA, USA) was used to transfect the exogenously constructed lentiviral vector and helper plasmid into 293T cells (Cell Bank of the Chinese Academy of Sciences, Shanghai, China). The pLKO.1-EGFP-shhPRMT7 lentivirus was produced in 293T cells and then concentrated in vitro by ultracentrifugation (100,000 g).

Developmentally arrested embryos were divided randomly into three groups, namely the control group (no injection), the empty lentivirus group (injected with pLKO.1-EGFP lentivirus), and the knockdown group (injected with pLKO.1-EGFP-shhPRMT7 lentivirus). In the experiment, the lentivirus was microinjected into the perivitelline space of the arrested embryos (lentivirus titer was 10⁸ TU/ml, and each embryo was injected with 3–5 nl of lentivirus). All injections were performed using an Eppendorf transferman NK2 micromanipulator (Eppendorf, Hamburg, Germany). Subsequently, embryos were cultured in G-2™ PLUS human embryos culture medium at 37°C with 6% CO₂. The morphology of embryos was examined using an Eclipse microscope (Nikon, Tokyo, Japan).

Statistical analysis

Data were presented as the mean ± standard deviation (SD), and experiments were repeated at least three times. Statistical analyses were performed by the Student’s t-test using the SPSS19.0 software. Difference was considered statistically significant at P < 0.05.

Results

PRMT7 transcript level was high in developmentally arrested embryos

In order to identify the transcript level of the PRMT7 gene at different developmental stages of human early embryos, we analyzed the PRMT7 mRNA level in human IVF embryos of the control group (the polyspermic embryos), developmentally arrested group, and blastocyst group by qRT-PCR. The transcript level of PRMT7 in the developmentally arrested embryo group was significantly increased compared with that in the control group (P < 0.05, Fig. 1). At the same time, the transcript level of PRMT7 was low in the blastocyst group, with significant difference between the developmentally arrested group and the blastocyst group (P < 0.05, Fig. 1). Collectively, qRT-PCR results revealed that human PRMT7 gene had a dynamical expression during embryonic development and showed a high expression level in developmentally arrested embryos.

PRMT7 protein level was increased in developmentally arrested embryos

To investigate whether the PRMT7 protein level is consistent with its mRNA level, we detected the PRMT7 protein level in human IVF embryos of the control group (the polyspermic embryos), developmentally arrested group, and blastocyst group by confocal microscopy. Confocal results showed that the PRMT7 protein level in the developmentally arrested embryo group was significantly increased compared with that in the control and blastocyst groups (Fig. 2). Significant difference was found between the control group, as well as the blastocyst group, versus the developmentally arrested group (P < 0.05, Fig. 2B). These results are consistent with the qRT-PCR results. As shown in the merged images (Fig. 2A), PRMT7 protein was ubiquitously expressed in the cytoplasm and nucleus of embryonic cells. These results demonstrated that the PRMT7 protein was highly expressed in developmentally arrested embryos.

H4R3me2s methylation level was high in developmentally arrested embryos

H4R3me2s usually exhibits a low level of methylation during the process of gene transcription activation, which is often regulated by PRMT7 [23], so we examined the methylation level of H4R3me2s in human IVF embryos of the control group (the polyspermic embryos), developmentally arrested group, and blastocyst group by confocal microscopy. Our results demonstrated that H4R3me2s had a very low methylation level in the control embryos, while the methylation level of H4R3me2s in the developmentally arrested group was significantly higher than that in the control group (Fig. 3A,B). When we compared the levels of H4R3me2s methylation in the developmentally arrested group and blastocyst group, we found that the level of H4R3me2s methylation in developmentally arrested group was higher than that in the blastocyst group, and significant difference was found between the blastocyst group and the developmentally arrested group (P < 0.05, Fig. 3A,B). Collectively, these results indicated that the methylation level of H4R3me2s was strongest in the developmentally arrested group, which might be related to the inhibition of gene expression.

Effect of PRMT7 knockdown in developmentally arrested embryos

To investigate whether the overexpression of PRMT7 in early embryos causes developmental arrest, we downregulated PRMT7 in developmentally arrested embryos to observe whether this treatment could rescue the developmental arrest defects. The confocal results showed that the expression of PRMT7 was significantly knocked down in embryos infected with pLKO.1-EGFP-shhPRMT7 lentivirus, compared with that in embryos injected with pLKO.1-EGFP lentivirus or in embryos with no injection (Fig. 4A,B). Interestingly, the cortical deposit of PRMT7 in Fig. 4F became thinner, which is consistent with the effect of the PRMT7 knockdown. These results confirmed the effectiveness and specificity of the recombinant PRMT7-knockdown lentivirus.

Then, the developmentally arrested embryos microinjected with pLKO.1-EGFP-shhPRMT7 lentivirus were cultured in G2 medium, and the morphology of embryos was examined by microscopy. The results demonstrated that most embryos in the control and the empty lentivirus group could not develop further; however, embryos of the PRMT7-knockdown group could develop further on the third day, and the number of embryonic cells was significantly increased compared with that on the first day (Fig. 5A). Surprisingly, these PRMT7-knockdown embryos were undergoing embryo compaction on the third day (black circle), and blastocyst formation could be observed on the fourth day (black square) (Fig. 5A). Meanwhile, about 10.35% of the arrested embryos in the PRMT7-knockdown group were able to regenerate to 32–64 cells on the fourth day (Fig. 5B), indicating that the suppression of PRMT7 overexpression in the arrested embryos could rescue some of the arrested embryos to restart developing. Moreover, in the control group or the empty lentivirus group, the embryos did not change much (Fig. 5B). Furthermore, most of the rescued embryos were blocked at about 32-cell embryos and could not continue to develop after prolonged cultivation and, eventually, died.

These data confirmed that reducing the overexpression of PRMT7 in arrested embryos could partially rescue the developmentally arrested embryos and restart the developmental process.

Discussion

When the early embryonic development undergoes transcriptional silence, it is accompanied by large-scale zygotic gene transcription activation to support the subsequent development of the embryo. Abnormal expression of some key genes for embryonic development will lead to early embryonic developmental arrest. At this time, there is also dynamic variation in histone methylated modification. There are some extracellular and intracellular signals working cooperatively to regulate the expression and repression of a series of genes, and many mechanisms participate in this regulation [27,28]. In recent years, many studies have showed that histone methylase modification plays an increasingly important role in this process, but its function and mechanism are still unclear [29–31]. As a type II arginine methylase, PRMT7 methylate different arginine residues on histone H4-Arg-3 and Arg-17 and Arg-19. H4R3me2s is a repressive mark whose level is positively regulated by PRMT7 [23,32]. Recent studies revealed that human PRMT7 transcripts were expressed in human preimplantation embryos from one-cell stage to blastocyst [33]. Our research results showed that PRMT7 mRNA and protein had significant high expression levels in developmentally arrested embryos; meanwhile, the methylation level of H4R3me2s in arrested embryos was also increased significantly. However, knockdown of PRMT7 in arrested embryos could partially rescue those developmentally arrested embryos.

Genes in different species have species specificity. For example, the expression pattern and function of PRMT7 gene in zebrafish are completely different from those in mice and humans [25,34,35]. Previous studies have suggested that PRMT7 gene has significantly high expression in some cancer cells, and it promotes the invasion function of cancer cells [36,37]. Some studies confirmed that PRMT7 is also one of the key genes for maintaining the stemness of mouse embryonic and muscle stem cells [38,39]. In recent years, studies have found that PRMT7 plays an important role in human early embryos. For example, embryos that are homozygous for the PRMT7 mutation have dysplasia in the uterus. Moreover, patients with such PRMT7 mutations suffered from sensorineural hearing loss, severe mental retardation, and physical defects such as delayed development after birth [40,41]. Our previous studies indicated that PRMT7 can affect the epiboly and gastrulation of zebrafish embryos by regulating its downstream genes, such as 2-OST and syntenin. At the same time, we also found that inhibiting the expression of PRMT7 in zebrafish embryos will lead to embryonic developmental arrest [25,26]. Therefore, we should clarify the role and function of PRMT7 gene in human early embryonic development. This will provide us with a theoretical basis for understanding the mechanism of embryonic developmental arrest in vitro.

Our previous research demonstrated that when the expression of PRMT7 is inhibited in zebrafish embryo, the development of embryos in the gastrulation stage will be arrested [25,26]. PRMT7 gene is widely expressed in zebrafish early embryos. However, we found that PRMT7 gene expression was very low in developmentally arrested embryos. When PRMT7 gene is overexpressed in these arrested embryos, these developmentally arrested embryos can be rescued and develop further [25]. One study showed that if the PRMT7 gene is knocked out, PRMT7^-/- mice will die within 10 days after birth [42]. Collectively, the above findings suggest that PRMT7 plays an important role in early embryonic development, but it has different physiological functions in different species. In the present study, we found that the PRMT7 transcript in the developmentally arrested embryo group was significantly increased compared with that in the control group (Fig. 1). The results of the expression level of PRMT7 in this study are different from those in our previous study carried out in zebrafish arrested embryos, indicating that the gene expression patterns of PRMT7 are different among different species [25].

Proteins are the performers of cell physiological functions [43]. Is the expression level of PRMT7 protein consistent with its transcript level in developmentally arrested embryos? Confocal results showed that the PRMT7 protein level in the developmentally arrested group embryos was significantly increased compared with that in the control group embryos or the blastocyst embryos (Fig. 2). These observations are consistent with the qPCR results (Fig. 1). PRMT7 usually catalyzes symmetric dimethylarginine of histone proteins to induce gene silencing by generating repressive histone marks, including H4R3me2s [44]. Our results demonstrated that the methylation level of H4R3me2s was the highest in the developmentally arrested group (Fig. 3). Perhaps the high methylation level of H4R3me2s inhibits the expression of development-related genes. The above results indicate that abnormal expression of PRMT7 may be one of the important reasons for early embryonic developmental arrest.

To investigate whether the overexpression of PRMT7 in early embryos causes developmental arrest, we down-regulated the PRMT7 protein level in developmentally arrested embryos to observe if it could rescue the developmental arrest defects. Our results demonstrated that most embryos in the control and the empty lentivirus group could not develop further; however, most embryos of the PRMT7-knockdown group could develop further (Fig. 5B). These data confirmed that reducing the overexpression of PRMT7 in arrested embryos could partially rescue the developmentally arrested embryos and restart the development process. Therefore, overexpression of PRMT7 disrupts the early embryo development process, leading to early embryo developmental arrest, but these development-arrest defects can be partially rescued by PRMT7 knockdown. Unlike other members of the PRMT family, PRMT7 can not only methylate histones but also methylate both peptides and proteins to form symmetric di-methylated arginine residues. In recent years, two specific cytosolic proteins have been proposed to be the physiological substrates of PRMT7-HSP70 and eIF2α [45,46]. Studies have shown that bulk of PRMT7 is in the cytoplasm and only weakly expressed in the nucleus of HEK293T and MCF7 cells. These results suggest that the major physiological substrate of PRMT7 may exist in the cytoplasm. If PRMT7 plays a physiological role in the nucleus, it may be transported to the nucleus by small amounts of enzyme or only under certain conditions. All these research results indicate that PRMT7 has a variety of physiological action modes and functions.

In summary, our study delineates that PRMT7 may play a crucial role in the early embryonic development process. Our data are important for understanding the functional relationship between developmental arrest and PRMT7. The next step is to explore what signaling pathways PRMT7 participates in, and how PRMT7 affects the process of early embryonic development by regulating those specific downstream molecular targets.

Acknowledgement

We thank all doctors and embryologists in our center for their contributions. At the same time, we would also like to thank all patients who agreed to participate in this study.

Funding

This work was supported by the grants from the National Natural Science Foundation of China (Nos. 81571442 and 81170571).

Conflict of Interest

The authors declare that they have no conflict of interest.

References

Author notes