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Two new sesquiterpenoids, including a kessane-type sesquiterpenoid ( 1 ) and one bisabolane derivative ( 2 ), together with fourteen known sesquiterpenoids ( 3 - 16 ), were isolated from the roots and rhizomes of Valeriana amurensis . The structures of new compounds were established on the basis of extensive spectroscopic analysis. All isolates were evaluated for their effects on nerve growth factor (NGF)-mediated neurite outgrowth in pheochromocytoma (PC12) cells. As a results, four compounds including 10-12 and 15 showed potent promoting effects at the concentration of 10 µM on NGF-induced neurite outgrowth in PC12 cells with the differentiation rate of 11.84%, 12.21%, 13.77% and 12.16%, respectively.

Objective: To establish a noninvasive method for measuring upper airway critical closing pressure (Pcrit), so as to evaluate collapsibility of the upper airway during sleep. Methods: Pcrit was determined through the use of a noninvasive positive/negative pressure (CPAP/CPNP) ventilator(with independent intellectual property rights) during stageⅡ of non-rapid eye movement sleep. For the direct measurement, Pcrit was the pressure below which the upper airway occluded. For the indirect measurement, nasal pressure was plotted against maximum inspiratory flow (V imax ), and linear regression was used to interpolate the pressure (i.e., Pcrit) at which zero flow occurred. Pcrit was attained from 19 subjects without obstructive sleep apnea syndrome(OSAS), and the correlation between direct and indirect measurement methods was analyzed. Results: Directly measured and indirectly measured Pcrit showed no significant difference [(-7.02±2.74 vs (-7.26±2.96) cmH 2 O, 1 cmH 2 O=0.098 kPa; t =1.667, P >0.05] and had a highly significant correlation ( r =0.986, P =0.000). Bland-Altman analysis revealed that the mean between-method difference was (0.24±0.53) cmH 2 O, and 95% limits of agreement ranged from -0.80 to 1.27 cmH 2 O, and all points except one were within limits of agreement. Conclusion: Pcrit derived from the direct and indirect measurement methods does not differ, and both methods could be used for evaluating the upper airway collapsibility.

Embryo implantation and decidualization are crucial steps during early pregnancy. We recently showed that nucleolar stress is involved in embryo implantation. This study was to explore whether nucleolar stress participates in mouse and human decidualization. Our data demonstrated that a low dose of actinomycin D (ActD) could induce nucleolar stress in stroma cells. Nucleolar stress promotes the stromal-epithelial transition during mouse in vitro decidualization through nucleophosmin1 (NPM1). Under nucleolar stress, Wnt family member 4 (Wnt4), a decidualization marker, is significantly increased, but decidua/trophoblast prolactin-related protein (Dtprp/Prl8a2) expression remains unchanged. For translational significance, we also examined the effects of nucleolar stress on human decidualization. Nucleolar stress stimulated by a low dose of ActD enhances human stromal-epithelial transition during human decidualization, but has no effects on the expression of insulin-like growth factor-binding protein 1 (IGFBP1). Our study indicates that nucleolar stress may promote only the mesenchymal-epithelial transition (MET), but not for all the molecular changes during decidualization.

Embryo implantation is a crucial step for the successful establishment of mammalian pregnancy. Cyclophilin A (CYPA) is a ubiquitously expressed intracellular protein and is secreted in response to inflammatory stimuli to regulate diverse cellular functions. However, there are currently no reports about the role of CYPA in embryo implantation. Here, we examine the expression pattern of CYPA during mouse early pregnancy and explore the potential role of CYPA during implantation. CYPA is expressed in the subluminal stroma surrounding the implanting blastocyst on day 5 of pregnancy, but not at inter-implantation sites. In ovariectomized mice, estrogen and progesterone significantly stimulate CYPA expression. When pregnant mice are injected intraperitoneally with CYPA inhibitor, the numbers of implantation sites are significantly reduced. Using an in vitro stromal cell culture system, Ppia siRNA knockdown of CYPA and CYPA-specific inhibitor treatment partially inhibits levels of CD147, MMP3 and MMP9. Decreased CYPA expression also significantly inhibits Stat3 activity and expands estrogen responsiveness. Taken together, CYPA may play an important role during mouse embryo implantation.

Embryo implantation is essential to the successful establishment of pregnancy. A previous study has demonstrated that actinomycin D (ActD) could initiate the activation of mouse delayed implantation. However, the mechanism underlying this activation remains to be elucidated. A low dose of ActD is an inducer of nucleolar stress. This study was to examine whether nucleolar stress is involved in embryo implantation. We showed that nucleolar stress occurred when delayed implantation was activated by ActD in mice. ActD treatment also stimulated the Lif-STAT3 pathway. During early pregnancy, nucleolar stress was detected in the luminal epithelial cells during the receptive phase. Blastocyst-derived lactate could induce nucleolar stress in cultured luminal epithelial cells. The inhibition of nucleophosmin1 (NPM1), which was a marker of nucleolar stress, compromised uterine receptivity and decreased the implantation rates in pregnant mice. To translate these mouse data into humans, we examined nucleolar stress in human endometrium. Our data demonstrated that ActD-induced nucleolar stress had positive effects on the embryo attachment by upregulating IL32 expression in non-receptive epithelial cells rather than receptive epithelial cells. Our data should be the first to demonstrate that nucleolar stress is present during early pregnancy and is able to induce embryo implantation in both mice and humans.

Orexin is a peptide neurotransmitter released in the globus pallidus. Morphological evidence reveals that both orexin 1 receptor (OX 1 R) and orexin 2 receptor (OX 2 R) exist in the globus pallidus. Here we showed that bilateral microinjection of both orexin-A and orexin-B into the globus pallidus alleviated motor deficits in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonian mice. Further in vivo extracellular single-unit recording revealed that the basal spontaneous firing rate of the globus pallidus neurons in MPTP parkinsonian mice was slower than that of normal mice. Application of orexin-A or orexin-B significantly increased the spontaneous firing rate of pallidal neurons. The influx of Ca 2+ through the L-type Ca 2+ channel is the major mechanism involved in orexin-induced excitation in the globus pallidus. Orexin-A-induced increase in firing rate of pallidal neurons in MPTP parkinsonian mice was stronger than that of normal mice. Orexin-A exerted both electrophysiological and behavioral effects mainly via OX 1 R, and orexin-B exerted the effects via OX 2 R. Endogenous orexins modulated the excitability of globus pallidus neurons mainly through OX 1 R. The present behavioral and electrophysiological results suggest that orexins ameliorate parkinsonian motor deficits through increasing the spontaneous firing of globus pallidus neurons.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies. However, the molecular mechanisms underlying PDAC are still not completely understood. Circular RNAs (circRNAs) are a unique class of RNA formed by special loop splicing. More and more researchers have paid attention to circRNAs., Material and Methods: In this study, we constructed a circRNA-mediated competing endogenous RNA (ceRNA) network in PDAC. Gene ontology (GO) analysis was performed to explore circRNAs' potential roles in PDAC progression. We also constructed an up-stream transcriptional network of circRNAs' parental genes and found that many transcription factors (TFs), such as tumor protein p53 (TP53) and MYC, could regulate their expression., Results: This study, which aimed to identify differentially expressed circRNAs in PDAC, suggested that circRNAs may also act as biomarkers for PDAC. We analyzed two public datasets (GSE69362 and GSE79634) to identify differentially expressed circRNAs in PDAC. Finally, we found that DExH-Box Helicase 9 (DHX9) may be a potential regulator of circRNA formation in PDAC. Genomic loci of four down-regulated circRNAs - hsa_circ_000691, hsa_circ_0049392, hsa_circ_0005203, and hsa_circ_0001626 - contained DHX9 binding sites, suggesting that they may be directly regulated by DHX9., Conclusions: Our study identified differentially expressed circRNAs in PDAC, suggesting that circRNAs may also act as biomarkers for PDAC. Additional investigations of function and up-stream regulation of differentially expressed circRNA in PDAC are still needed., Competing Interests: The authors declare no conflict of interest.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by progressive and selective death of dopaminergic neurons. Orexin-A is involved in many biological effects of the body. It has been reported that orexin-A has protective effects in cellular models of PD. However, little is known about the protective effects of orexin-A in animal parkinsonian models and the cellular mechanism has not yet been fully clarified. The aim of this study was to evaluate the effects of orexin-A in MPTP mice model of PD as well as the possible neuroprotective mechanisms of orexin-A on dopaminergic neurons. The results from animal experiments demonstrated that orexin-A attenuated the loss of dopaminergic neurons and the decrease of tyrosine hydroxylase (TH) expression in the substantia nigra, normalized the striatal dopaminergic fibers, and prevented the depletion of dopamine and its metabolites in the striatum. MPTP-treated mice showed cognitive impairments accompanied with significant motor deficiency. Orexin-A improved MPTP-induced impairments in both motor activity and spatial memory. Importantly, orexin-A increased the protein level of brain-derived neurotrophic factor (BDNF) in dopaminergic neurons of the substantia nigra. Furthermore, the protective effects of orexin-A on MPTP parkinsonian mice could be blocked by orexinergic receptor 1 (OX1R) antagonist, SB334867. In another set of experiments with SH-SY5Y dopaminergic cells, orexin-A significantly induced the expression of BDNF in a dose and time-dependent manner. The upregulation of BDNF is mainly concerned with PI3K and PKC signaling pathways via OX1R. The present study demonstrated that orexin-A exerted neuroprotective effects on MPTP parkinsonian mice, which may imply orexin-A as a potential therapeutic target for PD.

Orexin is a member of neuropeptides which is involved in the central motor control. The substantia nigra pars compacta (SNc) is an important nucleus participating in motor control under both physiological and pathological conditions. Morphological studies reveal that orexinergic neurons located in lateral hypothalamus innervate the SNc. Both orexin-1 receptors (OX 1 R) and orexin-2 receptors (OX 2 R) are expressed in the SNc. To investigate the effects of orexins on SNc, single unit in vivo extracellular recordings and behavioral tests were performed in this study. Micro-pressure administration of orexin A and orexin B significantly increased the spontaneous firing rate of nigral DAergic neurons by 65.87 ± 7.73% and 90.49 ± 17.83%, respectively. The excitatory effects of orexin A on nigral DAergic neurons were mainly mediated by OX 1 R, while OX 2 R were involved in the increase in firing rate induced by orexin B. Selectively blocking OX 1 R and OX 2 R significantly decreased the firing rate of nigral DAergic neurons by 36.77 ± 6.26% and 32.04 ± 6.12%, respectively, which suggested that endogenous orexins modulated the spontaneous firing activity of nigral DAergic neurons. Finally, both elevated body swing test and haloperidol-induced postural behavioral test showed that unilateral microinjection of orexin A and orexin B induced significantly contralateral-biased swing and deflection behavior. Meanwhile, the specific OX 1 R and OX 2 R antagonists produced opposite effects. The present electrophysiological and behavioral studies suggested that orexins increased the firing activity of nigral DAergic neurons and participated in central motor control. Open Practices Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/., (© 2018 International Society for Neurochemistry.)

The globus pallidus is a central nucleus in the basal ganglia motor control circuit. Morphological studies have revealed the expression of adenosine A 2A receptors in the globus pallidus. To determine the modulation of adenosine A 2A receptors on the activity of pallidal neurons in both normal and parkinsonian rats, in vivo electrophysiological and behavioral tests were performed in the present study. The extracellular single unit recordings showed that micro-pressure administration of adenosine A 2A receptor agonist, CGS21680, regulated the pallidal firing activity. GABAergic neurotransmission was involved in CGS21680-induced modulation of pallidal neurons via a PKA pathway. Furthermore, application of two adenosine A 2A receptor antagonists, KW6002 or SCH442416, mainly increased the spontaneous firing of pallidal neurons, suggesting that endogenous adenosine system modulates the activity of pallidal neurons through adenosine A 2A receptors. Finally, elevated body swing test (EBST) showed that intrapallidal microinjection of adenosine A 2A receptor agonist/antagonist induced ipsilateral/contralateral-biased swing, respectively. In addition, the electrophysiological and behavioral findings also revealed that activation of dopamine D 2 receptors by quinpirole strengthened KW6002/SCH442416-induced excitation of pallidal activity. Co-application of quinpirole with KW6002 or SCH442416 alleviated biased swing in hemi-parkinsonian rats. Based on the present findings, we concluded that pallidal adenosine A 2A receptors may be potentially useful in the treatment of Parkinson's disease.

Objective: To investigate the effect of BIX01294 (BIX), a methyltransferase inhibitor, on the migration and decidualization of the stromal cells in mouse uterus., Methods: Mouse endometrial stromal cells were isolated and cultured from the uterus of pregnant mice on day 3.5 of gestation. The migration and decidualization of mouse endometrial stromal cells treated with BIX at different concentrations were observed with wound healing assay and real-time PCR., Results: The migration distance of mouse endometrial stromal cells increased as the BIX concentration increased within the range below 15 µmol/L. Compared with the control cells, the cells treated with BIX (15 µmol/L) showed significantly increased migration distances, but increasing BIX concentration to 20 µmol/L did not further increase the cell migration distance and began to cause cell death. Compared with the control cells, the BIX-treated stromal cells exhibited significantly down-regulated expression of Ehmt2 mRNA, and 15 µmol/L BIX caused inhibition of decidualization in the stromal cells., Conclusion: Within a defined concentration range, BIX promotes the migration and inhibits decidualization of mouse uterine stromal cells by inhibiting the expression of Ehmt2 mRNA.

Objective: To detect the expression of FABP7 in the placenta of pregnant mice and in HTR-8/Svneo cells., Methods: Real-time PCR and immunofluorescence were used to detect FABP7 mRNA and protein expressions in the uterine and placental tissue of pregnant mice at different days of gestation. FABP7 expression was also detected in cultured HTR-8/Svneo cells using immunofluorescence assay. The mice were treated with E 2 , P 4 or their combination for 6 and 24 h and Fabp7 mRNA level in the uterus was detected with real-time PCR., Results: At 7.5-10.5 days of gestation, the pregnant mice showed positive expressions of Fabp7 mRNA in the uterus and placenta, and FABP7 protein was detected in the decidualized cells and trophoblast giant cells. The expressions of FABP7 were detected at both the mRNA and protein levels in cultured HTR-8/Svneo cells. In mice treated with P4 alone or with E 2 +P 4 for 6 and 24 h, the expression level of Fabp7 mRNA was upregulated in the uterus. Fabp7 upregulation was observed in mice at 24 h following E 2 treatment but not at 6 h., Conclusion: FABP7 is expressed in trophoblast giant cells and decidual cells in the placental tissue of mice and in cultured HTR-8/Svneo cells, suggesting the involvement of FABP7 in placental development and in maintenance of pregnancy. E 2 and P 4 can regulate the expression of FABP7 in mouse uterus.

Background: Excessive reactive oxygen species (ROS) may lead to a number of reproductive diseases such as polycystic ovary syndrome. This study aimed to establish an animal model of ovarian oxidative stress and to assess the protective effect of curcumin against oxidative injury., Methods: Ovarian oxidative stress was induced in female Kunming mice (n = 40) with intraperitoneal injection of 8 mg/kg sodium arsenite (As) once every other day for 16 days; meanwhile, they were, respectively, treated by intragastric administration of 0, 100, 150, or 200 mg/kg (n = 10/group) curcumin once per day for 21 days. Ten normal mice were used as control. Then, the mice were injected intraperitoneally with BrdU and sacrificed; the right ovaries were collected for hematoxylin and eosin (HE) staining and BrdU immunohistochemistry, and the left ovaries for enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses., Results: The ELISA results showed that ROS (11.74 ± 0.65 IU/mg in 8 mg/kg AS + 0 mg/kg curcumin group vs. 10.71 ± 0.91 IU/mg in control group, P= 0.021) and malondialdehyde (MDA) (0.32 ± 0.02 nmol/g in 8 mg/kg AS + 0 mg/kg curcumin group vs. 0.27 ± 0.02 nmol/g in control group, P= 0.048) increased while superoxide dismutase (SOD) (3.96 ± 0.36 U/mg in 8 mg/kg AS + 0 mg/kg curcumin group vs. 4.51 ± 0.70 U/mg in control group, P= 0.012) and glutathione peroxidase (17.36 ± 1.63 U/g in 8 mg/kg AS + 0 mg/kg curcumin group vs. 18.92 ± 1.80 U/g in control group, P= 0.045) decreased in the ovary after injection of As, indicating successful modeling of oxidative stress. Curcumin treatment could considerably increase SOD (4.57 ± 0.68, 4.49 ± 0.27, and 4.56 ± 0.25 U/mg in 100 mg/kg, 150 mg/kg, and 200 mg/kg curcumin group, respectively, allP < 0.05) while significantly reduce ROS (10.64 ± 1.38, 10.73 ± 0.71, and 10.67 ± 1.38 IU/mg in 100 mg/kg, 150 mg/kg, and 200 mg/kg curcumin group, respectively, allP < 0.05) and MDA (0.28 ± 0.02, 0.25 ± 0.03, and 0.27 ± 0.04 nmol/g in 100 mg/kg, 150 mg/kg, and 200 mg/kg curcumin group, respectively; bothP < 0.05) in the ovary. HE staining and BrdU immunohistochemistry of the ovarian tissues indicated the increased amount of atretic follicles (5.67 ± 0.81, 5.84 ± 0.98, and 5.72 ± 0.84 in 100 mg/kg, 150 mg/kg, and 200 mg/kg curcumin group, respectively, all P < 0.05), and the inhibited proliferation of granular cells under oxidative stress would be reversed by curcumin. Furthermore, the Western blotting of ovarian tissues showed that the p66Shc expression upregulated under oxidative stress would be lowered by curcumin., Conclusion: Curcumin could alleviate arsenic-induced ovarian oxidative injury to a certain extent.

The globus pallidus occupies a critical position in the indirect pathway of the basal ganglia circuit, which regulates movement under both normal and pathological conditions. Previous studies have shown that the globus pallidus receives dopaminergic innervation from the axonal collaterals of nigrostriatal fibers. Both dopamine D 1 and D 2 like receptors are expressed in the globus pallidus. The present study was aimed to investigate the direct in vivo electrophysiological effects of dopamine D 2 like receptors in the globus pallidus of both normal and parkinsonian rats. Extracellular recordings of multi-barreled microelectrode were used in the present study. In normal rats, micro-pressure ejection of dopamine D 2 like receptor agonist quinpirole induced different effects on the firing rate of globus pallidus neurons. In 24 out of the 61 pallidal neurons, quinpirole significantly increased the firing rate by (62.7 ± 11.2)%. In another 16 neurons, quinpirole decreased the spontaneous firing rate by (37.5 ± 2.9)%. Furthermore, co-application of dopamine D 2 like receptor antagonist, sulpride, blocked quinpirole-induced modulation of the firing rate of pallidal neurons. On the 6-hydroxydopamine (6-OHDA) lesioned side of parkinsonian rats, quinpirole increased the firing rate in 25 out of the 47 pallidal neurons by (64.2 ± 10.1)%, while decreased the firing rate in 11 neurons by (51.9 ± 6.2)%. Our findings suggest that activation of pallidal dopamine D 2 like receptors may bidirectionally modulate the spontaneous firing of globus pallidus neurons in both normal and parkinsonian rats.

Decidualization is an essential step in the establishment of pregnancy. However, the functional contributions of long intergenic noncoding RNAs (LincRNAs) to decidualization have not been explored. To explore the regulation and role of LincRNAs during human decidualization, human endometrial stromal cells (HESCs) are induced to undergo in vitro decidualization by treating with estradiol-17β, db-cAMP and medroxyprogesterone acetate. LINC00473 (LINC473) expression is highly induced in HESCs after decidual stimulus. We found that cAMP-PKA pathway regulates the expression of LINC473 through IL-11-mediated STAT3 phosphorylation. RNA interference-mediated down-regulation of LINC473 inhibits in vitro decidualization. These results suggested that LINC473 might be functionally required for human decidualization. This is the first report demonstrating the presence of LincRNA during human decidualization.

The asymmetric unit of the title compound, C(19)H(12)Cl(2)O, contains four independent mol-ecules, which can be divided into two pairs of mol-ecules with close values of the C-C(=O)-C=C torsion angles in each pair, viz. 165.12 (16) and 165.68 (15)° in one pair, and -164.66 (15) and -164.81 (15)° in the other pair. The crystal packing exhibits short inter-molecular Cl⋯Cl contacts of 3.362 (1) Å.

Objective: To examine the effects of androgen on implantation and decidualization in the mouse delayed-implantation model., Design: Experimental animal study., Setting: University research laboratory., Animal(s): Sexually mature female mice (Kunming White strain)., Intervention(s): Delayed and activated implantation; pseudopregnancy; embryo transfer (ET); E(2) assay; inhibitor., Main Outcome Measure(s): Effects of androgen on embryo implantation were determined by treating the mice under delayed implantation with different doses of testosterone propionate (TP); the effects of androgen on the expression of implantation-related genes were examined by in situ hybridization., Result(s): Delayed implantation could be initiated by TP. Dihydrotestosterone was also able to initiate implantation in the delayed-implantation model. The implantation window could be maintained for at least 48 hours by 5 mg TP per mouse. Prostaglandin endoperoxide synthase 2 (Ptgs2) and microsomal prostaglandin E synthase (mPtges) were aberrantly expressed in mouse uterus at implantation sites after delayed implantation was activated by high doses of TP., Conclusion(s): A low dose of TP led to a delay in embryo implantation, but a high dose caused aberrant expression of both Ptgs2 and mPtges at the implantation site. It is possible that high doses of TP may disturb peri-implantation development or may be involved in early pregnancy loss by disturbing the uterine prostaglandin system.

In the title compound, C(24)H(24)N(2)O(4), the pendant benzene and naphthalene ring systems make dihedral angles of 87.9 (3) and 19.2 (3)°, respectively, with the central pyrazoline ring. In the crystal structure, weak C-H⋯O inter-actions help to establish the packing.

Aim: To determine the exact roles of prolactin (PRL) in the pathogenesis of rheumatoid arthritis (RA) and supply experimental basis for clinical treatment of RA, and to investigate the expression of matrix metalloproteinase-9 (MMP-9) in the synovium of adjuvant arthritis rats., Methods: Forty rats were divided into four groups (n = 10): (1) Normal control group (group A); (2) Adjuvant arthritis control group (group B); (3) Hyperprolactinemic adjuvant arthritis group (group C); (4) Hypoprolactinemic adjuvant arthritis group (group D). The content of PRL in the serum was detected by radio-immunoassay method. The activity of MMP-9 was analyzed by gelatin zymography. The alteration of MMP-9 immunoreactivity were investigated by means of immunohistochemistry in the synovium of all groups. The expressions of MMP-9 were investigated by Western blot in the synovium of all groups., Results: Compared with group A, the activity and expression of MMP-9 of group B in the synovium were highly increased. The activity and expression of MMP-9 in the synovium were the most distinctive in group C. Compared with group B, the activity and expression of MMP-9 in the synovium were decreased in group D, but still higher than group A., Conclusion: The present results indicated that PRL might involved in the pathogenesis of RA by regulating the secretion of MMP-9 in the synovium.

Objective: To examine the spatiotemporal expression and regulation of prostaglandin transporter (PGT), 15-hydroxy-PG dehydrogenase (15-PGDH), and carbonyl reductase 1 (CBR1) messenger RNA (mRNA) and protein in the mouse uterus during embryo implantation and in related models., Design: Experimental animal study., Setting: University research laboratory., Animal(s): Sexually mature female Kunming strain white mice., Intervention(s): Delayed and activated implantation, artificial decidualization, and subcutaneous injection of progesterone (P) and E(2) in ovariectomized mouse., Main Outcome Measure(s): The expression of mRNA and protein were detected by in situ hybridization and immunohistochemistry in mouse uterus., Result(s): Prostaglandin transporter mRNA and protein were expressed in the subluminal stroma at implantation site on day 5 of pregnancy and then in decidua but were not detected at the interimplantation sites and in preimplantation or pseudopregnant uterus. The presence of an active blastocyst was required for PGT expression at the implantation site. Both 15-PGDH and CBR1 mRNA were detected in glandular epithelium on day 4 of pregnancies. The expression of 15-PGDH and CBR1 mRNA was also detected in postimplantation embryos., Conclusion(s): These data suggest that differentially expressed PGT and 15-PGDH may participate in PG signaling in mouse uterus during implantation and decidualization.

Although Cyclooxygenase-2 (COX-2) is essential for mouse ovulation, fertilization, implantation and decidualization, the regulation and function of COX-2 in rat reproduction are still unknown. This study was designed to examine the action of COX-2 in rat ovulation, implantation and decidualization by using two specific inhibitors of COX-2 (nimesulide and niflumic acid). Compared to control, either nimesulide or niflumic acid significantly inhibited the ovulation in the superovulated rats. Although nimesulide had no obvious effects on the number of implantation sites and the vascular permeability, the expression of PPARdelta, HB-EGF and vimentin proteins was down-regulated in the nimesulide-treated groups. COX-1 protein was upregulated by nimesulide treatment. Nimesulide also had an inhibitory effect on decidualization during early pregnancy and under artificial decidualization. Moreover, nimesulide caused the increase of the gestation period and the reduction of litter size and birth weight compared to controls. Based on our data, rat implantation and decidualization were delayed by nimesulide treatment, resulting in the reduction of litter size and birth weight and the prolongation of gestational length, suggesting that COX-2 plays an important role in implantation and decidualization.

Although implantation types differ between species, the interaction between blastocyst trophectoderm and apical surface of the uterine epithelium is a common event during the implantation process. In this study, uterine luminal epithelium at implantation and inter-implantation sites was isolated by enzymatic digestion and used for microarray analysis. In a mouse microarray containing 12 345 unigenes, we found 136 genes upregulated more than twofold at the implantation site, while 223 genes were downregulated by at least twofold. Reverse transcription-PCR was used to verify the differential expression of seven upregulated and six downregulated genes chosen randomly from our microarray analysis, and the expression trends were similar. The differential expression patterns of eight upregulated genes were verified by in situ hybridization. Compared with the inter-implantation site on day 5 of pregnancy and the uterus on day 5 of pseudopregnancy, protease, serine, 12 neurotrypsin, endothelin-1, gamma-glutamyl hydrolase, Ras homolog gene family, member U, T-cell immunoglobulin, and mucin domain containing 2, proline-serine-threonine phosphatase-interacting protein 2, 3-monooxgenase/tryptophan 5-monooxgenase activation protein, gamma-polypeptide, and cysteine-rich protein 61 (Cyr61) were upregulated in the luminal epithelium at implantation site on day 5 of pregnancy. These genes may be related to embryo apposition and adhesion during embryo implantation. Cyr61, a gene upregulated at the implantation site, was chosen to examine its expression and regulation during the periimplantation period by in situ hybridization. Cyr61 mRNA was specifically localized in the luminal epithelium surrounding the implanting blastocyst at day 4 midnight and on day 5 of pregnancy, and in the activated uterus, but not expressed in inter-implantation sites and under delayed implantation, suggesting a role for Cyr61 in mediating embryonic-uterine dialog during embryo attachment. Our data could be a valuable source for future study on embryo implantation.

Prostaglandin (PGE) 2 is the most common prostanoid and plays an important role in female reproduction. The aim of this study was to examine the expression and regulation of microsomal (m) PGE synthase (PGES)-1 and cytosolic (c) PGES in the mouse ovary during sexual maturation, gonadotropin treatment and luteal development by in situ hybridization and immunohistochemistry. Both mPGES-1 mRNA signals and immunostaining were localized in the granulosa cells, but not in the thecal cells and oocytes. cPGES mRNA signals were localized in both granulosa cells and oocytes, whereas cPGES immunostaining was exclusively localized in the oocytes. In our superovulated model of immature mice, there was a basal level of mPGES-1 mRNA signals in the granulosa cells at 48 h after equine chorionic gonadotropin (eCG) treatment. mPGES-1 mRNA level was induced by human chorionic gonadotropin (hCG) treatment for 0.5 h, whereas mPGES-1 immunostaining was slightly induced at 0.5 h after hCG treatment and reached a maximal level at 3 h after hCG treatment. eCG treatment had no obvious effects on either cPGES mRNA signals or immunostaining. A strong level of cPGES immunostaining was present in both unstimulated and eCG-treated groups. Both mPGES-1 mRNA signals and immunostaining were highly detected in the corpus luteum 2 days post-hCG injection and declined from days 3 to 7 post-hCG injection. cPGES immunostaining was at a basal level or not detectable from days 1 to 7 after hCG injection and was highly expressed in the corpus luteum from days 9 to 15 post-hCG injection. PGE2 biosynthesized through the mPGES-1 pathway may be important for follicular development, ovulation and luteal formation.

It has been shown that both prostaglandin I2 (PGI2) and PGE2 are essential for mouse implantation, whereas only PGE2 is required for hamster implantation. To date, the expression and regulation of cyclooxygenase (COX) and prostaglandin E synthase (PGES), which are responsible for PGE2 production, have not been reported in the rat. The aim of this study was to examine the expression pattern and regulation of COX-1, COX-2, membrane-associated PGES-1 (mPGES-1), mPGES-2 and cytosolic PGES (cPGES) in rat uterus during early pregnancy and pseudopregnancy, and under delayed implantation. At implantation site on day 6 of pregnancy, COX-1 immunostaining was highly visible in the luminal epithelium, and COX-2 immunostaining was clearly observed in the subluminal stroma. Both mPGES-1 mRNA and protein were only observed in the subluminal stroma surrounding the implanting blastocyst at the implantation site on day 6 of pregancy , but were not seen in the inter-implantation site on day 6 of pregnancy and on day 6 of pseudopregnancy. Our data suggest that the presence of an active blastocyst is required for mPGES-1 expression at the implantation site. When pregnant rats on day 5 were treated with nimesulide for 24 h, mPGES-1 protein expression was completely inhibited. cPGES immunostaining was clearly observed in the luminal epithelium and subluminal stromal cells immediately surrounding the implanting blastocyst on day 6 of pregnancy. mPGES-2 immunostaining was clearly seen in the luminal epithelium at the implantation site. Additionally, immunostaining for prostaglandin I synthase (PGIS) was also strongly detected at the implantation site. In conclusion, our results indicate that PGE2 and PGI2 should have a very important role in rat implantation.

PGE2 is essential for mammalian female reproduction. This study was to examine the expression of EP2 gene in the rat uterus during early pregnancy, delayed implantation and artificial decidualization by in situ hybridization and immunohistochemistry. There was no detectable EP2 mRNA expression in the uterus from days 1 to 4 of pregnancy (day 1 = day of vaginal sperm). A low level of EP2 immunostaining was observed in the luminal and glandular epithelium from days 1 to 4 of pregnancy. Both EP2 mRNA and protein expression were highly detected in the luminal epithelium at implantation sites on day 6 of pregnancy. EP2 expression decreased from day 7 of pregnancy and was undetectable on days 8 and 9 of pregnancy. After delayed implantation was terminated by estrogen treatment and the embryo implanted, both EP2 mRNA and protein expression were strongly observed in the luminal epithelium at the implantation site. There was no detectable EP2 expression in both control and decidualized uteri. In conclusion, these data suggest that EP2 expression at implantation site may play an important role during embryo implantation in rats.

Prostaglandin E2 (PGE2) is shown to be essential for female reproduction. Cyclooxygenase (COX) is a rate-limiting enzyme in prostaglandin synthesis from arachidonic acid and exists in two isoforms: COX-1 and COX-2. Prostaglandin E synthase (PGES) is a terminal prostanoid synthase and can catalyse the isomerization of the COX product PGH2 to PGE2, including microsomal PGES-1 (mPGES-1), cytosolic PGES (cPGES) and mPGES-2. This study examined the protein expression of COX-1, COX-2, mPGES-1, cPGES and mPGES-2 in preimplantation mouse embryos by immunohistochemistry. Embryos at different stages collected from oviducts or uteri were transferred into a flushed oviduct of non-pregnant mice. The oviducts containing embryos were paraffin-embedded and processed for immunostaining. COX-1 immunostaining was at a basal level in zygotes and a low level at the 2-cell stage, reaching a high level from the 4-cell to blastocyst stage. COX-2 immunostaining was at a low level at the zygote stage and was maintained at a high level from the 2-cell to blastocyst stages. A low level of mPGES-1 immunostaining was observed from the zygote to 8-cell stages. The signal for mPGES-1 immunostaining became stronger at the morula stage and was strongly seen at the blastocyst stage. cPGES immunostaining was strongly observed in zygotes, 2-cell and 8-cell embryos. There was a slight decrease in cPGES immunostaining at the 4-cell, morula and blastocyst stages. mPGES-2 immunostaining was at a low level from the zygote to morula stages and at a high level at the blastocyst stage. We found that the COX-1, COX-2, mPGES-1, cPGES and mPGES-2 protein signals were all at a high level at the blastocyst stage. PGE2 produced during the preimplantation development may play roles during embryo transport and implantation.

Signal transducer and activator of transcription (STATs) can be activated by many cytokines and growth factors. Stat3, a member of STAT family, is essential for embryonic development. Stat3 is specifically activated during mouse embryo implantation. This study was to investigate the expression, activation, and regulation of Stat3 in mouse uterus during early pregnancy, pseudopregnancy, delayed implantation, artificial decidualization, and hormonal treatments using in situ hybridization and immunohistochemistry. There was a strong level of Stat3 phosphorylation in the luminal epithelium only at the midnight of day 4 pregnancy, which coincides with attachment reaction between the blastocyst and luminal epithelium. However, there was no detectable Stat3 phosphorylation at the corresponding period during pseudopregnancy. On day 5 of pregnancy, Stat3 phosphorylation was strongly observed in the luminal epithelium and the stroma surrounding the implanting blastocyst at implantation sites, but not at the inter-implantation sites. Stat3 phosphorylation was also not detected on day 5 of pseudopregnancy. Stat3 phosphorylation was at a high level in the decidual cells on days 6-8 of pregnancy. Under artificial decidualization, Stat3 was also phosphorylated in the decidual cells. In the ovariectomized mice, there was no Stat3 expression and activation in the uterus. Progesterone had no obvious effects. However, Stat3 mRNA expression and phosphorylation were significantly stimulated by estrogen treatment. Our data suggest that Stat3 phosphorylation may be important for mouse embryo implantation and decidualization, and may also be regulated by maternal estrogen.

Signal transducer and activator of transcription 3 (Stat3), a member of the Stat family, is specifically activated during mouse embryo implantation. The aim of this study was to investigate the expression, activation and regulation of Stat3 in rat uterus during early pregnancy, pseudopregnancy, delayed implantation and artificial decidualization. Stat3 mRNA was highly expressed in the luminal epithelium on day 5 and in the luminal epithelium and underlying stromal cells at implantation sites on day 6 of pregnancy. There was a strong level of Stat3 protein expression and phosphorylation in the stromal cells near the lumen and in the luminal epithelium on day 5 of pregnancy, which was similar to day 5 of pseudopregnancy. In the afternoon of day 6, the strong level of Stat3 phosphorylation was detected only in the luminal epithelium. Stat3 was highly expressed and activated in the decidual cells from days 7 to 9 of pregnancy and under artificial decidualization in the present study. Our results suggest that the strong level of Stat3 activation in the luminal epithelium and underlying stromal cells during the pre-implantation period may be important for establishing uterine receptivity as in mice, and the high level of Stat3 expression and activation in decidual cells may play a role during decidualization., (Copyright 2004 Society for Reproduction and Fertility)

Cyclooxygenase (COX), a rate-limiting enzyme that produces prostaglandins (PGs) from arachidonic acid, exists in two isoforms, COX-1 and COX-2. PGE2 synthase (PGES) is a terminal prostanoid synthase and can enzymatically convert the cyclooxygenase product PGH2 to PGE2, including two isoforms: microsomal PGES (mPGES) and cytosolic PGES (cPGES). cPGES is predominantly linked with COX-1 to promote the immediate response. mPGES is preferentially coupled with the inducible COX-2 to promote delayed PGE2 generation. COX-2-deficient female mice are infertile with abnormalities in ovulation, fertilization, implantation and decidualization. The aim of this study was to examine immunohistochemically the expression pattern of COX-1, COX-2, mPGES and cPGES proteins in the endometrium of the rhesus monkey during the menstrual cycle. COX-1 immunostaining was mainly localized in the luminal epithelium and glandular epithelium near the lumen, and detected in all the stages during the menstrual cycle. COX-2 immunostaining was mainly localized in the luminal and glandular epithelium, and strongly shown during the mid-luteal phase (days 16 and 20) of the menstrual cycle. There was a strong cPGES immunostaining in the luminal and glandular epithelium on days 12, 16, 20 and 25 of the menstrual cycle. mPGES immunostaining was strongly detected in the glandular epithelium on days 20 and 25 of the menstrual cycle. These data suggest that the coupling of cPGES and COX-1 in the luminal epithelium may be responsible for the synthesis of PGE2 in monkey endometrium, and the coupling of mPGES and COX-2 in the glandular epithelium may be of importance for preparing the receptive endometrium.

Calcitonin has been shown to be a progesterone-regulated potential marker of the receptive endometrium in the rat and human. The present study was undertaken to immunohistochemically investigate the changes in calcitonin in the monkey uterus during the menstrual cycle and periimplantation period. Calcitonin immunostaining was primarily localized in the glandular epithelium on d 16, 20, and 25 of the menstrual cycle. During early pregnancy, calcitonin immunostaining was strongly observed in the glandular epithelium only on d 9 of pregnancy, the day before implantation. Since the high level of calcitonin immunostaining in the glandular epithelium during the luteal phase of the menstrual cycle and periimplantation period matched the high level of maternal progesterone during this period, the expression of calcitonin in monkey endometrium may be under the regulation of maternal progesterone.