Search

STUDY QUESTION What are some pathogenic mutations for non-obstructive azoospermia (NOA) and their effects on spermatogenesis? SUMMARY ANSWER Biallelic missense and frameshift mutations in ADAD2 disrupt the differentiation of round spermatids to spermatozoa causing azoospermia in humans and mice. WHAT IS KNOWN ALREADY NOA is the most severe cause of male infertility characterized by an absence of sperm in the ejaculate due to impairment of spermatogenesis. In mice, the lack of the RNA-binding protein ADAD2 leads to a complete absence of sperm in epididymides due to failure of spemiogenesis, but the spermatogenic effects of ADAD2 mutations in human NOA-associated infertility require functional verification. STUDY DESIGN, SIZE, DURATION Six infertile male patients from three unrelated families were diagnosed with NOA at local hospitals in Pakistan based on infertility history, sex hormone levels, two semen analyses and scrotal ultrasound. Testicular biopsies were performed in two of the six patients. Adad2 mutant mice (Adad2Mut/Mut) carrying mutations similar to those found in NOA patients were generated using the CRISPR/Cas9 genome editing tool. Reproductive phenotypes of Adad2Mut/Mut mice were verified at 2 months of age. Round spermatids from the littermates of wild-type (WT) and Adad2Mut/Mut mice were randomly selected and injected into stimulated WT oocytes. This round spermatid injection (ROSI) procedure was conducted with three biological replicates and >400 ROSI-derived zygotes were evaluated. The fertility of the ROSI-derived progeny was evaluated for three months in four Adad2WT/Mut male mice and six Adad2WT/Mut female mice. A total of 120 Adad2Mut/Mut , Adad2WT/Mut , and WT mice were used in this study. The entire study was conducted over 3 years. PARTICIPANTS/MATERIALS, SETTING, METHODS Whole-exome sequencing was performed to detect potentially pathogenic mutations in the six NOA-affected patients. The pathogenicity of the identified ADAD2 mutations was assessed and validated in human testicular tissues and in mouse models recapitulating the mutations in the NOA patients using quantitative PCR, western blotting, hematoxylin-eosin staining, Periodic acid-Schiff staining, and immunofluorescence. Round spermatids of WT and Adad2Mut/Mut mice were collected by fluorescence-activated cell sorting and injected into stimulated WT oocytes. The development of ROSI-derived offspring was evaluated in the embryonic and postnatal stages. MAIN RESULTS AND THE ROLE OF CHANCE Three recessive mutations were identified in ADAD2 (MT1: c.G829T, p.G277C; MT2: c.G1192A, p.D398N; MT3: c.917_918del, p.Q306Rfs*43) in patients from three unrelated Pakistani families. MT1 and MT2 dramatically reduced the testicular expression of ADAD2, likely causing spermiogenesis failure in the NOA patients. Immunofluorescence analysis of the Adad2Mut/Mut male mice with the corresponding MT3 mutation showed instability and premature degradation of the ADAD2 protein, resulting in the spermiogenesis deficiency phenotype. Through ROSI, the Adad2Mut/Mut mice could produce pups with comparable embryonic development (46.7% in Adad2Mut/Mut versus 50% in WT) and birth rates (21.45 ± 10.43% in Adad2Mut/Mut versus 27.5 ± 3.536% in WT, P  = 0.5044) to WT mice. The Adad2WT/Mut progeny from ROSI (17 pups in total via three ROSI replicates) did not show overt developmental defects and had normal fertility. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION This is a preliminary report suggesting that ROSI can be an effective treatment for infertile Adad2Mut/Mut mice. Further assisted reproductive attempts need to be carefully examined in humans during clinical trials. WIDER IMPLICATIONS OF THE FINDINGS Our work provides functional evidence that mutations in the ADAD2 gene are deleterious and cause consistent spermiogenic defects in both humans and mice. In addition, preliminary results show that ROSI can help Adad2Mut/Mut to produce biological progeny. These findings provide valuable clues for genetic counselling on the ADAD2 mutants-associated infertility in human males. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the National Natural Science Foundation of China (32000587, U21A20204, and 32061143006), and the National Key Research and Developmental Program of China (2019YFA0802600 and 2021YFC2700202). This work was also supported by Institute of Health and Medicine, Hefei Comprehensive National Science Center, Hefei, China. The authors declare no competing interests. [ABSTRACT FROM AUTHOR]

Plant mineral nutrition is critical for agricultural productivity and for human nutrition; however, the availability of mineral elements is spatially and temporally heterogeneous in many ecosystems and agricultural landscapes. Nutrient imbalances trigger intricate signalling networks that modulate plant acclimation responses. One signalling agent of particular importance in such networks is phytomelatonin, a pleiotropic molecule with multiple functions. Evidence indicates that deficiencies or excesses of nutrients generally increase phytomelatonin levels in certain tissues, and it is increasingly thought to participate in the regulation of plant mineral nutrition. Alterations in endogenous phytomelatonin levels can protect plants from oxidative stress, influence root architecture, and influence nutrient uptake and efficiency of use through transcriptional and post-transcriptional regulation; such changes optimize mineral nutrient acquisition and ion homeostasis inside plant cells and thereby help to promote growth. This review summarizes current knowledge on the regulation of plant mineral nutrition by melatonin and highlights how endogenous phytomelatonin alters plant responses to specific mineral elements. In addition, we comprehensively discuss how melatonin influences uptake and transport under conditions of nutrient shortage. [ABSTRACT FROM AUTHOR]

Study Question: Can whole-exome sequencing (WES) reveal pathogenic mutations in two consanguineous Pakistani families with infertile patients?Summary Answer: A homozygous spermatogenesis associated 22 (SPATA22) frameshift mutation (c.203del), which disrupts the interaction with meiosis specific with OB-fold (MEIOB), and a MEIOB splicing mutation (c.683-1G>A) that led to loss of MEIOB protein cause familial infertility.What Is Known Already: MEIOB and SPATA22, direct binding partners and functional collaborators, form a meiosis-specific heterodimer that regulates meiotic recombination. The protein stability and the axial localization of MEIOB and SPATA22 depend on each other. Meiob and Spata22 knockout mice have the same phenotypes: mutant spermatocytes can initiate meiotic recombination but are unable to complete DSB repair, leading to crossover formation failure, meiotic prophase arrest, and sterility.Study Design, Size, Duration: We performed WES for the patients and controls in two consanguineous Pakistani families to screen for mutations. The pathogenicity of the identified mutations was assessed by in vitro assay and mutant mouse model.Participants/materials, Setting, Methods: Two consanguineous Pakistani families with four patients (three men and one woman) suffering from primary infertility were recruited. SPATA22 and MEIOB mutations were screened from the WES data, followed by functional verification in cultured cells and mice.Main Results and the Role Of Chance: A homozygous SPATA22 frameshift mutation (c.203del) was identified in a patient with non-obstructive azoospermia (NOA) from a consanguineous Pakistani family and a homozygous MEIOB splicing mutation (c.683-1G>A) was identified in two patients with NOA and one infertile woman from another consanguineous Pakistani family. The SPATA22 mutation destroyed the interaction with MEIOB. The MEIOB splicing mutation induced Exon 9 skipping, which causes a 32aa deletion in the oligonucleotide-binding domain without affecting the interaction between MEIOB and SPATA22. Furthermore, analyses of the Meiob mutant mice modelling the patients' mutation revealed that the MEIOB splicing mutation leads to loss of MEIOB proteins, abolished SPATA22 recruitment on chromosome axes, and meiotic arrest due to meiotic recombination failure. Thus, our study suggests that SPATA22 and MEIOB may both be causative genes for human infertility.Limitations, Reasons For Caution: As SPATA22 and MEIOB are interdependent and essential for meiotic recombination, screening for mutations of SPATA22 and MEIOB in both infertile men and women in larger cohorts is important to further reveal the role of the SPATA22 and MEIOB heterodimer in human fertility.Wider Implications Of the Findings: These findings provide direct clinical and functional evidence that mutations in SPATA22 and MEIOB can cause meiotic recombination failure, supporting a role for these mutations in human infertility and their potential use as targets for genetic diagnosis of human infertility.Study Funding/competing Interest(s): This work was supported by the National Key Research and Developmental Program of China (2018YFC1003900, 2018YFC1003700, and 2019YFA0802600), the National Natural Science Foundation of China (31890780, 31630050, 32061143006, 82071709, and 31871514), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB19000000). The authors declare no conflicts of interest.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Study Question: Are mutations in the zinc finger SWIM domain-containing protein 7 gene (ZSWIM7) associated with human male infertility?Summary Answer: The homozygous frameshift mutation (c.231_232del) in ZSWIM7 causes decreased meiotic recombination, spermatogenesis arrest, and infertility in men.What Is Known Already: ZSWIM7 is a SWIM domain-containing Shu2/SWS1 protein family member and a subunit of the Shu complex. Zswim7 knockout mice were infertile due to impaired meiotic recombination. However, so far there is no direct evidence that mutations of ZSWIM7 cause human infertility.Study Design, Size, Duration: Screening for mutations of ZSWIM7 was performed using in-house whole-exome sequencing data from 60 men with non-obstructive azoospermia (NOA). Mice with a corresponding Zswim7 mutation were generated for functional verification.Participants/materials, Setting, Methods: Sixty Chinese patients, who were from different regions of China, were enrolled. All the patients were diagnosed with NOA owing to spermatocyte maturation arrest based on histopathological analyses and/or immunostaining of spermatocyte chromosome spreads. ZSWIM7 mutations were screened from the whole-exome sequencing data of these patients, followed by functional verification in mice.Main Results and the Role Of Chance: A homozygous frameshift mutation (c.231_232del) in ZSWIM7 was found in two out of the 60 unrelated NOA patients. Both patients displayed small testicular size and spermatocyte maturation arrest in testis histology. Spermatocyte chromosome spreads of one patient revealed meiotic maturation arrest in a pachytene-like stage, with incomplete synapsis and decreased meiotic recombination. Male mice carrying a homozygous mutation similar to that of our patients were generated and also displayed reduced recombination, meiotic arrest and azoospermia, paralleling the spermatogenesis defects in our patients.Limitations, Reasons For Caution: As Zswim7 is also essential for meiosis in female mice, future studies should evaluate the ZSWIM7 mutations more in depth and in larger cohorts of infertile patients, including males and females, to validate the findings.Wider Implications Of the Findings: These findings provide direct clinical and functional evidence that the recurrent ZSWIM7 mutation (c.231_232del) causes decreased meiotic recombination and leads to male infertility, illustrating the genotype-phenotype correlations of meiotic recombination defects in humans.Study Funding/competing Interest(s): This work was supported by the National Natural Science Foundation of China (31890780, 31630050, 32061143006, 82071709, and 31871514), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB19000000), and the National Key Research and Developmental Program of China (2018YFC1003900 and 2019YFA0802600).Trial Registration Number: Not applicable. [ABSTRACT FROM AUTHOR]

With the advances of next-generation sequencing technology, the field of disease research has been revolutionized. However, pinpointing the disease-causing variants from millions of revealed variants is still a tough task. Here, we have reviewed the existing linkage analysis tools and presented PedMiner, a web-based application designed to narrow down candidate variants from family based whole-exome sequencing (WES) data through linkage analysis. PedMiner integrates linkage analysis, variant annotation and prioritization in one automated pipeline. It provides graphical visualization of the linked regions along with comprehensive annotation of variants and genes within these linked regions. This efficient and comprehensive application will be helpful for the scientific community working on Mendelian inherited disorders using family based WES data. [ABSTRACT FROM AUTHOR]

Auxin and cytokinin are two kinds of important phytohormones that mediate outgrowth of axillary buds in plants. How nitric oxide and its regulator of S-nitrosoglutathione reductase (GSNOR) take part in auxin and cytokinin signaling for controlling axillary buds outgrowth remains elusive. We investigated the roles of GSNOR during tomato axillary bud outgrowth by using physiological, biochemical and genetic approaches. GSNOR negatively regulated NO homeostasis. Suppression of GSNOR promoted axillary bud outgrowth by inhibiting the expression of FZY in both apical and axillary buds. Meanwhile, AUX1 and PIN1 were down-regulated in apical buds but up-regulated in axillary buds in GSNOR -suppressed plants. Thus, reduced IAA accumulation was shown in both apical buds and axillary buds of GSNOR -suppressed plants. GSNOR -mediated changes of NO and auxin affected cytokinin biosynthesis, transport, and signaling. And a decreased ratio of auxin: cytokinin was shown in axillary buds of GSNOR -suppressed plants, leading to bud dormancy breaking. We also found that the original NO signaling was generated by nitrate reductase (NR) catalyzing nitrate as substrate. NR-mediated NO reduced the GSNOR activity through S -nitrosylation of Cys-10, then induced a further NO burst, which played the above roles to promote axillary buds outgrowth. Together, GSNOR -mediated NO played important roles in controlling axillary buds outgrowth by altering the homeostasis and signaling of auxin and cytokinin in tomato plants. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) impacts multiple developmental events and stress responses in plants. S-nitrosylation, regulated by S-nitrosoglutathione reductase (GSNOR), is considered as an important route for NO bioactivity. However, genetic evidence for GSNOR -mediated plant development and S-nitrosylation remains elusive in crop species. Genetic and site-specific nitrosoproteomic approach was used to obtain GSNOR -mediated phenotype and S-nitrosylated network. Knockdown of GSNOR increased the endogenous NO level and S-nitrosylation, resulting in higher germination rate, inhibition of root and hypocotyl growth, decreased photosynthesis, reduced plant growth, altered plant architecture, dysplastic pollen grains, and low fructification rate and fruit yield. For nitrosoproteomic analysis, 395 endogenously S-nitrosylated proteins with 554 S-nitrosylation sites were identified within a wide range of biological processes, especially for energy metabolism. Physiological and exogenous energy-support testing were consistent with the omic result, suggesting that GSNOR- mediated S-nitrosylation of energy metabolism plays key roles in impacting plant growth and development. Taken together, GSNOR is actively involved in the regulation of multiple developmental processes related to agronomically important traits. In addition, our results provide valuable resources and new clues for the study of S-nitrosylation-regulated metabolism in plants. [ABSTRACT FROM AUTHOR]

Melatonin plays important roles in multiple stress responses. However, the downstream signaling pathway and molecular mechanism are unclear until now. Here, we not only revealed the transcriptional control of melatonin-induced sodic alkaline stress tolerance, but also described a screen for key downstream transcriptional factors of melatonin through transcriptome analysis. The melatonin-induced transcriptional network of hormone, transcriptional factors and functional genes has been established under both control and stress conditions. Among these, six candidates of transcriptional factors have been identified via Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. Using the virus-induced gene silencing approach, we confirmed that DREB1α and IAA3 were key downstream transcriptional factors of melatonin-induced sodic alkaline stress tolerance at the genetic level. The transcriptions of DREB1α and IAA3 could be activated by melatonin or sodic alkaline treatment. Interestingly, we found that DREB1α could directly upregulate the expression of IAA3 by binding to its promoters. Moreover, several physiological processes of Na+ detoxification, dehydration resistance, high pH buffering and reactive oxygen species scavenging were confirmed to depend or partly depend on DREB1α and IAA3 pathway in melatonin-induced stress tolerance. Taken together, this study suggested that DREB1α and IAA3 are positive resistant modulators, and provided a direct link among melatonin, DREB1α and IAA3 in the sodic alkaline stress tolerance activating in tomato plants. [ABSTRACT FROM AUTHOR]

PIWI-interacting RNAs (piRNAs) are essential for transcriptional and post-transcriptional regulation of transposons and coding genes in germline. With the development of sequencing technologies, length variations of piRNAs have been identified in several species. However, the extent to which, piRNA isoforms exist, and whether these isoforms are functionally distinct from canonical piRNAs remain uncharacterized. Through data mining from 2154 datasets of small RNA sequencing data from four species (Homo sapiens, Mus musculus, Danio rerio and Drosophila melanogaster), we have identified 8 749 139 piRNA isoforms from 175 454 canonical piRNAs, and classified them on the basis of variations on 5′ or 3′ end via the alignment of isoforms with canonical sequence. We thus established a database named IsopiRBank. Each isoforms has detailed annotation as follows: normalized expression data, classification, spatiotemporal expression data and genome origin. Users can also select interested isoforms for further analysis, including target prediction and Enrichment analysis. Taken together, IsopiRBank is an interactive database that aims to present the first integrated resource of piRNA isoforms, and broaden the research of piRNA biology. IsopiRBank can be accessed at http://mcg.ustc.edu.cn/bsc/isopir/index.html without any registration or log in requirement. Database URL: http://mcg.ustc.edu.cn/bsc/isopir/index.html [ABSTRACT FROM AUTHOR]

Embryo implantation is a crucial step in mammalian reproduction. However, little is known regarding the physiological roles of microRNAs in the regulation of embryo implantation. Here we show that a minimum uterine expression of miR-181 is essential for the onset of embryo implantation. Both transient and prolonged transgenic expression of miR-181 led to impaired implantation, which can be rescued by exogenous administration of leukemia inhibitory factor (LIF). Mechanistically, miR-181 is able to directly target LIF and downregulate LIF expression, thereby inhibiting embryo implantation. We also show that miR-181 expression is regulated by the transcriptional factor Emx2, and the Emx2–miR-181 axis plays an important role in regulating embryo implantation. Taken together, these results reveal a novel function of miR-181 in embryo implantation through the regulation of LIF, and also indicate a potential link between miR-181 dysregulation and human embryo implantation defects. [ABSTRACT FROM PUBLISHER]

In mice, the occurrence of immunofluorescent foci for mismatch repair protein MLH1 correlates closely with the occurrence of crossovers, as detected genetically, and MLH1 foci represent virtually all prospective crossover positions. To examine the effects of γ-irradiation on meiotic crossovers in mouse spermatocytes, male mice were subjected to whole-body γ-irradiation at different sub-stages of meiotic prophase and crossovers on synaptonemal complexes (SCs) were analysed by visualising and quantifying the immunofluorescent MLH1 foci. At both 24 and 48 h after exposure, significant dose-dependent increases in the number of total MLH1 foci per spermatocyte were observed at late zygotene–early pachytene with the gradient increase of radiation dose from 0, 1.5, 3–6 Gy. Furthermore, irradiation at preleptotene–leptotene still led to significant dose-dependent increased meiotic crossovers in the spermatocytes analysed 120 h after exposure. In further analysis, these dose-dependent increases in the number of total MLH1 foci per cell were attributed to significant dose-dependent decreases in autosomal SCs with 0 MLH1 focus, and the dose-dependent increases in autosomal SCs with 2 MLH1 foci and the percentage of cells with MLH1 focus on XY bivalent. The increased number of cells with an MLH1 focus on the pseudoautosomal regions (PARs) may indicate that there is a delay in meiotic progression in the irradiated cells. Although significant dose-dependent increases in the number of total MLH1 foci per cell were examined 24, 48 or 120 h after exposure with the gradient increase of radiation doses, these increases were mild compared to the control groups. This suggests that there is tight control of crossover formation (at least with respect to MLH1 foci number). The mechanisms underlying irradiation-induced DNA lesion repair, cellular responses independent of DNA damage and meiotic crossover homeostasis in mammals will be the subjects of future study. [ABSTRACT FROM AUTHOR]

Although micronuclei (MNi) have been extensively used to evaluate genotoxic effects and chromosome instability, the most basic issue regarding their formation was not completely addressed until recently, due to limitations of traditional experimental methods. The development of live-cell imaging, combined with genetically engineered chromosome labelling techniques makes it possible to investigate the origin of a micronucleus in a single cell in a real-time and high-throughput manner. Here, we review all the available studies on the origins of MNi in live cells and discuss novel findings based on this recently emerged methodology. Some unsolved questions on MNi formation and limitations of live-cell imaging in the investigation of MNi have also been discussed. [ABSTRACT FROM AUTHOR]

Summary: Next generation sequencing (NGS) techniques have been widely used to document the small ribonucleic acids (RNAs) implicated in a variety of biological, physiological and pathological processes. An integrated computational tool is needed for handling and analysing the enormous datasets from small RNA deep sequencing approach. Herein, we present a novel web server, CPSS (a computational platform for the analysis of small RNA deep sequencing data), designed to completely annotate and functionally analyse microRNAs (miRNAs) from NGS data on one platform with a single data submission. Small RNA NGS data can be submitted to this server with analysis results being returned in two parts: (i) annotation analysis, which provides the most comprehensive analysis for small RNA transcriptome, including length distribution and genome mapping of sequencing reads, small RNA quantification, prediction of novel miRNAs, identification of differentially expressed miRNAs, piwi-interacting RNAs and other non-coding small RNAs between paired samples and detection of miRNA editing and modifications and (ii) functional analysis, including prediction of miRNA targeted genes by multiple tools, enrichment of gene ontology terms, signalling pathway involvement and protein–protein interaction analysis for the predicted genes. CPSS, a ready-to-use web server that integrates most functions of currently available bioinformatics tools, provides all the information wanted by the majority of users from small RNA deep sequencing datasets.Availability: CPSS is implemented in PHP/PERL+MySQL+R and can be freely accessed at http://mcg.ustc.edu.cn/db/cpss/index.html or http://mcg.ustc.edu.cn/sdap1/cpss/index.html.Contact: xueyu@mail.hust.edu.cn or qshi@ustc.edu.cnSupplementary information: Supplementary data are available at Bioinformatics online. [ABSTRACT FROM PUBLISHER]

Summary: High-throughput deep-sequencing technology has generated an unprecedented number of expressed short sequence reads, presenting not only an opportunity but also a challenge for prediction of novel microRNAs. To verify the existence of candidate microRNAs, we have to show that these short sequences can be processed from candidate pre-microRNAs. However, it is laborious and time consuming to verify these using existing experimental techniques. Therefore, here, we describe a new method, miRD, which is constructed using two feature selection strategies based on support vector machines (SVMs) and boosting method. It is a high-efficiency tool for novel pre-microRNA prediction with accuracy up to 94.0% among different species.Availability: miRD is implemented in PHP/PERL+MySQL+R and can be freely accessed at http://mcg.ustc.edu.cn/rpg/mird/mird.php.Contact: qshi@ustc.edu.cnSupplementary information: Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]