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American Journal of Translational... 2022To detect mRNA and protein expression of meiosis-specific genes in human umbilical cord mesenchymal stem cells (hUMSCs) in an co-culture microenvironment with mouse...
OBJECTIVE
To detect mRNA and protein expression of meiosis-specific genes in human umbilical cord mesenchymal stem cells (hUMSCs) in an co-culture microenvironment with mouse primordial germ cells (PGCs), and to further explore the effective potential of hUMSCs to differentiate into PGCs.
METHODS
HUMSCs were obtained from human Wharton's jelly fragments by adherent culture. PGCs were derived from 12.5 days post-coitum (dpc) BalbC mice. Then hUMSCs were co-cultured with PGCs in Matrigel, inside or outside of a culture chamber, respectively. The changes in morphology and cytogenetic characteristics were observed. SCP3 and DDX4 expression in hUMSCs were detected and analyzed using immunofluorescence staining. Oct-4, Stra8, Zp3 and Dmc1 gene expressions in PGCs, hUMSCs, and hUMSCs after co-culture with PGCs were analyzed by real time reverse transcription-polymerase chain reaction.
RESULTS
Both hUMSCs and PGCs expressed Oct-4 at different degrees. After co-culture with PGCs, hUMSCs became rounded and showed AKP activity. HUMSCs suspension-cultured in Matrigel or adherent cultured with cell chamber significantly expressed Stra8, DMC1, SCP3 and DDX4 genes.
CONCLUSION
HUMSCs can be induced to express PGC-specific genes Stra8 and DMC1, spermatogonium/oogonium-specific genes SCP3 and DDX4 that predict directed differentiation potential into early germ cells at a molecular level.
PubMed: 36628204
DOI: No ID Found -
Plant Disease Sep 2022Cork oak (Quercus suber L.) is an evergreen tree native to SW Europe and NW Africa. It covers 2·106 ha in the western Mediterranean basin, forms heterogeneous forest...
Cork oak (Quercus suber L.) is an evergreen tree native to SW Europe and NW Africa. It covers 2·106 ha in the western Mediterranean basin, forms heterogeneous forest ecosystems and represents an important source of income derived from cork production. While in Iberia, Italy, Tunisia and Algeria, drought and several endemic pathogens have been associated with cork oak decline (Moricca et al. 2016; Smahi et al. 2017), in Morocco there is no evidence, apart from overgrazing and human intervention (Fennane and Rejdali 2015), of a pathogen associated with oak decline. In December 2019, extensive dieback and mortality of 60-year-old cork oak trees were observed in a natural stand of ca 150 ha located 5 km east from Touazithe, in Maâmora forest, Morocco (34°13'38''N, 6°14'51''W - 87 m a.s.l.). Two years before, Q. suber seedlings from a local nursery were planted to increase tree density. Symptoms in trees and planted seedlings included chlorosis, reddish-brown discoloration of the whole crown and dieback starting in the upper crown. Root rot and lack of fine roots were observed. Tree mortality was estimated at ca 30%, and disease incidences of trees and seedlings were 45 and 70%, respectively. A Phytophthora species was consistently isolated from the rhizosphere of 3 symptomatic trees randomly selected at the site using leaves as bait (Jung et al. 1996). On carrot agar Phytophthora colonies were uniform and cottonwool-like. Sporangia were typically terminal, with ovoid, and obpyriform shape, mostly papillate, measuring 30.7 ± 4.7 µm length and 22.7 ± 4.1 µm wide. Oogonia were produced in single culture, and they were globose to subglobose, elongated to ellipsoid, 32.1 ± 2.9 µm in diameter and 46.1 ± 4.8 µm in length. Oospores were usually spherical, thick-walled, and measured 28.1 ± 2.4 µm. Antheridia were paragynous, mostly spherical, measuring 12.2 ± 1.4 µm. Isolates had minimum and maximum temperatures of 5 °C and 30 °C, respectively, and a growth optimum at 20 °C. Apart from the small size of sporangia, features were typical of Phytophthora quercina Jung. The identity of a representative strain (TJ1500) was corroborated by sequencing the ITS and mitochondrial cox1 gene regions, and BLAST search in GenBank showed 100% homology with sequences of the ex-type culture of P. quercina (KF358229 and KF358241 accessions, respectively). Both sequences of the representative isolate were submitted to GenBank (accessions OP086243 and OP290549). The strain TJ1500 is currently stored within the culture collections of the Mendel University in Brno and the University of Sassari. Its pathogenicity was verified and compared with a P. cinnamomi strain in a soil infestation test with one-year-old cork oak seedlings (Corcobado et al. 2017). Five months after inoculation, the symptoms described were observed in the seedlings, and fine root weight of plants inoculated with the TJ1500 strain and P. cinnamomi was reduced by 19 and 42%, respectively, in relation to non-inoculated controls. The pathogen was re-isolated from the necrotic roots, thus fulfilling Koch's postulates. So far, P. quercina has been reported associated with chronic mortality of cork oak in new plantations in Spain (Martín-García et al. 2015; Jung et al. 2016) and natural forests in Italy (Seddaiu et al. 2020). To our knowledge this is the first report of P. quercina in Morocco. Givenat Morocco is an important cork producing country, our finding warns about the risk this pathogen poses to Q. suber and other North African oaks.
PubMed: 36167516
DOI: 10.1094/PDIS-08-22-1795-PDN -
Reproduction (Cambridge, England) Mar 2021The germ cell lineage ensures the creation of new individuals and perpetuates the genetic information across generations. Primordial germ cells are pioneers of gametes...
The germ cell lineage ensures the creation of new individuals and perpetuates the genetic information across generations. Primordial germ cells are pioneers of gametes and exist transiently during development until they differentiate into oogonia in females, or spermatogonia in males. Little is known about the molecular characteristics of primordial germ cells in cattle. By performing single-cell RNA-sequencing, quantitative real-time PCR, and immunofluorescence analyses of fetal gonads between 40 and 90 days of fetal age, we evaluated the molecular signatures of bovine germ cells at the initial stages of gonadal development. Our results indicate that at 50 days of fetal age, bovine primordial germ cells were in the early stages of development, expressing genes of early primordial germ cells, including transcriptional regulators of human germline specification (e.g. SOX17, TFAP2C, and PRDM1). Bovine and human primordial germ cells also share expression of KIT, EPCAM, ITGA6, and PDPN genes coding for membrane-bound proteins, and an asynchronous pattern of differentiation. Additionally, the expression of members of Notch, Nodal/Activin, and BMP signaling cascades in the bovine fetal ovary, suggests that these pathways are involved in the interaction between germ cells and their niche. Results of this study provide insights into the mechanisms involved in the development of bovine primordial germ cells and put in evidence similarities between the bovine and human germline.
Topics: Animals; Cattle; Cell Differentiation; Cell Lineage; Female; Germ Cells; Gonads; Humans; Male; Sequence Analysis, RNA; Spermatogonia
PubMed: 33275120
DOI: 10.1530/REP-20-0313 -
Plant Disease May 2022Sanqi (Panax notoginseng (Burk.) F. H. Chen) is a precious traditional Chinese herbal medicine. During April of 2021, a root rot disease with approximate 15% incidence...
Sanqi (Panax notoginseng (Burk.) F. H. Chen) is a precious traditional Chinese herbal medicine. During April of 2021, a root rot disease with approximate 15% incidence was observed on 2-year-old Sanqi plants in a field of Zhouning (27º12' N, 119°33' E), Fujian Province of China. The disease symptoms included severe stunting, leaf chlorosis, root rotting and necrosis, as the disease progressed, the whole plant gradually wilted and died. To recover the causal agent, symptomatic roots were excised, surface sterilized in 75% alcohol for 1.5 min, rinsed in sterilized water three times, dried, and placed on PARP selective medium (Jeffers and Martin 1986), and incubated at 20°C in dark. After 5 days, total of 26 Pythium-like isolates were obtained, and one representative isolate Py21-6 (available from the Institute of Plant Protection, Fujian Academy of Agricultural Sciences) was selected for further identification. Colonies of Py21-6 on PARP plate were white with dense, cottony, aerial, and transparent mycelia. Sporangia were terminal or intercalary, non-papillate, spherical, pyriform or ovoid, measuring 21.7 ± 2.8 × 19.3 ± 2.3 μm (n = 30). Zoospores were saucer-like, released out of sporangium after maturation, and dispersed quickly by swimming. Oogonia were spherical, terminal or occasionally intercalary. Oospores were globose, smooth and aplerotic. The dimensions of zoospores, oogonia, and oospores were 6.8 ± 0.7 μm, 21.6 ± 2.2 μm and 18.2 ± 2.7 μm (n = 30), respectively. Antheridia were bell-shaped or irregular, terminal, monoclinous, and usually one per oogonium. According to the morphological characteristics the isolate was initially identified as Pythium spp. (Van der Plaats-Niterink 1981, Yong et al. 2016). For further identification, DNA extracted from Py21-6, the cytochrome c oxidase subunit I (COI) gene and internal transcribed spacer (ITS) region were amplified and sequenced with primers FM55/FM52R (Long et al. 2012) and ITS1 /ITS4 (White et al. 1990), respectively. BLAST analysis of 680-bp COI (OM688194) and 728-bp ITS (OM663703) sequences revealed 99.86% and 99.99% similarity to Pythium vexans in GenBank (HQ708995 [COI], GU133572 [ITS]). Therefore, the pathogen was identified as P. vexans. In order to fulfill Koch's postulates, isolate Py21-6 was grown on Martin's liquid medium (Martin 1992) for 72 h to produce a spore suspensions of 106 oospores/ml, and the pathogenicity test was conducted by root-dip method. Three groups of 2-year-old Sanqi (15 plants per group) with root soaked for 20 min in oospore suspension were used for pathogenicity, and the other three groups (15 plants per group) with root dipped in sterilized water as control. All treated plants were replanted in (15-cm-diameter) pots (2 plants/pot) filled with mixture of sterilized soil: vermiculite: pearlite (2:1:1, v/v), maintained in greenhouse under 60% black shade cloth at 20 to 26°C with 80% relative humidity, and watered once every three days. After 21days, all inoculated plants showed the same symptoms observed on the original diseased plants in the field, whereas, the control plants remained symptomless. The same pathogen was successfully re-isolated from the inoculated plants, and identical to those of the originals based on morphological and sequence data. To our knowledge, this is the first report of P. vexans causing root rot on Sanqi in China (Farr and Rossman 2022). Root rot is one of the destructive diseases in Sanqi production, identification of the pathogen will be useful to develop effective field management strategies to control this disease.
PubMed: 35581918
DOI: 10.1094/PDIS-04-22-0781-PDN -
Plants (Basel, Switzerland) Sep 2022Oedogoniales comprises the three genera , , and , which include more than 600 described species. The classification of Oedogoniaceae is currently based on morphology,...
Oedogoniales comprises the three genera , , and , which include more than 600 described species. The classification of Oedogoniaceae is currently based on morphology, and the complicated morphological characteristics make species identification difficult, with the limited molecular data also restricting the phylogenetic analysis. In the present study, we collected 47 specimens from China and sequenced 18S rDNA, ITS2, ITS (ITS1 + 5.8S + ITS2), and rbcL sequences to conduct phylogenetic analyses. We selected nine morphological characteristics, most of which were considered important in traditional systematics, for comparison with the molecular phylogeny results. All the topologies based on different datasets showed similar results; was a paraphyletic group, and and clustered with . The morphological characteristics matching the phylogenetic results showed that the types of sexual differentiation, characteristics of the oogonium (including shape, types of aperture, and ornamentation of oospore wall), division types of antheridial, and number of sperm of each antheridial, which are considered the most important morphological characteristics in traditional taxonomy of , did not form monophyletic lineages respectively, indicating that traditional systematics may not reflect the real phylogeny of the genus . In addition, a new taxonomical classification of the genus was presented according to the shapes of basal cells, which matched well with the phylogenetic topologies. In addition, we propose to divide the genus into two sections, section and section , representing the species with spherical or sub-hemispherical basal cells and elongated basal cells, respectively.
PubMed: 36145821
DOI: 10.3390/plants11182422 -
Zoology (Jena, Germany) Jun 2023There is a gap in our knowledge of microorganization and the functioning of ovaries in earthworms (Crassiclitellata) and allied taxa. Recent analyses of ovaries in...
There is a gap in our knowledge of microorganization and the functioning of ovaries in earthworms (Crassiclitellata) and allied taxa. Recent analyses of ovaries in microdriles and leech-like taxa revealed that they are composed of syncytial germline cysts accompanied by somatic cells. Although the pattern of cyst organization is conserved across Clitellata - each cell is connected via one intercellular bridge (ring canal) to the central and anuclear cytoplasmic mass termed the cytophore - this system shows high evolutionary plasticity. In Crassiclitellata, only the gross morphology of ovaries and their segmental localization is well known, whereas ultrastructural data are limited to lumbricids like Dendrobaena veneta. Here we present the first report about ovarian histology and ultrastructure in Hormogastridae, a small family of earthworms inhabiting the western parts of the Mediterranean sea basin. We analyzed three species from three different genera and showed that the pattern of ovary organization is the same within this taxon. Ovaries are cone-like, with a broad part connected to the septum and a narrow distal end forming an egg string. Ovaries are composed of numerous cysts uniting a small number of cells, eight in Carpetania matritensis. There is a gradient of cysts development along the long ovary axis, and three zones can be distinguished. In zone I, cysts develop in complete synchrony and unite oogonia and early meiotic cells (till diplotene). Then (zone II), the synchrony is lost, and one cell (prospective oocyte) grows faster than the rest (prospective nurse cells). In zone III, oocytes pass the growth phase and gather nutrients; at this time, their contact with the cytophore is lost. Nurse cells grow slightly, eventually die via apoptosis, and are removed by coelomocytes. The most characteristic feature of hormogastrid germ cysts is the inconspicuous cytophore in the form of thread-like thin cytoplasmic strands (reticular cytophore). We found that the ovary organization in studied hormogastrids is very similar to that described for D. veneta and propose the term "Dendrobaena" type of ovaries. We expect the same microorganization of ovaries will be found in other hormogastrids and lumbricids.
Topics: Female; Animals; Ovary; Oligochaeta; Oogenesis; Oocytes; Germ Cells
PubMed: 36871333
DOI: 10.1016/j.zool.2023.126081 -
Plant Disease May 2022Photinia × fraseri Dress is a hybrid species of Rosaceae and Photinia genus which is widely cultivated in China. During 2020 and 2021, approximately 80% of plants...
Photinia × fraseri Dress is a hybrid species of Rosaceae and Photinia genus which is widely cultivated in China. During 2020 and 2021, approximately 80% of plants growing in Xuanwu district of Nanjing, China, exhibited disease symptoms including blight, necrosis, and dieback of crowns and roots. Symptomatic root tissues collected from 2-year-old plants were rinsed with water, cut into 2-mm tissues which were surface-sterilized in 70% ethanol for 60 s, and plated onto 10% V8 PARP agar and incubated in the dark at 26°C for 3 days. Hyphae emerged from 70% of the samples. Two representative isolates (PF-he2, PF-he3) were obtained and deposited. Ten agar plugs (2×2 mm2) of each isolate were transferred into 10 mL of 10% V8 juice to produce mycelial mats. To stimulate sporangial production, 3-5 drops of soil extract solution (soil collected from healthy fields, immersed in sterile water, and filtered) were added to each plate. Sporangia were terminal, ovoid to globose or papillate. The zoospores were 7.1-9.3 µm in diameter. Each oogonium contained a single, smooth, spherical and aplerotic oospore with a diameter of 24.5-32.6 μm. These morphological properties resemble those of Phytopythium helicoides (CBS286.31 from S. F. Ashby). For molecular identification, the large subunit (LSU) rDNA, cytochrome c oxidase I (COXI) gene, and COXII gene were amplified using the primer pairs NL1/NL4 , FM55/FM52R , and FM66/FM58 . The LSU, COXI, and COXII sequences of isolate PF-he2 were 100% (763/763 nt), 98.07% (1066/1087 nt), 99.64% (561/563 nt) identical to isolate CBS 286.31 (AY598665.2), GDGJ6 (KT750956.1), and TC3 (MN952224.1), respectively. Based on the morphological and molecular analysis, the two isolates shared 100% homology were identified as Phytopythium helicoides. The pathogenicity of two isolates were tested on potted 2-yr-old (40-cm tall) P. × fraseri. Six plants were dug up to expose root balls which were wounded before inoculations with a sterile needle, and then inoculated with zoospore suspension (106 zoospore/mL). Controls were treated with ddH2O. Three seedlings/isolate were used for each treatment including controls. All plants were repotted using the original sterilized potting mix and pots. After inoculation, the plants were covered with plastic bags, and sterilized H2O was sprayed into the bags twice per day to maintain humidity and kept in a greenhouse at the day/night temperatures at 25/16 °C. All the inoculated plants showed lesions similar to those observed in the field after 23 days , whereas controls were asymptomatic. The isolates were reisolated from the lesions and sequenced as P. helicoides which has found causing root rot on Nelumbo nucifera, Rhododendron pulchrum, Zea mays in China, and also on Fragaria × ananassa in America, Peach Rootstock in California. Globally, this is the first report of P. helicoides causing crown blight and root rot of P. × fraseri. Management programs are under development to contain the spread of P. helicoides and treat diseased plants.
PubMed: 35581911
DOI: 10.1094/PDIS-03-22-0672-PDN -
The EMBO Journal Sep 2022In vitro oogenesis is key to elucidating the mechanism of human female germ-cell development and its anomalies. Accordingly, pluripotent stem cells have been induced...
In vitro oogenesis is key to elucidating the mechanism of human female germ-cell development and its anomalies. Accordingly, pluripotent stem cells have been induced into primordial germ cell-like cells and into oogonia with epigenetic reprogramming, yet further reconstitutions remain a challenge. Here, we demonstrate ex vivo reconstitution of fetal oocyte development in both humans and cynomolgus monkeys (Macaca fascicularis). With an optimized culture of fetal ovary reaggregates over three months, human and monkey oogonia enter and complete the first meiotic prophase to differentiate into diplotene oocytes that form primordial follicles, the source for oogenesis in adults. The cytological and transcriptomic progressions of fetal oocyte development in vitro closely recapitulate those in vivo. A comparison of single-cell transcriptomes among humans, monkeys, and mice unravels primate-specific and conserved programs driving fetal oocyte development, the former including a distinct transcriptomic transformation upon oogonia-to-oocyte transition and the latter including two active X chromosomes with little X-chromosome upregulation. Our study provides a critical step forward for realizing human in vitro oogenesis and uncovers salient characteristics of fetal oocyte development in primates.
Topics: Animals; Female; Humans; Macaca fascicularis; Meiosis; Mice; Oocytes; Oogenesis; Ovary
PubMed: 35912849
DOI: 10.15252/embj.2022110815 -
Comparative Biochemistry and... 2021microRNAs (miRNAs) are important components of non-coding RNAs that participate in diverse life activities by regulating gene expression at the post transcriptional...
microRNAs (miRNAs) are important components of non-coding RNAs that participate in diverse life activities by regulating gene expression at the post transcriptional level through base complementary pairing with 3'UTRs of target mRNAs. miR-133b is a member of the miR-133 family, which play important roles in muscle differentiation and tumorigenesis. Recently, miR-133b was reported to affect estrogen synthesis by targeting foxl2 in mouse, while its role in fish reproduction remains to be elucidated. In the present study, we isolated the complete sequence of miR-133b, which was highly expressed in tilapia ovary at 30 and 90 dah (days after hatching) and subsequently decreased at 120 to 150 dah by qPCR. Interestingly, only a few oogonia were remained in the antagomir-133b treated tilapia ovary, while phase I and II oocytes were observed in the ovaries of the control group. Unexpectedly, the expression of foxl2 and cyp19a1a, as well as estradiol levels in serum were increased in the treated group. Furthermore, tagln2, an important factor for oogenesis, was predicted as the target gene of miR-133b, which was confirmed by dual luciferase reporter vector experiments. miR-133b and tagln2 were co-expressed in tilapia ovaries. Taken together, miR-133b may be involved in the early oogenesis of tilapia by regulating tagln2 expression. This study enriches the understanding of miR-133b function during oogenesis and lays a foundation for further study of the regulatory network during oogenesis.
Topics: Animals; Female; Fish Proteins; Forkhead Box Protein L2; Gene Expression Profiling; Gene Expression Regulation, Developmental; MicroRNAs; Microfilament Proteins; Oogenesis; Ovary; Tilapia
PubMed: 34147671
DOI: 10.1016/j.cbpb.2021.110637 -
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