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1.
Genes (Basel) ; 15(8)2024 Aug 07.
Artículo en Inglés | MEDLINE | ID: mdl-39202402

RESUMEN

In Arabidopsis thaliana (Arabidopsis), microRNA160 (miR160) regulates the expression of AUXIN RESPONSE FACTOR10 (ARF10), ARF16 and ARF17 throughout development, including the development of the root system. We have previously shown that in addition to DOUBLE-STRANDED RNA BINDING1 (DRB1), DRB2 is also involved in controlling the rate of production of specific miRNA cohorts in the tissues where DRB2 is expressed in wild-type Arabidopsis plants. In this study, a miR160 overexpression transgene (MIR160B) and miR160-resistant transgene versions of ARF10 and ARF16 (mARF10 and mARF16) were introduced into wild-type Arabidopsis plants and the drb1 and drb2 single mutants to determine the degree of requirement of DRB2 to regulate the miR160 expression module as part of root development. Via this molecular modification approach, we show that in addition to DRB1, DRB2 is required to regulate the level of miR160 production from its precursor transcripts in Arabidopsis roots. Furthermore, we go on to correlate the altered abundance of miR160 or its ARF10, ARF16 and ARF17 target genes in the generated series of transformant lines with the enhanced development of the root system displayed by these plant lines. More specifically, promotion of primary root elongation likely stemmed from enhancement of miR160-directed ARF17 expression repression, while the promotion of lateral and adventitious root formation was the result of an elevated degree of miR160-directed regulation of ARF17 expression, and to a lesser degree, ARF10 and ARF16 expression. Taken together, the results presented in this study identify the requirement of the functional interplay between DRB1 and DRB2 to tightly control the rate of miR160 production, to in turn ensure the appropriate degree of miR160-directed ARF10, ARF16 and ARF17 gene expression regulation as part of normal root system development in Arabidopsis.


Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Regulación de la Expresión Génica de las Plantas , MicroARNs , Raíces de Plantas , Factores de Transcripción , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/genética , Ácidos Indolacéticos/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo
2.
Genes (Basel) ; 15(8)2024 Aug 03.
Artículo en Inglés | MEDLINE | ID: mdl-39202383

RESUMEN

MicroRNAs (miRNAs) are a class of small regulatory RNA that are generated via core protein machinery. The miRNAs direct gene-silencing mechanisms to mediate an essential role in gene expression regulation. In mollusks, miRNAs have been demonstrated to be required to regulate gene expression in various biological processes, including normal development, immune responses, reproduction, and stress adaptation. In this study, we aimed to establishment the requirement of the miRNA pathway as part of the molecular response of exposure of Biomphalaria glabrata (snail host) to Schistosoma mansoni (trematode parasite). Initially, the core pieces of miRNA pathway protein machinery, i.e., Drosha, DGCR8, Exportin-5, Ran, and Dicer, together with the central RNA-induced silencing complex (RISC) effector protein Argonaute2 (Ago2) were elucidated from the B. glabrata genome. Following exposure of B. glabrata to S. mansoni miracidia, we identified significant expression up-regulation of all identified pieces of miRNA pathway protein machinery, except for Exportin-5, at 16 h post exposure. For Ago2, we went on to show that the Bgl-Ago2 protein was localized to regions surrounding the sporocysts in the digestive gland of infected snails 20 days post parasite exposure. In addition to documenting elevated miRNA pathway protein machinery expression at the early post-exposure time point, a total of 13 known B. glabrata miRNAs were significantly differentially expressed. Of these thirteen B. glabrata miRNAs responsive to S. mansoni miracidia exposure, five were significantly reduced in their abundance, and correspondingly, these five miRNAs were determined to putatively target six genes with significantly elevated expression and that have been previously associated with immune responses in other animal species, including humans. In conclusion, this study demonstrates the central importance of a functional miRNA pathway in snails, which potentially forms a critical component of the immune response of snails to parasite exposure. Further, the data reported in this study provide additional evidence of the complexity of the molecular response of B. glabrata to S. mansoni infection: a molecular response that could be targeted in the future to overcome parasite infection and, in turn, human schistosomiasis.


Asunto(s)
Proteínas Argonautas , Biomphalaria , MicroARNs , Schistosoma mansoni , Animales , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Biomphalaria/parasitología , Biomphalaria/genética , Biomphalaria/metabolismo , Biomphalaria/inmunología , MicroARNs/genética , MicroARNs/metabolismo , Schistosoma mansoni/genética , Oocistos/metabolismo , Interacciones Huésped-Parásitos/genética
3.
Reprod Fertil ; 5(1)2024 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-38367345

RESUMEN

Abstract: Poly- and per-fluoroalkyl substances (PFAS) are synthetic environmentally persistent chemicals. Despite the phaseout of specific PFAS, their inherent stability has resulted in ubiquitous and enduring environmental contamination. PFAS bioaccumulation has been reported globally with omnipresence in most populations wherein they have been associated with a range of negative health effects, including strong associations with increased instances of testicular cancer and reductions in overall semen quality. To elucidate the biological basis of such effects, we employed an acute in vitro exposure model in which the spermatozoa of adult male mice were exposed to a cocktail of PFAS chemicals at environmentally relevant concentrations. We hypothesized that direct PFAS treatment of spermatozoa would induce reactive oxygen species generation and compromise the functional profile and DNA integrity of exposed cells. Despite this, post-exposure functional testing revealed that short-term PFAS exposure (3 h) did not elicit a cytotoxic effect, nor did it overtly influence the functional profile, capacitation rate, or the in vitro fertilization ability of spermatozoa. PFAS treatment of spermatozoa did, however, result in a significant delay in the developmental progression of the day 4 pre-implantation embryos produced in vitro. This developmental delay could not be attributed to a loss of sperm DNA integrity, DNA damage, or elevated levels of intracellular reactive oxygen species. When considered together, the results presented here raise the intriguing prospect that spermatozoa exposed to a short-term PFAS exposure period potentially harbor an alternate stress signal that is delivered to the embryo upon fertilization. Lay summary: PFAS are synthetic chemicals widely used in non-stick cookware, food packaging, and firefighting foam. Such extensive use has led to concerning levels of environmental contamination and reports of associations with a spectrum of negative health outcomes, including testicular cancer and reduced semen quality. To investigate the effects of PFAS on male reproduction, we incubated mouse sperm in a cocktail of nine PFAS at environmentally relevant concentrations before checking for a range of functional outcomes. This treatment strategy was not toxic to the sperm; it did not kill them or reduce their motility, nor did it affect their fertilization capacity. However, we did observe developmental delays among pre-implantation embryos created using PFAS-treated sperm. Such findings raise the intriguing prospect that PFAS-exposed sperm harbor a form of stress signal that they deliver to the embryo upon fertilization.


Asunto(s)
Fluorocarburos , Neoplasias de Células Germinales y Embrionarias , Enfermedades de los Roedores , Neoplasias Testiculares , Masculino , Ratones , Animales , Neoplasias Testiculares/veterinaria , Análisis de Semen/veterinaria , Especies Reactivas de Oxígeno/farmacología , Semen , Espermatozoides/fisiología , ADN/farmacología , Fluorocarburos/toxicidad
4.
Int J Mol Sci ; 24(15)2023 Aug 03.
Artículo en Inglés | MEDLINE | ID: mdl-37569766

RESUMEN

Our duty to conserve global natural ecosystems is increasingly in conflict with our need to feed an expanding population. The use of conventional pesticides not only damages the environment and vulnerable biodiversity but can also still fail to prevent crop losses of 20-40% due to pests and pathogens. There is a growing call for more ecologically sustainable pathogen control measures. RNA-based biopesticides offer an eco-friendly alternative to the use of conventional fungicides for crop protection. The genetic modification (GM) of crops remains controversial in many countries, though expression of transgenes inducing pathogen-specific RNA interference (RNAi) has been proven effective against many agronomically important fungal pathogens. The topical application of pathogen-specific RNAi-inducing sprays is a more responsive, GM-free approach to conventional RNAi transgene-based crop protection. The specific targeting of essential pathogen genes, the development of RNAi-nanoparticle carrier spray formulations, and the possible structural modifications to the RNA molecules themselves are crucial to the success of this novel technology. Here, we outline the current understanding of gene silencing pathways in plants and fungi and summarize the pioneering and recent work exploring RNA-based biopesticides for crop protection against fungal pathogens, with a focus on spray-induced gene silencing (SIGS). Further, we discuss factors that could affect the success of RNA-based control strategies, including RNA uptake, stability, amplification, and movement within and between the plant host and pathogen, as well as the cost and design of RNA pesticides.


Asunto(s)
Agentes de Control Biológico , Plaguicidas , Ecosistema , Interferencia de ARN , ARN Interferente Pequeño/genética , Productos Agrícolas/genética , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/prevención & control , Enfermedades de las Plantas/microbiología
5.
Plants (Basel) ; 12(5)2023 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-36903960

RESUMEN

Angiosperms form the largest phylum within the Plantae kingdom and show remarkable genetic variation due to the considerable difference in the nuclear genome size of each species. Transposable elements (TEs), mobile DNA sequences that can amplify and change their chromosome position, account for much of the difference in nuclear genome size between individual angiosperm species. Considering the dramatic consequences of TE movement, including the complete loss of gene function, it is unsurprising that the angiosperms have developed elegant molecular strategies to control TE amplification and movement. Specifically, the RNA-directed DNA methylation (RdDM) pathway, directed by the repeat-associated small-interfering RNA (rasiRNA) class of small regulatory RNA, forms the primary line of defense to control TE activity in the angiosperms. However, the miniature inverted-repeat transposable element (MITE) species of TE has at times avoided the repressive effects imposed by the rasiRNA-directed RdDM pathway. MITE proliferation in angiosperm nuclear genomes is due to their preference to transpose within gene-rich regions, a pattern of transposition that has enabled MITEs to gain further transcriptional activity. The sequence-based properties of a MITE results in the synthesis of a noncoding RNA (ncRNA), which, after transcription, folds to form a structure that closely resembles those of the precursor transcripts of the microRNA (miRNA) class of small regulatory RNA. This shared folding structure results in a MITE-derived miRNA being processed from the MITE-transcribed ncRNA, and post-maturation, the MITE-derived miRNA can be used by the core protein machinery of the miRNA pathway to regulate the expression of protein-coding genes that harbor homologous MITE insertions. Here, we outline the considerable contribution that the MITE species of TE have made to expanding the miRNA repertoire of the angiosperms.

6.
Int J Mol Sci ; 23(23)2022 Nov 22.
Artículo en Inglés | MEDLINE | ID: mdl-36498868

RESUMEN

Cannabis sativa (Cannabis) has recently been legalized in multiple countries globally for either its recreational or medicinal use. This, in turn, has led to a marked increase in the number of Cannabis varieties available for use in either market. However, little information currently exists on the genetic distinction between adopted varieties. Such fundamental knowledge is of considerable value and underpins the accelerated development of both a nascent pharmaceutical industry and the commercial recreational market. Therefore, in this study, we sought to assess genetic diversity across 10 Cannabis varieties by undertaking a reduced representation shotgun sequencing approach on 83 individual plants to identify variations which could be used to resolve the genetic structure of the assessed population. Such an approach also allowed for the identification of the genetic features putatively associated with the production of secondary metabolites in Cannabis. Initial analysis identified 3608 variants across the assessed population with phylogenetic analysis of this data subsequently enabling the confident grouping of each variety into distinct subpopulations. Within our dataset, the most diagnostically informative single nucleotide polymorphisms (SNPs) were determined to be associated with the long-terminal repeat (LTRs) class of retroelements, with 172 such SNPs used to fully resolve the genetic structure of the assessed population. These 172 SNPs could be used to design a targeted resequencing panel, which we propose could be used to rapidly screen different Cannabis plants to determine genetic relationships, as well as to provide a more robust, scientific classification of Cannabis varieties as the field moves into the pharmaceutical sphere.


Asunto(s)
Cannabis , Alucinógenos , Cannabis/genética , Cannabis/química , Filogenia , Secuencias Repetidas Terminales , Análisis de Secuencia de ADN , Polimorfismo de Nucleótido Simple , Agonistas de Receptores de Cannabinoides , Variación Genética
7.
Plants (Basel) ; 11(19)2022 Sep 28.
Artículo en Inglés | MEDLINE | ID: mdl-36235411

RESUMEN

Most flowering plant species contain at least two copies of the DEFECTIVE EMBRYO AND MERISTEMS (DEM) gene with the encoded DEM proteins lacking homology to proteins of known biochemical function. In tomato (Sl; Solanum lycopersicum), stable mutations in the SlDEM1 locus result in shoot and root meristem defects with the dem1 mutant failing to progress past the cotyledon stage of seedling development. Generation of a Somatic Mutagenesis of DEM1 (SMD) transformant line in tomato allowed for the characterization of SlDEM1 gene function past the seedling stage of vegetative development with SMD plants displaying a range of leaf development abnormalities. Further, the sectored or stable in planta expression of specific regions of the SlDEM1 coding sequence also resulted in the generation of tomato transformants that displayed a range of vegetative development defects, which when considered together with the dem1 mutant seedling and SMD transformant line phenotypic data, allowed for the assignment of SlDEM1 gene function to early embryo development, adaxial epidermis cell development, lateral leaf blade expansion, and mesophyll cell proliferation and differentiation.

8.
Data Brief ; 42: 108032, 2022 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-35392627

RESUMEN

This article reports the proteomic legacy of in vivo exposure to the xenobiotic, acrylamide, on the epithelial cell population of the proximal segments of the mouse epididymis. Specifically, adult male mice were administered acrylamide (25 mg/kg bw/day) or vehicle control for five consecutive days before dissection of the epididymis. Epididymal epithelial cells were isolated from the proximal (caput) epididymal segment and subjected to quantitative proteomic analysis using multiplexed tandem mass tag (TMT) labeling coupled to mass spectrometry. Here, we report the data generated by this strategy, including the identification of 4405 caput epididymal epithelial cell proteins, approximately 6.8% of which displayed altered expression in response to acrylamide challenge. Our interpretation and discussion of these data features in the article "Acrylamide modulates the mouse epididymal proteome to drive alterations in the sperm small non-coding RNA profile and dysregulate embryo development".

9.
Plants (Basel) ; 10(12)2021 Nov 24.
Artículo en Inglés | MEDLINE | ID: mdl-34961041

RESUMEN

In plant cells, the molecular and metabolic processes of nucleic acid synthesis, phospholipid production, coenzyme activation and the generation of the vast amount of chemical energy required to drive these processes relies on an adequate supply of the essential macronutrient, phosphorous (P). The requirement of an appropriate level of P in plant cells is evidenced by the intricately linked molecular mechanisms of P sensing, signaling and transport. One such mechanism is the posttranscriptional regulation of the P response pathway by the highly conserved plant microRNA (miRNA), miR399. In addition to miR399, numerous other plant miRNAs are also required to respond to environmental stress, including miR396. Here, we exposed Arabidopsis thaliana (Arabidopsis) transformant lines which harbor molecular modifications to the miR396 and miR399 expression modules to phosphate (PO4) starvation. We show that molecular alteration of either miR396 or miR399 abundance afforded the Arabidopsis transformant lines different degrees of tolerance to PO4 starvation. Furthermore, RT-qPCR assessment of PO4-starved miR396 and miR399 transformants revealed that the tolerance displayed by these plant lines to this form of abiotic stress most likely stemmed from the altered expression of the target genes of these two miRNAs. Therefore, this study forms an early step towards the future development of molecularly modified plant lines which possess a degree of tolerance to growth in a PO4 deficient environment.

10.
Cell Rep ; 37(1): 109787, 2021 10 05.
Artículo en Inglés | MEDLINE | ID: mdl-34610313

RESUMEN

Paternal exposure to environmental stressors elicits distinct changes to the sperm sncRNA profile, modifications that have significant post-fertilization consequences. Despite this knowledge, there remains limited mechanistic understanding of how paternal exposures modify the sperm sncRNA landscape. Here, we report the acute sensitivity of the sperm sncRNA profile to the reproductive toxicant acrylamide. Furthermore, we trace the differential accumulation of acrylamide-responsive sncRNAs to coincide with sperm transit of the proximal (caput) segment of the epididymis, wherein acrylamide exposure alters the abundance of several transcription factors implicated in the expression of acrylamide-sensitive sncRNAs. We also identify extracellular vesicles secreted from the caput epithelium in relaying altered sncRNA profiles to maturing spermatozoa and dysregulated gene expression during early embryonic development following fertilization by acrylamide-exposed spermatozoa. These data provide mechanistic links to account for how environmental insults can alter the sperm epigenome and compromise the transcriptomic profile of early embryos.


Asunto(s)
Acrilamida/farmacología , Desarrollo Embrionario/efectos de los fármacos , Epidídimo/metabolismo , Proteoma/efectos de los fármacos , ARN Pequeño no Traducido/metabolismo , Espermatozoides/efectos de los fármacos , Animales , Embrión de Mamíferos/citología , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/metabolismo , Epidídimo/citología , Vesículas Extracelulares/metabolismo , Femenino , Masculino , Ratones , MicroARNs/metabolismo , Proteoma/metabolismo , ARN de Transferencia/metabolismo , Espermatozoides/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
11.
Front Plant Sci ; 12: 639014, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33859660

RESUMEN

Safflower (Carthamus tinctorius) is a member of the Asteraceae family that is grown in temperate climates as an oil seed crop. Most commercially grown safflower varieties can be sown in late winter or early spring and flower rapidly in the absence of overwintering. There are winter-hardy safflower accessions that can be sown in autumn and survive over-wintering. Here, we show that a winter-hardy safflower possesses a vernalization response, whereby flowering is accelerated by exposing germinating seeds to prolonged cold. The impact of vernalization was quantitative, such that increasing the duration of cold treatment accelerated flowering to a greater extent, until the response was saturated after 2 weeks exposure to low-temperatures. To investigate the molecular-basis of the vernalization-response in safflower, transcriptome activity was compared and contrasted between vernalized versus non-vernalized plants, in both 'winter hardy' and 'spring' cultivars. These genome-wide expression analyses identified a small set of transcripts that are both differentially expressed following vernalization and that also have different expression levels in the spring versus winter safflowers. Four of these transcripts were quantitatively induced by vernalization in a winter hardy safflower but show high basal levels in spring safflower. Phylogenetic analyses confidently assigned that the nucleotide sequences of the four differentially expressed transcripts are related to FLOWERING LOCUS T (FT), FRUITFUL (FUL), and two genes within the MADS-like clade genes. Gene models were built for each of these sequences by assembling an improved safflower reference genome using PacBio-based long-read sequencing, covering 85% of the genome, with N50 at 594,000 bp in 3000 contigs. Possible evolutionary relationships between the vernalization response of safflower and those of other plants are discussed.

12.
Proteomics ; 21(13-14): e2000079, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33792189

RESUMEN

Spermatozoa transition to functional maturity as they are conveyed through the epididymis, a highly specialized region of the male excurrent duct system. Owing to their transcriptionally and translationally inert state, this transformation into fertilization competent cells is driven by complex mechanisms of intercellular communication with the secretory epithelium that delineates the epididymal tubule. Chief among these mechanisms are the release of extracellular vesicles (EV), which have been implicated in the exchange of varied macromolecular cargo with spermatozoa. Here, we describe the optimization of a tractable cell culture model to study the mechanistic basis of sperm-extracellular vesicle interactions. In tandem with receptor inhibition strategies, our data demonstrate the importance of milk fat globule-EGF factor 8 (MFGE8) protein in mediating the efficient exchange of macromolecular EV cargo with mouse spermatozoa; with the MFGE8 integrin-binding Arg-Gly-Asp (RGD) tripeptide motif identified as being of particular importance. Specifically, complementary strategies involving MFGE8 RGD domain ablation, competitive RGD-peptide inhibition and antibody-masking of alpha V integrin receptors, all significantly inhibited the uptake and redistribution of EV-delivered proteins into immature mouse spermatozoa. These collective data implicate the MFGE8 ligand and its cognate integrin receptor in the mediation of the EV interactions that underpin sperm maturation.


Asunto(s)
Factor de Crecimiento Epidérmico , Vesículas Extracelulares , Animales , Antígenos de Superficie , Epidídimo , Factor VIII , Glucolípidos , Glicoproteínas , Gotas Lipídicas , Masculino , Ratones , Proteínas de la Leche , Espermatozoides
13.
Biomedicines ; 9(3)2021 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-33652704

RESUMEN

Cannabis sativa (Cannabis) is one of the world's most well-known, yet maligned plant species. However, significant recent research is starting to unveil the potential of Cannabis to produce secondary compounds that may offer a suite of medical benefits, elevating this unique plant species from its illicit narcotic status into a genuine biopharmaceutical. This review summarises the lengthy history of Cannabis and details the molecular pathways that underpin the production of key secondary metabolites that may confer medical efficacy. We also provide an up-to-date summary of the molecular targets and potential of the relatively unknown minor compounds offered by the Cannabis plant. Furthermore, we detail the recent advances in plant science, as well as synthetic biology, and the pharmacology surrounding Cannabis. Given the relative infancy of Cannabis research, we go on to highlight the parallels to previous research conducted in another medically relevant and versatile plant, Papaver somniferum (opium poppy), as an indicator of the possible future direction of Cannabis plant biology. Overall, this review highlights the future directions of cannabis research outside of the medical biology aspects of its well-characterised constituents and explores additional avenues for the potential improvement of the medical potential of the Cannabis plant.

14.
Reprod Fertil Dev ; 2021 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-33743842

RESUMEN

Information on the morphology and histology of the male reproductive system of the Crocodylia species is necessary to determine the role of these tissues in the production of functional spermatozoa. Accordingly, in this study we examined the gross morphology and microanatomy of the testis and the male excurrent duct system through which spermatozoa pass before ejaculation. The data demonstrate that the reproductive system in male saltwater crocodiles comprises paired testes, which convey spermatozoa distally via the rete testis into an excurrent duct system comprising ductuli efferentes, ductuli epididymides, ductus epididymidis and ductus deferens. The epithelium delineating the male tract was dominated by non-ciliated and ciliated cells structured into a simple columnar lining of the ductuli efferentes and ductuli epididymides, through to the high pseudostratified columnar epithelium of the ductus epididymidis and ductus deferens. The morphology and histochemical staining of these ducts suggest their involvement in seminal fluid production and/or its modification, which likely contributes to the nourishment, protection and/or storage of crocodile spermatozoa. As a reflection of their common Archosaurs ancestry, the overall structural characteristics we describe for the crocodile male excurrent duct system share closer similarities to those of the Aves than other clades within the Reptilia class or Mammalia.

15.
Plant J ; 106(4): 1058-1074, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33650173

RESUMEN

Cell wall invertase (CWIN) hydrolyses sucrose into glucose and fructose in the extracellular matrix and plays crucial roles in assimilate partitioning and sugar signalling. However, the molecular regulators controlling CWIN gene transcription remain unknown. As the first step to address this issue, we performed bioinformatic and transgenic studies, which identified a cohort of transcription factors (TFs) modulating CWIN gene expression in Arabidopsis thaliana. Comprehensive bioinformatic analyses identified 18 TFs as putative regulators of the expression of AtCWIN2 and AtCWIN4 that are predominantly expressed in Arabidopsis reproductive organs. Among them, MYB21, ARF6, ARF8, AP3 and CRC were subsequently shown to be the most likely regulators of CWIN gene expression based on molecular characterization of the respective mutant of each candidate TF. More specifically, the obtained data indicate that ARF6, ARF8 and MYB21 regulate CWIN2 expression in the anthers and CWIN4 in nectaries, anthers and petals, whereas AP3 and CRC were determined primarily to regulate the transcriptional activity of CWIN4. TF-promoter interaction assays demonstrated that ARF6 and ARF8 directly control CWIN2 and CWIN4 transcription with AP3 activating CWIN4. The involvement of ARF8 in regulating CWIN4 expression was further supported by the finding that enhanced CWIN4 expression partially recovered the short silique phenotype displayed by the arf8-3 mutant. The identification of the five TFs regulating CWIN expression serves as a launching pad for future studies to dissect the upstream molecular network underpinning the transcription of CWINs and provides a new avenue, potentially, to engineer assimilate allocation and reproductive development for improving seed yield.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Regulación de la Expresión Génica de las Plantas/genética , Factores de Transcripción/metabolismo , beta-Fructofuranosidasa/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Pared Celular/enzimología , Biología Computacional , Mutación , Fenotipo , Factores de Transcripción/genética , beta-Fructofuranosidasa/genética
16.
Reprod Fertil Dev ; 2021 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-33631095

RESUMEN

Conservation efforts to secure the long-term survival of crocodilian species would benefit from the establishment of a frozen sperm bank in concert with artificial breeding technologies to maintain genetic diversity among captive assurance populations. Working towards this goal, our research has focused on the saltwater crocodile Crocodylus porosus as a tractable model for understanding crocodilian sperm physiology. In extending our systematic characterisation of saltwater crocodile spermatozoa, in this study we examined the development of motility during sperm transport through the excurrent duct system of the male crocodile. The results show that approximately 20% of crocodile testicular spermatozoa are immediately motile but experience a gradient of increasing motility (percentage motile and rate of movement) as they transit the male reproductive tract (epididymis). Moreover, we confirmed that, as in ejaculated crocodile spermatozoa, increased intracellular cAMP levels promoted a significant and sustained enhancement of sperm motility regardless of whether the cells were isolated from the testis or epididymis. Along with the development of artificial reproductive technologies, this research paves the way for the opportunistic recovery, storage and potential utilisation of post-mortem spermatozoa from genetically valuable animals.

17.
Plants (Basel) ; 10(1)2021 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-33435199

RESUMEN

In recent decades, the presence of cadmium (Cd) in the environment has increased significantly due to anthropogenic activities. Cd is taken up from the soil by plant roots for its subsequent translocation to shoots. However, Cd is a non-essential heavy metal and is therefore toxic to plants when it over-accumulates. MicroRNA (miRNA)-directed gene expression regulation is central to the response of a plant to Cd stress. Here, we document the miRNA-directed response of wild-type Arabidopsis thaliana (Arabidopsis) plants and the drb1, drb2 and drb4 mutant lines to Cd stress. Phenotypic and physiological analyses revealed the drb1 mutant to display the highest degree of tolerance to the imposed stress while the drb2 mutant was the most sensitive. RT-qPCR-based molecular profiling of miRNA abundance and miRNA target gene expression revealed DRB1 to be the primary double-stranded RNA binding (DRB) protein required for the production of six of the seven Cd-responsive miRNAs analyzed. However, DRB2, and not DRB1, was determined to be required for miR396 production. RT-qPCR further inferred that transcript cleavage was the RNA silencing mechanism directed by each assessed miRNA to control miRNA target gene expression. Taken together, the results presented here reveal the complexity of the miRNA-directed molecular response of Arabidopsis to Cd stress.

18.
Front Endocrinol (Lausanne) ; 12: 799043, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-35356147

RESUMEN

Per-fluoroalkyl and polyfluoroalkyl substances (PFAS) are a diverse group of synthetic fluorinated chemicals used widely in industry and consumer products. Due to their extensive use and chemical stability, PFAS are ubiquitous environmental contaminants and as such, form an emerging risk factor for male reproductive health. The long half-lives of PFAS is of particular concern as the propensity to accumulate in biological systems prolong the time taken for excretion, taking years in many cases. Accordingly, there is mounting evidence supporting a negative association between PFAS exposure and an array of human health conditions. However, inconsistencies among epidemiological and experimental findings have hindered the ability to definitively link negative reproductive outcomes to specific PFAS exposure. This situation highlights the requirement for further investigation and the identification of reliable biological models that can inform health risks, allowing sensitive assessment of the spectrum of effects of PFAS exposure on humans. Here, we review the literature on the biological effects of PFAS exposure, with a specific focus on male reproduction, owing to its utility as a sentinel marker of general health. Indeed, male infertility has increasingly been shown to serve as an early indicator of a range of co-morbidities such as coronary, inflammatory, and metabolic diseases. It follows that adverse associations have been established between PFAS exposure and the incidence of testicular dysfunction, including pathologies such as testicular cancer and a reduction in semen quality. We also give consideration to the mechanisms that render the male reproductive tract vulnerable to PFAS mediated damage, and discuss novel remediation strategies to mitigate the negative impact of PFAS contamination and/or to ameliorate the PFAS load of exposed individuals.


Asunto(s)
Fluorocarburos , Neoplasias de Células Germinales y Embrionarias , Neoplasias Testiculares , Fluorocarburos/toxicidad , Humanos , Masculino , Reproducción , Análisis de Semen
19.
Int J Mol Sci ; 21(21)2020 Oct 23.
Artículo en Inglés | MEDLINE | ID: mdl-33114207

RESUMEN

Arabidopsis thaliana (Arabidopsis) has been used extensively as a heterologous system for molecular manipulation to genetically characterize both dicotyledonous and monocotyledonous plant species. Here, we report on Arabidopsis transformant lines molecularly manipulated to over-accumulate the small regulatory RNA microRNA397 (miR397) from the emerging C4 monocotyledonous grass model species Setaria viridis (S. viridis). The generated transformant lines, termed SvMIR397 plants, displayed a range of developmental phenotypes that ranged from a mild, wild-type-like phenotype, to a severe, full dwarfism phenotype. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR)-based profiling of the SvMIR397 transformant population revealed a strong correlation between the degree of miR397 over-accumulation, repressed LACCASE (LAC) target gene expression, reduced lignin content, and the severity of the developmental phenotype displayed by SvMIR397 transformants. Further, exposure of SvMIR397 transformants to a 7-day regime of salt stress revealed the SvMIR397 transformant lines to be more sensitive to the imposed stress than were wild-type Arabidopsis plants. Taken together, the findings reported here via the use of Arabidopsis as a heterologous system show that the S. viridis miR397 small regulatory RNA is able to repress the expression of three Arabidopsis LAC genes which led to reduced lignin content and increased salt stress sensitivity.


Asunto(s)
Arabidopsis/genética , Lacasa/genética , MicroARNs/genética , Setaria (Planta)/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Lignina/metabolismo , Fenotipo , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , ARN de Planta/genética , Estrés Salino
20.
Mol Cell Endocrinol ; 517: 110955, 2020 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-32783903

RESUMEN

Competition to achieve paternity has coerced the development of a multitude of male reproductive strategies. In one of the most well-studied examples, the spermatozoa of all mammalian species must undergo a series of physiological changes as they transit the male (epididymal maturation) and female (capacitation) reproductive tracts prior to realizing their potential to fertilize an ovum. However, the origin and adaptive advantage afforded by these intricate processes of post-testicular sperm maturation remain to be fully elucidated. Here, we review literature pertaining to the nature and the physiological role of epididymal maturation and subsequent capacitation in comparative vertebrate taxa including representative species from the avian, reptilian, and mammalian lineages. Such insights are discussed in terms of the framework they provide for helping to understand the evolutionary significance of post-testicular sperm maturation.


Asunto(s)
Análisis Mutacional de ADN/métodos , Hipogonadismo/genética , Síndrome de Kallmann/genética , Técnicas de Diagnóstico Molecular , Mutación , Adolescente , Niño , Femenino , Heterogeneidad Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Hipogonadismo/congénito , Hipogonadismo/diagnóstico , Hipogonadismo/fisiopatología , Sistema Hipotálamo-Hipofisario/fisiopatología , Síndrome de Kallmann/fisiopatología , Masculino , Medicina de Precisión , Testículo/fisiopatología , Secuenciación del Exoma
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