Marine natural products are considered to be extremely valuable resources of natural products and are furnished with diverse chemical structures and various bioactivities.1 Many marine natural products have successfully advanced to the late stages of clinical trials and even clinical use for many years, for example, ara-A (vidarabine). Psammaplin A (PsA), a natural product isolated from the Fijian marine sponge Aplysinella rhax, is a type of bromotyrosine derivative containing the intramolecular disulfide bond. Bioactivities of PsA include antibacterial, antiviral, anticancer, etc. PsA could inhibit Gram-positive bacteria, such as Staphylococcus aureus2 and methicillin-resistant S aureus (MRSA),3,4 and Gram-negative Vibrio vulnificus.5 Although PsA has not given origin to a commercial medication or clinical use so far, it has great potential for drug development and is one of the most promising antibacterial substances from sponges.6 The potential antibacterial activity of PsA may depend on its ability in inhibiting DNA synthesis and gyrase activity. Also, PsA is a potential antiviral agent for inhibiting the hepatitis C virus.7 Moreover, PsA possesses antiproliferative activities against various cancer cell lines, including triple-negative breast (TNBC; MDA-MB-231), doxorubicin-resistant human breast (MCF-7/adr), colon (HCT15), ovarian (SK-OV-3), lung (A549, LM4175), bone (BoM1833), endometria, brain (BrM-2a), skin (SK-MEL-2), and central nervous system (XF498) cancer cell lines.8–12 The underlying anticancer properties of PsA may depend on its efficacy in regulating enzymes that regulate apoptosis, differentiation, invasion, proliferation, angiogenesis, and DNA replication.
Of these bioactivities, PsA has attracted much attention because of its strong inhibitory activity in the well-known enzyme histone deacetylases (HDACs). Epigenetic changes modify the chromatin structure and the accessibility of DNA, thereby regulating the patterns of gene expression. In general, histone acetylation benefits the dissociation of DNA and histone octamer and loosens the structure of the nucleosomes, which leads to combine specifically all sorts of transcription factors and collaborative transcription factors with DNA binding sites.13,14 The level of histone acetylation is controlled by histone acetyltransferases (HATs) and HDACs. HDACs control gene transcription by regulating the acetylation of DNA sequence-specific transcription factors.15,16 HDACs tighten chromatin structure and repress transcription and gene expression by removing acetyl groups from histone or nonhistone proteins. Thus, HDACs are emerging as key regulators of gene expression. Eighteen mammalian HDACs have been identified to date,17 grouped into 4 classes.18 HDAC1 belongs to class I HDACs and is the HDAC involved in cell cycle regulation, cellular proliferation, DNA repair, and so on. Germline deletion of HDAC1 results in an early embryonic lethal phenotype due to severe developmental problems in mice.19 Overexpression of class I HDAC isotypes occurs in aggressive subtypes of tumors and somatic cell nuclear transfer (SCNT) embryos.20,21 In recent years, HDACs, especially class I, have been used as highly attractive targets for developing cancer therapeutics and inefficient reprogramming correction.
A large group of small molecules has been developed as HDAC inhibitors (HDACi) for cancer therapy and clone embryo reprogramming correction, such as trichostatin A (TSA),22,23 valproic acid (VPA),24 scriptaid,25 oxamflatin,26 and sodium butyrate (NaBu).9 Among those previously reported HDACi, PsA, as a new HDACi, displays an excellent HDAC1 inhibitory activity. However, PsA has not been extended to clinical cancer therapy most likely because of the unconfirmed side effects (cytotoxic or genotoxic), especially reproductive toxicity. In addition, since bovine and human 8-cell embryos (embryonic gene activation stage) demonstrated remarkably similar patterns of sequence enrichment and similar blastocysts stage and pregnancy duration, cattle may be a more informative model for human preimplantation development than mice.27 Therefore, in the present study we envisaged the effects of PsA on the development, gene expression, and epigenetic modifications of bovine-preimplanted PA embryos to provide novel insight into HDACi on reproduction toxicity and evaluate the potential application in cancer therapeutics and cloning efficiency improvement.
Materials and Methods
Preparation and maturation of oocytes
Bovine ovaries were purchased from the Changchun local slaughterhouse and transported to the laboratory in a warm saline solution, complying with the University of Jilin Agricultural University’s ethical animal testing and research regulations. Cumulus-oocyte complexes (COCs) were aspirated from follicles measuring 2 to 8 mm by using a 10-ml syringe. Only COCs with healthy layers of cumulus cells were selected for maturation. The cells were washed using the collection medium M199 supplemented with 2% fetal bovine serum (FBS; Hyclone) and transferred to the maturation medium containing modified M199 medium supplemented with alanine-glutamine (0.1 mM), sodium pyruvate (0.2 mM), epidermal growth factor (EGF; 50 ng/ml), follicle-stimulating hormone (FSH; 10 μg/ml), luteinizing hormone (LH; 10 μg/ml), estradiol(1 μg/ml), cysteamine (0.1 mM), and 10% FBS and with or without melatonin.
Parthenogenetic activation of oocytes and embryos culture
After the maturation of the COCs for 18 to 22 h, cumulus cells were removed in hyaluronidase solution (1 mg/mL). Subsequently, the MII oocytes were transferred to the manipulation medium synthetic oviductal fluid (SOF)-HEPES (107.7 mM NaCl, 7.16 mM KCl, 1.19 mM KH2PO4, 0.49 mM MgCl2·6H2O, 1.17 mM CaCl2·2H2O, 5.3 mM sodium lactate, 4 mM NaHCO3, 0.33 mM sodium pyruvate, 0.5 mM fructose, 21 mM HEPES, 1X nonessential amino acids, 1X essential amino acid, and 1 mg/mL bovine serum albumin [BSA]) and rinsed with ionomycin (5 μM) for 5 minutes and activated for 4 h (38.5 °C, 5% CO2) in 6-dimethylaminopurine (6-DMAP; 2 mM) supplemented with or without the epigenetic modifier PsA. After activation, the embryos were washed extensively and then transferred to SOFaa (107.7 mM NaCl, 7.16 mM KCl, 1.19 mM KH2PO4, 0.49 mM MgCl2·6H2O, 1.17 mM CaCl2·2H2O, 4.19 mM sodium lactate, 30.94 mM NaHCO3, 0.33 mM sodium pyruvate, 2.38 mM glucose, 1X nonessential amino acids, 1X essential amino acid, 4 mg/mL fatty acid-free BSA, and 10% FBS.) with or without PsA or melatonin (10−7 M). The culture medium was supplemented with 5% FBS on the third day.
Epigenetic modification treatments
PsA (Santa Cruz Biotechnology) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 3-mM stock solution and was frozen till use. The final concentration of PsA was prepared by dilution of the stock solutions in the culture or activation media, depending on the experimental procedure. A starting PsA concentration of 5 μM was employed based on earlier reports,9,28 and the embryos were divided into 3 groups by random: a control group and 2 treated groups (5 μM and 10 μM PsA, respectively). Embryos were exposed to PsA for 4 h for activation, subsequently cultured for 2 h or 8 h, and finally washed in drops of SOFaa culture medium.
Measurement of intracellular reactive oxygen species level
Intracellular reactive oxygen species (ROS) levels in embryos were measured using the ROS detection kit (S0033S). Briefly, PA embryos treated with/without PsA (n ≥ 10 for each group, repeated 3 times) were incubated with 2,7-dichlorofuorescin diacetate (DCFH-DA) (10 mM) at 37 °C for 20 min. The embryos were checked using a fluorescence microscope (Olympus) with a filter at 460-nm excitation. All embryos were photographed in fluorescence images using a digital camera (Nikon 990). The fluorescence images were analyzed using ImageJ by Wayne Rasband from the National Institute of Health to analyze the fluorescence intensities of the embryos compared with that of the control after deducting the background value.
Measurement of mitochondrial membrane potential
Mitochondrial membrane potential (MMP [ΔΨm]) was determined using a Mitochondrial Membrane Potential Detection Kit (C1071; Beyotime). Briefly, PA embryos treated with/without PsA (n ≥ 10 for each group, repeated 3 times) were incubated in 188 μL buffer, 2 μL Mito-Tracker Red CMXRos, and 5 μL H33342 for 30 minutes at 25 °C and then placed in an ice bath. Finally, embryos were mounted on glass slides with the antifluorescence quenching agent and analyzed using a fluorescent microscope (Olympus BX51). The photographic system was cellSens Standard.
Immunofluorescence staining
PA embryos treated with/without PsA (n ≥ 10 for each group, repeated 3 times) were washed 3 times with the washing buffer of PBS containing 0.1% polyvinyl alcohol (PVA), 0.01% Triton X-100, and 0.1% BSA for 3 minutes each, fixed in 4% paraformaldehyde for 30 min, and permeabilized with 0.2% Triton X-100 in PBS solution for 30 min. After being washed 3 times, the embryos were blocked using 2% BSA for 1 h at room temperature. The primary antibody used was anti-H3K9me3 (anti-histone H3 lysine 9 trimethylation) antibody (dilution 1:400 vol/vol; Abcam) and anti-H3K9ac (anti-histone H3 acetyl-lysine 9; 1:400; Abcam) antibody for 12 h at 4 °C. The secondary antibody employed was Alexa Fluor 594 (goat anti-rabbit; 1:400 vol/vol; Invitrogen) for 1.5 h at 37 °C, which recognized the primary antibodies for H3K9me3 and H3K9ac. After DNA staining for 10 minutes with Hoechst 33342 (10 μg/ml; Sigma), the embryos were mounted on glass slides with an anti-fluorescence quenching agent and analyzed using a fluorescent microscope (Olympus BX51). The Photographic system employed was cellSens Standard.
RNA isolation, cDNA synthesis, and quantitative PCR
RNA was extracted from the bovine PA blastocysts (n ≥ 5 for each group, repeated 3 times) using the RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. Briefly, 350 μL Buffer RLT Plus and Carrier RNA (20 ng) was added to blastocysts, and then 1 volume (usually 350 µl) of 70% ethanol was added. The sample was transferred to an RNeasy MinElute spin column placed in a 2-ml collection tube, centrifuged for 15 seconds at ≥ 8,000 X g, and washed 3 times by addition of 700 µl Buffer RW1, 500 µl Buffer RPE, and 500 µl of 80% ethanol. The RNeasy MinElute spin column was then placed in a new 2-ml collection tube centrifuge at full speed for 5 minutes. Next, the RNeasy MinElute spin column was placed in a new 1.5-ml collection tube and 22 µl RNase-free water was added to elute the RNA. The One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen) was used to synthesize the first-strand cDNA. Briefly, 6 µl RNA, 1 µl anchored oligo(dt)18 primer, 1 µl random primer, 1 µl gDNA remover, and 1 µl 2X TS reaction mix were subjected to the following parameters: 25 °C for 10 minutes, 42 °C for 30 minutes, and 85 °C for 5 seconds. The primers used for HDAC1, DNMT1, Nanog, B-cell lymphoma-2 (Bcl2L1), superoxide dismutase 1 (SOD1), and 18S rRNA are listed in Supplementary Table S1. The quantitative PCR (qPCR) mix (20 μl) consisted of 2 μl of cDNA, 10 μl of SYBR green master mix, 0.4 μl of ROX reference dye, 6 μl of RNase-free water, and 0.8 μl each of forward and reverse primers (10 pmol) for each gene. The relative gene expression data were analyzed using qPCR and the 2−ΔΔCT method.
Statistical analysis
Data were analyzed by one-way ANOVA and least significant difference tests with the SPSS 18.0 software (SPSS). A value of P < .05 was considered to be significantly different.
Results
Effect of different concentrations and durations on the development of bovine preimplantation PA embryos
The development of bovine PA embryos exposed to different concentrations of PsA (5 and 10 μM) for different durations (6 h and 12 h after activation) was compared and analyzed (Supplementary Table S2). The cleavage rate of PA embryos was not significantly affected by the 5- and 10-μM PsA treatment for 6 h and 12 h. However, the number of embryos developed to the 8-cell stage was significantly decreased in the group treated with 10 μM for 12 h (P < .05); the other groups did not show any significant difference. The blastocyst rate was significantly decreased (P < .01) in all treated groups except the starting concentration (5 μM for 6 h). Therefore, the effect of PsA on early PA embryo development depended on the concentration and the treatment time.
Effect of PsA on the relative abundance of gene expression in PA embryos
To evaluate the effects of PsA on gene expression at different concentrations (5 and 10 μM) and durations (6 h and12 h after activation), the response studies were performed in bovine parthenogenetic embryos (blastocysts) by qPCR (Figure 1). The results showed that PsA significantly decreased the expression of HDAC1, Nanog, and SOD1 at all the concentrations (5 and 10 μM) and durations (6 h and 12 h after activation) that we employed (P < .05). Similar to the result of HDAC1, the expression levels of Dnmt1 were significantly lower than the control group (untreated group) except for the starting concentration and duration (5 μM for 6 h). The qPCR results of BCL2L1 showed that there was no difference in the BCL2L1 transcription levels in the group that was treated with 5 μM for 6 h or 10 μM for 6 h compared to the control group. However, in the 12-h groups (5 and 10 μM), BCL2L1 transcription levels were significantly decreased than those in the control group (P < .05).
Effects of different concentrations and duration of PsA on the expression of genes HDAC1 (A), DNMT1 (B), Nanog (C), BCL2L1 (D), and SOD1 (E) and of blastocysts in bovine PA embryos, assayed using quantitative real-time PCR. Data are the mean ± SD at 3 trials. a–cValues with different superscripts significantly differ from one another.
Citation: American Journal of Veterinary Research 2023; 10.2460/ajvr.22.09.0159
Effects of different concentrations and duration of PsA on the expression of genes HDAC1 (A), DNMT1 (B), Nanog (C), BCL2L1 (D), and SOD1 (E) and of blastocysts in bovine PA embryos, assayed using quantitative real-time PCR. Data are the mean ± SD at 3 trials. a–cValues with different superscripts significantly differ from one another.
Citation: American Journal of Veterinary Research 2023; 10.2460/ajvr.22.09.0159
Effects of different concentrations and duration of PsA on the expression of genes HDAC1 (A), DNMT1 (B), Nanog (C), BCL2L1 (D), and SOD1 (E) and of blastocysts in bovine PA embryos, assayed using quantitative real-time PCR. Data are the mean ± SD at 3 trials. a–cValues with different superscripts significantly differ from one another.
Citation: American Journal of Veterinary Research 2023; 10.2460/ajvr.22.09.0159
Effects of PsA on the oxidative resistance of bovine PA embryos
Since this study aims to evaluate the cytotoxic or genotoxic effects of PsA during early development, we selected the group that was treated with 10 μM for 6 h for further study. At this concentration, HDAC1 and DNMT1 have been inhibited efficiently, blastocysts decreased moderately. To analyze the reasons that affected the bovine embryo’s development, the intracellular ROS and MMP were measured in the PsA-treated group and control group in the 4-cell and 8-cell stages. Intracellular ROS levels were measured by assessing the DCFH fluorescence (Figure 2). Quantitative analysis showed that the relative intracellular ROS levels were significantly increased in the PsA-treated group compared with the control group (P < .05). Nevertheless, antioxidant melatonin can significantly prevent bovine PA embryos from oxidative stress induced by PsA (Figure 3, P < .05). Subsequently, the intracellular MMP was measured. As shown (Figure 4), the quantitative analysis showed that the relative intracellular MMP levels were significantly lower in the PsA treatment group than in the control group (P < .05), suggesting that PsA could lead to oxidative stress and decrease the oxidative resistance of embryos. The accumulation of oxidative stress in PsA-treated embryos caused a decrease in blastocyst formation.
Effects of PsA treatment on the intracellular ROS generation in bovine PA embryos. A— Representative fluorescence images showing intracellular ROS levels in 4-cell stage. B—Quantification of relative intracellular ROS levels in 4-cell stages from the control and PsA treatment groups. C—Representative fluorescence images showing intracellular ROS levels in 8-cell stage. D—Quantification of relative intracellular ROS levels in 4-cell stages from the control and PsA treatment groups. *Differences are statistically significant (P < .05). Scale bar = 50 µm.
Citation: American Journal of Veterinary Research 2023; 10.2460/ajvr.22.09.0159
Effects of PsA treatment on the intracellular ROS generation in bovine PA embryos. A— Representative fluorescence images showing intracellular ROS levels in 4-cell stage. B—Quantification of relative intracellular ROS levels in 4-cell stages from the control and PsA treatment groups. C—Representative fluorescence images showing intracellular ROS levels in 8-cell stage. D—Quantification of relative intracellular ROS levels in 4-cell stages from the control and PsA treatment groups. *Differences are statistically significant (P < .05). Scale bar = 50 µm.
Citation: American Journal of Veterinary Research 2023; 10.2460/ajvr.22.09.0159
Effects of PsA treatment on the intracellular ROS generation in bovine PA embryos. A— Representative fluorescence images showing intracellular ROS levels in 4-cell stage. B—Quantification of relative intracellular ROS levels in 4-cell stages from the control and PsA treatment groups. C—Representative fluorescence images showing intracellular ROS levels in 8-cell stage. D—Quantification of relative intracellular ROS levels in 4-cell stages from the control and PsA treatment groups. *Differences are statistically significant (P < .05). Scale bar = 50 µm.
Citation: American Journal of Veterinary Research 2023; 10.2460/ajvr.22.09.0159
Melatonin prevented bovine PA embryos from oxidative stress successfully induced by PsA treatment. A—Representative fluorescence images showing intracellular ROS levels in 4-cell stage. B—Quantification of relative intracellular ROS levels in 4-cell stages from the control and PsA treatment groups. C—Representative fluorescence images showing intracellular ROS levels in 8-cell stage. D—Quantification of relative intracellular ROS levels in 4-cell stages from the control and PsA treatment groups. a,bValues with different superscripts significantly differ from one another. Scale bar = 50 µm.
Citation: American Journal of Veterinary Research 2023; 10.2460/ajvr.22.09.0159
Melatonin prevented bovine PA embryos from oxidative stress successfully induced by PsA treatment. A—Representative fluorescence images showing intracellular ROS levels in 4-cell stage. B—Quantification of relative intracellular ROS levels in 4-cell stages from the control and PsA treatment groups. C—Representative fluorescence images showing intracellular ROS levels in 8-cell stage. D—Quantification of relative intracellular ROS levels in 4-cell stages from the control and PsA treatment groups. a,bValues with different superscripts significantly differ from one another. Scale bar = 50 µm.
Citation: American Journal of Veterinary Research 2023; 10.2460/ajvr.22.09.0159
Melatonin prevented bovine PA embryos from oxidative stress successfully induced by PsA treatment. A—Representative fluorescence images showing intracellular ROS levels in 4-cell stage. B—Quantification of relative intracellular ROS levels in 4-cell stages from the control and PsA treatment groups. C—Representative fluorescence images showing intracellular ROS levels in 8-cell stage. D—Quantification of relative intracellular ROS levels in 4-cell stages from the control and PsA treatment groups. a,bValues with different superscripts significantly differ from one another. Scale bar = 50 µm.
Citation: American Journal of Veterinary Research 2023; 10.2460/ajvr.22.09.0159
Effects of PsA treatment on the MMP in bovine PA embryos. A—Fluorescence images of 4-cell stages. B—Quantification of the relative fluorescence intensity in the control and PsA treatment group of 4-cell stages. C—Fluorescence images of 8-cell stage. D—Quantification of the relative fluorescence intensity in the control and PsA treatment group of 8-cell stage. Scale bar = 50 µm. *Differences are statistically significant (P < .05).
Citation: American Journal of Veterinary Research 2023; 10.2460/ajvr.22.09.0159
Effects of PsA treatment on the MMP in bovine PA embryos. A—Fluorescence images of 4-cell stages. B—Quantification of the relative fluorescence intensity in the control and PsA treatment group of 4-cell stages. C—Fluorescence images of 8-cell stage. D—Quantification of the relative fluorescence intensity in the control and PsA treatment group of 8-cell stage. Scale bar = 50 µm. *Differences are statistically significant (P < .05).
Citation: American Journal of Veterinary Research 2023; 10.2460/ajvr.22.09.0159
Effects of PsA treatment on the MMP in bovine PA embryos. A—Fluorescence images of 4-cell stages. B—Quantification of the relative fluorescence intensity in the control and PsA treatment group of 4-cell stages. C—Fluorescence images of 8-cell stage. D—Quantification of the relative fluorescence intensity in the control and PsA treatment group of 8-cell stage. Scale bar = 50 µm. *Differences are statistically significant (P < .05).
Citation: American Journal of Veterinary Research 2023; 10.2460/ajvr.22.09.0159
Effect of PsA on the global H3K9ac and H3K9me3 levels
After PsA treatment (6 h for 10 μM), the 4-cell embryos and blastocyst were fixed and subjected to immunofluorescence staining, to determine the change in the global H3K9ac and H3K9me3 levels. As shown (Figure 5), histone acetylation of H3K9 in 4-cell embryos and blastocyst exhibited dramatic changes after PsA treatment (P < .05). The global H3K9ac levels in the PsA-treated group was higher than those in the control group at both 4-cell and blastocyst stages (P < .05). However, as shown (Figure 6), there was no visible difference in the global H3K9me3 levels between the treated embryos and the untreated control group.
Global acetylation levels of H3K9ac in embryos at the 4-cell and blastocyst stages. A—Fluorescence images of H3K9ac (red) at 4-cell stage and blastocysts. B—Quantification of the relative fluorescence intensity in the control and PsA treatment group of 4-cell stage. C—Quantification of the relative fluorescence intensity in control and PsA treatment group of blastocysts. Scale bar = 20 µm. *Differences are statistically significant (P < .05).
Citation: American Journal of Veterinary Research 2023; 10.2460/ajvr.22.09.0159
Global acetylation levels of H3K9ac in embryos at the 4-cell and blastocyst stages. A—Fluorescence images of H3K9ac (red) at 4-cell stage and blastocysts. B—Quantification of the relative fluorescence intensity in the control and PsA treatment group of 4-cell stage. C—Quantification of the relative fluorescence intensity in control and PsA treatment group of blastocysts. Scale bar = 20 µm. *Differences are statistically significant (P < .05).
Citation: American Journal of Veterinary Research 2023; 10.2460/ajvr.22.09.0159
Global acetylation levels of H3K9ac in embryos at the 4-cell and blastocyst stages. A—Fluorescence images of H3K9ac (red) at 4-cell stage and blastocysts. B—Quantification of the relative fluorescence intensity in the control and PsA treatment group of 4-cell stage. C—Quantification of the relative fluorescence intensity in control and PsA treatment group of blastocysts. Scale bar = 20 µm. *Differences are statistically significant (P < .05).
Citation: American Journal of Veterinary Research 2023; 10.2460/ajvr.22.09.0159
Global acetylation levels of H3K9me3 in embryos at the 4-cell and blastocyst stages. A—Fluorescence images of H3K9me3 (red) at 4-cell stage and blastocysts. B—Quantification of the relative fluorescence intensity in the control and PsA treatment group of 4-cell stage. C—Quantification of the relative fluorescence intensity in control and PsA treatment group of blastocysts. Scale bar = 20 µm. *Differences are statistically significant (*P < .05).
Citation: American Journal of Veterinary Research 2023; 10.2460/ajvr.22.09.0159
Global acetylation levels of H3K9me3 in embryos at the 4-cell and blastocyst stages. A—Fluorescence images of H3K9me3 (red) at 4-cell stage and blastocysts. B—Quantification of the relative fluorescence intensity in the control and PsA treatment group of 4-cell stage. C—Quantification of the relative fluorescence intensity in control and PsA treatment group of blastocysts. Scale bar = 20 µm. *Differences are statistically significant (*P < .05).
Citation: American Journal of Veterinary Research 2023; 10.2460/ajvr.22.09.0159
Global acetylation levels of H3K9me3 in embryos at the 4-cell and blastocyst stages. A—Fluorescence images of H3K9me3 (red) at 4-cell stage and blastocysts. B—Quantification of the relative fluorescence intensity in the control and PsA treatment group of 4-cell stage. C—Quantification of the relative fluorescence intensity in control and PsA treatment group of blastocysts. Scale bar = 20 µm. *Differences are statistically significant (*P < .05).
Citation: American Journal of Veterinary Research 2023; 10.2460/ajvr.22.09.0159
Discussion
The present study investigated the effects of PsA on the development of bovine PA embryos. A negative effect of PsA on developmental rates was observed, which was specifically dose and time dependent. In addition, the PsA treatment altered the acetylation at certain concentrations (from 10 μM for 6 h). However, PsA led to oxidative stress and decreased the oxidative resistance of the embryos.
The decrease in the developmental rates was observed with an increase in the PsA concentration and treatment duration. PsA displayed the concentration and duration-dependent effects observed in earlier reports consisting of PsA-treated mouse SCNT embryos29 and breast tumor cells.9 These findings are all consistent with our results of decreased development of 8-cell embryos and blastocyst after PsA treatment. Moreover, our results also supplied data for the PsA clinical application concentration to avoid reproductive toxicity. In addition, Mallol et al30 reported that the PsA treatment for 16 h with a dose of 10 μM notably improved the blastocyst formation rate and quality of mouse SCNT embryos. However, in our study, bovine PA embryos were treated with 10 μM for 12 h, and the 8-cell and blastocyst rates decreased significantly. The difference in the results of our study could possibly be due to the differences in embryo activation protocol, culture medium, and especially the nature of the zygotic origin and/or the embryo’s species used in the different studies.31 This may also suggest that a different nuclear origin of the preimplantation embryos (in vivo fertilized embryos, in vitro fertilized embryos, PA embryos, and SCNT embryos) can have different epigenetic states, which are governed by the different regulatory mechanisms and thereby have different sensitivity to the HDACis (PsA, TSA, etc.).
The bovine PA embryos were sensitive to the PsA treatment. The blastocyst formation rate and the expression of Nanog and BCL2L1 were decreased in the bovine PA embryos. The pluripotency-related gene Nanog is essential for the establishment and maintenance of pluripotency, which is related to normal embryonic development.30 Although several studies31 have shown that the treatment of embryos with HDACis increases Nanog expression, we did not observe a significant increase in the Nanog expression levels. A similar result was obtained by Huang et al32 for Oct4 mRNA abundance in VPA-treated cloned porcine embryos. Moreover, with the increase in concentration and durations, the transcription level of the pluripotent gene Nanog decreased significantly in PA embryos (P < .05). Despite the well-documented role of HDAC as a corepressor complex, Sin3a/HDAC can also act as a transcriptional coactivator complex in promoting the final stage of Nanog-driven regulation reprogramming of embryonic stem cell pluripotency and somatic cell reprogramming.33 This may be associated with the downregulated expression of Nanog. In addition, PsA also shows anticancer activity by inducing cell cycle arrest and apoptosis,10,11,34,35 consistent with the decreased expression of Nanog and BCL2L1 in our study.
PsA-induced oxidative stress can be one of the reasons for the decline in the bovine PA embryo development. ROS generated at low levels during mitochondrial respiration are known to play a key role in several signaling pathways. Oxidative stress arises when the generation of ROS exceeds the cell’s antioxidant scavenging ability and leads to cell damage. As ROS levels and the intracellular MMP appear to be critical to embryonic development, the accumulation of ROS and the reduction of MMP in 4-cell and 8-cell PA embryos may be the reasons for the decreased developmental rates. Embryos exposed to PsA showed increased ROS levels and decreased MMP levels, which suggests that PsA exposure can lead to oxidative stress. The excessive intracellular ROS accumulation can induce cell cycle arrest and apoptosis in oocytes and embryos.36 Depolarization of MMP can lead to excessive ROS production. Our findings indicate that the oxidative stress of PsA-treated PA embryos is synchronized and can be rescued by melatonin. Al Mamun Bhuyan et al37 also found that PsA induced oxidative stress, which triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane. PsA may hurt the mitochondria’s electron transfer system, resulting in increased ROS, but the mechanism of PsA-mediated oxidative is unclear and needs further study. Oxidative stress may be an important negative effect that needs to be considered in PsA clinical application.
PsA is a potential inhibitor for both HDAC1 and DNMT1. PsA from the sponge Pseudoceratina purpurea exhibited dual activity for the inhibition of both HDAC1 and DNMT1.38 In our study, with the increase in the PsA concentration and duration, the inhibiting effects on HDAC1 and DNMT1 were enhanced in bovine PA embryos, especially for HDAC1, that were inhibited significantly from the starting concentration. Correspondingly, global H3K9ac levels were significantly higher in the PsA-treated group. Histone acetylation is associated with transcriptionally permissive states, which may be useful for cell reprogramming. HDACis, for example, TSA is known to induce increased histone acetylation, which can significantly enhance mouse, cattle, pig, and rabbit SCNT embryo viability.39 This indicates the potent HDACi activity of PsA, which may contribute to reprogramming via enhancing H3K9ac.
In conclusion, PsA inhibited both HDAC1 and DNMT1 and modified H3K9ac in bovine parthenogenetic embryos. However, PsA inhibits the development of early embryos to the blastocyst stage at a high concentration and long duration. In addition, a reproduction toxic effect of PsA may be modulated through increased oxidative stress on the bovine parthenogenetic embryo, suggesting that PsA in combination with antioxidants, for example, melatonin, might be an effective clinical application strategy.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
This work was supported by grants from the Science and Technology Development Project of Jilin Province (20210202046NC).
Xin Ma and Chenglin Zhan contributed equally to this work.
No competing financial interests exist.
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