Developmental Biology


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0 Q&A 3059 Views Oct 5, 2020
Oocyte maturation is a process wherein an oocyte arrested at prophase of meiosis I resumes meiosis to become a fertilizable egg. In starfish ovaries, a hormone released from follicle cells activates the oocytes, resulting in an increase in their intracellular pH (pHi), which is required for spindle assembly. Herein, we describe a protocol for pHi measurement in living oocytes microinjected with the pH-sensitive dye BCECF. For in vivo BCECF calibration, we treated oocytes with artificial seawater containing CH3COONH4 to clamp pHi, injected pH-standard solutions, and converted the BCECF fluorescence intensity ratios to pHi values. Of note, if the actual pHi is higher or lower than the known pH of injected standard solutions, the BCECF fluorescence intensity ratio will decrease or increase, respectively. On the other hand, the pH of the injected solution displaying no change in fluorescence intensity should be considered the actual pHi. These methods for pHi calibration and clamping are simple and reproducible.
0 Q&A 4709 Views Aug 20, 2019
Endogenous retroviruses (ERV) are transposable retroelements that form ~10% of the murine genome and whose family members are differentially expressed throughout embryogenesis. However, precise regulation of ERV in germ cells remains unclear. To investigate ERV expression in oocytes, we adapted a single-cell mRNA-sequencing library preparation method to generate bulk sequencing libraries from growing oocytes in a time- and cost-efficient manner. Here, we present a modified Smart-seq2 protocol that yields full-length cDNA libraries from purified RNA obtained from low numbers of pooled immature or mature oocytes. Using this method, RNA-sequencing libraries can be generated from any rare or difficult-to-isolate populations for subsequent sequencing and retroelement expression analysis.
0 Q&A 11017 Views Mar 5, 2018
Arrays of short, singly-labeled ssDNA oligonucleotides enable in situ hybridization with single molecule sensitivity and efficient transcript specific RNA capture. Here, we describe a simple, enzymatic protocol that can be carried out using basic laboratory equipment to convert arrays of PCR oligos into smFISH and RAP probesets in a quantitative, cost-efficient and flexible way.
0 Q&A 9345 Views Apr 5, 2017
Fertilization calcium waves are a conserved trigger for animal development; however, genetic analysis of these waves has been limited due to the difficulty of imaging in vivo fertilization. Here we describe a protocol to image calcium dynamics during in vivo fertilization in the genetic animal model Caenorhabditis elegans. This protocol consists of germline microinjection of a chemical calcium indicator, worm immobilization, live imaging, and image processing that quantifies the calcium fluorescence in the oocyte region moving in the field-of-view during ovulation. This imaging protocol can also be used to image other cellular processes during in vivo fertilization in C. elegans, such as membrane fusion and cytoskeletal dynamics.



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