Influence of Vitamin D supplementation on reproductive outcomes of infertile patients: a systematic review and meta-analysis.

BACKGROUND: Low vitamin D status has been associated with an increased risk for infertility. Recent evidence regarding the efficacy of vitamin D supplementation in improving reproductive outcomes is inconsistent. Therefore, this systematic review was conducted to investigate whether vitamin D supplementation could improve the reproductive outcomes of infertile patients and evaluate how the parameters of vitamin D supplementation affected the clinical pregnancy rate. METHODS: We searched seven electronic databases (CNKI, Cqvip, Wanfang, PubMed, Medline, Embase, and Cochrane Library) up to March 2022. Randomized and cohort studies were collected to assess the reproductive outcomes difference between the intervention (vitamin D) vs. the control (placebo or none). Mantel-Haenszel random effects models were used. Effects were reported as odds ratio (OR) and their 95% confidence interval (CI). PROSPERO database registration number: CRD42022304018. RESULTS: Twelve eligible studies (n = 2352) were included: 9 randomized controlled trials (RCTs, n = 1677) and 3 cohort studies (n = 675). Pooled results indicated that infertile women treated with vitamin D had a significantly increased clinical pregnancy rate compared with the control group (OR: 1.70, 95% CI: 1.24-2.34; I2 = 63%, P = 0.001). However, the implantation, biochemical pregnancy, miscarriage, and multiple pregnancy rates had no significant difference (OR: 1.86, 95% CI: 1.00-3.47; I2 = 85%, P = 0.05; OR: 1.49; 0.98-2.26; I2 = 63%, P = 0.06; OR: 0.98, 95% CI: 0.63-1.53; I2 = 0%, P = 0.94 and OR: 3.64, 95% CI: 0.58-11.98; I2 = 68%, P = 0.21). The improvement of clinical pregnancy rate in the intervention group was influenced by the vitamin D level of patients, drug type, the total vitamin D dosage, the duration, administration frequency, and daily dosage of vitamin D supplementation. The infertile women (vitamin D level < 30 ng/mL) treated with the multicomponent drugs including vitamin D (10,000-50,000 IU or 50,000-500,000 IU), or got vitamin D 1000-10,000 IU daily, lasting for 30-60 days could achieve better pregnancy outcome. CONCLUSION: To the best of our knowledge, this is the first meta-analysis systematically investigated that moderate daily dosing of vitamin D supplementation could improve the clinical pregnancy rate of infertile women and reported the effects of vitamin D supplementation parameters on pregnancy outcomes. A larger sample size and high-quality RCTs are necessary to optimize the parameters of vitamin D supplementation to help more infertile patients benefit from this therapy.

[Progress of non-genomic action of estrogen and its impact on female reproduction].

Estrogen is one of the steroid hormones. Besides the genomic action mediated by its intracellular receptor on target cells, there is now increasing body of evidence indicating that estrogen also has non-genomic action. For the non-genomic action, estrogen binds to its receptor on cell membrane, subsequently rapidly activates various intracellular signaling pathways, such as PLC/Ca(2+), ERK/MAPK, cAMP-PKA, PI3K-AKT-NOS, and finally induces biological effects. The non-genomic effects of estrogen on physiologic and pathologic processes have been found in many tissues within the reproductive, nervous and cardiovascular systems and bone etc. In reproductive system, it has been demonstrated that estrogen plays important roles in follicle development, fertilization and embryo implantation, and it is involved in the genesis and development of genital tract tumors and breast cancer. In this review, we focus on the general characteristics of non-genomic action of estrogen, its main nonnuclear signaling pathways and physiological and pathological significance, especially its influences in female reproductive functions.

Relocalisation and activation of integrins induced rapidly by oestrogen via G-protein-coupled receptor 30 in mouse blastocysts.

Integrins are the dominant and final adhesion molecules in the attachment process between the blastocysts and endometrium. It is necessary for oestrogen to rapidly activate mouse blastocysts so that they attach to the endometrial epithelium. Our previous study suggested that oestrogen can rapidly induce an increase in intracellular calcium in mouse blastocysts via G-protein-coupled receptor 30 (GPR30). Thus, we deduced that integrins may be involved in GPR30 mediation of the fast effect of oestrogen on mouse blastocysts in implantation. To prove our hypothesis, we used immunofluorescence staining and in vitro coculture of mouse blastocysts and endometrial epithelial cell line (EECs), Ishikawa cells, in the present study. We found that αv and ß1 integrin clustered in mouse blastocysts, and that ß3 integrin was relocalised to the apical membrane of blastocyst cells when embryos were treated with 1 µM 17ß-estradiol (E2), 1 µM E2 conjugated to bovine serum albumin (E2-BSA) and 1 µM G-1, a specific GPR30 agonist, for 30 min respectively, whereas pretreatment with 1 µM G15, a specific GPR30 antagonist, and 5 µM 1,2-Bis(2-aminophenoxy)ethane-N,N,N'',N''-tetraacetic acid tetrakis (acetoxymethyl ester)(BAPTA/AM), a cellular Ca2+ chelator, blocked the localisation of integrins induced by oestrogen via GPR30 in mouse blastocyst cells. E2, E2-BSA and G-1 increased the fibronectin (FN)-binding activity of integrins in blastocysts, whereas G15 and BAPTA/AM blocked the activation of integrins induced by oestrogen via GPR30 in mouse blastocysts. Inhibition of integrins by Arg-Gly-Asp peptide in blastocysts resulted in their failure to adhere to EECs in vitro, even if oestrogen or G-1 was provided. Together, the results indicate the fast effect of oestrogen via the GPR30 membrane receptor further induces relocalisation and activation of integrins in mouse blastocysts, which play important roles in the adhesion of blastocysts to EECs.

[Estrogen induced rapid increase of intracellular calcium plays important role in the implantation of mouse blastocysts].

OBJECTIVE: To study the roles of the increased intracellular calcium induced rapidly by estrogen in the implantation of mouse blastocysts. METHODS: The mouse blastocysts were collected from the female mice on the pregnant day 4, divided into 3 groups: control, E2-BSA and BAPTA +E2-BSA. Immunofluorescence staining, confocal microscopy, embryo and endometrial epithenial cells co-culture and embryo transfer were used to investigate the effect of increased intracellular calcium induced by E2-BSA on the expression and localization of integrins in blastocysts and their adhesion to endometrial epithenial calls (EECs) and implantation into the endometrium. RESULTS: The increase of intracellular calcium induced rapidly by estrogen could cause the cluster and relocation of integrin av and beta3, and BAPTA might block this effect, the adhesion rate of blastocysts in contol group was 35.5%, BAPTA +E2-BSA group was 26.7% and significantly lower than 65.6% of E2-BSA group (P<0.05), and the implantation rate in BAPTA+E2-BSA group was 11.8%, which was significantly lower than 52.9% of E2-BSA group (P<0.05). CONCLUSION: The rapid increase of intracellular calcium induced by estrogen may cause the relocalization of integrin in blastocysts and their adhesion to ECCs, which is important in the process of implantation.

Two new withanolides from Physalis minima L.

Two new withanolides named physaminilides L (1) and M (2), together with four known ones (3-6) were isolated from the Physalis minima L. The structures were established by analysis of the HR ESIMS, IR and NMR spectroscopic data. The absolute configurations were determined through NOESY and ECD spectra. For compounds 1-5 assayed at 20 µM and compound 6 at 10 µM, inhibition rates of hepatic fibrosis were 22.19%, 15.29%, 37.07%, 9.27%, 12.45%, and 37.03%, respectively.

Intrauterine administration of peripheral blood mononuclear cells activated by human chorionic gonadotropin in patients with repeated implantation failure: A meta-analysis.

The purpose of this study was to assess whether intrauterine administration of peripheral blood mononuclear cells (PBMCs) activated by human chorionic gonadotropin (hCG) could improve the pregnancy and live birth rates in women with repeated implantation failure (RIF), and whether the parameters of co-culture of hCG and PBMCs would affect the clinical outcomes. Six databases (PubMed, Ovid, Medline, NCBI, Cqvip and Wanfang) were searched up to October 2020 by two independent reviewers. Seven studies were included according to specific inclusion and exclusion criteria. A meta-analysis showed that the pregnancy and live birth rates were significantly increased in the case group compared with the control group (odds ratio [OR]: 3.43, 95 % confidence interval [CI]: 1.78-6.61; P = 0.0002 and OR: 2.79, 95 % CI: 1.09-7.15; P = 0.03), especially when hCG was cultured with PBMCs for 48 h or PBMCs administration was performed two or three days before embryo transfer (ET). Neither the dosage of the hCG co-cultured with PBMCs nor the mean concentration of the administered PBMCs appeared to influence the therapeutic efficiency. In conclusion, intrauterine administration of PBMCs co-cultured with hCG for 48 h, conducted two or three days before ET, could be an effective therapy for women experiencing RIF. Due to the limitations of sample size and quality of the included studies, further high-quality studies with large sample sizes are warranted to optimize the parameters of hCG and PBMC co-culture to help more RIF patients benefit from this therapy.

Expression of SGLT1 in the Mouse Endometrial Epithelium and its Role in Early Embryonic Development and Implantation.

Many functional activities of endometrium epithelium are energy consuming which are very important for maintaining intrauterine environment needed by early embryonic development and establishment of implantation window. Glucose is a main energy supplier and one of the main components of intrauterine fluid. Obviously, glucose transports in endometrium epithelium involve in for these activities but their functions have not been elucidated. In this research, we observed a spatiotemporal pattern of sodium glucose transporter 1 (SGLT1) expression in the mouse endometrium. We also determined that progesterone can promote the expression of SGLT1 in the mouse endometrial epithelium in response to the action of oestrogen. Treatment with the SGLT1 inhibitor phlorizin or small interfering RNA specific for SGLT1 (SGLT1-siRNA) altered glucose uptake in primary cultured endometrial epithelial cells, which exhibited reduced ATP levels and AMPK activation. The injection of phlorizin or SGLT1-siRNA into one uterine horn of each mouse on day 2 of pregnancy led to an increased glucose concentration in the uterine fluid and decreased number of harvested normal blastocysts and decreased expression of integrin αVß3 in endometrial epithelium and increased expression of mucin 1 and lactoferrin in endometrial epithelium and the uterine homogenates exhibited activated AMPK, a decreased ATP level on day 4, and a decreased number of implantation sites on day 5. In embryo transfer experiments, pre-treatment of the uterine horn with phlorizin or SGLT1-siRNA during the implantation window led to a decreased embryo implantation rate on day 5 of pregnancy, even when embryos from normal donor mice were used. In conclusion, SGLT1, which participates in glucose transport in the mouse endometrial epithelium, inhibition and/or reduced expression of SGLT1 affects early embryo development by altering the glucose concentration in the uterine fluid. Inhibition and/or reduced expression of SGLT1 also affects embryo implantation by influencing energy metabolism in epithelial cells, which consequently influences implantation-related functional activities.

The ionotropic γ-aminobutyric acid receptor gene family of the silkworm, Bombyx mori.

γ-Aminobutyric acid (GABA) is a very important inhibitory neurotransmitter in both vertebrate and invertebrate nervous systems. GABA receptors (GABARs) are known to be the molecular targets of a class of insecticides. Members of the GABAR gene family of the silkworm, Bombyx mori, a model insect of Lepidoptera, have been identified and characterized in this study. All putative silkworm GABAR cDNAs were cloned using the reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Bombyx mori appears to have the largest insect GABAR gene family known to date, including three RDL, one LCCH3, and one GRD subunit. The silkworm RDL1 gene has RNA-editing sites, and the RDL1 and RDL3 genes possess alternative splicing. These mRNA modifications enhance the diversity of the silkworm's GABAR gene family. In addition, truncated transcripts were found for the RDL1 and LCCH3 genes. In particular, the three RDL subunits may have arisen from two duplication events.

Characterization of a Bombyx mori nucleopolyhedrovirus with Bmvp80 disruption.

A BmNPV Bacmid with the Bmvp80 gene disrupted was constructed using the ET-recombination system in Escherichia coli to investigate the role of Bmvp80 during the baculovirus life cycle. Disruption of Bmvp80 resulted in single cell infection phenotype, whereas a rescue BmBacmid restored budded virus titers to wild type levels; however, the homologous gene Ac104 (Acvp80) from AcMNPV could not complement the BmBacmid lacking a functional Bmvp80 gene. Electron microscopy of cells transfected with BmNPV lacking functional Bmvp80 revealed that the number of nucleocapsids was markedly lower. These results suggest that Bmvp80 is essential for normal budded virus production and nucleocapsid maturation, and is functionally divergent between baculovirus species.

[The progesterone-induced expression of cyclin G1 and its effect on the proliferation of mouse uterine epithelial cells].

The aim of the present study is to investigate the effect of progesterone-induced expression of cyclin G1 on the proliferation of endometrial epithelial cells. To obtain mouse endometrial epithelial cells, the uteri were isolated from ovariectomized mice which were injected subcutaneously with 100 ng estradiol per day for two days. Then the uteri were digested by dispase and pancreatin respectively. Endometrial epithelial cells were cultured in DMEM/F12 containing 6% fetal bovine serum, and divided into four groups when they grew to confluence. Each of the groups was treated as follows: Group E was treated with 0.01 micromol/L estradiol only, group P was treated with 1 micromol/L progesterone, group EP was treated with both 0.01 micromol/L estradiol and 1 micromol/L progesterone, and group C was treated with 0.01% DMSO for control. Immunocytochemistry was used to examine the expression of cyclin G1 protein. MTT assay was used to evaluate metabolic activity of cells. Flow cytometry was used to check the number of cells distributing in each phase of the cell cycle. The result of immunocytochemistry showed that there was no expression of cyclin G1 protein in group C and group E, while cyclin G1 was obviously expressed in group P and group EP and localized in nucleus. In the MTT assay, compared with group C, the viability of group E significantly increased, while that of both group P and group EP decreased significantly. The results of flow cytometry were in accordance with those of MTT, which showed that compared with group C, group E had a higher proportion of cells in S phase, while group P, as well as group EP had a lower proportion of cells in S phase but a higher proportion in G1 phase and G2/M phase. These results indicate that progesterone could induce cyclin G1 expression in the primary culture of mouse endometrial epithelial cells, meanwhile inhibit the proliferation of cells and block the cell cycle progression. Thus, progesterone-induced expression of cyclin G1 is probably a negative factor in regulating cell cycle, which is involved in the inhibitory effect of progesterone on the proliferation of endometrial epithelial cells.

Bisphenol A Initiates Excessive Premature Activation of Primordial Follicles in Mouse Ovaries via the PTEN Signaling Pathway.

The essence of primary ovarian insufficiency (POI) is the premature exhaustion of primordial follicles in the follicle pool, which is caused by the excessive premature activation of primordial follicles after birth. Bisphenol A (BPA) exposure promotes the transition of primordial follicles to primary follicles, thus the number of primordial follicles in the primordial follicle pool decreases significantly. However, the molecular mechanisms underlying abnormal follicle activation are poorly understood. Phosphatase and tensin homologue (PTEN) signal system is a negative regulator of follicle activation, which is called the brake of follicle activation. Besides, BPA induces Michigan Cancer Foundation-7 breast cancer cells proliferation by dysregulating PTEN/serine/threonine kinase/p53 axis. Whether BPA initiates the excessive premature activation of primordial follicles in the mouse ovaries via PTEN signaling pathway is unclear. In this study, we treated 6-week-old female CD-1 mice with different concentrations of BPA to study the effect of BPA on follicular activation and development in vivo, as well as the role of PTEN signaling in this process. We observed that BPA in concentrations from 1 µg/kg to 10 mg/kg groups downregulated PTEN expression and initiated excessive premature activation of primordial follicles in the mouse ovaries, and this effect was partly reversible by PTEN overexpression. Our results improve the understanding of both the effect of BPA in occurrence of POI and molecular mechanisms underlying initiation of primordial follicle pool activation, thus providing insight for POI treatment and theoretical basis for reducing the risk of POI.

Complete sequence and organization of Antheraea pernyi nucleopolyhedrovirus, a dr-rich baculovirus.

BACKGROUND: The completion and reporting of baculovirus genomes is extremely important as it advances our understanding of gene function and evolution. Due to the large number of viral genomes now sequenced it is very important that authors present significantly detailed analyses to advance the understanding of the viral genomes. However, there is no report of the Antheraea pernyi nucleopolyhedrovirus (AnpeNPV) genome. RESULTS: The genome of AnpeNPV, which infects Chinese tussah silkworm (Antheraea pernyi), was sequenced and analyzed. The genome was 126,629 bp in size. The G+C content of the genome, 53.4%, was higher than that of most of the sequenced baculoviruses. 147 open reading frames (ORFs) that putatively encode proteins of 50 or more amino acid residues with minimal overlap were determined. Of the 147 ORFs, 143 appeared to be homologous to other baculovirus genes, and 4 were unique to AnpeNPV. Furthermore, there are still 29 and 33 conserved genes present in all baculoviruses and all lepidopteran baculoviruses respectively. In addition, the total number of genes common to all lepidopteran NPVs is sill 74, however the 74 genes are somewhat different from the 74 genes identified before because of some new sequenced NPVs. Only 6 genes were found exclusively in all lepidopteran NPVs and 12 genes were found exclusively in all Group I NPVs. AnpeNPV encodes v-trex(Anpe115, a 3' to 5' repair exonuclease), which was observed only in CfMNPV and CfDEFNPV in Group I NPVs. This gene potentially originated by horizontal gene transfer from an ancestral host. In addition, AnpeNPV encodes two conotoxin-like gene homologues (ctls), ctl1 and ctl2, which were observed only in HycuNPV, OpMNPV and LdMNPV. Unlike other baculoviruses, only 3 typical homologous regions (hrs) were identified containing 2~9 repeats of a 30 bp-long palindromic core. However, 24 perfect or imperfect direct repeats (drs) with a high degree of AT content were found within the intergenic spacer regions that may function as non-hr, ori-like regions found in GrleGV, CpGV and AdorGV. 9 drs were also found in intragenic spacer regions of AnpeNPV. CONCLUSION: AnpeNPV belongs to Group I NPVs and is most similar to HycuNPV, EppoNPV, OpMNPV and CfMNPV based on gene content, genome arrangement, and amino acid identity. In addition, analysis of genes that flank hrs supported the argument that these regions are involved in the transfer of sequences between the virus and host.

The Changes of Cytoskeletal Proteins Induced by the Fast Effect of Estrogen in Mouse Blastocysts and Its Roles in Implantation.

It is necessary for estrogen to activate mouse blastocysts, so that they can attach to endometrial epithelium in implantation and in our previous research, we have proved estrogen can induce a fast increase in intracellular calcium of mouse blastocysts through acting on G protein-coupled receptor 30 (GPR30), which further promotes their implantation. Moreover, there has been evidence that cytoskeletal proteins are involved in integrin-mediated adhesion of many kinds of cells, which also plays an important role in implantation. To prove estrogen induces rapidly the changes of cytoskeletal proteins in mouse blastocysts and its roles in implantation, we first used immunofluorescence staining and laser confocal microscopy to investigate the fast effect of estrogen on the expression and localization of cytoskeletal proteins in mouse blastocysts. Second, we used electroporation associated with RNA interference to knock down one of the important cytoskeletal proteins, talin, in the mouse blastocyst cells to investigate the fast effect of estrogen on the localization of integrins and the binding activity of integrins with their ligand fibronectin (FN). At last, mouse blastocysts with different treatments were cultured with FN or uterine epithelial cell line Ishikawa in vitro, respectively, and transferred into the bilateral uterine horns of recipient mice, to study the role of the fast effect of estrogen on cytoskeletal proteins in blastocysts adhesion and implantation. Our results indicated that estradiol (E2), E2 conjugated with bovine serum album (E2-BSA) and G-1 (a GPR30-specific agonist) could induce cytoskeletal protein talin, vinculin, and actin to cluster in the mouse blastocysts, while G15 (a GPR30-specific antagonist) and BAPTA (a calcium chelator) may block this effect induced by E2-BSA. Furthermore, E2-BSA could induce the clustering and relocalization of integrin ß1 and ß3 and increase the FN-binding activity of integrins in blastocyst cells, while E2-BSA could not induce these effects in the blastocysts pretreated with talin-small interfering RNA (siRNA). Meanwhile, the adhesion rate and implantation rate of blastocysts pretreated with talin-siRNA were significantly lower than those pretreated with control-siRNA. We provided the first evidence that the fast effect of estrogen might cause the clustering of the cytoskeletal proteins in mouse blastocyst cells and further induce the changes of localization and functional activity of integrins in the blastocyst cells, which play important roles in blastocyst implantation.

Identification and analysis of YELLOW protein family genes in the silkworm, Bombyx mori.

BACKGROUND: The major royal jelly proteins/yellow (MRJP/YELLOW) family possesses several physiological and chemical functions in the development of Apis mellifera and Drosophila melanogaster. Each protein of the family has a conserved domain named MRJP. However, there is no report of MRJP/YELLOW family proteins in the Lepidoptera. RESULTS: Using the YELLOW protein sequence in Drosophila melanogaster to BLAST silkworm EST database, we found a gene family composed of seven members with a conserved MRJP domain each and named it YELLOW protein family of Bombyx mori. We completed the cDNA sequences with RACE method. The protein of each member possesses a MRJP domain and a putative cleavable signal peptide consisting of a hydrophobic sequence. In view of genetic evolution, the whole Bm YELLOW protein family composes a monophyletic group, which is distinctly separate from Drosophila melanogaster and Apis mellifera. We then showed the tissue expression profiles of Bm YELLOW protein family genes by RT-PCR. CONCLUSION: A Bombyx mori YELLOW protein family is found to be composed of at least seven members. The low homogeneity and unique pattern of gene expression by each member among the family ensure us to prophesy that the members of Bm YELLOW protein family would play some important physiological functions in silkworm development.

Screening the active constituents of Chinese medicinal herbs as potent inhibitors of Cdc25 tyrosine phosphatase, an activator of the mitosis-inducing p34cdc2 kinase.

OBJECTIVE: To screen and evaluate the active constituents of Chinese medicinal herbs as potent inhibitors of Cdc25 phosphatase. METHODS: The affinity chromatography purified glutashione-S-transferase/Cdc25A phosphatase fusion protein and Cdc2/cyclin B from the extracts of starfish M phase oocytes are used as the cell cycle-specific targets for screening the antimitotic constituents. We tested 9 extracts isolated from the Chinese medicinal herbs and vegetables including the agents currently used in cancer treatment by measuring the inhibition of Cdc25A phosphatase and Cdc2 kinase activity. The antitumor activity of the extracts was also evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and flow cytometry. RESULTS: Cdc25A inhibitory activity and antitumor activity are detected in the extracts isolated from three Chinese medicinal herbs Agrimona pilosa; Herba solani lyrati; Galla chinesis. CONCLUSION: We found three extracts isolated from Chinese medicinal herbs have potential inhibitory activity of Cdc25 phosphatase using a highly specific mechanism-based screen assay for antimitotic drug discovery.

GPR30 Mediates the Fast Effect of Estrogen on Mouse Blastocyst and its Role in Implantation.

Our previous work demonstrated that estrogen could rapidly increase intracellular Ca(2+) in dormant mouse blastocysts. The purpose of the present study is to investigate the physiological relevance of G protein-coupled receptor 30 (GPR30) in the fast effect of estrogen on mouse blastocyst and in embryo implantation. We used reverse transcription-polymerase chain reaction, immunofluorescence, embryo coculture with Ishikawa uterine epithelial cell line, and embryo transfer technology to detect the expression of GPR30 in mouse embryos and the nongenomic effects of estrogen via GPR30 on blastocyst. We found that GPR30 is expressed in the mouse blastocyst, and its location is mostly consistent with the binding site of estrogen. Both estrogen and GPR30-specific agonist G-1 rapidly increase the intracellular Ca(2+) and phospholipase C activation in blastocyst cells, while GPR30-specific antagonist G-15 blocked this effect of estrogen. The pretreatment of G-15 on blastocysts lead to a lower attachment rate and implantation rate. Our data collectively suggested that GPR30 can mediate the fast effect of estrogen on blastocysts and play an important role in embryo implantation.

Identification and characterization of progesterone- and estrogen-regulated MicroRNAs in mouse endometrial epithelial cells.

In endometrial epithelial cells, progesterone (P4) functions in regulating the cell structure and opposing the effects of estrogen. However, the mechanisms of P4 that oppose the effects of estrogen remain unclear. MicroRNAs (miRNAs) are important posttranscriptional regulators that are involved in various physiological and pathological processes. Whether P4 directly induces miRNA expression to antagonize estrogen in endometrial epithelium is unclear. In this study, total RNAs were extracted from endometrial epithelium of ovariectomized mice, which were treated with estrogen alone or a combination of estrogen and P4. MicroRNA high-throughput sequencing with bioinformatics analysis was used to identify P4-induced miRNAs, predict their potential target genes, and analyze their possible biological functions. We observed that 146 mature miRNAs in endometrial epithelial cells were significantly upregulated by P4. These miRNAs were extensively involved in multiple biological processes. The miRNA-145a demonstrated a possible function in the antiproliferative action of P4 on endometrial epithelial cells.

[Expression of metallothionein 2 in middle ear cholesteatoma and its correlation with interleukin 1α and interleukin 6].

OBJECTIVE: To detect the expression and distribution of metallothionein 2 (MT-2) in middle ear cholesteatoma, and to explore the relationship of MT-2 with interleukin 1α (IL-1α) and interleukin 6 (IL-6), and well as to explore the role of MT-2 in the pathogenetic mechanism of middle ear cholesteatoma. METHODS: Using the immunohistochemistry and reverse transcription polymerase chain reaction to examine the expressions of MT-2, IL-1α and IL-6 protein and MT-2 mRNA in twenty-five middle ears' cholesteatoma and seven normal external ears' canal skin. The influence of cholesteatoma debris on the MT-2 mRNA and protein of HaCaT cell were further analyzed. RESULTS: According to immunohistochemistry research, the expressions of MT-2, IL-1α and IL-6 were extremely higher in middle ear cholesteatoma than those in normal external ears' canal skin (P < 0.05). The result of RT-PCR showed that there was significant difference between the expression of MT-2 mRNA of and middle ear cholesteatoma than those in normal external ear canal skin (t = 15.38, P < 0.05). There was a positive correlation between the expression of MT-2 and that of IL-1α (r = 0.856, P < 0.05). There was also positive correlation between the expression of MT-2 and that of IL-6 (r = 0.714, P < 0.05). MT-2 mRNA and protein of HaCaT cell significantly increased when exposed to cholesteatoma debris in a dose dependent manner. CONCLUSIONS: MT-2, IL-1α and IL-6 may play an important role in the pathogenetic process of middle ear cholesteatoma. Cholesteatoma debris may involve in the proliferation of middle ear cholesteatoma by activation of MT-2.

Molecular characterization of DSC1 orthologs in invertebrate species.

DSC1 and BSC1 are two founding members of a novel family of invertebrate voltage-gated cation channels with close structural and evolutionary relationships to voltage-gated sodium and calcium channels. In this study, we searched the published genome sequences for DSC1 orthologs. DSC1 orthologs were found in all 48 insect species, and in other invertebrate species belonging to phyla Mollusca, Cnidaria, Hemichordata and Echinodermata. However, DSC1 orthologs were not found in four arachnid species, Ixodes scapularis, Rhipicephalus microplus, Tetranychus urticae and Varroa destructor, two species in Annelida or any vertebrate species. We then cloned and sequenced NlSC1 and BmSC1 full-length cDNAs from the brown planthopper (Nilaparvata lugens) and the silkworm (Bombyx mori), respectively. NlSC1 and BmSC1 share about 50% identity with DSC1, and the expression of NlSC1 and BmSC1 transcripts was most abundant in the head and antenna in adults. All DSC1 orthologs contain a unique and conserved DEEA motif, instead of the EEEE or EEDD motif in classical calcium channels or the DEKA motif in sodium channels. Phylogenetic analyses revealed that DSC1 and its orthologs form a separate group distinct from the classical voltage-gated sodium and calcium channels and constitute a unique family of cation channels. The DSC1/BSC1-family channels could be potential targets of new and safe insecticides for pest control.