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1. Introduction

We tested this hypothesis by administering DCIns to wild-type female mice for three weeks, approximately corresponding to five ovulatory cycles, and analyzing the effects on ovarian histology/function, serum testosterone levels and expression of the ovarian enzyme aromatase. To this end, we employed three different DCIns formulations: 250 mg/kg/day, 500 mg/kg/day and 1000 mg/kg/day. These doses provide approximate daily amounts of 5 mg, 10 mg and 20 mg DCIns/mouse, which are comparable to human doses of 1200, 2400 and 4800 mg/day, respectively [].

2.3. Gross Morphology of Uteri/Ovaries and Histology of Ovaries at the End of the Treatment

Ovaries from DCIns 250-treated mice had apparently normal primary and secondary follicles but also cystic tertiary follicles, some of which contained an atretic oocyte, strongly resembling those found in human polycystic ovaries (B1,B2; C). Ovaries of letrozole-treated mice were similar but contained larger cystic follicles characterized by the absence of the oocyte (C1,C2; D). In some cases, secondary and tertiary follicles from these ovaries had a clearly atretic oocyte with cytosolic vacuolization, suggesting the progression of an apoptotic process.

2.3. Gross Morphology of Uteri/Ovaries and Histology of Ovaries at the End of the Treatment

Ovaries from DCIns 500- and DCIns 1000-treated mice had some primary and secondary follicles, a very limited number of tertiary follicles, no follicles at more advanced stages and no cystic follicles (D1,D2; E1,E2). These ovaries had large follicles/areas with diffused, aberrant cell proliferation (E). The typical structure of the ovary was lost, especially under the DCIns 1000 dose, and the presence of follicles was difficult to assess in many cases (F). In some sections, the presence of oedema foci was observed.

2.6. Testosterone Levels at the End of the Treatment

Under the DCIns 250 dose, levels of testosterone (1.42 ± 0.107 ng/mL) (mean ± SD) increased above those typical of negative control mice (0.37 ± 0.11 ng/mL) (p < 0.05, one-way ANOVA), similarly to those of letrozole-administered mice (1.29 ± 0.22 ng/mL). Levels of testosterone in the serum of DCIns 500- and DCIns 1000-treated mice were similar to or lower than those of negative control mice, being respectively 0.14 ± 011 and 0.27 ± 0.37 ng/mL.

2.7. Presence and Relative Amounts of Aromatase at the End of the Treatment

From a qualitative point of view, a single band of approximately 55 kDa molecular mass was detected in protein extracts from control mouse ovaries, as already reported []. A band of similar size was detected after all treatments, but additional bands were detected, as follows: protein extracts from DCIns 250-treated mice contained a band of smaller size (approximately 52 kDa), those from letrozole-treated mice had a band of closely smaller size (approximately 54 kDa), and extracts from DCIns 500- and DCIns 1000-treated mice had a band of much smaller size (approximately 50 kDa). Induction of different aromatase isoforms by various treatments in mouse ovarian cells appears reasonable in light of the observation that the control of aromatase expression in humans involves a complex mechanism of different promoter and downstream exons []. Densitometric measurements of the amounts of aromatase bands relative to GAPDH, showed a reduction in DCIns 250-ovaries (0.16 ± 0.02 relative units, RU) (mean ± SD) from those of negative control-ovaries (0.34 ± 0.03 RU) (p < 0.05); on the contrary, an increase was observed in letrozole-ovaries (0.56 ± 0.06 RU) (p < 0.05). No variations in the amounts of aromatase were observed in DCIns 500-ovaries (0.45 ± 0.07 RU) and DCIns 1000-ovaries (0.40 ± 0.08 RU) from either negative control- or letrozole-ovaries.