α₂-macroglobulin and tissue inhibitor of metalloproteinases: Collagenase inhibitors in human preovulatory ovaries

Extensive remodeling of the follicular extracellular matrix occurs during the process of ovulation. This remodeling involves the breakdown of collagen, which is regulated, in part, by the action of the metalloproteinase collagenase and its associated inhibitors. In the present study, follicular metalloproteinase inhibitors were characterized to determine whether they were serum-borne or of ovarian origin, possibly a tissue-derived inhibitor known as tissue inhibitor of metalloproteinase (TIMP). Human follicular fluid and granulosa cells were obtained from preovulatory follicles of patients in an in vitro fertilization program. Chromatographic separation of follicular fluid on Sepharose 6B resulted in two peaks of inhibitory activity. The large molecular radius (M_r) inhibitor was similar in size to the serum-borne metalloproteinase inhibitor α₂-macroglobulin (i.e. M_r 700,000) whereas the small M_r inhibitor approximated the size of TIMP (i.e. M_r29,000). Incubation of aliquots from either of the two peaks of inhibitor activity or an α₂-macroglobulin standard with an antibody to α₂-macroglobulin decreased the inhibitory activity in both the large M_r peak and the α₂-macroglobulin standard by 86.6 ± 1.7% and 71.5 ± 7.7% (n = 4, P < 0.005), respectively, implying cross-reactivity with the α₂-macroglobulin antibody. The inhibitory activity in the small M_r peak, however, was unchanged. Northern analysis of total granulosa cell RNA demonstrated TIMP messenger RNA (mRNA) in all eight granulosa cell samples examined whereas α₂-macroglobulin mRNA was virtually undetectable. A positive correlation (r = 0.85, P < 0.01) was observed between the levels of TIMP mRNA and the ratio of the follicular estradiol-progesterone concentration. However, inhibitor activity in the follicular fluid was not correlated with the levels of TIMP mRNA (r = 0.05). These findings confirm the presence of α₂-macroglobulin in follicular fluid and demonstrate that human preovulatory granulosa cells contain mRNA for TIMP, an inhibitor that regulates metalloproteinases such as collagenase, gelatinase, and proteoglycanase. Additionally, the expression of TIMP mRNA is steroid related and may be hormonally regulated. It is proposed that TIMP produced in the granulosa cell compartment in conjunction with α₂-macroglobulin from the serum may act to control the site and extent of ovarian connective tissue remodeling.