The antiandrogenic vinclozolin induces differentiation delay of germ cells and changes in energy metabolism in 3D cultures of fetal ovaries

Abstract

Vinclozolin is a pesticide with antiandrogenic activity as an endocrine disruptor compound. Its effects upon the progression of primordial follicles were assessed in cultures of mouse fetal ovaries from the onset of meiotic differentiation of germ cells (13.5 days post coitum) and from both in vivo exposed mice and in vitro exposed ovaries. Exposure of ovaries to vinclozolin-at in vitro dosages ranging from 10 to 200 μM and in 3D ex vivo culture following in vivo exposure to 50 mg/kg bw/day-showed delays in meiocyte differentiation and in follicle growth, even at the lowest in vitro dose exposure. Immunofluorescent analysis showed the presence of the proteins MSY2 and NOBOX in the primary follicles but no difference in the level of protein signals or in the number of follicles in relation to treatment. However, assessing the cytological differentiation of germ cells by detecting the synaptonemal complex protein SYCP3, the exposure to vinclozolin delayed meiotic differentiation from both in vitro- and in vivo-exposed ovaries. These effects were concomitant with changes in the energy metabolism, detected as a relative increase of glycolytic metabolism in live-cell metabolic assays in exposed ovaries.

Figure 1

In situ microscopy contrast phase images of the progress of follicles in control conditions from ovary cultures initiated at 13.5 dpc: after 2 (A), 9 (B), 11 (C), 13 (D), 15 (E), and 17 (F) days of culture. Bars represent 50 μm.

Figure 2

Follicle size at various days of VZN exposure. (A) In vitro exposure at various concentrations of VZN. Bars represent the mean of 2 to 6 replicates with each replicate containing 15–109 follicles in each experimental condition. (B). In vivo exposure to VZN. Bars represent the mean of 2 to 6 replicates with each replicate containing 39–152 follicles in each experimental condition. Statistical significant differences are indicated by * (p ≤ 0.05) or ** (p ≤ 0.01), in comparison to the respective DMSO controls.

Figure 3

Changes in the localization of MSY2 during follicular progression during culture, DMSO controls (first three columns), in in vivo and in vitro (10 μM) exposures to VZN (as VZN exposure is initiated at day 5 of culture the first image of in vitro exposed cultures corresponds to day 7 of culture). Bars represent 50 μm.

Figure 4

In toto immunostaining of cultured ovaries and analysis of meiotic progression. (A) Immunofluorescence localization of MSY2, SYCP3, and DAPI staining in meiocytes at day 5 of DMSO control culture. (B) Colocalization of SYCP3 and nucleolin proteins in meiotic cells SYCP3 staining. Discrete nuclear structures of lateral elements of the synaptonemal complexes are detected colocalizing with nucleoli staining during desynapsis. (C) Representative images of SYCP3 immunostaining of controls and VZN exposed cultures; as VZN exposure is initiated at day 5 of culture the image corresponds to day 7 of culture. Z = zygotene, LP = late pachytene and D/D = diplotene/diakinesis. Bar represent 25 μm. (D, E) Proportional distribution of different stages of meiotic cells in prophase I during the progress of ovarian cultures. (D) In vivo exposure to VZN. (E) In vitro exposure to VZN. Day 5 is not represented in this figure as the exposure to VZN in vitro began on day 5 of culture. Bars represent the mean of two biological replicates (ovaries) + SD [(C) 100–611 follicles per replicate; (D) 100–427 follicles per replicate]. Statistically significant differences respect the metabolic basal condition are indicated as: “a” correspond to p value ≤ 0.05 and “b” to p value ≤ 0.01.

Figure 5

Energetic metabolism analysis. (A) Scheme of the parameters evaluated in the 3D cultures of fetal ovaries by the Mito Stress Test. (B) Mitochondrial oxygen consumption rate (OCR) on key representative days during the progress of the culture in control conditions. (C, D) Mitochondrial oxygen consumption rate (OCR) in ovaries exposed in vitro or in vivo to VZN. (C) Basal rate. (D) Maximum rate. (E) Quantitative measurement of basal glycolytic rate using extracellular acidification rate (ECAR) parameter, in controls cultures and in VZN-exposed ovaries. (F) Ratio between mitochondrial respiration and basal glycolysis in controls and in ovaries exposed in vitro or in vivo to VZN. Bars indicate the mean of 3–6 biological replicates (ovaries) ± SD. Statistically significant differences respect the metabolic basal condition are indicated as: * correspond to p value ≤ 0.05 and ** to p value ≤ 0.01 (ANOVA, Tukey’s and Dunnette’s test); (basal conditions correspond to day 5 in in vivo exposures but basal condition for in vitro exposure is day 5 control ST since it is on day 5 when experimental in vitro exposure to VZN began). (G) NOBOX in toto immunostaining of oocytes on day 12 of culture in DMSO controls and in vitro exposed with the different VZN concentrations experimentally used. Bar represents 50 μm.