Coordinate induction and activation of metalloproteinase and ascorbate depletion in structural luteolysis

Structural luteolysis was found decades ago to be induced by PRL in the hypophysectomized rat, but the mechanisms of this process are unknown. To gain information on mechanisms of luteal involution, we developed an animal model that circumvented complex surgery and provided ample tissue for analyses. Gonadotropin-synchronized ovulation and luteinization were induced in immature rats, followed by treatment with ergot alkaloid and PRL. PRL-induced structural luteolysis, as shown by loss of luteal weight, protein, and DNA after pretreatment with ergot alkaloid, was evident after 36 h. Ascorbic acid depletion was rapid, severe, and lasting in luteal tissue during structural luteolysis, but lipid peroxidation or depletion of vitamin E was not evident. PRL treatment of animals with functional corpora lutea did not induce luteal involution. Significantly, after natural functional luteolysis occurred, PRL was highly effective in inducing structural luteolysis. Thus, either natural or ergot-induced functional luteolysis permitted the luteolytic expression of PRL. A greater depletion of protein than DNA was seen during PRL-induced structural luteolysis and was associated with a significant increase in neutral caseinase activity in luteal extracts. Caseinase activity was markedly reduced by calcium chelators and profoundly inhibited by the chelator orthophenanthroline; only slightly reduced activity was seen with serine, aspartate, or cysteine proteinase inhibitors. These findings implicate metalloproteinase (MMP) as the relevant caseinase that was increased during structural luteolysis. The major proteinase identified by zymography had apparent sizes of 72 and 66 kilodaltons (kDa), and slight but detectable activity was also seen at 92 and 84 kDa. Organomercurial treatment caused a major shift of the 72-kDa band to 66 kDa and the 92-kDa band to 84 kDa, confirming MMP-2 and MMP-9 by activation of latent activity of each MMP, respectively. Structural luteolysis caused a significant increase in the activated 66-kDa form and the latent 72-kDa form of MMP-2, which occurred before a loss of luteal weight or protein. As MMP-2 degrades collagen (type IV) in basement membranes, we conclude that an early event in PRL-induced structural luteolysis is the degradation of extracellular matrix. This conclusion is further emphasized by the marked and lasting depletion of ascorbic acid, a vitamin long known to serve an essential role in collagen synthesis.