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Germline and reproductive tract effects intensify in male mice with successive generations of estrogenic exposure [1]

['Tegan S. Horan', 'School Of Molecular Biosciences', 'Center For Reproductive Biology', 'Washington State University', 'Pullman', 'Washington', 'United States Of America', 'Alyssa Marre', 'Terry Hassold', 'Crystal Lawson']

Date: 2023-09

The hypothesis that developmental estrogenic exposure induces a constellation of male reproductive tract abnormalities is supported by experimental and human evidence. Experimental data also suggest that some induced effects persist in descendants of exposed males. These multi- and transgenerational effects are assumed to result from epigenetic changes to the germline, but few studies have directly analyzed germ cells. Typically, studies of transgenerational effects have involved exposing one generation and monitoring effects in subsequent unexposed generations. This approach, however, has limited human relevance, since both the number and volume of estrogenic contaminants has increased steadily over time, intensifying rather than reducing or eliminating exposure. Using an outbred CD-1 mouse model, and a sensitive and quantitative marker of germline development, meiotic recombination, we tested the effect of successive generations of exposure on the testis. We targeted the germline during a narrow, perinatal window using oral exposure to the synthetic estrogen, ethinyl estradiol. A complex three generation exposure protocol allowed us to compare the effects of individual, paternal, and grandpaternal (ancestral) exposure. Our data indicate that multiple generations of exposure not only exacerbate germ cell exposure effects, but also increase the incidence and severity of reproductive tract abnormalities. Taken together, our data suggest that male sensitivity to environmental estrogens is increased by successive generations of exposure.

Developmental exposure to manmade chemicals that interfere with endogenous hormones (endocrine disrupting chemicals) has been reported to adversely affect male reproductive health, increasing the incidence of reproductive tract abnormalities and reducing sperm production. Experimental evidence suggests that some exposure effects can persist in unexposed descendant males. To date, however, studies of these transgenerational effects have failed to accurately model human exposure, which spans multiple generations and involves an increasing number and diversity of endocrine disrupting chemicals. Using a quantitative measure of exposure effects on the germline, we assessed the effects of successive generations of estrogenic exposure in mice. We found that multiple generations of exposure not only exacerbated previously reported effects on the male germline, but elicited reproductive tract defects that increased in frequency and severity. These results have important implications for human reproductive health, suggesting that multiple generations of exposure to common endocrine disrupting chemicals may increase male sensitivity to exposure.

Funding: Grant support for these studies was provided by NIH grants to PAH (R01 HD083177 Male germline development and estrogenic exposures) and TH (R37 HD21341 A program of research in population cytogenetics). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2017 Horan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

(A) F0 males (designated E) were treated from 1–12 dpp with 0.25 ng/g ethinyl estradiol and bred with unexposed females to produce F1 males that received daily oral doses of either ethinyl estradiol (EE; red) or placebo (E0; blue) from 1–12 dpp. Representative F1 males chosen at random were bred with unexposed females to generate F2 generation males that, like the F1, were either exposed (E0E and EEE; red) or placebo treated (E00 and EE0; blue). After mating, all males were killed at 12 weeks of age for reproductive tract and testis analysis (B) Summary of abbreviations and specific exposure(s) represented by each. (C) Summary of animal numbers in each treatment group.

To our knowledge, the effects of successive generations of exposure on male reproduction have not been addressed. Thus, we decided to use a sensitive, quantitative measurement of exposure, meiotic recombination, to assess the effect of exposures spanning multiple generations. We utilized an outbred mouse model and a complex three-generation scheme ( Fig 1A ) involving low-dose, neonatal exposure to the synthetic estrogen, ethinyl estradiol. Our data not only demonstrate an increase in the severity of exposure-induced effects on meiotic recombination with successive generations of exposure, but also an unexpected increase in both the incidence and severity of male reproductive tract aberrations. Taken together, our findings suggest that continued exposure spanning several generations will have cumulative effects on male reproductive health.

Documenting transgenerational effects in humans is challenging. Assessing potential transgenerational transmission of DES-induced effects will require analysis of an additional generation of descendants and, for most common environmental chemical contaminants, assessment likely will never be possible due to the nature of human exposure: Typically, humans are not exposed for only a single generation. Instead, exposures persist over time or become more diverse as new chemical variants are introduced.

Evidence that the effects of exposure may be transmitted to subsequent, unexposed generations is accumulating. Because exposure not only can directly affect the exposed individual (F0), but also his or her germline, effects evident in generations derived from this germline (the F1 in the case of male exposure, but both the F1 and F2 generations in the case of fetal exposure involving the female) are said to be multigenerational. For an effect to be considered transgenerational, it must be evident in the first unexposed generation (F2 and F3 for male and female exposures, respectively). Transgenerational effects in mammals–presumably resulting from epigenetic changes to the germline–have been reported in numerous studies (e.g., [ 18 – 25 ]). Few studies, however, have focused on germ cells [ 26 – 28 ], and the evidence supporting the persistence and transmission of specific germline alterations remains insufficient to convince some skeptics (e.g. [ 29 – 31 ]). Direct effects on the developing male germline have been induced by perinatal exposure to exogenous estrogens in mice and rats, with adverse effects reported on both gonocyte number and adult sperm production [ 32 – 36 ]. In addition, we previously demonstrated an effect on the developing spermatogonial stem cell (SSC) induced by brief postnatal exposure coinciding with the formation of the SSC lineage in male mice and evident as a reduction in meiotic recombination levels in descendant spermatocytes [ 37 ].

The most compelling evidence of an effect of developmental estrogenic exposure on human male reproductive health comes from studies of diethylstilbestrol (DES) exposed sons. From the 1940s through the 1970s, DES was prescribed to millions of pregnant women to prevent miscarriage. This treatment not only was not efficacious, but increased the incidence of a variety of reproductive disorders, including cancers in both male and female offspring (reviewed in [ 12 ]). Although DES daughters have been studied more extensively, in DES sons and in male mice exposed prenatally to DES, the incidence of cryptorchidism, underdeveloped testes, and testicular cancer is increased, and sperm count and quality is decreased [ 13 – 16 ]. Further, although the lack of information on sources, levels and timing of exposure precludes systematic studies of other developmental estrogenic exposures in humans, epidemiological studies suggest etiological links between environmental exposures and changes in spermatogenesis and the incidence of testicular germ cell cancers of fetal origin (reviewed in [ 7 , 17 ]).

Data from human populations around the world provide evidence of a marked decline in male fertility during the past several decades. For example, a comprehensive analysis in 2000 of data from more than 100 studies in Western countries provided evidence of a decline in human spermatogenesis during the preceding 50 years [ 1 ]. More recent longitudinal cross-sectional studies suggest reductions in both sperm count and quality among young men (ages 18–37) in China (2001–2015;[ 2 ]), Spain (2001–2011;[ 3 ]), France (1989–2005;[ 4 ]), Denmark (1996–2010;[ 5 ]), and Finland (1998–2006; [ 6 ]). Changes in sperm production have coincided with increases in the incidence of other reproductive defects, including hypospadias, cryptorchidism, and testicular germ cell cancers (reviewed in [ 7 ]), and the combined spectrum of reproductive effects has been termed testicular dysgenesis syndrome (TDS; [ 8 ]). The observed changes correspond to the rapid introduction of manmade chemicals in the postwar era, and were originally hypothesized to result from exposure to maternally- or environmentally-derived estrogens [ 9 ]. Subsequent experimental data, however, have provided evidence that male reproductive abnormalities can be induced by developmental exposure to different types of endocrine disrupting chemicals (EDCs; reviewed in [ 10 , 11 ]). Given the rapid increase in the variety and ubiquity of EDCs in our environment and the adverse reproductive effects ascribed to some of these chemicals, the implications for humans are significant.

Results

We recently reported that neonatal estrogenic exposure induces permanent meiotic effects in adult outbred CD-1 and inbred C3H, but not C57BL/6J male mice [37]. Germ cell transplantation experiments demonstrated that the meiotic phenotype was due to alterations in the spermatogonial stem cells (SSCs) of the testis, a lineage thought to be determined during the window of exposure used in the study [38–40]. The SSC is many cell divisions upstream of meiotic entry; thus, rather than affecting the meiotic DNA double strand break (DSB) repair process per se, it is likely that changes induced in the SSC population altered the recombination set point. Consistent with this, we found no difference in DSB formation or synaptonemal complex length in exposed and control males [37]. Studies to determine how exposure alters the SSC epigenome are in progress, and ultimately will provide important insight to recombination control in male mammals. In the interim, because recombination provides a quantitative measure of an exposure effect on the germline, it provides a direct means of tracing effects through generations to determine if they are multi- or transgenerational.

Our previous studies demonstrated that exposure to either bisphenol A (BPA) or ethinyl estradiol from 1–12 days postpartum (dpp) significantly reduced meiotic recombination (as assessed by the number of foci of the DNA mismatch repair protein, MLH1 in pachytene spermatocytes) in adult males. Daily oral doses of 0.25 ng/g ethinyl estradiol (roughly equivalent to a daily oral contraceptive dose) exerted the strongest effect, causing a 5% reduction in MLH1 values in inbred C3H males [37]. Although this difference appears subtle, the direct biological consequence is the elimination of spermatocytes. Cells with one or more pairs of homologous chromosomes that fail to form a crossover site will not yield sperm, because the presence of unpaired chromosomes at the first meiotic division triggers checkpoint-induced spermatocyte elimination [41,42].

We were interested not only in analyzing second- and third-generation descendants of exposed males for the transgenerational persistence of meiotic effects, but also in assessing the effects of successive generations of exposure. Accordingly, we developed the three-generation exposure protocol outlined in Fig 1 and S1 Fig, and conducted all analyses on 12-week-old adult males. To track both generational and individual exposure history, F0 founder exposed males were designated as ‘E’, and ‘E’s and ‘0’s used to designate exposure or placebo treatment, respectively in subsequent generations (Fig 1B and 1C). For example, EE males represent F1 generation exposed sons with two generations of exposure; E00 males, F2 grandsons two generations removed from the founder exposure; and EEE males, F2 grandsons with three successive generations of exposure. In this paradigm, E0 and E00 males serve as important negative controls for EE and EEE exposure groups.

To eliminate genetic variability, we initiated our three-generation studies using inbred C3H males; however, four of the nine founder males proved infertile with orchitis. When we attempted the study using inbred 129 males orchitis was not observed, but only one of four exposed males proved fertile. We next turned to outbred CD-1 males.

Although the use of outbred animals introduces genetic variability, our previous studies demonstrated meiotic effects in neonatally exposed CD-1 males (i.e., an average decrease in adult males of 1.3 MLH1 foci for BPA and 2.5 for ethinyl estradiol) and suggested that exposed CD-1 males are fertile [37]. The highly significant difference between ethinyl estradiol and placebo exposed males suggested that, despite genetic variation, we would be able to discern generational differences using CD-1 males, thus, we conducted our studies on this outbred background.

Meiotic effects worsen with successive generations of exposure In addition to eliciting more severe reproductive tract aberrations, multiple generations of estrogenic exposure exacerbated the meiotic recombination phenotype that was the original focus of our analysis. As in our previous studies [37], we analyzed recombination in pachytene stage spermatocytes by counting MLH1 foci in preparations immunostained with antibodies to both SYCP3 (a component of the synaptonemal complex or SC) and MLH1, a mismatch repair protein that localizes to the majority of meiotic crossovers [43]. In our previous studies, the MLH1 mean for placebo treated males was 24.6 ± 0.3 and both BPA and EE exposure induced a significant decrease (i.e., 1–2.5 foci, depending upon the exposure) [37]. Thus, the means of F0 founder males (23.7 ± 0.3, 22.7 ± 0.4, and 22.1 ± 0.3 for family 1, 2, and 3, respectively) fell within the expected range for exposed males. To compare recombination levels across generations and among different categories of F1 and F2 males, mean MLH1 counts were derived by pooling cells from males of the same generation and exposure category. To assess the effect of successive generations of exposure, we used one-way ANOVA to compare mean MLH1 counts in exposed F0 males with those in F1 and F2 males exposed for two or three generations (Fig 4A; F = 29.4, p < 0.0001). Significant differences between groups were determined by a Tukey-Kramer post-hoc test. By comparison with F0 founders (22.8 ± 0.2) we found a small but nonsignificant decrease in mean MLH1 counts in EE sons (22.7 ± 0.1), but a significant reduction in EEE grandsons (21.8 ± 0.1; p < 0.05). In addition, MLH1 means were lower in F1 EE (22.7 ± 0.1) than E0 sons (23.1 ± 0.1, p < 0.05). Similarly, the mean was significantly lower in EEE by comparison with E00 F2 males (21.8 ± 0.1 and 22.8 ± 0.1, respectively; p < 0.05). PPT PowerPoint slide

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TIFF original image Download: Fig 4. Meiotic recombination levels decrease with successive generations of exposure. (A) Pooled data from exposed families. X-axis represents MLH1 mean of 3 F0 founder males (25–30 pachytene cells/male) and bars show mean ± SEM for E0 and EE F1 sons and E00 and EEE F2 grandsons. Bar color denotes individual exposure (red for exposed, blue for placebo) with increased intensity for successive generations of exposure or placebo. Each group represents data from 25–30 pachytene stage cells/male for 22–23 males. (B-D) Individual data for families 1, 2, and 3 (B, C, and D, respectively); each group represents 4–12 males. Groups were compared by one-way ANOVA; single asterisk denotes significant difference by comparison with founder and double asterisk denotes significance between indicated groups as determined by Tukey-Kramer post-hoc test (p < 0.05). https://doi.org/10.1371/journal.pgen.1006885.g004 Because family 1 exhibited the strongest effect, we assessed each family individually to determine if trends were consistent across families (Fig 4B–4D). In family 1, MLH1 means were significantly lower in both F1 EE sons (21.8 ± 0.1) and F2 EEE grandsons (22.0 ± 0.2) by comparison with the founder mean (23.7 ± 0.3; p < 0.05; Fig 4B). For families 2 and 3, reductions were evident in F2 EEE males, but the differences were not statistically significant (Fig 4C and 4D). Thus, all families exhibited the same trend; differences among them prompted us to consider a paternal effect on recombination.

Recombination exhibits a strong paternal effect As observed for vas deferens abnormalities, the recombination phenotype of offspring appeared to be influenced by paternal phenotype. Specifically, the extent to which the phenotype worsened with successive generations of exposure not only varied among families, but also among the offspring of males within a family, with a more pronounced effect in sons of males with higher mean MLH1 counts. For example, the founder of family 1 had the highest mean MLH1 level (23.7 ± 0.3), and his seven F1 sons (EE) all had lower mean values (ranging from 21.0 ± 0.3 to 23.4 ± 0.3; Fig 4B, S3 Fig). In contrast, in the other two families where founder MLH1 means were lower (22.7 ± 0.4, and 22.1 ± 0.3 for family 2 and 3, respectively), means in F1 EE sons (23.2 ± 0.1 and 22.8 ± 0.2, respectively) were not significantly different from the F0 founder mean (Fig 4C and 4D). A comparison of the F2 sons of F1 EE fathers provided further evidence of this paternal effect. For example, the two F1 EE males in family 2 that were mated to produce F2 EEE males had very different MLH1 means (25.1 ± 0.4 and 22.6 ± 0.3). Although the five F2 EEE offspring of each male had lower mean MLH1 counts than their fathers (23.0 ± 0.2, t = 4.6, p < 0.0001, and 21.4 ± 0.2, t = 3.1, p < 0.01 respectively; Fig 5), the means and ranges of the two groups of males were remarkably different. Importantly, the magnitude of the reduction was greater in F2 sons of the F1 male with the high MLH1 count. Similar paternal effects were observed among the offspring in all three families (S3 Fig); however, the impact of the paternal phenotype on the response to exposure was most pronounced in family 3, where the MLH1 mean of one F1 male was particularly low (20.7 ± 0.2). The mean for the F2 EEE sons of this male (20.1 ± 0.2) did not differ significantly from the F1 EE father, making this the only group of F2 EEE males that did not demonstrate an additional reduction in recombination levels by comparison with their father (Fig 5). PPT PowerPoint slide

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TIFF original image Download: Fig 5. Paternal phenotype affects meiotic recombination levels. Mean MLH1 ± SEM for F1 EE fathers (open circle) and their F2 EEE sons (closed circles). Each point represents the MLH1 mean ± SEM for 25–30 pachytene cells from a single male. Left and center panels show offspring data from family 2 for two F1 EE fathers with different means: 25.1 ± 0.4 (high paternal MLH1), and 22.6 ± 0.3 (low paternal MLH1). Right panel shows offspring data for EE father with a very low mean, 20.7 ± 0.2, from family 3. Fathers and offspring were compared by one-tailed t-test; for high paternal MLH1, p < 0.0001; for low paternal MLH1 p < 0.01. https://doi.org/10.1371/journal.pgen.1006885.g005

MLH1 null SCs increase in frequency with successive generations of exposure The variability induced by the use of outbred males, coupled with the male reproductive tract abnormalities we encountered, confounded the use of standard measurements of impaired male fertility. Thus, we elected to directly measure meiotic impairment by scoring cells with lethal defects, i.e., the frequency of pachytene stage cells containing one or more SCs lacking an MLH1 focus (MLH1 null SCs). As expected, exposure-induced reductions in meiotic recombination resulted in an increase in MLH1 null SCs (Fig 6A). A comparison of males exposed each generation (i.e. E, EE, and EEE) showed a significant increase in the incidence of these cells over three successive generations. Specifically, MLH1 null SCs were observed in 10.8% (9/83) of cells from F0 founder males, 14.2% (91/643) of cells from F1 EE sons, and 34.9% (248/710) of cells from F2 EEE grandsons (Χ2 = 87.9, p < 0.0001; Fig 6B). Although the difference in levels of SCs that fail to form an exchange between founders and EE sons was not significant, levels in EEE grandsons were significantly higher by comparison with both founders (Χ2 = 18.6, p < 0.0001) and EE fathers (Χ2 = 76.5, p < 0.0001). This trend held among father-son comparisons within individual families (S4 Fig). PPT PowerPoint slide

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TIFF original image Download: Fig 6. Cells with SCs lacking an MLH1 focus increase in frequency with successive generations of exposure. (A) Example of a pachytene spermatocyte immunostained with antibodies to SYCP3 (red) and MLH1 (green), and showing an SC lacking an MLH1 focus (white arrowhead). (B) Frequency of MLH1 null SCs in 3 founder males, 28 E0 and 24 EE F1 sons, and 32 E00 and 25 EEE F2 grandsons (25–30 cells analyzed per male). https://doi.org/10.1371/journal.pgen.1006885.g006 The MLH1 null phenotype also provided a means of assessing the transgenerational persistence of meiotic effects. A comparison of levels in E, E0, and E00 males not only did not reveal a decrease in the frequency of cells with MLH1 negative SCs levels in subsequent unexposed generations but provided evidence of a slight increase across generations (Χ2 = 11.2, p < 0.01; Fig 6B). However, the effect was only statistically significant in the pooled data and a definitive trend was not observed across all families (S4 Fig). Thus, although these data are consistent with transgenerational persistence of the phenotype, clearly additional analyses are warranted.

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[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1006885

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