Developmental programming: preconceptional and gestational exposure of sheep to biosolids on offspring ovarian dynamics†

Abstract

Developmental exposure to environmental chemicals perturbs establishment and maintenance of the ovarian reserve across the reproductive lifetime, leading to premature follicle depletion and ovarian aging. Considering humans are exposed to a complex mixture of environmental chemicals, real-life models assessing their cumulative impact on the ovarian reserve are needed. Biosolids are a source of a real-life mixture of environmental chemicals. While earlier studies demonstrated that grazing pregnant sheep on biosolids-treated pastures did not influence establishment of the ovarian reserve in fetal life, its impact on subsequent depletion of ovarian reserve during reproductive life of offspring is unknown. We hypothesized that developmental exposure to biosolids accelerates depletion of ovarian reserve. Ovaries were collected from F1 juveniles (9.5 weeks) and adults (2.5 years) born to F0 ewes grazed on control inorganic fertilizer pastures or biosolids-treated pastures from before conception and throughout gestation. The impact on follicular density, activation rate, and anti-Müllerian hormone (mediator of activation) expression by immunohistochemistry was determined. Activation rate was increased in F1 biosolids-treated pastures juveniles with a corresponding reduction in primordial follicle density. In contrast, activation rate and ovarian reserve were similar between control and F1 biosolids-treated pastures adults. The density of anti-Müllerian hormone-positive antral follicles was lower in biosolids-treated pastures juveniles, whereas anti-Müllerian hormone expression tended to be higher in antral follicles of biosolids-treated pastures adults, consistent with the changes in the ovarian reserve. These findings of detrimental effects of developmental exposure to biosolids during juvenile life that normalizes in adults is supportive of a shift in activation rate likely related to peripubertal hormonal changes.

Keywords: AMH; biosolids; environmental chemical mixtures; ovarian follicles; ovary.

Graphical Abstract

Figure 1

Experimental design. F0 ewes were maintained on either control pasture (inorganic fertilizer) or on BTP from before conception and throughout gestation. At lambing, BTP F0 ewes and F1 lambs were moved to control pastures. Ovaries were collected from F1 offspring at 9.5 weeks of age (juvenile, before puberty) and at 2.5 years of age (adult, after puberty).

Figure 2

Summary of ovarian morphometry. Overall ovarian weight (g), section area (mm²), and cortex area (mm²) are shown for juveniles and adults in Panel A. “Section area” refers to the average area across the five sections for juveniles and four sections for adults that were used for follicle counting. “Cortex area” refers to the average area of the cortex in each section, across the five sections for juveniles and four sections for adults that were used for follicle counting. The sum cortex area and section area were used to calculate follicle densities, for which the number of follicles are represented per unit area. Panel B shows representative images of juvenile and adult ovaries. Panel C shows ovary and cortex volumes (mm³) of the juvenile ovaries for which the full cortex was visible, which were used to calculate the total follicle counts in juveniles. Data are presented as means ± SEM with individual animals plotted. Juveniles: C, n = 10; BTP, n = 11. Adults: C, n = 10; BTP, n = 10 (all animals). Juveniles: C, n = 5; BTP, n = 7 (ovaries where full cortex was visible).

Figure 3

Total number of nest clusters in juvenile ovaries. Number of clusters were calculated after taking into account the cortex volume. Data are presented as means ± SEM with individual animals plotted. Juveniles: C, n = 10; BTP, n = 11.

Figure 4

Follicle densities and activation rate. Follicle densities from juvenile ovaries (left side of panels) and adult ovaries (right side of panels). Panel A—for both juvenile and adult ovaries, follicle densities were calculated as the sum number of follicles (n) per unit sum area (mm²) of the cortex (for primordial through secondary follicles, n/mm² cortex area) or the whole section (for antral follicles, n/mm² section area), across five sections used for juveniles and four sections used for adults. Panel B represents the density of all the follicle classes combined (total follicles). Panel C shows the density of activated follicles (transitory through antral; top) and the activation rate (bottom). Data are presented as means ± SEM with individual animals plotted. Juveniles: C, n = 10; BTP, n = 11. Adults: C, n = 10; BTP, n = 10.

Figure 5

Percentage of follicle counts in each class out of total number of follicles. The percentage of each class out of the total number of follicles is presented. Follicles at all stages were counted and the sum follicle count across five sections (juveniles) and four sections (adults) were used to determine the percentage of each stage out of the total number of follicles. Data are presented as means ± SEM with individual animals plotted. Juveniles: C, n = 10; BTP, n = 11. Adults: C, n = 10; BTP, n = 10.

Figure 6

AMH in juvenile and adult ovaries. Representative images for juvenile and adult ovaries are shown in Panels A and B, respectively. Panel C (juveniles) and Panel D (adults) show the density of AMH-positive follicles (top row), and the relative expression of AMH (bottom row) in primary, secondary, and antral follicles. Antral follicles were subclassified by the thickness of the granulosa cell layer, and the density of AMH-positive antral follicles (top row) and relative AMH expression (row) are shown in Panel E (juveniles) and Panel F (adults). Density is calculated as the sum number of follicles (n) immuno-positive for AMH, relative to the sum area (mm²) of the cortex (for primary and secondary follicles) or area of the section (for antral follicles), across five sections used in juveniles and four sections used in adults. IgG negative images are shown as insets for juveniles and adults. Data are presented as means ± SEM with individual animals plotted. Scale bar = 50 um. Juveniles: C, n = 10; BTP, n = 11. Adults: C, n = 10; BTP, n = 10.