Correction to: Accompaniment of Time-Lapse Parameters and Cumulus Cell RNA-Sequencing in Embryo Evaluation

Oocyte activation, the dynamic transformation of an oocyte into an embryo, is largely driven by Ca2+ oscillations that vary in duration and amplitude across species. Previous studies have analysed intraoocyte Ca2+ oscillations in the absence of the oocyte’s supporting cumulus cells. Therefore, it is unknown whether cumulus cells also produce an ionic signal that reflects fertilisation success. Time-lapse confocal microscopy and image analysis on abattoir-derived cattle cumulus–oocyte complexes coincubated with spermatozoa revealed a distinct discharge of fluorescence from the cumulus vestment. This study demonstrated that this Ca2+ fluorescence discharge was an artefact induced by the imaging procedure independently of oocyte activation success. The fluorescence discharge was a direct result of cumulus cell membrane integrity loss, and future studies should consider the long-term effect of fluorescent labels on cells in time-lapse imaging. However, this study also demonstrated that the distinctive pattern of a coordinated fluorescence discharge was associated with both the presence of spermatozoa and subsequent embryo development to the morula stage, which was affected by Ca2+ chelation and a reduction in the active efflux of the fluorophore. This indicates that the cumulus vestment may have a relationship with oocyte activation at and beyond fertilisation that requires further investigation.

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Lineage analysis reveals an endodermal contribution to the vertebrate pituitary

Science ◽

10.1126/science.aba4767 ◽

2020 ◽

Vol 370 (6515) ◽

pp. 463-467 ◽

Cited By ~ 1

Author(s):

Peter Fabian ◽

Kuo-Chang Tseng ◽

Joanna Smeeton ◽

Joseph J. Lancman ◽

P. Duc Si Dong ◽

...

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Endocrine Cell ◽

Time Lapse ◽

Cell Types ◽

Lineage Tracing ◽

Single Cell Rna Sequencing ◽

Growth Metabolism ◽

Time Lapse Imaging ◽

Lineage Analysis

Vertebrate sensory organs arise from epithelial thickenings called placodes. Along with neural crest cells, cranial placodes are considered ectodermal novelties that drove evolution of the vertebrate head. The anterior-most placode generates the endocrine lobe [adenohypophysis (ADH)] of the pituitary, a master gland controlling growth, metabolism, and reproduction. In addition to known ectodermal contributions, we use lineage tracing and time-lapse imaging in zebrafish to identify an endodermal contribution to the ADH. Single-cell RNA sequencing of the adult pituitary reveals similar competency of endodermal and ectodermal epithelia to generate all endocrine cell types. Further, endoderm can generate a rudimentary ADH-like structure in the near absence of ectodermal contributions. The fish condition supports the vertebrate pituitary arising through interactions of an ancestral endoderm-derived proto-pituitary with newly evolved placodal ectoderm.

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Combining Time-Lapse Imaging and Next Generation RNA-Sequencing to assess Equine Embryo Developmental Potential

Journal of Equine Veterinary Science ◽

10.1016/j.jevs.2016.04.081 ◽

2016 ◽

Vol 41 ◽

pp. 80-81 ◽

Cited By ~ 2

Author(s):

B.L. Daughtry ◽

K.R. Masterson ◽

E.S. Metcalf ◽

D. Battaglia ◽

S.S. Fei ◽

...

Keyword(s):

Rna Sequencing ◽

Time Lapse ◽

Next Generation ◽

Developmental Potential ◽

Time Lapse Imaging

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P–547 Single-cell RNA sequencing identifies molecular regulations associated with poor maturation performance on rescue in vitro matured oocytes

Human Reproduction ◽

10.1093/humrep/deab130.546 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

W T Lee ◽

K W Ng ◽

J Liao ◽

A C S Luk ◽

H C Suen ◽

...

Keyword(s):

Wnt Signaling ◽

Single Cell ◽

Rna Sequencing ◽

Maternal Age ◽

Polar Body ◽

Rna Extraction ◽

Cumulus Cell ◽

Single Cell Rna Sequencing

Abstract Study question What is the transcriptome signature associated with rescuein vitro matured (rIVM) oocytes? Summary answer GATA–1/CREB1/WNT signaling axis was repressed in rIVM oocytes of poor quality. What is known already rIVM aims to produce mature oocytes (MII) for in vitro fertilization (IVF) through IVM of immature oocytes collected from stimulated ovaries. It is less popular due to limited success rate in infertility treatment. Genetic aberrations, cellular stress, and the absence of cumulus cell support in oocytes could account for the failure of rIVM. Study design, size, duration We applied single-cell RNA sequencing (scRNA-seq) to capture the transcriptomes of human in vivo (IVO) oocytes (n = 10) from 7 donors and rIVM oocytes (n = 10) from 10 donors, followed by studying the maternal age effect and ovarian responses on rIVM oocyte transcriptomes. Participants/materials, setting, methods Human oocytes were collected from donors aged 28–41 years with a body mass index of < 30. RNA extraction, cDNA generation, library construction and sequencing were performed in one preparation. scRNA-seq data were then processed and analyzed. Selected genes in therIVM vs. IVO comparison were validated by quantitative real-time PCR. Main results and the role of chance The transcriptome profiles of rIVM/IVO showed distinctive differences. A total of 1559 differentially expressed genes (DEGs, genes with at least two-fold change and adjusted p < 0.05) were found to be enriched in metabolic processes, biosynthesis, and oxidative phosphorylation. Among these DEGs, we identified a repression of WNT/β-catenin signaling in rIVM when compared with IVO oocytes. We found that estradiol level exhibited a significant age-independent correlation with the IVO mature oocyte ratio (MII ratio). rIVM oocytes with higher MII ratio showed over-represented cellular processes such as anti-apoptosis. To further identify targets that contribute to the poor outcomes of rIVM, we compared oocytes collected from young donors with high MII ratio versus donors of advanced maternal age and revealed CREB1was an important regulator in rIVM. Our study identified GATA–1/CREB1/WNT signaling was repressed in both rIVM condition and rIVM oocytes of low-quality. Limitations, reasons for caution In the rIVM oocytes of high- and low-quality comparison, the number of samples was limited after data filtering with stringent selection criteria. For the oocyte stage identification, we were unable to predict the presence of oocyte spindle so polar body extrusion was the only indicator. Wider implications of the findings: This study showed that GATA–1/CREB1/WNT signaling and antioxidant actions were repressed in rIVM condition and was further downregulated in rIVM oocytes of low-quality, providing us the foundation of subsequent follow-up research on human subjects. Trial registration number Not applicable

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Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100084120 ◽

1978 ◽

Vol 36 (2) ◽

pp. 650-651

Author(s):

Raul I. Garcia ◽

Evelyn A. Flynn ◽

George Szabo

Keyword(s):

Direct Injection ◽

Skin Pigmentation ◽

Freeze Fracture ◽

Pigment Granule ◽

Time Lapse ◽

Ultrastructural Level ◽

Lanthanum Tracer ◽

A Cell

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.

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High-voltage Electron Microscopy of astroglial cell whole mounts

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100143201 ◽

1986 ◽

Vol 44 ◽

pp. 316-317

Author(s):

J.N. Turner ◽

W.G. Shain ◽

V. Madelian ◽

R.A. Grassucci ◽

D.L. Forman

Keyword(s):

Electron Microscopy ◽

High Voltage ◽

Time Lapse ◽

Astroglial Cells ◽

High Voltage Electron Microscopy ◽

Rat Glioma ◽

Voltage Electron ◽

High Voltage Electron ◽

Nervous System Function ◽

Beta Adrenergic Agonist

Homogeneous cultures of astroglial cells have proved useful for studying biochemical, pharmacological, and toxicological responses of astrocytes to effectors of central nervous system function. LRM 55 astroglial cells, which were derived from a rat glioma and maintained in continuous culture, exhibit a number of astrocyte properties (1-3). Stimulation of LRM 55s and astrocytes in primary cell cultures with the beta-adrenergic agonist isoproterenol results in rapid changes of morphology. Studies with time lapse video light microscopy (VLM) and high-voltage electron microscopy (HVEM) have been correlated to changes in intracellular levels of c-AMP. This report emphasizes the HVEM results.

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Measurement of spontaneous neuronal membrane activity by time lapse confocal microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100141238 ◽

1995 ◽

Vol 53 ◽

pp. 972-973

Author(s):

R H. Selinfreund ◽

A. H. Cornell-Bell

Keyword(s):

Membrane Potential ◽

Confocal Microscopy ◽

Patch Clamp ◽

Electrical Activity ◽

Time Lapse ◽

Neuronal Firing ◽

Neuronal Membrane ◽

Pituitary Cells ◽

Patch Clamp Recording ◽

Spontaneous Electrical Activity

Cellular electrophysiological properties are normally monitored by standard patch clamp techniques . The combination of membrane potential dyes with time-lapse laser confocal microscopy provides a more direct, least destructive rapid method for monitoring changes in neuronal electrical activity. Using membrane potential dyes we found that spontaneous action potential firing can be detected using time-lapse confocal microscopy. Initially, patch clamp recording techniques were used to verify spontaneous electrical activity in GH4\C1 pituitary cells. It was found that serum depleted cells had reduced spontaneous electrical activity. Brief exposure to the serum derived growth factor, IGF-1, reconstituted electrical activity. We have examined the possibility of developing a rapid fluorescent assay to measure neuronal activity using membrane potential dyes. This neuronal regeneration assay has been adapted to run on a confocal microscope. Quantitative fluorescence is then used to measure a compounds ability to regenerate neuronal firing.The membrane potential dye di-8-ANEPPS was selected for these experiments. Di-8- ANEPPS is internalized slowly, has a high signal to noise ratio (40:1), has a linear fluorescent response to change in voltage.

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Five-Dimensional Microscopy using Widefield-Deconvolution: Practical Considerations and Biological Applications

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100163800 ◽

1996 ◽

Vol 54 ◽

pp. 268-269

Author(s):

W.F. Marshall ◽

K. Oegema ◽

J. Nunnari ◽

A.F. Straight ◽

D.A. Agard ◽

...

Keyword(s):

Confocal Microscopy ◽

High Speed ◽

Laser Scanning ◽

Ccd Camera ◽

Image Plane ◽

Time Lapse ◽

Laser Scanning Confocal Microscopy ◽

Three Dimensions ◽

Excitation Light ◽

Cooled Ccd

The ability to image cells in three dimensions has brought about a revolution in biological microscopy, enabling many questions to be asked which would be inaccessible without this capability. There are currently two major methods of three dimensional microscopy: laser-scanning confocal microscopy and widefield-deconvolution microscopy. The method of widefield-deconvolution uses a cooled CCD to acquire images from a standard widefield microscope, and then computationally removes out of focus blur. Using such a scheme, it is easy to acquire time-lapse 3D images of living cells without killing them, and to do so for multiple wavelengths (using computer-controlled filter wheels). Thus, it is now not only feasible, but routine, to perform five dimensional microscopy (three spatial dimensions, plus time, plus wavelength).Widefield-deconvolution has several advantages over confocal microscopy. The two main advantages are high speed of acquisition (because there is no scanning, a single optical section is acquired at a time by using a cooled CCD camera) and the use of low excitation light levels Excitation intensity can be much lower than in a confocal microscope for three reasons: 1) longer exposures can be taken since the entire 512x512 image plane is acquired in parallel, so that dwell time is not an issue, 2) the higher quantum efficiently of a CCD detect over those typically used in confocal microscopy (although this is expected to change due to advances in confocal detector technology), and 3) because no pinhole is used to reject light, a much larger fraction of the emitted light is collected. Thus we can typically acquire images with thousands of photons per pixel using a mercury lamp, instead of a laser, for illumination. The use of low excitation light is critical for living samples, and also reduces bleaching. The high speed of widefield microscopy is also essential for time-lapse 3D microscopy, since one must acquire images quickly enough to resolve interesting events.

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