Downregulation of ADAMTS3 Suppresses Stemness and Tumorigenicity in Glioma Stem Cell.

AIMS: Glioblastoma multiforme (GBM) is the most aggressive type of human brain tumor, with a poor prognosis and a median overall survival of fewer than 15 months. Glioma stem cells (GSCs) have recently been identified as a key player in tumor initiation and therapeutic resistance in GBM. ADAMTS family of metalloproteinases is known to cleave a wide range of extracellular matrix substrates and has been linked to tissue remodeling events in tumor development. Here, we investigate that ADAMTS3 regulates GSC proliferation and self-renewal activities, and tumorigenesis in orthotopic xenograft models. METHODS: ADAMTS3 mRNA expression levels in normal human astrocyte (NHA), glioma, and GSCs cell lines were compared. After knockdown of ADAMTS3, alamarBlue assay, in vitro limiting dilution, and orthotopic xenograft assays were performed. To investigate the tumor-associated roles of ADAMTS3, several statistical assays were conducted using publicly available datasets. RESULTS: ADAMTS3 level was remarkably higher in GSCs than in NHA, glioma cell lines, and their matched differentiated tumor cells. Interestingly, knockdown of ADAMTS3 disrupted GSC's proliferation, self-renewal activity, and tumor formation in vivo. Furthermore, ADAMTS3 could be used as an independent predictor of malignancy progression in GBM. CONCLUSION: We identified ADAMTS3 as a potential therapeutic target for GBM.

Hypoxic regulation of ADAMTS-2 and -3 (a disintegrin and matrix metalloproteinase with thrombospondin motifs 2 and 3) procollagen N proteinases by HIF-1α in endothelial cells.

ADAMTS-2 and ADAMTS-3, known as procollagen amino proteases (PNP), are primarily responsible for processing the amino ends of the fibrillar collagen precursors. ADAMTS-2 is a highly expressed gene in type I collagen-rich tissues, such as skin, bones, tendons, and aorta. ADAMTS-3 is mainly expressed in cartilage, where it colocalizes with type II procollagen and in the nervous system. Studies about ADAMTS-2 and ADAMTS-3 enzymes primarily focused on their collagen processing activity. Knowledge about the transcriptional regulations of these genes is rather limited. Here we analyzed the transcriptional regulations of ADAMTS-2 and ADAMTS-3 genes under chemically induced hypoxic conditions in endothelial cell model, HUVECs. We elucidated that hypoxia is the potent positive regulator of ADAMTS-2 and ADAMTS-3 genes. qRT-PCR and western blotting studies revealed that ADAMTS-2 and ADAMTS-3 expressions were increased at mRNA and protein levels under chemically induced hypoxic conditions in HUVECs. In addition, Transient transfection experiments of ADAMTS-2 and ADAMTS-3 promoter-reporter constructs indicated that low oxygen conditions increased ADAMTS-2 and ADAMTS-3 promoter activities. Furthermore, the DNA-protein interaction assay provided evidence of the functional binding of HIF-1α on bioinformatically determined HRE regions on the ADAMTS-2 and ADAMTS-3 promoters.

Specific fibroblast subpopulations and neuronal structures provide local sources of Vegfc-processing components during zebrafish lymphangiogenesis.

Proteolytical processing of the growth factor VEGFC through the concerted activity of CCBE1 and ADAMTS3 is required for lymphatic development to occur. How these factors act together in time and space, and which cell types produce these factors is not understood. Here we assess the function of Adamts3 and the related protease Adamts14 during zebrafish lymphangiogenesis and show both proteins to be able to process Vegfc. Only the simultaneous loss of both protein functions results in lymphatic defects identical to vegfc loss-of-function situations. Cell transplantation experiments demonstrate neuronal structures and/or fibroblasts to constitute cellular sources not only for both proteases but also for Ccbe1 and Vegfc. We further show that this locally restricted Vegfc maturation is needed to trigger normal lymphatic sprouting and directional migration. Our data provide a single-cell resolution model for establishing secretion and processing hubs for Vegfc during developmental lymphangiogenesis.

Expression and Preparation of Recombinant Reelin and ADAMTS-3 Proteins.

Reelin is a large secreted protein that is essential for the brain development and function. Reelin is negatively regulated by the specific cleavage by a disintegrin and metalloproteinase with thrombospondin type 1 motifs 3 (ADAMTS-3) which is also secreted from neurons. It is likely that there are other proteases that can cleave Reelin. This chapter describes the protocol for expression and handling of recombinant Reelin and ADAMTS-3 proteins to facilitate investigation of these proteins.

Biallelic mutation of FBXL7 suggests a novel form of Hennekam syndrome.

Hennekam lymphangiectasia-lymphedema syndrome is an autosomal recessive disorder characterized by congenital lymphedema, intestinal lymphangiectasia, facial dysmorphism, and variable intellectual disability. Known disease genes include CCBE1, FAT4, and ADAMTS3. In a patient with clinically diagnosed Hennekam syndrome but without mutations or copy-number changes in the three known disease genes, we identified a homozygous single-exon deletion affecting FBXL7. Specifically, exon 3, which encodes the F-box domain and several leucine-rich repeats of FBXL7, is eliminated. Our analyses of databases representing >100,000 control individuals failed to identify biallelic loss-of-function variants in FBXL7. Published studies in Drosophila indicate Fbxl7 interacts with Fat, of which human FAT4 is an ortholog, and mutation of either gene yields similar morphological consequences. These data suggest that FBXL7 may be the fourth gene for Hennekam syndrome, acting via a shared pathway with FAT4.

An additional case of Hennekam lymphangiectasia-lymphedema syndrome caused by loss-of-function mutation in ADAMTS3.

Hennekam lymphangiectasia-lymphedema syndrome (HKLLS) is a genetically heterogeneous lymphatic dysplasia with characteristic of facial dysmorphism, neurocognitive impairments, and abnormalities of the pericardium, intestinal tract, and extremities. It is an autosomal recessive condition caused by biallelic mutations in CCBE1 (collagen- and calcium-binding epidermal growth factor domain-containing protein 1) (HKLLS1; OMIM 235510) or FAT4 (HKLLS2; OMIM 616006). CCBE1 acts via ADAMTS3 (a disintegrin and metalloprotease with thrombospondin motifs-3 protease) to enhance vascular endothelial growth factor C signaling. There is report of one family supporting mutations in ADAMTS3 as causative for the phenotype labeled as HKLLS3. Here, we report an additional case of HKLLS that appears to be associated with homozygous nonsense mutation of ADAMTS3.

SP1-mediated downregulation of ADAMTS3 gene expression in osteosarcoma models.

ADAMTS3 is a member of procollagen N-proteinase subfamily of ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family. It has an important function in the procollagen maturation process. The removal of N-peptidases is required for the accurate processing of fibrillar collagens. Otherwise, several disorders can occur that is related with the collagenous tissues. ADAMTS3 mainly maturates type II collagen molecule which is the main component of the bone and cartilage. There are several expression studies about ADAMTS3 gene however its transcriptional regulation has not been lightened up, yet. Here we first time cloned and functionally analyzed the promoter region of ADAMTS3 gene, approximately 1380 bp upstream of the transcription start site. Transient transfection experiments showed that all truncated promoter constructs are active and 171 bp fragment is sufficient to activate gene expression in both Saos-2 and MG63 cells. In silico analysis showed that ADAMTS3 has a TATA-less promoter and contains several SP1/GC box binding motifs and a CpG island. Therefore we mainly investigated the SP1 dependent regulation of ADAMTS3 promoter. SP1 downregulated ADAMTS3 transcriptional activity. As consistent with the transcriptional activity, mRNA, and protein expression levels were also decreased by SP1. On the other hand, functional binding of the SP1 on multiple regions of ADAMTS3 promoter was confirmed by EMSA studies. As ADAMTS3 is responsible for the collagen maturation and biosynthesis, further we investigated the effect of SP1 on type I-II and III collagen gene expressions. We point out that SP1 increased type II and III collagen expression and in contrast decreased type I collagen expression levels in Saos-2 cells. mRNA expression level was decreased for all collagen types in MG63 model. Decrease in the type II collagen expression was also demonstrated at the protein level by SP1. Collectively these results provide first findings for the SP1-related transcriptional regulation of ADAMTS3 and collagen genes in osteosarcoma cell lines.

Genetic variants in the metzincin metallopeptidase family genes predict melanoma survival.

Metzincins are key molecules in the degradation of the extracellular matrix and play an important role in cellular processes such as cell migration, adhesion, and cell fusion of malignant tumors, including cutaneous melanoma (CM). We hypothesized that genetic variants of the metzincin metallopeptidase family genes would be associated with CM-specific survival (CMSS). To test this hypothesis, we first performed Cox proportional hazards regression analysis to evaluate the associations between genetic variants of 75 metzincin metallopeptidase family genes and CMSS using the dataset from the genome-wide association study (GWAS) from The University of Texas MD Anderson Cancer Center (MDACC) which included 858 non-Hispanic white patients with CM, and then validated using the dataset from the Harvard GWAS study which had 409 non-Hispanic white patients with invasive CM. Four independent SNPs (MMP16 rs10090371 C>A, ADAMTS3 rs788935 T>C, TLL2 rs10882807 T>C and MMP9 rs3918251 A>G) were identified as predictors of CMSS, with a variant-allele attributed hazards ratio (HR) of 1.73 (1.32-2.29, 9.68E-05), 1.46 (1.15-1.85, 0.002), 1.68 (1.31-2.14, 3.32E-05) and 0.67 (0.51-0.87, 0.003), respectively, in the meta-analysis of these two GWAS studies. Combined analysis of risk genotypes of these four SNPs revealed a decreased CMSS in a dose-response manner as the number of risk genotypes increased (Ptrend < 0.001). An improvement was observed in the prediction model (area under the curve [AUC] = 81.4% vs. 78.6%), when these risk genotypes were added to the model containing non-genotyping variables. Our findings suggest that these genetic variants may be promising prognostic biomarkers for CMSS.

Loss of ADAMTS3 activity causes Hennekam lymphangiectasia-lymphedema syndrome 3.

Primary lymphedema is due to developmental and/or functional defects in the lymphatic system. It may affect any part of the body, with predominance for the lower extremities. Twenty-seven genes have already been linked to primary lymphedema, either isolated, or as part of a syndrome. The proteins that they encode are involved in VEGFR3 receptor signaling. They account for about one third of all primary lymphedema cases, underscoring the existence of additional genetic factors. We used whole-exome sequencing to investigate the underlying cause in a non-consanguineous family with two children affected by lymphedema, lymphangiectasia and distinct facial features. We discovered bi-allelic missense mutations in ADAMTS3. Both were predicted to be highly damaging. These amino acid substitutions affect well-conserved residues in the prodomain and in the peptidase domain of ADAMTS3. In vitro, the mutant proteins were abnormally processed and sequestered within cells, which abolished proteolytic activation of pro-VEGFC. VEGFC processing is also affected by CCBE1 mutations that cause the Hennekam lymphangiectasia-lymphedema syndrome syndrome type1. Our data identifies ADAMTS3 as a novel gene that can be mutated in individuals affected by the Hennekam syndrome. These patients have distinctive facial features similar to those with mutations in CCBE1. Our results corroborate the recent in vitro and murine data that suggest a close functional interaction between ADAMTS3 and CCBE1 in triggering VEGFR3 signaling, a cornerstone for the differentiation and function of lymphatic endothelial cells.

Rare and low-frequency coding variants alter human adult height.

Height is a highly heritable, classic polygenic trait with approximately 700 common associated variants identified through genome-wide association studies so far. Here, we report 83 height-associated coding variants with lower minor-allele frequencies (in the range of 0.1-4.8%) and effects of up to 2 centimetres per allele (such as those in IHH, STC2, AR and CRISPLD2), greater than ten times the average effect of common variants. In functional follow-up studies, rare height-increasing alleles of STC2 (giving an increase of 1-2 centimetres per allele) compromised proteolytic inhibition of PAPP-A and increased cleavage of IGFBP-4 in vitro, resulting in higher bioavailability of insulin-like growth factors. These 83 height-associated variants overlap genes that are mutated in monogenic growth disorders and highlight new biological candidates (such as ADAMTS3, IL11RA and NOX4) and pathways (such as proteoglycan and glycosaminoglycan synthesis) involved in growth. Our results demonstrate that sufficiently large sample sizes can uncover rare and low-frequency variants of moderate-to-large effect associated with polygenic human phenotypes, and that these variants implicate relevant genes and pathways.

ADAMTS-3, -13, -16, and -19 levels in patients with habitual abortion.

A disintegrin-like and metalloproteinase domain with thrombospondin-type 1 motifs (ADAMTS) protein superfamily includes 19 secreted metalloproteases. Proteolytic substrates of ADAMTS enzymes have been linked to reproductive function. The aim of this study was to investigate serum ADAMTS-3, -13, -16, and -19 levels in women with habitual abortions compared with those in healthy controls. A total of 86 women were enrolled in this prospective case-control study. ADAMTS-3, -13, -16, and -19 values were recorded and analyzed in association with demographic and clinical parameters. There were no statistically significant differences between the two groups in terms of demographics. No statistically significant differences were observed between the groups with regard to ADAMTS-13 and -19 levels (p>0.05). However, ADAMTS-3 and -16 were significantly higher in the study group than in the control group (p=0.004 and p=0.005, respectively). To estimate habitual abortions using an area under receiver operating characteristic curve analysis, the cutoff values for ADAMTS-3 and -16 were found to be 87.28 ng/mL (sensitivity, 64.44%; specificity 68.29%) and 15.75 ng/mL (sensitivity, 66.67%; specificity 68.29%), respectively. In conclusion, the pregnancy-loss rate seems to be affected by both ADAMTS-3 and -16.

Proteolytic activation defines distinct lymphangiogenic mechanisms for VEGFC and VEGFD.

Lymphangiogenesis is supported by 2 homologous VEGFR3 ligands, VEGFC and VEGFD. VEGFC is required for lymphatic development, while VEGFD is not. VEGFC and VEGFD are proteolytically cleaved after cell secretion in vitro, and recent studies have implicated the protease a disintegrin and metalloproteinase with thrombospondin motifs 3 (ADAMTS3) and the secreted factor collagen and calcium binding EGF domains 1 (CCBE1) in this process. It is not well understood how ligand proteolysis is controlled at the molecular level or how this process regulates lymphangiogenesis, because these complex molecular interactions have been difficult to follow ex vivo and test in vivo. Here, we have developed and used biochemical and cellular tools to demonstrate that an ADAMTS3-CCBE1 complex can form independently of VEGFR3 and is required to convert VEGFC, but not VEGFD, into an active ligand. Consistent with these ex vivo findings, mouse genetic studies revealed that ADAMTS3 is required for lymphatic development in a manner that is identical to the requirement of VEGFC and CCBE1 for lymphatic development. Moreover, CCBE1 was required for in vivo lymphangiogenesis stimulated by VEGFC but not VEGFD. Together, these studies reveal that lymphangiogenesis is regulated by two distinct proteolytic mechanisms of ligand activation: one in which VEGFC activation by ADAMTS3 and CCBE1 spatially and temporally patterns developing lymphatics, and one in which VEGFD activation by a distinct proteolytic mechanism may be stimulated during inflammatory lymphatic growth.

[Preparation of rabbit polyclonal antibody against mouse ADAMTS2].

OBJECTIVE: To obtain recombinant mouse collagenase ADAMTS2 (ADAM metallopeptidase with thrombospondin type 1 motif, 2) C terminal (1109-1213), and prepare the corresponding rabbit anti-ADAMTS2 polyclonal antibodies. METHODS: The recombinant expression plasmid pGEX-6p-1-ADAMTS2 (1109-1213) was transformed into E.coli. The target protein was induced by IPTG and identified by mass spectrometry following affinity purification. The expressed and purified ADAMTS2 (1109-1213) protein was used to immunize New Zealand rabbits to prepare anti-ADAMTS2 polyclonal antibodies. The antibody titers were detected by ELISA and the antibody specificity by Western blotting. RESULTS: The protein ADAMTS2 (1109-1213) was expressed in E.coli after IPTG induction, and with the purified protein, we prepared antiserum in the immunized rabbits. The titer of the antiserum reached over 1:160 000. The antiserum showed a good specificity. CONCLUSION: The high titer and specific rabbit anti-ADAMTS2 antibody has been prepared successfully.

Fluid-induced, shear stress-regulated extracellular matrix and matrix metalloproteinase genes expression on human annulus fibrosus cells.

BACKGROUND: Mechanical loading plays an important role in the regulation of extracellular matrix (ECM) homeostasis as well as pathogenesis of intervertebral disc (IVD) degeneration. The human annulus fibrosus (hAF) in the IVD is subjected to contact shear stress during body motion. However, the effects of shear stress on hAF cells remain unclear. This aim of the study was to investigate the expression of the ECM (COLI, COLIII and aggrecan) and matrix metalloproteinase (MMP-1, MMP-3 and ADAMTS-4) genes in hAF cells following fluid-induced shear stress in a custom-fabricated bio-microfluidic device. METHODS: hAF cells were harvested from degenerated disc tissues in routine spine surgery, staged by magnetic resonance imaging, expanded in monolayers and then seeded onto the bio-microfluidic device. The experimental groups were subjected to 1 and 10 dyne/cm(2) shear stress for 4 h, and no shear stress was applied to the control group. We used real time polymerase chain reaction for gene expression. RESULTS: Shear stress of 1 dyne/cm(2) exerted an anabolic effect on COLI and COLIII genes and catabolic effects on the aggrecan gene, while 10 dyne/cm(2) had an anabolic effect on the COLI gene and a catabolic effect on COLIII and aggrecan genes. The COLI gene was upregulated in a stress-dependent manner. Expression of MMP-1 was significantly higher in the 10 dyne/cm(2) group compared to the control group (P < 0.05), but was similar in the control and 1 dyne/cm(2) groups. Expression of MMP-3 and ADAMTS-4 were similar in all three groups. CONCLUSION: Taken together, hAF cells responded to shear stress. The findings help us understand and clarify the effects of shear stress on IVD degeneration as well as the development of a new therapeutic strategy for IVD degeneration.

Progesterone acts via the progesterone receptor to induce adamts proteases in ovarian cancer cells.

BACKGROUND: Ovarian carcinomas, usually associated with sex hormones dysregulation, are the leading cause of gynecological neoplastic death. In normal ovaries, hormones play a central role in regulating cell proliferation, differentiation, and apoptosis. On the other hand, hormonal alterations also play a variety of roles in cancer. Stimulation by sex hormones potentially affects gene expression, invasiveness, cell growth and angiogenesis. Proteases of the "a disintegrin and metalloproteinase with thrombospondin motifs" (ADAMTS) family are secreted by different cell types and become involved in collagen processing, cleavage of the proteoglycan matrix, and angiogenesis. We evaluated whether sex hormones affect ADAMTS 1 and 4 expression in ovarian cancer cells. METHODS: We analysed mRNA and protein levels in human ovarian tumor cells with different degrees of malignancy, NIH-OVCAR-3 and ES-2, that were treated or not with estrogen, testosterone and progesterone. RESULTS: Our results suggest that progesterone increases ADAMTS protein and mRNA levels in the lysates from ES-2 cells, and it increases ADAMTS protein in the lysates and conditioned media from NIH-OVCAR-3. Progesterone effects were reversed by RU486 treatment. CONCLUSION: We conclude that progesterone acts via the progesterone receptor to modulate ADAMTS 1 and 4 levels in ovarian cancer cell lines.

Determination of the substrate repertoire of ADAMTS2, 3, and 14 significantly broadens their functions and identifies extracellular matrix organization and TGF-ß signaling as primary targets.

A disintegrin and metalloproteinase with thrombospondin type I motif (ADAMTS)2, 3, and 14 are collectively named procollagen N-proteinases (pNPs) because of their specific ability to cleave the aminopropeptide of fibrillar procollagens. Several reports also indicate that they could be involved in other biological processes, such as blood coagulation, development, and male fertility, but the potential substrates associated with these activities remain unknown. Using the recently described N-terminal amine isotopic labeling of substrate approach, we analyzed the secretomes of human fibroblasts and identified 8, 17, and 22 candidate substrates for ADAMTS2, 3, and 14, respectively. Among these newly identified substrates, many are components of the extracellular matrix and/or proteins related to cell signaling such as latent TGF-ß binding protein 1, TGF-ß RIII, and dickkopf-related protein 3. Candidate substrates for the 3 ADAMTS have been biochemically validated in different contexts, and the implication of ADAMTS2 in the control of TGF-ß activity has been further demonstrated in human fibroblasts. Finally, the cleavage site specificity was assessed showing a clear and unique preference for nonpolar or slightly hydrophobic amino acids. This work shows that the activities of the pNPs extend far beyond the classically reported processing of the aminopropeptide of fibrillar collagens and that they should now be considered as multilevel regulators of matrix deposition and remodeling.-Bekhouche, M., Leduc, C., Dupont, L., Janssen, L., Delolme, F., Vadon-Le Goff, S., Smargiasso, N., Baiwir, D., Mazzucchelli, G., Zanella-Cleon, I., Dubail, J., De Pauw, E., Nusgens, B., Hulmes, D. J. S., Moali, C., Colige, A. Determination of the substrate repertoire of ADAMTS2, 3, and 14 significantly broadens their functions and identifies extracellular matrix organization and TGF-ß signaling as primary targets.

MiR-29a and MiR-140 Protect Chondrocytes against the Anti-Proliferation and Cell Matrix Signaling Changes by IL-1ß.

As a degenerative joint disease, osteoarthritis (OA) constitutes a major cause of disability that seriously affects the quality of life of a large population of people worldwide. However, effective treatment that can successfully reverse OA progression is lacking until now. The present study aimed to determine whether two small non-coding RNAs miR-29a and miR-140, which are significantly down-regulated in OA, can be applied together as potential therapeutic targets for OA treatment. MiRNA synergy score was used to screen the miRNA pairs that potentially synergistically regulate OA. An in vitro model of OA was established by treating murine chondrocytes with IL-1ß. Transfection of miR-29a and miR-140 via plasmids was investigated on chondrocyte proliferation and expression of nine genes such as ADAMTS4, ADAMTS5, ACAN, COL2A1, COL10A1, MMP1, MMP3, MMP13 and TIMP metal-lopeptidase inhibitor 1 (TIMP1). Western blotting was used to determine the protein expression level of MMP13 and TIMP1, and ELISA was used to detect the content of type II collagen. Combined use of miR-29a and miR-140 successfully reversed the destructive effect of IL-1ß on chondrocyte proliferation, and notably affected the MMP13 and TIMP1 gene expression that regulates extracellular matrix. Although co-transfection of miR-29a and miR-140 did not show a synergistic effect on MMP13 protein expression and type II collagen release, but both of them can significantly suppress the protein abundance of MMP13 and restore the type II collagen release in IL-1ß treated chondrocytes. Compared with single miRNA transfection, cotransfection of both miRNAs exceedingly abrogated the suppressed the protein production of TIMP1 caused by IL-1ß, thereby suggesting potent synergistic action. These results provided novel insights into the important function of miRNAs' collaboration in OA pathological development. The reduced MMP13, and enhanced TIMP1 protein production and type II collagen release also implies that miR-29a and miR-140 combination treatment may be a possible treatment for OA.

Suppressive effect of Sanmiao formula on experimental gouty arthritis by inhibiting cartilage matrix degradation: An in vivo and in vitro study.

Sanmiao formula (SM) is a compound prescription, which has been used in traditional Chinese medicine since the Ming Dynasty for gouty and rheumatoid arthritis treatments. However, no evidence has been unfolded to show the relationship between SM and gouty arthritis (GA), particularly inhibiting cartilage matrix degradation. In the present study, we undertook a characterization of anti-GA activity of SM using an in vivo rat model induced by potassium oxonate and cold bath together with in vitro studies with chondrocytes for further molecular characterization. Potassium oxonate and cold bath rats were treated with SM at doses of 7.2g/kg per day for 5days. SM treatments significantly suppressed the swelling rate and the severe pathologic changes in the joints of the animals in gout model. Inflammatory factors count by ELISA analysis, SM exhibited inhibition on IL-1ß and TNF-α. Moreover, histological analysis of the joints and SM-serum substantially interfered with the MSU-induced expression of glycosaminoglycans (GAG), up-regulated the content of proteoglycan. Importantly, SM interfered with GA-augmented expression of matrix metalloproteinases (MMPs) -3 and aggrecanases (ADAMTS)-4, which are considered to be key enzymes in cartilage matrix degradation, and simultaneously augmented GA-reduced tissue inhibitors of metalloproteinases (TIMPs) -1 and -3 expression in the joints and chondrocytes. Therefore, SM is looking forward to be a potential novel agent that could prevent cartilage matrix degradation effectively in gouty arthritis, and this provides a new target for development of new medicines.

Ephrin-A5 Is Required for Optimal Fertility and a Complete Ovulatory Response to Gonadotropins in the Female Mouse.

Follicle growth and ovulation involve the coordinated expression of many genes, driven by FSH and LH. Reports indicate that Eph receptors and ephrins are expressed in the ovary, suggesting roles in follicle growth and/or ovulation. We previously reported FSH-induced expression of ephrin-A5 (EFNA5) and 4 of its cognate Eph receptors in mouse granulosa cells. We now report that female mice lacking EFNA5 are subfertile, exhibit a compromised response to LH, and display abnormal ovarian histology after superovulation. Efna5(-/-) females litters were 40% smaller than controls, although no difference in litter frequency was detected. The ovarian response to superovulation was also compromised in Efna5(-/-) females, with 37% fewer oocytes ovulated than controls. These results corresponded with a reduction in ovarian mRNA levels of several LH-responsive genes, including Pgr, Ptgs2, Tnfaip6, Ereg, Btc, and Adamts4, suggesting that Efna5(-/-) ovaries exhibit a partially attenuated response to LH. Histopathological analysis indicated that superovulated Efna5(-/-) females exhibited numerous ovarian defects, including intraovarian release of cumulus oocyte complexes, increased incidence of oocytes trapped within luteinized follicles, granulosa cell and follicular fluid emboli, fibrin thrombi, and interstitial hemorrhage. In addition, adult Efna5(-/-) ovaries exhibited a 4-fold increase in multioocyte follicles compared with controls, although no difference was detected in 3-week-old mice, suggesting the possibility of follicle merging. Our observations indicate that loss of EFNA5 in female mice results in subfertility and imply that Eph-ephrin signaling may also play a previously unidentified role in the regulation of fertility in women.