The Involvement of Collagenolysis in Ovulation in the Rat*

Reuven Reich, A. Tsafriri, Gerald L. Mechanic
In: Endocrinology · 1985 · vol. 116(2) , pp. 522–527 · doi:10.1210/endo-116-2-522 · PMID:2981665 · W1983781550
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This study demonstrated collagenolysis in rat ovaries and follicles following ovulation stimulation, showing dose-dependent inhibition by cysteine and indomethacin, corroborating collagenolysis's role in ovulation.

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Abstract

Collagenolytic activity in ovarian follicles was previously demonstrated by using synthetic peptides and reconstituted collagen fibers. However, attempts to demonstrate degradation of ovarian collagen and to correlate collagenase activity with ovulation were not successful. By administration of L-(5-3H) proline, we have labeled ovarian and follicular collagen and followed collagenolytic activity by separation of 3H-hydroxyproline (3H-Hyp) from acid hydrolyzates of ovarian tissue by HPLC. The level of ovarian and follicular 3H-Hyp decreased by about 40% on the afternoon of proestrus or after exogenous stimulation of ovulation by human CG (hCG), and this decrease was abolished by blocking the surge of gonadotropins with Nembutal. To verify that the observed reduction in 3H-Hyp was due to the action of a typical collagenase, the collagenous fraction was prepared from ovarian tissue and from preovulatory follicles before and after the ovulatory stimulus. The extracts were treated with trypsin (25 min, 25 C, 0.01 mg/ml) plasmin and p-amino-phenyl-mercuric acetate to fully activate the collagenase extracted along with collagen. Both, enzymatic and chemical activation of collagenase in vitro resulted in degradation of collagen. This degradation could be inhibited by cysteine and EDTA; both are classic inhibitors of mammalian collagenases. The activity of ovarian collagenase increased within 3 h after hCG-stimulation, peaked at 5-fold 6 h after hCG, and declined afterwards. Administration of cysteine (0.001-0.01 mmol) into the bursal cavity of proestrous rats blocked ovulation and breakdown of ovarian collagen in a dose-dependent manner. Cysteine effectively inhibited ovulation even when injected 7 h after the hCG stimulus. Inhibitors of arachidonic acid metabolism prevent ovulation. Indomethacin (inhibitor of cyclooxygenase) and nordihydroguaiaretic acid (inhibitor of lipoxygenase) blocked ovulation and inhibited hCG-induced ovarian collagenolysis. Collectively, these results corroborate the essential role of collagenolysis in follicular rupture in mammals.

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