Regulation of the mammalian maternal-to-embryonic transition by eukaryotic translation initiation factor 4E.

Eukaryotic translation initiation factor 4E (eIF4E) mediates cap-dependent translation. Genetic and inhibitor studies show that eIF4E expression is required for the successful transition from maternal to embryonic control of mouse embryo development. eIF4E was present in the oocyte and in the cytoplasm soon after fertilization and during each stage of early development. Functional knockout (Eif4e-/-) by PiggyBac [Act-RFP] transposition resulted in peri-implantation embryonic lethality because of the failure of normal epiblast formation. Maternal stores of eIF4E supported development up to the two- to four-cell stage, after which new expression occurred from both maternal and paternal inherited alleles. Inhibition of the maternally acquired stores of eIF4E (using the inhibitor 4EGI-1) resulted in a block at the two-cell stage. eIF4E activity was required for new protein synthesis in the two-cell embryo and Eif4e-/- embryos had lower translational activity compared with wild-type embryos. eIF4E-binding protein 1 (4E-BP1) is a hypophosphorylation-dependent negative regulator of eIF4E. mTOR activity was required for 4E-BP1 phosphorylation and inhibiting mTOR retarded embryo development. Thus, this study shows that eIF4E activity is regulated at key embryonic transitions in the mammalian embryo and is essential for the successful transition from maternal to embryonic control of development.

Tamoxifen resistance and metastasis of human breast cancer cells were mediated by the membrane-associated estrogen receptor ER-α36 signaling in vitro.

The drug resistance and tumor metastasis have been the main obstacles for the longer-term therapeutic effects of tamoxifen (TAM) on estrogen receptor-positive (ER+) breast cancer, but the mechanisms underlying the TAM resistance are still unclear. Here, we demonstrated that the membrane-associated estrogen receptor ER-α36 signaling, but not the G protein-coupled estrogen receptor 1 (GPER1) signaling, might be involved in the TAM resistance and metastasis of breast cancer cells. In this study, a model of ER+ breast cancer cell MCF-7 that involves the up-regulated expression of ER-α36 and unchanged expression of ER-α66 and GPER1 was established via the removal of insulin from the cell culture medium. The mechanism of TAM resistance in the ER+ breast cancer cell line MCF-7 was investigated, and the results showed that the stimulating effect of insulin on susceptibility of MCF-7 to TAM was mediated by ER-α36 and that the expression level of ER-α36 in TAM-resistant MCF-7 cells was also significantly increased. Both TAM and estradiol (E2) could promote the migration of triple negative (ER-α66-/PR-/HER2-) and ER-α36+/GPER1+ breast cancer cells MDA-MB-231. The migration of MDA-MB-231 cells was inhibited by the down-regulated intracellular expression of ER-α36 by transient transfection of specific small interfering RNA, whereas no effect of GPER1 down-regulation was observed. Meanwhile, the effect of TAM on the migration of ER-α36-down-regulated MDA-MB-231 cells was also reduced. Furthermore, it was found that TAM enhanced the distribution of integrin ß1 on the cell surface but did not affect the expression of integrin ß1 in MDA-MB-231 cells. Collectively, these data suggested that ER-α36 signaling might play critical roles in acquired and de novo TAM resistance and metastasis of breast cancer, and ER-α36 might present a potential biomarker of TAM resistance in the clinical diagnosis and treatment of ER+ breast cancer.

Integrins ß1 and ß3 are biomarkers of uterine condition for embryo transfer.

BACKGROUND: Clinical ovulation induction induces blood estrogen (E2) in excess of physiological levels, which can hinder uterine receptivity. In contrast, progesterone produces the opposite clinical effect, suggesting that it might be capable of recovering the lost receptivity resulting from exposure to high estrogen levels. Integrins are the most widely used biological markers for monitoring uterine conditions. We studied progesterone-induced changes in integrin ß expression patterns as biomarkers for changes in uterine receptivity in response to increased estrogen levels. METHODS: Endometrial biopsy samples from patients were screened for their estrogen (E2) and progesterone (P4) content and expressing levels of integrin ß1 and ß3. Uterine receptivity was evaluated using human endometrial adenocarcinoma cells in an embryo attachment model. The respective and concatenated effects of embryo attachment and changes in the integrin ß1 and ß3 expression patterns on the adenocarcinoma cell plasma membranes in response to 100 nM concentrations of E2 and P4 were evaluated. RESULTS: Increased blood E2 concentrations were associated with significantly decreased the levels of integrin ß3 expression in uterine biopsy samples. In vitro experiments revealed that a 100 nM E2 concentration inhibited the distribution of integrin ß3 on the plasma membranes of human endometrial adenocarcinoma cells used in the embryo attachment model, and resulted in decreased rates of embryo attachment. In contrast, P4 enhanced the expression of integrin ß1 and promoted its distribution on the plasma membranes. Furthermore, P4 recovered the embryo attachment efficiency that was lost by exposure to 100 nM E2. CONCLUSIONS: Blood E2 and P4 levels and integrin ß3 and ß1 expression levels in uterine biopsy samples should be considered as biomarkers for evaluating uterine receptivity and determining the optimal time for embryo transfer. Trial registration Trial number: ChiCTR-TRC-13003777; Name of registry: Chinese Clinical Trial Registry; Date of registration: 4 September 2013; Date of enrollment of the first study participant: 15 October 2013.

Altered microRNAs expression profiling in cumulus cells from patients with polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common endocrine disorder in women of reproductive age, and oocyte developmental competence is altered in patients with PCOS. In recent years microRNAs (miRNAs) have emerged as important regulators of gene expression, the aim of the study was to study miRNAs expression patterns of cumulus cells from PCOS patients. METHODS: The study included 20 patients undergoing in vitro fertilization (IVF) and intra-cytoplasmic sperm injection (ICSI): 10 diagnosed with PCOS and 10 matching controls. We used deep sequencing technology to identify the miRNAs differentially expressed in the cumulus cells of PCOS. RESULTS: There were 17 differentially expressed miRNAs in PCOS cumulus cells, including 10 miRNAs increase and 7 miRNAs decrease. These miRNAs were predicted to target a large set of genes with different functions, including Wnt- and MAPK- signaling pathways, oocyte meiosis, progesterone-mediated oocyte maturation and cell cycle. Unsupervised hierarchical clustering analysis demonstrated that there was a specific miRNAs expression pattern in PCOS cumulus cells. CONCLUSION: We found that the miRNAs expression profile was different in cumulus cells isolated from PCOS patients compared with control. This study provided new evidence for understanding the pathogenesis of PCOS.

Application of genome tagging technology in elucidating the function of sperm-specific protein 411 (Ssp411).

The genome tagging project (GTP) plays a pivotal role in addressing a critical gap in the understanding of protein functions. Within this framework, we successfully generated a human influenza hemagglutinin-tagged sperm-specific protein 411 (HA-tagged Ssp411) mouse model. This model is instrumental in probing the expression and function of Ssp411. Our research revealed that Ssp411 is expressed in the round spermatids, elongating spermatids, elongated spermatids, and epididymal spermatozoa. The comprehensive examination of the distribution of Ssp411 in these germ cells offers new perspectives on its involvement in spermiogenesis. Nevertheless, rigorous further inquiry is imperative to elucidate the precise mechanistic underpinnings of these functions. Ssp411 is not detectable in metaphase II (MII) oocytes, zygotes, or 2-cell stage embryos, highlighting its intricate role in early embryonic development. These findings not only advance our understanding of the role of Ssp411 in reproductive physiology but also significantly contribute to the overarching goals of the GTP, fostering groundbreaking advancements in the fields of spermiogenesis and reproductive biology.

Fluorescent quenching probes based SAA 1 genotyping with a fully automated system.

OBJECTIVE: The aim of the present study is to develop and validate a reliable and simple application for genotyping serum amyloid A1 (SAA1). METHODS: The specific nested PCR was performed to amplify a product of SAA1 gene. Two quenching probes (QPs) were designed for detecting two single nucleotide polymorphism (SNP) sites, rs1136743(C/T) and rs1136747(C/T) respectively for SAA1 genotypes. The specific nested PCR and QPs of SAA1 genotying was introduced into a fully automated genotyping system (I-densy, ARKRAY, Inc.), which enables the genotyping of SAA1 from whole blood. RESULTS: Six genotypes of SAA1 (α+/+, ß+/+, γ+/+, αß, αγ and ßγ) could be determined by monitoring the fluorescence intensity of two QPs with melting temperature (TM) analysis. Total 121 clinical samples were SAA1 genotyped in the fluorescent quenching probes based method with a fully automated I-densy system and were further sequence confirmed with a PCR direct sequencing approach. CONCLUSION: This fully automated system is a rapid and reliable strategy for the SAA1 genotyping and for its future clinical application.

A triplex probe-based TaqMan qPCR assay for Calreticulin type I and II mutation detection.

BACKGROUND: Calreticulin (CALR) exon 9 frameshift mutations have recently been identified in 30-40% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF) without JAK2 or MPL mutations. We aimed to develop a qPCR assay to screen type I and II mutations of CALR. METHODS: Three different fluorescent-labeled hydrolysis probes and one pair of primers in a closed-tube system were developed to detect CALR type I and II mutations and distinguish them from wild-type. The sensitivity and specificity were validated using TA-cloning plasmids containing CALR wild-type and type I and II mutants, respectively. Fifty-nine ET and PMF specimens were screened by TaqMan qPCR and sequenced by Sanger sequencing. For intra-assay validation, 20 replicates of the assay were performed with each sample. For inter-assay validation, four replications of each sample were carried out and repeated continuously for 5 days. RESULTS: We found that triplex probe-based TaqMan qPCR was reliable in detecting CALR type I and II mutants within DNA that was diluted to 1% of total DNA with the wild-type DNA as background. In 59 patient specimens, six of the observed mutations of CALR were type I and five were type II. Genotyping results obtained from TaqMan qPCR were 100% concordant with Sanger sequencing. The intra- and inter-assay CVs of TaqMan qPCR were less than 3%, respectively. CONCLUSIONS: Triplex probe-based TaqMan qPCR is an accurate and sensitive method for screening ET or PMF patients with type I and II mutations in CALR.

Lectin binding of human sperm associates with DEFB126 mutation and serves as a potential biomarker for subfertility.

Coating on the sperm surface, glycocalyx, plays a key role in sperm motility, maturation and fertilization. A comprehensive profile of sperm surface glycans will greatly facilitate both basic researches and clinical studies. Because of the capability of recognizing different glycan moieties, lectins are widely used in glycobiology. However, lacking high-throughput technology, limited lectins have been reported for analyzing the glycan of human sperm. In this study, we employed a lectin microarray for profiling the surface glycans of human sperm, on which 54 out of 91 lectins showed positive binding. Based on this technique, we compared lectin binding profiling of sperm with homozygous DEFB126 mutation (del/del) with that of wild type (wt/wt). DEFB126 was reported to contribute to the sialylation on sperm surface and its homozygous mutation was related to male subfertility. Six lectins (Jacalin/AIA, GHA, ACL, MPL, VVL and ABA) were found to develop lower binding affinity to sperm with del/del. Further validation showed that these lectins, especially ABA and MPL, can be potential biomarkers for clinical diagnosis of subfertility due to the mutation of DEFB126. Our research provides insight into the detection of some unexplained male subfertility, and the lectin microarray is generally applicable for infertility/subfertility sperm biomarker discovery.

Adenovirus-Mediated th Gene Therapy of Parkinson's Disease in a Experimental Rat Model.

A recombinant adenovirus (AdCMV th) encoding tyrosine hydroxylase (TH) gene with CMV promoter was constructed and propagated. Southern blot analyses was used to identify positive plaques. Virus titer was about 1.4x10(14) pfu/L as determined by plaque forming assay. In glial cells infected with AdCMV th, the TH expression was demonstrated by immunohistochemical staining and HPLC-ECD. 678.8 ng DA was detected in the extract of 1x10(6) AdCMV th infected glial cells, but no detectable DA was found in AdCMVLacZ-infected glial cells. Injection of AdCMV th (1x10(7) pfu/rat) into the striatum of PD rats significantly improved the apomorphine-induced rotation movement(approximately 60%). The improvement in rotation movement remained up to 5 months after injection, and TH expression positive cells were found in the vicinity of injection. These results indicate that adenovirus may be a useful carrier for in vivo gene therapy in the PD patients.

[Effect of integral syndrome differentiation acupuncture on the tear film stability in the patient of xerophthalmia].

OBJECTIVE: To probe into a treatment method for increasing clinical therapeutic effect on xerophthalmia. METHODS: Forty-five cases were randomly divided into acupuncture group 1, acupuncture group 2 and control group. The acupuncture group 1 were treated with acupuncture based on syndrome types, and the acupuncture group 2 with acupuncture at local acupoints, and the control group with dripping of artificial tears. Local symptoms of eyes, S1T, BUT and FL were observed before and after treatment. RESULTS: S1T and BUT increased after treatment in the two groups (P < 0.01, P < 0.05), but did not significantly change in the control group (P > 0.05). There were significant differences in change of FL as the control group compared with the acupuncture group 1 and 2. The total effective rate was 86.7%, 66.7% and 33.3% in the acupuncture group 1, 2 and the control group, respectively. CONCLUSION: The integral syndrome differentiation acupuncture can improve secretion of tear, increase stability of tear film, improve local symptoms of eyes with a therapeutic effect better than acupuncture at local acupoints and artificial tears.

[Establishment and application of fluorescent quantitative PCR method for detecting human herpes virus type 6].

OBJECTIVE: To establish fluorescent quantitative PCR method for detecting human herpes virus type 6 (HHV6). METHODS: According to the specific sequence of human herpes virus type 6 genes, the primers and the fluorescent probe (TaqMan) were designed and synthesized. The fragment generated from HHV 6 gene as template was cloned into the pMD18-T vector which was constructed from the pUC 18. And the positive recombinant plasmid was 1:10 diluted and used as standard quantitative template to make the standard curve for sample detection. RESULTS: The standard curve indicated the linear relationship between Ct (cycle threshold) and template concentration. The clinical samples from 135 cases were detected by this system, 16 cases among 135 were positive. CONCLUSION: The fluorescent quantitative PCR method for the detection of human herpes virus type 6 is simple and accurate, and this method may be helpful to clinical diagnosis.