Name: isolation of small preantral follicles from the bovine ovary using a combination of fragmentation, homogenization, and serial filtration
Uploaded: 2022-09-27T15:00:00
Description: Watch this Scientific Journal Video about Isolation of Small Preantral Follicles from the Bovine Ovary Using a Combination of Fragmentation, Homogenization, and Serial Filtration at JoVE.com

This project was partially funded by USDA Multi-state project W4112 and UC Davis Jastro Shields award to SM.
The authors would like to extend their appreciation to Central Valley Meat, Inc. for providing the bovine ovaries used in all experiments. The authors also thank Olivia Silvera for assistance with ovary processing and follicle isolation.

Bovine (Bos taurus) ovaries were sourced from a local abattoir and transported to the laboratory within 6 h of collection. Due to the large number of animals processed in the facility, the age, breed, and stage of the estrus cycle of the animals are unknown. Because no live animals were used in these experiments, an approved animal care and use protocol was not required.
1. Preparation of equipment and reagents
<ol>
	<li>Cover a 2 ft wide section of a lab bench with...

The present protocol details a reproducible method to retrieve early stage preantral follicles, specifically at primary and early secondary stages, from the bovine ovary. This protocol builds on previous reports20,25,30,34,35,36 and provides optimizations that result in the isolation of a meaningful number of follicles from a...

Ovarian follicles are the functional units of the ovary, responsible for production of the gamete (oocyte) as well as hormones critical for reproductive function and overall health. Primordial follicles form in the ovary during fetal development or in the neonatal period depending on the species1, and they constitute a female&#39;s ovarian reserve. Follicular growth begins with the activation of primordial follicles that leave the resting pool and enter the growing phase. Preantral folliculogenesis, encompassing all follicle stages before antrum development, is a highly dynamic process that requires synchronous morphological and metabolic changes in the oocyte and the surrounding granulosa cells, driven by tight communication between these two cell types2,3. Preantral follicles constitute the majority of the follicular units found in the ovary at any given time4. Development through the preantral stages of folliculogenesis is estimated to be several weeks longer than antral development5,6, and this time is necessary for the oocyte and somatic cells to acquire sufficient maturity to enter the final stage of development (i.e., the antral stage), and prepare for ovulation, fertilization, and embryonic development7,8,9.
Much of the current knowledge about ovarian preantral folliculogenesis comes from mouse models10,11,12,13, due in part to the ease in recovering a large number of these follicles from a smaller and less fibrous ovary. Although reports of isolation of large numbers of preantral follicles from bovine ovaries date back approximately 30 years14, a more complete understanding about the processes regulating the development of these early-stage follicles has remained unrealized, largely due to the lack of optimized, efficient, and repeatable methods to retrieve sufficient numbers of viable preantral follicles, particularly at early stages of development. With the increasing interest in preserving the ovarian reserve for future use in assisted reproduction in humans, cows become an attractive model due to their more similar ovarian structure15. However, the bovine ovary is markedly richer in collagen compared to the mouse ovary16, making mechanical isolation using methods described for the mouse very inefficient. Efforts to expand fertility preservation techniques include complete in vitro growth of preantral follicles to the antral stage, followed by in vitro maturation (IVM) of the enclosed oocytes, in vitro fertilization (IVF), and embryo production and transfer17. Thus far, this entire process has only been achieved in mice18. In cattle, the progress toward follicle growth in vitro is limited to a few reports with variable follicle stages at the start of culture, as well as variable length of culture between protocols17,19.
The methods described in the literature for the harvest of preantral follicles from the bovine ovary have mostly used mechanical and enzymatic techniques, either isolated or in combination2,14,17,20. The first report of a protocol for bovine preantral follicle isolation used a tissue homogenizer and serial filtration to process whole ovaries20. This study was followed by reports combining mechanical and enzymatic procedures that utilized collagenase14. A recurrent theme when utilizing collagenase to digest the ovarian tissue is the potential risk for damage of the follicular basement membrane, which may compromise follicle viability14,21,22,23. Therefore, different combinations of mechanical methods have been employed, such as the use of a tissue chopper and repeated pipetting or a tissue chopper combined with homogenization20,24,25,26. Another mechanical technique that has been described utilizes needles to dissect preantral follicles directly from the ovarian tissue, which is especially useful for isolating larger (&#62;200 &#181;m) secondary follicles. However, this process is time-consuming, inefficient for isolating smaller preantral follicles, and is skillset-dependent when attempted in bovine ovaries19,27,28.
Taking advantage of the different techniques described in the literature, this protocol aimed to optimize the isolation of preantral follicles from single bovine ovaries in a simple, consistent, and efficient manner that avoids incubation in enzymatic solutions. Improving the methods to isolate preantral follicles will provide an opportunity to enhance the understanding of this stage of folliculogenesis and enable the development of effective culture systems to develop preantral follicles to the antral stage. The detailed procedures described herein for the isolation of preantral follicles from a large mammal such as the bovine species will be vital for researchers aiming to study early folliculogenesis in a non-murine species that is translatable to humans.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5-3/4&#34; Soda Lime Disposable Glass Pasteur Pipette</td>
			<td>Duran Wheaton Kimble</td>
			<td>63A54</td>
			<td>Pasteur pipette that can be used to dislodge follicles from debris while searching within the petri dish</td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Diluted to 4%; fixation of follicles for immunostaining</td>
		</tr>
		<tr>
			<td>20 mL Luer-lock Syringe</td>
			<td>Fisher Scientific</td>
			<td>Z116882-100EA</td>
			<td>Syringe used with the 18 G needle to dislodge follicles from the 40 &#956;m cell strainer</td>
		</tr>
		<tr>
			<td>#21 Sterile Scalpel Blade</td>
			<td>Fisher Scientific</td>
			<td>50-365-023</td>
			<td>Used to cut the ovaries and remove the medula</td>
		</tr>
		<tr>
			<td>40 &#956;m Cell Strainer</td>
			<td>Fisher Scientific&#160;</td>
			<td>22-363-547</td>
			<td>Used to filter the filtrate from the 300 &#956;m cell strainer</td>
		</tr>
		<tr>
			<td>104 mm Plastic Funnel</td>
			<td>Fisher Scientific</td>
			<td>10-348C</td>
			<td>Size can vary, but ensure the cheese cloth is cut appropriately and that the ovarian homogenate will not spill over</td>
		</tr>
		<tr>
			<td>300 &#956;m Cell Strainer</td>
			<td>pluriSelect&#160;</td>
			<td>43-50300-03</td>
			<td>Used to filter the filtrate from the cheese cloth&#160;</td>
		</tr>
		<tr>
			<td>500 mL Erlenmeyer Flask</td>
			<td>Fisher Scientific</td>
			<td>FB500500</td>
			<td>Funnel and flask used to catch filtrate from the cheese cloth&#160;</td>
		</tr>
		<tr>
			<td>Air-Tite Sterile Needles 18 G</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-817-151</td>
			<td>18 G offers enough pressure to dislodge follicles from the 40 &#956;m cell strainer</td>
		</tr>
		<tr>
			<td>Air-Tite Sterile Needles 27 G 13 mm</td>
			<td>Fisher Scientific</td>
			<td>14-817-171</td>
			<td>Needles that can be used to manipulate any debris in which follicles are stuck</td>
		</tr>
		<tr>
			<td>BD Hoechst 33342 Solution</td>
			<td>Fisher Scientific</td>
			<td>BDB561908</td>
			<td>Fluorescent DNA stain</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A7030-100G&#160;</td>
			<td>Component of follicle wash media</td>
		</tr>
		<tr>
			<td>Cheese Cloth</td>
			<td>Electron Microscopy Sciences</td>
			<td>71748-00</td>
			<td>First filtering step of the ovarian homogenate meant to remove large tissue debris</td>
		</tr>
		<tr>
			<td>Classic Double Edge Safety Razor Blades</td>
			<td>Wilkinson Sword</td>
			<td>N/A</td>
			<td>Razor blades that fit the best in the McIlwain Tissue Chopper and do not dull quickly</td>
		</tr>
		<tr>
			<td>Donkey-Anti-Rabbit Secondary Antibody, Alexa Fluor 488</td>
			<td>Fisher Scientific</td>
			<td>A-21206</td>
			<td>Secondary antibody for immunostaining</td>
		</tr>
		<tr>
			<td>Eisco Latex Pipette Bulbs</td>
			<td>Fisher Scientific</td>
			<td>S29388</td>
			<td>Rubber bulb to use with Pasteur pipettes</td>
		</tr>
		<tr>
			<td>HEPES Buffer</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>Component of follicle wash media</td>
		</tr>
		<tr>
			<td>Homogenizer</td>
			<td>VWR</td>
			<td>10032-336</td>
			<td>Homogenize the ovarian tissue to release follicles&#160;</td>
		</tr>
		<tr>
			<td>ImageJ/Fiji</td>
			<td>NIH</td>
			<td>v2.3.1</td>
			<td>Software used for analysis of fluorescence-immunolocalization</td>
		</tr>
		<tr>
			<td>McIlwain Tissue Chopper</td>
			<td>Ted Pella</td>
			<td>10184</td>
			<td>Used to cut ovarian tissue small enough for homogenization</td>
		</tr>
		<tr>
			<td>Microscope - Stereoscope</td>
			<td>Olympus</td>
			<td>SZX2-ILLT</td>
			<td>Dissection microscope used for searching and harvesting follicles from the filtrate</td>
		</tr>
		<tr>
			<td>Microscope - Inverted</td>
			<td>Nikon</td>
			<td>Diaphot 300</td>
			<td>Inverted microscope used for high magnification brightfield visualization of isolated follicles</td>
		</tr>
		<tr>
			<td>Microscope - Inverted</td>
			<td>ECHO</td>
			<td>Revolve R4</td>
			<td>Inverted microscope used for high magnification brightfield and epifluorescence visualization of isolated follicles</td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>Sigma-Aldrich</td>
			<td>M8410-1L</td>
			<td>Oil to cover the drops of follicle wash medium to prevent evaporation during searching</td>
		</tr>
		<tr>
			<td>Non-essential Amino Acids (NEAA)</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td>Component of follicle wash medium</td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Jackson ImmunoResearch</td>
			<td>017-000-001</td>
			<td>Reagent for immunostaining blocking buffer</td>
		</tr>
		<tr>
			<td>Nunc 4-well Dishes for IVF</td>
			<td>Thermo Fisher Scientific</td>
			<td>144444</td>
			<td>4-well dishes for follicle isolation and washing</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution 100x</td>
			<td>Gibco</td>
			<td>15-140-122</td>
			<td>Component of follicle wash medium</td>
		</tr>
		<tr>
			<td>Petri Dish 60 mm OD x 13.7 mm</td>
			<td>Ted Pella</td>
			<td>10184-04</td>
			<td>Petri dish that fits the best in the McIlwain Tissue Chopper</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>BP665-1</td>
			<td>Washing buffer for ovaries and follicles</td>
		</tr>
		<tr>
			<td>Plastic Cutting Board</td>
			<td>Fisher Scientific</td>
			<td>09-002-24A</td>
			<td>Cutting board of sufficient size to safely cut ovaries</td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone (PVP)</td>
			<td>Fisher Scientific</td>
			<td>BP431-100</td>
			<td>Addition of PVP (0.1% w/v) to PBS prevents follicles from sticking to the plate or each other&#160;</td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>Thermo Fisher Scientific</td>
			<td>P36930</td>
			<td>Mounting medium for fluorescently labeled cells or tissue</td>
		</tr>
		<tr>
			<td>Qiagen RNeasy Micro Kit</td>
			<td>Qiagen</td>
			<td>74004</td>
			<td>RNA column clean-up kit</td>
		</tr>
		<tr>
			<td>R</td>
			<td>The R Foundation</td>
			<td>v4.1.2</td>
			<td>Statistical analysis software</td>
		</tr>
		<tr>
			<td>Rabbit-Anti-Human Cx37/GJA4 Polyclonal Antibody</td>
			<td>Abcam</td>
			<td>ab181701</td>
			<td>Cx37 primary antibody for immunostaining</td>
		</tr>
		<tr>
			<td>RevertAid RT Reverse Transcription Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>K1691</td>
			<td>cDNA synthesis kit</td>
		</tr>
		<tr>
			<td>Rstudio</td>
			<td>RStudio, PBC</td>
			<td>v2021.09.2</td>
			<td>Statistical analysis software</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide Solution (1N/Certified)</td>
			<td>Fisher Scientific</td>
			<td>SS266-1</td>
			<td>Used to increase media pH to 7.6-7.8</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate (NaPyr)</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td>Component of follicle wash medium</td>
		</tr>
		<tr>
			<td>Square Petri Dish 100 mm x 15 mm&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>60872-310</td>
			<td>Gridded petri dishes allow for more efficient identification of follicles&#160;</td>
		</tr>
		<tr>
			<td>SsoAdvanced Universal SYBR Green Supermix</td>
			<td>BioRad</td>
			<td>1725271</td>
			<td>Mastermix for PCR reaction</td>
		</tr>
		<tr>
			<td>Steritop Threaded Bottle Top Filter</td>
			<td>Sigma-Aldrich</td>
			<td>S2GPT02RE</td>
			<td>Used to sterilize follicle wash medium</td>
		</tr>
		<tr>
			<td>SYBR-safe DNA gel stain</td>
			<td>Thermo Fisher Scientific</td>
			<td>S33102</td>
			<td>Staining to visual PCR products on agarose gel</td>
		</tr>
		<tr>
			<td>TCM199 with Hank&#8217;s Salts</td>
			<td>Gibco</td>
			<td>12-350-039</td>
			<td>Component of follicle wash medium</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-100</td>
			<td>Detergent for immunostaining permeabilization buffer</td>
		</tr>
		<tr>
			<td>Trizol reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>15596026</td>
			<td>RNA isolation reagent</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Gibco</td>
			<td>15-250-061</td>
			<td>Used for testing viability of isolated follicles</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td></td>
			<td></td>
			<td>Detergent for immunostaining wash buffer</td>
		</tr>
		<tr>
			<td>Warmer Plate Universal</td>
			<td>WTA</td>
			<td>20931</td>
			<td>Warm plate to keep follicles at 38.5 &#176;C while searching under the microscope</td>
		</tr>
		<tr>
			<td>Wiretrol II Calibrated Micropipets</td>
			<td>Drummond</td>
			<td>50002-005</td>
			<td>Glass micropipettes to manipulate follicles</td>
		</tr>
	</tbody>
</table>

isolation of small preantral follicles from the bovine ovary using a combination of fragmentation, homogenization, and serial filtration

Understanding the full process of mammalian folliculogenesis is crucial for improving assisted reproductive technologies in livestock, humans, and endangered species. Research has been mostly limited to antral and large preantral follicles due to difficulty in the isolation of smaller preantral follicles, especially in large mammals such as bovine species. This work presents an efficient approach to retrieve large numbers of small preantral follicles from a single bovine ovary. The cortex of individual bovine ovaries was sliced into 500 &#181;m cubes using a tissue chopper and homogenized for 6 min at 9,000-11,000 rpm using a 10 mm probe. Large debris was separated from the homogenate using a cheese cloth, followed by serial filtration through 300 &#181;m and 40 &#181;m cell strainers. The contents retained in the 40 &#181;m strainer were rinsed into a search dish, where follicles were identified and collected into a drop of medium. The viability of the collected follicles was tested via trypan blue staining. This method enables the isolation of a large number of viable small preantral follicles from a single bovine ovary in approximately 90 min. Importantly, this method is entirely mechanical and avoids the use of enzymes to dissociate the tissue, which may damage the follicles. The follicles obtained using this protocol can be used for downstream applications such as isolation of RNA for RT-qPCR, immunolocalization of specific proteins, and in vitro culture.

Overview and critical steps 
Using this protocol, small bovine preantral follicles can be reliably isolated from single ovaries in experimentally relevant numbers. From a total of 30 replicates, an average of 41 follicles were obtained per replicate, with a range of 11 to 135 follicles (Figure 4A). In 14 replicates, the follicles were characterized for stage of development as previously described26 by measuring the follicle diameter using a 1 &#1...

Watch this Scientific Journal Video about Isolation of Small Preantral Follicles from the Bovine Ovary Using a Combination of Fragmentation, Homogenization, and Serial Filtration at JoVE.com

Isolation of Small Preantral Follicles from the Bovine Ovary Using a Combination of Fragmentation, Homogenization, and Serial Filtration

Advancing the study of preantral folliculogenesis requires efficient methods of follicle isolation from single ovaries. Presented here is a streamlined, mechanical protocol for follicle isolation from bovine ovaries using a tissue chopper and homogenizer. This method allows collection of a large number of viable preantral follicles from a single ovary.

Understanding the full process of mammalian folliculogenesis is crucial for improving assisted reproductive technologies in livestock, humans, and ...

This protocol allows for the efficient and repeatable isolation of early stage pre-antral follicles from a single bovine ovary and it creates new opportunities for the study of ovarian folliculogenesis in a non-bovine species. By using a greater number of follicles from a single ovary, this technique enables in vivo treatments before ovary harvest, or obtention of multiple samples over time from the same animal via ovarian biopsy. Isolation and cryo-preservation of early stage pre-antral follicles can be a valuable option for fertility preservation in young women undergoing gonadotoxic treatment such as chemotherapy, as well as germplasm preservation from endangered species.Demonstrating the procedure will be Stephanie McDonnell, Amanda Morton, and Juliana Candelaria, PhD students from my laboratory. To begin, transfer ovaries in the laboratory to warm, sterile PBS containing Pen-Strep and select ovaries with many small antral follicles and no prominent corpus lutetium. Remove any excess connective tissue and fat from the ovaries using scissors.Transfer one ovary to the cutting board on the bench paper. Using a scalpel, cut the ovary in half longitudinally from one ligament attachment site to the opposite attachment site. Keep one half of the ovary on the cutting board to be processed and place the other half of the ovary back into warm PBS containing Pen-Strep.To remove the medulla, slice along the curvature of the ovary approximately 2 millimeter away from the surface without cutting through the cortex with the exposed medulla facing upward. Slice down through the cut to remove the majority of the medulla. Flip the ovary half over so that the epithelium faces upward and use the scalpel to finish cutting the medulla away from the cortex and trim away any remaining white connective tissue around the edge of the ovary piece that was connected to the ligaments.Once the majority of the medulla is removed, use the scalpel to cut the cortex to approximately 1-millimeter thickness while manipulating the scalpel with small back-and-forth motions to shave away the remainder of the medulla, then cut each ovary cortex half into two pieces. Keep the cortex pieces in warm PBS containing Pen-Strep until ready to chop. Transfer a single piece of the cortex to the Petri dish on the tissue chopper and wet the tissue with three or four drops of warm PBS containing Pen-Strep.While holding the piece of tissue steady with a pair of forceps, press the Reset button once to start the tissue chopper. After the entire piece of the cortex has been cut into strips, use the blade holder knob to lift the blade off the Petri dish and the forceps to remove any tissue from the blade. After rotating the plate holder by 90 degrees, again, press the Reset button and pass the blade entirely through the tissue strips.Use the blade holder knob to lift the blade off the Petri dish and the forceps to remove any tissue from the blade as demonstrated previously. Using a transfer pipette and warm PBS containing Pen-Strep, wash the chopped tissue into a pre-warmed 50-milliliter conical tube. Return the conical tube to the water or bead bath to keep the chopped tissue warm while repeating the chopping procedure with the other three halves of the ovary.For best cutting results, use the nut driver to remove the nut from the chopping arm and remove the washer and blade clasp. Using forceps, remove the blade from the chopping arm. Flip it over so that the unused edge is facing the Petri dish and place it back onto the chopping arm.After ensuring that the homogenizer unit is plugged in and the speed is set to the second bar, insert the 10-millimeter generator probe into the unit according to the manufacturer's specifications. Next, set a timer for 1 minute and insert the probe into the 50-milliliter conical tube containing the chopped cortex tissue from one ovary and enough PBS containing Pen-Strep to fill the tube to the 25 milliliter line. The depth to which the probe is inserted must be 1/3 of the liquid's height measured from the bottom of the chamber.Once the timer starts, turn on the homogenizer, ensuring that the bottom of the probe does not touch the tube and hold the tube still while the homogenizer is turned on. After 1 minute of homogenization, remove the probe from the tube, then remove any connective tissue clogging the venting holes in the space between the rotor knife and rotor tube or cortex pieces stuck in the probe using forceps and place them back into the tube. Pour the dispersed tissue into the cheesecloth-covered funnel, inserted into the Erlenmeyer flask and rinse the contents of the tube into the funnel using warm PBS.Finally, rinse the cheesecloth with warm PBS. Force the liquid in small fragments to pass through the holes of the cloth by twisting the cheesecloth around the tissue fragments down and squeezing until all excess fluid and tissue are removed from the cheesecloth. After reopening the cheesecloth over the funnel, rinse the cheesecloth with PBS containing Pen-Strep using a transfer pipette, and again, squeeze any residual tissue fragments through the cloth.Using a hemostat, hold the 300-micrometer cell strainer over a 200-milliliter beaker and pour the filtrate in the Erlenmeyer flask through the cell strainer. If the cell strainer becomes clogged with tissue, gently tap it against the beaker, then turn the strainer upside down and tap out the large tissue debris onto the bench paper. Rinse the contents of the flask using warm PBS containing Pen-Strep until no tissue fragments remain and pour it into the cell strainer.Next, while holding the 40-micrometer cell strainer with a hemostat over a second 200-milliliter beaker, pour all the initial filtrate present in the first 200-milliliter beaker through the strainer, then rinse the contents of the beaker using warm PBS containing Pen-Strep until no tissue fragments remain and pour it into the cell strainer. After fitting the 18 gauge needle to the 20-milliliter syringe, fill the syringe with follicle wash medium. With a hemostat, hold the 40-micrometer cell strainer upside down over a square Petri dish and use the syringe to wash out the contents of the cell strainer into the dish.Transfer the square Petri dish to a stereoscope with a warm stage set to 38.5 degrees Celsius with a magnification set between 1.25 and 3.2x. After identifying follicles from the square Petri dish, transfer the follicle to wash medium drops using the micropipette. The total number of isolated pre-antral follicles per replicate using 6-minute tissue homogenization shows an average of 41 follicles per replicate, ranging from 11 to 135 follicles.Assessment of the developmental stage of 476 isolated follicles showed that the majority were in the primary stage of development, measuring 40 to 79 micrometers in containing one layer of cuboidal granulosa cells. Homogenization of the ovarian cortex for 6 minutes yielded a greater number of pre-antral follicles compared to 5 minutes of homogenization at the same speed. Transcript expression of the granulosa cell marker FSH receptor and germ cell marker DAZL in the follicles was evaluated using reverse transcription quantitative PCR demonstrating that both primary and early secondary follicles expressed FSH receptor and DAZL transcripts.Immunofluorescent localization of CX37 in isolated pre-antral follicles at both the primary and early secondary stages shows that CX37 was localized to the O plasm, being absent from the nucleus of the O site and to the membrane of the granulosa cells. In our experience, proper dissection of a thin layer of cortical tissue, chopping the tissue to the right size, and proper homogenization are critical steps to ensure release of viable follicles from the tissue. Isolated pre-antral follicles can be used to answer questions such as regulation of pre-antral follicle growth and the nature of interactions between granulosa cells and the oocyte, thus facilitating the development of more efficient culture conditions.

isolation-small-preantral-follicles-from-bovine-ovary-using

Research

JoVE Journal

Biology

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse 

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid peptide (A&#946;) and intraneuronal accumulation of hyperphosphorylated tau protein. Because cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the brain, it provides a reflection of the biochemical changes in the brain in response to pathological processes. CSF from AD patients shows a decrease in the 42 amino-acid form of A&#946; (A&#946;42), and increases in total tau and hyperphosphorylated tau, though the mechanisms responsible for these changes are still not fully understood. Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses. Here, we demonstrate a refined cisterna magna puncture technique for CSF sampling from the mouse. This extremely gentle sampling technique allows serial CSF samples to be obtained from the same mouse at 2-3 month intervals which greatly minimizes the confounding effect of between-mouse variability in A&#946; or tau levels, making it possible to detect subtle alterations over time. In combination with A&#946; and tau ELISA, this technique will be useful for studies designed to investigate the relationship between the levels of CSF A&#946;42 and tau, and their metabolism in the brain in AD mouse models. Studies in Tg mice could provide important validation as to the potential of CSF A&#946; or tau levels to be used as biological markers for monitoring disease progression, and to monitor the effect of therapeutic interventions. As the mice can be sacrificed and the brains can be examined for biochemical or histological changes, the mechanisms underlying the CSF changes can be better assessed. These data are likely to be informative for interpretation of human AD CSF changes.

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses in human patients. Here, we demonstrate a refined cisterna magna puncture technique for serial CSF sampling from the mouse.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid ...

Small Volume (1-3L) Filtration of Coastal Seawater Samples

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For today s demonstration samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. This video documents small volume (~1 L) filtration of microbial biomass from the water column. The protocol is an extension of the large volume sampling protocol described earlier, with one major difference: here, there is no pre-filtration step, so all size classes of biomass are collected down to the 0.22 &mu;m filter cut-off. Samples collected this way are ideal for nucleic acid analysis. The set-up, filtration, and clean-up steps each take about 20-30 minutes. If using two peristaltic pumps simultaneously, up to 8 samples may be filtered at the same time. To prevent biofilm formation between sampling trips, all filtration equipment must be rinsed with dilute HCl and deionized water and autoclaved immediately after use.

Small Volume 1-3L Filtration of Coastal Seawater Samples

This video documents small volume (~1 L) filtration of microbial biomass from the water column.

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For today s ...

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

A Simple Benchtop Filtration Method to Isolate Small Extracellular Vesicles from Human Mesenchymal Stem Cells

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this isolation method is relatively lower, and these methods are inefficient in separating sEV subtypes. This study demonstrates a simple benchtop filtration method to isolate human umbilical cord-derived MSC small extracellular vesicles (hUC-MSC-sEVs), successfully separated by ultrafiltration from the conditioned medium of hUC-MSCs. The size distribution, protein concentration, exosomal markers (CD9, CD81, TSG101), and morphology of the isolated hUC-MSC-sEVs were characterized with nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively. The isolated hUC-MSC-sEVs&#39; size was 30-200 nm, with a particle concentration of 7.75 &#215; 1010 particles/mL and a protein concentration of 80 &#181;g/mL. Positive bands for exosomal markers CD9, CD81, and TSG101 were observed. This study showed that hUC-MSC-sEVs were successfully isolated from hUC-MSCs conditioned medium, and characterization showed that the isolated product fulfilled the criteria mentioned by Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV 2018).

This protocol demonstrates how to isolate human umbilical cord-derived mesenchymal stem cells&#39; small extracellular vesicles (hUC-MSC-sEVs) with a simple lab-scale benchtop setting. The size distribution, protein concentration, sEVs markers, and morphology of isolated hUC-MSC-sEVs are characterized by nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively.

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this ...

Extraction, Labeling, and Purification of Lineage-Specific Cells from Human Antral Follicles

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary but also across multiple points of control within the hypothalamic/pituitary/ovarian circuit. Within the ovary, multiple specialized cell types grow in close association with the oocyte within the ovarian follicles. The biology of these cells has been well described at the later stages, when they are easily recovered as byproducts of assisted reproductive treatments. However, the in-depth analysis of small antral follicles isolated directly from the ovary is not commonly carried out due to the scarcity of human ovarian tissue and the limited access to the ovary in patients undergoing assisted reproductive treatments. 
These methods for processing whole ovaries for the cryopreservation of cortical strips with the concurrent identification/isolation of ovary resident cells enable the high-resolution analysis of the early stages of antral follicle development. We demonstrate protocols for isolating discrete cell types by treating antral follicles enzymatically and separating the granulosa, theca, endothelial, hematopoietic, and stromal cells. The isolation of cells from the antral follicles at various sizes and developmental stages enables the comprehensive analysis of the cellular and molecular mechanisms that drive follicle growth and ovarian physiology and provides a source of viable cells that can be cultured in vitro to recapitulate the follicle microenvironment.

Here, we present protocols for the identification and purification of ovarian cells from antral follicles. We elaborate on methods for processing whole ovaries for the cryopreservation of cortical strips while also harvesting intact antral follicles that are treated enzymatically to liberate multiple follicle resident cell types, including granulosa, theca, endothelial, hematopoietic, and stromal cells.

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary ...

3D Culture of Organoids from Murine Intestinal Crypts and Single Stem Cell

The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine small intestinal crypts can form organoids that mimic the intestinal epithelium when cultured in a 3D extracellular matrix. The organoids are composed of all cell types that fulfill various intestinal homeostatic functions. These include Paneth cells, enteroendocrine cells, enterocytes, goblet cells, and tuft cells. Well-characterized molecules are added into the culture medium to enrich the intestinal stem cells (ISCs) labeled with leucine-rich repeats containing G protein-coupled receptor 5 and are used to drive differentiation down specific lineages; these molecules include epidermal growth factor, Noggin (a bone morphogenetic protein), and R-spondin 1. Additionally, a protocol to generate organoids from a single erythropoietin-producing hepatocellular receptor B2 (EphB2)-positive ISC is also detailed. In this methods article, techniques to isolate small intestinal crypts and a single ISC from tissues and ensure the efficient establishment of organoids are described.

Author Spotlight: The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research  

We describe a protocol to isolate murine small intestinal crypts and culture intestinal 3D organoids from the crypts. Additionally, we describe a method to generate organoids from a single intestinal stem cell in the absence of a sub-epithelial cellular niche.

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine ...