We are interested in the basic question of how the regulation of the enzymes involved with extracellular matrix turnover impacts ovarian function. Specifically we have begun to examine the involvement of a family of proteolytic enzymes, known as matrix metalloproteinases (MMPs), with follicular growth, ovulation, and corpus luteum function. The MMPs are responsible for connective tissue remodeling throughout the body. We have observed that the midcycle surge of LH, which sets in motion the ovulatory process, results in an increase in the expression of mRNA and activity for certain members of the MMP family prior to ovulation. When the LH surge is inhibited or certain downstream LH-induced signals are blocked, the increase in metalloproteinases does not occur. Further evidence for the importance of the MMPs in oocyte release is the finding that when the preovulatory increase in metalloproteinase activity is blocked by exogenous enzyme inhibitors, ovulation is inhibited. These observations, that LH induces the MMPs and that inhibition of metalloproteinase action blocks oocyte release, suggest a crucial role for the MMPs in the process of ovulation. Thus, one of the long term goals of this research program is to further understand the mechanisms which promote or inhibit oocyte release and thereby explore alternative methods for enhancing or prohibiting ovulation and thus fertility. Ongoing studies are focused on understanding the control of metalloproteinase induction, the site of cellular action, and the important members of this family in not only the ovulatory process but also follicular development and luteal function.
Of particular interest is the finding that enzyme activity in the extracellular space appears to be regulated by enzyme inhibitors present in the ovary and that LH stimulates an increase in both the MMPs and these inhibitors. Studies in the laboratory are aimed at characterizing the location and regulation of these enzymes' inhibitors known as the tissue inhibitors of metalloproteinases or TIMPs. This information will aid in our understanding of the interplay between the MMPs and TIMPs in regulating extracellular matrix remodeling during follicular growth, oocyte release, and luteal formation. Dr. Kristen Simpson has presented an example of some of the work performed in understanding the localization of the TIMPs in this area. Micrograph depicting presence of apoptosis in preovulatory follicle adjacent to healthy follicle with overlay of TIMP-3 in situ hybridization reaction product. The photomicrograph is a three-color composite image demonstrating TIMP-3 mRNA in rat ovaries collected on the day of estrous. The photomicrograph illustrates nuclear DNA by propidium iodide staining (shown in red), apoptosis of the granulosa cell layer using an ApoAlert procedure (indicated in green), and a colorized in situ hybridization reaction product for TIMP-3 mRNA (depicted in blue). TIMP-3 mRNA is highly abundant in the theca layer of the follicle. What is particularly exciting, however, is the observation of higher levels of expression of TIMP-3 mRNA in granulosa cells from the healthy follicle (H) whereas TIMP-3 expression in the adjacent atretic follicle (A) is diminished. This is a novel finding for TIMP-3 mRNA expression suggesting an association with follicular differentiation.
Aging and Ovarian Function
Another area of research emphasis in our laboratory is the exploration of aging and ovarian function. These studies are aimed at determining the interaction between the loss of reproductive function with aging, the cohort of ovarian follicles, and changes in the MMPs and TIMPs during follicular growth. These studies are being performed as part of a consortium project with other investigators at the University to explore the ovarian and neuroendocrine axis during reproductive aging in the rat. An example of some of the work performed exploring the follicular populations is seen here. Shown is a section of a rat ovary cut and stained by Tammie Tyler, one of our undergraduate BIOL 395 students. The photomicrograph clearly illustrates the different follicular compartments such as the oocyte, the granulosa layer, and the thecal layer in a secondary follicle. This information is being used to evaluate the follicular populations during the aging process in the rat.