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Spermatozoa cryopreservation alters pronuclear formation and zygotic DNA demethylation in mice
Theriogenology xxx (2014) 1–7
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Spermatozoa cryopreservation alters pronuclear formation and zygotic DNA demethylation in mice Gongxue Jia a, Xiangwei Fu a, Keren Cheng a, Mingxing Yue a, Baoyu Jia a, Yunpeng Hou b, Shien Zhu a, * a
National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing, China State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 August 2014 Received in revised form 18 November 2014 Accepted 30 November 2014
This study was conducted to investigate the effects of spermatozoa cryopreservation on DNA demethylation in mouse zygotes. Global methylation was studied in zygotes fertilized with cryopreserved sperm by immunostaining, and relative transcript abundance of Tet3, a key gene responsible for zygotic DNA demethylation, was examined by real-time quantitative polymerase chain reaction. Fresh sperm group served as control. Results indicated spermatozoa cryopreservation decreased fertilization rate (68.2% vs. 86.9%; P < 0.01) and delayed pronuclear formation (P < 0.05), compared with the control group. The percentages of embryos developed to cleavage and blastocyst stages in the freezing group (52.9% and 66.8%, respectively) were lower (P < 0.01 and P < 0.05, respectively) than those of the control group (83.4% and 81.1%, respectively). Furthermore, embryos obtained from cryopreserved sperm had higher relative methylation levels (P < 0.05) and less Tet3 mRNA concentrations (P < 0.01) in advanced pronuclear stages. Hence, we reported that spermatozoa cryopreservation disturbed the Tet3-mediated DNA demethylation progression in the zygotic paternal genome, which could be detrimental to the development of early mouse embryos, and most of the differences observed might be explained by delayed fertilization when using cryopreserved sperm. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Sperm Cryopreservation Zygote DNA methylation Tet3
1. Introduction The first successful spermatozoa cryopreservation was reported in 1949 [1]. Since then, this technology has been used in numerous mammalian species including humans [2–4]. Despite recent considerable progress, spermatozoa cryopreservation is still far from perfection [5,6]. For example, although the viability and fertility of frozen/ thawed sperm are acceptable, embryo implantation and normal fetus rates are significantly decreased [5,7,8]. Cryopreservation damages developmentally important organelles including plasma membrane [9], acrosome [10],
* Corresponding author. Tel./fax: þ86 (0) 10 62731979. E-mail address: [email protected] (S. Zhu). 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.11.036
and mitochondria [11], which may cause problems in embryonic development and fetal growth. The sperm delivers genetic and epigenetic information to the oocyte. External factors can have limited effects on the nuclear structure and integrity, owing to the special chromatin packaging in spermatozoa [12]. No obvious defect in the DNA integrity of frozen/thawed sperm has been detected, and only slight structural damages can be found [7,8]. Nevertheless, structural damages can be restored through DNA repair mechanisms after fertilization, ensuring safe delivery of the paternal genetic information [13,14]. In addition to DNA sequences, epigenetic modifications in the genome are also crucial for embryonic development in mammals [15]. DNA methylation, one of the major epigenetic modifications, plays a crucial role in gene expression regulation, zygotic genome activation, and embryonic
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development [16]. DNA methylation is sensitive to environmental insults, and various forms of assisted reproductive treatments can alter the methylome and impact the embryonic development [17]. It is well established that cryopreservation can change the global genomic methylation and specific gene expression in oocytes and embryos [18–21]. However, little is known about the effect of spermatozoa cryopreservation on DNA methylation in zygotes. The DNA methylation pattern changes dramatically during preimplantation development. To regain developmental totipotency, the zygotic genome must undergo DNA demethylation to erase the epigenetic memory inherited from the gametes. Usually, DNA demethylation is achieved passively in a replication-dependent manner through cell divisions. In the zygote, however, active DNA demethylation is observed, and this process has important implications for embryonic development [16,22]. In the paternal pronucleus (PN), 5-methylcytosine (5mC) is converted into 5-hydroxymethylcytosine (5hmC) by oxidation [23–25]. This reaction can be catalyzed by a family of dioxygenases, the ten-eleven translocation (TET) proteins [26,27]. Recently, works have established an essential role of TET3 in mouse zygotes. TET3 is specifically enriched in the paternal PN of the zygote, and its ablation by RNA knockdown or genetic deletion abolishes the generation of 5hmC to preclude complete DNA demethylation [24,25]. In the present study, we investigated the 5mC and 5hmC distribution patterns and the Tet3 mRNA abundances in mouse zygotes fertilized with cryopreserved sperm to determine the potential changes in zygotic global genomic methylation due to spermatozoa cryopreservation. 2. Materials and methods Unless otherwise stated, all chemicals were purchased from Sigma–Aldrich. All animals were maintained and handled in accordance with the requirements of the Institutional Animal Care and Use Committee of the China Agricultural University. 2.1. Oocyte collection Oocytes were collected from 8- to 12-week-old Kunming female mice (Academy of Military Medical Sciences, Beijing, China) as described previously [28]. Briefly, females were induced to superovulate with 10 IU eCG (Ningbo Hormone Products Company, Ningbo, China) followed by 10 IU hCG (Ningbo Hormone Products Company) 48 hours later. Cumulus–oocyte complexes were collected from oviducts at 14 hours after hCG treatment and recovered in M2 medium supplemented with 3 mg/mL BSA. Cumulus cells were dispersed with 0.1% hyaluronidase, then the cumulus-free oocytes were rinsed thoroughly and placed in drops of human tubal fluid medium (HTF; Millipore, MA, USA), which were covered with mineral oil for up to 1 hour in an incubator (at 37 C in an atmosphere of 5% CO2 and at maximum humidity) before IVF. 2.2. Spermatozoa preparation, freezing, and thawing Cauda epididymides were excised from 10- to 15-weekold Kunming male mice, and spermatozoa were collected
by squeezing them into drops of cryopreservative medium (R18S3, 18% raffinose and 3% skim milk [Becton Dickinson and Company, NJ, USA] in distilled water). To allow even dispersion, spermatozoa were incubated for 10 minutes at 37 C. Sperm freezing and thawing procedures were carried out as described previously with minor modifications [29]. The sperm suspension was divided into 10-mL aliquots and loaded into 0.25-mL plastic straws (IMV Technologies, L’Aigle, France). The straws were heat sealed, placed in liquid nitrogen vapor for 10 minutes, and then immediately plunged into liquid nitrogen before being stored for at least 1 week. Cryopreserved sperm were thawed in a water bath at 37 C for 5 minutes. Once thawed, spermatozoa were transferred into HTF medium and incubated for 30 minutes before insemination. 2.3. IVF and embryo culture The IVF procedures using fresh or frozen/thawed sperm were performed as described previously [29,30]. Briefly, spermatozoa were isolated from the cauda epididymides and capacitated by incubation for 1.5 hours in pregassed HTF medium before introducing to HTF-containing mature oocytes. Meanwhile, the frozen/thawed sperm were used for insemination in the same way. At 4 hours postfertilization (hpf), the eggs were removed from the fertilization drop, washed in KSOM medium (Millipore), transferred to drops of KSOM medium covered with mineral oil, and then cultured in an incubator. Samples were collected at several time points corresponding to embryo developmental period. The zygotes were recovered individually at 4, 6, 8, and 10 hpf for pronuclear morphometric analysis and immunostaining. The oocytes, zygotes of PN3 to PN4 stages or 2-cell embryos were harvested at 0, 8, or 24 hpf for real-time quantitative polymerase chain reaction (RT-qPCR). The proportions of the fertilized zygotes, 2-cell embryos, and blastocysts were assessed at 10, 24, and 96 hpf, respectively. 2.4. Pronuclear morphometric analysis The pronuclear morphologies of cells were examined on an inverted IX70 microscope (Olympus, Tokyo, Japan) immediately after staining with propidium iodide. The classification of zygotic PN1 to PN5 stages was performed according to previous studies, in which the pronuclear morphology and postfertilization timing were taken into consideration [24,31]. 2.5. Immunostaining The immunostaining analysis was carried out as described with modifications [32]. Immunostaining reagents for washing, blocking, antibody dilution, and mounting were purchased from Beyotime (Shanghai, China). After brief washing, the embryos were fixed in 3.7% paraformaldehyde for 40 minutes and permeabilized with 0.5% Triton X-100 for 10 minutes. Subsequently, the cells were denatured with 4N HCl for 10 minutes and
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neutralized with 100-mM Tris HCl (pH 8.5) for 20 minutes before blocking for 30 minutes. For quantitative analysis of 5mC, the samples were incubated with anti-5mC (diluted 1:500; Epigentek, NY, USA) overnight at 4 C. After several washes, the embryos were incubated with fluorescein isothiocyanate–conjugated antimouse immunoglobulin G (IgG; diluted 1:100; Santa Cruz Biotech, TX, USA) for 1.5 hours at 37 C. DNA was labeled with 10 mg/mL propidium iodide for 10 minutes. Finally, the cells were extensively rinsed again and mounted on slides with a drop of antifade mounting medium. For localization analyses of 5mC and 5hmC, the samples were incubated with anti-5mC and anti-5hmC (Active Motif, CA, USA) antibodies (diluted 1:500) overnight at 4 C. After several washes, the embryos were incubated with a 1:100 dilution of fluorescein isothiocyanate–conjugated antimouse IgG and Cy3-labeled antirabbit IgG (Beyotime) for 1.5 hours at 37 C. The cells were washed again before depositing them on slides. The immunostaining signal was examined on the Olympus FluoView FV1000 laser scanning confocal microscope. To construct three dimensional pictures of the zygotic PNs, we obtained images of planes at various depths within the stained embryos (also known as z-stacks). The ImageJ software (National Institutes of Health, MA, USA) was used to quantify the fluorescence signals of z-stack computed (with 2 mm per sample) fluorescence images. The relative methylation level (paternal PN to the maternal PN) was calculated as described previously [24,33]. The average methylation intensity for each PN was calculated using the ratio of the antibody signal to the DNA signal after subtraction of the cytoplasm background fluorescence. The relative methylation level in each zygote was the average methylation intensity in the paternal PN divided by the average methylation intensity in the maternal PN.
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2.7. Statistical analysis Statistical analysis was conducted by one-way ANOVA followed by Duncan’s test using SPSS statistical software (IBM, IL, USA). Data were expressed as the mean standard error of the mean, and P < 0.05 was considered significant. Oocytes were pooled and randomly distributed to two groups, and all experiments were repeated three times. 3. Results 3.1. Zygotic pronuclear formation and preimplantation embryonic development As shown in Figure 1, the fertilization and cleavage rates based on oocytes were decreased (P < 0.01) in the freezing group (68.2% and 52.9%, respectively) when compared with the control group (86.9% and 83.4%, respectively). Moreover, the blastocyst rate based on 2-cell embryos in the freezing group (66.8%) was lower (P < 0.05) than that of the control group (81.1%). To determine whether cryopreserved sperm affected the pronuclear formation, the pronuclear morphologies of fertilized zygotes were detected by DNA staining. The exact pattern of pronuclear development in zygotes was characterized in Figure 2. In the control group, most embryos were found in PN1 to PN2 stages (72.7%) at the earlier developmental period after sperm entry (4 hpf), and thereafter, the amount of PN4 to PN5 zygotes gradually increased up to a peak at 10 hpf (70.7%). In the freezing group, however, more embryos still stayed in the PN1 to PN2 stages (P < 0.05) at 6 and 8 hpf (53.1% vs. 33.2% and 28.0% vs. 17.5%, respectively), and PN4 to PN5 zygotes were reduced (P < 0.05) at 8 and 10 hpf (36.9% vs. 55.2% and 52.8% vs. 70.7%, respectively).
2.6. Real-time quantitative PCR
3.2. Dynamic distributions of 5mC and 5hmC
Total RNA was isolated from mouse oocytes (n ¼ 100), zygotes from fresh sperm (n ¼ 100), zygotes from freezing sperm (n ¼ 100), 2-cell embryos from fresh sperm (n ¼ 50), or 2-cell embryos from freezing sperm (n ¼ 50) using TRIzol reagent (Invitrogen, CA, USA) in each replication. The RNA was reverse transcribed into complementary DNA (cDNA) using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, CA, USA); then, the cDNA was quantified by RT-qPCR using a SYBR PrimeScript RT-PCR Kit (TaKaRa, Dalian, China) on a CFX96 Real-Time PCR Detection System (Bio-Rad, CA, USA) under standard conditions. The sizes of the PCR products were further confirmed using gel electrophoresis on a standard GelRed-stained (Biotium, CA, USA) 1.5% agarose gel and visualized after exposure to ultraviolet light. The cycle threshold (Ct) value used to calculate the relative expression was the average of three replicates and was normalized against that of the reference gene (Gapdh). The primers used are as follows: Gapdh Fd50 catggccttccgtgttccta-30 ; Gapdh Rd50 -gcctgcttaccaccttctt-30 ; Tet3 Fd50 -cccacaaggaccaacataac-30 ; Tet3 Rd50 -cctcgctgccaaa ctcat-30 . The mRNA expression levels were calculated using the 2ddCt method [34].
To determine whether zygotic DNA demethylation was altered after fertilization with cryopreserved sperm, we used anti-5mC to visualize the genomic methylation. As shown in Figure 3A for the control group, the intensities of the 5mC signals became increasingly different between paternal and maternal PNs. Although a similar downward trend was obtained, the relative methylation levels in the late (PN3–PN5) zygotes of the freezing group were higher (P < 0.05) than those of the control group (Fig. 3B). The tracing of 5hmC dynamics during zygotic development showed that the inverse correlation between the 5mC and 5hmC signals in the parental genomes persisted, even when cryopreserved sperm were used. However, the 5hmC signals seemed to decrease in zygotes of the freezing group (Fig. 3C). 3.3. Expression levels of Tet3 Because TET3 mediated 5mC oxidation in zygotes, we examined whether the Tet3 expression was disturbed in embryos derived from cryopreserved sperm. The RT-qPCR revealed that the Tet3 mRNA expression in PN3 to PN4 zygotes of the freezing group was lower (P < 0.01) than that
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Fig. 1. Fertilization ability and early embryo development. Shown is the reduced efficiency of fertilization and early embryo development with spermatozoa cryopreservation. The fertilization and cleavage rates are based on the number of surviving oocytes, whereas the blastocyst rate is based on the number of 2-cell embryos. Number of oocytes used in total: the freezing group, n ¼ 306; the control group, n ¼ 313. Different letters (a and b) indicate P < 0.01 compared with control group.
of the control group. No significant difference in Tet3 transcript level was detected at 2-cell stage (Fig. 4). 4. Discussion The understanding about the contribution of the sperm epigenome for the developing embryo remains rudimentary [35]. The once held dogma declares that sperm is only responsible for the delivery of a DNA blueprint and does not drive epigenetic changes in the embryo. However, recent evidence indicates the importance of the sperm epigenome during embryogenesis. For example, paternal PNs in mouse zygotes present different DNA methylation statuses after insemination with round spermatids or mature spermatozoa [36]. Furthermore, paternal diet can affect the development and health status of offspring by altering DNA methylation patterns in mice [37,38]. Transgenerational epigenetic inheritance is also largely mediated by changes in the sperm epigenome [39,40]. Despite these seminal findings, the epigenetic reprogramming of zygotes obtained from frozen/thawed sperm remains elusive. Bovine zygotes derived from sperm frozen by different methods show
different DNA methylation statuses [41]. As for the mouse, no abnormality is found in the epigenetic reprogramming of embryos derived from sperm frozen at 20 C [42], whereas delayed DNA demethylation is observed in the zygotes produced by intracytoplasmic sperm injection using frozen/ thawed sperm [43]. Taken together, the effect of spermatozoa cryopreservation on zygotic DNA demethylation still needs to be defined. Here, our results provide clear evidence that spermatozoa cryopreservation alters zygotic DNA demethylation, which could adversely affect in vitro development of mouse zygotes. Spermatozoa cryopreservation is one of the significant factors that could influence the embryonic development in assisted reproductive treatments [5,6]. In the present study, we observed significant declines in embryonic development after fertilization using cryopreserved sperm. This should be due to abnormal fertilization and delayed pronuclear formation. Previous studies have shown that spermatozoa cryopreservation can directly result in defective acrosome reaction [10], hyperactivated motility [44], and calcium equilibrium [45]; all these factors could endanger the normal fertilization process. The delayed
Fig. 2. Distributions of the pronuclear stages during zygotic development. Shown are the percentages of zygotes at paternal pronucleus (PN) 1 to PN2, PN3, and PN4 to PN5 stages in the freezing and control groups. Cells were sampled at 4, 6, 8, and 10 hours postfertilization (hpf), which permitted a profile of pronuclear maturation stages as a function of time to be constructed. Number of zygotes analyzed in total: in the freezing group, 4 hpf, 82; 6 hpf, 96; 8 hpf, 103; 10 hpf, 92; in the control group, 4 hpf, 96; 6 hpf, 108; 8 hpf, 120; 10 hpf, 107. Asterisk indicates P < 0.05 compared with control group.
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Fig. 3. Dynamic DNA methylation patterns during zygotic development. (A) Representative images of paternal pronucleus (PN) 1 to PN5 zygotes stained with 5-methylcytosine (5mC) antibody and propidium iodide. Scale bar ¼ 10 mm. (B) Comparison of paternal and maternal 5mC signals (maternal signal set to one) normalized against DNA signal in the two groups. Number of zygotes analyzed in total: in the freezing group, PN1, 24; PN2, 25; early PN3, 29; late PN3, 29; PN4, 33; PN5, 30; in the control group, PN1, 30; PN2, 31; early PN3, 35; late PN3, 30; PN4, 26; PN5, 28. Asterisk indicates P < 0.05 compared with control group. (C) Representative images of zygotes stained with 5mC and 5-hydroxymethylcytosine (5hmC) antibodies at 4 and 10 hours postfertilization (hpf) in the two groups. Scale bar ¼ 10 mm. (For interpretation of the references to color in this figure, the reader is referred to the Web version of this article.)
pronuclear formation that we observed might be attributed to the DNA repair machinery activated by sperm-derived DNA damage, which is thought to have the potential to disturb cell cycle check points and delays zygotic development [13,14]. Furthermore, this delay may be associated with DNA demethylation that accompanies pronuclear formation in zygotes. DNA methylation plays a crucial role in the control of gene expression and chromosomal structure during the early embryogenesis [16,46]. Any abnormality in DNA methylation could be deleterious to zygotic development. For example, global sperm DNA hypomethylation is correlated with poor pregnancy outcomes in IVF patients [47]. Additionally, aberrant DNA methylation found in cloned embryos is identified as one of the causes for the low cloning efficiency [48]. In this study, the PN3 to PN5 zygotes of the freezing group possessed more paternal 5mC, and the generation of 5hmC seemed to be weakened in paternal PNs compared with that of the control group. Therefore, we concluded that zygotic DNA demethylation could be changed by spermatozoa cryopreservation.
The compromised 5mC oxidation mediated by TET3 may be one of the major reasons for this altered DNA demethylation in zygotes. Before fertilization, Tet3 expression increases gradually during oocyte growth and reaches the highest level in fully grown oocytes [49]. One study indicates that Tet3 expression could continue to be enhanced after fertilization [23], which is consistent with our result, whereas another study shows merely a slight increase [24]. Furthermore, we detected the decreased expression of Tet3 mRNA during zygotic development in the freezing group, indicating that the transcription of Tet3 in zygotes was affected by cryopreserved sperm. It is reported that Tet3-deficient zygotes have reduced expression of OCT4 during the morula stage, and about half of these embryos show serious abnormalities by embryonic day E11.5 and fail to develop [25]. However, no proof of either mechanism for this change in Tet3 has been clearly reported [27]. It can be speculated that one of the candidate mechanisms could be DNA repair pathway that works together with TET3 to facilitate DNA demethylation in zygotes [50,51]. This pathway might be appropriated preferentially
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The authors also appreciate Nature Publishing Group Language Editing for proofreading this article. References
Fig. 4. Expression levels of Tet3 transcripts in embryonic development. Oocytes were used as the calibrator sample (expression set to one). There were 100 oocytes, 100 zygotes from fresh sperm, 100 zygotes from freezing sperm, 50 2-cell embryos from fresh sperm, or 50 2-cell embryos from freezing sperm used in each replication, which were harvested at 0, 8, 8, 24 or 24 hours postfertilization, respectively. Bars with different letters (a, b and c) were significantly different ( P < 0.05).
by sperm-derived DNA damage in mouse zygotes fertilized with cryopreserved sperm, similar to the situation simulated by H2O2 treatment [14]; thereby, DNA demethylation progression could become obstructed. Similarly, a recent study confirms that TET3 facilitates DNA demethylation largely by coupling with DNA replication [52], meaning that the obstruction of DNA replication could damage TET3 function. It is also possible that the sperm-derived residual nucleosomes that contribute to zygotic chromatin are damaged by cryopreservation and may affect the Tet3 function in zygotes [53,54]. 4.1. Conclusions In conclusion, this study reported that the paternal 5mC and 5hmC distributions and the Tet3 transcriptional program in the zygote were affected by spermatozoa cryopreservation. Furthermore, decreased fertilization capacity and delayed pronuclear formation were found in oocytes fertilized by cryopreserved sperm, and the subsequent embryo development was also clearly affected. Most of the differences observed might be explained by delayed fertilization when using cryopreserved sperm. Therefore, the abnormality in zygotic global genomic methylation due to spermatozoa cryopreservation may increase the risk of compromised in vitro developmental potential in early mouse embryos. Acknowledgments This study was funded by the National Natural Science Foundation of China (ID 31372307) and the Doctoral Program Foundation of the State Education Ministry (ID 20120008110001). The authors would like to thank Dr. Qien Yang (Washington State University) and Dr. Guangbin Zhou (Sichuan Agricultural University) for helpful discussions.
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[40] Guerrero-Bosagna C, Covert TR, Haque MM, Settles M, Nilsson EE, Anway MD, et al. Epigenetic transgenerational inheritance of vinclozolin induced mouse adult onset disease and associated sperm epigenome biomarkers. Reprod Toxicol 2012;34:694–707. [41] Abdalla H, Hirabayashi M, Hochi S. Demethylation dynamics of the paternal genome in pronuclear-stage bovine zygotes produced by in vitro fertilization and ooplasmic injection of freeze-thawed or freeze-dried spermatozoa. J Reprod Dev 2009;55:433–9. [42] Chao S, Li J, Jin X, Tang H, Wang G, Gao G. Epigenetic reprogramming of embryos derived from sperm frozen at 20 C. Sci China Life Sci 2012;55:349–57. [43] Fernandez-Gonzalez R, Moreira PN, Perez-Crespo M, SanchezMartin M, Ramirez MA, Pericuesta E, et al. Long-term effects of mouse intracytoplasmic sperm injection with DNA-fragmented sperm on health and behavior of adult offspring. Biol Reprod 2008;78:761–72. [44] Wang YP, Zhou GB, Zeng Y, Li JJ, Zhang QJ, Hou YP, et al. Impact on hyperactivated motility of cryopreserved mouse sperm from pretreatment with thimerosal. Asian J Anim Vet Adv 2011;6: 1052–60. [45] Meseguer M, Garrido N, Martinez-Conejero JA, Simon C, Pellicer A, Remohi J. Role of cholesterol, calcium, and mitochondrial activity in the susceptibility for cryodamage after a cycle of freezing and thawing. Fertil Steril 2004;81:588–94. [46] Kiefer JC. Epigenetics in development. Dev Dyn 2007;236:1144–56. [47] Benchaib M, Braun V, Ressnikof D, Lornage J, Durand P, Niveleau A, et al. Influence of global sperm DNA methylation on IVF results. Hum Reprod 2005;20:768–73. [48] Pick M, Stelzer Y, Bar-Nur O, Mayshar Y, Eden A, Benvenisty N. Clone- and gene-specific aberrations of parental imprinting in human induced pluripotent stem cells. Stem Cells 2009;27: 2686–90. [49] Sakashita A, Kobayashi H, Wakai T, Sotomaru Y, Hata K, Kono T. Dynamics of genomic 5-hydroxymethylcytosine during mouse oocyte growth. Genes Cells 2014;19:629–36. [50] Wossidlo M, Arand J, Sebastiano V, Lepikhov K, Boiani M, Reinhardt R, et al. Dynamic link of DNA demethylation, DNA strand breaks and repair in mouse zygotes. EMBO J 2010;29:1877–88. [51] Santos F, Peat J, Burgess H, Rada C, Reik W, Dean W. Active demethylation in mouse zygotes involves cytosine deamination and base excision repair. Epigenetics Chromatin 2013;6:39. [52] Shen L, Inoue A, He J, Liu Y, Lu F, Zhang Y. Tet3 and DNA replication mediate demethylation of both the maternal and paternal genomes in mouse zygotes. Cell Stem Cell 2014;15:459–70. [53] van der Heijden GW, Ramos L, Baart EB, van den Berg IM, Derijck AA, van der Vlag J, et al. Sperm-derived histones contribute to zygotic chromatin in humans. BMC Dev Biol 2008;8:34. [54] Flores E, Ramio-Lluch L, Bucci D, Fernandez-Novell JM, Pena A, Rodriguez-Gil JE. Freezing-thawing induces alterations in histone H1-DNA binding and the breaking of protein-DNA disulfide bonds in boar sperm. Theriogenology 2011;76:1450–64.
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Spermatozoa cryopreservation alters pronuclear formation and zygotic DNA demethylation in mice Gongxue Jia a, Xiangwei Fu a, Keren Cheng a, Mingxing Yue a, Baoyu Jia a, Yunpeng Hou b, Shien Zhu a, * a
National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing, China State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 August 2014 Received in revised form 18 November 2014 Accepted 30 November 2014
This study was conducted to investigate the effects of spermatozoa cryopreservation on DNA demethylation in mouse zygotes. Global methylation was studied in zygotes fertilized with cryopreserved sperm by immunostaining, and relative transcript abundance of Tet3, a key gene responsible for zygotic DNA demethylation, was examined by real-time quantitative polymerase chain reaction. Fresh sperm group served as control. Results indicated spermatozoa cryopreservation decreased fertilization rate (68.2% vs. 86.9%; P < 0.01) and delayed pronuclear formation (P < 0.05), compared with the control group. The percentages of embryos developed to cleavage and blastocyst stages in the freezing group (52.9% and 66.8%, respectively) were lower (P < 0.01 and P < 0.05, respectively) than those of the control group (83.4% and 81.1%, respectively). Furthermore, embryos obtained from cryopreserved sperm had higher relative methylation levels (P < 0.05) and less Tet3 mRNA concentrations (P < 0.01) in advanced pronuclear stages. Hence, we reported that spermatozoa cryopreservation disturbed the Tet3-mediated DNA demethylation progression in the zygotic paternal genome, which could be detrimental to the development of early mouse embryos, and most of the differences observed might be explained by delayed fertilization when using cryopreserved sperm. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Sperm Cryopreservation Zygote DNA methylation Tet3
1. Introduction The first successful spermatozoa cryopreservation was reported in 1949 [1]. Since then, this technology has been used in numerous mammalian species including humans [2–4]. Despite recent considerable progress, spermatozoa cryopreservation is still far from perfection [5,6]. For example, although the viability and fertility of frozen/ thawed sperm are acceptable, embryo implantation and normal fetus rates are significantly decreased [5,7,8]. Cryopreservation damages developmentally important organelles including plasma membrane [9], acrosome [10],
* Corresponding author. Tel./fax: þ86 (0) 10 62731979. E-mail address: [email protected] (S. Zhu). 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.11.036
and mitochondria [11], which may cause problems in embryonic development and fetal growth. The sperm delivers genetic and epigenetic information to the oocyte. External factors can have limited effects on the nuclear structure and integrity, owing to the special chromatin packaging in spermatozoa [12]. No obvious defect in the DNA integrity of frozen/thawed sperm has been detected, and only slight structural damages can be found [7,8]. Nevertheless, structural damages can be restored through DNA repair mechanisms after fertilization, ensuring safe delivery of the paternal genetic information [13,14]. In addition to DNA sequences, epigenetic modifications in the genome are also crucial for embryonic development in mammals [15]. DNA methylation, one of the major epigenetic modifications, plays a crucial role in gene expression regulation, zygotic genome activation, and embryonic
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development [16]. DNA methylation is sensitive to environmental insults, and various forms of assisted reproductive treatments can alter the methylome and impact the embryonic development [17]. It is well established that cryopreservation can change the global genomic methylation and specific gene expression in oocytes and embryos [18–21]. However, little is known about the effect of spermatozoa cryopreservation on DNA methylation in zygotes. The DNA methylation pattern changes dramatically during preimplantation development. To regain developmental totipotency, the zygotic genome must undergo DNA demethylation to erase the epigenetic memory inherited from the gametes. Usually, DNA demethylation is achieved passively in a replication-dependent manner through cell divisions. In the zygote, however, active DNA demethylation is observed, and this process has important implications for embryonic development [16,22]. In the paternal pronucleus (PN), 5-methylcytosine (5mC) is converted into 5-hydroxymethylcytosine (5hmC) by oxidation [23–25]. This reaction can be catalyzed by a family of dioxygenases, the ten-eleven translocation (TET) proteins [26,27]. Recently, works have established an essential role of TET3 in mouse zygotes. TET3 is specifically enriched in the paternal PN of the zygote, and its ablation by RNA knockdown or genetic deletion abolishes the generation of 5hmC to preclude complete DNA demethylation [24,25]. In the present study, we investigated the 5mC and 5hmC distribution patterns and the Tet3 mRNA abundances in mouse zygotes fertilized with cryopreserved sperm to determine the potential changes in zygotic global genomic methylation due to spermatozoa cryopreservation. 2. Materials and methods Unless otherwise stated, all chemicals were purchased from Sigma–Aldrich. All animals were maintained and handled in accordance with the requirements of the Institutional Animal Care and Use Committee of the China Agricultural University. 2.1. Oocyte collection Oocytes were collected from 8- to 12-week-old Kunming female mice (Academy of Military Medical Sciences, Beijing, China) as described previously [28]. Briefly, females were induced to superovulate with 10 IU eCG (Ningbo Hormone Products Company, Ningbo, China) followed by 10 IU hCG (Ningbo Hormone Products Company) 48 hours later. Cumulus–oocyte complexes were collected from oviducts at 14 hours after hCG treatment and recovered in M2 medium supplemented with 3 mg/mL BSA. Cumulus cells were dispersed with 0.1% hyaluronidase, then the cumulus-free oocytes were rinsed thoroughly and placed in drops of human tubal fluid medium (HTF; Millipore, MA, USA), which were covered with mineral oil for up to 1 hour in an incubator (at 37 C in an atmosphere of 5% CO2 and at maximum humidity) before IVF. 2.2. Spermatozoa preparation, freezing, and thawing Cauda epididymides were excised from 10- to 15-weekold Kunming male mice, and spermatozoa were collected
by squeezing them into drops of cryopreservative medium (R18S3, 18% raffinose and 3% skim milk [Becton Dickinson and Company, NJ, USA] in distilled water). To allow even dispersion, spermatozoa were incubated for 10 minutes at 37 C. Sperm freezing and thawing procedures were carried out as described previously with minor modifications [29]. The sperm suspension was divided into 10-mL aliquots and loaded into 0.25-mL plastic straws (IMV Technologies, L’Aigle, France). The straws were heat sealed, placed in liquid nitrogen vapor for 10 minutes, and then immediately plunged into liquid nitrogen before being stored for at least 1 week. Cryopreserved sperm were thawed in a water bath at 37 C for 5 minutes. Once thawed, spermatozoa were transferred into HTF medium and incubated for 30 minutes before insemination. 2.3. IVF and embryo culture The IVF procedures using fresh or frozen/thawed sperm were performed as described previously [29,30]. Briefly, spermatozoa were isolated from the cauda epididymides and capacitated by incubation for 1.5 hours in pregassed HTF medium before introducing to HTF-containing mature oocytes. Meanwhile, the frozen/thawed sperm were used for insemination in the same way. At 4 hours postfertilization (hpf), the eggs were removed from the fertilization drop, washed in KSOM medium (Millipore), transferred to drops of KSOM medium covered with mineral oil, and then cultured in an incubator. Samples were collected at several time points corresponding to embryo developmental period. The zygotes were recovered individually at 4, 6, 8, and 10 hpf for pronuclear morphometric analysis and immunostaining. The oocytes, zygotes of PN3 to PN4 stages or 2-cell embryos were harvested at 0, 8, or 24 hpf for real-time quantitative polymerase chain reaction (RT-qPCR). The proportions of the fertilized zygotes, 2-cell embryos, and blastocysts were assessed at 10, 24, and 96 hpf, respectively. 2.4. Pronuclear morphometric analysis The pronuclear morphologies of cells were examined on an inverted IX70 microscope (Olympus, Tokyo, Japan) immediately after staining with propidium iodide. The classification of zygotic PN1 to PN5 stages was performed according to previous studies, in which the pronuclear morphology and postfertilization timing were taken into consideration [24,31]. 2.5. Immunostaining The immunostaining analysis was carried out as described with modifications [32]. Immunostaining reagents for washing, blocking, antibody dilution, and mounting were purchased from Beyotime (Shanghai, China). After brief washing, the embryos were fixed in 3.7% paraformaldehyde for 40 minutes and permeabilized with 0.5% Triton X-100 for 10 minutes. Subsequently, the cells were denatured with 4N HCl for 10 minutes and
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neutralized with 100-mM Tris HCl (pH 8.5) for 20 minutes before blocking for 30 minutes. For quantitative analysis of 5mC, the samples were incubated with anti-5mC (diluted 1:500; Epigentek, NY, USA) overnight at 4 C. After several washes, the embryos were incubated with fluorescein isothiocyanate–conjugated antimouse immunoglobulin G (IgG; diluted 1:100; Santa Cruz Biotech, TX, USA) for 1.5 hours at 37 C. DNA was labeled with 10 mg/mL propidium iodide for 10 minutes. Finally, the cells were extensively rinsed again and mounted on slides with a drop of antifade mounting medium. For localization analyses of 5mC and 5hmC, the samples were incubated with anti-5mC and anti-5hmC (Active Motif, CA, USA) antibodies (diluted 1:500) overnight at 4 C. After several washes, the embryos were incubated with a 1:100 dilution of fluorescein isothiocyanate–conjugated antimouse IgG and Cy3-labeled antirabbit IgG (Beyotime) for 1.5 hours at 37 C. The cells were washed again before depositing them on slides. The immunostaining signal was examined on the Olympus FluoView FV1000 laser scanning confocal microscope. To construct three dimensional pictures of the zygotic PNs, we obtained images of planes at various depths within the stained embryos (also known as z-stacks). The ImageJ software (National Institutes of Health, MA, USA) was used to quantify the fluorescence signals of z-stack computed (with 2 mm per sample) fluorescence images. The relative methylation level (paternal PN to the maternal PN) was calculated as described previously [24,33]. The average methylation intensity for each PN was calculated using the ratio of the antibody signal to the DNA signal after subtraction of the cytoplasm background fluorescence. The relative methylation level in each zygote was the average methylation intensity in the paternal PN divided by the average methylation intensity in the maternal PN.
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2.7. Statistical analysis Statistical analysis was conducted by one-way ANOVA followed by Duncan’s test using SPSS statistical software (IBM, IL, USA). Data were expressed as the mean standard error of the mean, and P < 0.05 was considered significant. Oocytes were pooled and randomly distributed to two groups, and all experiments were repeated three times. 3. Results 3.1. Zygotic pronuclear formation and preimplantation embryonic development As shown in Figure 1, the fertilization and cleavage rates based on oocytes were decreased (P < 0.01) in the freezing group (68.2% and 52.9%, respectively) when compared with the control group (86.9% and 83.4%, respectively). Moreover, the blastocyst rate based on 2-cell embryos in the freezing group (66.8%) was lower (P < 0.05) than that of the control group (81.1%). To determine whether cryopreserved sperm affected the pronuclear formation, the pronuclear morphologies of fertilized zygotes were detected by DNA staining. The exact pattern of pronuclear development in zygotes was characterized in Figure 2. In the control group, most embryos were found in PN1 to PN2 stages (72.7%) at the earlier developmental period after sperm entry (4 hpf), and thereafter, the amount of PN4 to PN5 zygotes gradually increased up to a peak at 10 hpf (70.7%). In the freezing group, however, more embryos still stayed in the PN1 to PN2 stages (P < 0.05) at 6 and 8 hpf (53.1% vs. 33.2% and 28.0% vs. 17.5%, respectively), and PN4 to PN5 zygotes were reduced (P < 0.05) at 8 and 10 hpf (36.9% vs. 55.2% and 52.8% vs. 70.7%, respectively).
2.6. Real-time quantitative PCR
3.2. Dynamic distributions of 5mC and 5hmC
Total RNA was isolated from mouse oocytes (n ¼ 100), zygotes from fresh sperm (n ¼ 100), zygotes from freezing sperm (n ¼ 100), 2-cell embryos from fresh sperm (n ¼ 50), or 2-cell embryos from freezing sperm (n ¼ 50) using TRIzol reagent (Invitrogen, CA, USA) in each replication. The RNA was reverse transcribed into complementary DNA (cDNA) using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, CA, USA); then, the cDNA was quantified by RT-qPCR using a SYBR PrimeScript RT-PCR Kit (TaKaRa, Dalian, China) on a CFX96 Real-Time PCR Detection System (Bio-Rad, CA, USA) under standard conditions. The sizes of the PCR products were further confirmed using gel electrophoresis on a standard GelRed-stained (Biotium, CA, USA) 1.5% agarose gel and visualized after exposure to ultraviolet light. The cycle threshold (Ct) value used to calculate the relative expression was the average of three replicates and was normalized against that of the reference gene (Gapdh). The primers used are as follows: Gapdh Fd50 catggccttccgtgttccta-30 ; Gapdh Rd50 -gcctgcttaccaccttctt-30 ; Tet3 Fd50 -cccacaaggaccaacataac-30 ; Tet3 Rd50 -cctcgctgccaaa ctcat-30 . The mRNA expression levels were calculated using the 2ddCt method [34].
To determine whether zygotic DNA demethylation was altered after fertilization with cryopreserved sperm, we used anti-5mC to visualize the genomic methylation. As shown in Figure 3A for the control group, the intensities of the 5mC signals became increasingly different between paternal and maternal PNs. Although a similar downward trend was obtained, the relative methylation levels in the late (PN3–PN5) zygotes of the freezing group were higher (P < 0.05) than those of the control group (Fig. 3B). The tracing of 5hmC dynamics during zygotic development showed that the inverse correlation between the 5mC and 5hmC signals in the parental genomes persisted, even when cryopreserved sperm were used. However, the 5hmC signals seemed to decrease in zygotes of the freezing group (Fig. 3C). 3.3. Expression levels of Tet3 Because TET3 mediated 5mC oxidation in zygotes, we examined whether the Tet3 expression was disturbed in embryos derived from cryopreserved sperm. The RT-qPCR revealed that the Tet3 mRNA expression in PN3 to PN4 zygotes of the freezing group was lower (P < 0.01) than that
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Fig. 1. Fertilization ability and early embryo development. Shown is the reduced efficiency of fertilization and early embryo development with spermatozoa cryopreservation. The fertilization and cleavage rates are based on the number of surviving oocytes, whereas the blastocyst rate is based on the number of 2-cell embryos. Number of oocytes used in total: the freezing group, n ¼ 306; the control group, n ¼ 313. Different letters (a and b) indicate P < 0.01 compared with control group.
of the control group. No significant difference in Tet3 transcript level was detected at 2-cell stage (Fig. 4). 4. Discussion The understanding about the contribution of the sperm epigenome for the developing embryo remains rudimentary [35]. The once held dogma declares that sperm is only responsible for the delivery of a DNA blueprint and does not drive epigenetic changes in the embryo. However, recent evidence indicates the importance of the sperm epigenome during embryogenesis. For example, paternal PNs in mouse zygotes present different DNA methylation statuses after insemination with round spermatids or mature spermatozoa [36]. Furthermore, paternal diet can affect the development and health status of offspring by altering DNA methylation patterns in mice [37,38]. Transgenerational epigenetic inheritance is also largely mediated by changes in the sperm epigenome [39,40]. Despite these seminal findings, the epigenetic reprogramming of zygotes obtained from frozen/thawed sperm remains elusive. Bovine zygotes derived from sperm frozen by different methods show
different DNA methylation statuses [41]. As for the mouse, no abnormality is found in the epigenetic reprogramming of embryos derived from sperm frozen at 20 C [42], whereas delayed DNA demethylation is observed in the zygotes produced by intracytoplasmic sperm injection using frozen/ thawed sperm [43]. Taken together, the effect of spermatozoa cryopreservation on zygotic DNA demethylation still needs to be defined. Here, our results provide clear evidence that spermatozoa cryopreservation alters zygotic DNA demethylation, which could adversely affect in vitro development of mouse zygotes. Spermatozoa cryopreservation is one of the significant factors that could influence the embryonic development in assisted reproductive treatments [5,6]. In the present study, we observed significant declines in embryonic development after fertilization using cryopreserved sperm. This should be due to abnormal fertilization and delayed pronuclear formation. Previous studies have shown that spermatozoa cryopreservation can directly result in defective acrosome reaction [10], hyperactivated motility [44], and calcium equilibrium [45]; all these factors could endanger the normal fertilization process. The delayed
Fig. 2. Distributions of the pronuclear stages during zygotic development. Shown are the percentages of zygotes at paternal pronucleus (PN) 1 to PN2, PN3, and PN4 to PN5 stages in the freezing and control groups. Cells were sampled at 4, 6, 8, and 10 hours postfertilization (hpf), which permitted a profile of pronuclear maturation stages as a function of time to be constructed. Number of zygotes analyzed in total: in the freezing group, 4 hpf, 82; 6 hpf, 96; 8 hpf, 103; 10 hpf, 92; in the control group, 4 hpf, 96; 6 hpf, 108; 8 hpf, 120; 10 hpf, 107. Asterisk indicates P < 0.05 compared with control group.
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Fig. 3. Dynamic DNA methylation patterns during zygotic development. (A) Representative images of paternal pronucleus (PN) 1 to PN5 zygotes stained with 5-methylcytosine (5mC) antibody and propidium iodide. Scale bar ¼ 10 mm. (B) Comparison of paternal and maternal 5mC signals (maternal signal set to one) normalized against DNA signal in the two groups. Number of zygotes analyzed in total: in the freezing group, PN1, 24; PN2, 25; early PN3, 29; late PN3, 29; PN4, 33; PN5, 30; in the control group, PN1, 30; PN2, 31; early PN3, 35; late PN3, 30; PN4, 26; PN5, 28. Asterisk indicates P < 0.05 compared with control group. (C) Representative images of zygotes stained with 5mC and 5-hydroxymethylcytosine (5hmC) antibodies at 4 and 10 hours postfertilization (hpf) in the two groups. Scale bar ¼ 10 mm. (For interpretation of the references to color in this figure, the reader is referred to the Web version of this article.)
pronuclear formation that we observed might be attributed to the DNA repair machinery activated by sperm-derived DNA damage, which is thought to have the potential to disturb cell cycle check points and delays zygotic development [13,14]. Furthermore, this delay may be associated with DNA demethylation that accompanies pronuclear formation in zygotes. DNA methylation plays a crucial role in the control of gene expression and chromosomal structure during the early embryogenesis [16,46]. Any abnormality in DNA methylation could be deleterious to zygotic development. For example, global sperm DNA hypomethylation is correlated with poor pregnancy outcomes in IVF patients [47]. Additionally, aberrant DNA methylation found in cloned embryos is identified as one of the causes for the low cloning efficiency [48]. In this study, the PN3 to PN5 zygotes of the freezing group possessed more paternal 5mC, and the generation of 5hmC seemed to be weakened in paternal PNs compared with that of the control group. Therefore, we concluded that zygotic DNA demethylation could be changed by spermatozoa cryopreservation.
The compromised 5mC oxidation mediated by TET3 may be one of the major reasons for this altered DNA demethylation in zygotes. Before fertilization, Tet3 expression increases gradually during oocyte growth and reaches the highest level in fully grown oocytes [49]. One study indicates that Tet3 expression could continue to be enhanced after fertilization [23], which is consistent with our result, whereas another study shows merely a slight increase [24]. Furthermore, we detected the decreased expression of Tet3 mRNA during zygotic development in the freezing group, indicating that the transcription of Tet3 in zygotes was affected by cryopreserved sperm. It is reported that Tet3-deficient zygotes have reduced expression of OCT4 during the morula stage, and about half of these embryos show serious abnormalities by embryonic day E11.5 and fail to develop [25]. However, no proof of either mechanism for this change in Tet3 has been clearly reported [27]. It can be speculated that one of the candidate mechanisms could be DNA repair pathway that works together with TET3 to facilitate DNA demethylation in zygotes [50,51]. This pathway might be appropriated preferentially
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The authors also appreciate Nature Publishing Group Language Editing for proofreading this article. References
Fig. 4. Expression levels of Tet3 transcripts in embryonic development. Oocytes were used as the calibrator sample (expression set to one). There were 100 oocytes, 100 zygotes from fresh sperm, 100 zygotes from freezing sperm, 50 2-cell embryos from fresh sperm, or 50 2-cell embryos from freezing sperm used in each replication, which were harvested at 0, 8, 8, 24 or 24 hours postfertilization, respectively. Bars with different letters (a, b and c) were significantly different ( P < 0.05).
by sperm-derived DNA damage in mouse zygotes fertilized with cryopreserved sperm, similar to the situation simulated by H2O2 treatment [14]; thereby, DNA demethylation progression could become obstructed. Similarly, a recent study confirms that TET3 facilitates DNA demethylation largely by coupling with DNA replication [52], meaning that the obstruction of DNA replication could damage TET3 function. It is also possible that the sperm-derived residual nucleosomes that contribute to zygotic chromatin are damaged by cryopreservation and may affect the Tet3 function in zygotes [53,54]. 4.1. Conclusions In conclusion, this study reported that the paternal 5mC and 5hmC distributions and the Tet3 transcriptional program in the zygote were affected by spermatozoa cryopreservation. Furthermore, decreased fertilization capacity and delayed pronuclear formation were found in oocytes fertilized by cryopreserved sperm, and the subsequent embryo development was also clearly affected. Most of the differences observed might be explained by delayed fertilization when using cryopreserved sperm. Therefore, the abnormality in zygotic global genomic methylation due to spermatozoa cryopreservation may increase the risk of compromised in vitro developmental potential in early mouse embryos. Acknowledgments This study was funded by the National Natural Science Foundation of China (ID 31372307) and the Doctoral Program Foundation of the State Education Ministry (ID 20120008110001). The authors would like to thank Dr. Qien Yang (Washington State University) and Dr. Guangbin Zhou (Sichuan Agricultural University) for helpful discussions.
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