Murine uterine gland branching is necessary for gland function in implantation.

Uterine glands are branched, tubular structures whose secretions are essential for pregnancy success. It is known that pre-implantation glandular expression of leukemia inhibitory factor (LIF) is crucial for embryo implantation; however, the contribution of uterine gland structure to gland secretions, such as LIF, is not known. Here, we use mice deficient in estrogen receptor 1 (ESR1) signaling to uncover the role of ESR1 signaling in gland branching and the role of a branched structure in LIF secretion and embryo implantation. We observed that deletion of ESR1 in neonatal uterine epithelium, stroma, and muscle using the progesterone receptor PgrCre causes a block in uterine gland development at the gland bud stage. Embryonic epithelial deletion of ESR1 using a Müllerian duct Cre line, Pax2Cre, displays gland bud elongation but a failure in gland branching. Reduction of ESR1 in adult uterine epithelium using the lactoferrin-Cre (LtfCre) displays normally branched uterine glands. Unbranched glands from Pax2Cre Esr1flox/flox uteri fail to express glandular pre-implantation Lif, preventing implantation chamber formation and embryo alignment along the uterine mesometrial-antimesometrial axis. In contrast, branched glands from LtfCre Esr1flox/flox uteri display reduced expression of ESR1 and glandular Lif resulting in delayed implantation chamber formation and embryo-uterine axes alignment but mice deliver a normal number of pups. Finally, pre-pubertal unbranched glands in control mice express Lif in the luminal epithelium but fail to express Lif in the glandular epithelium, even in the presence of estrogen. These data strongly suggest that branched glands are necessary for pre-implantation glandular Lif expression for implantation success. Our study is the first to identify a relationship between the branched structure and secretory function of uterine glands and provides a framework for understanding how uterine gland structure-function contributes to pregnancy success.

Upregulation of FOXA2 in uterine luminal epithelium and vaginal basal epithelium of epiERα-/- (Esr1fl/flWnt7aCre/+) mice†.

Forkhead box protein A2 (FOXA2) is a pioneer transcription factor important for epithelial budding and morphogenesis in different organs. It has been used as a specific marker for uterine glandular epithelial cells (GE). FOXA2 has close interactions with estrogen receptor α (ERα). ERα binding to Foxa2 gene in the uterus indicates its regulation of Foxa2. The intimate interactions between ERα and FOXA2 and their essential roles in early pregnancy led us to investigate the expression of FOXA2 in the female reproductive tract of pre-implantation epiERα-/- (Esr1fl/flWnt7aCre/+) mice, in which ERα is conditionally deleted in the epithelium of reproductive tract. In the oviduct, FOXA2 is detected in the ciliated epithelial cells of ampulla but absent in the isthmus of day 3.5 post-coitum (D3.5) Esr1fl/fl control and epiERα-/- mice. In the uterus, FOXA2 expression in the GE appears to be comparable between Esr1fl/fl and epiERα-/- mice. However, FOXA2 is upregulated in the D0.5 and D3.5 but not PND25-28 epiERα-/- uterine luminal epithelial cells (LE). In the vagina, FOXA2 expression is low in the basal layer and increases toward the superficial layer of the D3.5 Esr1fl/fl vaginal epithelium, but FOXA2 is detected in the basal, intermediate, and superficial layers, with the strongest FOXA2 expression in the intermediate layers of the D3.5 epiERα-/- vaginal epithelium. This study demonstrates that loss of ERα in LE and vaginal basal layer upregulates FOXA2 expression in these epithelial cells during early pregnancy. The mechanisms for epithelial cell-type specific regulation of FOXA2 by ERα remain to be elucidated.

Visualization of preimplantation uterine fluid absorption in mice using Alexa Fluor™ 488 Hydrazide†.

Uterine fluid plays important roles in supporting early pregnancy events and its timely absorption is critical for embryo implantation. In mice, its volume is maximum on day 0.5 post-coitum (D0.5) and approaches minimum upon embryo attachment ~D4.0. Its secretion and absorption in ovariectomized rodents were shown to be promoted by estrogen and progesterone (P4), respectively. The temporal mechanisms in preimplantation uterine fluid absorption remain to be elucidated. We have established an approach using intraluminally injected Alexa Fluor™ 488 Hydrazide (AH) in preimplantation control (RhoAf/f) and P4-deficient RhoAf/fPgrCre/+ mice. In control mice, bulk entry (seen as smeared cellular staining) via uterine luminal epithelium (LE) decreases from D0.5 to D3.5. In P4-deficient RhoAf/fPgrCre/+ mice, bulk entry on D0.5 and D3.5 is impaired. Exogenous P4 treatment on D1.5 and D2.5 increases bulk entry in D3.5 P4-deficient RhoAf/fPgrCre/+ LE, while progesterone receptor (PR) antagonist RU486 treatment on D1.5 and D2.5 diminishes bulk entry in D3.5 control LE. The abundance of autofluorescent apical fine dots, presumptively endocytic vesicles to reflect endocytosis, in the LE cells is generally increased from D0.5 to D3.5 but its regulation by exogenous P4 or RU486 is not obvious under our experimental setting. In the glandular epithelium (GE), bulk entry is rarely observed and green cellular dots do not show any consistent differences among all the investigated conditions. This study demonstrates the dominant role of LE but not GE, the temporal mechanisms of bulk entry and endocytosis in the LE, and the inhibitory effects of P4-deficiency and RU486 on bulk entry in the LE in preimplantation uterine fluid absorption.

Varied effects of doxorubicin (DOX) on the corpus luteum of C57BL/6 mice during early pregnancy†.

Certain chemotherapeutic drugs are toxic to ovarian follicles. The corpus luteum (CL) is normally developed from an ovulated follicle for producing progesterone (P4) to support early pregnancy. To fill in the knowledge gap about effects of chemotherapy on the CL, we tested the hypothesis that chemotherapy may target endothelial cells and/or luteal cells in the CL to impair CL function in P4 steroidogenesis using doxorubicin (DOX) as a representative chemotherapeutic drug in mice. In both mixed background mice and C57BL/6 mice, a single intraperitoneal injection of DOX (10 mg/kg) on 0.5-day postcoitum (D0.5, postovulation) led to ~58% D3.5 mice with serum P4 levels lower than the serum P4 range in the phosphate buffer saline-treated control mice. Further studies in the C57BL/6 ovaries revealed that CLs from DOX-treated mice with low P4 levels had less defined luteal cords and disrupted collagen IV expression pattern, indicating disrupted capillary, accompanied with less differentiated luteal cells that had smaller cytoplasm and reduced StAR expression. DOX-treated ovaries had increased granulosa cell death in the growing follicles, reduced proliferating cell nuclear antigen-positive endothelial cells in the CLs, enlarged lipid droplets, and disrupted F-actin in the luteal cells. These novel data suggest that the proliferating endothelial cells in the developing CL may be the primary target of DOX to impair the vascular support for luteal cell differentiation and subsequently P4 steroidogenesis. This study fills in the knowledge gap about the toxic effects of chemotherapy on the CL and provides critical information for risk assessment of chemotherapy in premenopausal patients.

Chemotherapeutic agent doxorubicin alters uterine gene expression in response to estrogen in ovariectomized CD-1 adult mice†.

Chemotherapy can potentially impair fertility in premenopausal cancer patients. Female fertility preservation has been mainly focused on the ovarian aspects and benefited greatly from assisted reproductive technologies, such as in vitro fertilization (IVF). The rate-limiting step for the success of IVF is embryo implantation in the uterus. Doxorubicin (DOX) is a widely used chemotherapeutic agent with ovarian toxicity. It remains unknown if the uterus is a direct target of DOX. To circumvent the indirect uterine effect from ovarian toxicity of DOX and to investigate potential long-term impact of DOX on the uterus, young adult ovariectomized CD-1 mice were given an intraperitoneal injection once with PBS or DOX (10 mg/kg, a human relevant chemotherapeutic dose), and 30 days later, each set of mice was randomly assigned into three groups and subcutaneously injected with oil, 17ß-estradiol (E2, for 6 h), and progesterone (P4, for 54 h), respectively. Uterine transcriptomic profiles were determined using RNA-seq. Principal component analysis of the uterine transcriptomes revealed four clusters from the six treatment groups: PBS-oil & DOX-oil, PBS-P4 & DOX-P4, PBS-E2, and DOX-E2, indicating that DOX treatment did not affect the overall uterine transcriptomic profiles in the oil and P4-treated mice but altered uterine responses to E2 treatment. DAVID analysis indicated that the top-affected gene cluster was "Glycoprotein". These data demonstrate that DOX can directly target the uterus and has a long-term impact on uterine responses to E2.

Association of luteal cell degeneration and progesterone deficiency with lysosomal storage disorder mucolipidosis type IV in Mcoln1-/- mouse model†.

Transient receptor potential cation channel, mucolipin subfamily, member 1 (TRPML1) (MCOLN1/Mcoln1) is a lysosomal counter ion channel. Mutations in MCOLN1 cause mucolipidosis type IV (MLIV), a progressive and severe lysosomal storage disorder with a slow onset. Mcoln1-/- mice recapitulate typical MLIV phenotypes but roles of TRPML1 in female reproduction are unknown. Despite normal mating activities, Mcoln1-/- female mice had reduced fertility at 2 months old and quickly became infertile at 5 months old. Progesterone deficiency was detected on 4.5 days post coitum/gestation day 4.5 (D4.5). Immunohistochemistry revealed TRPML1 expression in luteal cells of wild type corpus luteum (CL). Corpus luteum formation was not impaired in 5-6 months old Mcoln1-/- females indicated by comparable CL numbers in control and Mcoln1-/- ovaries on both D1.5 and D4.5. In the 5-6 months old Mcoln1-/- ovaries, histology revealed less defined corpus luteal cord formation, extensive luteal cell vacuolization and degeneration; immunofluorescence revealed disorganized staining of collagen IV, a basal lamina marker for endothelial cells; Nile Red staining detected lipid droplet accumulation, a typical phenotype of MLIV; immunofluorescence of heat shock protein 60 (HSP60, a mitochondrial marker) and in situ hybridization of steroidogenic acute regulatory protein (StAR, for the rate-limiting step of steroidogenesis) showed reduced expression of HSP60 and StAR, indicating impaired mitochondrial functions. Luteal cell degeneration and impaired mitochondrial functions can both contribute to progesterone deficiency in the Mcoln1-/- mice. This study demonstrates a novel function of TRPML1 in maintaining CL luteal cell integrity and function.

Seipin deficiency leads to increased endoplasmic reticulum stress and apoptosis in mammary gland alveolar epithelial cells during lactation.

Seipin is an integral endoplasmic reticulum (ER) membrane protein encoded by Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2/Bscl2) gene. Most litters (59%) from Bscl2-/- dams mated with wild type (WT) (Bscl2+/+) males did not survive postnatal day 5 (PND5) and pups (Bscl2+/-) lacked milk in their stomachs. The survived litters had reduced pup survival rate at PND21. It was hypothesized that seipin was critical for lactation. Bscl2 was upregulated and highly detected in the lactation day 1 (LD1) WT mammary gland alveolar epithelial cells. LD1 Bscl2-/- mammary glands lacked adipocytes and alveolar clusters and had varied alveolar morphology: from interconnected mammary gland alveoli with dilated lumen and sloughed epithelial cells to undifferentiated mammary gland alveoli with unexpanded lumen. Comparable levels of whey acidic protein (WAP, a major component in rodent milk) staining and Nile Red lipid droplet staining between WT and Bscl2-/- LD1 alveolar epithelial cells indicated normal milk protein synthesis and lipid syntheses in LD1 Bscl2-/- mammary glands. Significantly reduced percentage of larger lipid droplets was detected in LD1 Bscl2-/- alveoli with unexpanded lumen. There was no obviously impaired proliferation detected by PCNA staining but increased apoptosis detected by cleaved caspase-3 staining in LD1 Bscl2-/- alveolar epithelial cells. Increased expression of protein disulfide isomerase and binding immunoglobulin protein in the LD1 Bscl2-/- mammary gland alveolar epithelial cells indicated increased ER stress. This study demonstrates increased ER stress and apoptosis in LD1 Bscl2-/- mammary gland alveolar epithelial cells and reveals a novel in vivo function of seipin in lactation.

Seipin deficiency leads to defective parturition in mice.

Seipin is an integral endoplasmic reticulum membrane protein encoded by Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2/Bscl2) gene. Seipin deficiency results in lipodystrophy, diabetes, muscle hypertrophy, and male infertility in both human and mouse. Seipin function in female reproduction is unknown. Bscl2-/- dams had normal embryo implantation and body weight gain during pregnancy but reduced delivery rates from 2nd to 4th pregnancies and reduced numbers of pups delivered from 1st to 4th pregnancies. Characterization of first pregnancy revealed increased gestation period and parturition problems, including uterine prolapse, difficulty in delivery, undelivered fetuses, and undelivered tissues in Bscl2-/- females. Bscl2-/- uterine weight was comparable to control at 3 weeks old but significantly increased with myometrial hypertrophy at 10 months old. In situ hybridization revealed relatively low level of Bscl2 mRNA expression in myometrium throughout pregnancy and postpartum but high level of expression in uterine luminal epithelium, suggesting that systemic effect (e.g. elevated glucose and insulin levels) rather than local seipin-deficiency in myometrium might be a main contributing factor to myometrial hypertrophy. On near-term gestation day 18.5 (D18.5), Bscl2-/- females had normal levels of serum progesterone and 17ß-estradiol, indicating functional ovary and placenta. Proliferating Cell Nuclear Antigen (PCNA) staining showed minimal myometrial cell proliferation in both D18.5 Bscl2+/+ and Bscl2-/- uteri. There was strong LC3 immunostaining in Bscl2+/+ and Bscl2-/- peripartum myometrium and increased LC3 staining in Bscl2-/- peripartum uterine luminal epithelium, suggesting a potential role of seipin in regulating autophagy in uterine luminal epithelium but not myometrium. This study demonstrates an association of seipin with myometrium and parturition.

Acidification of uterine epithelium during embryo implantation in mice.

Uterine luminal epithelium (LE) is essential for establishing uterine receptivity. Previous microarray analysis revealed upregulation of Atp6v0d2 in gestation day 4.5 (D4.5) LE in mice. Realtime PCR showed upregulation of uterine Atp6v0d2 starting right before embryo attachment ∼D4.0. In situ hybridization demonstrated specific uterine localization of Atp6v0d2 in LE upon embryo implantation. Atp6v0d2 encodes one subunit for vacuolar-type H+-ATPase (V-ATPase), which regulates acidity of intracellular organelles and extracellular environment. LysoSensor Green DND-189 detected acidic signals in LE and glandular epithelium upon embryo implantation, correlating with Atp6v0d2 upregulation in early pregnant uterus. Atp6v0d2-/- females had significantly reduced implantation rate and marginally reduced delivery rate from first mating only, but comparable number of implantation sites and litter size compared to control and comparable fertility to control from subsequent matings, suggesting a nonessential role of Atp6v0d2 subunit in embryo implantation. Successful implantation in both control and Atp6v0d2-/- females was associated with uterine epithelial acidification. No significant compensatory upregulation of Atp6v0d1 mRNA was detected in D4.5 Atp6v0d2-/- uteri. To determine the role of V-ATPase instead of a single subunit in embryo implantation, a specific V-ATPase inhibitor bafilomycin A1 (2.5 µg/kg) was injected via uterine fat pad on D3 18:00 h. This treatment resulted in reduced uterine epithelial acidification, delayed implantation, and reduced number of implantation sites. It also suppressed oil-induced artificial decidualization. These data demonstrate uterine epithelial acidification as a novel phenomenon during embryo implantation and V-ATPase is involved in uterine epithelial acidification and uterine preparation for embryo implantation.

Deletion of Lysophosphatidic Acid Receptor 3 (Lpar3) Disrupts Fine Local Balance of Progesterone and Estrogen Signaling in Mouse Uterus During Implantation.

Lpar3 encodes LPA3, the third G protein-coupled receptor for lysophosphatidic acid (LPA). Lpar3(-/-) female mice had delayed embryo implantation. Their serum progesterone and estrogen levels were comparable with control on Gestation Day 3.5 (D3.5) at 1100 h. There was reduced cell proliferation in D3.5 and D4.5 Lpar3(-/-) stroma. Progesterone receptor (PGR) disappeared from D4.5 Lpar3(+/+) uterine luminal epithelium (LE) but remained highly expressed in D4.5 Lpar3(-/-) LE. Pgr and PGR- target genes but not estrogen receptor alpha (ERalpha [Esr1]) or ESR target genes, were upregulated in D4.5 Lpar3(-/-) LE. It was hypothesized that suppression of PGR activity in LE could restore on-time uterine receptivity in Lpar3(-/-) mice. A low dose of RU486 (5 µg/mouse) given on D3.5 at 900 h rescued delayed implantation in all pregnant Lpar3(-/-) females and significantly increased number of implantation sites compared to vehicle-treated pregnant Lpar3(-/-) females detected on D4.5. E2 (25 ng/mouse) had a similar effect as 5 µg RU486 on embryo implantation in Lpar3(-/-) females. However, when the ovaries were removed on late D2.5 to create an experimentally induced delayed implantation model, 25 ng E2 activated implantation in Lpar3(+/+) but not Lpar3(-/-) females detected on D4.5. These results demonstrate that deletion of Lpar3 leads to an increased ratio of progesterone signaling/estrogen signaling that can be optimized by low doses of RU486 or E2 to restore on-time implantation in Lpar3(-/-) females.

[Effect of Naoshuantong capsule on change of SSQOL index in patients with ischemic stroke in six mouths follow-up].

To evaluate the effect of Naoshuantong capsule on the life quality of patients with ischemic stroke in six months of follow-up studies, and observe the adverse events. The results would provide reference for the secondary prevention on the recovery stage of ischemic stroke. 696 patients from 12 Class III Grade I hospitals nationwide were divided into 2 groups by central randomization system. The study group, 344 cases, were treated with Naoshuantong capsule plus Aspirin, and the control group, 352 cases, were treated with Aspirin. The patients were treated for 6 months. At the end of treatment, SS-QOL used for evaluating the quality of life was observed. The safety index was defined by adverse observation event. The incidence of adverse events and laboratory tests results were observed before and after treatment at the same time. The results indicated that compared to the control group, the treatment group had significant statistical difference in the impact of effort, self-care ability and the the work or labor ability of patients (P < 0.05). No serious adverse events were observed. Naoshuantong capsule showed some superiority to Asprin on improving the quality of life on patients with ischemic stroke, and it could be used in secondary prevention on the recovery stage of ischemic stroke. Naoshuantong capsule is safe and effective in the treatment of convalescence ischemic stroke.

Attenuated retinoic acid signaling is among the early responses in mouse uterus approaching embryo attachment.

The uterus is transiently receptive for embryo implantation. It remains to be understood why the uterus does not reject a semi-allogeneic embryo (to the biological mother) or an allogeneic embryo (to a surrogate) for implantation. To gain insights, we examined uterine early response genes approaching embryo attachment on day 3 post coitum (D3) at 22 hours when blue dye reaction, an indication of embryo attachment, had not manifested in mice. C57BL/6 pseudo-pregnant (control) and pregnant mouse uteri were collected on D3 at 22 hours for microarray analysis. The self-assembling-manifold (SAM) algorithm identified 21,858 unique probesets. Principal component analysis indicated a clear separation between the pseudo-pregnant and pregnant groups. There were 106 upregulated and five downregulated protein-coding genes in the pregnant uterus with fold change (fc) >1.5 and q value <5%. Gene ontology (GO) analysis of the 106 upregulated genes revealed 38 significant GO biological process (GOBP) terms (P <0.05), and 32 (84%) of them were associated with immune responses, with a dominant natural killer (NK) cell activation signature. Among the top eight upregulated protein-coding genes, Cyp26a1 inactivates retinoic acid (RA) while Lrat promotes vitamin A storage, both of which are expected to attenuate RA bioavailability; Atp6v0d2 and Gjb2 play roles in ion transport and transmembrane transport; Gzmb, Gzmc, and Il2rb are involved in immune responses; and Tdo2 is important for kynurenine pathway. Most of these genes or their related pathways have functions in immune regulations. RA signaling has been implicated in immune tolerance and immune homeostasis, and uterine NK cells have been implicated in immunotolerance at the maternal-fetal interface in the placenta. The mechanisms of immune responses approaching embryo attachment remain to be elucidated. The coordinated effects of the early response genes may hold the keys to the question of why the uterus does not reject an implanting embryo.

Broad gap junction blocker carbenoxolone disrupts uterine preparation for embryo implantation in mice.

Gap junctions have an important role in cell-to-cell communication, a process obviously required for embryo implantation. Uterine luminal epithelium (LE) is the first contact for an implanting embryo and is critical for the establishment of uterine receptivity. Microarray analysis of the LE from peri-implantation mouse uterus showed low-level expression of 19 gap junction proteins in preimplantation LE and upregulation of gap junction protein, beta 2 (GJB2, connexin 26, Cx26) in postimplantation LE. Time course study using in situ hybridization and immunofluorescence revealed upregulation of GJB2 in the LE surrounding the implantation site before decidualization. Similar dynamic expression of GJB2 was observed in the LE of artificially decidualized mice but not pseudopregnant mice. To determine the potential function of uterine gap junctions in embryo implantation, carbenoxolone (CBX), a broad gap junction blocker, was injected i.p. (100 mg/kg) or via local uterine fat pad (10 mg/kg) into pregnant mice on Gestation Day 3 at 1800 h, a few hours before embryo attachment to the LE. These CBX treatments disrupted embryo implantation, suggesting local effects of CBX in the uterus. However, i.p. injection of glycyrrhizic acid (100 mg/kg), which shares similar structure and multiple properties with CBX but is ineffective in blocking gap junctions, did not affect embryo implantation. Carbenoxolone also inhibited oil-induced artificial decidualization, concomitant with suppressed molecular changes and ultrastructural transformations associated with uterine preparation for embryo implantation, underscoring the adverse effect of CBX on uterine preparation for embryo implantation. These data demonstrate that uterine gap junctions are important for embryo implantation.

Murine uterine gland branching is necessary for gland function in implantation.

Uterine glands are branched, tubular structures whose secretions are essential for pregnancy success. It is known that pre-implantation glandular expression of leukemia inhibitory factor (LIF) is crucial for embryo implantation, however contribution of uterine gland structure to gland secretions such as LIF is not known. Here we use mice deficient in estrogen receptor 1 (ESR1) signaling to uncover the role of ESR1 signaling in gland branching and the role of a branched structure in LIF secretion and embryo implantation. We observed that deletion of ESR1 in neonatal uterine epithelium, stroma and muscle using the progesterone receptor PgrCre causes a block in uterine gland development at the gland bud stage. Embryonic epithelial deletion of ESR1 using a mullerian duct Cre line - Pax2Cre, displays gland bud elongation but a failure in gland branching. Surprisingly, adult uterine epithelial deletion of ESR1 using the lactoferrin-Cre (LtfCre) displays normally branched uterine glands. Intriguingly, unbranched glands from Pax2Cre Esr1flox/flox uteri fail to express glandular pre-implantation Lif, preventing implantation chamber formation and embryo alignment along the uterine mesometrial-antimesometrial axis. In contrast, branched glands from LtfCre Esr1flox/flox uteri display reduced expression of glandular Lif resulting in delayed implantation chamber formation and embryo-uterine axes alignment but deliver a normal number of pups. Finally, pre-pubertal unbranched glands in control mice express Lif in the luminal epithelium but fail to express Lif in the glandular epithelium even in the presence of estrogen. These data strongly suggest that branched glands are necessary for pre-implantation glandular Lif expression for implantation success. Our study is the first to identify a relationship between the branched structure and secretory function of uterine glands and provides a framework for understanding how uterine gland structure-function contributes to pregnancy success.

Altered spatiotemporal expression of collagen types I, III, IV, and VI in Lpar3-deficient peri-implantation mouse uterus.

Lpar3 is upregulated in the preimplantation uterus, and deletion of Lpar3 leads to delayed uterine receptivity in mice. Microarray analysis revealed that there was higher expression of Col3a1 and Col6a3 in the Preimplantation Day 3.5 Lpar3(-/-) uterus compared to Day 3.5 wild-type (WT) uterus. Since extracellular matrix (ECM) remodeling is indispensable during embryo implantation, and dynamic spatiotemporal alteration of specific collagen types is part of this process, this study aimed to characterize the expression of four main uterine collagen types: fibril-forming collagen (COL) I and COL III, basement membrane COL IV, and microfibrillar COL VI in the peri-implantation WT and Lpar3(-/-) uterus. An observed delay of COL III and COL VI clearance in the Lpar3(-/-) uterus may be associated with higher preimplantation expression of Col3a1 and Col6a3. There was also delayed clearance of COL I and delayed deposition of COL IV in the decidual zone in the Lpar3(-/-) uterus. These changes were different from the effects of 17beta-estradiol and progesterone on uterine collagen expression in ovariectomized WT uterus, indicating that the altered collagen expression in Lpar3(-/-) uterus is unlikely to be a result of alterations in ovarian hormones. Decreased expression of several genes encoding matrix-degrading metallo- and serine proteinases was observed in the Lpar3(-/-) uterus. These results demonstrate that pathways downstream of LPA3 are involved in the dynamic remodeling of ECM in the peri-implantation uterus.

LPA3-mediated lysophosphatidic acid signalling in embryo implantation and spacing.

Every successful pregnancy requires proper embryo implantation. Low implantation rate is a major problem during infertility treatments using assisted reproductive technologies. Here we report a newly discovered molecular influence on implantation through the lysophosphatidic acid (LPA) receptor LPA3 (refs 2-4). Targeted deletion of LPA3 in mice resulted in significantly reduced litter size, which could be attributed to delayed implantation and altered embryo spacing. These two events led to delayed embryonic development, hypertrophic placentas shared by multiple embryos and embryonic death. An enzyme demonstrated to influence implantation, cyclooxygenase 2 (COX2) (ref. 5), was downregulated in LPA3-deficient uteri during pre-implantation. Downregulation of COX2 led to reduced levels of prostaglandins E2 and I2 (PGE2 and PGI2), which are critical for implantation. Exogenous administration of PGE2 or carbaprostacyclin (a stable analogue of PGI2) into LPA3-deficient female mice rescued delayed implantation but did not rescue defects in embryo spacing. These data identify LPA3 receptor-mediated signalling as having an influence on implantation, and further indicate linkage between LPA signalling and prostaglandin biosynthesis.

[Post-marketed re-evaluation of fleabane injection and Dengzhan Shengmai capsule study on treatment in patients with ischemic stroke].

OBJECTIVE: To verify the efficacy and safety of post-marketed fleabane injection combined with Dengzhan Shengmai capsules in the treatment of ischemic stroke (IS). METHOD: A multicentre, prospective, practical, randomized controlled study was carried out to compare the efficacy and safety of Dengzhan group (n = 343) and western medicine group (n = 335), appling "clinical study central stochastic system". The treatment of Dengzhan group is using fleabane injection in acute stage and Dengzhan Shengmai capsules in convalescence. The primary indexes of effect evaluation are the important outcome events in 360 days' follow-up, including mortality, recurrence, disability and quality of life to reflect the effect of clinical study. The indexes of safety evaluation involve laboratory examination results and incidence of adverse events. RESULT: After 360 days' follow-up, 4 people died of IS in Dengzhan group, and the mortality rate of which is 1.17%, while 16 died in Western medicine group (WM group), and the mortality rate is 4.78%, suggesting that the mortality rate of Dengzhan group is significantly lower than WM group (P<0.05). Eleven cases recurred in Dengzhan group, and the recurrence rate of which is 3.21%, while 12 recurred in WM group, and the recurrence rate is 3.59%, indicating that the recurrence rate of Dengzhan group is slightly lower than WM group. The disability rate of Dengzhan group is 39.53%, among which the rate of severely disabled cases are 1.49%, while the disability rate of WM group is 40.13%, among which the rate of severely disabled cases are 3.13%, suggesting that the disability rate of Dengzhan group is lower and the severity of disability is also lighter than WM group. In the field of quality of life, the activity ability and the upper limb function store of stroke patients in Dengzhan group improved far much better than WM group (P<0.05). Analysis of safety suggested that, adverse events occurred in 11 cases in Dengzhan group, among which 4 cases is related with the drug treatment, the incidence of adverse events of which is 1.17%, and the main manifestations involve fever and chilling, rash, nausea, dizziness, palpitation, etc. which were all appeared after the treatment of fleabane injection, and disappeared 1 to 2 days after drug withdrawal. 13 cases occurred abnormal liver function and 2 cases abnormal kidney function in Dengzhan group. According to the judgment of clinical physicians, 3 case of ALT abnormality is possibly related to the treatment, the others are all unrelated with the treatment. CONCLUSION: Fleabane injection and Dengzhan Shengmai capsules are all safe and effective TCM in the treatment of ischemic stroke.

[Post-marketing re-evaluation of Kudiezi injection study on early treatment in patients with ischemic stroke].

OBJECTIVE: To study the effect and safety of Kudiezi injection on patients with acute ischemic stroke. METHOD: Seven hundreds patients were divided into two groups by central randomization system. The study group, 346 cases, was treated with kudiezi injection plus traditional Chinese medicine (TCM) synthesis rehabilitation project, and the control group, 354 cases, was treated with synthetic rehabilitation project. The patients were treated for 10 to 21 days. Before treatment and at the 7th, 14th and 21th day of treatment, the indexes include NIHSS used for evaluating the neurological deficit degree and the motor function score (Fugl-Meyer) for evaluating motor function were observed. The safety index is defined by adverse observation event and laboratory test. The incidence of adverse events and laboratory tests results were observed before and after treatment at the same time. RESULT: Application of generalized estimating equation model, we found that as the treatment time, NIHSS score and FMI score of the two groups showed a trend of improvement. And at the 14th days and 21th days of treatment, compared to the control group the treatment group showed significant statistical difference on the impact of NIHSS and FMI (P<0.05). No serious adverse events were observed. CONCLUSION: Kudiezi injection plus TCM rehabilitation project of ischemic stroke showed some superiority to western medicine rehabilitation program on improving the neurological deficit and motor function. Kudiezi injection is safe and effective in the treatment of acute ischemic stroke.

Synergistic effects of Co/CoO nanoparticles on imine-based covalent organic frameworks for enhanced OER performance.

The development of non-noble metal electrocatalysts toward the oxygen evolution reaction (OER) is a key challenge in advancing electrocatalytic water splitting, which is essential for the commercialization of clean and renewable energy. A covalent organic framework (COF) has a precise and controllable structure, high π-π conjugation, large surface area, and porosity and shows great potential as an OER electrocatalyst. However, the relative conductivity and inherent instability greatly limit the further improvement of its performance. Herein, imine-based COF-supported Co/CoO nanoparticles (Co/CoO@COF) were developed for the high-performance electrocatalytic OER. For the Co/CoO@COF catalyst, Co/CoO could form a conjugation effect with the COF, which can increase the electron cloud density of the delocalized large π bond, then improve the conductivity. The combination of Co/CoO and COF effectively enhances the structural stability of the catalyst and enriches the catalytic active sites. Under alkaline conditions, the Co/CoO@COF shows a very low overpotential of 278 mV at a current density of 10 mA cm-2, and a Tafel slope of 80.11 mV dec-1 which is better than that of commercial RuO2.