CONTEXT:Embryo implantation and decidualization are essential to the establishment and maintenance of pregnancy. However, the underlying mechanism on embryo implantation and decidualization remains unclear. Although the regulation and role of hypothalamic corticotropin-releasing hormone (CRH) during early pregnancy have been extensively studied, uterine CRH is still poorly defined. AIMS:This study examined CRH expression in mouse uterus during early pregnancy and evaluated the regulation and role of CRH during embryo implantation and decidualization. METHODS:Immunofluorescence was used to show CRH protein concentrations in mouse uterus. The inhibitor for CRH receptor CRHR1 and recombinant CRH protein were applied to analyze the function of CRH on mouse in vitro decidualization. KEY RESULTS:CRH is expressed in luminal epithelium of mouse uterus from Day 1 to Day 4 of pregnancy. At implantation site on Day 5 of pregnancy, CRH is strongly localized in primary decidua. Verucerfont, a CRHR1 inhibitor, significantly suppresses mouse in vitro decidualization. CONCLUSIONS:Our results indicated that CRH is strong expressed in primary decidua and should play a role during mouse decidualization. IMPLICATIONS:This result should shed lights on understanding the underlying mechanism during embryo implantation and decidualization.

The supply of amino acids is essential to embryo survival and successful pregnancy. The accumulating evidence indicates that arginine, a semi-essential amino acid, plays a key role during early pregnancy. Arginase (ARG2) is a key enzyme for catalyzing arginine into ornithine and urea. However, the expression, regulation, and role of arginase during early mouse pregnancy are still unknown. In our study, ARG1 immunofluorescence is mainly detected in uterine epithelium and gradually decreases from days 1 to 5 of pregnancy. From days 1 to 4 of pregnancy, there is no detectable ARG2 immunofluorescence in the mouse uterus. On day 5 of pregnancy, ARG2 signals are strongly seen in the primary decidua surrounding the implanting blastocyst at the implantation site, but not at the inter-implantation site. There is a temporary increase for ARG2 levels under mouse in vitro decidualization, suggesting ARG2 may be involved in the initiation of mouse decidualization. Prl8a2, a marker of mouse in vitro decidualization, is significantly decreased after ARG levels are suppressed. However, Arg2 overexpression obviously increases Prl8a2 levels. Mouse in vitro decidualization is downregulated by arginine and ornithine but stimulated by a low dose of urea. Urea has a beneficial effect on uterine receptivity and antioxidative enzymes. Our results indicate that ARG2 plays an important role during mouse decidualization by balancing the levels of arginine, ornithine, and urea.

Acrylamide is a class 2A carcinogen with neurotoxicity and genotoxicity. In addition to industrial production, it is ubiquitous in high-temperature heated high-carbohydrate foods. Numerous studies have confirmed the toxicity of ACR on reproduction. Implantation and decidualization are crucial processes during the establishment of pregnancy in rodents and humans. However, its effect on uterine implantation and decidualization remains poorly understood. The objective of this study is to elucidate the mechanism by which ACR affects implantation and decidualization in mice. ACR is exposed in the daily drinking water of female mice, and the dose is calculated according to the body weight of the mice. After 3 months of administration at concentrations of 0, 20, and 30 mg ACR/kg/d, female mice are mated with male mice to induce pregnancy. Compared to the control group, ACR treatment significantly reduces the number of embryo implantations and litter size. ACR treatment leads to abnormal expression of endometrial receptivity-related molecules in the luminal epithelium on day 4 of pregnancy, including a decrease in p-STAT3 level and an increase in MUC1 and MSX1 levels. The level of decidualization-related molecules is obviously downregulated by ACR. Furthermore, ACR treatment results in abnormality of oxidative stress- and ferroptosis-related protein levels at the implantation site on day 5. In conclusion, acrylamide can impair mouse implantation and decidualization by disrupting oxidative stress and ferroptosis.

Primary cilia are solitary, non-motile, microtubule-based organelles that protrude from the surface of most vertebrate cells, functioning as highly specialized sensory and signaling compartments. Architecturally, they comprise the basal body, transition zone, and 9 + 0 axoneme, which together establish a biochemically distinct and selectively permeable domain, spatially segregated from the cytoplasm. This compartmentalization enables primary cilia to integrate and modulate diverse signaling cascades, including Hedgehog, Wnt, Notch, TGF-β/BMP, Hippo, cGAS-STING, calcium, GPCR, and phosphoinositide cascades, thereby coordinating developmental programs, tissue patterning, and homeostatic regulation. Ciliogenesis proceeds through basal body docking to the plasma membrane, vesicle fusion, and axoneme elongation, a sequence precisely orchestrated by bidirectional trafficking machinery of intraflagellar transport (IFT). The dynamic equilibrium between ciliary assembly and disassembly is closely coupled to cell cycle progression and signaling flux. Within the confined ciliary compartment, molecular gating at the transition zone and the polarized trafficking of receptors and effectors confer stringent control over pathway specificity and signal fidelity. Disruption of primary cilia perturbs this spatiotemporal precision, resulting in defective signal integration and a broad spectrum of disorders collectively termed ciliopathies, which range from congenital malformations to metabolic and neoplastic diseases. This review summarizes recent advances in elucidating the structural architecture, biogenesis, and signaling functions of primary cilia, highlighting their critical roles in vertebrate biology and disease.

The corpus luteum is the principal progesterone-secreting gland, while primary cilia function as pivotal organelles in intercellular signal transduction. Together, they play an essential role in the establishment and maintenance of pregnancy. However, the mechanisms underlying the role of primary cilia in granulosa cell luteinization in mouse ovaries remain poorly understood. This study discovered the regularity of primary cilia in mouse ovaries and revealed the role of primary cilia in regulating progesterone synthesis in luteinized granulosa cells. In vivo test results showed that the expression of primary cilia was obvious in the corpus luteum. The secretion of P4 in mice was significantly increased at 6, 12, 24, 48, and 72 h. The secretion of P4 and the expressions of luteinization markers (STAR, 3β-HSD) and primary ciliate proteins (IFT88, Arl13B) were significantly up-regulated at different time points (6, 12, 24 h and 24, 48, 72 h), and the INS group was significantly higher than the LH group and the control. In vitro test results showed that the follicular granulosa cells were luteinized under INS, the length and number of primary cilia increased, and the secretion of progesterone increased. The expression levels of STAR and 3β-HSD of the primary cilia marker Arl13B and luteinization markers were increased, while the expression levels of CYP19A1 were decreased. Ciliobrevin A (CBA) and Y-27632 2HCl were used to regulate the expression of primary cilia. The results showed that after CBA treatment, the expression level of cilia protein Arl13B decreased, and the secretion level of P4 and the expression levels of STAR and 3β-HSD decreased, indicating that the level of luteinization decreased. Conversely, after inducing ciliogenesis with Y-27632 2HCl, the results were the opposite of those observed with CBA treatment. In conclusion, our study demonstrates that primary cilia regulate the expression of steroidogenic enzymes, thereby promoting progesterone secretion by granulosa cells in mice and ensuring proper luteinization.

Embryo implantation and decidualization are crucial for a successful pregnancy. How the inflammatory response is regulated during these processes is undefined. Pyroptosis is an inflammatory form of cell death mediated by gasdermin D (GSDMD). Through in vivo, cultured epithelial cells and organoids, it is shown that pyroptosis occurs in epithelial cells at the implantation site. Compared with those on day 4 of pseudopregnancy and delayed implantation, pyroptosis-related protein levels are significantly increased on day 4 of pregnancy and activated implantation, suggesting that blastocysts are involved in regulating pyroptosis. Blastocyst-derived cathepsin B (CTSB) is stimulated by preimplantation estradiol-17β and induces pyroptosis in epithelial cells. Pyroptosis-induced IL-18 secretion from epithelial cells activates a disintegrin and metalloprotease 12 (ADAM12) to process the epiregulin precursor into mature epiregulin. Epiregulin (EREG) enhances in vitro decidualization in mice. Pyroptosis-related proteins are detected in the mid-secretory human endometrium and are elevated in the recurrent implantation failure endometrium. Lipopolysaccharide treatment in pregnant mice causes implantation failure and increases pyroptosis-related protein levels. Therefore, the data suggest that modest pyroptosis is beneficial for embryo implantation and decidualization. Excessive pyroptosis can be harmful and lead to pregnancy failure.

The establishment of pregnancy depends on interactions between the epithelial and stromal cells of the endometrium that drive the decidual reaction that remodels the stroma and enables embryo implantation. Decidualization in mice also depends on ovarian hormones and the presence of a blastocyst. Hedgehog signaling is transduced by primary cilia in many tissues and is involved in epithelial-stromal cross-talk during decidualization. We found that primary cilia on mouse uterine stromal cells increased in number and length during early pregnancy and were required for decidualization. In vitro and in vivo, progesterone promoted stromal ciliogenesis and the production of Indian hedgehog (IHH) in the epithelium and Sonic hedgehog (SHH) in the stroma. Blastocyst-derived TNF-α also induced epithelial IHH, which stimulated the production of SHH in the stroma through a mechanism that may involve the release of arachidonic acid from epithelial cells. In the stroma, SHH activated canonical Hedgehog signaling through primary cilia and promoted decidualization through a mechanism that depended on interleukin-11 (IL-11) and primary cilia. Our findings identify a primary cilia–dependent network that controls endometrial decidualization and suggest primary cilia as a candidate therapeutic target for endometrial diseases.

Decidualization is essential to the establishment of pregnancy in rodents and primates. Laminin A5 (encoding by Laminin α5) is a member of the laminin family, which is mainly expressed in the basement membranes. Although laminins regulate cellular phenotype maintenance, adhesion, migration, growth, and differentiation, the expression, function, and regulation of laminin A5 during early pregnancy are still unknown. Therefore, we investigated the expression and role of laminin A5 during mouse and human decidualization. Laminin A5 is highly expressed in mouse decidua and artificially induced deciduoma. Laminin A5 is significantly increased under in vitro decidualization. Laminin A5 knockdown significantly inhibits the expression of Prl8a2, a marker for mouse decidualization. Progesterone stimulates the expression of laminin A5 in ovariectomized mouse uterus and cultured mouse stromal cells. We also show that progesterone regulates laminin A5 through the PKA-CREB-C/EBPβ pathway. Laminin A5 is also highly expressed in human pregnant decidua and cultured human endometrial stromal cells during in vitro decidualization. Laminin A5 knockdown by siRNA inhibits human in vitro decidualization. Collectively, our study reveals that laminin A5 may play a pivotal role during mouse and human decidualization via the PKA-CREB-C/EBPβ pathway.

Background: There are around 300 million adolescent pregnancies worldwide, accounting for 11% of all births worldwide. Accumulating evidence demonstrates that many adverse perinatal outcomes are associated with adolescent pregnancies. However, how and why these abnormalities occur remain to be defined. Methods: To compare uterine maturity, implantation, decidualization and placental development between 25-30 days old and 3 months old mature female CD-1 strain mice. Both in vivo mouse pregnancy and in vitro cell culture were used. Western blot, real time RT-PCR, immunostaining and immunofluorescence were used to study the progress of pregnancy during two groups.Results: We found the litter size of adolescent pregnancy is significantly decreased from F1 to F3 generations compared to mature pregnancy. On days 8 and 12 of pregnancy, multiple abnormalities in placental and decidual developments appear in F3 adolescent pregnancy. On days 5 and 8, uterine endoplasmic reticulum stress is dysregulated compared to mature pregnancy. Embryo implantation and decidualization are also compromised in adolescent pregnancy. Many genes are abnormally expressed in adolescent estrous uteri. Conclusion: The uterine immaturities and abnormal implantation may cause multiple pregnancy failures in adolescent pregnancy.

There are around 300 million adolescent pregnancies worldwide, accounting for 11% of all births worldwide. Accumulating evidence demonstrates that many adverse perinatal outcomes are associated with adolescent pregnancies. However, how and why these abnormalities occur remain to be defined. In this study, pregnancy at different stages was compared between 25- and 30- day-old and mature female mice. We found that the litter size of adolescent pregnancy is significantly decreased from F1 to F3 generations compared to mature pregnancy. On days 8 and 12 of pregnancy, multiple abnormalities in decidual and placental development appear in F3 adolescent pregnancy. On days 5 and 8, uterine endoplasmic reticulum stress is dysregulated in F3 adolescent pregnancy. Embryo implantation and decidualization are also compromised in adolescent pregnancy. Many genes are abnormally expressed in adolescent estrous uteri. The abnormal endocrine environment and abnormal implantation from uterine immaturity may result in multiple pregnancy failures in adolescent pregnancy. The aim of this study is to shed light on human adolescent pregnancy.

Maintenance of a suitable uterine milieu is important for embryo development and subsequent implantation during early pregnancy. High estrogen level in proestrous and estrous stages is essential for uterine anti-bacterial activity during preimplantation period. Lipocalin-2 is an essential molecule which prevents bacterial infection by sequestering iron. In this study, the highest expression of lipocalin-2 is observed in the endometrial epithelium on day 1 of normal pregnancy and pseudopregnancy, which exhibit a similar hormone scenario. By injecting the agonists for estrogen receptor α and estrogen receptor β in ovariectomized mice, we found estrogen receptor α is the dominant member for estrogen regulation on lipocalin-2 expression. Estrogen treatment in estrogen receptor α-knockout mice further confirmed the role of estrogen receptor α. Using published data from whole-genome estrogen receptor α binding site assay, significant estrogen receptor α recruitment peaks are found at the downstream of lipocalin-2 gene after estrogen treatment. Furthermore, to study the anti-bacterial activity of lipocalin-2 in uterus, Escherichia coli is injected to mimic bacterial infection. Our results showed an obvious induction of lipocalin-2 in Escherichia coli-treated group. Taken together, this study indicates estrogen regulation of lipocalin-2 in uterine epithelium is mediated by estrogen receptor α, and lipocalin-2 may have anti-bacterial activity during early pregnancy.

Receptivity is a limited time in which uterine endometrium can establish a successful dialogue with blastocyst. This study was to investigate the effect of asynchronous embryo transfer on uterine receptivity in mice. Embryos under different stages were transferred into two oviduct sides of a recipient mouse on day 1 of pseudopregnancy. Our results showed the asynchronously transferred embryos can implant in all groups. Compared to zygote-transfer group, the length of implanted embryos is longer in 8-cell embryo- or blastocyst-transfer group. The levels of Snail and COX-2 immunostaining in blastocyst-transfer group are significantly stronger than that in zygote-transfer group. Embryos in blastocyst-transfer group migrate faster than that in zygote-transfer group within uterus. Blastocysts are in a state of developmental delay after they are transferred into oviducts and they are reactivated and implanted rapidly in uterus. The developmental rate to newborn in zygote-transfer group is obviously higher than that in blastocyst-transfer group, suggesting that a delay in embryo development and implantation will lead to a decrease of litter size. These results indicated that the window of implantation is differentially regulated in two uterine horns of a recipient by embryos at different stages.

Thymosin β4 (TMSB4X) belongs to a class of highly conserved small proteins that are present in a high abundance in immune tissues, where it participates in various biological activities, including anti-inflammation, wound healing, apoptosis, and cell survival. However, little is known about the expression and regulation of TMSB4X in reproductive tissues. The aim of this study was to examine the expression of rat Tmsb4x and chicken TMSB4X genes during testicular and epididymal development. Rat Tmsb4x was strongly detected in the spermatogonia and spermatocytes of testis at early postnatal development and transmitted to Leydig cells at sexual maturation. Also, rat Tmsb4x was detected at an increased level in the epididymis during postnatal development. When compared to the rat, expression of the chicken TMSB4X gene was low in the testis and epididymal region, and the mRNA localization was also unexpected. Three experiments were performed to examine the regulation of rat Tmsb4x in the epididymis: after elimination of Leydig cells using ethylene dimethane sulfonate (EDS); after destruction of the testis by cryptorchidism; and after castration. EDS-treated and castrated rats were injected with testosterone propionate. The expression of Tmsb4x was significantly reduced in the epididymis of EDS-treated, and castrated rats. In contrast, Tmsb4x was significantly enhanced in the epididymis after testosterone treatment. The expression of rat Tmsb4x was also regulated in the epididymis after cryptorchidism. Collectively, the expression of Tmsb4x was strongly detected in the testis and epididymis of rats, and was highly regulated in the epididymis by testosterone.

Luminal closure and embryo apposition are essential for blastocyst attachment during early pregnancy. In our preliminary microarray results (unpublished data), sodium-potassium adenosine triphosphatase (Na/K-ATPase) β1 (Atp1b1) was highly expressed in mouse uterus on Days 3 and 4 of pregnancy. However, expression and regulation of Atp1b1 in the mammalian uterus during early pregnancy are unknown. Using in situ hybridization, a strong level of Atp1b1 mRNA was detected in luminal epithelial cells on Days 3 and 4 of pregnancy (Day 1 = day of vaginal plug). The expression pattern of FXYD domain-containing ion transport regulator 4 (Fxyd4) was similar to that of Atp1b1. Real-time reverse transcription polymerase chain reaction confirmed the high expression level of Atp1b1 mRNA. Compared with Day 1, the mRNA level of Atp1b1 on Days 3 and 4 increased by 3.5 ± 0.5 and 4.5 ± 0.5 fold, respectively. When the embryo invaded through epithelial cells into the maternal stromal compartment on day 5, Atp1b1 expression decreased to a basal level. Progesterone stimulated Atp1b1 expression by 2.8 ± 1 fold compared with oil in ovariectomized mice at 24 hours after treatment. Expression of Atp1b1 was further upregulated to 4 ± 0.4 fold by estrogen and progesterone. Based on time-course study, progesterone rapidly induced Atp1b1 expression at 6 and 12 hours (13.7 ± 0.5 and 16.6 ± 1.4, respectively); furthermore, this upregulation was blocked by RU486 (progesterone receptor antagonist). Transcription activity of the Atp1b1 promoter was (Day 1 = day of vaginal plug) stimulated by CCAAT/enhancer binding protein beta (Cebpb). In conclusion, Atp1b1 was highly expressed in luminal epithelium during peri-implantation and upregulated by progesterone.

Objective: To investigate the role of arachidonic acid (AA) in mouse endometrial stromal cells.Design: Experimental animal study.Setting: University research laboratory.Animal(s): Sexually mature female CD1-strain mice.Intervention(s): Primary culture of endometrial stromal cells.Main Outcome Measure(s): Western blot and real-time polymerase chain reaction for gene expression and/or phophorylation analysis. Luciferase assay for Cox-2 promoter analysis.Result(s): AA-derived prostaglandins play important roles during embryo implantation and decidualization. However, the function of AA itself in reproduction is largely unknown. In this study, exogenous AA stimulated cPLA(2 alpha) phosphorylation and COX-2 expression, mainly through ERK1/2 in mouse endometrial stromal cells, and p38 inhibitor modestly inhibited cPLA(2 alpha) phosphorylation induced by AA. The induction of COX-2 by AA was diminished by short interfering RNA against C/EBP beta and inhibitory C/EBP beta (LIP). C/EBPb binding site at -872- -864 of Cox-2 promoter contributes to Cox-2 promoter activation induced by C/EBPb transfection. The expression of C/EBPb protein induced by AA was inhibited by p38 inhibitor, and the phosphorylation of C/EBP beta induced by AA was inhibited by p38 inhibitor and ERK1/2 inhibitor. A nonmetabolized analogue of AA (ETYA) also enhanced cPLA(2 alpha) phosphorylation and COX-2 expression. The activation of cPLA(2 alpha)/COX-2 by AA was not inhibited by COX inhibitor indomethacin.Conclusion(s): AA can induce cPLA(2 alpha)/COX-2 pathway activation in mouse endometrial stromal cells. (Fertil Steril (R) 2012;97:1199-1205. (C) 2012 by American Society for Reproductive Medicine.)

Embryo implantation into the maternal uterus is a crucial step for the successful establishment of mammalian pregnancy. Following the attachment of embryo to the uterine luminal epithelium, uterine stromal cells undergo steroid hormone-dependent decidualization, which is characterized by stromal cell proliferation and differentiation. The mechanisms underlying steroid hormone-induced stromal cell proliferation and differentiation during decidualization are still poorly understood. Ribonucleotide reductase, consisting of two subunits (RRM1 and RRM2), is a rate-limiting enzyme in deoxynucleotide production for DNA synthesis and plays an important role in cell proliferation and tumorgenicity. Based on our microarray analysis, Rrm2 expression was significantly higher at implantation sites compared with interimplantation sites in mouse uterus. However, the expression, regulation, and function of RRM2 in mouse uterus during embryo implantation and decidualization are still unknown. Here we show that although both RRM1 and RRM2 expression are markedly induced in mouse uterine stromal cells undergoing decidualization, only RRM2 is regulated by progesterone, a key regulator of decidualization. Further studies showed that the induction of progesterone on RRM2 expression in stromal cells is mediated by the AKT/c-MYC pathway. RRM2 can also be induced by replication stress and DNA damage during decidualization through the ATR/ATM-CHK1-E2F1 pathway. The weight of implantation sites and deciduoma was effectively reduced by specific inhibitors for RRM2. The expression of decidual/trophoblast prolactin-related protein (Dtprp), a reliable marker for decidualization in mice, was significantly reduced in deciduoma and steroid-induced decidual cells after HU treatment. Therefore, RRM2 may be an important effector of progesterone signaling to induce cell proliferation and decidualization in mouse uterus.

Background: Junctional adhesion molecule 2 (Jam2) is a member of the JAM superfamily. JAMs are localized at intercellular contacts and participated in the assembly and maintenance of junctions, and control of cell permeability. Because Jam2 is highly expressed in the luminal epithelium on day 4 of pregnancy, this study was to determine whether Jam2 plays a role in uterine receptivity and blastocyst attachment in mouse uterus.Methodology/Principal Findings: Jam2 is highly expressed in the uterine luminal epithelium on days 3 and 4 of pregnancy. Progesterone induces Jam2 expression in ovariectomized mice, which is blocked by progesterone antagonist RU486. Jam2 expression on day 4 of pregnancy is also inhibited by RU486 treatment. Leukemia inhibitory factor (LIF) up-regulates Jam2 protein in isolated luminal epithelium from day 4 uterus, which is blocked by S3I-201, a cell-permeable inhibitor for Stat3 phosphorylation. Under adhesion assay, recombinant Jam2 protein increases the rate of blastocyst adhesion. Both soluble recombinant Jam2 and Jam3 can reverse this process.Conclusion: Jam2 is highly expressed in the luminal epithelium of receptive uterus and up-regulated by progesterone and LIF via tyrosine phosphorylation of Stat3. Jam2 may play a role in the interaction between hatched blastocyst and receptive uterus.

Epithelial-mesenchymal transition(EMT) is a process that epithelial cells lose polarity,become mesenchymal cells and acquire the ability of migration and invasion.It exists in many physiological and pathological processes.EMT is involved in a number of signal transduction pathways and performs different physiological functions.During the early stages of embryonic development,both EMT and MET(mesenchymal-epithelial transition) contribute to the formation and development of organs.Moreover,EMT can promote tumor metastasis.EMT also occurs in female mammalian reproduction.In the ovary,EMT is beneficial for repairing process following ovulation.During decidualization,MET may be required for successful uterine anchorage of the embryo.Placental development undergoes an EMT in order to facilitate nutrient and gas exchange between mother and fetus.The failure of EMT process may cause related reproductive diseases.

The establishment of endometrial receptivity is a prerequisite for successful pregnancy, which is controlled by a complex mechanism. MicroRNAs (miRNAs) are small non-coding RNAs that have emerged as important regulators of gene expression. However, the contribution of miRNAs in endometrial receptivity is still unknown. Here we used rhesus monkey as an animal model and compared the endometrial miRNA expression profiles during early-secretory (pre-receptive) phase and mid-secretory (receptive) phase by deep sequencing. A set of differentially expressed miRNAs were identified, 8 of which were selected and validated using quantitative RT-PCR. To facilitate the prediction of their target genes, the 3'-UTRome was also determined using tag sequencing of mRNA 3'-termini. Surprisingly, about 50% of the 10,677 genes expressed in the rhesus monkey endometrium exhibited alternative 3'-UTRs. Of special interest, the progesterone receptor (PGR) gene, which is necessary for endometrial receptivity, processes an ultra long 3'-UTR (~10 kb) along with a short variant (~2.5 kb). Evolutionary analysis showed that the 3'-UTR sequences of PGR are poorly conserved between primates and rodents, suggesting a species-biased miRNA binding pattern. We further demonstrated that PGR is a valid target of miR-96 in rhesus monkey and human but not in rodents, whereas the regulation of PGR by miR-375 is rhesus monkey-specific. Additionally, we found that miR-219-5p regulates PGR expression through a primate-specific long non-coding RNA immediately downstream of the PGR locus. Our study provides new insights into the molecular mechanisms underlying endometrial receptivity and presents intriguing species-specific regulatory roles of miRNAs.