Analysis of the transgenerational actions of glyphosate used outbred Sprague Dawley female rats (F0 generation) transiently exposed (25 mg/kg body weight glyphosate daily) during days 8 to 14 of gestation. This is half the NOAEL exposure of 50 mg/kg/day10, and due to rapid metabolism turnover would lead to a decreased (5–10 mg/kg) dose during the transient exposure period. The F1 generation animals (direct fetal exposure) were bred within the lineage to generate the F2 generation (direct germline exposure), which were bred to generate the F3 generation (transgenerational, no direct exposure). A control lineage used F0 generation gestating females administered vehicle control dimethyl sulfoxide (DMSO) or phosphate buffered saline (PBS). The control and glyphosate lineages were aged to 1 year and euthanized for pathology and sperm epigenetic analysis. No sibling or cousin breeding (crosses) was used in order to avoid any inbreeding artifacts in either the control or glyphosate lineages. Generally, 6–8 founder gestating females from different litters were bred, and 5 animals of each sex from each litter used to generate 25–50 individuals of each sex for each generation for analysis, as previously described41. Therefore, litter bias was negligible, and the full spectrum of pathology within the generation and lineage was assessed.

Pathology Analysis

Upon dissection at one year of age, abdominal and thoracic organs were briefly examined for obvious gross abnormalities and pathologies. No pathologies or remarkable abnormalities were observed with the exceptions of some animals showing enlarged roughened kidneys (associated with histological evidence of renal disease), and some female animals showing enlarged fluid-filled uteri. All animals that died or were euthanized for welfare reasons prior to 1 year of age were submitted for necropsy and examined for gross and histologic pathologies by the Washington Animal Disease Diagnostic Laboratory (WADDL) at Washington State University College of Veterinary Medicine. For the eleven animals so submitted there were three F3 generation glyphosate control rats that showed aspiration pneumonia, dermal necrosis, or hepatic centrolobar necrosis. There were four F2 generation glyphosate lineage rats showing metritis, dystocia, hepatic necrosis, or adrenal cortical necrosis. There were four F3 generation glyphosate lineage rats showing granulomatous furunculosis, ulcerative balanoposthitis, or seizures for which the underlying diagnosis was open.

Upon dissection at 1 year of age the testis, prostate, kidney, and ovary were collected and examined for histopathologies, Supplemental Figure S2. Stained paraffin sections of isolated tissues were examined by three different trained pathology observers blinded to the exposure lineages to assess the presence of specific histological abnormalities as described in the Methods29, (Supplemental Figure S2). The male and female pathologies are summarized in Figs 1 and 2, respectively, with the diseased individuals per total number of individuals presented for each generation and lineage, Supplemental Tables S1–S3. For the purposes of this paper an animal was considered to have a diseased tissue if the number of histological abnormalities was markedly increased (i.e. greater than two standard deviations) compared to that of the controls for that tissue, as described in Methods. Previously we have confirmed with apoptosis analysis an increase in spermatogenic cell death in testes36,37. Testis disease was characterized by the presence of histopathologies including azoospermia, atretic seminiferous tubules, presence of vacuoles in basal regions of the seminiferous tubules, sloughed germ cell in the lumen of tubules, and lack of tubule lumen29, (Supplemental Figure S2). The most common histology abnormalities were atrophy and vacuoles, followed by sloughed cells and debris in the tubule lumen. The frequency or incidence of testis disease was found to be significantly elevated in the F2 generation glyphosate lineage, but no effect was observed in the direct exposure F1 generation or transgenerational F3 generation at one year of age, Fig. 1a. The different mechanisms and exposures for each generation (F1 generation direct somatic exposure, F2 generation direct germline exposure and F3 generation no exposure) can generate distinct pathologies for each generation29. No gross abnormalities were observed in the corresponding epididymis at the time of dissection.

Figure 1 Male pathology analysis in F1, F2 and F3 generation control and glyphosate linage 1 year-old rats. (a) testis disease frequency, (b) prostate disease frequency, (c) male kidney disease frequency, (d) average puberty age for males, (e) average weaning weight for males, (f) male obesity frequency, (g) frequency of one disease in males, and (h) frequency of multiple disease in males. The pathology number ratio with total animal number is listed for each bar graph (a–f), or mean ± SEM (d,e), presented with asterisks indicating a statistical difference (*)p < 0.05, (**)p < 0.01, and (***)p < 0.001 in comparison with control lineage animals. Full size image

Figure 2 Female pathology analysis in F1, F2 and F3 generation control and glyphosate linage 1-yearr old rats. (a) ovary disease frequency, (b) female kidney disease frequency, (c) tumor frequency (males and females), (d) parturition abnormalities, (e) average weaning weight for females, (f) average age of puberty for females, (g) female obesity frequency, and (h) frequency of multiple disease in females. The pathology number ratio with total animal number is listed for each bar graph or mean ± SEM (e,f), presented with asterisks indicating a statistical difference (*)p < 0.05, (**)p < 0.01, and (***)p < 0.001 in comparison with control lineage animals. Full size image

Prostate disease was characterized by atrophic or hyperplastic prostate glandular epithelium, and the presence of vacuole spaces in the epithelium as previously described46 (Supplemental Figure S2). The most common histological abnormalities were epithelial cell atrophy and vacuoles, followed by hyperplasia. The prostate atrophy and regions with the presence of vacuoles are generally distinct from the regions showing hyperplasia. The frequency or incidence of prostate disease was similar for the control and glyphosate F1 and F2 generation males at one year of age, Fig. 1b. Interestingly, there was an increased frequency of prostate disease observed in the F3 generation glyphosate lineage males (p < 0.01). Therefore, one of the transgenerational pathologies (F3 generation) observed was prostate disease in approximately 30% of glyphosate lineage males, a three-fold increase in disease rate over controls, Fig. 1b.

Kidney disease was characterized by the presence of an increased number of proteinaceous fluid filled cysts, reduction in size of glomeruli, and thickening of Bowman’s capsules, as previously described37,47 (Supplemental Figure S2). The most common histological abnormalities were cysts, presumably derived from dilated tubules, and thickened Bowman capsules, followed by reduced glomerular areas. There was an increase in kidney disease frequency in the glyphosate lineage males in the F2 generation, but not F1 or F3 generations, Fig. 1c. The frequency of kidney disease was found to be similar for both the F1 and F2 generation between the control and the glyphosate lineage females. There was an increased incidence of kidney disease observed in the F3 generation glyphosate lineage females affecting nearly 40% of females (a four-fold increase in disease rate) compared to the F3 generation control females, Fig. 2b.

Ovarian disease was characterized by the development of polycystic ovaries with an increase in the number of small and large cysts showing negligible granulosa cells, as previously described48 (Supplemental Figure S2). The most common histological abnormalities were small cysts followed by large cysts. In addition, follicle counts were performed to determine any changes in the primordial follicle pool size, as previously described48,49. The frequency of ovarian disease was not significantly different between control and glyphosate lineages in the F1 generation. However, there was a significant increase in ovarian disease observed in the F2 and F3 generation glyphosate lineage females when compared to the control lineage, Fig. 2a.

Tumor development was also monitored in males and females, and found to increase in the F2 generation glyphosate female lineage, but not the F1 or F3 generation glyphosate lineages, Fig. 2c. The most predominant tumors to develop in the male and female were mammary adenomas, as previously described37,41. One mammary fibrosarcoma, one lymphoma, one trichoepithelioma and one aural fibrosarcoma were also identified. Tumor histopathology analysis was performed by WADDL.

Pubertal analysis revealed delayed pubertal onset in males in the F1 and F2 generation glyphosate lineage, but no effects in the F3 generation, Fig. 1d. Female pubertal onset was delayed in the F2 generation glyphosate lineage, and no effects were observed in the F1 or F3 generations, Fig. 2f.

Analysis of potential direct fetal exposure toxicity effects of glyphosate in the F1 generation and subsequent F2 and F3 generations included evaluation of litter sizes, sex ratios and weaning body weights of pups. There was no effect on litter size or sex ratio observed for any generation, Supplemental Figure S3. There was significantly lower weaning body weight observed for the F1 generation in the glyphosate lineage for both males (p < 0.01), Fig. 1e, and females (p < 0.01), Fig. 2e. In the F3 generation, there was no statistical difference in weaning weights for females, but an increase in males between the control and glyphosate lineages (Figs 1e and 2e).

A parturition (birth) abnormality was observed, and involved either the death of the late stage gestating mother or her pups immediately after or during birth. This phenotype was not observed in the F0 generation breeding to produce the F1 generation in either the glyphosate or control lineages. In F1 breeding to produce F2 generation offspring there was one instance in the control population where a parturition abnormality was observed, and no such instance occurred in that generation in the glyphosate lineage, Fig. 2d. However, during the gestation of F2 generation mothers with the F3 generation fetuses, dramatic parturition abnormalities were observed in the glyphosate lineage. The frequency of unsuccessful parturition was 35% (p < 0.03). Out of the 7 cases that were classified as unsuccessful pregnancies, Fig. 2d, there were 5 maternal mortalities observed. Necropsy of these animals by WADDL diagnosed 2 cases of dystocia, 1 case of severe rhinitis, 1 case of adrenal gland necrosis, and 1 case in which the cause was unknown. To further investigate the parturition abnormalities an outcross of F3 generation glyphosate lineage males with a wildtype female was performed. There were parturition abnormalities observed with a frequency of 30% (p < 0.04), Fig. 2d. In the paternal outcross generation, there were 3 cases of maternal mortality. The causes of maternal death confirmed by WADDL were 2 cases in which the cause of death was identified as dystocia, and 1 case of hyperplasia-mastitis. In order to quantify the rates of initial successful pregnancies, fertility rates of the females were compared between the control and the glyphosate lineages. Fertility rate was defined as the number of pregnancies divided by the number of breedings. Results showed no significant difference in the comparison of glyphosate and control lineage fertility rates in any generation, Supplemental Figure S3a.

The weight, body mass index (BMI), abdominal adiposity, and adipocyte cell size were analyzed in order to assess the frequency of obesity in glyphosate and control lineage males and females, as described in the Methods45 (Supplemental Figure S2). Analysis of potential obese phenotypes in the F1, F2, and F3 generation glyphosate and control lineages identified a significant increase in the obese phenotype of the F2 and F3 glyphosate lineage males and females, Figs 1f and 2g. The frequency of obesity was not found to be different between the control versus glyphosate lineage F1 generation males and females. Therefore, a transgenerational (F3 generation) obese phenotype was observed in approximately 40% of the glyphosate lineage females and 42% of the glyphosate lineage males, Figs 1f and 2g.

Direct exposure studies to glyphosate have been shown to induce behavioral abnormalities in the exposed F0 generation50,51,52,53. Behavioral analysis of the glyphosate and control lineage transgenerational F3 generation at 11 months of age was done. Both a light and dark box (LDB) and elevated plus maze (EPM) were used to assess potential anxiety behavior50. The F3 generation glyphosate lineage males and females had fewer total light side attempts and fewer total attempts compared to the controls in the light and dark box, Supplemental Figure S4a–e. No changes in other light and dark box parameters were observed. For the elevated plus maze with an open and closed arm results indicate that there was no behavioral difference observed (p > 0.05) for the control or glyphosate (open arm time or closed arm time per total time ratio) lineage F3 generation females or males, Supplemental Figure S4g and i. None of the other parameters of the EPM analysis were found to be altered, Supplemental Figure S4f–j. Although there was a reduced number of light and total attempts in the LDB by the glyphosate lineage F3 generation males, none of the other LDB or EPM parameters supported a behavioral effect, Supplemental Figure S4. Therefore, no major behavioral effects were observed in the F3 generation glyphosate lineage males or females.

The incidence of disease and abnormalities in all F1, F2 and F3 generation control and glyphosate lineage males and females is presented in Figs 1 and 2 and Supplemental Tables S1–S3 (a–d). The specific diseases associated with each individual animal are shown in Tables S1 (a–d), S2 (a–d) and S3 (a–d). This information was used for the analysis of one (≥1) disease and multiple (≥2) disease incidence, Figs 1 and 2. The frequency of one (≥1) disease in F1, F2 or F3 generation glyphosate lineage females was not statistically different from control lineage animals. In males, the frequency rate of one (≥1) male disease did not differ from the controls in the F1 generation, but increased significantly in the F2 and F3 generations, Fig. 1g. The frequency of multiple diseases (≥2) for females was not significant for the F1 generation, but the frequency increased for glyphosate lineage females in the F2 generation (p < 0.01) and F3 generation (p < 0.01) (Fig. 2h). Over 40% of the F3 generation glyphosate lineage females (2-fold increase) developed disease and abnormalities when compared to the controls. The frequency of multiple disease in the F1 and F3 generation males was not statistically different from controls, but an increase in multiple disease frequency was observed in the F2 generation males (p < 0.01) (Fig. 1h). Therefore, the F3 generation glyphosate lineage females had a significant increase in multiple diseases, suggesting a transgenerational increase in disease susceptibility.

Sperm Epigenetic Analysis

Glyphosate induced transgenerational inheritance of disease and pathology requires the germline (sperm or egg) transmission of epigenetic information between generations29. Therefore, sperm was collected from the control and glyphosate lineage F1, F2 and F3 generation males for epigenetic analysis. Potential differential DNA methylation regions (DMRs) in the sperm were identified using a comparison between the control and glyphosate lineage, as described in the Methods45. The sperm DNA was isolated, fragmented and the methylated DNA immunoprecipitated (MeDIP) with a methyl-cytosine antibody. The MeDIP DNA fragments were sequenced for an MeDIP-Seq analysis as described in the Methods54. The sperm DMR numbers are presented in Fig. 3 for a variety of p-value cutoff thresholds, and p < 10−6 was selected as the threshold for all subsequent analyses. The total number of DMRs for the control versus glyphosate lineage F1 generation is 264 with 40 of them having multiple neighboring 100 bp windows, Fig. 3a. The F2 generation had 174 DMRs with 6 of them with two multiple windows detected, Fig. 3b. The transgenerational F3 generation sperm were found to have 378 total DMRs with 31 of these having multiple neighboring windows, Fig. 3c. Therefore, the glyphosate lineage sperm were found to have altered DNA methylation in direct exposure F1 and F2 generations, as well as the transgenerational F3 generation55. Interestingly, there was negligible overlap of the sperm DMRs between each generation, Fig. 3d. Previous studies have observed that direct exposure and transgenerational generation DMRs are distinct, apparently due to the unique mechanisms for direct exposure toxicity and transgenerational actions of environmental exposures29,55. Observations indicate glyphosate can promote germline epigenetic alterations in DNA methylation.

Figure 3 Epigenetic analysis and DMR identification. The number of DMRs found using different p-value cutoff thresholds. The All column shows all DMRs. The Multiple Window column shows the number of DMRs containing at least two significant windows. The number of DMR with each specific number of significant windows at a p-value threshold of 1e-06 is shown below each table. (a) DMR F1 Generation. (b) DMR F2 Generation. (c) DMR F3 Generation. (d) DMR overlap Venn diagram. Full size image

The chromosomal locations of the DMRs for each generation are presented in Fig. 4. Nearly all chromosomes had DMRs for the F1, F2 and F3 generations, indicated by arrowhead, along with clusters of DMRs indicated by black boxes, Fig. 4. Therefore, the DMR identified were genome-wide on all chromosomes. The genomic features of the DMRs were investigated and shown to have a low CpG density “CpG deserts”56, and be predominantly 1 kb in length, Supplemental Figure S5. Similar DMR genomic factors were observed for the F1, F2 and F3 generations. The F3 generation DMR data was used in a permutation analysis to show the number of DMRs identified (red line) is not due to random variation in the control and glyphosate data, Fig. 5a, and correlated with the false discovery rate (FDR) analysis performed. In addition, a principle component analysis (PCA), with the DMRs not whole genome, of the F3 generation DMRs showed efficient separation of the control versus glyphosate DMR data and clustering of control DMR data, Fig. 5b. Similar observations were made with the F1 and F2 generation DMR PCA analysis, Supplemental Figure S6. These data demonstrate that statistically significant DMRs are observed for the F1, F2 and F3 generations sperm.

Figure 4 Chromosomal location of DMRs at a p-value threshold of 1e-06. (a) Chromosome locations of F1 generation DMRs. (b) Chromosome location F2 generation DMRs. (c) Chromosome location F3 generation DMRs. Triangles indicate DMRs. Rectangles indicate clusters of DMRs. Full size image

Figure 5 Permutation and principle component DMR analysis (PCA). (a) The number of F3 generation DMR for all permutation analyses. The vertical red line shows the number of DMR found in the original analysis. All DMRs are defined using an edgeR p-value threshold of 1e-06. (b) DMR PCA using DMRs and not whole genome for F3 generation control and glyphosate DMR analysis with legend insert. The control (PBS), control (DMSO) and glyphosate F3 generation lineage (GF3) indicated. Full size image

The DMR associated genes were identified for DMRs within 10 kb of a gene to include gene promoters and listed in Supplemental Figures S4, S5 and S6 for the F1, F2 and F3 generation DMR lists, respectively. The majority of DMRs were not associated with genes. The genes and associated gene categories for each DMR and associated genes are provided. A summary of the DMR associated gene categories indicates transcription, signaling, metabolism, receptors, and cytokines are predominant, Fig. 6a. A summary of DMR associated gene categories are presented for the F1, F2 and F3 generation gene categories. The DMR associated gene pathways are presented in Fig. 6b. The top five KEGG (Kyoto Encyclopedia of Genes and Genomes) gene pathways for the F1, F2 and F3 generations are listed with number of DMR associated genes involved in the pathway shown in brackets. The only pathway that overlaps in all three generations is the metabolic pathway, but this pathway involves hundreds of genes and sub-pathways so is anticipated. Other common pathways between the F1 and F2 generations and F2 and F3 generations are present. Various signaling pathways are the most common pathways identified.