Name: phosphopeptide analysis of rodent epididymal spermatozoa
Uploaded: 2014-12-30T13:00:00
Duration: 9 min 30 s
Description: Watch this Scientific Journal Video about Phosphopeptide Analysis of Rodent Epididymal Spermatozoa at JoVE.com

The authors would like to acknowledge the NHMRC for career development award and grant support APP1066336 which supported this work.

1. Preparation of Culture Media and Dishes
<ol>
	<li>Make 200 ml&#160;of Biggers Whitten and Whitten (BWW)17 working solution by adding 5.54 g NaCl, 0.356 g KCl, 0.25 g CaCl2, 0.162 g H2KO4P, and 0.294 g MgSO4 to 1 L of Milli-Q water. This is the stock solution and can be kept at 4 &#176;C&#160;for up to a month.</li>
	<li>From the stock solution, add 420 mg of NaHCO3-, 200 mg glucose, 6 mg sodium pyruvate, 600 mg Bovine Serum Albumin, 0.74 ml&#160;sodium lactate, and 4.0 ml&#160;of HEPES buffer to 193 ml&#160;of the BWW stock. This is the working solution and is always made fresh on the day and equilibrated to 37 &#176;C before using.</li>
	<li>To make the cannula, take polyethylene (PE) tubing with an internal diameter of 0.4 mm and an external diameter of 1.1 mm and hold over a low heat (typically methanol flame) such that the tubing begins to melt. Immediately pull the tubing outward to stretch and make the outer diameter narrower.</li>
	<li>Cut to produce a narrowing of one end which allows for easier cannulation of the vas deferens.</li>
	<li>Cut the other end of the cannula to about 15 cm. Insert a 30 G&#160;needle into the blunt end, and attach a 3 ml&#160;syringe to it (fully retracted).</li>
	<li>Make a suction mouthpiece by cutting a 20 cm length of PE tubing (4.2 mm internal diameter, 6.4 mm outer diameter). Insert a mouthpiece into one end.</li>
	<li>Insert the micro-capillary glass tube holder and the glass micro-capillary itself (typically 3 &#181;l&#160;for a mouse, 40 &#181;l&#160;for a rat).</li>
</ol>
2. Removal of Epididymides from Mouse
<ol>
	<li>Euthanize mice according to IACUC-approved procedures specific to each institution.</li>
	<li>Take the animal and make a small incision in the scrotum to expose the epididymis. Pull the testis and epididymis out of the cavity using a pair of watchmaker&#8217;s #5 forceps.</li>
	<li>Cut the vas deferens such that at least 1-2 cm remain attached to the cauda epididymis. In addition, cut the proximal efferent ducts and tissue connecting the epididymis to the testis and remove the entire male reproductive track.</li>
	<li>Place the entire male reproductive track under a dissecting microscope with a magnification range in the order of 5-40X.</li>
</ol>
3. Cannulating the Epididymis
<ol>
	<li>Tape down the cannula to the microscope, such that 1-2 cm of the narrowed (cone-shaped) end is free and visible through the lens.</li>
	<li>Take a pair of watchmaker&#8217;s #5 forceps and gently clasp each side of the vas deferens and pull the vas deferens onto the cannula, such that the cannula goes into the vas deferens.</li>
	<li>Take a length of non-absorbable black braided treated silk (size 5-0) and tie a knot around the cannulated vas deferens. Ensure the knot is pulled tightly to hold the cannula inside the vas deferens when air pressure from the syringe is applied.</li>
</ol>
4. Retrograde or Backflushing of Caudal Epididymal Spermatozoa
<ol>
	<li>Using watchmakers #5 forceps, grab the distal end of the cauda epididymides and remove the tunica albuginea in order to expose a single epididymal tubule.</li>
	<li>Using the forceps, gently pull the tubule out to expose and then tease apart so as to create an opening for the sperm to be released.</li>
	<li>Gently push on the 3 ml&#160;(mouse) or 20 ml&#160;(rat) syringe so as to expel air from the syringe into the vas deferens. At the appropriate pressure, spermatozoa will start to slowly come out of the broken tubule at the distal end of the cauda epididymis. At this time, apply suction to the mouthpiece to draw the spermatozoa into the glass capillary. Note: Typically in a mouse, 2-3 &#181;l&#160;of spermatozoa can be obtained depending on the age of the animal. A rat will, on average yield 30-40 &#181;l.</li>
</ol>
5. Washing and Lysing the Spermatozoa in Preparation for Proteomics
<ol>
	<li>Expel the spermatozoa from the glass capillary into 1 ml&#160;solution of BWW solution (37 &#176;C) by gently blowing into the mouthpiece or attaching a syringe and expelling air so as to push the spermatozoa back out of the capillary.</li>
	<li>Once the sperm cells are diffused, wash 3x&#160;(300 x g, 5 min) with 1 ml&#160;BWW to remove contaminating proteins. After the final wash, remove the supernatant. Note: At this point, the sperm cells can be frozen for later use.</li>
	<li>Solubilize proteins using 4% CHAPS, 2 M thiourea, and&#160;50 mM Tris, pH 7.4 for 1 hr with intermittent vortexing.</li>
	<li>After lysing the cells, centrifuge (10,000 x g, 20 min), take supernatant and transfer to a new tube. Note: At this point, protein quantification can be performed.</li>
</ol>
6. Disulfide Bond Reduction and Alkylation
<ol>
	<li>Add 10 mM DTT as a final concentration to lysed proteins, vortex and incubate at RT for 30 min.</li>
	<li>Add 50 mM idodoacetamide as a final concentration to lysate, vortex and incubate at RT for 30 min in the dark.</li>
</ol>
7. Precipitation
<ol>
	<li>Methanol chloroform precipitate the sample by adding 1 volume of lysate (400 &#181;l&#160;as an example), add one volume (400 &#181;l) of methanol and 0.5 volume (200 &#181;l) chloroform.</li>
	<li>Vortex the sample and spin (10,000 x g, 2 min).</li>
	<li>Note that two phases will appear following centrifugation. Discard all but 2 mm of the top layer (being careful not to disturb the interface).</li>
	<li>Add 1 volume (400 &#181;l) of methanol, invert tube gently 1-2x&#160;to mix and again spin (10,000 x g, 15 min). Discard supernatant to waste and air dry pellet for 3-4 min.</li>
</ol>
8. Trypsin Digestion
<ol>
	<li>Reconstitute trypsin in 25 mM ammonium bicarbonate containing 1 M urea to a ratio of 50:1 (W/W; protein:trypsin) and incubated overnight at 37 &#176;C preferably at 700 rpm on a thermomixer.</li>
	<li>Spin (10,000 x g, 15 min) to pellet undigested material. Transfer supernatant to new tube.</li>
</ol>
9. Phosphopeptide Enrichment
<ol>
	<li>Perform purification and enrichment of phosphopeptides from the tryptic digest as previously described18. Dilute tryptic peptides 10-fold in DHB buffer [DHB buffer consists of: 350 mg/ml&#160;2,5 dihydrobenzesulfonic acid (DHB), 80% (v/v) ACN (acetonitrile), 2% (v/v) TFA (trifluoroacetic acid)] and apply to dry TiO2 beads (200 &#181;g).</li>
	<li>Leave on a rotator at room temperature for 1 hr.</li>
	<li>Wash the sample with DHB buffer, spin and remove supernatant. Then wash the sample three times with wash buffer [80% ACN (v/v), 2% TFA (v/v)] to remove the DHB.</li>
	<li>After the final spin, directly elute the phosphopeptides using elution buffer by adding 25 &#181;l&#160;of a 2.5% ammonium hydroxide, pH &#8805; 10.5 solution. Spin and remove the supernatant. Immediately neutralize with ~0.3 &#181;l&#160;formic acid.</li>
</ol>

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The authors declare that they have no competing financial interests.

The critical steps for a successful and reproducible proteomic analysis of spermatozoa are: 1) purity of the starting material; 2) removal of unwanted salts and detergents; 3) denaturing proteins to their full extent so as to allow trypsin to digest a high yield of proteins and 4) minimizing sample handling to reduce loss of peptide.
In order to successfully backflush the cauda epididymis, it is essential to locate the area from which the spermatozoa will exit. In the case of both rat and mice, this is at the apex of the concave area, in the middle of the caudal region of the epididymis (see Figure&#160;2A). If one comes further toward the vas deferens, backflushing is easier and the success rate is generally higher. However, this comes at the loss of sperm numbers. Alternatively, if one attempts to move more proximal to the corpus, then the amount of pressure required to push the spermatozoa back through the epididymal ducts is often so high, that damage to the epididymis inevitably occurs.
Backflushing of the epididymis is traditionally performed using water saturated mineral oil and a balanced salt solution in the syringe itself as a medium to remove the spermatozoa22-25. Both procedures are potentially problematic of LC-MS. Firstly, mineral is likely to block the nano-C18 nano-columns that are basically used worldwide for proteomic analysis and care must be taken so that none is carried forward in the procedure. If this occurs, it is impossible to continue and the sample is essentially lost. This can be overcome by the use of BWW or other balanced salt solutions in the syringe, however, although this is successful, we soon recognized that many of the spermatozoa become motile as soon as the BWW solution came into contact and mixed with the caudal epididymal cells. To circumvent this problem we simply backflush the spermatozoa with air. Not only is the quality of spermatozoa comparable to that of liquid-based methods, but the quantity is identical.
Phosphoproteomics is perhaps one of the only ways to establish which signaling pathways are occurring in spermatozoa post-ejaculation. One of the major pathways we are investigating is the process of capacitation. Spermatozoa must undergo &#8220;capacitation&#8221; before it is capable of binding to an egg. In practice, this is achieved basically by incubating spermatozoa for a period of time (mouse 40 min; rat 1.5 hr; human 3-24 hr) in BWW solution with serum albumin. Previously, we and others have shown a role for several kinases involved in capacitation. Of interest, deletion of the PKA&#181;II produce mice whose spermatozoa swim spontaneously in vitro, but cannot undergo hyperactivation26. The latter is a hallmark of capacitation, whereby spermatozoa change their swimming pattern from a high velocity, low amplitude, to a low velocity, high amplitude beat frequency. We have shown downstream kinases involved in this process include pp60-cSRC (SRC)13,27 c-yes28 and c-ABL14. Interestingly, inhibition of SRC stops capacitation-dependent tyrosine phosphorylation13. However, this can be overcome with okadaic acid, suggesting that SRC is not directly involved in the general onset of tyrosine phosphorylation 29 but may regulate a phosphatase29. The problem with using BWW as a medium to force spermatozoa out of the epididymis is that once activated, mouse spermatozoa only take approximately 40 min to capacitate. Given that isolation of spermatozoa may take 5 min/mouse and often several mice are used in an experiment, then spermatozoa will be at different stages of maturity at the start of the experiment. To overcome this, air pressure can be used to push the spermatozoa from the caudal epididymis into a glass cannula. Not only are all the sperm inactive and essentially as they would be found in the caudal milieu, it makes it possible to compare non-motile and motile phosphoproteomics.
Sample handling for phosphoproteomics should be kept to a minimum when comparing spermatozoa in two different functional states. The use of methanol chloroform over other traditional methods of protein precipitation 1) decreases the need for extra washing steps, 2) removes almost all traces of salts and fats and 3) has a proven ability to precipitate low abundance proteins over TCA19. Protein precipitation prior to trypsin digestion is recommended since not only does this help to denature protein (which aids trypsin digestion), but removes many of the MS-incompatible metabolites present in the cell.
The comparison of sperm phosphopeptides can be done in a number of ways. At the basic level, a simple comparison of the identified phosphopeptide in one sample, to that in another sample in the process known as &#8220;spectral counting&#8221; can be done. However criticism has been at this approach basically because early proteomic studies were using low replicates (for a fuller discussion see Lundren&#160;et al.30). A more sophisticated approach is to look at the intensity of the peptide parent mass and compare this with the other samples (label-free comparison). In the example shown in Figure 4, a peptide absent from the from non-motile (Figure&#160;4 top) but present in the motile (Figure 4 bottom) spermatozoa from m/z 650-670 can be seen. This process, referred to as MS-based label free quantification is a label-free strategy.
An alternative strategy commonly used to reduce the amount of runs required for proteomic quantification is to use isotopes. As the mass of an isotope is different, the mass spectrometer can be used to compare the intensities of the eluting peptides. However, unlike most other cell types, some isotopic labeling cannot be applied to spermatozoa. For example, the addition of stable isotopes (one heavy isotope gets added to one sample, whilst a lighter version gets added to another) can be used in tissue culture. When the isotopes get incorporated into the protein, a proteomics analysis can be performed by combining the two samples (multiplexing). However in the case of spermatozoa, this cannot be done, simply by virtue of the fact that 1) isotope labeling requires several passages (up to 8) in tissue culture and 2) the sperm cells has no nuclear gene transcription and translation and hence, they cannot incorporate the isotopes into all their protein anyway. One way around this is to order radiolabelled mice, however, these are notoriously expensive. An alternative approach is to use a chemical tag. This has been done when non-capacitated mice were compared with capacitated mice. In this case, a D0 and a D3-label was used to distinguish between one sample and another in the mass spectrometer31. Other approaches can include the use of iTRAQ (isobaric tag for relative and absolute quantitation), whereby lysines are labeled chemically with different mass isotopes; iCAT (isotope coded affinity tag) whereby cysteines are labeled with different mass isotopes and heavy and light water labeling.
In each and every case, however, one thing needs to be kept in mind. The MS only reports what is present in a sample, and this is a reflection of everything that has happened to that sample up to that point. Minimizing sample handling whilst maximizing the yield at each step is necessary for a successful proteomic analysis.

Having emerged from the testis, spermatozoa are highly differentiated yet remarkably, these cells are immature1,2. As such, they lack complete functionality, including the ability to swim and bind to an oocyte3. Although further maturation of the sperm cell is required, this cannot take place via canonical routes, since at this stage of their cell differentiation process, they are incapable of gene transcription and further protein biosynthesis4. Biological competence is conferred upon spermatozoa as they leave the testis and enter a part of the male reproductive system known as the epididymis5. The epididymis is a tightly coiled, highly differentiated tube that connects the efferent ducts (of the testis) to the vas deferens and is present in all male mammals6. Spermatozoa progressively acquire their fertilizing potential during epididymal descent, relying on the ever-changing luminal environment which is created by the local epididymal epithelial cells7. The various secretory and re-absorptive activities present in the epididymal milieu act to modify the sperm itself, changing its protein, carbohydrate and lipid composition6. The importance of the epididymis, particularly the initial &#8216;caput&#8217; region of this structure has been exemplified using surgical ligation techniques8. Spermatozoa retained within the testis by efferent duct ligation are completely lacking in any capacity to fertilize the oocyte9-11.
In addition to the epididymal environment, signal transduction pathways within the spermatozoa work to post-translationally modify the existing sperm cell complement. For example, spermatozoa from the early regions of the epididymis display different patterns of protein phosphorylation compared to those latter regions5,12. This is not surprising since there are vast differences in the capability of the sperm from these regions. For example, spermatozoa from the early, caput epididymis are immotilite. In contrast, sperm cells from the cauda region will undergo forward progressive motility once placed in isotonic medium. Since epididymal sperm cells are incapable of de-novo protein biosynthesis, all the intrinsic pathways regulating sperm function must be through post-translational modifications (PTM). Hence, it stands to reason that proteomics, and in particular the investigation of PTMs such as phosphorylation must be a major thrust if we are to understand sperm cell maturation.
An alternative method to obtain spermatozoa from the epididymidies to use retro-backflushing13-16. Although this technique is certainly more time consuming and takes a greater degree of skill by the operator, the spermatozoa obtained consistently demonstrate purity in excess of 99.99%. In addition, unlike all the other techniques, spermatozoa can be isolated in a quiescent state, making it possible to study how sperm motility is initiated. As sample preparation is the most important aspect for proteomic analysis, isolation of spermatozoa has become one of the most important aspects of sperm-proteomic studies. This protocol provides an explanation on how spermatozoa are isolated from the cauda epididymis. Following this, the TiO2 phosphopeptide enrichment procedure is outlined, with specific reference on how to extract peptides from the highly differentiated sperm cells. The MS approach can be used to distinguish changing phosphopeptides if one were to compare the caudal epididymal spermatozoa in one state (immotile or non-capacitated) to another (motile, capacitated, acrosome reacted, etc.) making this a powerful approach to study sperm function.

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			<td>195464</td>
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			<td height="21" style="height:21px;">2-D Quantitation Kit</td>
			<td>VWR (GE Healthcare)</td>
			<td>80-6483-56</td>
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			<td height="21" style="height:21px;">Albumin from bovine serum</td>
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			<td>A7030</td>
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			<td height="21" style="height:21px;">Ammonia Solution 25%</td>
			<td>Merck</td>
			<td>1.05432</td>
			<td></td>
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			<td height="21" style="height:21px;">Ammonium Bicarbonate</td>
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			<td>A6141</td>
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			<td height="21" style="height:21px;">Calcium Chloride</td>
			<td>Sigma</td>
			<td>C5080</td>
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			<td height="21" style="height:21px;">CHAPS 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate</td>
			<td>Research Organics</td>
			<td>1300C</td>
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			<td height="21" style="height:21px;">Chloroform</td>
			<td>BDH</td>
			<td>10077.6B</td>
			<td></td>
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			<td height="21" style="height:21px;">di-sodium hydrogen phosphate</td>
			<td>Merck</td>
			<td>1.06580</td>
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			<td height="21" style="height:21px;">Dithiothreitol (DTT)</td>
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			<td>D9779</td>
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			<td>O6440</td>
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			<td>G6152</td>
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			<td height="21" style="height:21px;">Glutamine-L</td>
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			<td height="21" style="height:21px;">Hepes (free acid)</td>
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			<td>I4386</td>
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			<td>L1750</td>
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			<td height="21" style="height:21px;">Magnesium Chloride</td>
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			<td>0090M</td>
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			<td height="21" style="height:21px;">Methanol</td>
			<td>Merck</td>
			<td>10158.6B</td>
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			<td height="21" style="height:21px;">Percoll (sterile)</td>
			<td>VWR (GE Healthcare)</td>
			<td>GEHE17-0891-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphatase Inhibitor Cocktail Halt</td>
			<td>ThermoFisher Scientific (Pierce)</td>
			<td>PIE78420</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Chloride</td>
			<td>Sigma</td>
			<td>P9333</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Hydrogen Carbonate</td>
			<td>Medical Store</td>
			<td>CH 600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>S3817</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride&#160;</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium di-hydrogen phosphate</td>
			<td>Merck</td>
			<td>1.06346</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydrogen carbonate</td>
			<td>BDH</td>
			<td>10247.4V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Pyruvate</td>
			<td>Sigma</td>
			<td>S8636</td>
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			<td height="21" style="height:21px;">Tris Ultra Pure Grade</td>
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			<td>AM0497</td>
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			<td>Promega</td>
			<td>V5280</td>
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			<td>U5128</td>
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			<td height="21" style="height:21px;">Zinc Chloride</td>
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			<td>Z0173</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.4 mm polyethylene tubing</td>
			<td style="width:175px;">Goodfellow</td>
			<td style="width:129px;">ET317100</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">30 gauge needle</td>
			<td style="width:175px;">Terumo</td>
			<td style="width:129px;">NN2038R</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">3 mL syringe</td>
			<td style="width:175px;">Terumo</td>
			<td style="width:129px;">SS035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4.2 mm polyethylene tubing</td>
			<td style="width:175px;">Goodfellow</td>
			<td style="width:129px;">ET317640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Braided silk (5-0)</td>
			<td style="width:175px;">Dynek</td>
			<td style="width:129px;">CSS0100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trichloroacetic acid</td>
			<td>Sigma</td>
			<td>299537</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TiO2 beads (from disassembled column)</td>
			<td style="width:175px;">Titansphere</td>
			<td style="width:129px;">6HT68003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Eppendorf Tube</td>
			<td style="width:175px;">Eppendorf</td>
			<td style="width:129px;">22431081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C7B30</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:129px;">C0856</td>
			<td></td>
		</tr>
	</tbody>
</table>

phosphopeptide analysis of rodent epididymal spermatozoa

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once the pre-cursor round cell begins to elongate and differentiate into what is morphologically recognized as a spermatozoon. However, the spermatozoon is very immature, having no ability for motility or egg recognition. Both of these events occur once the spermatozoa transit a secondary organ known as the epididymis. During the ~12 day passage that it takes for a sperm cell to pass through the epididymis, post-translational modifications of existing proteins play a pivotal role in the maturation of the cell. One major facet of such is protein phosphorylation. In order to characterize phosphorylation events taking place during sperm maturation, both pure sperm cell populations and pre-fractionation of phosphopeptides must be established. Using back flushing techniques, a method for the isolation of pure spermatozoa of high quality and yield from the distal or caudal epididymides is outlined. The steps for solubilization, digestion, and pre-fractionation of sperm phosphopeptides through TiO2 affinity chromatography are explained. Once isolated, phosphopeptides can be injected into MS to identify both protein phosphorylation events on specific amino acid residues and quantify the levels of phosphorylation taking place during the sperm maturation processes.

The quality of the results from any proteomic analysis is heavily dependent on the starting material. With modern MS, slight contaminations in sample preparations are easily picked up. Therefore, it is critical, in the case of sperm cell proteomics, to choose a method that gives highly pure spermatozoa. As illustrated in Figure 1, retrograde backflushing is used to retrieve sperm cells from the cauda epididymis. This involves cannulating the vas deferens with PE tubing which allows one to slowly apply air pressure from the syringe attached. Figure 1A demonstrates the cauda epididymis together with the vas deferens. Figure 1B is a close up shot of how the vas deferens is tied where the cannula is inserted. In doing, so, spermatozoa are released at the excised end of one tubule from the caudal epididymides. As shown in Figure&#160;2A, the spermatozoa are extracted from the apex regions of the cauda epididymis and are sucked up into a glass capillary in a quiescent state (Figure&#160;2B). This has several advantages over traditional &#8220;swim-up&#8221; methods, not the least of which is the ability to perform a phoshoproteomic analysis of spermatozoa before and after the activation of motility. The spermatozoa can then be expelled into media of one&#8217;s choice. Figure 2C (left hand side) demonstrates how the sperm look immediately after expulsion into BWW media. Many of the cells are clumped together. However, after 10 min the competence of the cells is demonstrated because they swim out to homogeneity into the solution (Figure&#160;2C, right hand side cells). Since the backflushing technique has very little impact on the sperm cells, we obtain close to 100% motility and the cells disperse rapidly into the media without external provocation. In a typical backflushing experiment, sperm recovered from the Swiss-mouse contains between 1-5 x 106 cells. This is highly dependent on the age of the animal and can vary from different strains of mice. In a Norwegian Rat, we typically obtain 200 x 106 spermatozoa, &#177; 20%. Figure 2D represents the purity of spermatozoa obtained from the rat cauda epididymis.
Besides the sample itself, one of the major issues concerning sample preparation is the yield of trypsin digestion. Protein precipitation plays a major role, not only in removing unwanted detergents and salts, both of which are incompatible with MS, but also in denaturing proteins. We have found the methanol-chloroform precipitation to work best. Not only has this procedure been reported to precipitate low abundant proteins better than others including TCA precipitation19, but it has additional advantages. Following TCA precipitation, it is often necessary to readjust the pH after suspension of the TCA pellet which can remain quite acidic. Although the precipitated pellet of TCA can be washed in high organic solutions to remove the acid, this adds to sample handling and irreproducibility of results. Failure to neutralize the acid will result in poor tryptic peptide yields. Methanol-chloroform precipitation is not only quick but will not acidify the sample. Figure 3 illustrates the protein pellet typically seen from 100 &#181;g of sample between the lower and upper interphase.
The isolation of phosphopeptides can occur in a number of ways; however reproducibility depends on how the sample has been handled up to and including this stage. One of the more common, developing methods is TiO2, originally developed by the group of Martin Larsen18,20,21. Although described as a process to use in microcolumns, we can obtain reproducible data using batch chromatography. The success of the process is heavily dependent on the elution buffer, which must be made with the correct composition; otherwise phosphopeptides are not eluted at all.
The reproducibility of the protocol and representative data from TiO2 enriched phosphopeptides from cauda epididymal sperm in a non-motile (Figure&#160;4 top) and motile (Figure 4 bottom) state from m/z 650-670 can be seen. We have demonstrated that this is an extremely reproducible technique even when using biological replicates17. The blue streaks appearing over time are peptides eluting from a C18 nano-column as the concentration of acetonitrile increases. Clearly, in the motile population (n=3 shown, n=8 typically run), there is a peptide cluster, with a monoisotopic mass around the 651.5 Da range that is completely absent in the non-motile range. Tandem mass spectrometry is then used to identify this peptide.
<img alt="Figure 1" src="/files/ftp_upload/51546/51546fig1highres.jpg" /> 
Figure 1: Cannulation of the cauda epididymis. (A) Picture of the cauda epididymis. The cannula itself is taped down to expose approximately 1-2 cm of the pre-pulled end. This is inserted into the vas deferens using fine #5watchmakers forceps. (B) The cannula is held in place by tying fine silk around a segment containing both the vas deferens and the cannula. From the mouse, approximately 2-3 &#181;l&#160;of caudal epididymal cells and fluid are collected at a concentration of 1 x 106/&#181;l. From the rat (shown), approximately 30-40 &#181;l&#160;of fluid are obtained at similar concentrations.
<img alt="Figure 2" src="/files/ftp_upload/51546/51546fig2highres.jpg" /> 
Figure 2: Glass capillary used to collect competent epididymal spermatozoa. (A) One tubule from the apex of the cauda epididymis is isolated and broken. The approximate position from which this occurs is shown. (B) The top of the image shows an empty glass capillary tube and the bottom shows one from a previously backflushed rat. There is no blood contamination and minimal epithelial cell contamination. (C) As the spermatozoa are still undiluted in the epididymal fluid, they have not begun to activate. When the spermatozoa are initially expelled into a BWW solution, they come out as a compact &#8220;string&#8221; (Left hand side). The competence of the cell is easily established, since after 10 min most of the spermatozoa swim into solution without any external disturbance (Right hand side). (D) A picture of an aliquot of caudal epididymal cells immediately after they have been left to swim out demonstrates the purity of the cells.
<img alt="Figure 3" src="/files/ftp_upload/51546/51546fig3highres.jpg" /> 
Figure 3: Methanol-chloroform precipitation. Once spermatozoa have been solubilized, the soluble fraction is methanol-chloroform precipitated to ensure denaturation of the proteins and removal of contaminating substances. The protein pellet appears on the interface between the methanol/water and chloroform phases. The top layer is removed carefully so as not to disturb this pellet. It is common to leave about 2-3 mm of the upper phase so as no protein is lost.
<img alt="Figure 4" fo:content-width="6in" src="/files/ftp_upload/51546/51546fig4highres.jpg" width="600" /> 
Figure 4: Typical TiO2-enriched phosphopeptide profile. Using the described protocol, caudal epididymal spermatozoa were isolated in either immotile (top, N = 3) or motile states (bottom, N = 3). A peptide cluster with a mono-isotopic mass of ~651.5 Da is shown to be present in the motile populations but completely absent in the immotile ones (arrow). Comparisons of this nature are referred to as &#8220;label-free (MS-based)&#8221;. The peptide mass and retention time are then used to target the compound for tandem-mass spectrometry and identify the protein from which the peptide was derived.

Watch this Scientific Journal Video about Phosphopeptide Analysis of Rodent Epididymal Spermatozoa at JoVE.com

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

Proteomic analysis of any cell type is highly dependent on both purity and pre-fractionation of the starting material in order to de-complexify the sample prior to liquid chromatography mass spectrometry (MS). By using back-flushing techniques, pure spermatozoa can be obtained from rodents. Following digestion, phosphopeptides can be enriched using TiO2.

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once ...

phosphopeptide-analysis-of-rodent-epididymal-spermatozoa

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Biology

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

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High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

Principles of Rodent Surgery for the New Surgeon

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform surgery on a rodent. Students, post-doctoral scholars, and others interested in performing surgery on rodents as part of a research protocol may not have had formal surgical training as part of their required coursework. Surgery itself is a technical skill, and one that will improve with practice. The principles of aseptic technique, however, often remain unexplained or untaught. For most new surgeons, this vital information is presented in piecemeal fashion or learned on the job, neither of which is ideal. It may also make learning how to perform a particular surgery difficult, as the new surgeon is learning both a surgical technique and the principles of asepsis at the same time. This article summarizes and makes recommendations for basic surgical skills and techniques necessary for successful rodent surgery. This article is designed to supplement hands-on training by the user's institution.

Before attempting surgery, a new surgeon should have training in basic surgical techniques and concepts. This article will present basic surgical considerations with an emphasis on rodents.

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform ...

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency

De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for assessing the induction of germ cell mutations require the use of large numbers of animals, making them impractical. As such, germ cell mutagenicity is rarely assessed during chemical testing and risk assessment. Herein, we describe an in vivo male germ cell mutation assay using a transgenic rodent model that is based on a recently approved Organisation for Economic Co-operation and Development (OECD) test guideline. This method uses an in vitro positive selection assay to measure in vivo mutations induced in a transgenic &#955;gt10 vector bearing a reporter gene directly in the germ cells of exposed males. We further describe how the detection of mutations in the transgene recovered from germ cells can be used to characterize the stage-specific sensitivity of the various spermatogenic cell types to mutagen exposure by controlling three experimental parameters: the duration of exposure (administration time), the time between exposure and sample collection (sampling time), and the cell population collected for analysis. Because a large number of germ cells can be assayed from a single male, this method has superior sensitivity compared with traditional methods, requires fewer animals and therefore much less time and resources.

De novo mutations in the male germline may contribute to adverse health outcomes in subsequent generations. Here we describe a protocol for the use of a transgenic rodent model for quantifying mutations in male germ cells induced by environmental agents.

De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for ...

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. 
In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident adipose progenitor cells (APCs) play a key role in adipose tissue homeostasis and can contribute to the tissue pathology. The growing use of single cell analysis technologies &#8211; including single-cell RNA-sequencing and single-cell proteomics &#8211; is transforming the stem/progenitor cell field by permitting unprecedented resolution of individual cell expression changes within the context of population- or tissue-wide changes. In this article, we provide detailed protocols to dissect mouse epididymal adipose tissue, isolate single adipose tissue-derived cells, and perform fluorescence activated cell sorting (FACS) to enrich for viable Sca1+/CD31-/CD45-/Ter119- APCs. These protocols will allow investigators to prepare high quality APCs suitable for downstream analyses such as single cell RNA sequencing.

We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting.

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

Rodent Estrous Cycle Monitoring Utilizing Vaginal Lavage: No Such Thing As a Normal Cycle

The current methodology establishes a reproducible, standardized, and cost-effective approach to monitoring the estrous cycle of female Sprague Dawley (SD) adolescent rats. This study demonstrates the complexity of hormonal cycles and the broad spectrum of understanding required to construct a reliable and valid monitoring technique. Through an in-depth examination of principal experimental design and procedural elements,&#160;this description of the cycle and its fundamental principles provides a framework for further understanding and deconstructs misconceptions for future replication.
Along with an outline of the sample collection process employing vaginal lavage, the procedure describes the mechanism of data categorization into the four-stage model of proestrus, estrus, metestrus, and diestrus. These stages are characterized by a new proposed approach, utilizing the 4 categorizing determinants of vaginal fluid condition, cell type(s) present, cell arrangement, and cell quantity at the time of collection. Variations of each stage, favorable and unfavorable samples, the distinction between cyclicity and acyclicity, and graphic depictions of the collected categorizing components are presented alongside effective interpretive and organizational practices of the data. Overall, these tools allow for the publication of quantifiable data ranges for the first time, leading to the standardization of categorization factors upon replication.

This study details the crucial factors to consider in experimental designs involving female rats. In a larger sense, these data serve to decrease stigma and assist in the development of more inclusive diagnostic and intervention tools.

The current methodology establishes a reproducible, standardized, and cost-effective approach to monitoring the estrous cycle of female Sprague Dawley ...