Ovarian ferroptosis induced by androgen is involved in pathogenesis of PCOS.

STUDY QUESTION: Does ovarian ferroptosis play an active role in the development of polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: Increased ovarian ferroptosis was present in PCOS ovaries and the inhibition of ferroptosis with ferrostatin-1 (Fer-1) ameliorated polycystic ovary morphology and anovulation. WHAT IS KNOWN ALREADY: Programmed cell death plays a fundamental role in ovarian follicle development. However, the types and mechanisms of cell death involved in the ovary are yet to be elucidated. Ferroptosis is a recently discovered iron-dependent programmed cell death. Impaired iron metabolism and cell death have been observed in women with PCOS, the main cause of anovulatory infertility. Additionally, previous studies reported that an abnormal expression of noncoding RNA may promote ferroptosis in immortalized ovarian granulosa cell lines. However, little is known about whether ovarian ferroptosis is increased in PCOS, and there is insufficient direct evidence for a role of ferroptosis in PCOS, and the underlying mechanism. Moreover, the effect of the inhibition of ferroptosis with Fer-1 in PCOS remains unclear. STUDY DESIGN SIZE DURATION: Ferroptosis was evaluated in human granulosa cells (hGCs) from non-PCOS (n = 6-16) and PCOS (n = 7-18) patients. The experimental study was completed in vitro using primary hGCs from women undergoing IVF. Improvements in PCOS indicators following ferroptosis inhibition with Fer-1 were investigated in a dehydroepiandrosterone (DHEA)-induced PCOS rat model (n = 8 per group). PARTICIPANTS/MATERIALS SETTING METHODS: Ovarian ferroptosis was evaluated in the following ways: by detecting iron concentrations via ELISA and fluorescent probes; measuring malondialdehyde (MDA) concentrations via ELISA; assessing ferroptosis-related protein abundance with western blotting; observing mitochondrial morphology with transmission electron microscopy; and determining cell viability. Primary hGCs were collected from women undergoing IVF. They were treated with dihydrotestosterone (DHT) for 24 h. The effect of DHT on ferroptosis was examined in the presence or absence of small interfering RNA-mediated knockdown of the putative receptor coregulator for signaling molecules. The role of ovarian ferroptosis in PCOS progression was explored in vivo in rats. The DHEA-induced PCOS rat model was treated with the ferroptosis inhibitor, Fer-1, and the oocytes and metaphase II oocytes were counted after ovarian stimulation. Additionally, rats were treated with the ferroptosis inducer, RSL3, to further explore the effect of ferroptosis. The concentrations of testosterone, FSH, and LH were assessed. MAIN RESULTS AND THE ROLE OF CHANCE: Increased ferroptosis was detected in the ovaries of patients with PCOS and in rats with DHEA-induced PCOS. Increased concentrations of Fe2+ (P < 0.05) and MDA (P < 0.05), and upregulated nuclear receptor coactivator 4 protein levels, and downregulated ferritin heavy chain 1 (FTH1) and glutathione peroxidase 4 (GPX4) proteins were observed in the hGCs in patients with PCOS and ovaries of PCOS rats (P < 0.05 versus control). DHT was shown to induce ferroptosis via activation of NOCA4-dependent ferritinophagy. The inhibition of ferroptosis with Fer-1 in rats ameliorated a cluster of PCOS traits including impaired glucose tolerance, irregular estrous cycles, reproductive hormone dysfunction, hyperandrogenism, polycystic ovaries, anovulation, and oocyte quality (P < 0.05). Treating rats with RSL3 resulted in polycystic ovaries and hyperandrogenism (P < 0.05). LARGE-SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: Although ovarian-targeted ferroptosis inhibition may be a more targeted treatment for PCOS, the underlying mechanisms in the cycle between ferroptosis and hyperandrogenism require further exploration. Additionally, since PCOS shows high heterogeneity, it is important to investigate whether ferroptosis increases are present in all patients with PCOS. WIDER IMPLICATIONS OF THE FINDINGS: Androgen-induced ovarian ferroptosis appears to play a role in the pathogenesis of PCOS, which potentially makes it a promising treatment target in PCOS. STUDY FUNDING/COMPETING INTERESTS: This study was supported by the National Key R&D Program of China (2023YFC2705500, 2023YFC2705505, 2019YFA0802604), National Natural Science Foundation of China (No. 82130046, 82320108009, 82101708, 82101747, and 82001517), Shanghai leading talent program, Innovative research team of high-level local universities in Shanghai (No. SHSMU-ZLCX20210201, No. SSMU-ZLCX20180401), Shanghai Jiaotong University School of Medicine, Affiliated Renji Hospital Clinical Research Innovation Cultivation Fund Program (RJPY-DZX-003) and Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (No. 20161413), Shanghai's Top Priority Research Center Construction Project (2023ZZ02002), and Three-Year Action Plan for Strengthening the Construction of the Public Health System in Shanghai (GWVI-11.1-36). The authors report no competing interests.

Chronic stress induces meiotic arrest failure and ovarian reserve decline via the cAMP signaling pathway.

Chronic stress is suspected to be a causal factor of female subfertility; however, the underlying mechanisms remain unclear. Here, we found that chronic stress inhibited the cyclic adenosine 3',5'-monophosphate (cAMP) signaling pathway, leading to ovarian reserve decline in mice. A chronic stress model was constructed using restraint stress for 8 weeks. An elongated estrous cycle and a significant increase in the number of atretic follicles were observed in the stress group. We identified a significant increase in meiotic arrest failure (MAF) in oocytes in the stress group, characterized by condensed metaphase chromosomes, assembled spindles, or polar bodies in the oocytes. Whole-mount ovarian reserve estimation at the single-oocyte level using the CUBIC method (clear, unobstructed brain/body imaging cocktails and computational analysis) revealed a significant decrease in quiescent oocytes from 2,261/ovary in the control group to 1,373/ovary in the stress group. The number of growing oocytes also significantly decreased from 220/ovary in the control group to 150/ovary in the stress group. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of the meiotic arrest maintenance pathways revealed significant downregulation of Gpr3, Nppc, and Npr2 in the stress group. These results indicate that blocking cAMP production contributes to MAF and a decline in ovarian reserve. Overall, we present new insights into the mechanisms underlying chronic-stress-induced oocyte loss and potential targets for ovarian reserve preservation.

Novel mutations in PLCZ1 lead to early embryonic arrest as a male factor.

Early embryonic arrest is one of the causes of assist reproduction technology (ART) failure. We have previously reported that the first sperm-derived genetic factor, ACTL7a mutations, could lead to early embryonic arrest. However, whether there are other male genetic factors associated with early embryonic arrest remains elusive. Here, we reported bi-allelic mutations in PLCZ1, a well-known causal gene of total fertilization failure, in four infertile males. Among these mutations, p.403_404del, p.I489S, and p.W536X were newly reported in this study. Histological and Western blotting analysis of the patients' sperm indicated these variants as loss-of-function mutations. These patients manifested normal conventional semen parameters and ultra-structures in sperm heads. However, among four in vitro fertilization (IVF) cycles, 81.8% (18/22) of the oocytes were polyspermic fertilized, which was rarely reported in PLCZ1-related male patients. In the following six ICSI cycles, artificial oocyte activation (AOA) was applied and successfully rescued the fertilization failure and polyspermy phenotypes, with 31.3% (15/48) of the MII oocytes normally fertilized. However, 60.0% (9/15) of these normally fertilized zygotes were arrested at 2-5-cell stage, with one failing to cleave, indicating that PLCZ1 was not only necessary for fertilization, but also crucial for early embryonic development. However, these rescued zygotes showed a lower potential in developing into blastocysts when cultured in vitro. Thus, fresh cleavage transfer was tried and two live births were successfully achieved thereafter. In conclusion, this study provided novel mutations in PLCZ1 gene to expand the pathogenic mutational spectrum in male infertility and demonstrated that PLCZ1 was a crucial sperm-related genetic factor for early embryonic arrest. We also proposed that cleavage transfer after ICSI and AOA treatment could be a potential treatment method for male patients carrying bi-allelic mutations in PLCZ1.

Identification, characterization and expression analysis of rLcn13, an epididymal lipocalin in rats.

As the essential tissue for sperm maturation and storage, the epididymis secretes a number of tissue-specific proteins to exert its functions. Among these proteins, epididymal lipocalins have been intensively studied because of their epididymis-specific expression pattern and clustered genomic organization. In this study, rLcn13, a member of the rat epididymal lipocalin family, is identified and elaborately characterized. The cDNA sequence of rLcn13 consists of 719 nucleotides and encodes a 176 amino-acid protein with a predicted N-terminal signal peptide of 19 amino acids. rLcn13 shares a similar genomic structure and predicted 3D protein structure with other lipocalin family members. A recombinant rLCN13 mature peptide of 157 amino acids is expressed and purified, which is used to raise a polyclonal antibody against rLCN13 with high specificity and sensitivity. Northern blot, western blot, and immunohistochemistry assays reveal that rLcn13 is an epididymis-specific gene which is expressed predominantly in the initial segment and proximal caput epididymis and influenced by androgen. The rLCN13 protein is modified by N-glycosylation and secreted into the epididymal lumen, and then binds to the acrosome region of the sperm. Our data demonstrate that rLcn13 exhibits a specific temporospatial expression pattern and androgen dependence, indicating its potential roles in sperm maturation.

iTRAQ-based Proteomic Analysis Unveils ACSL4 as a Novel Potential Regulator of Human Endometrial Receptivity.

Competent endometrial receptivity is a prerequisite for successful embryo implantation. Identification of novel key molecules involved in endometrial receptivity is essential to better interpret human implantation and improve pregnancy rates in assisted reproduction treatment. Isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics was performed to profile the proteomes of the prereceptive (luteinizing hormone [LH] + 2, n = 4) and receptive (LH + 7, n = 4) endometrial tissues. A total of 173 differentially expressed proteins (DEPs) between LH + 2 and LH + 7 endometrial samples were identified. Integrated analysis of the proteomic data and published transcriptomic data was performed to identify the concordant DEPs with differential expression at both the messenger RNA and protein levels. Protein-protein interaction (PPI) network analysis was performed on concordant DEPs. We first identified 63 novel concordant DEPs and 5 hub proteins (ACSL4, ACSL5, COL1A1, PTGS1, and PLA2G4F) between LH + 2 and LH + 7 endometrial samples. ACSL4 was predominantly expressed in endometrial epithelial cells and its expression was significantly upregulated by progesterone in the LH + 7 endometrium and significantly downregulated in repeated implantation failure patients. Knockdown of ACSL4 in endometrial epithelial cells induced the downregulation of endometrial receptivity markers (HOXA10, COX2, and LIF) and the significant decrease of implantation rate during in vitro implantation analysis. This study provides the first gel-independent quantitative proteomes of the LH + 2 and LH + 7 human endometrium using iTRAQ technology. The identified concordant DEPs and hub proteins open a new avenue for future studies aimed at elucidating the underlying mechanisms governing endometrial receptivity. ACSL4 was identified as a novel regulatory molecule in the establishment of endometrial receptivity and might play important roles during implantation.

Comprehensive study of the occurrence and characteristics of organic matter, nitrogen, and phosphorus in sediments and riparian soils of a large drinking water reservoir.

The contamination of nutrients has caused considerable worry about the environment, resource value, and ecological worth of drinking water reservoirs. Therefore, we comprehensively studied the abundance, sources, distribution, and environmental behavior of carbon, nitrogen, and phosphorus in Fengshuba Reservoir (FSBR) (a large drinking reservoir, China). A graded leaching technique (introduced in 2003), the European Standard, Measurement and Testing (SMT) protocol, and spectrometry combined with parallel factor analysis (EEMs-PARAFAC) were used to assess nitrogen, phosphorus forms, and spectra data in the sediment and soil phases, respectively. The study demonstrates that seasonal hydrological variation had no significant effect on the nutrient abundance and nutrient structure composition of the FSBR, while different environmental media (e.g., sediment and soil phase) exhibited considerable differences in nutrient abundance, composition, and environmental behavior. The abundance of colored dissolved organic matter (CDOM), as well as molecular weight, aromatization degree, and humification degree of dissolved organic matter (DOM) were all lower in sediments than in soils, whereas the authigenic component was greater than in soils. Microbial-derived humus (C1), terrestrial-derived humus (C2), and protein-like tryptophan (C3) were identified as the three primary fluorescence components. Principal component analysis indicated that three components were closely associated with phosphorus in the sediment phase, whereas nitrogen and phosphorus in the soil phase were mainly related to C1 and C2. In summary, soil media (drawdown area) must be carefully considered in the management and control of water environment nutrients in reservoirs.

Increased expression of HOXA11-AS attenuates endometrial decidualization in recurrent implantation failure patients.

Endometrial decidualization is a prerequisite for implantation, and impaired decidualization is associated with recurrent implantation failure (RIF). Coding genes of the HOX family have been clarified as critical regulators in endometrial decidualization, but the role of long non-coding RNAs (lncRNAs) in the HOX gene family has yet to be determined. The aim of this study was to clarify the possible roles of lncRNAs in the HOX gene family in decidualization. In this study, we identified that HOXA11-AS was the most reduced lncRNA in the HOX family in the human endometrium during the window of implantation, and it was elevated in RIF patients. Mechanistically, HOXA11-AS negatively regulated decidualization through competitive interaction with PTBP1, an RNA-binding protein. Binding of PTBP1 to HOXA11-AS limited PTBP1 availability to regulate PKM1/2 alternative splicing, resulting in enhanced PKM1 and diminished PKM2 expression, thus attenuating decidualization. The pattern of high HOXA11-AS expression and impaired PKM2 splicing was consistently observed in RIF patients. Collectively, our study indicates that the increase of HOXA11-AS is detrimental to endometrial decidualization, likely contributing to RIF. Our study may shed light on the pathogenesis and treatment of RIF.

Addressing the role of 11ß-hydroxysteroid dehydrogenase type 1 in the development of polycystic ovary syndrome and the putative therapeutic effects of its selective inhibition in a preclinical model.

BACKGROUND: Polycystic ovary syndrome (PCOS) is the most common metabolic and endocrine disorder among reproductive-age women, and the leading cause of anovulatory infertility. 11ß-hydroxysteroid dehydrogenases-1 (11ß-HSD1) catalysing the conversion of inactive cortisone to active cortisol plays a crucial role in various metabolic diseases. However, whether 11ß-HSD1 is associated with the pathogenesis of PCOS and whether 11ß-HSD1 can be a treating target of PCOS remain unknown. METHODS: This study was first designed to explore the role of 11ß-HSD1 in PCOS development and the effect of selective 11ß-HSD1 inhibitor administration on PCOS treatment. Follicular fluid and granulosa cells (GCs) were collected from 32 non-PCOS patients and 37 patients with PCOS to measure cortisol and 11ß-HSDs levels. Female Sprague-Dawley rats (3-week-old) were injected with dehydroepiandrosterone (DHEA) to induce PCOS and their ovaries were collected to measure the abundance of corticosterone (CORT) and 11ß-HSDs. To determine the role of 11ß-HSD1 in PCOS development, we overexpressed 11ß-HSD1 in the ovaries of female rats (5-week-old) or knocked down the expression of 11ß-HSD1 in the ovaries from PCOS rats via lentivirus injection. After lentivirus infection, the body weights, ovarian weights, estrous cycles, reproductive hormones and morphology of the ovary were analysed in rats from different experimental groups. Then to figure out the translational potential of the selective 11ß-HSD1 inhibitor in treating PCOS, PCOS rats were treated with BVT.2733, a selective 11ß-HSD1 inhibitor and a cluster of PCOS-like traits were analysed, including insulin sensitivity, ovulatory function and fertility of rats from the Control, PCOS and PCOS+BVT groups. Rat ovarian explants and human GCs were used to explore the effect of CORT or cortisol on ovarian extracellular matrix remodelling. RESULTS: The elevated expression of 11ß-HSD1 contributed to the increased cortisol and corticosterone (CORT) concentrations observed in the ovaries of PCOS patients and PCOS rats respectively. Our results showed that ovarian overexpression of 11ß-HSD1 induced a cluster of PCOS phenotypes in rats including irregular estrous cycles, reproductive hormone dysfunction and polycystic ovaries. While knockdown of ovarian 11ß-HSD1 of PCOS rats reversed these PCOS-like changes. Additionally, the selective 11ß-HSD1 inhibitor BVT.2733 alleviated PCOS symptoms such as insulin resistance (IR), irregular estrous cycles, reproductive hormone dysfunction, polycystic ovaries, ovulatory dysfunction and subfertility. Moreover, we showed that cortisol target ovarian insulin signalling pathway and ovarian extracellular matrix (ECM) remodelling in vivo, in ovarian explants and in GCs. CONCLUSION: Elevated 11ß-HSD1 abundance in ovarian is involved in the pathogenesis of PCOS by impairing insulin signalling pathway and ECM remodelling. Selective inhibition of 11ß-HSD1 ameliorates a cluster of PCOS phenotypes. Our study demonstrates the selective 11ß-HSD1 inhibitor as a novel and promising strategy for the treatment of PCOS.

Identification of HOXA10 target genes in human endometrial stromal cells by RNA-seq analysis.

Homeobox A10 (HOXA10) is a transcription factor belonging to the homeobox gene family. It is highly expressed in endometrial stromal cells (ESCs) and plays essential roles in the proliferation and differentiation of endometrium, the establishment of endometrial receptivity and embryo implantation. However, little is known about the target genes and signaling pathways regulated by HOXA10 in ESCs. In this study, we identified 1830 transcripts regulated by HOXA10 in ESCs by RNA interference (RNAi) and RNA-sequencing (RNA-seq) analysis, of which 980 were positively regulated by HOXA10 and 850 were negatively regulated by HOXA10. Interestingly, matrix metallopeptidase-11 was downregulated by HOXA10 in stromal cells verified by quantitative real-time polymerase chain reaction and western blot analysis. Pathway analysis demonstrated that the target genes were enriched in various pathways, including cellular metabolism, DNA replication and repair, cell junction, and lysosome and signal transduction. The results of the present study provide novel insights into the mechanism underlying HOXA10 regulation in ESCs and may identify novel targets for the diagnosis and treatment of endometrium-related infertility.

A Novel Molecule in Human Cyclic Endometrium: LncRNA TUNAR Is Involved in Embryo Implantation.

Embryo implantation rate remains an inefficient process in in vitro fertilization and embryo transfer (IVF-ET) cycles. The role long non-coding RNA (lncRNA) plays in embryo implantation remains unclear. We aimed to investigate the expression pattern of lncRNA TCL1 upstream neural differentiation-associated RNA (TUNAR) in human cyclic endometrium and clarify the role of TUNAR in the development of endometrial receptivity. Endometrial biopsies were collected at the late proliferative phase, luteinizing hormone (LH) + 2 and LH + 7, from patients with or without recurrent implantation failure (RIF). Real-time RT PCR was performed to detect the level of lncRNAs. After pZW1-snoVector-TUNAR transfection, multiple function of TUNAR in endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs) was investigated. The expression of TUNAR in endometrium was found down-regulated at LH + 7 and up-regulated in RIF patients. In proliferative phase, TUNAR was overwhelmingly more abundant in ESCs and regulated its proliferation. In LH + 7, the difference in the expression of TUNAR between ESCs and EECs was narrowed. Overexpression of TUNAR not only impaired spheroid attachment to EECs, but also inhibited decidualization of ESCs. TUNAR was found expressed in human endometrium for the first time, which might be involved in embryo implantation by modulating the blastocyst attachment to the endometrial epithelium and regulating the proliferation and decidualization of ESCs. Our study helps us to better understand the molecular mechanisms of embryo implantation and may provide a promising biomarker of endometrial receptivity.

Systematic prediction of the biological functions of TAS2R10 using positive co-expression analysis.

Type 2 taste receptor 10 (TAS2R10), belonging to the TAS2R family of bitter receptors, is widely expressed in extra-oral tissues. However, its biological roles beyond bitterness sensing in the tongue have remained largely elusive. The present study aimed to perform a positive co-expression analysis using 60,000 Affymetrix expression arrays and 5,000 The Cancer Genome Atlas datasets to uncover such roles. Based on the functional enrichment analysis, it was indicated that in the Gene Ontology (GO) category biological process, TAS2R10 was mostly involved in 'cellular protein metabolic process', 'protein modification process', 'cellular protein modification process' and 'cellular component assembly'. In the GO category cellular component, the co-expressed genes were accumulated in 'Spt-Ada-Gcn5 acetyltransferase (SAGA)-type complex' and 'SAGA complex', and in the category molecular function, they were concentrated in 'hexosaminidase activity', 'cytoskeletal adaptor activity', 'cyclin binding' and 'ß-N-acetylhexosaminidase activity'. Of note, it was indicated that TAS2R10 may be involved in 'ubiquitin-mediated proteolysis', which may provide a starting point to fully investigate the detailed functions of TAS2R10 in the future. TAS2R10 was also indicated to be associated with human diseases, i.e. 'Salmonella infection'. Overall, the present study was the first to perform a comprehensive bioinformatics analysis of the functions of TAS2R10 and provide insight regarding the notion that this gene may have crucial roles beyond bitterness sensing.

Specific detection of hypochlorite based on the size-selective effect of luminophore integrated MOF-801 synthesized by a one-pot strategy.

Hypochlorous acid (HClO), as one of the reactive oxygen species, plays a key role in a variety of physiological and pathological processes, while its accurate and specific in vitro monitoring remains a profound challenge. Herein, a novel luminescent metal-organic framework with high chemical stability has been designed for the specific detection of intracellular ClO-. The specificity was realized by the size-selective effect of MOF-801 with an ultra-small aperture, which can inhibit the entry of large-sized interferents into the cages of MOFs. A universal "ship in a bottle" approach has been proposed to construct this novel sensory platform, in which a large class of luminescent molecules containing carboxylic groups serve as modulators and combine with Zr6 clusters, eventually becoming the luminescent genes of these novel designed MOF-801. Luminescent molecules were readily locked in the framework since they were larger than the small pore entrance of MOF-801, skillfully solving the possible issue of dye leakage. By introducing active sites of 5-aminofluorescein (AF) into MOF-801 (AF@MOF-801) as an example, an excellent ClO- sensing probe was fabricated, which showed strong reliability and excellent sensing performance toward intracellular ClO- with an ultrahigh linear correlation of the Stern-Volmer equation, a rapid response time as short as 30 s and a limit of detection (LOD) as low as 0.05172 µM. Compared with the free AF molecular probe, the specificity of AF@MOF-801 NPs toward ClO- was scarcely affected by other possibly coexistent large-sized interferents in biosystems. The in vitro monitoring of ClO- was also tested with these newly developed AF@MOF-801 NPs, prefiguring their great promise as a robust imaging tool to disclose the complexities of ClO- homeostasis and its pathophysiological contributions.

The expression of the new epididymal luminal protein of PDZ domain containing 1 is decreased in asthenozoospermia.

Spermatozoa are not mature until they transit the epididymis where they acquire motility and the ability to fertilize an egg through sequential modifications. The epididymis has three functional regions, caput, corpus, and cauda, and the luminal proteins of the epididymis play important roles in the above modifications. However, the proteins with differential enrichment between the caput and cauda are still largely unknown. To reveal the functions of the caput and cauda during sperm maturation, luminal proteins from caput and cauda of mice were analyzed by isobaric tag for relative and absolute quantitation (iTRAQ). Overall, 128 differentially enriched proteins were found, of which 46 were caput enriched and 82 were cauda enriched. Bioinformatic analysis showed that lipid metabolism was active in the caput; while anion- and cation-binding activity and phosphorus and organophosphate metabolism were active in the cauda. A new epididymal luminal protein, the caput-enriched PDZ domain containing 1 (Pdzk1), also named Na+/H+ exchange regulatory cofactor 3 (NHERF3), which plays a critical role in cholesterol metabolism and carnitine transport, was found in the lipid metabolism. Western blotting and immunofluorescence analyses showed that Pdzk1 was expressed in the epididymis but not in the testis, and localized at the middle piece of the sperm tail. Pdzk1 protein level was also reduced in the spermatozoa in case of asthenozoospermic patients compared with that in normozoospermic men, suggesting that Pdzk1 may participate in sperm maturation regulation and may be associated with male infertility. These results may provide new insights into the mechanisms of sperm maturation and male infertility.

Genome-Wide Mapping of In Vivo ERα-Binding Sites in Male Mouse Efferent Ductules.

As an important nuclear hormone receptor, estrogen receptor α (ERα), which is encoded by the Esr1 gene, regulates the expression of hundreds of genes in a stimulus-specific, temporal, and tissue-specific fashion, mainly by binding to specific DNA sequences called estrogen response elements (EREs). As an important estrogen target tissue in males, the function of the efferent ductules relies on the presence of the ERα protein, but the underlying regulatory mechanisms are poorly illustrated. In this study, genome-wide ERα-binding sites in mouse efferent ductules were mapped by chromatin immunoprecipitation sequencing. In total, 12,105 peaks were identified, and a majority of them were located far from the annotated gene transcription start site. Motif analysis revealed that ∼80% of the ERα-binding peaks harbored at least one ERE, whereas androgen response element-like sequences were the most overrepresented motif in the peaks without any EREs. A number of candidate transcription factor motifs adjacent to the EREs were significantly enriched, including AP2 and GRE, implying the involvement of these putative adjacent factors in the global regulation of ERα target genes. Unexpectedly, more than 50% of the ERα-binding peaks in mouse efferent ductules overlapped with those binding peaks previously identified in mouse uterus, suggesting the conserved mechanism of ERα action in these two tissues. Cobinding of ERα target genes by androgen receptor was further confirmed for Slc9a3 gene, which was responsible for fluid resorption in the efferent ductules. Taken together, our study provides a useful reference set for future work aimed at exploring the mechanism of ERα action in physiological conditions.

Emerging roles for PIWI proteins in cancer.

It is generally accepted that PIWI proteins are predominately expressed in the germline but absent in somatic tissues. Their best-characterized role is to suppress transposon expression, which ensures genomic stability in the germline. However, increasing evidence has suggested that PIWI proteins are linked to the hallmarks of cancer defined by Weinberg and Hanahan, such as cell proliferation, anti-apoptosis, genomic instability, invasion and metastasis. This provides new possibilities for anticancer therapies through the targeting of PIWI proteins, which may have fewer side effects due to their potential classification as a CTA (cancer/testis antigen). Furthermore, PIWI has been proposed to act as a diagnostic and prognostic marker for many types of cancer, and even to differentiate early- and late-stage cancers. We herein summarize the latest progress in this exciting field, hoping to encourage new investigations of PIWIs in cancer biology that will help to develop new therapeutics for clinical application.

Epididymal Region-Specific miRNA Expression and DNA Methylation and Their Roles in Controlling Gene Expression in Rats.

Region-specific gene expression is an intriguing feature of the mammalian epididymis. This unique property is essential for sperm maturation and storage, and it also implicates stringent and multi-level regulations of gene expression. Over the past decade, the androgen-driven activation of epididymal gene transcription has been extensively studied. However, it still remains largely unexplored whether and how other regulatory mechanisms, such as miRNAs and DNA methylation, are involved in controlling regional gene expression in the epididymis. Using microarray-based approaches, we studied the regional miRNA expression and DNA methylation profiles in 4 distinct epididymal regions (initial segment, caput, corpus and cauda) of rats. We found that the miR-200 family members were more expressed in caput, compared with cauda. By GSEA analysis, the differential expression of miR-200 family between caput and cauda was shown to be negatively correlated with their predicted target genes, among which 4 bona fide targets were verified by luciferase reporter assay. Predicted target genes of miR-200 family have enriched functions in anti-apoptosis, cell transportation and development, implying the regional diversity in epididymal functions. On the other hand, we revealed epididymal DNA methylation of 2002 CpG islands and 2771 gene promoters (-3.88-0.97 kb), among which 1350 (67.43%) CpG islands and 2095 (75.60%) promoters contained region-specific DNA methylation. We observed significant and distinct functional enrichment in genes with specifically methylated promoters in each epididymal regions, but these DNA methylations did not show significant correlation with repressed gene transcription in the mature epididymis. Conclusively, we investigated the regional miRNA expression and DNA methylation in the rat epididymis and revealed a potential role of miR-200 family in gene expression regulation between caput and cauda. This may contribute to the distinct physiological function in sperm maturation / storage of caput / cauda epididymis.