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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 5  |  Issue : 1  |  Page : 9-14

Effects of rosmarinic acid on DNA integrity and H19 differentially methylated region methylation levels in human sperm preserved by freeze-drying


Department of Developmental and Regenerative Biology, College of Life Science and Technology, Jinan University, Guangzhou 510632, China

Date of Submission15-Oct-2020
Date of Decision16-Nov-2020
Date of Acceptance23-Dec-2020
Date of Web Publication19-Feb-2021

Correspondence Address:
Wei-Jie Zhu
Department of Developmental and Regenerative Biology, College of Life Science and Technology, Jinan University, No. 601, West Huangpu Avenue, Guangzhou 510632
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2096-2924.309790

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  Abstract 


Objective: To investigate the effects of rosmarinic acid (RA) on the DNA integrity and methylation levels of the H19 differentially methylated region (DMR) of freeze-dried human sperm after 1 week and 6 months of storage at 4°C.
Methods: Semen samples from 15 healthy normospermic donors were used in this study. The samples were divided into five groups, including the control group with fresh sperm and four experimental groups with freeze-dried sperm (1-week storage with EGTA buffer solution, Group A; 1-week storage with EGTA buffer solution containing 105 μmol/L RA, Group B; 6-month storage with EGTA buffer solution, Group C; and 6-month storage with EGTA buffer solution containing 105 μmol/L RA, Group D). DNA integrity was evaluated using the sperm chromatin dispersion test. H19 DMR methylation levels were detected by bisulfite sequencing polymerase chain reaction.
Results: After 1 week of storage, no differences in sperm DNA integrity were observed among Groups A, B, and controls (P > 0.05). After 6 months of storage, the sperm DNA integrity of Group D did not change significantly compared with that of the control group (P > 0.05), whereas that of Group C decreased significantly (P < 0.05). There were no differences in H19 DMR methylation levels among the five groups (P > 0.05).
Conclusions: The DNA integrity of freeze-dried human sperm can be effectively protected by adding RA within 6 months, and the H19 DMR methylation level of human sperm can be maintained for 6 months after freeze-drying.

Keywords: DNA Integrity; DNA Methylation; Freeze-Drying; Human Sperm; Rosmarinic Acid


How to cite this article:
Wang YY, Zhu WJ. Effects of rosmarinic acid on DNA integrity and H19 differentially methylated region methylation levels in human sperm preserved by freeze-drying. Reprod Dev Med 2021;5:9-14

How to cite this URL:
Wang YY, Zhu WJ. Effects of rosmarinic acid on DNA integrity and H19 differentially methylated region methylation levels in human sperm preserved by freeze-drying. Reprod Dev Med [serial online] 2021 [cited 2021 Jun 22];5:9-14. Available from: https://www.repdevmed.org/text.asp?2021/5/1/9/309790




  Introduction Top


Freeze-drying (lyophilization) is a technology that is in the exploration stage for male gametes stored for long periods and allows sperm preservation in a refrigerator and translation at ambient temperature.[1],[2] Offspring derived from intracytoplasmic injection of freeze-dried mouse sperm were first reported in 1998.[3] This method has been applied to various mammals.[4],[5],[6] However, spermatozoa can be damaged during freeze-drying. A previous study by Zhu et al. showed that preservation by freeze-drying resulted in damage to the human sperm membrane and ultrastructure.[7] It has been recognized that damage to sperm DNA can be caused by endonuclease activity or oxidative stress during freeze-drying and preservation.[8],[9] Endonucleases can be inactivated in freeze-drying buffer solution by adding chelating agents, which provide necessary protection to sperm DNA from degradation.[10] In addition, reactive oxygen species (ROS) can induce sperm DNA strand cross-linking, base modification, and breakage.[11] A buffer solution with rosmarinic acid (RA) protected sperm DNA against oxidative damage in freezing and freeze-drying buffer solution.[12],[13] However, the effects of RA on freeze-dried human sperm have not been understood to date.

DNA methylation is a significant mechanism of epigenetics, a genetic phenomenon that alters gene expression and can be inherited by the progeny without sequence changes in DNA.[14] The methylation of cytosines at CpG dinucleotides is the main mechanism of imprinted genes, which typically results in gene silencing from one of two parental alleles. Aberrant DNA methylation patterns of sperm can cause abnormal fetal development and human genetic diseases.[15],[16] Imprinted genes are known to be affected by various assisted reproductive technology manipulations in animal studies.[17],[18] The H19 imprinted gene is more susceptible to environmental stress than other imprinted genes.[19] Evidence suggests that embryo vitrification can decrease the methylation levels of H19 differentially methylated region (DMR) in mice.[20] However, it is unclear whether preservation by freeze-drying influences the DNA methylation levels of sperm.

This study assessed the effects of RA on human sperm DNA integrity and methylation levels of the H19 DMR after preservation by freeze-drying for 1 week and 6 months at 4°C. These findings might provide valuable insights on how to improve the quality of freeze-dried sperm preservation and provide a reference for the application and safety of human sperm preservation by freeze-drying.


  Methods Top


Semen samples

Semen samples were obtained from 15 normospermic donors by masturbation after 2–7 days of abstinence. According to the World Health Organization semen analysis manual,[21] semen samples were subjected to routine semen analysis. The modified Papanicolaou staining method was used to evaluate sperm morphology. The freeze-drying buffer solutions were EGTA (10 mmol/L Tris-HCl, 50 mmol/L EGTA, 50 mmol/L NaCl, pH 8.0) and EGTAR (EGTA supplemented with 105 μmol/L RA).[12] The samples were divided into five groups: a control group and four experimental groups (1-week storage with EGTA buffer solution, Group A; 1-week storage with EGTAR buffer solution, Group B; 6-month storage with EGTA buffer solution; Group C, 6-month storage with EGTAR buffer solution, Group D). The final pH of the buffer solutions was adjusted to 8.2. Informed consent was obtained from each donor. This study was approved by the local research ethics committee.

Sperm freeze-drying

The semen sample was washed three times using phosphate buffer solution (PBS), followed by the addition of 1.5 mL buffer solution into a 1.5-mL centrifuge tube to resuspend the sperm. One hundred microliters of sperm suspension was placed into a 1.5-mL centrifuge tube and immersed in liquid nitrogen for 30 s. The samples of frozen sperm were moved to a precooled (−50°C) freeze-drying machine (CT 60E, Heto, Denmark). After vacuuming at a pressure of 0.06 mbar for 4 h, the tubes were removed from the machine and firmly sealed with parafilm in a nitrogen gas atmosphere. Samples of freeze-dried human sperm were stored in a 4°C refrigerator. After storage for 1 week and for 6 months, samples of freeze-dried human sperm were rehydrated using 100 μL Milli-Q water into the tubes to assess the sperm DNA integrity and DNA methylation of H19 DMR.

DNA integrity analysis

Sperm DNA integrity was assessed using the sperm chromatin dispersion (SCD) test.[22],[23] Briefly, each sample was diluted in PBS to obtain sperm concentrations from to 5 to 15 × 106/mL, followed by mixing 30 μL of the diluent with 70 μL of 1% low-melting-point agarose. Twenty microliters of the mixture was placed onto a precooled glass slide precoated with 0.65% standard agarose, covered with 20 mm × 20 mm glass coverslips, and left in a 4°C refrigerator for 5 min. Coverslips were gently removed; the slides were immediately immersed in fresh acid denaturation solution (0.08 mol/L HCl) for 7 min and then moved to lysing solution A (0.4 mol/L/Tris, 0.8 mol L-1 DTT, 50 mmol/L EDTA, and 1% SDS, pH 7.5) for 10 min and lysing solution B (2 mol/L NaCl, 0.4 mol/L Tris and 1% SDS, pH 7.5) for 5 min at room temperature. Slides were washed in Milli-Q water for 5 min and dehydrated in a series of increasing concentrations of ethanol (70%, 90%, and 100%) for 2 min each. Slides were air-dried and stained with Wright's stain (Jiancheng Technology Co., Ltd., Nanjing, China). The slides were examined under a light microscope at ×400, and at least 400 spermatozoa were scored per sample.

DNA methylation analysis

Sperm DNA methylation levels of 10 freeze-dried sperm samples were evaluated by bisulfite sequencing polymerase chain reaction (BSP). Sperm genomic DNA was extracted using the traditional phenol-chloroform method.[24] Bisulfite conversion of genomic DNA was performed as previously reported.[25] Briefly, 50 μL of genomic DNA (100–200 ng/μL) was mixed with NaOH (5.5 μL, 3 mol/L) for 20 min at 55°C. Hydroquinone (666 μL, 40 mmol/L) and NaOH (400 μL, 10 mol/L) were added to the bisulfite solution (10 mL, 5.2 mol/L). Bisulfite solution (150 μL) was then added to the genomic DNA solution, with the reaction mixture covered with 100 μL of mineral oil to inhibit air contact. The DNA/bisulfite solution was incubated at 55°C for 6 h in the dark. Converted DNA was desalted using the DNA cleanup Wizard (Promega, Madison, WI, USA) according to the manufacturer's instructions. NaOH (4 μL, 3 mol/L) was then added to the eluted DNA solution to desulfonate the DNA for 15 min at 37°C, followed by the addition of ammonium acetate (22 μL, 6 mol/L) and 250 μL of absolute ethanol and DNA precipitation at −20°C for at least 1 h. DNA was washed twice using 70% ethanol, and after complete removal of ethanol, it was resuspended in 10 μL of deionized water. The region of interest to be amplified was the imprinted gene H19 (AF087017, 6098–6328 bp). Primers specific for bisulfite-converted DNA were as follows: forward, 5'-GTATAGTATATGGGTATTTTTGGAGGTTTT-3'; reverse: 5'-TAAATATCCCAAATAACCC-3'. Primers were designed using the available MethPrimer online software (http://www.urogene. org/methprimer/).[26] The polymerase chain reaction (PCR) protocol was as follows: 5 min of denaturation at 96°C followed by 25 cycles of 30 s at 94°C, 30 s at 58°C, and 30 s at 72°C, and a final extension at 72°C for 7 min. PCR products were purified using 2% agarose gel electrophoresis, and the bands corresponding to 220 bp were excised using a TIANgel Midi Purification Kit (Tiangen Biotech Co., Ltd., Beijing, China) and cloned into the pMD19-T vector (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's instructions. In total, 7–10 clones were sequenced (Synbio Technologies Co., Ltd., Suzhou, China) for each PCR product.

Statistical analysis

Sequencing results were analyzed by QUMA software for DNA methylation analysis (http://quma.cdb.riken.jp). Data concerning DNA integrity and H19 DMR methylation levels were expressed as mean ± standard deviation (x¯± s) and analyzed using one-way ANOVA followed by multiple-comparison post-hoc tests. Differences were considered significant if P < 0.05. Statistical analysis was performed using IBM SPSS version 23.0 for Windows.


  Results Top


DNA integrity by sperm chromatin dispersion test

Analyses of sperm DNA integrity are shown in [Figure 1]. [Table 1] shows the sperm DNA integrity before and after preservation by freeze-drying for 1 week and for 6 months; a significant difference was observed between group C and the control (P < 0.05).
Figure 1: Freeze-dried human sperm DNA integrity analyzed using the sperm chromatin dispersion test. Intact sperm DNA (black arrow), broken sperm DNA (red arrow).

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Table 1: DNA integrity of human sperm before and after freeze-drying (x¯ ± s)

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DNA methylation analysis by bisulfite sequencing polymerase chain reaction

Eighteen CpG sites were analyzed in the 230-bp fragment of H19 DMR containing the CTCF-binding site (CpGs 4, 5, 6, 7, and 8). The CTCF-binding site harbors a single nucleotide polymorphism T/C at the seventh CpG site that is uninformative after bisulfite conversion and was not counted. Methylated CpG sites of each group are shown in [Figure 2]. [Table 2] shows the H19 DMR methylation levels of human sperm before and after preservation by freeze-drying for 1 week and for 6 months. The data showed no significant difference among the five groups (P > 0.05).
Figure 2: Methylation status of the H19 differentially methylated region of freeze-dried human sperm. ([a] 1 week; [b] 1 week with AR; [c] 6 months; [d] 6 months with AR; [e] control), 1–18 represent 18 CpG islands. P represents 10 samples. C-represents the clones of each sample. • and ○ represent methylated and unmethylated CpG islands, respectively.

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Table 2: H19 DMR methylation levels of human sperm before and after freeze-drying

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  Discussion Top


Sperm motility is lost after rehydration by freeze-drying; however, viable offspring have been reported to be born from intracytoplasmic sperm injection (ICSI) with freeze-dried sperm in several mammalian species.[4],[5],[6] Therefore, freeze-drying could be an alternative method to sperm cryopreservation in the future.

Intact DNA of the sperm nucleus is necessary to fertilize oocytes, and embryos were obtained through ICSI.[8] Thus far, most studies on sperm preservation by freeze-drying have focused on optimization of the freeze-drying process to increase its protective effect on DNA integrity. Sperm DNA can be degraded by endogenous nucleases when the sperm plasma membrane is damaged.[10] Previous studies have demonstrated that a freeze-drying buffer solution containing chelating agents, such as EGTA and EDTA, can inactivate endogenous nucleases, protecting sperm DNA from degradation.[8],[10],[27] On the other hand, sperm DNA can be attacked by oxidative stress during freeze-drying and rehydration procedures.[8],[9] Luño et al.[12] reported that RA, an antioxidant, was involved in the protection of boar sperm against oxidative stress during cryopreservation. Several reports have shown that freeze-dried sperm in boars, rams, rabbits, and dogs can be protected when the detrimental effects of ROS are decreased by adding RA to the freeze-drying buffer solution.[13],[28],[29],[30] Consistent with previous research,[31] the DNA integrity of freeze-dried human sperm did not change significantly after preservation for 1 week in the current study. However, results suggested that the percentage of freeze-dried human sperm DNA integrity decreased significantly without RA after 6 months of storage, indicating that the DNA integrity of freeze-dried human sperm was damaged by the period of storage and could be protected by using freeze-drying buffer solutions with RA.

The H19 imprinted gene is hypermethylated on the silent paternal allele and is expressed from the maternal allele, which is involved in fetal and postnatal growth.[32] There is substantial evidence indicating that oxidative attack results in breakage of the DNA chain and covalent modification of the DNA base in somatic cells, which interferes with the regulation of DNA methyltransferases and can ultimately result in CpG island hypomethylation by restraining the methylation of cytosine residues.[33] Previous studies have suggested that sperm DNA integrity is positively correlated with DNA methylation.[34],[35] Tunc and Tremellen[34] showed that sperm DNA damage caused by oxidative stress inhibited DNA methylation, while sperm DNA integrity was increased and DNA methylation level was restored by adding antioxidants. Furthermore, animal studies revealed that the H19 imprinted gene was sensitive to manipulations.[17],[19] It has been reported that embryo cryopreservation can decrease the methylation level of H19 DMR.[20] However, a previous study on cryopreservation of human sperm revealed that DNA methylation, including that of the H19 imprinted gene, was unaffected after cryopreservation for 4 weeks.[36] Lu et al.[37] suggested that the human sperm DNA methylation level of H19 DMR was not affected after cryopreservation for 2 months. In this study, no obvious variation was observed in the methylation level of the H19 DMR of human sperm after lyophilization, demonstrating that the methylation level of human sperm H19 DMR remained stable after freeze-drying for 6 months. This might be owing to the special compact nuclear chromatin structure of the sperm, which completely restricts the replication and transcription of sperm DNA.[38]

In summary, the current study shows, for the first time, that RA exerts protective effects on human sperm DNA integrity and that H19 DMR methylation levels are not altered after preservation by freeze-drying for 6 months. However, the limitations of this study include short-term preservation and focus on the analysis of H19 methylation only. It might be interesting to evaluate global DNA methylation or other gene-specific DNA methylation patterns. Further studies need to be conducted to optimize the freeze-drying protocol to improve the quality of sperm preservation by freeze-drying. In addition, more imprinted genes should be analyzed to evaluate the safety of human sperm preservation by freeze-drying.

Financial support and sponsorship

This study was supported by the Science and Technology Planning Project of Guangdong Province, China (No. 2014A020213007).

Conflicts of interest

There are no conflicts of interest.



 
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