Effect of mitotane on mouse ovarian follicle development and fertility

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   Figure 1

   Effects of mitotane on follicular growth and maturation in ovaries removed from untreated (Ctrl) or treated animals (MTT). (A) Representative histological sections of ovaries removed after 18 days of treatment from vehicle alone- (Ctrl) and mitotane-treated (MTT) mice. Histological evaluation of ovaries showed no evident morphological changes in treated animals compared to controls. In MTT-treated animals, we observed numerous preantral follicles (arrow heads), a significant decrease in the number of early antral follicles (arrows) and no antral follicles (asterisk). Scale bar = 200 µm. (B) Counts of follicles at different stages of maturation, categorized as follows: mono-bilaminar follicles with one/two layers of cuboidal GCs, preantral follicles with several layers of GCs and no antrum, early antral follicles with vacuolization of the intercellular spaces among GCs, antral follicles with a fluid-filled cavity, the antrum. Data are expressed as mean ± s.d. from three independent experiments carried out on a total animal number of Ctrl = 18 and MTT = 18. In MTT-treated animals, we observed a decrease in the number of early antral follicles (19% in treated vs 28% in untreated mice, P = 0.05), a corresponding increase in the number of preantral follicles (57% in treated mice vs 38% in untreated, P < 0.001), and no antral follicles. Statistical analysis was performed using ANOVA followed by the Tukey–Kramer test. *P < 0.001 vs respective Ctrl.
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   Figure 2

   Effects of mitotane on cell proliferation in ovaries removed from untreated (Ctrl) or treated animals (MTT). Effect of MTT treatment on PCNA immunoreactivity in control and treated ovaries. Representative images of immunofluorescent PCNA staining in sections of ovaries from mice untreated (Ctrl) and MTT treated (MTT). Scale bar = 100 µm. The graph shows the mean ± s.d. of PCNA positive GCs per field. In the presence of MTT treatment, PCNA immunoreactivity was significantly decreased, whereas control ovaries showed a higher number of PCNA positive cells in almost all the growing preantral, early antral follicles. Statistical analysis was performed using the Student’s t-test; **P < 0.01 vs Ctrl.
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   Figure 3

   Effects of mitotane on gene expression in ovaries removed from untreated (Ctrl) or treated animals (MTT). (A) Ovaries were collected from control mice; GCs and TI cells were separated as described in ‘Materials and methods’ section. Total RNA extracted from isolated granulosa and TI cells was subject to real-time PCR using primers specific for Fsh-R, and Cyp17a1, specific markers for GCs and TI cells, respectively. Each sample was normalized to its β-actin content. Final results are expressed as arbitrary units (a.u.) and are represented as the mean ± s.d. of three independent cell preparations. Statistical analysis was performed using the Student’s t-test; *P < 0.01 and **P < 0.001 vs respective Ctrl. (B) Expression of Fsh-R and Amh mRNA in mouse GCs and (C) Cyp17a1 and Cyp11a1 in TI cells, detected by real-time PCR. Ovaries were collected from control (Ctrl) or MTT-treated (MTT) mice, GCs and TI cells were separated as described in ‘Materials and methods’ section. Total RNA extracted from isolated granulosa and TI cells, was subject to real-time PCR using primers specific for Fsh-R, Amh, and P-450scc (Cyp11a1) and 17α-hydroxylase (Cyp17a1), respectively. Each sample was normalized to its β-actin content. Final results are expressed as arbitrary units (a.u.) and are represented as the mean ± s.d. of three independent experiments with a total animal number of Ctrl = 15 and MTT = 18. Statistical analysis was performed using the Student’s t-test; *P < 0.05, **P < 0.01, and ***P < 0.001 vs respective Ctrl. We observed significantly lower levels of Fsh-R mRNA and higher levels of Amh in GCs obtained from MTT-treated mice while the expression levels of both Cyp11a1 and Cyp17a1 mRNA were significantly decreased in TI cells from the same animals.
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   Figure 4

   Effect of mitotane treatment on female fertility. Prepubertal mice were treated for 18 days with vehicle alone (Ctrl, n = 10) or mitotane (MTT, n = 12) before PMSG/hCG induction of ovulation. (A) Mean ± s.d. of numbers of ovulated oocytes/mouse. Midline histological sections of ovaries removed from control (B) and treated (C) animals stained with carmalum and showing corpora lutea (examples of corpora lutea in each section are marked with asterisks) and antral follicles (arrows). Scale bars, 200 µm. *P < 0.01 vs Ctrl.
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   Figure 5

   Effect of mitotane treatment on reproduction. (A) Graph of the housing time with the male required to yield pregnancy in control (Ctrl, n = 10) and mitotane-treated (MTT, n = 10) mice. (B) Mean litter size per pregnant mouse in control (Ctrl) and mitotane-treated (MTT) females. Values are expressed as mean ± s.d. Statistical significance was calculated by Student’s t-test. *P < 0.05, **P < 0.01 vs respective Ctrl.
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   Figure 6

   Effect of mitotane withdrawn on female fertility. Prepubertal mice were treated for 18 days with vehicle alone (Ctrl, n = 12) or mitotane (MTT, n = 12). At the end of treatment, 5 mice/group were injected with PMSG/hCG to induce ovulation. While the remaining mice (Ctrl, n = 7; MTT, n = 7) were left with no treatment and 25 days later ovulation was induced. (A) Mean ± s.d. of numbers of ovulated oocytes/mouse after 18 days of treatment. (B) Mean ± s.d. of numbers of ovulated oocytes/mouse 25 days after drug withdrawn.
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   Figure 7

   Effect of mitotane treatment on COV-434 cell growth. (A) MTT induces cell growth inhibition on COV-434 cells. The cells were treated with MTT at the indicated concentrations (5, 15, 25 and 50 µM) for different times. Cell number was determined at each time point as described in ‘Materials and methods’ section. The figure shows a dose- and time-dependent inhibitory effect on cell number. Cell growth inhibition progressively increases and, at the highest concentration at 72 h, reaches 72% of inhibition after MTT treatment. The results represent the mean ± s.d. of three independent experiments done in duplicate. A comparison of the individual treatment was conducted by using ANOVA followed by the Tukey–Kramer post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001 vs Ctrl. (B) Western blot analyses of cell cycle regulatory proteins, cyclin E and Cdk2, were performed on 50 µg of total proteins extracted from COV 434 cells treated with vehicle alone (Ctrl) or with 50 µM MTT for 48 h and 72 h. Vinculin was used as a loading control. Densitometric absorbance values were normalized by their respective vinculin values and are presented as arbitrary units (a.u.). Values are expressed as mean ± s.d. of three independent experiments. Statistical significance was calculated by Student’s t-test. The results of Western blots show a downregulation of cyclin E and Cdk2 at both indicated times. *P < 0.05, **P < 0.01 vs respective Ctrl.
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   Figure 8

   Induction of apoptosis by mitotane treatment in COV-434 cells. (A) Cells were treated for 24 and 48 h with 50 µM mitotane (MTT) or vehicle alone (Ctrl), the apoptotic process was examined using the TUNEL assay (terminal deoxynucleotidyl transferase dUTP nick end labeling) and the quantification of the TUNEL labeling was evaluated by flow cytometric analysis as described in ‘Materials and methods’ section. After MTT treatment, the percentage of apoptotic cells was 19% at 24 h and the cell death increased at 48 h reaching 60% dUTP (Terminal deoxynucleo­tidyltransferase-mediated deoxyuridine 5-triphosphate). (B) Western blot analyses of apoptotic proteins pro-caspase 3 and PARP were performed on 50 µg of total proteins extracted from COV 434 cells treated with vehicle alone (Ctrl) or with 50 µM MTT for 24 h and 48 h. Vinculin was used as a loading control. The figure shows the decrease of pro-caspase3 content and the cleavage of PARP protein after MTT treatment at all the indicated times. *P < 0.05, **P < 0.01 vs respective Ctrl.

• © 2017 Society for Endocrinology