Role of PCI in fertility

Protein C inhibitor (PCI) is a nonspecific, heparin-binding serine protease inhibitor (serpin) that inactivates many plasmatic and extravascular serine proteases by forming stable inactive 1:1 complexes. Proteases inhibited by PCI include the anticoagulant activated protein C, the plasminogen activators urokinase (uPA) and tissue plasminogen activator (tPA), and the sperm protease acrosin. In humans PCI circulates as a plasma protein but is also present at high concentrations in organs of the male reproductive tract. The co-localization of high concentrations of PCI together with several of its target proteases in the male reproductive tract suggests a role of PCI in reproduction. Within our previous FWF project (P13452-BIO) we generated mice lacking PCI (PCI -/- mice). PCI-/- mice were apparently healthy but the males were infertile. The Sertoli cell barrier in these mice was disrupted and the spermatogenesis was abnormal. The resulting sperm were malformed and were morphologically similar to abnormal sperm seen in some cases of human male infertility. Within the proposed project we would like to further elucidate reasons for this infertility. In order test the possibility that observed changes in the PCI deficient males were caused by the unopposed proteolytic activity, established double knockout mice lacking both PCI and one of its target proteases, e.g. uPA, tPA or acrosin, respectively, will be tested with respect to their fertilities. Additionally, their reproductive organs will be characterized with respect to their morphology and proteolytic activities. Since lately accomplished histological analyses revealed that PCI during the embryonic development is not expressed in testes (in contrast to the adult mice), we shall correlate the observed changes in morphology of the testes and sperms in the PCI knockouts with the developmental expression of PCI. In adult mice, precise localization of the PCI in the mice male reproductive tract of the wild-types will be determined by immunohistochemistry. Taking into account a disruption of a blood testis-barrier in PCI-knockouts, PCI knockout males will be checked on the presence/absence of the anti-sperm antibodies. Additionally, forms of PCI in the testis (active, cleaved, creating complexes with proteases) will be analyzed within a proposal. The analysis of the interaction of germ cells with Sertoli cells ex vivo will be performed in order to analyze the mechanism(s) responsible for the disruption of the Sertoli cell barrier and the Sertoli cell-germ cell junctions in PCI-deficient mice. Successful solution of the proposed project should further elucidate the mechanism of the male fertility due to PCI deficiency. We believe that the outcome of this animal model might be relevant for analyzing the molecular bases of human infertility using a large population of infertile patients in the future.

Since many physiological processes in mice and human are analogous, the knowledge obtained in a mouse model can be applied to human physiology and pathological conditions. Using an embryonic stem (ES) cells gene technology, it became possible to deactivate specific genes in mice, thus obtaining relevant information about the function of the gene of interest. Previously we generated mice lacking PCI gene, designated as PCI-/- mice. Such mice are apparently healthy but that males of this genotype are infertile. As we could show, infertility of these mice is apparently caused by abnormal development of the sperm due to destruction of the blood-testis cell barrier, perhaps due to unopposed proteolytic activity of proteases present in the testis. Such results are consistent with previously accomplished studies showing that assembly and disassembly of Sertoli cells is associated with transient induction of proteases, such as urokinase (uPA) and tissue plasminogen activators (tPA) and protease inhibitors, and that their activities must be coordinated to maintain the integrity of the blood-testis barrier. Therefore we generated double knockout mice lacking PCI and either uPA or tPA and analyzed them in detail. We found that the absence of uPA or tPA did not revert infertility of PCI-/- males. Additionally, we accomplished histological analyses revealing that PCI during the embryonic development is not expressed in testes (in contrast to the adult mice) and we correlated the observed morphological changes of the testes and sperms in the PCI knockouts with the developmental expression of PCI. We also determined subcellular the localization of PCI antigen in the male reproductive tract of wild-type mice. The pattern of PCI expression suggested involvement in migration of spermatogenic cells in the basal compartment (i.e. where the blood testis barrier is located) and in maturation of spermatids in the apical compartment of the walls of seminiferous tubules. In summary, generated animal model might be useful for analyzing the molecular bases of human infertilities and elucidation of the role of PCI in human reproduction might lead to the development of new strategies to treat such human infertilities.