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Cell Death Discovery Jul 2023A faithful reconstitution of the complete process of oogenesis in vitro is helpful for understanding the molecular mechanisms, genetics, and epigenetic changes related...
A faithful reconstitution of the complete process of oogenesis in vitro is helpful for understanding the molecular mechanisms, genetics, and epigenetic changes related to gametogenesis; it can also be useful for clinical drug screening, disease research, and regenerative medicine. To this end, given the consensus that murine female germ cells initiate meiosis at E13.5, substantial works have reported the successful generation of fertile oocytes using E12.5 female gonads as starting materials. Nevertheless, our data demonstrated that murine germ cells at E12.5 have heterogeneously initiated a meiotic transcriptional program based on a measurement of pre-mRNAs (unspliced) and mature mRNAs (spliced) at a single-cell level. Therefore, to establish a platform that faithfully recapitulates the entire process in vitro (from premeiotic murine germ cells to fully developed oocytes), we here report a novel three-dimensional organoid culture (3-DOC) system, which successfully induced fully developed oocytes from E11.5 premeiotic female germ cells (oogonia). Compared with 2D culture and other 3D culture methods, this new culture system is more cost-effective and can create high-quality oocytes similar to in vivo oocytes. In summary, our new culture platform provides an experimental model for future research in regenerative medicine and reproductive biology.
PubMed: 37518361
DOI: 10.1038/s41420-023-01577-w -
Plant Disease Apr 2024Pythium-like species cause damping-off symptoms of various hosts, including umbelliferous crops. In April 2023, parsley plantlets (), showing stunted growth, yellowing,...
Pythium-like species cause damping-off symptoms of various hosts, including umbelliferous crops. In April 2023, parsley plantlets (), showing stunted growth, yellowing, decayed roots and damping-off, were obtained from a nursery in central Slovenia, where parsley was grown in polystyrene trays in a greenhouse. Nearly 30% of plants were symptomatic. Sampled roots of ten plants contained ornamented oogonia (avg. 33.3 ± 1.4 µm in diam) with conical projections (5.2 ± 0.5 µm long) (Figure S1 A, B) in microscopically analyzed squash mounts. The pathogen was isolated from root pieces treated for surface disinfection with 0.5% sodium hypochlorite for 30 s, and washed with sterile water. Four 1-2 mm root pieces were taken from each of 10 plants, plated on the selective medium P5ARP, and incubated at 21 °C. Mycelia emerging from root pieces were transferred to carrot piece agar (CPA). Twenty-two equally looking oomycetous colonies were obtained; all sampled plants were infested. Oogonia formed by all colonies were similar to those observed on decayed roots and suggested that is the causal disease agent. Analyses of partial β-tubulin (Kroon et al. 2004) and mitochondrial cytochrome c oxidase I (COI) gene sequences (Robideau et al. 2011) confirmed the identification. Obtained COI (Genbank accession number OR725417) sequence was 100% identical to that from strain CBS 375.72 (EU350523), whereas the β-tubulin sequence (OR725416) corresponded to 99.6 % pairwise identity (KJ595502). Further, pathogenicity of an obtained isolate was tested on 4 wk-old curly leaf (cv. Petra F1) parsley. Half of a 7 d-old CPA culture, consisting of mycelium and oogonia, was finely cut and mixed with ca 50 ml of nonsterile commercial substrate (Potgrond H, AGRO-FertiCrop) in each of six 400 ml pots. Pots were filled with ca 300 ml additional substrate, into which 5 parsley seedlings were planted. Control plants were treated equally but with sterile CPA. Plantlets were watered with sterile tap water and held at ambient light conditions and temperature (night 18 °C - day 23 °C). After 14 d, inoculated plants started wilting and yellowing and showed stunted growth. After 21 d, roots were severely decayed and the seedlings damped-off (Figure S1 C). Four pieces each from 10 decayed roots were plated. Thirty-one pieces revealed pythium-like colonies. Obtained isolates were morphologically identical to the strain used for inoculation and identified as . Control plants developed no foliar or root symptoms and no pythium-like species was obtained. Agricultural advisors observed occurrence of parsley damping-off also in other nurseries in Slovenia what may lead to spreading the pathogen to parsley in production fields and private gardens. The case emphasizes the need for implementing phytosanitary measures in order to eliminate primary inoculum. Reports from field-infected plants showed that is a pathogen of parsley in Australia (Petkowski et al. 2013) and the USA (Tsuchida et al. 2018), and celery in the Czech Republic (Šafránková and Holková 2017). Others isolated from parsley in The Netherlands (online database of the Westerdijk Fungal Biodiversity Institute, strain CBS 243.86). However, the here described case is, to the best of our knowledge, one of the rare documentations of damping-off due to in Europe (Šafránková and Holková 2017) and the first in Slovenia. Funding: The work was funded by the Ministry of Agriculture, Forestry and Food of Slovenia, and Slovenian Research and Innovation Agency (ARIS Programs P4-0431 and P4-0072).
PubMed: 38625690
DOI: 10.1094/PDIS-01-24-0246-PDN -
Communications Biology Apr 2024The cellular and molecular mechanisms governing sexual reproduction are conserved across eukaryotes. Nevertheless, hybridization can disrupt these mechanisms, leading to...
The cellular and molecular mechanisms governing sexual reproduction are conserved across eukaryotes. Nevertheless, hybridization can disrupt these mechanisms, leading to asexual reproduction, often accompanied by polyploidy. In this study, we investigate how ploidy level and ratio of parental genomes in hybrids affect their reproductive mode. We analyze the gametogenesis of sexual species and their diploid and triploid hybrids from the freshwater fish family Cobitidae, using newly developed cytogenetic markers. We find that diploid hybrid females possess oogonia and oocytes with original (diploid) and duplicated (tetraploid) ploidy. Diploid oocytes cannot progress beyond pachytene due to aberrant pairing. However, tetraploid oocytes, which emerge after premeiotic genome endoreplication, exhibit normal pairing and result in diploid gametes. Triploid hybrid females possess diploid, triploid, and haploid oogonia and oocytes. Triploid and haploid oocytes cannot progress beyond pachytene checkpoint due to aberrant chromosome pairing, while diploid oocytes have normal pairing in meiosis, resulting in haploid gametes. Diploid oocytes emerge after premeiotic elimination of a single-copied genome. Triploid hybrid males are sterile due to aberrant pairing and the failure of chromosomal segregation during meiotic divisions. Thus, changes in ploidy and genome dosage may lead to cyclical alteration of gametogenic pathways in hybrids.
Topics: Animals; Female; Male; Triploidy; Tetraploidy; Gametogenesis; Haploidy; Cypriniformes
PubMed: 38589507
DOI: 10.1038/s42003-024-05948-6 -
Plant Disease Feb 2024⨯ 'Silver Star' (a cross between and ) from the Crassulaceae family, are an evergreen succulent with lotus constellation-shaped flowers, making it consumer favorite...
⨯ 'Silver Star' (a cross between and ) from the Crassulaceae family, are an evergreen succulent with lotus constellation-shaped flowers, making it consumer favorite ornamental plant in Korea. In 2019, Korea's ornamental production was estimated at KRW 517.4 billion (EUR 382 million), from 4,244 ha of farming area according to the Ministry of Agriculture, Food and Rural Affairs of Korea. In July 2023, ⨯ 'Silver Star' plants with chlorotic leaves, root and collar rot were observed in a greenhouse in Yongin (37°14'27.9"N, 127°10'39.19"E), Korea. To isolate the causal agent, small pieces (1 mm) of symptomatic tissues were surface-sterilized using 1% NaOCl for 1 min, then put onto a water agar (WA) plate and incubated in the dark at 25℃ for five days. Two isolates (FD00202, FD00203) were obtained from diseased leaves, stem and roots by isolating single sporangium. To investigate the morphological characteristics of the isolates, the mycelium from potato dextrose agar (PDA) were transferred to V8 agar (V8A) followed by incubation at 25°C in the dark for 7 days. The isolates produced dense cottony mycelium, with slightly petaloid and light rossette pattern, with coralloid edges measuring 70 to 83 mm diameter. Sporangium were spheroid (30.0-48.0 µm long, 25.0-35.0 µm wide) with globose chlamydospores (17.0-50.0 µm long, 18.0-38.0 µm wide). Oogonia were not observed. Morphological and cultural characteristics of these isolates were phenotypically similar to that of (Faedda et al. 2013; Abad et al. 2023). For molecular identification, genomic DNA was extracted from 5 days old cultures using the Maxwell RSC PureFood GMO and Authentication Kit (Promega). Two gene regions, the rDNA-ITS, COX I were amplified and sequenced using primers ITS1/ITS4 and FM83/FM84, respectively (White et al. 1990; Martin and Tooley 2003). The resulting sequences were deposited in GenBank with accession no. LC783858 to LC783861. A BLASTn search of the DNA sequences from ITS, COX I showed 99.81 and 98.94% identity to isolate IMI 398853, respectively. Maximum likelihood phylogenetic analyses were performed for the combined data set with ITS, COX I using MEGA7 under the Tamura-Nei model (Kumar et al. 2016). The isolates formed a monophyletic group with isolate IMI 398853, CPHST BL162, and CPHST BL 44. Based on morphological characteristics and molecular analysis, the isolates were identified as . T confirm their pathogenicity, inoculum was prepared in accordance with Ann (2000). Artificially wounded healthy plant roots were dipped in zoospore suspension (3.0 × 10 zoospore/ml) for 24 hours, with mock-treated plants (control) dipped in sterile distilled water (Ann. 2000). Thereafter, the plants were transplanted into new medium and kept under high humidity. Symptoms were observed after 10 days of incubation. The plants inoculated with showed similar symptoms of chlorotic leaves with root and collar rot, while control remained symptomless. The pathogen was re-isolated from all inoculated plants and confirmed as by morphological and molecular analysis. but not from controls, fulfilling Koch's postulates. was previously report on and causing brown spot on stems and roots in California and Korea, respectively (French 1989; Oh and Son 2008). To best of our knowledge, this is the first report of causing root and collar rot on ⨯ 'Silver Star' plants in the Korea.
PubMed: 38422436
DOI: 10.1094/PDIS-11-23-2283-PDN -
International Journal of Molecular... Jan 2024is gonochoristic and sexually dimorphic. The male prawn grows faster and usually has a larger size than the female. Therefore, a higher male proportion in stock usually...
is gonochoristic and sexually dimorphic. The male prawn grows faster and usually has a larger size than the female. Therefore, a higher male proportion in stock usually results in higher yield. To investigate the impact of temperature on sexual differentiation in , two temperature treatments (26 °C and 31 °C) were conducted. The results showed that compared to the 31 °C treatment (3.20 ± 0.12), the 26 °C treatment displayed a lower female/male ratio (2.20 ± 0.11), which implied that a lower temperature could induce masculinization in The temperature-sensitive sex differentiation phase was 25-35 days post hatching (DPH) at 26 °C while 15-20 DPH at 31 °C. Transcriptome and qPCR analysis revealed that a lower temperature up-regulated the expression of genes related to androgen secretion, and down-regulated the expressions of genes related to oogonia differentiation. Thirty-one temperature-regulated sex-differentiation genes were identified and the molecular mechanism of temperature-regulated sex differentiation was suggested. The finding of this study indicates that temperature regulation can be proposed as an innovative strategy for improving the culture yield of .
Topics: Animals; Male; Female; Palaemonidae; Sex Differentiation; Temperature; Transcriptome; Penaeidae; Arthropod Proteins
PubMed: 38279207
DOI: 10.3390/ijms25021207 -
Animals : An Open Access Journal From... Feb 2024Zygote arrest-1 (Zar1) and Wilms' tumor 1 (Wt1) play an important role in oogenesis, with the latter also involved in testicular development and gender differentiation....
Zygote arrest-1 (Zar1) and Wilms' tumor 1 (Wt1) play an important role in oogenesis, with the latter also involved in testicular development and gender differentiation. Here, and were identified in Asian seabass (), a hermaphrodite fish, as the valuable model for studying sex differentiation. The cloned cDNA fragments of were 1192 bp, encoding 336 amino acids, and contained a zinc-binding domain, while those of cDNA were 1521 bp, encoding a peptide of 423 amino acids with a Zn finger domain belonging to Wt1b family. RT-qPCR analysis showed that mRNA was exclusively expressed in the ovary, while mRNA was majorly expressed in the gonads in a higher amount in the testis than in the ovary. In situ hybridization results showed that mRNA was mainly concentrated in oogonia and oocytes at early stages in the ovary, but were undetectable in the testis. mRNA was localized not only in gonadal somatic cells (the testis and ovary), but also in female and male germ cells in the early developmental stages, such as those of previtellogenic oocytes, spermatogonia, spermatocytes and spermatids. These results indicated that and possibly play roles in gonadal development. Therefore, the findings of this study will provide a basis for clarifying the mechanism of and in regulating germ cell development and the sex reversal of Asian seabass and even other hermaphroditic species.
PubMed: 38338151
DOI: 10.3390/ani14030508 -
Plant Disease May 2024Kousa dogwood () is an economically important woody ornamental crop that exhibits creamy, white, pointed bracts in late spring, and reddish to pink drupe fruits in late...
Kousa dogwood () is an economically important woody ornamental crop that exhibits creamy, white, pointed bracts in late spring, and reddish to pink drupe fruits in late summer and fall. It bears shiny dark green leaves that become reddish-purple to scarlet in the fall. In August of 2023, 3-year-old container grown var. plants in a commercial nursery in Warren Co., Tennessee, exhibited severe yellowing, dieback and root rot symptoms (Fig. 1a and 1b). Dark brown to black lesions were observed in the root and crown region of the plants. Disease severity was 40% to 60% of root area affected, and disease incidence was approximately 40% of 1,000 plants. Surface-sterilized (10% NaOCl: 1 min) symptomatic root tissues were plated on V8-PARPH and incubated at 25°C. Sparse aerial mycelium, showing a distinct rosette or faint radiate to chrysanthemum colony pattern, was observed within four days of incubation (Fig. 2). All isolates produced ovoid or subglose, papillate, and proliferating sporangia in grass blade water cultures (Derviş et al. 2020). Sporangia measured as 19.18 to 24.80 µm X 18.08 to 22.16 µm (n = 50) with a length/width ratio of 1.06 to 1.11. Zoospores observed were between 7.07 to 9.98 µm in diameter (n = 50). Oogonia and oospores were not produced. The ribosomal internal transcribed spacer () and large subunit (), as well as mitochondrial cytochrome oxidase subunit II (-II) genetic markers were amplified and sequenced using primer pairs ITS1/ITS4 (White et al. 1990), NL1/NL4 (Baten et al. 2014), and cox-F/cox-RC4 (Choi et al. 2015), respectively. The , , and -II sequences of isolates FBG6343, FBG6344 (: PP458373 and PP461387; : PP461390 and PP461391; II: PP477112 and PP477113) were 100% identical to those of MN306118, HQ643386, and MN206732, respectively. Based on the morphology (Nechwatal and Mendgen 2006) and sequence data, the isolates were identified as (Nechw.) Abad, De Cock, Bala, Robideau, Lodhi & Lévesque. The pathogenicity test was performed on 3-year-old var. plants grown in a 3-gal container to fulfill Koch's postulates. Kousa dogwood plants were drench inoculated (800 ml/plant) with a pathogen slurry (two plates of 7-day-old culture/liter) of isolates FBG6343 and FBG6364 (five plants per isolate). Control plants were drenched with agar slurry without the pathogen. The study was conducted in a greenhouse maintained at 21 to 23°C and 70% relative humidity with a 16-h photoperiod and irrigated twice a day for 2 min using an overhead irrigation system. Forty-five days after inoculation, plants showed dieback symptoms, and dark brown lesions developed in the roots of all inoculated plants. Isolates with morphology and sequences identical to those of FBG6343 and FBG6364 were recovered from root tissues of all inoculated plants. All control plants remained symptom-free, and was not isolated from the root tissue. Previously, was reported to cause disease on apple, kiwi, planatus, and rhododendron (Derviş et al. 2020; Li et al. 2021; Mert et al. 2020; Polat et al. 2023). To our knowledge, this is the first report of causing root rot of kousa dogwood in Tennessee and the United States. Identification of this pathogen as the causal agent is crucial to developing timely management practices.
PubMed: 38764336
DOI: 10.1094/PDIS-03-24-0616-PDN -
Animals : An Open Access Journal From... Aug 2023The following paper gives a detailed description of the oogenesis cycle for the European Plaice (), from oogonia to post-ovulatory follicle, including ovarian follicle...
The following paper gives a detailed description of the oogenesis cycle for the European Plaice (), from oogonia to post-ovulatory follicle, including ovarian follicle and sizes. Noteworthy particularities were the difficulty in identifying cortical alveoli due to their very small size. Quantitative histology (stereology) on histological slides was used to determine a first size at maturity for females from the English Channel, which was found to be smaller compared to the literature (19 cm). Stereology also determined a first spawning event starting in January, with a peak in February and ongoing until March. Moreover, the use of stereology showed misclassifications for individuals categorized into a maturity phase using a macroscopic visual method. Misclassifications were found with individuals that had spawned (D) but were put under the immature (A) phase, and individuals in development (B) classified under D.
PubMed: 37570314
DOI: 10.3390/ani13152506 -
Animal Cells and Systems 2024The system forming ovarian follicles is developed to investigate folliculogenesis in a confined environment to obtain functional oocytes. Several studies have reported...
The system forming ovarian follicles is developed to investigate folliculogenesis in a confined environment to obtain functional oocytes. Several studies have reported the successful generation of fully functional oocytes using mouse-induced pluripotent stem cells (iPSCs) and mouse female germline stem cells (fGSCs) as sources of stem cells for gametogenesis models. In addition, human oogonia have been generated through heterologous co-culture of differentiated human primordial germ cell-like cells (hPGCLCs) with mouse germline somatic cells, although oocyte formation remains challenging. Thus, studies on ovarian formation in other species are utilized as an introductory approach for mammalian gametogenesis by understanding the differences in culture systems between species and underlying mechanisms. In this study, we optimized the method of the entire oogenesis process from rat embryonic gonads. We identified well-maturated MII oocytes from rat gonads using our constructed method. Moreover, we generated the first successful reconstitution of xenogeneic follicles from mouse primordial germ cells (PGCs) and rat somatic cells. We also established an appropriate culture medium and incubation period for xenogeneic follicles. This method will be helpful in studies of xenogeneic follicular development and oocyte generation.
PubMed: 38868077
DOI: 10.1080/19768354.2024.2363601 -
Animals : An Open Access Journal From... Feb 2024is one of the earliest expressed genes in the oocyte during ovarian development. In this study, was characterized in . , one of the main aquaculture species in China,...
is one of the earliest expressed genes in the oocyte during ovarian development. In this study, was characterized in . , one of the main aquaculture species in China, and designated as . The length of cDNA was 1303 bp, encoding 197 amino acids that contained a conserved bHLH domain. revealed a female-biased expression patterns in the gonads of adult fish, and expression was far higher in ovaries than that in testes at all gonadal development stages, especially at 60~180 days post-fertilization (dpf). Furthermore, a noteworthy inverse relationship was observed between expression and the methylation of its promoter in the adult gonads. Gonads at 90 dpf were used for in situ hybridization (ISH), and transcripts were mainly concentrated in oogonia and the primary oocytes in ovaries, but undetectable in the testes. These results indicated that Figla would play vital roles in the ovarian development in . Additionally, the frame-shift mutations of were successfully constructed through the CRISPR/Cas9 system, which established a positive foundation for further investigation on the role of in the ovarian development of . Our study provides valuable clues for exploring the regulatory mechanism of in the fish ovarian development and maintenance, which would be useful for the sex control and reproduction of fish in aquaculture.
PubMed: 38338134
DOI: 10.3390/ani14030491