Abstract
A predominant gelatinolytic enzyme with approximately 26 kDa was observed in gelatin zymogram of immature mice uterine luminal fluid (ULF). Size exclusion analysis revealed that the native size of this enzyme was close to that of human α2-macroglobulin (α2-MG), a 725 kDa protein. This large protease was isolated by a series of chromatographic steps on the Sephacryl S-400 and DEAE-Sepharose columns. The results from gelatin zymography and SDS-PAGE analysis supported that this large protease consists of gelatinolytic enzyme and a 360 kDa protein. Through tandem mass spectrometry analysis followed by MASCOT database search, the 360 kDa protein was identified as ovostatin homolog (accession: NP_001001179.2) assigned as a homolog of chicken ovostatin, a protease inhibitor. The co-fractionation analysis by gel filtration and mouse ovostatin homolog (mOH) co-immunoprecipitation experiments demonstrated that the mOH formed a complex with three gelatinolytic enzymes in immature mice ULF. Substrate zymography analysis revealed that the mOH-associated gelatinolytic enzymes were suitable to digest type I collagen rather than type IV collagen. In addition, the refolded mOH-associated 26 kDa gelatinolytic enzyme displayed the type I collagen-digesting activity in the assay, but the other two enzymes did not have this function. RT-PCR analysis showed that mOH gene was abundantly expressed in brain, spinal cord, lung, uterus, and in 17-day embryo. Taken together, our data suggest that mOH/cognate protease system may play a potential role in regulation of tissue remodeling and fetal development.
Highlights
In mice, the uterine endometrium undergoes extensive remodeling during estrous cycle
To ask whether extracellular matrix (ECM)-degrading enzymes are secreted by endometrial cells for tissue remodeling, gelatin zymography of immature mice uterine luminal fluid (ULF) was performed (Fig. 1A)
The understanding of estrogen influences on endometrial remodeling in mice was based on the study of immature mice ULF gelatinolytic enzymes
Summary
The uterine endometrium undergoes extensive remodeling during estrous cycle. Several lines of evidence had exhibited to support the contention that MMPs were key players at endometrial breakdown and repair[19,20] This argument was not supported by mouse model studies[21], raising the possibility that the unidentified collagen-degrading enzymes may exist in mouse endometrium to www.nature.com/scientificreports/. The collection of a great deal of mature mouse ULF from the specific stages was difficult It is well-known that the stimulation of immature mice by estrogen or its analogues results in endometrial growth accompanying with a remarkable increase in ULF26–28, providing a good experimental system to isolate or purify the estrogen-stimulated gelatinolytic enzymes for functional characterization and antibody production. Our findings provided the basis for unveiling and understanding the function of mOH in ECM remodeling
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
