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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 -
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 -
Nature Biotechnology Jan 2019
Topics: Animals; Cell Culture Techniques; Cell Differentiation; Epigenesis, Genetic; Female; Germ Cells; Humans; Mice; Oocytes; Oogonia; Ovary; Pluripotent Stem Cells
PubMed: 30605165
DOI: 10.1038/nbt.4329 -
Fish Physiology and Biochemistry Dec 2023Primordial germ cells (PGCs) are embryonic pluripotent cells that can differentiate into spermatogonia and oogonia, and therefore, PGCs are a genetic source for...
Primordial germ cells (PGCs) are embryonic pluripotent cells that can differentiate into spermatogonia and oogonia, and therefore, PGCs are a genetic source for germplasm conservation through cryobanking and the generation of germline chimeras. The knowledge of PGC migration routes is essential for transplantation studies. In this work, the mRNA synthesized from the ddx4 3'UTR sequence of Pseudopimelodus mangurus, in fusion with gfp or dsred, was microinjected into zygotes of three neotropical species (P. mangurus, Astyanax altiparanae, and Prochilodus lineatus) for PGC labeling. Visualization of labeled PGCs was achieved by fluorescence microscopy during embryonic development. In addition, ddx4 and dnd1 expressions were evaluated during embryonic development, larvae, and adult tissues of P. mangurus, to validate their use as a PGC marker. As a result, the effective identification of presumptive PGCs was obtained. DsRed-positive PGC of P. mangurus was observed in the hatching stage, GFP-positive PGC of A. altiparanae in the gastrula stage, and GFP-positive PGCs from P. lineatus were identified at the segmentation stage, with representative labeling percentages of 29% and 16% in A. altiparanae and P. lineatus, respectively. The expression of ddx4 and dnd1 of P. mangurus confirmed the specificity of these genes in germ cells. These results point to the functionality of the P. mangurus ddx4 3'UTR sequence as a PGC marker, demonstrating that PGC labeling was more efficient in A. altiparanae and P. lineatus. The procedures used to identify PGCs in P. mangurus consolidate the first step for generating germinal chimeras as a conservation action of P. mangurus.
PubMed: 38060079
DOI: 10.1007/s10695-023-01279-1 -
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 -
Methods in Molecular Biology (Clifton,... 2018The cohesin complex is essential for chromosome segregation in mitosis and meiosis. Cohesin is a tripartite protein complex that holds sister chromatids together from...
The cohesin complex is essential for chromosome segregation in mitosis and meiosis. Cohesin is a tripartite protein complex that holds sister chromatids together from DNA replication until anaphase. In mammals, meiotic DNA replication occurs in oogonia of embryos and chromosome segregation occurs in oocytes of sexually mature females. Sister chromatid cohesion establishment and chromosome segregation are thus separated by months in the mouse and decades in the human. The meiotic cohesin complex that maintains sister chromatid cohesion must therefore hold replicated sisters together for a long time in oocytes. Remarkably, this is achieved by establishing cohesion exclusively in prenatal oocytes. Meiotic cohesion in females is maintained without detectable turnover and cohesin is therefore thought to be a long-lived protein complex. Nevertheless, the lifespan of cohesin molecules is limited as chromosomal cohesin levels decline with maternal age. The age-related loss of cohesin and weakened cohesion correlate with an age-related increase in chromosome missegregation of meiosis I oocytes that results in aneuploid eggs. Therefore, loss of chromosomal cohesin has been proposed to be a leading cause of the maternal age effect. To better understand cohesin deterioration in oocytes, it is crucial to gain insights into mammalian cohesion establishment and maintenance mechanisms by manipulating cohesin in live oocytes.This chapter describes techniques that address the manipulation of meiotic cohesin levels in mouse oocytes. First, we describe how cohesin can be efficiently removed from meiotic chromosomes by injecting mRNA encoding TEV protease in live oocytes expressing cohesin with engineered TEV recognition sites, followed by imaging. Secondly, we describe how cohesin expression can be induced during different stages of oocyte development using genetically modified mouse strains. In particular, we describe how to determine the deletion timing of germline-specific Cre recombinases using β-galactosidase staining of fetal ovaries. Lastly, we provide guidance on how to quantify cohesin levels on metaphase I chromosome spreads.
Topics: Animals; Cell Cycle Proteins; Cells, Cultured; Chromosomal Proteins, Non-Histone; Chromosome Segregation; Endopeptidases; Female; Fetus; Gene Expression Regulation, Developmental; Integrases; Meiosis; Metaphase; Mice; Oocytes; beta-Galactosidase; Cohesins
PubMed: 29961260
DOI: 10.1007/978-1-4939-8603-3_12 -
Journal of Morphology May 2022The main goal of the article is to describe the ovary organization and oogenesis in Peristodrilus montanus, an aquatic oligochaete of the subfamily Rhyacodrilinae. The...
The main goal of the article is to describe the ovary organization and oogenesis in Peristodrilus montanus, an aquatic oligochaete of the subfamily Rhyacodrilinae. The presented analysis will not only enrich the knowledge about how eggs are formed but, because of the suggested conservatism of ovary organization in clitellate annelids, can contribute to disentangling the complex phylogenetic relationships of the rhyacodrilines within Naididae. The paired, conically shaped ovaries are located in segment XI. They are composed of a dozen or so syncytial germ-line cysts, which are associated with somatic cells. Each germ cell in a cyst has one intercellular bridge that joins it to a central and anuclear cytoplasmic mass, the cytophore. This pattern of cyst organization is typical for all clitellates that have been studied to date. Initially, the germ cells in a cyst undergo a synchronous development, however, there is no synchrony between cysts, and therefore there is a developmental gradient (oogonia, pre-diplotene germ cells, germ cells in diplotene) of oogenesis along the long ovary axis. The cysts are composed of a maximum of 32 cells. Cysts with cells in diplotene detach from the ovaries and the extraovarian phase of oogenesis begins. The developmental synchrony is lost, one cell (an oocyte) per cyst starts to gather cell components and yolk and grows considerably. The remaining cells grow to some extent and function as nurse cells. Like in other microdriles, P. montanus oocytes are rich in yolk; other features of oogenesis are also similar to those that are known from other microdrile taxa. The system of ovary organization found in the studied species is broadly similar to the corresponding features known from Naidinae and Phreodrilidae and, to some extent, in Enchytraeidae. However, this system is different from the one that is known in Tubificinae, Limnodriloidinae and Branchiurinae.
Topics: Animals; Cysts; Female; Oligochaeta; Oocytes; Oogenesis; Ovary; Phylogeny
PubMed: 35150164
DOI: 10.1002/jmor.21461