Developmental Origins of Transgenerational Sperm DNA Methylation Epimutations Following Ancestral DDT Exposure

Introduction

Numerous environmental factors have been shown to promote the epigenetic transgenerational inheritance of disease such as the agricultural fungicide vinclozolin [1] and the pesticide DDT (dichlorodiphenyltrichloroethane) [2]. Caloric restriction, high fat diets, stress and a variety of different toxicants have also been linked to the transgenerational epigenetic inheritance phenomenon [3–5]. This non-genetic form of inheritance involves epigenetic modifications of the germline (sperm and egg) to pass an altered epigenome to the early embryo that can then impact the transcriptomes and epigenetics of all subsequently derived somatic cells [1, 5]. Different epigenetic processes are involved in the transgenerational germline transmission where the environment can impact the health and evolution of species [3, 4, 6].

Epigenetics is defined as “molecular factors and processes around the DNA that regulate genome activity independent of DNA sequence and that are mitotically stable” [7]. Transgenerational epigenetic inheritance requires the germline transmission of epigenetic information. Different epigenetic processes have been shown to be involved in the transgenerational phenomenon such as DNA methylation [8–10], non-coding RNA [11, 12], and histone modifications and retention [10, 13, 14]. Recently, concurrent alterations in all three of these processes have been observed in transgenerational sperm transmission after exposure to DDT and vinclozolin [15, 16]. Most previous studies have been conducted in sperm due to the ability to isolate large numbers of cells and the inability to isolate large numbers of eggs, but experiments have shown that the female germline also has the capacity to transmit epigenetic inheritance [2, 17]. The current study investigated the transgenerational sperm transmission of DNA methylation alterations after DDT exposure.

The primordial germ cells (PGCs), which give rise to spermatogenic (or oogenic) cells, develop and migrate down the genital ridge to colonize the indifferent gonad [18]. Upon sex determination and depending on the chromosomal sex, the PGCs differentiate into the male or female germline lineage [19]. In the rat gonad, the early developing testis germ cells will become prospermatogonia cells during the male gonadal sex determination period by embryonic day 16 (E16) [20, 21]. By postnatal day 10 (P10) the germline develops into spermatogonia. Following the onset of puberty the initial wave of spermatogenesis begins in the testis where the spermatocytes, including the meiotic stage of pachytene spermatocytes, develop. After meiosis the round spermatid stage differentiates, then in the final spermatozoa stage the cells are released into the lumen of the seminiferous tubules [22], Fig 1A. After entering the caput region of the epididymis the spermatozoa undergo further differentiation. During the transit in the epididymis the spermatozoa mature. When the spermatozoa reach the final caudal stage of the epididymis, the sperm have developed the capacity for motility and fertility [23, 24]. Prior to ejaculation or degradation the mature caudal epididymal sperm will be stored in the vas deferens. In the current study we used several male germline developmental periods (E16 prospermatogonia, P10 spermatogonia, adult pachytene spermatocytes, round spermatids, caput epididymal spermatozoa, and caudal epididymal sperm), Fig 1A, to investigate the developmental origins of the transgenerational sperm DMRs following DDT exposure in the F0 generation.

Epigenetic programming occurs during these male germline developmental periods. The migrating PGCs have an erasure of DNA methylation that then re- methylate during gonadal sex determination to create the male germline and prospermatogonia [25]. In the adult testis, during spermatogenesis some epigenetic programming events occur [26, 27]. The first examples of this male germline epigenetic programming were observed with imprinted genes [28, 29]. Imprinted genes can develop either early in the embryonic period or develop during spermatogenesis. Although previous studies have discussed the germline developmental origins of epigenetic inheritance [5, 30–34], none of these studies identified the developmental programming origins of the transgenerational epigenetic alterations observed in sperm.

The current study was designed to investigate the developmental origins of the DDT induced transgenerational sperm DMRs termed epimutations. The hypothesis tested is that a cascade of epigenetic changes occurs during development such that the majority of DMRs develop throughout gametogenesis and spermatogenic stages. It has been previously demonstrated that epigenetic alterations occur in the PGC [35]. However, due to this being the earliest stem cell stage of the germline, the PGCs DMRs were found to be different from the epimutations observed in sperm [35]. These observations suggest that the epimutations in sperm develop at later stages of epigenetic programming. The present study shows the majority of sperm DMRs originate during spermatogonia development and spermatogenesis, but some also do originate during epididymal maturation. This is one of the first genome-wide analyses of epigenetic programming during gametogenesis for transgenerational sperm epimutations [36].

Results

F3 generation control and DDT lineage male rats were used in this experimental design to isolate various germ cell stages for epigenetic analysis. The F0 generation gestating female rats at 90 days of age were transiently exposed to DDT or a vehicle control during gestational days 8–14. This stage of fetal development corresponds to when the PGCs migrate to colonize the indifferent gonad through the early stage of gonadal sex determination as previously described [18, 25, 35]. The F1 generation offspring were obtained and when they reached 90 days of age they were bred within the control and DDT lineages to generate the F2 generation (grand offspring) which were then bred at 90 days of age to obtain the transgenerational F3 generation for each control and DDT lineage. No cousin or sibling breedings were used to avoid any inbreeding artifacts. Only the F0 generation gestating females were exposed to DDT, which also directly exposed the F1 generation fetus and the germline within the F1 generation fetus that will generate the F2 generation. Therefore, the F3 generation is the first transgenerational generation not directly exposed [37] and was used to study the developmental origins of the transgenerational sperm epimutations. The F3 generation control and DDT lineage male rats were aged to the embryonic day 16 (E16) fetal stage for prospermatogonia cell isolation, to postnatal day 10 (P10) for spermatogonial cell isolation, and to 120 days of age for pachytene spermatocyte isolation, round spermatid isolation, caput spermatozoa isolation, and cauda epididymal sperm isolation. A gravity sedimentation StaPut protocol, as described in the Methods [38, 39], was used to isolate the prospermatogonia, spermatogonia, pachytene spermatocytes and round spermatids cells. Three different pools each with different individual sets of animals from different litters were obtained with 5–6 males per pool for the E16 stage, 6–7 males per pool for the P10 stage, 3 adult males per pool for the adult stages. Therefore, 3–7 different males were used in the analysis within each of the three different pools analyzed for both the control and DDT lineages. Genomic DNA was isolated from each purified germ cell stage pool and from individual animals for epididymal caput spermatozoa and cauda sperm [8, 13] and then three pools generated for subsequent epigenetic analysis.

The analysis of differential DNA methylation regions (DMRs) was performed with a methylated DNA immunoprecipitation (MeDIP) procedure followed by next generation sequencing for an MeDIP-Seq protocol described in the Methods [8]. The F3 generation control versus DDT lineage pools were compared to identify the DMRs at each developmental stage of the male germline. The DMRs at various p-value thresholds are presented at each developmental stage in Fig 2. An EdgeR p-value of p<1e-05 was selected at all stages for further analysis of the DMRs. This correlated with an FDR (false discovery rate) value of <0.1 for all except the E16 prospermatogonia which was more variable. The All Windows is all DMRs with at least one 100 bp region (window) with statistical significance and the Multiple Windows being ≥2 significant 100 bp windows. The number of DMRs with multiple windows is also presented in Fig 2. Most developmental stages showed approximately 100 – 300 DMRs. The epigenetic alterations were higher at the pachytene spermatocyte and round spermatid stages which showed more than 300 DMRs. The chromosomal locations of the DMRs at each developmental stage are presented in Fig 3. The red arrowheads indicate the location of a DMR with the black boxes indicating clusters of DMRs. In most stages, all the chromosomes, except the Y and mitochondrial DNA (MT), were found to contain DMRs. The genomic features of the DMRs at each stage of development were investigated. The CpG density of the DMRs at all stages was 1–5 CpG per 100 bp with 1 CpG per 100 bp being predominant, Fig 4. This is characteristic of a low density CpG desert [40] which has been observed with previous transgenerational DMRs. The length of the DMRs at all the developmental stages were between 1–4 kb with 1 kb length being predominant, Fig 5. Therefore, the DMRs are generally 1 kb in size with around 10 CpGs as previously observed [40]. The overlap between the various developmental stage DMRs was investigated and is presented in Fig 6A. The DMRs were found to be primarily stage specific with a very low number of DMRs overlapping between the stages with a p<1e-05. The cauda sperm and the caput spermatozoa had the highest level of overlap with 88 DMRs, Fig 6B and S7 Table, comprising over 45% of the caput epididymal spermatozoa DMRs. These observations suggest a cascade of epigenetic programming occurs throughout male germline development.

The DNA methylation alterations of the DMRs at each stage of development were investigated. For the developmental time-course the top 100 statistically significant DMRs at each stage of development were examined regarding the scaled mean read depth between the control versus DDT lineage DMR data. The scaled mean read depths at each DMR was separated into control greater than DDT data (i.e. decrease in DNA methylation) and DDT greater than control data (i.e. increase in DNA methylation) which is presented as a scaled mean read depth, Figs 7 & 8. The E16 prospermatogonia DMRs had 28 DMR with a decrease in methylation and 72 DMR with an increase in methylation, Figs 7A & 8A. For both data sets the DMR methylation alterations generally dropped by the P10 spermatogonia stage and became mixed with no distinct patterns in later stages. Although a few of both populations stayed elevated, the majority had an alteration in DNA methylation at later stages of development. The P10 spermatogonia DMRs had 38 DMRs with a decrease in methylation and 62 with an increase in methylation in regard to % mean read depth, Figs 7B & 8B. A dramatic change in methylation between the prospermatogonia to spermatogonia stage and from the spermatogonia to later stages of development were observed. Therefore, the E16 prospermatogonia and P10 spermatogonia stage DMRs are for the most part unique at these stages of development. The pachytene spermatocyte DMRs had predominantly a decrease in methylation with 63 DMRs and 37 DMRs with an increase, Figs 7C & 8C. Therefore, DNA methylation was primarily decreased in the pachytene spermatocyte DMRs. The round spermatid DMRs had a higher number of 72 DMR with an increase and 28 DMR with a decrease, Figs 7D & 8D. The caput epididymal spermatozoa DMRs had 46 DMR with a decrease and 54 DMR with an increase in methylation, Figs 7E & 8E. The cauda epididymal sperm DMRs had 49 DMR with a decrease and 51 DMR with an increase, Figs 7F & 8F. The caput spermatozoa and cauda epididymal sperm have relatively consistent DNA methylation characteristics for both data sets. Therefore, the cauda sperm show more consistent DNA methylation profiles during spermatogenesis and epididymal sperm maturation, but are distinct in the spermatogonial stem cell stages.

Genes associated with the DMRs were identified and compared at each of the developmental stages. The DMRs that had a gene within 10 kb distance, so the promoter is considered, were identified and the associated genes and gene functional categories determined, S1-S6 Tables. The DMRs with a p<1e-05 were used in this analysis at all the developmental stages. The number of genes associated with specific gene functional categories is presented in Fig 9A. Each of the developmental stages are presented to compare the major gene categories. The signaling, metabolism, transcription and receptor gene categories were the major categories present in the different developmental stages. Additional categories were cytoskeleton, development, transport and immune system, that were present in all the developmental stages, Fig 9A. The DMR associated genes at each developmental stage were also used in a gene pathway analysis applying the KEGG pathway association as described in the Methods. The number of DMR associated genes involved with the major pathways is presented in Fig 9B. The most predominant pathways present in at least two different developmental stages are shown. The E16 prospermatogonia, round spermatids, caput spermatozoa and cauda sperm had DMR associated genes in the metabolic pathways. The E16 prospermatogonia, P10 spermatogonia, pachytene spermatocytes had DMR associated genes in the pathways in cancer in common. Therefore, the only pathways that were present in nearly all the stages are metabolism and pathways in cancer. The caput epididymal spermatozoa and cauda sperm were more consistent than the others which were more distinct.

The final aspect of the study involved an investigation of the developmental origins of the cauda sperm DMRs, meaning when the differential DNA methylation alterations appeared within the developmental stages examined. In the cauda sperm there are 265 DMRs at p<1e-05. For the analysis of developmental origins the 265 DMRs were examined individually with a reduced statistical stringency of p<0.05 to see the first stage the DMR appears or originates. Fig 1B demonstrates 70 DMRs were developed in the E16 prospermatogonia stage, 65 DMRs in the P10 spermatogonia stage, 47 DMRs in the spermatocyte stage, 14 DMRs in the round spermatid stage, 33 DMRs in the caput epididymal spermatozoa stage, and 36 DMRs in the cauda sperm stage, S8 Table. The cauda sperm had a total of 265 DMRs at p<1e-05 with the majority of DMRs originating in the prospermatogonia and spermatogonia. Fewer were observed in the round spermatids and caput epididymal spermatozoa stages, Fig 1B. The percentage of the total DMR origins and schematic of the different stage cells during development is presented in Fig 1A. Therefore, the majority of the transgenerational sperm DMRs were developed during the prospermatogonia and spermatogonia stages with a small number developed in the epididymal maturation stages. This correlated to a cascade of epigenetic and transcriptome changes during these stages of male germline development to generate the transgenerational sperm epimutations, Fig 1A.

Discussion

The aim of the current study was to identify the developmental origins of the cauda sperm transgenerational DMRs that transmit the epigenetic transgenerational inheritance of disease and phenotypic variation. During embryonic day 8–14 in the rat, when the primordial germ cells (PGCs) migrate to colonize the indifferent fetal gonad, the F0 generation gestating female exposure to DDT was performed [18, 25, 35]. Five different stages of male germ cell development were investigated: E16 prospermatogonia, P10 spermatogonia, and adult pachytene spermatocytes, round spermatids, caput epididymal spermatozoa and cauda sperm, Fig 1A. The functional considerations of these cell populations involve the prospermatogonia precursor stem cell population, spermatogonia stem cell population in the testis, the meiotic pachytene spermatocyte cell population, the post-meiotic spermatid population and the spermatozoa present in the caput epididymis, and mature sperm in the cauda epididymis. The caput spermatozoa and cauda epididymal sperm were directly isolated as described in the Methods then sonicated and washed to remove any contaminating somatic cells, so are pure sperm cell preparations. For the other cell populations a gravity sedimentation on a StaPut apparatus procedure was used to isolate the specific cell populations as previously described [39, 40]. The E16 prospermatogonia and P10 spermatogonia were found to have a greater than 85% purity. The pachytene spermatocytes were greater than 95% spermatocytes with two-thirds of these cells pachytene spermatocytes and one-third other spermatocyte stages. This is due to the longer developmental life span or developmental period of pachynema compared to the other stages of first meiotic prophase [41] and to the efficiency of the StaPut gradient protocol. For the isolated round spermatids 95% of them were spermatids with 90% of them being round spermatids and 10% being elongate spermatids and other spermatogenic cell stages [42]. Despite obtaining a high purity for these developmental stage male germline cell populations, cell purity needs to be considered in the data interpretations. The embryonic day 16 (E16) prospermatogonia were isolated from the fetal gonad, the postnatal day 10 (P10) from the early pubertal age, the pachytene spermatocytes, round spermatids, caput epididymal spermatozoa and cauda sperm were all isolated from postnatal day 120 (P120) age adult male rats. DDT has been previously shown to induce transgenerational inheritance of disease between 6–12 months of age [5]. Therefore, by choosing the postnatal day 120 or earlier with negligible disease present, no disease artifacts are anticipated in the current study. This is consistent with the primary objective of the study to investigate the developmental origins of the sperm DMR, but future studies can now consider disease correlations.

The transgenerational F3 generation control and DDT lineage germline samples were compared at each of the developmental stages. The F1 and F2 generations have somatic cell and germline epigenetic alterations due to the F1 generation fetal direct exposure and F1 generation fetal germline direct exposure that will generate the F2 generation. Therefore, the F3 generation is the first transgenerational generation not having any direct exposures, so was the focus of the current study [37]. A comparison of the F1 generation direct exposure sperm DMR development with the transgenerational F3 generation observations will be an interesting future study to consider. All F3 generation developmental samples had differential DNA methylation regions (DMRs). Excluding the caput spermatozoa and cauda epididymal sperm with common DMRs, all the other stage DMRs were found to be primarily distinct from each other, Fig 6. The majority of the epigenome was not altered (i.e. potentially millions of CpG regions) and the current study focused on the transgenerational alterations at each stage of development. The distinct DMRs at each stage indicate a cascade of epigenetic changes occur to program the sperm transgenerational epigenome.

A comparison of the different developmental stage germline populations was conducted in regard to DNA methylation alterations for the top 100 most statistically significant DMRs at each developmental stage separately. Analysis of these DNA methylation alterations for the different developmental stage DMRs revealed that the E16 prospermatogonia and P10 spermatogonia presented distinct patterns of DNA methylation compared to the other stages, Figs 7 & 8. The statistically significant top 100 DMRs at both these developmental stages altered greatly with adjacent stages and often lost their statistical significance. In contrast, the pachytene spermatocytes, round spermatids, caput epididymal spermatozoa and caudal sperm were more consistent between each other regarding the DMRs maintaining similar DNA methylation alterations and statistical significance, Figs 7 & 8. This suggests that the developing stem cells and spermatogonia have principally unique DNA methylation profiles associated with the cascade of epigenetic programming of the cells. When the spermatogonia initiate the spermatogenesis process there is a cascade of epigenetic programming and DNA methylation observed between the spermatogenic cell populations, as well as between spermatozoa undergoing epididymal maturation. Observations suggest a dynamic epigenetic programming of the male germline in the testis and epididymis. Epididymal maturation has been shown to promote structural and molecular alterations in the developing sperm, which also appear to be involved in the transgenerational sperm DMR programming [32].

Significant epigenetic reprogramming in the primordial germ cells (PGCs) has already been described. DNA methylation erasure during migration and colonization of the fetal gonad has been shown to occur [18]. This process enables the germline stem cell population to facilitate the generation of the male or female germ lines following gonadal sex determination [18, 25]. A stable epigenetic cascade of events occurs to produce the spermatogonial stem cell population in the adult testis. Studies have shown that environmental toxicants have the ability to promote epigenetic DNA methylation alterations in the PGCs and prospermatogonia [35]. These transgenerational DMRs observed in the PGCs were found to be distinct from the caudal epididymal sperm DMRs [35]. The current study supports this observation and determines that sperm DMRs not only originate in the fetal period, but trigger a cascade of epigenetic alterations that eventually impact the mature sperm epigenome, Fig 1B. Therefore, the developmental cascade of epigenetic programming initiated by fetal exposure induces a number of DMRs that are also maintained throughout development.

In the cauda sperm, the origins of the DMRs were identified. The majority of them originate throughout the development of prospermatogonia, spermatogonia and spermatocytes, Fig 1B and S9 Table. A smaller number of DMRs also arose in the round spermatids and cauda epididymal sperm. The highest number of DMRs was observed in the prospermatogonia, the stage of development during which the initial F0 generation gestating female and F1 generation developing fetus were exposed. The DMRs in the cauda sperm originated in all the earlier developmental spermatogenic stages and through epididymal maturation. These observations correlated with the DNA methylation alteration data for DMRs between the different developmental stages shown in Figs 7 & 8. Thus, the primary origin of the transgenerational sperm DMR epimutations/DMRs does not occur during early PGC development or in the embryonic stem cell population, but throughout development and spermatogenesis in the testis, as well as during epididymal maturation. Although the induction of a cascade of epigenetic programming in the PCGs is critical as previously suggested [7, 35], there is a continual cascade of epigenetic alterations throughout spermatogenesis to give rise to the transgenerational sperm DMRs, Fig 1A. Another recent study investigating vinclozolin induced sperm DMR origins found similar observations, but the highest level of DMRs were developed at the pachytene spermatocyte stage [36]. Therefore, an exposure specificity may exist in the developmental epigenetics observed.

The DMRs associated genes were identified for DMRs at p<1e-05 at each stage of development. The analysis of associated gene functional categories identified signaling, transcription, metabolism and receptor as the major categories at all stages of development. The pathway analysis also identified a number of stage specific pathways with negligible overlap except in metabolism and pathways in cancer. Gene pathways more specific to testis development such as meiosis or the pyruvate pathway did not contain more than a few DMR associated genes. Therefore, for all the stages of development for the DMR-associated genes involved similar gene categories, but different pathways. Future studies are needed to correlate the DMRs with data identified from transcriptome and altered non-coding RNA expression to provide a better understanding as to how the DMRs may regulate genome activity.

The caput epididymal spermatozoa displayed unique DMRs and others that were in common with the cauda sperm. Interestingly, the cauda epididymal sperm did acquire DMRs that were not present in earlier stages of development. Even though the majority of the cauda epididymal sperm DMRs were obtained during prior developmental stages, 36 DMRs were acquired at the cauda epididymal sperm stage of maturation only, S7 and S8 Tables. Two studies have previously suggested that epididymal maturation (epididymosomes) may be involved with transgenerational sperm maturation regarding epigenetic alterations, but no direct data have been provided [43–45]. The current study supports a role for epididymal maturation altering sperm DMRs. However, the majority of DMRs originated during earlier developmental stages and spermatogenesis in the testis and not the epididymis, Fig 1B. A combination of early development, spermatogenesis and epididymal mutation appears to be involved in the development of the transgenerational sperm epimutations. Further investigation is needed to elucidate the molecular mechanisms in the epididymis that alter sperm epigenetics.

Conclusions

The current study was designed to investigate the developmental origins of transgenerational sperm DMRs induced by the pesticide DDT. Results show that each stage of male germ cell development examined, including the E16 prospermatogonia, P10 spermatogonia, pachytene spermatocytes, round spermatids, caput epididymal spermatozoa and cauda sperm, has distinct and unique DMRs when the DDT lineage and control lineage F3 generation samples are compared. Both the prospermatogonia and spermatogonia display unique DNA methylation alterations in comparison with later stages that are more consistent. The origins of the cauda sperm DMRs developed throughout the earlier developmental stages with the highest number during prospermatogonia, spermatogonia and pachytene spermatocytes, and fewer originating during epididymal maturation. In conclusion, a developmental cascade of epigenetic programming occurs from the PGCs to the sperm with epimutations developing throughout the different stages of development, Fig 1A. The initial speculation that the DMRs may develop in the PGCs is not the case, even though the developmental cascade initiated is important. Recently, sperm carrying transgenerational epimutations were found to have alterations in DNA methylation, ncRNAs and histone retention, such that all epigenetic factors are involved in the epigenetic transgenerational inheritance phenomenon [15, 16]. Future studies will now need to investigate these other epigenetic processes and correlated gene expression to develop a more systems biology assessment of the molecular mechanism involved.

Methods

Supplementary Material