Implantation-competent Blastocyst Research Articles

Basic Research Advances in China on Embryo Implantation, Placentation, and Parturition

Abstract This review aimed to summarize the major progress in maternal-fetal medicine achieved by Chinese scientists in recent years. PubMed was systematically searched from January 2020 to November 2023. Publications that reported the progress in embryo implantation, placentation, and parturition made by Chinese scientists in the last 3 years were selected. The milestone events during gestation, embryo implantation, endometrial decidualization, placentation, and parturition are pivotal to a successful pregnancy. Embryo implantation requires intricate interactions between implantation-competent blastocysts and receptive endometrium. To adapt to pregnancy, endometrial stromal cells transform into specialized decidual cells, which occur spontaneously under the influence of ovarian hormones in humans but require the presence of embryos in mice. With embryonic development, the placenta forms to support fetal growth until parturition. The maternal-fetal interface is composed of diverse cell types, including endometrial decidual cells, placental trophoblast cells, endothelial cells, and various immune cells, a sophisticated interplay among which contributes to the maintenance of pregnancy. Near term, the uterus transitions from quiescence to contractility, in preparation for delivery. Disruptions to these events lead to pregnancy-related disorders such as repeated implantation failure, recurrent pregnancy loss, preeclampsia, fetal growth restriction, preterm birth, and infertility. In recent years, Chinese scientists have made prominent achievements in basic research on the aforementioned pregnancy events. Chinese scientists have made remarkable contributions to reproductive biology and maternal-fetal medicine research in recent years, highlighting future research directions in this field.

Protease secretions by the invading blastocyst induce calcium oscillations in endometrial epithelial cells via the protease-activated receptor 2

BackgroundEarly embryo implantation is a complex phenomenon characterized by the presence of an implantation-competent blastocyst and a receptive endometrium. Embryo development and endometrial receptivity must be synchronized and an adequate two-way dialogue between them is necessary for maternal recognition and implantation. Proteases have been described as blastocyst-secreted proteins involved in the hatching process and early implantation events. These enzymes stimulate intracellular calcium signaling pathways in endometrial epithelial cells (EEC). However, the exact molecular players underlying protease-induced calcium signaling, the subsequent downstream signaling pathways and the biological impact of its activation remain elusive.MethodsTo identify gene expression of the receptors and ion channels of interest in human and mouse endometrial epithelial cells, RNA sequencing, RT-qPCR and in situ hybridization experiments were conducted. Calcium microfluorimetric experiments were performed to study their functional expression.ResultsWe showed that trypsin evoked intracellular calcium oscillations in EEC of mouse and human, and identified the protease-activated receptor 2 (PAR2) as the molecular entity initiating protease-induced calcium responses in EEC. In addition, this study unraveled the molecular players involved in the downstream signaling of PAR2 by showing that depletion and re-filling of intracellular calcium stores occurs via PLC, IP3R and the STIM1/Orai1 complex. Finally, in vitro experiments in the presence of a specific PAR2 agonist evoked an upregulation of the ‘Window of implantation’ markers in human endometrial epithelial cells.ConclusionsThese findings provide new insights into the blastocyst-derived protease signaling and allocate a key role for PAR2 as maternal sensor for signals released by the developing blastocyst.Graphical

DNA Methylation and Histone Modification Are the Possible Regulators of Preimplantation Blastocyst Activation in Mice.

Under ovarian hormone control, dormant blastocysts obtain implantation capacity (known as blastocyst activation) through their global gene expression. After the activated blastocysts communicate with the receptive uterus, the implantation-competent blastocysts start the implantation. Although dormant and activated blastocysts have different gene expression levels, the regulatory mechanisms underlying these transcriptions remain unclear. Hence, this study aimed to analyze epigenetic marks in dormant and activated blastocysts. In mice, blastocyst dormancy is artificially induced by daily progesterone injection without estrogen supplementation after peri-implantation ovariectomy; when estrogen is administered concomitantly, blastocyst activation and implantation occur. These phenomena demonstrate a mouse model of delayed implantation. We collected dormant and activated blastocysts from a delayed implantation mouse model. RNA-seq, methylated DNA immunoprecipitation (MeDIP)-seq, and chromatin immunoprecipitation (ChIP)-seq for H3K4 me3 and H3K27 me3 were performed using dormant and activated blastocysts. Cell cycle-related transcripts were affected during blastocyst activation. DNA methylations were accumulated in downregulated genes in the activated blastocysts. Histone H3 trimethylations were globally altered between the dormant and activated blastocysts. Dormant and activated blastocysts have unique methylation patterns on DNA and histone H3, with high correlation to gene expression. DNA methylation and histone modification can regulate preimplantation blastocyst activation.

Uterine Epithelial LIF Receptors Contribute to Implantation Chamber Formation in Blastocyst Attachment.

Recent studies have demonstrated that the formation of an implantation chamber composed of a uterine crypt, an implantation-competent blastocyst, and uterine glands is a critical step in blastocyst implantation in mice. Leukemia inhibitory factor (LIF) activates signal transducer and activator of transcription 3 (STAT3) precursors via uterine LIF receptors (LIFRs), allowing successful blastocyst implantation. Our recent study revealed that the role of epithelial STAT3 is different from that of stromal STAT3. However, both are essential for blastocyst attachment, suggesting the different roles of epithelial and stromal LIFR in blastocyst implantation. However, how epithelial and stromal LIFR regulate the blastocyst implantation process remains unclear. To investigate the roles of LIFR in the uterine epithelium and stroma, we generated Lifr-floxed/lactoferrin (Ltf)-iCre (Lifr eKO) and Lifr-floxed/antimüllerian hormone receptor type 2 (Amhr2)-Cre (Lifr sKO) mice with deleted epithelial and stromal LIFR, respectively. Surprisingly, fertility and blastocyst implantation in the Lifr sKO mice were normal despite stromal STAT3 inactivation. In contrast, blastocyst attachment failed, and no implantation chambers were formed in the Lifr eKO mice with epithelial inactivation of STAT3. In addition, normal responsiveness to ovarian hormones was observed in the peri-implantation uteri of the Lifr eKO mice. These results indicate that the epithelial LIFR-STAT3 pathway initiates the formation of implantation chambers, leading to complete blastocyst attachment, and that stromal STAT3 regulates blastocyst attachment without stromal LIFR control. Thus, uterine epithelial LIFR is critical to implantation chamber formation and blastocyst attachment.

Metabolic Changes of Maternal Uterine Fluid, Uterus, and Plasma during the Peri-implantation Period of Early Pregnancy in Mice.

Embryo implantation is a complex process which involves biochemical and physiological interactions between an implantation-competent blastocyst and a receptive uterus. However, the exact biochemical changes of uterine fluid, uterus, and plasma during peri-implantation remain unclear. This study aims to characterize the biochemical and metabolic changes that occur during the peri-implantation period of early pregnancy, using mice as an animal model. Gas chromatography-mass spectrometry was used to analyze the metabolite profiles of the uterus, uterine fluid, and maternal plasma at pre-implantation and implantation. The multivariate analyses, ANOVA and Tukey's HSD test, were applied to detect significant changes in metabolites and metabolic pathways. The metabolic networks were reconstructed in silico based on the identified metabolites and KEGG metabolic framework. Between pre-implantation day 1 and day 4, dramatic metabolic changes were observed in the uterine fluid that could be important for blastocyst development and protection against the harsh uterine environment. Palmitoleic acid, fumaric acid, and glutaric acid changed levels at day 4 in the uterus, suggesting that they may be associated with endometrial receptivity. Both the uterus and maternal plasma showed profound changes in cellular metabolism at the early implantation period, including upregulation of branched-chain amino acids and intermediates of one-carbon metabolism, an upregulation of glyoxylate and dicarboxylate metabolism, and downregulation of aerobic respiration; all of which could be involved in the regulation of the maternal-fetal interface, alternative nutrient utilization, and energy preservation for implantation as well as later placentation and fetal development to ensure successful embryo implantation.

Uterine deficiency of high-mobility group box-1 (HMGB1) protein causes implantation defects and adverse pregnancy outcomes.

A reciprocal communication between the implantation-competent blastocyst and the receptive uterus is essential to successful implantation and pregnancy success. Progesterone (P4) signaling via nuclear progesterone receptor (PR) is absolutely critical for pregnancy initiation and its success in most eutherian mammals. Here we show that a nuclear protein high-mobility group box-1 (HMGB1) plays a critical role in implantation in mice by preserving P4-PR signaling. Conditional deletion of uterine Hmgb1 by a Pgr-Cre driver shows implantation defects accompanied by decreased stromal cell Hoxa10 expression and cell proliferation, two known signatures of inefficient responsiveness of stromal cells to PR signaling in implantation. These mice evoke inflammatory conditions with sustained macrophage accumulation in the stromal compartment on day 4 of pregnancy with elevated levels of macrophage attractants Csf1 and Ccl2. The results are consistent with the failure of exogenous P4 administration to rescue implantation deficiency in the mutant females. These early defects are propagated throughout the course of pregnancy and ultimately result in substantial subfertility. Collectively, the present study provides evidence that nuclear HMGB1 contributes to successful blastocyst implantation by sustaining P4-PR signaling and restricting macrophage accumulation to attenuate harmful inflammatory responses.

Role of maternal folic acid on implantation of embryos and on maternal body weight & offspring's birth weight

Introduction There has been a much debate on the role of pre-/periconceptional and prenatal folic acid (FA) supplements for the long-term health of their offspring. Maternal deficiency of FA or excess of FA would likely to affect the developing fetus at critical stages of development to cause long-lasting health consequences in their offspring. The present study aimed to know the effect of folic acid supplementation and folic acid absence diet during pregnancy on the number of implantation, endometrial changes at the implantation site and on maternal body weight & offspring's birth weight. Material & Methods Thirty-Six Wistar strained female rats were approved for experimental use by the Institutional Animal Ethical Committee. Rats were randomly divided into two groups with 18 rats in each group. Further, each group has been divided into three sets with 6 rats in each. The control set was fed a normal diet, the second set was fed a diet containing no FA. 1% Succinyl sulfathiazole was added to the diet with no FA content to reduce/ inhibit the gut flora responsible for the synthesis of FA in their body. The third set was given diet with an excess of FA (40mg/kg body weight/day). Rats were given respective diet for 4 weeks before mating, continued throughout their pregnancy and 5 weeks after the delivery of pups (weening period). All female rats were paired with males of their own strain overnight at room temperature (25°C). To check whether they mated or not, the vaginal smear was taken in the early morning and stained with methylene blue and confirmed the presence of sperm plug under microscopy. On confirmation of sperm plug, the gestational day was termed as day 1 (GD1). Rats have remained in their cage and diets until they were sacrificed during gestation from day GD6-GD9. Rats were remained in their cage and with their respective diets until they delivered the pups. Delivered pups were counted for each rat and gross malformation in each pup was noted. Immediately after the delivery, all the pups born to each rat were weighed individually in the digital weighing machine. Results Average number of pups born to pregnant dams fed with FA supplementation, FA absent diet, and in normal diet was found to be 8,8, and 9 respectively. Regarding the birth weight of pups born to pregnant dams fed with FA supplementation the average weight was found to be 6.01-5.09 gm, with evidence diet it was 5.51-5.08 gm, and least weight was observed in pups born with FA absent diet (4.74-3.31 gm). Pair-wise comparison was done to correlate the significant association between the different groups. It showed significant difference when compared with pups born to FA supplementation diet to pups born to FA absent diet. Also, significant differences were noted when compared pups born to FA absent diet mother with pups born to FA supplementation and with pups born to normal diet. Conclusion Our study provides clear evidence that FA is essential in successful implantation by providing favorable receptive environment to receive the implantation-competent blastocyst for a successful pregnancy. Birth weight of pups born to pregnant dams fed with FA supplementation was found to be significantly increased when compared to those with the diet with normal amount of FA, and least weight wasobserved in pups born with FA absent diet. As the fetal birth weight is the best predictor of pregnancy outcome as the growth restriction has been associated with high morbidity and mortality. FA supplementation is essential during the period of pregnancy to prevent untoward effects on pregnancy-related complications in mother and to prevent birth defects in fetuses. Further research from longitudinal studies is warranted to confirm these results before its recommendation to the public health.

Role of folic acid supplementation and/ or its absence during pregnancy on implantation of embryos – An experimental study of Wistar rats

IntroductionThe present study was undertaken to know the effect of folic acid (FA) supplementation and FA absence diet during pregnancy on the number of implantation as well as their endometrial changes. MethodsEighteen Wistar strain Albino rats were randomly divided into three groups and given different diets: Control group with normal diet, group with FA supplementation diet, and group with FA absent diet (with added Succinyl Sulfathiazole) for 5 weeks. The number of coloured thickenings along the uterine horns were identified by injecting 1% Evan’s blue due solution. The sites of embryo implantation were fixed in 10% buffered formalin for 8 hours and embedded. ResultAverage number of implantation observed in pregnant dams fed with FA supplementation diet was 10 in contrast it was 7 in diet with FA absent diet and 8 in control groups. Histologically, implantation site in FA supplemented group showed favorable endometrial environment than the other groups. DiscussionFA is essential in successful implantation by providing favorable receptive environment to receive the implantation-competent blastocyst for a successful pregnancy.

Tridimensional visualization reveals direct communication between the embryo and glands critical for implantation

Embryo implantation is central to pregnancy success. Our previous understanding is limited by studying this phenomenon primarily in two dimensions. Here we employ 3D visualization, revealing that epithelial evaginations that form implantation chambers (crypts) consistently arise with preexisting glands, suggesting direct access of glands to embryos within the chamber. While the lobular domains of the glands become more developed, the ductal regions continue to elongate and progressively stretch following implantation. Using diapausing mice and mice with deletion of the planar cell polarity gene Vangl2 in uterine epithelial cells, we show that dynamic changes in gland topography depend on implantation-competent blastocysts and planar cell polarity. By transferring blastocyst-size beads preloaded with HB-EGF in pseudopregnant mice, we found that HB-EGF is a trigger for the communication between embryos and glands. Glands directly connecting the crypt encasing the embryo during implantation are therefore fundamental to pregnancy success.

Sequence analysis of microRNAs during pre-implantation between Meishan and Yorkshire pigs

Embryonic implantation in sows is a coordinated interaction between the implantation-competent blastocyst and receptive uterus. In addition, microRNAs are small endogenous non-coding RNAs which are involved in post-transcriptional gene regulation of several biological processes including embryonic implantation. However, the mechanisms of miRNAs involved in embryonic implantation of sows remain largely unknown. Here, we analyzed miRNAome of endometrium on day 9, 12 and 15 of pregnancy and on day 12 of non-pregnancy in Meishan and Yorkshire pigs by Illumina sequencing. From 24 libraries, we identified 312 known microRNAs and 211 potential novel miRNAs. Bioinformatics analysis showed that differentially expressed microRNAs on day 12 of pregnancy between the two breeds may play critical roles by involving “p53 signaling pathway” and “Wnt signaling pathway”. Furthermore, our results demonstrated that ssc-miR-21, ssc-miR-451, ssc-miR-204, ssc-miR-199a-5p and ssc-miR-199b-5p would play crucial roles for implantation. The data generated in this study were expected to elucidate the influence of microRNAs during pre-implantation in pigs.

Roles of DEK in the endometrium of mice in early pregnancy

Embryo implantation is a complex process requiring reciprocal interactions between implantation-competent blastocysts and receptive uteri. Accumulating evidence from Digital Protein Expression Profiling indicates that DEK protein expression at implantation sites (ISs) was much higher than that at inter-implantation sites (IISs). In this study, we investigated the expression of DEK in mouse uterus by immunohistochemistry (IHC), Western blotting. We explored its function during decidualization of uterine stromal cells by inhibiting the expression of DEK. In further study of mechanism, the cell proliferation, apoptosis and DNA damage were detected after inhibiting DEK during decidualization of stromal cells. The results suggest that DEK participates in decidualization of stromal cells through mediating cell proliferation, apoptosis and DNA repair.

Integrative Analyses of Uterine Transcriptome and MicroRNAome Reveal Compromised LIF-STAT3 Signaling and Progesterone Response in the Endometrium of Patients with Recurrent/Repeated Implantation Failure (RIF).

Intimate two-way interactions between the implantation-competent blastocyst and receptive uterus are prerequisite for successful embryo implantation. In humans, recurrent/repeated implantation failure (RIF) may occur due to altered uterine receptivity with aberrant gene expression in the endometrium as well as genetic defects in embryos. Several studies have been performed to understand dynamic changes of uterine transcriptome during menstrual cycles in humans. However, uterine transcriptome of the patients with RIF has not been clearly investigated yet. Here we show that several signaling pathways as well as many genes and microRNAs are dysregulated in the endometrium of patients with RIF (RIFE). Whereas unsupervised hierarchical clustering showed that overall mRNA and microRNA profiles of RIFE were similar to those of endometria of healthy women, many genes were significantly dysregulated in RIFE (cut off at 1.5 fold change). The majority (~75%) of differentially expressed genes in RIFE including S100 calcium binding protein P (S100P), Chemokine (C-X-C motif) ligand 13 (CXCL13) and SIX homeobox 1 (SIX1) were down-regulated, suggesting that reduced uterine expression of these genes is associated with RIF. Gene Set Enrichment analyses (GSEA) for mRNA microarrays revealed that various signaling pathways including Leukemia inhibitory factor (LIF) signaling and a P4 response were dysregulated in RIFE although expression levels of Estrogen receptor α (ERα) and Progesterone receptor (PR) were not significantly altered in RIFE. Furthermore, expression and phosphorylation of Signal transducer and activator of transcription 3 (STAT3) are reduced and a gene set associated with Janus kinase (JAK)-STAT signaling pathway is systemically down-regulated in these patients. Pairwise analyses of microRNA arrays with prediction of dysregulated microRNAs based on mRNA expression datasets demonstrated that 6 microRNAs are aberrantly regulated in RIFE. Collectively, we here suggest that dysregulation of several major signaling pathways and genes critical for uterine biology and embryo implantation may lead to uterine abnormalities in patients with RIF.

Update of Wnt signaling in implantation and decidualization

Embryonic development into an implantation-competent blastocyst, synchronized uterine transformation into a receptive stage, and an intimate cross-talk between the activated blastocyst and the receptive uterus are essential for successful implantation, and therefore for subsequent pregnancy outcome. Evidence accumulating during recent years has underlined the importance of the Wnt signaling pathway in mammalian implantation and decidualization. Herein, this review focuses on the current state of knowledge regarding Wnt signaling in multiple implantation and decidualization events: pre-implantation embryo development, blastocyst activation for implantation, uterine development, and decidualization.

Costimulatory Molecule CD28 Participates in the Process of Embryo Implantation in Mice

Embryo implantation is a complex process requiring reciprocal interactions between implantation-competent blastocysts and receptive uteri. Accumulating literatures have indicated that T cells are involved in this process. The first signal mediated by T-cell receptor/CD3 complex and the second signal delivered by costimulatory molecules are essential for the differentiation of T cell into an effector cell. Expression and function of CD28, an important costimulatory molecule, during early pregnancy in mice is still unclear. In the present study, we investigated the expression pattern of CD28 in mouse uterus during early pregnancy and pseudopregnancy by real-time quantitative polymerase chain reaction, Western blotting, in situ hybridization, and immunohistochemistry (IHC). We found that injection of the uterine horn with CD28 antisense oligodeoxynucleotides leads to a decreased number of implantation sites. The expression pattern of CD3 protein examined by IHC is similar to that of CD28. These findings suggest that CD28 participates in the process of embryo implantation in mice, which might play its role through delivering the second costimulatory signal.

Distribution of tubulointerstitial nephritis antigen-like 1 and structural matrix proteins in mouse embryos during preimplantation developmentin vivoandin vitro

Summary Tubulointerstitial nephritis antigen-like 1 (TINAGL1) is a novel matricellular protein that interacts with structural matrix proteins and promotes cell adhesion and spreading. We have previously reported unique localization of TINAGL1 to the trophectoderm (TE) of mouse blastocysts. TINAGL1 was found to be upregulated in implantation-competent blastocysts after estrogen treatment using progesterone-treated delayed-implantation models. Moreover, colocalization of TINAGL1 and extracellular matrix (ECM) protein laminin 1 was detected in the Reichert membrane on embryonic days 6.5 and 7.5. Although these data suggested a role for TINAGL1 in the embryo development at postimplantation, its relevance to other ECM proteins during preimplantation development is not clear. In this study, we examined the expression of TINAGL1 and its relevance to other ECM proteins fibronectin (FN) and collagen type IV (ColIV) during in vivo development of preimplantation embryos, particularly at blastocyst stage in detail. Localizations of TINAGL1, FN, and ColIV were similar. In 1-cell to 8-cell embryos, they were expressed in cytoplasm of blastomeres, and in morulae they were localized in the outer cells. FN and ColIV were expressed primarily on outer surface of the cells. In blastocysts, FN and ColIV were distributed in the cytoplasm of TE, but, just prior to implantation, they became localized uniquely to the blastocoelic surface of TE. In in vitro fertilized (IVF) blastocysts, expression levels of TINAGL1 and FN were lower than in in vivo blastocysts. These results suggest that, during preimplantation development, TINAGL1 may be involved in roles of structural matrix proteins, whose expression in blastocysts may be affected by in vitro culture.

Conditional Deletion of MSX Homeobox Genes in the Uterus Inhibits Blastocyst Implantation by Altering Uterine Receptivity

An effective bidirectional communication between an implantation-competent blastocyst and the receptive uterus is a prerequisite for mammalian reproduction. The blastocyst will implant only when this molecular cross-talk is established. Here we show that the muscle segment homeobox gene (Msh) family members Msx1 and Msx2, which are two highly conserved genes critical for epithelial-mesenchymal interactions during development, also play crucial roles in embryo implantation. Loss of Msx1/Msx2 expression correlates with altered uterine luminal epithelial cell polarity and affects E-cadherin/β-catenin complex formation through the control of Wnt5a expression. Application of Wnt5a in vitro compromised blastocyst invasion and trophoblast outgrowth on cultured uterine epithelial cells. The finding that Msx1/Msx2 genes are critical for conferring uterine receptivity and readiness to implantation could have clinical significance, because compromised uterine receptivity is a major cause of pregnancy failure in IVF programs.

Breast Cancer 1 (Brca1) Is a Potential Marker of Implantation-Competent Blastocysts in Mouse.

Breast Cancer 1 (Brca1) Is a Potential Marker of Implantation-Competent Blastocysts in Mouse.

Temporal and Spatial Regulation of miR-320 in the Uterus during Embryo Implantation in the Rat

The implantation process is complex, requiring reciprocal interactions between implantation-competent blastocysts and the receptive uterus. There were reports to show that some microRNAs (miRNAs) may play a key role during embryo implantation in mouse. However, the miR-320 expression profiles in the rat uterus during peri-implantation are unknown. In the present study, we found that the expression level of miR-320 was lower on day 5 of gestation (g.d. 5) in rats than g.d.3 and g.d.4 and restored gradually from g.d.6. MiR-320 was specifically localized in glandular and luminal epithelia and decidua. The expression of miR-320 was not significantly different in the pseudopregnant uterus and decreased in the uteri of rats subjected to activation of delayed implantation. Artificial decidualization and treatment with progesterone increased the miR-320 expression. Thus, miR-320 was differentially expressed in the rat uterus during implantation. The expression level was affected by active blastocysts and decidualization during the window of implantation. Steroid hormones, progesterone stimulated miR-320 expression.

Temporal and Spatial Regulation of Let-7a in the Uterus During Embryo Implantation in the Rat

Mammalian embryonic implantation requires reciprocal interactions between implantation-competent blastocysts and a receptive uterus. Some microRNAs might play a key role during embryo implantation in the mouse, but the let-7a expression profiles in the rat uterus during peri-implantation are unknown. In the study, the expression of let-7a in the uterus during early pregnancy, pseudopregnancy, artificial decidualization and activation of delayed implantation was detected by Northern blotting and in situ hybridization. The effect of steroid hormones on let-7a expression was also detected by Northern blotting and in situ hybridization. Here, we found that the expression level of let-7a was higher on gestation day 6-7 (g.d. 6-7) in rats than on g.d.4-5 and g.d.8-9. Let-7a was specifically localized in glandular and luminal epithelia and decidua. The expression of let-7a was not significantly different in the pseudopregnant uterus and increased significantly in the uteri of rats subjected to artificial decidualization and activation of delayed implantation. Treatment with estradiol-17beta or progesterone significantly increased let-7a expression. Thus, let-7a expression was significantly induced by the process of embryo invasion, and this increased expression level was mainly induced by active blastocysts and decidualization during the window of implantation, implying that let-7a may participate in endometrial decidualization. Steroid hormones, estradiol-17beta or progesterone stimulated let-7a expression.

Tubulointerstitial Nephritis Antigen-Like 1 Is Expressed in Extraembryonic Tissues and Interacts with Laminin 1 in the Reichert Membrane at Postimplantation in the Mouse1

Tubulointerstitial nephritis antigen-like 1 (Tinagl1, also known as adrenocortical zonation factor 1 [AZ-1] or lipocalin 7) has been cloned from mouse adrenocortical cells and is known to be closely associated with zonal differentiation of adrenocortical cells. In cell culture systems, TINAGL1 is a matricellular protein that interacts with both structural matrix proteins and cell surface receptors. However, the physiological roles of TINAGL1 and regulation of its expression are still not clearly understood. In the present study, the expression and localization of TINAGL1 in peri-implantation mouse embryos was examined. During preimplantation, the expression of both Tinagl1 mRNA and TINAGL1 protein was increased just prior to implantation. In blastocysts, TINAGL1 expression was localized to the trophectoderm. Using a progesterone-treated, delayed-implantation model, TINAGL1 was found to be upregulated in implantation-competent blastocysts after estrogen treatment. During postimplantation, TINAGL1 expression was restricted to extraembryonic regions. Marked expression was detected in the Reichert membrane on Embryonic Days 6.5 (E6.5) and E7.5. Colocalization of laminin 1 and TINAGL1 was also examined. Using an anti-LAMA1 antibody, colocalization of LAMA1 and TINAGL1 was observed in postimplantation embryos. Colocalization was also detected in the Reichert membrane. Immunoprecipitation analysis determined that LAMA1 and TINAGL1 interact in embryos on E7.5. These results demonstrate that after implantation, TINAGL1 is an extraembryonic tissue-specific protein. In particular, TINAGL1 is a novel component of the Reichert membrane that interacts with laminin 1. These results suggest that TINAGL1 most likely plays a physical and physiological role in embryo development at postimplantation.