Name: isolation of small preantral follicles from the bovine ovary using a combination of fragmentation, homogenization, and serial filtration
Uploaded: 2022-09-27T15:00:00.000+00:00
Duration: 9 min 11 s
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.

The authors have nothing to disclose.

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 an individual ovary. Preantral follicles isolated using this method are viable and can be used for downstream applications such as immunolocalization of proteins and RNA extraction for analysis of gene expression. Culturing of the isolated follicles was not attempted in this study. However, efforts are currently underway to achieve successful long-term culture of small bovine preantral follicles, which will be an important downstream application of this protocol that will help improve the understanding of ovarian folliculogenesis, perform toxicology tests, and devise ways to successfully manipulate follicles for fertility preservation.
Data from these experiments show that the 6 min homogenization is critical for proper dispersion of the ovarian cortical tissue and follicle release (Figure 4). Additional steps that contribute to thorough homogenization of the tissue are the initial dissection of the cortex from the medulla ensuring proper thickness of the cortex (~1 mm), trimming away of excess connective tissue, and chopping the cortex into small fragments. These steps prevent clogging of the homogenizer probe and unnecessary debris in the search plate. Cell strainer pore size is important for excluding debris and follicles of undesired size and stage; in the case of this protocol, the final step is collecting the contents of a 40 &#181;m cell strainer so as to exclude primordial follicles, which are typically &#60;40 &#181;m in diameter in cattle26. Finally, using a Pasteur pipette to dislodge follicles from debris in the search dish is recommended over attempting to manually dissect follicles out of the debris, as the latter technique requires more technical skill and, if done improperly, can damage the follicles.
While this protocol presents improvements compared to existing methods, there are limitations that must be considered. First, due to the heterogeneous nature of the ovarian follicle population15 and unknown history of the animals from which ovaries were retrieved, the number of follicles isolated after performing this protocol were highly variable. One factor that must be considered is animal age, as the ovarian reserve is known to decrease with age37.
Isolation of small preantral follicles from the bovine ovary provides opportunities to investigate basic scientific questions at the single-sample level, an approach that has been difficult to conduct previously. For example, all preantral follicle stages evaluated here showed expression of CX37, a critical protein for oocyte-granulosa cell communication. Similarly, expression of FSH receptor has been previously demonstrated in individually isolated preantral follicles32. Other specific markers of granulosa cells, such as the steroidogenic enzymes CYP17 and CYP1938, would be relevant additions to the gene/protein expression panel of preantral follicles, and will be the subject of future projects. The visualization of the entire follicular structure via regular or confocal microscopy is more comprehensive compared to histological examination, where only one segment of the follicle can be analyzed at a time. This is especially advantageous in studies that may want to utilize immunostaining or in situ hybridization, where detailed spatial localization of expression is required. Additionally, isolated preantral follicles can be used to investigate single-follicle gene expression by using sensitive kits for RT-qPCR30. The ability to analyze follicles individually and without influence of the ovarian environment is a critical step forward in understanding the complexity of preantral folliculogenesis.
Cryopreservation of the ovarian cortex tissue has become a promising technique for safeguarding fertility in female mammals39, including women40. The preservation and restoration of fertility using preantral follicles presents an attractive assisted reproductive technology as well as a promising method for studying folliculogenesis41. Following freezing and thawing, the tissue can be subjected to this isolation protocol for retrieval of viable preantral follicles. Although the potential ability of this protocol to retrieve follicles from previously cryopreserved tissue was not specifically assessed, others have demonstrated success and used viable small preantral follicles isolated from frozen-thawed tissue for in vitro culture42,43,44,45. Furthermore, freshly isolated preantral follicles can be individually cryopreserved via vitrification and maintain structural integrity36.
An update on details for the efficient isolation of preantral follicles from a large mammalian species such as the bovine has been missing in the literature. Moreover, a visual and explicit guide on how to isolate bovine preantral follicles is much needed, as the lack of reproducibility has been a bottleneck in the field. Recently, Bus et al.36 described the isolation of bovine preantral follicles using a mechanical method, with a calculated average yield of 6.1 primary follicles per ovary. The mechanical isolation method described here results in the isolation of an average of 41 preantral follicles per ovary, the majority being at the primary stage. Therefore, this technique is especially useful for studies focused on primary follicle physiology and development. Primary follicles represent the first stage of the growing pool and are abundant in the ovarian cortex. Therefore, the use of the described technique, which is optimized to harvest this developmental stage, is novel and may be particularly desirable to take advantage of the ovarian reserve while avoiding the more difficult manipulation of primordial follicles. Another advantage of isolating primary follicles would be to associate ovarian tissue culture for the initial activation of primordial follicles with subsequent isolation of primary follicles for further culture. Given the interest in culturing follicles from the more abundant primary stage, the method presented here can incentivize researchers to pursue avenues they may have previously avoided due to the challenge of isolating primary follicles.

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><tbody><tr><td>5-3/4&quot; 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 &amp;mu;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 &amp;mu;m Cell Strainer</td><td>Fisher Scientific&amp;nbsp;</td><td>22-363-547</td><td>Used to filter the filtrate from the 300 &amp;mu;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 &amp;mu;m Cell Strainer</td><td>pluriSelect&amp;nbsp;</td><td>43-50300-03</td><td>Used to filter the filtrate from the cheese cloth&amp;nbsp;</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&amp;nbsp;</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 &amp;mu;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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>60872-310</td><td>Gridded petri dishes allow for more efficient identification of follicles&amp;nbsp;</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&amp;rsquo;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 &amp;deg;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 &#181;m microscope calibration slide under the stereomicroscope. Using this method, a total of 476 follicles were classified as either primary (follicles measuring 40-79 &#181;m), early secondary (80-119 &#181;m), or secondary (&#8805;120 &#181;m) follicles (Figure 4B). Primordial follicles were excluded due to the 40 &#181;m filtration step. Follicle viability was evaluated using the trypan blue exclusion test as described previously30. Isolated follicles were briefly incubated for 1 min in 0.05% (v/v) trypan blue in PBS containing 0.2% PVP, followed by three washes with PBS + PVP and examination under a stereoscope. Using the procedure described, 100% of isolated follicles were viable in all replicates. Overall, the mechanical isolation process, from ovary collection until immediately prior to searching the Petri dish, takes approximately 30-45 min. The time spent for searching and identifying follicles takes no longer than 30 min, resulting in a process that takes a total of 1-1.25 h per ovary.
The most critical step of this protocol is ensuring that the ovarian tissue is properly homogenized. Increasing the homogenization time from 5 min to 6 min led to an increase in the number of follicles isolated (P &#60; 0.05). An average of 41 follicles were isolated per ovary from 30 trials using a 6 min homogenization, which is a nearly threefold increase over the average of 13.8 follicles per ovary obtained from 22 trials with a 5 min homogenization time (Figure 5). Another important step is the homogenization speed, which must remain within 9,000-11,000 rpm. Higher speeds lead to decreased yields, presumably due to excessive damage and/or rupture of follicles. Follicle isolation can be further improved after the filtration steps by passing the contents of the search dish through a glass Pasteur pipette with a rubber bulb multiple times. By using the Pasteur pipette, follicles are dislodged from debris, and more follicles can be seen in the dish.
If the follicles are to be cultured after isolation, it is important to mitigate microbial contamination. Contamination can be prevented by trimming any excess connective tissue and fat from the whole ovary and then performing a series of wash steps. The trimmed ovaries should be briefly washed in 70% ethanol to reduce the microbial load, followed by several washes in warm (38.5 &#176;C) PBS + PenStrep solution to wash away the 70% ethanol and further remove any potential microorganisms. These washes can be done without compromising follicle viability. To further prevent contamination, it is possible to perform the entire isolation protocol inside a biosafety cabinet or clean bench with sterilized equipment.
RNA isolation and RT-qPCR 
Follicles of the same stage and from separate isolations were pooled, and a total of 46 primary and 34 early secondary follicles were used for analysis of mRNA expression. RNA was isolated from the pooled follicles (primary follicles = 259 ng of total RNA and early secondary follicles = 91 ng of total RNA) and used for cDNA synthesis. Transcript expression of the granulosa cell marker FSHR19&#160;and germ cell marker DAZL in the follicles was then evaluated using RT-qPCR. As expected, both primary and early secondary follicles expressed FSHR and DAZL transcripts, confirming a previous report in human follicles31&#160;and demonstrating that the FSHR is expressed in bovine follicles at the primary stage of development32 (Figure 6A,B).
Immunoflourescent localization of Connexin 37 
Immunofluorescent localization of proteins can be performed in whole follicles. Here, this technique was used to localize the gap junction protein Connexin 37 (CX37) in isolated preantral follicles at both the primary and early secondary stages. CX37 is critical for communication between the oocyte and granulosa cells of the follicle and has been identified in both cell types at all stages of follicle development in bovine species in histological analyses33. After the trypan blue exclusion test, viable follicles were fixed in PFA and incubated with rabbit anti-human CX37 antibody or rabbit isotype IgG (negative control), followed by incubation with donkey-anti-rabbit AlexaFluor 488 secondary antibody. Hoechst 33342 was used to label DNA. The follicles were imaged using an inverted epifluorescence microscope under DAPI and FITC filters, and the signal intensity was quantified. CX37 was found to localize to the ooplasm, although notably absent from the oocyte nucleus, and to the membrane of the granulosa cells (Figure 7). These results are consistent with the known localization of CX37 to the oocyte and membrane of the granulosa cells33. The immunolocalization of proteins known to be critical for cell communication and follicle growth, such as CX37 (shown here) and FSHR32 is a vital tool to study the development of preantral follicles, effects of interventions on follicle growth, structure, and quality, as well as to assess culture effectiveness by comparing in vivo-obtained and in vitro-cultured follicles.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Gene</td>
			<td colspan="3">Forward sequence (5&#39;-3&#39;)</td>
			<td colspan="3">Reverse sequence (5&#39;-3&#39;)</td>
			<td>Product size (bp)</td>
		</tr>
		<tr>
			<td>FSHR</td>
			<td colspan="3">CCC AAC TCG ATG AGC TGA ATC T</td>
			<td colspan="3">CAT AGC TAG GCA GGG AAC GG</td>
			<td>132</td>
		</tr>
		<tr>
			<td>DAZL</td>
			<td colspan="3">GCC CAC AAA AGA AAT CTG TGG A</td>
			<td colspan="3">ACT TAA GCA CTG CCC GAC TT</td>
			<td>156</td>
		</tr>
		<tr>
			<td>H2A</td>
			<td colspan="3">GAG GAG CTG AAC AAG CTG TTG</td>
			<td colspan="3">TTG TGG TGG CTC TCA GTC TTC</td>
			<td>104</td>
		</tr>
		<tr>
			<td>ACTB</td>
			<td colspan="3">CTC TTC CAG CCT TCC TTC CT</td>
			<td colspan="3">GGG CAG TGA TCT CTT TCT GC</td>
			<td>178</td>
		</tr>
	</tbody>
</table>
Table 1: List of primers used for RT-qPCR.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64423/64423fig04v4.jpg" /> 
Figure 4: Number and developmental stage of isolated preantral follicles. (A) Total number of isolated preantral follicles per replicate using 6 min tissue homogenization (n = 30 replicates). The average number of follicles is represented by the red line across the bars. (B) In 14&#160;replicates, the developmental stage of the isolated follicles was recorded. The pie chart shows the distribution of developmental stages as a proportion of the total follicles isolated (n = 476). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64423/64423fig04largev4.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64423/64423fig05v4.jpg" /> 
Figure 5: Comparison of 5- versus 6-min homogenization. Homogenization of the ovarian cortex for 6 min yielded a greater number of preantral follicles compared to homogenization at the same speed for 5 min (n = 22 replicates for 5 min and 30 replicates for 6 min; P &#60; 0.01). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64423/64423fig05largev4.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64423/64423fig06v5.jpg" /> 
Figure 6: Reverse-transcription quantitative PCR of transcripts expressed in isolated preantral follicles. Primary and early secondary preantral follicles expressed mRNA transcripts for markers of granulosa cells (FSHR), germ cells (DAZL), and reference genes (H2A and ACTB). (A) Agarose gel of PCR products for each gene with respective base pair (bp) product sizes. (B) Expression of FSHR and DAZL transcripts was quantified relative to that of the reference gene ACTB. NTC = non-template control, CT = cycle threshold. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64423/64423fig06largev5.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/64423/64423fig07v5.jpg" /> 
Figure 7: Immunofluorescent localization of CX37 in isolated preantral follicles. Representative images of CX37 immunolocalization in a primary&#160;(A) and early secondary&#160;(B) bovine preantral follicle.&#160;(C) Representative image of negative control rabbit isotype labeling in an early secondary bovine preantral follicle. Left panel: DNA labeling (DAPI). Middle panel: primary antibody rabbit anti-human CX37 labeling (FITC). Right panel: DAPI and FITC merged. Scale bars = 50 &#181;m. (D) Average CX37 signal intensity according to follicle developmental stage (n = 21 primary and 11 early secondary follicles in two replicates).<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64423/64423fig07largev5.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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Nanyang Technological University

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JoVE Journal

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

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

Bioengineering

In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that during the reproductive life span only about 1% of the oocytes in an ovary mature and ovulate, several techniques have been developed to increase the exploitation of the ovarian reserve to the growing population of non-ovulatory follicles. Such technologies have allowed interventions of fertility preservation, selection programs in livestock, and conservation of endangered species. However, the vast potential of the ovarian reserve is still largely unexploited. In cows, for instance, some attempts have been made to support in vitro culture of oocytes at specific developmental stages, but efficient and reliable protocols have not yet been developed. Here we describe a culture system that reproduce the physiological conditions of the corresponding follicular stage, defined to develop in vitro growing oocytes collected from bovine early antral follicles to the fully-grown stage, corresponding to the medium antral follicle in vivo. A combination of hormones and a phosphodiesterase 3 inhibitor was used to prevent untimely meiotic resumption and to guide oocyte&#39;s differentiation.

We describe the procedures for isolation of growing oocytes from ovarian follicles at early stages of development, as well as the setup of an in vitro culture system which can support the growth and differentiation up to the fully-grown stage. &#160;

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that ...

Developmental Biology

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.

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 ...

Use of Bisection to Reduce Mitochondrial DNA in the Bovine Oocyte

Interspecies somatic cell nuclear transfer (iSCNT) may be used to rescue endangered species, but two distinct populations of mitochondrial DNA (mtDNA) exist within the reconstructed embryo: one within the recipient ooplasm and one within the donor somatic cell. This mitochondrial heteroplasmy can lead to developmental issues in the embryo and the fetus. Handmade cloning protocols include oocyte bisection, which can be used to decrease the mtDNA copy number, reducing the degree of mitochondrial heteroplasmy in a reconstructed embryo. Centrifugation of denuded, mature bovine oocytes produced a visible mitochondria-dense fraction at one pole of the oocyte. Oocytes&#39; zonae pellucidae were removed by exposure to a pronase solution. Bisection was performed using a microblade to remove the visible mitochondria fraction. qPCR was used to quantify the mtDNA present in DNA samples extracted from whole oocytes and bisected ooplasts, providing a comparison of mtDNA copy numbers before and after bisection. Copy numbers were calculated using cycle threshold values, a standard curve&#39;s regression line formula, and a ratio that included the respective sizes of mtDNA PCR products and genomic PCR products. One bovine oocyte had an average mtDNA copy number (&#177; standard deviation) of 137,904 &#177; 94,768 (n = 38). One mitochondria-depleted ooplast had an average mtDNA copy number of 8,442 &#177; 13,806 (n = 33). Average mtDNA copies present in a mitochondria-rich ooplast were 79,390 &#177; 58,526 mtDNA copies (n = 28). The differences between these calculated averages indicate that the centrifugation and subsequent bisection can significantly decrease the mtDNA copy numbers present in the mitochondria-depleted ooplast when compared to the original oocyte (P &#60; 0.0001, determined by one-way ANOVA). The reduction in mtDNA should decrease the degree of mitochondrial heteroplasmy in a reconstructed embryo, possibly fostering standard embryonic and fetal development. Supplementation with mitochondrial extract from the somatic donor cell may also be essential to achieve successful embryonic development.

Here, we present a protocol to significantly reduce the mitochondrial DNA copy numbers in a bovine oocyte (P &lt; 0.0001). This method utilizes centrifugation and bisection to substantially reduce oocyte mitochondria and may allow for an increased chance of development in the reconstructed interspecies somatic cell nuclear transfer embryos.

Interspecies somatic cell nuclear transfer (iSCNT) may be used to rescue endangered species, but two distinct populations of mitochondrial DNA (mtDNA) ...

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 ...

Improved Procedure to Obtain Granulosa Cell-Free Stage I Zebrafish Oocytes

Rapid Isolation of Stage I Oocytes in Zebrafish Devoid of Granulosa Cells

The study of oocyte development holds significant implications in developmental biology. The zebrafish (Danio rerio) has been extensively used as a model organism to investigate early developmental processes from oocyte to embryo. In zebrafish, oocytes are surrounded by a single layer of somatic granulosa cells. However, separating granulosa cells from oocytes poses a challenge, as achieving pure oocytes is crucial for precise analysis. Although various methods have been proposed to isolate zebrafish oocytes at different developmental stages, current techniques fall short in removing granulosa cells completely, limiting the accuracy of genome analysis focused solely on oocytes. In this study, we successfully developed a rapid and efficient process for isolating pure stage I oocytes in zebrafish while eliminating granulosa cell contamination. This technique facilitates biochemical and molecular analysis, particularly in exploring epigenetic and genome structure aspects specific to oocytes. Notably, the method is user-friendly, minimizes oocyte damage, and provides a practical solution for subsequent research and analysis.

Author Spotlight: Enhanced Method for Isolating Pure Zebrafish Stage I Oocytes

This protocol describes a modified procedure for rapidly isolating clean stage I oocytes in zebrafish devoid of granulosa cells, thereby providing a convenient method for oocyte-specific research.

The study of oocyte development holds significant implications in developmental biology. The zebrafish (Danio rerio) has been extensively used as a model ...

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila follicles provides a rich source of information into widely conserved processes of development and morphogenesis.

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Stage-specific isolation of mid-to-late Drosophila follicles is useful for a variety of purposes. Such follicles develop in culture, which allows for genetic and/or pharmacologic manipulations to be coupled with in vitro development assays and live imaging. Additionally, follicles can be used for molecular studies, such as isolating mRNA and protein.

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. 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 ...

Separation of Hen Ovarian Follicle Layers for Reproductive Research Applications

Separation of Avian Preovulatory Follicle Granulosa and Theca Cell Layers for Downstream Applications

Layer hens (egg-laying chickens) and broiler breeders (breeding stock for meat-producing chickens) are crucial to the world's food supply as a reliable source of protein. They are also an emerging animal model for the study of human reproductive disease. As the field of poultry research develops, the health and function of the layer hen and broiler breeder ovary will be an important point of study for both agricultural and biomedical researchers. One of the challenges presented by this emerging interest is the need for replicable techniques that all researchers can employ in ovarian specimen collection. In particular, a detailed visual process must be established to define the proper separation of the specialized granulosa and theca cell layers from hen follicles to achieve agreement and consistency among researchers. 
This study describes the extraction of preovulatory follicles and ovary tissue in white leghorn hens of prime reproductive age. The separation of these follicles is performed under cold, liquid conditions to congeal the yolk for easier manipulation and to prevent the follicle's own weight from tearing apart cell layers during the separation process. Once the separation is complete, the desired cell layers can be further digested for tissue culture approaches or can be cryopreserved for genomic and proteomic analyses.

Here, we describe a protocol for separating yolk, granulosa cells, and theca cells in avian preovulatory follicles. This precision handling enables critical investigations into the role of these layers in reproductive function, aiding the understanding of follicular development, hormonal regulation, and disease research for enhanced agricultural yield and biomedical insights.

Layer hens (egg-laying chickens) and broiler breeders (breeding stock for meat-producing chickens) are crucial to the world's food supply as a reliable ...

Mouse Antral Oocyte Isolation: A Method to Isolate Antral Oocytes from Freshly Harvested Mouse Ovaries

Source: Monti, M. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/53616" target="_blank">Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization</a>. J. Vis. Exp. (2016)
This video describes the isolation of antral oocytes from freshly harvested mouse ovaries. These antral oocytes can be used for further characterization and developmental studies.

Source: Monti, M. et al. Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization. J. Vis. Exp. (2016)
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Isolation of Small Preantral Follicles from the Bovine Ovary Using a Combination of Fragmentation, Homogenization, and Serial Filtration

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