Inhibition of matrix metalloproteinases in Siberian hamsters impedes photostimulated recrudescence of ovaries

Abstract

Exposure of Siberian hamsters to short photoperiod for 14 weeks induces ovarian regression. Subsequent transfer to long photoperiod restores ovarian function, and 2 weeks of photostimulation increases plasma estradiol (E(2)), antral follicles, and corpora lutea (CL). Because tissue remodeling involved with photostimulated ovarian recrudescence is associated with differential expression of matrix metalloproteinases (MMPs), we hypothesized that inhibiting MMP activity using a broad-spectrum in vivo MMP inhibitor, GM6001, would curtail recrudescence. One group of hamsters was placed in long days (LD; 16 h light:8 h darkness) for 16 weeks. Another group was placed in inhibitory short days (SD; 8 h light:16 h darkness) for 14 weeks. A third group was placed in SD for 14 weeks and transferred to LD for 2 weeks to stimulate recrudescence. During weeks 14-16, animals were either not treated or treated daily with i.p. injections of GM6001 (20 mg/kg) or vehicle (DMSO). GM6001 reduced gelatinase activity and decreased immunohistochemical staining for MMP1, MMP2, and MMP3 compared with vehicle. No differences between controls, vehicle, or GM6001 treatment were observed among LD animals, despite a trend toward reduction in CL and E(2) with GM6001. Although SD reduced ovarian function, photostimulation of transferred controls increased uterine mass, plasma E(2), appearance of antral follicles, and CL. With GM6001 treatment, photostimulation failed to increase uterine mass, plasma E(2), antral follicles, or CL. These data show, for the first time, that in vivo GM6001 administration inhibits MMP activity in hamster ovaries during photostimulation, and indicate that this inhibition may impede photostimulated recrudescence of ovaries. This study suggests an intriguing link between MMP activity and return to ovarian function during photostimulated recrudescence.

Fig. 1

Paired ovary mass (A) and paired uterine horn mass (B). Mean ± SEM (A) ovarian mass (g) and (B) uterine mass (g) in Siberian hamsters exposed to long day (LD, n=9; LDV, n=9; LDG, n=9); animals transferred from short day to long day photoperiod (PT, n=4; PTV, n=7); animals exposed to short day photoperiod alone (SD, n=4) and animals treated with GM6001 during exposure to long day photoperiod (PTG, n=9). Groups with different letters are significantly different (p < 0.05).

Fig. 2

Representative cross sections of ovaries from Siberian hamsters stained with hematoxylin and eosin. (A) Long day (LD) control. (B) LD with GM6001 treatment (LDG). (C) Short day (SD). (D) Post-transfer with vehicle treatment (PTV). (E) Post-transfer with GM6001 treatment (PTG). CL, corpus luteum; P, pre-antral follicles; A, antral follicles; T, terminal atretic follicles, At, atretic follicles. N per group the same as listed for Fig. 1.

Fig. 3

Mean ± SEM number of (A) pre-antral follicles, (B) antral follicles, (C) corpora lutea, and (D) terminal atretic follicles per ovarian cross section in Siberian hamsters exposed to long day (LD), short day (SD), or post-transfer (PT) from short to long day photoperiod with no treatment (LD, SD, PT), or with treatment with vehicle (LDV, PTV) or GM6001 (LDG, PTG). Groups with different letters are significantly different (p < 0.05). N per group the same as listed for Fig. 1.

Fig. 4

Plasma estradiol concentrations (pg/mL) in recrudescing Siberian hamsters. Mean ± SEM plasma estradiol concentrations (pg/mL) in Siberian hamsters exposed to long day (LD), short day (SD), or post-transfer (PT) from short to long day photoperiod with no treatment (LD, SD, PT), or with treatment with vehicle (LDV, PTV) or GM6001 (LDG, PTG). Groups with different letters are significantly different (p < 0.05). N per group the same as listed for Fig. 1.

Fig. 5

Protease activity of MMP-2 and MMP-9 in uterine protein extracts in recrudescing Siberian hamsters. (A) Gelatin Zymogram. Active MMP-9 (80–82 kd), proMMP-2 (72kd), and active MMP-2 (60kd) bands are labeled. (B) Mean ± SEM active MMP-2 and (C) MMP-9 activity in Siberian hamsters exposed to long day with vehicle treatment (LDV), post-transfer with vehicle treatment (PTV), and post-transfer with GM6001 treatment (PTG). Groups with different letters are significantly different (P<0.05). N per group the same as listed for Fig. 1.

Fig. 6

Extent and intensity of immunostaining for MMPs. Mean ± SEM immunostaining index levels (scores of 0–3) indicating extent and intensity of red/pink stained cells in Siberian hamsters post-transfer from short to long day photoperiod with vehicle (PTV) or GM6001 (PTG) treatment. (A) MMP-1 staining index, typical (B) high and (C) low MMP-1 immunostaining, (D) MMP-2 staining index, typical (E) high and (F) low MMP-2 immunostaining, (G) MMP-1 staining index, typical (H) high and (I) low MMP-1 immunostaining. Insets depict negative controls processed without primary antibody. N = 4–9/group. Groups with different letters are significantly different (P< 0.05).