Coordinate Expression of Anticoagulant Heparan Sulfate Proteoglycans and Serine Protease Inhibitors in the Rat Ovary: A Potent System of Proteolysis Control1

Abstract

During the reproductive cycle, ovarian follicles undergo major tissue-remodeling involving vascular changes and proteolysis. Anticoagulant heparan sulfate proteoglycans (aHSPGs) are expressed by granulosa cells during the development of the ovarian follicle. The function of aHSPGs in the ovary is unknown, but they might be involved in proteolysis control through binding and activation of serine protease inhibitors. To identify functional interactions between aHSPGs and heparin-binding protease inhibitors in the follicle, we have coordinately localized aHSPGs, antithrombin III, protease nexin-1, and plasminogen activator inhibitor-1 in the rat ovary during natural and gonadotropin-stimulated cycles. Anticoagulant HSPGs were visualized by autoradiography of cryosections incubated with 125I-antithrombin III, and protease inhibitors were assessed by immunohistochemistry and Northern blot hybridization. Anticoagulant HSPGs were expressed in follicles before ovulation, were transiently decreased in postovulatory follicles, and were abundant in the corpus luteum, mainly on capillaries. Anticoagulant HSPGs were colocalized with protease nexin-1 in follicles from the early antral stage until ovulation, with antithrombin III in the preovulatory stage and after ovulation, and with plasminogen activator inhibitor-1 in the corpus luteum. These data demonstrate that aHSPGs are critically expressed in the ovary to interact sequentially with protease nexin-1, antithrombin III, and plasminogen activator inhibitor-1 during the cycle. The specificity of these inhibitors is shifted toward thrombin inhibition in the presence of heparin, suggesting that aHSPGs direct their action to control fibrin deposition in the follicle. The occupation of aHSPGs antithrombin-binding sites by mutant R393C antithrombin III, injected in the ovarian bursa, decreased ovulation efficiency, further supporting the involvement of aHSPGs in the ovulation process.

Expression of protease nexin-1 and plasminogen activators during follicular growth and the periovulatory period in cattle

“…We demonstrate that during the periovulatory period the expression of PN-1 is initially upregulated by hCG, and then declines in a pattern similar to the expression of PAI-1 and tPA. This profile of PN-1 expression appears to differ from that observed in rodents, where no change in expression was observed until the onset of ovulation (Hägglund et al 1996, Hasan et al 2002, although these are in situ studies that do not lend themselves well to quantification. We also demonstrate a potential role for PN-1 in dominant follicular growth, as follicular protein levels are higher in non-atretic than in early-atretic and atretic follicles.…”

“…In mice, granulosa cell PN-1 expression generally did not vary throughout the periovulatory period (Hägglund et al 1996), although a decrease in immunostaining was observed 12 h after hCG injection in PMSG-stimulated rats (Hasan et al 2002). The present data clearly show a transient upregulation of PN-1 expression within 6 h of hCG treatment, indicating that PN-1 regulation in cattle is different from that of rodents.…”

“…PN-1 is also expressed in preovulatory follicles, although its role is not clear. PN-1 expression decreased following an ovulatory dose of hCG in rats (Hasan et al 2002) but not in mice (Hägglund et al 1996). How the proteolytic cascade is controlled in cattle is not well understood, as tPA activity increases prior to ovulation, whereas PAI-1 activity does not change (Dow et al 2002a(Dow et al , 2002b.…”

“…The expression and regulation of PN-1 has been examined in ovarian follicles. In contrast to PAI-1, which is predominantly expressed in thecal-interstitial cell layers, PN-1 was exclusively expressed in granulosa cells in mice (Häg-glund et al 1996), rats (Hasan et al 2002) and cattle (Bédard et al 2003).…”

Production and binding of endothelin-2 (EDN2) in the rat ovary: endothelin receptor subtype A (EDNRA)-mediated contraction

“…Again, no effect on the rate of ovulation was observed. Because there are a multitude of proteolytic enzymes present within the ovary during the periovulatory period (Chun et al 1992;Liu et al 1997;Hasan et al 2002), it is likely that the lack of any effect of these antagonists on oocyte release is due to rapid degradation of these peptide-based antagonists in vivo. However, given the results reported herein, the requirement for EDNRB-mediated signalling in the process of ovulation needs to be re-evaluated.…”