ABSTRACT
This study evaluated whether the treatment of pseudopregnancy in bitches with vitamin B6 modulates uterine expression of receptors for progesterone (PR), oestrogen (ERα), androgen (AR), thyroid hormone (TRα) and the kisspeptin/Kiss1r system. Eighteen pseudopregnant bitches were treated for 20 days in groups receiving placebo (n = 6); cabergoline (5 µg/kg/day; n = 6); or vitamin B6 (50 mg/kg/day; n = 6). Blood was collected on the 1st day of drug administration and 120 h later to measure serum prolactin (PRL). After treatment, they were ovariohysterectomized and uterine fragments were collected for histomorphometry and immunohistochemical evaluation of PR, ERα, AR, TRα, Kiss1 and Kiss1r. After 120 h of cabergoline or vitamin B6 treatment, PRL levels were reduced in the bitches, confirming the antiprolactinemic effect of these drugs. Furthermore, regardless of treatment, the animals exhibited uterine histomorphometry consistent with dioestrus. The PR showed strong immunostaining in all regions and an increase in scores was observed for this receptor in animals treated with vitamin B6 in deep glands. In contrast, ERα and Kiss1R receptors showed weak to no immunostaining in all uterine regions and no changes between groups. Regarding AR, most animals treated with vitamin B6 showed increased trends in the deep gland and myometrium marking scores. In contrast, in both vitamin B6 and cabergoline treatments, a reduction in TRα marking scores was observed compared to the control group. In addition, on the endometrial surface, a reduction was observed in the marked area of Kiss1 after administration of cabergoline when compared to the pseudopregnant control group. These findings shed valuable insight into the use of vitamin B6 as a drug with actions similar to cabergoline in reducing PRL and uterine modulation in bitches.
Subject(s)
Cabergoline , Kisspeptins , Prolactin , Pseudopregnancy , Uterus , Animals , Female , Dogs , Kisspeptins/pharmacology , Kisspeptins/metabolism , Uterus/drug effects , Uterus/metabolism , Cabergoline/pharmacology , Prolactin/metabolism , Pseudopregnancy/veterinary , Pseudopregnancy/metabolism , Receptors, Progesterone/metabolism , Receptors, Androgen/metabolism , Ergolines/pharmacologyABSTRACT
It has been well established that uterine function during the peri-implantation period is precisely regulated by ovarian estrogen and progesterone. The embryo enters the uterine cavity before implantation. However, the impact of pre-implantation embryo on uterine function is largely unknown. In the present study, we performed RNA-seq analysis of mouse uterus on day 4 morning of natural pregnancy (with embryos in the uterus) and pseudo-pregnancy (without embryos in the uterus). We found that 146 genes were upregulated, and 77 genes were downregulated by the pre-implantation embryo. Gene ontology and gene network analysis highlighted the activation of inflammatory reaction in the uterus. By examining the promoter region of differentially expressed genes, we found that NF-kappaB was a causal transcription factor. Finally, we validated 4 inflammation-related genes by quantitative RT-PCR. These 4 genes are likely the main mediators of the inflammatory reaction in the uterus triggered by the pre-implantation embryo. Our data indicated that the pre-implantation embryo causes uterine inflammatory reaction, which in turn might contribute to the establishment of uterine receptivity and embryo implantation.
Subject(s)
Blastocyst/metabolism , Embryo Implantation , Inflammation Mediators/metabolism , NF-kappa B/metabolism , Uterus/metabolism , Animals , Blastocyst/immunology , Chemokines, CXC/genetics , Chemokines, CXC/metabolism , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Female , Gene Regulatory Networks , Interleukins/genetics , Interleukins/metabolism , Mice , NF-kappa B/genetics , Pregnancy , Pseudopregnancy/genetics , Pseudopregnancy/immunology , Pseudopregnancy/metabolism , RNA-Seq , Uterus/immunologyABSTRACT
This study was conducted to evaluate the changing concentrations of metabolic hormones and metabolites in pregnant (P) and pseudopregnant (PP) rabbit does. Twenty-five New Zealand White rabbit does were submitted to artificial insemination (AI) and then classified as P (n = 15) or PP (n = 10). Blood samples were collected weekly until day 32 post AI. During pregnancy, leptin concentrations were greater on Days 14 and 21 (P < 0.05), while insulin was greater on days 21 and 32 post AI (P < 0.05) compared to PP does. The triiodothyronine/thyroxine (T3/T4) ratio was greater in the first and last week (P < 0.001); whereas, cortisol concentrations were greater in the last week of pregnancy and after parturition (P < 0.01) compared with that of PP does. Non-esterified fatty acids (NEFA) concentrations increased from day 7 until day 32 post AI (P < 0.05). Glucose concentrations were unchanged throughout pregnancy although concentrations were positively associated with litter size. These results indicate concentrations of hormones and metabolites change during pregnancy to ensure energy requirements are met for both the foetuses and the maternal tissues. Physiological hyperleptinemia, hyperinsulinemia, and changes in cortisol as well as thyroid hormones indicate there is an adaptation of metabolic functions induced by pregnancy. These adaptations could be mediated by gonadal steroids because changes mainly occur in the second half of pregnancy when the profile of the sex hormones differs between P and PP does.
Subject(s)
Energy Metabolism/physiology , Homeostasis/physiology , Pregnancy, Animal , Pseudopregnancy/veterinary , Rabbits/physiology , Adaptation, Physiological , Animals , Female , Hydrocortisone/blood , Insulin/blood , Insulin Resistance , Leptin/blood , Pregnancy , Pregnancy, Animal/metabolism , Pseudopregnancy/metabolism , Triiodothyronine/bloodABSTRACT
Decidualization is a critical process for successful embryo implantation and subsequent placenta formation. The characterization and physiological function of lncRNA during decidualization remain largely unknown. In the present study, we conducted RNA-sequencing analysis to compare gene expression between decidua of days 6 and 8, and normal pregnant endometrium (day 4). A total of 2332 high-confidence putative lncRNA transcripts were expressed. Functional clustering analysis of cis and trans lncRNA targets showed that differentially expressed lncRNAs may regulate multiple gene ontology terms and pathways that have important functions in decidualization. Subsequent analyses using qRT-PCR validated that eight of all lncRNAs were differentially regulated in mice uteri during decidualization, both in vivo and in vitro. Furthermore, we showed that differentially expressed lncRNA of Hand2os1 was specifically detected in stromal cells on days 2 to 5 of pregnancy and was strongly upregulated in decidual cells on days 6-8 of pregnancy. Similarly, Hand2os1 expression was also strongly expressed in decidualized cells following artificial decidualization, both in vivo and in vitro. In uterine stromal cells, P4 was able to significantly upregulate the expression of Hand2os1, but upregulation was impeded by RU486, whereas E2 appeared to have no regulating effect on Hand2os1 expression. Concurrently, Hand2os1 significantly promoted the decidual process in vitro and dramatically increased decidualization markers Prl8a2 and Prl3c1. Our results provide a valuable catalog for better understanding of the functional roles of lncRNAs in pregnant mouse uteri, as it relates to decidualization.
Subject(s)
Endometrium/metabolism , Pregnancy, Animal/metabolism , Progesterone/metabolism , RNA, Long Noncoding/metabolism , Receptors, Progesterone/metabolism , Animals , Female , Gene Expression Profiling , Gonadal Steroid Hormones/metabolism , Male , Mice , Pregnancy , Pseudopregnancy/metabolismABSTRACT
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 ß 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.
Subject(s)
Estrogen Receptor alpha/metabolism , Lipocalin-2/metabolism , Pregnancy, Animal/metabolism , Uterus/metabolism , Animals , Epithelium/metabolism , Escherichia coli , Female , Lipopolysaccharides , Mice , Mice, Knockout , Pregnancy , Pseudopregnancy/metabolismABSTRACT
The study investigated the effect of normal and supraphysiological (resulting from gonadotropin-dependent ovarian stimulation) levels of estradiol (E2) and progesterone (P4) on mouse uterine aquaporin gene/protein (Aqp/AQP) expression on Day 1 (D1) and D4 of pregnancy. The study also examined the effect of ovarian stimulation on uterine luminal closure and uterine receptivity on D4 of pregnancy and embryo implantation on D5 and D7 of pregnancy. These analyses revealed that the expression of Aqp3, Aqp4, Aqp5 and Aqp8 is induced by E2 while the expression of Aqp1 and Aqp11 is induced by P4. Additionally, P4 inhibits E2 induction of Aqp3 and Aqp4 expression while E2 inhibits Aqp1 and Aqp11 expression. Aqp9, however, is constitutively expressed. Ovarian stimulation disrupts Aqp3, Aqp5 and Aqp8 expression on D4 and AQP1, AQP3 and AQP5 spatial expression on both D1 and D4, strikingly so in the myometrium. Interestingly, while ovarian stimulation has no overt effect on luminal closure and uterine receptivity, it reduces implantation events, likely through a disruption in myometrial activity and embryo development. The wider implication of this study is that ovarian stimulation, which results in supraphysiological levels of E2 and P4 and changes (depending on the degree of stimulation) in the E2:P4 ratio, triggers abnormal expression of uterine AQP during pregnancy, and this is associated with implantation failure. These findings lead us to recognize that abnormal expression would also occur under any pathological state (such as endometriosis) that is associated with changes in the normal E2:P4 ratio. Thus, infertility among these patients might in part be linked to abnormal uterine AQP expression.
Subject(s)
Aquaporins/physiology , Embryo Implantation/drug effects , Estradiol/physiology , Ovulation Induction , Progesterone/physiology , Animals , Aquaporins/biosynthesis , Aquaporins/genetics , Embryo Implantation/physiology , Embryo Transfer , Estradiol/pharmacology , Female , Gene Expression Regulation, Developmental , Mice , Mice, Inbred C57BL , Mifepristone/pharmacology , Pregnancy , Progesterone/pharmacology , Pseudopregnancy/metabolism , Uterus/physiopathology , Water/metabolismABSTRACT
Recent studies indicate that FoxO1 has roles in female reproductive system, especially in maternal endometrium. Although various cellular aspects and molecular pathways have been identified, the exact molecular characteristics of embryo implantation are still not completely understood. In this study, we aimed to investigate uterine expression and regulation of FoxO1 during peri-implantation period in mice. Experimental mouse models including, normal pregnancy, pseudopregnancy, artificial decidualization, and delayed implantation and activation were performed. Our results showed that FoxO1 expression was spatiotemporal in mouse endometrial tissue throughout peri-implantation period and its expression was significantly upregulated in luminal and glandular epithelium at the time of implantation. Moreover, on day 5 morning (09:00 AM) of pregnancy, expression of FoxO1 was cytoplasmic in endometrial luminal epithelial cells where embryo homing takes place. With progressing time on day 5 evening (19:00 PM) of pregnancy FoxO1 expression was nuclear in luminal epithelium at implantation site. Pseudopregnancy and artificial decidualization models indicated that FoxO1 expression was regulated by pregnancy hormones. Delayed implantation and activation model indicated that FoxO1 expression at the time of implantation is dependent upon activation status of blastocyst due to E2 induction and uterine sensitivity to implantation. In conclusion, our findings highlight a perspective for FoxO1 expression and regulation in mouse uterus during peri-implantation period indicating that its expression is regulated by implanting embryo and pregnancy hormones.
Subject(s)
Decidua/metabolism , Embryo Implantation, Delayed/physiology , Forkhead Box Protein O1/biosynthesis , Gene Expression Regulation/physiology , Pregnancy/physiology , Pseudopregnancy/metabolism , Animals , Blastocyst/metabolism , Female , Mice , Mice, Inbred BALB CABSTRACT
Kisspeptin (KiSS) and its related receptors (KiSS1R) have a critical role in the reproduction of mammals. The KiSS/KiSS1R system is expressed in numerous reproductive organs including the ovary. Here, we studied the expression of the KiSS/KiSS1R system and its functional role in rabbit corpora lutea (CL) at days 4 (early-), 9 (mid-), and 13 (late-stage) of pseudopregnancy. In vitro progesterone, prostaglandin (PG) F2α (PGF2α) and E2 (PGE2) productions and prostaglandin-endoperoxide synthase 1 (PTGS1) and 2 (PTGS2) activities were evaluated. Immune reactivity (IR) for KiSS and KiSS1R were detected in luteal cells at nuclear and cytoplasmic level at all luteal stage for KiSS and only at early- and mid-stage for KiSS1R; IR decreased from early- to later stages of pseudopregnancy. The KiSS-10 augmented progesterone and PGE2 and diminished PGF2α secretions by early- and mid-CL; KiSS-10 reduced PTGS2 activity at early- and mid-stages, but did not affect PTGS1 at any luteal stages. The antagonist KiSS-234 counteracted all KiSS-10 effects. This study shows that the KiSS/KiSS1R system is expressed in CL of pseudopregnant rabbits and exerts a luteotropic action by down-regulating PTGS2, which decreases PGF2α and increases PGE2 and progesterone.
Subject(s)
Kisspeptins/biosynthesis , Luteal Cells/metabolism , Pseudopregnancy/metabolism , Receptors, Kisspeptin-1/biosynthesis , Animals , Cyclooxygenase 2/metabolism , Dinoprost/metabolism , Dinoprostone/metabolism , Female , Luteal Cells/pathology , Pregnancy , Pseudopregnancy/pathology , RabbitsABSTRACT
Embryonic signals can affect the spatiotemporal-specific expression of the uterus to establish a successful pregnancy. Our previous study has found that talin1 underwent dynamic changes in the mouse endometrium during peri-implantation period. However, whether talin1 is affected by the embryo signals is not clear. In order to investigate the effect of embryonic signals, especially human chorionic gonadotropin (HCG) on talin1, we have designed mouse models of pseudopregnancy, delayed implantation and activation, and HCG treatment. Using these models, the expression of talin1 in the mouse endometrium was determined by immunohistochemistry and Western blotting. In the pseudopregnancy model, an increased expression of talin1 was found from day 3 to day 5, whereas the talin1 protein was decreased on day 5 in the normal pregnant mice. In the delayed implantation model, a strong cytoplasmic staining of talin1 was found, especially in stromal cells. However, after activation of the implantation, the expression of talin1 decreased (P < .05). Furthermore, a significantly lower expression of talin1 was found at the implantation site when compared to the interimplantation sites (P < .05). In the HCG treatment model, an intrauterine perfusion of 10u HCG significantly reduced the expression of talin1 in both stromal and epithelial cells in pseudopregnant mice (P < .05), although further increase in the HCG concentration did not have additional effect on expression of talin1. Taken together, our data suggest that the presence of embryos can affect expression of talin1 in the mouse endometrium, and a certain concentration of HCG can regulate its expression.
Subject(s)
Chorionic Gonadotropin/pharmacology , Endometrium/metabolism , Gene Expression Regulation/physiology , Pseudopregnancy/metabolism , Stromal Cells/metabolism , Talin/metabolism , Animals , Embryo Implantation/physiology , Endometrium/drug effects , Female , Gene Expression Regulation/drug effects , Mice , Progesterone/pharmacology , Pseudopregnancy/genetics , Stromal Cells/drug effects , Talin/geneticsABSTRACT
Tubulin polymerization promoting protein 3 (TPPP3) is known to be expressed in the endometrium in a cyclic manner, and its functional role in the physiology of implantation remains unknown. Here we demonstrate a novel function of TPPP3 during the window of implantation and in the establishment of pregnancy using a mouse model. The increased protein expression of TPPP3 and ß-catenin during peri-implantation period, i.e. D5 (receptive phase, 0800 h), was observed as compared to that on D1 (nonreceptive phase, 0800 h). SiRNATPPP3-mediated knockdown of uterine TPPP3 resulted in implantation failure and inhibited the expression of receptivity markers: LIF, Integrin-ß3, IHH, and Wnt4. TPPP3 silencing in mouse endometrial epithelial cells also prevented blastocyst attachment and the adhesion reaction. In delayed implantation experiment, expression of TPPP3 was increased in active implantation group (E2 + P4) compared to delayed implantation group (P4). The increased expression of TPPP3 in E2 + P4-treated Ishikawa cells compared to vehicle or P4 or E2 alone-treated Ishikawa cells also revealed its upregulation by E2. The suppression of ß-catenin in uterus under the condition of transient knockdown of TPPP3 and the co-immunoprecipitation experiment revealed that regulation of ß-catenin was mediated via TPPP3 during implantation. Additionally, in order to gain insight into TPPP3 collaborators, we identified TPPP3 interacting proteins by nanoLC-MS analysis in mouse uterus which might be involved during implantation. In conclusion, our study suggests that TPPP3 is important for embryo implantation and for the establishment of early pregnancy through modulation of ß-catenin.
Subject(s)
Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/physiology , Embryo Implantation/genetics , Embryo Implantation/physiology , Uterus/metabolism , beta Catenin/metabolism , Animals , Blastocyst , Cell Line, Tumor , Estradiol/pharmacology , Female , Gene Knockdown Techniques , Mice , Mice, Inbred BALB C , Pregnancy , Pseudopregnancy/genetics , Pseudopregnancy/metabolismABSTRACT
Prion protein (PrP) is encoded by a single copy gene Prnp in many cell and tissue types. PrP is very famous for its infectious conformers (PrPSC) resulting in transmissible spongiform encephalopathies. At present, physiological functions of its cellular isoform (PrPC) remain ambiguous. Although PrPC expression has been found in uterus, whether it functions in maternal-fetal dialogue during early pregnant is unknown. In this study, we examined PrPC mRNA and protein in the uterus of peri-implantation mice, and found that they were expressed with a spatiotemporal dynamic pattern. Interestingly, PrPC was significantly increased in the decidual zones around the implanting embryos at the implantation window stage. To further demonstrate that PrPC is involved in the decidualization of mouse uterus during embryo implantation, we constructed the artificial decidualization models and the delayed implantation models. Once the pseudopregnant mice were artificially induced to decidualization, the PrPC expression then increased significantly in the decidua zone. And also, if the delayed implantation embryos were allowed to implant, PrPC protein was also simultaneously improved in stromal cells surrounding the implanting embryos. Moreover, PrPC expression can be inhibited by progesterone but upregulated by estrogen in mouse uterus. These results suggest that PrPC may play an important role in embryo implantation and decidualization.
Subject(s)
Embryo Implantation/physiology , Prion Proteins/metabolism , Uterus/metabolism , Animals , Decidua/drug effects , Decidua/metabolism , Embryo Implantation/drug effects , Embryo Implantation, Delayed/drug effects , Embryo Implantation, Delayed/physiology , Estradiol/pharmacology , Female , Mice , Progesterone/pharmacology , Pseudopregnancy/metabolism , Stromal Cells/drug effects , Stromal Cells/metabolism , Uterus/drug effectsABSTRACT
Embryo implantation is a complex process involving synchronised crosstalk between a receptive endometrium and functional blastocysts. Apoptosis plays an important role in this process as well as in the maintenance of pregnancy. In this study, we analysed the expression pattern of programmed cell death 4 (Pdcd4), a gene associated with apoptosis in the mouse endometrium, during early pregnancy and pseudopregnancy by real-time quantitative polymerase chain reaction, in situ hybridisation, Western blotting and immunohistochemistry. The results showed that Pdcd4 was increased along with days of pregnancy and significantly reduced at implantation sites (IS) from day 5 of pregnancy (D5). The level of Pdcd4 at IS was substantially lower than that at interimplantation sites (IIS) on D6 and D7. In addition, Pdcd4 expression in the endometrium was reduced in response to artificially induced decidualisation in vivo and in vitro Downregulation of Pdcd4 gene expression in cultured primary stromal cells promoted decidualisation, while upregulation inhibited the decidualisation process by increasing apoptosis. These results demonstrate that Pdcd4 is involved in stromal cell decidualisation by mediating apoptosis and therefore plays a role in embryo implantation in mice.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Blastocyst/metabolism , Embryo Implantation , Endometrium/metabolism , Gene Expression Regulation, Developmental , Pseudopregnancy/metabolism , RNA-Binding Proteins/metabolism , Stromal Cells/metabolism , Animals , Apoptosis Regulatory Proteins/genetics , Blastocyst/cytology , Endometrium/cytology , Female , Mice , Pregnancy , RNA-Binding Proteins/genetics , Stromal Cells/cytologyABSTRACT
Vaginocervical stimulation (VCS) induces twice-daily prolactin (PRL) surges resulting in pseudopregnancy in the rat. Furthermore, activation of the extracellular signal-regulated kinase-1/2 (Erk-1/2) is involved in the effect of estradiol (E2) on the Prl gene expression in pituitary cells. Herein, we investigated whether Erk-1/2 signaling is involved in the control of Prl expression in the pituitary of VCS rats and whether VCS regulates the effect of E2 on Erk-1/2 and Prl in the pituitary. Estrous rats were assigned as control or VCS groups and 0, 6, 12 or 24h later the levels and localization of phosphorylated Erk-1/2 (p-Erk-1/2) were analyzed in the pituitary. The effect of an Erk-1/2 inhibitor PD98059 on the Prl level in the pituitary of control or VCS rats was also analyzed. Other control or VCS rats were treated with E2 and the level of p-Erk-1/2 and Prl were measured in the pituitary. In control rats, p-Erk-1/2 decreased at 6 and 12h and increased at 24h while Erk-1/2 was phosphorylated at all time points in VCS rats. p-Erk-1/2 was localized only in the anterior pituitary. PD98059 decreased Prl level in VCS, but not in control rats. Estradiol decreased Erk-1/2 phosphorylation although did not change Prl level in the pituitary of control or VCS rats. These findings show that prolonged activation of Erk-1/2 is necessary to induce Prl expression in the pituitary of VCS rats; however, VCS does not influence the role of E2 on the activation of Erk-1/2 and Prl expression the pituitary.
Subject(s)
Gene Expression , MAP Kinase Signaling System/physiology , Pituitary Gland/metabolism , Prolactin/genetics , Pseudopregnancy/genetics , Animals , Female , Phosphorylation , Prolactin/metabolism , Pseudopregnancy/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
Early preimplantation embryo-maternal communication is crucial for the establishment and development of pregnancy. Though the involvement of several candidate genes and proteins in this complex event has been described, the hierarchy of molecular networks governing this communication remains unknown. The primary objective of this study was to determine whether the presence of embryos in the uterine lumen stimulated or inhibited gene expression in the uterine tissue on day 3.5 post coitum. To answer this question, we investigated the gene expression of dedicated signal transduction pathways in the uterus of CD-1 mice during the preimplantation stage of pregnancy and compared this expression to mice with induced pseudopregnancy. The expression levels of 84 genes assigned to nine intracellular signalling pathways were investigated by real-time PCR. The results demonstrated down-regulation of the uterine gene expression in the majority of pathways. Among target genes, 27 were significantly (p<0.05) down-regulated, and only three were significantly up-regulated. A majority of the down-regulated genes were found to be regulated by the TGFB and NFKB pathways, which suggests that the presence of the embryo selectively regulates signalling within signal transduction pathways. One of the up-regulated genes crucial for early pregnancy was Ptgs2 (p<0.05). The increased amount of both Ptgs2 gene and protein products indicates that Ptgs2 expression may be the earliest positive embryo signal for implantation and pregnancy recognition in mice. In conclusion, our results not only underline which signalling pathways are regulated in embryo-maternal communication before implantation but also support "the quiet state hypothesis" of silencing gene expression.
Subject(s)
Blastocyst/physiology , Cyclooxygenase 2/metabolism , Enzyme Induction , Gene Expression Regulation, Developmental , Pregnancy/metabolism , Pseudopregnancy/metabolism , Uterus/metabolism , Animals , Animals, Outbred Strains , Cyclooxygenase 2/genetics , Embryo Implantation , Female , Gene Expression Profiling , Mice , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Specific Pathogen-Free Organisms , Uterus/enzymologyABSTRACT
BACKGROUND: Cyclooxygenase (COX)-derived prostanoids (PGE2, PGI2) are important contributors to the process of decidualization. Previous studies showed the presence of Ang-(1-7) in the primary and secondary decidualized zones of the implantation site at early pregnancy. Decreased concentrations of Ang-(1-7) were found in the decidualized uterus compared to the non-decidualized uterus of pseudopregnant rats, suggesting that low levels of Ang-(1-7) are required for successful decidualization at early pregnancy. METHODS: To understand the role of Ang-(1-7) in prostaglandin production in a decidualized uterus, induced by a bolus injection of sesame oil, Ang-(1-7) (24 µg/kg/h) or vehicle was then infused directly into the decidualized uterine horn using an osmotic minipump. The right horns were not injected or infused and served as non-decidualized uterine horns in both groups of animals. RESULTS: Decidualization increased PGE2 concentration in the uterus (0.53 ± 0.05 vs. 12.0 ± 3.2 pmol/mg protein, p < 0.001, non-decidualized vs. decidualized horns); Ang-(1-7) infusion attenuated the increase of PGE2 (12.0 ± 3.2 vs. 5.1 ± 1.3 pmol/mg protein, p < 0.01 control vs. Ang-(1-7) treated decidualized horns). The stable metabolite of PGI2 (6-keto PGF1α) was increased with decidualization (0.79 ± 0.17 vs. 3.5 ± 0.82 pmol/mg protein, p < 0.001, non-decidualized vs. decidualized horns). Ang-(1-7) infusion attenuated the increase in 6-keto PGF1α in the decidualized horn (3.5 ± 0.82 vs 1.8 ± 0.37 pmol/mg protein, p < 0.05 control vs. Ang-(1-7) treated decidualized horns). The circulating levels of 6-keto-PGF1a and TXB2 were decreased by Ang-(1-7) infusion, while no difference was observed in circulating PGE2. Although the global assessment of cleaved caspase 3 immunostaining, a marker of apoptosis, was unchanged within the Ang-(1-7) decidualized horn, there were localized decreases in cleaved caspase 3 staining in the luminal region in the decidualized uterus of Ang-(1-7)-treated rats. CONCLUSIONS: These studies show that increased local uterine Ang-(1-7) alters the uterine prostaglandin environment, possibly leading to disruptions of early events of decidualization.
Subject(s)
6-Ketoprostaglandin F1 alpha/metabolism , Angiotensin I/administration & dosage , Dinoprostone/metabolism , Peptide Fragments/administration & dosage , Pseudopregnancy/metabolism , Uterus/metabolism , 6-Ketoprostaglandin F1 alpha/antagonists & inhibitors , Animals , Decidua/drug effects , Decidua/metabolism , Dinoprostone/antagonists & inhibitors , Female , Pregnancy , Rats , Rats, Sprague-Dawley , Uterus/drug effectsABSTRACT
BACKGROUND: ATF6α, one of the sensor proteins in the stress signaling pathway of the endoplasmic reticulum, is located in the membrane of the endoplasmic reticulum. To date, the physiological function of ATF6α in the process of embryo implantation has not been reported. METHODS: In this study, the expression pattern of ATF6α in the mouse uterus during peri-implantation and the estrous cycle was detected by real-time PCR, western blot and immunohistochemistry. RESULTS: ATF6α mRNA and protein levels were higher in the uterus near the implantation site on day 5 and were intensely expressed in the secondary decidual zone (SDZ) on days 7-8. In the uteri of pseudopregnant mice, ATF6α mRNA and protein levels were lower on day 5 than on other days. The activating blastocyst and artificial decidualization had an obvious effect of increasing the expression of ATF6α. In addition, the expression of ATF6α was affected by progesterone (P4) and estrogen (E2) in ovariectomized mice. This finding is further supported by evidence from mice during the estrous cycle. CONCLUSIONS: Thus, we have concluded that ATF6α may play an important role during embryo implantation and decidualization.
Subject(s)
Activating Transcription Factor 6/biosynthesis , Embryo Implantation/physiology , Gene Expression Regulation, Developmental , Uterus/metabolism , Activating Transcription Factor 6/genetics , Animals , Estrous Cycle/genetics , Estrous Cycle/metabolism , Female , Male , Mice , Pregnancy , Pseudopregnancy/genetics , Pseudopregnancy/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/geneticsABSTRACT
Successful implantation of an embryo requires adequate depth of invasion in the endometrium, which depends upon decidualization. The aim of the present study was to elucidate why humans experience spontaneous decidualization and menstruation while most other mammals do not. We established a spontaneous decidualization model in pseudopregnant rats with vitamin E deficiency (VED) to investigate mechanisms associated with spontaneous decidualization. Vaginal smears were used to monitor bleeding while vitamin E levels were analyzed with a commercial vitamin E assay kit. Trypan blue staining was used to observe the implantation site at 5.5 days post-coitum (dpc). Uterine morphology, estradiol (E2) and progesterone levels, and the anti-oxidation system were evaluated at 5.5, 7.5, and 9.5 dpc. The proportion of rats in the VED group exhibiting endometrial bleeding gradually increased (5.9%, 32.3%, and 50%) over three consecutive cycles of pseudopregnancy. Vitamin E levels in the VED group were markedly lower compared to the control group in both the plasma and uterus, while the level of vitamin E in the liver did not differ between the control and VED groups. Spontaneous decidualization in the VED group was validated by histological examination and immunohistochemistry. At 5.5 dpc, the mean serum E2 level in the VED group was more than twice that of the control group. The mean total anti-oxidizing capability, catalase level, and glutathione peroxidase activity were significantly reduced in the decidualized portion of the VED group compared to controls, while the malondialdehyde level was also significantly higher in the decidualized portion of the VED group. We hypothesize that the E2 surge at 5.5 dpc and increasing levels of reactive oxygen species are responsible for spontaneous decidualization in VED rats.
Subject(s)
Deciduoma/physiopathology , Estradiol/metabolism , Pregnancy Complications/metabolism , Pseudopregnancy/metabolism , Reactive Oxygen Species/metabolism , Vitamin E Deficiency/complications , Vitamin E Deficiency/metabolism , Animals , Female , Pregnancy , Pseudopregnancy/complications , Rats, WistarABSTRACT
Previous studies have shown that microRNAs are involved in the process of implantation. They play an important role in cell growth and proliferation. DROSHA is the microRNA-processing enzyme and is required for the maturation of microRNAs. However, its expression and function during early pregnancy in mice still remain unclear. In the present study, we analyzed the expression pattern of DROSHA in the mouse uterus during early pregnancy, pseudopregnancy, artificially induced decidualization, and in the ovariectomized mouse uterus using real-time quantitative polymerase chain reaction, Western blotting analyses, and immunohistochemistry. We found that DROSHA was spatiotemporally expressed in decidualizing stromal cells during early pregnancy and in pseudopregnant mice in which decidualization was artificially induced. In the ovariectomized mouse uterus, the expression of DROSHA was upregulated after progesterone treatment. In a stromal cell culture model, the expression of DROSHA gradually increased with the progression of stromal decidualization. Taken together, our findings suggest that DROSHA is involved in stromal decidualization and may play an important role in embryo implantation in mice.
Subject(s)
Embryo Implantation/genetics , Gene Expression Regulation , Ribonuclease III/metabolism , Uterus/metabolism , Animals , Decidua/metabolism , Female , Mice , Ovariectomy , Pregnancy , Progesterone/pharmacology , Pseudopregnancy/metabolism , Ribonuclease III/genetics , Stromal Cells/drug effects , Stromal Cells/metabolism , Up-Regulation/drug effects , Uterus/drug effectsABSTRACT
To determine the function of Annexin A2 (Axna2) in mouse embryo implantation in vivo, experimental manipulation of Axna2 activities was performed in mouse endometrial tissue in vivo and in vitro. Histological examination of endometrial tissues was performed throughout the reproduction cycle and after steroid treatment. Embryo implantation was determined after blockage of the Axna2 activities by siRNA or anti-Axna2 antibody. The expression of Axna2 immunoreactivies in the endometrial luminal epithelium changed cyclically in the estrus cycle and was upregulated by estrogen. After nidatory estrogen surge, there was a concentration of Axna2 immunoreactivities at the interface between the implanting embryo and the luminal epithelium. The phenomenon was likely to be induced by the implanting embryos as no such concentration of signal was observed in the inter-implantation sites and in pseudopregnancy. Knockdown of Axna2 by siRNA reduced attachment of mouse blastocysts onto endometrial tissues in vitro. Consistently, the number of implantation sites was significantly reduced after infusion of anti-Axna2 antibody into the uterine cavity. Steroids and embryos modulate the expression of Axna2 in the endometrial epithelium. Axna2 may function as an adhesion molecule during embryo implantation in mice.
Subject(s)
Annexin A2/metabolism , Cell Adhesion Molecules/metabolism , Embryo Implantation/physiology , Endometrium/metabolism , Epithelium/metabolism , Animals , Blastocyst/metabolism , Estrogens/metabolism , Female , Male , Mice , Mice, Inbred ICR , Pseudopregnancy/metabolismABSTRACT
The corpus luteum is a temporary endocrine structure in mammals that plays an important role in the female reproductive cycle and is formed from a ruptured and ovulated follicle with rapid angiogenesis. Vascular endothelial growth factor (VEGF) is thought to be vital in normal and abnormal angiogenesis in the ovary, but the molecular regulation of luteal VEGF expression during corpus luteum development in vivo is still poorly understood at present. Therefore, we examined whether hypoxia-inducible factor-1a (HIF-1a) is induced and regulates VEGF expression and luteal function in vivo using a pseudopregnant rat model treated with a small-molecule inhibitor of HIF-1a, echinomycin. Corpus luteum development in the pseudopregnant rat ovary was determined after measuring plasma progesterone concentration and ovarian prostaglandin F2a content to reflect changes in HIF-1a and VEGF on different days of this developmental process. At day 7, the corpus luteum was formed and the expression of HIF- 1a/VEGF reached a maximum, while a significant decrease in HIF-1a/ VEGF expression was observed when luteolysis occurred at day 13. Additionally, echinomycin blocked luteal development by inhibiting VEGF expression mediated by HIF-1a and following luteal function by detecting the progesterone changes at day 7. These results demonstrated that HIF-1a-mediated VEGF expression might be an important mechanism regulating ovarian luteal development in mammals in vivo, which may provide new strategies for fertility control and for treating some types of ovarian dysfunction, such as polycystic ovarian syndrome, ovarian hyperstimulation syndrome, and ovarian neoplasia.