Early Pregnancy In Mice Research Articles

Exposure to Benzo(a)pyrene damages mitochondrial function via suppressing mitochondrial melatonin receptors in ovarian corpus luteum during early pregnancy

Benzo(a)pyrene (BaP) is a well-known environmental endocrine pollutant, which has ovarian toxicity in mammals. Ovarian corpus luteum (CL), as the main source of progesterone synthesis in early pregnant female, requires a large number of mitochondria for energy supply. We previously demonstrated that BaP and its metabolite benzo(a)pyren-7, 8-dihydrodiol-9, 10-epoxide (BPDE) inhibited the ovarian melatonin receptors (MTRs) expression and decreased the levels of estrogen and progesterone during early pregnancy in mice. Emerging researches show that MTRs also exist on mitochondrial membrane and participate in the regulation of mitochondrial function. However, the relationship between BaP, MTRs on mitochondrial membrane and mitochondrial function remains unknown. Consequently, this study focuses on the effect and potential mechanism of BaP on ovarian luteal mitochondrial function during early pregnancy. We found that BaP and its metabolite BPDE decreased MTRs in early pregnant CL and luteinized KGN cells, especially in mitochondria. Furthermore, BaP or BPDE up-regulated the expression of SIRT3, Mfn2 and Drp-1, damaged mitochondrial morphology and decreased the MMP and the ATP levels, thereby causing mitochondrial dysfunction. Notably, activation of the MTRs on mitochondrial membrane by MTRs agonist ramelteon partially alleviated BPDE-induced up-regulation of SIRT3, Mfn2 and Drp-1, reduced mitochondrial fragmentation and enhanced the MMP and the ATP levels, thus restoring the expression of steroid rate-limiting enzymes. Together, these findings firstly proved that BaP and BPDE down-regulate MTRs on mitochondrial membrane, and further injure mitochondrial function in early pregnant rats’ CL, which provides a new insight for understanding the exact mechanism of the BaP-induced ovarian toxicity.

Endometrial glycogen metabolism during early pregnancy in mice.

Glucose is critical during early pregnancy. The uterus can store glucose as glycogen but uterine glycogen metabolism is poorly understood. This study analyzed glycogen storage and localization of glycogen metabolizing enzymes from proestrus until implantation in the murine uterus. Quantification of diastase-labile periodic acid-Schiff (PAS) staining showed glycogen in the glandular epithelium decreased 71.4% at 1.5 days postcoitum (DPC) and 62.13% at DPC 3.5 compared to proestrus. In the luminal epithelium, glycogen was the highest at proestrus, decreased 46.2% at DPC 1.5and 63.2% at DPC 3.5. Immunostaining showed that before implantation, glycogen metabolizing enzymes were primarily localized to the glandular and luminal epithelium. Stromal glycogen was low from proestrus to DPC 3.5. However, at the DPC 5.5 implantation sites, stromal glycogen levels increased sevenfold. Similarly, artificial decidualization resulted in a fivefold increase in glycogen levels. In both models, decidualization increased expression of glycogen synthase as determine by immunohistochemistry and western blot. In conclusion, glycogen levels decreased in the uterine epithelium before implantation, indicating that it could be used to support preimplantation embryos. Decidualization resulted in a dramatic increase in stromal glycogen levels, suggesting it may have an important, but yet undefined, role in pregnancy.

Acbp is essential for decidualization during early pregnancy in mice.

Decidualization of uterine stromal cells plays an important role in the establishment of normal pregnancy. Previous studies have demonstrated that Acyl-CoA binding protein (Acbp) is critical to cellular proliferation, differentiation, mitochondrial functions, and autophagy. The characterization and physiological function of Acbp during decidualization remain largely unknown. In the present study, we conducted the expression profile of Acbp in the endometrium of early pregnant mice. With the occurrence of decidualization, the expression of Acbp gradually increased. Similarly, Acbp expression was also strongly expressed in decidualized cells following artificial decidualization, both in vivo and in vitro. We applied the mice pseudopregnancy model to reveal that the expression of Acbp in the endometrium of early pregnant mice was not induced by embryonic signaling. Moreover, P4 significantly upregulated the expression of Acbp, whereas E2 appeared to have no regulating effect on Acbp expression in uterine stromal cells. Concurrently, we found that interfering with Acbp attenuated decidualization, and that might due to mitochondrial dysfunctions and the inhibition of fatty acid oxidation. The level of autophagy was increased after knocking down Acbp. During induced decidualization, the expression of ACBP was decreased with the treatment of rapamycin (an autophagy inducer), while increased with the addition of Chloroquine (an autophagy inhibitor). Our work suggests that Acbp plays an essential role in the proliferation and differentiation of stromal cells during decidualization through regulating mitochondrial functions, fatty acid oxidation, and autophagy.

Cytochrome P450 26A1 regulates the clusters and killing activity of NK cells during the peri-implantation period.

Cytochrome P450 26A1 (CYP26A1) plays a vital role in early pregnancy in mice. Our previous studies have found that CYP26A1 affects embryo implantation by modulating natural killer (NK) cells, and that there is a novel population of CYP26A1+ NK cells in the uteri of pregnant mice. The aim of this study was to investigate the effects of CYP26A1 on the subsets and killing activity of NK cells. Through single‐cell RNA sequencing (scRNA‐seq), we identified four NK cell subsets in the uterus, namely, conventional NK (cNK), tissue‐resident NK (trNK) 1 and 2, and proliferating trNK (trNKp). The two most variable subpopulations after uterine knockdown of CYP26A1 were trNKp and trNK2 cells. CYP26A1 knockdown significantly downregulated the expression of the NK cell function‐related genes Cd44, Cd160, Vegfc, and Slamf6 in trNK2 cells, and Klra17 and Ogn in trNKp cells. Both RNA‐seq and cytotoxicity assays confirmed that CYP26A1+ NK cells had low cytotoxicity. These results indicate that CYP26A1 may affect the immune microenvironment at the maternal‐foetal interface by regulating the activity of NK cells.

The expression of lincRNA AC027700.1 in mouse decidualization.

Long non-coding RNAs (lncRNAs), which belong to the non-protein-coding RNAs, are greater than 200 nt in length. Although they have been found to play crucial roles in the regulation of cell growth and development, cell metabolism and the development of diseases, they are rarely reported in decidualization. The objective of our study is to explore the expression of lincRNA AC027700.1 in the endometrium of early pregnant mice and its role in decidualization. The expression of AC027700.1 in uterine tissues at implantation sites and inter implantation sites on the 6th day of pregnancy were detected by qRT-PCR. The relative expression of AC027700.1 in an in vivo model of induced decidualization in pseudopregnant mice and in in vitro model of induced decidualization in primary stromal cells and nucleus/cytoplasmic fractions were detected by qRT-PCR. GO and KEGG analysis of downstream target genes were performed by GOseq and KOBAS, respectively. The results show that AC027700.1 expression is significantly increased in tissues at implantation sites on the 6th day of pregnancy and in decidualized endometrial tissues and stromal cells. Furthermore, AC027700.1 localizes in the nuclear fraction and the downstream targeted genes are mainly involved in autophagy, cell cycle and RNA transport pathways. This study revealed that lincRNA AC027700.1 may be involved in decidualization of endometrium in early pregnancy, but the specific role and regulatory mechanism remain to be further studied.

The role of IP-10 and its receptor CXCR3 in early pregnancy

The local immune microenvironment of the uterus plays an important role in a successful pregnancy. IP-10 (CXCL10) has been extensively studied in many immune-related diseases. However, the immune role of IP-10 in early pregnancy has not been fully recognized. This study mainly investigated the role of pro-inflammatory chemokine IP-10 in pregnancy. The levels of IP-10 and its receptor chemokine receptor 3 (CXCR3) were lower in the decidual tissues of an abortion-prone mice than in normal pregnant mice. Meantime, the expression of IP-10 and CXCR3 was higher in the decidual tissues of early pregnant women than in the endometrial tissues of non-pregnant women. IP-10 promoted the production of interleukin 17 (IL-17) and interferon gamma (IFN-γ), and also promoted the migration and differentiation of uterine decidual T cells to type 1 T helper (Th1) cells and Th17 cells. The abortion rate of early pregnant mice increased but the number of CD49b+, CD11b+, and CD3ε+ cells in the decidual tissues decreased upon treatment with anti-IP-10 antibody. Moreover, anti IP-10 antibody decreased the expression of RANTES but increased the expression of anti-inflammatory cytokines IL-6 and IL-10. A successful pregnancy requires the participation of IP-10. IP-10 participates in formation of the pro-inflammatory immune microenvironment during early pregnancy by regulating the distribution of immune cells and promoting the production of pro-inflammatory cytokines.

Carnitine palmitoyltransferase 1A is essential for decidualization in mice

Decidualization accompanies with extensive stromal cell proliferation and differentiation, is a crucial step in early pregnancy. Aberrant decidualization is linked to infertility and miscarriage but the mechanisms remain unclear. Carnitine palmitoyltransferase 1A (CPT1A) is an enzyme catalyzing key steps in the fatty acid beta-oxidation pathway. The objective of this study was to investigate the role of CPT1A in decidualization during early pregnancy. An increased expression of CPT1A was found both in Days 6 and 7 as compared with in Days 1, 4 and 5. Further examination showed that on days 5–7 of pregnancy, the protein level of CPT1A was strongly up-regulated at implantation sites compared with inter-implantation sites, the location of CPT1A protein was distributed in the decidual zone. Upon further exploration, CPT1A expression was significantly increased in response to artificially induced decidualization both in vivo and in vitro. After down-regulating CPT1A expression by CPT1A-small interfering RNA (siCPT1A) in primary mouse endometrial stromal cells, expressions of decidualization markers and cell proliferation markers were decreased. After siCPT1A was transfected into the mouse uterus, decidualization impaired and then led to the loss of the implanted embryos. Thus, CPT1A is important for decidualization in mice and it may regulate the stromal cell proliferation progress. It is worth noting that the expression of CPT1A protein of human decidua was significantly decreased in spontaneous abortion groups compared to normal pregnancy groups. Collectively, CPT1A is essential for endometrium of early pregnant mice and humans.

A reduction in voluntary physical activity in early pregnancy in mice is mediated by prolactin.

As part of the maternal adaptations to pregnancy, mice show a rapid, profound reduction in voluntary running wheel activity (RWA) as soon as pregnancy is achieved. Here, we evaluate the hypothesis that prolactin, one of the first hormones to change secretion pattern following mating, is involved in driving this suppression of physical activity levels during pregnancy. We show that prolactin can acutely suppress RWA in non-pregnant female mice, and that conditional deletion of prolactin receptors (Prlr) from either most forebrain neurons or from GABA neurons prevented the early pregnancy-induced suppression of RWA. Deletion of Prlr specifically from the medial preoptic area, a brain region associated with multiple homeostatic and behavioral roles including parental behavior, completely abolished the early pregnancy-induced suppression of RWA. As pregnancy progresses, prolactin action continues to contribute to the further suppression of RWA, although it is not the only factor involved. Our data demonstrate a key role for prolactin in suppressing voluntary physical activity during early pregnancy, highlighting a novel biological basis for reduced physical activity in pregnancy.

Extrathymic Aire-expressing cells support maternal-fetal tolerance.

Healthy pregnancy requires tolerance to fetal alloantigens as well as syngeneic embryonic and placental antigens. Given the importance of the autoimmune regulator (Aire) gene in self-tolerance, we investigated the role of Aire-expressing cells in maternal-fetal tolerance. We report that maternal ablation of Aire-expressing (Aire +) cells during early mouse pregnancy caused intrauterine growth restriction (IUGR) in both allogeneic and syngeneic pregnancies. This phenotype is immune mediated, as IUGR was rescued in Rag1-deficient mice, and involved a memory response, demonstrated by recurrence of severe IUGR in second pregnancies. Single-cell RNA sequencing demonstrated that Aire + cell depletion in pregnancy results in expansion of activated T cells, particularly T follicular helper cells. Unexpectedly, selective ablation of either Aire-expressing medullary thymic epithelial cells or extrathymic Aire-expressing cells (eTACs) mapped the IUGR phenotype exclusively to eTACs. Thus, we report a previously undescribed mechanism for the maintenance of maternal-fetal immune homeostasis and demonstrate that eTACs protect the conceptus from immune-mediated IUGR.

Exposure to ethylparaben and propylparaben interfere with embryo implantation by compromising endometrial decidualization in early pregnant mice.

Ethylparaben (EtP) and propylparaben (PrP) are common preservatives and well-known endocrine-disrupting chemicals. Studies have demonstrated that they can reduce female fertility, but the underlying mechanism, especially that on embryo implantation, is still poorly understood. Endometrial decidualization is a critical event for embryo implantation. In this study, we aimed to explore the effects of EtP/PrP on endometrial decidualization. Pregnant mice were dosed daily by oral gavage with EtP at 0, 400, 800 and 1600 mg/kg or with PrP at 0, 625, 1250 and 2500 mg/kg from Day 1 of pregnancy until sacrifice. The results showed that the rate of pregnant mice with impaired embryo implantation, whose number of implantation sites was less than 7, was significantly increased after exposure to 1600 mg/kg EtP or 2500 mg/kg PrP. Further study found that the expression of endometrial decidualization markers HOXA10, MMP9 and PR was significantly downregulated in 1600 mg/kg EtP group and 2500 mg/kg PrP group. Notably, serum oestrogen and progesterone levels were significantly increased, whereas the expression of uterine oestrogen receptor and progesterone receptor was decreased following 1600 mg/kg EtP or 2500 mg/kg PrP exposure. In the breeding test, fewer offspring were found after females were exposed to 1600 mg/kg EtP or 2500 mg/kg PrP in early pregnancy. This demonstrated that exposure to EtP/PrP interfered with embryo implantation by compromising endometrial decidualization in early-stage pregnant mice. Disorders of reproductive hormones and hormone receptor signals could be responsible for impaired decidualization. This study broadened the understanding on the biological safety of EtP and PrP.

AMPK/mTOR downregulated autophagy enhances aberrant endometrial decidualization in folate-deficient pregnant mice.

Existing evidence suggests that adverse pregnancy outcomes are closely related to dietary factors. Folate plays an important role in neural tube formation and fetal growth, folate deficiency is a major risk factor of birth defects. Our early studies showed that folate deficiency could impair enddecidualization, however, the mechanism is still unclear. Dysfunctional autophagy is associated with many diseases. Here, we aimed to evaluate the adverse effect of folate deficiency on endometrial decidualization, with a particular focus on endometrial cell autophagy. Mice were fed with no folate diet in vivo and the mouse endometrial stromal cell was cultured in a folate-free medium in vitro. The decrease of the number of endometrial autophagosomes and the protein expressions of autophagy in the folate-deficient group indicated that autophagosome formation, autophagosome-lysosome fusion, and lysosomal degradation were inhibited. Autophagic flux examination using mCherry-GFP-LC3 transfection showed that the fusion of autophagosomes with lysosomes was inhibited by folate deficiency. Autophagy inducer rapamycin could reverse the impairment of folate deficiency on endometrial decidualization. Moreover, folate deficiency could reduce autophagy by disrupting AMPK/mTOR signaling, resulting in aberrant endometrial decidualization and adverse pregnancy outcomes. Further co-immunoprecipitation examination showed that decidual marker protein Hoxa10 could interact with autophagic marker protein Cathepsin L, and the interaction was notably reduced by folate deficiency. In conclusion, AMPK/mTOR downregulated autophagy was essential for aberrant endometrial decidualization in early pregnant mice, which could result in adverse pregnancy outcomes. Thisprovided some new clues for understanding the causal mechanisms of birth defects induced by folate deficiency.

The effect of reproductive toxicity induced by ZnO NPs in mice during early pregnancy through mitochondrial apoptotic pathway.

The potential toxicity of Zinc oxide nanoparticles (ZnO NPs) to human beings has become a widespread concern. This study explored the reproductive toxicity and the mechanism of toxicity of ZnO NPs in early pregnant mice. The results showed that abnormal weight changes, induced inflammation, reduced level of serum sex hormones, damaged uterus, increased abortion, and abnormal development of fetus. In the uterus, the transcription levels of ZnT-1, HO-1, Bax, Bax/Bcl-2, JNK, and Caspase-3 were significantly up-regulated while Bcl-2, ER-1 and PR were significantly down-regulated. The TUNEL-positive cells increased that were exposed to high levels of ZnO NPs. In summary, those results indicated that Zn from high levels of exposure to ZnO NPs accumulated in the uterus that could have caused the formation of ROS that led to oxidative stress, which might have activated the mitochondrial apoptotic pathway that could have caused the uterine injury which induced the observed reproductive toxicity.

Effects of exogenous adiponectin supplementation in early pregnant PCOS mice on the metabolic syndrome of adult female offspring

ObjectivePCOS is a heterogeneous endocrine disorder with both reproductive and metabolic abnormalities. At present, PCOS has been confirmed to have a certain genetic background. Compared with healthy women, the vast majority of PCOS patients have hyperandrogenemia, and this excessive androgen exposure during pregnancy may affect the development of female fetuses. The aim of the current study was to investigate the effect of adiponectin intervention during early pregnancy of obese mice with PCOS on the metabolic phenotype of adult female offspring.MethodsAfter the PCOS model was established, C57BL/6J mice were divided into maternal-control, maternal-PCOS, and maternal-PCOS + APN groups. DHEA-induced PCOS mice were supplemented with adiponectin (10 mg/kg/day) in the early pregnancy in order to eliminate adverse hormone exposure and then traced for endocrine indicators in their adult female offspring, which were observed for metabolism syndrome or endocrine disturbance and exhibited the main effects of APN. To further explore the underlying mechanism, the relative expressions of phosphorylated AMPK, PI3K, and Akt were detected in the ovaries of offspring mice.ResultsThe serum testosterone level of the maternal-PCOS + APN group in early pregnancy was significantly lower than that of the maternal-PCOS group (p < 0.01). The serum testosterone level in the offspring-PCOS + APN group was significantly lower than in the offspring-PCOS group (p <0.05), the diestrus time characterized by massive granulocyte aggregation in the estrus cycle was significantly shorter than in the offspring-PCOS group (p<0.05), and the phenotypes of PCOS-like reproductive disorders and metabolic disorders, such as obesity, insulin resistance, impaired glucose tolerance, and hyperlipidemia, were also significantly improved in the offspring-PCOS + APN group (p < 0.05). Compared with the control group, the expression levels of phosphorylated AMPK, PI3K, and Akt in the offspring-PCOS group were significantly decreased (p < 0.05), while those in the offspring-PCOS + APN group were significantly increased (p < 0.05).ConclusionsAPN intervention in early pregnancy significantly reduced the adverse effects of maternal obesity and high androgen levels during pregnancy on female offspring and corrected the PCOS-like endocrine phenotype and metabolic disorders of adult female offspring. This effect may be caused by the activation of the AMPK/PI3K-Akt signaling pathway in PCOS offspring mice.

Hyaluronan control of the primary vascular barrier during early mouse pregnancy is mediated by uterine NK cells.

Successful implantation is associated with a unique spatial pattern of vascular remodeling, characterized by profound peripheral neovascularization surrounding a periembryo avascular niche. We hypothesized that hyaluronan controls the formation of this distinctive vascular pattern encompassing the embryo. This hypothesis was evaluated by genetic modification of hyaluronan metabolism, specifically targeted to embryonic trophoblast cells. The outcome of altered hyaluronan deposition on uterine vascular remodeling and postimplantation development were analyzed by MRI, detailed histological examinations, and RNA sequencing of uterine NK cells. Our experiments revealed that disruption of hyaluronan synthesis, as well as its increased cleavage at the embryonic niche, impaired implantation by induction of decidual vascular permeability, defective vascular sinus folds formation, breach of the maternal-embryo barrier, elevated MMP-9 expression, and interrupted uterine NK cell recruitment and function. Conversely, enhanced deposition of hyaluronan resulted in the expansion of the maternal-embryo barrier and increased diffusion distance, leading to compromised implantation. The deposition of hyaluronan at the embryonic niche is regulated by progesterone-progesterone receptor signaling. These results demonstrate a pivotal role for hyaluronan in successful pregnancy by fine-tuning the periembryo avascular niche and maternal vascular morphogenesis.

Особенности морфофункционального состояния маточно-плацентарной единицы и эмбриона мыши на ранних сроках экспериментального невынашивания беременности

Introduction. The majority of cases of habitual miscarriage are closely related to impaired maternal immune tolerance. Experimental mice models of abortion, where the critical time points are established to monitor pregnancy progression, are widely used to study the pathogenesis of early reproductive loss. However, the relationship between the embryonic development and extraembryonic structures with the immune microen-vironment is not well understood. The aim of the study is to compare the morphofunctional characteristics of the uteroplacental unit and embryo in early pregnancy in mice with experimental spontaneous, induced, and potentiated abortions. Materials and methods. Allogeneic physiological pregnancy was modeled using ♀CBA × ♂BALB/c mice mating. Spontaneous abortions were reproduced by mating ♀CBA × ♂DBA/2 mice. Induced abortions and potentiated abortions were triggered by intraperitoneal administration of the immunomodulator ß-heptylglycoside muramyl dipeptide (MDP) to pregnant females on gestation day (gd) 5 and gd 7 mated in following combinations ♀CBA × ♂BALB/c and ♀CBA × ♂DBA/2, respectively. Mice were withdrawn from the experiment on gd 8, then uterine horns and uteroplacental units were isolated. The material was fixed in 10% neutral formalin, and standard processing was performed for histological examination. In equatorial sections, the cross-sectional area of uteroplacental unit was measured, and maternal, embryonic, as well as extraembryonic structures were analyzed. An immunohistochemical assay with rabbit polyclonal anti-CD68 antibodies was conducted to detect macrophages, then the density of CD68+ cells per 1 mm2 of the decidual area was determined. Results. On day 8 of pregnancy, in the groups of induced and potentiated abortions cross-sectional area of the uteroplacental units was decreased compared with the groups of physiological pregnancy and sponta-neous abortions. Induced and potentiated abortions demonstrated vasodilation in the mesometric decidua zone and stagnation of blood in the blood vessels, compaction of trophoblast, and hemorrhage in decidua basalis whether spontaneous abortions showed occluded maternal vessels surrounded by edematous and vacuolated trophoblast cells.At the early stages of modeling of spontaneous and immuno-dependent abortions, disturbances in the devel-opment of the embryo and extraembryonic structures were revealed, ranging from initial signs of resorption to the destruction of the embryo and membranes.In viable embryos, density indices of CD68+ macrophages in the decidual membrane of the uteroplacental units did not differ significantly in all miscarriage cases, CD68+ cells were not detected in cases of embryo-nal resorption. Conclusion. The MDP treatment in a period after completion of implantation and before forming a mature placenta (gd 5–7) results in the inducing or the potentiating abortions in CBA females mated with by Balb/c or DBA/2 males, respectively. In both cases, we revealed disturbed chorion morphogenesis and embryonic development to the extent of its complete resorption and the absence of CD68+ macrophages in resorbed embryos’ microenvironment on day 8 of pregnancy. Keywords: miscarriage, models, muramyl dipeptide β-heptylglycoside, uteroplacental unit, macrophages, immunotolerance

Endothelial Jagged1 Antagonizes Dll4/Notch Signaling in Decidual Angiogenesis during Early Mouse Pregnancy.

Maternal spiral arteries and newly formed decidual capillaries support embryonic development prior to placentation. Previous studies demonstrated that Notch signaling is active in endothelial cells of both decidual capillaries and spiral arteries, however the role of Notch signaling in physiologic decidual angiogenesis and maintenance of the decidual vasculature in early mouse pregnancy has not yet been fully elucidated. We used the Cdh5-CreERT2; Jagged1(Jag1)flox/flox (Jag1∆EC) mouse model to delete Notch ligand, Jag1, in maternal endothelial cells during post-implantation, pre-placentation mouse pregnancy. Loss of endothelial Jag1 leads to increased expression of Notch effectors, Hey2 and Nrarp, and increased endothelial Notch signaling activity in areas of the decidua with remodeling angiogenesis. This correlated with an increase in Dll4 expression in capillary endothelial cells, but not spiral artery endothelial cells. Consistent with increased Dll4/Notch signaling, we observed decreased VEGFR2 expression and endothelial cell proliferation in angiogenic decidual capillaries. Despite aberrant Dll4 expression and Notch activation in Jag1∆EC mutants, pregnancies were maintained and the decidual vasculature was not altered up to embryonic day 7.5. Thus, Jag1 functions in the newly formed decidual capillaries as an antagonist of endothelial Dll4/Notch signaling during angiogenesis, but Jag1 signaling is not necessary for early uterine angiogenesis.

Timing of exposure to gonadotropins has differential effects on the conceptus: evidence from a mouse model†.

Superovulation with gonadotropins alters the hormonal milieu during early embryo development and placentation, and may be responsible for fetal and placental changes observed after in vitro fertilization (IVF). We hypothesized that superovulation has differential effects depending on timing of exposure. To test our hypothesis, we isolated the effect of superovulation on pre- and peri-implantation mouse embryos. Blastocysts were obtained from either natural mating or following superovulation and mating, and were transferred into naturally mated or superovulated pseudopregnant recipient mice. Fetal weight was significantly lower after peri-implantation exposure to superovulation, regardless of preimplantation exposure (p=0.006). Placentas derived from blastocysts exposed to superovulation pre- and peri-implantation were larger than placentas derived from natural blastocysts that are transferred into a natural or superovulated environment (p<0.05). Fetal-to-placental weight ratio decreased following superovulation during the pre- or peri-implantation period (p=0.05, 0.01, respectively) and these effects were additive. Peg3 DNA methylation levels were decreased in placentas derived from exposure to superovulation both pre- and peri-implantation compared with unexposed embryos and exposure of the preimplantation embryo only. Through RNA sequencing on placental tissue, changes were identified in genes involved in immune system regulation, specifically interferon signaling, which has been previously implicated in implantation and maintenance of early pregnancy in mice. Overall, we found that the timing of exposure to gonadotropin stimulation can have differential effects on fetal and placental growth. These findings could impact clinical practice and underscores the importance of dissecting the role of procedures utilized during IVF on pregnancy complications.

CDC20 inhibitor Apcin inhibits embryo implantation in vivo and in vitro.

For successful implantation, endometrial receptivity must be established. The high expression of CDC20 in many kinds of malignant tumours has been reported, and it is related to the occurrence and development of tumours. According to these functions, we think that CDC20 may also play important roles in the process of embryo implantation. To prove our hypothesis, we observed the distribution and expression of CDC20 in mouse and human early pregnancy. The effect of E2 and/or P4 on the expression of CDC20 in human endometrial cells was detected by Western blot. To further explore whether CDC20 is an important factor in adhesion and proliferation. The results showed that the expression of CDC20 in the uterus and menstrual cycle of early pregnant mice was spatiotemporal. E2 can promote the expression of CDC20. On the contrary, P4 and E2 + P4 inhibited the expression of CDC20. We also detected the proliferation and adhesion of human endometrial cells. We found that the inhibition of CDC20 with its inhibitor Apcin could reduce the adhesion rate and proliferation ability to RL95‐2 and HEC‐1A cells, respectively. Inhibiting CDC20 by Apcin could interfere the embryo implantation of mouse. It is suggested that CDC20 may play an important role in the process of embryo implantation.Significance of the studyEmbryo implantation is an extremely complex and delicate process, including identification, localisation, adhesion and invasion between embryo and endometrium. Studies have shown the process of embryo implantation is very similar to that of tumour invasion. CDC20 is a cancer‐promoting factor. We found CDC20 is spatially and spatially expressed in mouse and human menstrual cycles and is regulated by oestrogen and progesterone. Apcin can inhibit the adhesion of JAR cells and embryo implantation of mouse. CDC20 may provide a new way to improve the success rate of assisted reproduction.

Functions of Lysosomes in Mammalian Female Reproductive System

The lysosome is the most acidic membrane-bound intracellular organelle. Lysosomal acidity is primarily maintained by vacuolar H+-ATPase (V-ATPase) and counter ion channels. There are >60 hydrolytic enzymes in the lysosome for its fundamental digestive role. Lysosomes also play important roles in endocytosis, exocytosis, autophagy, and cell death. Studies that have implicated roles of lysosomes in the female reproductive system are reviewed here. In the ovary, lysosomes are implicated in the preparation of free cholesterol for steroidogenesis and degradation of regulators of steroidogenesis, regulation of follicular atresia, follicle rupture during ovulation, luteal cell survival, and luteal regression. In the oviduct, lysosomes are involved in endocytosis of both serum and oviductal luminal components. In the uterus during the menstrual/estrous cycle, lysosomes are associated with endometrial secretion, apoptosis, and menstruation. In the uterus during early pregnancy, lysosomes are involved in the temporal and directional changes of endocytosis, uterine epithelial acidification upon embryo implantation initiation, and embryo-maternal mutual communications via extracellular vesicles. In the placenta, lysosomes are implicated in nutrient transport and placental separation from the uterus for parturition. In the mammary gland, lysosomes are important for mammary gland development and involution. These findings suggest/demonstrate functions of lysosomes in multiple processes of female reproduction, from ovulation to ovarian steroidogenesis for pregnancy maintenance, and from essential in utero nurturing of developing embryos/fetuses via embryo/fetal-maternal communications, to optional postpartum nurturing of newborns via lactation. Future studies using genetically or modified animal models and pharmacological approaches will provide novel insights into the functions and mechanisms of lysosomes in the mammalian female reproductive system.

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.