Methoxychlor-Induced Atresia: RESULTS(2)

Effect of MXC on Bcl-2 and Bax Levels

Using immunohistochemical analysis, Bcl-2 staining was found in the oocyte, granulosa, and thecal cells of the follicles in sesame oil- and MXC-treated ovaries. MXC (64 mg kg—1 day1 for 20 days) did not affect the expression of Bcl-2 in the antral follicles of the ovary. There was no difference with regard to follicle staining whether the follicles were healthy or atretic. Small (primordial and primary) follicles were unstained, although staining was present in some larger preantral follicles (Fig. 2, A-D). Image analysis confirmed there was no difference in staining in either the granulosa or thecal cell layers between the sesame oil- and MXC-treated ovaries (n = 3, P = 0.5).

Immunohistochemical staining for Bax was very light in the granulosa and thecal cells of follicles from the sesame oil-treated mice; however, the staining in the granulosa and especially in the thecal cells was very intense in the follicles and interstitium of the MXC-treated (64 mg kg-1 day-1 for 20 days) mice in both healthy and atretic follicles. As with the Bcl-2 staining, the primordial and primary follicles were largely unstained, although the larger preantral follicles did show Bax staining (Fig. 3, A-D). Image analysis confirmed that there was greater staining in both the granulosa and thecal cell layers. Mean pixel density in the granulosa cell layer was 0.31 ± 0.01 and 0.36 ±0.01 in the sesame oil and the MXC-treated animals, respectively (n = 3; P < 0.001). Mean pixel density in the thecal cell layer was 0.31 ± 0.01 and 0.38 ± 0.01 in the sesame oil- and MXC-treated animals, respectively (n = 3; P < 0.001).
Fig1Methoxychlor-Induced Atresia-1
FIG. 1. Effect of MXC on the percentage of atretic antral follicles per ovary. Ovaries were collected after 20 days of dosing from sesame oil-and 8, 16, 32, or 64 mg kg—1 day—1 MXC-treated mice. Complete serial sections were prepared and subjected to histological examination for atresia. Bars represent means ± SEM; *significant difference from control, P s 0.001, n = 3-9.

Fig2Methoxychlor-Induced Atresia-2
FIG. 2. Effect of MXC on Bcl-2 protein levels. A) An ovarian section from a sesame oil-treated animal at X40 magnification, (C) the same section at X10 magnification. B) An ovarian section from an animal treated with 64 mg kg-1 day-1 MXC at X40 magnification, (D) shows the same section at X10 magnification. E) A representative ovarian section from a representative negative control. Red, Bcl-2 staining; blue, counterstain; black arrows, stain in follicles; CL, corpora lutea.

Fig3Methoxychlor-Induced Atresia-3
FIG. 3. Effect of MXC on Bax protein levels. A) An ovarian section from a sesame oil-treated animal at X40 magnification, (C) the same section at X10 magnification. B) An ovarian section from an animal treated with 64 mg kg-1 day-1 MXC at X40 magnification, (D) shows the same section at X10 magnification. E) An ovarian section from a representative negative control. Red, Bax staining; blue, counterstain; black arrows, stain in follicles; CL, corpora lutea.