Ruizhi Feng

Identification of MicroRNAs in Human Follicular Fluid: Characterization of MicroRNAs That Govern Steroidogenesis in Vitro and Are Associated With Polycystic Ovary Syndrome in Vivo

CONTEXT Human follicular fluid is a combination of proteins, metabolites, and ionic compounds that is indicative of the general state of follicular metabolism and is associated with maturation and quality of oocytes. Deviations in these components are often associated with reproductive diseases. There has been no report of microRNAs (miRNAs) in human follicular fluids. OBJECTIVE We hypothesized that human follicular fluid may contain miRNAs. We sought to identify cell-free miRNAs in human follicular fluid and to investigate the function of these miRNAs in vitro and any roles they play in polycystic ovary syndrome (PCOS). DESIGN Genome-wide deep sequencing and TaqMan miRNA arrays were used to identify miRNAs, and the roles of the highly expressed miRNAs in steroidogenesis were investigated in KGN cells. Quantification of candidate miRNAs in follicular fluids of PCOS and controls was performed using TaqMan miRNA assays. RESULTS We identified miRNAs in microvesicles and the supernatant of human follicular fluid. Bioinformatics analysis showed that the most highly expressed miRNAs targeted genes associated with reproductive, endocrine, and metabolic processes. We found that miR-132, miR-320, miR-520c-3p, miR-24, and miR-222 regulate estradiol concentrations and that miR-24, miR-193b, and miR-483-5p regulate progesterone concentrations. Finally, we showed that miR-132 and miR-320 are expressed at significantly lower levels in the follicular fluid of polycystic ovary patients than in healthy controls (P = .005 and P = .0098, respectively). CONCLUSION These results demonstrate that there are numerous miRNAs in human follicular fluids, some of which play important roles in steroidogenesis and PCOS. This study substantially revises our understanding of the content of human follicular fluid and lays the foundation for the future investigation of the role of miRNAs in PCOS.

Mutations in TUBB8 and Human Oocyte Meiotic Arrest

Background Human reproduction depends on the fusion of a mature oocyte with a sperm cell to form a fertilized egg. The genetic events that lead to the arrest of human oocyte maturation are unknown. Methods We sequenced the exomes of five members of a four-generation family, three of whom had infertility due to oocyte meiosis I arrest. We performed Sanger sequencing of a candidate gene, TUBB8, in DNA samples from these members, additional family members, and members of 23 other affected families. The expression of TUBB8 and all other β-tubulin isotypes was assessed in human oocytes, early embryos, sperm cells, and several somatic tissues by means of a quantitative reverse-transcriptase-polymerase-chain-reaction assay. We evaluated the effect of the TUBB8 mutations on the assembly of the heterodimer consisting of one α-tubulin polypeptide and one β-tubulin polypeptide (α/β-tubulin heterodimer) in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes. Results We identified seven mutations in the primate-specific gene TUBB8 that were responsible for oocyte meiosis I arrest in 7 of the 24 families. TUBB8 expression is unique to oocytes and the early embryo, in which this gene accounts for almost all the expressed β-tubulin. The mutations affect chaperone-dependent folding and assembly of the α/β-tubulin heterodimer, disrupt microtubule behavior on expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle-assembly defects and maturation arrest on expression in mouse and human oocytes. Conclusions TUBB8 mutations have dominant-negative effects that disrupt microtubule behavior and oocyte meiotic spindle assembly and maturation, causing female infertility. (Funded by the National Basic Research Program of China and others.).

MiRNA-320 in the human follicular fluid is associated with embryo quality in vivo and affects mouse embryonic development in vitro

Previous work from our laboratory demonstrated the existence of miRNAs in human follicular fluid. In the current study, we have sought to identify miRNAs that might affect oocyte/embryo quality in patients undergoing intracytoplasmic sperm injection and to investigate their roles in in vitro fertilization outcomes in mouse oocytes. 53 samples were classified as Group 1 (high quality) if the day-3 embryos had seven and more cells or as Group 2 (low quality) if the embryos had six and fewer cells. TaqMan Human microRNAs cards and qRT-PCR were performed to verify differently expressed miRNAs. The function of the corresponding miRNA was investigated in mouse oocytes by injecting them with miRNA-inhibitor oligonucleotides. We found that hsa-miR-320a and hsa-miR-197 had significantly higher expression levels in the Group 1 follicular fluids than in Group 2 (p = 0.0073 and p = 0.008, respectively). Knockdown of mmu-miR-320 in mouse oocytes strongly decreased the proportions of MII oocytes that developed into two-cell and blastocyst stage embryos (p = 0.0048 and p = 0.0069, respectively). Wnt signaling pathway components had abnormal expression level in miR-320 inhibitor-injected oocytes. This study provides the first evidence that miRNAs in human follicular fluid are indicative of and can influence embryo quality.

A Possible New Mechanism in the Pathophysiology of Polycystic Ovary Syndrome (PCOS): The Discovery That Leukocyte Telomere Length Is Strongly Associated With PCOS

CONTEXT Telomeres are specialized chromatin structures located at the ends of eukaryotic chromosomes, and telomere length plays a clear role in various diseases. However, it is not known whether telomere length is related to polycystic ovary syndrome (PCOS). OBJECTIVE We hypothesized that leukocyte telomere length (LTL) plays an important role in the pathophysiology of PCOS. DESIGN We used an established and validated quantitative PCR technique to measure the mean LTL in a large sample of PCOS patients and controls. We used logistic regression and multiple linear regression to analyze the association of PCOS and several related clinical parameters with the age-adjusted ratio of the telomere repeat length to the copy number of a single-copy gene (T/S). RESULTS Individuals with PCOS (n = 698) exhibited significantly shorter LTLs than the controls (n = 611) after adjusting for age (0.764 ± 0.016 vs 0.876 ± 0.023; P = .001; odds ratio = 1.403; 95% confidence interval, 1.150-1.712). The mean telomere length in the leukocytes of PCOS patients was comparable to that of control individuals who were on average 6.16 years older. Individuals having shorter telomere lengths (middle and lowest tertile) had significantly higher disease risk than those having the longest telomere length (highest tertile) (odds ratio = 1.614; 95% confidence interval, 1.262-2.066; P = .0001) after adjusting for age. In addition, a significant correlation between the LTL and the level of dehydroepiandrosterone sulfate was observed in controls (r = -0.185; P = .01). CONCLUSION We provide the first report that LTL is strongly associated with PCOS. This study suggests a new role for LTL in the pathophysiology of PCOS and might have important implications for our understanding of the etiology of the disease.

Mutations in TUBB8 cause a multiplicity of phenotypes in human oocytes and early embryos

Background TUBB8 is a primate-specific β-tubulin isotype whose expression is confined to oocytes and the early embryo. We previously found that mutations in TUBB8 caused oocyte maturation arrest. The objective was to describe newly discovered mutations in TUBB8 and to characterise the accompanying spectrum of phenotypes and modes of inheritance. Methods and results Patients with oocyte maturation arrest were sequenced with respect to TUBB8. We investigated the effects of identified mutations in vitro, in cultured cells and in mouse oocytes. Seven heterozygous missense and two homozygous mutations were identified. These mutations cause a range of folding defects in vitro, different degrees of microtubule disruption upon expression in cultured cells and interfere to varying extents in the proper assembly of the meiotic spindle in mouse oocytes. Several of the newly discovered TUBB8 mutations result in phenotypic variability. For example, oocytes harbouring any of three missense mutations (I210V, T238M and N348S) could extrude the first polar body. Moreover, they could be fertilised, although the ensuing embryos became developmentally arrested. Surprisingly, oocytes from patients harbouring homozygous TUBB8 mutations that in either case preclude the expression of a functional TUBB8 polypeptide nonetheless contained identifiable spindles. Conclusions Our data substantially expand the range of dysfunctional oocyte phenotypes incurred by mutation in TUBB8, underscore the independent nature of human oocyte meiosis and differentiation, extend the class of genetic diseases known as the tubulinopathies and provide new criteria for the qualitative evaluation of meiosis II (MII) oocytes for in vitro fertilization (IVF).

Quantitative Methylation Level of the EPHX1 Promoter in Peripheral Blood DNA Is Associated with Polycystic Ovary Syndrome

Steroid synthesis and metabolic pathways play important roles in the pathophysiology of PCOS, but until now there have been no studies on the methylation profiles of specific genes in steroid synthesis pathways that are known to be associated with PCOS. Here we used MassARRAY quantitative methylation analysis to determine the methylation levels of each CpG site or cluster in the promoters of EPHX1, SRD5A1, and CYP11A1 in 64 peripheral blood samples. We further examined the methylation level of EPHX1 in an independent cohort consisting of 116 people. Finally, we investigated the role of EPHX1 in steroidogenesis in the KGN cell line. For SRD5A1 and CYP11A1, there was no significant difference in methylation level between patients and controls. For EPHX1, however, the methylation levels of a few consecutive CpG sites and clusters were found to be significantly associated with PCOS. The methylation levels of a number of CpG clusters or sites were significantly lower in patients than in controls in the first cohort consisting of 64 people, such as clusters 13–14 (P<0.05), 15–16 (P<0.001), and 19–24 (P<0.001) and sites CpG_53 (P<0.01) and CpG_54 (P<0.05). Among differentiated methylation sites and clusters, the methylation levels of the CpG cluster 13–14 and CpG cluster 19–24 in PCOS patients were significantly lower than in controls in the second cohort of 116 people (P<0.05 for both). In addition, knockdown and overexpression experiments in KGN cells showed that EPHX1 can regulate estradiol concentrations, and this indicates a role for EPHX1 in steroidogenesis. Our study has demonstrated that methylation of the EPHX1 promoter might be associated with PCOS. This study provides direct evidence that methylation plays an important role in PCOS and demonstrates a novel role for EPHX1 in female reproduction.

Identification and Functional Study of a New Missense Mutation in the Motor Head Domain of Myosin VIIA in a Family with Autosomal Dominant Hearing Impairment (DFNA11)

The MYO7A encodes a protein classified as an unconventional myosin. Here, we present a family with non-syndromic autosomal dominant hearing impairment that clinically resembles other previously published DFNA11 families. Affected members of the family present with an ascending audiogram affecting low and middle frequencies at young ages and then affecting all frequencies with increasing age. Genome-wide linkage analysis using Illumina Cyto-12 Chip mapped the disease locus to the DFNA11 interval in the family. A c.2003G→A (p.R668H) mutation of the MYO7A, is heterozygous in all affected family members and absent in 100 healthy individuals. Arg668His is located in a region of the myosin VIIA motor domain that is highly conserved among different species. Molecular modeling predicts that the conserved R668 residue plays important structural role in linking different lobes of motor domain together. In the actin-activated ATPase activity assay, the rate of NADH oxidation was higher in the wild-type myosin VIIA, indicating that the ATPase activity in the p.R668H mutant myosin VIIA was significantly destroyed.

Ildr1b is essential for semicircular canal development, migration of the posterior lateral line primordium, and hearing ability in zebrafish: Implications for a role in the recessive hearing impairment DFNB42

Immunoglobulin-like domain containing receptor 1 (ILDR1) is a poorly characterized gene that was first identified in lymphoma cells. Recently, ILDR1 has been found to be responsible for autosomal recessive hearing impairment DFNB42. Patients with ILDR1 mutations cause bilateral non-progressive moderate-to-profound sensorineural hearing impairment. However, the etiology and mechanism of ILDR1-related hearing loss remains to be elucidated. In order to uncover the pathology of DFNB42 deafness, we used the morpholino injection technique to establish an ildr1b-morphant zebrafish model. Ildr1b-morphant zebrafish displayed defective hearing and imbalanced swimming, and developmental delays were seen in the semicircular canals of the inner ear. The gene expression profile and real-time PCR revealed down-regulation of atp1b2b (encoding Na(+)/K(+) transporting, beta 2b polypeptide) in ildr1b-morphant zebrafish. We found that injection of atp1b2b mRNA into ildr1b-knockdown zebrafish could rescue the phenotype of developmental delay of the semicircular canals. Moreover, ildr1b-morphant zebrafish had reduced numbers of lateral line neuromasts due to the disruption of lateral line primordium migration. In situ hybridization showed the involvement of attenuated FGF signaling and the chemokine receptor 4b (cxcr4b) and chemokine receptor 7b (cxcr7b) in posterior lateral line primordium of ildr1b-morphant zebrafish. We concluded that Ildr1b is crucial for the development of the inner ear and the lateral line system. This study provides the first evidence for the mechanism of Ildr1b on hearing in vivo and sheds light on the pathology of DFNB42.

ILDR1 deficiency causes degeneration of cochlear outer hair cells and disrupts the structure of the organ of Corti: a mouse model for human DFNB42.

Immunoglobulin-like domain containing receptor 1 (ILDR1) is a poorly characterized gene that was first identified in lymphoma cells. Mutations in ILDR1 are responsible for DFNB42, but the pathogenesis of hearing loss caused by ILDR1 mutations remains to be elucidated. To explore the role of ILDR1 in hearing, we created Ildr1 knockout mice. In heterozygous mice, ILDR1 expression was found in outer hair cells (OHCs) and inner hair cells (IHCs) of the organ of Corti. ILDR1-deficient mice are profoundly deaf by postnatal day 21 (P21). No significant difference was observed in the supporting cells and IHCs of ILDR1-deficient mice, but progressive degeneration of OHCs occurred at P15 and disruption of the tunnel running through the organ of Corti was noticeable at P21. By P28, there were no OHCs visible in any of the turns of the organ of Corti, and the tunnel of the organ of Corti was entirely destroyed. ILDR1 deficiency affects expression of tricellulin in vivo, and this provides a possible explanation to hearing loss. To further elucidate the mechanism of deafness related to ILDR1 deficiency, we pursued a differential proteomic approach to comprehensively assess differential protein expression in the cochleae of Ildr1+/− and Ildr1−/− mice at P21. Altogether, 708 proteins were up-regulated (fold change >1.5) and 114 proteins were down-regulated (fold change <0.5) in the Ildr1−/− mice compared with Ildr1+/− mice. Gene ontology classification indicated that a number of differentially expressed proteins are involved in cell adhesion, protein and vesicle-mediated transport, cell death, membrane organization, and cellular homeostasis. A few of these proteins are closely related to hearing development. Taken together, our data suggest that ILDR1 is important for the survival of OHCs and provide novel insights into the pathogenesis of human deafness DFNB42 deafness.

Identification of a novel homozygous mutation in MYO3A in a Chinese family with DFNB30 non-syndromic hearing impairment

INTRODUCTION Hearing loss is a common sensory impairment. Several genetic loci or genes responsible for non-syndrome hearing loss have been identified, including the well-known deafness genes GJB2, MT-RNR1 and SLC26A4. MYO3A belongs to the myosin superfamily. Previously only three mutations in this gene have been found in an Isreali family with DFNB30, in which patients demonstrated progressive hearing loss. METHODS In this study, we characterized a consanguineous Kazakh family with congenital hearing loss. By targeted sequence capture and next-generation sequencing, we identified a homozygous mutation and did bioinformatics analysis to this mutation. RESULTS A homozygous mutation, MYO3A:c.1841C>T (p.S614F), was identified to be responsible for the disease. Ser614 is located in the motor domain of MYO3A that is highly conserved among different species. Molecular modeling predicts that the conserved Ser614 may play an important role in maintaining the stability of β-sheet and the interaction between neighboring β-strand. CONCLUSIONS This is the second report on MYO3A mutations in deafness and the first report in China. The finding help facilitate establishing a better relationship between MYO3A mutation and hearing phenotypes.