EXPRESSION OF MMP-2, -9 AND TIMP-2 IN EQUINE GRANULOSA CELLS AND THE IMPACT OF ELEVATED INSULIN IN VITRO

Abstract

Matrix metalloproteinases (MMPs), and tissue inhibitors of matrix metalloproteinases (TIMPs) play significant roles in follicular development and ovulation. The horse is distinctive in that the dominant follicle must migrate to the ovulation fossa for ovulation to occur, and it is probable that MMPs and TIMPs facilitate this process. In humans, chronic insulin resistance and hyperinsulinemia are frequently associated with increased duration of the follicular phase of the menstrual cycle especially in females with polycystic ovarian syndrome. Similarly, obesity and insulin resistance in horses are associated with hyperinsulinemia, lengthened interovulatory intervals, together with failure to ovulate, characterized by luteinization of dominant follicles associated with circulating progesterone. Hyperinsulinemia has been associated with alterations in MMP and TIMP expression. The objective of these studies was to better understand the relationship between insulin, MMPs and TIMPs and their effects on follicular development and ovulation in the mare. Granulosa cells from medium (16 to 31mm) and large (>31mm) follicles were collected via ultrasound guided transvaginal aspiration. In the first study, cells were collected and either immediately snap frozen for analysis of expression in vivo or cultured for 48 hours to determine in vitro gene expression of MMP-2, MMP-9 and TIMP-2. A second study was conducted to test the hypothesis that elevated insulin disrupts expression of MMPs and TIMPs in cultured equine granulosa cells. Cells were pooled according to aspirated follicle size and then separated into four treatment groups: control (no insulin), 10 ng/ml, 100 ng/ml, or 1000 ng/ml of insulin. Following a 48-hour incubation, RNA was isolated and gene expression of MMP-2, MMP-9 and TIMP-2 was analyzed using real-time RT-PCR. In vivo TIMP-2 expression was higher in cells collected from large follicles compared to cells from medium follicles (P<0.05). There was a trend towards decreased MMP-2 (P=0.051) and MMP-9 (P=0.066) expression in cells from large versus medium follicles. Ratios of both MMP-2/TIMP-2 and MMP-9/TIMP-2 were significantly higher in cells from medium versus large follicles (P<0.05) in vivo. However, in vitro there were no significant differences in these same parameters. Exposure to 100 ng/ml or 1000 ng/ml insulin significantly decreased TIMP-2 expression as compared to cells treated with 10 ng/ml insulin in cells harvested from large follicles (P<0.05). Cells collected from medium follicles displayed no change in TIMP-2 expression. There were no changes in expression of MMP-2 or MMP-9 in cells collected from either medium or large follicles. However, in cells from large follicles the ratio of MMP2/TIMP2 increased from 5% in control samples to over 30% in samples treated with both 100 ng/ml and 1000 ng/ml insulin. In conclusion, changes in MMP-2, MMP-9 and TIMP-2 expression in granulosa cells may play a role in follicular development and ovulation in the mare. Additionally, exposure to elevated concentrations of insulin disrupts expression of TIMP-2, thereby modifying the ratio of MMP/TIMP expression by equine granulosa cells from large follicles. This alteration in expression may lead to the inability of follicle wall remodeling necessary for follicular development, migration, and ovulation. (poster)