Oocyte Development and Quality in Young and Old Mice following Exposure to Atrazine

Abstract

Background: Egg development has unique features that render it vulnerable to environmental perturbation. The herbicide atrazine is an endocrine disruptor shown to have detrimental effects on reproduction across several vertebrate species.

Objectives: This study was designed to determine whether exposure to low levels of atrazine impairs meiosis in female mammals, using a mouse model; in particular, the study's researchers sought to determine whether and how the fidelity of oocyte chromosome segregation may be affected and whether aging-related aneuploidy is exacerbated.

Methods: Female C57BL/6J mice were exposed to two levels of atrazine in drinking water: The higher level equaled aqueous saturation, and the lower level corresponded to detected environmental contamination. To model developmental exposure, atrazine was ingested by pregnant females at 0.5 d post coitum and continued until pups were weaned at 21 d postpartum. For adult exposure, 2-month-old females ingested atrazine for 3 months. Following exposure, various indicators of oocyte development and quality were determined, including: a) chromosome synapsis and crossing over in fetal oocytes using immunofluorescence staining of prophase-I chromosome preparations; b) sizes of follicle pools in sectioned ovaries; c) efficiencies of in vitro fertilization and early embryogenesis; d) chromosome alignment and segregation in cultured oocytes; e) chromosomal errors in metaphase-I and -II (MI and MII) preparations; and f) sister-chromatid cohesion via immunofluorescence intensity of cohesin subunit REC8 on MI-chromosome preparations, and measurement of interkinetochore distances in MII preparations.

Results: Mice exposed to atrazine during development showed slightly higher levels of defects in chromosome synapsis, but sizes of initial follicle pools were indistinguishable from controls. However, although more eggs were ovulated, oocyte quality was lower. At the chromosome level, frequencies of spindle misalignment and numerical and structural abnormalities were greater at both meiotic divisions. In vitro fertilization was less efficient, and there were more apoptotic cells in blastocysts derived from eggs of atrazine-exposed females. Similar levels of chromosomal defects were seen in oocytes following both developmental and adult exposure regimens, suggesting quiescent primordial follicles may be a consequential target of atrazine. An important finding was that defects were observed long after exposure was terminated. Moreover, chromosomally abnormal eggs were very frequent in older mice, implying that atrazine exposure during development exacerbates effects of maternal aging on oocyte quality. Indeed, analogous to the effects of maternal age, weaker cohesion between sister chromatids was observed in oocytes from atrazine-exposed animals.

Conclusion: Low-level atrazine exposure caused persistent changes to the female mammalian germline in mice, with potential consequences for reproductive lifespan and congenital disease. https://doi.org/10.1289/EHP11343.

Figure 1.

Analysis of meiotic prophase I, ovarian reserves, and MOFs following atrazine exposure during development. (A) Schematic of developmental atrazine exposure regimen. (B) Representative images of prophase-I oocyte chromosome preparations from E18.5 embryos immunostained for SYCP3 (gray), to label homolog axes, and CREST (magenta), to label centromeres. Examples of pachytene-stage nuclei with normal synapsis, nonhomologous pairing, broken axes, and asynapsis are shown. Yellow arrows indicate synaptic defects. Scale bars represent $10\mathrm{\mu }\mathrm{m}$. (C) Quantification of synaptic defects across litters (numbers of animals and litters used to generate data: control, 6 animals and $4\text{\hspace{0.17em}litters}$; low dose, 6 and 4; high dose 9 and 5; also see Excel Table S1). (D) Representative pachytene-stage oocyte chromosomes immunostained for SYCP3 (gray), CREST (magenta) and the crossover marker MLH1 (green). A single synapsed chromosome pair is magnified in the bottom panels. Scale bars represent $10\mathrm{\mu }\mathrm{m}$ (main panels) and $1\mathrm{\mu }\mathrm{m}$ (magnified panels). (E) Quantification of MLH1 foci per nucleus (also see Excel Table S2). (F) Distributions of MLH1 focus numbers per chromosome (also see Excel Table S3). Percentages of chromosomes with 0, 1, and $\ge 2$ MLH1 foci are plotted for the three exposure groups [2,860, 2,740, and 2,560 chromosomes in control, low, and high groups, respectively; numbers of animals and litters used to generate the data in (E) and (F): control, 10 and 4; low, 8 and 4; high 6 and 3]. (G) Total oocyte counts per ovary from 18 dpp females (numbers of animals and litters used to generate the data: control, 7 and 6; high 6 and 2. Both ovaries from individual animals were analyzed; also see Excel Table S4). (H) Representative ovary section from an 18 dpp female exposed to a high dose of atrazine, immunostained for p63 to mark oocyte nuclei and counterstained with hematoxylin. The white caret indicates an MOF containing two oocytes that is magnified in the right panel. Scale bars represent $200\mathrm{\mu }\mathrm{m}$ (left panel) and $100\mathrm{\mu }\mathrm{m}$ (right panel). (I) Numbers of MOFs per ovary (numbers of animals and litters used to generate the data: control, 4 and 2; high 6 and 2. Both ovaries from individual animals were analyzed; also see Excel Table S5). Bidirectional error bars in (C), (E), (G), and (I) represent $\text{mean}\pm \text{standard\hspace{0.17em}deviation}$; unidirectional error bars in (F) represent standard error of a proportion. Data were analyzed with ordinary one-way ANOVA Dunnett’s multiple comparisons tests (C) and (E), unpaired t test (G) and Mann-Whitney test (I). The chi-square test was applied in (F) to compare distributions of MLH1 focus numbers. Low, $100\mathrm{\mu }\mathrm{g}/\mathrm{L}$; High, $33\mathrm{mg}/\mathrm{L}$. Note: ANOVA, analysis of variance; dpp, days postpartum; E0.5, embryonic day 0.5; MOF, multi-oocyte follicle; ns, not significant. *$p<0.05$; **$p<0.01$.

Figure 2.

In vitro fertilization and preimplantation embryo development following atrazine exposure during development. (A) Numbers of MII eggs collected from superovulated females (also see Excel Table S6). (B) Fractions of MII eggs that were fragmented [numbers of fragmented and unfragmented eggs provided in Excel Table S7; representative image is shown below; numbers of animals and litters used to generate data in (A) and (B): control, 7 animals and 4 litters; low dose, 7 and 3; high dose, 7 and 3]. (C) Efficiencies of in vitro fertilization (2-cell embryos) and preimplantation embryo development (actual numbers of embryos in each stage of development are provided in Excel Table S8. Representative images are shown below. Efficiencies of 4-cell embryo and blastocyst formation are expressed as percentages of 2-cell embryos; number of animals and litters to generate data for 2-cell embryos are 5 and 3, 6 and 3, 5 and 2 in each exposure group; number of animals and litters to generate data for 4-cell embryos and blastocysts are 3 and 2 in each exposure group). (D) Representative images of single z-sections of blastocysts derived from in vitro fertilization, stained for DNA (Hoechst; blue) and TUNEL (green). (E) Total cell numbers per blastocyst (also see Excel Table S9). (F) Percentage of blastocyst cells that were TUNEL positive. Numbers of animals and litters used to generate data in (E) and (F) were 3 and 2, 2 and 2, and 3 and 2 in control, low, and high exposure groups, respectively; also see Excel Table S10. Numbers of eggs (B) or embryos (C) analyzed are indicated in parentheses above the bars. Bidirectional error bars represent $\text{mean}\pm \text{standard\hspace{0.17em}deviation}$ (A,E,F) or standard error of a proportion (B,C). Data in A, E, and F were analyzed with one-way ANOVA and Dunnett’s tests; B and C were analyzed with Fisher’s exact tests. Scale bars in B–D represent $50\mathrm{\mu }\mathrm{m}$. Low, $100\mathrm{\mu }\mathrm{g}/\mathrm{L}$ atrazine; High, $33\mathrm{mg}/\mathrm{L}$. Note: ANOVA, analysis of variance; MII, metaphase II; ns, not significant; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. *$p<0.05$; **$p<0.01$.

Figure 3.

Chromosome alignment and segregation in meiosis-I oocytes following atrazine exposure during development. (A) Representative images of MI oocytes, stained for spindles ($\mathrm{\alpha }-\text{tubulin}$, green) and DNA (Hoechst, white), illustrating classes of chromosome misalignment. (B) Quantification of chromosome misalignment in the MI oocytes represented in panel A (data generated from 5 animals from $3\text{\hspace{0.17em}litters}$ in each exposure group; see Excel Table S11). (C) Live-cell images from selected timepoints of meiosis-I stage oocytes showing examples of normal chromosome segregation, NDJ, and lagging segregation. Arrows highlight a NDJ event. The arrowhead highlights lagging chromosomes. (D) Quantification of chromosome missegregation in meiosis-I oocytes. Data generated from 7 animals and $5\text{\hspace{0.17em}litters}$ (unexposed control); and 8 animals and $4\text{\hspace{0.17em}litters}$ (high-dose atrazine), respectively (also see Excel Table S12 for summary data). Numbers of oocytes examined in (B) and (D) are indicated in parentheses above the bars. Error bars represent standard error of a proportion; unidirectional bars are shown for the individual classes to avoid overlaps. Data in (B) and (D) were analyzed with Fisher’s exact tests. Statistical analysis performed in (B) compares the distributions of the three alignment classes shown in (A). All missegregation types were combined for the statistical analysis in (D). Scale bars in (A) and (C) represent $10\mathrm{\mu }\mathrm{m}$. Low, $100\mathrm{\mu }\mathrm{g}/\mathrm{L}$ atrazine; High, $33\mathrm{mg}/\mathrm{L}$. Note: MI, metaphase I; NDJ, nondisjunction. *$p<0.05$; **$p<0.01$.

Figure 4.

Analysis of chromosomal abnormalities and alignment in metaphase-II eggs following atrazine exposure during development. (A) Representative images of MII oocyte chromosomes showing normal euploid (20 pairs of sister chromatids) and aneuploid (21 pairs) nuclei, and a cell with a single free chromatid (20’; magnified in the right-hand side panels) indicative of a premature separation event (predivision). Centromeres (green) were immunostained with CREST, and chromosomes (magenta) were counterstained with DAPI. Scale bars represent $10\mathrm{\mu }\mathrm{m}$ (main panels) and $1\mathrm{\mu }\mathrm{m}$ (magnified panel). (B) Quantification of chromosomal abnormalities in MII eggs from 3-month-old mice. Numbers of animals used were 8 (from $7\text{\hspace{0.17em}litters}$; unexposed control), 4 (from $3\text{\hspace{0.17em}litters}$; low dose) and 5 (from $3\text{\hspace{0.17em}litters}$; high dose), respectively (also see Excel Table S13 for the summary data). (C) Representative images of MII eggs, stained for spindles ($\mathrm{\alpha }-\text{tubulin}$, green) and DNA (Hoechst, white), illustrating classes of chromosome misalignment. PB1, indicates the position of the first polar body. Scale bars represent $10\mathrm{\mu }\mathrm{m}$ (top panels) and $5\mathrm{\mu }\mathrm{m}$ (lower panels). (D) Quantification of chromosome misalignment in the metaphase-II eggs represented in (C). 3 animals were used for each exposure group, from 3 (control), 3 (low), and 2 (high) litters, respectively (also see Excel Table S14 for summary data). Numbers of eggs examined in (B) and (D) are indicated in parentheses above the bars. Error bars represent standard error of a proportion; unidirectional bars are shown for the individual classes to avoid overlaps. Data in (B) and (D) were analyzed with Fisher’s exact tests. All missegregation types were combined for the statistical analysis in (B). Statistical analysis performed in (D) compares the distributions of the three alignment classes shown in (C). Low, $100\mathrm{\mu }\mathrm{g}/\mathrm{L}$ atrazine; High, $33\mathrm{mg}/\mathrm{L}$. Note: MII, metaphase II; NDJ, nondisjunction. *$p<0.05$; **$p<0.01$.

Figure 5.

Analysis of chromosomal abnormalities and sister-chromatid cohesion in oocytes of aged mice following atrazine exposure during development. (A) Quantification of chromosomal abnormalities in MII eggs from 15-month-old mice. Numbers of animals used were 8 (from $4\text{\hspace{0.17em}litters}$; unexposed control), 6 (from $2\text{\hspace{0.17em}litters}$; low dose), and 5 (from $2\text{\hspace{0.17em}litters}$; high dose), respectively (also see Excel Table S15 for summary data). (B) Representative image of MII chromosomes illustrating the measurement of IKD for a single chromatid pair (magnified panels). Kinetochores (green) were immunostained with CREST, and chromosomes (magenta) were counterstained with DAPI. Scale bars represent $10\mathrm{\mu }\mathrm{m}$ (main panel) and $2\mathrm{\mu }\mathrm{m}$ (magnified panels). (C) Average IKDs per nucleus for MII eggs from 15-month-old mice (also see Excel Table S16). (D) Rank distributions of IKDs for individual sister-chromatid pairs of MII eggs from 15-month-old mice. Numbers of sister-chromatid pairs examined in each group were 546 (28 cells, unexposed control), 422 (22 cells, low dose), and 575 (30 cells, high dose), respectively. Numbers of animals used in (C) and (D) were 8 (from $4\text{\hspace{0.17em}litters}$), 6 (from $2\text{\hspace{0.17em}litters}$), and 5 (from $2\text{\hspace{0.17em}litters}$) in control, low-, and high-dose groups, respectively; also see Excel Table S17. (E) Representative image of MI oocyte chromosomes, immunostained for meiosis-specific cohesin component REC8 (green), centromere marker CREST (magenta) and counterstained with DAPI (blue). The right-hand side panels show a single homolog pair; the dashed line highlights the area in which fluorescent intensity was measured to quantify chromosome-associated REC8 (details in the “Materials and Methods” section). Scale bars represent $10\mathrm{\mu }\mathrm{m}$ (main panel) and $2\mathrm{\mu }\mathrm{m}$ (magnified panel). (F) Quantification of average chromosomal REC8 level per MI oocyte nucleus from 3-month-old mice. Numbers of animals used were 4 (from $3\text{\hspace{0.17em}litters}$; unexposed control), 4 (from $3\text{\hspace{0.17em}litters}$; low dose), and 5 (from $3\text{\hspace{0.17em}litters}$; high dose), respectively; also see Excel Table S18. Numbers of eggs examined in (A) are indicated in parentheses above the bars; error bars in (A) show standard error of a proportion; unidirectional bars are shown to avoid overlaps. Bidirectional error bars in (C) and (F) indicate $\text{mean}\pm \text{standard\hspace{0.17em}deviation}$. Data were analyzed with Fisher’s exact tests (A), and one-way ANOVA and Dunnett’s tests (C,D,F), respectively. MII chromosomal abnormalities were combined for statistical analysis in (A). Low, $100\mathrm{\mu }\mathrm{g}/\mathrm{L}$ atrazine; High, $33\mathrm{mg}/\mathrm{L}$. Note: ANOVA, analysis of variance; IKD, interkinetochore distance; MI, metaphase I; MII, metaphase II; NDJ, nondisjunction. *$p<0.05$; **$p<0.01$; ***$p<0.001$; ****$p<0.0001$.

Figure 6.

Chromosomal abnormalities and alignment in oocytes following atrazine exposure during adulthood. (A) Schematic of atrazine exposure regimen in adult females. (B) Representative images of MI oocyte chromosome preparations showing a normal nucleus with 20 bivalents and a nucleus containing 19 bivalents and 2 univalents (yellow arrows). Chromosomes shown in insets are from the same cell but were in different fields of view. DNA is colored magenta, and centromeres are green. Scale bars represent $10\mathrm{\mu }\mathrm{m}$. (C) Quantification of unconnected univalent chromosomes in chromosome preparations from MI oocytes. Numbers of animals used were 6 (unexposed control), 5 (low dose), and 5 (high dose), respectively (also see Excel Table S19 for summary data). (D) Quantification of chromosome misalignment in metaphase-I oocytes. Numbers of animals used were 4, 3, and 4, respectively (also see Excel Table S20 for summary data). (E) Quantification of chromosomal abnormalities in MII eggs. Numbers of animals used were 7, 6, and 6, respectively (also see Excel Table S21 for summary data). (F) Quantification of chromosome misalignment in metaphase-II eggs. Numbers of animals used were 4, 3, and 4, respectively (also see Excel Table S22 for summary data). (G) Schematic of the discontinuous atrazine exposure regimen. (H) Quantification of chromosomal abnormalities in MII eggs following discontinuous atrazine exposure. Numbers of animals used were 9, 4, and 8, respectively (also see Excel Table S23 for summary data). Numbers of MI oocytes (C,D) and MII eggs (E,F,H) examined are indicated in parentheses above the bars. Error bars represent standard error of a proportion; unidirectional bars are shown in (D–F), and (H) to avoid overlaps. Data were analyzed with Fisher’s exact tests. Statistical analysis performed in (D) and (F) compares the distributions of the three alignment classes. MII chromosomal abnormalities were combined for statistical analyses in (E) and (H). Low, $100\mathrm{\mu }\mathrm{g}/\mathrm{L}$ atrazine; High, $33\mathrm{mg}/\mathrm{L}$. Note: MI, metaphase I; MII, metaphase II; ns, not significant. *$p<0.05$; **$p<0.01$.

Figure 7.

Summary of the long-term effects of atrazine exposure during development on oocyte quality suggested by this study. First row: timeline of mouse development and window of atrazine exposure (developmental exposure regimen) and ages at which parameters of oocyte quality were assessed. Second row: timeline of oocyte development. The events of meiotic prophase-I occur in fetal oocytes, which then arrest around birth and assemble into primordial follicles. With each estrous cycle, cohorts of follicles grow, and meiosis resumes in dominant follicles that are about to be ovulated. The meiosis-I division ensues, and eggs then arrest at MII until fertilization triggers division. Third row: The chromosomal events of meiosis begin in fetal oocytes with chromosome pairing, synapsis (synaptonemal complex indicated by a thick dashed line) and crossing over, shown here for a single pair of homologs (dark and light blue lines). Each homolog comprises a pair of sister chromatids connected by cohesins (black rings); consequently, crossing over results in the connection of homologs, which enables their bipolar alignment on the MI spindle and accurate segregation (disjunction) at meiosis-I (gray disks, kinetochores; orange lines, microtubules). Thus, connections must be maintained in primordial follicles throughout their arrest period. Connections are resolved at anaphase I by the cleavage of cohesins, allowing homologs to separate. Cohesins that connect sister centromeres are protected from cleavage until anaphase II, allowing accurate congression and segregation of sister chromatids. Fourth row: chromosomal errors in oocytes following atrazine exposure. In fetal oocytes, synaptic errors were modestly higher, but numbers of crossovers were not significantly changed. We favor the possibility that primordial follicles are the consequential target of atrazine exposure, which mimics and exacerbates the effects of maternal age by weakening sister-chromatid cohesion, indicated by fewer cohesin complexes (black rings). Weakening of cohesion between centromeres can result in merotelic attachment (i), misalignment at MI, and chromosome lagging at anaphase I. If sister chromatids prematurely separate in anaphase I, the free chromatids are prone to misalign in MII (ii) and missegregate in anaphase II, leading to aneuploidy. Alternatively, sister chromatids may prematurely separate in MII and missegregate in anaphase II. Loss of cohesion distal to crossover points results in prematurely separated univalents (iii), which can co-orient at MI and cosegregate at anaphase I (NDJ), resulting in aneuploidy in MII (iv) and in the ovum pronucleus following fertilization. Alternatively, the sister chromatids of a univalent may undergo premature segregation in anaphase I (reverse segregation^,). Note: MI, metaphase I; MII, metaphase II; NDJ, nondisjunction.