Inhibition of embryo implantation in mice through vaginal administration of a proprotein convertase 6 inhibitor

Uterine proprotein convertase 6 (PC6) plays a critical role in embryo implantation in both mice and women. It was hypothesized that inhibiting uterine PC6 could prevent pregnancy. Vaginal administration of a PC6 inhibitor presents the ideal route for local drug delivery. A peptide-based PC6 inhibitor, C-30k-PEG Poly R that was previously shown to have properties of increased vaginal absorption and penetration was tested for its contraceptive potential in mice following vaginal administration. The study demonstrated that this approach could inhibit embryo implantation in some mice (24% completely and 47% partially inhibited).     

Subtilisin proprotein convertase-6 expression in the mouse uterus during implantation and artificially induced decidualization

During implantation, a balance of factors regulates the invasive properties of the embryo and the anti-invasive properties of uterine decidua. Although antiproteinases such as the metalloproteinase inhibitor TIMP-3 are thought to play critical roles in preventing the overaggressive invasion of trophoblasts, the mechanism of antiproteinase regulation is unknown. Recently, the prohormone convertase SPC-6 has been found to be co-expressed in embryo-proximal decidua in association with TIMP-3. As members of this serine proteinase family are known to activate latent TGFbeta family members which regulate decidual TIMP-3 levels, we sought to characterize the expression of SPC-6 during pregnancy and artificial decidualization. In this study, we demonstrate that the zone of SPC-6 gene expression exhibits a great degree of temporal and spatial overlap with TIMP-3 gene expression in uterine decidua from E5.5 through to E8.5. Like TIMP-3, we demonstrate that SPC-6 expression is induced during the decidual cell response using an in vivo model of artificial decidualization. Both the secreted and membrane bound forms of SPC-6 are expressed throughout the period of decidualization, suggesting that SPC-6 may play multiple roles during this developmental period. This is confirmed by our observation of the movement of SPC-6 expression to the presumptive placental region, as TIMP-3 expression regresses at the implantation site.     

Inhibiting uterine PC6 blocks embryo implantation: an obligatory role for a proprotein convertase in fertility

Successful embryo implantation involves complex interactions between the embryo and the uterus and is critical in establishing pregnancy. Proprotein convertase (PC) 6 (PC6) is one of the PC endoproteases regulating protein function through posttranslational activation of precursor proteins, including growth and differentiation factors. Here we show that PC6 protein is induced in the uterine stromal cells specifically at the site of embryo attachment during early pregnancy in mice. In vivo blocking of uterine production of PC6 protein using morpholino antisense oligonucleotides in mice resulted in total inhibition of implantation, revealing a vital role for PC6 in modulating the uterus for embryo implantation. Studies in primates (rhesus monkey and human) showed a dramatic upregulation of endometrial PC6 during the phase of uterine receptivity and at implantation, particularly during a critical uterine cell differentiation process termed decidualization. Thus, the current studies have demonstrated that PC6 is an essential molecule in modulating uterine function to support the establishment of embryo implantation. Interestingly, PC6 is one of the PCs identified to be important in processing the coat protein of HIV; inhibition of PCs has been suggested to be an effective approach to reduce HIV transmission. We therefore propose the novel concept that PC6 could be a potential nonhormonal target in the female reproductive tract for dual protection for women, both in preventing pregnancy and reducing HIV infection.     

Embryo implantation is closely associated with dynamic expression of proprotein convertase 5/6 in the rabbit uterus

Background  

Proprotein convertase 5/6 (PC5/6) is critical for embryo implantation in women, regulating both uterine epithelial receptivity and stromal cell decidualization. PC5/6 is likewise essential for implantation in mice, but involved only in decidualization. An alternative animal model is required to address the function of PC5/6 in the uterine epithelium. This study aimed to establish whether PC5/6 is associated with embryo implantation in rabbits.     

MicroRNA expression and regulation in mouse uterus during embryo implantation

MicroRNAs (miRNAs) are 21-24-nucleotide non-coding RNAs found in diverse organisms. Although hundreds of miRNAs have been cloned or predicted, only very few miRNAs have been functionally characterized. Embryo implantation is a crucial step in mammalian reproduction. Many genes have been shown to be significantly changed in mouse uterus during embryo implantation. However, miRNA expression profiles in the mouse uterus between implantation sites and inter-implantation sites are still unknown. In this study, miRNA microarray was used to examine differential expression of miRNAs in the mouse uterus between implantation sites and inter-implantation sites. Compared with inter-implantation sites, there were 8 up-regulated miR-NAs at implantation sites, which were confirmed by both Northern blot and in situ hybridization. miR-21 was highly expressed in the subluminal stromal cells at implantation sites on day 5 of pregnancy. Because miR-21 was not detected in mouse uterus during pseudopregnancy and under delayed implantation, miR-21 expression at implantation sites was regulated by active blastocysts. Furthermore, we showed that Reck was the target gene of miR-21. Our data suggest that miR-21 may play a key role during embryo implantation.     

GRP78 expression and regulation in the mouse uterus during embryo implantation

The aim of this study was to investigate the spatiotemporal expression and regulation of GRP78 in the mouse uterus during the peri-implantation period. The GRP78 protein was mainly detected in the luminal and glandular epithelia on days 1–4 of pregnancy. On day 5 of pregnancy, the GRP78 protein was more highly observed around the implanted embryo at the implantation site. There was no detectable GRP78 protein signal on day 5 of pseudopregnancy. GRP78 mRNA and protein levels gradually increased on days 6–8 of pregnancy, and the expression pattern was also expanded, coinciding with the development of decidua. Similarly, GRP78 expression was also strongly expressed in decidualised cells following artificial decidualisation. Compared with the results obtained with the delayed uterus, a high level of GRP78 expression was detected in the implantation-activated uterus. In the uteri of ovariectomised mice, GRP78 expression increased and reached its highest level after injection of oestrogen, and progesterone seemed to have an antagonistic effect on oestrogen up-regulation of GRP78 expression. Our data indicate that GRP78 might play an important role during the process of mouse embryo implantation, and GRP78 expression was mainly regulated by active blastocysts and maternal oestrogen.     

Limited redundancy of the proprotein convertase furin in mouse liver

Furin is an endoprotease of the family of mammalian proprotein convertases and is involved in the activation of a large variety of regulatory proteins by cleavage at basic motifs. A large number of substrates have been attributed to furin on the basis of in vitro and ex vivo data. However, no physiological substrates have been confirmed directly in a mammalian model system, and early embryonic lethality of a furin knock-out mouse model has precluded in vivo verification of most candidate substrates. Here, we report the generation and characterization of an interferon inducible Mx-Cre/loxP furin knock-out mouse model. Induction resulted in near-complete ablation of the floxed fur exon in liver. In sharp contrast with the general furin knock-out mouse model, no obvious adverse effects were observed in the transgenic mice after induction. Histological analysis of the liver did not reveal any overt deviations from normal morphology. Analysis of candidate substrates in liver revealed complete redundancy for the processing of the insulin receptor. Variable degrees of redundancy were observed for the processing of albumin, alpha(5) integrin, lipoprotein receptor-related protein, vitronectin and alpha(1)-microglobulin/bikunin. None of the tested substrates displayed a complete block of processing. The absence of a severe phenotype raises the possibility of using furin as a local therapeutic target in the treatment of pathologies like cancer and viral infections, although the observed redundancy may require combination therapy or the development of a more broad spectrum convertase inhibitor.     

Identification of a functional proprotein convertase cleavage site in microfibril-associated glycoprotein 2

Microfibril-associated glycoprotein 2 (MAGP2) is a secreted protein associated with multiple cellular activities including the organization of elastic fibers in the extracellular matrix (ECM), angiogenesis, as well as regulating Notch and integrin signaling. Importantly, increases in MAGP2 positively correlate with poor prognosis for some ovarian cancers. It has been assumed that full-length MAGP2 is responsible for all reported effects; however, here we show MAGP2 is a substrate for the proprotein convertase (PC) family of endoproteases. Proteolytic processing of MAGP2 by PC cleavage could serve to regulate secretion and thus, activity and function as reported for other extracellular and cell-surface proteins. In support of this idea, MAGP2 contains an evolutionarily conserved PC consensus cleavage site, and amino acid sequencing of a newly identified MAGP2 C-terminal cleavage product confirmed functional PC cleavage. Additionally, mutagenesis of the MAGP2 PC consensus cleavage site or treatment with PC inhibitors prevented MAGP2 proteolytic processing. Finally, both cleaved and uncleaved MAGP2 were detected extracellularly and MAGP2 secretion appeared independent of PC cleavage, suggesting that PC processing occurs mainly outside the cell. Our characterization of alternative forms of MAGP2 present in the extracellular space not only enhances diversity of this ECM protein but also provides a previously unrecognized molecular mechanism for regulation of MAGP2 biological activity.     

Peroxisome proliferator-activated receptor delta expression and regulation in mouse uterus during embryo implantation and decidualization

The aim of this study was to examine the expression and regulation of peroxisome proliferator-activated receptor (PPAR) PPARdelta gene in mouse uterus during early pregnancy by in situ hybridization and immunohistochemistry. PPARdelta expression under pseudopregnancy, delayed implantation, hormonal treatment, and artificial decidualization was also investigated. There was a very low level of PPARdelta expression on days 1-4 of pregnancy. On day 5 when embryo implanted, PPARdelta expression was exclusively observed in the subluminal stroma surrounding the implanting blastocyst. No corresponding signals were seen in the uterus on day 5 of pregnancy. There was no detectable PPARdelta signal under delayed implantation. Once delayed implantation was terminated by estrogen treatment and embryo implanted, a strong level of PPARdelta expression was induced in the subluminal stroma surrounding the implanting blastocyst. Estrogen treatment induced a moderate level of PPARdelta expression in the glandular epithelium, while progesterone treatment had no effects in the ovariectomized mice. A strong level of PPARdelta expression was seen in the decidua on days 6-8 of pregnancy. PPARdelta expression was also induced under artificial decidualization. These data suggest that PPARdelta expression at implantation sites require the presence of an active blastocyst and may play an essential role for blastocyst implantation.     

Alpha-tocopherol transfer protein is specifically localized at the implantation site of pregnant mouse uterus

Alpha-tocopherol transfer protein (alpha-TTP) was first described to play a major role in maintaining alpha-tocopherol levels in plasma, while alpha-tocopherol was primarily reported to be a factor relevant for reproduction. Expression of alpha-TTP is not only seen in the liver, from where it was first isolated, but also in mouse uterus, depending on its state of pregnancy, stressing the importance of alpha-TTP for embryogenesis and fetal development. The cellular localization of alpha-TTP in mouse uterus is reported here. By immunohistochemistry, alpha-TTP could be localized in the secretory columnar epithelial cells of the pregnant uterus on Days 4.5 and 6.5 postcoitum as well as in the glandular epithelial cells and the inner decidual reaction zone surrounding the implantation site. On Days 8.5 and 10.5 postcoitum (midterm of mouse pregnancy), alpha-TTP could still be detected in the uterine secretory columnar epithelial cells, while in alpha-TTP knockout mice, no immunostaining was visible. It is suggested that alpha-TTP plays a major role in supplying the placenta and consecutively the fetus with alpha-tocopherol throughout pregnancy. We conclude that alpha-tocopherol plays a role in the process of implantation and that alpha-TTP may be necessary for adequate alpha-tocopherol status of the fetus.     

Expression of Ly-6A/E in the mouse uterus during implantation period

To investigate the molecular mechanisms of implantation, we constructed a complementary DNA library of mouse uterus enriched with pregnancy-induced genes by subtractive hybridization and polymerase chain reaction. One of the isolated clones was a part of complementary DNA for the Ly-6A/E. Ly-6A/E is reported to be differentially expressed on hematopoietic stem cells and some lymphoid and nonlymphoid tissues, mediate cell-cell adhesion on lymphoid cells, and associate with cell proliferation and angiogenesis of tumor cells. Northern blot, in situ hybridization, and immunohistochemical analyses demonstrated that the Ly-6A/E mRNA and protein were expressed in the endometrial epithelial cells as well as myometrial cells and vascular endothelial cells in the uterus of nonpregnant mouse. The expression was downregulated in luminal epithelial cells during pregnancy days 1-5, while it was upregulated in decidualized stromal cells around the implanted embryo at the time of implantation. The signals were primarily localized in stromal cells at the mesometrial pole on day 9. The increased expression was also observed in stromal cells of the embryo-transferred uterus and artificially-induced deciduoma, indicating that the expression of Ly-6A/E in the endometrial cells is concurrent with decidualization. These findings suggest that Ly-6A/E plays a role in embryo implantation.     

Imaging proprotein convertase activities and their regulation in the implanting mouse blastocyst

Axis formation and allocation of pluripotent progenitor cells to the germ layers are governed by the TGF-β–related Nodal precursor and its secreted proprotein convertases (PCs) Furin and Pace4. However, when and where Furin and Pace4 first become active have not been determined. To study the distribution of PCs, we developed a novel cell surface–targeted fluorescent biosensor (cell surface–linked indicator of proteolysis [CLIP]). Live imaging of CLIP in wild-type and Furin- and Pace4-deficient embryonic stem cells and embryos revealed that Furin and Pace4 are already active at the blastocyst stage in the inner cell mass and can cleave membrane-bound substrate both cell autonomously and nonautonomously. CLIP was also cleaved in the epiblast of implanted embryos, in part by a novel activity in the uterus that is independent of zygotic Furin and Pace4, suggesting a role for maternal PCs during embryonic development. The unprecedented sensitivity and spatial resolution of CLIP opens exciting new possibilities to elucidate PC functions in vivo.