Differential expression of matrix metalloproteinases during stimulated ovarian recrudescence in Siberian hamsters (Phodopus sungorus)

Abstract

The matrix metalloproteinases (MMPs) are a family of extracellular matrix-cleaving enzymes involved in ovarian remodeling. In many non-tropical species, including Siberian hamsters, ovarian remodeling is necessary for the functional changes associated with seasonal reproduction. We evaluated MMPs and their endogenous inhibitors (TIMPs), during photoperiod-induced ovarian recrudescence in Siberian hamsters. Hamsters were transferred from long day (LD; 16:8) to short day (SD; 8:16) photoperiods for 14weeks, and then returned to LD for 0, 1, 2, 4, or 8weeks for collection of ovaries and plasma. Post-transfer (PT) LD exposure increased body and ovarian mass. Number of corpora lutea and antral, but not preantral follicles increased in PT groups. Plasma estradiol concentrations were lower in PT weeks 0-4, and returned to LD levels at PT week 8. No change was observed in relative MMP/TIMP mRNA levels at PT week 0 (SD week 14) as compared to LD. Photostimulation increased MMP-2 mRNA at PT week 8 as compared to PT weeks 0-1. MMP-14 mRNA expression peaked at PT weeks 1-2 as compared to LD levels, while MMP-13 expression was low during this time. TIMP-1 mRNA peaked at PT week 8 as compared to PT weeks 0-4. No changes were noted in MMP-9 and TIMP-2 mRNA expression. In general, MMP/TIMP protein immunodetection followed the same patterns with most staining occurring in granulosa cells of follicles and corpora lutea. Our data suggest that mRNA and protein for several members of the MMP/TIMP families are expressed in Siberian hamster ovaries during recrudescence. Because of the variation observed in expression patterns, MMPs and TIMPs may be differentially involved with photostimulated return to ovarian function.

Figure 1. Body mass (A) and paired ovary mass (B)

Mean ± S.E.M. A) body mass (g) and B) ovarian mass (g) in Siberian hamsters exposed to long day (LD; n=18)), or post-transfer (PT) from short to long day photoperiod for 0 (n=10), 1 (n=10), 2 (n=10), 4 (n=11), and 8 (n=11) weeks. Groups with different letters are significantly different (P<0.05).

Figure 2. Representative cross sections of Siberian hamster ovaries stained with hematoxylin and eosin

A) Long day (LD) control (n=18). B) Post-transfer (PT) week 0 (same as SD week 14). C) PT week 1. D) PT week 2. E) PT week 4. F) PT week 8. CL, corpus luteum; AF, antral follicles; PAF, preantral follicles; → indicate terminal atretic follicles. All photos taken at 4x magnification; scale bar pertains to all sections and represents 1.0mm. N per group the same as listed for Figure 1.

Figure 3. Number of corpora lutea (A), antral follicles (B), and preantral follicles (C) per section

Mean ± S.E.M. A) corpora lutea, B) antral follicles, and C) pre-antral follicles per ovarian section in Siberian hamsters exposed to long day (LD), or post-transfer (PT) from short to long day photoperiod for 0, 1, 2, 4, and 8 weeks. Groups with different letters are significantly different (P<0.05). N per group the same as listed for Figure 1.

Figure 4. Plasma estradiol concentrations (pg/mL) in recrudescing Siberian hamsters

Mean ± S.E.M. plasma estradiol concentrations (pg/mL) in Siberian hamsters exposed to long day (LD), or post-transfer (PT) from short to long day photoperiod for 0, 1, 2, 4, and 8 weeks. Groups with different letters are significantly different (P<0.05). N per group the same as listed for Figure 1.

Figure 5. mRNA expression for MMPs and TIMPs

Semi-quantitative RT-PCR expression of MMP and TIMP mRNA in Siberian hamsters exposed to long day (LD), or post-transfer (PT) from short to long day photoperiod for 0, 1, 2, 4, and 8 weeks. A) MMP-2, B) MMP-9, C) MMP-13, D) MMP-14 (mt-MMP-1), E) TIMP-1, F) TIMP-2, and G) β-actin used as a control gene for all RT-PCR reactions.

Figure 6. Relative mRNA expression levels for MMPs and TIMPs

Mean ± S.E.M. relative levels mRNA expression in Siberian hamsters exposed to long day (LD), or post-transfer (PT) from short to long day photoperiod for 0, 1, 2, 4, and 8 weeks. A) MMP-2:β-actin mRNA expression. B) MMP-9:β-actin mRNA expression. C) MMP-13:β-actin mRNA expression. D) MMP-14:β-actin mRNA expression. E) TIMP-1:β-actin mRNA expression. F) TIMP-2:β-actin mRNA expression. Groups with different letters are significantly different (P<0.05). LD (n=14), PT 0 (n=5), PT 1 (n=9), PT 2 (n=7), PT 4 (n=11), PT 8 (n=9).

Figure 7. MMP and TIMP protein immunodetection during recrudescence in Siberian hamsters

Immunohistochemical staining (red/pink stain on purple background) for MMPs and TIMPs. Scale bars (A, inset B) depict 1.0mm with photographs taken at 4× magnification unless stated otherwise (micrographs in F and K). A) Tissue section from post-transfer (PT) week 2 demonstrating diffuse, cytoplasmic immunodetection of MMP-2 typical of PT weeks 0, 1, and 2. B) MMP-2 immunolabeling was stronger during PT weeks 4 and 8, and strongly abundant in granulosa cells (PT week 8 shown). C) Section depicting MMP-9 protein levels during ovarian recrudescence; cytoplasmic MMP-9 staining was consistent among all tested groups (PT week 2 shown). D) Typical lower levels of punctate, peri-nuclear MMP-13 staining observed in PT week 2. E) Ovary section illustrating high levels of punctate, peri-nuclear MMP-13 staining observed in PT week 8. F) Detail of puntate, perinuclear staining observed with the MMP-13 antibody; photo taken at 20×, scale bar indicates 200μm. G) Cytoplasmic MMP-14 immunolabeling in the LD control group. H) MMP-14 staining in the recrudescing ovary; MMP-14 protein levels appeared elevated in post-transfer weeks 0, 1, 2, and 4 (PT week 2 shown). I) Tissue section showing cytoplasmic TIMP-1 immunolabeling typical of the recrudescing ovary during weeks 0, 1, 2, and 4, post-transfer (PT week 2 shown). J) TIMP-1 labeling was more extensive at PT week 8. K) Cytoplasmic diffuse staining typical of MMPs-2, -9, -14 and TIMPs-1 and -2, shown here in a 40× magnification photo of TIMP-1 at PT week 8. Scale bar indicates 100μm. L) Section illustrating cytoplasmic TIMP-2 immunolabeling in the ovary during recrudescence, TIMP-1 intensity did not appear to change in response to stimulatory photoperiod. Insets on B, C, E, H, J, and L depict negative control sections processed without primary antibody.

Figure 8. Extent and Intensity of Immunostaining for MMPs and TIMPs

Mean ± S.E.M. immunostaining index levels (scores of 0−3) indicating extent and intensity of red/pink stained cells in Siberian hamsters exposed to long day (LD), or post-transfer (PT) from short to long day photoperiod for 0, 1, 2, 4, and 8 weeks. A) MMP-2. B) MMP-9. C) MMP-13. D) MMP-14. E) TIMP-1. F) TIMP-2. N=5/group. Groups with different letters are significantly different (P<0.05).