Name: transgenic rodent assay for quantifying male germ cell mutant frequency
Uploaded: 2014-08-06T15:30:00
Duration: 14 min 45 s
Description: Watch this Scientific Journal Video about Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency at JoVE.com

This research was funded by the Canadian Regulatory System for Biotechnology (CRSB) and Chemicals Management Plan (CMP) initiatives.

All protocols involving animal husbandry, maintenance and handling were approved by Health Canada&#8217;s animal care committee.
1. Animal Exposures
<ol>
	<li>Randomly distribute transgenic male mice (8-12 weeks old) to control groups and treatment groups (min = 5 per group).Treat mice with test compound and relevant control by an appropriate exposure route for the selected administration time. Choose the appropriate sampling time according to the spermatogenic cell type of interest (Figure 3).</li>
	<li>After the sampling time, euthanize mice by cervical dislocation under isofluorane anesthesia (or other appropriate method). Carefully draw the testes from an incision in the abdomen or scrotum and excise the cauda epididymides.(Figure 4, to view a detailed video of epididymis collection see Duselis&#160;et al.24). Alternatively, collect the testes if analyzing seminiferous tubules cells. Freeze in liquid nitrogen and store at -80 &#176;C for later use.</li>
</ol>
2. Isolation and Digestion of Cauda Sperm
<ol>
	<li>Defrost cauda epididymis on ice. Transfer thawed cauda to a Petri dish and thoroughly mince with a scalpel or razor blade.</li>
	<li>Add 700 &#181;l of room temperature D-PBS to the Petri dish. Using a wide-bore 1,000 &#181;l pipette tip, release sperm from the cauda by drawing and releasing the suspension until the D-PBS becomes cloudy with sperm (approximately 10 times). NOTE: To prepare wide-bore pipette tip cut 2-3 mm from the end of a plastic pipette tip.</li>
	<li>Filter suspension through a stainless steel mesh filter into a fresh 1.5 ml tube. Wash the Petri dish with an additional 700 &#181;l of D-PBS and transfer to same 1.5 ml tube through the mesh filter. Remove mesh, place tube on ice.</li>
	<li>Repeat steps 2.1-2.3 for remaining samples.</li>
	<li>Spin samplesat 11,000 x g for 3 min. Carefully decant supernatant. Avoid disturbing the pellet.</li>
	<li>Add 1.0 ml cold 1x saline sodium citrate (SSC). Vortex until pellet is completely re-suspended. This will sometimes take several rounds of vortexing.</li>
	<li>Add 15 &#181;l of 10% SDS. Invert/shake vigorously for 30 sec to disrupt non-sperm cells. Shaking too gently will result in inadequate disruption and the pellet will not form properly in the following step.</li>
	<li>Spin at 11,000 x g for 2 min. A loose, &#8220;fluffy&#8221; pellet is indicative of incomplete disruption of somatic cells due to insufficient shaking in step 2.7. If this occurs, simply shake the sample again, and respin until a tight pellet is formed. Carefully decant supernatant. Avoid disturbing the pellet. Briefly spin again and remove remaining supernatant with 200 &#181;l pipette.</li>
	<li>Add 940 &#181;l 0.2x cold SSC and vortex until pellet is resuspended. This pellet may be very difficult to re-suspend and may take several rounds of vortexing. Sometimes clumps of sperm are unavoidable.</li>
	<li>Add 120 &#181;l &#946;-mercaptoethanol, 100 &#181;l 10% SDS, 20 &#181;l 0.5M EDTA, pH 8, and 20 &#181;l proteinase K (60 mg/ml, prepared fresh). Mix well and digest overnight with rotation at 37 &#176;C. Proceed to phenol/chloroform extraction.</li>
</ol>
3. Phenol/chloroform Extraction of DNA from Cauda Sperm
NOTE: Because the nuclear DNA in late phase spermatids and mature sperm is complexed with protamines, and is highly condensed compared to the DNA of somatic cells, conventional nucleic acid isolation methods will not generate DNA of sufficient yield and purity for the mutation assay to work efficiently. Multiple phenol-chloroform extractions after an aggressive digestion are required to release and purify sperm DNA (based on methods from15).
<ol>
	<li>Transfer sperm cell digest to a 15 ml polypropylene tube.</li>
	<li>Add 2 ml of phenol:chloroform mixture (1:1). Rotate tube at 22 rpm for 3 min.</li>
	<li>Centrifuge at 1,600 x g for 10 min and transfer aqueous top layer along with the fuzzy interface layer to a fresh 15 ml tube.</li>
	<li>Repeat steps 3.1.2 and 3.1.3 3x, but changing the rotation times to 3 min, 4 min, and 6 min, respectively. On the final repeat, avoid transferring any of the &#8220;fuzzy&#8221; interface layer.</li>
	<li>After the 4th extraction, add 70 &#181;l of 3M NaAc, pH 5.2 per 1 ml of aqueous extract and 2 ml of chloroform:isoamyl alcohol (24:1). Rotate tube at 22 rpm for 12 min.</li>
	<li>Centrifuge at 1,600 x g for 10 min and transfer aqueous top layer to a fresh 15 ml tube.</li>
	<li>Precipitate DNA by adding 2 volumes of absolute ethanol and gently rotating the tube on its side with gentle rocking.</li>
	<li>Collect DNA by spooling onto the tip of a heat-sealed pasture pipette. Rinse DNA by swirling the pipet tip in 70% ethanol and air dry for 5 min.</li>
	<li>After extraction, dissolve DNA precipitate in 40 - 100 &#181;l Tris-EDTA buffer, pH 8. Store at 4 &#176;C. Allow the DNA to dissolve at 4 &#176;C for a minimum of two days before proceeding to lacZ mutation assay. If solubility issues are encountered, DNA can be further dissolved at 65 &#176;C for 15 min before use. Determine the concentration of the DNA with a spectrophotometre at A260 and ensure that the concentration of the dissolved DNA is between 200-2,000 ng/&#181;l.</li>
</ol>
4. Isolation and Digestion of Germ Cells from Seminiferous Tubules
<ol>
	<li>If frozen, defrost testis on ice (approximately 1 hr). Transfer testis to a ground glass plate.</li>
	<li>Hold one end of the testis with a pair of forceps. At the other end of the testis, puncture a hole in the epithelial capsule using another pair of forceps or a pair of dissection scissors (Figure 5A). Squeeze the seminiferous tubules through the puncture and discard the epithelial capsule (Figure 5B).</li>
	<li>Add 500 &#181;l of room temperature D-PBS to the decapsulated seminiferous tubules.</li>
	<li>Angle a tissue roller (silicone rubber tightly fitted over a freely rotating 5 mm diameter stainless steel tube, or similar apparatus) so that one end is in contact with the plate at an approximate angle of 5-10&#176; (Figure 5C). Without applying any pressure, gently move the roller back and forth across the tubules until they are flattened and the D-PBS becomes cloudy with released cells (approximately 5-10x).</li>
	<li>Add another 500 &#181;l of D-PBS over the tubules and gently roll over the tubules a few additional times.</li>
	<li>Transfer the cell suspension to a 1.5 ml microfuge tube while minimizing the amount of detached tubules being transferred (Figure 5D).</li>
	<li>Repeat steps 4.5-4.6 to collect more cells if necessary.</li>
	<li>Allow 1 - 2 min for any accidentally collected tubules to settle to the bottom of the tube. Transfer the D-PBS to a fresh 1.5 ml tube leaving behind the settled tubules (approximately 100 &#181;l of D-PBS). A small aliquot of this suspension can be checked under a microscope (phase contrast) to assess the composition of the cell population.</li>
	<li>Spin down the cells at 11,000 x g for 30 sec. Carefully decant the supernatant without disturbing the pellet. The cell pellet can be frozen at -80 &#176;C at this point if needed.</li>
	<li>Thaw cells if required. Transfer to a 15 ml polypropylene tube and resuspend cells in 5 ml of lysis buffer (10 mM Tris pH 7.6, 10 mM EDTA, 100 mM NaCl, 1 mg/ml proteinase K, 1% SDS). Digest overnight in incubator with rotation at 37 &#176;C.</li>
	<li>Proceed to phenol/chloroform extraction.</li>
</ol>
5. Phenol/Chloroform Extraction of DNA from Seminiferous Tubule Germ Cells
NOTE: A less aggressive extraction is used to isolate seminiferous tubules germ cell DNA, since these cell types have not yet progressed through nuclear condensation.
<ol>
	<li>Add 5 ml of phenol:chloroform mixture (1:1) to overnight seminiferous tubules cell digestion. Rotate tube at 22 rpm for 20 min.</li>
	<li>Centrifuge at 1,600 x g for 10 min and transfer aqueous top layer, while avoiding the &#8220;fuzzy&#8221; interface layer, to a fresh 15 ml tube.</li>
	<li>Add 100 &#181;l of 5 M NaCl per 5 ml aqueous extract (usually 5 ml is recovered)</li>
	<li>Add 5 ml of chloroform:isoamylalcohol (24:1). Rotate tube at 22 rpm for 12 min.</li>
	<li>Centrifuge at 1,600 x g for 10 min and transfer aqueous top layer to a fresh 15 ml tube.</li>
	<li>Precipitate DNA by adding 2 volumes of absolute ethanol and gently rotating and inverting tubes.</li>
	<li>Collect DNA by spooling onto the tip of a sealed off pasture pipette. Rinse DNA by swirling the pipet tip in 70% ethanol and air dry for 5 min.</li>
	<li>Dissolve DNA in 40-100 &#181;l Tris-EDTA buffer, pH 8. Store at 4 &#176;C. Allow DNA to dissolve at 4 &#176;C for a minimum of two days before proceeding to lacZ mutation assay (see step 3.9). If solubility issues are encountered, DNA can be further dissolved at 65 &#176;C for 15 min before use. Determine the concentration of the DNA with a spectrophotometre at A260 and ensure that the concentration is between 200 and 2,000 ng/&#181;l.</li>
</ol>
6. LacZ mutation Assay
Bacterial or fungal contamination can interfere with packaging efficiency, as well as plaque growth and scoring. It is therefore critical to perform the lacZ assay using the appropriate aseptic measures to prevent contamination of the packaging reaction, host culture and growth media.
<ol>
	<li>Day Before Assay: Prepare Bottom Agar and Overnight Culture
	<ol>
		<li>Eight plates (4 plates to score mutants, 4 plates to count titer) containing 8 ml bottom agar each are required per sample (i.e., 64 ml per sample). The bottom agar is identical for both the mutant and titer&#160;count plates. Prepare sufficient bottom agar for the number of samples being processed. Aseptically pour into 90 mm Petri dishes (8 ml per dish) and allow agar to solidify. NOTE: Bottom agar plates can be prepared up to 1 week in advance.</li>
		<li>In a 50 ml tube add 10 ml LB broth, 100 &#181;l of 20 % maltose solution, 25 &#181;l ampicillin (20 mg/ml) and 20 &#181;l kanamycin (5 mg/ml). Inoculate with E. coli (lacZ-/galE-)25 and grow overnight at 37 &#176;C with shaking at 240 rpm.</li>
	</ol>
	</li>
	<li>Day 1: Sub-culture Cells
	<ol>
		<li>Sub-culture cells by preparing a 1:100 dilution of the overnight culture in fresh LB (no antibiotics). A volume of 8 ml of subculture is needed per sample. Incubate at 37 &#176;C with shaking at 240 rpm for about 3.5 hr until OD600 = 1.</li>
		<li>When OD600 = 1, divide cell suspension evenly into 50 ml tubes and centrifuge at 1,300 x g at 15 &#176;C for 10 min. Remove supernatant and re-suspend cells in half of the original volume (i.e., 4 ml per sample) of LB containing 10 mM MgSO4. Put cells aside until needed [step 6.2.4.3].</li>
	</ol>
	</li>
	<li>Day 1: Packaging DNA in Lambda Phage Particles
	<ol>
		<li>Warm up a water bath to 30 &#176;C.</li>
		<li>Using a wide-bore 10 &#181;l pipette tip, transfer 4 &#181;l DNA to a 1.5 ml tube. NOTE: This step can be performed a day or more in advance to reduce preparation time.</li>
		<li>Warm up the first tube (red) from a phage packaging extract kit (1 tube for every 2 samples). Briefly spin to collect extract at bottom of tube.</li>
		<li>Transfer 4.8 &#181;l of packaging extract from the first tube to the DNA sample and mix by gentle stirring with the pipette tip. Briefly spin down samples in tubes. Incubate for 1.5 hr in 30 &#176;C water bath.</li>
		<li>Warm up the second (blue) packaging extract tube (1 tube for ~15 samples). Briefly spin to collect extract at bottom of tube.</li>
		<li>Transfer 4.8 &#181;l of packaging extract from the second tube to the DNA sample and mix by gentle stirring. Briefly spin down samples in tubes. Incubate for an additional 1.5 hr in 30 &#176;C water bath.</li>
		<li>Re-suspend packaged phage particles in 500 &#181;l SM buffer and mix by rotating for 30 min at 20 rpm.</li>
		<li>After rotating, briefly vortex samples and centrifuge at 11,000 x g for 30 sec to collect samples at bottom of tubes. Phage particles are ready for infection [step 6.2.4].</li>
	</ol>
	</li>
	<li>Day 1: Prepare Top Agar
	<ol>
		<li>Prepare separate top agar for the titer&#160;plates and mutant selection plates. Each sample requires 4 titer&#160;plates, and 4 mutant plates. Each plate requires 8 ml top agar. Add the selective agent phenyl-&#946;-D-galactopyranoside (P-Gal) to the mutant selection top agar only. Prepare both top agars in advance (day of assay) and maintain at 50 &#176;C before adding MgSO4 to both top agars, and P-Gal to mutant selection agar.</li>
	</ol>
	</li>
	<li>Day 1: Infecting Cells with Packaged Phage and Plating
	<ol>
		<li>Label two 50 ml tubes per sample: 1 &#8220;mutant&#8221; tube per sample and 1 &#8220;titer&#8221; tube per sample</li>
		<li>Label 8 agar plates per sample: 4 &#8220;mutant&#8221; plates per sample and 4 &#8220;titer&#8221; plates per sample</li>
		<li>Aliquot 2 ml of resuspended cells [from step 6.2.1.2] to each tube.</li>
		<li>Add 500 &#181;l of packaged phage particles [from step 6.2.2.8] to &#8220;mutant&#8221; 50 ml tube (containing cells). Gently mix and allow phage particles to infect cells for 30 min at room temperature.</li>
		<li>After 30 min, briefly vortex infected cells and transfer 15 &#181;l of infected cells to the appropriate 50 ml &#8220;titer&#8221; tube (containing cells).</li>
		<li>To plate titer&#160;sample, add 30 ml of warm (50 &#176;C) &#8220;titer&#8221; top agar (NOT containing P-Gal) to &#8220;titer&#8221; 50 ml tube. Immediately distribute agar/cell mixture among the 4 &#8220;titer&#8221; plates (~8 ml per plate). Work quickly so that the top agar does not cool in the pipettes, and try not to introduce air bubbles.</li>
		<li>Plate the &#8220;mutant&#8221; samples next. Ensure P-Gal is added to &#8220;mutant selection&#8221; top agar. Add 30 ml of warm &#8220;mutant selection&#8221; agar (containing P-Gal) to &#8220;mutant&#8221; 50 ml tube. Immediately distribute agar/cell mixture among the 4 &#8220;mutant&#8221; plates (~8 ml per plate).</li>
		<li>Allow plates to solidify (~15 min) then invert and incubate at 37 &#176;C overnight.</li>
	</ol>
	</li>
	<li>Day 2: Counting Plaques
	<ol>
		<li>After overnight incubation, count the number of plaques on the mutant and titer&#160;plates. For large numbers of plaques, count only a portion of the plate to estimate the total count (e.g., often &#188; of the titer plate will have between 100-200 plaques). A minimum of 100 plaques should be counted per plate when counting a portion of the plate.</li>
		<li>Calculate the number of plaque forming units (PFUs) per &#181;l of cells. This is done by dividing the number of plaques on the &#8220;titer&#8221; plates by the volume of cells plated (15 &#181;l).</li>
		<li>Use the number of PFUs/&#181;l to estimate the total number of PFUs in the total volume of infected cells plated on the &#8220;mutant&#8221; plates (PFUs/&#181;l *[2 ml cells + 0.5 ml packaged phage particles &#8211; 15 &#181;l for titer&#160;plates]).</li>
		<li>Estimate the MF by dividing the total number of mutant plaques counted on the 4 &#8220;mutant&#8221; plates by the estimated total number of PFUs in the total volume of infected cells determined from the &#8220;titer&#8221; plates.</li>
	</ol>
	</li>
	<li>When the spontaneous lacZ MF is in the order of 3 x 10-5 in the control group, as it for MutaMouse germ cells, the OECD guideline recommends a minimum of 125,000 to 300,000 non-mutant PFUs per animal be screened for mutation in order to obtain a reliable baseline signal. Other transgenic models may have lower spontaneous MF, in which case a greater number of PFUs would be required. A statistical power test can be performed in order to determine the minimum number of PFUs and animals required to obtain the desired resolution. Data from multiple replicates may be pooled to satisfy this minimum PFU requirement, provided they do not produce significantly different mutant frequencies.</li>
</ol>
7. Statistics
The experimental unit for the analysis is the mouse. Data produced from this assay are generally not normally distributed. As such, select the statistical method for analysis based on the distribution characteristic of the data.
NOTE: Standard parametric analyses (e.g., ANOVA) may be employed if appropriate data transformation is applied to equalize the variance of the response across the range of observation. Poisson or binomial regression analyses are often more appropriate. Nonparametric analysis may also be employed. We routinely employ Poisson regression using the generalized linear model procedure (i.e., Proc GENMOD) in SAS v.9.2 (SAS Institute, Cary, NC) as described by Lemieux et al26.

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R., Vijg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+galactose-sensitive+E.+coli+strains+as+selective+hosts+for+LacZ-+plasmids.">Application of galactose-sensitive E. coli strains as selective hosts for LacZ- plasmids.</a> Nucleic Acids Res. 20 (12), 3254 (1992).</li><li>Lemieux, C. L., 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=Simultaneous+measurement+of+benzo[a]pyrene-induced+pig-a+and+lacZ+mutations,+micronuclei+and+DNA+adducts+in+muta+mouse.">Simultaneous measurement of benzo[a]pyrene-induced pig-a and lacZ mutations, micronuclei and DNA adducts in muta mouse.</a> Environ. Mol. Mutagen. 52 (9), 756-765 (2011).</li><li>Douglas, G. R., Jiao, J., Gingerich, J. D., Gossen, J. A., Soper, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+molecular+characteristics+of+mutations+induced+by+ethylnitrosourea+in+germ+cells+isolated+from+seminiferous+tubules+and+in+spermatozoa+of+lacZ+transgenic+mice.">Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice.</a> Proc. Natl. Acad. Sci. U.S.A. 92 (16), 7485-7489 (1995).</li><li>Russell, W. L., Hunsicker, P. R., Raymer, G. D., Steele, M. H., Stelzner, K. F., Thompson, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dose--response+curve+for+ethylnitrosourea-induced+specific-locus+mutations+in+mouse+spermatogonia.">Dose--response curve for ethylnitrosourea-induced specific-locus mutations in mouse spermatogonia.</a> Proc. Natl. Acad. Sci. U.S.A. 79 (11), 3589-3591 (1982).</li><li>Swiger, R. R., Cosentino, L., Shima, N., Bielas, J. H., Cruz-Munoz, W., Heddle, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cII+locus+in+the+MutaMouse+system.">The cII locus in the MutaMouse system.</a> Environ. Mol. Mutagen. 34 (2-3), 201-207 (1999).</li><li>Singer, T. M., Lambert, I. B., Williams, A., Douglas, G. R., Yauk, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+induced+male+germline+mutation:+Correlations+and+comparisons+between+traditional+germline+mutation+assays,+transgenic+rodent+assays+and+expanded+simple+tandem+repeat+instability+assays.">Detection of induced male germline mutation: Correlations and comparisons between traditional germline mutation assays, transgenic rodent assays and expanded simple tandem repeat instability assays.</a> Mutat. Res. 598 (1-2), 164-193 (2006).</li></ol>

The authors declare that they have no competing financial interests.

Compared to traditional methods, the TGR germ cell mutation assay provides a faster, more economical, and more sensitive means of quantifying induced in vivo germ cell mutations. By assessing transgene MF directly in sperm, as opposed to offspring, the number of animals, time and resources required to assess the germline mutagenicity of any single compound is significantly reduced. In terms of sensitivity, we were able to detect a significant 2.6 fold increase in spermatogonia stem cell MF following exposure to 25 mg/kg ENU using only 5 animals per dose group. In contrast, the specific locus assay was unable to detect any significant change in MF at this same dose using &#62;3,000 mice in the exposed group and &#62;500,000 control mice28.
In addition to its suitability for both mechanistic and regulatory investigations, this method provides an opportunity for comparative studies between somatic and germ line mutation rates. Recent evidence suggests that for some agents germ cell mutations may be induced at lower concentration than required for somatic mutation. For example, prolonged exposure to N-hydroxymethylacrylamide, a metabolite of the food carcinogen acrylamide12, increases the frequency of dominant lethal germ cell mutations in mice without affecting the micronucleus frequency in red blood cells, a traditional measure of somatic cell cytogenetic damage13. Additionally, exposure of mice to both mainstream and sidestream tobacco smoke causes elevated mutation frequencies at tandem repeat DNA loci in sperm at doses that do not increase blood micronucleus frequency14. These findings challenge the assumption that somatic genotoxicity testing is always protective of the germline, and reinforce the demand for a more efficient and cost-effective means to quantify germ cell mutation frequency. However, the evidence for preferential germ cell mutagens is still weak, largely due to the lack of available data for comparing mutation rates in somatic and germ cell tissues. The TGR mutation assay allows parallel testing and comparison of induced mutation rates in multiple tissues using the same transgene. Thus, comparative mutation testing using the TGR assay would help fill data gaps surrounding the possibility of preferential germ cell effects.
Simultaneous assessment of somatic and germ cell mutation for regulatory testing would also improve efficiency by reducing the number of animals required. The OECD guideline for somatic mutation recommends a 28 day administration time, followed by a three day sampling time (28+3). Analysis of cauda sperm may offer poor sensitivity at this time point, since it targets cells exposed mostly during the spermatocyte and spermatid phases of spermatogenesis (Figure 3). Cells in these phases do not synthesize DNA and progressively lose their capacity for DNA repair6. Furthermore, sampling cauda sperm at this time point would fail to detect mutations occurring in spermatogonia and stem cells. Thus, for integration into the 28+3 design, the OECD guideline recommends collecting germ cells from the seminiferous tubules. This mixed population contains cells derived from DNA synthesis and repair proficient cell types, including stem cells, and are exposed across the majority of spermatogenic phases. However, due to the mixed nature of these cells, seminiferous tubules cell analysis does not provide phase-specific information. Furthermore, there is concern that the presence of non-target cells can influence the observed MF (e.g. false positive germ cell mutagen calls due to contamination of mutated somatic cells, or dilution of a mutated germ cell signal from DNA repair-deficient germ cells). Currently there is insufficient data to conclude whether results from seminiferous tubules cells at 28+3 offer the same sensitivity and specificity as cauda sperm at later time points. Our laboratory is currently comparing induced MFs in seminiferous tubules cells and cauda sperm collected after various sampling times to address this point. We note that the OECD guideline suggests an alternative sampling time of 28 days for slowly dividing tissues such as the liver that may also be suitable for germ cell analysis. Nevertheless, available data is still insufficient and we are currently unable to recommend one single experimental design for the simultaneous analysis of somatic cells and germ cells using the TGR mutation assay for regulatory testing.
One characteristic of this assay that should be noted is that mutational events are assessed on a non-murine transgene. However, there is ample evidence to suggest that the transgene responds to environmental mutagens in a similar fashion to endogenous genes20. In addition, because the precise origins of independent mutational events are difficult to resolve, results are generally reported as a mutant frequency (in contrast to a mutation frequency). The actual mutation frequency can be resolved if results are corrected for clonal expansion (i.e. the division and multiplication of a single mutated cell that can contribute to the observed frequency of transgene mutants) by DNA sequencing. Sequencing of the mutant transgenes may be performed to characterize the lacZ mutations, and identify mutants that may be derived from clonal expansion events, although this adds significantly to the time and cost of the analysis. In addition to the lacZ gene, the&#955;gt10 transgenic vector harbors an alternative temperature-dependent mutation-reporter gene: a variant of the &#955; cII gene, which is shorter (294 bp vs the 3021 bp lacZ, Figure 1) and easier to sequence29. Sequencing also permits the analysis of the induced mutation spectrum, providing insight into the mutational mechanism of the compound in question. An extreme example of clonal expansion is the occurrence of a &#8220;jackpot&#8221; mutation (i.e., transgene mutations at a very early stage of an organ&#39;s development that contribute to a dramatically elevated MF, sometimes hundreds to thousands of times greater than background). Animals or tissues with a &#8220;jackpot&#8221; mutation should be removed from the analysis.
The assay that we have described is broadly applicable to other TGR models such as the BigBlue mouse and rat, and the lacZ plasmid mouse, all of which harbor similar mutation reporter vectors (reviewed in20). The vast majority of germ cell studies conducted to date that employ similar methods have focused almost exclusively on well characterized mutagens such as ENU and radiation (reviewed in30). It is expected that with the recent release of the OECD test guideline for the TGR assay, this assay will be increasingly popular for chemical screening and regulatory evaluation. Incorporation of the TGR germ cell mutation assay into a regulatory testing battery will fill the existing gap by permitting efficient assessment of mutation induction in germ cells11. Moreover, this assay can be used to measure MF in virtually any tissue, providing a suitable means for comparing the relative sensitivities of somatic cells and germ cells to the induction of mutations by environmental agents at identical genetic endpoints.

Sporadic DNA mutations in the germline can lead to reduced reproductive success and, if inherited, may cause genetic disease or heightened predisposition to cancer in the offspring1-3. Substantial evidence demonstrates that a large proportion of de novo mutations are inherited from the paternal germline4, and that the number of mutations in the offspring is positively correlated with paternal age at the time of conception5. The higher proportion of male mutations is believed to be a result of the difference in age during gametogenesis between the sexes, the greater number of spermatogenic cell divisions compared with the number of oogenic cell divisions in the female germline2, and a progressive decline in DNA repair efficiency with age in males. All of these factors contribute to an increased probability of replication errors in the male germline6. However, the impact of paternal exposure to environmental factors on the frequency of de novo mutations remains uncertain. Nevertheless, a large number of environmental agents are known to induce germ cell mutations in rodents7, and there is mounting evidence that some of these agents can also affect the human germline8. Despite these concerns, chemicals are routinely tested for their ability to induce mutations in somatic cells for regulatory purposes and it is generally assumed that somatic tests are sufficient to protect the germline. Therefore, chemicals are only rarely assessed for their ability to induce germ cell mutations.
One reason germ cell mutagenicity testing has been largely omitted from the regulatory decision making process is a lack of practical methodologies. Traditional rodent-based methods, such as the dominant lethal9 and specific locus10 tests, estimate germ cell mutation rates by scoring mutant phenotypes in embryos or offspring of exposed parents. These assays require the use of a very large number of animals, time and resources to acquire statistically meaningful results.
Although several modern methods for quantifying germ cell mutation have recently emerged, many suffer shortcomings in terms of their practicality, efficiency, and biological relevance. For example, repeat length mutations at expanded simple tandem repeat (ESTR) loci can be quantified in male germ cells using a single molecule PCR approach15. However, execution of this method can be technically challenging and laborious, and unlike point mutations, the biological and health significance of changes in the repeat length of the highly unstable ESTR loci remain unclear16. Modern whole genome sequencing technologies can provide a wealth of biologically meaningful data when applied to the problem of heritable mutations4,17, but the high cost, high error rates, associated validation required to confirm mutations, and bioinformatics challenges still limit the routine application of this option in a regulatory testing capacity18.
Herein, we describe a practical method for quantifying induced mutations directly in the germ cells of transgenic male mice. This protocol is described for the transgenic MutaMouse model, which has multiple concatenated copies of a recombinant &#955;gt10 phage vector containing an Escherichia coli lacZ reporter gene integrated into both copies of chromosome 319 (Figure 1).
This protocol is also relevant to other transgenic rodent (TGR) models based on the same principles (BigBlue mouse and rat, or lacZ plasmid mouse, etc.) or slightly different reporter genes (gpt delta mouse and rat, TGR models reviewed in Lambert&#160;et al.20). This method is based on the TGR mutation assay described in a recently released and revised OECD test guideline21 and we elaborate upon the special considerations required to accommodate assessment of mutations in the male germline because of the unique characteristics of spermatogenesis. Briefly, the assay involves exposing transgenic male mice to a mutagenic substance, followed by a sampling time where pre-mutational lesions are fixed into stable mutations. At the chosen sampling time, mice are euthanized and germ cells are collected from either the cauda epididymis or the seminiferous tubules. As discussed below, mutagenic effects at different phases of spermatogenesis can be determined by selecting the time between exposure and sample collection. Transgenic inserts, comprising multiple copies of the &#955; phage genome per cell, are isolated from germ cell genomic DNA and packaged into empty &#955; phage capsids creating infectious &#955; phage particles that are then used to infect an E. coli host. The infected bacteria are grown on selective media that can distinguish cells containing a vector with a mutated copy of lacZ from cells harboring wild-type lacZ. The mutagenic effect of exposure on the male germline is determined by comparing the frequency of mutant transgenes between control and treated mice (Figure 2, reviewed in Lambert&#160;et al.20). A large number of germ cells can be assayed from a single mouse, giving this assay superior sensitivity over traditional methods, while reducing the number of animals required. And because no specialized equipment or training is required, this assay provides a practical and efficient option for germ cell mutation testing in most modern toxicology/molecular biology laboratories.
One essential requirement for the effective application of the TGR germ cell mutation assay is an understanding of the spermatogenic cycle (Figure 3). The time for mouse germ cells to progress from stem cells in the seminiferous tubules to spermatogonia, spermatocytes, spermatids, and finally to mature sperm in the epididymis (i.e. spermatogenesis) is approximately 49 days. Mutation can occur at various phases of this cycle and is often compound specific. Two key features that are of particular relevance to mutagenesis in male germ cells are the cessation of DNA synthesis during early meiosis, and the progressive loss of DNA repair capacity6 during late post-meiosis, two processes that are required for the induction and fixation of most mutations.
Because of these unique characteristics of spermatogenesis, there are three critical experimental variables for the conduct of the TGR germ cell mutation assay: (1) the test compound administration time; (2) the sampling time; and (3) the selection of the germ cell population to collect for analysis (Figure 3 and Table 1). Administration time is the experimental variable that determines how long target cells are exposed to the test compounds. The length of the administration time can also be used to target exposures to specific cell types or phases of spermatogenesis. For example, a single day administration could be used to determine the effects of an acute exposure on one particular cell type. Similarly, exposure can be focused to an entire spermatogenic phase, for example by targeting only meiotically dividing spermatocytes, or mitotically dividing spermatogonia using a 2 week administration time and an appropriate sampling time. Chronic and sub-chronic administration times are used to assess the effects of long term exposure, to ensure sufficient pharmacokinetic distribution of the test compound, or permit sufficient accumulation of mutations from weak mutagens (for example the 28 day administration time recommended in the OECD test guideline).
Sampling time is the critical variable for determining at which phase of spermatogenesis the target cells were in at the time of exposure. The sampling time dictates how much time, and therefore how much further along the spermatogenic cycle, cells pass through after exposure. For example, to investigate effects in stem cell spermatogonia, a sampling time &#62;49 days is required if collecting fully matured sperm, or &#62;42 days if collecting immature germ cells from the seminiferous tubules, to ensure that all collected cells have had enough time to develop from exposed stems cells. It is important to note that a sampling time of at least 70 days would be preferable to demonstrate a true stem cell effect to provide sufficient time for pharmacokinetic distribution of the toxicant, for elimination of cells exposed at later phases of spermatogenesis, and to account for a period of temporary sterility that may occur ~6 weeks after exposure to highly mutagenic compounds22. Similarly, a sampling time of 21 days would ensure that sperm collected from the cauda epididymis would have just completed meiosis on the final day of exposure.
Germ cells can be collected as mature sperm from the cauda epididymis, or as a mixture of various spermatogenic cell types from the seminiferous tubules. Mature sperm remain in the cauda for ~3 days, making it possible to determine with relative accuracy the cell type or phase of spermatogenesis from which the sperm originated for any given experimental design. Thus, analysis of cauda sperm permits highly targeted investigations of stage-specific mutational effects. On the other hand, cell suspensions collected from the seminiferous tubules contain a mixture of various germ cell types in different phases of development, and thus offer poorer resolution of the spermatogenic phase in which mutations originated. In addition, cell suspensions recovered from the seminiferous tubules tend to contain an over-representation of spermatids, followed by spermatocytes, and very few spermatogonia and stem cells (these proportions are represented by graduated white bars in Figure 3). Moreover, suspensions prepared from the seminiferous tubules may also contain various somatic cells. Thus, because so many cells types are present, mutational effects can be influenced by a variety of non-target cells. However, collecting samples from seminiferous tubules offers an economical option for simultaneously screening multiple germ cell types, and easy integration of germ cell analysis into the standard OECD test protocol for somatic mutation.
To reiterate, depending on the needs of the investigator, administration time, sampling time and the collected cell population can be adjusted to interrogate the effects of exposure in various cell types and at different phases of spermatogenesis. By carefully selecting these variables, experiments can be designed for targeted mechanistic studies, or for more generalized regulatory testing purposes.
To attain proficiency in the assay, we recommend the use of an acute oral administration of 100 mg/kg N-ethyl-N-nitrosourea (ENU), followed by a 70 day sampling time as positive control. Analysis of cauda sperm thus targets spermatogonial stem cells (Figure 3), which typically exhibit a 4-5 fold increase in mutant frequency (MF) over controls following this highly mutagenic dose of ENU. It should be noted that this dose is known to induce sterility 6 weeks post exposure, thus it may not be a suitable control dose for shorter sampling times. This dose will also produce a detectable increase in MF in most somatic tissues20. The representative results presented below were generated following an acute +70 exposure regimen using three doses of ENU up to and including 100 mg/kg.

<table border="0" cellpadding="0" cellspacing="0" width="1326">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Name of Reagent/Material</td>
			<td style="width:145px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td style="width:664px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MutaMouse</td>
			<td>Covance</td>
			<td>-</td>
			<td style="width:664px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">E.coli (lacZ-/galE-)</td>
			<td>Covance</td>
			<td>-</td>
			<td style="width:664px;">See reference 25 in manuscript</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloroform</td>
			<td>Caledon</td>
			<td>3001-2-40</td>
			<td style="width:664px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s phosphate-buffered saline (D-PBS)</td>
			<td>Gibco</td>
			<td>14190-250</td>
			<td style="width:664px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylenediaminetetraacetic acid (EDTA), 0.5M, pH 8</td>
			<td>Sigma-Aldrich</td>
			<td>03690 FLUKA</td>
			<td style="width:664px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoamyl alcohol</td>
			<td>Caledon</td>
			<td>2/10/7900</td>
			<td style="width:664px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lennon LB broth base</td>
			<td>Invitrogen</td>
			<td>12780-029</td>
			<td style="width:664px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stainless steel mesh filter</td>
			<td>Sigma-Aldrich</td>
			<td>S3770</td>
			<td style="width:664px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transpack packaging extract</td>
			<td>Agilent Technologies</td>
			<td>200220</td>
			<td style="width:664px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148</td>
			<td style="width:664px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethyl alcohol, anhydrous (EtOH)</td>
			<td>Commercial Alcohols</td>
			<td>-</td>
			<td style="width:664px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Ampicilin</td>
			<td>Gibco</td>
			<td>11593-027</td>
			<td style="width:664px;">prepare 20 mg/ml in dH2O</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Kanamycin</td>
			<td>Gibco</td>
			<td>11815-024</td>
			<td style="width:664px;">prepare 5 mg/ml in dH2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenol</td>
			<td>Invitrogen</td>
			<td>15509-097</td>
			<td style="width:664px;">Saturate in 0.1 M Tris-HCl as per manufacturers direction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenyl-&#946;-D-galactopyranoside (P-Gal)</td>
			<td>Sigma-Aldrich</td>
			<td>P6501</td>
			<td style="width:664px;">dissolve 3 g per 10 ml of dimethylformamide</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Proteinase K</td>
			<td>Invitrogen</td>
			<td>25530-031</td>
			<td style="width:664px;">prepare 60 mg/ml solution dH2O just before use, 20 &#181;l per sample</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium acetate (NaAc)</td>
			<td>Fisher Scientific</td>
			<td>BP333-500</td>
			<td style="width:664px;">prepare 3M solutution, pH 5.2</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td>L4390</td>
			<td style="width:664px;">prepare 10% solution in dH2O</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">1 M MgSO4</td>
			<td></td>
			<td></td>
			<td style="width:664px;">24.6 g MgSO4&#183;7H2O per 100 ml dH2O, autoclave, store at room temperature up to 1 year</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">20% maltose solution</td>
			<td></td>
			<td></td>
			<td style="width:664px;">20.0 g maltose, 24.6 g MgSO4&#183;7H2O, per 100 ml dH2O, filter sterilize, sore at 4&#7506;C up to 6 months</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;">Bottom agar</td>
			<td></td>
			<td></td>
			<td style="width:664px;">Prepare 64 ml per sample (8 pitri plates per sample X&#160; 8 ml per plate): 5.0 g LB base, 6.4 g NaCl, 7.5 g agar, per 1L dH2O. Autoclave and cool to 50&#7506;C before pouring into Petri plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LB Broth</td>
			<td></td>
			<td></td>
			<td style="width:664px;">5.0 g LB base, 6.4 g NaCl per 1L dH2O. Autoclave and cool</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saline sodium citrate (SSC)</td>
			<td></td>
			<td></td>
			<td style="width:664px;">150 mM NaCl, 15 mM trisodium citrate, pH 7.0&#160;</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;">SM Buffer</td>
			<td></td>
			<td></td>
			<td style="width:664px;">5.8 g NaCl, 2.0 g MgSO4&#183;7H2O, 50 ml 1 M Tris-HCl (pH 7.5), 5.0 ml of gelatin (2% w/v), per 1L dH2O, autoclave, store at room temperature up to 1 year</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TE buffer</td>
			<td></td>
			<td></td>
			<td style="width:664px;">10 mM Tris, 1 mM EDTA, pH 8</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;">Top agar, mutant plates</td>
			<td></td>
			<td></td>
			<td style="width:664px;">Prepare 32 ml per sample (4 mutant plates per sample X 8 ml per plate): 5.0 g LB base, 6.4 g NaCl, 7.5 g agar, per 1L dH2O. Autoclave and cool to 50&#7506;C. Add MgSO4 to 10 mM</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;">Top agar, titre plates</td>
			<td></td>
			<td></td>
			<td style="width:664px;">Prepare 32 ml per sample (4 mutant plates per sample X 8 ml per plate): 5.0 g LB base, 6.4 g NaCl, 7.5 g agar, per 1L H2O. Autoclave and cool to 50&#7506;C. Add MgSO4 to 10 mM and P-Gal to 3 g/L</td>
		</tr>
	</tbody>
</table>

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.

With an average plaque count of 200,000 plaques per animal, we typically observe a mean background MF of approximately 2.8 x 10-5 in male germ cells with a standard deviation of 1.7 x 10-5 in our control groups (based on data from eight independent experiments). With this plaque count, background level and variance, dose groups with n = 5 animals each are sufficient to detect a 2 fold increase in MF with power &#62;0.8.
Results are typically reported in tabular or graphical formats. Table 2 and Figure 6 show representative results from cauda sperm of MutaMouse males (n = 5 per group) exposed to a single acute oral dose of 0, 25, 50, and 100 mg/kg N-ethyl-N-nitrosourea (ENU) followed by a 70 day sampling time. This 70 day period permits the measurement of mutational events that occurred in sperm that were spermatogonial stem cells at the time of exposure (Figure 3). Typical plaque densities on mutant and titer plates are shown in Figure 7. As depicted in Table 2 and Figure 6, acute ENU exposure induced a significant dose-dependent increase in the MF of spermatogonial stem cells. The low dose induced a significant 2.6 fold increase over controls, which had a baseline MF of 2.6 x 10-5. Maximum induction occurred in the high dose, which elicited a 4.4 fold increase over controls.
An example of this assay performed on seminiferous tubule germ cells can be found in Douglas et al.27, where MF was determined in tubule germ cells at various time intervals following a 5 day repeat intraperitoneal injection of 50 mg/kg ENU. In that study, mutant frequencies in seminiferous cells increased up to 15 days after treatment and remained constant thereafter.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr tr="">
			<td>Exposure regimen</td>
			<td>Collected tissue</td>
			<td>Collected cell type (target cell)</td>
			<td>Phase of target cell at the beginning of exposure</td>
			<td>Phases passed during exposure</td>
		</tr>
		<tr>
			<td>Acute+70</td>
			<td>Cauda</td>
			<td>Mature sperm</td>
			<td>Stem cell</td>
			<td>Stem cell</td>
		</tr>
		<tr>
			<td>14+21</td>
			<td>Cauda</td>
			<td>Mature sperm</td>
			<td>Spermatogonia</td>
			<td>Spermatid</td>
		</tr>
		<tr>
			<td>14+35</td>
			<td>Cauda</td>
			<td>Mature sperm</td>
			<td>Stem cell</td>
			<td>Spermatocyte</td>
		</tr>
		<tr>
			<td>28+3</td>
			<td>Cauda</td>
			<td>Mature sperm</td>
			<td>Spermatocyte</td>
			<td>Spermatocyte 
			Spermatid 
			Mature sperm</td>
		</tr>
		<tr>
			<td></td>
			<td>Tubules</td>
			<td>Stem cell</td>
			<td>Stem cell</td>
			<td>Stem cell</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Spermatogonia</td>
			<td>Spermatogonia</td>
			<td>Spermatogonia</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Spermatocyte</td>
			<td></td>
			<td>Spermatocyte</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Spermatid</td>
			<td></td>
			<td>Spermatid</td>
		</tr>
		<tr>
			<td>28+49</td>
			<td>Cauda</td>
			<td>Mature sperm</td>
			<td>Stem cel</td>
			<td>Stem cell</td>
		</tr>
		<tr>
			<td></td>
			<td>Tubules</td>
			<td>Stem cell 
			Spermatogonia 
			Spermatocyte 
			Spermatid</td>
			<td>Stem cell</td>
			<td>Stem cell</td>
		</tr>
	</tbody>
</table>
Table 1. Cell types and phases of spermatogenesis that are targeted by the transgenic rodent mutation assay by various experimental designs.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Dose group</td>
			<td>Animal #</td>
			<td>Mutant PFU</td>
			<td>Total PFU</td>
			<td>MF (x 10-5)</td>
			<td>Avg MF (x 10-5)</td>
			<td>Fold change</td>
			<td>p-value</td>
		</tr>
		<tr>
			<td>Control</td>
			<td>1</td>
			<td>5</td>
			<td>180245</td>
			<td>2.8</td>
			<td>2.6</td>
			<td>1.0</td>
			<td>-</td>
		</tr>
		<tr>
			<td></td>
			<td>2</td>
			<td>4</td>
			<td>137835</td>
			<td>2.9</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>3</td>
			<td>11</td>
			<td>385672</td>
			<td>2.9</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>4</td>
			<td>2</td>
			<td>431396</td>
			<td>0.5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>5</td>
			<td>6</td>
			<td>152413</td>
			<td>3.9</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25 mg/kg</td>
			<td>6</td>
			<td>17</td>
			<td>162353</td>
			<td>10.5</td>
			<td>6.9</td>
			<td>2.6</td>
			<td>0.0002</td>
		</tr>
		<tr>
			<td></td>
			<td>7</td>
			<td>14</td>
			<td>150094</td>
			<td>9.3</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>8</td>
			<td>4</td>
			<td>154401</td>
			<td>2.6</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>9</td>
			<td>9</td>
			<td>154401</td>
			<td>5.8</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>10</td>
			<td>12</td>
			<td>196978</td>
			<td>6.1</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 mg/kg</td>
			<td>11</td>
			<td>17</td>
			<td>155727</td>
			<td>10.9</td>
			<td>9.3</td>
			<td>3.6</td>
			<td>&#60;0.0001</td>
		</tr>
		<tr>
			<td></td>
			<td>12</td>
			<td>11</td>
			<td>135847</td>
			<td>8.1</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>13</td>
			<td>25</td>
			<td>193499</td>
			<td>12.9</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>14</td>
			<td>12</td>
			<td>133859</td>
			<td>9.0</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>15</td>
			<td>14</td>
			<td>252807</td>
			<td>5.5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mg/kg</td>
			<td>16</td>
			<td>26</td>
			<td>170968</td>
			<td>15.2</td>
			<td>11.5</td>
			<td>4.4</td>
			<td>&#60;0.0001</td>
		</tr>
		<tr>
			<td></td>
			<td>17</td>
			<td>28</td>
			<td>234584</td>
			<td>11.9</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>18</td>
			<td>10</td>
			<td>145289</td>
			<td>6.9</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>19</td>
			<td>35</td>
			<td>292236</td>
			<td>12.0</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>20</td>
			<td>22</td>
			<td>190848</td>
			<td>11.5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2. The lacZ mutant frequency in cauda sperm of transgenic male mice exposed acutely to ENU when they were spermatogonial stem cells (administration time = 1 day, sampling time= 70 days). PFU = plaque forming unit, MF = mutant frequency.
<img alt="Figure 1" fo:content-width="6in" width="600" src="/files/ftp_upload/51576/51576fig1highres.jpg" /> 
Figure 1. A schematic representation of the &#955;gt10 phage construct.
<img alt="Figure 2" fo:content-width="6in" width="600" src="/files/ftp_upload/51576/51576fig2highres.jpg" /> 
Figure 2. An outline of the transgenic rodent germ cell mutation assay.
<img alt="Figure 3" fo:content-width="6in" width="600" src="/files/ftp_upload/51576/51576fig3highres.jpg" /> 
Figure 3. A schematic diagram of spermatogenesis in the mouse and the cell types and phases that are targeted by the transgenic rodent mutation assay by various experimental designs. NOTE: The graduated white bars represent the relative proportion of cell types present in cell suspensions prepared from the seminiferous tubules (i.e. spermatids &#62; spermatocytes &#62; spermatogonia &#62; stem cells). <a href="https://www.jove.com/files/ftp_upload/51576/51576fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" fo:content-width="6in" width="600" src="/files/ftp_upload/51576/51576fig4highres.jpg" /> 
Figure 4. Collection of the mouse cauda epididymis. A) Make an incision in the abdomen towards the scrotum. B) Identify the epididymal fat pad. C) Gently pull the fat pad to draw out the testes and epididymis. D) Locate and excise the cauda epididymis.
<img alt="Figure 5" fo:content-width="6in" width="600" src="/files/ftp_upload/51576/51576fig5highres.jpg" /> 
Figure 5. Preparation of suspension of germ cells from the seminiferous tubules. A) An incision is made in the epithelial capsule of the testis, exposing the seminiferous tubules. B) The seminiferous tubules are squeezed out of the capsule. C) A tissue roller is gently passed over the tubules at a 5-10&#176; angle to release the germ cells contained within. D) The germ cell suspension is collected for further processing.
<img alt="Figure 6" fo:content-width="6in" width="600" src="/files/ftp_upload/51576/51576fig6highres.jpg" /> 
Figure 6. Graphical representation of lacZ mutant frequency in MutaMouse sperm exposed acutely as stem cells to ENU (n = 5). Each triangle represents the MF of one animal.* p &#60;0.05 as determined by Poisson regression.
<img alt="Figure 7" fo:content-width="6in" width="600" src="/files/ftp_upload/51576/51576fig7highres.jpg" /> 
Figure 7. Representative agar plates from the transgenic rodent mutation assay with plaques formed on a lawn of E. coli host bacteria. A) The &#8220;mutant&#8221; plates will have very few plaques (marked with arrows), especially in the control dose groups, which will occasionally have zero plaques on some plates. B) The &#8220;titer&#8221; plates can have hundreds of plaques.

Watch this Scientific Journal Video about Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency at JoVE.com

Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency

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 ...

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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.

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as tumor-propagating cells, or tumor-initiating cells. It is thought that targeting these self-renewing cells for destruction will block tumor progression and stop relapse, greatly improving patient prognosis1. The most common way to determine the frequency of self-renewing cells within a tumor is a limiting dilution cell transplantation assay, in which tumor cells are transplanted into recipient animals at increasing doses; the proportion of animals that develop tumors is used the calculate the number of self-renewing cells within the original tumor sample2, 3. Ideally, a large number of animals would be used in each limiting dilution experiment to accurately determine the frequency of tumor-propagating cells. However, large scale experiments involving mice are costly, and most limiting dilution assays use only 10-15 mice per experiment. 
Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease4. While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression5. The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies 6-8. Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency9, 10. Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish8 and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency.
Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish.

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as ...

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies showed that microinjection of germ cells from fertile donor mice into the seminiferous tubules of infertile recipient mice results in donor-derived spermatogenesis and sperm production by the recipient animal2. The use of donor males carrying the bacterial &beta;-galactosidase gene allowed identification of donor-derived spermatogenesis and transmission of the donor haplotype to the offspring by recipient animals1. Surprisingly, after transplantation into the lumen of the seminiferous tubules, transplanted germ cells were able to move from the luminal compartment to the basement membrane where spermatogonia are located3. It is generally accepted that only SSCs are able to colonize the niche and re-establish spermatogenesis in the recipient testis. Therefore, germ cell transplantation provides a functional approach to study the stem cell niche in the testis and to characterize putative spermatogonial stem cells. To date, germ cell transplantation is used to elucidate basic stem cell biology, to produce transgenic animals through genetic manipulation of germ cells prior to transplantation4,5, to study Sertoli cell-germ cell interaction6,7, SSC homing and colonization3,8, as well as SSC self-renewal and differentiation9,10.
Germ cell transplantation is also feasible in large species11. In these, the main applications are preservation of fertility, dissemination of elite genetics in animal populations, and generation of transgenic animals as the study of spermatogenesis and SSC biology with this technique is logistically more difficult and expensive than in rodents. Transplantation of germ cells from large species into the seminiferous tubules of mice results in colonization of donor cells and spermatogonial expansion, but not in their full differentiation presumably due to incompatibility of the recipient somatic cell compartment with the germ cells from phylogenetically distant species12. An alternative approach is transplantation of germ cells from large species together with their surrounding somatic compartment. We first reported in 2002, that small fragments of testis tissue from immature males transplanted under the dorsal skin of immunodeficient mice are able to survive and undergo full development with the production of fertilization competent sperm13. Since then testis tissue xenografting has been shown to be successful in many species and emerged as a valuable alternative to study testis development and spermatogenesis of large animals in mice14.

Protocols for germ cell transplantation and testis tissue xenografting are described. Germ cell transplantation results in donor-derived spermatogenesis in recipient testes and represents a functional reconstitution assay for identification of spermatogonial stem cells (SSCs). Testis tissue xenografting reproduces testis development and spermatogenesis of various donor species in recipient mice.

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies ...

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell migration, gene expression, cell cycle progression and cell adhesion 1. Rho proteins are molecular switches that are active in GTP-bound and inactive in GDP-bound state. Their activation is mediated by a family of Guanine-nucleotide Exchange Factor (GEF) proteins. Rho-GEFs constitute a large family, with overlapping specificities 2. Although a lot of progress has been made in identifying the GEFs activated by specific signals, there are still many questions remaining regarding the pathway-specific regulation of these proteins. The number of Rho-GEFs exceeds 70, and each cell expresses more than one GEF protein. In addition, many of these proteins activate not only Rho, but other members of the family, contributing further to the complexity of the regulatory networks. Importantly, exploring how GEFs are regulated requires a method to follow the active pool of individual GEFs in cells activated by different stimuli. Here we provide a step-by-step protocol for a method used to assess and quantify the available active Rho-specific GEFs using an affinity precipitation assay. This assay was developed a few years ago in the Burridge lab 3,4 and we have used it in kidney tubular cell lines 5,6,7. The assay takes advantage of a "nucleotide free" mutant RhoA, with a high affinity for active GEFs. The mutation (G17A) renders the protein unable to bind GDP or GTP and this state mimics the intermediate state that is bound to the GEF. A GST-tagged version of this mutant protein is expressed and purified from E. coli, bound to glutathione sepharose beads and used to precipitate active GEFs from lysates of untreated and stimulated cells. As most GEFs are activated via posttranslational modifications or release from inhibitory bindings, their active state is preserved in cell lysates, and they can be detected by this assay8. Captured proteins can be probed for known GEFs by detection with specific antibodies using Western blotting, or analyzed by Mass Spectrometry to identify unknown GEFs activated by certain stimuli.

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein GST-RhoAG17A from Epithelial Cell Lysates

The method presented here describes an assay to follow activation of RhoA specific GDP/GTP Exchange Factors (GEFs) in cultured cells by making use of a mutant RhoA GST fusion protein that has high affinity for activated GEFs. GEFs are precipitated from cell lysates, detected by Western blotting and quantified by densitometry.

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell ...

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes. Currently, there is no reliable cell culture system allowing for spermatogenic differentiation in vitro, and most biological studies of spermatogenic cells require tissue harvest from animal models like the mouse and rat. Because the testis contains numerous cell types - both non-spermatogenic (Leydig, Sertoli, myeloid, and epithelial cells) and spermatogenic (spermatogonia, spermatocytes, round spermatids, condensing spermatids and spermatozoa) - studies of the biological mechanisms involved in spermatogenesis require the isolation and enrichment of these different cell types. The STA-PUT method allows for the separation of a heterogeneous population of cells - in this case, from the testes - through a linear BSA gradient. Individual cell types sediment with different sedimentation velocity according to cell size, and fractions enriched for different cell types can be collected and utilized in further analyses. While the STA-PUT method does not result in highly pure fractions of cell types, e.g. as can be obtained with certain cell sorting methods, it does provide a much higher yield of total cells in each fraction (~1 x 108 cells/spermatogenic cell type from a starting population of 7-8 x 108 cells). This high yield method requires only specialized glassware and can be performed in any cold room or large refrigerator, making it an ideal method for labs that have limited access to specialized equipment like a fluorescence activated cell sorter (FACS) or elutriator.

The STA-PUT method allows for the separation of different populations of spermatogenic cells based on size and density.

Mammalian spermatogenesis is a complex  differentiation process that occurs in several stages in the seminiferous  tubules of the testes. Currently, there ...

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin&#8212;the histone-to-protamine switch.
Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.
The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes ...

Generation of Transgenic Hydra by Embryo Microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

Generation of Transgenic Hydra by Embryo Microinjection

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among ...

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and ...

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm ...