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 brine shrimp.</li>
	</ol>
	</li>
	<li>Pulled glass capillary
	<ol>
		<li>Use a 15 cm glass capillary and place its head in an alcohol burner to heat it. Hold one end of glass capillary by hand and hold the other end with forceps.</li>
		<li>After heating 5-10 seconds, the glass on the fire of alcohol burner become red and soft. Stretch the head of the glass capillary with forceps to make the capillary opening with required diameter. 
		NOTE: The diameter depends on the follicle diameters: previtellogenic (PV; about 0.30 mm in diameter), early vitellogenic (EV; about 0.40 mm in diameter), midvitellogenic (MV; about 0.50 mm in diameter), late vitellogenic (LV; about 0.60 mm in diameter) and full grown but immature (FG; about 0.65 mm in diameter).</li>
		<li>Stretch the opening of glass capillary to the appropriate diameter, which is slightly smaller the size of separated follicles. The glass capillary with an opening much bigger than the size of ovarian follicles cannot perform separation, but the much smaller ones would break the follicles during separation. Practice the stretching of the glass capillary several times. 
		NOTE: Heating time depends on the size of glass capillary. Do not directly stretch the glass capillary on the fire of alcohol burner. It will break the glass capillary.</li>
		<li>Cut the glass capillary with an ampoule cutter and break the glass capillary with forceps to obtain a smooth incision. 
		NOTE: A sharp or broken opening of the glass capillary would damage the follicles.</li>
		<li>Using a 30 cm plastic tube, insert a 1 mL pipette tip with a filter element at one end, insert the pulled glass capillary at the end of the pipette, and connect a 200 &#181;L pipette tip at another end.</li>
	</ol>
	</li>
</ol>
2. Separation of zebrafish oocytes and follicular cells at different stages
<ol>
	<li>Dissection of the zebrafish ovary
	<ol>
		<li>Fill an ice bucket to 4/5 full with slurry ice and add sufficient fish water to let slurry ice float. Wait 2-5 minutes, check the water temperature, and make sure the temperature is between 2-4 &#176;C.</li>
		<li>Anesthetize adult females by placing fish in the ice water for at least 2-5 minutes, until fish stop gill movement. Decapitate the fish by severing the spinal cord using a sharp scissors to ensure death.</li>
		<li>Place the fish on the dissecting plate with forceps.</li>
		<li>Use dissecting scissors to cut as follows. Dissect from the cloaca to the gills along the midline of the abdomen. Cut from the back of the cloaca. Cut from the anterior tip to the dorsal side. Gently remove the skin and muscles on one side of the body. Expose the internal organs and take out the entire ovary with forceps.</li>
		<li>Immediately, place the ovaries gently into a 100 mm culture dish containing 60% Leibovitz&#39;s L-15 (L-15) medium.</li>
	</ol>
	</li>
	<li>Isolation of ovarian follicles 
	NOTE: The staging system that we have adopted is based on the original definition of Selman et al.4.
	<ol>
		<li>Manually isolate follicles of different stages using a pair of fine forceps as described previously8.</li>
		<li>Group the ovarian follicles into the following stages: PV, EV, MV, LV and FG stages.</li>
	</ol>
	</li>
	<li>Separation of follicular cells and oocyte from ovarian follicles
	<ol>
		<li>Put the ovarian follicles at different stages into a clean Petri dish containing 60% L-15 medium.</li>
		<li>Using the pulled glass capillary from step 1.2, suck the follicles into the glass capillary 2-3 cm and blow them out. 
		NOTE: When the opening diameter of the glass capillaries is appropriate, the follicle can be separated from the oocyte by inhalation once.
		<ol>
			<li>If the follicular cell layer is attached to the oocyte firmly, repeat 2-3 times to accomplish a complete separation. Avoid multiple attempts to force a follicle through a smaller capillary, which would damage the follicle. 
			NOTE: In the process of inhalation and blowing out, the follicular layer falls off and separates from the oocyte, and finally, follicular cells and denuded oocytes are separated (Figure 1).</li>
		</ol>
		</li>
		<li>Pool the denuded but intact oocytes and collect the surviving denuded oocytes for further assays.</li>
		<li>To investigate whether the follicular cells were separated from intact follicles, stain the intact follicles and separated oocytes with 4&#39;,6-diamidino-2-phenylindole (DAPI).
		<ol>
			<li>Fix the samples in 4% buffered paraformaldehyde at room temperature (RT) for 1 h. Wash several times by PBS.</li>
			<li>Stain by DAPI at RT for 30 min. Washed several times with PBS. View and photograph with a fluorescence microscope (e.g., Leica DFC7000 T).</li>
		</ol>
		</li>
		<li>To confirm the completed separation, fix the intact follicles and separated oocytes in 4% buffered paraformaldehyde overnight at 4 &#176;C, and embed intissue-freezing medium (e.g., Leica) at -25 &#176;C.
		<ol>
			<li>Cut the fixed tissues on a microtome, and mount onto glass slides.</li>
			<li>Wash the sections in PBS and visualize the cell nuclei with DAPI. View and photograph on a fluorescence microscope.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. In vitro maturation (IVM) and in vitro fertilization (IVF)
NOTE: The procedure of IVM of IVF was followed as described previously with minor modification 11,12,13.
<ol>
	<li>Prepare fresh maturation medium (+DHP medium) before ovary dissection from adult zebrafish female. To prepare the fresh maturation medium, add 9 mL of Leibovitz&#39;s L-15 medium, pH 9.0, to 15 mL conical tubes. Add 10 &#181;L of 17&#945;-20&#946;-dihydroxy-4 pregnen-3-one (DHP) (5 mg/mL), 490 &#181;L of dH2O, and 500 &#181;L of 10% bovine serum albumin (BSA).</li>
	<li>Prepare Hank&#39;s solution and E3 solution in advance. Prepare Hanks&#39; solution as described by Baars et al.14. Prepare E3 medium: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, and 1-5% Methylene Blue.</li>
	<li>Perform dissection of zebrafish ovary and isolation of ovarian follicles as in steps step 2.</li>
	<li>Collect full grown stage follicles and use as immature oocytes. For each adult female zebrafish, at least 150-200 full grown stage follicles can be isolated. Select the intact and healthy follicles for IVM.</li>
	<li>Incubate the full-grown stage follicles in +DHP medium in 12-well plates, checking periodically to ensure that the oocytes remain intact. Remove any lysing oocytes with a Pasteur pipette. 
	NOTE: During the oocyte maturation, the oocytes will become progressively translucent. A majority of the oocytes become translucent after treatment of DHP for 2 h. This can be observed under a dissecting microscope with transmitted light optics.</li>
	<li>Remove the outermost follicular cells from each matured oocyte as described in step 2.3.</li>
	<li>Transfer the denuded oocytes to a Petri dish with a few drops of culture medium and proceed to fertilization.</li>
	<li>Prepare the fresh sperm solution using testes dissected from at least three males in 500 &#181;L of Hanks&#39; solution. Put the sperm solution on ice, where it can keep its potency of fertilization for up to 2-3 h.</li>
	<li>Add 100 &#181;L of sperm solution to denuded &#38; matured oocytes on a Petri dish. Gently add the sperm solution among the eggs, and swirl the sperm and eggs together using a pipette tip.</li>
	<li>Immediately add 1 mL of E3 medium solution to activate the eggs and again gently swirl eggs and sperm using a pipette tip.</li>
	<li>After around 1 minute post-fertilization (mpf), flood the plate with E3 medium solution. Full chorion expansion can be observed within 10-15 mpf.</li>
	<li>Between 35-45 mpf, select the embryos that are undergoing symmetrical cleavage into the 2-cell stage, and which are therefore fertilized. Remove embryos that are not undergoing cell cleavage.</li>
	<li>Allow embryos to develop in the Petri dish at 28 &#176;C, with a limit of 80 embryos per 10 cm plate. View and photograph the embryo development.</li>
</ol>

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The authors have nothing to disclose.

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 ...

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7.4K Views.  College of Life Sciences, Northwest Normal University. 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.

Video: Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

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 ...

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix (ECM) remodeling. In vivo, ECM-rich environment consisting of fibrillar collagens provides a structural support to adipose tissues during the progression and regression of obesity. Physiological ECM remodeling mediated by matrix metalloproteinases (MMPs) plays a major role in regulating adipose tissue size and function1,2. The loss of physiological collagenolytic ECM remodeling may lead to excessive collagen accumulation (tissue fibrosis), macrophage infiltration, and ultimately, a loss of metabolic homeostasis including insulin resistance3,4. When a phenotypic change of the adipose tissue is observed in gene-targeted mouse models, isolating primary ASCs from fat depots for in vitro studies is an effective approach to define the role of the specific gene in regulating the function of ASCs. In the following, we define an immunomagnetic separation of Sca1high ASCs.

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

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 ...

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 ...