Name: a seminiferous tubule squash technique for the cytological analysis of spermatogenesis using the mouse model
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This work was supported by NIGMS (R01GM11755) to P.W.J. and by a training grant fellowship from the National Cancer Institute (NIH) (CA009110) to S.R.W. and J.H. 
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The use of mice was approved by the Institutional Animal Care and Use Committee of Johns Hopkins University. Experiments were performed on juvenile (20 - 26 days post-partum, dpp) C57BL/6J mice, taking advantage of the semi-synchronous first wave of spermatogenesis. However, this technique can also be performed using adult mice.
1. Dissection and isolation of mouse seminiferous tubules
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	<li>Prepare the fix/lysis solution and antibody dilution buffer (ADB) described in Table 1</s...

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Mice have proven to be a useful model organism for studying the cellular events that govern meiotic progression during spermatogenesis. Further, it is necessary to develop tools catered to the study of spermatogenesis because many events, such as exit from meiotic prophase I, are sexually dimorphic. This protocol describes a seminiferous tubule squash method for visualization and study of different stages of mouse spermatogenesis. This method preserves cellular integrity and thereby allows detailed analyses of nuclear an...

Meiosis is a complex cellular event in which a single round of DNA replication is followed by two successive rounds of cell division. Several meiosis-specific events must be coordinated during the initial stages of meiosis to ensure accurate chromosome segregation. These events include the completion of homologous recombination, co-orientation of sister kinetochores during the first meiotic division, and the stepwise loss of cohesin complexes to resolve chiasmata between homologs. Precise regulation of these processes is necessary to maintain fertility and to prevent chromosome missegregation events that can lead to genetic developmental disorders and spontaneous miscarriage1.
While the key events of meiosis take place in both males and females, significant temporal and mechanistic differences exist between spermatogenesis and oogenesis2. For example, during female meiosis, prophase I occurs during embryonic development and arrests at the dictyate stage until puberty. In contrast, spermatogenesis commences at puberty and progresses in waves throughout adult life without arrest. The differences between male and female meiosis emphasizes the need to develop methods that are specifically catered towards assessing these processes in both spermatocytes and oocytes. Currently, assessing meiotic progression largely relies on the use of chromatin spreads3,4,5. While chromatin spreads are useful for studying meiotic chromosomes, they fail to preserve cellular integrity, preventing evaluation of cellular structures such as spindle microtubules, centrosomes, the nuclear envelope, and telomere attachments. Live imaging and long-term culturing techniques have greatly advanced our understanding of female meiosis; similar approaches to visualize the entire intact cell, however, are less frequently implemented for the study of spermatogenesis6,7. In order to visualize dynamic events throughout male meiosis, we have adapted established tubule squash techniques to rapidly assess the cytological features of developing mouse spermatocytes8,9. The method described here maintains the integrity of the cell, enabling the study of multiple cellular structures during different stages of spermatogenesis.
This tubule squash technique is a whole cell approach, which allows for the assessment of cellular structures via immunofluorescence microscopy. Common histological approaches to visualize meiotic progression in male mice such as haematoxylin and eosin staining of paraffin embedded testes, and immunofluorescent labeling of cryosections allow for a broad overview of meiotic progression. However, these techniques fail to resolve single cells to the extent necessary for detailed analysis of the events occurring throughout meiosis10,11. Alternative techniques to visualize meiotic processes rely on significant chemiosmotic disruption to the spermatocyte to isolate and fix nuclear materials3,4,5. These chemical treatments hinder the observation of cell types other than primary spermatocytes. A recently described method by Namekawa has enabled the research community to preserve the nuclear architecture of isolated spermatocytes, but requires the use of a cytospin and accessories that may not be readily available to some laboratories4. In contrast, the tubule squash technique only requires equipment that is generally standard in most cell biology laboratories.
The tubule squash method described here can be used to visualize the diverse cell types found within the seminiferous tubule, including sertoli cells, spermatogonia, primary and secondary spermatocytes, and spermatids. By coupling this technique with the near-synchronous first wave of spermatogenesis in juvenile mice, it is possible to obtain enriched populations of spermatogenic cells as they progress through meiosis12. This process permits the detailed analysis of processes throughout spermatogenesis, such as early prophase events, the G2/MI and metaphase to anaphase transitions, and spermiogenesis. Furthermore, tubule squash preparations can be used to visualize cytological features of the chromosomes (e.g. interchromatid domains (ICDs) and kinetochores) and centrosomes (centrioles and pericentriolar material/matrices). The squash method can be readily performed in parallel with other experimental approaches, such as chromatin spreads and protein extraction. In addition, this technique has been successfully modified to deposit living spermatogenic cells on slides for direct visualization13.
The method described here involves a whole cell seminiferous tubule squash technique to analyze the G2/MI transition in wild-type C57BL/6J mice. The cytological features of primary spermatocytes entering the first meiotic division were visualized with immunofluorescence microscopy to observe the meiotic spindle. This versatile technique can be easily modified to visualize other meiotic stages and different cell types. The technique is also amenable to alternative visualization strategies, such as DNA and RNA FISH approaches.

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			<td height="21" style="height:21px;width:251px;">16% Paraformaldehyde Aqueous</td>
			<td style="width:247px;">Electron Microscopy Sciences (EMS)</td>
			<td style="width:137px;">15710</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">10x PBS</td>
			<td style="width:247px;">Quality Biological</td>
			<td style="width:137px;">119-069-161</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Triton X-100</td>
			<td style="width:247px;">Sigma</td>
			<td style="width:137px;">T8787</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:251px;">BSA</td>
			<td style="width:247px;">Sigma</td>
			<td style="width:137px;">A1470</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:251px;">Horse Serum</td>
			<td style="width:247px;">Sigma</td>
			<td style="width:137px;">H-1270</td>
			<td style="width:167px;"></td>
		</tr>
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			<td height="42" style="height:42px;width:251px;">35mm x 10mm Petri Dish, Sterile, non-treated</td>
			<td style="width:247px;">CellTreat</td>
			<td style="width:137px;">P886-229638</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Poly-L-lysine coated glass slides</td>
			<td style="width:247px;">Sigma</td>
			<td style="width:137px;">P0425-72EA</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Liquid Blocker Pen</td>
			<td style="width:247px;">Electron Microscopy Sciences (EMS)</td>
			<td style="width:137px;">71310</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Humid Box</td>
			<td style="width:247px;">Evergreen</td>
			<td style="width:137px;">240-9020-Z10</td>
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			<td height="42" style="height:42px;width:251px;">Wheaton Coplin Glass Staining Dish for 5 or 10 Slides</td>
			<td style="width:247px;">Fisher</td>
			<td style="width:137px;">08-813E</td>
			<td style="width:167px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:251px;">VECTASHIELD Antifade Mounting Medium with DAPI</td>
			<td style="width:247px;">Vector Labs</td>
			<td style="width:137px;">H-1200</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Microscope Cover Slides (22mmx60mm)</td>
			<td style="width:247px;">Fisher</td>
			<td style="width:137px;">12-544-G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Clear Nail Polish</td>
			<td style="width:247px;">Amazon</td>
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			<td height="26" style="height:26px;width:251px;">Microsopes</td>
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			<td height="21" style="height:21px;width:251px;">SteREO Discovery.V8</td>
			<td style="width:247px;">Zeiss</td>
			<td style="width:137px;">495015-0001-000&#160;</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Observer Z1</td>
			<td style="width:247px;">Zeiss</td>
			<td style="width:137px;">4109431007994000</td>
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			<td height="42" style="height:42px;width:251px;">Zeiss ZEN 2012 blue edition image software</td>
			<td style="width:247px;">Zeiss</td>
			<td style="width:137px;"></td>
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			<td height="21" style="height:21px;width:251px;">Primary Antibodies</td>
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			<td height="21" style="height:21px;width:251px;">Name</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Mouse anti-SYCP3</td>
			<td style="width:247px;">Santa Cruz</td>
			<td style="width:137px;">sc-74569</td>
			<td style="width:167px;">1 in 50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Rabbit anti-SYCP3</td>
			<td style="width:247px;">Fisher (Novus)</td>
			<td style="width:137px;">NB300-231</td>
			<td style="width:167px;">1 in 1000</td>
		</tr>
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			<td height="21" style="height:21px;width:251px;">Goat anti-SCP3</td>
			<td style="width:247px;">Santa Cruz</td>
			<td style="width:137px;">sc-20845</td>
			<td style="width:167px;">1 in 50</td>
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			<td height="21" style="height:21px;width:251px;">Human anti-Centromere Protein</td>
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			<td style="width:137px;">15-235</td>
			<td style="width:167px;">1 in 100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Mouse anti-alpha tubulin</td>
			<td style="width:247px;">Sigma</td>
			<td style="width:137px;">T9026</td>
			<td style="width:167px;">1 in 1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Mouse anti-AIM1</td>
			<td style="width:247px;">BD Biosciences</td>
			<td style="width:137px;">611082</td>
			<td style="width:167px;">1 in 200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Mouse anti-&#947;H2AX</td>
			<td style="width:247px;">Thermo Fisher</td>
			<td style="width:137px;">MA1-2022</td>
			<td style="width:167px;">1 in 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Mouse anti-CENT3</td>
			<td style="width:247px;">Abnova</td>
			<td style="width:137px;">H00001070-M01</td>
			<td style="width:167px;">1 in 200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Rabbit anti-pericentrin</td>
			<td style="width:247px;">Abcam</td>
			<td style="width:137px;">ab4448</td>
			<td style="width:167px;">1 in 200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Rabbit anti-REC8</td>
			<td style="width:247px;">Courtesy of&#160; Dr. Karen Schindler</td>
			<td style="width:137px;">N/A</td>
			<td style="width:167px;">1 in 1000</td>
		</tr>
	</tbody>
</table>

a seminiferous tubule squash technique for the cytological analysis of spermatogenesis using the mouse model

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during meiosis can lead to infertility, pregnancy loss or genetic defects. Commencing at the onset of puberty and continuing throughout adulthood, continuous semi-synchronous waves of spermatocytes undergo spermatogenesis and ultimately form haploid sperm. The first wave of mouse spermatocytes undergoing meiotic initiation appear at day 10 post-partum (10 dpp) and are released into the lumen of seminiferous tubules as mature sperm at 35 dpp. Therefore, it is advantageous to utilize mice within this developmental time-window in order to obtain highly enriched populations of interest. Analysis of rare cell stages is more difficult in older mice due to the contribution of successive spermatogenic waves, which increase the diversity of the cellular populations within the tubules. The method described here is an easily implemented technique for the cytological evaluation of the cells found within the seminiferous tubules of mice, including spermatogonia, spermatocytes, and spermatids. The tubule squash technique maintains the integrity of isolated male germ cells and allows examination of cellular structures that are not easily visualized with other techniques. To demonstrate the possible applications of this tubule squash technique, spindle assembly was monitored in spermatocytes progressing through the prophase to metaphase I transition (G2/MI transition). In addition, centrosome duplication, meiotic sex chromosome inactivation (MSCI), and chromosome bouquet formation were assessed as examples of the cytological structures that can be observed using this tubule squash method. This technique can be used to pinpoint specific defects during spermatogenesis that are caused by mutation or exogenous perturbation, and thus, contributes to our molecular understanding of spermatogenesis.

Here, we have used the tubule squash method to visualize transitory cell populations undergoing the prophase to metaphase I (G2/MI) transition, which were enriched by harvesting testes from juvenile wild-type mice undergoing the first wave of spermatogenesis (24 dpp). Figure 1 depicts representative images of the various cell stages that can be visualized using the tubule squash method. Enriched populations of metaphase I spermatocytes were visualized using a...

Watch this Scientific Journal Video about A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model at JoVE.com

A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model

The goal of this tubule squash technique is to rapidly assess cytological features of developing mouse spermatocytes while preserving cellular integrity. This method allows for the study of all stages of spermatogenesis, and can be easily implemented alongside other biochemical and molecular biological approaches for the study of mouse meiosis.

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during ...

The overall goal of this seminiferous tubule squash technique is to visualize the cytological features of mouse spermatogonia, spermatocytes, and spermatids while preserving their cellular integrity. For this demonstration, we focus on the analysis of spermatocytes undergoing meiosis. This method can help answer key questions in the field of male reproductive biology, such as spermatogonial stem cell differentiation, meiotic chromosome segregation, and post-meiotic differentiation of round spermatids.The main advantage of this technique is that the cellular integrity of male germ cells is maintained, which allows examination of cellular structures not easily visualized with other techniques. This method can be used to improve our understanding of the paternal causes of infertility in any platy. And it can be applied to mammalian systems such as mouse or organ donor derived testes.Generally, individuals new to this method will struggle with making a good squash. Applying sufficient force and properly arranging the tubules are important factors to producing an even monolayer of cells. For each mouse to be dissected, load two 35 millimeter Petri dishes, one with three milliliters of PBS and the other with two milliliters of freshly made Fixative Lysis solution.Select mice undergoing the first wave of spermatogenesis to observe an enriched population of interest. After sacrificing the mouse, clean the abdomen with 70%alcohol and then proceed with the surgery. Make a V-shaped opening into the abdominal pelvic cavity and remove the testes by pulling on the epididymal fat pad using forceps, but without disturbing the tunica albuginea.Transfer the testes to the dish containing PBS. There, remove the testicular tunica albuginea by puncturing the tissue with sharp forceps and collect the loose seminiferous tubules. Next transfer the tubules into the dish with Fixative Lysis solution and let the reaction proceed for five minutes at room temperature.During the incubation, prepare a poly-L-lysine coated glass slide by outlining the edges of the slide with a liquid blocker pen and then filling the slide with 100 microliters of Fixative Lysis solution within the borders. After the five minute incubation, use sterile forceps and scissors to gently tease apart and cut the seminiferous tubules so that 20 millimeter individual segments are obtained. Next, transfer five 20 millimeter tubule segments to the prepared slide.Then continue to break up the segments into 1.5 to three millimeter lengths using scissors. Now with the forceps, arrange the segments so that they do not overlap. Distribute them evenly across the slide.Ideally, 20 to 40 short lengths will occupy one slide. Begin by removing excess liquid on the slide preparation using an absorbent tissue. Now, apply a cover slip and squash the tubules using pressure from the heel of the palm for 10 to 20 seconds.The goal is to apply enough force to disperse the spermatocytes from the seminiferous tubules. Using a dissection microscope, check that the segments were disrupted by the squash. Then, pour a little liquid nitrogen into a small Dewar and flash freeze the slide for 15 seconds or until the liquid ceases to bubble.To proceed with amino blotting, immediately remove the cover slip using a sharp point or edge. Although not ideal, frozen slides can be immediately preserved at minus 80 degrees Celsius and saved for up to two weeks. To remove the cover slip later, immerse the slides in liquid nitrogen for 15 seconds and pry off the cover slip.To proceed with the amino labeling, wash the slides three times in PBS for five minutes per wash using a 50 milliliter Coplin jar. After the washes, apply one milliliter of antibody dilution buffer onto each slide to serve as a block. Allow the block reaction to run for one to two hours in a humidified chamber.Never let the slides dry out during this procedure. Next, tap the slides onto a paper towel to gently remove the buffer and return them to the humidified chamber. Then, apply 100 microliters of diluted primary antibody to each slide and transfer them to 40 degrees Celsius where the binding reaction can run overnight.If antibody solution is at a premium, half the volume can be used if it is covered with a cover slip or Parafilm. The next day, use three washes in PBS to remove the primary antibody. Next, apply 100 microliters of diluted secondary antibody and allow this reaction to go for one to one and a half hours at room temperature.To avoid photobleaching, keep the slides in a dark humidified chamber during this incubation. Next, wash the slides twice with PBS. Now, dot the slides using a mounting medium containing DAPI.Then seal on cover slips using nail polish and proceed with imaging. To take images, use an epifluorescence microscope with an automated stage that enables precise z-axis movement. Also use a high resolution camera to capture images.Now, assess the slides using a 20x objective. Determine the quality of the squash preparation. A good quality squash will have a monolayer of nuclei that are evenly distributed.Some regions of the squash may be better than others. Note the coordinates of the best regions to easily find them under higher magnification. Now, increase the magnification to up to 100x.Then capture images from noted regions that have a consistent monolayer of nuclei. Set the upper and lower z-stacks to ensure that all of the in-focus light is captured. The optimal number of z-stacks is generally designated by the image acquisition software and is different for each objective.From the images, compile an extended depth image using processing software. The software will combine the in-focus light from each z-stack, which is essential for optimal imaging of tubule squash preparations. Using the described tubule squash method, transitory cell populations undergoing the prophase to metaphase one transition were visualized.Enriched populations of metaphase one spermatocytes were visualized using the antibodies against alpha tubulin. To visualize the cells in prophase one, tubule squash preparations were immunolabeled with an antibody to the Synaptonemal Complex Protein 3. Leptotene, zygotene, and pachytene diplotene staged spermatocytes were both identified.Antibodies against the cell cycle kinase Aurora B were used to visualize later events of meiosis one. This kinase localizes to the inner centromere during metaphase, then relocalizes to the spindle mid zone during anaphase, and finally to the cleavage furrow during cytokinesis. By maintaining cellular integrity, the tubule squash technique allows for the visualization of subcellular organization in structures associated with prophase one.Chromosome bouquet dynamics can be followed through prophase one. Centriole duplication in centrosome disjunction can also be visualized. In early diplotene stage spermatocyte undergoing centrosome disjunction following centriole duplication was visualized with antibodies against pericentrin, a protein component of the pericentriolar matrix and antibodies against Centrin-3 to mark centrioles.After watching this video, you should have a good understanding of how to rapidly assess the cytological features of developing mouse spermatocytes while preserving cellular integrity. Once mastered, dissection through squashing can be accomplished in under one hour per mouse. While attempting this procedure, it's important to remember to apply enough force to disperse the spermatocytes from the seminiferous tubules.In addition, the Fixative concentration and incubation time can be optimized depending on the cellular structure to be studied. Alternative visualization strategies, such as DNA and RNA fish approaches, can be easily implemented with this technique. By complementing tubule squashes with downstream biochemical assays, it is possible to assess the mechanistic determinants of successful meiotic progression.

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