Polycomb proteins RING1A/B promote H2A monoubiquitination to regulate female gametophyte development in Arabidopsis.

A functional female gametophyte is the basis of successful sexual reproduction in flowering plants. During female gametophyte development, the megaspore mother cell (MMC), differentiated from a single subepidermal somatic cell in the nucellus, undergoes meiosis to produce four megaspores; only the one at the chalazal end, referred to as functional megaspore (FM), undergoes three rounds of mitosis and develops into a mature embryo sac. Here, we reported that RING1A and RING1B (RING1A/B), two functionally redundant Polycomb proteins in Arabidopsis, are critical for female gametophyte development. The mutations of RING1A/B resulted in defects in MMC and FM's specification and subsequent mitosis of FM, thereby leading to aborted ovules. Gene expression analysis revealed several genes essential for female gametophyte development, including Argonaute (AGO) family genes and critical transcription factors, were ectopically expressed in ring1a ring1b. Furthermore, RING1A/B bound some of these genes to promote H2A monoubiquitination (H2Aub) deposition. Together, RING1A/B promote H2Aub modification at genes essential for female gametophyte development, suppressing their expression to ensure the progression of female gametophyte development.

Yeast sexes: mating types do not determine the sexes in Metschnikowia species.

Although filamentous Ascomycetes may produce structures that are interpreted as male and female gametangia, ascomycetous yeasts are generally not considered to possess male and female sexes. In haplontic yeasts of the genus Metschnikowia, the sexual cycle begins with the fusion of two morphologically identical cells of complementary mating types. Soon after conjugation, a protuberance emerges from one of the conjugants, eventually maturing into an ascus. The originating cell can be regarded as an ascus mother cell, hence as female. We tested the hypothesis that the sexes, female or male, are determined by the mating types. There were good reasons to hypothesize further that mating type α cells are male. In a conceptually simple experiment, we observed the early stages of the mating reaction of mating types differentially labeled with fluorescent concanavalin A conjugates. Three large-spored Metschnikowia species, M. amazonensis, M. continentalis, and M. matae, were examined. In all three, the sexes were found to be independent of mating type, cautioning that the two terms should not be used interchangeably.

Characterization of a novel cell wall hydrolase CwlE involved in Bacillus thuringiensis subsp. israelensis mother cell lysis.

Cell wall hydrolases are ubiquitous among spore-form bacteria and essential for mother cell lysis. In this study, a novel cell wall hydrolase gene cwlE involved in mother cell lysis was characterized from Bacillus thuringiensis subsp. israelensis (Bti) strain Bt-59. cwlE was specifically expressed in Bti and located in the large plasmid carrying the insecticidal genes. The encoded CwlE protein consists of a MurNAc-LAA domain and two highly conserved catalytic residues (E26 and E151). The recombinant CwlE-His protein was able to digest the cell wall of Bti, indicating that CwlE is an N-acetylmuramoyl-L-alanine amidase. Transcriptional analysis indicated that cwlE began to express at the early stage of stationary phase and was controlled by SigE. Single mutation of cwlE gene delayed Bti mother cell lysis, while double mutation of cwlE and sigK completely blocked Bti mother cell lysis. After exposure to UV light to deactivate the crystal proteins, the level of decrease of insecticidal activity against mosquito larvae of Bt-59 (ΔcwlE-sigK) was less than that observed for Bt-59. This study elucidates the mechanism of Bti mother cell lysis and provides an effective strategy for mosquito control using Bt products with increased persistence.

Insights into the role of phytohormones in plant female germline cell specification.

Germline specification is a fundamental process in plant reproduction, and the Megaspore Mother Cell (MMC), is a critical cell that differentiates and develops into the female gametophyte. While numerous studies have investigated the molecular mechanisms underlying female germline specification, previous reviews have mainly focused on gene regulatory networks, epigenetic pathways, and small RNAs, neglecting the potential contribution of phytohormones to this process. This review aims to address this gap by highlighting recent advances in MMC formation and discussing the roles of specific phytohormones in female germline specialization. Here, we provide a comprehensive overview of the functions of phytohormones in the formation of MMC and their effects on female gametophyte development. Specifically, it examines the roles of gibberellins (GAs), brassinosteroids (BRs), auxins, and cytokinin, in MMC development. Understanding the function of phytohormones in MMC development is essential for comprehending the complex mechanisms underlying plant reproduction. This review adds valuable insights to the existing knowledge on MMC development, providing a new perspective for future research in the field of plant reproduction.

The role of mitosis in generating fitness heterogeneity.

Cancer cells have heterogeneous fitness, and this heterogeneity stems from genetic and epigenetic sources. Here, we sought to assess the contribution of asymmetric mitosis (AM) and time on the variability of fitness in sister cells. Around one quarter of sisters had differences in fitness, assessed as the intermitotic time (IMT), from 330 to 510 min. Phenotypes related to fitness, such as ERK activity (herein referring to ERK1 and ERK2, also known as MAPK3 and MAPK1, respectively), DNA damage and nuclear morphological phenotypes were also asymmetric at mitosis or turned asymmetric over the course of the cell cycle. The ERK activity of mother cell was found to influence the ERK activity and the IMT of the daughter cells, and cells with ERK asymmetry at mitosis produced more offspring with AMs, suggesting heritability of the AM phenotype for ERK activity. Our findings demonstrate how variabilities in sister cells can be generated, contributing to the phenotype heterogeneities in tumor cells.

Deletion of the novel gene mother cell lysis X results in Cry1Ac encapsulation in the Bacillus thuringiensis HD73.

The novel protein MclX (mother cell lysis X) in Bacillus thuringiensis subsp. kurstaki strain HD73 (B. thuringiensis HD73) was characterized in this work. MclX has no known domain and its gene deletion in HD73 resulted in Cry1Ac encapsulation in the mother cell and did not influence Cry1Ac protein production or insecticidal activity. In vitro cell wall hydrolysis experiments showed that MclX cannot hydrolyze the cell wall. In mclX deletion mutants, the expression of cwlC (which encodes a key cell wall hydrolase) was significantly decreased, as shown by the ß-galactosidase activity assay. MclX cannot directly bind to the cwlC promoter, based on the electrophoretic mobility shift assay (EMSA). The cwlC was reported to be regulated by σK and GerE. However, the transcriptional activities of sigK and gerE showed no difference between HD73 and the mclX deletion mutant. It is indicated that MclX influenced cwlC expression independently of σK or GerE, through a new pathway to regulate cwlC expression. mclX deletion could be a new approach for insecticidal protein encapsulation in Bacillus thuringiensis.

Characterization of CwlC, an autolysin, and its role in mother cell lysis of Bacillus thuringiensis subsp. israelensis.

Bacillus thuringiensis subsp. israelensis (Bti) has been proven to efficiently control mosquitoes, of which many species are important vectors of human disease. The larvicidal action is attributed to the parasporal crystals formed in the sporulating cells and released upon cell autolysis. In this study, a sporulation-specific cwlC gene that encodes an N-acetylmuramoyl-L -alanine amidase was characterized in Bti strain Bt-59. CwlC was the only cell wall hydrolase in Bti found to contain both MurNAc-LAA and Amidase02_C domains. A recombinant CwlC-His protein was able to digest the Bacillus cell wall. Deletion of the cwlC gene delayed Bti mother cell lysis without impacting vegetative growth or insecticidal efficacy. Transcriptional analyses indicated that cwlC was expressed at the late sporulation stage and was controlled by SigK. Two other cell wall hydrolase genes, cwlB and cwlE, with high expression levels at T14 in Bt-59, were also identified. Like cwlC, cwlB expression was controlled by SigK; in contrast, cwlE was found not to be under the control of this sigma factor and unlike the other two, its gene was found to be plasmid encoded.

The Inactivation of Arabidopsis UBC22 Results in Abnormal Chromosome Segregation in Female Meiosis, but Not in Male Meiosis.

Protein ubiquitination is important for the regulation of meiosis in eukaryotes, including plants. However, little is known about the involvement of E2 ubiquitin-conjugating enzymes in plant meiosis. Arabidopsis UBC22 is a unique E2 enzyme, able to catalyze the formation of ubiquitin dimers through lysine 11 (K11). Previous work has shown that ubc22 mutants are defective in megasporogenesis, with most ovules having no or abnormally functioning megaspores; furthermore, some mutant plants show distinct phenotypes in vegetative growth. In this study, we showed that chromosome segregation and callose deposition were abnormal in mutant female meiosis while male meiosis was not affected. The meiotic recombinase DMC1, required for homologous chromosome recombination, showed a dispersed distribution in mutant female meiocytes compared to the presence of strong foci in WT female meiocytes. Based on an analysis of F1 plants produced from crosses using a mutant as the female parent, about 24% of female mutant gametes had an abnormal content of DNA, resulting in frequent aneuploids among the mutant plants. These results show that UBC22 is critical for normal chromosome segregation in female meiosis but not for male meiosis, and they provide important leads for studying the role of UBC22 and K11-linked ubiquitination.

The Role of Grass MUTE Orthologs in GMC Progression and GC Morphogenesis.

Stomata arose about 400 million years ago when plants left their aquatic environment. The last step of stomatal development is shared by all plant groups, and it implies a symmetrical cell division from the guard mother cell (GMC) to produce two guard cells (GCs) flanking a pore. In Arabidopsis, the basic helix-loop-helix transcription factor MUTE controls this step, upregulating cell-cycle regulators of the GMC division, and immediately afterward, repressors of theses regulators like FAMA and FOUR LIPS. Recently, three grass MUTE orthologs (BdMUTE from Brachypodium distachyon, OsMUTE from rice, and ZmMUTE from maize) have been identified and characterized. Mutations in these genes disrupt GMC fate, with bdmute also blocking GC morphogenesis. However, because these genes also regulate subsidiary cell recruitment, which takes place before GMC division, their functions regulating GMC division and GC morphogenesis could be an indirect consequence of that inducing the recruitment of subsidiary cells. Comprehensive data evaluation indicates that BdMUTE, and probably grass MUTE orthologs, directly controls GMC fate. Although grass MUTE proteins, whose genes are expressed in the GMC, move between cells, they regulate GMC fate from the cells where they are transcribed. Grass MUTE genes also regulate GC morphogenesis. Specifically, OsMUTE controls GC shape by inducing OsFAMA expression. In addition, while SCs are not required for GMC fate progression, they are for GC maturation.

Stomata and Sporophytes of the Model Moss Physcomitrium patens.

Mosses are an ancient land plant lineage and are therefore important in studying the evolution of plant developmental processes. Here, we describe stomatal development in the model moss species Physcomitrium patens (previously known as Physcomitrella patens) over the duration of sporophyte development. We dissect the molecular mechanisms guiding cell division and fate and highlight how stomatal function might vary under different environmental conditions. In contrast to the asymmetric entry divisions described in Arabidopsis thaliana, moss protodermal cells can enter the stomatal lineage directly by expanding into an oval shaped guard mother cell (GMC). We observed that when two early stage P. patens GMCs form adjacently, a spacing division can occur, leading to separation of the GMCs by an intervening epidermal spacer cell. We investigated whether orthologs of Arabidopsis stomatal development regulators are required for this spacing division. Our results indicated that bHLH transcription factors PpSMF1 and PpSCRM1 are required for GMC formation. Moreover, the ligand and receptor components PpEPF1 and PpTMM are also required for orientating cell divisions and preventing single or clustered early GMCs from developing adjacent to one another. The identification of GMC spacing divisions in P. patens raises the possibility that the ability to space stomatal lineage cells could have evolved before mosses diverged from the ancestral lineage. This would have enabled plants to integrate stomatal development with sporophyte growth and could underpin the adoption of multiple bHLH transcription factors and EPF ligands to more precisely control stomatal patterning in later diverging plant lineages. We also observed that when P. patens sporophyte capsules mature in wet conditions, stomata are typically plugged whereas under drier conditions this is not the case; instead, mucilage drying leads to hollow sub-stomatal cavities. This appears to aid capsule drying and provides further evidence for early land plant stomata contributing to capsule rupture and spore release.

Global identification and analysis revealed differentially expressed lncRNAs associated with meiosis and low fertility in autotetraploid rice.

BACKGROUND: Autotetraploid rice is a useful germplasm for polyploid rice breeding. Our previous research showed that non-coding RNAs might be associated with low fertility in autotetraploid rice. However, little information is available on long non-coding RNAs (lncRNAs) involved in the low fertility of autotetraploid rice. In the present study, RNA-seq was employed to detect the differentially expressed meiosis-related lncRNAs in autotetraploid rice, and gene overexpression and knock out experiments were used to validate the potential function of candidate lncRNA. RESULTS: A total of 444 differentially expressed lncRNAs (DEL) were detected during anther and ovary meiosis in autotetraploid rice. Of these, 328 DEL were associated with the transposable elements, which displayed low expression levels during meiosis in autotetraploid rice. We used rapid amplification of cDNA ends (RACE) assay to validate 10 DEL and found that the lncRNAs were not assembly artifacts, and six of them were conserved in tetraploid rice. Moreover, 237 and 20 lncRNAs were associated with pollen mother cell (PMC) and embryo sac mother cell (EMC) meiosis in autotetraploid rice, respectively. The differential expressions of some meiosis-related targets and its DEL regulator, including MEL1 regulated by TCONS_00068868, LOC_Os12g41350 (meiotic asynaptic mutant 1) by TCONS_00057811 in PMC, and LOC_Os12g39420 by TCONS_00144592 in EMC, were confirmed by qRT-PCR. TCONS_00057811, TCONS_00055980 and TCONS_00130461 showed anther specific expression patterns and were found to be highly expressed during meiosis. CRISPR/Cas9 editing of lncRNA57811 displayed similar morphology compared to wild type. The overexpression of lncRNA57811 resulted in low pollen fertility (29.70%) and seed setting (33%) in rice. CONCLUSION: The differential expression levels of lncRNAs, associated with transposable elements and meiosis-regulated targets, might be endogenous noncoding regulators of pollen/embryo sac development that cause low fertility in autotetraploid rice. The results enhance our understanding about rice lncRNAs, and facilitate functional research in autotetraploid rice.

Effect of the spoIIID mutation on mother cell lysis in Bacillus thuringiensis.

SpoIIID is a small, sequence-specific DNA-binding protein which can direct many genes' transcription and has an effect on spore formation in Bacillus subtilis. We investigated the role of SpoIIID in mother cell lysis in Bacillus thuringiensis. A ß-galactosidase assay based on the promoter fusions with lacZ indicated that the sigK gene was positively regulated by SpoIIID and σK negatively regulated the expression of sigE. The spoIIID mutant strain exhibited no mother cell lysis in Schaeffer's sporulation medium (SSM) but did in ½ Luria-Bertani (LB) medium. cwlC is an essential hydrolase gene for mother cell lysis. Moreover, the expression of a PcwlC-lacZ fusion in spoIIID mutant was proved to be higher in ½ LB medium than in SSM. HD (ΔspoIIID)(ΔcwlC) mutant was obtained by knocking out the cwlC gene in HD(ΔspoIIID) and displayed no mother cell lysis in both SSM and ½ LB mediums. The deletion of spoIIID decreased the crystal protein production in HD73. The expression of Porf1cry8E and P5014 promoter fusions with lacZ gene in the acrystalliferous HD-(ΔspoIIID) mutant showed similar activity to that in the acrystalliferous HD73- strain before T7 and slightly higher than that in the acrystalliferous HD73- after T7. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that Cry1Ac production in HD-(ΔspoIIID) directed by the Porf1cry8E and P5014 promoters was at a similar level as that in HD73 wild strain. Altogether, these results suggested that the spoIIID mutant with Porf1cry8E or P5014 promoters could be an alternative delivery system for cry gene expression with no mature spore formation and medium-dependent mother cell lysis.

Cytosolic calcium localization and dynamics during early endosperm development in the genus Agave (Asparagales, Asparagaceae).

Calcium is a secondary messenger that regulates and coordinates the cellular responses to environmental cues. Despite calcium being a key player during fertilization in plants, little is known about its role during the development of the endosperm. For this reason, the distribution, abundance, and dynamics of cytosolic calcium during the first stages of endosperm development of Agave tequilana and Agave salmiana were analyzed. Cytosolic calcium and actin filaments detected in the embryo sacs of Agave tequilana and A. salmiana revealed that they play an important role during the division and nuclear migration of the endosperm. After fertilization, a relatively high concentration of cytosolic calcium was located in the primary nucleus of the endosperm, as well as around migrating nuclei during the development of the endosperm. Cytosolic calcium participates actively during the first mitosis of the endosperm mother cell and interacts with the actin filaments that generate the motor forces during the migration of the nuclei through the large cytoplasm of the central cell.

Immunolabeling Protocols for Studying Meiosis in Plant Mutants Defective for Nuclear Envelope Components.

The nuclear envelope (NE) is a dynamic boundary that allows the communication between nuclear and cytoplasmic components. It has essential roles in a variety of physiological processes including cell division. The linker of nucleoskeleton and cytoskeleton (LINC) complexes span the NE and are important during meiosis, the specialized cell division needed for sexual reproduction. During this division, the LINC complex proteins AtSUN1 and AtSUN2, located in the inner nuclear membrane (INM), are involved in tethering telomeres to the NE. This attachment promotes chromosome movements by the forces that are generated in the cytoplasmic face. In Arabidopsis, the double mutant Atsun1 Atsun2 exhibits a delayed prophase I meiotic progression, partial synapsis, and recombination defects that lead to the formation of unbalanced gametes and sterility. In meiocytes from these mutants, immunolabeling can be applied to analyze possible changes in the dynamics of different meiotic proteins. In addition, if the specific antibodies are available, this technique is an easy and useful tool to determine the spatial distribution of NE proteins.

The Canonical E2Fs Are Required for Germline Development in Arabidopsis.

A number of cell fate determinations, including cell division, cell differentiation, and programmed cell death, intensely occur during plant germline development. How these cell fate determinations are regulated remains largely unclear. The transcription factor E2F is a core cell cycle regulator. Here we show that the Arabidopsis canonical E2Fs, including E2Fa, E2Fb, and E2Fc, play a redundant role in plant germline development. The e2fa e2fb e2fc (e2fabc) triple mutant is sterile, although its vegetative development appears normal. On the one hand, the e2fabc microspores undergo cell death during pollen mitosis. Microspores start to die at the bicellular stage. By the tricellular stage, the majority of the e2fabc microspores are degenerated. On the other hand, a wild type ovule often has one megaspore mother cell (MMC), whereas the majority of e2fabc ovules have two to three MMCs. The subsequent female gametogenesis of e2fabc mutant is aborted and the vacuole is severely impaired in the embryo sac. Analysis of transmission efficiency showed that the canonical E2Fs from both male and female gametophyte are essential for plant gametogenesis. Our study reveals that the canonical E2Fs are required for plant germline development, especially the pollen mitosis and the archesporial cell (AC)-MMC transition.

Novel Cell Wall Hydrolase CwlC from Bacillus thuringiensis Is Essential for Mother Cell Lysis.

In this study, a sporulation-specific gene (tentatively named cwlC) involved in mother cell lysis in Bacillus thuringiensis was characterized. The encoded CwlC protein consists of an N-terminal N-acetylmuramoyl-l-alanine amidase (MurNAc-LAA) domain and a C-terminal amidase02 domain. The recombinant histidine-tagged CwlC proteins purified from Escherichia coli were able to directly bind to and digest the B. thuringiensis cell wall. The CwlC point mutations at the two conserved glutamic acid residues (Glu-24 and Glu-140) shown to be critical for the catalytic activity in homologous amidases resulted in a complete loss of cell wall lytic activity, suggesting that CwlC is an N-acetylmuramoyl-l-alanine amidase. Results of transcriptional analyses indicated that cwlC is transcribed as a monocistronic unit and that its expression is dependent on sporulation sigma factor K (σK). Deletion of cwlC completely blocked mother cell lysis during sporulation without impacting the sporulation frequency, Cry1Ac protein production, and insecticidal activity. Taken together, our data suggest that CwlC is an essential cell wall hydrolase for B. thuringiensis mother cell lysis during sporulation. Engineered B. thuringiensis strains targeting cwlC, which allows the crystal inclusion to remain encapsulated in the mother cell at the end of sporulation, may have the potential to become more effective biological control agents in agricultural applications since the crystal inclusion remains encapsulated in the mother cell at the end of sporulation.IMPORTANCE Mother cell lysis has been well studied in Bacillus subtilis, which involves three distinct yet functionally complementary cell wall hydrolases. In this study, a novel cell wall hydrolase, CwlC, was investigated and found to be essential for mother cell lysis in Bacillus thuringiensis CwlC of B. thuringiensis only shows 9 and 21% sequence identity with known B. subtilis mother cell hydrolases CwlB and CwlC, respectively, suggesting that mechanisms of mother cell lysis may differ between B. subtilis and B. thuringiensis The cwlC gene deletion completely blocked the release of spores and crystals from the mother cell without affecting insecticidal activity. This may provide a new effective strategy for crystal encapsulation against UV light inactivation.

KLU suppresses megasporocyte cell fate through SWR1-mediated activation of WRKY28 expression in Arabidopsis.

Germ-line specification is essential for sexual reproduction. In the ovules of most flowering plants, only a single hypodermal cell enlarges and differentiates into a megaspore mother cell (MMC), the founder cell of the female germ-line lineage. The molecular mechanisms restricting MMC specification to a single cell remain elusive. We show that the Arabidopsis transcription factor WRKY28 is exclusively expressed in hypodermal somatic cells surrounding the MMC and is required to repress these cells from acquiring MMC-like cell identity. In this process, the SWR1 chromatin remodeling complex mediates the incorporation of the histone variant H2A.Z at the WRKY28 locus. Moreover, the cytochrome P450 gene KLU, expressed in inner integument primordia, non-cell-autonomously promotes WRKY28 expression through H2A.Z deposition at WRKY28. Taken together, our findings show how somatic cells in ovule primordia cooperatively use chromatin remodeling to restrict germ-line cell specification to a single cell.

3D Imaging of Whole-Mount Ovules at Cellular Resolution to Study Female Germline Development in Rice.

Recent advances in fluorescence-based staining of cellular compartments coupled with confocal microscopy imaging have allowed the visualization of three-dimensional (3D) structures with cellular resolution in various intact plant tissues and species. Such approaches are of particular interest for the analysis of the reproductive lineage in plants including the meiotic precursor cells deeply embedded within the ovary of the gynoecium enclosed in the flower. Yet, their relative inaccessibility and the lack of optical clarity of plant tissues prevent robust staining and imaging across several cell layers. Several whole-mount tissue staining and clearing techniques are available. One of them specifically allows staining of cellular boundaries in thick tissue samples while providing extreme optical clarity, using an acidic treatment followed by a modified Pseudo-Schiff propidium iodide (mPS-PI) method. While commonly used for Arabidopsis tissues, its application to other species like the model crop rice required protocol adaptations for obtaining robust staining that we present here. The procedure comprises six steps: (a) Material sampling; (b) Material fixation; (c) Tissue preparation; (d) Staining; (e) Sample mounting; and (d) Microscopy imaging. Particularly, we use ethanol and acetic anhydride as fixative reagents. A modified enzymatic treatment proved essential for starch degradation influencing optical clarity hence allowing acquisition of images at high resolution. This improved protocol is efficient for analyzing the megaspore mother cells in rice (Oryza sativa) ovary but is broadly applicable to other crop tissues of complex composition, without the need for tissue sectioning.

The THO Complex Non-Cell-Autonomously Represses Female Germline Specification through the TAS3-ARF3 Module.

In most sexually reproducing plants, a single somatic, sub-epidermal cell in an ovule is selected to differentiate into a megaspore mother cell, which is committed to giving rise to the female germline. However, it remains unclear how intercellular signaling among somatic cells results in only one cell in the sub-epidermal layer differentiating into the megaspore mother cell. Here we uncovered a role of the THO complex in restricting the megaspore mother cell fate to a single cell. Mutations in TEX1, HPR1, and THO6, components of the THO/TREX complex, led to the formation of multiple megaspore mother cells, which were able to initiate gametogenesis. We demonstrated that TEX1 repressed the megaspore mother cell fate by promoting the biogenesis of TAS3-derived trans-acting small interfering RNA (ta-siRNA), which represses ARF3 expression. The TEX1 protein was present in epidermal cells, but not in the germline, and, through TAS3-derived ta-siRNA, restricted ARF3 expression to the medio domain of ovule primordia. Expansion of ARF3 expression into lateral epidermal cells in a TAS3 ta-siRNA-insensitive mutant led to the formation of supernumerary megaspore mother cells, suggesting that TEX1- and TAS3-mediated restriction of ARF3 expression limits excessive megaspore mother cell formation non-cell-autonomously. Our findings reveal the role of a small-RNA pathway in the regulation of female germline specification in Arabidopsis.

The changes of mitochondrial membrane potential detected by confocal microscopy in spermatocyte induced by bisphenol A / 中国医学装备

Objective:To detect changes in mitochondrial membrane potential after sperm mother cell damage induced by bisphenol A by laser scanning confocal microscopy(LSCM) and underline its potential action mechanism.Methods: The cultured spermatogenic cells were divided into 3 groups, respectively. Then 0, 10μmol/L, 100μmol/L of Bisphenol A(BPA) were added into the culture, after 3 hours culture, fluorescence probe JC-1 was used to lable the three groups. The fluorescence intensity of JC-1 in mitochondrial was then detected by LSCM. LSCM software was used to analyze the fluorescence intensity. Change of the mitochondrial membrane potential was represented by the change of fluorescence colors (relative proportion of red and green fluorescence is commonly used to measure mitochondrial depolarization ratio). Results:The ratio between the red and green fluorescence in the control group, low dose group and high dose group had significant difference. Intracellular mitochondrial membrane potential of Bisphenol A treatment group was lower than that of the control group, while mitochondrial membrane potential of high dose group was lower than that of the low dose group. Conclusion: Bisphenol A could damage spermatocytes, and as the dose increased, the more serious the injury. The method could real-time monitor with high sensitivity the change of intracellular mitochondrial membrane potential.