Mechanism Of Male Sterility Research Articles

Integrated cytological, physiological, and comparative transcriptome profiling reveals the regulatory network for male sterility in Welsh onion (Allium fistulosum L.)

Welsh onion (Allium fistulosum L.) is a specialty crop with value as a vegetable, condiment, and medicine. However, the molecular mechanisms related to pollen abortion and cytoplasmic male sterility are poorly understood, limiting the heterosis utilization and hybrid production of A. fistulosum. This study investigated the dynamic differences between male-sterile (MS) plants and male-fertile (MF) plants at different anther developmental stages based on integrated cytological, physiological, and transcriptomic analyses. Cytological observations revealed that pollen abortion in the MS plants began during the tetrad stage and was caused by vacuolated microspores, abnormal degradation of the tapetum, and inadequate nutrient supply. The physiological results showed that MS plants exhibited low total soluble sugar and starch contents and decreased antioxidant enzyme activities (peroxidase and catalase). Comparative transcriptome analysis identified 7005 differentially expressed genes (DEGs) and functional enrichment analysis indicated that significant transcriptomic differences were related to oxidative phosphorylation, starch and sucrose metabolism, and cutin, suberine, and wax biosynthesis. The relative expression levels of key DEGs in these three important metabolic pathways were determined using quantitative real-time polymerase chain reaction. On this basis, a core transcriptome-mediated MS network was proposed for MS plants with four functional components: inhibited sucrose synthesis and transport; blocked cutin, suberine, and wax synthesis; accelerated reactive oxygen species generation; and abnormal tapetal programmed cell death. These results provide theoretical guidance for further exploring the molecular mechanisms of male sterility in A. fistulosum.

A narrative review of mitochondrial dysfunction and male infertility.

Recent investigations have highlighted mitochondrial dysfunction as a major component in reduced sperm function and male infertility. The creation of energy, control of reactive oxygen species (ROS), apoptosis, and sperm motility are all critically dependent on mitochondria. The health of the male reproductive system may be significantly impacted by any alteration of mitochondrial structure, function, or integrity. This review intends to open the door to better diagnostic methods, novel therapy strategies, and improved reproductive outcomes for infertile couples by clarifying the crucial function of mitochondria. We searched PubMed, Google Scholar, and others for articles related to male infertility and mitochondrial dysfunction from 2014 to 2023. The articles related to the theme were preliminarily screened by abstract, and then the selected literature was read and summarized. In this essay, we examine the research on male infertility and mitochondrial malfunction. We investigate the intricate connection between sperm quality, deoxyribonucleic acid damage, oxidative stress (OS), and mitochondrial bioenergetics. We discuss about how spermatogenesis and sperm function are affected by mitochondrial mutations, deletions, and single nucleotide polymorphisms. We also explore the impact of age-related changes, lifestyle choices, and environmental factors on mitochondrial function and male fertility. This review also clarifies the mechanisms by which mitochondrial dysfunction impacts the viability, morphology, and capacitation of sperm, among other aspects of male reproductive health. Furthermore, we go over the recently developed field of mitochondrial treatments and possible therapeutic approaches that target mitochondrial malfunction to enhance male fertility. Mitochondria are important for sperm: The control of sperm motility, capacitation, and general quality is largely dependent on mitochondria. Deterioration of sperm motility and male infertility may result from disruption of the structure, function, or integrity of the mitochondria. Future studies should focus on figuring out the processes underlying mitochondrial dysfunction as fertility and reproductive health are significantly impacted by it. We discuss the evaluation of infertile men mitochondrial function defects and difficulties, and make recommendations for further study in this area. This article provides a thorough resource for clinicians, researchers, and reproductive biologists to understand the underlying mechanisms of male infertility and explore potential therapeutic interventions.

In-Depth Understanding of Cytoplasmic Male Sterility by Metabolomics in Spring Stem Mustard (Brassica juncea var. tumida Tsen et Lee)

Male sterility (MS) caused by aborted pollen is a vital germplasm resource. In this study, metabolomic, transcriptomic, and proteomic analyses were performed to investigate the molecular regulatory mechanism of cytoplasmic male sterility (CMS) in the flower buds of the male sterile line 09-05A and its maintainer line, 09-05B, of Brassica juncea. Our metabolomic analysis revealed that 41 metabolites involved in pollen development and male fertility formation were differentially accumulated between 09-05A and 09-05B at the bi-nucleate stage of B. juncea. Integrated omics indicated that the expression of key genes and proteins in the amino acid and phenylpropanoid metabolic pathways was remarkably downregulated in the flower buds of 09-05A. Furthermore, the abnormal expression of key transcription factor (TF) genes related to tapetum development and pollen wall formation may affect pollen development in the 09-05A CMS line. The results indicated that the downregulated expression level of critical genes and proteins in amino acid metabolism; phenylpropanoid synthesis; and TF genes, such as shikimate kinase, phenylalanine ammonia-lyase, and MYB103, may have led to impaired tapetum and pollen wall development, thereby causing pollen abortion in 09-05A. This study provides new insights into the metabolic and molecular regulatory mechanisms underlying the formation of CMS in B. juncea and lays a foundation for detailed studies on the identity and characteristics of MS-related genes.

Genome-Wide Identification and Male Sterility-Related Expression Analysis of Papain-like Cysteine Protease Gene Family in Capsicum annuum

PLCPs (papain-like cysteine proteases) are one of the most abundant groups of cysteine proteases and play vital roles in multiple processes. The pepper (Capsicum annuum) is an important Solanaceae vegetable crop; its commercial hybrid seeds are widely used in production. Male sterility is a valuable trait for hybrid seed production. However, the function of PLCPs and the underlying mechanisms of male sterility in peppers remain unclear. In this study, we comprehensively identified the PLCP gene family in peppers, identifying 31 CaPLCPs. A phylogenetic analysis classified 31 members into eight clades. These CaPLCPs were unevenly distributed across eight chromosomes, and five segmental duplicated pairs were observed. The promoter cis-acting element analysis indicated that CaPLCP promoters contained abundant hormone-responsive and stress-responsive cis-elements, suggesting that CaPLCPs may play important roles in responding to abiotic stress, such as drought and low temperatures, as well as in plant immunity. The qRT-PCR analysis demonstrated that the expression levels of CaPLCP1, CaPLCP5, CaPLCP11, CaPLCP12, CaPLCP13, CaPLCP17, CaPLCP19, and CaPLCP21 were significantly reduced in the flowers of MS (male sterile pepper) at least at one stage, indicating their potential roles as regulatory factors in pepper male sterility. These findings provide important insights into the functional analysis of the PLCP gene family in peppers and other species, laying a crucial foundation for understanding the mechanisms of male sterility in peppers.

Morphological and cytological assessments reveal pollen degradation causes pollen abortion in cotton cytoplasmic male sterility lines

BackgroundUnderstanding the mechanism of male sterility is crucial for producing hybrid seeds and developing sterile germplasm resources. However, only a few cytoplasmic male sterility (CMS) lines of cotton have been produced due to several challenges, like inadequate variation of agronomic traits, incomplete sterility, weak resilience of restorer lines, and difficulty in combining strong dominance. Therefore, the morphological and cytological identification of CMS in cotton will facilitate hybrid breeding.ResultsTwo F2 segregating populations of cotton were constructed from cytoplasmic male sterile lines (HaA and 01A, maternal) and restorer lines (HaR and 26R, paternal). Genetic analysis of these populations revealed a segregation ratio of 3:1 for fertile to sterile plants. Phenotypic analysis indicated no significant differences in traits of flower bud development between sterile and fertile plants. However, sterile plants exhibited smaller floral organs, shortened filament lengths, and anther atrophy on the flowering day in comparison with the fertile plants. When performed scanning electron microscopy (SEM), the two F2 populations revealed morphological variations in the anther epidermis. Cellular analysis showed no significant differences in pollen development before pollen maturation. Interestingly, between the pollen maturation and flowering stages, the tapetum layer of sterile plants degenerated prematurely, resulting in abnormal pollen grains and gradual pollen degradation.ConclusionThe results of this study suggest that fertility-restoring genes are controlled by a single dominant gene. Sterile plants exhibit distinctive floral morphology, which is characterized by stamen atrophy and abnormal anthers. Pollen abortion occurs between pollen maturity and flowering, indicating that premature tapetum degradation may be the primary cause of pollen abortion. Overall, our study provides a theoretical basis for utilizing CMS in hybrid breeding and in-depth investigation of the dominant configuration of cotton hybrid combinations, mechanisms of sterility, and the role of sterile and restorer genes.

Mechanisms of Male Reproductive Sterility Triggered by Dysbiosis of Intestinal Microorganisms.

The intestinal microbiota, comprised of bacteria, archaea, and phages, inhabits the gastrointestinal tract of the organism. Male reproductive sterility is currently a prominent topic in medical research. Increasing research suggests that gut microbiota dysbiosis can result in various reproductive health problems. This article specifically investigates the impact of gut microbiota dysbiosis on male reproductive infertility development. Gut microbiota imbalances can disrupt the immune system and immune cell metabolism, affecting testicular growth and sperm production. This dysfunction can compromise the levels of hormones produced and secreted by the endocrine glands, affecting male reproductive health. Furthermore, imbalance of the gut microbiota can disrupt the gut-brain-reproductive axis, resulting in male reproductive infertility. This article explores how the imbalance of the gut microbiota impacts male reproductive infertility through immune regulation, endocrine regulation, and interactions of the gut-brain-reproductive axis, concluding with recommendations for prevention and treatment.

The Roles of N6-Methyladenosine Modification and Its Regulators in Male Reproduction

Infertility affects an estimated 10 to 15 percent of couples worldwide, with approximately half of the cases attributed to male-related issues. Most men diagnosed with infertility exhibit symptoms such as oligospermia, asthenospermia, azoospermia, and compromised sperm quality. Spermatogenesis is a complex and tightly coordinated process of germ cell differentiation, precisely regulated at transcriptional, posttranscriptional, and translational levels to ensure stage-specific gene expression during the development of spermatogenic cells and normal spermiogenesis. N6-methyladenosine (m6A) stands out as the most prevalent modification on eukaryotic mRNA, playing pivotal roles in various biological processes, including mRNA splicing, transportation, and translation. RNA methylation modification is a dynamic and reversible process primarily mediated by "writers", removed by "erasers", and recognized by "readers". In mammals, the aberrant methylation modification of m6A on mRNA is associated with a variety of diseases, including male infertility. However, the precise involvement of disrupted m6A modification in the pathogenesis of human male infertility remains unresolved. Intriguingly, a significant correlation has been found between the expression levels of m6A regulators in the testis and the severity of sperm concentration, motility, and morphology. Aberrant expression patterns of m6A regulatory proteins have been detected in anomalous human semen samples, including those of oligospermia, asthenozoospermia, and azoospermia. Furthermore, the examination of both sperm samples and testicular tissues revealed abnormal mRNA m6A modification, leading to reduced sperm motility and concentration in infertile men. Consequently, it is hypothesized that dysregulation of m6A modification might serve as an integral link in the mechanism of male infertility. This paper presents a comprehensive review of the recent discoveries regarding the spatial and temporal expression dynamics of m6A regulators in testicular tissues and the correlation between deregulated m6A regulators and human male infertility. Previous studies predominantly utilized constitutive or conditional knockout animal models for testicular phenotypic investigations. However, gene suppression in additional tissues could potentially influence the testis in constitutive knockout models. Furthermore, considering the compromised spermatogenesis observed in constitutive animals, distinguishing between the indirect effects of gene depletion on testicular development and its direct impact on the spermatogenic process is challenging, due to their intricate relationship. Such confounding factors might compromise the validity of the findings. To address this challenge, an inducible and conditional gene knockout model may serve as a superior approach. To date, nearly all reported studies have concentrated solely on the level changes of m6A and its regulators in germs cells, while the understanding of the function of m6A modification in testicular somatic cells remains limited. Testicular somatic cells, including peritubular myoid cells, Sertoli cells, and Leydig cells, play indispensable roles during spermatogenesis. Hence, comprehensive exploration of m6A modification within these cells as an additional crucial regulatory mechanism is warranted. In addition, exploration into the presence of unique methylation mechanisms or m6A regulatory factors within the testes is warranted. To elucidate the role of m6A modification in germ cells and testicular somatic cells, detailed experimental strategies need to be implemented. Among them, manipulation of the levels of key enzymes involved in m6A methylation and demethylation might be the most effective approach. Moreover, comprehensive analysis of the gene expression profiles involved in various signaling pathways, such as Wnt/β-catenin, Ras/MAPK, and Hippo, in m6A-modified germ cells and testicular somatic cells can provide more insight into its regulatory role in the spermatogenesis process. Further research in this area could provide valuable insights for developing innovative strategies to treat male infertility. Finally, considering the mitigation impact of m6A imbalance regulation on disease, investigation concerning whether restoring the equilibrium of m6A modification regulation can restore normal spermatogenesis function is essential, potentially elucidating the pivotal clinical significance of m6A modulation in male infertility.

The Expression and Epigenetic Characteristics of the HSF2 Gene in Cattle-Yak and the Correlation with Its Male Sterility.

Aberrant expression of the heat shock proteins and factors was revealed to be closely associated with male reproduction. Heat shock factor 2 (HSF2) is a transcription factor that is involved in the regulation of diverse developmental pathways. However, the role and the corresponding molecular mechanism of HSF2 in male cattle-yak sterility are still poorly understood. Therefore, the aim of this study was to obtain the sequence and the biological information of the cattle-yak HSF2 gene and to investigate the spatiotemporal expression profiles of the locus during the development of cattle-yak testes. Additionally, the differential expression was analyzed between the cattle-yak and the yak, and the methylation of corresponding promoter regions was compared. Our results showed an additional 54 bp fragment and a missense mutation (lysine to glutamic acid) were presented in the cattle-yak HSF2 gene, which correlated with enriched expression in testicular tissue. In addition, the expression of the HSF2 gene showed dynamic changes during the growth of the testes, reaching a peak in adulthood. The IHC indicated that HSF2 protein was primarily located in spermatocytes (PS), spermatogonia (SP), and Sertoli cells (SC) in cattle-yak testes, compared with the corresponding cells of cattle and the yak. Furthermore, bisulfite-sequencing PCR (BSP) revealed that the methylated CpG sites in the promoter region of the cattle-yak HSF2 were more numerous than in the yak counterpart, which suggests hypermethylation of this region in the cattle-yak. Taken together, the low expression abundance and hypermethylation of HSF2 may underpin the obstruction of spermatogenesis, which leads to male cattle-yak infertility. Our study provided a basic guideline for the HSF2 gene in male reproduction and a new insight into the mechanisms of male cattle-yak sterility.

Analysis of the Candidate Genes and Underlying Molecular Mechanism of P198, an RNAi-Related Dwarf and Sterile Line.

The genome-wide long hairpin RNA interference (lhRNAi) library is an important resource for plant gene function research. Molecularly characterizing lhRNAi mutant lines is crucial for identifying candidate genes associated with corresponding phenotypes. In this study, a dwarf and sterile line named P198 was screened from the Brassica napus (B. napus) RNAi library. Three different methods confirmed that eight copies of T-DNA are present in the P198 genome. However, only four insertion positions were identified in three chromosomes using fusion primer and nested integrated polymerase chain reaction. Therefore, the T-DNA insertion sites and copy number were further investigated using Oxford Nanopore Technologies (ONT) sequencing, and it was found that at least seven copies of T-DNA were inserted into three insertion sites. Based on the obtained T-DNA insertion sites and hairpin RNA (hpRNA) cassette sequences, three candidate genes related to the P198 phenotype were identified. Furthermore, the potential differentially expressed genes and pathways involved in the dwarfism and sterility phenotype of P198 were investigated by RNA-seq. These results demonstrate the advantage of applying ONT sequencing to investigate the molecular characteristics of transgenic lines and expand our understanding of the complex molecular mechanism of dwarfism and male sterility in B. napus.

Different mechanisms of X-ray irradiation-induced male and female sterility in Aedes aegypti

BackgroundAedes aegypti (Ae. aegypti) is the major vector that transmits many diseases including dengue, Zika, and filariasis in tropical and subtropical regions. Due to the growing resistance to chemical-based insecticides, biological control methods have become an emerging direction to control mosquito populations. The sterile insect technique (SIT) deploys high doses of ionizing radiation to sterilize male mosquitoes before the release. The Wolbachia-based population suppression method of the incompatible insect technique (IIT) involves the release of Wolbachia-infected males to sterilize uninfected field females. Due to the lack of perfect sex separation tools, a low percentage of female contamination is detected in the male population. To prevent the unintentional release of these Wolbachia-infected females which might result in population replacement, a low dose of X-ray irradiation is deployed to sterilize any female escapees. However, it remains unclear whether these irradiation-induced male and female sterilizations share common mechanisms.ResultsIn this work, we set out to define the minimum dose of X-ray radiation required for complete female sterilization in Ae. aegypti (NEA-EHI strain). Further results showed that this minimum dose of X-ray irradiation for female sterilization significantly reduced male fertility. Similar results have been reported previously in several operational trials. By addressing the underlying causes of the sterility, our results showed that male sterility is likely due to chromosomal damage in the germ cells induced by irradiation. In contrast, female sterility appears to differ and is likely initiated by the elimination of the somatic supporting cells, which results in the blockage of the ovariole maturation. Building upon these findings, we identified the minimum dose of X-ray irradiation on the Wolbachia-infected NEA-EHI (wAlbB-SG) strain, which is currently being used in the IIT-SIT field trial. Compared to the uninfected parental strain, a lower irradiation dose could fully sterilize wAlbB-SG females. This suggests that Wolbachia-carrying mosquitoes are more sensitive to irradiation, consistent with a previous report showing that a lower irradiation dose fully sterilized Wolbachia-infected Ae. aegypti females (Brazil and Mexican strains) compared to those uninfected controls.ConclusionsOur findings thus reveal the distinct mechanisms of ionizing X-ray irradiation-induced male or female sterility in Ae. aegypti mosquitoes, which may help the design of X-ray irradiation-based vector control methods.

Aberrant protamination in sperm correlates to anomalous nuclear and cytoplasmic architectures in infertile males with sperm dysmorphology.

Aberrant sperm protamination is linked to sperm dysmorphology and nuclear chromatin condensation. Yet, its effects on sperm cytoplasmic maturation remain largely unexplored. The relationships of protamines, sperm morphology, DNA damage, and cytoplasmic remodeling were illustrated in this study to provide fresh perspectives on the mechanisms of male infertility. A total of 205 infertile males were allocated into 5 groups according to the percentage of spermatozoa exhibiting abnormal morphology within their samples. Sperm concentration, motility, abnormal sperm morphology, cytoplasmic droplets (CDs), and excess residual cytoplasm (ERC) were analyzed according to the World Health Organization manual (2010). Sperm nuclear vacuoles (NVs) were determined by propidium iodide (PI) staining. Sperm protamine expressions (P1 and P2) were detected by western blot. DNA damage was measured by acridine orange test (AOT) to calculate the proportion of sperm with single-strand DNA breaks (SSBs). Our data showed that sperm concentration and motility in infertile males significantly decreased with the severity of abnormal sperm morphology (both P < 0.01). P1 level, P1/P2 ratio, and SSB rate increased with the severity of sperm dysmorphology, whilst the P2 level decreased (all P < 0.01). NVs, CDs, and ERC were more common in males with sperm dysmorphology and positively correlated with the SSB rate (all P < 0.01). The relationships between the SSB rate and the P1/P2 ratio were also significant ( P < 0.01). Aberrant protamination may cause sperm dysmorphology and compromise male fertility by impairing sperm's nucleus and cytoplasm maturation, with the P1/P2 ratio potentially serving as a valuable indicator of sperm quality and male fertility.

Molecular Mechanism of Male Sterility Induced by 60Co γ-Rays on Plutella xylostella (Linnaeus).

Plutella xylostella (Linnaeus) is one of the notorious pests causing substantial loses to numerous cruciferous vegetables across many nations. The sterile insect technique (SIT) is a safe and effective pest control method, which does not pollute the environment and does not produce drug resistance. We used proteomics technology and bioinformatics analysis to investigate the molecular mechanisms responsible for the effects of different doses of radiation treatment on the reproductive ability of male P. xylostella. A total of 606 differentially expressed proteins (DEPs) were identified in the 200 Gy/CK group, 1843 DEPs were identified in the 400 Gy/CK group, and 2057 DEPs were identified in the 400 Gy/200 Gy group. The results showed that after 200 Gy irradiation, the testes resisted radiation damage by increasing energy supply, amino acid metabolism and transport, and protein synthesis, while transcription-related pathways were inhibited. After 400 Gy irradiation, the mitochondria and DNA in the testis tissue of P. xylostella were damaged, which caused cell autophagy and apoptosis, affected the normal life activities of sperm cells, and greatly weakened sperm motility and insemination ability. Meanwhile, Western blotting showed that irradiation affects tyrosine phosphorylation levels, which gradually decrease with increasing irradiation dose.

High-Quality Genome Assembly and Genome-Wide Association Study of Male Sterility Provide Resources for Flax Improvement.

Flax is an economic crop with a long history. It is grown worldwide and is mainly used for edible oil, industry, and textiles. Here, we reported a high-quality genome assembly for "Neiya No. 9", a popular variety widely grown in China. Combining PacBio long reads, Hi-C sequencing, and a genetic map reported previously, a genome assembly of 473.55 Mb was constructed, which covers ~94.7% of the flax genome. These sequences were anchored onto 15 chromosomes. The N50 lengths of the contig and scaffold were 0.91 Mb and 31.72 Mb, respectively. A total of 32,786 protein-coding genes were annotated, and 95.9% of complete BUSCOs were found. Through morphological and cytological observation, the male sterility of flax was considered dominant nuclear sterility. Through GWAS analysis, the gene LUSG00017705 (cysteine synthase gene) was found to be closest to the most significant SNP, and the expression level of this gene was significantly lower in male sterile plants than in fertile plants. Among the significant SNPs identified in the GWAS analysis, only two were located in the coding region, and these two SNPs caused changes in the protein encoded by LUSG00017565 (cysteine protease gene). It was speculated that these two genes may be related to male sterility in flax. This is the first time the molecular mechanism of male sterility in flax has been reported. The high-quality genome assembly and the male sterility genes revealed, provided a solid foundation for flax breeding.

Potential candidate genes and pathways related to cytoplasmic male sterility in Dianthus spiculifolius as revealed by transcriptome analysis.

We introduced the candidate gene DsHSP70 into Arabidopsis thaliana, resulting in male gametophyte sterility and abnormal degeneration of sepals and petals. Cytoplasmic male sterility (CMS) is a useful tool for hybrid production. However, the regulatory mechanism of CMS in Dianthus spiculifolius remains unclear. In this study, we investigated whether male-sterile line of D. spiculifolius has a malformed tapetum and fails to produce normal fertile pollen. RNA sequencing technology was used to compare the gene expression patterns of the D. spiculifolius male-sterile line and its male fertility maintainer line during anther development. A total of 12,365 differentially expressed genes (DEGs) were identified, among which 1765 were commonly expressed in the S1, S2 and S3 stages. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that these DEGs were mainly involved in oxidation-reduction processes, signal transduction and programmed cell death. Additionally, weighted correlation network analysis (WGCNA) showed that three modules may be related to male sterility. A putative regulatory pathway for the male sterility traits was constructed based on the reproductive development network. After introducing the candidate DsHSP70 gene into Arabidopsis thaliana, we found that overexpressing plants showed anther abortion and shorter filaments, and accompanied by abnormal degeneration of sepals and petals. In summary, our results identified potential candidate genes and pathways related to CMS in D. spiculifolius, providing new insights for further research on the mechanism of male sterility.

A review of rice male sterility types and their sterility mechanisms

Male sterility plays an important role in the utilization of heterosis in rice. The establishment of male sterile lines in rice is one of the key technologies in hybrid rice production systems. The currently widely used male sterile line breeding systems mainly include: three-line hybrid rice based on cytoplasmic male sterility, two-line hybrid rice based on environmental sensitive gene male sterility, and third-generation hybrid rice based on nuclear gene male sterility Seed production system. This study reviewed the types and mechanisms of male sterility in rice, and looked forward to the development direction of hybrid rice.

Spatiotemporal profiles of gene activity in stamen delineate nucleo-cytoplasmic interaction in a male-sterile somatic cybrid citrus.

Cytoplasmic male sterility (CMS) has long been used to produce seedless fruits in perennial woody crops like citrus. A male-sterile somatic cybrid citrus (G1 + HBP) was generated by protoplast fusion between a CMS callus parent 'Guoqing No. 1' Satsuma mandarin (Citrus unshiu, G1) and a fertile mesophyll parent Hirado Buntan pummelo (Citrus grandis, HBP). To uncover the male-sterile mechanism of G1 + HBP, we compared the transcriptome profiles of stamen organ and cell types at five stages between G1 + HBP and HBP, including the initial stamen primordia, enlarged stamen primordia, pollen mother cells, tetrads, and microspores captured by laser microdissection. The stamen organ and cell types showed distinct gene expression profiles. A majority of genes involved in stamen development were differentially expressed, especially CgAP3.2, which was downregulated in enlarged stamen primordia and upregulated in tetrads of G1 + HBP compared with HBP. Jasmonic acid- and auxin-related biological processes were enriched among the differentially expressed genes of stamen primordia, and the content of jasmonic acid biosynthesis metabolites was higher in flower buds and anthers of G1 + HBP. In contrast, the content of auxin biosynthesis metabolites was lower in G1 + HBP. The mitochondrial tricarboxylic acid cycle and oxidative phosphorylation processes were enriched among the differentially expressed genes in stamen primordia, meiocytes, and microspores, indicating the dysfunction of mitochondria in stamen organ and cell types of G1 + HBP. Taken together, the results indicate that malfunction of mitochondria-nuclear interaction might cause disorder in stamen development, and thus lead to male sterility in the citrus cybrid.

Identification of miRNAs Involved in Male Fertility and Pollen Development in Brassica oleracea var. capitata L. by High-Throughput Sequencing

MicroRNAs (miRNAs) are endogenously expressed, non-coding RNAs that play essential roles in numerous developmental and physiological processes in plants. However, there is a lack of data available on regulating fertility in cabbage (Brassica oleracea var. capitata L.). This study examined the expression of miRNAs during cabbage pollen development using high-throughput sequencing. Two small RNA libraries were constructed from flower buds from the cytoplasmic male-sterile (CMS) line and its maintainer fertile (MF) linee of cabbage, resulting in over 10 million sequence reads. A total of 211 new miRNAs and 7 conserved miRNAs were discovered. Eight randomly chosen miRNAs were found to have differential expression between CMS and MF, as determined by stem-loop quantitative real-time PCR (qRT-PCR). The expression patterns were similar to those detected with Illumina sequencing. Moreover, the possible targets of these putative miRNAs were inferred using a BlastX search and Gene Ontology (GO) analysis. The majority of targets have been indicated to be essential for the development of pollen or other plant parts. The discovery and characterization of miRNAs involved in pollen development in CMS and MF cabbage lines might drive research on the regulatory mechanisms of male sterility, leading to a better understanding of the crucial roles performed by miRNAs in this process.

Evaluation of MYBL1 as the master regulator for pachytene spermatocyte genes dysregulated in interspecific hybrid dzo

Misregulation of spermatogenesis transcription factors (TF) in hybrids can lead to misexpression, which is a mechanism for hybrid male sterility (HMS). We used dzo (male offspring of Bos taurus ♂ × Bos grunniens ♀) in bovines to investigate the relationship of the key TF with HMS via RNA sequencing and assay for transposase-accessible chromatin with high-throughput sequencing analyses. RNA sequencing showed that the widespread misexpression in dzo was associated with spermatogenesis-related genes and somatic or progenitor genes. The transition from leptotene or zygotene spermatocytes to pachytene spermatocytes may be the key stage for meiosis arrest in dzo. The analysis of TF-binding motif enrichment revealed that the male meiosis-specific master TF MYB proto-oncogene like 1 (MYBL1, known as A-MYB) motif was enriched on the promoters of downregulated pachytene spermatocyte genes in dzo. Assay for transposase-accessible chromatin with high-throughput sequencing revealed that TF-binding sites for MYBL1, nuclear transcription factor Y, and regulatory factor X were enriched in the low-chromatin accessibility region of dzo. The target genes of the MYBL1-binding motif were associated with meiosis-specific genes and significantly downregulated in dzo testis. The transcription factor MYBL1 may be the candidate master regulator for pachytene spermatocyte genes dysregulated in interspecific HMS dzo. This study reported that a few upstream TF regulation changes might exert a cascading effect downstream in a regulatory network as a mechanism for HMS.

Cloning and Expression Analysis of Onion (Allium cepa L.) MADS-Box Genes and Regulation Mechanism of Cytoplasmic Male Sterility.

Flower organ development is one of the most important processes in plant life. However, onion CMS (cytoplasmic male sterility) shows an abnormal development of floral organs. The regulation of MADS-box transcription factors is important for flower development. To further understand the role of MADS-box transcription factors in the regulation of cytoplasmic male sterility onions. We cloned the full-length cDNA of five MADS-box transcription factors from the flowers of onion using RACE (rapid amplification of cDNA ends) technology. We used bioinformatics methods for sequence analysis and phylogenetic analysis. Real-time quantitative PCR was used to detect the expression patterns of these genes in different onion organs. The relative expression levels of five flower development genes were compared in CMS onions and wild onions. The results showed that the full-length cDNA sequences of the cloned MADS-box genes AcFUL, AcDEF, AcPI, AcAG, and AcSEP3 belonged to A, B, C, and E MADS-box genes, respectively. A phylogenetic tree construction analysis was performed on its sequence. Analysis of MADS-box gene expression in wild onion and CMS onion showed that the formation of CMS onion was caused by down-regulation of AcDEF, AcPI, and AcAG gene expression, up-regulation of AcSEP3 gene expression, and no correlation with AcFUL gene expression. This work laid the foundation for further study of the molecular mechanism of onion flower development and the molecular mechanism of CMS onion male sterility.

MKRN2 knockout causes male infertility through decreasing STAT1, SIX4, and TNC expression.

Makorin-2 (Mkrn2) is an evolutionarily conserved gene whose biological functions are not fully known. Although recent studies have shed insights on the potential causes of male infertility, its underlining mechanisms still remain to be elucidated. We developed a Mrkn2 knockout mice model to study this gene and found that deletion of Mkrn2 in mice led to male infertility. Interestingly, the expression level of signal transducer and activator of the transcription (STAT)1 was significantly decreased in MKRN2 knockout testis and MEF cells. Co-IP assay showed an interaction between MKRN2 and STAT1. Moreover, our results further indicated that MKRN2 regulated the expression level of SIX4 and tenascin C (TNC) via the EBF transcription factor 2 (EBF2) in mice. The results of our study will provide insights into a new mechanism of male infertility.