Abstract
Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, can significantly improve the reprogramming efficiency of somatic cells. However, whether TSA has a detrimental effect on other kinds of embryos is largely unknown because of the lack of integrated analysis of the TSA effect on natural fertilized embryos. To investigate the effect of TSA on mouse embryo development, we analyzed preimplantation and post-implantation development of in vivo, in vitro fertilized, and parthenogenetic embryos treated with TSA at different concentrations and durations. In vivo fertilized embryos appeared to be the most sensitive to TSA treatment among the three groups, and the blastocyst formation rate decreased sharply as TSA concentration and treatment time increased. TSA treatment also reduced the livebirth rate for in vivo fertilized embryos from 56.59 to 38.33% but did not significantly affect postnatal biological functions such as the pups’ reproductive performance and their ability for spatial learning and memory. Further analysis indicated that the acetylation level of H3K9 and H4K5 was enhanced by TSA treatment at low concentrations, while DNA methylation appeared to be also disturbed by TSA treatment only at high concentration. Thus, our data indicates that TSA has different effects on preimplantation embryonic development depending on the nature of the embryo’s reproductive origin, the TSA concentration and treatment time, whereas the effect of TSA at the indicated concentration on postnatal function was minor.
Introduction
The low full-term development rate of cloned embryos from somatic cell nuclear transfer (SCNT) is the result of inefficient reprogramming, as reflected by abnormal DNA hypermethylation and histone modifications. Although many efforts were applied to improve the success rate of SCNT in different species, the efficiency still remains low (Wakayama et al. 1998, 2003, Ogura et al. 2000, Wakayama & Yanagimachi 2001, Zhou et al. 2003, Zhao et al. 2009, Kim et al. 2012, 2019, Chen et al. 2015, Huang et al. 2016, Wang et al. 2017, Gao et al. 2018, Chang et al. 2019). However, when the cloned embryos were treated with the histone deacetylase inhibitor trichostatin A (TSA) after nuclear transfer, their preimplantation development as measured by the blastocyst rate, and the full-term development of these embryos was dramatically improved, indicating an important role of TSA in correcting reprogramming of the acetylation pattern, and converting a somatic nucleus from a differentiated state into a totipotent state (Kishigami et al. 2006a, Rybouchkin et al. 2006, Hai et al. 2011, Saini et al. 2014, Inoue et al. 2015, Qiu et al. 2017, Azuma et al. 2018).
In general, histone acetylation is associated with 'open' chromatin and active transcription, whereas deacetylation is associated with 'closed' transcriptionally silent chromatin (Marks 2003). The level of acetylation and deacetylation of histone is modulated by the two types of enzymes, histone acetyltransferase (HAT) and histone deacetylase (HDAC) (Mei et al. 2004). TSA has been shown to reversibly alter the balance between HAT and HDAC activities (Tsuji et al. 1976, Tsuji & Kobayashi 1978), therefore providing a useful tool to investigate the role of histone acetylation/deacetylation in the regulation of transcription and chromatin function. It has been demonstrated in various cancer cell lines that low concentrations of TSA can adversely affect a variety of cellular functions and biological processes, including the induction of cell cycle arrest, differentiation and apoptosis (Yoshida et al. 1990, Kelly et al. 2002, Frew et al. 2009, Falkenberg & Johnstone 2014, Song et al. 2018, Morales Torres et al. 2020). A detailed analysis of TSA’s effect on specific mouse tissues revealed its teratogenic potential, with a significant impact on the developing axial skeleton at term when pregnant mice were treated with TSA at E8 (Menegola et al. 2005). In xenopus and zebrafish embryos, TSA can cause growth retardation, axial malformations (crooked tail and shortened AP axis), neural patterning defects, cardiac malformation, and transcriptional changes (Gurvich et al. 2005). TSA treatment at the one- and two-cell stage also causes inhibition of subsequent embryonic cleavage (Ma et al. 2001).
Despite our appreciation of TSA’s teratogenic potential in vivo, and how it impacts the proliferation, differentiation and apoptosis of many different cancer cell lines, there exists a serious data gap that can only be filled by a more comprehensive, detailed study of TSA’s effect on normal mouse embryo development. Such a study would include an analysis of the biological functions of the offsprings derived from TSA-treated embryos at preimplantation period. Interestingly, in order to use the TSA to promote cell reprogramming more safely, many researchers investigated the biological effect of TSA on embryos derived from different nuclear origins. It was reported that pregnant CD mice were i.p. treated with TSA on day 8 post coitum caused teratogenic effects on axial skeleton of fetuses (Menegola et al. 2005). The embryos generated by ICSI, ROSI and parthenogenesis were treated by 50 nM TSA for 20 h decreased the blastocyst rate and offsprings production rate of ICSI embryos but not ROSI or parthenogenetic embryos. Furthermore, TSA treatment also reduced the body mass of offsprings derived from ICSI and ROSI embryos (Kishigami et al. 2006b). TSA treatment for GV stage oocytes followed by in vitro fertilization dramatically reduced the litter size and elevated the embryonic death in utero (Akiyama et al. 2006). Mechanically, DNA methylation of spermatid-derived paternal genomes and H3 and H4 acetylation in ROSI and ICSI zygotes were dysregulated by TSA treatment (Kishigami et al. 2006a). However, all the studies reported here did not use the TSA treatment strategies in SCNT reported by Ogura et al. (Wakayama et al. 1998, 2003, Ogura et al. 2000, Wakayama & Yanagimachi 2001, Zhou et al. 2003, 2009, Kim et al. 2012, Chen et al. 2015, Huang et al. 2016, Wang et al. 2017, Gao et al. 2018, Chang et al. 2019, Kim et al. 2019 ) and also did not perform comprehensive study for the effect of TSA on in vivo fertilized embryos.
Therefore, our study filled the gap between the effect of TSA on SCNT embryos and on fertilized embryos. We conducted a comprehensive study to explore the exact effect of TSA on embryos derived from in vivo/in vitro fertilization and parthenogenesis which included the index of preimplantation, post-implantation and postnatal stage embryos and their adult offsprings. More importantly, the possible underlying epigenetic mechanisms such as DNA methylation and histone acetylation were also investigated.
In this study, we investigated the potential roles of TSA on mouse embryogenesis and fetal development. We found that TSA is very effective in inducing hyperacetylation, and its toxicity on preimplantation development depends on the nature and origin of the early embryo and the concentration and treatment time of TSA exposure. However, the indicated concentration of TSA did not have any profound effects, on postnatal function and behavioral development.
Materials and methods
Culture medium
The embryos were cultured in CZB medium and transferred to CZB supplemented with 5.5 mM glucose after fourcell stage in a humidified atmosphere, 37°C, 5% CO2. Activation medium was calcium-free CZB supplemented with 10 mM SrCl2 and 5 μg/mL CB. α-MEM (Sigma, M4526) supplemented with 3 mg/mL BSA, 25 mg/L sodium pyruvate and 3.8 mg/L EDTA was used for preincubation of sperm and in vitro fertilization.
Animal and production of in vivo/in vitro fertilized embryos
All the animals used here are from the CD-1 strain. All animals are reared under the guidelines of the Guide for the Care and Use of Laboratory Animals. All procedures were approved by the Jilin University Animal Care and Use Committee. Females (8–12 weeks old) were superovulated with 10 IU PMSG (Ningbo hormone factory, G024, China), followed by 10 IU hCG (Ningbo hormone factory, GN026, China) 48 h later. For one-cell embryos (zygotes) collection, these mice were mated to male mice immediately after hCG injection. Oocytes and zygotes were collected with HEPES-buffered CZB medium from the oviducts approximately 13 and 16 h after hCG injection, respectively, and then treated with 300 IU/mL hyaluronidase (Sigma, H3506) until the cumulus cells dispersed. After three times washing with HEPES-buffered CZB medium the oocytes were then activated by a 6 h incubation in activation medium (parthenogenesis group) or mixed with spermatozoa in α-MEM (in vitro fertilization group) and the zygotes were transferred to CZB medium (in vivo fertilization group), covered with paraffin oil (Sigma, 18512) at 37°C and 5% CO2.
For in vitro fertilization, spermatozoa were collected from the caudal epididymis of adult male mice and capacitated by preincubation for 1 h in fertilization medium (modified α-MEM). Cumulus oocyte complexes (COCs) were collected in αMEM medium 13 h after hCG injection and inseminated with capacitated spermatozoa in a humidified atmosphere of 5% CO2, at 37°C. Three hours after insemination (16 h after hCG), the fertilized oocytes were washed and cultured in CZB medium.
Embryo transfer
The two-cell stage embryos were transferred into the oviducts (six to nine embryos per oviduct) of E0.5 pseudo-pregnant surrogate mothers. At E19.5, the number of pups born was recorded and live pups were nursed by their mothers. Recipient females that did not give birth naturally until noon on day 20 were sacrificed to record the implantation sites and number of fetuses. The body mass of the pups was measured weekly. The sex ratio and death rate of the pups were also recorded 21 days after birth.
On day 11 of pregnancy, some recipients were sacrificed to assess the number of embryos lost, the implantation sites and number of fetuses.
TSA treatment program
TSA was dissolved in DMSO and stored in −80°C (1 mM or 40 mM). Working concentration of TSA was prepared with activation and culture medium immediately before use, depending on the experiment design of each group (Table 1). Activation medium and CZB medium contain different concentrations of TSA: 0.005 μM TSA, 0.050 μM TSA, 0.100 μM TSA, 1 μM TSA, 40 μM TSA, 250 μM TSA. Control group includes two groups: CZB and CZB with DMSO (<1/1000, v/v).
TSA treatment program.
| Time after hCG | In vivo fertilization | In vitro fertilization | Parthenogenesis |
|---|---|---|---|
| 0 h | hCG, mate with male mice | hCG | hCG |
| 13 h | – | Oocyte collection, IVF | Oocyte collection, activation and TSA treatment |
| 16 h | Zygote collection, TSA treatment | Zygote collection, TSA treatment | – |
| 19 h | – | – | CZB+TSA culture |
| 23 h* | – | – | Half of the oocytes were transferred to CZB |
| 26 h* | Half of the oocytes were transferred to CZB | Half of the oocytes were transferred to CZB | – |
| 37 h† | – | – | CZB without TSA |
| 40 h† | CZB without TSA | CZB without TSA | - |
*10 h group; †24 h group.
Morris water maze (MWM) experiment
The effect of TSA on mice learning and memory ability was tested using a Morris water maze, as previously described (Morris 1984, Tonegawa et al. 1995). The MWM apparatus was a 100-cm diameter black, circular tank. Within the tank, an 18-cm height, 10-cm diameter platform submerged ~1 cm beneath the 24 ± 1°C water. During the test, the platform is hidden in opaque water, but distal visual cues in room are available for the mice to use for navigation purposes. To efficiently find the platform, the mice must develop a spatial map of the platform’s location using the distal visual cues provided. For acquisition, the mice were given six trials per day (1 min maximum/trial) separated by an interval of 15 s over 3 days, for a total of 18 trials. A probe test on the fourth day, with the platform removed, served to ascertain that the mice were using a spatial learning strategy that involved multiple, specific, distal cues, or some other strategy which was used to determine if the mice were selectively swimming in the quadrant in which the platform had previously been located. Immediately following the probe trial, six reversal trials were administered, in which the platform was placed in the opposite quadrant at the same distance from the pool wall as in the acquisition trials. These reversal trials continued on the fifth day for a total of 12 trials. Time spent and performance in each of the four quadrants was recorded using a camera located over the tank and attached to a computer.
Reproductive performance of offspring derived from TSA-treated embryos
The reproductive performance of adult (8–9 weeks) female offspring derived from embryos treated with TSA was examined. The female mice were mated with normal male mice, and the subsequent litter was examined at 21 days post coitum. At this time, all live births and dead pups were recorded. Similar data was collected for the performance of male mice derived from embryos treated with TSA.
Differential staining of blastocysts for cell counting
The numbers of cells in the inner cell mass (ICM) and trophectoderm (TE) of blastocysts were determined as previously described (Papaioannou & Ebert 1988). In brief, the blastocysts were denuded with acidic PBS, rinsed in HEPES-CZB medium and exposed to a 1:10 dilution of rabbit anti-mouse whole serum (Sigma, M5774) for 30 min. They were briefly washed in HEPES-CZB medium and incubated in a 1:10 dilution of guinea pig complement (Sigma, A5545) with 10 μg/mL propidium iodide (PI) (Sigma, 81845) and 1 μg/mL Hoechst 33342 (Sigma, 14533) for 30 min at 37°C, and immediately examined using an inverted NIKON fluorescent microscope (E600, Japan). Cell counting was performed directly under the microscope.
Apoptosis assays
Detection and quantification of apoptosis at single-cell level were achieved by TUNEL and the in situ Cell Death Detection Kit (Roche, 12156792910) according to the manufacturer’s instructions. Embryos were observed with Zeiss LSM 510 META confocal microscope (Zeiss).
Immunofluorescence staining
Indirect immunofluorescence was performed as previously described with some modifications (Santos et al. 2005). All steps were performed at room temperature unless otherwise mentioned. Embryos were fixed with 4% paraformaldehyde overnight at 4°C, and then permeabilized with 0.5% Triton X-100 in PBS for 30 min. (For the detection of 5-methyl-cytosine (5-MeC), embryos were treated with 4N HCl at room temperature for 1 h after permeabilization.) After washing three times, all samples were blocked in 2% BSA in PBS for 1 h. Embryos were incubated with the first antibodies to H3K9 acetylation (1:200, Cell Signaling Technology, 9927), H4K5 acetylation (1:200, Upstate, 04-118, Lake Placid, NY), H3K18 acetylation (1:200, Upstate, 07-354), Lamin B (1:200, Santa Cruz Biotechnology, sc-374015) and anti-5-methyl-cytosine antibody (1:4000, gift from Nathalie Beaujean) for 1 h at 37°C. After three washes, the embryos were incubated with a FITC second antibody (Santa Cruz Biotechnology, sc-2359) for 1 h at 37°C. Finally, the DNA was stained with propidium iodide (PI, 10 μg/mL) for 5 min at 37°C and after three times washing all the samples were mounted and observed under Zeiss LSM 510 META confocal microscope (Zeiss). The immunofluorescence signal was captured by confocal microscope using the same parameters. The average fluorescence intensity of the interested region of embryos was utilized to indicate the fluorescence intensity of each signal. Quantification of immunofluorescence intensity was performed with ImageJ software (NIH).
Statistical analysis
Statistical analysis was performed using SPSS 13.0 statistical software. One-way ANOVA and Fisher's exact test were used for statistical analysis. For all statistical analyses, a value of P < 0.05 was considered to be statistically significant.
Results
Embryos preimplantation development was impaired by increased concentration and duration of TSA
The in vitro development of the in vivo/in vitro fertilized zygotes and diploid parthenogenetic embryos exposed to different concentration of TSA were compared and analyzed (Tables 2 and 3). The blastocyst rate of in vivo fertilized embryos was significantly decreased (P < 0.01), and that of in vitro and parthenogenetic embryos was not significantly affected by the 0.005–1.0 μM TSA treatment for 10 h (Table 2). However, there was a significant decrease in the blastocyst rate for all three groups when the in vivo/in vitro fertilized and parthenogenetic embryos were treated with 0.05–1 μM TSA for 24 h (Table 3, P < 0.01).
Preimplantation development of embryos treated with 0–1 μM TSA for 10 h.
| TSA concentration | No. (%) of two-cell blastocysts | ||
|---|---|---|---|
| In vivo fertilization | In vitro fertilization | Parthenogenesis | |
| CZB con. | 58 (82.01 ± 12.55)A | 43 (81.74 ± 2.86)a | 38 (52.05 ± 7.91)a |
| CZB+DMSO | 58 (78.52 ± 14.62)A | 32 (76.57 ± 7.34)ab | 41 (56.22 ± 12.55)a |
| 0.005 μM | 38 (58.85 ± 12.07)B | 37 (81.38 ± 6.09)a | 40 (54.75 ± 3.62)a |
| 0.05 μM | 32 (46.83 ± 9.03)B | 30 (75.31 ± 4.59)ab | 33 (48.05 ± 5.97)a |
| 0.1 μM | 44 (58.92 ± 8.89)B | 26 (61.37 ± 7.79)b | 42 (58.33 ± 7.26)a |
| 1 μM | 44 (61.76 ± 17.06)B | 32 (70.56 ± 5.58)ab | 40 (44.94 ± 3.77)a |
Values with different superscripts are significantly different in one column at P < 0.05 (lowercases) or P <0.01 (capital letters).
Preimplantation development of embryos treated with 0–1 μM TSA for 24 h.
| TSA concentration |
No. (%) two-cell blastocysts | ||
|---|---|---|---|
| In vivo fertilization | In vitro fertilization | Parthenogenesis | |
| CZB con. | 58 (82.01 ± 12.55)A | 43 (81.74 ± 2.86)Aa | 38 (52.05 ± 7.91)aA |
| CZB+DMSO | 55 (67.05 ± 25.23)Aa | 33 (80.09 ± 3.79)Aa | 33 (46.53 ± 5.77)aA |
| 0.005 μM | 46 (58.32 ± 17.53)aC | 40 (77.49 ± 6.42)Aab | 33 (45.18 ± 4.6)a |
| 0.05 μM | 38 (37.88 ± 16.32)Bb | 26 (56.34 ± 10.54)b | 30 (43.03 ± 4.57)ab |
| 0.1 μM | 26 (29.92 ± 21.74)B | 26 (60.21 ± 6.01)ab | 21 (28.54 ± 4.24)Bbc |
| 1 μM | 19 (27.13 ± 16.25)B | 15 (33.50 ± 11.69)Bc | 15 (21.68 ± 4.14)Bc |
Values with different superscripts are significantly different in one column at P < 0.05 (lowercases) or P <0.01 (capital letters).
A closer analysis of the effects of TSA concentration was performed, and the developmental rate of in vivo fertilized embryos at different preimplantation stages were compared (Table 4). As shown in Table 4, the embryos showed a significant decrease in blastocyst rate when treated with 0.1–1 μM TSA for 24 h compared to the embryos exposed to TSA only for 10 h. However, when the TSA concentration was low enough (0.005–0.05 μM), the treatment time had no significant effect on the development of treated embryos.
Effects of TSA treatment duration on preimplantation development of mouse embryos.
| TSA concentration /treatment time (h) | PN | Two-cell | Four-cell | Morula, n (%) | No. (%) of blastocysts |
|---|---|---|---|---|---|
| 0.005 μM | |||||
| 10 | 66 | 65 | 65 | 53 (81.54)a | 38 (58.85 ± 12.07)a |
| 24 | 81 | 81 | 78 | 58 (71.60)a | 46 (58.32 ± 17.53)a |
| 0.05 μM | |||||
| 10 | 69 | 69 | 62 | 49 (71.01)a | 32 (46.83 ± 9.03)a |
| 24 | 98 | 97 | 81 | 62 (63.92)a | 38 (37.88 ± 16.32)a |
| 0.1 μM | |||||
| 10 | 75 | 75 | 73 | 62 (82.67)A | 44 (58.92 ± 8.89)A |
| 24 | 83 | 82 | 66 | 41 (50.00)B | 26 (29.92 ± 21.74)B |
| 1 μM | |||||
| 10 | 74 | 73 | 70 | 59 (80.82)a | 44 (61.76 ± 17.06)A |
| 24 | 74 | 74 | 56 | 46 (62.16)b | 19 (27.13 ± 16.25)B |
Values with different superscripts are significantly different in one column at P < 0.05 (lowercases) or P <0.01 (capital letters).
Therefore, the effect of TSA on early embryo development depends on the nature of the embryo origin, the TSA concentration and treatment time.
The ICM cell number and apoptosis of embryos were not significantly affected by TSA treatment
In view of the low percentage of blastocyst formation from in vivo fertilized embryos after TSA treatment, we decided to check the quality of these embryos by examining cell numbers and apoptosis, two important parameters in assessing blastocyst quality (van Soom et al. 1997), using differential staining and TUNEL assays, respectively. There were no significant differences in ICM cells number and percentage of ICM cells in blastocysts treated with 0.005–1 μM TSA compared with that of DMSO control (Table 5), although the total cells number of the blastocysts treated by 1 μM TSA was significantly increased compared with that of the control.
Effects of TSA treatment (10 h) on cell number of mouse blastocysts.
| TSA concentration |
No. of blastocysts | Total cells | ICM cells | Percentage of ICM cells (%) |
|---|---|---|---|---|
| CZB+DMSO | 19 | 54.37 ± 2.36a | 11.21 ± 0.70a | 20.56 ± 0.99ab |
| 0.005 μM | 45 | 51.00 ± 2.09a | 11.33 ± 0.63a | 22.36 ± 0.94b |
| 1 μM | 39 | 56.51 ± 2.73b | 10.46 ± 0.75a | 18.76 ± 0.98a |
Values with different superscripts are significantly different in one column. P < 0.05.
To examine the effect of TSA treatment on apoptosis in the blastocysts, TUNEL assays were conducted. As shown in Table 6 and Fig. 1, the TSA treatment appeared to have no significant effect on apoptosis, as calculated by the percentage of apoptotic blastocysts and the percent of apoptotic cells in blastocysts.
Effects of TSA treatment on apoptosis of mouse blastocysts. Fluorescent micrographs demonstrating apoptotic morphology in mice embryos treated with TSA are shown in A, B and C: CZB+DMSO (A), 0.005 μM TSA (B), 1 μM TSA (C). PI staining was used to visualize nuclei (red color). Condensed red nuclei are morphological signs without TUNEL labeling (M), apoptotic cells are green after TUNEL labeling. After overlapped with the signal for nuclei, the apoptotic cells are yellow in color (T+M). Bar 10 μm.
Citation: Reproduction 162, 1; 10.1530/REP-21-0020
Effects of TSA treatment on apoptosis of mouse blastocysts. Fluorescent micrographs demonstrating apoptotic morphology in mice embryos treated with TSA are shown in A, B and C: CZB+DMSO (A), 0.005 μM TSA (B), 1 μM TSA (C). PI staining was used to visualize nuclei (red color). Condensed red nuclei are morphological signs without TUNEL labeling (M), apoptotic cells are green after TUNEL labeling. After overlapped with the signal for nuclei, the apoptotic cells are yellow in color (T+M). Bar 10 μm.
Citation: Reproduction 162, 1; 10.1530/REP-21-0020
Effects of TSA treatment on apoptosis of mouse blastocysts. Fluorescent micrographs demonstrating apoptotic morphology in mice embryos treated with TSA are shown in A, B and C: CZB+DMSO (A), 0.005 μM TSA (B), 1 μM TSA (C). PI staining was used to visualize nuclei (red color). Condensed red nuclei are morphological signs without TUNEL labeling (M), apoptotic cells are green after TUNEL labeling. After overlapped with the signal for nuclei, the apoptotic cells are yellow in color (T+M). Bar 10 μm.
Citation: Reproduction 162, 1; 10.1530/REP-21-0020
Effects of TSA treatment (10 h) on apoptosis of mouse blastocysts.
| TSA concentration |
No. of blastocysts | No. (%) of apoptotic blastocysts | Total cells | Apoptotic cells (%) |
|---|---|---|---|---|
| CZB+DMSO | 14 | 7 (50.00)a | 1062 | 19 (1.79)ab |
| 0.005 μM | 18 | 10 (55.56)a | 1555 | 16 (1.03)a |
| 1 μM | 28 | 21 (75.00)a | 2155 | 47 (2.18)b |
Values with different superscripts are significantly different in one column. P < 0.05.
These results indicated that the blastocyst quality, evaluated by both the rate of apoptosis and the ratio of ICM cells, is not affected significantly by TSA treatment.
Post-implantation development at E11.5 was not significantly affected by TSA treatment
Post-implantation development of TSA-treated embryos were then further investigated. Two-cell stage embryos treated with or without TSA were transferred into E0.5 pseudo-pregnant recipients and the pups were delivered by caesarian section at E11.5 (Table 7). The implantation sites, normal embryos and absorbed embryos were recorded. There was a tendency of decline in implantation site and the percentage of normal embryos after TSA treatment. The percentage of absorbed embryos also increased more than two-fold compared with the control embryos (10.26% vs 28.00% vs 22.22% for control, low and high concentration of TSA treatment, respectively), but the difference was not statistically significant.
Effects of TSA treatment on mice post-implantation development.
| TSA concentration |
No. of two- cell/ female mice |
Implantation (% of embryos transferred) |
Embryos recovered | Normal embryos (% of embryos collected) | Absorbed embryos (% of embryos recovered) |
|---|---|---|---|---|---|
| CZB+DMSO | 74/8 | 39 (52.70)a | 39 | 35 (89.74)a | 4 (10.26)a |
| 0.005 μM | 55/6 | 25 (45.45)a | 25 | 18 (72.00)a | 7 (28.00)a |
| 1 μM | 70/7 | 27 (38.37)a | 27 | 21 (77.78)a | 6 (22.22)a |
Values with different superscripts are significantly different in one column. P < 0.05.
The sex ratio, postnatal death rate and body mass were not significantly affected by low dose TSA treatment
At 21 days after birth, the death rate, sex ratio and body mass of pups were recorded and analyzed (Table 8) to estimate the long-term effect of TSA on the embryos development. One micrometer TSA treatment resulted in a significant reduction in the livebirth rate from 56.59 to 38.33%, while the sex ratio, death rate, and body mass of male and female pups were not significantly affected.
Effects of TSA treatment (10 h) on pups’ body mass and death rate.
| TSA concentration |
Two- cell/ mice | No. (%) of pups | No. (%) of male pups | Dead pups (%) |
Body mass of male pups (g) | Body mass of female pups (g) |
|---|---|---|---|---|---|---|
| CZB | 56/4 | 31 (55.36)ac | 16 (51.61)a | 0 (0.00)a | 16.61 ± 1.27a | 15.09 ± 1.23a |
| CZB+DMSO | 129/9 | 73 (56.59)Aa | 44 (60.27)a | 2 (2.74)a | 15.14 ± 0.81a | 14.25 ± 0.694a |
| 0.005 μM TSA | 296/20 | 121 (40.88)Bbc | 55 (45.45)a | 3 (2.48)a | 14.05 ± 1.97a | 13.51 ± 1.57a |
| 1 μM TSA | 287/18 | 110 (38.33)Bb | 69 (62.73)a | 1 (0.91)a | 17.29 ± 0.79a | 15.87 ± 0.49a |
Values with different superscripts are significantly different in one column at P < 0.05 (lowercases) or P <0.01 (capital letters).
The reproductive performance of offsprings was not significantly affected by TSA treatment
The reproductive performance of adult female and male mice derived from TSA-treated embryos was compared (Tables 9 and 10). Eight TSA-treated female mice and nine DMSO control mice were randomly chosen to mate with normal male mice, and the pregnancy rate, litter size (pups per female), death rate and sex ratio of pups were recorded and analyzed. Although the death rate at 21 days post-parturition was slightly elevated (3.75% vs 0.00%) after TSA treatment, there were no significant differences between the two groups in the pregnancy rate, litter size and sex ratio of the pups. Results listed in Table 10 demonstrate that the performance of male, the litter size and sex ratio are nearly equal between the TSA and DMSO control group.
Effects of TSA treatment (10 h) on the reproductive performance of females derived from TSA-treated embryos.
| TSA concentration |
No. of tested females | Pregnant mice (%) | No. of pups (pups per female) | No. (%) of male pups | Dead pups (%) |
No. of female pups |
|---|---|---|---|---|---|---|
| CZB+DMSO | 9 | 9 (100.00)a | 87 (9.67 ± 1.33)a | 49 (60.91 ± 6.07)a | 0 (0.00)a | 38 |
| 1 μM TSA | 8 | 7 (87.50)a | 80 (10.00 ± 1.85)a | 37 (48.19 ± 3.89)a | 3 (3.75)a | 40 |
Values with same superscripts are not significantly different in one column. P > 0.05.
Effects of TSA treatment (10 h) on the reproductive performance of males derived from TSA-treated embryos.
| TSA concentration |
No. of tested males | No. of used females | No. of pups (pups per female) | No. (%) of male pups | No. of female pups |
|---|---|---|---|---|---|
| CZB+DMSO | 4 | 8 | 82 (10.25 ± 1.06)a | 39 (49.91 ± 8.52)a | 43 |
| 1 μM TSA | 5 | 10 | 103 (10.30 ± 1.09)a | 52 (51.74 ± 6.66)a | 51 |
Values with different superscripts are not significantly different in one column. P > 0.05.
TSA treatment did not significantly affect the ability of spatial learning and memory of pups
In order to study the influence of TSA treatment on the ability of spatial memory and learning, the Morris water maze experiment was performed (Fig. 2). There was no significant difference in learning ability between the pups derived from the embryos treated with 0.005–1 μM TSA and DMSO. The time spent in finding the platform became shorter in each group, with a lapse of the trials. Although the 0.005 μM TSA group showed a shorter time in searching for the platform in all the trials, there were no statistically significant differences between the groups (P > 0.05).
Effects of TSA treatment for 10 h on the ability of spatial learning and memory. (A) Average latency of male mice to find hidden platform-acquisition trials. (B) Average latency of male mice to find hidden platform-reversal trials. (C) Average time spent of male mice to search quadrant in which trained vs other quadrant during a Morris water task. (D) Average latency of female mice to find hidden platform-acquisition trials. (E) Average latency of female mice to find hidden platform-reversal trials. (F) Average time spent of female mice to search quadrant in which trained vs other quadrant during a Morris water task.
Citation: Reproduction 162, 1; 10.1530/REP-21-0020
Effects of TSA treatment for 10 h on the ability of spatial learning and memory. (A) Average latency of male mice to find hidden platform-acquisition trials. (B) Average latency of male mice to find hidden platform-reversal trials. (C) Average time spent of male mice to search quadrant in which trained vs other quadrant during a Morris water task. (D) Average latency of female mice to find hidden platform-acquisition trials. (E) Average latency of female mice to find hidden platform-reversal trials. (F) Average time spent of female mice to search quadrant in which trained vs other quadrant during a Morris water task.
Citation: Reproduction 162, 1; 10.1530/REP-21-0020
Effects of TSA treatment for 10 h on the ability of spatial learning and memory. (A) Average latency of male mice to find hidden platform-acquisition trials. (B) Average latency of male mice to find hidden platform-reversal trials. (C) Average time spent of male mice to search quadrant in which trained vs other quadrant during a Morris water task. (D) Average latency of female mice to find hidden platform-acquisition trials. (E) Average latency of female mice to find hidden platform-reversal trials. (F) Average time spent of female mice to search quadrant in which trained vs other quadrant during a Morris water task.
Citation: Reproduction 162, 1; 10.1530/REP-21-0020
Histone acetylation of lysine residues 18 of H3 (H3K18), lysine residues 9 of H3 (H3K9), lysine residues 5 of H4 (H4K5) in early preimplantation embryos were differentially affected by TSA treatment
After TSA treatment, the zygotes or two-cell embryos were fixed and subjected to immunofluorescence staining to determine the change of their histone acetylation status. Initially, there was no visible difference in H3K18 acetylation between embryos treated with 0.005–1 μM TSA for 10 h and the untreated control zygotes. This was indicative of the fact that TSA treatment did not significantly modify the H3K18 acetylation at the indicated concentration and treatment duration (Fig. 3, Supplementary Fig. 1C and E, see section on supplementary materials given at the end of this article). Secondly, histone acetylation of H3K9 in zygote and 2-cell embryos exhibited dramatic changes after TSA treatment in a dose-dependent manner (Fig. 4, Supplementary Fig. 1F and G). Thirdly, as shown in Fig. 5 and Supplementary Fig. 1H and I, the H4K5 acetylation signal cannot be detected in the untreated controls as well as in the 0.005 μM TSA-treated metaphase and anaphase zygotes, but the H4K5 was highly acetylated in the embryos of 1 μM TSA treatment group. However, the polar body was acetylated in all of the control embryos, in the 0.005 μM and 1 μM TSA-treated zygotes. This result suggested that the ability of TSA to induce the H4K5 acetylation is clearly concentration dependent.
The DNA methylation and H3K18 acetylation patterns of mice zygote treated with TSA for 10 h. Immunofluorescent staining of DNA methylation and H3K18 acetylation are portrayed (green), while the pronuclei and polar body were revealed by PI staining (red). In mice, the pronuclei can be easily assigned as the female pronucleus is the smaller one of the two pronuclei and the male pronucleus is the larger one of the two pronuclei. A, B, C, D and E: DNA methylation. The difference in intensity of DNA methylation is evident between the female and male pronucleus (higher in female pronucleus than in male pronucleus). A’, B’, C’, D’ and E’: H3K18 acetylation pattern. The intensity of H3K18 acetylation is nearly the same in both of the pronuclei. (A, A’) CZB+DMSO, (B, B’) 0.005 μM TSA, (C, C’) 0.050 μM TSA, (D, D’) 0.100 μM TSA, (E, E’) 1 μM TSA. There was no significant difference in DNA methylation and H3K18 acetylation between the TSA-treated and control zygotes. Bars 20 μm.
Citation: Reproduction 162, 1; 10.1530/REP-21-0020
The DNA methylation and H3K18 acetylation patterns of mice zygote treated with TSA for 10 h. Immunofluorescent staining of DNA methylation and H3K18 acetylation are portrayed (green), while the pronuclei and polar body were revealed by PI staining (red). In mice, the pronuclei can be easily assigned as the female pronucleus is the smaller one of the two pronuclei and the male pronucleus is the larger one of the two pronuclei. A, B, C, D and E: DNA methylation. The difference in intensity of DNA methylation is evident between the female and male pronucleus (higher in female pronucleus than in male pronucleus). A’, B’, C’, D’ and E’: H3K18 acetylation pattern. The intensity of H3K18 acetylation is nearly the same in both of the pronuclei. (A, A’) CZB+DMSO, (B, B’) 0.005 μM TSA, (C, C’) 0.050 μM TSA, (D, D’) 0.100 μM TSA, (E, E’) 1 μM TSA. There was no significant difference in DNA methylation and H3K18 acetylation between the TSA-treated and control zygotes. Bars 20 μm.
Citation: Reproduction 162, 1; 10.1530/REP-21-0020
The DNA methylation and H3K18 acetylation patterns of mice zygote treated with TSA for 10 h. Immunofluorescent staining of DNA methylation and H3K18 acetylation are portrayed (green), while the pronuclei and polar body were revealed by PI staining (red). In mice, the pronuclei can be easily assigned as the female pronucleus is the smaller one of the two pronuclei and the male pronucleus is the larger one of the two pronuclei. A, B, C, D and E: DNA methylation. The difference in intensity of DNA methylation is evident between the female and male pronucleus (higher in female pronucleus than in male pronucleus). A’, B’, C’, D’ and E’: H3K18 acetylation pattern. The intensity of H3K18 acetylation is nearly the same in both of the pronuclei. (A, A’) CZB+DMSO, (B, B’) 0.005 μM TSA, (C, C’) 0.050 μM TSA, (D, D’) 0.100 μM TSA, (E, E’) 1 μM TSA. There was no significant difference in DNA methylation and H3K18 acetylation between the TSA-treated and control zygotes. Bars 20 μm.
Citation: Reproduction 162, 1; 10.1530/REP-21-0020
H4K5 acetylation pattern of mice metaphase and anaphase stage embryos treated with TSA for 10 h. (A, A’) CZB+DMSO, (B, B’) 0.005 μM TSA, (C, C’) 1 μM TSA. The signal of H4K5 acetylation was not detected in the control and 0.005 μM TSA-treated embryos at metaphase and anaphase stage, but can be detected in the 1 μM TSA-treated embryos at metaphase and anaphase stage. Bars 20 μm.
Citation: Reproduction 162, 1; 10.1530/REP-21-0020
H4K5 acetylation pattern of mice metaphase and anaphase stage embryos treated with TSA for 10 h. (A, A’) CZB+DMSO, (B, B’) 0.005 μM TSA, (C, C’) 1 μM TSA. The signal of H4K5 acetylation was not detected in the control and 0.005 μM TSA-treated embryos at metaphase and anaphase stage, but can be detected in the 1 μM TSA-treated embryos at metaphase and anaphase stage. Bars 20 μm.
Citation: Reproduction 162, 1; 10.1530/REP-21-0020
H4K5 acetylation pattern of mice metaphase and anaphase stage embryos treated with TSA for 10 h. (A, A’) CZB+DMSO, (B, B’) 0.005 μM TSA, (C, C’) 1 μM TSA. The signal of H4K5 acetylation was not detected in the control and 0.005 μM TSA-treated embryos at metaphase and anaphase stage, but can be detected in the 1 μM TSA-treated embryos at metaphase and anaphase stage. Bars 20 μm.
Citation: Reproduction 162, 1; 10.1530/REP-21-0020
High concentration of TSA differentially affected the DNA methylation and H3K18 acetylation in early preimplantation stage embryos
The epigenetic profile of TSA-treated embryos were further analyzed with respect to DNA methylation and H3K18 acetylation following the various TSA treatments on the early preimplantation embryos. Our results indicated acetylation of H3K18 did not change following the low concentration TSA treatment (Fig. 3, Supplementary Fig. 1B and E). However, DNA methylation in male pronucleus was decreased upon TSA treatment, but that of female pronucleus was not significantly affected at the indicated concentration (Fig. 3, Supplementary Fig. 1A, C and D).
In order to investigate whether TSA could affect H3K18 acetylation and DNA methylation in a dose-dependent manner, we treated the zygotes using a little higher concentration of TSA. Interestingly, when TSA concentration was increased to 200 μM, H3K18 acetylation change cannot be detected in one-cell stage embryos (Fig. 6A’ and Supplementary Fig. 1J); however, the localization of DNA methylation in the heterochromatin was apparently disturbed. As show in Fig. 6B (arrow), the integrity of heterochromatin ring was disturbed, and a disarray of heterochromatin dots was displayed (Fig. 6B, arrow).
The DNA methylation, H3K18 acetylation and Lamin B immunofluorescent staining of mice zygotes treated by 200 μM TSA for 10 h. The zygotes were stained for 5-MeC and H3K18 distribution using an FITC-conjugated secondary antibody and the Lamin B distribution was visualized with a TRITC-conjugated secondary antibody. DNA was visualized by a red staining (PI staining). (A, A’) CZB+DMSO, (B, B’) 200 μM TSA. The localization of DNA methylation in the heterochromatin is disturbed and heterochromatin ring is not integrated which displays a disarray of heterochromatin dots (B, arrow). Bar 20 μm.
Citation: Reproduction 162, 1; 10.1530/REP-21-0020
The DNA methylation, H3K18 acetylation and Lamin B immunofluorescent staining of mice zygotes treated by 200 μM TSA for 10 h. The zygotes were stained for 5-MeC and H3K18 distribution using an FITC-conjugated secondary antibody and the Lamin B distribution was visualized with a TRITC-conjugated secondary antibody. DNA was visualized by a red staining (PI staining). (A, A’) CZB+DMSO, (B, B’) 200 μM TSA. The localization of DNA methylation in the heterochromatin is disturbed and heterochromatin ring is not integrated which displays a disarray of heterochromatin dots (B, arrow). Bar 20 μm.
Citation: Reproduction 162, 1; 10.1530/REP-21-0020
The DNA methylation, H3K18 acetylation and Lamin B immunofluorescent staining of mice zygotes treated by 200 μM TSA for 10 h. The zygotes were stained for 5-MeC and H3K18 distribution using an FITC-conjugated secondary antibody and the Lamin B distribution was visualized with a TRITC-conjugated secondary antibody. DNA was visualized by a red staining (PI staining). (A, A’) CZB+DMSO, (B, B’) 200 μM TSA. The localization of DNA methylation in the heterochromatin is disturbed and heterochromatin ring is not integrated which displays a disarray of heterochromatin dots (B, arrow). Bar 20 μm.
Citation: Reproduction 162, 1; 10.1530/REP-21-0020
Furthermore, DNA methylation and H3K18 acetylation were examined in two-cell embryos derived from the zygotes treated with TSA at a high concentration, and a significant change of DNA methylation and H3K18 acetylation was observed (Fig. 7, Supplementary Fig. 1K and L). These two-cell embryos were fixed at 60 h after 40 μM TSA treatment for 10 h (Fig. 7B and B’) and 24 h (Fig. 7C and C’). The level of DNA methylation and H3K18 acetylation in the whole nucleus of these two-cell stage embryos was dramatically decreased and increased, respectively. Notably, the development of these two-cell stage embryos was delayed by the TSA treatment (Fig. 7). The Lamin B staining that marks the intact nucleus membrane indicated that these embryos were delayed before nucleus membrane breakdown.
The DNA methylation (A, B, C), H3K18 acetylation (A’, B’, C’) and Lamin B (A’’, B’’, C’’) of developmentally arrested two-cell stage embryos (60 h after hCG injection) treated with 40 μM TSA for 10 h and 24 h. DNA was revealed by red staining (PI staining) and 5-MeC and H3K18 acetylation were revealed by green staining (FITC-conjugated secondary antibody) but the Lamin B distribution was stained by a TRITC-conjugated secondary antibody. (A, A’, A’’) CZB+DMSO, (B, B’, B’’) 40 μM TSA treatment for 10 h, (C, C’, C’’) 40 μM TSA treatment for 24 h. Compared to the control two-cell embryos (A, A’), DNA methylation (B, C) and H3K18 acetylation (B’, C’) exhibit an evident difference in the TSA-treated two-cell embryos. The intensity of DNA methylation and H3K18 acetylation of two-cell embryos treated with TSA for 10 and 24 h are decreased and increased, respectively. The immunofluorescent staining of Lamin B in the developmentally arrested two-cell embryos shows that the membrane of nuclei is integrated which indicates the two-cell embryos are blocked before the prometaphase. Bars 20 μm.
Citation: Reproduction 162, 1; 10.1530/REP-21-0020
The DNA methylation (A, B, C), H3K18 acetylation (A’, B’, C’) and Lamin B (A’’, B’’, C’’) of developmentally arrested two-cell stage embryos (60 h after hCG injection) treated with 40 μM TSA for 10 h and 24 h. DNA was revealed by red staining (PI staining) and 5-MeC and H3K18 acetylation were revealed by green staining (FITC-conjugated secondary antibody) but the Lamin B distribution was stained by a TRITC-conjugated secondary antibody. (A, A’, A’’) CZB+DMSO, (B, B’, B’’) 40 μM TSA treatment for 10 h, (C, C’, C’’) 40 μM TSA treatment for 24 h. Compared to the control two-cell embryos (A, A’), DNA methylation (B, C) and H3K18 acetylation (B’, C’) exhibit an evident difference in the TSA-treated two-cell embryos. The intensity of DNA methylation and H3K18 acetylation of two-cell embryos treated with TSA for 10 and 24 h are decreased and increased, respectively. The immunofluorescent staining of Lamin B in the developmentally arrested two-cell embryos shows that the membrane of nuclei is integrated which indicates the two-cell embryos are blocked before the prometaphase. Bars 20 μm.
Citation: Reproduction 162, 1; 10.1530/REP-21-0020
The DNA methylation (A, B, C), H3K18 acetylation (A’, B’, C’) and Lamin B (A’’, B’’, C’’) of developmentally arrested two-cell stage embryos (60 h after hCG injection) treated with 40 μM TSA for 10 h and 24 h. DNA was revealed by red staining (PI staining) and 5-MeC and H3K18 acetylation were revealed by green staining (FITC-conjugated secondary antibody) but the Lamin B distribution was stained by a TRITC-conjugated secondary antibody. (A, A’, A’’) CZB+DMSO, (B, B’, B’’) 40 μM TSA treatment for 10 h, (C, C’, C’’) 40 μM TSA treatment for 24 h. Compared to the control two-cell embryos (A, A’), DNA methylation (B, C) and H3K18 acetylation (B’, C’) exhibit an evident difference in the TSA-treated two-cell embryos. The intensity of DNA methylation and H3K18 acetylation of two-cell embryos treated with TSA for 10 and 24 h are decreased and increased, respectively. The immunofluorescent staining of Lamin B in the developmentally arrested two-cell embryos shows that the membrane of nuclei is integrated which indicates the two-cell embryos are blocked before the prometaphase. Bars 20 μm.
Citation: Reproduction 162, 1; 10.1530/REP-21-0020
Discussion
In this study, we investigated the effect of TSA treatment on mouse embryogenesis from both fertilized and parthenogenetic embryos and their subsequent development and certain neurodevelopmental functional endpoints. The effect of TSA on the preimplantation development of fertilized and parthenogenetic embryos appear to be dependent upon the origin of the embryos, the TSA concentration, as well as the duration of TSA treatment. A negative effect of TSA on post-implantation and full-term developmental rates was also observed, but the reproduction performance of adult offspring derived from TSA-treated group was not significantly different from that of untreated controls. In addition, TSA treatment can alter DNA methylation, acetylation of H3K9 and K4K5 in treated embryos at certain TSA concentrations.
Our results strongly suggest that TSA treatment has various effects on the preimplantation and post-implantation development of embryos with different reproductive origins (in vivo/in vitro fertilized embryos and parthenogenetic embryos) (Tables 2 and 3). This is consistent with previously reported observations that TSA treatment decreased the offspring production rate of the embryos derived from intracytoplasmic sperm injection (ICSI) but had no significant deleterious effects on round spermatid injection (ROSI) and parthenogenetic embryos. This suggests that the effect of TSA treatment on embryonic development depends upon the nature of the zygotic origin (Kishigami et al. 2006b). TSA at low concentrations is known to affect many biological processes, including the induction of cell cycle arrest, differentiation and apoptosis (Yoshida et al. 1990, Kelly et al. 2002, Falkenberg & Johnstone 2014, Song et al. 2018, Morales Torres et al. 2020). Similarly, histone hyperacetylation secondary to TSA treatment can disturb the cell cycle and inhibit embryonic development (Aoki & Schultz 1999). Furthermore, TSA treatment at the one- and two-cell stages lead to inhibition of subsequent embryonic cleavage (Ma et al. 2001). These findings are all consistent with our finding of delayed development of two-cell embryos after TSA treatment (Fig. 7). Akiyama et al.reported that TSA treatment could result in embryonic death in utero at an early stage of development due to the induction of aneuploidy in the oocyte (Akiyama et al. 2006). This is also in accordance with our finding that more fetuses treated with TSA were resorbed at E11.5 (Table 7). Interestingly, we found that 0.005 μM TSA treatment resulted in a significantly decreased livebirth rate, but cannot obviously reduce the implantation sites calculated at E11.5. The possible reason might be that we counted the implantation sites and livebirth rate at E11.5 and E19.5 post coitum, respectively. Therefore, during the time from E11.5 to E19.5, more embryos were absorbed again in the uterus in the 0.005 μM TSA treatment group than that of the control group. Because we did not count the absorbed embryos at birth so this data is lost. However, as we can see from Tables 7 and 8, there’s a same decline tendency for the implantation site and livebirth rate after TSA treatment. Teratogenic effects on axial skeleton were observed when TSA was introduced to the pregnant CD mice at day 8 post coitum (Menegola et al. 2005). However, our results indicated that the surviving liveborn pups did not show a significant difference in terms of their sex ratio, death rate, body mass, productive performance and biological function, such as the ability of spatial learning and memory compared to that of control group. This indicated the period of mouse development that is most affected by TSA treatment is limited to early preimplantation stage embryos.
For the normal development of in vivo fertilized embryos, the first major preimplantation developmental event, zygotic gene activation (ZGA), occurs at the one- to two-cell stages. Unique to this critical period of development is a global genome activation under the influence of selected genes involved in transcription and RNA processing being preferentially expressed (Zeng et al. 2004). All of this is superimposed with the development of a chromatin-based transcriptionally repressive state that can be relieved by inducing histone acetylation or by inhibiting the second round of DNA replication. These two processes function in concert to establish the appropriate pattern of gene expression required for further appropriate embryonic development. Large-scale microarray studies have identified a single HDAC1, which appeared to be one of the important candidate genes critical for sustaining the ZGA, as well as the development of the transcriptionally repressive state at the onset of ZGA (Zeng & Schultz 2005). The role of HDAC1 was further verified that it is likely to be a major deacetylase in preimplantation embryos, with expression inversely correlated with changes in the acetylation state of histone H4K5. This relationship appears to be responsible for the development of a transcriptionally repressive state that begins at two-cell embryos (Zeng & Schultz 2005, Ma & Schultz 2008). Thus, it is understandable that TSA treatment during this critical period of development would induce hyperacetylation such that it distorts the normal process of zygotic gene activation. Clearly, the inappropriate expression of genes may lead to developmental delay and differential effect on embryogenesis depending on the precise nature of the early embryo. The in vitro fertilized and the parthenogenetic embryos may already be in a differential state with respect to histone acetylation. Therefore DNA methylation would be more resistant to TSA’s adverse developmental effects at a certain concentration/length of treatment than for those embryos developing in vivo. Thus, the effects on different residues might also be different. The specific effects of TSA on reprogramming through HDAC1 can be further investigated through manipulation of HDAC1 using knockdown or overexpression approaches, which in turn, may elucidate the underlying mechanism for the regulation of early embryo development. In other studies, TSA treatment of SCNT, ICSI or ROSI embryos was shown to lead to hyperacetylated histone H3 and H4 (Spinaci et al. 2004, Kishigami et al. 2006a, Rybouchkin et al. 2006, Dai et al. 2010), and might trigger selective DNA demethylation relying on the cell type and genomic region in mammalian cells and Neurospora (Szyf 2005). For example, DNA methylation level in ROSI and ICSI zygotes, specifically on spermatid-derived paternal genomes but not maternal ones, is significantly decreased upon TSA treatment (Kishigami et al. 2006a), but no significant changes were observed with SCNT embryos (Enright et al. 2003). This may suggest different epigenetic states of preimplantation embryos carrying unique nuclear types that is governed by different mechanisms of regulation that initiate zygotic gene activation in these early embryos, reflected by their differences in histone acetylation (at different amino acid residues) as well as DNA methylation after TSA treatments. These effects could also sustain into the post-implantation stage in treated mice, but with minimum effect on postnatal functions once born.
Detailed molecular mechanisms underlying the effect of TSA on different types of embryonic development needs to be further elucidated. Our preliminary findings demonstrated that TSA’s effect is dependent upon the types of preimplantation embryos, TSA concentration and the duration of TSA treatment. With further understanding of the regulation of the histone acetylation/deacetylation and DNA methylation on mouse embryonic and postnatal development, we should be able to optimize the TSA treatment program for future applications that will greatly improve therapeutic cloning, among many other applications, including the cloned transgenic porcine.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/REP-21-0020.
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 work was supported in part by the National Natural Science Foundation of China (No. 81972558) and the Startup Funding of First Hospital, JLU (No. 04035020001).
Author contribution statement
Z Q and X D conceived the project, designed the experiments, and interpreted the data. C P and X D performed the majority of the experiments with crucial help from Z L and T H, X D and Z Q wrote the manuscript.
The H3K9 acetylation pattern of mouse zygotes treated with TSA for 10 h. Immunofluorescent staining of H3K9 acetylation in zygotes (A–C: 27 h after hCG) and two-cell stage embryos (A’–C’: 44 h after hCG) are portrayed (green), and the pronuclei and polar body were revealed by PI (red). (A, A’) CZB+DMSO, (B, B’) 0.005 μM TSA, (C, C’) 1 μM TSA. The H3K9 acetylation in zygote and two-cell stage embryo treated by TSA is increased compared with that of the controls. Bars 20 μm.
Citation: Reproduction 162, 1; 10.1530/REP-21-0020
The H3K9 acetylation pattern of mouse zygotes treated with TSA for 10 h. Immunofluorescent staining of H3K9 acetylation in zygotes (A–C: 27 h after hCG) and two-cell stage embryos (A’–C’: 44 h after hCG) are portrayed (green), and the pronuclei and polar body were revealed by PI (red). (A, A’) CZB+DMSO, (B, B’) 0.005 μM TSA, (C, C’) 1 μM TSA. The H3K9 acetylation in zygote and two-cell stage embryo treated by TSA is increased compared with that of the controls. Bars 20 μm.
Citation: Reproduction 162, 1; 10.1530/REP-21-0020
The H3K9 acetylation pattern of mouse zygotes treated with TSA for 10 h. Immunofluorescent staining of H3K9 acetylation in zygotes (A–C: 27 h after hCG) and two-cell stage embryos (A’–C’: 44 h after hCG) are portrayed (green), and the pronuclei and polar body were revealed by PI (red). (A, A’) CZB+DMSO, (B, B’) 0.005 μM TSA, (C, C’) 1 μM TSA. The H3K9 acetylation in zygote and two-cell stage embryo treated by TSA is increased compared with that of the controls. Bars 20 μm.
Citation: Reproduction 162, 1; 10.1530/REP-21-0020
Acknowledgements
The authors thank the members of Dai and Zhou laboratories for useful discussions.
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