RESUMEN
Embryo implantation is a key step in establishing pregnancy and a major limiting factor in IVF. Implantation requires a receptive endometrium but the mechanisms governing receptivity are not well understood. We have recently discovered that podocalyxin (PCX or PODXL) is a key negative regulator of human endometrial receptivity. PCX is expressed in all endometrial epithelial cells in the non-receptive endometrium but selectively down-regulated in the luminal epithelium at receptivity. We have further demonstrated that this down-regulation is essential for implantation because PCX inhibits embryo attachment and penetration. However, how PCX confers this role is unknown. In this study, through RNAseq analysis of Ishikawa cell line stably overexpressing PCX, we discovered that PCX suppresses expression of genes controlling cell adhesion and communication, but increases those governing epithelial barrier functions, especially the adherens and tight junctions. Moreover, PCX suppresses multiple factors such as LIF and signaling pathways including Wnt and calcium signaling that support receptivity but stimulates anti-implantation genes such as LEFTY2. Functional studies confirmed that PCX promotes epithelial barrier functions by increasing key epithelial junction proteins such as E-cadherin and claudin 4. PCX thus promotes an anti-adhesive and impermeable epithelium while impedes pro-implantation factors to negatively control endometrial receptivity for implantation.
Asunto(s)
Endometrio/metabolismo , Células Epiteliales/metabolismo , Sialoglicoproteínas/metabolismo , Antígenos CD/metabolismo , Cadherinas/metabolismo , Implantación del Embrión , Femenino , Humanos , Inflamación/metabolismo , Factores de Determinación Derecha-Izquierda/metabolismo , EmbarazoRESUMEN
STUDY QUESTION: How is endometrial epithelial receptivity, particularly adhesiveness, regulated at the luminal epithelial surface for embryo implantation in the human? SUMMARY ANSWER: Podocalyxin (PCX), a transmembrane protein, was identified as a key negative regulator of endometrial epithelial receptivity; specific downregulation of PCX in the luminal epithelium in the mid-secretory phase, likely mediated by progesterone, may act as a critical step in converting endometrial surface from a non-receptive to an implantation-permitting state. WHAT IS KNOWN ALREADY: The human endometrium must undergo major molecular and cellular changes to transform from a non-receptive to a receptive state to accommodate embryo implantation. However, the fundamental mechanisms governing receptivity, particularly at the luminal surface where the embryo first interacts with, are not well understood. A widely held view is that upregulation of adhesion-promoting molecules is important, but the details are not well characterized. STUDY DESIGN, SIZE, DURATION: This study first aimed to identify novel adhesion-related membrane proteins with potential roles in receptivity in primary human endometrial epithelial cells (HEECs). Further experiments were then conducted to determine candidates' in vivo expression pattern in the human endometrium across the menstrual cycle, regulation by progesterone using cell culture, and functional importance in receptivity using in vitro human embryo attachment and invasion models. PARTICIPANTS/MATERIALS, SETTING, METHODS: Primary HEECs (n = 9) were isolated from the proliferative phase endometrial tissue, combined into three pools, subjected to plasma membrane protein enrichment by ultracentrifugation followed by proteomics analysis, which led to the discovery of PCX as a novel candidate of interest. Immunohistochemical analysis determined the in vivo expression pattern and cellular localization of PCX in the human endometrium across the menstrual cycle (n = 23). To investigate whether PCX is regulated by progesterone, the master driver of endometrial differentiation, primary HEECs were treated in culture with estradiol and progesterone and analyzed by RT-PCR (n = 5) and western blot (n = 4). To demonstrate that PCX acts as a negative regulator of receptivity, PCX was overexpressed in Ishikawa cells (a receptive line) and the impact on receptivity was determined using in vitro attachment (n = 3-5) and invasion models (n = 4-6), in which an Ishikawa monolayer mimicked the endometrial surface and primary human trophoblast spheroids mimicked embryos. Mann-Whitney U-test and ANOVA analyses established statistical significance at *P ≤ 0.05 and **P ≤ 0.01. MAIN RESULTS AND THE ROLE OF CHANCE: PCX was expressed on the apical surface of all epithelial and endothelial cells in the non-receptive endometrium, but selectively downregulated in the luminal epithelium from the mid-secretory phase coinciding with the establishment of receptivity. Progesterone was confirmed to be able to suppress PCX in primary HEECs, suggesting this hormone likely mediates the downregulation of luminal PCX in vivo for receptivity. Overexpression of PCX in Ishikawa monolayer inhibited not only the attachment but also the penetration of human embryo surrogates, demonstrating that PCX acts as an important negative regulator of epithelial receptivity for implantation. LIMITATIONS, REASONS FOR CAUTION: Primary HEECs isolated from the human endometrial tissue contained a mixture of luminal and glandular epithelial cells, as further purification into subtypes was not possible due to the lack of specific markers. Future study would need to investigate how progesterone differentially regulates PCX in endometrial epithelial subtypes. In addition, this study used primary human trophoblast spheroids as human embryo mimics and Ishikawa as endometrial epithelial cells in functional models, future studies with human blastocysts and primary epithelial cells would further validate the findings. WIDER IMPLICATIONS OF THE FINDINGS: The findings of this study add important new knowledge to the understanding of human endometrial remodeling for receptivity. The identification of PCX as a negative regulator of epithelial receptivity and the knowledge that its specific downregulation in the luminal epithelium coincides with receptivity development may provide new avenues to assess endometrial receptivity and individualize endometrial preparation protocols in assisted reproductive technology (ART). The study also discovered PCX as progesterone target in HEECs, identifying a potentially useful functional biomarker to monitor progesterone action, such as in the optimization of progesterone type/dose/route of administration for luteal support. STUDY FUNDING/COMPETING INTEREST(S): Study funding was obtained from ESHRE, Monash IVF and NHMRC. LR reports potential conflict of interests (received grants from Ferring Australia; personal fees from Monash IVF Group and Ferring Australia; and non-financial support from Merck Serono, MSD, and Guerbet outside the submitted work. LR is also a minority shareholder and the Group Medical Director for Monash IVF Group, a provider of fertility preservation services). The remaining authors have no potential conflict of interest to declare. TRIAL REGISTRATION NUMBER: NA.
Asunto(s)
Implantación del Embrión , Células Endoteliales , Australia , Endometrio , Células Epiteliales , Femenino , Humanos , SialoglicoproteínasRESUMEN
Embryo implantation requires a healthy embryo and a receptive endometrium (inner lining of the uterus); endometrial receptivity acquisition involves considerable epithelial surface remodeling. Dystroglycan (DG), a large cell surface glycoprotein, consists of α- and ß-subunits; ß-DG anchors within the plasma membrane whereas α-DG attaches extracellularly to ß-DG. The glycosylated central α-DG mediates adhesion, but it is obstructed by its large N terminus (α-DG-N); α-DG-N removal enables DG's adhesive function. We demonstrate here that full-length α-DG in the human endometrial epithelium is a barrier for embryo attachment and that removal of α-DG-N by proprotein convertase 5/6 (PC6; a protease critical for implantation) regulates receptivity. This was evidenced by: 1) α-DG contains a PC6-cleavage site near α-DG-N, and PC6 cleaves a peptide harboring such a site; 2) PC6 knockdown reduces α-DG-N removal from endometrial epithelial cell surface and blastocyst adhesion; 3) mutating the PC6-cleavage site prevents α-DG-N removal, causing cell surface retention of full-length α-DG and loss of adhesiveness; 4) α-DG-N is removed from endometrial tissue in vivo for receptivity and uterine fluid α-DG-N reflects tissue removal and receptivity. We thus identified α-DG-N removal as an important posttranslational control of endometrial receptivity and uterine fluid α-DG-N as a potential biomarker for receptivity in women.
Asunto(s)
Distroglicanos/metabolismo , Implantación del Embrión/fisiología , Endometrio/metabolismo , Proproteína Convertasa 5/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Proteolisis , Blastocisto/citología , Blastocisto/metabolismo , Línea Celular , Distroglicanos/genética , Endometrio/citología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Epitelio/metabolismo , Femenino , Humanos , Proproteína Convertasa 5/genética , Estructura Terciaria de ProteínaRESUMEN
Decidualization is a tissue remodelling process within the uterus in preparation for embryo implantation and pregnancy. In this study we isolated primary human endometrial stromal cells and stimulated decidualization with cAMP. We then used 2D- differential in-gel electrophoresis (DIGE) to identify proteins induced by decidualization. Eighty-eight out of 2714 spots were differentially regulated, 18 of which were assigned clear identities by mass spectrometry. Many of these are proteins known to be associated with cell structure and cytoskeletal remodelling. We validated five of these proteins by Western blot and immunohistochemistry on human endometrial tissue. The validated proteins are caldesmon 1, src substrate contactin 8, tropomyosin alpha-4 chain, protein disulfide isomerase 1A, and LIM and SH3 domain protein. With the exception of caldesmon 1, none of the identified proteins have previously been associated with decidualization. This study provides insight into our understanding of decidualization, which is important for successful embryo implantation and establishment of pregnancy.
Asunto(s)
Proteínas del Citoesqueleto/análisis , Endometrio/citología , Proteómica/métodos , Células del Estroma/química , Células Cultivadas , AMP Cíclico/farmacología , Proteínas del Citoesqueleto/metabolismo , Electroforesis en Gel Bidimensional , Femenino , Humanos , Espectrometría de Masas , Embarazo , ÚteroRESUMEN
GHTD-amide is a tetrapeptide originally isolated from human urine that has hypoglycemic activity. Insulin occurs in secretory granules of beta cells as zinc-stabilized hexamers and must disperse to monomeric form in order to bind to its receptor. The aim of this study was to identify whether GHTD-amide and an analog called ISF402 (VHTD-amide) reduce blood glucose through enhancement of insulin activity by dispersing oligomers of insulin. Peptides containing the HTD-amide sequence and a free alpha-amino group were optimal at binding Zn(2+) and adopting secondary structure in the presence of Zn(2+). Binding was concentration dependent and resulted in a 1:1 Zn:peptide complex. In vitro the tetrapeptides dispersed hexameric insulin to dimers and monomers. GHTD-amide and ISF402 potentiated the activity of hexameric insulin when co-injected into insulin resistant Zucker rats. Injection of peptides with insulin caused reductions in blood glucose and C-peptide significantly larger than achieved with insulin alone, and serum insulin time profiles were also altered consistent with a reduced clearance or enhanced dispersal of the injected insulin. Insulin potentiation by ISF402 was reduced when lispro insulin, which does not form zinc-stabilized hexamers, was used in place of hexameric zinc insulin. In conclusion, GHTD-amide and ISF402 are zinc binding peptides that disperse hexameric insulin in vitro, and potentiate the activity of hexameric insulin more so than monomeric lispro insulin. These results suggest that dispersal of hexameric insulin through chelation of Zn(2+) contributes to the hypoglycemic activity of these tetrapeptides.