-
Theriogenology Apr 2015We report, for the first time, a series of baseline techniques comprising isolation and transplantation of female and male early-stage germ cells in sturgeon to generate...
We report, for the first time, a series of baseline techniques comprising isolation and transplantation of female and male early-stage germ cells in sturgeon to generate a germline chimera as a potential tool for surrogate reproduction and gene banking. Cells were dissociated from testis, characterized by mostly spermatogonia, and from ovary, exclusively comprising oogonia and previtellogenic oocytes, of Acipenser baerii, using 0.3% trypsin (2 hours, 23 °C) dissolved in PBS, isotonic with blood plasma. The dissociated germ cells were sorted by Percoll gradient centrifugation followed by immunolabeling with germ cell-specific vasa antibody DDX4, while 10% to 30% Percoll solution contained 79.4% and 70.8% labeled testicular and ovarian cells. Sorted germ cells were transplanted into a cavity close to a presumptive genital ridge of newly hatched heterospecific Acipenser ruthenus larvae with fluorescein isothiocyanate-labeled endogenous primordial germ cells. The transplanted germ cells were randomly distributed in the body cavity through 30-day posttransplantation (dpt). Subsequently, the cells were organized into genital ridges 50 dpt and proliferated 90 dpt. The number of both transplanted and endogenous germ cells significantly increased from 18.1, 22.2, and 29.1 (30 dpt) to 108.5, 90.8, and 118.5 (90 dpt) in ovarian, testicular, and endogenous germ cells, respectively (P < 0.05). The efficiency of transplantation was 60% (counted 90 dpt).
Topics: Animals; Female; Fishes; Germ Cells; Male; Ovary; Sexual Maturation; Testis
PubMed: 25559841
DOI: 10.1016/j.theriogenology.2014.12.010 -
Plant Disease Sep 2023Tobacco (Nicotiana tabacum L.) is an important economic crop that is widely grown around the world. Its annual production in China is estimated at 2.2 million tons...
Tobacco (Nicotiana tabacum L.) is an important economic crop that is widely grown around the world. Its annual production in China is estimated at 2.2 million tons (Berbeć and Matyka 2020). Since 2022, a root rot disease was sporadically observed on tobacco seedlings on cultivar Yunyan 87 in cultivated tobacco fields in the Hunan province of China. A disease incidence of about 10% occurred across 48 ha of tobacco fields. The affected tobacco plants had slow and stunted growth with yellowing leaves. The roots turned grayish brown, decayed, and died. Diseased roots were collected from six fields and cut into small pieces (5 mm ×5 mm) from the edge of the rotted portions, and then sterilized with 70% ethanol for 10 s, 0.1% HgCl2 for 1 min, and washed in sterilized water three times. All the sterilized tissue were placed on potato dextrose agar (PDA) medium and cultured at 26 ℃ in the dark. About 3 days later, colonies with similar morphology were removed and sub-cultured on fresh PDA. A total of six strains were obtained from six tobacco samples. Strains were white and had radial growth on PDA. Hyphae were aseptate and the sporangia were filamentous. The oogonia were subglobose, smooth, 16.04 ± 0.25 µm (n=50) in diameter, and developed on unbranched stalks. The antheridia were barrel shaped and clavate. Oospores were globose, aplerotic or nearly plerotic, measuring 6.62 ± 0.33 µm (n=50). These morphological characteristics were consistent with the description of Pythium spp. (van der Plaats-Niterink 1981). For molecular identification, the internal transcribed spacer (ITS) region of rDNA and cytochrome c oxidase subunit I (Cox I) of a representative isolate, GF-3, were amplified and sequenced (GenBank accession nos. OR228424 for ITS and OR237556 for Cox I) using universal primers ITS1/ITS4 (White et al. 1990) and FM58/FM66, respectively (Villa et al. 2006). BLASTn analysis revealed that the ITS and Cox I sequences were 99.76 % (838/840 bp) and 99.85% (671/672 bp) identical to the corresponding sequences of P. dissotocum strain CBS 166.68 (AY598634.2) and UM982 (MT981147.1), respectively. A neighbor-joining phylogenetic tree based on the Cox I sequence showed that GF-3 grouped in the P. dissotocum branch. Based on morphological and molecular characteristics, GF-3 was identified to be P. dissotocum. For pathogenicity testing, four- to five-leaf-old healthy potted tobacco seedlings of the Yunyan 87 cultivar were inoculated with a zoospore suspension (1 × 105 zoospores/ml), which was induced on V8-juice medium. The zoospore suspension was introduced into the soil around plant roots and 10 mL of inoculum was used for each plant. In the control group, plants were inoculated with sterilized water. All of the treated plants were kept in humid chambers at 26°C under a 12 h/12 h photoperiod. The pathogenicity assays were performed twice, with each treatment having three replicated plants. After 5 days, tobacco seedlings inoculated with P. dissotocum showed symptoms resembling that observed in the field. However, the control plants remained healthy. Pythium dissotocum was re-isolated from the infected plants and identified by morphological and molecular methods, thus confirming Koch's postulates. Pythium dissotocum has been reported causing root rot in other plants, including hydroponic lettuce (McGehee et al. 2018) and spinach (Huo et al. 2020). Also, many Pythium species have recently been recovered from float-bed tobacco transplant production greenhouses (Zhang et al. 2022). However, to our knowledge, this is the first report of root rot on tobacco caused by P. dissotocum in China. Since this disease could greatly affect tobacco seedling establishment in the field, appropriate management strategies need to be developed to reduce further losses in tobacco planting fields.
PubMed: 37732900
DOI: 10.1094/PDIS-07-23-1303-PDN -
Biomolecules Jun 2023, a proto-oncogene, regulates oocyte maturation by activating the classical MAPK pathway in cells. To examine the function of in , was identified in this study. The...
, a proto-oncogene, regulates oocyte maturation by activating the classical MAPK pathway in cells. To examine the function of in , was identified in this study. The full-length cDNA of was 2213 bp, including 144 bp in the 5' UTR, 923 bp in 3' the UTR, and 1146 bp in the open reading frame (ORF) region. During early gonad development, the expression of from 4 to 6 months of age in was significantly higher than that in other months, with the highest expression in 6-month-old , suggesting that may be involved in early gonadal development in . Clear hybridization signals were found by in situ hybridization in the oocytes, oocyte nucleus and oogonium, and a small number of hybridization signals were found in the follicular wall of the male gonads. In addition, the RNA interference (RNAi) assay results showed that the knockdown of caused a down-regulation of and . In summary, these results indicate that has a crucial part to play in gonadal development in .
Topics: Male; Animals; Amino Acid Sequence; Base Sequence; Unionidae; Bivalvia; Gonads
PubMed: 37371511
DOI: 10.3390/biom13060931 -
Life (Basel, Switzerland) Aug 2022Spinyhead croaker () is an economically important fish suffering from population decline caused by overfishing and habitat destruction. Researches on the development of...
Spinyhead croaker () is an economically important fish suffering from population decline caused by overfishing and habitat destruction. Researches on the development of primordial germ cell (PGC) and reproduction biology were an emergency for the long-term conservation of the involved species. () gene plays an indispensable role in PGC specification, maintenance, and development. In the current study, we report the cloning and expression patterns of in (). RT-PCR analysis revealed that was specifically expressed in both sexual gonads. In the ovary, RNA was uniformly distributed in the oocytes and abundant in oogonia, and gradually decreased with oogenesis. A similar expression pattern was also detected in testis. Dual fluorescent in situ hybridization of and demonstrated that they almost had the same distribution except in oocytes at stage I, in which the RNA aggregated into some particles. Furthermore, 3' UTR was sufficient to guide the Green Fluorescent Protein (GFP) specifically and stably expressed in the PGCs of medaka. These findings offer insight into that is an evolutionarily conserved germline-specific gene and even a potential candidate for PGC manipulation in .
PubMed: 36013405
DOI: 10.3390/life12081226 -
Archives of Razi Institute Jun 2022Dexamethasone (DEX), which is a corticosteroid hormone (glucocorticoid), has been used to treat different conditions, such as immune system disorders,...
Dexamethasone (DEX), which is a corticosteroid hormone (glucocorticoid), has been used to treat different conditions, such as immune system disorders, certain skin and eye disorders, as well as breathing problems. Cefotaxime sodium, also called Claforan, is synthesized from a naturally occurring material (semisynthetic). It is a broad-spectrum cephalosporin antibiotic that could be utilized for parenteral administration. The present study aimed to investigate histological changes occurring in the tissues and cells of the rats' ovary (primordial, primary, secondary, antral, and mature follicle) treated with Cefotaxime sodium, as well as DEX, and evaluate the impacts of these medications on animals' fertility. In total, 40 female adult Wistar rats were divided into four groups (n=10). The control group received 0.5 ml/kg of distilled water daily for five days as a placebo. The second group was injected with 0.5 mg/kg of DEX daily for five days. The same amount of Claforan (0.5 mg/kg) was injected into the third group daily for five days, and the fourth group received 0.5 mg/kg of both Claforan and DEX daily for five days. Afterward, the ovaries were prepared for histological examination. The ImageJ image analysis system was used to detect morphometric parameters and calculate the area of these organs. The findings of the present study showed that the DEX and Claforan brought changes to the ovarian area and the number of follicles. The ovarian area significantly increased (<0.007) in the DEX-treated group (mean±SEM=7.3±0.5 mm), compared to the control group (mean±SEM=4.6±0.20 mm). However, DEX was found to decrease body weight. Furthermore, the ovarian area significantly increased in the Claforan-treated group (mean±SEM=8.6±0.6 mm); however, their body weight significantly decreased (<0.008), in comparison with the control and DEX-treated groups. The combination treatment (i.e., DEX + Cefotaxime sodium) significantly increased (<0.009) the area of ovaries even more, compared to single treatments (mean±SEM=9.6±0.4 mm). Overall, both DEX and Claforan brought histological changes to ovaries. However, the effect of DEX on ovaries was less than that of Claforan. The concurrent administration of both medications was found to have more significant effects on rats' ovaries.
Topics: Rats; Female; Animals; Ovary; Cefotaxime; Rats, Wistar; Ovarian Follicle; Dexamethasone
PubMed: 36618313
DOI: 10.22092/ARI.2022.357511.2050 -
Plant Disease May 2022Rice ( L.) is the principle staple crops in the World and its production can be severely damaged by species. Several species including , , , , , have been recorded to...
Rice ( L.) is the principle staple crops in the World and its production can be severely damaged by species. Several species including , , , , , have been recorded to cause rice seedling root rot in Taiwan (List of Plant Diseases in Taiwan edited by Tzean et al., 2019). During the survey of rice seedling diseases, we identified a new species of that causes seedling root rot on rice in commercial nursery trays in two nursery fields in 2019 in Taichung, Taiwan. Stunting and root rot symptom were found on the affected plants and up to 20% seedlings in a nursery tray showed similar symptoms. To isolate the pathogen, symptomatic roots were surface sterilized with 75% ethanol for 1 min and rinsed in sterile water. The margin of lesion was cut off, placed on 1.5% water agar and incubated at 28 ℃. After 24 h, the hyphal tips of a white colony growing from the diseased region were transferred to potato dextrose agar (PDA) medium. Koch's postulates were fulfilled by inoculating the germinated rice seeds with mycelia. Rice seeds of var. Tainan11 (TN11) were treated with 75% ethanol and then 1.2% NaOCl for 15 min. The sterilized seeds were soaked in sterile water under dark condition for 3 days and the water was replaced every day. Five of the pre-germinated seeds with 2~5 mm embryonic shoot were placed in a sterile petri-dish and inoculated with 3-ml mycelial suspension (OD = 0.045) prepared by blending the mycelia of a 3-days PDA culture using an Oster 10 speed blender 6640 (Oster, USA). The seeds-mycelia were then covered with sterilized soil mixture of Akadama soil and rice husk (1:1, volume to volume) and incubated in a growth chamber at 28 ℃. Seven days post-inoculation, the inoculated seedlings showed stunting with short and necrotic roots (Fig. S1). The pathogen was reisolated from the diseased seedlings and identified with morphology and molecular methods. For morphological characterization, the pathogen was cultured on V8 agar to produce oogonia and zoospore (Chamswarng and Cook 1985). Globose oogonia with multiple antheridia (1-5 per oogonium), inflated filamentous sporangia, vesicle with abundant zoospores, main hypha with up to 6.57 μm wide and mature aplerotic oospores with diameter 24.35-30.81 μm (average= 27.22 μm; n=20) were observed (Fig. S1) that are similar to the descriptions for (van der Plaats-Niterink 1981). Genomic DNA was extracted with CTAB method (Wang and White 1997) and the sequences of the internal transcribed spacer (ITS) region and gene region of β-tubulin () and cytochrome c oxidase subunit II ( II) were amplified with published primers (Villa et al., 2006). The obtained sequences were submitted to GenBank (accession nos: OL701302 (ITS), OL763269 (tub), and OL763270 (cox II); Fig. S2). Phylogenetic relationships between this pathogen and other 55 isolates, including the type species of (ATCC11101), were conducted with the concatenated sequences of tub and cox II and analyzed by Bayesian interference (Fig. S3). Based on the tree built with and II sequences, this pathogen was identified as that has not been reported in rice and other plants in Taiwan. It was observed in laboratory assays that this pathogen caused significant root-rot symptoms on several major rice varieties grown in Taiwan, including TN11, Tainung67 and Kaoshiung139. It may potentially cause severe crop loss in rice production, especially in nurseries. This identification provides important information on rice disease management.
PubMed: 35596245
DOI: 10.1094/PDIS-01-22-0092-PDN -
Plant Disease Mar 2021Corn ( L.) stalk rot, caused by various pathogens, is one of the most prevalent corn diseases worldwide. In October 2019, a survey was carried out to determine...
Corn ( L.) stalk rot, caused by various pathogens, is one of the most prevalent corn diseases worldwide. In October 2019, a survey was carried out to determine pathogenic fungi causing corn stalk rot in 3 fields (~120 ha) in Harbin city (44.04°N 125.42°E), Heilongjiang Province, China. In each field, 100 plants at 5 sampling points were assessed at the milk stage (R3) of development. Disease incidence was 12%. Symptomatic plants showed rapid death of the upper leaves, drooping ears and stalks were soft, hollow, watersoaked with white hyphae present on teh outside of the stalk. Pieces of tissue (0.25 cm) from 15 individual diseased stalks (5 plants/field) were surface disinfested in 0.5% NaOCl for 5 min, rinsed three times in sterile distilled water and cultured on potato dextrose agar (PDA) containing streptomycin (50 μg/mL). After three days of incubation, a total of twelve fungal cultures with uniform characteristics were isolated and subcultured by transferring hyphal tips onto V8. Colonies on V8 selective medium were creamy white and floccus, with a growth rate of 20 mm/day at 26°C in darkness. Oospores were mostly plerotic, and oogonia walls were 1.3 to 2.7 μm thick ( = 50); globose oogonia, 23.9 to 30.5 μm in diameter ( = 50), and had 1 to 8 antheridia. Based on these characteristics, the isolates were identified as sp. (van der Plaats-Niterink 1981). Genomic DNA was extracted from single conidial cultures of representative isolates (MZYJF1, MZYJF3 and MZYJF7), and the internal transcribed spacer () region and cytochrome coxidase subunit II () gene were amplified and sequenced using the primers ITS1/ITS4 (Yin et al. 2012) and COX2f/COX2r (Hudspeth et al. 2000), respectively. Partial nucleotide sequences of 796 bp and 573 bp for the and amplicons, respectively, were obtained and deposited in GenBank (accession no. MW447501 for , and MW471006 for ). MegaBLAST analysis of the and sequences of MZYJF1 isolate showed 100% similarity with sequences from strain ATCC 11101. The isolates were identified as based on the fact that P. aristosporum has aplerotic oospores and less antheridia per oogonium than (van der Plaats-Niterink 1981). A pathogenicity test was performed on corn cv. Xianyu 335 at tasseling stage (VT) in the field. An oospore suspension, obtained from isolate MZYJF1 grown on V8 agar media for 4 weeks (Green and Jensen, 2000) and diluted to 1×10 oospores/mL using blood cell counting method, was injected into the base of the maize stems of 6 healthy plants (1.5 ml/plant ) using a syringe. Control plants were injected with distilled sterile water. All inoculated plants showed symptoms 25 days after inoculation that were similar to those observed in the field. The oomycete of was reisolated from symptomatic plants on V8 agar media and identified according to morphological and molecular characteristics. No symptoms were observed on the control plants. has previously been reported on causing damping-off of pea in the Columbia basin of Central Washington (Alcala et al. 2016) and on soybean in North Dakota (Zitnick-Anderson and Nelson 2015). To our knowledge, this is the first report of causing corn stalk rot in China. Corn stalk rot caused by poses a threat to significantly reduce the quality of corn. Thus, its distribution needs to be investigated and effective disease management strategies developed.
PubMed: 33728961
DOI: 10.1094/PDIS-01-21-0164-PDN -
Plant Disease Feb 2023Globisporangium sylvaticum (syn. Pythium sylvaticum), is an oomycete that causes root rot and damping off of field crops, ornamentals, and vegetables. Several species in...
Globisporangium sylvaticum (syn. Pythium sylvaticum), is an oomycete that causes root rot and damping off of field crops, ornamentals, and vegetables. Several species in Pythiaceae are associated with black root rot of strawberry [(Fragaria × ananassa) Duchesne] (Millner 2006). Mature, stunted 'Chandler' strawberry plants, with reduced shoot vigor and black necrotic roots, were collected from Rhea County (June 2018) and Cumberland County, TN (May 2019). Aboveground symptoms occurred in low incidence (<5% of plants) in the fields. Plant roots were rinsed with tap water, cut into 1 to 3 cm pieces, and surface-disinfested (70% ethanol, 1 min) followed by a sterile water rinse. Root segments were crushed, placed on 20% V8 juice agar, and incubated in the dark at 21°C for 3 days. White fluffy mycelia grew from a majority of roots and coenocytic hyphae with globose hyphal swellings, delimited from hyphae by septa, were observed with microscopy. Hyphae were initially branched, curled, hyaline, and aseptate; however, septations were observed in older cultures. Globose structures (terminal and intercalary) were identified as sporangia [11 to 32 (avg. 22.1) µm diameter] when zoospores were observed (Parikh et al. 2022). Oospores [9 to 21 (avg. 16) μm diameter] were globose, smooth, aplerotic, and thick-walled. Oogonia, with or without one or more inflated antheridia, were observed when isolates were paired in culture, characteristics consistent with descriptions of Campbell and Hendrix (1967), Pratt and Green (1971), van der Plaats-Niterink (1981), and Uzuhashi et al. (2010). Genomic DNA was extracted (Extract-N-Amp™; Sigma-Aldrich, MO) for PCR amplification of internal transcribed spacer (ITS) regions of rDNA with primers ITS1/ITS4 (White et al. 1990); ITS and large subunit rRNA regions with primers UN-up18S42/UN-lo28S22 (Robideau et al. 2011); and cytochrome c oxidase subunit I (COI) mitochondrial DNA with primers OomCoxI-Levup/OomCoxI-Levlo (Robideau et al. 2011). Primers ITS1/ITS4 were used to amplify isolate TN (GenBank Accession MW386310, which had 100% homology with reference isolate MK326528). Primers UN-up18S42/UN-lo28S22 amplified isolates SAP18 and OO1 (Accessions MZ881935 and MZ881936, which had 99.8% homology with HQ665236), and COI primers amplified isolate SAP18 (Accession OK020192, which had 100% homology with GU071816 and KT692835). To satisfy Koch's postulates, inoculum of G. sylvaticum grown on autoclaved wheat seeds was added (5% w/v) to planting mix (1 peat:1 sand, v/v). Young, rooted strawberry plants were planted in 1.2-L pots with infested (n = 6) and control (no pathogen, n = 6) mixes, which was saturated with deionized water. Pots were covered with clear plastic for 48 h to maintain high humidity. Plants were grown in a greenhouse (24°C avg.) for 8 weeks. The disease assay was repeated. All plants in infested mix died, with black, necrotic roots. Plants in the control mix were healthy and well-established. The pathogen was reisolated from roots of all inoculated plants and confirmed to be G. sylvaticum based on morphology and molecular analyses. Root disease of strawberry caused by G. sylvaticum has been reported in the USA (Campbell and Hendrix 1967; Nemec and Sanders 1970; Pratt and Green 1971). This is the first report of G. sylvaticum causing root rot of strawberry in Tennessee. With the loss of methyl bromide, sustainable disease control strategies are needed to provide effective management options for strawberry black root rot.
PubMed: 36825322
DOI: 10.1094/PDIS-01-23-0007-PDN -
Ecotoxicology and Environmental Safety Mar 2021This study aimed to explore the toxicity of environmental residues of graphene oxide nanoparticles (GONPs) to reproduction of Lepidopteron insects using both ovary cell...
This study aimed to explore the toxicity of environmental residues of graphene oxide nanoparticles (GONPs) to reproduction of Lepidopteron insects using both ovary cell line (BmN) and individual female Bombyx mori as the research subjects. The results showed that GONPs dose dependently affect BmN cells. At higher concentrations (>25 mg/L), GONPs led to oxidative stress, ROS accumulation and DNA damage in BmN cells and significantly reduced their survival rate (p ≤ 0.05). Moreover, feeding female B. mori larvae with mulberry leaves treated with 25 mg/L GONPs significantly decreased their gonadosomatic index (GSI) by 40.84%, and increased oxidation levels and antioxidant enzyme activity in silkworm ovary tissues. Pathological analysis found that exposure to GONPs decreased the numbers of both oogonia and oocytes in ovarian tissues, increased the formation of peroxisome and vacuoles in follicle cells, reduced the transcription of genes (Vg, Ovo, Sxl-s, Sxl-l, and Otu) related to ovarian development in B. mori by 0.61, 0.65, 0.75, 0.72, and 0.42-fold, respectively, and lowered the amount of spawning by 52.25%. Overall, these results revealed that GONPs exposure is toxic to the reproduction of B. mori. The underlying mechanism is that oxidative stress due to GONPs causes oxidative damage to DNA, damages ovarian tissues, as well as hinders B. mori development and spawning. Thus, this study provides important experimental data for safety evaluation of reproductive toxicity due to GONPs exposure.
Topics: Animals; Bombyx; Cell Line; DNA Damage; Female; Graphite; Male; Nanoparticles; Oocytes; Oxidative Stress; Reactive Oxygen Species; Reproduction
PubMed: 33421719
DOI: 10.1016/j.ecoenv.2020.111888 -
International Journal of Molecular... Sep 2021, a de novo methyltransferase, is essential for mammalian germ line DNA methylation. Only one is identified in mammals, and homozygous mutants of are lethal, while two...
, a de novo methyltransferase, is essential for mammalian germ line DNA methylation. Only one is identified in mammals, and homozygous mutants of are lethal, while two paralogs, and , are identified in teleosts due to the third round of genome duplication, and homozygous mutants of and are viable in zebrafish. The expression patterns and roles of and in gonadal development remain poorly understood in teleosts. In this study, we elucidated the precise expression patterns of and in tilapia gonads. was highly expressed in oogonia, phase I and II oocytes and granulosa cells in ovaries and spermatogonia and spermatocytes in testes, while was mainly expressed in ovarian granulosa cells and testicular spermatocytes. The mutation of and was achieved by CRISPR/Cas9 in tilapia. Lower gonadosomatic index (GSI), increased apoptosis of oocytes and spermatocytes and significantly reduced sperm quality were observed in mutants, while normal gonadal development was observed in mutants. Consistently, the expression of apoptotic genes was significantly increased in mutants. In addition, the 5-methylcytosine (5-mC) level in gonads was decreased significantly, compared with that of and wild type (WT) gonads. Taken together, our results suggest that , not , plays important roles in maintaining gametogenesis in teleosts.
Topics: Animals; Cichlids; DNA Methylation; DNA Modification Methylases; Female; Gene Expression Regulation, Developmental; Male; Ovary; Testis
PubMed: 34576333
DOI: 10.3390/ijms221810170