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Following meiotic recombination, each pair of homologous chromosomes acquires at least one crossover, which ensures accurate chromosome segregation and allows reciprocal exchange of genetic information. Recombination failure often leads to meiotic arrest, impairing fertility, but the molecular basis of recombination remains elusive. Here, we report a homozygous M1AP splicing mutation (c.1074 + 2T C) in patients with severe oligozoospermia owing to meiotic metaphase I arrest. The mutation abolishes M1AP foci on the chromosome axes, resulting in decreased recombination intermediates and crossovers in male mouse models. M1AP interacts with the mammalian ZZS (an acronym for yeast proteins Zip2-Zip4-Spo16) complex components, SHOC1, TEX11, and SPO16. M1AP localizes to chromosomal axes in a SPO16-dependent manner and colocalizes with TEX11. Ablation of M1AP does not alter SHOC1 localization but reduces the recruitment of TEX11 to recombination intermediates. M1AP shows cytoplasmic localization in fetal oocytes and is dispensable for fertility and crossover formation in female mice. Our study provides the first evidence that M1AP acts as a copartner of the ZZS complex to promote crossover formation and meiotic progression in males.

Polyploidy is a common mode of evolution in flowering plants. Both the natural tetraploid Thinopyrum elongatum and the diploid one from the same population show a diploid-like pairing in meiosis. However, debate on the chromosome composition and origin of the tetraploid Th. elongatum is ongoing. In the present study, we obtained the induced tetraploid Th. elongatum and found that the induced and natural tetraploids are morphologically close, except for slower development and lower seed setting. Using probes developed from single chromosome microdissection and a Fosmid library, obvious differentiations were discovered between two chromosome sets (E

Family with sequence similarity 181 (Fam181) is a gene family with two paralogues (Fam181a and Fam181b) found among vertebrates. Fam181a exhibits dynamic and stage-specific expression during murine embryo development. Furthermore, searching in the National Center for Biotechnology Information database revealed predominant expression of Fam181a in mouse and human testes, implying that it may have essential roles in spermatogenesis. In this study we investigated the in vivo function of Fam181a in mouse spermatogenesis and fertility by generating Fam181a–/– mice using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9 genome editing technology. The resulting Fam181a–/– mice exhibited normal growth and development. In addition, the mice were completely fertile, with no obvious differences in the testis-to-bodyweight ratio, epididymal sperm count or sperm motility compared with wild-type mice. Further examination of testicular and epididymal histology of Fam181a–/– mice found an intact seminiferous tubule structure and the presence of all types of germ cells, from spermatogonia to mature spermatozoa, similar to wild-type littermates. Similarly, analysis of meiotic prophase I progression revealed normal populations of each substage of prophase I in Fam181a+/+ and Fam181a–/– testes, suggesting that this gene is dispensable for male fertility. These negative findings will help avoid research overlap, save time and resources and allow researchers to concentrate on genes that are critical for male fertility and spermatogenesis.

Testis-specifically expressed genes are important for male reproduction according to their unique expression patterns. However, the functions of most of these genes in reproduction are unclear. Here, we showed that mouse 4930590J08Rik was a testis-specifically expressed gene. 4930590J08Rik knockout mice exhibited a delay in the first wave of spermatogenesis and a reduction of cauda epididymal sperm. Furthermore, knockout spermatozoa exhibited defective acrosome reactions and decreased progressive motility, which led to impaired in vivo fertilization. Transcriptome analysis of testes revealed that most of the differentially expressed genes in knockout testes were associated with metabolic processes. 4930590J08Rik knockout sperm exhibited oxidative phosphorylation deficiency and were highly dependent on increased anaerobic glycolysis to compensate for ATP demands. Taken together, the 4930590J08Rik-disrupted mouse partially mimics the phenotypes of human asthenospermia and oligozoospermia, which provides a new model for further understanding the pathogenesis of idiopathic male infertility.

The WRKY family is a large group of transcription factors found in higher plants; it plays an important role in many aspects of biological processes. However, there is very little information about this family in melon (Cucumis melo L.). In our research, 57 candidate WRKY genes in the melon genome were identified. According to their structural and phylogenetic features, the 57 CmWRKY genes were classified into three groups, I, II, and III, and the group II was further divided into five subgroups. Group I included 11 members that all have two conservative WRKY domains and a C2H2-type zinc finger motif; Group II contains 41 WRKY gene family members, that all have a WRKY domain and a C2H2-type zinc finger motif. Five members that all have a conservative WRKY domain and a C2HC-type zinc finger motif are classified as Group III. The expression of 16 selected melon WRKY genes was detected by quantitative real-time PCR after sprayed with salicylic acid (SA) or powdery mildew infection. qRT-PCR analysis showed that 16 CmWRKY genes exhibited distinct expression patterns upon powdery mildew infection, and the expression levels of nine genes were inhibited, and seven genes were induced. After being sprayed with SA, the expression levels of 11 genes were inhibited, and five genes were induced. The data here provide an important basis for further functional studies of the WRKY gene in melon resistance.

Multiple morphological abnormalities of the sperm flagella (MMAF) is a specific type of asthenoteratozoospermia, presenting with multiple morphological anomalies in spermatozoa, such as absent, bent, coiled, short, or irregular caliber flagella. Previous genetic studies revealed pathogenic mutations in genes encoding cilia and flagella-associated proteins (CFAPs; e.g., CFAP43, CFAP44, CFAP65, CFAP69, CFAP70, and CFAP251) responsible for the MMAF phenotype in infertile men from different ethnic groups. However, none of them have been identified in infertile Pakistani males with MMAF. In the current study, two Pakistani families with MMAF patients were recruited. Whole-exome sequencing (WES) of patients and their parents was performed. WES analysis reflected novel biallelic loss-of-function mutations in CFAP43 in both families (Family 1: ENST00000357060.3, p.Arg300Lysfs*22 and p.Thr526Serfs*43 in a compound heterozygous state; Family 2: ENST00000357060.3, p.Thr526Serfs*43 in a homozygous state). Sanger sequencing further confirmed that these mutations were segregated recessively in the families with the MMAF phenotype. Semiquantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) was carried out to detect the effect of the mutation on mRNA of the affected gene. Previous research demonstrated that biallelic loss-of-function mutations in CFAP43 accounted for the majority of all CFAP43-mutant MMAF patients. To the best of our knowledge, this is the first study to report CFAP43 biallelic loss-of-function mutations in a Pakistani population with the MMAF phenotype. This study will help researchers and clinicians to understand the genetic etiology of MMAF better.

Background Low temperature (LT) often occurs at the seedling stage in the early rice-growing season, especially for direct seeded early-season indica rice, and using flooding irrigation can mitigate LT damage in rice seedlings. The molecular mechanism by which flooding mitigates the damage induced by LT stress has not been fully elucidated. Thus, LT stress at 8 °C, LT accompanied by flooding (LTF) and CK (control) treatments were established for 3 days to determine the transcriptomic, proteomic and physiological response in direct seeded rice seedlings at the seedling stage. Results LT damaged chloroplasts, and thylakoid lamellae, and increased osmiophilic bodies and starch grains compared to CK, but LTF alleviated the damage to chloroplast structure caused by LT. The physiological characteristics of treated plants showed that compared with LT, LTF significantly increased the contents of rubisco, chlorophyll, PEPCK, ATP and GA3 but significantly decreased soluble protein, MDA and ABA contents. 4D-label-free quantitative proteomic profiling showed that photosynthesis-responsive proteins, such as phytochrome, as well as chlorophyll and the tricarboxylic acid cycle were significantly downregulated in LT/CK and LTF/CK comparison groups. However, compared with LT, phytochrome, chlorophyllide oxygenase activity and the glucan branching enzyme in LTF were significantly upregulated in rice leaves. Transcriptomic and proteomic studies identified 72,818 transcripts and 5639 proteins, and 4983 genes that were identified at both the transcriptome and proteome levels. Differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were significantly enriched in glycine, serine and threonine metabolism, biosynthesis of secondary metabolites, glycolysis/gluconeogenesis and metabolic pathways. Conclusion Through transcriptomic, proteomic and physiological analyses, we determined that a variety of metabolic pathway changes were induced by LT and LTF. GO and KEGG enrichment analyses demonstrated that DEGs and DEPs were associated with photosynthesis pathways, antioxidant enzymes and energy metabolism pathway-related proteins. Our study provided new insights for efforts to reduce the damage to direct seeded rice caused by low-temperature stress and provided a breeding target for low temperature flooding-resistant cultivars. Further analysis of translational regulation and metabolites may help to elucidate the molecular mechanisms by which flooding mitigates low-temperature stress in direct seeded early indica rice at the seedling stage.

Asthenoteratozoospermia characterized by multiple morphological abnormalities of the flagella (MMAF) has been identified as a sub-type of male infertility. Recent progress has identified several MMAF-associated genes with an autosomal recessive inheritance in human affected individuals, but the etiology in approximately 40% of affected individuals remains unknown. Here, we conducted whole-exome sequencing (WES) and identified hemizygous missense variants in the X-linked CFAP47 in three unrelated Chinese individuals with MMAF. These three CFAP47 variants were absent in human control population genome databases and were predicted to be deleterious by multiple bioinformatic tools. CFAP47 encodes a cilia- and flagella-associated protein that is highly expressed in testis. Immunoblotting and immunofluorescence assays revealed obviously reduced levels of CFAP47 in spermatozoa from all three men harboring deleterious missense variants of CFAP47. Furthermore, WES data from an additional cohort of severe asthenoteratozoospermic men originating from Australia permitted the identification of a hemizygous Xp21.1 deletion removing the entire CFAP47 gene. All men harboring hemizygous CFAP47 variants displayed typical MMAF phenotypes. We also generated a Cfap47-mutated mouse model, the adult males of which were sterile and presented with reduced sperm motility and abnormal flagellar morphology and movement. However, fertility could be rescued by the use of intra-cytoplasmic sperm injections (ICSIs). Altogether, our experimental observations in humans and mice demonstrate that hemizygous mutations in CFAP47 can induce X-linked MMAF and asthenoteratozoospermia, for which good ICSI prognosis is suggested. These findings will provide important guidance for genetic counseling and assisted reproduction treatments.

Meiosis is pivotal for sexual reproduction and fertility. Meiotic programmed DNA double-strand breaks (DSBs) initiate homologous recombination, ensuring faithful chromosome segregation and generation of gametes. However, few studies have focused on meiotic DSB formation in human reproduction. Here, we report four infertile siblings born to a consanguineous marriage, with three brothers suffering from non-obstructive azoospermia and one sister suffering from unexplained infertility with normal menstrual cycles and normal ovary sizes with follicular activity. An autosomal recessive mutation in TOP6BL was found co-segregating with infertility in this family. Investigation of one male patient revealed failure in programmed meiotic DSB formation and meiotic arrest prior to pachytene stage of prophase I. Mouse models carrying similar mutations to that in patients recapitulated the spermatogenic abnormalities of the patient. Pathogenicity of the mutation in the female patient was supported by observations in mice that meiotic programmed DSBs failed to form in mutant oocytes and oocyte maturation failure due to absence of meiotic recombination. Our study thus illustrates the phenotypical characteristics and the genotype-phenotype correlations of meiotic DSB formation failure in humans.

Thousands of genes are involved in spermatogenesis, however, the functional roles of most these genes for male fertility remain to be discovered. This research focused to explore the function of evolutionarily conserved and testis-specific expressed gene 4930524B15Rik, which is known as C5orf47 in human. We generated 4930524B15Rik knockout mice by CRISPR/Cas9 technology and found 4930524B15Rik-/- mice were fertile. Furthermore, no averted abnormalities were observed in testis morphology, epididymal sperm contents and sperm morphology in 4930524B15Rik knockout mice. Subsequently, histological analysis of testicular tissue revealed intact structure of seminiferous tubules along with the presence of all types of germ cells in 4930524B15Rik-/- mice similar to wild type. Additionally, cytological analysis of spermatocytes displayed no significant differences in the prophase I progression of meiosis, further indicating that 4930524B15Rik have no essential function in mammalian spermatogenesis. Altogether, these results indicated that 4930524B15Rik is dispensable for fertility of male mice and these findings will help researchers to avoid future research overlap and to focus on genes that are crucial for spermatogenesis and reproduction.

To better understand cytokinin signaling in melon (Cucumis melo L.), one of the most important fruit crops in the Cucurbitaceae family, we identified and characterized melon two-component system (TCS) genes in this study. The results showed that there were 51 genes encoding putative TCS proteins in melon, and these TCS genes were classified into 3 subgroups, with 17 HK(L)s (histidine kinase/histidine-kinase like; 9 HKs and 8 HKLs), 9 HPs (histidine phosphotransfer proteins; 6 authentic and 3 pseudo), and 25 RRs (response regulators; 8 Type-A, 11 Type-B and 6 pseudo). The identity values of these cytokinin signaling proteins were revealed by analyzing their conserved motifs, domains and amino acid sequences. By analyzing TCS genes in different plant species, we found that melon HK(L)s, HPs and RRs had closer phylogenetic relationships with cucumber genes than with the genes of other plants, and the expansion of melon cytokinin signaling genes might be attributed to segmental duplication events. Analysis of the putative promoter regions (2-kb upstream regions of the start codon) revealed the enrichment of stress- and hormone-response cis-elements. The involvement of these putative TCS genes in melon cytokinin signaling was further supported by qRT-PCR data.

Description, RAD51AP2 is required specifically for efficient meiotic recombination to form crossover between X and Y chromosomes., Faithful segregation of X and Y chromosomes requires meiotic recombination to form a crossover between them in the pseudoautosomal region (PAR). Unlike autosomes that have approximately 10-fold more double-strand breaks (DSBs) than crossovers, one crossover must be formed from the one or two DSBs in PARs, implying the existence of a sex chromosome–specific recombination mechanism. Here, we found that RAD51AP2, a meiosis-specific partner of RAD51, is specifically required for the crossover formation on the XY chromosomes, but not autosomes. The decreased crossover formation between X and Y chromosomes in Rad51ap2 mutant mice results from compromised DSB repair in PARs due to destabilization of recombination intermediates rather than defects in DSB generation or synapsis. Our findings provide direct experimental evidence that XY recombination may use a PAR-specific DSB repair mechanism mediated by factors that are not essential for recombination on autosomes.

Fascins belong to a family of actin-bundling proteins that are involved in a wide range of biological functions. FSCN3, a newly identified testis-specific actin-bundling protein, is specifically expressed in elongated spermatids. However, its in vivo function in mouse spermiogenesis remains unknown.We generated Fscn3 knockout mice through CRISPR/Cas9 gene-editing technology. Fscn3Our study provides the first evidence that FSCN3 is a testis-specific actin-bundling protein that is not required for mouse spermatogenesis. Our results will help reproductive biologists focus their efforts on genes that are crucial for fertility and avoid research duplication.

Premature ovarian insufficiency (POI) is a major cause of reduced female fertility and affects approximately 1% women under 40 years of age. Recent advances emphasize the genetic heterogeneity of POI. Fanconi anemia (FA) genes, traditionally known for their essential roles in DNA repair and cytogenetic instability, have been demonstrated to be involved in meiosis and germ cell development. Here, we conducted whole-exome sequencing (WES) in 50 Han Chinese female patients with POI. Rare missense variants were identified in FANCA (Fanconi anemia complementation group A): c.1772G > A (p.R591Q) and c.3887A > G (p.E1296G). Both variants are heterozygous in the patients and very rare in the human population. In vitro functional studies further demonstrated that these two missense variants of FANCA exhibited reduced protein expression levels compared with the wild type, suggesting the partial loss of function. Moreover, mono-ubiquitination levels of FANCD2 upon mitomycin C stimulation were significantly reduced in cells overexpressing FANCA variants. Furthermore, a loss-of-function mutation of Fanca was generated in C57BL/6 mice for in vivo functional assay. Consistently, heterozygous mutated female mice (Fanca+/−) showed reduced fertility and declined numbers of follicles with aging when compared with the wild-type female mice. Collectively, our results suggest that heterozygous pathogenic variants in FANCA are implicated in non-syndromic POI in Han Chinese women, provide new insights into the molecular mechanisms of POI and highlight the contribution of FANCA variants in female subfertility.

As the major somatic cell type, Sertoli cells undergo active proliferation and play essential roles to establish testis cord at fetal stage. They also function to maintain germ cell development throughout the life of testicular development. However, the significance of Sertoli cell number for testis cord development and gonocyte fate is still unclear. Nuclear protein ataxia-telangiectasia (NPAT, also known as p220), a substrate of cyclin E/cyclin-dependent kinase 2, is well known as a regulator of cell proliferation through regulating histone expression. To study the role of NPAT during Sertoli cell development, we generated a mouse strain carrying conditional floxed Npat alleles, when crossing with anti-Mullerian hormone-cre, leading to the specific deletion of Npat in Sertoli cells. Npat disruption in Sertoli cells inhibited the programmed proliferation of fetal Sertoli cells resulting in disruption of developing testis cords, and subsequent postnatal mutant testes were severely hypoplastic. Germ cells, which are presumed to be in quiescent status during perinatal stage, exited G0 phase arrest and re-enter mitotic cell cycle prematurely. Of particular note, some germ cells possessed the meiotic signal in Npat-deficient testes. Our data thus indicates that the function of Npat-dependent Sertoli cells is essential at multiple steps in testis development, and this study also identifies Sertoli cells as a major regulator of germ cell development, which are required to maintain a local growth niche to repress premature mitosis and meiosis of gonocytes.-Jiang, X., Yin, S., Fan, S., Bao, J., Jiao, Y., Ali, A., Iqbal, F., Xu, J., Zhang, Y., Shi, Q. Npat-dependent programmed Sertoli cell proliferation is indispensable for testis cord development and germ cell mitotic arrest.

Background Elytrigia lolioides (Kar. et Kir.) Nevski, which is a perennial, cross-pollinating wheatgrass that is distributed in Russia and Kazakhstan, is classified into Elytrigia, Elymus, and Lophopyrum genera by taxonomists on the basis of different taxonomic classification systems. However, the genomic constitution of E. lolioides is still unknown. To identify the genome constitution and evolution of E. lolioides, we used single-copy nuclear genes acetyl-CoA carboxylase (Acc1) and elongation factor G (EF-G), multi-copy nuclear gene internal transcribed space (ITS), chloroplast gene trnL-F together with fluorescence and genomic in situ hybridization. Results Despite the widespread homogenization of ITS sequences, two distinct lineages (genera Pseudoroegneria and Hordeum) were identified. Acc1 and EF-G sequences suggested that in addition to Pseudoroegneria and Hordeum, unknown genome was the third potential donor of E. lolioides. Data from chloroplast DNA showed that Pseudoroegneria is the maternal donor of E. lolioides. Data from specific FISH marker for St genome indicated that E. lolioides has two sets of St genomes. Both genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) results confirmed the presence of Hordeum genome in this species. When E genome was used as the probe, no signal was found in 42 chromosomes. The E-like copy of Acc1 sequences was detected in E. lolioides possibly due to the introgression from E genome species. One of the H chromosomes in the accession W6–26586 from Kazakhstan did not hybridize H genome signals but had St genome signals on the pericentromeric regions in the two-color GISH. Conclusions Phylogenetic and in situ hybridization indicated the presence of two sets of Pseudoroegneria and one set of Hordeum genome in E. lolioides. The genome formula of E. lolioides was designed as StStStStHH. E. lolioides may have originated through the hybridization between tetraploid Elymus (StH) and diploid Pseudoroegneria species. E and unknown genomes may participate in the speciation of E. lolioides through introgression. According to the genome classification system, E. lolioides should be transferred into Elymus L. and renamed as Elymus lolioidus (Kar. er Kir.) Meld.

Summary Human infertility is a multifactorial disease that affects 8%–12% of reproductive-aged couples worldwide. However, the genetic causes of human infertility are still poorly understood. Synaptonemal complex (SC) is a conserved tripartite structure that holds homologous chromosomes together and plays an indispensable role in the meiotic progression. Here, we identified three homozygous mutations in the SC coding gene C14orf39/SIX6OS1 in infertile individuals from different ethnic populations by whole-exome sequencing (WES). These mutations include a frameshift mutation (c.204_205del [p.His68Glnfs∗2]) from a consanguineous Pakistani family with two males suffering from non-obstructive azoospermia (NOA) and one female diagnosed with premature ovarian insufficiency (POI) as well as a nonsense mutation (c.958G>T [p.Glu320∗]) and a splicing mutation (c.1180−3C>G) in two unrelated Chinese men (individual P3907 and individual P6032, respectively) with meiotic arrest. Mutations in C14orf39 resulted in truncated proteins that retained SYCE1 binding but exhibited impaired polycomplex formation between C14ORF39 and SYCE1. Further cytological analyses of meiosis in germ cells revealed that the affected familial males with the C14orf39 frameshift mutation displayed complete asynapsis between homologous chromosomes, while the affected Chinese men carrying the nonsense or splicing mutation showed incomplete synapsis. The phenotypes of NOA and POI in affected individuals were well recapitulated by Six6os1 mutant mice carrying an analogous mutation. Collectively, our findings in humans and mice highlight the conserved role of C14ORF39/SIX6OS1 in SC assembly and indicate that the homozygous mutations in C14orf39/SIX6OS1 described here are responsible for infertility of these affected individuals, thus expanding our understanding of the genetic basis of human infertility.

X-derived retrogenes contribute to genetic diversity in evolution and are usually specifically expressed in testis and perform important functions during spermatogenesis. Ubl4b is an autosomal retrogene with testis-specific expression derived from Ubl4a, an X-linked housekeeping gene. In the current study, we performed phylogenetic analysis and revealed that Ubl4a and Ubl4b are subject to purifying selection and may have conserved functions in evolution. Ubl4b was knocked out in mice using CRISPR/Cas9 genome editing technology and interestingly, we found no alterations in reproductive parameters of Ubl4b–/– male mice. To get insights into whether Ubl4a could compensate the absence of Ubl4b in vivo, we further obtained Ubl4a–/Y; Ubl4b–/– mice that lack both Ubl4a and Ubl4b, and the double knockout (dKO) mice also displayed normal spermatogenesis, showing that Ubl4a and Ubl4b are both dispensable for spermatogenesis. Thus, through the in vivo study of UBL4A and UBL4B, we provided a direct evidence for the first time that some X chromosome-derived autosomal retrogenes can be unfunctional in spermatogenesis, which represents an additional evolutionary type of X-derived retrogenes.

Serine/threonine kinases domain-containing proteins are known to play important functions in sperm flagella and male fertility. However, the roles of these proteins in human reproduction remain poorly understood and whether their variants are associated with human asthenozoospermia have not been reported. Here, we recruited a Pakistani family having four infertile patients diagnosed with idiopathic asthenozoospermia without any ciliary-related symptoms. Whole-exome sequencing identified a novel homozygous frameshift mutation (c.1235del, p.T412Kfs*14) in serine/threonine kinase 33 (STK33), which displays a highly conserved and predominant expression in testis in humans. This variant led to a dramatic reduction of STK33 messenger RNA (mRNA) in the patients. Patients homozygous for the STK33 variant presented reduced sperm motility, frequent morphological abnormalities of sperm flagella and completely disorganized flagellar ultrastructures, which are typical for multiple morphological abnormalities of the flagella (MMAF) phenotypes. Overall, these findings present evidence establishing that STK33 is an MMAF-related gene and provide new insights for understanding the role of serine/threonine kinases domain-containing proteins in human male reproduction.

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.

Several genes have been reported to be involved in spermatogenesis but their functional importance in male fertility is yet needed to be elucidated. Therefore, in current research, we focused to explore the in vivo role of evolutionary conserved and testis-specifically expressed, C4orf46, gene in male mouse fertility and spermatogenesis. The expression profile of C4orf46 is specific to testes and expressed in testes from 7 days of postpartum to onward. Thus, we generated the C4orf46 knockout mice by utilizing CRISPR/Cas9 genome editing technology and examined gene function in spermatogenesis and fertility. Surprisingly, C4orf46 knockout mice were completely fertile, displayed normal testes morphology, however, higher sperm contents were observed in knockout mice compared to wild type (WT) littermates. Subsequently, intact testis histology and architecture of seminiferous tubules were observed in C4orf46 knockout and WT mice. Similarly, sperm morphology and swimming velocity of C4orf46 knockout mice were comparable with the WT littermates. Furthermore, all type of germ cells ranging from spermatogonia to mature spermatozoa were observed in the testes and epididymis sections of C4orf46 knockout mice suggesting that disruption of C4orf46 did not impact spermatogenesis. Moreover, meiotic prophase I progression was normal, and each type of cell population was comparable between knockout and WT mice. Overall, finding from this research indicates that C4orf46 is not an essential gene for fertility in mice. This study will help researchers to avoid the repetition and duplication of efforts, and to explore the genes that are indispensable for spermatogenesis and male fertility.

Research question Male infertility is a global issue worldwide and multiple morphological abnormalities of the sperm flagella (MMAF) is one of the most severe forms of the qualitative sperm defects with a heterogeneous genetic cause that has not been completely understood. Can whole-exome sequencing (WES) reveal novel genetic causes contributing to MMAF in a consanguineous Pakistani family, comprising three infertile brothers? Design WES and bioinformatic analysis were conducted to screen potential pathogenic variants. The identified variant was validated by Sanger sequencing in all available family members Transmission electron microscopy analyses was carried out to examine the flagella ultrastructure of spermatozoa from patient. Results WES and Sanger sequencing identified a novel homozygous stop-gain mutation (ENST00000392644.4, c.182C>G, p.S61X) in ARMC2, which is expected to lead to loss of protein functions. Transmission electron microscopy analyses revealed that the flagellar ultrastructure of the patient's spermatozoa was disorganized along with a complete absence of central pair complex (CPC), suggesting that ARMC2 is involved in the assembly, stability of the axonemal complex, or both, particularly the CPC. Conclusion We report that a familial stop-gain mutation in ARMC2 is associated with male infertility in humans caused by MMAF accompanied with loss of CPCs and axonemal disorganization. We provide genetic evidence that ARMC2 is essential for human spermatogenesis and its mutation may be pathogenic for MMAF. These findings will improve the knowledge about the genetic basis of MMAF and provide information for genetic counselling of this disease.

The lack of resistance resources is a major bottleneck for wheat Fusarium head blight (FHB) resistance breeding. Three wheat-Th. elongatum FHB resistant translocation lines have been developed and used for wheat breeding without yield penalty. Transcriptomic analysis identified a derivative glutathione S-transferase transcript T26102, which was homologous to Fhb7 and induced dramatically by Fusarium graminearum. Unlike other studies, Fhb7 homologs were detected not only in Thinopyrum but also in Elymus, Leymus, Pseudoroegeria and Roegeria. We also found that several wheat-Th. ponticum derivatives carrying Fhb7 and its homologs were highly susceptible to FHB. Moreover, the transgenic plants expressing Fhb7 and its homolog on different backgrounds did not improve the FHB resistance.One Sentence SummaryThe GST-encoding Fhb7 candidate cannot improve Fusarium head blight resistance in wheat breeding.

Powdery mildew caused by Podosphaera xanthii (P. xanthii) severely endangers melon (Cucumis melo L.) production, while the mechanistic understanding about its resistance to powdery mildew remains largely limited. In this study, we integrated transcriptomic and methylomic analyses to explore whether DNA methylation was involved in modulating transcriptional acclimation of melon to P. xanthii infection. Net photosynthetic rate (Pn), stomatal conductance (Gs), actual photochemical efficiency (ФPSII) and maximum PSII quantum yield (Fv/Fm) were significantly decreased in P. xanthii-infected plants relative to uninfected ones (Control), revealing apparent physiological disorders. Totally 4808 differentially expressed genes (DEGs) were identified by global analysis of gene expression in Control and P. xanthii-infected plants. Comparative methylome uncovered that 932 DEGs were associated with hypermethylation, while 603 DEGs were associated with hypomethylation in melon upon P. xanthii infection. Among these differential methylation-involved DEGs, a set of resistance-related genes including R genes and candidate genes in metabolic and defense pathways were further identified, demonstrating that DNA methylation might function as a new regulatory layer for melon resistance to P. xanthii infection. Altogether our study sheds new insights into the molecular mechanisms of melon against powdery mildew and provides some potential targets for improving melon disease resistance in future.

Centromeres are specialized chromosomal domains involved in kinetochore formation and faithful chromosome segregation. Despite a high level of functional conservation, centromeres are not identified by DNA sequences, but by epigenetic means. Universally, centromeres are typically formed on highly repetitive DNA, which were previously considered to be silent. However, recent studies have shown that transcription occurs in this region, known as centromeric-derived RNAs (cenRNAs). CenRNAs that contribute to fundamental aspects of centromere function have been recently investigated in detail. However, the distribution, behavior and contributions of centromeric transcripts are still poorly understood. The aim of this article is to provide an overview of the roles of cenRNAs in centromere formation and function. We describe the structure and DNA sequence of centromere from yeast to human. In addition, we briefly introduce the roles of cenRNAs in centromere formation and function, kinetochore structure, accurate chromosome segregation, and pericentromeric heterochromatin assembly. Centromeric circular RNAs (circRNAs) and R-loops are rising stars in centromere function. CircRNAs have been successfully identified in various species with the assistance of high-throughput sequencing and novel computational approaches for non-polyadenylated RNA transcripts. Centromeric R-loops can be identified by the single-strand DNA ligation-based library preparation technique. But the molecular features and function of these centromeric R-loops and circRNAs are still being investigated. In this review, we summarize recent findings on the epigenetic regulation of cenRNAs across species, which would provide useful information about cenRNAs and interesting hints for further studies.

Multiple morphological abnormalities of the flagella (MMAF) is a genetically heterogeneous disorder leading to male infertility. Recent studies have revealed that DNAH17 variants are associated with MMAF, yet there is no functional evidence in support of their pathnogenicity. Here, we recruited two consanguineous families of Pakistani and Chinese origins, respectively, diagnosed with MMAF. Whole-exome sequencing identified novel homozygous DNAH17 variants, which led to loss of DNAH17 proteins, in the patients. Transmission electron microscope analyses revealed completely disorganized axonemal structure as the predominant anomaly and increased frequencies of missings of microtubule doublet(s) 4-7 in sperm flagella of patients. Similar to those found in patients, Dnah17-/- mice also displayed MMAF phenotype along with completely disorganized axonemal structures. Clusters of disorganized microtubules and outer dense fibers were observed in developing spermatids, indicating impaired sperm flagellar assembly. Besides, we also noticed many elongating spermatids with a deformed nuclear shape and abnormal step 16 spermatids that failed to spermiate, which subsequently underwent apoptosis in Dnah17-null mice. These findings present direct evidence establishing that DNAH17 is a MMAF-related gene in humans and mice, extend the clinical interpretations of DNAH17 variants, and highlight an essential and complex role of DNAH17 in spermatogenesis.

Reduced fertility is a common clinical feature of the individuals with Fanconi anemia (FA), a rare autosomal recessive disorder due to deficiency in FA pathway during DNA repair. Our previous study reported that the heterozygous pathogenic variants in FANCA (Fanconi anemia complementation group A) induced premature ovarian insufficiency (POI). However, the genotype-phenotype correlation in POI caused by FANCA variants remains considerably uncertain. Herein, a heterozygous non-frameshift Fanca-mutated mouse strain (Fanca+/hypo) carrying a 9-bp deletion (c.3581del9, p.QEA1194-1196del) was generated. The mutant mice exhibited slightly decreased Fanca protein level in ovaries, suggesting the non-frameshift deletion mutant is hypomorphic. Female fertility test showed decreased number of litters, litter sizes and prolonged litter interval time in the female Fanca+/hypo mice compared to wild-type mice. Follicle counting revealed a consistent decreasing pattern of follicle numbers in Fanca+/hypo females compared to that in wild-type mice with aging. Furthermore, embryonic fibroblasts of Fanca+/hypo mice were hyper-responsive to Mitomycin C in vitro, demonstrating a partial loss of function of this hypomorphic Fanca mutant in DNA repair. Collectively, our experimental observations suggest that the hypomorphic Fanca allele is sufficient to reduce female fertility in mice, providing new insights into the genetic counseling of FANCA variants in subfertile women.

s Dwarf mutants are the crucial resources for molecular biology research and rice breeding. Here, a rice mutant, dwarf and deformed flower3 (ddf3), was identified in tissue culture of Oryza sativa cv. Dongjin. Compared with wild type, the ddf3 mutant exhibited severe dwarfism, a greater number of tillers and significantly decreased fertility. In addition, leaf length, panicle length, and grain length, were significantly shorter. All internodes of ddf3 were shorter than those of wild type, and histological analysis revealed that internode cell elongation was significantly inhibited in ddf3. In the ddf3 mutant, pollen activity was significantly decreased, and the development of most stigmas was abnormal. Genetic analysis indicated that the ddf3 mutant phenotypes are controlled by a single or tightly linked nuclear genes. Using an F2 mapping population generated from a cross between ddf3 and Yangdao 6 (9311), the DDF3 gene was mapped to a 45.21-kb region between insertion-deletion (InDel) markers M15 and M16 on the long arm of chromosome 7. Sequencing revealed a 13.98-kb-deletion in this region in the ddf3 mutant genome that resulted in the complete or partial deletion of ZF (DHHC type zinc finger protein), EP (expressed protein), and FH2 (actin-binding FH2 domain-containing protein) genes. Quantitative RT-PCR analyses revealed that in wild type, the transcript levels of FH2 were almost the same in all organs, while ZF was mainly expressed in the panicle, and no expression of EP was detected in any organ. Based on these results, ZF and FH2 could be potential DDF3 candidate genes involved in the regulation of rice morphology and flower organ development. Our work has laid the foundation for future functional analysis of these candidate genes and has provided a profitable gene resource for rice breeding for increased fertility in the future.

There are more than 2300 genes that are predominantly expressed in mouse testes. The role of hundreds of these genes has been studied in mouse spermatogenesis but still there are many genes whose function is unknown. Gene knockout (KO) strategy in mice is widely used for in vivo study of gene function. The present study was designed to explore the function of the four genes: Tex37, Ccdc73, Prss55 and Nxt2, which were evolutionarily conserved in eutherians. We found that these genes had a testis-enriched expression pattern in mice except Nxt2. We knocked out these genes by CRISPR/Cas9 individually and found that all the KO mice had normal fertility with no detectable difference in testis/body weight ratios, epididymal sperm counts, as well as testicular and epididymal histology from wild type mice. Although these genes are evolutionarily conserved in eutherians including human and mouse, they are not individually essential for spermatogenesis, testis development and male fertility in mice in laboratory conditions. Our report of these fertile KO data could avoid the repetition and duplication of efforts which will help in prioritizing efforts to focus on genes that are indispensable for male reproduction.

We evaluated the effect of different light combinations on powdery mildew resistance and growth of melon seedlings. Light-emitting diodes were used as the light source and there were five light combinations: white light (420–680 nm); blue light (460 nm); red light (635 nm); RB31 (ratio of red and blue light, 3: 1); and RB71 (ratio of red and blue light, 7: 1). Compared with other treatments, blue light significantly decreased the incidence of powdery mildew in leaves of melon seedlings. Under blue light, H2O2 showed higher accumulation, and the content of phenolics, flavonoid and tannins, as well as expression of the genes involved in synthesis of these substances, significantly increased compared with other treatments before and after infection. Lignin content and expression of the genes related to its synthesis were also induced by blue light before infection. Melon irradiated with RB31 light showed the best growth parameters. Compared with white light, red light and RB71, RB31 showed higher accumulation of lignin and lower incidence of powdery mildew. We conclude that blue light increases melon resistance to powdery mildew, which is dependent on the induction of secondary metabolism that may be related to H2O2 accumulation before infection. Induction of tolerance of melon seeds to powdery mildew by RB31 is due to higher levels of sucrose metabolism and accumulation of lignin.

Two-component system (TCS) is responsible for cytokinin signaling, which plays critical roles in plant development and physiological process. This system is generally composed of two signaling factors, a histidine kinase (HK) and a response regulator (RR) that is associated with a histidine phosphotransfer (HP) protein. In this study, we performed systematic investigation on TCS genes in melon (Cucumis melon L.). We identified 44 TCS genes in melon, including 18 HK(L)s (9 HKs and 9 HKLs), 5 HPs (4 authentic and 1 pseudo), and 21 RRs (7 Type-A, 8 Type-B, and 6 pseudo). The classification and structure of these melon TCS members were introduced in detail as well. Our results provided new insights into the characteristics of the melon TCS genes and might benefit their functional study in future.

The St genome is one of the most fundamental genomes in Triticeae. Repetitive sequences are widely used to distinguish different genomes or species. The primary objectives of this study were to (i) screen a new sequence that could easily distinguish the chromosome of the St genome from those of other genomes by fluorescence in situ hybridization (FISH) and (ii) investigate the genome constitution of some species that remain uncertain and controversial. We used degenerated oligonucleotide primer PCR (Dop-PCR), Dot-blot, and FISH to screen for a new marker of the St genome and to test the efficiency of this marker in the detection of the St chromosome at different ploidy levels. Signals produced by a new FISH marker (denoted St2-80) were present on the entire arm of chromosomes of the St genome, except in the centromeric region. On the contrary, St2-80 signals were present in the terminal region of chromosomes of the E, H, P, and Y genomes. No signal was detected in the A and B genomes, and only weak signals were detected in the terminal region of chromosomes of the D genome. St2-80 signals were obvious and stable in chromosomes of different genomes, whether diploid or polyploid. Therefore, St2-80 is a potential and useful FISH marker that can be used to distinguish the St genome from those of other genomes in Triticeae.

Meiosis is a distinctcell division pathway in gamete formation in sexually reproducing organisms. During meiosis, the chromosomes undergo only one round of replication, followed by two rounds of cell division to produce haploid gametes. The behaviorsof chromosomes during meiosis I,including pairing, synapsis, recombination, and segregation of homologous chromosomes, are the most distinguishing features of meiosis. These behaviors arebased on the formation and repair of programmed DNA double-strand breaks in early meiotic prophase I. In this paper, we summarize the most important chromosome behaviors during meiosis and discuss the possible molecular mechanisms underlying these behaviors.

Testis cords are the embryonic precursors of the seminiferous tubules. Development of testis cords is a key event during embryonic testicular morphogenesis and is regulated by multiple signaling molecules produced by Sertoli cells. However, the exact nature and the cascade of molecular events underlying testis cord development remain to be uncovered. In the current study, we explored the role of DNA damage binding protein 1 (DDB1) in Sertoli cells during mouse testis cord development. The genetic ablation of Ddb1 specifically in Sertoli cells resulted in the compromised Sertoli cell proliferation and disruption of testis cord remodeling in neonatal mice. This testicular dysgenesis persisted through adulthood, resulting in smaller testis and low sperm production. Mechanistically, we observed that the DDB1 degradation can stabilize SET domain-containing lysine methyltransferase 8 (SET8), which subsequently decreases the phosphorylation of SMAD2, an essential intracellular component of transforming growth factor beta (TGF&beta, ) signaling. Taken together, our results suggest an essential role of Ddb1 in Sertoli cell proliferation and normal remodeling of testis cords via TGF&beta, pathway. To our knowledge, this is the first upstream regulators of TGF&beta, pathway in Sertoli cells, and therefore it furthers our understanding of testis cord development.

Exploring the genetic basis of human infertility is currently under intensive investigation. However, only a handful of genes have been validated in animal models as disease-causing genes in infertile men. Thus, to better understand the genetic basis of human spermatogenesis and bridge the knowledge gap between humans and other animal species, we construct the FertilityOnline, a database integrating the literature-curated functional genes during spermatogenesis into an existing spermatogenic database, SpermatogenesisOnline 1.0. Additional features, including the functional annotation and genetic variants of human genes, are also incorporated into FertilityOnline. By searching this database, users can browse the functional genes involved in spermatogenesis and instantly narrow down the number of candidates of genetic mutations underlying male infertility in a user-friendly web interface. Clinical application of this database was exampled by the identification of novel causative mutations in synaptonemal complex central element protein 1 (SYCE1) and stromal antigen 3 (STAG3) in azoospermic men. In conclusion, FertilityOnline is not only an integrated resource for spermatogenic genes but also a useful tool facilitating the exploration of the genetic basis of male infertility. FertilityOnline can be freely accessed at http://mcg.ustc.edu.cn/bsc/spermgenes2.0/index.html.

More than 2300 genes have been reported to be involved in spermatogenesis but the functional roles of most genes in male fertility remain to be elucidated. In this study, we explored the function of dipeptidase 3 (Dpep3), a gene predicted to be testis-specific, in male fertility of mice. We showed that Dpep3 is evolutionarily conserved in human and mouse along with other eutherians. Its mRNA was exclusively detected in testicular tissue and expressed in testes from 7 days postpartum. To further explore its role in male fertility, we generated Dpep3 knockout mice (Dpep3−/−) using the CRISPR/Cas9 technology and found that the male Dpep3−/− mice are fertile despite a significant reduction in sperm count. Histology of testis and progression of meiotic prophase I showed no obvious difference between wild-type and Dpep3−/− mice. All these findings indicate that Dpep3 is not essential for male fertility in mice. These findings will help other researchers to avoid research duplication, save their time and resources to focus on the genes that are indispensable for male fertility.

Centromeres mediate the pairing of homologous chromosomes during meiosis; this pairing is particularly challenging for polyploid plants such as hexaploid bread wheat (Triticum aestivum), as their meiotic machinery must differentiate homologs from similar homoeologs. However, the sequence compositions (especially functional centromeric satellites) and evolutionary history of wheat centromeres are largely unknown. Here, we mapped T. aestivum centromeres by chromatin immunoprecipitation sequencing using antibodies to the centromeric-specific histone H3 variant (CENH3); this identified two types of functional centromeric satellites that are abundant in two of the three subgenomes. These centromeric satellites had unit sizes greater than 500 bp and contained specific sites with highly phased binding to CENH3 nucleosomes. Phylogenetic analysis revealed that the satellites have diverged in the three T. aestivum subgenomes, and the more homogeneous satellite arrays are associated with CENH3. Satellite signals decreased and the degree of satellites variation increased from diploid to hexaploid wheat. Moreover, several T. aestivum centromeres lack satellite repeats. Rearrangements, including local expansion and satellite variations, inversions, and changes in gene expression, occurred during the evolution from diploid to tetraploid and hexaploid wheat. These results reveal the asymmetry in centromere organization among the wheat subgenomes, which may play a role in proper homolog pairing during meiosis.