Nuclear progesterone receptor regulates blood coagulation facilitating ovulation in zebrafish

Ovulation is a remodeling process including blood capillary rupture and coagulation. Until now, there is no regulation and functional studies of coagulation factors in ovulation. Here, we report dramatic increases of coagulation factors (f5, f3a) in zebrafish preovulatory follicles. This upregulation was induced by progestin (DHP: 17α, 20β-dihydroxy-4-pregnen-3-one), a native ligand for nuclear progestin receptor (Pgr) that is essential for ovulation in zebrafish; but was abolished in pgr-/-. In addition, promoter activities of f5 and f3a were significantly enhanced by progestin via zebrafish Pgr. Similarly, we found promoter activities of human F5 were significantly stimulated by progesterone (P4) via human PGRB. Moreover, a dramatic increase of erythrocyte numbers in capillaries on ovarian follicles was associated with ovulation. Importantly, heparin, an anticoagulant, inhibited ovulation. Furthermore, reduced fecundity and impaired ovulation were observed in f5+/- female zebrafish. Together, our results provide plausible evidence for an exceptional function of coagulation factors in ovulation.

Progestin type affects the increase of heparanase level and procoagulant activity mediated by the estrogen receptor

STUDY QUESTION Does progestin have an effect on heparanase level and procoagulant activity? SUMMARY ANSWER Progestin increases the heparanase level and procoagulant activity via the estrogen receptor and the magnitude of the effect depends on the progestin type. WHAT IS KNOWN ALREADY Users of combined oral contraceptives (COCs) containing third- and fourth-generation progestins have a higher risk of venous thrombosis compared to those employing second-generation progestins. Heparanase protein is capable of degrading heparan sulfate (HS) chains and enhancing activation of the coagulation system. We have previously demonstrated that estrogen enhances the expression and procoagulant activity of heparanase. STUDY DESIGN, SIZE, DURATION Estrogen and progestin receptor positive breast carcinoma cell lines: EMT6, T47D and MCF-7 were compared to the MDA-231 breast carcinoma cell line devoid of these receptors. This observational study incorporated 45 users of third-generation COCs progestins, 21 users of fourth-generation COCs progestins and 28 individuals not using hormonal therapy and not pregnant per history. PARTICIPANTS/MATERIALS, SETTING, METHODS Second-generation progestin—levonorgestrel, third-generation progestin—desogastrel (DSG), an estrogen receptor antagonist—ICI 182.780 and a progestin receptor antagonist—mifepristone, were added to cell lines. Heparanase level and procoagulant activity, HS levels, tissue factor (TF) activity and factor Xa levels were evaluated in the plasma of the study group. MAIN RESULTS AND THE ROLE OF CHANCE Levonorgestrel and DSG increased heparanase levels in the cells and medium. The effect of DSG was more prominent and additive to that of estrogen. The effect was inhibited by ICI 182.780. In the plasma of COC users, heparanase procoagulant activity, HS levels, TF activity and factor Xa levels were significantly higher compared to controls. In COC pills containing the same dose of estrogen, the procoagulant effect of drospirenone was significantly stronger than that of DSG and gestodene. LIMITATIONS, REASONS FOR CAUTION The limitations of the study include a small number of participants in each study group, although the results are statistically significant and evaluated by several different coagulation parameters. WIDER IMPLICATIONS OF THE FINDINGS The study demonstrates a new mechanism through which progestin affects coagulation system activation and shows that this effect is progestin type-dependent. Development of a progestin derivative with an attenuated effect on heparanase procoagulant activity may reduce thrombotic risk. STUDY FUNDING/COMPETING INTEREST(S) No external funding was sought for this study. Y.N. is named in a European patent application No. IL201200027 filed on 18 January 2012. Other authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER N/A.

Nuclear Progestin Receptor Phosphorylation by Cdk9 Is Required for the Expression of Mmp15, a Protease Indispensable for Ovulation in Medaka

Ovulation denotes the discharge of fertilizable oocytes from ovarian follicles. Follicle rupture during ovulation requires extracellular matrix (ECM) degradation at the apex of the follicle. In the teleost medaka, an excellent model for vertebrate ovulation studies, LH-inducible matrix metalloproteinase 15 (Mmp15) plays a critical role during rupture. In this study, we found that follicle ovulation was inhibited not only by roscovitine, the cyclin-dependent protein kinase (CDK) inhibitor, but also by CDK9-inhibitor II, a specific CDK9 inhibitor. Inhibition of follicle ovulation by the inhibitors was accompanied by the suppression of Mmp15 expression in the follicle. In follicles treated with the inhibitors, the formation of the phosphorylated nuclear progestin receptor (Pgr) was inhibited. Roscovitine treatment caused a reduction in the binding of Pgr to the promoter region of mmp15. The expression of Cdk9 and cyclin I (Ccni), and their association in the follicle was demonstrated, suggesting that Cdk9 and Ccni may be involved in the phosphorylation of Pgr in vivo. LH-induced follicular expression of ccni/Ccni was also shown. This study is the first to report the involvement of CDK in ECM degradation during ovulation in a vertebrate species.