Nitrite exposure may induce infertility in mice

Abstract

In the present study, we investigated the potential of nitrite exposure to induce infertility in mice. Adult female C57BL/6J mice were randomly divided into control and nitrite exposure groups. Subsequently, the rate of mouse infertility was calculated, and pathological changes in ovarian tissues were examined using hematoxylin and eosin staining. In addition, TUNEL staining, immunofluorescent labeling, and western blotting were performed to assess cell apoptosis and oxidative stress response in ovarian tissues from various groups. We observed that nitrite exposure could induce infertility (p<0.05) in mice. High-dose nitrite exposure caused infertility in a time-dependent manner, and two-round exposure induced higher infertility than that one-round exposure (p<0.01). In addition, a higher number of atretic follicles were detected in the ovaries of nitrite-exposed groups than in the control group. Furthermore, TUNEL-positive cells were observed in granulosa cells of atretic follicles, and overexpression of caspase 8, c-Fos, and inducible nitric oxide synthase (iNOS) was detected in ovaries after nitrite exposure (p<0.01), suggesting that cell apoptosis and oxidative stress response were induced following nitrite exposure. Collectively, these findings suggest that nitrite exposure can induce mouse infertility in a time-dependent manner. Oxidative stress response and cell apoptosis are involved in mediating nitrite-induced infertility.

Keywords: atretic follicle; cell apoptosis; infertility; nitrite exposure; oxidative stress response.

Fig. 1.

Pathological and chemical changes in ovaries after nitrite exposure. A–E; Pathological alterations in control and treatment groups (H&E staining). A–B: Low magnification of control and treatment ovaries. The control ovary is plump, with some developing follicles (A, arrow). Conversely, the treatment group ovary shows notable atrophy and congested blood vessels (B, arrow). C–E: High magnification of control and treatment ovaries. Various follicles, including secondary follicles (arrow) and some antral follicles (*), can be observed in the control ovary (C). Corpora lutea with loose connective tissue and vessels inside can be observed in the control group tissues (D, *). After nitrite exposure, mature follicles are decreased, but atretic follicles with morphological deformation (arrow), such as oocyte autolysis, zona pellucida collapse, follicular wall collapse, are markedly increased (E). F–G: Nitrite exposure and cell apoptosis in granulosa follicular cells (TUNEL staining and DAPI counterstaining). In the control group, DAPI counterstaining (blue) shows numerous granulosa cells (blue) surrounding oocytes in the follicles, with few apoptotic cells (green, arrow) in granulosa cells (F). After high-dose nitrite exposure, numerous apoptotic cells (arrow) can be observed in granulosa cells (G). Interestingly, the matrix in the follicular cavity appears to be TUNEL-positive. H–J: Nitrite exposure and iNOS expression in granulosa cells (iNOS immunofluorescent labeling and DAPI counterstaining). In the control group (H), few iNOS positive cells with weak fluorescence (arrow) can be seen in granulosa cells (blue). The matrix in the follicular cavity exhibits iNOS-positivity. After nitrite exposure, numerous iNOS positive cells (arrow) can be observed in follicular granulosa cells (J). It should be noted that although there the iNOS positive matrix in the follicular cavity of the control group is dense, the iNOS positive granulosa cells are few. In contrast, the iNOS positive granulosa cells are enriched in treatment groups (arrow, with DAPI and iNOS double labeling). The control and high-dose exposure groups are indicated as Cont. and Treat. in the upper-left corner, respectively. Scale bar: A-B, 20 μm; C & E, 50 μm; D, 100 μm; F-J, 200 μm. H&E, hematoxylin-eosin; NOS, nitric oxide synthase; iNOS, inducible nitric oxide synthase.

Fig. 2.

Western blotting of activated-caspase-8 and c-Fos expressions in ovaries of various experimental groups. Nitrite exposure increases the expression of activated-caspase-8 and c-Fos in ovaries (A). The histogram shows that the differential expressions of caspase-8 and activated c-Fos in the control and high dose exposure group were statistically significant. The grayscale ratio is the relative gray value of target bands vs. internal reference bands, where β-actin was used as the internal reference. Mean ± standard deviation (SD), n=5. _*: p<0.01, if treatment vs. control.