Name: separation of follicular cells and oocytes in ovarian follicles of zebrafish
Uploaded: 2021-04-18T14:00:00
Description: Watch this Scientific Journal Video about Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish at JoVE.com

This research work was supported by the National Natural Science Foundation of China [32060170, 31601205 and 31560334], visiting scholar project supported by China Scholarship Council and the fund of State Key Laboratory of Freshwater Ecology and Biotechnology [2020FB05].

All of the procedures performed in fish experiments are in accordance with the regulations of the Animal Experimentation Ethics Committee of Northwest Normal University.
1. Preparations
<ol>
	<li>Animals
	<ol>
		<li>Use adult female zebrafish with a body length of 4-6 cm. 
		NOTE: We used zebrafish from a local market.</li>
		<li>Keep the zebrafish in a circulated water system with a 14 h light and 10 h dark cycle at about 28 &#176;C.</li>
		<li>Feed fish twice daily with newly hatched ...

<ol>
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W., Ge, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pituitary+adenylate+cyclase-activating+polypeptide+(PACAP)+and+its+receptors+in+the+zebrafish+ovary:+evidence+for+potentially+dual+roles+of+PACAP+in+controlling+final+oocyte+maturation.">Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors in the zebrafish ovary: evidence for potentially dual roles of PACAP in controlling final oocyte maturation.</a> Biology of Reproduction. 85, 615-625 (2011).</li><li>Li, J., Liu, Z., Wang, D., Cheng, C. H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-like+growth+factor+3+is+involved+in+oocyte+maturation+in+zebrafish.">Insulin-like growth factor 3 is involved in oocyte maturation in zebrafish.</a> Biology of Reproduction. 84, 476-486 (2011).</li><li>Pang, Y., Thomas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+G+protein-coupled+estrogen+receptor+1,+GPER,+in+inhibition+of+oocyte+maturation+by+endogenous+estrogens+in+zebrafish.">Role of G protein-coupled estrogen receptor 1, GPER, in inhibition of oocyte maturation by endogenous estrogens in zebrafish.</a> Developmental Biology. 342, 194-206 (2010).</li><li>Peyton, C., Thomas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+epidermal+growth+factor+receptor+signaling+in+estrogen+inhibition+of+oocyte+maturation+mediated+through+the+G+protein-coupled+estrogen+receptor+(Gper)+in+zebrafish+(Danio+rerio).">Involvement of epidermal growth factor receptor signaling in estrogen inhibition of oocyte maturation mediated through the G protein-coupled estrogen receptor (Gper) in zebrafish (Danio rerio).</a> Biology of Reproduction. 85, 42-50 (2011).</li><li>Welch, E. L., Eno, C. C., Nair, S., Lindeman, R. E., F, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+manipulation+of+maternal+gene+products+using+in+vitro+oocyte+maturation+in+zebrafish.">Functional manipulation of maternal gene products using in vitro oocyte maturation in zebrafish.</a> Journal of Visualized Experiments. (122), e55213 (2017).</li><li>Nair, S., Lindeman, R. E., Pelegri, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+oocyte+culture-based+manipulation+of+zebrafish+maternal+genes.">In vitro oocyte culture-based manipulation of zebrafish maternal genes.</a> Developmental Dynamics. 242, 44-52 (2013).</li><li>Seki, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+reliable+in+vitro+maturation+system+for+zebrafish+oocytes.">Development of a reliable in vitro maturation system for zebrafish oocytes.</a> Reproduction. 135, 285-292 (2008).</li><li>Baars, D. L., Takle, K. A., Heier, J., Pelegri, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ploidy+manipulation+of+zebrafish+embryos+with+heat+shock+2+treatment.">Ploidy manipulation of zebrafish embryos with heat shock 2 treatment.</a> Journal of Visualized Experiments. , e54492 (2016).</li><li>Xie, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+technique+based+on+in+vitro+oocyte+injection+to+improve+CRISPR/Cas9+gene+editing+in+zebrafish.">A novel technique based on in vitro oocyte injection to improve CRISPR/Cas9 gene editing in zebrafish.</a> Scientific Reports. 6, 34555 (2016).</li><li>Li, J., Bai, L., Liu, Z., Wang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+roles+of+PDE9a+in+meiotic+maturation+of+zebrafish+oocytes.">Dual roles of PDE9a in meiotic maturation of zebrafish oocytes.</a> Biochemical and Biophysical Research Communications. , (2020).</li></ol>

We describe here a novel method for the simple and rapid separation of follicular cells and oocytes from zebrafish ovarian follicles. This method has several advantages over the conventional method. Primary amongst these is the greatly increased ease of separation with high efficiency and effectiveness, as only a single and external manipulation is required. This point increases the applicability to researchers who are not good at microscopic anatomy. According to our experience, one can successfully separate follicular ...

Zebrafish is a major model organism for the study of vertebrate development and physiology. The zebrafish can serve as a good model for studying the molecular mechanisms of ovarian development1,2,3. Many features of ovarian development are much conserved during evolution from fish to mammals1,2. Similar to the other vertebrates,zebrafish adults have asynchronous ovaries, containing ovarian follicles of all developmental stages4. The follicle is the fundamental reproductive element of the ovary. The follicle consists of the oocyte that is surrounded by one or several layers of somatic cells called follicular cells. The development of follicles depends on the bidirectional communication between oocytes and follicular cells5. It is vital to separate follicular cells and oocytes from ovarian follicles for different research purposes such as follicular cell primary culture, gene expression analysis, oocyte maturation, and in vitro fertilization.
Traditional separation methods include mechanical separation by forceps and enzymatic digestion6,7,8,9,10. However, the mechanical separation by forceps is time-consuming and laborious. It will also cause the high damage to the oocyte during separation. Although the enzyme digestion method is simple to operate and requires a short time, the treatment time and enzyme concentration should be validated, and the integrity and survival rate of the isolated oocytes are not ideal.Therefore, we have established a simple method to separate both compartments at different developmental stages using pulled glass capillary tubes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >17&#945;,20&#946;-DHP</td>
			<td >Cayman</td>
			<td >16146-5 (5 mg)</td>
			<td ></td>
		</tr>
		<tr >
			<td >24-well plate</td>
			<td >Corning</td>
			<td >3524</td>
			<td></td>
		</tr>
		<tr >
			<td >Ampoule cutter</td>
			<td >AS ONE</td>
			<td >5-124-22 1 bag (100 pieces)</td>
			<td></td>
		</tr>
		<tr >
			<td >Anhydrous Na2HPO4</td>
			<td >Kaixin Chemical</td>
			<td >500 g</td>
			<td></td>
		</tr>
		<tr >
			<td >Brine shrimp</td>
			<td >Hongjie</td>
			<td >250 g</td>
			<td></td>
		</tr>
		<tr >
			<td >CaCl2</td>
			<td >Beichen Fangzheng</td>
			<td >500 g</td>
			<td></td>
		</tr>
		<tr >
			<td >Culture dish</td>
			<td >Biosharp</td>
			<td >BS-90-D (10PCS/PK)</td>
			<td></td>
		</tr>
		<tr >
			<td >DAPI</td>
			<td >Solarbio</td>
			<td >S2110 (25mL)</td>
			<td></td>
		</tr>
		<tr >
			<td >Dissecting Microscope</td>
			<td >ZEISS</td>
			<td >Stemi 305</td>
			<td></td>
		</tr>
		<tr >
			<td >Dissection forcep</td>
			<td >VETUS</td>
			<td >HRC30</td>
			<td></td>
		</tr>
		<tr >
			<td >Dissection scissor</td>
			<td >Kefu</td>
			<td >160 mm&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >Fluorescence Stereomicroscope&#160;</td>
			<td >Leica</td>
			<td >M205C</td>
			<td></td>
		</tr>
		<tr >
			<td >Glass capillary</td>
			<td >IWAKI</td>
			<td >IK-PAS-5P (200 pcs/PACK)</td>
			<td></td>
		</tr>
		<tr >
			<td >Hoechst 33342</td>
			<td >Solarbio</td>
			<td >C0031 (1 mg)</td>
			<td></td>
		</tr>
		<tr >
			<td >KCl</td>
			<td >Beichen Fangzheng</td>
			<td >500 g</td>
			<td></td>
		</tr>
		<tr >
			<td >KH2PO4</td>
			<td >Kaixin Chemical</td>
			<td >500 g</td>
			<td></td>
		</tr>
		<tr >
			<td >Leibovitz&#8217;s L-15 medium</td>
			<td >Gibco</td>
			<td >41300-039 (10&#215;1L)</td>
			<td></td>
		</tr>
		<tr >
			<td >MgSO4&#8226;7H2O</td>
			<td >Beichen Fangzheng</td>
			<td >500 g</td>
			<td></td>
		</tr>
		<tr >
			<td >Micropipette tips</td>
			<td >Axygen</td>
			<td >MCT-150-C</td>
			<td></td>
		</tr>
		<tr >
			<td >NaCl</td>
			<td >Beichen Fangzheng</td>
			<td >500 g</td>
			<td></td>
		</tr>
		<tr >
			<td >NaHCO3</td>
			<td >Beichen Fangzheng</td>
			<td >500 g</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicilia-streptomycia</td>
			<td >Gibco</td>
			<td >#15140122 (100 mL)</td>
			<td></td>
		</tr>
		<tr >
			<td >Stereomicroscope</td>
			<td >ZEISS</td>
			<td >Discover.v20</td>
			<td></td>
		</tr>
	</tbody>
</table>

separation of follicular cells and oocytes in ovarian follicles of zebrafish

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of oocytes and surrounding follicular cells. It is vital to separate both follicular cells and oocytes for various research purposes such as for primary culture of follicular cells, analysis of gene expression, oocyte maturation and in vitro fertilization, etc. The conventional method uses forceps to separate both compartments, which is laborious, time consuming and has high damage to the oocyte. Here, we have established a simple method to separate both compartments using a pulled glass capillary. Under a stereomicroscope, oocytes and follicular cells can be easily separated by pipetting in a pulled fine glass capillary (the diameter depends on the follicle diameter). Compared with the conventional method, this new method has high efficiency in separating both oocytes and follicular cells and has low damage to the oocytes. More importantly, this method can be applied to early-stage follicles including at the pre-vitellogenesis stage. Thus, this simple method can be used to separate follicular cells and oocytes of zebrafish.

This method can be used to separate follicular cells and oocytes at different stages of ovarian follicle development in zebrafish. Figure 1 shows the separation of zebrafish oocytes and follicular cells from ovarian follicles using a capillary glass tube (Figure 1). To investigate whether the follicular cells were separated from intact follicles, the intact follicles and separated oocytes from different stages of follicles from the PV stage to the FG stage were ...

Watch this Scientific Journal Video about Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish at JoVE.com

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

Here, we present a simple method for separating follicular cells and oocytes in zebrafish ovarian follicles, which will facilitate investigations of ovarian development in zebrafish.

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of ...

The overall goal of this procedure is to separate follicular cells and Oocytes in ovarian follicular of Zebrafish. The follicular is the basic unit of the ovary, which consists of the Oocytes and the surrounding follicular cells. It is the wide tool to separate both the follicular cell and outside compartments for well-research policies including primary capture of follicular cells and rises on gene expression, all saturation and in vitro fertilization.Recently, the Zebrafish has become a powerful model of Nissan, which studies a control of already development. Here, we present a simple method, so separates follicular cells and oocytes of Zebrafish follicular which will facilitate investigations of already development in Zebrafish The commercial, no mass or the two separates about follicular cells and all sides of spins the are using post which has a labyrinth, time-consuming and had high damage to the outside. Here, we have established a simple method to two separate compartments using or part glasses Tamela rate under a pure reef.Our Metro scope, both outside and the follicular cells can be easily fabricated by typing the pulled glass capillary. Firstly, prepare a poet glass capillary. Hit the head of glass capillary on the alcohol burner.Use a four sides to stretch the glass capillary to the rave card diameter. Secondly, desect that overate from the adult female fish, isolate ovarian follicles from the ovary. Finally use a polled glass capillary to separate follicular cell and Oocyte from ovarian follicles.Prepare the glass capillary, sharps container, ample cutter, alcohol burner, and a forceps. Lie the alcohol burner, take home a glass of capillary and heat one end of glass capillary on the fire of alcohol burner. Use a forceps to hold another end of glass capillary, stress the glass capillary quickly and gently.Put the glass waste to the sharps container. Use the ample cutter Gretch on the pool with glass. Then break off by the forceps Gently to make a smooth opening of the port glance quickly burn the end of the glass capillary on alcohol burner several times and rub the open end by ample cutter carefully.Take how the fish tank from the cultivated water system. And now the ties adult females by placing fish on the ice water for at least two to five minutes until fish stop Gill movement. Take out the fish from the ice box and remove the remaining water fish body by towel, placed a fish on the D section plate with four sets.Fish were decapitated by serving Spang chord using their sharp scissors to ensure the death. Use dissecting scissors to cut as follows. Dissect from the Chloe cut to the gills along the midline of the abdomen, cut from the bank of the Chloe, cut from the anterior tip to the dorsal side.Gently removed the skin and muscles on one side of the body, expose the internal organs and take healthy entire ovary with four steps. The ovaries were gently placed immediately in the 100 millimeter in culture, dish container 60%L 50 median. Sent a dissecting microscope with transmitted light optics based on the size and morphology of ovarian follicles of different stages.Isolate these ovarian follicles manually using a beer of vying four sets. Collect the isolated ovarian follicles and put them in another cultured dish containing 60%L 50 medium, nice isolated ovarian follicles will be used to for separating follicular cells and Oocytes. Then set a device for separation.Use a 30 centimeters plastic tube. One end is connected with a one milliliter pipette tip with a filter element. Another end is connected with a 200 microliter pipette tip.Under the disecting microscope, use mouth to control the airflow to let the faulty goes moving to the pool with glass capillary then blow the folly goes out from the glass capillary. During this process, follicular cell and Oocytes of ovarian follicles can be separated. This is a separation process for a mature stage follicle.During the process of inhalation, the follicular cell layer is detached from the Oocyte, does after blowing out the follicular cell and Oocyte can be separated. This is a full ground stage follicle. We can see the follicular cell layer is detached from the Oocyte.This method can also be applied in the early stage ovarian follicles. This is a follicle at middle Latella Genesis stage. This is a follicle at early vatella Genesis stage.This is a follicle at a pre-vatalla Genesis stage. From this picture, you can see the process of separation. Panel one shows a intact follicle at full grown stage panel.Panel two shows the angulation of this follicle into pulled glass capillary. Panel three shows the follicular was blown out from the pulled glass capillary. Panel four indicates the successful separation F denuded Oocyte and follicular cell.Even this method, separation of follicular cell in Oocyte can be performed at different stage follicles. Investigate whether the follicular cells were separated from the intact follicles. Intact a follicle and is separated Oocyte from different stages of follicles from BV stage to FG stage were stained with DAPI.The nucleus of follicular cells can be stained by DAPI. The blue signal of DAPI was observed in intact follicles but not denuded Oocytes at these different stage follicles. To further confirm the clear separation, the intact follicles and separated Oocytes were gutting to the histological sections.DAPI stating results showed that follicular cells were clearly removed and denuded Oocytes. Using this method, denuded Oocytes separated from full grown stage follicles were found to undergo the spontaneous Oocyte maturation. without any treatment after four hours incubation.After activation by water, these matured Oocytes can undergo the full chorion expansion. This result indicates the low damage to the Oocyte using this method. Thus, this mustard can be used to obtain the denuded Oocyte for studying the Oocyte matured ration of Zebra fish.The follicular layer of material to follicles can be removed by a capillary glass tube. These can be fertilized and devolve into the hatching stage. The results suggest that this method can be used in for In Vitro fertilization in Zebra fish.We describe here a novel method for the rapid and easy separation of follicular cell and Oocyte from Zebra fish ovarian Follicles. This method has several significant advantages, overturned methodologies. Primary amongst these is to greatly increase the ease of separation as only a single and external manipulation is required.The increased ease of separation increases the availability of this method to those researchers not skilled in microdissection. Second, this method can be used to separate follicular cell and Oocyte of ovarian follicles and early developmental stages, including PV, EBV MV stage, such separation can not be performed at early stage follicle using current methodologies. Third, this new method leads to less damage to the Oocytes.The denuded Oocytes separated by this method can undergo the Oocyte maturation and can be fertilized. In addition, this method can also be applied in other fish.

separation-follicular-cells-oocytes-ovarian-follicles

Research

JoVE Journal

Developmental Biology

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Culture and Transfection of Zebrafish Primary Cells

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in an intact and developing vertebrate. However, the detailed imaging of the morphologies of distinct cell types and subcellular structures is limited in whole mounts. Therefore, we established an efficient and easy-to-use protocol to culture live primary cells from zebrafish embryos and adult tissue.
In brief, 2 dpf zebrafish embryos are dechorionated, deyolked, sterilized, and dissociated to single cells with collagenase. After a filtration step, primary cells are plated onto glass bottom dishes and cultivated for several days. Fresh cultures, as much as long term differenciated ones, can be used for high resolution confocal imaging studies. The culture contains different cell types, with striated myocytes and neurons being prominent on poly-L-lysine coating. To specifically label subcellular structures by fluorescent marker proteins, we also established an electroporation protocol which allows the transfection of plasmid DNA into different cell types, including neurons. Thus, in the presence of operator defined stimuli, complex cell behavior, and intracellular dynamics of primary zebrafish cells can be assessed with high spatial and temporal resolution. In addition, by using adult zebrafish brain, we demonstrate that the described dissociation technique, as well as the basic culturing conditions, also work for adult zebrafish tissue.

We present an efficient and easy-to-use protocol for preparing primary cell cultures of zebrafish embryos for transfection and live cell imaging as well as a protocol to prepare primary cells from adult zebrafish brain.

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in ...

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies have demonstrated the wide differentiation capacity that these cells possess. The ovary represents a promising candidate for cell-based therapies due to the fact that it is rich in MSCs and that it is frequently discarded after ovariectomy surgeries as biological waste. This article describes procedures for the isolation, expansion, and differentiation of MSCs derived from the canine ovary, without the necessity of cell-sorting techniques. This protocol represents an important tool for regenerative medicine because of the broad applicability of these highly differentiable cells in clinical trials and therapeutic uses.

Herein, we describe a method for the isolation, expansion, and differentiation of mesenchymal stem cells from canine ovarian tissue.

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Vitrification of In Vitro Matured Oocytes Collected from Adult and Prepubertal Ovaries in Sheep

In livestock, in vitro embryo production systems can be developed and sustained thanks to the large number of ovaries and oocytes that can be easily obtained from a slaughterhouse. Adult ovaries always bear several antral follicles, while in pre-pubertal donors the maximal numbers of oocytes are available at 4 weeks of age, when ovaries bear peak numbers of antral follicles. Thus, 4 weeks old lambs are considered good donors, even if the developmental competence of prepubertal oocytes is lower compared to their adult counterpart. 
Basic research and commercial applications would be boosted by the possibility of successfully cryopreserving vitrified oocytes obtained from both adult and prepubertal donors. The vitrification of oocyte collected from prepubertal donors would also allow shortening the generation interval and thus increasing the genetic gain in breeding programs. However, the loss of developmental potential after cryopreservation makes mammalian oocytes probably one of the most difficult cell types to cryopreserve. Among the available cryopreservation techniques, vitrification is widely applied to animal and human oocytes. Despite recent advancements in the technique, exposures to high concentrations of cryoprotective agents as well as chilling injury and osmotic stress still induce several structural and molecular alterations and reduce the developmental potential of mammalian oocytes. Here, we describe a protocol for the vitrification of sheep oocytes collected from juvenile and adult donors and matured in vitro prior to cryopreservation. The protocol includes all the procedures from oocyte in vitro maturation to vitrification, warming and post-warming incubation period. Oocytes vitrified at the MII stage can indeed be fertilized following warming, but they need extra time prior to fertilization to restore damage due to cryopreservation procedures and to increase their developmental potential. Thus, post-warming culture conditions and timing are crucial steps for the restoration of oocyte developmental potential, especially when oocyte are collected from juvenile donors.

The protocol aims at providing a standard method for the vitrification of adult and juvenile sheep oocytes. It includes all the steps from the preparation of the in vitro maturation media to the post-warming culture. Oocytes are vitrified at the MII stage using Cryotop to ensure the minimum essential volume.

In livestock, in vitro embryo production systems can be developed and sustained thanks to the large number of ovaries and oocytes that can be easily ...

Gene Expression Analyses in Human Follicles

The ovary is a heterogeneous organ composed of different cell types. To study the molecular mechanisms occurring during folliculogenesis, the localization of proteins and gene expression can be performed on fixed tissue. However, to properly assess gene expression levels in a human follicle, this complex and delicate structure must be isolated. Hence, an adapted protocol previously described by Woodruff&#39;s laboratory has been developed to separate follicles (the oocyte and the granulosa cells) from their surrounding environment. The ovarian cortical tissue is first manually processed to obtain small fragments using two tools: a tissue slicer and a tissue chopper. The tissue is then enzymatically digested with 0.2% collagenase and 0.02% DNase for at least 40 min. This digestion step is performed at 37 &#176;C and 5% CO2 and is accompanied by mechanical pipetting of the medium every 10 min. After incubation, the isolated follicles are collected manually using a calibrated microcapillary pipette under microscope magnification. If follicles are still present in the pieces of tissue, the procedure is completed with manual microdissection. The follicles are collected on ice in a culture medium and are rinsed twice in droplets of phosphate-buffered saline solution. This digestion procedure must be carefully controlled to avoid follicle deterioration. As soon as the structure of the follicles appears to be compromised or after a maximum of 90 min, the reaction is stopped with a 4 &#176;C blocking solution containing 10% fetal bovine serum. A minimum of 20 isolated follicles (sized under 75 &#181;m) should be collected to obtain an adequate amount of total RNA after RNA extraction for real-time quantitative polymerase chain reaction (RT-qPCR). After extraction, the quantification of total RNA from 20 follicles reaches a mean value of 5 ng/&#181;L. The total RNA is then retrotranscribed into cDNA, and the genes of interest are further analyzed using RT-qPCR.

Here, we describe a protocol outlining how to isolate human ovarian follicles from frozen-thawed cortical tissue to perform gene expression analyses.

The ovary is a heterogeneous organ composed of different cell types. To study the molecular mechanisms occurring during folliculogenesis, the localization ...

Identifying DNA Damage in Prophase-Arrested Oocytes of Mice

Detection of DNA Double-Stranded Breaks in Mouse Oocytes

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain arrested at the prophase of meiosis I. The quiescent state may last for years until the oocytes receive a stimulus to grow and obtain the competency to resume meiosis. This protracted state of arrest makes them extremely susceptible to accumulating DNA-damaging insults, which affect the genetic integrity of the female gametes and, therefore, the genetic integrity of the future embryo. 
Consequently, the development of an accurate method to detect DNA damage, which is the first step for the establishment of DNA damage response mechanisms, is of vital importance. This paper describes a common protocol to test the presence and progress of DNA damage in prophase-arrested oocytes during a period of 20 h. Specifically, we dissect mouse ovaries, retrieve the cumulus-oocyte complexes (COCs), remove the cumulus cells from the COCs, and culture the oocytes in &#924;2 medium containing 3-isobutyl-1-methylxanthine to maintain the state of arrest. Thereafter, the oocytes are treated with the cytotoxic, antineoplasmic drug, etoposide, to engender double-strand breaks (DSBs). 
By using immunofluorescence and confocal microscopy, we detect and quantify the levels of the core protein &#947;H2AX, which is the phosphorylated form of the histone H2AX. H2AX becomes phosphorylated at the sites of DSBs after DNA damage. The inability to restore DNA integrity following DNA damage in oocytes can lead to infertility, birth defects, and increased rates of spontaneous abortions. Therefore, the understanding of DNA damage response mechanisms and, at the same time, the establishment of an intact method for studying these mechanisms are essential for reproductive biology research.

Author Spotlight: Understanding DNA Damage Response in Mammalian Oocytes and Preimplantation Embryos 

Maintaining oocyte genome integrity is necessary to ensure the genetic fidelity in the resulting embryo. Here, we present an accurate protocol for detecting DNA double-strand breaks in mammalian female germ cells.

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain ...