How to Create an Embryo Penetration Route
How to Create an Embryo Penetration Route
Hiroshi Uchida,Tetsuo Maruyama,Hirotaka Masuda,Sayaka Uchida,Fumie Miki,Hanako Hihara, Satomi Katakura,Yushi Yoshimasa,Mamoru Tanaka
Department of Obstetrics and Gynecology,Keio University School of Medicine,Tokyo,Japan
Keywords
Apoptosis,epithelial to mesenchymal transition,endometrial epithelial cell,motility, steroid hormone
Correspondence
Hiroshi Uchida,Department of Obstetrics and Gynecology,Keio University School of Medicine,35Shinanomachi,Shinjuku,Tokyo 160-8582,Japan.
E-mail:uchida@a5.keio.jp
Submission October1,2015;
accepted December8,2015.
Citation
Uchida H,Maruyama T,Masuda H,Uchida S, Miki F,Hihara H,Katakura S,Yoshimasa Y, Tanaka M.How to create an embryo penetration route.Am J Reprod Immunol 2016
doi:10.1111/aji.12476Numerous past investigations into human implantation have tended to pay attention to the mechanism of embryo adhesion to endometrial cells or embryo invasion into endometrial stromal tissue.For successful preg-nancy,however,embryo penetration through the endometrial epithelial cell(EEC)sheet is also absolutely required.To improve the performance of assisted reproductive technology,implantation studies should also focus on EEC dynamics,in particular,the action of EECs during embryo penetration.Although only EEC apoptosis has been proposed as a mech-anism for the formation of the embryo penetration route,we have also recently reported that characteristic EEC collective motion,regulated by the epithelial to mesenchymal transition,also plays a key role in permit-ting embryo penetration.We review here how EECs form embryo pene-tration routes in light of our?ndings.
Introduction
During the four decades since the?rst successful pregnancy and delivery from in vitro fertilization and embryo transfer(IVF-ET)in1978,1the number of cases of IVF-ET has increased every year in Japan.2 Despite the tremendous progress in the advancement of IVF techniques,the pregnancy rate from IVF has plateauefd at approximately30–35%on average for all ages,largely because of major loss at the embryo transfer step.Unveiling the mechanisms of coordi-nated dynamics between embryo and maternal uter-ine endometrium is therefore valuable key to improve implantation rate.Past implantation studies have tended to focus on adhesion between the embryo and endometrial tissue or embryo invasion into endometrial stromal tissue.The embryo is required to pass through the endometrial epithelial barrier for successful pregnancy;however,the question of how the embryo penetration route is created is still not clearly solved.To avoid the ethical and technical limitations in investigation of human implantation,an assay with which to observe and analyze implantation in vitro has been developed.3,4 This assay is based on the co-culture of embryonic and endometrial cells.Although human blastocysts cannot form spheroids,human chorionic gonadotro-pin-secreting choriocarcinoma cell lines,such as JAR,Jeg-3,and BeWo,form spheroids in shake cul-ture and are used as models for embryos.3Con?uent monolayers of human endometrial adenocarcinoma cell lines including Ishikawa,RL95-2,and Hec1B are used as endometrial epithelial cell(EEC)models.4,5 Among them,Ishikawa cells6are the most relevant model due to their expression of ovarian steroid hor-mone receptors for both of estrogen and proges-terone,and appropriate polar structure.5The in vitro implantation assays using cell lines are a quantitative
and high throughput tool and,moreover,are also adequate for real-time observation and recording because the dynamic change can be traced if?uores-cent dyes are used.7–9We review here how EECs form embryo penetration routes in light of our?nd-ings using in vitro implantation assays.
Embryo penetration
Human implantation initially involves crosstalk between the embryo and the EEC sheet,and the sequential embryo-driven events are divided into four steps as follows:(i)apposition against EECs; (ii)adhesion to EECs;(iii)penetration through the EEC barrier;and(iv)invasion to endometrial stro-mal cell(ESC)-containing tissue(Fig.1a).For suc-cessful pregnancy,embryo penetration through the EEC sheet is obviously required.The EEC sheet is also important as a barrier in the defense against pathogen invasion and for maintenance of the tis-sue structure.Therefore,an artful and carefully regulated mechanism is necessary to deconstruct the EEC sheet and create a route for embryo pen-etration.In fact,the step-by-step sequence of human implantation can be reconsidered from the viewpoint of EEC:(i)apposition and(ii)adhesion to blastocyst,(iii)local destruction of the EEC sheet,and(iv)reconstruction of the EEC barrier sheet(Fig.1a).Although numerous studies on human implantation have already focused on embryo adhesion to EECs and ESCs,or embryo invasion into ESCs,it is thought that the detailed investigation into EEC sheet dynamics during embryo penetration will provide keys to the improvement of pregnancy rates using assisted reproductive technologies.
Implantation is frequently compared to the sequential process of leukocyte rolling and penetra-tion through blood vessel epithelial cell sheets (Fig.1b).10,11There is certainly a lot of overlap in the mechanisms,including,for instance,the multi-ple steps from apposition to penetration utilizing paracrine chemokine networks,and integrin-based adhesion systems.Note that leukocyte penetration is a simple single-cell migration,but embryo penetra-tion is massive corrective cell migration.It is pre-dicted that specialized mechanisms exist to provide a route for embryo penetration through the EEC sheet,unlike the paracellular or transcellular migra-tion of leukocytes(Fig.1).Apoptosis
Animal studies demonstrate that EEC apoptosis occurs at the embryo–EEC interface in mouse,12 rat,13and monkey.14In human EECs,15,16it has also been reported that apoptosis is provoked by embryo implantation through binding between trophoblast FasL and EEC FasR.17Moreover,using in vitro implantation assays,the human endometrial adeno-carcinoma cell line RL95-2(EEC model)has been found to induce apoptosis,in accordance with adhe-sion to spheroids of the human choriocarcinoma cell line BeWo(embryo model).17
Apoptosis of EECs is meaningful not only for the creation of embryo penetration route through cell death,but also because it provides additional effects through destruction of the EEC body.During apop-tosis,self-derived components including nucleotides are released from degraded apoptotic cells,and the immune system is activated via sensors such as Toll-like receptors,through interaction with host nucleo-tides.18The host cell-derived nucleotide UDP-glucose and its receptor P2RY14,which is present on the surface of EECs,play important roles in the protec-tion of female reproductive organs through activa-tion of the immune system.19Human EEC or Ishikawa cells secrete IL-8through the stimulation of UDP-glucose via its receptor P2RY14in a time-and dose-dependent manner in vitro.Furthermore, neutrophil migration increases in response to the addition of UDP-glucose and/or co-culture with Ishi-kawa cells,and this increase is completely abrogated by anti IL-8antibody.19
Salamonsen and colleagues20reported that IL-8is expressed in human EECs but not ESCs and the expression level of IL-8peaks at menstruation phase and mentioned that IL-8is important for reconstruc-tion of endometrial tissue.IL-8is expressed in human EECs,but not ESCs in vivo,and is secreted from human endometrium,but not human embryo in in vitro co-culture.21Furthermore,the expression of IL-8in EECs is upregulated and the secretion from EECs is not changed by the addition of proges-terone or co-cultured with embryo.21
Taken together,UDP-glucose diffused from the degraded host cell binds to its receptor P2Y14 expressed in neighboring live EECs and induces secretion of IL-8from the living EECs,?nally recruit-ing immune cells to the destroyed EEC sheet in situ. This chain reaction is adequate for endometrial
American Journal of Reproductive Immunology(2016)
tissue repair in the case of menstruation and the initial step in immunological defense against destruc-tion of the EEC barrier sheet by mechanical or biological attack;at the same time,it is also under-stood as an ‘apoptosis-linked implantation assistance system’.Cell motion
Is apoptosis the most effective system for enabling embryos to penetrate the EEC barrier?Even if EECs undergo apoptosis,each apoptotic EEC occupies its original space.After the apoptotic phase in the local EEC barrier,it is probably necessary that the appro-priate innate immune system is activated and that dead EECs are cleared out by degradation and phagocytosis.For effective formation of an embryo penetration route,instead of an apoptosis-based sys-tem,including removal of apoptotic cells,it would appear that a more ef?cient system would be operat-ing in the EEC sheet during implantation.
Given that EECs characteristically move away from the site of adhesion to the embryo,EEC sheets can prepare an embryo penetration route more quickly than does apoptosis.We have indeed
succeeded in real-time recording of EEC motion away from embryo model in in vitro implantation assay.9The human genome is packaged with histone proteins.22–24The acetylation status of histone pro-teins is reversibly regulated by histone acetylases and histone deacetylases.Histone acetylation creates an electrical repulsion force between histone pro-teins because of negatively charged acetylated resi-dues and permits easy access of transcription factors to gene with reduced histone density;therefore,his-tone deacetylases inhibitors can arti?cially affect gene transcription and cell functions,including cell proliferation,22differentiation,25adhesion,8and motility.26Treatment with ovarian steroid hormones (17b estradiol plus progesterone)or one of the his-tone deacetylase inhibitors,such as suberoylanilide hydroxamic acid (SAHA),enhances single-cell migration and collective cell migration of EECs 26in association with unique motion away from the embryo.9After penetration,the embryo invades endometrial stromal tissue.Like EECs,ESCs also migrate away from the implantation site in vitro .27Characteristic descending migration of EECs (and ESCs)is a rational dynamic pattern for assistance with cell apoptosis to aid embryo
penetration.
Embryo
Endometrium
EECs
ESCs Embryo
Embryo
Embryo
Embryo
Reconstruction
Destruction
Apposition Adhesion Leukocyte
Leukocyte
Leukocyte
Leukocyte Leukocyte
Endothelial cells
Adhesion Apposition Invasion
Penetration Rolling
Apposition
Crawling
Adhesion
Transendothelial Migration Paracellular
Transcellular
a
b
Fig.1Sequential steps of adhesion and migration between ?oating cells and cell sheets.Apicobasally polarized cells generally do not adhere to any other cells at their apical sides.Both endometrial epithelial cells (a)and epithelial cells of blood vessels (b)are exceptions,permitting adherence and migrating in response to incoming ?oating cells,such as embryos and leukocytes.The sequential step-by-step mechanism from apposition to invasion is similar in both cases;however,it should be noted that the migrating masses are of different sizes (embryo as collective cell mass versus leukocyte as a single cell).
EMT
To maintain homeostasis and to enable development and function of tissues,transitions between immotile (epithelial)and motile(mesenchymal)states are essential.In general,epithelial cells are thought to be immotile,whereas steady state cells and mes-enchymal cells are motile.In addition to cell motil-ity,there are discriminating differences between epithelial and mesenchymal cells.The epithelial to mesenchymal transition(EMT)is a phenomenon in which epithelial cells lose cell polarity,change char-acteristic cytoskeleton structures,and acquire mes-enchymal properties,involving a highly motile character,and is observed in limited situations,such as tissue remodeling in primary development and wound repair(Fig.2).EMT can be seen in patholog-ical conditions,such as malignant cell invasion or metastasis.28,29Mesenchymal epithelial transition (MET)is almost the reverse phenomenon. Epithelial cells contact neighboring cells laterally with a variety of cell–cell junctions,such as tight junctions,adherens junctions,gap junctions,and desmosomes.In particular,at adherens junctions,epithelial cells connect to neighboring cells with homodimeric binding of the adhesion molecule E-cadherin(epithelial type cadherin).During EMT, the major adhesion molecule at adherens junctions is changed from E-cadherin to N-cadherin(neuron type cadherin),and this characteristic alteration in EMT is called the‘cadherin switch’.As a result of the dissolution of cell–cell contact structures,includ-ing the cadherin switch,epithelial cells lose cell polarity(Fig.2).Although epithelial cells create tis-sue structure via cell–cell contacts at their lateral sides and cell-extracellular matrix contacts at their basal sides,connection to other cells at the apical surface is not permitted because of poor expression of adhesion molecules.28,29Loss of polarity causes redistribution of adhesion molecules at cell surfaces and makes adhesion between EECs and the embryo possible.For EECs,loss of polarity is necessary for acquiring receptivity.Through the EMT,cytoskeletal structures,which de?ne cell shape and polarity,are also dramatically changed.The major intermediate ?laments are cytokeratin in epithelial cells and vimentin in mesenchymal cells.Apicobasally polar-ized?lamentous actin cytoskeleton(cortical actin)
in
Tight junction
Adherens junction
(E-cadherin-dependent)
Desmosome
Focal adhesion
Cortical actin
Actin stress fiber
Epithelial Mesenchymal Transition
(EMT)
Epithelial cells Mesenchymal cells
Adherens junction
(N-cadherin-dependent)
Fig.2Characteristic change during the epithelial mesenchymal transition.Barrier-forming epithelial cells and motile mesenchymal cells are compared.Characteristic differences in cell polarity,junction structures,and cytoskeletons are shown as a pattern diagram.During the epithelial to mesenchymal transition,these cell characteristics are switched.
American Journal of Reproductive Immunology(2016)
epithelial cells is broken in association with the destruction of cell –cell contact structures,and bun-dled actin stress ?bers are formed and anchored at focal adhesions,as in mesenchymal cells,during EMT (Fig.2).28,29Ishikawa cells are actually observed to ?atten and undergo widespread mor-phological change 25and actin rearrangement 9in response to ovarian steroid hormones or SAHA,even without embryo implantation.Loss of polarity and actin rearrangement is also observed in in vitro implantation assays using Ishikawa cells.30
Despite the fact that the EEC sheet is a rigid barrier and a mechanical and immunological defensive structure,real-time microscopy reveals that EECs temporarily and continuously change cell form and exhibit swing-like motion within con?uent monolayer cultures.Although cytoker-atin and vimentin are frequently used as marker proteins for EECs and ESCs,respectively,endome-trial adenocarcinoma cells and normal EECs both express vimentin at various levels according to the stage of the menstrual cycle.31,32From these obser-vations,it seems that EMT that transiently and locally occurs in EECs needs to be rearranged,besides primary development,wound repair,and cancer metastasis.
Endometrial epithelial cell motion is needed dur-ing implantation to generate an embryo penetration route,as mentioned above.Time-course microscopic observation can detect EEC migration away from the embryo implantation point,and the descending
motion of EEC is enhanced by treatment with ovar-ian steroid hormones and SAHA.9
The expression of vimentin in Ishikawa cells is upregulated by ovarian steroid hormones or SAHA,and the upregulation is signi?cantly ampli?ed by co-culture with JAR spheroids (embryo model),in particular around spheroids.9In cultured Ishikawa cells,upregulation of E-cadherin expression and downregulation of N-cadherin (cadherin switch)can also be detected.9Immuno?uorocytochemistry stud-ies reveal that the cadherin switch phenomenon is signi?cantly provoked near JAR spheroids.9More-over,induced and enhanced cadherin switching is abrogated by the addition of N-cadherin function-blocking antibody.9Conclusions
Taking these results together,we can create the fol-lowing story of human implantation (Fig.3).(i)Prior to arrival of the embryo,ovarian steroid hor-mones trigger EMT in EECs and cause loss of polar-ity and rearrangement of cell surface adhesion molecules to acquire receptivity.(ii)Apposition and adhesion of the embryo to EECs accelerates and maintains the EMT via an unknown outside-in sig-nal (probably binding of adhesion molecules).(iii)EMT results in EEC motility in a descending direc-tion from the implantation site and forms the embryo penetration route in association with cell apoptosis.(iv)Motile EECs resulting from the
EMT
Fig.3Hypothetical model showing Epithelial to mesenchymal transition (EMT)-based regulation of human implantation.Reversible EMT-derived dynamic regulation of endometrial epithelial cells (EECs)is depicted.The EMT can purposely provide receptivity and motility to EEC and can reasonably assist in creating the embryo penetration route.The numbers in the ?gure support it in the ‘Conclusion’part of the text.
move in the ascending direction to occupy the vacant space after embryo penetration.(v)After completion of embryo penetration,the EMT is grad-ually dissolved in accordance with the disappearance of the EMT-maintaining signal from the embryo, and MET occurs in EECs.After MET,to complete reconstruction of the EEC sheet,immotile EECs pro-liferate.Although the EMT is probably essential for implantation,it is still not known why EECs can migrate away,avoiding the rule of contact inhibition of cell locomotion that motile cells stop moving when in contact with other cells,and what mecha-nisms regulate the reversible direction of EEC motion.
Acknowledgements
A part of this review article was presented at the Delhi congress on‘Embryo Implantation and Preg-nancy:Intricacies and Strategies for its Success’.We thank Prof.Satish Gupta(Deputy Director,National Institute of Immunology,New Delhi,India)for pro-viding the chance to present this work as a guest speaker.
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