This work was made possible through Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS and supported by funding from National Health and Medical Research Council (ALW #1120300) and Australian Research Council (KJH #FT190100265). The authors would like to acknowledge the technical support of the Monash Animal Research Platform, Monash Histology Platform and Monash Micro Imaging facility.

Ovaries were collected from female C57BL6J mice. All animal procedures and experiments were performed in accordance with the NHMRC Australian Code of Practice for the Care and Use of Animals and approved by the Monash Animal Research Platform Animal Ethics Committee.
NOTE: A chemotherapy agent shown to deplete primordial follicle oocytes, as determined using stereology11 and direct counts12,13 was used in this r...

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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+PUMA+protects+the+ovarian+reserve+during+DNA-damaging+chemotherapy+and+preserves+fertility.">Loss of PUMA protects the ovarian reserve during DNA-damaging chemotherapy and preserves fertility.</a> Cell Death and Disease. 9 (6), 618 (2018).</li><li>Meirow, D., Assad, G., Dor, J., Rabinovici, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GnRH+antagonist+cetrorelix+reduces+cyclophosphamide-induced+ovarian+follicular+destruction+in+mice.">The GnRH antagonist cetrorelix reduces cyclophosphamide-induced ovarian follicular destruction in mice.</a> Human Reproduction. 19 (6), 1294-1299 (2004).</li><li>Winship, A. 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The authors have nothing to disclose.

This article provides a step-by-step protocol for the gold standard technique for enumerating mouse primordial follicles, stereology, and the more commonly employed method of direct follicle counting. Chemotherapy treatment was used to compare and contrast the results obtained from these two different methods within the left and right ovary from the same animal. Both methods revealed high inter-animal variability in primordial follicle numbers. A significant depletion of the ovarian reserve was recorded using stereology,...

The immature, meiotically-arrested oocytes stored within primordial follicles in the ovary are the storage unit for the female germline and comprise an individual&#8217;s lifetime ovarian reserve. Primordial follicle numbers decline naturally with age1, or alternatively, can be prematurely depleted following exposure to exogenous chemicals, including some pharmaceuticals and environmental toxicants in air, food and water2. Given that the primordial follicle number is finite, the quantity and quality of follicles present within the ovary largely determine female fertility and offspring health. Thus, accurate quantification of primordial follicle number in women is essential for evaluating the off-target impacts of exogenous insults on the ovarian reserve.
In women, analysis of the whole ovary is generally not possible, thus non-invasive surrogate measures of the ovarian reserve must be utilized in a clinical setting. Anti-M&#971;llerian hormone (AMH) is the most widely used surrogate biomarker clinically3. Serum AMH levels are often measured in women of advanced maternal age, or before and after cancer treatment, such as chemotherapy. However, AMH is produced by growing follicles and not by primordial follicles, and thus, serum levels do not inform on absolute primordial follicle number.
With the absence of methods to accurately determine primordial follicle number in women in situ, counting ovarian follicles in small animal models, such as rodents, remains an essential research tool to assess the degree by which exogenous insults impact on primordial follicles and thus, fertility. Unfortunately though, reports throughout the literature of primordial follicle numbers in rodent models are highly variable4. A major reason for this is widely reported technical differences in the counting method employed. Predominately, there are two different techniques described in the literature for enumerating primordial follicles in mice. These include stereology, which employs the fractionator optical dissector method, and direct follicle counts.
Stereology is widely regarded the gold standard as it uses systematic uniform random sampling5, making it the most accurate method of quantifying primordial follicle number in whole mouse, or rat ovaries4,6,7. Stereology is unbiased, as it accounts for the three-dimensional structure of the object of interest8. Using an optical dissector/fractionator method, three levels of sampling are applied to quantify primordial follicles using thick tissue sections (e.g., 20 &#181;m) within a known fraction of the total mouse ovary. Firstly, the sampling interval is chosen (e.g., every 3rd section) at a random start (sampling fraction 1, f1)4. Sections are then sampled in a systematic, uniform manner from this point through the whole ovary. Then, an unbiased counting frame is superimposed over the ovarian section and progressively moved along a defined, randomized counting grid (sampling fraction 2, f2)8. Lastly, a known fraction of the section thickness is optically sampled (e.g., 10 &#181;m) and follicles within this area are counted (sampling fraction 3, f3)4. The raw follicle number is multiplied by the inverse of these sampling fractions to obtain the final value. This method requires expert training and equipment, including a microscope with a motorized stage driven by stereological software. Tissues should be preserved in a specialized Bouin&#8217;s fixative, and embedded in glycolmethacrylate resin to allow for thick tissue sections to be cut using a glycolmethacrylate resin microtome with a glass knife. This method is designed to account for tissue shrinkage and deformation, to best preserve the three-dimensional morphological structure of the ovary and follicles9.
Direct follicle counting is the most frequently used method for counting follicles10. More common fixatives (i.e., formalin) can be used, followed by paraffin wax embedding and exhaustive serial sectioning using a standard microtome at a thickness of between 4-6 &#181;m. Follicles are systematically counted in the entire tissue section at a defined interval, and then multiplied by the inverse of the sampling interval to obtain the total follicle estimate. This method is quick, easy, can be performed using archived tissues, and prepared using standard histological techniques. It requires only a light microscope with standard imaging capabilities. However, despite these advantages, direct follicle counting lacks the accuracy and strict counting parameters of stereology, making it more prone to investigator bias. Additionally, tissues may undergo shrinkage and deformation during processing, disrupting the integrity and morphology of the ovary and thus making follicle classification and quantification difficult.
The aim of this article is to describe two commonly-used methods to quantitatively assess primordial follicle number in mouse ovaries: stereology and direct follicle counting. We will provide detailed protocols for these two methods and highlight some of their strengths and weaknesses, in order to enhance reproducibility in our field and allow researchers to make an informed decision of the most appropriate counting method for their studies.

<table>
	<tbody>
		<tr>
			<td>1-Butanol (HPLC)</td>
			<td>Fisher Chemical</td>
			<td>#A383-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Acid alcohol</td>
			<td>Amber Scientific</td>
			<td>#ACDL</td>
			<td></td>
		</tr>
		<tr>
			<td>Bouin&#8217;s fixative</td>
			<td>Sigma-Aldrich</td>
			<td>#HT10132</td>
			<td>Picric acid 0.9% (w/v), formaldehyde 9% (v/v), acetic acid 5% (w/v)</td>
		</tr>
		<tr>
			<td>Cyclophosphamide</td>
			<td>Sigma-Aldrich</td>
			<td>#C0768-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dibutylphthalate Polystyrene Xylene (DPX)</td>
			<td>Sigma-Aldrich</td>
			<td>#06522</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Amber Scientific</td>
			<td>#ETH</td>
			<td>Ethanol 100%</td>
		</tr>
		<tr>
			<td>Micro Feather opthalmic scalpel with aluminium handle</td>
			<td>Designs for Vision</td>
			<td>#FEA-745-SR</td>
			<td>Feather blade for dissections (seen in Figure 1)</td>
		</tr>
		<tr>
			<td>Formalin fixative</td>
			<td>Australian Biostain</td>
			<td>#ANBFC</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslip</td>
			<td>Thermo Scientific</td>
			<td>#MENCS22501GP</td>
			<td>22 mm x 50 mm</td>
		</tr>
		<tr>
			<td>Glycomethacrylate resin RM2165 microtome</td>
			<td>Leica Microsystems</td>
			<td>#RM2165</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycolmethacrylate DPX</td>
			<td>*made in house</td>
			<td></td>
			<td>*Mix 1.5 L Xylene; 800 g polystyrene pellets; 100mL Dibutyl phthalate for 3 weeks</td>
		</tr>
		<tr>
			<td>Histolene</td>
			<td>Trajan</td>
			<td>#11031</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayer&#8217;s haematoxylin</td>
			<td>Amber Scientific</td>
			<td>#MH</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus BX50 microscope</td>
			<td>Olympus</td>
			<td>#BX50</td>
			<td>Brightfield microscope fitted with 10x dry &#38; 100x oil immersion objective (numerical aperture 1.3)</td>
		</tr>
		<tr>
			<td>Olympus immersion oil type-F</td>
			<td>Olympus</td>
			<td>#IMMOIL-F30CC</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus TH4-200 light source</td>
			<td>Olympus</td>
			<td>#TH4-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin wax</td>
			<td>Sigma-Aldrich</td>
			<td>#03987</td>
			<td></td>
		</tr>
		<tr>
			<td>Periodic acid</td>
			<td>Trajan</td>
			<td>#PERI1%</td>
			<td>Periodic acid 1%</td>
		</tr>
		<tr>
			<td>Rotary Microtome CUT 4060</td>
			<td>MicroTec</td>
			<td>#4060R/F</td>
			<td>Used to cut paraffin sections</td>
		</tr>
		<tr>
			<td>Schiff&#8217;s reagent</td>
			<td>Trajan</td>
			<td>#SCHF</td>
			<td></td>
		</tr>
		<tr>
			<td>Scott&#39;s tap water</td>
			<td>Amber Scientific</td>
			<td>#SCOT</td>
			<td>Potassium carbonate, magnesium sulphate, water</td>
		</tr>
		<tr>
			<td>StereoInvestigator Stereological System</td>
			<td>MBF Bioscience</td>
			<td></td>
			<td>Includes StereoInvestigator software, multi-control unit, automatic stage and joystick</td>
		</tr>
		<tr>
			<td>Superfrost microscope slides</td>
			<td>Thermo Scientific</td>
			<td>#MENSF41296SP</td>
			<td>1 mm, 72 pcs</td>
		</tr>
		<tr>
			<td>Technovit 7100 Plastic embedding system</td>
			<td>Emgrid Australia</td>
			<td>#64709003</td>
			<td>500 mL/5 x 1 g/40 mL</td>
		</tr>
		<tr>
			<td>Technovit 3040 yellow</td>
			<td>Emgrid Australia</td>
			<td>#64708805</td>
			<td>100 g/80 mL</td>
		</tr>
	</tbody>
</table>

accurate follicle enumeration in adult mouse ovaries

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial follicles, the storage unit of the female germline. They are non-renewable, thus their number at birth and subsequent rate of loss largely dictates the female fertile lifespan. Accurate quantification of primordial follicle numbers in women and animals is essential for determining the impact of medicines and toxicants on the ovarian reserve. It is also necessary for evaluating the need for, and success of, existing and emerging fertility preservation techniques. Currently, no methods exist to accurately measure the number of primordial follicles comprising the ovarian reserve in women. Furthermore, obtaining ovarian tissue from large animals or endangered species for experimentation is often not feasible. Thus, mice have become an essential model for such studies, and the ability to evaluate primordial follicle numbers in whole mouse ovaries is a critical tool. However, reports of absolute follicle numbers in mouse ovaries in the literature are highly variable, making it difficult to compare and/or replicate data. This is due to a number of factors including strain, age, treatment groups, as well as technical differences in the methods of counting employed. In this article, we provide a step-by-step instructional guide for preparing histological sections and counting primordial follicles in mouse ovaries using two different methods: [1] stereology, which relies on the fractionator/optical dissector technique; and [2] the direct count technique. Some of the key advantages and drawbacks of each method will be highlighted so that reproducibility can be improved in the field and to enable researchers to select the most appropriate method for their studies.

A well-characterized model of follicle depletion was used, whereby young adult female mice were administered a single dose of cyclophosphamide chemotherapy, or saline vehicle control (n=5/group) and both ovaries were harvested from each animal after 48 hours. One ovary per animal was prepared as described in Step 1 for each of the two methods: stereology or direct counts. The left and right ovary from each animal was randomly assigned to each group. These data show that when using stereology, a significant depletion of m...

Watch this Scientific Journal Video about Accurate Follicle Enumeration in Adult Mouse Ovaries at JoVE.com

Accurate Follicle Enumeration in Adult Mouse Ovaries

Here, we describe, compare, and contrast two different techniques for accurate follicle counting of fixed mouse ovarian tissues.

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial ...

We hope these in-depth protocols will help to enhance reproducibility in our field while also allowing researchers to make an informed decision when selecting accounting method for their studies. As it uses several sampling parameters, stereology is highly sensitive and accurate. However, direct counts are quicker and easier and can be performed on samples prepared with standard histological techniques.Stereology is used in studies of ovarian biology for evaluating the impact of toxicants on ovarian reserves. It can also be applied to life sciences for study of the kidney and the brain. Familiarizing yourself with the morphology of the follicles requires experience.Using previous studies to define the morphology along with establishing standardized criteria of classification can help with consistency. To estimate the primordial follicle number by stereology using an optical fractionator, turn on the computer, multi control unit, camera, and light source within the stereology setup, and set the microscope objective to a low magnification. Open the stereology software and place the first slide securely onto the microscope stage.Use the joystick to locate the first tissue sample, and bring the sample into focus. In the camera settings panel under exposure, click and drag the sliding scale to adjust the light exposure. In the camera settings panel, click the white balance button and click a blank area of the section to adjust the white balance.Open the probes menu to select the optical fractionator workflow, then click start new subject, and okay. If an existing sampling configuration has been saved, click yes under sampling parameters, and select the desired sampling configuration. Click next, set the microscope to low magnification, and select the 10x magnification.Click next and left click to trace the entire ovarian section. Right click to end the selection and select close contour. Click next, set the microscope to high magnification, and select the 100x oil magnification.After placing a drop of oil on the tissue section on the slide, adjust the light exposure as demonstrated, refocus the sample, and click next. Next, click start counting, focus at the top of the sample, click okay, and begin counting. Use the focusing knob to move through the 10 micron sampling depth, and look within the counting frame for any follicles in which the oocyte nucleus comes into focus.Then, click next to move to the next site. Classify a follicle as primordial if the oocyte is surrounded by flat squamous granulosa cells but no cuboidal granulosa cells. Count only follicles in which the oocyte nucleus is visible.The nucleus must appear within the counting frame or be touching the green inclusion lines of the counting frame. If the oocyte nucleus falls outside the counting frame or touches the red exclusion lines of the counting frame, do not count this follicle. When assessing primordial follicle depletion in response to an exogenous chemical, ensure that all of the counted follicles are healthy with a normal morphology.Count any abnormal or atretic follicles separately. When all of the sites in the section have been counted, click add new section to set up and count the follicles in the next section, or click I finished counting to end the session and exit the software. Then, obtain the sum raw follicle number from each tissue section sampled from the entire ovary, and use the formula as indicated to calculate the final value for each replicate animal.To estimate primordial follicle numbers by direct follicle counting, obtain multiple stitched high-power photomicrographs of the entire ovarian tissue section. To analyze the images, open them in ImageJ software. To classify the follicles as primordial, oocytes must be clearly visible and surrounded by squamous granulosa cells, but not cuboidal granulosa cells.Use the counting tool to obtain a raw follicle number of the primordial follicles with each section sampled from the tissue. Once the sum raw follicle number from each tissue section sampled has been obtained, multiply this number by the inverse of the sampling fraction to obtain the final value for each animal as demonstrated. When using stereology, a significant depletion of the mouse primordial follicles can be detected following chemotherapy treatment compared to that detected in control animals.In contrast, using the contralateral ovaries from the same animals, direct follicle counts failed to detect a significant reduction of the ovarian reserve after chemotherapy compared to controls. Notably, it is clear that the primordial follicle number in young adult wild type animals is variable as the distribution of the saline treated animals is wider compared to the cyclophosphamide groups even when the counts are performed by stereology. It is important to be able to accurately classify primordial follicles to differentiate between healthy and atretic follicles, and to appropriately include or exclude individual follicles from the overall count.These growing follicle and corpora lutea quantification techniques can be used in conjunction with immunofluorescence and cell viability marker staining to quantify the number of follicles expressing specific markers of interest. Stereology and direct counting are used to evaluate how disease and exogenous insults can alter the ovarian reserve, leading to a more comprehensive evaluations of toxicants and their impacts on female fertility.

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Research

JoVE Journal

Developmental Biology

7.8K vistas.  Monash University. We hope these in-depth protocols will help to enhance reproducibility in our field while also allowing researchers to make an informed decision when selecting accounting method for their studies. As it uses several sampling parameters, stereology is highly sensitive and accurate. However, direct counts are quicker and easier and can be performed on samples prepared with standard histological techniques.Stereology is used in studies of ovarian biology for evaluating the impact of toxica...